Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

The loss of glycocalyx integrity impairs complement factor H binding and contributes to cyclosporine-induced endothelial cell injury.

Background: Calcineurin inhibitors (CNIs) are associated with nephrotoxicity, endothelial cell dysfunction, and thrombotic microangiopathy (TMA). Evolving evidence suggests an important role for complement dysregulation in the pathogenesis of CNI-induced TMA. However, the exact mechanism(s) of CNI-induced TMA remain(s) unknown. Methods: Using blood outgrowth endothelial cells (BOECs) from healthy donors, we evaluated the effects of cyclosporine on endothelial cell integrity. Specifically, we determined complement activation (C3c and C9) and regulation (CD46, CD55, CD59, and complement factor H [CFH] deposition) as these occurred on the endothelial cell surface membrane and glycocalyx. Results: We found that exposing the endothelium to cyclosporine resulted in a dose- and time-dependent enhancement of complement deposition and cytotoxicity. We, therefore, employed flow cytometry, Western blotting/CFH cofactor assays, and immunofluorescence imaging to determine the expression of complement regulators and the functional activity and localization of CFH. Notably, while cyclosporine led to the upregulation of complement regulators CD46, CD55, and CD59 on the endothelial cell surface, it also diminished the endothelial cell glycocalyx through the shedding of heparan sulfate side chains. The weakened endothelial cell glycocalyx resulted in decreased CFH surface binding and surface cofactor activity. Conclusion: Our findings confirm a role for complement in cyclosporine-induced endothelial injury and suggest that decreased glycocalyx density, induced by cyclosporine, is a mechanism that leads to complement alternative pathway dysregulation via decreased CFH surface binding and cofactor activity. This mechanism may apply to other secondary TMAs-in which a role for complement has so far not been recognized-and provide a potential therapeutic target and an important marker for patients on calcineurin inhibitors.

Emo-mirror: a proposal to support emotion recognition in children with autism spectrum disorders.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder defined as persistent difficulty in maturing the socialization process. Health professionals have used traditional methods in the therapies performed on patients with the aim of improving the expression of emotions by patients. However, they have not been sufficient to detect the different emotions expressed in the face of people according to different sensations. Therefore, different artificial intelligence techniques have been applied to improve the results obtained in these therapies. In this article, we propose the construction of an intelligent mirror to recognize five basic emotions: angry, scared, sad, happy and neutral. This mirror uses convolutional neural networks to analyze the images that are captured by a camera and compare it with the one that the patient should perform, thus supporting the therapies performed by health professionals in children with ASD. The proposal presents the platform and computer architecture, as well as the evaluation by specialists under the technology acceptance model.

Histamine H3 Receptor Activation Modulates Glutamate Release in the Corticostriatal Synapse by Acting at CaV2.1 (P/Q-Type) Calcium Channels and GIRK (KIR3) Potassium Channels.

The striatum is innervated by histaminergic fibers and expresses a high density of histamine H3 receptors (H3Rs), present on medium spiny neurons (MSNs) and corticostriatal afferents. In this study, in sagittal slices from the rat dorsal striatum, excitatory postsynaptic potentials (EPSPs) were recorded in MSNs after electrical stimulation of corticostriatal axons. The effect of H3R activation and blockers of calcium and potassium channels was evaluated with the paired-pulse facilitation protocol. In the presence of the H3R antagonist/inverse agonist clobenpropit (1 µM), the H3R agonist immepip (1 µM) had no effect on the paired-pulse ratio (PPR), but in the absence of clobenpropit, immepip induced a significant increase in PPR, accompanied by a reduction in EPSP amplitude, suggesting presynaptic inhibition. The blockade of CaV2.1 (P/Q-type) channels with ω-agatoxin TK (400 nM) increased PPR and prevented the effect of immepip. The CaV2.2 (N-type) channel blocker ω-conotoxin GVIA (1 µM) also increased PPR, but did not occlude the immepip action. Functional KIR3 channels are present in corticostriatal terminals, and in experiments in which immepip increased PPR, the KIR3 blocker tertiapin-Q (30 nM) prevented the effect of the H3R agonist. These results indicate that the presynaptic modulation by H3Rs of corticostriatal synapses involves the inhibition of Cav2.1 calcium channels and the activation of KIR3 potassium channels.

