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Revisiting the theory of auditory displays based on the virtual sphere model : Virtual Auditory Displays (VADs) are used to present realistic spatial sound. High-quality VADs must account for three factors: individuality (Head-Related Transfer Function), room acoustics (Room Transfer Function) and freedom of motion (active listening). The Auditory Display based on the VIrtual SpherE model (ADVISE) was proposed to simplify the problem by dividing it, through the Kirchhoff-Helmholtz integral theorem, into 1) a listener-free room acoustics simulation and 2) a free-field VAD using HRTFs. Users of ADVISE can move freely within the free-field region, thus accounting for active listening. This paper revisits the classic theory of ADVISE and identifies three oversights in the original proposal: 1) The ADVISE formulation suffers from non-unique boundary conditions at some frequencies. 2) The original proposal re-creates a set of boundary conditions using secondary sources that diverge on the boundary itself. 3) Considerations for sound propagation are absent in the original formulation. Two new formulations that retain the philosophy of ADVISE but are free from these problems are presented. The first one is based on the theory of Boundary Matching Filters, while the second is inspired by High-Order Ambisonics. The latter is found to be better suited for applications where freedom of motion is important since the presented sound field can be shifted by a translation matrix. INTRODUCTION The demand for realistic spatial sound has increased with the advent of virtual reality applications. Virtual auditory displays (VADs), and in particular headphonebased solutions, play an important role in conveying a natural and | 3,068,700 | 213544617 | 0 | 16 |
believable experience which allows the users of modern VR systems to become immersed in the presented environment. To achieve their goal, VADs often model the acoustic characteristics of the path between a given sound source and the listener's ears as a head-related transfer function (HRTF) [1]. The HRTF can realistically convey the illusion of sound arriving from a given direction; however, it varies between individuals and thus requires some degree of personalization. A limitation of the HRTF is that, while it accounts for the acoustic effects due to the listener's body, it does not take into account the properties of the environment. Sound reflected from walls or obstructed by objects can drastically alter how we listen to an environment. The accurate recreation of these effects is fundamental to achieve a believable experience leading to a high sense of presence. It is common to model environmental characteristics as a room transfer function (RTF) or, in combination with the HRTF, as a binaural room transfer function (BRTF). Unfortunately, the RTF and BRTF vary widely for different positions in a given environment. Simple VADs may combine a single RTF with a set of HRTFs for multiple directions. This allows for the accurate presentation of direct sounds with the addition of coarse room reverberation effects. Modern virtual reality applications, however, give the users a high level of freedom to move around and explore the presented environment. This freedom of motion can considerably enhance the spatial perception of sound [2,3] if the VAD can render consistent auditory cues. Summarizing, a realistic | 3,068,701 | 213544617 | 0 | 16 |
VAD must account for three fundamental factors: (1) the acoustic effects due to the listener's body, head and pinnae encoded in the HRTF, (2) the effects that reflecting surfaces and obstacles present in the environment have on sound propagation, modeled by a RTF and (3) the ability of the listener to move around and explore the environment resulting in an accurate active listening experience. In theory, if the geometries and acoustic properties of the environment and listener's body are known, it is possible to calculate the sound pressure at the listener's ears due to an arbitrary sound source. Unfortunately this requires an impractically large computation making it unsuitable for real-time applications such as VR, where freedom of motion and interactivity are essential. The problem can be divided into two simpler ones by making use of the Kirchhoff-Helmholtz integral theorem [4]. This observation led to the proposal of the Auditory Display based on the VIrtual SpherE model (ADVISE) [5,6]. The fundamental idea of ADVISE involves defining a spherical boundary that separates the listener from the environment (i.e. sound sources, walls and other objects that may affect sound propagation) as seen in Fig. 1. This way, the room acoustic characteristics can be calculated up to a fixed spherical boundary. Sound propagation inside the sphere obeys simple free-field conditions and, once the listener is introduced, are fully accounted for by a set of HRTFs covering all directions. The HRTFs can be swapped in real-time as the listener moves within the sphere, eliminating the need to update the room simulation | 3,068,702 | 213544617 | 0 | 16 |
part. This paper revisits the theory of ADVISE as proposed in [5,6]. Section 2 summarizes the original formulation of ADVISE. While the idea of separating the problem through a boundary as described above is effective, some oversights are identified in Sect. 3. Finally, Sect. 4 applies the theory of acoustic holography, and in particular high-order Ambisonics (HOA), to overcome these issues. AUDITORY DISPLAY BASED ON THE VIRTUAL SPHERE MODEL The Auditory Display based on the VIrtual SpherE model (ADVISE) seeks to present realistic spatial sound including room effects to a potentially moving listener. This requires real-time computation of the sound pressure at the listener's ears due to any number of sound sources, their reflections and scattering throughout the environment and over the listener's body, head and pinnae. To reduce computational costs, ADVISE introduces a spherical boundary which separates a free-field region, for the listener to move freely, from the environment. A room acoustics model can be solved outside the boundary through numerical methods such as finite-difference timedomain method (FDTD). This is computationally expensive, but only needs to be done once as long as the environment and the position of the boundary remain fixed. Free-field conditions inside the boundary make binaural rendering using a collection of HRTFs possible. These can be swapped easily in real-time as the listener moves within the sphere, providing accurate binaural signals for an active listening experience. The task of ADVISE is to match these two solutions at the boundary. The original formulation of ADVISE proposes to treat the sphere as the boundary | 3,068,703 | 213544617 | 0 | 16 |
in the Kirchhoff-Helmholtz integral theorem. The theorem states that the sound pressure inside a bounded region is fully determined by the pressure and its derivative at the boundary [4]: Here, the sound pressure p at a position r inside the boundary À is given for angular frequency ! as a surface integral. Position vectors r lie on the boundary. The derivatives are taken with respect to the boundary surface's normal. The Green's functions G model sound propagation in the free-field and correspond to monopoles: We use the timelike sign convention so that solutions to the wave equation depend on þct À r. Numerical implementations require discretization of the boundary À in order to replace the integral of Eq. (1) by a sum. In addition to this, the original ADVISE proposal propagation from the sound source, throughout the room and up to the listener's ears is modeled in two steps by introducing a spherical boundary. Room acoustic effects need to be calculated only outside of this boundary. On the other hand, free-field HRTFs are enough to render spatial sound anywhere inside the sphere. This allows the user to move around while retaining a stable spatial sound presentation without the need to continuously update the room acoustics model. relies on a first-order difference approximation to the normal derivatives. The approach is shown in Fig. 2 and summarized by the following equation: The boundary is sampled at N points À i with quadratures ÁÀ i . The normal derivatives are approximated as the firstorder difference between points r þ | 3,068,704 | 213544617 | 0 | 16 |
i and r À i , respectively outside and inside the boundary and separated by distance i . Binaural rendering from the formulation of Eq. (3) is achieved by multiplying the three Green's functions at r i , r þ i and r À i by the corresponding HRTFs. The original ADVISE proposal does not detail any conditions for the sampling À i ; it simply takes the sound pressure calculated by the room acoustics model at the positions required by Eq. (3) and renders it binaurally. In this sense, it does not involve an inverse acoustic problem and is similar to some early attempts at reproducing sound fields by directly feeding microphone recordings into loudspeaker arrays [7]. PROBLEMS WITH THE ORIGINAL FORMULATION OF ADVISE At first glance, ADVISE seems to deliver correct results as long as the boundary is sampled uniformly and the distance i used to approximate the normal derivatives is properly chosen. An example of a plane wave being recreated inside the spherical boundary is shown in Fig. 3. There are, however, some problems with the formulation detailed in Sect. 2. The first problem that Eq. (3) does not consider is related to the use of a spherical boundary to approximating the Kirchhoff-Helmholtz integral. This is known to lead to non-unique boundary conditions at specific wavenumbers kr ¼ m% with m being any integer [8]. This problem is present also in open microphone array recordings, such as those used in Boundary Surface Control (BoSC) [9]. A typical solution in sound field recording applications | 3,068,705 | 213544617 | 0 | 16 |
is to use a rigid baffle, as done in the SENZI binaural recording system [10] or to take multiple measurements with spheres of varying radii [11]. However, it is not immediately evident how could a rigid baffle or multiple concentric spheres may fit in the formulation of ADVISE. Another problem is that, through Eq. (3), ADVISE attempts to re-create a finite sound pressure distribution over À using point sources located directly over it. Equation (2) shows that a collection of point sources on the boundary surface will instead result in an arbitrarily large sound pressure. This can be seen indirectly as the abrupt change in sound pressure that Fig. 3 exhibits at the boundary. A final issue arises from how Eq. (3) in combination with the HRTF will simply render sound pressure measurements as virtual point sources. This approach disregards sound propagation since the effects that a point source at r i have on other sampling positions r j are ignored. The consequence of this is shown in Fig. 4, where the sound intensity vectors for a point source and the result of rendering by ADVISE differ substantially. APPLICATION OF ACOUSTIC HOLOGRAPHY TO ADVISE The theory of acoustic holography can be used to Fig. 2 The numerical implementation of ADVISE requires sampling the boundary surface and approximating the normal derivatives using a first-order difference. Fig. 3 Sound pressure field for a plane wave reproduced inside the ADVISE boundary. Computer simulations with properly tuned parameters appear to yield very high quality results at first glance. overcome the | 3,068,706 | 213544617 | 0 | 16 |
problems identified in Sect. 3. To this end, we go back to the basic task of matching two sound field models at a boundary surface. Sound field simulations in this Section are for a monopole source radiating at a single frequency of 1 kHz. The source is located 1.5 meters away from the origin at an azimuth angle of 60 degrees. The virtual sphere boundary is set at a radius of 1 meter and sampled at the 252 directions used in the SENZI binaural recording system [10]. Boundary Matching Filters An immediate solution is the use of the theory of boundary matching filters (BMFs) [12]. These are filters that transform sound pressure recordings on a boundary to single layer potentials on another. To avoid non-uniqueness of the boundary conditions, it is convenient to assume an acoustically rigid surface; this conditions are illustrated in Fig. 5. The choice calls for the use of rigid to open BMFs. The full procedure, described in [12], involves (1) the spherical harmonic decomposition of the measurements on the rigid sphere, (2) filtering each order's components using the respective BMF and (3) evaluation of the reproduction signals from the filtered spherical harmonic components. The use of BMFs can eliminate all of the problems identified in Sect. 3. The rigid baffle prevents non-unique boundary conditions, the monopoles used to reproduce the recorded field do not lie on the recording surface and sound propagation is fully considered in the formulation of these filters. Figure 6 shows the performance of a BMF-based version of ADVISE | 3,068,707 | 213544617 | 0 | 16 |
when reproducing a point source with the same parameters as the example of Fig. 4. The sound intensity vectors in the BMF-based revision of ADVISE show a maximum deviation from their ideal direction of under 4 degrees within 20 cm of the origin. Meanwhile, the error in their magnitude across the same region remained under À14 dB. Sound pressure is only accurate close to the center of the reproduction surface, as opposed to the original ADVISE formulation; however, within this region, the ideal and reproduced sound intensity vectors are correctly aligned. High-order Ambisonics One problem with using BMFs is the need for recordings on a rigid sphere. Since the sphere must be included in the room acoustics computation, its numerical complexity increases. The rigid sphere was introduced to remove nonunique boundary conditions. An alternative is to take sound field measurements at multiple concentric spheres [11]. However, the BMFs are not designed to match multiple boundaries into a single one. An alternative is to use high-order Ambisonics (HOA) [13] to reproduce the recorded sound field. In this approach, multiple layer recordings are integrated into a single spherical harmonic representation which can then be decoded for a virtual loudspeaker array. The spherical harmonic coefficients for an arbitrary sound field can be derived from free-field sound pressure measurements on a spherical boundary of radius a using the formula [13]: Here, k ¼ !=c stands for the wavenumber. The integral covers all solid angles. Functions j n and Y nm are, respectively, the spherical Bessel and spherical harmonic functions of | 3,068,708 | 213544617 | 0 | 16 |
order n and degree m. The division by the spherical Bessel functions in Eq. (4) may be undefined at certain frequencies and orders for which the functions are zero. This is similar to the issue identified in Sect. 3 when using an open recording boundary. However, this can be avoided by choosing the radius a appropriately so as to avoid the zeros. To avoid problems of numerical precision, it is possible to choose a for each wavenumber k and order n so as to maximize j n ðkaÞ. The results of this approach are shown in Fig. 7. The sound intensity vectors in the HOA-based revision of ADVISE show a maximum deviation from their ideal direction of under 0.1 degrees within 40 cm of the origin. Meanwhile, the error in their magnitude across the same region remained under 30 dB. The region over which sound pressure is accurately reproduced is larger than that of the BMF-based approach, but still narrower than the original ADVISE implementation. Nevertheless, sound intensity vectors in the reproduced field are an almost perfect match to the ideal intensity vectors for the target sound source. There is another advantage to the HOA-based approach. The coefficients obtained from Eq. (4) can be shifted using a translation matrix for spherical harmonic expansion coefficients [14]. When the listener moves in some direction, rather than updating the HRTFs used to render the binaural signals, it is possible to shift the sound field in the opposite direction. This way, the reproduction boundary can remain fixed, reducing the need | 3,068,709 | 213544617 | 0 | 16 |
to measure or compute a large number of HRTFs. SUMMARY AND CONCLUSIONS The present paper reviews the theory behind auditory displays based on the virtual sphere model. The original idea of joining a room acoustics model and a set of freefield HRTFs through a virtual boundary surface is a promising approach to rendering realistic spatial sound in real-time. However, the original implementation of ADVISE is not suitable for correct binaural presentation since it does not account for sound propagation between measurement and reproduction points. Two alternative ADVISE implementations, inspired by acoustic holography were introduced. One relies on boundary matching filters and requires the inclusion of a rigid spherical boundary in the room acoustics simulation. The second proposal, based on high-order Ambisonics, does not have this requirement. Both proposed approaches result in accurate sound pressure reproduction over a region that is narrower compared to the original ADVISE implementation; however, both present accurate sound intensity vectors pointing in the direction of intended sound propagation. The HOA approach seems to be the most promising as it results in larger listening regions. Further, it simplifies the task of updating the binaural signals to match listener movement. | 3,068,710 | 213544617 | 0 | 16 |
A short-term non-randomized controlled study of ultrasound-guided microwave ablation and parathyroidectomy for secondary hyperparathyroidism Abstract Background To compare the short-term clinical outcomes of ultrasound-guided microwave ablation (MWA) and parathyroidectomy (PTX) for severe secondary hyperparathyroidism(SHPT). Methods In a prospective multi-center study, we compared the outcomes of MWA and PTX for severe SHPT. The outcome measures were case rate of successful treatment, improvement of clinical symptoms, incidence of complications, and differences in treatment parameters and costs between the two groups. Results A total of 167 eligible patients were included in the study, of which 79 underwent MWA and 88 underwent PTX. There was no significant difference in rate of successful treatment between the MWA and PTX groups (χ2=2.299, p = 0.125). However, the MWA group showed significantly lower range of intact parathyroid hormone (iPTH) decrease than the PTX group (t=−2.352, p = 0.023). Postoperative clinical symptoms improved in both groups, with no significant difference between the two groups (p > 0.05). Postoperative hypocalcemia was significantly more common in the PTX group (p < 0.05). The operative time, incision and postoperative pain of the MWA group were significantly better than those of the PTX group (p < 0.05), while postoperative recurrent laryngeal nerve injury and hematoma showed no significant difference between the two groups (p > 0.05). The cost of MWA was significantly less than PTX (p = 0.000). Conclusions Both MWA and PTX are effective and safe for severe secondary hyperparathyroidism. PTX is more thorough and traumatic, while MWA is minimally invasive and postoperative iPTH is more consistent | 3,068,711 | 240421846 | 0 | 16 |
with the Kidney Disease: Improving Global Outcomes (KDIGO) recommendation. Introduction End-stage renal disease (ESRD) is a global health problem, with the annual incidence rate of 1425 per million people [1], and the prevalence rate is steadily increasing [2,3]. Secondary hyperparathyroidism (SHPT) is a common and serious complication of dialysis patients with chronic renal failure (CRF) [4,5], which can lead to multiple organ system involvement and death [6]. Surgical treatment is necessary for patients with intractable hyperparathyroidism, and the reported surgical resection rate was 7-8 per 1000 personyears, which has remained unchanged for many years [7]. In 2018, there were about 520,000 renal dialysis patients in China [8]. About 60% of maintenance dialysis patients had different degrees of SHPT, and the compliance rate of intact parathyroid hormone (iPTH) control in these patients was only about 55%. Renal failure and its associated complications resulted in poor physical condition, such as cardiopulmonary dysfunction, and the patient's tolerance to surgery was poor, so the surgical rate of the remaining patients was only 2% (Dialysis Outcomes and Practice Patterns Study, DOPPS). Thermal ablation therapy has become a research hotspot since it is minimally invasive and has short operation time (more than 10 min). The preliminary results showed that microwave ablation (MWA) of SHPT has a significant effect [9]. This study aimed to compare the outcomes of the two methods through a multi-center, prospective cohort study. Materials and methods The authors had complete control over the data and methods of the study. This prospective trial was approved by the ethics committee of | 3,068,712 | 240421846 | 0 | 16 |
