Zika virus non-coding RNAs antagonize antiviral responses by PKR-mediated translational arrest.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that causes severe outbreaks in human populations. ZIKV infection leads to the accumulation of small non-coding viral RNAs (known as sfRNAs) that are crucial for evasion of antiviral responses and for viral pathogenesis. However, the mechanistic understanding of how sfRNAs function remains incomplete. Here, we use recombinant ZIKVs and ribosome profiling of infected human cells to show that sfRNAs block translation of antiviral genes. Mechanistically, we demonstrate that specific RNA structures present in sfRNAs trigger PKR activation, which instead of limiting viral replication, enhances viral particle production. Although ZIKV infection induces mRNA expression of antiviral genes, translation efficiency of type I interferon and interferon stimulated genes were significantly downregulated by PKR activation. Our results reveal a novel viral adaptation mechanism mediated by sfRNAs, where ZIKV increases its fitness by repurposing the antiviral role of PKR into a proviral factor.

[Mental and nutritional habits at the beginning of the COVID-19 pandemic: impact, perspectives and recommendations]. / Hábitos de salud mental y nutricional a inicios de la pandemia por COVID-19: impacto, perspectivas y recomendaciones.

Besides the severe impact on the mortality rate in the world, the COVID-19 pandemic demonstrated the importance of having an optimal physical and mental health, since subjects with chronic diseases were the most affected. The aim of this review was to describe the consequences of nutritional and mental health during the beginning of the pandemic. In developing countries, such as Mexico, the prevalence of mental illnesses including depression, anxiety, stress, and suicide increased considerably. In addition, it was found an increase rate of sedentary lifestyles due to the confinement established as a preventive measure to avoid COVID-19 infections. Concerning eating habits, it was reported a decrease in fruits and vegetables consumption and an increase in energy-dense foods, as well as alcohol consumption or smoking, as compensatory measures for mental disorders of anxiety and depression. Based on the information we look up on the impact of COVID-19 on human health, we suggest as prevention measures multidisciplinary care strategies aimed at promoting mental health, diet, and physical activity habits in a comprehensive context. Likewise, information it is highlighted the importance of establishing care and monitoring programs for patients with chronic diseases, because this factor was decisive for mortality after COVID-19 infection.

Además de su impacto en la tasa de mortalidad en el mundo, la pandemia por COVID-19 demostró la importancia de un estado de salud física y mental óptimo, ya que los sujetos con enfermedades crónicas fueron los más afectados. El objetivo de esta revisión fue describir las consecuencias de la salud nutricional y mental durante los inicios de la pandemia. En los países en vías de desarrollo, como México, la prevalencia de enfermedades mentales como la depresión, la ansiedad, el estrés y los suicidios aumentaron. Además, incrementaron las cifras de sedentarismo debido al confinamiento. Con relación a los hábitos de alimentación, diversos autores reportaron una disminución en el consumo de frutas, verduras, y un aumento de alimentos densamente energéticos, así como el consumo de alcohol o tabaquismo como medidas compensatorias en relación con los trastornos mentales de ansiedad y depresión. A partir de la información consultada en torno al impacto de la COVID-19 en la salud humana, se sugieren como medidas de prevención estrategias de atención multidisciplinaria encaminadas a promover hábitos de salud mental, alimentación y actividad física en un contexto integral. De igual forma, destaca la importancia de establecer programas de atención y monitoreo de pacientes con enfermedades crónicas, debido a que este factor fue determinante para la mortalidad tras la infección por COVID-19.

Dengue virus NS5 degrades ERC1 during infection to antagonize NF-kB activation.

Dengue virus (DENV) is the most important human virus transmitted by mosquitos. Dengue pathogenesis is characterized by a large induction of proinflammatory cytokines. This cytokine induction varies among the four DENV serotypes (DENV1 to 4) and poses a challenge for live DENV vaccine design. Here, we identify a viral mechanism to limit NF-κB activation and cytokine secretion by the DENV protein NS5. Using proteomics, we found that NS5 binds and degrades the host protein ERC1 to antagonize NF-κB activation, limit proinflammatory cytokine secretion, and reduce cell migration. We found that ERC1 degradation involves unique properties of the methyltransferase domain of NS5 that are not conserved among the four DENV serotypes. By obtaining chimeric DENV2 and DENV4 viruses, we map the residues in NS5 for ERC1 degradation, and generate recombinant DENVs exchanging serotype properties by single amino acid substitutions. This work uncovers a function of the viral protein NS5 to limit cytokine production, critical to dengue pathogenesis. Importantly, the information provided about the serotype-specific mechanism for counteracting the antiviral response can be applied to improve live attenuated vaccines.

