FIB-SEM imaging of carbon nanotubes in mouse lung tissue.
Ultrastructural characterisation is important for understanding carbon nanotube (CNT) toxicity and how the CNTs interact with cells and tissues. The standard method for this involves using transmission electron microscopy (TEM). However, in particular, the sample preparation, using a microtome to cut thin sample sections for TEM, can be challenging for investigation of regions with agglomerations of large and stiff CNTs because the CNTs cut with difficulty. As a consequence, the sectioning diamond knife may be damaged and the uncut CNTs are left protruding from the embedded block surface excluding them from TEM analysis. To provide an alternative to ultramicrotomy and subsequent TEM imaging, we studied focused ion beam scanning electron microscopy (FIB-SEM) of CNTs in the lungs of mice, and we evaluated the applicability of the method compared to TEM. FIB-SEM can provide serial section volume imaging not easily obtained with TEM, but it is time-consuming to locate CNTs in the tissue. We demonstrate that protruding CNTs after ultramicrotomy can be used to locate the region of interest, and we present FIB-SEM images of CNTs in lung tissue. FIB-SEM imaging was applied to lung tissue from mice which had been intratracheally instilled with two different multiwalled CNTs; one being short and thin, and the other longer and thicker. FIB-SEM was found to be most suitable for detection of the large CNTs (Ø ca. 70 nm), and to be well suited for studying CNT agglomerates in biological samples which is challenging using standard TEM techniques.


Microemulsion-enhanced remediation of soils contaminated with organochlorine pesticides.
Soil contaminated by organic pollutants, especially chlorinated aromatic compounds such as DDT (1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane), is an environmental concern because of the strong sorption of organochlorine pesticide onto the soil matrix and persistence in the environment. The remediation of organochlorine pesticide contaminated soils through microemulsion is an innovative technology to expedite this process. The remediation efficiency was evaluated by batch experiments through studying the desorption of DDT and hexachlorocyclohexane (y-HCH) and sorption of microemulsion composed of Triton X-100, 1-pentanol and linseed oil in the soil-surfactant-water suspension system. The reduction of desorption efficiency caused by the sorption loss of microemulsion components onto the soil could be corrected by the appropriate adjustment of C/S (Cosurfactant/Surfactant) and O/S (Oil/Surfactant) ratio. The C/S and O/S ratios of 1:2 and 3:20 were suitable to desorb DDT and gamma-HCH from the studied soils because of the lower sorption of Triton X-100 onto the soil. Inorganic salts added in microemulsion increased the pesticides desorption efficiency of pesticides and calcium chloride has a stronger ability to enhance the desorption of DDT than sodium chloride. From the remediation perspective, the balance of surfactant or cosurfactant sorbed to soil and desorption efficiency should be taken into consideration to enhance the remediation of soils contaminated by organochlorine pesticides.


Supercritical CO2 Plant Extracts Show Antifungal Activities against Crop-Borne Fungi.
Fungal infections of cultivated food crops result in extensive losses of crops at the global level, while resistance to antifungal agents continues to grow. Supercritical fluid extraction using CO2 (SFE-CO2) has gained attention as an environmentally well-accepted extraction method, as CO2 is a non-toxic, inert and available solvent, and the extracts obtained are, chemically, of greater or different complexities compared to those of conventional extracts. The SFE-CO2 extracts of Achillea millefolium, Calendula officinalis, Chamomilla recutita, Helichrysum arenarium, Humulus lupulus, Taraxacum officinale, Juniperus communis, Hypericum perforatum, Nepeta cataria, Crataegus sp. and Sambucus nigra were studied in terms of their compositions and antifungal activities against the wheat- and buckwheat-borne fungi Alternaria alternata, Epicoccum nigrum, Botrytis cinerea, Fusarium oxysporum and Fusarium poae. The C. recutita and H. arenarium extracts were the most efficacious, and these inhibited the growth of most of the fungi by 80% to 100%. Among the fungal species, B. cinerea was the most susceptible to the treatments with the SFE-CO2 extracts, while Fusarium spp. were the least. This study shows that some of these SFE-CO2 extracts have promising potential for use as antifungal agents for selected crop-borne fungi.


[First experience with the laryngeal intubation mask].
We report our initial experience with an improved model of the laryngeal mask airway, the intubating laryngeal mask airway (ILMA, commercial name Fastrach), which was designed by A.I.J. Brain to improve blind endotracheal intubation through a laryngeal mask. In the ILMA, a number of construction details were newly designed compared to a standard laryngeal mask airway (SLM): 1) the angle between the shaft and the mask plane is changed, and also the radius of the shaft, 2) the internal diameter of the shaft is increased to allow the passage of an 8.0 mm cuffed endotracheal tube, resulting in an outer diameter of 2.0 cm, 3) a stable rubber lip (epiglottic elevating bar) is incorporated instead of the gills to clear the epiglottis out of the lumen during the passage of the endotracheal tube, 4) the shaft is manufactured out of stainless steel covered by silicone with a handle attached for more precise guidance. We prospectively used the ILMA in 80 patients for blind intubation. 51 had normal anatomy (group I), 19 were difficult to intubate (Cormack grade 3-4; group II), 18 had a reduced mouth opening (< or = 2.5 cm awake; group III), 8 of them also were difficult to intubate. Insertion of the ILMA and ventilation was possible in all patients but one with a mouth opening of 1.3 cm during anaesthesia. 4 patients with difficult intubation and one with normal anatomy could not be ventilated with a face mask but could be ventilated with the ILMA. Blind intubation was successful in 69 out of 80 patients (global success rate 87%; group I: 84%; II: 95%; III: 83%), in 38 during the first attempt (initial success rate 48%; group I: 45%; II: 63%; III: 44%), showing no difference for patients with normal anatomy, difficult intubation or reduced mouth opening. For 82 successful intubations, 157 intubation attempts were performed (success rate per attempt 52%; group I: 48%; II: 67%; III: 54%). Success rate per attempt decreased to 42%, if intubation was not successful during the first attempt (106 intubation attempts resulting in 44 successful intubations, including 31 intubation attempts for 11 failures; group I: 35%; II: 56%; III: 38%). Intubation through the ILMA was not possible in 11 patients (14%). Failures to intubate were caused by a reduced mouth opening in 1 patient, an unsuited endotracheal tube in 1 patient, a wrong size of the ILMA may have been the cause in 6 patients, in the remaining 3, lacking personal skill may have been responsible. Endotracheal tubes suited to be used with the ILMA are straight or preformed Woodbridge tubes, whereas standard plastic tubes are too stiff. Manouvres facilitating blind intubation though the ILMA were careful alignment of the ILMA with the handle, an up and down manouvre, rotation of the tube or head movements. The ILMA improved ventilation compared to a face mask and almost doubled the success rate of blind intubation compared to a SLM in our hands in a variety of intubation situations. The ILMA has the potential to be useful in difficult to intubate patients--except those with cervical pathology--or in emergency medicine. Handling can be trained during every day routine. Experienced judgement of definite endotracheal tube placement is mandatory.


Effectiveness of nanoemulsions of clove and lemongrass essential oils and their major components against Escherichia coli and Botrytis cinerea.
Nanoemulsions exhibit a number of advantages to carry and deliver lipophilic compounds such as essential oils (EOs) due to their good stability and high surface area per volume unit. The purpose of this work was to assess the long-term stability of nanoemulsions of clove and lemongrass (LG) EOs and their principal components eugenol and citral (CI), respectively, at 3 different concentrations (2, 5 or 10 times their respective minimum inhibitory concentrations) and at two storage temperatures (1 °C and 21 °C). The initial droplet size of LG and CI-loaded nanoemulsions was below 100 nm and most of them kept droplet sizes in the nano-range until the end of storage at both temperatures. The ζ-potential was lower than - 40 mV, but it increased through storage, indicating a weaker alginate adsorption at the oil surface at both temperatures. The antimicrobial activity increased with the EOs concentration and was negatively affected by the highest storage temperature. Nanoemulsions containing CI and LG were able to significantly decrease Escherichia coli counts during storage, particularly at 1 °C. Nanoemulsions containing 1.0 and 2.0% w/w CI and 2.5% w/w LG were the most efficient in reducing Botrytis cinerea growth through storage, mainly at 1 °C. The nanoemulsions containing 1.0 and 2.0% w/w CI, as well as, 1.25 and 2.5% w/w LG better maintained their stability and antimicrobial effect along 6-months storage mainly when at 1 °C, making those nanoemulsions suitable as edible coatings for food preservation. Future studies should be oriented to evaluate the impact of these nanoemulsions on the organoleptic properties of coated foods and their potential toxicity.


Structural basis of latrophilin-FLRT interaction.
Latrophilins, receptors for spider venom α-latrotoxin, are adhesion type G-protein-coupled receptors with emerging functions in synapse development. The N-terminal region binds the endogenous cell adhesion molecule FLRT, a major regulator of cortical and synapse development. We present crystallographic data for the mouse Latrophilin3 lectin and olfactomedin-like (Olf) domains, thereby revealing the Olf β-propeller fold and conserved calcium-binding site. We locate the FLRT-Latrophilin binding surfaces by a combination of sequence conservation analysis, point mutagenesis, and surface plasmon resonance experiments. In stripe assays, we show that wild-type Latrophilin3 and its high-affinity interactor FLRT2, but not the binding-impaired mutants we generated, promote HeLa cell adhesion. In contrast, cortical neurons expressing endogenous FLRTs are repelled by wild-type Latrophilin3 and not by the binding-impaired mutant. Taken together, we present molecular level insights into Latrophilin structure, its FLRT-binding mechanism, and a role for Latrophilin and FLRT that goes beyond a simply adhesive interaction.