the Beijing Friendship Hospital and registered at www.chictr.org.cn (ChiCTR1800018156) prior to patient recruitment. All patients provided signed informed consent before the study. Participants The patients presenting with SHPT to the Department of interventional ultrasound and surgical ward at the Beijing Friendship Hospital, China-Japan Friendship Hospital and People's Liberation Army General Hospital between June 2018 and May 2020 were enrolled in this study. Inclusion and exclusion criteria The inclusion criteria according to the consensus of clinical practice experts in surgical treatment of SHPT to CRF [5] were: 1. iPTH >800 pg/ml; 2. Clinical manifestations: severe osteodynia, itchy skin and other symptoms affecting the quality of life, such as severe anemia and persistence of erythropoietin resistance; 3. Patients who met the diagnosis of SHPT, were resistant to calcium-sensitive receptor agonists, vitamin D and its analogues, hypercalcemia (serum calcium >4 mmol/L) or hyperphosphatemia (serum phosphorus >1.94 mmol/L); 4. Ultrasound indicated that at least one or more parathyroid glands had hyperplasia and diameter >1 cm, with abundant blood flow. The exclusion criteria were: 1. Severe skeletal deformity and osteoporosis; 2. Combined with systemic diseases such as severe cardiopulmonary and brain dysfunction; 3. Severe coagulation dysfunction; 4. Ultrasound or radionuclide scans suggested ectopic parathyroid glands behind the sternum. 5. Patients with hyperplasia of parathyroids less than 4. All patients first met with the clinic general surgeon and ablation practitioner, and the patients who met the inclusion criteria were divided into the ablation group and surgery group based on their choice. Data extraction and quality assessment The following data were extracted: efficacy, | 3,068,713 | 240421846 | 0 | 16 |
adverse events/ complications, cost analysis, and social experience. Efficacy included changes in iPTH, alkaline phosphatase (ALP), serum calcium (Ca), phosphorous (P) and clinical symptoms after treatment. According to expert consensus on surgical practice of SHPT to CRF [5], postoperative iPTH levels <80% before surgery are considered to be completely removed. As a reference, 80% reduction of iPTH after ablation is considered complete ablation, less than 80% and greater than 50%, considered effective ablation. Evaluation scales such as visual analogue scale (VAS) for pain (ranging from 1 to 10) [10], Athens Insomnia Scale (AIS) (ranging from 0 to 24) [11], Sinicization of Four-item Itch Questionnaire (FIIQ) (ranging from 3 to 19) [12], Quality of life VAS (that includes 12 items related to physical health, economic status, psychological status, etc.) were used to evaluate the clinical symptoms and social experience. Before the start of the project, three centers jointly studied and determined the Case Report Form (CRF) tables and standard data determination tables. Data were independently extracted by one investigator at each center using standard data extraction forms. The lead center researcher managed and analyzed the data in a unified way. Cost analysis included the procedure (ablation or surgery) as well as pre-procedural and post-procedural exams. Direct costs were related to hospitalization for MWA or surgery plus any costs associated with adverse events. Pricing was obtained from the hospital database. Indirect costs were evaluated using the human capital approach according to the Chinese guidelines for cost-effectiveness studies, and using the standard formula as follows: Indirect costs ¼ Gross | 3,068,714 | 240421846 | 0 | 16 |
Domestic Product (GDP) per capita  day  productive weight Where productive weight is based on the internationally weighted value [13]. The value of GDP per capita was based on the Beijing, China regional value estimate of Yuan 292 per day. Pretreatment evaluation Pre-operative procedures included recording of disease history focusing on history of kidney disease and dialysis, symptoms. Laboratory tests of iPTH, ALP, Ca and P concentrations were performed. Routine blood work and clotting tests were also verified to be normal. Parathyroid (Figures 1(A,B) and 2A) dimensions were measured with an ultrasound machine (Ascendus Color Doppler Ultrasound Diagnostic, Hitachi Medical Systems, Tokyo, Japan) using a 5-18 MHz linear matrix transducer to measure the length and diameter of the parathyroid. Radionuclide imaging (99mTc-MIBI; Figure 1(C)) was used to confirm the number of parathyroid. Video laryngoscopy was used to check the vocal cords to judge the function of recurrent laryngeal nerve. MWA procedure MWA was performed in an outpatient setting by an experienced physician, as previously reported [9]. Microwave therapeutic equipment (KY-2000; Kangyou Medical, Nanjing, China) was used to administer microwave energy at a frequency of 2450 MHz. The microwave generator is capable of producing up to 100 W of power (30 W power was used in the study), with an antenna diameter of 2 mm, a total length of 100 mm and transmit segment length of 5 mm. Before ablation, intravenous access was achieved via the antecubital vein, and the patient's partial pressure of oxygen (PO 2 ), heart rhythm, respiratory rate and blood pressure were | 3,068,715 | 240421846 | 0 | 16 |
continuously monitored. SonoVue (Bracco, Italy) was used as the ultrasound contrast agent. The parathyroid blood supply was confirmed with contrast-enhanced ultrasound (CEUS) ( Figure 2(C)) and the ablation pathway was decided. Patients were placed in the supine position with their necks fully exposed. Routine neck disinfection was performed and 2% lidocaine was used for topical anesthesia. Key aspects of MWA technique included the following [1]: To protect the carotid artery, recurrent nerve and other adjacent structures, a liquid insulation zone was used as a hydrodissection technique. Specifically, physiological saline refrigerated at 4-7 C was injected into the area around the parathyroid as an isolation fluid to create a heat insulation layer (Figure 3(A)), so that the parathyroid glands exist as "an island" (Figure 3(B)). During ablation, the isolation liquid was continuously injected to ensure that the width of the isolation band was >1 cm, and the low-temperature isolation liquid could offset the diffusion of the ablation heat and ensure the safety of ablation [2]. After creating the isolation zone, ablation process was started (Figure 2(B)). Mobile ablation method was used. The ablation was conducted from deep to shallow, and from far to near. The ablation needle tip began to move when hyperechoic bubbles appeared. When the ablation bubble high echo completely covered the parathyroid gland, the ablation was terminated. The patient's voice was monitored during the operation, and the procedure was halted if dysphonia occurred [3]. CEUS was performed, and if there was no enhancement of the entire parathyroid gland ( Figure 2(D)), the procedure was | 3,068,716 | 240421846 | 0 | 16 |
discontinued. Surgical procedures Three surgical options are available for parathyroidectomy as follows: subtotal parathyroidectomy (sPTX), total parathyroidectomy with autotransplantation (tPTX þ AT), and total parathyroidectomy (tPTX). Currently, there is no consensus among endocrine surgeons on which operation is superior [14,15]. tPTX þ AT involves removing all parathyroid tissue and re-implanting small pieces of tissue at a distance in wellvascularized muscular structures [16,17], which was performed in this study. The surgeries were performed under general anesthesia. As a general principle, we constantly dissected and protected the recurrent laryngeal nerves in the procedures. If four parathyroid glands were definitively identified, the thymectomy was not performed. The parathyroid tissue was completely removed and non-nodular parathyroid tissues 30-60 mg (cut into 1 mm 3 ) were implanted in 3-4 "muscle sacs" of sternocleidomastoid muscle that did not have hematoma. The transplantation site was marked with metal clip or other non-absorbable materials. All parathyroid glands were weighed, measured, and sent for pathological testing. Follow-up evaluation Laboratory tests of iPTH, ALP, Ca and P were performed at 2 h, 1 week, 1 month, and 3 months after the operation. Clinical symptoms, such as bone pain and itching, were reassessed at 2 h, 1 week, 1 month and 3 months, and insomnia and social experience that improved slowly were reassessed at 3 months after treatment. Each complication was recorded during and after treatment. Statistical analysis Data were analyzed using Excel for Windows, and statistical analysis was performed using SPSS version 22. Continuous variables were presented as mean ± SD or median (interquartile range), | 3,068,717 | 240421846 | 0 | 16 |
and categorical variables were presented as frequencies. The Student's t-test (for normal distribution data) or Mann-Whitney U test (for skewed data) was used to compare numerical data, and chi-square analysis was used to compare categorical data. A p-value <0.05 was considered statistically significant, unless stated otherwise. Data were expressed as mean ± SEM for all continuous variables. Clinical data A total of 167 eligible patients were included in the study, of which 79 underwent MWA and 88 underwent PTX. The study included 69 males and 98 females, with an age range of 25-79 years. The average period of hemodialysis was 7.92 years, with a range of 3.512 years. The operation was smoothly performed in all cases (Table 1). Efficacy comparison 1. The successful treatment cases in the MWA group was 86.08%, and that in the PTX group was 93.18%, with no statistical difference between the two groups (v 2 ¼2.299, p ¼ 0.125). However, the range of iPTH decline two hours after the operation in the MWA group was significantly lower than that in the PTX group (t¼À2.352, p ¼ 0.023) (Tables 2 and 3). 2. The symptoms of bone pain, itching and insomnia in the two groups were significantly improved after the operation, with no significant difference between the two groups (p > 0.05). Hypocalcemia There were 21 cases of postoperative hypocalcemia in the ablation group and 45 cases in the surgery group, and the difference was statistically significant (p < 0.01) ( Table 4). Recurrent laryngeal nerve palsy (RLNP) Regardless of TRLNP or PRLNP, | 3,068,718 | 240421846 | 0 | 16 |
there was no significant difference in RLNP between the ablation group and the surgery group (p > 0.05). Postoperative hematoma There was no case of postoperative bleeding in the ablation group, and five cases of postoperative hematoma in the surgical group, but the difference was not statistically significant (p > 0.05). Postoperative pain The ablation group had a surgical incision of about 0.2 cm and a pain score of about 1.8points (1 d after treatment), while the surgical group had a surgical incision of 5 cm and a pain score of 8.00, with a statistical difference (p < 0.01). Treatment procedures The duration of hospitalization and operation time of MWA group was shorter than that of the surgery group (p < 0.01). The postoperative scar length of the MWA group was smaller than that of the surgery group (p < 0.01). Patient's sense of social experience (Quality of life VAS) and treatment cost Postoperative quality of life VAS were significantly improved, with no statistical difference between the two groups (p > 0.05). However, there was significant difference in treatment costs between the two groups (p ¼ 0.001) ( Table 5). Discussion Refractory hyperparathyroidism seriously affects patients' quality of life and increases mortality [18]. Numerous studies have shown that hyperplastic parathyroidectomy can significantly improve clinical symptoms and reduce the risk of death [19,20], which was also recommended by KDIGO and Kidney Disease Outcomes Quality Initiative (KDOQI). But many dialysis patients suffer from cardiopulmonary dysfunction due to basic diseases, they cannot tolerate the trauma of surgery. Thermal ablation | 3,068,719 | 240421846 | 0 | 16 |
is widely used because of its advantages of less trauma, simple operation, and repeatability. Radiofrequency ablation, laser ablation and microwave ablation had been reported for the treatment of secondary hyperparathyroidism, and the efficacy were significant [9,21,22]. Our data also showed that MWA could reduce iPTH and improve clinical symptoms as effectively as PTX þ AT. Although the postoperative iPTH in MWA was higher than that in PTX, it met the KDIGO recommendation that the iPTH of CKD-5 patients should be maintained at 2-9 times the upper limit of normal iPTH (0-65 pg/ml) [23]. This was different from the surgery group wherein the parathyroid tissue was completely excised under direct vision, and the ablation was only to inactivate the parathyroid tissue. Although the CEUS showed no enhancement of the nodules, some residual nodules may have been present at the edges that could not be detected by ultrasound since the surrounding structures and organs could not be damaged. However, the research team found that with the proficiency and improvement of the ablation technology, the ablation effect was significantly improved compared to before, especially in the same institution [24,25]. KDOQI suggests not to reduce PTH to twice or less of the upper limit of iPTH detection (grade 2C), because over-inhibition of iPTH can lead to an increase in the prevalence of adynamic bone disease (ABD) [26]. Many patients with ABD are asymptomatic, but some patients can have bone pain [27], and these patients have an increased risk of fracture (possibly caused by the impairment of the ability to repair | 3,068,720 | 240421846 | 0 | 16 |
micro-injury), hypercalcemia and vascular calcification (visible on imaging), which are associated with increased mortality [28,29]. In this study, 80% of PTX cases were lower than the normal two-fold level, and SPTX was reported to have a high incidence of hypoparathyroidism [30,31], while some studies showed that the risk was the lowest when the PTH concentration was about 300 pg/ml [32]. Based on these results, ablation treatment was recommended. MWA and PTX are effective in the treatment of severe SHPT, but each operation has its complications or limitations. Hypocalcemia is a common complication of PTX and MWA. In patients with surgically removed or thermally ablated parathyroid glands, circulating iPTH suddenly drops, and the digestive tract shows insufficient calcium absorption, while the bones of SHPT patients are in the state of "bone hunger", so a large amount of calcium and phosphorus need to be absorbed from the blood for component compensation, resulting in the rapid transfer of a large amount of blood calcium to the previously decalcified bone, and the blood calcium level decreases rapidly. Our findings revealed that hypocalcemia occurred earlier and more seriously in the PTX group, especially in the older patients and those with higher iPTH before operation, and this phenomenon was related to the complete removal of parathyroid tissue by PTX. Hoarseness is another common complication of treatment. Temporary hoarseness was caused by the compression of the nerves by the isolation fluid or temporary paralysis of lidocaine in the ablation group, and intraoperative nerve pulling in the surgical group. Generally, no treatment was required | 3,068,721 | 240421846 | 0 | 16 |
and both groups recovered within three months. Permanent hoarseness was caused by negative nerve damage leading to more than one year of hoarseness due to heat damage to the recurrent laryngeal nerve in the ablation group, and unintended cutting of the nerve in the surgery group. Transient hoarseness is more common in the ablation group, because the recurrent laryngeal nerve cannot be identified by ultrasound, in order to clearly separate the nerve from the parathyroid gland. So when the isolation fluid is injected, the nerve is affected by compression and traction, and lidocaine will also flow into the interstitial anesthesia nerve. Therefore, there is transient hoarseness in many cases, but no substantial damage to the nerve. There was no difference in the incidence of hoarseness between the two groups in this study. Postoperative hemorrhage did not occur in the ablation group, while there were five cases in the surgery group, including two cases of hemostasis after compression, two cases of compression-induced hemostasis after suction under the guidance of ultrasound, one case continued to increase after aspiration and hemostasis was treated after surgical incision. The positive pain, numbness and appearance of the wound were much better in the ablation group than in the surgery group, and there was no psychological impact on the patients. Recurrent hyperparathyroidism after treatment was a serious complication related to clinical efficacy [33]. Given the short follow-up time in this study, no recurring cases were seen in the two groups, but we will continue to follow-up the patients. In terms of treatment costs, | 3,068,722 | 240421846 | 0 | 16 |
we found that the cost of MWA was less than that of surgical resection. Perhaps, the occasional medical expense had no significant impact on the quality of life for most families. However, for long-term dialysis patients, treatment costs accounted for a certain proportion of family income, and led to a heavy economic burden. In this study, there were 36 low-income patients (Ministry of Civil Affairs of the People's Republic of China 2019 standard, the monthly income per person in Beijing was less than 1,100 RMB, and in Hebei and other provinces it was about 600 yuan), of which 23 were in the ablation group, which indicated that the patients also considered economic costs in the selection of treatment. This study had some limitations. Although both methods were proven to be effective for severe SHPT, the study did not follow the randomization principle, which inevitably led to certain deviations in the results. In addition, the ablation technique has relatively high requirements for surgeons to master the operational skills, which limits its widespread use. Conclusion In summary, both MWA and PTX are effective and safe for SHPT. PTX can remove the parathyroid more thoroughly, but is more traumatic. MWA is less traumatic and postoperative IPTH is more consistent with the KDIGO recommendation, but cannot completely inactivate tissue. Based on the findings of this study, in the selection of treatment options, it is necessary to meticulously consider the individual differences of patients to make the most suitable treatment choice. Ethical approval Informed consent was obtained from all the study | 3,068,723 | 240421846 | 0 | 16 |
participants. | 3,068,724 | 240421846 | 0 | 16 |
10 Years of Experience with the First Thawed Plasma Bank in Germany Background: Plasma is stored at –30°C, which requires thawing before transfusion, causing a time delay between ordering and issuing of at least 30 min. In case of bleeding emergencies, guidelines strongly recommend a 2:1 transfusion ratio of RBCs and plasma. In addition, each minute delay in issuing of blood products in bleeding emergencies increases the mortality risk. To provide plasma in time in bleeding emergencies, a thawed plasma bank was introduced in 2011. Summary: The thawed plasma bank of University Medicine Greifswald has provided 18,924 thawed stored plasma units between 2011 and 2020. The workflow in the laboratory as well as in the emergency room, the operating room, and the intensive care unit have been optimized by thawed stored plasma. In case of emergencies, the stress factor for the transfusion medicine laboratory staff has been reduced substantially. The thawed plasma bank allows to transfuse patients with massive transfusion demand at a 2:1 ratio of RBCs and plasma according to guidelines. To reduce storage time, we issue all plasma requests from the thawed plasma bank except for pediatric patients. This results in a median storage time in the thawed plasma bank of 24 h. The “just in time” availability of plasma within the entire hospital based on the thawed plasma bank has reduced precautionary ordering of plasma, and hereby the unnecessary use of plasma. After introduction of the thawed plasma bank, plasma usage decreased substantially by 24% within the first year and by 60% compared | 3,068,725 | 241541066 | 0 | 16 |
