Genomic epidemiology of superspreading events in Austria reveals mutational dynamics and transmission properties of SARS-CoV-2.
Superspreading events shaped the coronavirus disease 2019 (COVID-19) pandemic, and their rapid identification and containment are essential for disease control. Here, we provide a national-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading during the first wave of infections in Austria, a country that played a major role in initial virus transmissions in Europe. Capitalizing on Austria's well-developed epidemiological surveillance system, we identified major SARS-CoV-2 clusters during the first wave of infections and performed deep whole-genome sequencing of more than 500 virus samples. Phylogenetic-epidemiological analysis enabled the reconstruction of superspreading events and charts a map of tourism-related viral spread originating from Austria in spring 2020. Moreover, we exploited epidemiologically well-defined clusters to quantify SARS-CoV-2 mutational dynamics, including the observation of low-frequency mutations that progressed to fixation within the infection chain. Time-resolved virus sequencing unveiled viral mutation dynamics within individuals with COVID-19, and epidemiologically validated infector-infectee pairs enabled us to determine an average transmission bottleneck size of 103 SARS-CoV-2 particles. In conclusion, this study illustrates the power of combining epidemiological analysis with deep viral genome sequencing to unravel the spread of SARS-CoV-2 and to gain fundamental insights into mutational dynamics and transmission properties.


Women and other risk factors for chronic kidney disease of unknown etiology in Thailand: National Health Examination V Survey.
There are limited data on chronic kidney disease of unknown etiology (CKDu) from Southeast Asia. Initially described in working age men, a common approach to detect CKDu that includes all adults has recently been proposed. We determined the prevalence, and risk factors for CKDu using data from a cross-sectional, nationally representative survey of the adult population of Thailand. We used a proxy for CKDu as age < 70 with impaired kidney function (eGFR < 60) in the absence of diabetes and hypertension (CKDu1) and heavy proteinuria (CKDu2). Prevalence estimates were probability-weighted for the Thai population. The associations between risk factors and CKDu or elderly subjects with eGFR < 60 without traditional causes were assessed by multivariable logistic regression. Of 17,329 subjects, the prevalence were: eGFR < 60, 5.3%; CKDu1 0.78%; CKDu2, 0.75%. CKDu differed by 4.3-folds between regions. Women, farmers/laborers, older age, gout, painkillers, rural area, and stones were independent risk factors for CKDu. Women, age, rural, gout, painkillers were significant risk factors for both CKDu and elderly subjects. These data collected using standardized methodology showed that the prevalence of CKDu in Thailand was low overall, although some regions had higher risk. Unlike other countries, Thai women had a two-fold higher risk of CKDu.


Impairment of male rat reproductive function in F1 offspring from dams exposed to 2-bromopropane during gestation and lactation.
The toxic effects of 2-bromopropane (2-BP) on the reproductive tracts of male F1 offspring from dams exposed to 2-BP during gestation and lactation were investigated. Ten pregnant (sperm-positive) Sprague-Dawley rats per group were exposed sc to 2-BP at 135, 405, and 1215 mg/kg/day from gestation day (GD) 6 to postnatal day (PND) 20. 2-BP decreased the proportion of dams littering at the two highest doses. At the highest dose, the rate of delivery and surviving pups were significantly lower than in the controls (P < or = 0.05). The relative weights of testes vs. brain were significantly lower than the controls (P < or = 0.05) on PND 33 and 63 at 405 mg/kg/day, and on PND 90 at 1215 mg/kg/day in the F1 rats. Seminiferous tubule atrophy, germ cell loss, and increased Leydig cell proliferation were observed at the highest dose by histopathologic examination. Female offspring has a decrease in all follicle types at the high dose. These results suggest that gestational and lactational exposure to 2-BP at a high maternally toxic dose impairs the development of the reproductive organs of the offspring.


Effectiveness of pharmacist intervention for deprescribing potentially inappropriate medications: a prospective observational study.
Potentially inappropriate medications (PIMs) and polypharmacy in older adults lead to increase the risk of adverse drug events. This study aimed to evaluate the effectiveness of pharmacist intervention combining the criteria for detecting PIMs with the deprescribing algorithm on correcting PIMs, reducing the number of medications, and readmissions. A prospective observational study was conducted at a Japanese University Hospital enrolling new inpatients aged ≥65 years prescribed ≥1 daily medication. Pharmacists detected PIMs based on the criteria combined the screening tool of older persons' potentially inappropriate prescriptions criteria version 2 with the screening tool for older persons' appropriate prescriptions for Japanese, examined changes using the deprescribing algorithm, and suggested changes to the physician. The proportion of patients whose number of medications was reduced at discharge and the rate of readmissions within 30 and 90 days were compared between patients without PIMs (without PIMs group), patients who were not suggested to change PIMs (no suggestions group), and patients who were suggested to change PIMs (suggested group). The study enrolled 544 patients (median age 75.0 years, 54.4% males, median number of medications 6.0/patient). The number of patients with PIMs was 240 (44.1%), and 304 patients had no PIMs (without PIMs group). Among the patients with PIMs, 125 (52.1%) patients received pharmacist suggestions to change ≥1 PIMs (suggested group), and 115 patients received no suggestions for change (no suggestions group). The total number of PIMs was 432, of which changes were suggested for 189 (43.8%). Of these 189 cases, 172 (91.0%) were changed. The proportion of patients whose number of medications was reduced was significantly higher in the suggested group than in the without PIMs group and the no suggestions group [56.8% (71/125) vs. 26.6% (81/304) and 19.1% (22/115), respectively; P < 0.001 in both comparisons]. There were no significant differences in the rates of readmissions within 30 and 90 days among the three groups. Pharmacist intervention combining the criteria for detecting PIMs with the deprescribing algorithm was effective for correcting PIMs and may be associated with a reduction in the number of medications.


A computerized approach to statistical quality control for radioimmunoassays in the clinical chemistry laboratory.
Methods for statistical quality control for the clinical laboratory in general, and radioimmunoassay in particular, have been proposed for many years. Unfortunately, only a very small number of laboratories have adapted these procedures. By use of teletypes and other remote terminals, it is possible for all laboratories to access centralized computers where a general purpose quality control program can be stored. This relieves each laboratory of the costly task of developing software, provides some degree of inter-laboratory standardization and facilitates comparison of precision and accuracy between laboratories. A prototype program for this purpose is described. This program evaluates within-assay and between-assay variability, by means of an analysis of variance for a one-way classification random-effects model, and can monitor any assay parameter by use of control chart techniques. In addition, several tests are provided to evaluate the temporal stability of the assay system, and appropriate tests for outliers are included. Also, methods are described for combination of information from several quality control samples. This provides a valid basis for adjustment of assay results or for outright rejection of an assay. For convenience, this program is designed for output on a teletype or similar terminal located in the laboratory. Simplified versions of this program can be readily adapted to desk-top calculators. The original purpose for developing this system was to provide the clinical laboratory with a simple, general, and flexible method for assessing the performance of radioimmunoassays, but its usefulness should extend to virtually all assay methods.


Early captopril treatment inhibits DNA synthesis in endothelial cells and normalization of maximal coronary flow in infarcted rat hearts.
Cardiac remodeling due to myocardial infarction (MI) includes myocyte hypertrophy, collagen deposition, a rise in DNA synthesis, and normalization of initially diminished maximal coronary bloodflow. Previously, we demonstrated that early captopril treatment can prevent the rise in total DNA synthesis, collagen deposition and hypertrophy. In the present experiments, we investigated the effects of captopril or perindoprilat treatment on cardiac endothelial cell proliferation and maximal coronary flow. MI was induced by ligation of the left coronary artery in Wistar rats. Sham-operated and infarcted rats were treated with captopril (12 mg/kg.d s.c.) from either day 0-21 (early) or day 21-35 (late) after surgery. In isolated retrogradely perfused rat hearts, maximal coronary flow was determined following maximal dilatation with nitroprusside and adenosine (1 mM each). In separate groups, sections of hearts of sham-operated and MI rats treated with BrdU (day 7-14) and either captopril or perindoprilat (1 mg/kg.d s.c.; day 0-14) were double stained with a monoclonal anti-BrdU antibody and the lectin GSI. The total fraction of DNA synthesizing cells and its proportion of endothelial cells was determined. Maximal coronary flow was completely normalized in MI hearts within three weeks after surgery. Early captopril, but not late captopril, inhibited the normalization of maximal coronary flow in MI hearts (Early: sham, 27.4 +/- 1.0; MI, 21.2 +/- 1.4 ml/min; P < 0.05; mean +/- SEM) without affecting the hypertrophic response. The total fraction of DNA synthesizing cells was significantly increased in MI hearts (sham: 7.6 +/- 1.9; MI: 14.9 +/- 2.2%). The proportion of endothelial cells, however, was comparable in sham-operated and infarcted hearts (sham: 30 +/- 3; MI: 33 +/- 3%). Both early captopril and perindoprilat treatment inhibited total DNA synthesis in MI hearts. Only in captopril pre-treated hearts, this inhibition was associated with a disproportionate inhibition of the endothelial cell proliferation (10.3 +/- 2.0%). Early captopril treatment inhibits endothelial cell proliferation and coronary vessel growth following MI, which seems to be partly due to inhibition of the renin angiotensin system.


Integration of an Intensive Care Unit Visualization Dashboard (i-Dashboard) as a Platform to Facilitate Multidisciplinary Rounds: Cluster-Randomized Controlled Trial.
Multidisciplinary rounds (MDRs) are scheduled, patient-focused communication mechanisms among multidisciplinary providers in the intensive care unit (ICU). i-Dashboard is a custom-developed visualization dashboard that supports (1) key information retrieval and reorganization, (2) time-series data, and (3) display on large touch screens during MDRs. This study aimed to evaluate the performance, including the efficiency of prerounding data gathering, communication accuracy, and information exchange, and clinical satisfaction of integrating i-Dashboard as a platform to facilitate MDRs. A cluster-randomized controlled trial was performed in 2 surgical ICUs at a university hospital. Study participants included all multidisciplinary care team members. The performance and clinical satisfaction of i-Dashboard during MDRs were compared with those of the established electronic medical record (EMR) through direct observation and questionnaire surveys. Between April 26 and July 18, 2021, a total of 78 and 91 MDRs were performed with the established EMR and i-Dashboard, respectively. For prerounding data gathering, the median time was 10.4 (IQR 9.1-11.8) and 4.6 (IQR 3.5-5.8) minutes using the established EMR and i-Dashboard (P<.001), respectively. During MDRs, data misrepresentations were significantly less frequent with i-Dashboard (median 0, IQR 0-0) than with the established EMR (4, IQR 3-5; P<.001). Further, effective recommendations were significantly more frequent with i-Dashboard than with the established EMR (P<.001). The questionnaire results revealed that participants favored using i-Dashboard in association with the enhancement of care plan development and team participation during MDRs. i-Dashboard increases efficiency in data gathering. Displaying i-Dashboard on large touch screens in MDRs may enhance communication accuracy, information exchange, and clinical satisfaction. The design concepts of i-Dashboard may help develop visualization dashboards that are more applicable for ICU MDRs. ClinicalTrials.gov NCT04845698; https://clinicaltrials.gov/ct2/show/NCT04845698.


