Functional antibodies exhibit light chain coherence.

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies that bind particular antigens1. In most mammals, antibodies are composed of heavy and light chains that are generated sequentially by recombination of V, D (for heavy chains), J and C gene segments. Each chain contains three complementarity-determining regions (CDR1-CDR3), which contribute to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-22. Here we consider pairs of B cells that share the same heavy chain V gene and CDRH3 amino acid sequence and were isolated from different donors, also known as public clonotypes23,24. We show that for naive antibodies (those not yet adapted to antigens), the probability that they use the same light chain V gene is around 10%, whereas for memory (functional) antibodies, it is around 80%, even if only one cell per clonotype is used. This property of functional antibodies is a phenomenon that we call light chain coherence. We also observe this phenomenon when similar heavy chains recur within a donor. Thus, although naive antibodies seem to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

Gene expression differences associated with intrinsic hindfoot muscle loss in the jerboa, Jaculus jaculus.

Vertebrate animals that run or jump across sparsely vegetated habitats, such as horses and jerboas, have reduced the number of distal limb bones, and many have lost most or all distal limb muscle. We previously showed that nascent muscles are present in the jerboa hindfoot at birth and that these myofibers are rapidly and completely lost soon after by a process that shares features with pathological skeletal muscle atrophy. Here, we apply an intra- and interspecies differential RNA-Seq approach, comparing jerboa and mouse muscles, to identify gene expression differences associated with the initiation and progression of jerboa hindfoot muscle loss. We show evidence for reduced hepatocyte growth factor and fibroblast growth factor signaling and an imbalance in nitric oxide signaling; all are pathways that are necessary for skeletal muscle development and regeneration. We also find evidence for phagosome formation, which hints at how myofibers may be removed by autophagy or by nonprofessional phagocytes without evidence for cell death or immune cell activation. Last, we show significant overlap between genes associated with jerboa hindfoot muscle loss and genes that are differentially expressed in a variety of human muscle pathologies and rodent models of muscle loss disorders. All together, these data provide molecular insight into the process of evolutionary and developmental muscle loss in jerboa hindfeet.

Obstructive cardiac myxosarcoma of the right ventricular outflow tract with pulmonary embolism and concurrent right atrial hemangiosarcoma in a dog.

A 13-year-old spayed female rottweiler crossbreed dog was presented with an 8-day history of abnormal gait and collapse associated with excitement or physical activity. A cardiac gallop was noticed on thoracic auscultation, and a 1st-degree atrioventricular block and sinus tachycardia were noted on an electrocardiogram. Echocardiography identified a hypoechoic, irregularly marginated luminal mass in the right ventricle at the level of the pulmonic valves. Postmortem gross examination confirmed the presence of a soft, polypoid, and botryoid mass (9 × 3 × 3 cm) with a smooth and glistening surface attached to the endocardium of the right ventricular outflow tract and extending to the pulmonary artery. The histological findings were consistent with the diagnosis of myxosarcoma with pulmonary embolism. In addition, the dog in this report had a right atrial hemangiosarcoma and a cutaneous hemangioma unrelated to her clinical findings. Key clinical message: Cardiac myxosarcomas are very rare neoplasms in dogs and concomitant primary heart tumors of different histogenesis are even rarer in dogs. To the authors' knowledge, this is the first report of coexistent myxosarcoma and hemangiosarcoma in the heart of a dog. Cardiac myxosarcomas should be considered in the differential diagnosis of intracavitary heart masses associated with signs of cardiac obstruction and failure.