The impact of preterm birth on the executive functioning of preschool children: A systematic review.

This paper presents a systematic review of the impact of preterm childbirth on the later executive functioning of preschool-aged children. A systematic search for studies published between 2014 and 2019 was performed using the following keywords: executive funct* AND preterm AND child. The methodological quality of the reports was examined using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement. Thirty-two studies were reviewed and scored at least 67% on the methodological quality assessment. In comparison to children born full-term, preschool children born preterm exhibit executive functioning deficits in the dimensions of the global index, inhibitory control, cognitive flexibility, working memory, and planning/executive functioning. These findings are independent of the degree of prematurity at birth. Since executive functioning has many complex components, future studies should assess the dimensions of executive functioning separately in preschool-aged children born preterm, rather than as a single measure.

Shiga Toxin 2a Induces NETosis via NOX-Dependent Pathway.

Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is the most common cause of hemolytic uremic syndrome (HUS), one of the main causes of acute kidney injury in children. Stx plays an important role in endothelium damage and pathogenesis of STEC-HUS. However, the effects of Stx on neutrophils and neutrophil extracellular trap (NET) formation are not well understood. In this study, we investigated how Stx2a affects NET formation and NETotic pathways (NADPH or NOX-dependent and -independent) using neutrophils isolated from healthy donors and patients with STEC-HUS, during the acute and recovery phase of the disease. Stx2a dose-dependently induced NETosis in neutrophils isolated from both healthy controls and STEC-HUS patients. NETosis kinetics and mechanistic data with pathway-specific inhibitors including diphenyleneiodonium (DPI)-, ERK-, and P38-inhibitors showed that Stx2a-induced NETosis via the NOX-dependent pathway. Neutrophils from STEC-HUS patients in the acute phase showed less ROS and NETs formation compared to neutrophils of the recovery phase of the disease and in healthy controls. NETs induced by Stx2a may lead to the activation of endothelial cells, which might contribute to the manifestation of thrombotic microangiopathy in STEC-HUS.

Primary Human Derived Blood Outgrowth Endothelial Cells: An Appropriate In Vitro Model to Study Shiga Toxin Mediated Damage of Endothelial Cells.

Hemolytic uremic syndrome (HUS) is a rare disease primarily characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. Endothelial damage is the hallmark of the pathogenesis of HUS with an infection with the Shiga toxin (Stx) producing Escherichia coli (STEC-HUS) as the main underlying cause in childhood. In this study, blood outgrowth endothelial cells (BOECs) were isolated from healthy donors serving as controls and patients recovered from STEC-HUS. We hypothesized that Stx is more cytotoxic for STEC-HUS BOECs compared to healthy donor control BOECs explained via a higher amount of Stx bound to the cell surface. Binding of Shiga toxin-2a (Stx2a) was investigated and the effect on cytotoxicity, protein synthesis, wound healing, and cell proliferation was studied in static conditions. Results show that BOECs are highly susceptible for Stx2a. Stx2a is able to bind to the cell surface of BOECs with cytotoxicity in a dose-dependent manner as a result. Pre-treatment with tumor necrosis factor alpha (TNF-α) results in enhanced Stx binding with 20-30% increased lactate dehydrogenase (LDH) release. Endothelial wound healing is delayed in a Stx2a-rich environment; however, this is not caused by an effect on the proliferation rate of BOECs. No significant differences were found between control BOECs and BOECs from recovered STEC-HUS patients in terms of Stx2a binding and inhibition of protein synthesis.

The RNA-binding protein SERBP1 functions as a novel oncogenic factor in glioblastoma by bridging cancer metabolism and epigenetic regulation.

BACKGROUND: RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. RESULTS: We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. CONCLUSIONS: SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

Vascular endothelial cells evade complement-mediated membrane injury via Weibel-Palade body mobilization.