Caseating cutaneous granulomas in a child with common variable immunodeficiency.
Cutaneous, noninfectious, granulomatous lesions have been reported occasionally in different types of immunodeficiencies, including common variable immunodeficiency (CVD). We present a child with CVID and cutaneous granulomas with a strikingly prominent caseating necrosis. We think that such granulomatous lesions constitute a distinctive manifestation of immunodeficiency, and may reflect a altered immune response. Corticosteroids have been of benefit to our patient, as well as in similar cases.


Fall frequency and risk assessment in early Parkinson's disease.
We sought to define the frequency of falls in early PD and assess potential risk factors for falls in this population. We analyzed the data from two randomized, placebo controlled trials (NET-PD FS1 and FS-TOO) of 413 individuals with early PD over 18 months of follow-up in FS1 and 12 months in FS-TOO. Falls were defined as any report of falls on the UPDRS or the adverse event log. We assessed the frequency of falls overall and by age. The relationship between prespecified fall risk markers and the probability of falling was assessed using logistic and multiple logistic regression. A hurdle Poisson model was used to jointly model the probability of remaining fall-free and the number of falls. During the follow-up period, 23% of participants fell, and 11% were habitual fallers. In a multiple logistic regression model, age, baseline UPDRS Falling score, and baseline PDQ-39 scores were associated with subsequent fall risk (p < 0.001). Similarly, in a hurdle Poisson regression model, age, baseline UPDRS falling item, and baseline PDQ-39 were all significantly related to the probability of falling, but only UPDRS falling >0 was associated with the number of falls. Falls are frequent and are associated with impaired quality of life, even in early PD. Current standard rating scales do not sufficiently explain future fall risk in the absence of a prior fall history. New assessment methods for falls and postural instability are required to better evaluate this important problem in clinical trials and clinical practice.


Ectopic blood supply of hepatocellular carcinoma as depicted by angiography with computed tomography: associations with morphological features and therapeutic history.
To investigate the associations of ectopic blood supply of hepatocellular carcinoma (HCC) with its morphological features and therapeutic history. Three hundred and six patients with 373 HCC lesions were enrolled in this study, and underwent biphasic contrast-enhanced scans on a 64-section MDCT. The anatomy of ectopic blood supply, morphological characteristics of HCC including the size, location and pseudocapsule, and history of transcatheter arterial chemoembolization (TACE) therapy were quantitively assessed and statistically analyzed. Ectopic blood supply was found in 30.8% (115/373) lesions. The ectopic arteries were predominantly composed of inferior phrenic artery (86/115) followed by left and right gastric artery (25/115). Tumor size, location, status of pseudocapsule, and history of TACE therapy could impact the origination of ectopic arteries (all p<0.05). The ectopic feeding arteries of HCC predominantly composed of the perihepatic arteries are associated with the morphological features of the tumor and therapeutic history.


Pharmacological characterization of isoproterenol-treated medaka fish.
The transparent bodies of see-through medaka fish has led to their use as models for in vivo cell and tissue imaging. However, these fish may also prove useful as models for pathological processes. Accumulating reports show that pathology in fish is similar to that in mammals, including humans. In this study, pathological characterization, comparison of data from rodents and in vivo pharmacological analyses of isoproterenol (Iso)-treated fish were performed. Larval fish showed cardiac hypertrophy-like changes after exposure to > 10 microg/ml Iso for more than one day. Hearts of Iso-treated fish had a larger size and a thicker wall, especially in the ventricle, compared with normal hearts. Several messenger RNA levels dose-dependently increased in Iso-treated fish in a manner similar to that in rodents. Symptoms that accompany mammalian cardiac hypertrophy, such as congestion, were also observed. Propranolol (Pro), a non-selective beta-adrenoceptor blocker (AR), significantly protected the increase in heart rate and volume and other simultaneous symptoms induced by Iso exposure. Athletic capability, measured by optomotor response, showed a dose-dependent decrease after Iso treatment, but was protected by Pro. In contrast, metoprolol (Met) and butoxamine (Btx), selective beta1- and beta2-AR blockers, had little effect in this analysis. These results show that Iso-treated fish develop pathologies analogous to those found in mammalian systems, suggesting that medaka fish can be used for investigation of cardiac hypertrophy; however, drug affinity differences should be considered between species.


[Clinical value of positron emission tomography in neuromedicine. Position paper on results of an interdisciplinary consensus conference].
To date, positron emission tomography (PET) is the most powerful method for the in-vivo investigation of human brain metabolism. Besides extensive application of this technology in the neurosciences, PET is also being increasingly used as a clinical tool. However, despite its acceptance in clinical practice a major obstacle is its high costs. The present article reviews the literature on the clinical use of PET in neurology, neurosurgery, and psychiatry in order to substantiate the clinical indications for PET in these specialties as established by an interdisciplinary conference.


Coordinated Regulation Among Progesterone, Prostaglandins, and EGF-Like Factors in Human Ovulatory Follicles.
In animal models, the luteinizing hormone surge increases progesterone (P4) and progesterone receptor (PGR), prostaglandins (PTGs), and epidermal growth factor (EGF)-like factors that play essential roles in ovulation. However, little is known about the expression, regulation, and function of these key ovulatory mediators in humans. To determine when and how these key ovulatory mediators are induced after the luteinizing hormone surge in human ovaries. Timed periovulatory follicles were obtained from cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. The in vivo and in vitro expression of PGR, PTG synthases and transporters, and EGF-like factors were examined at the level of messenger RNA and protein. PGR binding to specific genes was assessed. P4 and PTGs in conditioned media were measured. PGR, PTGS2, and AREG expressions dramatically increased in ovulatory follicles at 12 to 18 hours after human chorionic gonadotropin (hCG). In human granulosa/lutein cell cultures, hCG increased P4 and PTG production and the expression of PGR, specific PTG synthases and transporters, and EGF-like factors, mimicking in vivo expression patterns. Inhibitors for P4/PGR and EGF-signaling pathways reduced hCG-induced increases in PTG production and the expression of EGF-like factors. PGR bound to the PTGS2, PTGES, and SLCO2A1 genes. This report demonstrated the time-dependent induction of PGR, AREG, and PTGS2 in human periovulatory follicles. In vitro studies indicated that collaborative actions of P4/PGR and EGF signaling are required for hCG-induced increases in PTG production and potentiation of EGF signaling in human periovulatory granulosa cells.


Dihydromyricetin attenuates hypertrophic scar formation by targeting activin receptor-like kinase 5.
Hypertrophic scar (HPS) is a manifestation of abnormal tissue repair, representing excessive extracellular matrix production and abnormal function of fibroblasts, for which no satisfactory treatment is available at present. Here we identified a natural product of flavonoid, dihydromyricetin, could effectively attenuate HPS formation. We showed that local intradermal injection of dihydromyricetin (50 μM) reduced the gross scar area, cross-sectional size of the scar and the scar elevation index in a mechanical load-induced mouse model. In addition, dihydromyricetin treatment also markedly decreased collagen density of the scar tissue. Furthermore, both in vitro and in vivo study both demonstrated that dihydromyricetin inhibited the proliferation, activation, contractile and migration abilities of hypertrophic scar-derived fibroblasts (HSFs) but did not affect HSFs apoptosis. Western blot analysis revealed that dihydromyricetin could down-regulate the phosphorylation of Smad2 and Smad3 of TGF-β signaling. Such bioactivity of dihydromyricetin may result from its selective binding to the catalytic region of activin receptor-like kinase 5 (ALK5), as suggested by the molecular docking study and kinase binding assay (12.26 μM). Above all, dihydromyricetin may prove to be a promising agent for the treatment of HPS and other fibroproliferative disorders.


Characterization and Optimization of the Tumor Microenvironment in Patient-Derived Organotypic Slices and Organoid Models of Glioblastoma.
While glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs (peripheral blood mononuclear cells) and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future.


Three-Dimensional Free-Breathing Whole-Heart Coronary Magnetic Resonance Angiography at 1.5 T: Gadobutrol-Enhanced Gradient-Echo Acquisition Sequence Versus Non-Contrast-Enhanced Steady-State Free Precession Sequence.
The objective of this study was to compare gadobutrol-enhanced gradient-echo sequence (GRE) acquisition with T2-prepared non-contrast-enhanced steady-state free precession (SSFP) in coronary magnetic resonance angiography at 1.5 T. Twenty-one subjects successfully completed GRE and SSFP acquisition. Signal-to-noise ratio (SNR), contrast-to-noise ratio, image quality, sharpness, visibility, length, and lumen diameter of vessels were analyzed by 2 experienced radiologists. The SNR at whole left circumflex artery, left main artery, and proximal left descending artery (LAD) was significantly higher in SSFP acquisition (P < 0.05). The SNR of distal LAD was slightly higher in GRE acquisition (P < 0.05). The contrast-to-noise ratio at distal LAD, proximal and distal RCA were significantly higher with GRE acquisition (P < 0.05). Double-dose gadobutrol-enhanced GRE and unenhanced SSFP coronary magnetic resonance angiography at 1.5 T have their own characteristics, and the combined use of the 2 methods may be taken into consideration.