Myxosarcome cardiaque obstructif de la voie d'éjection du ventricule droit avec embolie pulmonaire et hémangiosarcome auriculaire droit concomitant chez un chien. Une chienne croisée rottweiler stérilisée âgée de 13 ans a été présentée avec une histoire de démarche anormale et d'effondrement associés à l'excitation ou à l'activité physique depuis 8 jours. Un galop cardiaque a été noté à l'auscultation thoracique, un bloc auriculo-ventriculaire du 1er degré et une tachycardie sinusale ont été notés à l'électrocardiogramme. L'échocardiographie a permis d'identifier une masse luminale hypoéchogène et irrégulièrement marginalisée dans le ventricule droit au niveau des valvules pulmonaires. L'examen macroscopique post-mortem a confirmé la présence d'une masse molle, polypoïde et botryoïde (9 × 3 × 3 cm) avec une surface lisse et brillante attachée à l'endocarde de la voie d'éjection du ventricule droit et s'étendant jusqu'à l'artère pulmonaire. Les résultats histologiques concordaient avec le diagnostic de myxosarcome avec embolie pulmonaire. De plus, la chienne dans ce rapport présentait un hémangiosarcome auriculaire droit et un hémangiome cutané sans rapport avec ses résultats cliniques.Message clinique clé :Les myxosarcomes cardiaques sont des néoplasmes très rares chez le chien et les tumeurs cardiaques primaires concomitantes d'histogenèse différente sont encore plus rares chez le chien. À la connaissance des auteurs, il s'agit du premier rapport de myxosarcome et d'hémangiosarcome coexistant dans le cœur d'un chien. Les myxosarcomes cardiaques doivent être pris en compte dans le diagnostic différentiel des masses cardiaques intracavitaires associées à des signes d'obstruction et d'insuffisance cardiaque.(Traduit par Dr Serge Messier).

Functional characterization of xanthorhodopsin in Salinivibrio socompensis, a novel halophile isolated from modern stromatolites.

A putative xanthorhodopsin-encoding gene, XR34, was found in the genome of the moderately halophilic gammaproteobacterium Salinivibrio socompensis S34, isolated from modern stromatolites found on the shore of Laguna Socompa (3570 m), Argentina Puna. XR-encoding genes were clustered together with genes encoding X-carotene, retinal (vitamin-A aldehyde), and carotenoid biosynthesis enzymes while the carotene ketolase gene critical for the salinixanthin antenna compound was absent. To identify its functional behavior, we herein overexpressed and characterized this intriguing microbial rhodopsin. Recombinant XR34 showed all the salient features of canonical microbial rhodopsin and covalently bound retinal as a functional chromophore with λmax = 561 nm (εmax ca. 60,000 M-1 cm-1). Two canonical counterions with pK values of around 6 and 3 were identified by pH titration of the recombinant protein. With a recovery time of approximately half an hour in the dark, XR34 shows light-dark adaptation shifting the absorption maximum from 551 to 561 nm. Laser-flash induced photochemistry at pH 9 (deprotonated primary counterion) identified a photocycle starting with a K-like intermediate, followed by an M-state (λmax ca. 400 nm, deprotonated Schiff base), and a final long wavelength-absorbing N- or O-like intermediate before returning to the parental 561 nm-state. Initiating the photocycle at pH 5 (protonated counterion) yields only bathochromic intermediates, due to the lacking capacity of the counterion to accept the Schiff base proton. Illumination of the membrane-embedded protein yielded a capacitive transport current. The presence of the M-intermediate under these conditions was demonstrated by a blue light-induced shunt process.

Dissecting Light Sensing and Metabolic Pathways on the Millimeter Scale in High-Altitude Modern Stromatolites.

Modern non-lithifying stromatolites on the shore of the volcanic lake Socompa (SST) in the Puna are affected by several extreme conditions. The present study assesses for the first time light utilization and functional metabolic stratification of SST on a millimeter scale through shotgun metagenomics. In addition, a scanning-electron-microscopy approach was used to explore the community. The analysis on SST unveiled the profile of a photosynthetic mat, with cyanobacteria not directly exposed to light, but placed just below a high-UV-resistant community. Calvin-Benson and 3-hydroxypropinate cycles for carbon fixation were abundant in upper, oxic layers, while the Wood-Ljungdahl pathway was dominant in the deeper anoxic strata. The high abundance of genes for UV-screening and oxidant-quenching pigments and CPF (photoreactivation) in the UV-stressed layers could indicate that the zone itself works as a UV shield. There is a remarkable density of sequences associated with photoreceptors in the first two layers. Also, genetic evidence of photosynthesis split in eukaryotic (layer 1) and prokaryotic (layer 2). Photoheterotrophic bacteria, aerobic photoautotrophic bacteria, and anaerobic photoautotrophic bacteria coexist by selectively absorbing different parts of the light spectrum (blue, red, and IR respectively) at different positions of the mat. Genes for oxygen, nitrogen, and sulfur metabolism account for the microelectrode chemical data and pigment measurements performed in previous publications. We also provide here an explanation for the vertical microbial mobility within the SST described previously. Finally, our study points to SST as ideal modern analogues of ancient ST.