BACKGROUND: Defective complement inhibition can lead to the formation of membrane attack complexes (MAC; C5b-9) on the plasma membranes of vascular endothelial cells, resulting in injury that drives the progression of thrombotic microangiopathy (TMA), a key pathology in kidney disease. OBJECTIVE/METHODS: We examined the response of human endothelial cells to complement-mediated damage using blood outgrowth endothelial cells (BOECs) derived from healthy donors. BOECs were sensitized to complement factors present in normal human serum to induce the formation of C5b-9 on their plasma membranes. RESULTS: This triggered an expected abrupt rise in intracellular Ca2+ reflecting membrane leakage. Remarkably, while intracellular Ca2+ remained elevated, membrane leakage ceased within 30 minutes, and cells did not show significant death. Extensive mobilization of Weibel-Palade bodies (WPBs) was observed along with secretion of von Willebrand factor (VWF). The potential role of WPBs and VWF in mitigating complement-mediated damage was examined by comparing the effects of C5b-9 on BOECs derived from von Willebrand disease (VWD) patients expressing reduced amounts of VWF, lacking expression of functional VWF, or lacking both VWF and WPBs. BOECs lacking WPBs were not resistant to complement-mediated damage, but became resistant when transfected to express VWF (and thus WPBs). CONCLUSION: We conclude that BOECs exposed to C5b-9 attack respond by mobilizing WPBs, which mitigate and repair damage by fusing with the plasma membrane. We propose that a similar cell-specific response may protect the vascular endothelium from complement-mediated damage in vivo.

Differentiation of adipose-derived stem cells to functional CD105neg CD73low melanocyte precursors guided by defined culture condition.

BACKGROUND: The generation of functional human epidermal melanocytes (HEM) from stem cells provides an unprecedented source for cell-based therapy in vitiligo. Despite the important efforts exerted to obtain melanin-producing cells from stem cells, pre-clinical results still lack the safety and scalability characteristics essential for their translational application. METHODS: Here, we report a rapid and efficient protocol based on defined culture conditions capable of differentiating adult adipose-derived stem cells (ADSC) to scalable amounts of proliferative melanocyte precursors (PreMel) within 30 days. PreMel were characterized in vitro through qPCR, Western blot, flow cytometry, biochemical assays, and in vivo assays in immunocompromised mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, or NSG). RESULTS: After 30 days of differentiation, the stem cell-derived PreMel were defined as CD105neg CD73low according to immunophenotypic changes in comparison with parental stem cell markers. In addition, expression of microphthalmia-associated transcription factor (MITF), active tyrosinase (TYR), and the terminal differentiation-involved premelanosome protein (PMEL) were detected. Furthermore, PreMel had the potential to synthesize melanin and package it into melanosomes both in vitro and in vivo in NSG mice skin. CONCLUSIONS: This study proposes a rapid and scalable protocol for the generation of proliferative melanocyte precursors (PreMel) from ADSC. These PreMel display the essential functional characteristics of bona fide HEM, opening a new path for an autologous cellular therapy for vitiligo patients.

Author Correction: Caveolin-1 impairs PKA-DRP1-mediated remodelling of ER-mitochondria communication during the early phase of ER stress.

Following publication of the article, the authors wrote to point out the following mistake.

Caveolin-1 impairs PKA-DRP1-mediated remodelling of ER-mitochondria communication during the early phase of ER stress.

Close contacts between endoplasmic reticulum and mitochondria enable reciprocal Ca2+ exchange, a key mechanism in the regulation of mitochondrial bioenergetics. During the early phase of endoplasmic reticulum stress, this inter-organellar communication increases as an adaptive mechanism to ensure cell survival. The signalling pathways governing this response, however, have not been characterized. Here we show that caveolin-1 localizes to the endoplasmic reticulum-mitochondria interface, where it impairs the remodelling of endoplasmic reticulum-mitochondria contacts, quenching Ca2+ transfer and rendering mitochondrial bioenergetics unresponsive to endoplasmic reticulum stress. Protein kinase A, in contrast, promotes endoplasmic reticulum and mitochondria remodelling and communication during endoplasmic reticulum stress to promote organelle dynamics and Ca2+ transfer as well as enhance mitochondrial bioenergetics during the adaptive response. Importantly, caveolin-1 expression reduces protein kinase A signalling, as evidenced by impaired phosphorylation and alterations in organelle distribution of the GTPase dynamin-related protein 1, thereby enhancing cell death in response to endoplasmic reticulum stress. In conclusion, caveolin-1 precludes stress-induced protein kinase A-dependent remodelling of endoplasmic reticulum-mitochondria communication.

The role of Ca2+ -dependent K+ - channels at the rat corticostriatal synapses revealed by paired pulse stimulation.