to 2019/2020. However, as the overall approach to using blood products has changed over the last 10 years due to the patient blood management initiative, quantification of the effects of the thawed plasma bank in reduction of plasma transfusion is difficult. Key Messages: (1) A thawed plasma bank for the routine supply of blood products in a large hospital is feasible in Germany. (2) The thawed plasma bank allows to supply RBCs and plasma in a 2:1 ratio in bleeding emergencies. (3) The beneficial logistical effects of the thawed plasma bank are optimal if all plasma requests are supplied from the thawed plasma bank. This results in a median storage time of 24 h for thawed plasma. Introduction Major blood loss is one of the few indications to transfuse plasma [1]. Further indications are deficiency in clotting factors for which no factor concentrates are available like for factor V, improvement of hemostasis in coagulopathy, e.g., in case of liver impairment, increased general clotting factor consumption, plasma exchange for thrombotic thrombocytopenic purpura, and other rare indications [1]. In case of massive blood loss, it becomes increasingly clear that early plasma transfusion and maintenance of a red cells to plasma and single donor buffy coat platelets transfusion ratio of at least 2:1:1 improve patient outcome [2][3][4][5][6][7]. In this situation, plasma transfusion should be started as early as possible. In a prospective trial, each minute from activation of a massive transfusion protocol to arrival of the cooling container at the patient's bedside increased the odds of mortality by 5% | 3,068,726 | 241541066 | 0 | 16 |
[3]. Plasma is stored at <-30°C and has to be thawed before transfusion usually at 37-40°C. Thawing of plasma usually requires approximately 30 min using a conventional water-based plasma thawing device, e.g., plasmatherm (Barkey, Leopoldshöhe, Germany). The use of specially designed microwave ovens for thawing introduced in the late 1980s or thawing at higher temperatures can reduce thawing time [8][9][10][11][12]. Thawing at temperatures of 45°C is safe if the plasma is removed before thawing is completed. Otherwise the hemostatic capacity of the plasma is reduced [13]. Options to provide plasma for transfusion are summarized in Table 1: Primarily used is fresh frozen plasma (FFP) after quarantine storage and thawing at the time of transfusion request. Storage of plasma directly after blood donation without freezing is approved by the Food and Drug Administration (FDA) in the USA, called liquid plasma, with a shelf life up to 26 days [14]. Liquid plasma is not an option in Germany because of the risk of pathogen transmission. As an alternative it is accepted by the FDA to store FFP at 1-6°C for up to 24 h after thawing and to relabel it as thawed FFP (tFFP) for further storage for up to 4 days at 1-6°C [14] based on data by Downes et al. [15] and others [16][17][18][19][20]. Freeze-dried (lyophilized) plasma can be used after reconstitution directly before transfusion [21]. With Lyoplas ® (German Red Cross NSTOB, Germany), such a product is available in Germany, but reconstitution takes time and the development of foam during reconstitution seems to be sometimes | 3,068,727 | 241541066 | 0 | 16 |
a problem for the transfusing physician. Spray-dried plas-ma, also called on-demand plasma, is under investigation but not yet available [22]. The German hemotherapy guidelines accept for FFP a factor VIII activity after thawing of at least 70% of the value before freezing [23]. There are no definitions for minimal clotting factor activities in the product for plasma therapy, and no data from clinical studies are available assessing the efficacy of FFP or liquid plasma transfused after variable days of storage after thawing. Based on the experiences in treatment of severely injured military casualties [24], the recommendation for development and use of a massive transfusion protocol rose [25]. All massive transfusion protocols require the immediate availability of plasma for transfusion. Under conditions of quarantine storage of plasma to reduce pathogen transmission, thawing of plasma and storage of thawed plasma for up to 7 days has been established by University Medicine Greifswald in 2011. Here we report our experience of working with this thawed plasma bank. Organization of the Thawed Plasma Bank Plasma is produced and stored by the manufacturer according to specifications approved by the regulatory body Paul Ehrlich Institute. Hemotherapy-qualified physicians (transfusion committee of the hospital with its head "Transfusionsverantwortlicher") establish and supervise the organizational structure of hemotherapy in the hospital, including management of the blood bank according to the German hemotherapy guidelines [23]. The hemotherapy guidelines recommend storage of plasma after validation of specified conditions and immediate transfusion after release of thawed plasma from the blood bank to the recipient [23]. This allows storage of | 3,068,728 | 241541066 | 0 | 16 |
thawed plasma as part of the quality management system of hemotherapy. Specific conditions require individualized regulations for each hospital, including validation and standard operation for plasma processing and transfusion. Accordingly, we performed extensive validation of clotting factor activities using 50 thawed apheresis plasmas to evaluate the hemostatic quality of thawed FFP over 7 days [16,17]. Plasma units were divided into three biologically identical subunits and either stored for 7 days at 4°C, at room temperature, or at 4°C after methylene blue/ light treatment. Single clotting factor activities (II, V, VII, VIII, IX, X, XI, XII, XIII, fibrinogen, antithrombin, von Willebrand factor antigen, protein C and S) and functional hemostasis assays (aPTT, PT/INR, endogenous thrombin generation, and ProteinC ® Global) were assessed after thawing and on days 3, 5, and 7. Thawed FFP stored at 4°C for 7 days revealed major changes (activities outside the reference range) only for factor VIII (median 56%, range 33-114%) and protein S (median 51%, range 20-88%). The most pronounced decrease in factor VIII and protein S activity occurred within the first 3 days after thawing (about 50% and 15%, respectively). Thereafter, a further decrease in activity occurred within a 10% range compared to day 3. The storage for 7 days at room temperature affected activities of all clotting factors and inhibitors except for protein C [16]. The endogenous thrombin generation potential remained stable after storage at 4°C (1,287 ± 283 nmol vs. 1,260 ± 278 nmol; p = not significant; a representative example is shown in Fig. 1) [17]. The change | 3,068,729 | 241541066 | 0 | 16 |
in procoagulatory factors is commpensated by the concomitant decline of the anticoagulatory factors resulting in a new balance. INR and aPTT showed only modest alterations. Although reports about clotting factor activities after storage of thawed plasma at 1-6°C for several days are sometimes controversial [15,18,[26][27][28][29][30][31][32][33], a general finding of these studies is that storage is feasible without relevant impairment of hemostatic activity of plasma. Published data have been generated with thawed apheresis as well as with whole-blood (recovered) plasma. Recovered plasma is often later shock-frozen than apheresis plasma, and clotting factor activities might therefore be lower. Whether this has any impact on clinical efficacy is unresolved. No clinical studies or prospective trials evaluating the clinical efficacy of tFFP (apheresis or recovered) in relation to storage time are available. Patient Populations to Whom Thawed Plasma Is Issued Situations of massive transfusion are relatively rare. Maintaining a thawed plasma bank for massive transfusion only would result in a major wastage rate. In this case, we expected that the majority of plasmas would be stored up to the end of the storage period (7 days). We therefore provide thawed plasma to all patients for whom plasma is requested, with the exception of pediatric pa- tients and patients with known single clotting factor deficiencies for whom clotting factor concentrates are available. All other patients, including patients with liver disease, receive thawed plasma. Importantly, patients do not receive more than 4 units of thawed stored plasma because not more than 4 units are thawed in advance. All additional plasma demand is | 3,068,730 | 241541066 | 0 | 16 |
supplied by freshly thawed FFP. Impact on Blood Bank Management The use of the thawed plasma bank has reduced workload and stress factors for the technologists of the blood bank. In case of emergencies, plasma can be issued immediately, and also regular requests from the operation theater, the intensive care unit, or any ward can be handled without time delay. This allows the staff to focus on organizational issues associated with the management of bleeding emergencies. In addition, storage of thawed plasma allowed to introduce a trauma management policy at our hospital, including early plasma transfusion and achievement of an RBC:plasma transfusion ratio of at least 2:1 immediately after hospital admission of the patient. With activation of this massive transfusion protocol a "trauma box" is issued to the emergency room containing a defined number of blood products shown in Table 2. Special in-hospital transport staff is activated by phone call (the number is unique for this emergency situation), who then transports the trauma boxes and blood samples of the patient between the blood bank and the emergency room. The thawed plasma bank has also improved the workflow in the operating room. Plasma can now be ordered when transfusion is definitely indicated. Before the availability of thawed plasma, the anesthetist had to plan always 30-60 min in advance and to order plasma prospectively in case plasma transfusion might be needed; if not needed, the thawed plasma was wasted or transfused based on a weak indication. Furthermore, for surgery with high risk of acute major blood loss (risk | 3,068,731 | 241541066 | 0 | 16 |
of arterial bleeding), we now provide RBCs and thawed plasma in a stand-by cooling container in the operating room. If the RBCs and plasma are not needed, they can be returned to the blood bank and further stored for the next transfusion request. For reintegration of the blood products, the cooling chain must not have been interrupted (monitored by a minimum-maximum thermometer). The annual plasma consumption has declined from a mean of 4,386 units between 2010 and 2016 to 1,779 units in 2020 (Fig. 2). However, due to the parallel establishment of patient blood management with stricter consideration of plasma transfusion indication and use of blood products, it is difficult to differentiate the effects of the patient blood management program and of the thawed plasma bank. Nevertheless, plasma wastage rate has to be evaluated and weighed against the advantages of a thawed plasma bank. Storage time of the thawed plasma is often a matter of concern. To keep storage times short, we issue all plasma requests from the thawed plasma bank. When plasma is issued from the blood bank, FFP is immediately thawed and the plasma bank is restocked. This results in short Table 2. Trauma management policy of University Medicine Greifswald includes consecutively issued trauma boxes by the blood bank, which contain a defined number of blood products for massively bleeding patients Blood type of the patient is unknown (admission of a severely bleeding patient with unknown blood type to the emergency room) First medical care supported by a sub-depot of blood products in the | 3,068,732 | 241541066 | 0 | 16 |
emergency room storage times of the thawed plasma units. During the 10year period most units (81%) were issued within the first 3 days after thawing (shown in Fig. 3). The median storage time for thawed plasma was 1 day, the mean was 1.81 ± 1.88 days. The overall wastage rate was 6.26% (Fig. 2). This is higher compared to the plasma wastage rate of about 3% (2010: 3.13%; 2018: 3.17%; 2019: 2.92%) reported to the Paul Ehrlich Institute by blood product users in Germany [34], but considering the decline in plasma consumption (by 24.5% within the first year and by 66.7% in 2020 compared to 2011), the introduction of the thawed plasma bank reduced the overall usage of plasma due to the advantages of "just in time" availability. Mostly plas-ma of blood type B (17.06%) and blood type AB (8.13%) expired in the thawed plasma bank. The lowest wastage rate and close to the overall wastage rate in German hospitals was plasma of blood type A (3.49%). Blood type B patients are rare, and in case of transfusion requests for type B patients, type AB plasma can be used. Therefore, we stopped using type B plasma for the thawed plasma bank in 2015. The ideal product for the thawed plasma bank would be "universal plasma" without isoagglutinins, which can be transfused independently of the patient's blood group. We are currently developing such a product independently of blood type AB donors [35]. Additional Advantages of the Liquid Plasma Bank A spinoff of our thawed plasma bank is | 3,068,733 | 241541066 | 0 | 16 |
the blood product supply for the helicopter emergency medical service (HEMS). Only with the background of a thawed plasma bank, the plasma supply for the helicopter allows an acceptable wastage rate of plasma because of the rare prehospital transfusion frequency. We provide type A thawed plasma for the HEMS to reduce AB plasma shortage. Since we introduced blood supply for the HEMS, the challenge for the management of the thawed plasma bank was to integrate the returned HEMS plasma without expanding the number of thawed plasmas and increasing their wastage rate. Possible Risks of the Thawed Plasma Bank There is the theoretical risk of bacterial growth in plasma during storage at 2-6°C. This risk is not expected to be different from that of red cell concentrates for which 15-20 products are checked monthly after >7 days of storage (49 ± 3 days) [23]. Within 10 years we have not received a single report about a septic transfusion reaction after transfusion of tFFP. However, we have established an additional safety step. After quarantine storage, the frozen plasma bags are labeled and put into a vacuumsealed plastic bag. The thawed plasma is maintained in this plastic bag to reduce any risk of contamination from the outside in case of microlesions in the plastic bag, which may occur during handling of frozen plasma bags. This overpacking also allows easy discovery of minor leakage. There could be a risk of providing insufficient clotting factors to a bleeding patient. However, the patient receives a maximum of four plasmas from the thawed | 3,068,734 | 241541066 | 0 | 16 |
plasma bank. Thereafter, freshly thawed FFP is issued. Thus, in any situation where more than four plasmas are transfused, there is no risk of clotting factor activity reduction. In situations where a maximum of four plasmas are transfused, the reduction in factor VIII of the 4 plasma units is barely relevant. This is especially true in patients with liver impairment who typically have elevated factor VIII levels. As the situation is different in newborns and children, we issue thawed plasma only for adult patients. An additional potential risk of the thawed plasma bank is an increase in plasticizers dissolved from the blood bag into the lipids of the plasma [36]. Higher levels of diethylhexyl phthalate are present in plasma after 5 days of storage than in RBC units after 30 days of storage [36]. But again, the maximal number of tFFP transfused is limited to 4 units. With the introduction of new diethylhexyl phthalate-free whole-blood bag systems, e.g., 1,2-cyclohexane dicarboxylic acid diisononyl ester-containing blood bags, the risk of transmission of plasticizers can be reduced. Conclusion The thawed plasma bank of the University Medicine Greifswald has provided 18,924 thawed stored plasma units between 2011 and 2020. The workflow in the laboratory as well as in the emergency room, the operating room, and the intensive care unit have been optimized by thawed stored plasma. In case of emergencies, the stress factor for the transfusion medicine laboratory staff has been reduced substantially. The thawed plasma bank allows to transfuse patients with massive transfusion demand at the 2:1 ratio of | 3,068,735 | 241541066 | 0 | 16 |
RBCs and plasma according to guidelines. The "just in time" availability of plasma within the entire hospital has reduced precautionary ordering of plasma and hereby unnecessary use of plasma or wastage. As the overall approach to using blood products has changed over the last 10 years due to the patient blood management initiative, quantification of the effects of the thawed plasma bank in reduction of plasma units by a before-after comparison is problematic. | 3,068,736 | 241541066 | 0 | 16 |
Comprehensive Multi-Omic Evaluation of the Microbiota and Metabolites in the Colons of Diverse Swine Breeds Simple Summary Pigs play a crucial role in human sustenance, with their intestinal microbiota composition significantly influencing their nutrition and metabolism. Recent research has focused on understanding these differences among various swine breeds to attain improved food quality and safety. We sought the microbial evidence of differential performances of Tibetan pigs. In this study, six Duroc × Landrace × Yorkshire (DLY) pigs and six Tibetan pigs at 200 days of age were examined. Analysis revealed distinct microbial compositions in DLY pigs, with higher levels of Alloprevotella and Prevotellaceae_UCG-003. This resulted in variations in short-chain fatty acids (SCFAs) concentrations, contributing to enhanced growth performance. Tibetan pigs exhibited increased abundance of NK4A214_group, leading to higher L-cysteine levels and the subsequent elevation of taurine in the colon and plasma. Taurine influences microbiota dynamics and metabolism, particularly in bile acid metabolism, showcasing Tibetan pigs’ proficiency in this area. Overall, DLY pigs excel in SCFA metabolism, while Tibetan pigs exhibit competence in bile acid metabolism. Leveraging these breed-specific differences could improve production performance in these pig breeds. Abstract Pigs stand as a vital cornerstone in the realm of human sustenance, and the intricate composition of their intestinal microbiota wields a commanding influence over their nutritional and metabolic pathways. We employed multi-omic evaluations to identify microbial evidence associated with differential growth performance and metabolites, thereby offering theoretical support for the implementation of efficient farming practices for Tibetan pigs and establishing a robust foundation for enhancing pig growth | 3,068,737 | 269251312 | 0 | 16 |
and health. In this work, six Duroc × landrace × yorkshi (DLY) pigs and six Tibetan pigs were used for the experiment. Following humane euthanasia, a comprehensive analysis was undertaken to detect the presence of short-chain fatty acids (SCFAs), microbial populations, and metabolites within the colonic environment. Additionally, metabolites present within the plasma were also assessed. The outcomes of our analysis unveiled the key variables affecting the microbe changes causing the observed differences in production performance between these two distinct pig breeds. Specifically, noteworthy discrepancies were observed in the microbial compositions of DLY pigs, characterized by markedly higher levels of Alloprevotella and Prevotellaceae_UCG-003 (p < 0.05). These disparities, in turn, resulted in significant variations in the concentrations of acetic acid, propionic acid, and the cumulative SCFAs (p < 0.05). Consequently, the DLY pigs exhibited enhanced growth performance and overall well-being, which could be ascribed to the distinct metabolite profiles they harbored. Conversely, Tibetan pigs exhibited a significantly elevated relative abundance of the NK4A214_group, which consequently led to a pronounced increase in the concentration of L-cysteine. This elevation in L-cysteine content had cascading effects, further manifesting higher levels of taurine within the colon and plasma. It is noteworthy that taurine has the potential to exert multifaceted impacts encompassing microbiota dynamics, protein and lipid metabolism, as well as bile acid metabolism, all of which collectively benefit the pigs. In light of this, Tibetan pigs showcased enhanced capabilities in bile acid metabolism. In summation, our findings suggest that DLY pigs excel in their proficiency in short-chain fatty acid metabolism, | 3,068,738 | 269251312 | 0 | 16 |