A Comparison of Total and Plasma Membrane Abundance of Transporters in Suspended, Plated, Sandwich-Cultured Human Hepatocytes Versus Human Liver Tissue Using Quantitative Targeted Proteomics and Cell Surface Biotinylation.
Suspended (SH), plated (PH), and sandwich-cultured hepatocytes (SCH) are commonly used models to predict in vivo transporter-mediated hepatic uptake (SH or PH) or biliary (SCH) clearance of drugs. When doing so, the total and the plasma membrane abundance (PMA) of transporter are assumed not to differ between hepatocytes and liver tissue (LT). This assumption has never been tested. In this study, we tested this assumption by measuring the total and PMA of the transporters in human hepatocyte models versus LT (total only) from which they were isolated. Total abundance of OATP1B1/2B1/1B3, OCT1, and OAT2 was not significantly different between the hepatocytes and LT. The same was true for the PMA of these transporters across the hepatocyte models. In contrast, total abundance of the sinusoidal efflux transporter, MRP3, and the canalicular efflux transporters, MRP2 and P-gp, was significantly greater (P < 0.05) in SCH versus LT. Of the transporters tested, only the percentage of PMA of OATP1B1, P-gp, and MRP3, in SCH (82.8% ± 7.3%, 57.5% ± 10.9%, 69.3% ± 5.7%) was significantly greater (P < 0.05) than in SH (73.3% ± 6.4%, 27.4% ± 6.4%, 53.6% ± 4.1%). If the transporters measured in the plasma membrane are functional and the PMA in SH is representative of that in LT, these data suggest that SH, PH, and SCH will result in equal prediction of hepatic uptake clearance of drugs mediated by the transporters tested above. However, SCH will predict higher sinusoidal efflux and biliary clearance of drugs if the change in PMA of these transporters is not taken into consideration.


Identification of proteins in human substantia nigra.
Characterization of the human brain proteome is a critical area of research. While examination of the human cortex has provided some insight, very little is known about the proteome of the human midbrain, which demonstrates substantial loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson's disease (PD). Therefore, characterization of this region is essential to a better understanding of the pathogenesis of PD. This dataset paper reports two separate studies, where human SNpc was collected from PD and control subjects and 1263 proteins were identified using MALDI-TOF/TOF as well as linear ion trap MS platforms. With gene ontology analysis, the proteins were categorized according to their biological processes, as well as cellular components. These data were also compared with previous proteomic characterization of the human frontal and temporal cortex, and cerebrospinal fluid to establish shared proteins of relevance. The present dataset is the most extensive survey of the human SNpc proteome, to date. Further characterization of the SNpc proteome will significantly facilitate our understanding of the function and expression of proteins involved in PD, as well as provide potential proteins that may be utilized as biomarkers.


Linkages of acute care and EMS to state and local public health programs: application to public health programs.
Response to terrorism and mass casualty incidents has become a focal point for many public service agencies. Public health agencies and the emergency response community must work together to effectively and efficiently respond to any future incidents. Historically, collaboration has been a challenge since these agencies have functioned independently from one another, maintaining separate infrastructures that are not adequately interoperable. This article will summarize the consensus achieved during a meeting of multidisciplinary stakeholders held to discuss linkages between acute care, emergency medical services, and public health. The relevancy of these findings to public health, as well as the benefits from development of an interoperable infrastructure to public health, will be opined.


Amplitude/frequency differences in single-lead ECG of normal versus coronary heart diseased males.
A resting "normal" ECG can coexist with known coronary heart disease (CHD). Combined sensitivity and specificity errors of at least 10% in exercise ECGs are not unusual. Improved screening for CHD was attempted using amplitude/frequency analysis of ECG recordings. Thirty normal males and 30 with documented CHD were selected. The ECGs were recorded on electromagnetic tape during supine rest. Analysis provided digital conversion, selection of four ECG segments, time-normalization and amplitude/frequency analysis. Analyses provided one digital, plot per each segment and one per each 30-subject average. The results from one ECG lead are presented. Significant differences (p less than or equal to 0.05) appeared in the comparisons between the normal and CHD groups. Retrospectively, amplitude criteria individually screened normal from CHD males to an improved degree compared with exercise ECGs.


Diagnosis of COVID-19 with simultaneous accurate prediction of cardiac abnormalities from chest computed tomographic images.
COVID-19 has potential consequences on the pulmonary and cardiovascular health of millions of infected people worldwide. Chest computed tomographic (CT) imaging has remained the first line of diagnosis for individuals infected with SARS-CoV-2. However, differentiating COVID-19 from other types of pneumonia and predicting associated cardiovascular complications from the same chest-CT images have remained challenging. In this study, we have first used transfer learning method to distinguish COVID-19 from other pneumonia and healthy cases with 99.2% accuracy. Next, we have developed another CNN-based deep learning approach to automatically predict the risk of cardiovascular disease (CVD) in COVID-19 patients compared to the normal subjects with 97.97% accuracy. Our model was further validated against cardiac CT-based markers including cardiac thoracic ratio (CTR), pulmonary artery to aorta ratio (PA/A), and presence of calcified plaque. Thus, we successfully demonstrate that CT-based deep learning algorithms can be employed as a dual screening diagnostic tool to diagnose COVID-19 and differentiate it from other pneumonia, and also predicts CVD risk associated with COVID-19 infection.


Latino Families' Experiences With Autism Services: Disparities, Capabilities, and Occupational Justice.
This article examines six cases of publicly funded Applied Behavior Analysis (ABA) therapy for Latino children with autism spectrum disorder (ASD) to contribute to thinking about occupational justice. In this ethnographic study of six Latino families of children with ASD in Los Angeles County, all families were offered ABA for their children, but five families experienced occupational challenges leading them to insist on modifications of ABA or to opt out of the service. We consider in each case (a) how the families' experiences can be understood occupationally, (b) how ABA affected the functionings and capabilities of the children and their families, and (c) how the parents' accounts relate to occupational justice. Applying the capabilities approach can help operationalize the concept of occupational justice as a tool to evaluate social policy across cases.


Imaging fluid flow and cilia beating pattern in Xenopus brain ventricles.
Brain development and health depends upon the efficient movement of the cerebrospinal fluid inside of brain ventricles. When disrupted either through mutation, disease, or physiological damage, brain function becomes significantly impaired. Here I present a simple method of following cerebrospinal fluid circulation in Xenopus tadpoles using fluorescent microspheres which can be applied to imaging fluid circulation in any transparent embryo. In particular, cilia may be labeled with these microspheres to study their dynamics and movement patterns in vivo while simultaneously measuring bulk fluid flow. This technique will facilitate the analysis of fluid dynamics in developing embryos and aid in understanding the regulation of cilia dependent fluid flow in vivo.


[Effect of adenosine and ATP on electrogenesis and contraction in the smooth muscles of the cerebral arteries].
The effects of adenosine and ATP on electrogenesis and contraction in smooth muscles of the cattle basilar and middle cerebral arteries was studied with a modified sucrose gap technique. Adenosine (10(-8) - 10(-3) M) and ATP (10(-8) - 10(-6) M) induced relaxation of the muscle strips with no change in resting potential of the muscle cells. ATP (10(-5) - 10(-3) M) led to depolarization of the cell membrane followed by a hyperpolarization which was accompanied by the complex contractile reaction of muscle strips. Adenosine (10(-3) M) caused relaxation comparable to the relaxation induced by Ca++-free solution or by Mn++ ions. This suggests that extracellular Ca++ ions which participate in maintaining of the blood vessel tone enter the smooth muscle cells mainly through the stationary open adenosinesensitive Ca channels of the membrane. Ca++ ions entering the smooth muscle cells through voltage-dependent noninactivating Ca channels contribute to maintaining of cerebral arteries' basal tone. Adenosine does not affect the voltage-dependent influx of extracellular Ca++ ions through the slow noninactivating Ca channels of the cell membrane or through fast inactivating Ca channels, participating in the AP generation.


Absorption reconstruction improves biodistribution assessment of fluorescent nanoprobes using hybrid fluorescence-mediated tomography.
Fluorescence-mediated tomography (FMT) holds potential for accelerating diagnostic and theranostic drug development. However, for proper quantitative fluorescence reconstruction, knowledge on optical scattering and absorption, which are highly heterogeneous in different (mouse) tissues, is required. We here describe methods to assess these parameters using co-registered micro Computed Tomography (µCT) data and nonlinear whole-animal absorption reconstruction, and evaluate their importance for assessment of the biodistribution and target site accumulation of fluorophore-labeled drug delivery systems. Besides phantoms with varying degrees of absorption, mice bearing A431 tumors were imaged 15 min and 48 h after i.v. injection of a fluorophore-labeled polymeric drug carrier (pHPMA-Dy750) using µCT-FMT. The outer shape of mice and a scattering map were derived using automated segmentation of the µCT data. Furthermore, a 3D absorption map was reconstructed from the trans-illumination data. We determined the absorption of five interactively segmented regions (heart, liver, kidney, muscle, tumor). Since blood is the main near-infrared absorber in vivo, the absorption was also estimated from the relative blood volume (rBV), determined by contrast-enhanced µCT. We compared the reconstructed absorption with the rBV-based values and analyzed the effect of using the absorption map on the fluorescence reconstruction. Phantom experiments demonstrated that absorption reconstruction is possible and necessary for quantitative fluorescence reconstruction. In vivo, the reconstructed absorption showed high values in strongly blood-perfused organs such as the heart, liver and kidney. The absorption values correlated strongly with the rBV-based absorption values, confirming the accuracy of the absorption reconstruction. Usage of homogenous absorption instead of the reconstructed absorption map resulted in reduced values in the heart, liver and kidney, by factors of 3.5, 2.1 and 1.4, respectively. For muscle and subcutaneous tumors, which have a much lower rBV and absorption, absorption reconstruction was less important. Quantitative whole-animal absorption reconstruction is possible and can be validated in vivo using the rBV. Usage of an absorption map is important when quantitatively assessing the biodistribution of fluorescently labeled drugs and drug delivery systems, to avoid a systematic underestimation of fluorescence in strongly absorbing organs, such as the heart, liver and kidney.