Gestational Insulin Resistance Is Mediated by the Gut Microbiome-Indoleamine 2,3-Dioxygenase Axis.

BACKGROUND & AIMS: Normal gestation involves a reprogramming of the maternal gut microbiome (GM) that contributes to maternal metabolic changes by unclear mechanisms. This study aimed to understand the mechanistic underpinnings of the GM-maternal metabolism interaction. METHODS: The GM and plasma metabolome of CD1, NIH-Swiss, and C57 mice were analyzed with the use of 16S rRNA sequencing and untargeted liquid chromatography-mass spectrometry throughout gestation. Pharmacologic and genetic knockout mouse models were used to identify the role of indoleamine 2,3-dioxygenase (IDO1) in pregnancy-associated insulin resistance (IR). Involvement of gestational GM was studied with the use of fecal microbial transplants (FMTs). RESULTS: Significant variation in GM alpha diversity occurred throughout pregnancy. Enrichment in gut bacterial taxa was mouse strain and pregnancy time point specific, with the species enriched at gestation day 15/19 (G15/19), a point of heightened IR, being distinct from those enriched before or after pregnancy. Metabolomics revealed elevated plasma kynurenine at G15/19 in all 3 mouse strains. IDO1, the rate-limiting enzyme for kynurenine production, had increased intestinal expression at G15, which was associated with mild systemic and gut inflammation. Pharmacologic and genetic inhibition of IDO1 inhibited kynurenine levels and reversed pregnancy-associated IR. FMT revealed that IDO1 induction and local kynurenine level effects on IR derive from the GM in both mouse and human pregnancy. CONCLUSIONS: GM changes accompanying pregnancy shift IDO1-dependent tryptophan metabolism toward kynurenine production, intestinal inflammation, and gestational IR, a phenotype reversed by genetic deletion or inhibition of IDO1. (Gestational Gut Microbiome-IDO1 Axis Mediates Pregnancy Insulin Resistance; EMBL-ENA ID: PRJEB45047. MetaboLights ID: MTBLS3598).

Longitudinal Study after Sputnik V Vaccination Shows Durable SARS-CoV-2 Neutralizing Antibodies and Reduced Viral Variant Escape to Neutralization over Time.

Recent studies have shown a temporal increase in the neutralizing antibody potency and breadth to SARS-CoV-2 variants in coronavirus disease 2019 (COVID-19) convalescent individuals. Here, we examined longitudinal antibody responses and viral neutralizing capacity to the B.1 lineage virus (Wuhan related), to variants of concern (VOC; Alpha, Beta, Gamma, and Delta), and to a local variant of interest (VOI; Lambda) in volunteers receiving the Sputnik V vaccine in Argentina. Longitudinal serum samples (N = 536) collected from 118 volunteers obtained between January and October 2021 were used. The analysis indicates that while anti-spike IgG levels significantly wane over time, the neutralizing capacity for the Wuhan-related lineages of SARS-CoV-2 and VOC is maintained within 6 months of vaccination. In addition, an improved antibody cross-neutralizing ability for circulating variants of concern (Beta and Gamma) was observed over time postvaccination. The viral variants that displayed higher escape to neutralizing antibodies with respect to the original virus (Beta and Gamma variants) were the ones showing the largest increase in susceptibility to neutralization over time after vaccination. Our observations indicate that serum neutralizing antibodies are maintained for at least 6 months and show a reduction of VOC escape to neutralizing antibodies over time after vaccination. IMPORTANCE Vaccines have been produced in record time for SARS-CoV-2, offering the possibility of halting the global pandemic. However, inequalities in vaccine accessibility in different regions of the world create a need to increase international cooperation. Sputnik V is a recombinant adenovirus-based vaccine that has been widely used in Argentina and other developing countries, but limited information is available about its elicited immune responses. Here, we examined longitudinal antibody levels and viral neutralizing capacity elicited by Sputnik V vaccination. Using a cohort of 118 volunteers, we found that while anti-spike antibodies wane over time, the neutralizing capacity to viral variants of concern and local variants of interest is maintained within 4 months of vaccination. In addition, we observed an increased cross-neutralization activity over time for the Beta and Gamma variants. This study provides valuable information about the immune response generated by a vaccine platform used in many parts of the world.