whereas Tibetan pigs exhibit a more pronounced competence in the realm of bile acid metabolism. These insights underscore the potential for future studies to leverage these breed-specific differences, thereby contributing to the amelioration of production performance within these two distinct pig breeds. Introduction The intestines of mammals harbor a substantial population of microorganisms, with reported microbial numbers reaching up to 10 13 -10 14 [1].As such, the intestinal ecosystem stands as one of the most extensive microbial habitats, playing an integral role in myriad physiological functions including digestion, absorption, and metabolic processes [2].These microorganisms actively engage in interactions with the host, exerting a considerable influence over both disease susceptibility and normal physiological functions [3].Furthermore, the establishment and diversity of these microbial communities are influenced by a variety of factors, including genetic lineage, age, gender, and feeding patterns.Furthermore, the gut microbiota serves as a producer of various metabolites, encompassing bile acids, fatty acids such as butyrate and other short chain fatty acids, and vitamins such as thiamine, folate, biotin, riboflavin, and pantothenic acid [4].These compounds are generated through the co-metabolism of microorganisms and the host, ultimately impacting the overall health status of the host organism [5]. Metabolites encompass tangible compounds generated during the metabolic process, serving as both substrates and outcomes of metabolic reactions [6].These compounds wield the capacity to instigate alterations in cellular functionalities, encompassing signal transduction, energy conversion, and cellular apoptosis [6].In parallel, metabolomics stands as a discerning analytical methodology capable of holistically scrutinizing and contrasting end-stage products.This approach provides an intuitive depiction of the | 3,068,739 | 269251312 | 0 | 16 |
molecular metabolic profiles that arise from the metabolism of individuals, organ tissues, and cellular entities, thereby establishing a link between metabolic pathways and underlying biological mechanisms.Furthermore, it offers the ability to delineate an organism's well-being by assessing the comprehensive metabolic network and conducting an exhaustive exploration of the organism's metabolic processes [7]. Recent investigations have unveiled substantial disparities in the fecal microbial composition across distinct swine breeds [8][9][10].Metabolites emerge as pivotal mediators that forge a connection between gut microorganisms and the physiological well-being of the host organism.Research has elucidated marked distinctions in both plasma and colonic metabolomes among diverse breeds of fattening pigs [11].Furthermore, Yan et al. (2017) established significant discrepancies in microbiota in the gut makeup between Landrace and Meihua pigs, accompanied by notable differentials in microbial metabolite profiles [12].Nevertheless, the specific differences between different pig breeds' microbial communities and the metabolites that go along with them are yet unclear. The commercial swine known as Duroc × Landrace × Yorkshire (DLY) pigs are distinguished by their excellent market retention rate.They exhibit advantages such as rapid growth, high lean meat percentage, and superior feed conversion efficiency [13,14].On the other hand, the Tibetan pig is a local swine breed predominantly inhabiting the Qinghai-Tibet Plateau region of China.It demonstrates robust environmental adaptability and disease resistance.However, it is also burdened with disadvantages including low reproductive capacity and slow growth rate [15].This study aimed to investigate the relationships between intestinal microbiota, metabolites, and plasma metabolic profiles in DLY pigs and Tibetan pigs, elucidating the differences in gut ecological niches and | 3,068,740 | 269251312 | 0 | 16 |
metabolic patterns.Additionally, it sought to unveil the microbial evidence associated with differential growth performance and metabolites between these two groups.The findings of this study offer a novel perspective for the swine industry to consider the Tibetan pig breed, thereby presenting prospects for the future scale-up of Tibetan pig farming, precision nutrition formulation, and improvement in husbandry practices. Experimental Section 2.1. Animals Twelve pigs, six of which were Tibetan and Duroc × Landrace × Yorkshire (DLY) pigs, were selected at random to be fed the same commercial diet when they were 200 days old (Zhengda, Tianjin, China).The animals were accommodated in pens furnished with plastic slatted flooring at the Tibet Liuya Agro-Pastoral Development Co., Ltd.(Shannan, China).The animals were randomly distributed into 2 experimental groups, and all animals were raised in the same house and fed the same diet, and were free to eat and drink during the experiment.The preliminary experiment lasted for 3 days and the formal experiment lasted for 5 weeks.No antibiotics were administered to the pigs throughout the 5-week experiment.The dietary composition adhered to the nutritional requirements outlined in the National Research Council (NRC) guidelines from 2012 (Table 1). Sample Collection At the final day, blood samples were collected via jugular venipuncture from each pig in an EDTA anticoagulation tube.After blood samples were centrifuged at 12,000 rpm for 10 min, they were quickly frozen in liquid nitrogen, and kept at −80 • C. Subsequently, samples of the colon digesta were taken out, quickly frozen in liquid nitrogen, and kept at −80 • C. The Tibet | 3,068,741 | 269251312 | 0 | 16 |
Academy of Agriculture and Animal Husbandry Science's Institutional Animal Care and Use Committee (TAHS2021-15) approved all animal procedures, and all methods were performed in accordance with the relevant guidelines and regulations. Untargeted Metabolomics Research Method and Parameter Setting A 60 mg sample of colon digesta was meticulously weighed and subsequently transferred into a 1.5 mL Eppendorf tube.To the same tube, two diminutive steel balls were introduced.Following this, a solution of L-2-chlorophenylalanine (0.06 mg/mL) dissolved in methanol, serving as an internal standard, was added at 20 µL.Additionally, a composite mixture of methanol and water (4:1, v/v) totaling 650 µL, was incorporated into each individual sample.The samples were then subjected to cold storage at −20 • C for a duration of 2 min.Thereafter, they were subjected to grinding at 60 Hz for a span of 2 min. The subsequent step involved subjecting the entire set of samples to a 10 min extraction process using ultrasonication within an ice water bath, followed by further storage at −20 • C for a period of 5 h.Subsequently, the extracts were subjected to centrifugation at 4 • C (13,000 rpm) for a duration of 10 min.From each tube, supernatants amounting to 150 µL were meticulously collected using crystal syringes.These collected supernatants were then passed through sterile 0.22 µm microfilters and subsequently transferred into liquid chromatography vials.The vials, in turn, were stored at an ultra-low temperature of −80 • C until they were subjected to GC-MS analysis. Thawing was performed on plasma samples stored at −80 • C, allowing them to reach room | 3,068,742 | 269251312 | 0 | 16 |
temperature.A 150 µL aliquot of each sample was subsequently introduced into a 1.5 mL Eppendorf tube, wherein 10 µL of L-2-chlorophenylalanine (0.06 mg/mL) dissolved in methanol, serving as an internal standard, was meticulously added.The tube contents were then subjected to vortexing for a span of 10 s.Following this, a solvent mixture consisting of methanol and acetonitrile (2:1, v/v) totaling 450 µL was carefully introduced, and the resultant mixtures were subjected to vortexing for a duration of 1 min.Subsequently, the entire set of samples underwent a 10 min ultrasonic extraction within an ice water bath, followed by storage at −20 °C for a period of 30 min.The subsequent step encompassed centrifugation at 4 • C (13,000 rpm) for a duration of 10 min.From each glass vial, a 150 µL supernatant fraction was subjected to drying within a freeze concentration centrifugal dryer.Subsequently, a mixture of methanol and water (1:4, v/v) totaling 50 µL was introduced to each sample, followed by 30 s of vortexing.The samples were then subjected to a 3 min ultrasonic extraction within an ice water bath, followed by storage at −20 °C for a duration of 2 h.Thereafter, centrifugation was conducted at 4 • C (13,000 rpm) for a duration of 10 min.The resulting supernatants from each individual tube were meticulously collected through the utilization of crystal syringes.These collected supernatants were subsequently passed through 0.22 µm microfilters before being transferred into GC vials.The vials were subsequently stored at an ultra-low temperature of −80 • C in anticipation of GC-MS analysis [16,17]. Processing and Multivariate Analysis | 3,068,743 | 269251312 | 0 | 16 |
of Metabolomic Data The initial GC-MS data underwent processing using Progenesis QI V2.3 software (Nonlinear Dynamics, Newcastle, UK) for tasks including baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization.Subsequently, following the description by Chawes (2019), parameters were established, and an analysis was conducted utilizing pertinent databases including the Human Metabolome Database (HMDB), Lipidmaps (V2.3),Metlin, EMDB, PMDB, and custom-built databases.This analytical process encompassed data processing, analysis, and comparative evaluation [16]. Detection of Short-Chain Fatty Acid Content in Samples The extraction and quantification of SCFAs in the colonic chyme were conducted following established methodologies as outlined in a prior study [18].Samples were extracted from approximately 0.5 g of colon content using ultrapure water.The resultant extract underwent centrifugation at 11,000× g rpm, followed by mixing with metaphosphoric acid (25%, w/v).A subsequent round of centrifugation at 12,500× g rpm was performed, and the resulting supernatant was filtered using a 0.45 µm Milled-LG filter (Millipore, Billerica, MA, USA).Subsequently, the analysis was carried out using an Agilent 7890 N gas chromatograph (Agilent, Santa Clara, CA, USA). Quantitative Real-Time (qRT) PCR Analysis Total RNA was extracted from the colon mucosa, using the RNeasy Mini Kit (GeneBetter, Beijing, China).The quantification of RNA sample concentrations was achieved using the NanoDrop 2000 spectrophotometer.Subsequently, cDNA synthesis was performed at 37 • C for 15 minutes, followed by a brief incubation at 85 • C for 5 seconds using the PrimeScriptTM RT reagent kit with gDNA Eraser (Thermo Fisher, Waltham, MA, USA).Detailed methods are outlined in Luo et.al (2021) [19].The PCR primers used are | 3,068,744 | 269251312 | 0 | 16 |
shown in Supplementary Table S1. Microbial 16S rRNA Gene Sequencing Analysis Total genomic DNA was extracted using the manufacturer's protocol with the EZ-NATM Soil DNA kit.For bacterial diversity analysis, The V3-V4 hypervariable regions of the bacterial 16S rDNA were amplified by a two-step PCR method using primers 338F (5 ′ -ACTCCTRCGGGAGGCAGCAG-3 ′ ) and 806R (5 ′ -GGACTACCVGGGTATCTAAT-3 ′ ) with unique 8-bp barcodes to facilitate multiplexing, and library sequencing and data processing were conducted by OE biotech Co., Ltd.(Shanghai, China).Sequences were analyzed and classified into operational taxonomic units (OTUs; 97% identity).Sequence data were analyzed with quantitative insights into microbial ecology (QIIME) package version 1.8.0 [20], using the Silva 138 reference database as a reference template [21].The low abundant operational taxonomic units (OTU), identified by filtering OTU that had <10% of samples below 10 read counts, were removed.Tax4fun was used to predict functional profiles of microbial communities.Using BLAST, each representative read was annotated and tested against the Unite database (ITSs rDNA) (Blast 2015) (PRJNA872017). Statistical Analysis Data conforming to normal distribution were compared using Student t-test, while those with non-normal distribution were tested using the Kruskal-Wallis test.These analyses were performed using the JMP software (JMP R version 10.0.0,SAS Institute, Cary, NC, USA) for Windows.The correlation between the outcomes was examined using the psych package (Version 2.3.6)within the R environment (Version 4.3.1).The visual representation of the results was generated utilizing the pheatmap package (Version 1.0.12)within the same R environment (Version 4.3.1).All data were presented as mean ± standard error of the mean (SEM).Acceptable significance levels were at | 3,068,745 | 269251312 | 0 | 16 |
* p < 0.05, ** p < 0.01, and *** p < 0.001. Variations in Colonic Luminal Microbiome between DLY and Tibetan Pigs Following size filtering, quality control, and chimera checking, 16S rRNA amplicon sequencing results revealed a total of 901,467 reads ranging from 73,049 to 77,051.Reads per sample were used to examine the species of pig breed in terms of microbial population in the colon.Sequencing counts were normalized to acquire normalized reads for each sample in operational taxonomic units (OTUs) based on 97% identity.As indicated in Figure 1, a Venn diagram was utilized to reveal the common and unique OTUs in two groups.In pigs from two groups, there were 2397 types of identical OTUs from over 3300 OTUs (Figure 1A).Regarding α-diversity, the microbiota diversity in DLY pigs was found to be notably higher when contrasted with that of Tibetan pigs (p < 0.05) (Figure 1B,C).In terms of β-diversity, there was a discernible differentiation in the microbiota composition between the two groups (Figure 1D,E). The Differential Metabolites in Colon between DLY and Tibetan Pigs The disparities in colonic metabolites between the two pig breeds are depicted in Figure 2. The utilization of PCA score-plots and OPLS-DA highlighted distinct metabolomic profiles for both pig breeds (Figure 2A,B).Furthermore, a comparison between Tibetan and DLY pigs revealed 60 up-regulated and 10 down-regulated metabolites in Tibetan pigs (VIP ≥ 1, p value ≤ 0.05) (Figure 2C,D).The primary differential metabolites are presented in Supplementary Table S2.In comparison to DLY pigs, Tibetan pigs had considerably reduced relative levels of three carbohydrates (maltotriose, | 3,068,746 | 269251312 | 0 | 16 |
D-tagatose, and methyl beta-d-glucopyranoside), two amino acids (tranexamic acid and ornithine), and five additional substances.Meanwhile, in Tibetan pigs, the relative levels of 13 amino acids, two benzoic acids, one bile acid, three carbohydrates, 13 fatty acids, one organosulfonic acid, one cholestane steroids and 26 other compounds were significantly higher than those in DLY pigs. The Differential Metabolites in Colon between DLY and Tibetan Pigs The disparities in colonic metabolites between the two pig breeds are depicted in Figure 2. The utilization of PCA score-plots and OPLS-DA highlighted distinct metabolomic profiles for both pig breeds (Figure 2A,B).Furthermore, a comparison between Tibetan and DLY pigs revealed 60 up-regulated and 10 down-regulated metabolites in Tibetan pigs (VIP ≥ 1, p value ≤ 0.05) (Figure 2C,D).The primary differential metabolites are presented in Supplementary Table S2.In comparison to DLY pigs, Tibetan pigs had considerably reduced relative levels of three carbohydrates (maltotriose, D-tagatose, and methyl beta-d-glucopyranoside), two amino acids (tranexamic acid and ornithine), and five additional substances.Meanwhile, in Tibetan pigs, the relative levels of 13 amino acids, two benzoic acids, one bile acid, three carbohydrates, 13 fatty acids, one organosulfonic acid, one cholestane steroids and 26 other compounds were significantly higher than those in DLY pigs. These differential metabolites were related to various pathways based on KEGG analysis (Figure 2E), including differential metabolites mainly enriched in protein and bile acid metabolism.These differential metabolites were related to various pathways based on KEGG analysis (Figure 2E), including differential metabolites mainly enriched in protein and bile acid metabolism. Differential Plasma Metabolites between DLY and Tibetan | 3,068,747 | 269251312 | 0 | 16 |
Pigs The distinctive plasma metabolomic profiles between the two pig breeds are illustrated in Figure 3. PCA score-plots and OPLS-DA show that both breeds of pigs had differential metabolites (Figure 3A,B).Compared with DLY pigs, Tibetan pigs had 39 up-regulated and 37 down-regulated metabolites (VIP ≥ 1, p value ≤ 0.05) (Figure 3C,D).Notably, the key differential metabolites are detailed in Supplementary Table S3.In Tibetan pigs, the relative levels of five amino acids, eight carbohydrates, two fatty acids, one organosulfonic acid, one cholestane steroid, and 22 other compounds were significantly higher compared to those in DLY pigs.Moreover, Tibetan pigs displayed significantly lower relative levels of six amino acids, eight carbohydrates, two fatty acids and 21 other compounds in comparison to DLY pigs.All these differential metabolites exhibited enrichment in diverse pathways, including purine, galactose, cysteine metabolism, and the TCA cycle, as indicated by KEGG analysis (Figure 3E). Differential Plasma Metabolites between DLY and Tibetan Pigs The distinctive plasma metabolomic profiles between the two pig breeds are illustrated in Figure 3. PCA score-plots and OPLS-DA show that both breeds of pigs had differential metabolites (Figure 3A,B).Compared with DLY pigs, Tibetan pigs had 39 up-regulated and 37 down-regulated metabolites (VIP ≥ 1, p value ≤ 0.05) (Figure 3C,D).Notably, the key differential metabolites are detailed in Supplementary Table S3.In Tibetan pigs, the relative levels of five amino acids, eight carbohydrates, two fatty acids, one organosulfonic acid, one cholestane steroid, and 22 other compounds were significantly higher compared to those in DLY pigs.Moreover, Tibetan pigs displayed significantly lower relative levels of six amino | 3,068,748 | 269251312 | 0 | 16 |
acids, eight carbohydrates, two fatty acids and 21 other compounds in comparison to DLY pigs.All these differential metabolites exhibited enrichment in diverse pathways, including purine, galactose, cysteine metabolism, and the TCA cycle, as indicated by KEGG analysis (Figure 3E). In the differential metabolites, only three metabolites showed the same trends (increased) in both colonic digesta and plasma, which were hexadecanedioic acid, linoleic acid, and taurine.In the differential metabolites, only three metabolites showed the same trends (increased) in both colonic digesta and plasma, which were hexadecanedioic acid, linoleic acid, and taurine. The same Metabolism Pathways in Both Plasma and Colon There were six pathways of KEGG enrichment found in the analysis results of differential metabolites that were the same in both plasma and colonic contents, which were ABC transporters, glucagon signaling pathway, alanine, aspartate and glutamate metabolism, central carbon metabolism in cancer, citrate cycle (TCA cycle), and cysteine and methionine metabolism.In these six pathways, there were 13 metabolites in the colon (pyruvic acid, L-valine, L-aspartic acid, serine, taurine, 4-aminobutyric acid, L-methionine, 4-hydroxyproline, L-lactic acid, L-cysteine, phthalic acid, isocitric acid, 2-ketobutyric acid, and digalacturonic acid) and 12 metabolites in the plasma (glucose-1-phosphate, sulfuric acid, glycerol, fumaric acid, L-histidine, malic acid, N-carbamoylaspartate, L-asparagine, homocystine, taurine, L-cystine, and erythritol).Compared with the three common differential metabolites showing the same trends in both plasma and colon, only taurine was enriched in the pathways based on KEGG analysis.While DLY pigs also demonstrated higher butyric acid concentrations, this difference did not reach statistical significance (p = 0.078) (Figure 4C).In the case of other SCFAs, | 3,068,749 | 269251312 | 0 | 16 |
no statistically significant differences were observed (p > 0.05); however, there were discernible trends indicating higher concentrations of isobutyric acid and hexanoic acid in DLY pigs compared to Tibetan pigs, as illustrated in Supplementary Figure S1.The proportions of SCFA concentrations displayed similar patterns in both breeds.Acetic acid was present at 65.22% and 61.72%, propionic acid at 16.50% and 16.59%, and butyric acid at 10.58% and 11.39% in the DLY pigs, while other SCFAs' concentrations were all lower than 5% (Figure 4E,F). Gene Expression Levels Related to Metabotropic Receptors in Mucosal Tissues Figure 5 displays the gene expressions associated with metabotropic receptors.The expressions of the genes TGR5 (p = 0.031) and FXR5 (p = 0.0004) were much higher in Tibetan pigs (Figure 5A,B).On the other hand, DLY pigs exhibited significantly higher gene expressions of GPR41 (p = 0.0039) and GPR43 (p = 0.0065) (Figure 5C,D).In terms of SLC5A8 expression, Tibetan pigs exhibited a higher level, although the difference was not statistically significant (Figure 5F).As for the expressions of other genes, no significant differences were observed (p > 0.05) (Figure 5E,G). Gene Expression Levels Related to Metabotropic Receptors in Mucosal Tissues Figure 5 displays the gene expressions associated with metabotropic receptors.The expressions of the genes TGR5 (p = 0.031) and FXR5 (p = 0.0004) were much higher in Tibetan pigs (Figure 5A,B).On the other hand, DLY pigs exhibited significantly higher gene expressions of GPR41 (p = 0.0039) and GPR43 (p = 0.0065) (Figure 5C,D).In terms of SLC5A8 expression, Tibetan pigs exhibited a higher level, although the difference was | 3,068,750 | 269251312 | 0 | 16 |