Origin and evolution of invasive naturalized material of Rhododendron ponticum L. in the British isles.
Information concerning the area of origin, genetic diversity and possible acquisition of germplasm through hybridization is fundamental to understanding the evolution, ecology and possible control measures for an introduced invasive plant species. Rhododendron ponticum is extensively naturalized in the British Isles, but it is not known whether native material in Turkey, Spain or Portugal gave rise to the naturalized material, or to what extent introgression has affected this material. Chloroplast (cp) and nuclear ribosomal DNA (rDNA) restriction fragment length polymorphisms (RFLPs) were sought which could distinguish between native material of R. ponticum, and between 15 other Rhododendron species including R. ponticum's closest relatives. Thereafter, a total of 260 naturalized accessions of R. ponticum from throughout the British Isles was examined with respect to informative polymorphisms. It was found that 89% of these accessions possessed a cpDNA haplotype that occurred in native material of R. ponticum derived almost entirely from Spain, while 10% of accessions had a haplotype unique to Portuguese material. These results therefore indicated an Iberian origin for British material. rDNA or cpDNA evidence of introgression from R. catawbiense was found in 27 British accessions of R. ponticum, and such accessions were significantly more abundant in Britain's coldest region, eastern Scotland, than elsewhere. This could indicate that introgression from R. catawbiense confers improved cold tolerance. Introgression from R. maximum and an unidentified species was also detected.


Malignant dysembryoplastic neuroepithelial tumour in a zebrafish (Danio rerio).
Neuroectodermal tumours in man, including medulloblastoma, medulloepithelioma, neuroblastoma, esthesioneuroblastoma, primitive neuroectodermal tumour and dysembryoplastic neuroepithelial tumour, typically occur in children and young adults. These tumour types are occasionally observed in juvenile and adult zebrafish (Danio rerio) either as induced tumours in carcinogen-exposed zebrafish or as an incidental finding in zebrafish≥2years of age. An adult zebrafish submitted for routine histological examination was sent for a second opinion consultation after an uncharacteristic brain mass was identified. Microscopically, the expansile and infiltrative extracortical mass arising from the cerebellum had a diffuse microcystic pattern with solid hypercellular regions occupying 80% of the extrameningeal space and effacing the endomeninx and significantly displacing the metencephalon. The mass was composed of dense sheets of oligodendrocyte-like cells, random neurons and pseudocysts containing 'floating neurons' within a scant mucinous matrix. Neoplastic cells demonstrated positive perinuclear and intracytoplasmic expression of S-100. Malignant dysembryoplastic neuroepithelial tumour was diagnosed based on the histological features of the brain mass, which were indistinguishable from the human tumour. To our knowledge, this is the first report of a dysembryoplastic neuroepithelial tumour in a zebrafish.


Genetic variability in G2 and F2 region between biological clones of human respiratory syncytial virus with or without host immune selection pressure.
Human respiratory syncytial virus (HRSV) is an important respiratory pathogens among children between zero-five years old. Host immunity and viral genetic variability are important factors that can make vaccine production difficult. In this work, differences between biological clones of HRSV were detected in clinical samples in the absence and presence of serum collected from children in the convalescent phase of the illness and from their biological mothers. Viral clones were selected by plaque assay in the absence and presence of serum and nucleotide sequences of the G2 and F2 genes of HRSV biological clones were compared. One non-synonymous mutation was found in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymous mutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-B sample. Only one of these clones was obtained after treatment with the child's serum. In addition, some synonymous mutations were determined in two clones of the HRSV-A samples. In conclusion, it is possible that minor sequences could be selected by host antibodies contributing to the HRSV evolutionary process, hampering the development of an effective vaccine, since we verify the same codon alteration in absence and presence of human sera in individual clones of BR-85 sample.


The dual-targeted plant sulfiredoxin retroreduces the sulfinic form of atypical mitochondrial peroxiredoxin.
Sulfiredoxin (Srx) couples the energy of ATP hydrolysis to the energetically unfavorable process of reducing the inactive sulfinic form of 2-cysteine peroxiredoxins (Prxs) to regenerate its active form. In plants, Srx as well as typical 2-cysteine Prx have been considered as enzymes with exclusive chloroplast localization. This work explores the subcellular localization of Srx in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana). Immunocytochemistry, analysis of protein extracts from isolated intact organelles, and cell-free posttranslational import assays demonstrated that plant Srx also localizes to the mitochondrion in addition to plastids. The dual localization was in line with the prediction of a signal peptide for dual targeting. Activity tests and microcalorimetric data proved the interaction between Srx and its mitochondrial targets Prx IIF and thioredoxin. Srx catalyzed the retroreduction of the inactive sulfinic form of atypical Prx IIF using thioredoxin as reducing agent. Arabidopsis Srx also reduced overoxidized human Prx V. These results suggest that plant Srx could play a crucial role in the regulation of Prx IIF activity by controlling the regeneration of its overoxidized form in mitochondria, which are sites of efficient reactive oxygen species production in plants.


Concomitant airway expression of granulocyte-macrophage colony-stimulating factor and decorin, a natural inhibitor of transforming growth factor-beta, breaks established inhalation tolerance.
We previously showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) breaks tolerance induction. The objective of this study was to determine whether GM-CSF breaks established inhalation tolerance. To induce tolerance, BALB/c mice were exposed to aerosolized ovalbumin (OVA) for 10 consecutive days. A control group was exposed to saline. Subsequently, tolerant and control animals were exposed to OVA in a GM-CSF-enriched airway microenvironment. Tolerant animals, unlike control animals, did not develop airway and peripheral blood eosinophilia, had diminished levels of OVA-specific IgE, and reduced airway hyper-responsiveness. While tolerant animals did not express IL-4, IL-5 and IL-13, levels of the regulatory cytokines IL-10, IFN-gamma and transfoming growth factor (TGF)-beta were similar between tolerant and non-tolerant animals. Lung CD4+ T cells were activated according to CD69, CD25 and T1/ST2 expression, but systemic responses characterized by splenocyte proliferation and Th2 effector function were dramatically reduced. Concurrent expression of GM-CSF and decorin, a natural inhibitor of TGF-beta, reversed eosinophilic unresponsiveness. Our study suggests that the breakdown of tolerance and, by extension, the emergence of eosinophilic inflammation, requires two signals: one that triggers sensitization and one that interferes with negative regulation. Moreover, our study shows that dysregulated expression of an extracellular matrix protein may break established tolerance and lead to eosinophilic airway inflammation.


Expression of Human Placenta-specific 1 (PLAC1) in CHO-K1 Cells.
Placenta-specific 1 (PLAC1), as a new Cancer/Testis Antigen (CTA), is frequently expressed in a variety of cancers and localized to cytoplasm and plasma membrane. Surface expression of cancer target antigens is of great importance that enables antibody-mediated cancer immunotherapy. The aim of the current study was to express the intact human PLAC1 protein on plasma membrane of a eukaryotic cell as a model for future anti-PLAC1-based cancer immunotherapy. In the first approach, entire human PLAC1 gene including its own Signal Peptide (SP) was cloned into pIRES2-EGFP and LeGO-iG2 vectors and expressed in CHO-K1 cells. In the second approach, cytosolic and Signal-Anchor (SA) sequence of Transferrin Receptor Protein 1 (TFR1) were fused to extracellular portion of PLAC1 and expressed as above. Expression of PLAC1 was then assessed using Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western Blot (WB), Immunocytochemistry (ICC), Immunofluorescence (IF) and Flow Cytometry (FC). The first approach resulted in the expression of PLAC1 in submembranous but not in the surface of transfected CHO-K1 cells. Using the chimeric human PLAC1 construct, the same intracellular expression pattern was observed. These results indicated that there are some yet unknown PLAC1 localization signals employed by cancer cells for surface expression of PLAC1.


Potential independent action of sigma receptor ligands through inhibition of the Kv2.1 channel.
The sigma-1 receptor (σ1-R) and sigma-2 receptor (σ2-R) are potential drug targets for treatment of cancer, pain, depression, retinal degeneration and other neuronal diseases. Previous reports show that sigma-1 receptor modulates the activities of multiple channels. We are interested in possible sigma receptor modulation of Kv2.1, a K+ channel abundant in retinal photoreceptors. We tested the effect of established sigma receptor ligands on Kv2.1 channels which were stably expressed in HEK293 cells. Surprisingly, σ1-R antagonists inhibited Kv2.1 currents in both wild type and σ1-R knockout HEK293 cells that we engineered using the CRISPR/Cas9 technology. Moreover, PB28, a σ1-R antagonist and also σ2-R agonist, inhibited Kv2.1 in σ1-R knockout cells, but this action was not blocked by the σ2-R antagonists that did not have an effect on Kv2.1. We also observed inhibition of electroretinogram by PB28 in wild type as well as σ1-R knockout mice. Thus, the results in this study indicate that the Kv2.1-inhibiting function of the sigma ligands is not sigma receptor dependent, suggesting a direct effect of these ligands on the Kv2.1 channel.


Primary clear cell adenocarcinoma of the pancreas: a case report and literature update.
Primary clear cell carcinoma of pancreas is extremely rare. We present a case of a 64-year-old male with a mass in the distal body and tail of the pancreas. He underwent a distal pancreatectomy. The histopathology of tumor cells showed features with abundant clear cytoplasm and prominent cell boundaries. Immunohistochemical analysis of neoplastic cells showed reactions to antibodies against cytokeratin-7 and showed no reactions to antibodies against hepatocyte nuclear factor-1β, carbonic anhydrase 9, synaptophysin, and chromogranin A. The patient was subsequently diagnosed with a primary clear cell carcinoma of the pancreas. This is the first time we have encountered it. We report this rare case and update the current literature of this tumor.


Sustainability of a gender-focused HIV and alcohol risk-reduction intervention in usual care settings in South Africa: a mixed methods analysis.
Intervention sustainability is a critical yet understudied aspect of implementation science research. To address this gap, we examined the sustainability of the Women's Health CoOp (WHC), a gender-focused, evidence-based, HIV and alcohol risk-reduction intervention, after an implementation trial. We used a mixed methods design consisting of questionnaires (n = 12), 3 focus groups (n = 11), and a semistructured interview conducted with interventionists implementing the WHC in clinics and substance use treatment programs in Cape Town, South Africa. Five out of 8 facilities implemented the WHC beyond the 6-month implementation period, and 4 were still implementing the WHC as of October 2019. Sustainability ranged from approximately 8 months to more than 3 years. At the most recent assessment, interventionists delivered the intervention to 0-20 participants in the past month. Qualitative findings indicate that long-term sustainability would require support from upper management, staff dedicated to the WHC, and booster trainings. The WHC was sustained postimplementation. Integrating the program into usual care would be feasible; however, human resources, financial, and institutional support would be needed for sustainability. To move implementation science forward, it is essential to determine sustainability beyond the presence and involvement of researchers.