Dengue Virus Capsid-Protein Dynamics in Live Infected Cells Studied by Pair Correlation Analysis.

It has become increasingly evident that unveiling the mechanisms of virus entry, assembly, and virion release is fundamental for identifying means for preventing viral spread and controlling viral disease. Due to virus mobility and structural and/or functional heterogeneity among viral particles, high spatiotemporal resolution single-virus/single-particle techniques are required to capture the behavior of viral particles inside infected cells.In this chapter, we present fluorescence imaging analysis methods for studying the mobility of fluorescently labeled dengue virus (DENV) proteins in live infected cells. Some of the most recent Fluorescence Fluctuation Spectroscopy (FFS) methods will be presented and, in particular, the pair Correlation Functions (pCF) approach will be discussed. The pCF method does not require individual molecule isolation, as in a particle-tracking experiment, to capture single viral protein behavior. In this regard, image acquisition is followed by the spatiotemporal cross-correlation function at increasing time delays, yielding a quantitative view of single-particle mobility in intact live infected cells.We provide a general overview and a practical guidance for the implementation of advanced FFS techniques, and the pair Correlation Functions analysis, as quantitative tools to reveal insights into previously unreported DENV mechanisms. We expect this protocol report will serve as an incentive for further applying correlation imaging studies in virology research.

In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells.

Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow mobilities. While in the cytoplasm the correlation times were in the 2-6 and 40-110 ms range for the fast and slow mobility populations respectively, in the cell nucleus they were 1-10 and 25-140 ms range, respectively. The fast mobility of DENV C in cytoplasmic and nuclear regions agreed with the diffusion coefficients from Brownian motion previously reported from correlation analysis. These studies provide the first evidence of DENV C shuttling from and to the nucleus in infected cells, opening new venues for antiviral interventions.

Dengue and Zika virus capsid proteins bind to membranes and self-assemble into liquid droplets with nucleic acids.

Dengue virus (DENV) and Zika virus (ZIKV) capsid proteins efficiently recruit and surround the viral RNA at the endoplasmic reticulum (ER) membrane to yield nascent viral particles. However, little is known either about the molecular mechanisms by which multiple copies of capsid proteins assemble into nucleocapsids (NCs) or how the NC is recruited and wrapped by the ER membrane during particle morphogenesis. Here, we measured relevant interactions concerning this viral process using purified DENV and ZIKV capsid proteins, membranes mimicking the ER lipid composition, and nucleic acids in in vitro conditions to understand the biophysical properties of the RNA genome encapsidation process. We found that both ZIKV and DENV capsid proteins bound to liposomes at liquid-disordered phase regions, docked exogenous membranes, and RNA molecules. Liquid-liquid phase separation is prone to occur when positively charged proteins interact with nucleic acids, which is indeed the case for the studied capsids. We characterized these liquid condensates by measuring nucleic acid partition constants and the extent of water dipolar relaxation, observing a cooperative process for the formation of the new phase that involves a distinct water organization. Our data support a new model in which capsid-RNA complexes directly bind the ER membrane, seeding the process of RNA recruitment for viral particle assembly. These results contribute to our understanding of the viral NC formation as a stable liquid-liquid phase transition, which could be relevant for dengue and Zika gemmation, opening new avenues for antiviral intervention.

Sputnik V vaccine elicits seroconversion and neutralizing capacity to SARS-CoV-2 after a single dose.