not statistically significant (Figure 5F).As for the expressions of other genes, no significant differences were observed (p > 0.05) (Figure 5E,G). Correlations between Microbial Communities, Colonic Metabolites, and Plasmatic Metabolites The interactions between differentially abundant microbes, differential colonic metabolites, and differential plasmatic metabolites are shown in Figure 6.Streptococcus, UCG−002, UCG-005 and NK4A214_group showed significantly high correlations with most metabolites in either the colon or plasma (Figure 6A,B).In particular, the four microbes were all significantly positively correlated with L-cysteine and taurine in the colon, in which NK4A214_group showed the highest correlation (Figure 6B).Furthermore, only NK4A214_group and Streptococcus showed significantly positive correlations with taurine in plasma (Figure 6A).Meanwhile, taurine in either plasma or colon showed positive correlations with amino acids (Figure 6C), but the correlation between taurine in two tissues was not significant; instead, they were all positively correlated with L-cysteine (Figure 6D). Correlations between Microbial Communities, Colonic Metabolites, and Plasmatic Metabolites The interactions between differentially abundant microbes, differential colonic metabolites, and differential plasmatic metabolites are shown in Figure 6.Streptococcus, UCG−002, UCG-005 and NK4A214_group showed significantly high correlations with most metabolites in either the colon or plasma (Figure 6A,B).In particular, the four microbes were all significantly positively correlated with L-cysteine and taurine in the colon, in which NK4A214_group showed the highest correlation (Figure 6B).Furthermore, only NK4A214_group and Streptococcus showed significantly positive correlations with taurine in plasma (Figure 6A).Meanwhile, taurine in either plasma or colon showed positive correlations with amino acids (Figure 6C), but the correlation between taurine in two tissues was not significant; instead, they were all positively | 3,068,751 | 269251312 | 0 | 16 |
correlated with L-cysteine (Figure 6D). Correlations between Lithocholic Acid and Microbial Communities, Metabolites, or Receptor Related Genes Then, we analyzed the relationships between lithocholic acid and colonic microbes (Figure 7A), colonic metabolites (Figure 7B), plasmatic metabolites (Figure 7C), and related genes (Figure 7D).As shown in Figure 7, we found that lithocholic acid did not have a significant correlation with taurine in the colon (Figure 7C), but it was significantly positively correlated with NK4A214_group (Figure 7A), taurine in plasma (Figure 7B), and L-cysteine in colon (Figure 7C).Finally, it had highly significantly positive correlations with FXR5 and TGR5 (Figure 7D). Correlations between Lithocholic Acid and Microbial Communities, Metabolites, or Receptor Related Genes Then, we analyzed the relationships between lithocholic acid and colonic microbes (Figure 7A), colonic metabolites (Figure 7B), plasmatic metabolites (Figure 7C), and related genes (Figure 7D).As shown in Figure 7, we found that lithocholic acid did not have a significant correlation with taurine in the colon (Figure 7C), but it was significantly positively correlated with NK4A214_group (Figure 7A), taurine in plasma (Figure 7B), and L-cysteine in colon (Figure 7C).Finally, it had highly significantly positive correlations with FXR5 and TGR5 (Figure 7D). Correlations between Short Chain Fatty Acids, Microbial Communities, and Receptor-Related Genes Figure 8 shows the associations between SCFAs, colonic microbes and receptor genes.For significantly different concentrations of SCFAs, which were acetic acid, propionic acid and total SCFAs, they were all significantly positively correlated with three less abundant microbes in Tibetan pigs (Prevotellaceae_UCG-003, Alloprevotella, and Lachnospiraceae_NK4A136_group) (Figure 8A) and two related genes (GPR41 and GPR43) (Figure | 3,068,752 | 269251312 | 0 | 16 |
8B), in which Prevotellaceae_UCG-003 showed the highest correlations, and it also had positive correlations with the other three SCFAs (isobutyric acid, butyric acid, and isovaleric acid).However, only Prevotellaceae_UCG-003 and Alloprevotella showed significantly positive correlations with GPR41 and GPR43 (Figure 8C).Meanwhile, SCFAs (acetic acid, propionic acid and total SCFAs) were significantly negatively correlated with four more abundant microbes in Tibetan pigs (UCG−002, NK4A214_group, dgA−11_gut_group, and Streptococcus) (Figure 8A), and except for Correlations between Short Chain Fatty Acids, Microbial Communities, and Receptor-Related Genes Figure 8 shows the associations between SCFAs, colonic microbes and receptor genes.For significantly different concentrations of SCFAs, which were acetic acid, propionic acid and total SCFAs, they were all significantly positively correlated with three less abundant microbes in Tibetan pigs (Prevotellaceae_UCG-003, Alloprevotella, and Lach-nospiraceae_NK4A136_group) (Figure 8A) and two related genes (GPR41 and GPR43) (Figure 8B), in which Prevotellaceae_UCG-003 showed the highest correlations, and it also had positive correlations with the other three SCFAs (isobutyric acid, butyric acid, and isovaleric acid).However, only Prevotellaceae_UCG-003 and Alloprevotella showed significantly positive correlations with GPR41 and GPR43 (Figure 8C).Meanwhile, SCFAs (acetic acid, propionic acid and total SCFAs) were significantly negatively correlated with four more abundant microbes in Tibetan pigs (UCG−002, NK4A214_group, dgA−11_gut_group, and Streptococcus) (Figure 8A), and except for dgA−11_gut_group, the correlations between the other three more abundant microbes and related genes (GPR41 and GPR43) were significantly negative (Figure 8C).dgA−11_gut_group, the correlations between the other three more abundant microbes and related genes (GPR41 and GPR43) were significantly negative (Figure 8C). Discussion In our study, we aimed to explore the differences | 3,068,753 | 269251312 | 0 | 16 |
in gut microbiome composition and metabolites among pigs of different genetic backgrounds and their implications for host physiology.Previous research has underscored variations among pig breeds [22][23][24].Building on this foundation, our findings demonstrate significant disparities in metabolites, short-chain fatty acids (SCFAs), and receptor expression levels in both colonic and plasma environments across distinct pig breeds.Notably, these differences correlate with divergent production performances observed in these breeds. Discussion In our study, we aimed to explore the differences in gut microbiome composition and metabolites among pigs of different genetic backgrounds and their implications for host physiology.Previous research has underscored variations among pig breeds [22][23][24].Building on this foundation, our findings demonstrate significant disparities in metabolites, short-chain fatty acids (SCFAs), and receptor expression levels in both colonic and plasma environments across distinct pig breeds.Notably, these differences correlate with divergent production performances observed in these breeds. Numerous studies have shown that intestinal function is positively correlated with gut microbial abundance, and that higher gut microbial diversity and richness lead to better nutrient absorption [25,26].In essence, microbes can be regarded as an additional organ within the host organism.Noteworthy parallels can be drawn between our findings and previous research, which has identified divergent microbial profiles in different pig breeds [12,24].Our experimental outcomes further underscore these similarities.Specifically, our investigation revealed notable discrepancies in colonic microbial compositions, notably a diminished α-diversity index in Tibetan pigs.Among the six microbial strains exhibiting higher abundances in Tibetan pigs, UCG-002 and UCG-005 have been linked to cholesterol levels [27]; a finding consonant with our observation of elevated cholesterol levels in | 3,068,754 | 269251312 | 0 | 16 |
Tibetan pigs.Elevated cholesterol concentrations are associated with several diseases, including diabetes and cardiovascular disorders [28].Concurrently, the presence of Lachnospiraceae_NK4A136_group has been associated with the fortification of the host's intestinal barrier [29].This observation implies that DLY pigs may possess superior gut health in comparison to Tibetan pigs. The variances observed in colon and plasma metabolites can be attributed to multiple factors, including microbes, dietary factors, and age, collectively impacting various physiological functions.In this study, the two groups of experimental animals were selected at similar ages and fed the same commercial diet.Our findings suggest that certain alterations may be linked to microbial influences, with the resultant metabolic shifts offering insights into the disparities between the two breeds.Notably, elevated levels of specific amino acids, particularly essential amino acids such as methionine and threonine, may facilitate enhanced efficiency in utilizing other amino acids among pigs [30].In our research, the KEGG enrichment result shows that different metabolites were enriched in cysteine and methionine metabolism and alanine, aspartate and glutamate metabolism in either the plasma or colon, and the concentrations of most enriched metabolites were higher in Tibetan pigs in either plasma (four higher level vs. two lower level) or colon (seven higher level vs. zero lower level).Especially in the colons of Tibetan pigs, the concentrations of all enriched metabolites were higher, which indicates that Tibetan pigs have a better protein metabolism capacity.Moreover, for the energy and carbohydrate metabolism, ABC transporters, Glucagon signaling pathway, and TCA cycle were enriched in both plasma and colon KEGG enrichment results.Most metabolites enriched in these three | 3,068,755 | 269251312 | 0 | 16 |
pathways were increased in Tibetan pigs (six higher level vs. three lower level in plasma, nine higher level vs. zero lower level in colon).Therefore, Tibetan pigs have a better energy metabolism capacity.Zeng et al. (2020) also found that energy metabolism, amino acid metabolism, and carbohydrate metabolism were consistently enriched at high altitudes in both Tibetans and Tibetan pigs.High altitude will result in unique gut bacteriomes and functions [31].Therefore, these two better capacities may be related to the fact that the microbes are affected by high altitude.Afterwards, the colons of Tibetan pigs had two different types of higher-level benzoic acids and derivatives, which reduced the microbiota's variety [32], which might explain the result whereby Tibetan pigs had lower α-diversity in the colon.Furthermore, two kinds of fatty acids were increased in both plasma and colon (hexadecanedioic acid and linoleic acid), which supply energy to animals.Nevertheless, excessive fatty acid concentrations cause a number of illnesses and increase fat tissue, which explains why Tibetan pigs have higher adipose tissue [33]. In Tibetan pigs, taurine was increased in both the plasma and colon, which indicates that taurine is a key metabolite supporting the differences between the two breeds of pigs.Firstly, UCG-005 was significantly positively correlated with taurine in the colon.Therefore, it can enhance the concentration of taurine in the colon.However, the correlations between taurine in plasma and microbes or taurine in colon were weak based on the correlation analysis; on the other hand" L-cysteine in the colon was significantly positively correlated with plasma (r = 0.82, p = 0.007) and colon | 3,068,756 | 269251312 | 0 | 16 |
(r = 0.74, p = 0.007), respectively, which indicates that taurine in the colon does not directly affect the concentration of taurine in plasma, and L-cysteine is the main factor affecting the concentrations of taurine in two tissues.This is because L-cysteine is the upstream metabolite of taurine, which can enhance the production of taurine [34].Then, we analyzed the correlations between L-cysteine and microbes; we found that four microbes (Streptococcus, UCG−002, UCG-005 and NK4A214_group) were significantly positively correlated with L-cysteine, especially NK4A214_group, which showed the highest correlation.Therefore, although microbes can affect the concentration of taurine in colon, the main factor affecting taurine metabolism in both the colon and plasma is L-cysteine, which is enhanced by NK4A214_group.Furthermore, taurine was found to increase the abundance of microbes in the colon, such as NK4A214_group and Ruminococcus [35].Therefore, taurine conversely maintains microbial homeostasis.Finally, taurine was found to maintain the protein metabolism, which is similar to our result that taurine showed positive correlations with amino acids [36].Therefore, Tibetan pigs with a higher level of taurine can support better protein metabolism to maintain growth and health. Elevated taurine levels may improve the bile acid metabolic system, which would help pigs by protecting the body by providing microbes energy and warding off pathogens [37].In our result, Tibetan pigs also had higher level of lithocholic acid in the colon.Therefore, we further analyzed the interactions between lithocholic and microbes or metabolites.We found that in only the NK4A214_group, taurine in plasma and L-cysteine in colon showed significant correlations with lithocholic acid, which indicates that taurine in the | 3,068,757 | 269251312 | 0 | 16 |
plasma, and not in the colon, directly affects the concentration of lithocholic acid.Furthermore, lithocholic acid can protect intestinal mucosa by inhibiting epithelial apoptosis [38].Additionally, by sensitizing BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide, which may cause cell death, lithocholic acid can kill gliomas without endangering normal neurocytes.Normal neuronal cells are resistant to this kind of cell death [39,40].Therefore, lithocholic acid can support Tibetan pigs in maintaining health.Furthermore, taurine can affect lipid metabolism by conjugating bile acids [41], which proves that Tibetan pigs have more adipose tissue.Finally, to prove the increase in lithocholic acid in Tibetan pigs, we analyzed two related genes' expressions (TGR5 and FXR5).These two genes were expressed significantly more highly in Tibetan pigs.FXR is a nuclear receptor highly expressed in the liver and the intestine [42].TGR5 and FXR5, which maintain glucose homeostasis, are impacted by bile acid [43].Specifically, bile acid stimulates FXR to suppress proglucagon transcription and GLP-1 synthesis, and stimulates TGR5 to increase GLP-1 secretion [44].Thus, the increased lithocholic acid in Tibetan pigs can be explained by the higher level of mRNA expression of TGR5 and FXR5.In our correlation result, lithocholic acid also showed highly significantly positive correlations with these two genes. The pivotal role of short-chain fatty acids (SCFAs) as key mediators connecting disease, nutritional aspects, and gut microbiota hinges on their concentrations, which are highly influenced by multifactorial determinants, including microbes, age, and dietary constituents.Our findings highlight microbes as the primary determinants shaping SCFA concentrations.In particular, diminished levels of SCFA-producing microbes within the colons of Tibetan pigs contribute to lower SCFA | 3,068,758 | 269251312 | 0 | 16 |
concentrations, corroborating findings from prior research [15].Fundamentally, elevated levels of total SCFAs within the colons of DLY pigs are indicative of enhanced growth performance.[45].More specifically, the decreased contents of acetic acid and propionic acid in Tibetan pigs correlate with compromised growth and health, aligning with other studies [46].Moreover, the presence of butyric acid, an SCFA, not only provides energy for the host, but also bolsters host defense by promoting the production of defense peptides [47].Therefore, higher concentrations of butyric acid could support DLY pigs' health.Moreover, many of the regulatory properties of SCFAs require signaling through GPRs, including GPR43, GPR41, and GPR109 [48].These receptors are expressed across various cell types, including the intestinal epithelium and immune cells.Their interaction with GPRs fortifies the intestinal epithelial barrier and upholds intestinal equilibrium [49].In Tibetan pigs, the decreasing expressions of GPR41 and GPR43 can explain the lower concentrations of SCFAs, and our correlation results also show that there were significantly positive correlations with SCFAs (acetic acid, propionic acid, butyric acid, and total SCFAs) and related genes (GPR41 and GPR43). To further identify correlations between microbes and SCFAs metabolism, we analyzed the relations between microbes and SCFAs or related genes.We found that, in Tibetan pigs, three more abundant microbes (UCG−002, NK4A214_group, and Streptococcus), especially Streptococcus, were significantly negatively correlated with SCFAs (acetic acid, propionic acid and total SCFAs) and related genes (GPR41 and GPR43), which indicates that SCFAs may affect the abundances of some of these three microbes.Another study also found that SCFAs can inhibit the biofilm formation of Streptococcus gordonii, which | 3,068,759 | 269251312 | 0 | 16 |
supports our results [50].Meanwhile, higher abundances of these microbes may lead to lower abundances of SCFA producers, thus reducing SCFAs.In our results, the abundances of two other SCFA producers were decreased in Tibetan pigs, which were Prevotellaceae_UCG-003 and Alloprevotella.All of them were significantly positively correlated with SCFAs (acetic acid, propionic acid and total SCFAs) and related genes (GPR41 and GPR43), which indicates that these two microbes can enhance the production of SCFAs by stimulating the expression of GPR41 and GPR43.Other researchers also attained similar results, finding that Alloprevotella and Prevotellaceae_UCG-003 can produce SCFAs or affect SCFAs production [51,52].While our study has provided valuable insights, it is not without limitations.A larger sample size of experimental animals would enhance the generalizability of our findings.Additionally, this study only compared Tibetan pigs with the common commercial breed DYL for reference.In consequence, in subsequent research, we will expand the sample size of experimental pigs based on the results of this study, delve deeper into the nutritional requirements of Tibetan pigs, and conduct comparative studies involving more breeds of pigs. Conclusions In our experiment, we found that both breeds of pigs had differential microbe, metabolite, SCFA and related gene expression levels.In DLY pigs, higher relative abundances of microbes containing Alloprevotella and Prevotellaceae_UCG-003 resulted in higher concentrations of SCFAs in acetic acid and propionic acid, as well as of total SCFAs, thus benefiting pigs.In Tibetan pigs, higher relative abundances of the NK4A214_group resulted in higher levels of L-cysteine, which enhance the production of taurine, and higher levels of taurine enhance the gut microbiota, | 3,068,760 | 269251312 | 0 | 16 |