Genomic epidemiology reveals multiple introductions of Zika virus into the United States.

Zika virus (ZIKV) is causing an unprecedented epidemic linked to severe congenital abnormalities. In July 2016, mosquito-borne ZIKV transmission was reported in the continental United States; since then, hundreds of locally acquired infections have been reported in Florida. To gain insights into the timing, source, and likely route(s) of ZIKV introduction, we tracked the virus from its first detection in Florida by sequencing ZIKV genomes from infected patients and Aedes aegypti mosquitoes. We show that at least 4 introductions, but potentially as many as 40, contributed to the outbreak in Florida and that local transmission is likely to have started in the spring of 2016-several months before its initial detection. By analysing surveillance and genetic data, we show that ZIKV moved among transmission zones in Miami. Our analyses show that most introductions were linked to the Caribbean, a finding corroborated by the high incidence rates and traffic volumes from the region into the Miami area. Our study provides an understanding of how ZIKV initiates transmission in new regions.

Protective Effect of Quercetin 3-O-Glucuronide against Cisplatin Cytotoxicity in Renal Tubular Cells.

Quercetin, a flavonoid with promising therapeutic potential, has been shown to protect from cisplatin nephrotoxicity in rats following intraperitoneal injection, but its low bioavailability curtails its prospective clinical utility in oral therapy. We recently developed a micellar formulation (P-quercetin) with enhanced solubility and bioavailability, and identical nephroprotective properties. As a first aim, we herein evaluated the oral treatment with P-quercetin in rats, which displayed no nephroprotection. In order to unravel this discrepancy, quercetin and its main metabolites were measured by HPLC in the blood and urine after intraperitoneal and oral administrations. Whilst quercetin was absorbed similarly, the profile of its metabolites was different, which led us to hypothesize that nephroprotection might be exerted in vivo by a metabolic derivate. Consequently, we then aimed to evaluate the cytoprotective capacity of quercetin and its main metabolites (quercetin 3-O-glucoside, rutin, tamarixetin, isorhamnetin and quercetin 3-O-glucuronide) against cisplatin toxicity, in HK-2 and NRK-52E tubular cell lines. Cells were incubated for 6 h with quercetin, its metabolites or vehicle (pretreatment), and subsequently 18 h in cotreatment with 10-300 µM cisplatin. Immediately after treatment, cell cultures were subject to the MTT technique as an index of cytotoxicity and photographed under light microscopy for phenotypic assessment. Quercetin afforded no direct cytoprotection and quercetin-3-O-glucuronide was the only metabolite partially preventing the effect of cisplatin in cultured tubule cells. Our results identify a metabolic derivative of quercetin contributing to its nephroprotection and prompt to further explore exogenous quercetin-3-O-glucuronide in the prophylaxis of tubular nephrotoxicity.

Genomic insights into an andean multiresistant soil actinobacterium of biotechnological interest.

Central-Andean Ecosystems (between 2000 and 6000 m above sea level (masl) are typical arid-to-semiarid environments suffering from the highest total solar and ultraviolet-B radiation on the planet but displaying numerous salt flats and shallow lakes. Andean microbial ecosystems isolated from these environments are of exceptional biodiversity enduring multiple severe conditions. Furthermore, the polyextremophilic nature of the microbes in such ecosystems indicates the potential for biotechnological applications. Within this context, the study undertaken used genome mining, physiological and microscopical characterization to reveal the multiresistant profile of Nesterenkonia sp. Act20, an actinobacterium isolated from the soil surrounding Lake Socompa, Salta, Argentina (3570 masl). Ultravioet-B, desiccation, and copper assays revealed the strain's exceptional resistance to all these conditions. Act20's genome presented coding sequences involving resistance to antibiotics, low temperatures, ultraviolet radiation, arsenic, nutrient-limiting conditions, osmotic stress, low atmospheric-oxygen pressure, heavy-metal stress, and toxic fluoride and chlorite. Act20 can also synthesize proteins and natural products such as an insecticide, bacterial cellulose, ectoine, bacterial hemoglobin, and even antibiotics like colicin V and aurachin C. We also found numerous enzymes for animal- and vegetal-biomass degradation and applications in other industrial processes. The resilience of Act20 and its biotechnologic potential were thoroughly demonstrated in this work.