Bio-inspired self-assembled bacteriochlorin nanoparticles for superior visualization and photothermal ablation of tumors.
Although hyperthermia-based photothermal therapy (PTT) has achieved great success in the battle against malignant tumors, various commonly used photothermal sensitizers still suffer from non-selective tumor accumulation, limited photothermal conversion efficiency, potential toxicity and side effects, as well as complex and low cost-effective preparation process. Therefore, novel photothermal sensitizers are urgently required. The well-organized self-assembling of natural bacteriochlorophylls with superior photothermal property may provide an interesting option for the engineering of ideal PTS. Inspired by the self-assembly peripheral light-harvesting antennas of natural bacteriochlorin in microorganisms, a biomimetic light-harvesting nanosystem (Nano-Bc) was developed via bacteriochlorophylls self-arranging in aqueous phase. The characterization of Nano-Bc were measured using DLS, TEM, UV-vis-near-infrared spectroscopy and preclinical PA imaging system. The cytotoxicity of Nano-Bc was quantitatively evaluated via a standard MTT assay using mouse breast cancer 4T1 cells, and the in vivo photothermal eradication of tumor was investigated in the 4T1 breast tumor-bearing mouse model. The obtained bacteriochlorin nanoparticles (Nano-Bc) exhibited ultra-high photothermal performance within the biological transparent window, showing superior heating capacity compared to commonly used photothermal sensitizers of organic dye indocyanine green and inorganic gold nanorods. Guiding by the inherent photoacoustic imaging of Nano-Bc, complete tumor elimination in vitro and vivo was evidenced upon laser irradiation. The green and facile preparation, ultra-high photothermal effect in the transparent window, excellent photoacoustic imaging capacity, and great biosafety prompt, the bio-inspired Nano-Bc as a promising theranostic platform against cancer in the areas of healthcare.


Technology induced error and usability: the relationship between usability problems and prescription errors when using a handheld application.
This paper describes an innovative approach to the evaluation of a handheld prescription writing application. Participants (10 physicians) were asked to perform a series of tasks involving entering prescriptions into the application from a medication list. The study procedure involved the collection of data consisting of transcripts of the subjects who were asked to "think aloud" while interacting with the prescription writing program to enter medications. All user interactions with the device were video and audio recorded. Analysis of the protocols was conducted in two phases: (1) usability problems were identified from coding of the transcripts and video data, (2) actual errors in entering prescription data were also identified. The results indicated that there were a variety of usability problems, with most related to interface design issues. In examining the relationship between usability problems and errors, it was found that certain types of usability problems were closely associated with the occurrence of specific types of errors in prescription of medications. Implications for identifying and predicting technology-induced error are discussed in the context of improving the safety of health care information systems.


Biochar regulates bacterial-fungal diversity and associated enzymatic activity during sheep manure composting.
Aimed to evaluate the coexistence of bacterial and fungal diversity and their correlation with enzymatic activity in response to biochar. This study performed aerobic composting based on typical agricultural wastes of sheep manure with additive apple tree branch biochar at distinct concentration (0, 2.5, 5, 7.5, 10 and 12.5% corresponding from T1 to T6). The result demonstrated that appropriate amendment of biochar enriched bacterial diversity (1646-1686 OTUs) but interestingly decreased fungal diversity (542-630 OTUs) compared to control (1444 and 682 OTUs). Biochar addition enhanced all enzymatic activities and its correlation with bacterial was more complex than fungal community (786 and 359 connect edges). The dominant microbes comprised of Firmicutes (45.2-35.2%), Proteobacteria (14.0-17.5%), Basidiomycota (32.4-49.5%) and Ascomycota (11.3-37.5%) among all the treatments. Overall, biochar regulates the composting microenvironment by influencing the microbial diversity and associated enzymatic activities.


Joint Initiative Between Infectious Diseases and Podiatry in Outpatient Settings Improves Outcomes and Adherence to Treatment.
A joint infectious disease-podiatry clinic (JIDPC) in which an infectious diseases physician and a podiatrist see patients with diabetic foot infections together once a week was initiated in January 2017. This study was designed to investigate if the JIDPC can improve patient adherence and reduce recurrent infections. A retrospective analysis of patients with diabetic foot infection admitted to Wheeling Hospital from March 2013 to December 2018 was performed. Initially, the patients were followed by infectious diseases and podiatry in their clinics separately (preintervention group). Beginning January 2017, they were followed together at the JIDPC (postintervention group). Recurrent infection, mortality, and loss to follow-up were compared using logistic regression models. Surgeries were performed in 52.5% of preintervention group participants (n = 99) and 81.9% of postintervention group participants (n = 55; P < .001). The preintervention group was more likely to be lost to follow-up (30.3% vs 9.1%; odds ratio [OR], 4.35 [confidence interval (CI), 1.58-11.99]), but the association was attenuated with further adjustment for surgery (OR 3.35 [CI, 1.17-9.62]). The risk of infection recurrence in 6 months was significantly higher in the preintervention group (36.1% vs 20.8%; OR, 2.16 [CI, 0.99-4.71]), but with further adjustment for surgery, this was not significant (P = .067; OR, 2.17 [CI, 0.95-4.94]). Mortality and 90-day readmission were not significantly different. Implementation of JIDPCs may decrease the incidence of recurrent infections among patients with diabetic foot infections.


Turning intermittent regimens into daily regimens using blister-packs. An exploration in murine tuberculosis.
Blisterpacks might be used to present low cost intermittent regimens while maintaining an easily remembered daily frequency of opening blisters, by alternating blisters containing 2 of the drugs of a 4-drug regimen with blisters containing the remaining 2 drugs. The efficacy of the alternating regimens was examined in murine tuberculosis. 2 weeks after infection with Mycobacterium tuberculosis H37Rv, groups of mice were treated with rifampicin (R) 15 mg/kg, isoniazid (H) 25 mg/kg, pyrazinamide (Z) 300 mg/kg, ethambutol (E) 100 mg/kg three times weekly (RHZE3); RH/ZE, an alternating regimen of RH on days 1, 3 and 5 of the week and ZE on days 2, 4 and 6, RZ/HE or RE/HZ. Spleen and lung bacillary counts at 7 and 12 weeks indicated large differences in the efficacy of the regimens: RZ/HE > RE/HZ > RHZE3 > RH/ZE. Serum assays showed that Rifampicin (RMP) levels were much lower after HRZE and slightly lower after RZ, RE and RH than after R alone, whereas levels were similar when R was given before the remaining drugs; also, the absorption of Z was slightly increased by R. A second experiment used the same 4 regimens but gave R before other drugs. The organ colony forming units counts at 6 and 12 weeks were then similar. A third experiment examined continuation phase regimens of R3, R2, R2H2 and R2H6 given after daily RHZ treatment for 4 weeks. It found R2H2 only slightly superior to R2H6 and R3 much better than R2. 1. Alternating initial phase regimens were as effective as conventional intermittent regimens. 2. R3H6 might be an optimal continuation phase regimen for blisterpacks.


Genetic variation in wild and hatchery stocks of Suminoe Oyster (Crassostrea ariakensis) assessed by PCR-RFLP and microsatellite markers.
Genetic variation in wild Asian populations and U.S. hatchery stocks of Crassostrea ariakensis was examined using polymerase chain reactions with restriction fragment length polymorphism (PCR-RFLP) analysis of both the mitochondrial COI gene and the nuclear internal transcribed spacer (ITS) 1 region and using 3 microsatellite markers. Hierarchical analysis of molecular variance and pairwise comparisons revealed significant differentiation (P < 0.05) between samples from the northern region, represented by collections from China and Japan, and 2 of 3 samples from southern China. PCR-RFLP patterns were identified that were diagnostic for the northern (N-type) and southern (S-type) groups. Microsatellite marker profiles were used to assign each oyster to one of the two northern or two southern populations. Results for more than 97% of the oysters were consistent with the PCR-RFLP patterns observed for each individual in that oysters with N-type patterns were assigned to one of the northern populations and those with S-type patterns to one of the southern populations. At one site of the Beihai (B) region in southern China a mix of individuals with either the N-type or S-type PCR-RFLP genotypes was found. No heterozygotes at the nuclear ITS-1 locus were found in the sample, possibly indicating reproductive isolation in sympatry. Microsatellite assignment test results of the B individuals were also consistent with identifications as either the N-type or S-type based on PCR-RFLP patterns. The parental population for one hatchery stock was this B sample, which initially was composed of almost equal numbers of northern and southern genetic types. After hatchery spawns, however, more than 97% of the progeny fell into the northern genetic group by PCR-RFLP and microsatellite assignment test analyses, indicating that the individuals with the southern genotype contributed little to the spawn, owing to gametic incompatibility, differential larval survival, or a difference in timing of sexual maturity. Overall, results suggested that oysters collected as C. ariakensis in this study, and likely in other studies as well, include two different sympatric species with some degree of reproductive isolation.


The clinical implications of non-idiopathic intussusception.
A pathological lesion can be identified at the leadpoint of intussusception in about 6% of episodes. Occasionally, general manifestations of an underlying disease indicate the specific cause of an intussusception (e.g., perioral pigmentation in Peutz-Jeghers syndrome), but usually the clinical features provide no clues as to the aetiology. Neonatal intussusception may be caused by a duplication cyst or Meckel's diverticulum. Beyond 12 months, the proportion of intussusceptions due to a pathological lesion at the leadpoint increases with age. There is an identifiable lesion in the majority of children over 5 years of age. Postoperative intussusception accounts for between 0.5% and 16% of intussusceptions, although it has a variety of causes; it typically follows retroperitoneal dissection. It is unusual for an intussusception due to a pathological lesion at the leadpoint to be reduced by enema. If it is reduced, the lesion may be seen at the time of reduction or fluoroscopy, or subsequently on ultrasonography.


The Association of Mental Illness With Outcomes of Emergency Surgery for Bowel Obstruction.
Bowel obstruction is one of the most common surgical emergencies. The management of SBO is variable and influenced by numerous confounding factors. Recent studies have identified mental health as a health disparity that affects surgical outcomes. We aim to assess whether mental illness is a health disparity and its association with postoperative complications and secondary outcomes for bowel obstruction in Emergency General Surgery (EGS). This was a retrospective study utilizing the National Inpatient Sample. Individuals aged 18-64 who underwent emergency adehesiolysis or bowel resection from 2015 to 2017 were identified. Postoperative complications, in-hospital mortality, length of stay, and total cost for surgical patients with and without mental illness were recorded. Univariate and multivariate analyses were used to evaluate the association between mental health and bowel obstruction. 20,574 patients who underwent surgery for bowel obstruction were identified. 3756 of these patients had mental illness and 16,998 patients did not. Patients with mental illness did not have significantly worse outcomes compared to patients without mental illness. Among 3576 patients with mental illness, sex, race, patient location, insurance, location/teaching status of hospital, hospital control and procedure type were significant predictors of prolonged length of stay, higher cost, and increased postoperative complications. Mental health does not appear to be a health disparity in outcomes for bowel obstruction procedures. However, the intersection of mental health with race and insurance status predicts worse outcomes. This essential area should be further explored to determine how marginalized populations are affected in emergency surgical care.