Endoplasmic reticulum stress and C/EBP homologous protein-induced Bax translocation are involved in angiotensin II-induced apoptosis in cultured neonatal rat cardiomyocytes.
The aim of this study was to identify the roles and potential mechanisms of endoplasmic reticulum stress (ER stress), proapoptotic transcription factor C/EBP homologous protein (CHOP) and Bax in angiotensin II (Ang II)-induced cardiomyocyte apoptosis. Cultured neonatal rat cardiomyocytes were incubated with Ang II or antisense CHOP oligonucleotide which was used to inhibit CHOP expression. Expressions of ER chaperone immunoglobulin heavy chain-binding protein (BiP), CHOP and cytochrome c were examined by Western blotting. Mitochondrial membrane potential (MMP) was detected by a spectrofluorimeter. Apoptosis was analyzed with flow cytometry. Bax translocation was determined by double-labeling of immunofluorescence and Western blotting. Our results showed that Ang II-induced cardiomyocyte apoptosis was associated with the upregulations of BiP and CHOP, Bax translocation, MMP deplorization and cytochrome c release. These above effects were suppressed by antisense CHOP oligonucleotide. Furthermore, BiP and CHOP expressions, reactive oxygen species (ROS) production and cardiomyocyte apoptosis, which were upregulated by Ang II, were depressed by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. From our results, ROS, ER stress and CHOP-mediated Bax translocation may be involved in Ang II-induced cardiomyocyte apoptosis.


Transposable elements: targets for early nutritional effects on epigenetic gene regulation.
Early nutrition affects adult metabolism in humans and other mammals, potentially via persistent alterations in DNA methylation. With viable yellow agouti (A(vy)) mice, which harbor a transposable element in the agouti gene, we tested the hypothesis that the metastable methylation status of specific transposable element insertion sites renders them epigenetically labile to early methyl donor nutrition. Our results show that dietary methyl supplementation of a/a dams with extra folic acid, vitamin B(12), choline, and betaine alter the phenotype of their A(vy)/a offspring via increased CpG methylation at the A(vy) locus and that the epigenetic metastability which confers this lability is due to the A(vy) transposable element. These findings suggest that dietary supplementation, long presumed to be purely beneficial, may have unintended deleterious influences on the establishment of epigenetic gene regulation in humans.


Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence.
The two prohibitin proteins, Phb1p and Phb2p(BAP37), have been ascribed various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging. We show that the mammalian prohibitins are present in the inner mitochondrial membrane and are always bound to each other, with no free protein detectable. They are coexpressed during development and in adult mammalian tissues, and expression levels are indicative of a role in mitochondrial metabolism, but are not compatible with roles in the regulation of cellular proliferation or apoptosis. High level expression of the proteins is consistently seen in primary human tumors, while cellular senescence of human and chick fibroblasts is accompanied by heterogeneous decreases in both proteins. The two proteins are induced by metabolic stress caused by an imbalance in the synthesis of mitochondrial- and nuclear-encoded mitochondrial proteins, but do not respond to oxidative stress, heat shock, or other cellular stresses. The gene promoter sequences contain binding sites for the Myc oncoprotein and overexpression of Myc induces expression of the prohibitins. The data support conserved roles for the prohibitins in regulating mitochondrial respiratory activity and in aging.


Approaching rice domestication in South Asia: New evidence from Indus settlements in northern India.
The nature and timing of rice domestication and the development of rice cultivation in South Asia is much debated. In northern South Asia there is presently a significant gap (c.4200 years) between earliest evidence for the exploitation of wild rice (Lahuradewa c.6000 BCE) and earliest dated evidence for the utilisation of fully domesticated rice (Mahagara c.1800 BCE). The Indus Civilisation (c.3000-1500 BCE) developed and declined during the intervening period, and there has been debate about whether rice was adopted and exploited by Indus populations during this 'gap'. This paper presents new analysis of spikelet bases and weeds collected from three Indus Civilisation settlements in north-west India, which provide insight into the way that rice was exploited. This analysis suggests that starting in the period before the Indus urban phase (Early Harappan) and continuing through the urban (Mature Harappan/Harappan), post-urban (Late Harappan) and on into the post-Indus Painted Grey Ware (PGW) period, there was a progressive increase in the proportion of domesticated-type spikelet bases and a decrease in wild-types. This pattern fits with a model of the slow development of rice exploitation from wild foraging to agriculture involving full cultivation. Importantly, the accompanying weeds show no increased proportions of wetland species during this period. Instead a mix of wetland and dryland species was identified, and although these data are preliminary, they suggest that the development of an independent rice tradition may have been intertwined with the practices of the eastern most Indus peoples. These data also suggest that when fully domesticated Oryza sativa ssp. japonica was introduced around 2000 BCE, it arrived in an area that was already familiar with domesticated rice cultivation and a range of cultivation techniques.


Dual individualization of intravenous ciprofloxacin in patients with nosocomial lower respiratory tract infections.
Dual individualization is the integration of patient-specific pharmacokinetic parameters with the pharmacodynamics (concentration versus response) of the infecting pathogen. This technique allows description of the time of in vivo bacterial eradication, and allows estimation of optimal dosages using small numbers of seriously ill patients. In an ongoing study, 11 patients with nosocomial lower respiratory tract infections were given 200 mg of intravenous ciprofloxacin every 12 hours. Ten blood samples were taken after the first dose, with additional peaks and troughs measured on Day 4 and at the end of treatment. Bacterial isolates had minimal inhibitory concentrations (MICs) determined by standard microdilution techniques. In the 11 patients, there were 14 bacterial isolates, of which seven were Pseudomonas aeruginosa and the remainder were other pathogens. Ciprofloxacin MICs ranged from 0.008 to 1.0 microgram/ml. The pharmacokinetics of ciprofloxacin in these patients varied with renal function, and average peak serum concentrations ranged from 1.7 to 4.9 micrograms/ml. Eradication of bacteria from tracheal aspirates occurred between Days 1 and 7, except in four patients in whom the organism persisted. Correlations were observed between the day of eradication and the length of time ciprofloxacin concentrations remained above the minimal inhibitory concentration (MIC). Essentially all bacteria with MICs of less than 0.25 were eradicated. Of the non-eradicated bacteria, most had either MICs of more than 0.25, or less than 100 percent time above the MIC. The clinical response was satisfactory. It is concluded that 200 mg of intravenous ciprofloxacin every 12 hours is highly effective for bacteria with MICs less than 0.25 microgram/ml, but higher dosages may be required to eradicate organisms with higher MICs.


Fast-atom-bombardment mass spectrometry and electron-impact direct-probe mass spectrometry in the structural analysis of oligosaccharides with special reference to the poly(N-acetyl-lactosamine) series.
A comparison has been made of positive- and negative-ion fast-atom-bombardment (FAB) and electron-impact (EI) mass spectrometry for analysis of oligosaccharides and alditols containing alternating and consecutive sequences of neutral and acetamido sugars. Among these were novel chemically synthesized tetrasaccharides with Ii antigen activities. FAB ionization has the advantage that it is applicable to non-derivatized oligosaccharides and it can determine Mr. However, the abundance of fragment ions providing structural information and the amount of material required for analysis (1-50 nmol) varied from sample to sample. In contrast, EI mass spectrometry of 5 nmol of permethylated or peracetylated oligosaccharides reliably gives all the fragment ions formed by cleavage across the glycosidic bonds.


Second-line antiretroviral treatment successfully resuppresses drug-resistant HIV-1 after first-line failure: prospective cohort in Sub-Saharan Africa.
Little is known about the effect of human immunodeficiency virus type 1 (HIV-1) resistance mutations present at time of regimen switch on the response to second-line antiretroviral therapy in Africa. In adults who switched to boosted protease inhibitor-based regimens after first-line failure, HIV-RNA and genotypic resistance testing was performed at switch and after 12 months. Factors associated with treatment failure were assessed using logistic regression. Of 243 participants, 53% were predicted to receive partially active second-line regimens due to drug resistance. The risk of treatment failure was, however, not increased in these participants. In this African cohort, boosted protease inhibitors successfully resuppressed drug-resistant HIV after first-line failure.


Medical cannabis and cannabidiol: A new harvest for Malawi.
In February 2020 parliament passed the Cannabis Regulation Bill (2020) which regulates the cultivation and production of industrial hemp and medical cannabis. The country will only fully benefit from this development if the medical and scientific community can take the lead in enabling the country to exploit the plant's potential to help address some of our economic and public health challenges. This special communication provides some basic information on cannabis and discusses its history and medical uses. Cannabidiol (CBD) has emerged as one of the most important cannabis-derived phytochemicals and has formed the basis for the growth of the medical cannabis industry. The scientific data on the mechanisms of the effects of CBD on the human neuroendocrine-immune network is reviewed and the first effective cannabis-based FDA-approved treatment for epilepsy discussed. Some clinical research that is being done on the antipsychotic and neuroprotective properties of CBD is also reviewed. A case is made for the potential of CBD as a neuroprotective adjunctive therapy for the prevention of neuropsychological sequelae associated with complicated malaria. The safety profile of CBD is reviewed and finally, the potential importance of the re-medicalization of cannabis-based therapies for the broader field of phytomedicine is pointed out.