Potassium channels play an important role in modulating synaptic activity both at presynaptic and postsynaptic levels. We have shown before that presynaptically located KV and KIR channels modulate the strength of corticostriatal synapses in rat brain, but the role of other types of potassium channels at these synapses remains largely unknown. Here, we show that calcium-dependent potassium channels BK-type but not SK-type channels are located presynaptically in corticostriatal synapses. We stimulated cortical neurons in rat brain slices and recorded postsynaptic excitatory potentials (EPSP) in medium spiny neurons (MSN) in dorsal neostriatum. By using a paired pulse protocol, we induced synaptic facilitation before applying either BK- or SK-specific toxins. Thus, we found that blockage of BKCa with iberiotoxin (10 nM) reduces synaptic facilitation and increases the amplitude of the EPSP, while exposure to SK-blocker apamin (100 nM) has no effect. Additionally, we induced train action potentials on striatal MSN by current injection before and after the exposure to KCa toxins. We found that the action potential becomes broader when the MSN is exposed to iberiotoxin, although it has no impact on frequency. In contrast, exposure to apamin results in loss of afterhyperpolarization phase and an increase of spike frequency. Therefore, we concluded that postsynaptic SK channels are involved in afterhyperpolarization and modulation of spike frequency while the BK channels are involved on the late repolarization phase of the action potential. Altogether, our results show that calcium-dependent potassium channels modulate both input towards and output from the striatum.

Arachidonic acid triggers [Ca2+]i increases in rat round spermatids by a likely GPR activation, ERK signalling and ER/acidic compartments Ca2+ release.

Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca2+ from internal stores in round spermatids and pachytene spermatocytes. In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca2+ stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca2+ using fluorescent Ca2+ probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca2+ stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca2+ release from intracellular Ca2+ stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis.

Rab32 connects ER stress to mitochondrial defects in multiple sclerosis.

BACKGROUND: Endoplasmic reticulum (ER) stress is a hallmark of neurodegenerative diseases such as multiple sclerosis (MS). However, this physiological mechanism has multiple manifestations that range from impaired clearance of unfolded proteins to altered mitochondrial dynamics and apoptosis. While connections between the triggering of the unfolded protein response (UPR) and downstream mitochondrial dysfunction are poorly understood, the membranous contacts between the ER and mitochondria, called the mitochondria-associated membrane (MAM), could provide a functional link between these two mechanisms. Therefore, we investigated whether the guanosine triphosphatase (GTPase) Rab32, a known regulator of the MAM, mitochondrial dynamics, and apoptosis, could be associated with ER stress as well as mitochondrial dysfunction. METHODS: We assessed Rab32 expression in MS patient and experimental autoimmune encephalomyelitis (EAE) tissue, via observation of mitochondria in primary neurons and via monitoring of survival of neuronal cells upon increased Rab32 expression. RESULTS: We found that the induction of Rab32 and other MAM proteins correlates with ER stress proteins in MS brain, as well as in EAE, and occurs in multiple central nervous system (CNS) cell types. We identify Rab32, known to increase in response to acute brain inflammation, as a novel unfolded protein response (UPR) target. High Rab32 expression shortens neurite length, alters mitochondria morphology, and accelerates apoptosis/necroptosis of human primary neurons and cell lines. CONCLUSIONS: ER stress is strongly associated with Rab32 upregulation in the progression of MS, leading to mitochondrial dysfunction and neuronal death.

TMX1 determines cancer cell metabolism as a thiol-based modulator of ER-mitochondria Ca2+ flux.

The flux of Ca(2+) from the endoplasmic reticulum (ER) to mitochondria regulates mitochondria metabolism. Within tumor tissue, mitochondria metabolism is frequently repressed, leading to chemotherapy resistance and increased growth of the tumor mass. Therefore, altered ER-mitochondria Ca(2+) flux could be a cancer hallmark, but only a few regulatory proteins of this mechanism are currently known. One candidate is the redox-sensitive oxidoreductase TMX1 that is enriched on the mitochondria-associated membrane (MAM), the site of ER-mitochondria Ca(2+) flux. Our findings demonstrate that cancer cells with low TMX1 exhibit increased ER Ca(2+), accelerated cytosolic Ca(2+) clearance, and reduced Ca(2+) transfer to mitochondria. Thus, low levels of TMX1 reduce ER-mitochondria contacts, shift bioenergetics away from mitochondria, and accelerate tumor growth. For its role in intracellular ER-mitochondria Ca(2+) flux, TMX1 requires its thioredoxin motif and palmitoylation to target to the MAM. As a thiol-based tumor suppressor, TMX1 increases mitochondrial ATP production and apoptosis progression.