Massive vaccination offers great promise for halting the global COVID-19 pandemic. However, the limited supply and uneven vaccine distribution create an urgent need to optimize vaccination strategies. We evaluate SARS-CoV-2-specific antibody responses after Sputnik V vaccination of healthcare workers in Argentina, measuring IgG anti-spike titers and neutralizing capacity after one and two doses in a cohort of naive or previously infected volunteers. By 21 days after receiving the first dose of the vaccine, 94% of naive participants develop spike-specific IgG antibodies. A single Sputnik V dose elicits higher antibody levels and virus-neutralizing capacity in previously infected individuals than in naive ones receiving the full two-dose schedule. The high seroconversion rate after a single dose in naive participants suggests a benefit of delaying administration of the second dose to increase the number of people vaccinated. The data presented provide information for guiding public health decisions in light of the current global health emergency.

Emergency response for evaluating SARS-CoV-2 immune status, seroprevalence and convalescent plasma in Argentina.

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used for clinical trials and therapies across the country. Using this protocol, about 80% of convalescent donor plasmas were potentially suitable for therapies. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs.

Zika Virus Subgenomic Flavivirus RNA Generation Requires Cooperativity between Duplicated RNA Structures That Are Essential for Productive Infection in Human Cells.

Zika virus (ZIKV) is an emerging flavivirus, mainly transmitted by mosquitoes, which represents a global health threat. A common feature of flavivirus-infected cells is the accumulation of viral noncoding subgenomic RNAs by partial degradation of the viral genome, known as sfRNAs, involved in immune evasion and pathogenesis. Although great effort is being made to understand the mechanism by which these sfRNAs function during infection, the picture of how they work is still incomplete. In this study, we developed new genetic tools to dissect the functions of ZIKV RNA structures for viral replication and sfRNA production in mosquito and human hosts. ZIKV infections mostly accumulate two kinds of sfRNAs, sfRNA1 and sfRNA2, by stalling genome degradation upstream of duplicated stem loops (SLI and SLII) of the viral 3' untranslated region (UTR). Although the two SLs share conserved sequences and structures, different functions have been found for ZIKV replication in human and mosquito cells. While both SLs are enhancers for viral infection in human cells, they play opposite roles in the mosquito host. The dissection of determinants for sfRNA formation indicated a strong cooperativity between SLI and SLII, supporting a high-order organization of this region of the 3' UTR. Using recombinant ZIKV with different SLI and SLII arrangements, which produce different types of sfRNAs or lack the ability to generate these molecules, revealed that at least one sfRNA was necessary for efficient infection and transmission in Aedes aegypti mosquitoes. Importantly, we demonstrate an absolute requirement of sfRNAs for ZIKV propagation in human cells. In this regard, viruses lacking sfRNAs, constructed by deletion of the region containing SLI and SLII, were able to infect human cells but the infection was rapidly cleared by antiviral responses. Our findings are unique for ZIKV, since in previous studies, other flaviviruses with deletions of analogous regions of the genome, including dengue and West Nile viruses, accumulated distinct species of sfRNAs and were infectious in human cells. We conclude that flaviviruses share common strategies for sfRNA generation, but they have evolved mechanisms to produce different kinds of these RNAs to accomplish virus-specific functions.IMPORTANCE Flaviviruses are important emerging and reemerging human pathogens. Understanding the molecular mechanisms for viral replication and evasion of host antiviral responses is relevant to development of control strategies. Flavivirus infections produce viral noncoding RNAs, known as sfRNAs, involved in viral replication and pathogenesis. In this study, we dissected molecular determinants for Zika virus sfRNA generation in the two natural hosts, human cells and mosquitoes. We found that two RNA structures of the viral 3' UTR operate in a cooperative manner to produce two species of sfRNAs and that the deletion of these elements has a profoundly different impact on viral replication in the two hosts. Generation of at least one sfRNA was necessary for efficient Zika virus infection of Aedes aegypti mosquitoes. Moreover, recombinant viruses with different 3' UTR arrangements revealed an essential role of sfRNAs for productive infection in human cells. In summary, we define molecular requirements for Zika virus sfRNA accumulation and provide new ideas of how flavivirus RNA structures have evolved to succeed in different hosts.

Dengue Virus Capsid Protein Dynamics Reveals Spatially Heterogeneous Motion in Live-Infected-Cells.