Circadian profiling of the transcriptome in immortalized rat SCN cells.
Endogenous oscillations in gene expression are a prevalent feature of the circadian clock in the mammalian suprachiasmatic nucleus (SCN) and similar timekeeping systems in other organisms. To determine whether immortalized cells derived from the rat SCN (SCN2.2) retain these intrinsic rhythm-generating properties, oscillatory behavior of the SCN2.2 transcriptome was analyzed and compared with that found in the rat SCN in vivo using rat U34A Affymetrix GeneChips. In SCN2.2 cells, 116 unique genes and 46 ESTs or genes of unknown function exhibited circadian fluctuations with a 1.5-fold or greater difference in their mRNA abundance for two cycles. Many (35%) of these rhythmically regulated genes in SCN2.2 cells also exhibited circadian profiles of mRNA expression in the rat SCN in vivo. Functional analyses and cartography indicate that a diverse set of cellular pathways are strategically regulated by the circadian clock in SCN2.2 cells and that the largest categories of rhythmic genes are those involved in cellular and systems-level communication or in metabolic processes like cellular respiration, fatty acid recycling, and steroid synthesis. Because many of the same genes or nodes within these functional categories were rhythmically expressed in both SCN2.2 cells and the rat SCN, the circadian regulation of these pathways may be important in modulating input to or output from the SCN clock mechanism. In summary, global expression and circadian regulation of the SCN2.2 transcriptome retain many SCN-like properties, suggesting that genes displaying rhythmic profiles in both experimental models may be integral to their function as both circadian oscillators and pacemakers.


Orthostatic challenge does not alter skin sympathetic nerve activity in heat-stressed humans.
Perturbations that load or unload baroreceptors do not alter skin sympathetic nerve activity (SSNA) in normothermic individuals. However, in pronounced heat-stressed individuals, when a significant component of the SSNA signal is sudomotor and possibly vasodilator in origin, the effects of baroreceptor unloading via an orthostatic stress on SSNA remain unclear. The purpose of the present study was to test the hypothesis that low and moderate levels of orthostatic stress via lower body negative pressure (LBNP) alter SSNA in pronounced heat-stressed individuals. In both normothermic and heat-stressed conditions, progressive LBNP at -3, -6, -9, -12, -15, -18, -21 and -40 mm Hg were applied to 11 subjects for 2 min per stage. Whole-body heating increased sublingual temperature by 0.7+/-0.1 degrees C, heart rate by 28+/-2.1 bpm, SSNA by 259+/-76 %, forearm skin blood flow by 631+/-142% and forearm sweat rate to 0.68+/-0.14 mg/cm(2)/min (all p<0.005), but did not change mean arterial blood pressure (MAP) (p>0.05). LBNP did not change total SSNA in normothermic or heat-stressed conditions (both p>0.05), although skin blood flow and sweat rate decreased during moderate levels of LBNP while heat stressed. These data suggest that in pronounced heat-stressed individuals, when a significant component of the SSNA signal contains sudomotor and possibly cutaneous active vasodilator activities, low and moderate levels of baroreceptor unloading via LBNP do not alter total SSNA. This observation, coupled with reductions in skin blood flow and sweating during moderate levels of LBNP, suggests that integrated SSNA should not be used as an indicator of baroreflex modulation of the cutaneous vasculature or sweat rate in heat-stressed subjects.


Nonlinear responses of consumption to wealth, income, and interest rate shocks.
Nonlinear adjustments of consumption to housing prices, stock prices, income, and interest rates were investigated by employing panel data from 25 countries, spanning the period 2000 to 2016. This is the first study which STAR family models and nonlinear impulse response functions based on the local projections employed alternatively. We present three main pieces of evidence: (1) housing prices, stock prices, interest rates, and income exposures of consumption show time-varying and asymmetric behaviours across all countries, (2) housing wealth effects show stronger persistency and are generally larger than financial wealth effects in most of the countries, and (3) time-varying housing and financial wealth effects are high (low) during expansionary (recessionary) periods across all countries. We suggest to consider both monetary and fiscal policies, as well as the asymmetric and time-varying nature of house prices, stock prices, income, and interest rates on the top of any potential impact of the level of transition in these variables.


Topographic organization of convergent projections to the thalamus from the inferior colliculus and spinal cord in the rat.
The purpose of this study was to identify thalamic areas receiving convergent sensory inputs from acoustic and spinal projection systems in the rat. The topographic distribution of afferents to the thalamus from the inferior colliculus and spinal cord was examined by using WGA-HRP as an anterograde axonal tracer. Following injections in the inferior colliculus, terminal labeling was present in ventral, medial, and dorsal divisions of the medial genicuate body (MGB) and in adjacent areas of the posterior thalamus, including the posterior limitans nucleus, the posterior intralaminar nucleus, the marginal zone, the peripeduncular region, the lateral or parvicellular part of the subparafascicular nucleus, and a region intercalated between the posterior limitans nucleus and the suprageniculate nucleus. In the caudal thalamus spinal projections remained in the reticular formation medial to the collicular terminal field. At intermediate levels of the MG, however, the spinal projection began to overlap the collicular field, terminating in the medial division of the MG and in the posterior intralaminar nucleus, the marginal zone, the lateral subparafascicular nucleus, and the area between the suprageniculate and posterior limitans nuclei. More rostrally, the convergent field expanded to include aspects of the dorsal MG division. The extent to which afferent projections to the thalamus from the inferior colliculus and spinal cord converge is thus graded in the caudorostral plane, with the greatest overlap occurring at the level of the rostral third of the MGB. These observations identify potential areas of acoustic and somesthetic integration and may account for observations of neuronal plasticity in the thalamus in response to the pairing of acoustic and somesthetic inputs.


Olanzapine for cachexia in patients with advanced cancer: an exploratory study of effects on weight and metabolic cytokines.
Olanzapine is used for treatment of psychiatric conditions but causes substantial weight gain. This study assessed safety, efficacy, and changes in metabolic cytokines associated with olanzapine administration in patients with cachexia due to advanced cancer. Patients with cancer-related cachexia were treated with olanzapine (doses ranging from 2.5 to 20 mg daily by mouth). Patients also received anti-neoplastic treatments. Serum samples were collected at baseline and after weeks 1, 2, 4, and 8 for analysis of levels of leptin, growth hormone, ghrelin, and interleukin-6 (IL-6). Of the 39 participants, 31 were evaluable for weight change (N = 6 excluded for new ascites; N = 2, incomplete body weight of data). Toxicities related to olanzapine were somnolence (n = 1), pancreatitis (n = 1), extrapyramidal symptoms (n = 1), and nausea/vomiting (n = 1) (all grade 2). The recommended dose of Olanzapine is 20 mg PO daily for cancer patients (same as FDA approved dose for psychiatric conditions). Samples from 29 patients were eligible for analysis of serum cytokine levels. Mean values of leptin, ghrelin, and growth hormone did not change on treatment, though IL-6 levels increased, perhaps due to tumor progression. There was no association between changes in cytokines and weight. The mean change in slope of weight loss before versus after therapy was 0.24 (95 % CI, -0.08, 0.56; p = 0.13) indicating a trend, albeit not reaching statistical significance, toward attenuation of weight loss. Changes in metabolic cytokines and body weight did not correlate. Treatment with olanzapine had only a modest effect in altering the trajectory of weight loss.


Mechanism of volume adaptation in the awake early pregnant rat.
The objective of the present study was to determine whether the increase in plasma volume (PV) during pregnancy is established by fluid retention or by a shift within the extracellular fluid volume (ECFV) from the interstitium toward the intravascular compartment. To this end, we simultaneously, measured total body water, (TBW), ECFV, and PV together with the hematocrit (Hct) and plasma osmolality 4, 8, and 12 days postsurgery in chronically instrumented pregnant (P) and nonpregnant (NP) rats. The P rats were instrumented with a catheter in the femoral artery on day 1 postconception. In the NP group, neither TBW nor ECFV and PV had changed consistently on days 8 and 12 postsurgery relative to day 4. In contrast, in the P animals, TBW, ECFV, and PV had increased by 16, 24, and 20%, respectively, by day 12 relative to day 4. To evaluate whether PV had increased in concert with an overall rise in TBW or as a result of a fluid shift at the cost of the interstitial fluid volume, we calculated the relative size of each fluid compartment on three consecutive measurement sessions. In the NP group, TBW, presented as percentage of maternal weight (%MW) as well as ECFV (%TBW) and PV (%ECFV) had not changed consistently throughout the measurement period. In the P animals, TBW (%MW) was slightly higher on day 12 compared with day 4, but ECFV (%TBW) and PV (%ECFV) had not changed significantly. Finally, in the NP group, Hct had not changed, whereas, in the P animals, Hct was 10% lower on days 8 and 12 compared with day 4. Plasma osmolality did not change consistently in either group during the course of the experimental period. The gradual synchronous increase in all fluid compartments, without consistent change in their relative distribution, suggests that, in normal rat pregnancy, PV expansion is primarily achieved by fluid retention rather than by a redistribution of the ECFV.


Multiple myeloma: 2012 update on diagnosis, risk-stratification, and management.
Multiple myeloma accounts for ∼10% of all hematologic malignancies. The diagnosis requires 10% or more clonal plasma cells on bone marrow examination or a biopsy proven plasmacytoma plus evidence of end-organ damage felt to be related to the underlying plasma-cell disorder. Patients with 17p deletion, t(14;16), t(14;20), or high-risk gene expression profiling signature have high-risk myeloma. Patients with t(4;14) translocation, karyotypic deletion 13, or hypodiploidy are considered to have intermediate-risk disease. All others are considered to have standard-risk myeloma. Standard-risk patients are treated with nonalkylator-based therapy such as lenalidomide plus low-dose dexamethasone (Rd) followed by autologous stem-cell transplantation (ASCT). An alternative strategy is to continue initial therapy after stem-cell collection, reserving ASCT for first relapse. Intermediate-risk and high-risk patients are treated with a bortezomib-based induction followed by ASCT and then bortezomib-based maintenance. Patients not eligible for ASCT can be treated with Rd for standard risk disease, or with a bortezomib-based regimen if intermediate-risk or high-risk features are present. To reduce toxicity, when using bortezomib, the once-weekly subcutaneous dose is preferred; similarly, when using dexamethasone, the low-dose approach (40 mg once a week) is preferred, unless there is a need for rapid disease control. Patients with indolent relapse can be treated first with two-drug or three-drug combinations. Patients with more aggressive relapse often require therapy with a combination of multiple active agents. The most promising new agents in development are pomalidomide and carfilizomib.