Surgical clearance for the patient with chronic liver disease.
Patients with chronic liver disease face greater risk of perioperative morbidity and mortality, with the greatest risk among patients with cirrhosis. Both the Child-Pugh score and the Model for End-Stage Liver Disease have been evaluated as predictors of postoperative mortality. Other comorbidities, age, and American Society of Anesthesiologists physical status classification are also important predictors of these outcomes. In patients with liver disease, elective surgeries should be delayed to allow complete evaluation of the severity of liver disease, including the role of transplantation in the event of hepatic decompensation postoperatively.


Safety and Tolerability of Lisdexamfetamine: A Retrospective Cohort Study.
Attention-deficit hyperactivity disorder (ADHD) is a common neurobehavioural disorder in children. Pharmacotherapy plays a main role in multimodal treatment, albeit adverse effects are a concern. Lisdexamfetamine is a newer pharmacological option and post-marketing studies on adverse events are limited. The aim of this study was to investigate the treatment-emergent adverse events (TEAEs) in patients receiving lisdexamfetamine in a clinical setting. We performed a retrospective cohort study at the Department of Child and Adolescent Psychiatry of Glostrup Hospital in Copenhagen, Denmark. We included all consecutive patients >6 years old, with an ICD-10 diagnosis of ADHD who were initiated on lisdexamfetamine between May 2013 and July 2014. TEAEs were assessed by a clinician and chart audit. Forty-three patients (91 % male) with a median age of 11 (range 8-15) years were included and received lisdexamfetamine for a median of 188 days (range 3-433). In total, 23.3 % of the patients discontinued treatment due to a TEAE. 88 % of the patients experienced at least one TEAE and the time to first TEAE ≤4 weeks in 83.8 % of the patients. A new TEAE was experienced by 39.5 % of the patients compared with the TEAEs that patients had experienced when taking previous ADHD medication. The most common TEAEs (≥ 5 %) were decreased appetite, difficulty falling asleep, tics, stomach ache and weight loss. A subjectively assessed good or good but time-limited (during the day only) effect was observed in 62.7 %. Lisdexamfetamine treatment in this small group of patients who had received previous stimulant medication for ADHD was well tolerated and the TEAEs were consistent with findings in previous trials, although more than one third of the patients experienced TEAEs not observed with previously taken ADHD medication. Both the number of patients experiencing TEAEs and the rates of discontinuation due to TEAEs were higher than previously reported.


GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function.
The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.


Speciation of nickel in surface waters measured with the Donnan membrane technique.
The evaluation of the ecotoxicological risk of nickel (Ni) in surface water is hampered by a lack of speciation data. Six surface waters were sampled and speciation of Ni(II) was measured by the Donnan membrane technique (DMT) combined with radiochemical determination of 63Ni. The free Ni2+ ion fraction in the dissolved (<0.45 microm) phase was determined at background Ni concentration ((4-8) x 10(-8) M) and at concentrations in the range of toxicity thresholds for the Ni sensitive species Cerodaphnia dubia (5 x 10(-8) to 2 x 10(-6) M). The free ion fraction ranged from 4 to 45% at background Ni and increased with increasing Ni concentration and water hardness and with decreasing pH. The equilibration time after addition of Ni2+ (3h-7d) did not significantly change the measured free ion fraction. Predictions of the Humic-Ion Binding Model WHAM (Windermere Humic Aqueous Model) VI overestimated the observed free Ni2+ fraction (median>two-fold), even when assuming that all dissolved organic matter (DOM) was present as fulvic acid (FA). The impact of several model parameters affecting the prediction of Ni speciation were evaluated, including the solubility product of Fe(OH)3, which affects the Fe competition for complexation by DOM. The best fit (R2=0.88) was obtained by increasing only the distribution term DeltaLK2, which modifies the binding strength of multi-dentate sites, to accommodate the observed dependence of free ion fraction on Ni concentration.


Influence of age and gender on the 28-joint Disease Activity Score (DAS28) in rheumatoid arthritis.
To investigate the influence of age and gender on the components of the 28-joint Disease Activity Score (DAS28) in patients with rheumatoid arthritis (RA), and to clarify whether a high DAS28 can be equally interpreted in all age groups, independent of gender. A prospective cohort of 553 patients with RA was studied for approximately 20 years after diagnosis. The single measures of disease activity and the share of different components of the DAS28 (eg, erythrocyte sedimentation rate; ESR) were analysed and compared between three age groups (<45, 45-65 and >65 years) and per gender, using analysis of variance (ANOVA). The performance of the DAS28 and its components was explored in moderate to high and low DAS28 categories. Linear mixed model analysis was used to design the models best predicting ESR and the share of ESR. ESR significantly increased with age, independent of other variables of disease activity. This increase was more pronounced in male than in female patients. Nevertheless, the share of ESR increased with age only in male patients with a low DAS28 (<3.2). If the DAS28 score was >3.2, age and gender did not have a significant effect on any components of the DAS28. C-reactive protein (CRP) and DAS28(CRP) were not influenced by age. A high DAS28 was found to perform equally in all age groups, in men and women, despite the elevating effect of age on ESR. In elderly men with low disease activity, remission rate could be underestimated by an elevated ESR.


Meningitis Caused by Streptococcus agalactiae in Nile Tilapia (Oreochromis niloticus): Infection and Inflammatory Response.
Streptococcus agalactiae (Sta) of Lancefield group B is the primary etiological agent of bacterial meningitis in Nile tilapia and newborn humans. Thus, the study of this disease is of fundamental importance for aquaculture and human medicine. Additionally, elucidation of the mechanisms involved in the host-pathogenic response is important for the success of new therapies. In the present study, we elucidated important aspects of the innate immune response in the brain tissue of Nile tilapia (Oreochromis niloticus) infected by Sta. The neuroinflammatory process in the meninges started with the migration of MHC class II and CD68 + cells, production of TNF-alpha, and the effective immune response to Sta was mediated by the increased iNOs+. In conclusion, the present study brings a partial understanding of the pathophysiological and neuroinflammatory mechanisms in meningitis in Sta infected tilapia, enabling important advances in the therapy of this disease as well as the possibility of using this biological model to understand human meningitis.


The regulated-breathing method for stuttering: an experimental evaluation.
The regulated-breathing method of Azrin and Nunn (1974) appears to be one of the most promising treatments of stuttering in adults. Their study, however, has some methodological limitations. In particular no operational definition of stuttering is provided, and all measures of stuttering are based on self-report. The present study is an experimental evaluation of the regulated-breathing method. Thirty-two subjects were randomly assigned either to treatment or to a waiting-list control group. The treatment was completed during one single session of 2-3 hours. Both frequency of stuttering and rate of speech were measured before and after treatment, and on follow-ups 2, 3 and 8 months later. The dependent variables were assessed under conditions of both phrase reading and spontaneous speech. Both obtrusive and unobtrusive measures of speech were recorded. At the 8 month follow-up, stuttering in the treatment group was significantly less than in the control group, and also significantly less than before treatment.


Acute Liver Dysfunction Criteria in Critically Ill Children: The PODIUM Consensus Conference.
Develop evidence-based criteria for individual organ dysfunction. Evaluate current evidence and develop contemporary consensus criteria for acute liver dysfunction with associated outcomes in critically ill children. Electronic searches of PubMed and Embase conducted from January 1992 to January 2020, used medical subject heading terms and text words to characterize acute liver dysfunction and outcomes. Studies evaluating critically ill children with acute liver dysfunction, assessed screening tools, and outcomes were included. Studies evaluating adults, infants ≤36 weeks gestational age, or animals or were reviews/commentaries, case series with sample size ≤10, or non-English language studies were excluded. Data were abstracted from each eligible study into a data extraction form along with risk of bias assessment by a task force member. The systematic review supports criteria for acute liver dysfunction, in the absence of known chronic liver disease, as having onset of symptoms <8 weeks, combined with biochemical evidence of acute liver injury, and liver-based coagulopathy, with hepatic encephalopathy required for an international normalized ratio between 1.5 and 2.0. Unable to assess acute-on-chronic liver dysfunction, subjective nature of hepatic encephalopathy, relevant articles missed by reviewers. Proposed criteria identify an infant, child, or adolescent who has reached a clinical threshold where any of the 3 outcomes (alive with native liver, death, or liver transplant) are possible and should prompt an urgent liaison with a recognized pediatric liver transplant center if liver failure is the principal driver of multiple organ dysfunction.


Limitations of fine-needle aspiration and core needle biopsies in the diagnosis of tall cell variant of papillary thyroid carcinoma.
The tall cell variant papillary thyroid carcinoma (TCV-PTC) shows aggressive behaviour. Thus far, the diagnosis of TCV-PTC can only be confirmed using the postoperative specimen. This study aims to evaluate whether fine-needle aspiration (FNA) or core needle biopsy (CNB) could diagnose TCV-PTC preoperatively. This is a retrospective cohort study. We included adult patients diagnosed with TCV-PTC or PTC with tall cell features (TCF) at final surgical pathology between January 2015 and December 2018. Preoperative histology was reviewed for six cytomorphologic features suggesting TCV-PTC in FNA or the percentage of tall cells in the CNB specimen. The postoperative pathology was also reviewed to confirm the percentage of tall cells. A total of 119 patients were included in this study; 35 (29%) patients with PTC with TCF served as controls. The most frequent cytomorphological feature in FNA samples of TCV-PTC was tall columnar cells, including single tombstone-like cells (70%). Among 43 TCV-PTC evaluated by FNA, 3 FNA (7%) revealed the absence of any of the six cytomorphologic features suggesting TCV-PTC. When we defined 30% of tall cells in CNB specimens as a cutoff suggesting TCV-PTC, only 16 (41%) TCV-PTCs could be preoperatively detected, and 3 (7%) TCV-PTCs did not have any tall cells. The proportion of tall cells was not associated with the postoperative percentage of tall cells. Both cytomorphologic features in FNA and the percentage of tall cells in CNB present limitations for use as accurate preoperative diagnostic tools of TCV-PTC.