protein metabolism, and lipid metabolism of pigs.Meanwhile, lithocholic acid, which is affected by taurines, also maintains pig health.Therefore, it is possible to use these differences to benefit either DLY pigs or Tibetan pigs, such as by using fecal microbiota transplantation to alter Tibetan pigs' microbes and thus improve their growth performance.This study not only elucidates the metabolic differences in intestinal microorganisms between two pig breeds in the plateau region, but also provides a theoretical basis for future research on local pig breeds. Data Availability Statement: The data presented in this study are available on request from the corresponding author.The data are not publicly available due to specific ethical and privacy considerations. Conflicts of Interest: The authors declare no conflict of interest. Figure 1 . Figure 1.Differential microbiome in colon of two breeds of pigs.(A) Venn diagrams of two groups.(B) Chao1 index of two groups.(C) Shannon index of two groups.(D) NMDS results of two groups.(E) PCoA results of two groups.(F) Community analysis of two groups at the phylum level.(G) Top 10 differential microbes of two groups at phylum level.(H) Community analysis of two groups at genus level.(I) Top 10 differential microbes of two groups at genus level.Data are presented as mean ± SD and statistical significance was determined by the Wilcoxon rank-sum test; DLY pigs; TP, Tibetan pig; * p ≤ 0.05; n = 6. ( B) Chao1 index of two groups.(C) Shannon index of two groups.(D) NMDS results of two groups.(E) PCoA results of two groups.(F) Community analysis of two groups at the phylum level.(G) Top | 3,068,761 | 269251312 | 0 | 16 |
10 differential microbes of two groups at phylum level.(H) Community analysis of two groups at genus level.(I) Top 10 differential microbes of two groups at genus level.Data are presented as mean ± SD and statistical significance was determined by the Wilcoxon rank-sum test; DLY pigs; TP, Tibetan pig; * p ≤ 0.05; n = 6. Figure 2 . Figure 2. Differential metabolites in colon of two breeds of pigs.(A) OPLS-DA results of two groups.(B) PCA results of two groups.(C) Volcano plot of two groups.(D) Number of differential metabolites of two groups.(E) Top 27 possible pathways of metabolites of two groups.DLY pigs; TP, Tibetan pig; n = 6. Animals 2024 , 20 Figure 2 . Figure 2. Differential metabolites in colon of two breeds of pigs.(A) OPLS-DA results of two groups.(B) PCA results of two groups.(C) Volcano plot of two groups.(D) Number of differential metabolites of two groups.(E) Top 27 possible pathways of metabolites of two groups.DLY pigs; TP, Tibetan pig; n = 6. Figure 3 . Figure 3. Differential metabolites in plasma of two breeds of pigs.(A) OPLS-DA results of two groups.(B) PCA results of two groups.(C) Volcano plot of two groups.(D) Number of differential Figure 3 . Figure 3. Differential metabolites in plasma of two breeds of pigs.(A) OPLS-DA results of two groups.(B) PCA results of two groups.(C) Volcano plot of two groups.(D) Number of differential metabolites of two groups.(E) Top 10 possible pathways of metabolites of two groups.DLY pigs; TP, Tibetan pig; n = 6. Figure 4 Figure 4 illustrates the concentrations of SCFAs | 3,068,762 | 269251312 | 0 | 16 |
within the colons of the two pig groups.In comparison to Tibetan pigs, DLY pigs exhibited notably elevated concentrations of acetic acid (p < 0.05), propionic acid (p < 0.01), and total SCFAs (p < 0.05) (Figure 4A,B,D).While DLY pigs also demonstrated higher butyric acid concentrations, this difference did not reach statistical significance (p = 0.078) (Figure4C).In the case of other SCFAs, no statistically significant differences were observed (p > 0.05); however, there were discernible trends indicating higher concentrations of isobutyric acid and hexanoic acid in DLY pigs compared to Tibetan pigs, as illustrated in Supplementary FigureS1.The proportions of SCFA concentrations displayed similar patterns in both breeds.Acetic acid was present at 65.22% and 61.72%, propionic acid at 16.50% and 16.59%, and butyric acid at 10.58% and 11.39% in the DLY pigs, while other SCFAs' concentrations were all lower than 5% (Figure4E,F). Figure 7 . Figure 7. Correlations between lithocholic acid and microbial communities, metabolites, or receptor related genes.(A) Interactions between lithocholic acid and colonic microbes.(B) Interactions between lithocholic acid and plasmatic metabolites.(C) Interactions between lithocholic acid and colonic metabolites.(D) Interactions between lithocholic acid and related genes.Correlations were determined by the spearman test, blue represents a negative correlation, red represents a positive correlation, n = 6.* p < 0.05, ** p < 0.01, and *** p < 0.001. Figure 7 . Figure 7. Correlations between lithocholic acid and microbial communities, metabolites, or receptor related genes.(A) Interactions between lithocholic acid and colonic microbes.(B) Interactions between lithocholic acid and plasmatic metabolites.(C) Interactions between lithocholic acid and colonic metabolites.(D) Interactions between | 3,068,763 | 269251312 | 0 | 16 |
lithocholic acid and related genes.Correlations were determined by the spearman test, blue represents a negative correlation, red represents a positive correlation, n = 6.* p < 0.05, ** p < 0.01, and *** p < 0.001. Figure 8 . Figure 8. Correlations between short-chain fatty acids, microbial communities, and receptor-related genes.(A) Interactions between colonic microbes and SCFAs.(B) Interactions between SCFAs and related genes.(C) Interactions between colonic microbes and related genes.Correlations were determined by the spearman test, blue represents a negative correlation, red represents a positive correlation, n = 6.* p < 0.05, ** p < 0.01, and *** p < 0.001. Figure 8 . Figure 8. Correlations between short-chain fatty acids, microbial communities, and receptor-related genes.(A) Interactions between colonic microbes and SCFAs.(B) Interactions between SCFAs and related genes.(C) Interactions between colonic microbes and related genes.Correlations were determined by the spearman test, blue represents a negative correlation, red represents a positive correlation, n = 6.* p < 0.05, ** p < 0.01, and *** p < 0.001. Author Contributions: Y.Z. and W.B. designed the experiment; G.S. and Y.C. carried out the experiment; Y.Z., Z.T. and J.Z. wrote the manuscript; B.S., Y.Z. and W.B. revised the manuscript.All authors have read and agreed to the published version of the manuscript.Funding: This research was supported by the Key Research and Transformation of Pure Breeding and Expansion Technology of Excellent Animal Breeds in Tibet (XZ-2019-NK-NS-003), for the project led by Yanbin Zhu.Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by | 3,068,764 | 269251312 | 0 | 16 |
the Institutional Review Board (or Ethics Committee) of the Institute of Animal Husbandry and Veterinary Medicine, Tibet Academy of Agriculture and Animal Husbandry Science (TAHS2021-15).Informed Consent Statement: Not applicable. Table 1 . Composition and nutrient levels of basal diets (basis). | 3,068,765 | 269251312 | 0 | 16 |
Ligand- and weak base-induced redistribution of asialoglycoprotein receptors in hepatoma cells. The receptor for asialoglycoproteins (ASGPR) was localized in human hepatoma Hep G2 cells by means of quantitative immunoelectron microscopy. Without ligand added to the culture medium, we found 34% of the total cellular receptors on the plasma membrane, 37% in compartment of uncoupling receptor and ligand (CURL), and 21% in a trans-Golgi reticulum (TGR) that was defined by the presence of albumin after immuno-double labeling. A small percent of the ASGPR was associated with coated pits, the Golgi stacks, and lysosomes. After incubation of the cells with saturating concentrations of the ligand asialo-orosomucoid (ASOR), the number of cell surface receptors decreased to 20% of total cellular receptors, whereas the receptor content of CURL increased by a corresponding amount to 50%. The ASGPR content of TGR remained constant. In contrast, after treatment of the cells with 300 microM of the weak base primaquine (PMQ), cell surface ASGPR had decreased dramatically to only 4% of total cellular receptors whereas label in the TGR had increased to 42%. ASGPR labeling of CURL increased only to 47%. The labeling of other organelles remained unchanged. This affect of PMQ was independent of the presence of additional ASOR. Implications for the intracellular pathway of the ASGPR are discussed. Abstract. The receptor for asialoglycoproteins (ASGPR) was localized in human hepatoma Hep G2 cells by means of quantitative immunoelectron microscopy. Without ligand added to the culture medium, we found 34 % of the total cellular receptors on the plasma membrane, 37 % in | 3,068,766 | 16662624 | 0 | 16 |
compartment of uncoupling receptor and ligand (CURL), and 21% in a trans-Golgi reticulum (TGR) that was defined by the presence of albumin after immuno-double labeling. A small percent of the ASGPR was associated with coated pits, the Golgi stacks, and lysosomes. After incubation of the cells with saturating concentrations of the ligand asialo-orosomucoid (ASOR), the number of cell sur-face receptors decreased to 20% of total cellular receptors, whereas the receptor content of CURL increased by a corresponding amount to 50%. The ASGPR content of TGR remained constant. In contrast, after treatment of the cells with 300 ~tM of the weak base primaquine (PMQ), cell surface ASGPR had decreased dramatically to only 4% of total cellular receptors whereas label in the TGR had increased to 42 %. ASGPR labeling of CURL increased only to 47 %. The labeling of other organelles remained unchanged. This affect of PMQ was independent of the presence of additional ASOR. Implications for the intracellular pathway of the ASGPR are discussed. IVER parenchymal cells possess cell surface receptors that function in the clearance of galactose-terminated glycoproteins from the circulation (1,17). In addition, these asialoglycoprotein receptors (ASGPR) 1 are present in abundance on the human hepatoma cell line Hep G2 that contains •225,000 high affinity ligand-binding sites per cell (22,23). In Hep G2 cells not exposed to ligand a considerable fraction of the functional ligand-binding sites is located on the cell surface (21). However, the presence of ligand (e.g., asialoorosomucoid [ASOR]) in the extracellular fluid promotes a relative increase in the fraction of receptors | 3,068,767 | 16662624 | 0 | 16 |
located intracellularly (4,23). Lysosomotropic amines, which accumulate within intracellular acidic organelles (5) thereby neutralizing the internal pH (18,33), alter the intraceUular distribution of several receptor species. The number of surface-binding sites for LDL (2), mannose-(29) and mannose-6-phosphate-(13) terminated ligands, alpha-2 macroglobulin (15), and asialoglycoproteins (21,28) is markedly reduced after incubation with these agents. This reduction was seen in both the presence and absence of added ligand. However, in several cases the 1. Abbreviations used in this paper: ASGPR, asialoglycoprotein receptor; ASOR, asialo-orosomucoid; CURL, compartment of uncoupling receptor and ligand; MVB, multi-vesicular body; PMQ, primaquine; TGR, trans-Golgi reticulum. effect was enhanced in the presence ofligand (2,15,21). This loss of cell surface-binding sites is reversible since after removal of the agent the cell surface ligand-binding capacity is rapidly and fully restored (21,29). These observations suggest that the receptors accumulate intracellularly in a nonlysosomal compartment. The aim of this study is to establish the morphologic identity of this compartment after treatment of the cells with ligand, a lysosomotropic amine, or with both. We have reported a dose-dependent and reversible loss of surface ASGPR in Hep G2 cells treated with primaquine (PMQ) or other lysosomotropic amines (21,28). The cells were found to possess a well-developed endocytotic apparatus including compartment of uncoupling receptor and ligand (CURL) (8, 11). Using immunoelectron microscopy and antibodies against PMQ, we found that PMQ accumulates in CURL, including multivesicular bodies (MVB), lysosomes, and in the Golgi complex (25). In the present study we used quantitative immunoelectron microscopy to describe the intracellular localization of ASGPR in Hep | 3,068,768 | 16662624 | 0 | 16 |
G2 cells, and examined the effects of the ligand ASOR and of PMQ on receptor distribution. We found that ASOR induced a partial redistribution of receptors from the plasma membrane to CURL. PMQ however, caused a dramatic disappearance of receptors from the cell surface in favor of trans-Golgi reticulum (TGR). Materials Human orosomucoid was provided by the American Red Cross and desialylated as described earlier (24). PMQ as the biphosphate was obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit antibody to the human ASGPR was affinity purified as described earlier (20). Rabbit anti-human albumin (Nordic Immunology, Tilburg, The Netherlands) was affinity purified on an albumin ultrogel column. Rabbit anti-cathepsin D (14) was a kind gift of Drs. K. von Figura and A. Hasilik (University of Miinster, Federal Republic of German!c). Uniformly-sized protein A-gold complexes of 8 and 5 nm were prepared according to the tannic-acid procedure as described (27). Cells Human hepatoma Hep G2 cells (16), clone a 16, were maintained as described previously (22). Confluent cultures were used in all experiments. Experimental Procedures Hep G2 cells were washed twice in MEM and cultured in MEM containing 10% normal rabbit serum for 4 h at 37°C. All cells were washed again and preincubated for 30-60 min at 37°C in MEM supplemented with 20 mM Hepes (pH 7.4). Thereafter, control cells that received no additional supplements were maintained in MEM-Hepes for 40 min at 37°C. Cells exposed to ligand only were incuba~d with medium containing 200 Ixg ASOR/ml for 40 min at 37°C. Cells exposed to | 3,068,769 | 16662624 | 0 | 16 |
PMQ only were incubated in medium containing 300 I~M PMQ for 30 rain at 37°C. Cells exposed to both ligand and PMQ were first incubated for 10 min at 37°C with ASOR alone, after which PMQ was added for the final 30 min at 37°C. Processing for Immunocytochemistry and Morphology After the appropriate incubations the cells were fixed in 2 % glutaraldehyde in 0.05 M phosphate buffer at pH 7.4 for 4 h at room temperature. Cells were then scraped from the dish and embedded in gelatin as previously described (6). The gelatin blocks were stored at 4°C for several weeks in 0.1 M phosphate buffer containing 0.02 % sodium azide. Blocks were immersed in 2.3 M sucrose and frozen in liquid nitrogen. Cryosections were prepareti according to Tokuyasu (32). Immuno-double labeling of sections was performed as previously described in detail (12). Anti-human albumin antiserum was used in the first step, followed by protein A-gold. Anti-ASGPR was used as the second antibody, followed by protein A-gold of a different diameter. In double labeling of the ASGPR with cathepsin D, anti-ASGPR was used in the first step. Sections were stained with uranyl acetate and embedded in methyl cellulose according to Tokuyasu (31). Normal cells and cells treated with 300 IxM PMQ for 30 min were fixed with 2% glutaraldehyde in 0.1 M cacodylate buffer, embedded in 1% agar in distilled water at 70°C, and postfixed in 1% osmiumtetroxyde in distilled water. Agar blocks were dehydrated in alcohol and embedded in epon. Ultrathin sections were stained with 7 | 3,068,770 | 16662624 | 0 | 16 |
% uranylacetate in methanol and 0.4 % leadcitrate in 0.1 M sodiumhydroxide. Quantitation of Receptors Three independent experiments were performed. Of several series of grids, sections were selected that exhibited good morphology. Approximately 1,000-2,000 gold particles labeling the ASGPR were counted for each of the three experiments. This represented ,vl00 cells per experiment, distributed over at least three grids. The particles were counted directly from the microscope screen. At low magnification the electron beam was placed in the top left comer of a grid mesh. After enlarging the magnification to 15,000× the grid was moved in one direction to the other side of the mesh. Thus the cells were encountered in a nonselective manner. The numbers of gold particles within each cell compartment (Table I) were expressed as the percentage of the total number of gold particles counted. The number of measurements required for a statistically reliable sample was determined by progressive mean analysis (3). 15 cell cross sections appeared to represent a reliable sample. Limits of confidence were 5 % or less for at least the final three samples. Determination of ASOR-binding Sites Quantitation of specific cell surface ~25I-ASOR binding was determined at 4°C as described previously (22). The total cell number of ligand-binding sites was determined by specific ~25I-ASOR binding to cells at 4°C after permeabilization with saponin for 30 min at 4°C (26). In experiments in which cells were preincubated with nonradioactive ASOR, the cells were incubated for 5 rain at 4°C with PBS containing no calcium but containing 5 mM EDTA at | 3,068,771 | 16662624 | 0 | 16 |
pH 5 to remove ligand from the receptor before saturation binding with 125I-ASOR (26). Localization of ASGPR in Ligand-Naive Hep G2 Cells The normal localization of ASGPR was studied in "ligandnaive" cells, i.e., cells that had been cultured in medium without additional serum or ligand, and is shown in Figs. 1 and 2. ASGPR labeling occurred diffusely along the entire plasma membrane but was most abundant at the surface facing the culture medium. Receptors were concentrated in peripheral coated pits and vesicles. CURL was heavily labeled (Fig. 2). Double labeling with cathepsin D showed that some ASGPR-positive MVBs were poorly labeled for cathepsin D while others showed abundant cathepsin D labeling but did not label significantly for the ASGPR (not shown). This is in accordance with our observation that ASGPRs are present in newly formed CURL vacuoles but sort out from older ones into the CURL tubules (7). Typical secondary lysosomes showed only an occasional ASGPR label. In contrast to the situation in rat liver (9), only a little ASGPR was found in the Golgi stack ( Figs. 1 and 2). TGR showed moderate ASGPR labeling ( Figs. 1 and 2). In general CURL and TGR could easily be distinguished morphologically. Although both contain anastomosing tubules and vesicles, TGR displayed a more heterogenous appearance and electron density, and showed more coated buds than did CURL (see also 8). Generally CURL occupied a peripheral position in the cells (Fig. 2) whereas TGR occurred adjacent to the Golgi complex deeper in the cell. However, some CURL elements could | 3,068,772 | 16662624 | 0 | 16 |
be detected close to a Golgi complex in the vicinity of the plasma membrane. This hampered a sharp distinction between CURL and TGR. Therefore an additional parameter was used, the presence of secretory albumin in TGR but not in CURL (8). Under the incubation conditions used, possible endocytosed albumin was not detected in CURL. Only the biosynthetic pathway including TGR was labeled for albumin. In the sections double labeled with anti-albumin and anti-ASGPR, ASGPR was distributed heterogeneously in the TGR. Some elements were enriched in ASGPR whereas others were devoid of it ( Figs. 1 and 2). ASGPR-labeling of dense-coated Figures 1 and 2. (Fig. 1) Immunoelectron micrograph of ligand-naive Hep G2 cell incubated without PMQ. The cryosection was immunodouble labeled for albumin with 10 nm gold and ASGPR with 6 nm gold particles. Both albumin and ASGPR are present in the stack of Golgi cisternae (G) and TGR elements (T). Note the heterogenous distribution of the receptors in TGR. Bar, 0.25 ~tm. (Fig. 2) Like Fig. 1. The Golgi cisternae (G) and TGR (T) contain both albumin and ASGPR in contrast to a CURL vacuole (C) that characteristically shows ASGPR only. P, plasma membrane. Bar, 0.25 Ixm. buds at the TGR was low which was different from what we have previously found with the receptor for mannose-6phosphate residues (8). Quantitative Distribution of ASGPR in Ligand-Naive Cells To determine the quantitative distribution of the receptors, we counted the number of gold particles confined to the various organelles as described in Materials and Methods. Particle counts were | 3,068,773 | 16662624 | 0 | 16 |
expressed as a percentage of the total number of gold particles evaluated. In ligand-naive cells (first column of Table I) the three plasma membrane domains together (lateral, bile canalicular, and those facing the culture medium and dish bottom) accounted for "ol/3 of the cells' ASGPR label. Coated pits of the plasma membrane were evaluated separately. No distinction was made between coated pits and vesicles. Together they contained "o5 % of the label. CURL and TGR contained 37 and 21% of the total cellular receptor, respectively. Lysosomes contained <1% of the ASGPR labeling. The Golgi stack represented only a few percent of the label. The rough endoplasmic reticulum, the nuclear envelope, the mitochondria, and the nuclei were unlabeled. Control sections incubated with antibodies to rat amylase and rat IgA or with protein A-gold alone, gave negligible background values. Effect of Ligand Administration on ASGPR Distribution We compared ligand-naive cells with cells exposed to ligand. For this purpose Hep G2 cells were washed and preincubated for 30-60 min in medium without additional ligand or serum. Thereafter cells were incubated for 40 min at 37°C in the continuous presence of receptor-saturating concentrations of ASOR. Cell morphology was not affected by treatment with ASOR. However receptor distribution was altered. The second column of Table I shows that receptor labeling of the plasma membrane declined from 34 to 20%. Labeling of peripheral coated pits and vesicles was not affected. The receptors lost from the surface appeared to accumulate exclusively on the endocytotic pathway, i.e., 13 % in CURL, of which 5 | 3,068,774 | 16662624 | 0 | 16 |