Rapid Screening of Forskolin-Type Diterpenoids of Blumea aromatica DC Using Ultra-High-Performance Liquid Chromatography Tandem Quadrupole Time-of-Flight Mass Spectrometry Based on the Mass Defect Filtering Approach.
The discovery of new active compounds of natural products tends to be increasingly more challenging due to chemical complexity and unpredictable matrices. Forskolin is an active natural labdane-type diterpenoid ingredient widely used worldwide for the treatment of glaucoma, heart failure, hypertension, diabetes, and asthma, and is expected to be a promising anticancer, anti-inflammation, and anti-HIV agent. In recent years, demand for forskolin in the medicine market has increased dramatically. However, natural forskolin originates exclusively from traditional Indian herb medicine Coleus forskohlii (Willd.) Briq. In a previous study, we isolated a series of diterpenoids including an 8,13-epoxy-14ene labdane carbon skeleton from Blumea aromatica DC. In order to identify alternative plant resources, a novel and effective strategy was proposed for the screening of potential forskolin-type diterpenoids (FSKD) compounds obtained from B. aromatica, using the mass defect filtering (MDF) strategy via ultra-high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) approach. Within a narrow, well-defined mass defect range, the strategy developed could significantly improve the detection efficiency of selected FSKD compounds by filtering out certain major or moderate interference compounds. Additionally, the MS/MS cleavage behavior and the characteristic diagnostic ions of the FSKD compounds were proposed to be used in aiding structural identification of the filtration compounds. As a result, a total of 38 FSKD of B. aromatica were filtered out and tentatively identified. To the best of our knowledge, it was the first time that these forskolin-type diterpenoids were identified in B. aromatica, which significantly expands our understanding of the chemical constituents of Blumea species, and allows B. aromatica to be used as a potential alternative plant resource that contains these forskolin-type active compounds. The strategy proposed was proven efficient and reliable for the discovery of novel compounds of herbal extracts.


Collective roles of molecular chaperones in protein degradation pathways associated with neurodegenerative diseases.
The homeostasis of the protein synthesis and degradation is crucial for cell survival. Most age-related neurodegenerative diseases are characterized by accumulation of aberrant protein aggregates in affected brain regions. The principal routes of intracellular protein metabolism are the ubiquitin proteasome system (UPS) and the autophagy-lysosome pathway (ALP). They collaborate to degrade wasted proteins and interact each other to cope with the pathological conditions, in which molecular chaperones play collective roles by assisting the protein targeting to the proteasome or autophagy. It is known that intracellular protein degradation functions are decreased with aging in many tissues and organs. Failure to perform their functions could underlie the inability of cells to adapt to stress conditions, lead to accelerated course of misfolding protein deposit and the inclusion body formation, and eventually result in neurodegeneration.One of the functions of the molecular chaperones is to help the new synthesized or the misfolding toxic proteins fold to their native and nontoxic formation, as our common conception. In this review, we analyze the recent perceptions and findings of molecular chaperones biology in the two degradation pathways and their pathological attribution in several neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and others. It is worthy noticing that some of the HSPs can not only block the protein aggregation in the early stages, but also have promising effect on attenuating the formation of fibrils. Further more, when the degradation pathways are too weak to degrade all the toxic soluble proteins, molecular chaperones can also help to sequenstrate the toxic proteins into inclusion bodies. However, whether it is good or bad is still unclear. Therefore, the study of HSPs might shed new light on not only the mechanisms of protein synthesis and degradation, but also the possible therapeutic targets of fibril formation associating diseases.


Host cell-dependent differences in the oligosaccharide moieties of the VSV G protein.
The oligosaccharide moieties of vesicular stomatitis virus glycoprotein from virus grown in four different cell lines have been characterized by sequential enzymic degradation followed by ion-exchange chromatography and analytical gel filtration. Whilst the same two peptide sites are glycosylated in all cell lines, the extent of sialylation of the oligosaccharides is, however, a function of the cell line in which the virus is produced. Using specific purified glycosidases for sequential degradation of glycopeptides obtained after Pronase digestion, the oligosaccharide structures from the different host cell lines appear similar. However, differential sensitivity of the glycopeptides to treatment with a partially purified mixture of endo- and exoglycosidases indicates that the oligosaccharide structures are not identical.


Moxibustion versus diclofenac sodium gel for the treatment of knee osteoarthritis: a study protocol for a double-blinded, double-placebo, randomised controlled trial.
Knee osteoarthritis is a common form of arthritis in elderly patients that is characterised by pain and functional limitation. Moxibustion has been employed to relieve chronic pain as an alternative therapy for knee osteoarthritis. However, the evidence of its efficacy is equivocal due to the low methodological quality in most clinical studies. Therefore, we are performing a double-blinded, double-placebo, randomised controlled trial to evaluate the efficacy of moxibustion in participants with knee osteoarthritis. This is a multicentre, double-blinded, double-placebo, randomised controlled clinical trial. 144 eligible participants with knee osteoarthritis will be randomly assigned to two different groups in a 1:1 ratio. Participants in the moxibustion group will undergo active moxibustion plus placebo gel, whereas participants in the control group will receive diclofenac sodium gel plus placebo moxibustion. Each participant will receive 12 sessions of active/placebo moxibustion at three acupoints (ST35, ST36 and EX-LE4) as well as 2 months of follow-up. Diclofenac sodium gel or placebo gel at a dose of 4 g per knee will be applied three times per day for 4 weeks. The primary outcome measure will be the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score change at the end of the intervention period from baseline. The secondary outcome measures include changes of other subscales (pain, stiffness and function) of WOMAC, visual analogue scale and patient globalassessment. The safety of moxibustion and diclofenac sodium gel will be assessed at every visit. This trial has been approved by the Sichuan Regional Ethics Review Committee (permission number: 2015KL-014). The results of this study are expected to provide clinical evidence on the efficacy of moxibustion for pain relief and physical function improvement in patients with knee osteoarthritis. The findings will be submitted for publication in peer-reviewed medical journals and presented at relevant academic conferences. NCT02769572.


A national survey of videolaryngoscopes and alternative intubation devices in Hungary.
Videolaryngoscopy (VL) as a new airway management technique has evolved in recent decades, and a large number of videolaryngoscopes are now available on the market. Most recent major guidelines already recommend the immediate availability and use of VL in difficult airway management scenarios. However, national data on the availability of VL, introduction into practice and patterns of use are rarely published. Therefore, the current study aimed to provide data on VL in Hungary. An electronic survey was designed and popularized with the help of the Hungarian Society of Anaesthesiology and Intensive Therapy to explore the availability, use, and practice of and attitudes toward VL among Hungarian anesthesiologists. The survey was conducted between 01.01.2018 and 31.12.2018. In total, 324 duly completed forms were returned and analyzed. Responders were mainly males (58%), specialists (80%) and those involved mainly in anesthesia practice (68%) in the public sector. Two hundred and ten (65%) responders had access to various videolaryngoscopes and were mainly from surgery, intensive care and traumatology units. No responders reported the availability of eight videolaryngoscopes out of the eighteen listed devices, and 32% of the responders had never used any videolaryngoscope in clinical settings. The most commonly available devices were KingVision, MacGrath Mac and Airtraq. Most of the responders reported using videolaryngoscopes mainly for difficult airway management and reported using a fiberscope as the first alternative device. Popular methods for selecting videolaryngoscopes included the following: short clinical trial (n = 67/324), decision of the departmental lead (n = 65/324) and price (n = 54/324). The majority of responders had some training prior to clinical application, but training was mainly voluntary. Overall, 98% of the responders considered videolaryngoscopes beneficial. Approximately two-thirds of Hungarian anesthesiologists have immediate access to videolaryngoscopes, which are used mainly for difficult airway management. The overall attitude towards VL is positive, and many videolaryngoscopes are known and have been used by Hungarian anesthesiologists. However, only a few devices on the market are used commonly. Based on the results, further improvement might be recommended regarding VL training and availability.


Nurses' perceptions of ethical issues related to patients' rights law.
August 2006 marked the 10th anniversary of landmark legislation when Israel's parliament passed the unique Patient's Rights Law. This law underscores the importance of medical ethics in Israeli society. During a seminar at the Shaare Zedek School of Nursing, third-year students performed a qualitative research study investigating ethical issues arising in the field of nursing, and how nursing staff dealt with these issues in relation to the law. The research was conducted using semistructured questionnaires. The results showed that the staff participants knew the law, but did not differentiate between legal and ethical problems. The establishment of a framework for dealing with these issues would help to promote professional ethics, encourage broad-based agreements related to ethical decisions, reduce ethical conflict, and increase implementation of the law on patients' rights.


Seroprevalence of anti-SARS-CoV-2 IgG at the first epidemic peak in French Guiana, July 2020.
While Latin America has been heavily affected by the pandemic, only a few seroprevalence studies have been conducted there during the first epidemic wave in the first half of 2020. A cross-sectional survey was performed between 15 July 2020 and 23 July 2020 among individuals who visited 4 medical laboratories or 5 health centers for routine screening or clinical management, with the exception of symptomatic suggestive cases of covid-19. Samples were screened for the presence of anti-SARS-CoV-2 IgG directed against domain S1 of the SARS-CoV-2 spike protein using the anti-SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA) from Euroimmun. The overall seroprevalence was 15.4% [9.3%-24.4%] among 480 participants, ranging from 4.0% to 25.5% across the different municipalities. The seroprevalence did not differ according to gender (p = 0.19) or age (p = 0.51). Among SARS-CoV-2 positive individuals, we found that 24.6% [11.5%-45.2%] reported symptoms consistent with COVID-19. Our findings revealed high levels of infection across the territory but a low number of resulting deaths, which can be explained by French Guiana's young population structure.