Netrin-1 attenuates hepatic steatosis via UNC5b/PPARγ-mediated suppression of inflammation and ER stress.
The current study investigated whether netrin-1 can attenuate hepatic steatosis through PPARγ/autophagy-mediated suppression of inflammation and endoplasmic reticulum (ER) stress in experimental animal models. Hepatic steatosis was induced by a high-fat diet in experimental mice. Recombinant mouse netrin-1 was administered via the tail vein (1 μg/mouse, once every two days). Serum inflammatory cytokines and hepatic inflammatory and ER stress markers were determined in mice using ELISA and western blotting protocol. We found that netrin-1 expression was significantly increased (P < 0.05) in cultured macrophages treated with supernatants of subcutaneous adipocytes in the presence of palmitate and subcutaneous fat of obese mice. Recombinant netrin-1 treatment promoted PPARγ expression and autophagy, thereby attenuating inflammation and ER stress, lipid accumulation, and the expression of lipogenic proteins in mouse primary hepatocytes. High-fat diet (HFD) treatment increased hepatic inflammation and ER stress, causing hepatic steatosis in experimental mice. However, administration of netrin-1 reversed the effects of HFD on hepatic ER stress and lipid deposition. These results suggest that subcutaneous adipose macrophage-derived netrin-1 ameliorates inflammation and ER stress in the liver, which in turn alleviates hepatic steatosis by enhancing basal PPARγ/autophagy-dependent signaling. The current study sheds light on the pathogenesis of hepatic steatosis in obesity and provides a promising therapeutic approach for treating metabolic-associated fatty liver disease (MAFLD).


Analysis of hepatitis C virus (HCV) RNA load in platelets of HCV-monoinfected patients receiving antiviral therapy.
Detection of hepatitis C virus (HCV) has been reported in extrahepatic sites such as peripheral blood mononuclear cells and platelets. Quantitation of HCV-RNA in platelets from patients under antiviral therapy has not been reported. HCV-RNA levels in paired serum and platelet samples of 17 chronically HCV-infected patients were determined at baseline, week 12, end-of-treatment, and 24 weeks after completion of treatment with pegylated interferon plus ribavirin. Quantitation of HCVRNA load was performed using COBAS® TaqMan® HCV Test v 2.0 (lower limit of detection, 25 IU/mL). The cohort predominantly consisted of female (59%) with a mean age of 50.7 ± 10.0 years. Measurements of HCV-RNA in relation to different timepoints of therapy revealed baseline viral load was most frequently detected in higher levels in serum than in platelets (5.6 x 104 IU/mL vs. 379.0 IU/mL; p = 0.0002), a trend also demonstrated in most samples throughout the study. HCV-RNA was also found at low levels (< 25.0-314.0 UI/mL) persistently in platelets of three patients who have lost detectable HCV-RNA in serum during antiviral therapy, resulting in virological relapse. HCV-RNA levels are most frequently detected in higher levels in serum than in platelets, independent of timepoint of antiviral therapy. Further studies with an increase in size of the samples are needed to better evaluate whether or not patients who presented HCV-RNA at low levels in platelets after having lost detectable HCV-RNA in serum during antiviral therapy are at increased risk of relapse of HCV infection during follow-up evaluation.


Synthesis, characterization, and surface wettability properties of amine functionalized graphene oxide films with varying amine chain lengths.
Surface functionalization of graphene oxide (GO) an important graphene precursor using alkylamines of varying chain lengths followed by thermal treatment resulted in the formation of superhydrophobic surfaces. Alkylamines consisting of hydrophobic long chain alkyl groups and hydrophilic amine groups were chemically reacted to the GO surface via two types of reactions viz. (i) amidation reaction between amine groups and carboxylic acid sites of GO and (ii) nucleophilic substitution reactions between amine and epoxy groups on GO surface. Successful grafting of alkylamines was confirmed using Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance ((1)H NMR), and thermogravimetric analysis (TGA). Alkylamine-modified GO surfaces showed enhanced roughness, and this effect was more pronounced with increasing amine chain length. Water contact angle measurements revealed that the hydrophobic nature of graphene depended on the chain length of the grafted alkylamines, and this fact may be corroborated to the decrease in the surface energy values. Our results indicate that superhydrophobic graphene films can be produced by thermal treatment of hexadecylamine- and octadecylamine-grafted GO films. These results will provide valuable guidance for the design and manufacture of graphene-based biomaterials, medical instruments, structural composites, electronics, and renewable energy devices.


Novel cytokine release inhibitors. Part I: Triterpenes.
Tripterine and closely related triterpenoid derivatives as IL-1 beta release inhibitors are discussed.


A study on the postulated transoptic regulation of the intraocular pressure.
The possibility that fibers regulating the intraocular pressure originate in the hypothalamus and course in the optic nerve, has been investigated in albino rabbits with different techniques. By means of horseradish perixodase tracing-technique, oculohypothalamic but no hypothalamoocular connections were observed. The water-loading effect on the intraocular pressure was studied after unilateral optic nerve transection. The transection was performed in three experimental groups in the following way: Intracranial transection of one optic nerve, retrobulbar transection of one optic nerve and sham operation on the other side, retrobulbar transection of one optic nerve without sham operation on the other side. In contrast to previous reports, we found no significant side difference in intraocular pressure after water-loading in any of these groups.


Circulating tumor DNA is a prognostic marker of tumor recurrence in stage II and III colorectal cancer: multicentric, prospective cohort study (ALGECOLS).
In non-metastatic colorectal cancer (CRC), we evaluated prospectively the pertinence of longitudinal detection and quantification of circulating tumor DNA (ctDNA) as a prognostic marker of recurrence. The presence of ctDNA was assessed from plasma collected before and after surgery for 184 patients classified as stage II or III and at each visit during 3-4 years of follow-up. The ctDNA analysis was performed by droplet-based digital polymerase chain reaction, targeting mutation and methylation markers, blindly from the clinical outcomes. Multivariate analyses were adjusted on age, gender, stage, and adjuvant chemotherapy. Before surgery, 27.5% of patients were positive for ctDNA detection. The rate of recurrence was 32.7% and 11.6% in patients with or without detectable ctDNA respectively (P = 0.001). Time to recurrence (TTR) was significantly shorter in patients with detectable ctDNA before (adjusted hazard ratio [HR] = 3.58, 95% confidence interval [CI] 1.71-7.47) or immediately after surgery (adjusted HR = 3.22, 95% CI 1.32-7.89). The TTR was significantly shorter in patients with detectable ctDNA during the early postoperative follow-up (1-6 months) (adjusted HR = 5, 95% CI 1.9-12.9). Beyond this period, ctDNA remained a prognostic marker with a median anticipated diagnosis of recurrence of 13.1 weeks (interquartile range 28 weeks) when compared to imaging follow-up. The rate of ctDNA+ might be underestimated knowing that consensus pre-analytical conditions were not described at initiation of the study. This prospective study confirms the relevance of ctDNA as a recurrence risk factor in stage II and III CRC before surgery and as a marker of minimal residual disease after surgery that may predict recurrence several months before imaging techniques.


Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIβ alleles of New World ranid frogs.
Genes encoded by the major histocompatibility complex (MHC) play key roles in the vertebrate immune system. However, our understanding of the evolutionary processes and underlying genetic mechanisms shaping these genes is limited in many taxa, including amphibians, a group currently impacted by emerging infectious diseases. To further elucidate the evolution of the MHC in frogs (anurans) and develop tools for population genetics, we surveyed allelic diversity of the MHC class II β1 domain in both genomic and complementary DNA of seven New World species in the genus Rana (Lithobates). To assign locus affiliation to our alleles, we used a "gene walking" technique to obtain intron 2 sequences that flanked MHC class IIβ exon 2. Two distinct intron sequences were recovered, suggesting the presence of at least two class IIβ loci in Rana. We designed a primer pair that successfully amplified an orthologous locus from all seven Rana species. In total, we recovered 13 alleles and documented trans-species polymorphism for four of the alleles. We also found quantitative evidence of selection acting on amino acid residues that are putatively involved in peptide binding and structural stability of the β1 domain of anurans. Our results indicated that primer mismatch can result in polymerase chain reaction (PCR) bias, which influences the number of alleles that are recovered. Using a single locus may minimize PCR bias caused by primer mismatch, and the gene walking technique was an effective approach for generating single-copy orthologous markers necessary for future studies of MHC allelic variation in natural amphibian populations.


The Optimal Dose of Intraoperative Dexmedetomidine for Antiemetic Effects of Post-operative Nausea and Vomiting in Patients Undergoing Elective Thoracic Surgery: A Retrospective Cohort Study.
Dexmedetomidine (DEX) administration decreases post-operative nausea and vomiting (PONV), but it is a lack of large-scale retrospective cohort study and is unclear whether there is a dose-relationship and optimal dose for antiemetic effects between DEX and PONV. We performed a large-scale retrospective cohort study to explore the optimal dose of intraoperative DEX for antiemetic effects of PONV. A total of 5,310 patients aged ≥18 who underwent elective thoracic surgery from January 2016 to March 2020 under total intravenous anesthesia (TIVA) or combined intravenous and inhalation anesthesia in Henan Provincial People's Hospital. Patients were divided into two groups, those who received DEX intraoperatively and those who did not receive DEX. Patients who received DEX after surgery were excluded. Our primary outcomes were the association, the dose-response relationship, and the optimal dose for antiemetic effects between intraoperative DEX and PONV. Among the 3,878 patients enrolled, 2,553 patients received DEX and 1,325 patients did not receive DEX. The incidence of PONV in patients who received DEX was 21.3%, and the incidence of PONV in patients who did not receive DEX was 46.5% (P = 0.001). After the matched-pairs cohort consisted of 1,325 patients, the incidence of PONV in patients who received DEX was 23.6%, and the incidence of PONV in patients who did not receive DEX was 46.5% (P = 0.001). We analyzed three different models after propensity matching to validate the stability of the prediction model between intraoperative DEX and PONV. A dose-response relationship between intraoperative DEX and PONV was observed. The optimal dose range of intraoperative DEX for antiemetic effects of PONV is 50-100 μg in elective thoracic surgery. Intraoperative DEX was associated with a decreased incidence of PONV in the large-scale retrospective cohort study. A dose-response relationship between intraoperative DEX and PONV was observed. The optimal dose range of intraoperative DEX for antiemetic effects of PONV is 50-100 μg in elective thoracic surgery.