CDrift: An Algorithm to Correct Linear Drift From A Single High-Resolution STEM Image.

In this work, a new method to determine and correct the linear drift for any crystalline orientation in a single-column-resolved high-resolution scanning transmission electron microscopy (HR-STEM) image, which is based on angle measurements in the Fourier space, is presented. This proposal supposes a generalization and the improvement of a previous work that needs the presence of two symmetrical planes in the crystalline orientation to be applicable. Now, a mathematical derivation of the drift effect on two families of asymmetric planes in the reciprocal space is inferred. However, though it was not possible to find an analytical solution for all conditions, a simple formula was derived to calculate the drift effect that is exact for three specific rotation angles. Taking this into account, an iterative algorithm based on successive rotation/drift correction steps is devised to remove drift distortions in HR-STEM images. The procedure has been evaluated using a simulated micrograph of a monoclinic material in an orientation where all the reciprocal lattice vectors are different. The algorithm only needs four iterations to resolve a 15° drift angle in the image.

[Structured clinical examinations with simulated patients during internal medicine clerkship]. / Descripción y análisis de ECOE con pacientes simulados en internado de Medicina Interna 2016-2017 en Facultad de Medicina Universidad de Chile.

BACKGROUND: Practice is essential in clinical training and can be carried out with simulations. The medical school of the University of Chile performs Objective Structured Clinical Examinations (OSCE) using standardized simulated patients for interns. AIM: To report the experience with OSCE in an environment with a high number of students. MATERIAL AND METHODS: Four hundred sixty-two students, participated in three OSCEs during 2016 and 2017, during their internal medicine clerkship. Each OSCE consisted of five scenarios of ambulatory care patients with prevalent medical conditions. Every student had to perform history taking, physical examination and deliver a diagnostic hypothesis, and a plan for management and counseling. Simulated patients provided feedback. To assess validity and reliability, in an argument-based model, planning and activity development were declared. The results were evaluated according to students' and organization variables, internal consistency, interobserver and theoretical concordance tests. RESULTS: No significant bias was found by demographic features or evaluation moment. Internal consistency was good (Cronbach 0.8). Simulated patient evaluators had a high correlation with medical evaluators (r = 0.7). There was a low correlation between OSCE results and theoretical tests. CONCLUSIONS: OSCE's continuously assess competences during internal medicine clerkship in an environment with a high number of students.

Transverse-Momentum-Dependent Distributions with Jets.

We investigate the use of jets to measure transverse-momentum-dependent distributions (TMDs). The example we use to present our framework is the dijet momentum decorrelation at lepton colliders. Translating this momentum decorrelation into an angle θ≪1, we analyze the factorization of the cross section for the cases θ≫R, θ∼R, and θ≪R, where R is the jet radius. Critically, for the winner-take-all axis, the jet TMD has the same double-scale renormalization group evolution as TMD fragmentation functions for all radii R. TMD fragmentation functions in factorization theorems may then simply be replaced by the jet TMDs we calculate, and all ingredients to perform the resummation to next-to-next-to-leading logarithmic accuracy are available. Our approach also applies to semi-inclusive deep inelastic scattering, where a jet instead of a hadron is measured in the final state, and we find a clean method to probe the intrinsic transverse momentum of quarks and gluons in the proton that is less sensitive to final-state nonperturbative effects.

Total liver fat quantification using three-dimensional respiratory self-navigated MRI sequence.