A novel trans-spliced mRNA from Onchocerca volvulus encodes a functional S-adenosylmethionine decarboxylase.
Complete cDNA and genomic sequences encoding the Onchocerca volvulus S-adenosylmethionine decarboxylase (SAMDC), a key enzyme in polyamine biosynthesis, have been isolated and characterized. The deduced amino acid sequence encodes a 42 kDa proenzyme with a moderate level of sequence homology to eukaryotic SAMDCs. Enzymically active O. volvulus SAMDC was expressed at a high level in an Escherichia coli mutant strain lacking endogenous SAMDC. The recombinant enzyme was purified to homogeneity using DEAE-cellulose, methylglyoxal bis(guanylhydrazone)-Sepharose and Superdex S-200 chromatography. It was determined that the recombinant proenzyme is cleaved to produce 32 and 10 kDa subunits. The sequence of the N-terminal portion of the large subunit was determined and comparison with the sequence of the proenzyme revealed that the precise cleavage site lies between Glu86 and Ser87. Gel-filtration experiments demonstrated that these two subunits combine to form an active heterotetramer. Comparison of the cDNA and genomic sequences revealed that the SAMDC mRNA undergoes both cis- and trans-splicing in its 5'-untranslated region (UTR). Anchored PCR on O. volvulus mRNA confirmed the cDNA sequence and identified two distinct trans-spliced products, a 22-nucleotide spliced-leader sequence and a 138 bp sequence containing the 22 nucleotide spliced-leader sequence. Genomic Southern-blot analysis suggests that the O. volvulus SAMDC is encoded by a single-copy gene. This gene spans 5.3 kb and is comprised of nine exons and eight introns. The first intron is located in the 5'-UTR and processing of this intron has a potential regulatory function. The 5'-flanking region of the gene contains potential transcriptional regulatory elements such as a TATA box, two CAAT boxes and AP-1-, C/EBP-, ELP-, H-APF-1-, HNF-5- and PEA3-binding sites.


High-temperature actuation performance of BiScO3-PbTiO3 ceramics and their multilayer configuration.
Electric-field-induced strains of BiScO3-PbTiO3 (BS-PT) ceramics and actuation performance in multilayer configuration were investigated in the temperature range from 25°C to 250°C. Experimental results confirmed that BS-PT ceramics showed better piezoelectric temperature stability than PZT-5H ceramics, and larger strain than PZT-4 and PZT-8 ceramics at temperatures above 50°C. The strain of a BSPT multilayer actuator prepared by the silicate-cement-glue method was comparable to that of a single-layer BS-PT ceramic. Under ±7.5 kV/cm electric field, the strain of a BS-PT multilayer actuator reached 0.115% at 200°C. Our work shows that BS-PT ceramics are suitable for high-temperature actuation applications.


Interdental cleaning behaviour and its relationship with psychological constructs based on the transtheoretical model.
The aim of this study was to test the applicability of the Transtheoretical Model (TTM) to gain an understanding of interdental cleaning behaviour change in senior high school students (12th grade) in Iran. Multistage cluster sampling was employed to recruit 361 senior high school students from 8 schools in Yazd City, Iran. This cross-sectional study took place from November 2008 to March 2009. Appropriate instruments were used to identify the stages of interdental cleaning behaviour and psychological attributes, including interdental cleaning behaviour, decisional balance, and self-efficacy. The statistical analysis of the data included descriptive statistics, t-test and ANOVA. Of the 361 students, nearly 12.5% were in the maintenance stage, while 49.6% were in the pre-contemplation stage, with the rest distributed among the other stages of interdental cleaning behaviour change. There was a statistically significant difference in the stages of interdental cleaning behaviour change by gender. Self-efficacy and decisional balance differed significantly across the stages of interdental cleaning behaviour change. The TTM was found to be useful in determining the stages of interdental cleaning behaviour change among students. This study suggests the development of theory-based and empirically supported intervention strategies and programs to improve interdental cleaning behaviour with an emphasis on improving students' self-efficacy in this area.


Flexible additive free H2V3O8 nanowire membrane as cathode for sodium ion batteries.
Sodium ion batteries (SIBs) have emerged as a potential candidate to succeed lithium ion batteries (LIBs), because of the abundant sodium resources on earth. Layered vanadium oxides are regarded as the promising candidates for SIBs because of their large interlayer spacing, high theoretical specific capacity, abundant sources and low cost. In this paper, a vanadium oxide hydrate (H2V3O8) nanowire membrane is presented as a flexible cathode for SIBs without addition of any other additives (binders or conductive compounds). Such a freestanding flexible membrane exhibits a high specific capacity of 168 mA h g(-1) at 10 mA g(-1), and its high capacity is maintained well after 100 cycles. It is found that the capacitive charge storage accounts for a relatively large proportion of the total capacity, whereas the crystal structure of H2V3O8 is highly reversible during the sodiation/desodiation processes. This research demonstrates that the H2V3O8 nanowire is an exceptional candidate for SIBs.


Pathogen manipulation of chloroplast function triggers a light-dependent immune recognition.
In plants and animals, nucleotide-binding leucine-rich repeat (NLR) proteins are intracellular immune sensors that recognize and eliminate a wide range of invading pathogens. NLR-mediated immunity is known to be modulated by environmental factors. However, how pathogen recognition by NLRs is influenced by environmental factors such as light remains unclear. Here, we show that the agronomically important NLR Rpi-vnt1.1 requires light to confer disease resistance against races of the Irish potato famine pathogen Phytophthora infestans that secrete the effector protein AVRvnt1. The activation of Rpi-vnt1.1 requires a nuclear-encoded chloroplast protein, glycerate 3-kinase (GLYK), implicated in energy production. The pathogen effector AVRvnt1 binds the full-length chloroplast-targeted GLYK isoform leading to activation of Rpi-vnt1.1. In the dark, Rpi-vnt1.1-mediated resistance is compromised because plants produce a shorter GLYK-lacking the intact chloroplast transit peptide-that is not bound by AVRvnt1. The transition between full-length and shorter plant GLYK transcripts is controlled by a light-dependent alternative promoter selection mechanism. In plants that lack Rpi-vnt1.1, the presence of AVRvnt1 reduces GLYK accumulation in chloroplasts counteracting GLYK contribution to basal immunity. Our findings revealed that pathogen manipulation of chloroplast functions has resulted in a light-dependent immune response.


Nucleation of iron oxide nanoparticles mediated by Mms6 protein in situ.
Biomineralization proteins are widely used as templating agents in biomimetic synthesis of a variety of organic-inorganic nanostructures. However, the role of the protein in controlling the nucleation and growth of biomimetic particles is not well understood, because the mechanism of the bioinspired reaction is often deduced from ex situ analysis of the resultant nanoscale mineral phase. Here we report the direct visualization of biomimetic iron oxide nanoparticle nucleation mediated by an acidic bacterial recombinant protein, Mms6, during an in situ reaction induced by the controlled addition of sodium hydroxide to solution-phase Mms6 protein micelles incubated with ferric chloride. Using in situ liquid cell scanning transmission electron microscopy we observe the liquid iron prenucleation phase and nascent amorphous nanoparticles forming preferentially on the surface of protein micelles. Our results provide insight into the early steps of protein-mediated biomimetic nucleation of iron oxide and point to the importance of an extended protein surface during nanoparticle formation.


[Analysis of polymorphism of the 5'-terminal region of apolipoprotein B gene in patients with ischemic heard disease].
Comparative analysis of polymorphism of two polymorphic regions in the 5'-end of the apolipoprotein B gene-insertion/ deletion polymorphism of a signal peptide of apo B and microsatellite (CA)n repeat-was performed in a random sample of men from the Moscow population aged 49-51 and in heart patients. No associations among the gene polymorphic regions, development of coronary heart disease, and certain changes in the lipid spectrum of plasma are revealed. This indicates that the structural organization of the 5'-region of apolipoprotein gene B (Moscow population) has no substantial role in determining the risk of coronary heart disease.


AT(1) receptor regulation in salt-sensitive hypertension.
The molecular events governing salt-sensitive hypertension are currently unknown. Because the renin-ANG system plays a central role in blood pressure regulation and electrolyte balance, it may be closely involved in the phenomenon of salt sensitivity. Therefore, we examined the effect of a high-salt diet (8%) and a low-salt diet (0.4%) on ANG II-caused vascular constriction and ANG II type 1 (AT(1)) receptor expression in aorta, brain, and kidney of Dahl S (salt-sensitive) and Dahl R (salt-resistant) rats by means of radioligand binding assays and quantitative PCR. NaCl diet at 8% led to a significant increase of blood pressure in Dahl S but not in Dahl R rats. High-sodium intake caused a profound decrease of ANG II-induced aortic vasoconstriction in both Dahl R and Dahl S rats. The underlying mechanism was a downregulation of aortic AT(1) receptor density and AT(1) receptor mRNA. AT(1) receptor mRNA was downregulated to 57.8% in Dahl R and 59.0% in Dahl S rats by an 8% NaCl diet compared with a 0.4% NaCl diet (P < 0.05). There was a similar decrease in aortic AT(1) receptor density. Additionally, AT(1) receptor mRNA was also downregulated in the kidney but upregulated the brain of Dahl R and S rats on a high-salt diet. Thus high NaCl intake causes organ-specific AT(1) receptor regulation in Dahl R and in Dahl S rats despite the differential blood pressure regulation in these animal models in response to a high-salt diet. These findings suggest that the regulation of vascular AT(1) receptors is influenced by numerous factors such as the renin-ANG system and obviously by various other events that are currently only partly understood.


A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome.
Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population.


Changes of Conformation in Albumin with Temperature by Molecular Dynamics Simulations.
This work presents the analysis of the conformation of albumin in the temperature range of 300 K - 312 K , i.e., in the physiological range. Using molecular dynamics simulations, we calculate values of the backbone and dihedral angles for this molecule. We analyze the global dynamic properties of albumin treated as a chain. In this range of temperature, we study parameters of the molecule and the conformational entropy derived from two angles that reflect global dynamics in the conformational space. A thorough rationalization, based on the scaling theory, for the subdiffusion Flory-De Gennes type exponent of 0 . 4 unfolds in conjunction with picking up the most appreciable fluctuations of the corresponding statistical-test parameter. These fluctuations coincide adequately with entropy fluctuations, namely the oscillations out of thermodynamic equilibrium. Using Fisher's test, we investigate the conformational entropy over time and suggest its oscillatory properties in the corresponding time domain. Using the Kruscal-Wallis test, we also analyze differences between the root mean square displacement of a molecule at various temperatures. Here we show that its values in the range of 306 K - 309 K are different than in another temperature. Using the Kullback-Leibler theory, we investigate differences between the distribution of the root mean square displacement for each temperature and time window.