Mechanisms responsible for long-term survival of adult rat hepatocytes in the presence of phenobarbital in primary culture.
The mechanisms, by which phenobarbital (PB) supports the survival of adult rat hepatocytes in primary culture, were investigated. PB altered the shape of rat erythrocytes to produce cup-formed cells and protected them from hypotonic hemolysis. Anesthetics (ketamine, lidocaine, mepivacaine, and bupivacaine) and an anti-inflammatory agent (indomethacin), which are also known to protect erythrocytes from hypotonic hemolysis by stabilizing their membranes, efficiently supported the survival of hepatocytes in primary culture. Furthermore, the well-known biological membrane stabilizers, such as cholesterol and vitamin E, also showed the maintenance effect on primary cultured hepatocytes. PB effectively reduced the leakage of lactate dehydrogenase from hepatocytes caused by chenodeoxycholic acid in primary culture. Rotenone and amobarbital, which act repressively on the PB-sensitive site in the respiratory chain and are known to inhibit the mitochondrial formation of active oxygen species with NAD-linked substances, effectively prolonged the hepatocyte survival in primary culture. Elevation of oxygen tension in primary culture remarkably decreased the hepatocyte survival rate, which was preserved by addition of antioxidant substances, such as vitamin C, vitamin E, bifemelane, selenite, and superoxide dismutase. On the other hand, in the presence of PB, the hepatocyte survival rate hardly changed with the elevation of oxygen tension. From these findings, it seems that PB stabilizes the hepatocyte membranes and reduces the mitochondrial formation of active oxygen species and that the stabilized functions of membrane and the reduction of oxidative stress result in the prolonged survival of hepatocytes in primary culture.


Protease-activated receptor-2 induces proinflammatory cytokine and chemokine gene expression in canine keratinocytes.
Although the molecular basis of the allergenicity remains to be fully elucidated, the ability of allergens to elicit allergic responses is at least partly attributed to their proteolytic activity. Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by site-specific proteolysis by serine proteases and is known to mediate inflammatory processes in various tissues. In this study, we investigated the effects of trypsin, a major serine protease, and a human PAR-2 agonist peptide (SLIGKV-NH2) on proinflammatory cytokine and chemokine gene expression in the canine keratinocyte cell line CPEK. The expression of PAR-2 mRNA and protein in CPEK cells was detected by RT-PCR and Western blotting, respectively. The localization of PAR-2 in CPEK was examined by immunofluorescence. The mRNA expression levels of proinflammatory cytokines and chemokines were quantified by real-time RT-PCR. The free intracellular Ca(2+) concentration was measured using the Ca(2+)-sensitive fluorescent dye. CPEK cells constitutively expressed PAR-2 mRNA and protein. Stimulation of CPEK cells with trypsin induced significant upregulation of the mRNA expression levels of tumor necrosis factor alpha (TNF-α, P<0.05), granulocyte-macrophage colony-stimulating factor (GM-CSF, P<0.01), thymus and activation regulated chemokine (TARC/CCL17, P<0.01), and interleukin 8 (IL-8/CXCL8, P<0.01). Similarly, the PAR-2 agonist peptide increased the mRNA expression levels of TNF-α (P<0.05), GM-CSF (P<0.05), TARC/CCL17 (P<0.05), and IL-8/CXCL8 (P<0.05) in CPEK cells. Both trypsin and the PAR-2 agonist peptide increased the intracellular Ca(2+) concentration and PAR-2 internalization. These results suggest that PAR-2 activation can augment inflammatory cytokine and chemokine expression in canine keratinocytes, and it may initiate allergic inflammation through the proteolytic activity of allergens in canine atopic dermatitis.


Influence of maturation on ulcer-development and immunodeficiency induced by activity-stress in rats.
Young adult rats housed in the activity-wheel cages and fed only 1 hr daily, have ulcers in the glandular stomach and reveal immunodeficiency. The present study was attempted to investigate how to separate the ulceration and immunodeficiency in order to utilize the activity-stress (A-S) model as an animal model of the human stress ulcer. Six and 10 weeks old rats were used as subjects in this study. They were stressed for 3, 5, or 7 days. In the younger rats, the longer stress exposure caused to deteriorate more both the ulcer and immunodeficiency. The ulceration was found in all of the three stress periods, while the immunodeficiency was recognized in the 5- and 7-day stressed rats. In the elder rats, the ulceration was found in the 5- and 7-day stressed rats, whereas the immunodeficiency was seen only in the 7-day stressed rats. In the free-feeding control rats, relative weights of the thymus and spleen in the elder rats were smaller than those of the younger rats. In the adrenal weight, there was no difference between two age rats. The thymus and spleen of rats exposed to the stress revealed atrophy in both ages. These results suggest that immature rats are more susceptible for the stress than young mature rats, and application of mature rats to the A-S experiment appears to be available in the case of utilizing A-S rats as the stress ulcer model.


Employment Standards for Australian Urban Firefighters: Part 2: The Physiological Demands and the Criterion Tasks.
The physiological demands of 15 essential, physically demanding fire-fighting tasks were investigated to identify criterion tasks for bona fide recruit selection. A total of 51 operational firefighters participated in discrete, field-based occupational simulations, with physiological responses measured throughout. The most stressful tasks were identified and classified according to dominant fitness attributes and movement patterns. Three movement classes (single-sided load carriage [5 tasks], dragging loads [4 tasks], and overhead pushing and holding objects [2 tasks]) and one mandatory strength task emerged. Seven criterion tasks were identified. Load holding and carriage dominated these movement patterns, yet no task accentuated whole-body endurance. Material handling movements from each classification must appear within a physical aptitude (selection) test for it to adequately represent the breadth of tasks performed by Australian urban firefighters.


Emulsifying properties of wheat germ protein: Effect of different ultrasonic treatment.
The effect of ultrasonic treatment on emulsifying properties of wheat germ protein (WGP) was studied in this paper. WGP was subjected to low frequency (20 kHz), high intensity ultrasonic treatment at different power (200, 400, 600, 800 W) for 10 min, or different time (2, 4, 6, 8, 10, 15, 20 min) at 400 W. The emulsifying activity index and emulsion stability index of WGP were significantly improved, and the emulsion droplet was smaller and more uniform after ultrasound treatment. Ultrasound increased the adsorbed WGP concentration at the oil-water interface and reduced the interfacial tension, which explained the improved emulsifying properties of WGP. The investigation on molecular properties and protein conformation showed that ultrasound processing increased solubility, but decreased particle size and surface charge of WGP. Ultrasound processing resulted in the unfolding of the protein molecular structure indicated by the increase of surface hydrophobicity and surface free sulfhydryl group levels, and the decrease of intrinsic fluorescence intensity. Correlation analysis showed that the changes in WGP solubility, particle size, and surface hydrophobicity were the main driven factors for the improved emulsifying properties of WGP.


Supravalvular aortic stenosis with parafollicular cell (C-cell) hyperplasia.
Autopsy examination of a patient with well-documented supravalvular aortic stenosis and other characteristic features of the idiopathic infantile hypercalcemia syndrome revealed previously unreported hyperplasia of parafollicular cells (C cells). Immunohistochemical analysis demonstrated up to 30 calcitonin-containing cells per high power field, whereas normal glands contain only 4 to 10 cells per low power field in areas of highest concentration. The parathyroid glands were found to be normal both grossly and microscopically, whereas the bones showed thickened trabeculas, normal Haversian canals and no apparent increase in osteoblast or osteoclast activity, suggesting normal parathyroid hormone, but increased thyrocalcitonin activity. We suggest that C-cell hyperplasia has occurred in response to a persistent, rather than transient, elevation in serum calcium levels and that thyrocalcitonin function is augmented, rather than impaired in this disorder. The primary biochemical defect promoting hypercalcemia remains to be clarified, as well as the role, if any, such a defect plays in producing significant pathology in the central nervous system and in the cardiovascular, renal and skeletal systems.


Detailed muscular structure and neural control anatomy of the levator ani muscle: a study based on female human fetuses.
Injury to the levator ani muscle or pelvic nerves during pregnancy and vaginal delivery is responsible for pelvic floor dysfunction. We sought to demonstrate the presence of smooth muscular cell areas within the levator ani muscle and describe their localization and innervation. Five female human fetuses were studied after approval from the French Biomedicine Agency. Specimens were serially sectioned and stained by Masson trichrome and immunostained for striated and smooth muscle, as well as for somatic, adrenergic, cholinergic, and nitriergic nerve fibers. Slides were digitized for 3-dimensional reconstruction. One fetus was reserved for electron microscopy. We explored the structure and innervation of the levator ani muscle. Smooth muscular cell beams were connected externally to the anococcygeal raphe and the levator ani muscle and with the longitudinal anal muscle sphincter. The caudalmost part of the pubovaginal muscle was found to bulge between the rectum and the vagina. This bulging was a smooth muscular interface between the levator ani muscle and the longitudinal anal muscle sphincter. The medial (visceral) part of the levator ani muscle contained smooth muscle cells, in relation to the autonomic nerve fibers of the inferior hypogastric plexus. The lateral (parietal) part of the levator ani muscle contained striated muscle cells only and was innervated by the somatic nerve fibers of levator ani and pudendal nerves. The presence of smooth muscle cells within the medial part of the levator ani muscle was confirmed under electron microscopy in 1 fetus. We characterized the muscular structure and neural control of the levator ani muscle. The muscle consists of a medial part containing smooth muscle cells under autonomic nerve influence and a lateral part containing striated muscle cells under somatic nerve control. These findings could result in new postpartum rehabilitation techniques.


Brain abnormalities in schizophrenia. A qualitative comparative study of schizophrenic patients and control individuals assessed by magnetic resonance imaging.
The present study examined, by means of Magnetic Resonance Imaging (MRI), the qualitative brain abnormalities in a group of 58 schizophrenic patients compared to a group of 58 matched control individuals. The possible relationships between these abnormalities and the demographic and clinical features of the participants in the study were also investigated. Schizophrenic patients presented a higher percentage of bland-moderate enlargement of the periencephalic-subarachnoid spaces (p=0.01) and a widespread cerebral atrophy, the latter below the threshold of significance (p=0.06). In the subset of patients with ventricular asymmetry (right larger than left) the age was significantly lower compared to the age of patients without this abnormality (p=0.04). In the subset of patients with cerebellar cisterns enlargement the age as well as the age of onset was higher in comparison to the one of patients without this abnormality (p=0.02; p=0.006). Taking together with previous studies, these findings underline the importance of qualitative assessment of brain morphology in research and clinical evaluation of patients with schizophrenia.