% was in MVBs. Receptor labeling of the Golgi complex and lysosomes remained unaltered. Cells were washed and precincubated in serum-free medium for 30-60 min at 37°C. ASOR was added to -+ and ++ while the cells were kept at 37°C, 10 min later PMQ was added to +-and ++. Incubation continued for 30 min. Cells were fixed and treated for immunoelectron microscopy. The numbers of gold particles present in the various cellular compartments were counted and expressed as percentages of the total number of gold particles. The values are the mean of three experiments. The limits of confidence are 5 % as determined by progressive mean analysis. Effect of PMQ Treatment on ASGPR Distribution We have previously demonstrated (21) that incubation of Hep G2 cells with PMQ results in a considerable and reversible loss of cell surface ASOR-binding sites. In the present study we incubated cells with 300 gM PMQ for 30 min as described in Materials and Methods and quantitated the receptor redistribution. Morphologically PMQ treatment was manifested in a disappearance of most of the microvilli on the cell surface (Figs. 5 and 6). The resulting change in cell shape, probably from the loss of surface membrane, was accompanied by an impressive increase in the number of intracellular tubules and vesicles, especially in the Golgi region. The Golgi complexes were more irregularly shaped and less contiguous. Golgi cisternae and TGR were often swollen (Figs. 3 and 4). MVBs and lysosomes were enlarged and more numerous. The MVBs showed a more electron-lucent appearance and were | 3,068,775 | 16662624 | 0 | 16 |
negative for cathepsin D. As a result of the increase in the number of tubules and vesicles, the morphological distinction between the tubules of CURL and TGR was less clear than in controls. More frequently than in controls, albumin-negative but receptor-containing CURL tubules occurred in the Golgi region close to the albumin-positive TGR elements. After PMQ incubation the distribution of the ASGPR over the cell organelles changed dramatically as shown in the third column of Table I. ASGPR surface label decreased to <5 % of the control value and a significant decrease of the labeling of peripheral coated pits and vesicles was found. This was accompanied by a considerable increase of ASGPR in TGR from 21% in control cells to 42%. As in control cells, the distribution of ASGPR in TGR was not homogeneous (Fig. 5) and coated buds of TGR were not labeled. Labeling of CURL increased by 10 %. Double labeling with cathepsin D showed that lysosomes were not involved in this increase. Labeling of the Golgi stack had not changed significantly. The combined effects of 300 ~tM PMQ and saturating amounts of ligand on ASGPR distribution are shown in the fourth column of Table I. There was no significant difference in relative distribution of ASGPR between ceils exposed to PMQ alone and cells exposed to both PMQ and ligand. Effects of PMQ on Distribution of ASOR-binding Sites We next determined biochemically the number of ASORbinding sites at the cell surface relative to the total cellular ASOR-binding sites in control cells or cells exposed | 3,068,776 | 16662624 | 0 | 16 |
to either ligand, or PMQ, or both (Table II). In eight independent experiments the surface ASOR-binding sites were found to be 41 + 11% (SD) (range 26-64%) of total cell ASOR-binding sites. As seen in Table II, the total cell receptor content was unaltered by incubation with either 300 gM PMQ, or 200 ~tg/ ml ASOR, or both. However, the amount of cell surface receptor was reduced significantly in all three cases. In the presence of ASOR alone, surface receptor was reduced by 50 to 22 % of total cell receptor. In the presence of PMQ alone, surface receptor was reduced by 65 to 15 % of total cell receptor. Discussion We have recently described the localization of the ASGPR in rat liver hepatocytes and quantified the receptor distribu- Figures 3 and 4. (Fig. 3) Golgi stack (G) and TGR (T) of Hep G2 cell incubated in the presence of 300 ~tM PMQ and saturating concentration of ASOR for 30 min. Label of both ASGPR and albumin are present in the swollen TGR profiles (T). G, Golgi cisternae. Bar, 0.25 lam. (Fig. 4) Like Fig. 3 but single labeled with 10 Ixm gold for the presence of ASGPR to show the intense labeling ofTGR (T) as compared with the Golgi cisternae (G). As in many cells, the Golgi cisternae are swollen as a result of PMQ treatment. Bar, 0.25 I.tm. tion (9). There is a close similarity in distribution between hepatocytes and Hep G2 cells: in hepatocytes 36% of total cellular receptors is present at the | 3,068,777 | 16662624 | 0 | 16 |
cell surface and 39% in Hep G2. CURL contains 31 and 37%, respectively, and the total Golgi complex, including TGR, contains 23 % in both cases. However the amount of receptors present in the Golgi cisternae differ remarkably. In Hep G2 cells, the stacks of Golgi cisternae contain only a small fraction (2%) of the cell's receptors, whereas the Golgi stacks in hepatocytes contain a major pool of receptors (14). A corresponding difference between hepatocytes and Hep G2 cells was found for the mannose-6-phosphate receptor (8, 10). The significance of this difference is presently unknown. We were previously unable to detect an effect of added ligand on ASGPR distribution in rat liver parenchymal cells after infusion with asialofetuin in vivo for 60 min (9). However, as the physiological concentration of ASGPs present in the serum is unknown, it is possible that the rate of ligand administration in vivo was far below that necessary for saturation. This problem has been overcome in the present cell culture system. Using ASOR, which is a higher affinity ligand than asialofetuin (19), we found that the continuous administration of a saturating amount of ligand for 40 min resulted in a 14% decrease of cell surface receptors and a corresponding increase of receptors in CURL. Strikingly, the receptor content of TGR remained unchanged, as was the case for all other organelles. Previous biochemical studies on the ASGPR in Hep G2 cells (4) have shown that the receptors are able to internalize ligand and recycle back to the cell surface every 7 min. | 3,068,778 | 16662624 | 0 | 16 |
In the present study we exposed Hep G2 cells to ASOR for 40 min. Thus most of the receptors on the cell surface had ample time to complete several cycles of ligand internalization. A passage of the 14 % surface receptors through TGR would have lead to an increase in TGR receptors during this time period. Since the receptor content of TGR had not significantly increased, in contrast to CURL, these results suggest that a major fraction of the surface ASGPRs recycle directly from CURL, bypassing the TGR. That a minority of recycling surface receptors travel through TGR may well be possible since a small increase in TGR receptors may have been overlooked. A direct CURL to plasma membrane recycling pathway raises the question as to the possible function of the relatively large fraction of one-fifth of total cellular receptors present in TGR under normal and ligand stimulated conditions. It is unlikely that the TGR receptors only represent newly synthesized molecules in transit to the cell surface. On the basis of life time (tt/2 = 30 h) (20) and transit time (45-60 min) (20) one can estimate that only 2.5-3.5 % of the cell's receptors are present in the entire biosynthetic pathway. The TGR population alone exceeds this figure 10-fold. Therefore, TGR receptors likely belong to other pools. Thus far only the "slowly dissociating pool" of ASGPR-ligand complexes (34) and the "long transit pool" of ASGPR-ligand complexes (26) have been described. Whether these pools are localized to TGR has yet to be investigated. In the present | 3,068,779 | 16662624 | 0 | 16 |
study we wished to compare our morphologic quantitation of receptors with the biochemical determination of ligand binding sites. Previous biochemical studies were limited by the inability to determine total cell receptor content. In the present study we permeabilized cells with saponin and determined the number of cell surface ASORbinding sites relative to total cell-binding sites. We found that the total number of cell receptors remained unchanged under the various conditions considered (Table II). The ratio between cell surface and intracellular ligand-binding sites was similar to the surface to internal receptor ratio determined immunocytochemically. Thus Hep G2 cells expose 40 % of their ASGPRs at the cell surface. Studies on the effects of weak bases such as chloroquine, PMQ, and NHaC1 have shown that these drugs reduce the number of a variety cell surface receptors by inhibiting the rate of receptor recycling from intracellular pools of a number of receptor species (2,13,21,29,30). Kinetic studies demonstrated that these effects are rapidly reversible after removal of the agents (21). This suggested that the receptors accumulate intracellularly in a nonlysosomal compartment. We confirmed the dramatic decrease in the number of cell surface receptors by PMQ treatment of the cells. Accordingly, we found no significant increase in the receptor content of lysosomes that were identified by the presence of cathepsin D in immuno-double labeling. The most striking intracellular accumulation of ASGPRs after PMQ treatment occurred in TGR. As defined in this study, TGR is part of the pathway of biosynthesis and secretion. CURL, including MVBs, displayed a less dramatic accumulation of | 3,068,780 | 16662624 | 0 | 16 |
receptors (Table I). The effect of PMQ appeared not to be influenced by the presence of additional ASOR. Besides its effect on surface and TGR receptors, PMQ induced an impressive de-crease in cell surface area (Figs. 5 and 6) and a simultaneous expansion of TGR. The effects of ligand and PMQ on intracellular receptor distribution thus differed remarkably. Ligand challenge shifted a fraction of the surface receptors to CURL, whereas PMQ treatment almost depleted the cell surface from receptors in favor of TGR in particular. A possible explanation is that PMQ not only inhibited receptor recycling from CURL but also induced surface receptors and/or receptors in CURL to be transferred to TGR. If so, receptor recycling from TGR is apparently blocked as well, resulting in a receptor accumulation in TGR and an extension of this compartment. Whether such a connection between the endocytotic and biosynthetic pathways exists under normal conditions is currently under study in our laboratory. It remains to be established whether the PMQ effects were the result of neutralization of acidic compartments or whether additional factors were involved. | 3,068,781 | 16662624 | 0 | 16 |
Angiotensin-(1–7) Expressed From Lactobacillus Bacteria Protect Diabetic Retina in Mice Purpose A multitude of animal studies substantiates the beneficial effects of Ang-(1–7), a peptide hormone in the protective axis of the renin angiotensin system, in diabetes and its associated complications including diabetic retinopathy (DR). However, the clinical application of Ang-(1–7) is limited due to unfavorable pharmacological properties. As emerging evidence implicates gut dysbiosis in pathogenesis of diabetes and supports beneficial effects of probiotics, we sought to develop probiotics-based expression and delivery system to enhance Ang-(1–7) and evaluate the efficacy of engineered probiotics expressing Ang-(1–7) in attenuation of DR in animal models. Methods Ang-(1–7) was expressed in the Lactobacillus species as a secreted fusion protein with a trans-epithelial carrier to allow uptake into circulation. To evaluate the effects of Ang-(1–7) expressed from Lactobacillus paracasei (LP), adult diabetic eNOS−/− and Akita mice were orally gavaged with either 1 × 109 CFU of LP secreting Ang-(1–7) (LP-A), LP alone or vehicle, 3 times/week, for 8 and 12 weeks, respectively. Results Ang-(1–7) is efficiently expressed from different Lactobacillus species and secreted into circulation in mice fed with LP-A. Oral administration of LP-A significantly reduced diabetes-induced loss of retinal vascular capillaries. LP-A treatment also prevented loss of retinal ganglion cells, and significantly decreased retinal inflammatory cytokine expression in both diabetic eNOS−/− and Akita mice. Conclusions These results provide proof-of-concept for feasibility and efficacy of using engineered probiotic species as live vector for delivery of Ang-(1–7) with enhanced bioavailability. Translational Relevance Probiotics-based delivery of Ang-(1–7) may hold important therapeutic potential for the | 3,068,782 | 229298312 | 0 | 16 |
treatment of DR and other diabetic complications. Introduction Diabetic retinopathy (DR) is a major microvascular complication of diabetes mellitus and the leading cause of blindness in the working-age population. 1,2 A large body of experimental and clinical evidence has demonstrated that dysregulation of the renin angiotensin system (RAS), resulting in elevated concentrations of Angiotensin II (Ang II) systemically and locally at tissue level, contributes to increased oxidative stress, inflammation, development of metabolic syndrome, obesity, diabetes, and its associated complications. 3−7 Angiotensin-(1-7) (Ang-(1-7)) is a peptide hormone in the protective axis of the RAS, generated through cleavage of Ang II by the angiotensin converting enzyme 2 (ACE2). 8,9 Ang-(1-7) binds to the G protein coupled receptor Mas and activate signaling pathways that counteract the deleterious effects of Ang II. 10−12 Ang-(1-7) has been shown beneficial in improving metabolic dysfunction and diabetic complications by inhibiting apoptosis, oxidative stress, and inflammation, 12−17 and is also protective in diabetic retina in rodents. 18 However, clinical application of Ang-(1-7) is limited due to its extreme short half-life and rapid clearance in circulation and tissues. 19,20 As emerging evidence indicates that impaired gut microbiota contributes to the pathogenesis of both type 1 and type 2 diabetes 21−27 and probiotic supplements are beneficial in managing diabetes and other metabolic diseases, 28−34 we sought to develop an expression and delivery system based on the use of recombinant probiotic species of Lactobacillus bacteria to serve as a live vector for the oral delivery of Ang-(1-7) and evaluate the efficacy of recombinant probiotics expressing Ang- (1)(2)(3)(4)(5)(6)(7) | 3,068,783 | 229298312 | 0 | 16 |
in improving diabetes-induced retinal damage in animal models of diabetes. Lactobacillus bacteria are components of the normal gut microbiota 35 and are also commonly used in production of fermented food and beverages in the food industry. Many species of Lactobacillus bacteria are also used as probiotic supplements with beneficial effects in humans. 36−38 As ingested bacteria can survive both gastric acid and bile to reach the small intestine and colon, where they exert their effects, making them ideal vehicle for delivery of protein and peptide drugs. We generated an expression system based on the use of recombinant Lactobacillus paracasei (LP) to serve as a live vector for the oral delivery of the Ang-(1-7) using the strategy that we have reported previously. 39 The Ang-(1-7) peptide is expressed as a secreted fusion protein with the chorea toxin binding protein subunit B (CTB), which facilitates the transmucosal transport into circulation and tissue uptake by GM1 receptor mediated endocytosis. We show that oral administration of LP expressing Ang-(1-7) significantly attenuated diabetesinduced loss of retinal capillaries and retinal ganglion cells (RGCs), and reduced the inflammatory cytokine expression in diabetic animals. These results provide proof-of concept for the feasibility of using recombinant probiotic species as live vector for delivery of Ang-(1-7) and this approach may have important therapeutic potential for treating metabolic diseases and diabetic complications. Vector Construction to Express Secreted Ang-(1-7) in Lactobacillus Species and In Vivo Characterization in Mice As reported previously, 39 the backbone Lactobacillus shuttle plasmid containing a GFP reporter gene driven by the lactate dehydrogenase (LDH) | 3,068,784 | 229298312 | 0 | 16 |
promoter from Lactobacillus acidophilus was from Addgene (Plasmid #27167). 40 The original GFP reporter gene was replaced by a synthetic gene -7) expression is under the control of the LDH promoter and expressed as a secreted fusion protein to the non-toxic subunit of cholera toxin B (CTB), separated by a furin cleavage site. Ang-(1-7) is efficiently expressed and secreted into circulation in mice fed with these bacteria. N = 4. *P < 0.001 (versus unfed control). Error bars represent standard deviation. construct in which the Ang-(1-7) peptide is expressed as a secreted fusion protein with the CTB (Fig. 1A), which facilitates transepithelial transport into circulation and tissue uptake. 39 The CTB is separated by a furin cleavage site to release Ang-(1-7) once it is secreted. The expression of fusion protein (CTB-Ang-1-7) in Lactobacillus strains was confirmed by Western blotting (data not shown). The ability of the Lactobacillus-expressed proteins to enter the circulation and uptake by different tissues following oral administration in mice was evaluated by ELISA. Six week old C57Bl/6J mice were orally fed with Lactobacillus expressing Ang-(1-7) at 1 × 10 10 cfu/mouse daily for 3 days. Mice were then euthanized, and serum and tissue samples were collected 6 hours after the last oral gavage. Ang-(1-7) levels were determined by a commercial enzyme immunoassay (EIA) kit (Peninsula Laboratories International, Inc., San Carlos, CA). As shown in Figure 1B, serum levels of Ang-(1-7) in mice fed with these bacteria were approximately five-fold higher than unred control animals, suggesting that Ang-(1-7) is efficiently expressed from three probiotic | 3,068,785 | 229298312 | 0 | 16 |
species and secreted into circulation. Oral Administration of Recombinant L. paracasei Expressing Ang-(1-7) did not Affect Body Weight Oral feeding of L. paracasei expressing Ang-(1-7) (LP-A) did not have any effects on blood glucose levels (data not shown). Non-diabetic animals gained approximately 20% body weight during the period of the study. The body weight was significantly reduced in all diabetic animals. Treatment with LP or LP-A did not affect the body weight (Fig. 2). Oral Administration of Recombinant L. paracasei Expressing Ang-(1-7) (LP-A) Prevents Diabetes-Induced Retinal Capillary Loss in Mice The protective effect of Ang-(1-7) in DR has been demonstrated previously using AAV vector mediated gene delivery. 18 To evaluate the efficacy of Ang-(1-7) expressed from LP-A in diabetic retinopathy, we used two mouse models of diabetes: the streptozotocin (STZ)-induced diabetes in eNOS −/− mice, which develop an accelerated time course and increased severity of retinopathy; 18,39,41 and the Akita mice, which carry a mutation in the insulin 2 gene resulting in mice exhibiting reduced ß cell mass and reduced insulin secretion. 42 The Akita mice develop progressive retinal abnormalities, including increased vascular permeability, apoptosis, and inner retinal thinning as early as 12 weeks after the onset of hyperglycemia. 43 Mice were gavaged 3 times/week with either 1 × 10 9 CFU of LP-A, wild-type bacteria (LP), or vehicle (PBS) for 8 weeks after STZ-induced diabetes in eNOS −/− mice. The Akita mice were treated with the same dose for 12 weeks beginning at 6 weeks of age. Ang-(1-7) level in serum and retinal samples collected | 3,068,786 | 229298312 | 0 | 16 |