[Primary HIV resistance in Buenos Aires metropolitan area]. / Resistencia primaria de HIV en el área metropolitana de Buenos Aires.

Surveillance of primary drug resistance is critical to optimize antiretroviral therapy (ART) for HIV. Mutations to be monitored are defined in a reference list of the World Health Organization (WHO), which does not include mutations for new drugs, such as rilpivirine. We undertook a retrospective analysis of medical records of ART naive patients treated at a specialized HIV/AIDS center, evaluating the prevalence of WHO mutations and mutations specific for rilpivirine. Ninety-one patients were included during 2011-2013, being male 71 (78.0%), and men who have sex with men 46 (50.5%). The median values for age, viral load, and CD4 counts were 33 years, 62 100 copies/mL, and 548 cells/µl, retrospectively; 34 (37.3%) had early infection and 60 (65.9%) were asymptomatic. WHO mutations were found in 11 (12.1%) patients, two of whom presented multiple mutations. Seven mutations corresponded to non-nucleoside reverse transcriptase inhibitors, four to nucleoside analogues, and two to protease inhibitors. The most frequent mutations were K103N and M41L. No differences in mutation frequencies were found when compared by time post-infection, gender, sexual orientation, or CD4 count. Mutations conferring low-level resistance to rilpivirine were found in 3 (3.3%) patients; such mutations were E138A and E138G. The overall moderate primary resistance levels found in this study highlight the value of performing a resistance test before ART initiation in the served population. The observed prevalence of primary resistance to rilpivirine was low.

Resistencia primaria de HIV en el área metropolitana de Buenos Aires

La vigilancia de resistencia primaria de HIV es fundamental para optimizar el tratamiento antirretroviral (TARV) de la infección. Las mutaciones a vigilar están definidas en una lista de referencia de la Organización Mundial de la Salud (OMS), que no incluye mutaciones para drogas nuevas como la rilpivirina. Revisamos retrospectivamente las historias clínicas de pacientes naive de TARV asistidos en 2011-2013 en un centro privado de derivación de HIV/Sida, pesquisando mutaciones según criterios de OMS y mutaciones específicas de resistencia a rilpivirina. Incluimos 91 pacientes; 71 (78.0%) eran hombres y 46 (50.5%) eran hombres que tenían sexo con hombres; 34 (37.4%) presentaban infección temprana y 60 (65.9%) estaban asintomáticos. Los valores medianos de edad, carga viral y recuento de CD4 fueron 33 años, 62 100 copias/ml y 548 células/μl, respectivamente. Encontramos mutaciones de lista OMS en 11 (12.1%) pacientes, dos de ellos presentaron mutaciones a dos familias de drogas. Siete mutaciones correspondieron a inhibidores no nucleosídicos de la retrotranscriptasa, cuatro a análogos nucleosídicos y dos a inhibidores de la proteasa; las más frecuentes fueron K103N y M41L. No hubo mayor frecuencia de mutaciones en pacientes con infección temprana, ni diferencias según sexo, orientación sexual o recuento de CD4. Tres pacientes (3.3%) presentaron mutaciones asociadas a bajos niveles de resistencia a rilpivirina (E138A, E138G). Los niveles de resistencia primaria observados en este estudio evidencian la importancia de determinar resistencia previo al inicio de TARV en la población asistida en nuestro centro. La prevalencia observada de resistencia primaria a rilpivirina fue baja.