PURPOSE: MRI can produce quantitative liver fat fraction (FF) maps noninvasively, which can help to improve diagnoses of fatty liver diseases. However, most sequences acquire several two-dimensional (2D) slices during one or more breath-holds, which may be difficult for patients with limited breath-holding capacity. A whole-liver 3D FF map could also be obtained in a single acquisition by applying a reliable breathing-motion correction method. Several correction techniques are available for 3D imaging, but they use external devices, interrupt acquisition, or jeopardize the spatial resolution. To overcome these issues, a proof-of-concept study introducing a self-navigated 3D three-point Dixon sequence is presented here. METHODS: A respiratory self-gating strategy acquiring a center k-space profile was integrated into a three-point Dixon sequence. We obtained 3D FF maps from a water-fat emulsions phantom and fifteen volunteers. This sequence was compared with multi-2D breath-hold and 3D free-breathing approaches. RESULTS: Our 3D three-point Dixon self-navigated sequence could correct for respiratory-motion artifacts and provided more precise FF measurements than breath-hold multi-2D and 3D free-breathing techniques. CONCLUSION: Our 3D respiratory self-gating fat quantification sequence could correct for respiratory motion artifacts and yield more-precise FF measurements. Magn Reson Med 76:1400-1409, 2016. © 2015 International Society for Magnetic Resonance in Medicine.

Poly(ADP-ribose) polymerase 1 promotes transcriptional repression of integrated retroviruses.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a cellular enzyme with a fundamental role in DNA repair and the regulation of chromatin structure, processes involved in the cellular response to retroviral DNA integration. However, the function of PARP-1 in retroviral DNA integration is controversial, probably due to the functional redundancy of the PARP family in mammalian cells. We evaluated the function of PARP-1 in retroviral infection using the chicken B lymphoblastoid cell line DT40. These cells lack significant PARP-1 functional redundancy and efficiently support the postentry early events of the mammalian-retrovirus replication cycle. We observed that DT40 PARP-1(-/-) cells were 9- and 6-fold more susceptible to infection by human immunodeficiency virus type 1 (HIV-1)- and murine leukemia virus (MLV)-derived viral vectors, respectively, than cells expressing PARP-1. Production of avian Rous-associated virus type 1 was also impaired by PARP-1. However, the susceptibilities of these cell lines to infection by the nonretrovirus vesicular stomatitis virus were indistinguishable. Real-time PCR analysis of the HIV-1 life cycle demonstrated that PARP-1 did not impair reverse transcription, nuclear import of the preintegration complex, or viral DNA integration, suggesting that PARP-1 regulates a postintegration step. In support of this hypothesis, pharmacological inhibition of the epigenetic mechanism of transcriptional silencing increased retroviral expression in PARP-1-expressing cells, suppressing the differences observed. Further analysis of the implicated molecular mechanism indicated that PARP-1-mediated retroviral silencing requires the C-terminal region, but not the enzymatic activity, of the protein. In sum, our data indicate a novel role of PARP-1 in the transcriptional repression of integrated retroviruses.

Household management of childhood diarrhoea: a population-based study in Nicaragua.

Diarrhoea remains an important cause of mortality and morbidity among children in Nicaragua. As the majority of diarrhoeal cases are treated at home and appropriate household management can lessen severity of diarrhoea, the objective of this study was to examine household management of childhood diarrhoea. A simple random sample of households was selected from the Health and Demographic Surveillance Site-León. Parents or caretakers of children below five years of age, who developed diarrhoea (n = 232), were surveyed about household diarrhoea management practices in 2011. Fifty-seven percent of children received oral rehydration therapy (ORT) in the home prior to visiting any health facility. We encountered certain practices in contradiction with WHO recommendations for the management of diarrhoea in communities: 41% of children were offered protein-rich foods less frequently during diarrhoeal episodes, 20% of children were nursed less frequently or not at all during diarrhoeal episodes, and zinc supplementation was recommended at only 39% of visits with healthcare providers. Our findings provide insights for efforts to improve the household management of childhood diarrhoea in Nicaragua.

Performance of convolutional neural networks for the classification of brain tumors using magnetic resonance imaging.