Potential of Chlorella as a Dietary Supplement to Promote Human Health.
Chlorella is a green unicellular alga that is commercially produced and distributed worldwide as a dietary supplement. Chlorella products contain numerous nutrients and vitamins, including D and B12, that are absent in plant-derived food sources. Chlorella contains larger amounts of folate and iron than other plant-derived foods. Chlorella supplementation to mammals, including humans, has been reported to exhibit various pharmacological activities, including immunomodulatory, antioxidant, antidiabetic, antihypertensive, and antihyperlipidemic activities. Meta-analysis on the effects of Chlorella supplementation on cardiovascular risk factors have suggested that it improves total cholesterol levels, low-density lipoprotein cholesterol levels, systolic blood pressure, diastolic blood pressure, and fasting blood glucose levels but not triglycerides and high-density lipoprotein cholesterol levels. These beneficial effects of Chlorella might be due to synergism between multiple nutrient and antioxidant compounds. However, information regarding the bioactive compounds in Chlorella is limited.


Clostridioides difficile: diagnosis and treatments.
Clostridioides difficile (formerly Clostridium) is a major cause of healthcare associated diarrhea, and is increasingly present in the community. Historically, C difficile infection was considered easy to diagnose and treat. Over the past two decades, however, diagnostic techniques have changed in line with a greater understanding of the physiopathology of C difficile infection and the use of new therapeutic molecules. The evolution of diagnosis showed there was an important under- and misdiagnosis of C difficile infection, emphasizing the importance of algorithms recommended by European and North American infectious diseases societies to obtain a reliable diagnosis. Previously, metronidazole was considered the reference drug to treat C difficile infection, but more recently vancomycin and other newer drugs are shown to have higher cure rates. Recurrence of infection represents a key parameter in the evaluation of new drugs, and the challenge is to target the right population with the adapted therapeutic molecule. In multiple recurrences, fecal microbiota transplantation is recommended. New approaches, including antibodies, vaccines, and new molecules are already available or in the pipeline, but more data are needed to support the inclusion of these in practice guidelines. This review aims to provide a baseline for clinicians to understand and stratify their choice in the diagnosis and treatment of C difficile infection based on the most recent data available.


Effect of inactivation of the Arg- and/or Lys-gingipain gene on selected virulence and physiological properties of Porphyromonas gingivalis.
Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis. Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis, inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis, including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects.


[One-stage repair and reconstruction of knee anterior cruciate ligament, posterior cruciate ligament, and medial collateral ligament].
To investigate the effectiveness of anterior cruciate ligament (ACL) and posterior cruciate ligament (PCL) reconstruction combined with limited open repair of medial collateral ligament (MCL) in recovering the stability and the function of the knee. Between April 2003 and October 2010, 14 patients (14 knees) with multiple injuries of ACL, PCL, and MCL were treated. There were 10 males and 4 females with an average age of 41 years (range, 21-71 years). Injury was caused by traffic accident in 11 cases and falling in 3 cases. The average time from injury to admission was 2 days (range, 1-4 days). Lysholm score was 17.00 +/- 8.29, and the International Knee Documentation Committee (IKDC) score was 20.93 +/- 8.28. The complicated injuries included dislocation of the knee joint in 9 cases and meniscus injury in 5 cases. Allogeneic tendons (2 cases) and autologous harmstring tendon (12 cases) were used to reconstruct ACL and PCL under arthroscopy, and all cases underwent limited open repair of MCL. All incisions healed by first intention. Numbness of the lower limb occurred in 3 cases and alleviated spontaneously. All patients were followed up 14 months on average (range, 12-18 months). The knee flexion was 120 degrees and extension was 0 degrees at 3 months of follow-up. After 1 year of follow-up, IKDC score and Lysholm score were 89.93 +/- 6.26 and 88.93 +/- 4.82, respectively, showing significant differences when compared with preoperative scores (P < 0.01). For multiple injuries of the knee ligaments, an arthroscope with limited open repair and reconstruction of the knee ligament can avoid open joint chamber, reduce postoperative articular adhesion, and encourage the joint function recovery.


Trends in lower limb amputation in the Veterans Health Administration, 1989-1998.
To assess trends in lower limb amputation performed in Veterans Health Administration (VHA) facilities. All lower limb amputations recorded in the Patient Treatment File for 1989-1998 were analyzed using the hospital discharge as the unit of analysis. Age-specific rates were calculated using the VHA user-population as the denominator. Frequency tables and linear, logistic, and Poisson regression were used respectively to assess trends in amputation numbers, reoperation rates, and age-specific amputation rates. Between 1989-1998, there were 60,324 discharges with amputation in VHA facilities. Over 99.9% of these were in men and constitute 10 percent of all US male amputations. The major indications were diabetes (62.9%) and peripheral vascular disease alone (23.6%). The age-specific rates of major amputation in the VHA are higher than US rates of major amputation. VHA rates of major and minor amputation declined an average of 5% each year, while the number of diabetes-associated amputations remained the same. The number and age-specific rates of amputations decreased over 10 years despite an increase in the number of veterans using VHA care.


Is publication bias present in gastroenterological research? An analysis of abstracts presented at an annual congress.
Publication bias is the tendency of investigators, reviewers, and editors to submit or accept manuscripts for publication based on their direction or strength of findings. In this study, we investigated if publication bias was present in gastroenterological research by evaluating abstracts at Americas Hepato-Pancreato-Biliary Congresses from 2011 to 2013. We searched Google, Google Scholar, and PubMed to locate the published reports of research described in these abstracts. If a publication was not found, a second investigator searched to verify nonpublication. If abstract publication status remained undetermined, authors were contacted regarding reasons for nonpublication. For articles reaching publication, the P value, study design, time to publication, citation count, and journals in which the published report appeared were recorded. Our study found that of 569 abstracts presented, 297 (52.2%) reported a P value. Of these, 254 (85.5%) contained P values supporting statistical significance. The abstracts reporting a statistically significant outcome were twice as likely to reach publication than abstracts with no significant findings (OR 2.10, 95% CI [1.06-4.14]). Overall, 243 (42.7%) abstracts reached publication. The mean time to publication was 14 months and a median time of nine months. In conclusion, we found evidence for publication bias in gastroenterological research. Abstracts with significant P values had a higher probability of reaching publication. More than half of abstracts presented from 2011 to 2013 failed to reach publication. Readers should take these findings into consideration when reviewing medical literature.


Does the Intake of Ethanol Affect Oral Absorption of Poorly Soluble Drugs?
The presence of ethanol in gastrointestinal (GI) fluids may increase the solubility of poorly water-soluble drugs. This suggests that intake of ethanol with such compounds could result in increased drug absorption in the stomach and duodenum because of the greater concentration gradient present. To test this hypothesis, in vitro dissolution of 2 poorly soluble compounds (indomethacin and felodipine) was studied in simulated GI rat fluids in the presence or absence of ethanol. Results were used to predict plasma exposure of the compounds using the software PK-Sim. Finally, in vivo plasma exposure in rats was investigated after oral dosing followed by immediate administration of water or ethanol. Despite increased solubility in GI fluids in the presence of ethanol, simulations predicted a negligible effect on absorption. This was confirmed in the rat study where oral intake of indomethacin or felodipine with ethanol did not increase in vivo plasma exposure. A possible explanation for the lack of an effect may be that dilution, absorption, and transfer of ethanol upon arrival in the stomach resulted in intragastric and intraduodenal ethanol concentrations that did not reach the levels required to affect local solubility.


Cytokine gene expression in normal human lymphocytes in response to stimulation.
Sequential gene expression of two type 1 cytokines (interleukin 2 [IL-2] and gamma interferon), one type 2 cytokine (IL-10), two monokines (IL-6 and tumor necrosis factor alpha), and one cytokine receptor (IL-2 receptor [IL-2R]) in normal human peripheral blood mononuclear cells (PBMC) following in vitro stimulation was investigated by reverse transcription-PCR methods. Two stimuli were utilized: phytohemagglutinin (PHA), which acts on the CD2 molecule and T-cell receptors, and anti-CD3 monoclonal antibody, which acts on the CD3 molecule and on T-cell receptors. Increased expression of all studied genes occurred between 1 and 4 hours after stimulation, except for that of the gene encoding IL-10, which was delayed. Expression of all but one of the genes was transient, with a maximal mRNA accumulation at about 8 h on average. IL-2R mRNA expression was an exception, showing a prolonged increase (72 h). The general profiles of expression of the five cytokine genes were similar but not identical, suggesting some shared regulatory mechanisms. When responses to four additional stimuli (pokeweed mitogen, Candida albicans, and IL-2 at high and low doses) were compared, similar profiles of cytokine gene expression were found. Thus, the various stimuli caused induction of all cytokines with quantitative, not qualitative, differences. Altogether, the present data are useful for defining the kinetics of gene expression for key cytokines in response to standard immune-cell stimuli.


In situ Raman spectroscopy: a process analytical technology tool to monitor a de-protection reaction carried out in aqueous solution.
The application of in situ Raman spectroscopy at small scale (maximum 80 mL) during the development of a manufacturing process is disclosed. The reaction was run in aqueous solution between ambient and 100 °C. Raman spectroscopy has proven to be a viable method to track the reaction. Three distinct phases could be followed: dissolution of the starting material, production of a reactive intermediate, and then subsequent conversion of that intermediate to form product. The objective of the work was to confirm the presence of a reactive intermediate and this could only be carried out via in situ spectroscopy as the intermediate was known to be unstable. Toward the end, the reaction passes though several neutralization points and these are consistent with changes in the spectra. Comparison of data obtained at an illumination wavelength of 998 nm with that obtained at 785 nm is also disclosed. The data obtained at shorter wavelength was contaminated by reasonably strong fluorescence, whereas the data obtained at 998 nm was free of fluorescence. An unexpected observation from this work was that the reaction time was much shorter than expected and this work was key in showing that a reduction in batch cycle time was possible during commercial manufacture.