at the end points was measured by EIA using a commercial kit. Both serum and retinal Ang-(1-7) levels were significantly reduced in diabetic eNOS −/− and Akita mice compared with non-diabetic controls and were restored to normal levels by LP-A treatment (Fig. 3). Diabetes resulted in increased capillary loss in eNOS −/− (Fig. 4A) as reported previously. 18,39,41 Akita mice also showed increased retinal acellular capillaries compared with age-matched littermate controls (Fig. 4C). LP-A treatment significantly reduced the number of acellular capillaries in both diabetic eNOS −/− (approximately 29% reduction) and Akita (approximately 40% reduction) mice compared with untreated diabetic animals (Fig. 4). Wild type LP alone also showed small but insignificant reduction of capillary loss in both diabetic eNOS −/− and Akita mice. Oral Administration of L. paracasei Ang-(1-7) Reduce Diabetes-Induced Retinal Ganglion Cell Loss In additional to microvascular change, diabetic retina showed considerable loss of retinal ganglion cells (RGCs), as detected by Brn3a immunostaining, a specific marker for RGCs, 44 in both diabetic eNOS −/− and Akita mice (Fig. 5), as reported previously. 39 RGCs loss also occurs in human patients with diabetes as well as other diabetic rodents. 45−50 LP-A treatment, but not wild-type LP, prevented RGCs loss in both diabetic eNOS −/− and Akita mice (Fig. 5). Oral Administration of L. paracasei-Ang-(1-7) Reduces Diabetes-Induced Expression of Inflammatory Cytokines in Diabetic Retina in Mice Diabetes is associated with increased inflammatory responses in the retina. The expression level of proinflammatory cytokines and chemokines was evaluated by real-time RT-polymerase chain reaction (PCR) in the retina | 3,068,787 | 229298312 | 0 | 16 |
from each experimental group. LP-A treated animals show significantly decreased retinal expression of all these cytokines and chemokines in both diabetic eNOS −/− mice (Fig. 6A) and Akita mice (Fig. 6B). LP alone did not show any effect on the expression of these genes in diabetic eNOS −/− mice (Fig. 6A), however, the expression levels of MCP-1 and VEGF were slightly but significantly reduced in Akita mice (Fig. 6B). There was also a slight decrease in ICAM-1 expression in LP treated Akita mice, but not statistically significant. The expression of Iba-1, a marker of microglia, was also elevated in diabetic retina of both eNOS −/− and Akita mice, and was significantly reduced by LP-A treatment but not affected by LP alone (Fig. 6). Elevated expression of Iba-1 in diabetic retina was also detected by immunofluorescence. Diabetic eNOS −/− mouse retina showed increased number of Iba-1 positive microglial cells, which was significantly reduced in LP-A treated mice (Fig. 7). Bacterial Strains and Growth Conditions The Lactobacillus strains used in this study were from American Type Culture Collection (ATCC, Manassas, VA): L. paracasei (LP) (ATCC 27092), L. gasseri (ATCC 4963), and L. plantarum (ATCC 8014) and were cultured in de Man, Rogosa, and Sharpe (MRS) broth (Thermo Fisher Scientific, #DF0881-17-5) at 37°C for 18 hours without shaking. The plasmid pTRKH3-ldhGFP (Addgene, plasmid #27170) was used as a backbone for cloning of secreted Ang-(1-7) in fusion with the CTB, which allows for uptake of the protein into the enterocytes via its binding to the GM1 receptor. A mutant form | 3,068,788 | 229298312 | 0 | 16 |
of CTB, which retains the binding to GM1-ganglioside for cellular uptake but lacks immunomodulatory and toxic activity 51,52 was used as reported previously 39 to construct the fusion construct. The resulting plasmid was electroporated into different Lactobacillus species by electroporation as described by Welker et al. 53 Recombinant Lactobacillus bacteria expressing Ang-(1-7) were grown in the MRS media supplemented with 5 μg/mL erythromycin (Sigma-Aldrich, St. Louis, MO). For oral gavage of mice, bacteria were harvested by centrifugation at 5,000 × g for 20 minutes and resuspended in sterile PBS. Animals and Experimental Procedures Wild-type C57Bl/6J (Stock Number: 000664), eNOS −/− (Stock Number: 002684) and Akita mice (Stock Number: 003548) were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained in the Animal Care Service at the University of Florida. All procedures adhered to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research, and the protocol was approved by the Animal Care and Use Committee of the University of Florida. The animals were fed standard laboratory chow and allowed free access to water in an air-conditioned room with a ELISA Analysis Serum and retinal level of Ang-(1-7) was measured using a commercial EIA kit (Bachem, San Carlos, CA), following the manufacturer's instructions. All measurements were performed in duplicate and the data represent the mean of two separate assay results. Retinal Vascular Preparation by Trypsin-Digestion Retinal vasculature was prepared using trypsin digest, as described previously. 41 Briefly, eyes were fixed in 4% paraformaldehyde freshly made in PBS overnight. Retinas were dissected out from the | 3,068,789 | 229298312 | 0 | 16 |
eyecups and digested in 3% trypsin (GIBCO-BRL) for 2-3 hours at 37°C. Retinal vessels were separated from other retinal neuronal cells by gentle shaking and manipulation under a dissection microscope. The vessels were then mounted on a clean slide, allowed to dry, and stained with Periodic Acid Solution hematoxylin and eosin, Gill No. 3 (PAS-H&E; , Sigma, St. Louis, MO) according to the instruction manual. After staining and washing in water, the tissue was dehydrated and mounted using Permount mounting media (Sigma). Immunofluorescence and Immunocytochemistry For immunofluorescence studies, eyes were fixed in 4% paraformaldehyde overnight at 4°C and subsequently processed for either quick freezing in optical cutting temperature (OCT) compound or paraffin embedding. For OCT embedding, the eyes were cryoprotected in 30% sucrose/PBS for several hours or overnight before quick freezing in OCT compound, then 12-μm-thick sections were cut at −20 to -22 degrees. For paraffin embedded eyes, 4 μm-thick paraffin sections were cut and mounted on Superfrost Plus slides. The paraffin sections were first deparaffinized followed by antigen retrieval in low pH citric acid buffer for 20 minutes. The sections were then incubated in blocking solution (5% BSA + 0.3% Triton X-100 in PBS) for 1 hour. This was followed by incubation overnight at 4°C with primary antibody (mouse anti-Brn3a, 1:200, MAB1585; Millipore, Billerica, MA). Iba-1 (1:500, 019-19741; Wako, Richmond, VA) immunostaining was performed using OCTembedded frozen sections without antigen retrieval. After washing, secondary antibody conjugated to Alexa 488 (Molecular Probes/Invitrogen, Carlsbad, Real Time RT-PCR analysis Total RNA was isolated from freshly enucleated eyes using | 3,068,790 | 229298312 | 0 | 16 |
Trizol Reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Reverse transcription was performed using Enhanced Avian HS RT-PCR kit (Sigma-Aldrich, Inc., St. Louis, MO) following manufacturer's instructions. Real time PCR was carried out on real time thermal cycler (iCycler; Bio-Rad Life Sciences, Hercules, CA) using iQTM Sybr Green Supermix (Bio-Rad Life Sciences). The threshold cycle number (Ct) for real-time PCR was set by the cycler software. Optimal primer concentration for PCR was determined separately for each primer pair. Each reaction was run in duplicate or in triplicate, and reaction tubes with target primers and those with Actin primers were always included in the same PCR run. Primer sequences used in this study are shown in the Table. All the reactions were repeated at least twice. Statistical Analysis Data are expressed as the mean + SD of at least two independent experiments. Differences between mean values of multiple groups were analyzed by 1-way ANOVA with Dunnett's test for post hoc comparisons. A P value < 0.05 was considered statistically significant. Discussion Dysregulation of RAS, resulting in elevated Ang II, contributes diabetes and diabetic complications, including DR. The protective axis of RAS, involving ACE2/Ang-(1-7)/Mas, opposes these effects by degradation of Ang II to generate Ang-(1-7), which binds to a G-protein coupled receptor, Mas, and activates signaling pathways that counteract the effects of Ang II. 10−12 We have previously demonstrated that increased expression of ACE2 or Ang-(1-7) diminishes diabetes-induced retinal pathophysiology 18,54 and ocular inflammation 55−57 ; providing the "proof-ofprinciple" that enhancing the ACE2/Ang-(1-7) axis is a promising approach | 3,068,791 | 229298312 | 0 | 16 |
for treating DR. Moreover, a large number of studies have shown that enhanced expression of Ang-(1-7) and ACE2 reduces inflammation 58−61 and oxidative damage, 62−65 increases glucose uptake, 63 improves lipid and glucose metabolism, 66,67 ameliorates insulin resistance and dyslipidemia, 14,67−69 improves pancreatic β-cell function, 70−72 improves the reparative function of dysfunctional endothelial cells and progenitors, 73−75 and confers protection against a variety of pathological conditions including diabetesinduced nephropathy 76−83 and cardiovascular dysfunction. 83−88 Despite the mounting evidence for beneficial effects of Ang-(1-7), its clinical studies and applications are limited, largely due to extreme short halflife and rapid clearance in circulation and tissues, 19,20 making it challenging to develop a pharmaceutical composition of Ang-(1-7) that delivers the peptide to target tissues with sufficient bioavailability. In this study, we designed an expression and delivery system based on the use of Lactobacillus bacteria to serve as a live vector for the oral delivery of Ang-(1-7) peptide. Using the similar strategy as reported previously, 39 the expression of Ang-(1-7) is driven by the LDH promoter from Lactobacillus acidophilus, a strong promoter that is active in different bacterial hosts. 40,89 The peptide is expressed as a secreted fusion protein with the CTB, which facilitates the transmucosal transport into circulation and tissue uptake by monosialotetrahexosylganglioside (GM1) mediated endocytosis. The Usp45 secretion signal peptide, CTB, and furin cleavage site enable Ang-(1-7) to be secreted into the gut lumen and its transepithelial transport into circulation as described previously. 39 We show that the Ang-(1-7) peptide is efficiently expressed from three different Lactobacillus species | 3,068,792 | 229298312 | 0 | 16 |
and secreted into circulation in mice fed with these bacteria. Treatment with L. paracasei expressing Ang-(1-7) reduced diabetes-induced increase in retinal acellular capillaries, prevented RGC loss and decreased inflammatory cytokine expression in the retina in both diabetic eNOS −/− and Akita mice. We sought to develop probiotics-based expression and delivery system to enhance Ang-(1-7) based on the following rationale. First, diabetes inflicts multiple tissues including the retina and Ang-(1-7) has been shown to be beneficial in many tissues. Second, increasing evidence implicates the gut and its microbiota in pathogenesis of diabetes and its associated complications, 21−24,26,90−104 including DR, 105−107 and probiotics are beneficial in management of diabetes. 108−112 The probiotic-based delivery of Ang-(1-7) offers many advantages. First, probiotics have been consumed by humans for thousands of years, are generally recognized as safe (GRAS) to consume and offer many beneficial effects on their own. 113 Probiotics are known to promote host health by modulating immune system, interfere with potential pathogens, improving intestinal barrier function, positively modulating the composition and function of the commensal microbiota, as well as many other protective actions. 114−121 Second, ingested probiotics can survive both gastric acid and bile to reach the small intestine and colon, where they exert their effects, making them an ideal vehicle for delivery of protein drugs. Third, unlike conventional approaches, the probiotics-based oral delivery system, using a carrier fused with the therapeutic protein, facilitates efficient transmucosal transport into the circulation, increases half-life and target tissue uptake, thus enhancing bioavailability. 39,122,123 Moreover, probiotic bacteria are inexpensive to produce and oral | 3,068,793 | 229298312 | 0 | 16 |
delivery of therapeutics is patient-friendly, thus probioticsbased approach is more cost-effective. Thus, a probiotic enriched with Ang-(1-7) represent potential therapy to improve metabolism, and intestinal and immune functions, thereby preventing DR and other diabetic complications. Our results provide proof-of concept for feasibility using probiotics-based oral delivery of Ang-(1-7) with enhanced bioavailability and efficacy in two mouse models of DR. In current study, we have used LP expressing Ang-(1-7) to test the efficacy in animal models of DR. The rationale for choosing LP is that, first, it is part of the healthy gut microbiota 124 and is widely used as a probiotic 125−127 and, second, LP has been shown to be beneficial in colitis-induced gut inflammation and barrier dysfunction, 128−130 as well as improving diabetes in experimental models. 131−134 LP-A treatment protected development of DR in both models. However, wild-type LP alone did not show protective effects, as reported previously, 39 this is likely due to the strain used in this study. A number of experimental and clinical studies has demonstrated that probiotics supplements of several Lactobacillus species (including LP) are beneficial in management of both type 1 and type 2 diabetes and other metabolic diseases; however, these various beneficial effects are strain and species-specific. 109 132 It would be interesting to determine whether Ang-(1-7) expressed from this strain would provide better protection against DR. Moreover, our results showed that Ang-(1-7) can be expressed from several species of Lactobacillus, each of which may offer different beneficial effects, future studies will be required to determine whether specific probiotics | 3,068,794 | 229298312 | 0 | 16 |
species or strains can be selected for oral delivery to optimally target specific patient populations to achieve personalized precision medicine paradigm. It is interesting to note that although LP alone did not provide significant protection against diabetes-induced loss of retinal capillaries and RGCs, but it did slightly reduce the expression of some inflammatory cytokines (e.g. MCP-1 and VEGF), but not other cytokines measured in this study. The reduction of these cytokines was only detected in Akita mice but not in diabetic eNOS −/− . This likely due to the fact that diabetic eNOS −/− mice not only develop more severe retinopathy, but also other tissue damage and impaired immune functions compared with Akita mice. Ang-(1-7) has been shown to be beneficial under various pathologic conditions in various tissues, including diabetes and diabetic complications, by improving metabolism and insulin sensitivity, inhibiting apoptosis, oxidative stress, and inflammation. 12−17 Here, we showed that Ang-(1-7) is increased in both circulation and retina in mice fed with LP-A, suggesting that the protective effects of LP-A in diabetic retina may be mediated by both local effects of Ang- (1)(2)(3)(4)(5)(6)(7) in the retina as well as general beneficial effects in other tissues of diabetic animals. It is also possible that LP-A may provide protective effects by modulating gut microbiome. A previous study showed that oral administration of LP-A in rats increased abundance of gut Akkermansia muciniphila (A. muciniphila). 123 As decreased abundance of A. muciniphila has been associated with increased prevalence of metabolic disorders, such as obesity and type 2 diabetes, 135−137 such | 3,068,795 | 229298312 | 0 | 16 |
increase in A. muciniphila by LP-A treatment, which remains to be confirmed in diabetic mice by ongoing study, may provide additional beneficial effects in diabetic animals. More comprehensive studies will be required to further understand the effects and underlying mechanisms of LP-A treatment in various tissues. In conclusion, Ang-(1-7) is efficiently expressed from different probiotic species and secreted into circulation in mice fed with these bacteria. Oral administration of L. paracasei secreting Ang-(1-7) provides protection against diabetes-induced DR. Thus, LPbased delivery of Ang-(1-7) may hold important therapeutic potential for the treatment of DR and other complications. | 3,068,796 | 229298312 | 0 | 16 |
Lateral electromigration and diffusion of Fc epsilon receptors on rat basophilic leukemia cells: effects of IgE binding. We have used in situ electromigration and post-field relaxation (Poo, M.-m., 1981, Annu. Rev. Biophys. Bioeng., 10:245-276) to assess the effect of immunoglobulin E (IgE) binding on the lateral mobility of IgE-Fc receptors in the plasmalemma of rat basophilic leukemia (RBL) cells. Bound IgE sharply increased the receptor's electrokinetic mobility, whereas removal of cell surface neuraminic acids cut it to near zero. In contrast, we found only a small difference between the lateral diffusion coefficients (D) of vacant and IgE-occupied Fc receptors (D: 4 vs. 3 X 10(-10) cm2/s at 24 degrees C). This is true for monomeric rat IgE; with mouse IgE, the difference in apparent diffusion rates was slightly greater (D: 4.5 vs. 2.3 X 10(-10) cm2/s at 24 degrees C). This range of D values is close to that found in previous photobleaching studies of the IgE-Fc epsilon receptor complex in RBL cells and rat mast cells. Moreover, enzymatic depletion of cell coat components did not measurably alter the diffusion rate of IgE-occupied receptors. Thus, binding of fluorescent macromolecular probes to cell surface proteins need not severely impede lateral diffusion of the probed species. If the glycocalyx of RBL cells does limit lateral diffusion of the Fc epsilon receptor, it must act primarily on the receptor itself, rather than on receptor-bound IgE. M-m. Poo, unpublished results). Using PFR, Tank (8) found that uncomplexed low-density lipoprotein receptors on an internalization-defective human fibroblast cell line diffuse at 1.1 | 3,068,797 | 7602999 | 0 | 16 |
x 10 -9 cm2/s; but using FRAP a D value of 1.4 x 10 -~ cm2/s was observed for the low-density lipoprotein receptor complex (9). The goal of our present work was to use one technique, namely, PFR, to compare the diffusion coefficients of one receptor in ligand-free and ligand-bound states. The choice of IgE-Fc receptors on rat basophilic leukemia (RBL) cells offers two advantages in this regard. First, the lateral diffusion rates of IgE-Fc receptor complexes on RBL cells (and rat mast cells) have already been measured with FRAP (2, 10), so one can compare the results from two different methods for one receptor on one cell type. Second, the interaction of IgE with its receptor is strong yet univalent. By using multimer-free IgE one can therefore maintain a high molecular weight (185,000) ligand for long periods on the cell surface without inducing receptor cross-linkage or the active cellular responses of anchorage modulation and internalization that can attend the binding of lectins, F(ab')2 fragments, or other multivalent ligands. The present study therefore provides a direct test of the idea that nonspecific interaction of a macromolecular label (IgE) with the cell coat retards lateral diffusion of the IgE-bound Fc receptor. Cells: The 2H3 sublin¢ of RBL-IV cells (1 l) was provided by Dr. Henry Metzger, National Institutes of Health. Cell monolayers were grown and passaged as described in (12). Stock cultures were passaged weekly into 75-cm 2 flasks (Coming Glass Works, Coming, NY), and at this time aliquots of 1.5 x l06 cells were seeded into | 3,068,798 | 7602999 | 0 | 16 |
6-cm-diam tissue culture dishes (Coming), using the same medium as for the stock culture. In pilot experiments, we observed a systematic increase in the starting asymmetry index (see below) with culture age; in the diffusion studies we therefore only used cultures 3-4 d old. The medium was decanted, and the monolayer was rinsed twice in liquid policeman (0.6 mM Na2 EDTA, 0.14 M NaCl, 2.7 mM KCI, 9.5 mM NaKP~, pH 7.2), and incubated for 12 rain at 37°C in 4 ml of the same buffer. Cells were dislodged with a gentle stream from a pasteur pipette, diluted to 25-50 ml with Tyrode's buffer (0.137 M NaCl, 2.7 mM KCI, 1.5 mM CaCl2, 0.4 mM P~, l mM MgCl2, 5.6 mM glucose, 0.2% BSA, 0.02 M HEPES, pH 7.4) at room temperature, and harvested by centrifugation for 4-5 min at 200 g in polypropylene tubes. For pre-field labeling experiments cell pellets were resuspended in Tyrode's plus 0.2% BSA (Sigma Chemical Co., St. Louis, MO), and for post-field labeling in cell buffer with azide (CBA) (10 mM HEPES, 28 mM NaNs, 1 mM NaPi, 109 mM NaCl, 2 mM CaCl2, 1.5 mM MgCl2, 5.4 mM KCI, 0.1% glucose, pH 7.3). Cell viability was checked with trypan blue, and cells were diluted to 1-2 x 106/ml and then either plated directly in electromigration chambers (postfield labeling) or labeled in suspension with fluorescent lgE (pre-field labeling). In the former case cells were used within 2 h of isolation, and most data is for those used within 30 min. In | 3,068,799 | 7602999 | 0 | 16 |
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