γ-Cyclodextrin-polyurethane copolymer adsorbent for selective removal of endotoxin from DNA solution.
Copolymer particles for removal of endotoxins (lipopolysaccharides, LPSs) were prepared by suspension copolymerization of γ-cyclodextrin (CyD) and 1,6-hexamethylenediisocyanate. The LPS-removing activity of the copolymer particles was compared with that of poly(ε-lysine)-immobilized Cellufine (cationic adsorbent) or polystyrene particles (hydrophobic adsorbent) by a batch method. When DNA was present in solution with LPSs under physiological conditions (pH 6.0, ionic strength of μ = 0.05-0.8), LPS-removing activity of the cationic or hydrophobic adsorbent was unsatisfactory because both the DNA and the LPSs were adsorbed onto each adsorbent. By contrast, the copolymer particles with γ-CyD cavity (CyD content: 14-20 mol%) could selectively remove LPSs from a DNA solution (50 μg ml(-1), pH 6.0, and μ = 0.05-0.2) containing LPSs (15 EU ml(-1)) without the adsorption of DNA. The residual concentration of LPSs in the treated DNA solution was below 0.1 EU ml(-1), and the recovery of DNA was 99%.


Identification of key genes for hypertrophic cardiomyopathy using integrated network analysis of differential lncRNA and gene expression.
Hypertrophic cardiomyopathy (HCM) is a complex heterogeneous heart disease. Recent reports found that long non-coding RNAs (lncRNAs) play an important role in the progression of cardiovascular diseases. The present study aimed to identify the novel lncRNAs and messenger RNAs (mRNAs) and determine the key pathways involved in HCM. The lncRNA and mRNA sequencing datasets of GSE68316 and GSE130036 were downloaded from the Gene Expression Omnibus (GEO) database. An integrated co-expression network analysis was conducted to identify differentially expressed lncRNAs and differentially expressed mRNAs in patients with HCM. Then, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were explored to identify the biological functions and signaling pathways of the co-expression network. Protein-protein interaction (PPI) and hub gene networks were constructed by using Cytoscape software. Plasma samples of patients with HCM and the GSE89714 dataset were used to validate the bioinformatics results. A total of 1,426 differentially expressed long non-coding RNAs (lncRNAs) and 1,715 differentially expressed mRNAs were obtained from GSE68316, of which 965 lncRNAs and 896 mRNAs were upregulated and 461 lncRNAs and 819 mRNAs were downregulated. A total of 469 differentially expressed lncRNAs and 2,407 differentially expressed mRNAs were screened from GSE130036, of which 183 lncRNAs and 1,283 mRNAs were upregulated and 286 lncRNAs and 1,124 mRNAs were downregulated. A co-expression network was constructed and contained 30 differentially expressed lncRNAs and 63 differentially expressed mRNAs, which were primarily involved in 'G-protein beta/gamma-subunit complex binding,' 'polyubiquitin modification-dependent protein binding,' 'Apelin signaling pathway,' and 'Wnt signaling pathway.' The 10 hub genes in the upregulated network [G Protein Subunit Alpha I2 (GNAI2), G Protein Subunit Alpha I1 (GNAI1), G Protein Subunit Alpha I3 (GNAI3), G Protein Subunit Gamma 2 (GNG2), G Protein Subunit Beta 1 (GNB1), G Protein Subunit Gamma 13 (GNG13), G Protein Subunit Gamma Transducin 1 (GNGT1), G Protein Subunit Gamma 12 (GNG12), AKT Serine/Threonine Kinase 1 (AKT1) and GNAS Complex Locus (GNAS)] and the 10 hub genes in the downregulated network [Nucleotide-Binding Oligomerization Domain Containing Protein 2 (NOD2), Receptor-Interacting Serine/Threonine Kinase 2 (RIPK2), Nucleotide-Binding Oligomerization Domain Containing Protein 1 (NOD1), Mitochondrial Antiviral Signaling Protein (MAVS), Autophagy Related 16-Like 1 (ATG16L1), Interferon Induced With Helicase C Domain 1 (IFIH1), Autophagy Related 5 (ATG5), TANK-Binding Kinase 1 (TBK1), Caspase Recruitment Domain Family Member 9 (CARD9), and von Willebrand factor (VWF)] were screened using cytoHubba. The expression of LA16c-312E8.2 and RP5-1160K1.3 in the plasma of patients with HCM was elevated, and the expression of the MIR22 host gene (MIR22HG) was decreased, which was consistent with our analysis, while the expression of LINC00324 and Small Nucleolar RNA Host Gene 12 (SNHG12) was not significantly different between the two groups. Verification analyses performed on GSE89714 showed the upregulated mRNAs of Chloride Voltage-Gated Channel 7 (CLCN7), N-Acetylglucosamine-1-Phosphate Transferase Subunit Gamma (GNPTG), Unk Like Zinc Finger (UNKL), Adenosine Monophosphate Deaminase 2 (AMPD2), GNAI3, WD Repeat Domain 81 (WDR81), and Serpin Family F Member 1 (SERPINF1) and downregulated mRNAs of TATA-Box Binding Protein Associated Factor 12 (TAF12) co-expressed with five crucial lncRNAs. Moreover, GNAI2, GNAI3, GNG12, and vWF were upregulated and GNAS was downregulated in the top 10 hub genes of upregulated and downregulated PPI networks. These findings from integrative biological analysis of lncRNA-mRNA co-expression networks explored the key genes and pathways and provide new insights into the understanding of the mechanism and discovering new therapeutic targets for HCM. Three differentially expressed pivotal lncRNAs (LA16c-312E8.2, RP5-1160K1.3, and MIR22HG) in the co-expression network may serve as biomarkers and intervention targets for the diagnosis and treatment of HCM.


Inhibition of cell growth and induction of apoptosis via inactivation of NF-kappaB by a sulfurcompound isolated from garlic in human colon cancer cells.
Compounds such as S-allylmercaptocysteine, diallyl disulfide, and S-trityl-L-cysteine isolated from garlic have been known to be effective in chemoprevention. Nuclear transcription factor-kappaB (NF-kappaB) has been known to be an implicated factor in apoptotic cell death of several cancer cells. In this study, we investigated whether a sulfurcompound (named thiacremonone) isolated from garlic could modulate NF-kappaB activity and thereby induce apoptotic cell death of colon cancer cells. Treatment with different concentrations (30 - 150 microg/ml) of thiacremonone for various periods (0 - 48 h) inhibited colon cancer cell (SW620 and HCT116) growth followed by induction of apoptosis in a dose-dependent manner. We also found that thiacremonone modulated tumor necrosis factor-alpha (TNF-alpha) and tetradeanoyl phorbol acetate (TPA)-induced NF-kappaB transcriptional and DNA binding activity. Moreover, thiacremonone suppressed NF-kappaB target anti-apoptotic genes (Bcl-2, cIAP1/2, and XIAP) and inflammatory genes (iNOS and COX-2), whereas it induced apoptotic genes (Bax, cleaved caspse-3, and cleaved PARP) expression. These results suggest that a novel sulfurocompound from garlic inhibited colon cancer cell growth through induction of apoptotic cell death by modulating of NF-kappaB.


The Management of Atrial Fibrillation in a Patient with Unrepaired Atrial Septal Defect.
Atrial fibrillation (AF) is a frequent comorbidity in adults with atrial septal defect (ASD), one of the most common congenital heart defects. However, there are currently limited recommendations for the management of AF associated with ASD. This article describes a case using a planned approach of catheter ablation followed by transcatheter device closure and discusses management options.


[How can the haematopoietic stem cells from the umbilical cord blood be de-differentiated in vitro? Our first results using the co-cultivation systems].
Aim of this study was to de-differentiate the haematopoietic stem cells (HSCs) that originated from the umbilical cord blood. One of the ways to do it is to use a co-cultivation system. Prospective experimental study. Laboratory study - Institute of reproductive medicine and endocrinology, Pilsen. HSCs were co-cultivated with mouse embryonic stem cells (mESC) with and without feeder cells. After co-cultivation HSCs were analyzed using flow-cytometry for presence of haematopoietic markers (CD34, CD45, CD133) and using immunohistochemistry for presence of embryonic stem cell markers (SSEA-4, Tra-1-60, Tra-1-81). No de-differentiation was detectable in any our experiment, only the intensity of the HSC cell markers decreased. We suppose that there were two major reasons for the experiment failure: there was no direct cell to cell contact and there was a mixture of cell types that originated from two different species. To reach our goal of in vitro de-differentiation we will need to change our strategy towards a pure human culture system without any animal additives and with cell to cell contact.


Methanol-acetic anhydride: an efficient blocking agent for electron microscope cytochemistry. Its application to mouse testis and other tissues.
A mixture of absolute methanol and acetic anhydride (MA) (5:1, v/v; 24 h at 25 degrees C) which is known to methylate and acetylate--respectively--free carboxyl and amino groups in proteins, was tested for its effectiveness as a blocking agent in glutaraldehyde-fixed mouse testis and skeletal muscle. In young spermatids the staining of the acrosome with either uranyl acetate (UA) or ethanolic phosphotungstic acid (PTA) was completely prevented by prior treatment with MA. Esterification of carboxyl groups with 0.1 N HCl in methanol (24 h at 30 degrees C) eliminated the UA staining without affecting that due to PTA. It is concluded that--COOH groups are responsible for UA binding and that acetylation (of amino groups) prevented PTA binding. MA also abolishes the strong affinity of PTA to the lateral elements of the synaptonemal complex in meiotic chromosomes, the axoneme and fibrous sheath of the spermatid tail and the Z band in skeletal muscle. Reactivity was diminished in nucleoli and remained unaffected in chromatin, the outer coarse fibers of the flagellum and collagen fibrils. Different functional groups may participate in PTA staining. The ultrastructure was well preserved in all cases.


Extinction in multiple contexts does not necessarily make extinction less vulnerable to relapse.
Three fear-conditioning experiments with rat subjects examined the effects of extinction in multiple contexts on a final relapse (renewal) effect that occurred when the extinguished fear cue was tested in a new context (Experiments 1 and 3) or in the context in which fear conditioning had first occurred (Experiment 2). Rats that received extinction in three contexts demonstrated more fear during extinction than rats that received the same number and temporal distribution of extinction trials in one context; extinction was partially lost with each context switch. Although extinction in multiple contexts thus had an impact on extinction behavior, it did not reduce the size of the final renewal effect. Fear during extinction was occasionally positively correlated with fear during final testing, but the two were never negatively correlated. The results suggest that extinction in multiple contexts does not necessarily weaken fear renewal, and that extinction procedures that generate high levels of responding in extinction do not necessarily make extinction learning less context-specific.