Brain tumors are a diverse group of neoplasms that are challenging to detect and classify due to their varying characteristics. Deep learning techniques have proven to be effective in tumor classification. However, there is a lack of studies that compare these techniques using a common methodology. This work aims to analyze the performance of convolutional neural networks in the classification of brain tumors. We propose a network consisting of a few convolutional layers, batch normalization, and max-pooling. Then, we explore recent deep architectures, such as VGG, ResNet, EfficientNet, or ConvNeXt. The study relies on two magnetic resonance imaging datasets with over 3000 images of three types of tumors -gliomas, meningiomas, and pituitary tumors-, as well as images without tumors. We determine the optimal hyperparameters of the networks using the training and validation sets. The training and test sets are used to assess the performance of the models from different perspectives, including training from scratch, data augmentation, transfer learning, and fine-tuning. The experiments are performed using the TensorFlow and Keras libraries in Python. We compare the accuracy of the models and analyze their complexity based on the capacity of the networks, their training times, and image throughput. Several networks achieve high accuracy rates on both datasets, with the best model achieving 98.7% accuracy, which is on par with state-of-the-art methods. The average precision for each type of tumor is 94.3% for gliomas, 93.8% for meningiomas, 97.9% for pituitary tumors, and 95.3% for images without tumors. VGG is the largest model with over 171 million parameters, whereas MobileNet and EfficientNetB0 are the smallest ones with 3.2 and 5.9 million parameters, respectively. These two neural networks are also the fastest to train with 23.7 and 25.4 seconds per epoch, respectively. On the other hand, ConvNext is the slowest model with 58.2 seconds per epoch. Our custom model obtained the highest image throughput with 234.37 images per second, followed by MobileNet with 226 images per second. ConvNext obtained the smallest throughput with 97.35 images per second. ResNet, MobileNet, and EfficientNet are the most accurate networks, with MobileNet and EfficientNet demonstrating superior performance in terms of complexity. Most models achieve the best accuracy using transfer learning followed by a fine-tuning step. However, data augmentation does not contribute to increasing the accuracy of the models in general.

Synthesis and Characterization of Multilayered CrAlN/Al2O3 Tandem Coating Using HiPIMS for Solar Selective Applications at High Temperature.

The effect of applying a negative bias during deposition of a previously designed multilayer solar selective absorber coating was studied on two types of substrates (316L stainless steel and Inconel 625). The solar selective coating is composed of different chromium aluminum nitride layers deposited using a combination of radiofrequency (RF), direct current (DC), and high-power impulse magnetron sputtering (HiPIMS) technologies. The chemical composition is varied to generate an infrared reflective/absorber layer (with low Al addition and N vacancies) and two CrAlN intermediate layers with medium and high aluminum content (Al/Cr = 0.6 and 1.2). A top aluminum oxide layer (Al2O3) is deposited as an antireflective layer. In this work, a simultaneous DC-pulsed bias (-100 V, 250 kHz) was applied to the substrates in order to increase the film density. The optical performance, thermal stability, and oxidation resistance was evaluated and compared with the performance obtained with similar unbiased coating and a commercial Pyromark paint reference at 600, 700, and 800 °C. The coating remained stable after 200 h of annealing at 600 °C, with solar absorptance (α) values of 93% and 92% for samples deposited on stainless steel and Inconel, respectively, and a thermal emittance ε25°C of 18%. The introduction of additional ion bombardment during film growth through bias assistance resulted in increased durability, thermal stability, and working temperature limits compared with unbiased coatings. The solar-to-mechanical energy conversion efficiency at 800 °C was found to be up to 2 times higher than Pyromark at C = 100 and comparable at C = 1000.

Laser Engineering Nanocarbon Phases within Diamond for Science and Electronics.