Surveillance of primary drug resistance is critical to optimize antiretroviral therapy (ART) for HIV. Mutations to be monitored are defined in a reference list of the World Health Organization (WHO), which does not include mutations for new drugs, such as rilpivirine. We undertook a retrospective analysis of medical records of ART naive patients treated at a specialized HIV/AIDS center, evaluating the prevalence of WHO mutations and mutations specific for rilpivirine. Ninety-one patients were included during 2011-2013, being male 71 (78.0%), and men who have sex with men 46 (50.5%). The median values for age, viral load, and CD4 counts were 33 years, 62 100 copies/mL, and 548 cells/l, retrospectively; 34 (37.3%) had early infection and 60 (65.9%) were asymptomatic. WHO mutations were found in 11 (12.1%) patients, two of whom presented multiple mutations. Seven mutations corresponded to non-nucleoside reverse transcriptase inhibitors, four to nucleoside analogues, and two to protease inhibitors. The most frequent mutations were K103N and M41L. No differences in mutation frequencies were found when compared by time post-infection, gender, sexual orientation, or CD4 count. Mutations conferring low-level resistance to rilpivirine were found in 3 (3.3%) patients; such mutations were E138A and E138G. The overall moderate primary resistance levels found in this study highlight the value of performing a resistance test before ART initiation in the served population. The observed prevalence of primary resistance to rilpivirine was low.

Resistencia primaria de HIV en el área metropolitana de Buenos Aires

La vigilancia de resistencia primaria de HIV es fundamental para optimizar el tratamiento antirretroviral (TARV) de la infección. Las mutaciones a vigilar están definidas en una lista de referencia de la Organización Mundial de la Salud (OMS), que no incluye mutaciones para drogas nuevas como la rilpivirina. Revisamos retrospectivamente las historias clínicas de pacientes naive de TARV asistidos en 2011-2013 en un centro privado de derivación de HIV/Sida, pesquisando mutaciones según criterios de OMS y mutaciones específicas de resistencia a rilpivirina. Incluimos 91 pacientes; 71 (78.0%) eran hombres y 46 (50.5%) eran hombres que tenían sexo con hombres; 34 (37.4%) presentaban infección temprana y 60 (65.9%) estaban asintomáticos. Los valores medianos de edad, carga viral y recuento de CD4 fueron 33 años, 62 100 copias/ml y 548 células/μl, respectivamente. Encontramos mutaciones de lista OMS en 11 (12.1%) pacientes, dos de ellos presentaron mutaciones a dos familias de drogas. Siete mutaciones correspondieron a inhibidores no nucleosídicos de la retrotranscriptasa, cuatro a análogos nucleosídicos y dos a inhibidores de la proteasa; las más frecuentes fueron K103N y M41L. No hubo mayor frecuencia de mutaciones en pacientes con infección temprana, ni diferencias según sexo, orientación sexual o recuento de CD4. Tres pacientes (3.3%) presentaron mutaciones asociadas a bajos niveles de resistencia a rilpivirina (E138A, E138G). Los niveles de resistencia primaria observados en este estudio evidencian la importancia de determinar resistencia previo al inicio de TARV en la población asistida en nuestro centro. La prevalencia observada de resistencia primaria a rilpivirina fue baja.(AU)

Surveillance of primary drug resistance is critical to optimize antiretroviral therapy (ART) for HIV. Mutations to be monitored are defined in a reference list of the World Health Organization (WHO), which does not include mutations for new drugs, such as rilpivirine. We undertook a retrospective analysis of medical records of ART naive patients treated at a specialized HIV/AIDS center, evaluating the prevalence of WHO mutations and mutations specific for rilpivirine. Ninety-one patients were included during 2011-2013, being male 71 (78.0%), and men who have sex with men 46 (50.5%). The median values for age, viral load, and CD4 counts were 33 years, 62 100 copies/mL, and 548 cells/l, retrospectively; 34 (37.3%) had early infection and 60 (65.9%) were asymptomatic. WHO mutations were found in 11 (12.1%) patients, two of whom presented multiple mutations. Seven mutations corresponded to non-nucleoside reverse transcriptase inhibitors, four to nucleoside analogues, and two to protease inhibitors. The most frequent mutations were K103N and M41L. No differences in mutation frequencies were found when compared by time post-infection, gender, sexual orientation, or CD4 count. Mutations conferring low-level resistance to rilpivirine were found in 3 (3.3%) patients; such mutations were E138A and E138G. The overall moderate primary resistance levels found in this study highlight the value of performing a resistance test before ART initiation in the served population. The observed prevalence of primary resistance to rilpivirine was low.(AU)

A role for the ancient SNARE syntaxin 17 in regulating mitochondrial division.

Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.