Selective expression of vacuolar H+-ATPase subunit d2 by particular subsets of dendritic cells among leukocytes.
Dendritic cells (DC) are far more potent to activate T cells than other antigen presenting cells (e.g., macrophages) and distributed to many organs where DC develop to functionally and phenotypically distinctive subsets. To isolate DC-differentially expressed genes, we used a subtractive cDNA cloning (XS52 DC minus J774 macrophages), resulting in the identification of d2 isoform of vacuolar (V) H+-ATPase subunit d. Unlike the ubiquitously expressed isoform (d1), d2 mRNA manifested expression restricted to particular subsets of DC (e.g., skin- and bone marrow-derived DC) among leukocytes and encoded two transcripts (1.6 and 3.0 kb) that differed in the length of the 3'-untranslated region. The d2 protein displayed association with membranes and the localization in lysosomes and antigen-containing endosomes. Interestingly, XS52 DC expressed seven-fold higher V-ATPase proton-pump activity than J774 macrophages and distinguished from the macrophage by high levels of isoforms a1 and a2 expression among V-ATPase subunits. These results indicated that d2 is a new marker for DC and it may, co-operatively with subunit a isoforms, regulate V-ATPase activity.


Immune-mediated pathways in chronic inflammatory arthritis.
Rheumatoid arthritis (RA) is the prototype chronic inflammatory arthropathy; its precise aetiology is unknown. Over recent years, a number of crucial advances in our understanding of the disease have had a major impact on the treatment of patients with RA.


Increased aortic blood pressure contributes to potentiated dobutamine inotropic responses after systemic NO synthase inhibition in sheep.
To determine whether inotropic responses to the beta-adrenergic agonist dobutamine are potentiated by systemic inhibition of nitric oxide synthase (NOS) with the L-arginine analogue N omega-nitro-L-arginine (L-NNA), and to establish to what extent any observed responses are related to the increase in aortic blood pressure accompanying systemic NOS inhibition. Dobutamine was infused incrementally at rates of 1, 2.5, 5 and 10 micrograms/kg/min in 15 open-chest, anaesthetised ewes before and after inhibition of NO synthesis with i.v. L-NNA (n = 8), or elevation of mean aortic blood pressure to the same extent as attained with NOS inhibition using proximal arterial occlusion (n = 7). By the peak infusion rate, dobutamine increased the maximal rate of rise of left ventricular pressure (LV dP/dtMAX) by 100% (p < 0.001) and reduced LV stroke work by 18% (p < 0.01). L-NNA and arterial occlusion increased resting mean aortic blood pressure by 55 +/- 4 and 51 +/- 3 mmHg respectively. Compared to dobutamine alone, subsequent peak dobutamine-related increases in LV dP/dtMAX were augmented by 76% after L-NNA and by 88% after arterial occlusion (both p < 0.001). Moreover, dobutamine increased LV stroke work by 23% at infusion rates of 1-5 micrograms/kg/min (p < 0.001) after L-NNA, and by 17% at an infusion rate of 1 microgram/kg/min (p < 0.01) after arterial occlusion. Systemic NOS inhibition potentiates the effects of dobutamine on LV isovolumic and pumping performance in the intact circulation, but this potentiation is in large part related to the increase in arterial blood pressure accompanying NOS inhibition.


Detection of antinuclear antibodies (ANA), antibodies to double stranded DNA (anti-dsDNA) and antibodies to extractable nuclear antigens (anti-ENA) in Greek patients.
The diagnosis of autoimmune diseases depends on clinical history, physical examination, and laboratory detection of specific autoantibodies directed against nuclear or cytoplasmic antigens. The aim of this study was to investigate the types and prevalence of serum ANA, anti-dsDNA, and anti-ENA antibodies in a population examined at the Immunology Laboratory of the Naval Hospital of Athens and their correlation with patient age and gender. We evaluated the sera of 3000 patients, both male and female, aged between 18 and 75 years old, born in different parts of Greece. All requests for ANA detection were performed by enzyme linked immunoassay (ELI SA). All ELISA borderline, weak positive and pbsitive results were run on the indirect immunofluorescence assay (IFA) on Hep-2 cells. Anti-dsDNA antibodies were detected by IFA on Crithidia luciliae substrate slides. Antibodies to Sm, RNP, SS-A(Ro), SS-B(La), Jo-1, and Scl-70 were determined by an immunodot qualitative test. 206 patients were positive for ANA, representing a prevalence of 6.87%. The positive samples demonstrated the expected variety of titers and reactivity patterns. 9 samples (4.37%) presented anti-dsDNA positive result and 44 (21.36%) presented reactivity to various ENA autoantibodies. All the examined autoantibodies presented a higher prevalence among women. In this study we estimated the prevalence of ANA, anti-dsDNA, and anti-ENA antibodies in samples of 3000 sera. We followed the strategy of performing immunofluorescence testing in order to confirm positive ELISA results, proposed by many scientists. We also evaluated autoantibody titers and fluorescence patterns, and we examined the correlation of autoantibody presence with patient age and gender.


Muscarinic cholinergic binding sites on rat lymphocytes.
Receptors for neurotransmitters in blood cells could serve as useful markers for the same receptors in solid tissues. Muscarinic receptors have been identified in human, rat and mouse lymphocytes by binding of [3H]quinuclidinyl benzilate (3H-QNB); however, the biochemical and pharmacological characterization of such binding sites has not been complete. Spleen lymphocytes were isolated on a histopaque gradient and incubated in Hank's buffer with 3H-QNB. Binding of 3H-QNB was linear with increasing protein concentrations and was saturable. Binding constants were Bmax = 111 +/- 10.5 fmol/10(6) cells, and Kd = 29.7 +/- 3.9 nM (n = 7). An extensive pharmacological analysis of these binding sites indicated that several cholinergic muscarinic drugs were capable of inhibiting 3H-QNB binding. Muscarinic antagonists were more potent than agonists, and lipophilic drugs were more potent than hydrophilic drugs. Several non-cholinergic drugs were also capable of inhibiting 3H-QNB binding; however, they did so also in brain membranes, while a third group of non-cholinergic drugs and neurotransmitters were inactive. Similar results were also obtained in circulating lymphocytes and in lymphocyte membranes. These results suggest that lymphocytes possess muscarinic receptors which share several, although not all, characteristics of the same receptors in brain and other tissues. Measurement of these binding sites could be useful to monitor the status of muscarinic receptors in solid tissues.


Lymphocyte blast transformation responses to mitogens and specific antigens in different clinical phases of multiple sclerosis: a follow-up study.
28 MS patients were studied at least 5 times for lymphocyte blast transformation responses to several mitogens (PHA, Con, PWM) and specific antigens (PPD, herpes simplex, measles, mumps, rubella), the mean duration of the follow-up being 15.3 months. Comparison of responses during remissions, exacerbations and ACTH treatment revealed no straight-forward association between the strength of response and clinical disease activity. Mitogen responses did not differ among various disease phases, but the PPD response was significantly lower during exacerbations than during remissions and still lower during ACTH treatment (P less than 0.05, Wilcoxon paired test). In responses to viral antigens, there was the same trend as in the PPD response. However, there were great individual differences in the behaviour of all antigen and mitogen induced lymphocyte responses. As a group, MS patients also showed more changes in their PHA mitogen responses during the follow-up than 8 control subjects followed similarly (P less than 0.005, F-test). The variation coefficient of the PHA response was also correlated with the maximal difference in the Fog neurologic deficit scale during the follow-up time (r = 0.460, P less than 0.05). However, patients with greatest clinical changes most often also received ACTH treatment, which may affect the results. The follow-up results of individual patients revealed that some had quite regular patterns of decreasing responses during disease relapses, whereas others had more irregular wide fluctuations of responses.


Optimization of a method for collecting infant and toddler urine for non-target analysis using cotton pads and commercially available disposable diapers.
Urine is an abundant and useful medium for measuring biomarkers related to chemical exposures in infants and children. Identification of novel biomarkers is greatly enhanced with non-targeted analysis (NTA), a powerful methodology for broad chemical analysis of environmental and biological specimens. However, collecting urine in non-toilet trained children presents many challenges, and contamination from specimen collection can impact NTA results. We optimized a caregiver-driven method for collecting urine from infants and children using cotton pads and commercially available disposable diapers for NTA and demonstrate its applicability to various children biomonitoring studies. Experiments were first performed to evaluate the effects of processing method (i.e., centrifuge vs. syringe), storage temperature, and diaper brand on recovery of urine absorbed to cotton pads. Caregivers of 11 children (<2 years) used and retained diapers (with cotton pads) to collect their child's urine for 24 h. Specimens were analyzed via a NTA method implementing an exclusion list of ions related to contamination from collection materials. Centrifuging cotton pads through a small-pore membrane, compared to a manual syringe method, and storing diapers at 4 °C, compared to room temperature, resulted in larger volumes of recovered sample. This method was successfully implemented to recover urine from cotton pads collected in the field; between 5-9 diapers were collected per child in 24 h, and the total mean volume of urine recovered was 44.7 (range 26.7-71.1) mL. NTA yielded a list of compounds present in urine and/or stool that may hold promise as biomarkers of chemical exposures from a variety of sources. Infant and children urine is a valuable matrix for studies of the early life exposome, in that numerous biological markers of exposure and outcome can be derived from a single analysis. Depending on the nature of the exposure study, it may be the case that a simple collection method that can be facilitated by caregivers of young children is desirable, especially when time-integrated samples or large volumes of urine are needed. We describe the process for development and results of an optimized method for urine collection and analysis using commercially available diapers and non-target analysis.


Bone mineral content and mechanical strength. An ex vivo study on human femora at autopsy.
Bone mineral content as determined by computed tomography (CT) and mechanical strength on axial loading were compared in 36 cadaveric femur specimens. In axial loading, 34 femora fractured vertically in the neck, one femur fractured subtrochanterically, and one in the shaft. Based on the CT measurements of density and area, the mass of a transverse slice of the femur was estimated. Highly significant correlations were demonstrated between strength and cancellous bone density. Even higher correlations were revealed when the bone masses of the proximal and distal femoral areas were calculated. Based on these findings, an equal distribution of the effective mass of the femur was postulated.