Diamond, as the densest allotrope of carbon, displays a range of exemplary material properties that are attractive from a device perspective. Despite diamond displaying high carbon-carbon bond strength, ultrashort (femtosecond) pulse laser radiation can provide sufficient energy for highly localized internal breakdown of the diamond lattice. The less-dense carbon structures generated on lattice breakdown are subject to significant pressure from the surrounding diamond matrix, leading to highly unusual formation conditions. By tailoring the laser dose delivered to the diamond, it is shown that it is possible to create continuously modified internal tracks with varying electrical conduction properties. In addition to the widely reported conducting tracks, conditions leading to semiconducting and insulating written tracks have been identified. High-resolution transmission electron microscopy (HRTEM) is used to visualize the structural transformations taking place and provide insight into the different conduction regimes. The HRTEM reveals a highly diverse range of nanocarbon structures are generated by the laser irradiation, including many signatures for different so-called diaphite complexes, which have been seen in meteorite samples and seem to mediate the laser-induced breakdown of the diamond. This work offers insight into possible formation methods for the diamond and related nanocarbon phases found in meteorites.

Involvement of aquaporins in Shiga toxin-induced swelling and water transport dysfunction in human renal microvascular endothelial cells.

One of the hallmarks of Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS) is kidney damage. Our previous research demonstrated that Shiga toxin type 2 (Stx2a) decreases cell viability and induces swelling of human glomerular endothelial cells (HGEC). However, Stx2a can disrupt net water transport across HGEC monolayers without affecting cell viability. This work aimed to elucidate the possible mechanisms involved in the water transport disruption caused by Stx2a across HGEC monolayers. We investigated paracellular and transcellular water transfer across HGEC by analyzing the passage of FITC-Dextran and the hydrostatic pressure (Phydr) and measuring the osmotic pressure (Posm), respectively. Stx2a selectively affected the transcellular pathway without impacting the paracellular route. Furthermore, Stx2a cell swelling was prevented by pretreatment with aquaporin inhibitors tetraethylammonium chloride (TEA), Mercury (II) chloride (HgCl2) or TGN-020, suggesting aquaporin involvement in this process. Confocal microscopy revealed that Stx2a increased HGEC total volume, which TEA and TGN-020 counteracted. Additionally, we identified in HGEC not only the expression of aquaporin-1 (AQP1) but also the expression of aquaporin-4 (AQP4). Surprisingly, we observed a decrease in the expression of both AQPs after Stx2a exposure. Our findings suggest that Stx2a may induce water movement into HGEC via AQP1 and AQP4, increasing total cell volume. Subsequently, decreased AQP1 and AQP4 expression could inhibit transcellular water transfer, potentially as a protective mechanism against excessive water entry and cell lysis.

Function and expression of N-acetyltransferases 1 and 2 are altered in lymphocytes in type 2 diabetes and obesity.

The cytosolic enzymes N-Acetyl Transferases 1 and 2 (NATs) transfer an acetyl group from acetyl-CoA to a xenobiotic substrate. NATs are regulated at the genetic and epigenetic levels by deacetylase enzymes such as sirtuins. The enzymatic expression of NAT1, NAT2, and SIRT1 was evaluated by flow cytometry, as well as the enzymatic activity of NATs by cell culture and HPLC analysis. Six SNPs were determined through genotyping. T2D patients (n = 29) and healthy subjects (n = 25) with a median age of 57 and 50, respectively, were recruited. An increased enzyme expression and a diminished NAT2 enzymatic activity were found in cells of T2D patients compared to the control group, while NAT1 was negatively correlated with body fat percentage and BMI. In contrast, Sirtuin inhibition increased NAT2 activity, while Sirtuin agonism decreased its activity in both groups. The analysis of NAT2 SNPs showed a higher frequency of rapid acetylation haplotypes in T2D patients compared to the control group, possibly associated as a risk factor for diabetes. The enzymatic expression of CD3+NAT2+ cells was higher in the rapid acetylators group compared to the slow acetylators group. The levels and activity of NAT1 were associated with total cholesterol and triglycerides. Meanwhile, CD3+NAT2+ cells and NAT2 activity levels were associated with HbA1c and glucose levels. The results indicate that NAT2 could be involved in metabolic processes related to the development of T2D, due to its association with glucose levels, HbA1c, and the altered SIRT-NAT axis. NAT1 may be involved with dyslipidaemias in people who are overweight or obese.