Dengue is the single most important human viral infection transmitted by insects. The function of the viral proteins andtheir interactions with the host cell is under exhaustive investigation with the aim of identifying antiviral strategies. Here,using recombinant full-length dengue virus genomes, carrying a fluorescent mCherry fused to capsid, we studied biophysicalproperties of the viral protein during one infectious cycle in living cells. Dengue virus capsid protein associates to differentcellular compartments but its function in these locations is largely unknown. We evaluated the diffusion of capsid inside the celland determined a higher effective diffusion coefficient in the cytoplasm than in the nucleus. Using advanced fluorescencecorrelation methods, including the recently developed two-dimensional pair correlation analysis, we constructed for the first timehigh resolution maps of capsid mobility in an infected cell. We observed that the motion of capsid in the nucleoplasm-nucleolusinterface was highly organized, indicating an obstacle in this interface. Although nucleoli are membraneless structures, theydisplayed liquid-liquid phase separation. Once inside nucleoli, the protein showed isotropic mobility, indicating free diffusion orimmobilized capsid inside these structures. This is the first study presenting spatial and temporal dynamics of the dengue viruscapsid protein during infection.

Association of the genetic variants (-794 CATT5-8 and -173 G > C) of macrophage migration inhibitory factor (MIF) with higher soluble levels of MIF and TNFα in women with breast cancer.

BACKGROUND: Functional variants -173 G > C (rs755622) and -794CATT5-8 (rs5844572) MIF gene have been associated with the risk in several types of cancer, as well as with the increase of soluble levels of MIF and TNFα. However, in previous studies contradictory and uncertain results have been presented on the implication of MIF polymorphisms with the association in cancer, specifically in breast cancer (BC). We investigated whether the variants are associated with the susceptibility to develop BC and the soluble levels of MIF and TNFα in women with BC from western Mexico. MATERIALS AND METHODS: A total of 152 women with BC and 182 control subjects (CS) were enrolled in this study. The determination of genotypes -173 G > C and -794 CATT5-8 MIF polymorphisms was performed by PCR-RFLP and PCR, respectively. In addition, the soluble levels of MIF and TNFα in both studied groups were quantified by ELISA and MILLIPLEX assay, respectively. RESULTS: The most frequent allele found in BC was the G (74.3%) and 6 (54%) in the variants -173G > C and -794 CATT5-8 , respectively, without significant differences in both groups. Nevertheless, the women with BC carriers -173*C and -794CATT7 have higher levels of MIF in comparison with CS. An increase of MIF (BC: 11.1 ng/mL vs CS: 5.2 ng/mL, P < .001) and TNFα (BC: 24.9 ng/mL vs CS: 9.9 pg/mL, P < .001) was found. CONCLUSION: The functional variants of MIF are not genetic susceptibility markers for BC. Nevertheless, the alleles -173*C and -794CATT7 are associated with the increase of MIF circulating in women with BC.

Circulating soluble levels of MIF in women with breast cancer in the molecular subtypes: relationship with Th17 cytokine profile.

Breast cancer (BC) is a health problem worldwide; there is evidence that inflammatory cytokines are increased in BC. Macrophage migration inhibitory factor (MIF) has multiple effects on immune cells, inflammation and cancer. Besides, in previous studies, contradictory and uncertain results have been presented on the implication of Th17 cytokine profile in BC. The aim of this study was to evaluate the plasma levels of MIF and the Th17 cytokine profile in BC and their association with their molecular subtypes and clinical stage. A total of 150 women with BC of Ella Binational Breast Cancer Study and 60 healthy women (HW) were evaluated in cross-sectional study. The molecular subtypes were identified by immunohistochemistry. The plasma levels of MIF were quantified by ELISA and Th17 cytokine profile by multiplex system. MIF and IL-17 were significantly increased in BC versus HW (11.1 vs. 5.2 ng/mL and 14.8 pg/mL vs. 2.5 pg/mL p < 0.001, respectively). Our analysis showed that both MIF and IL-17A were associated with increased risk of breast cancer (OR 3.85 CI 95% 1.98-7.50 and OR 4.51 95% 1.83-11.15, respectively), higher in aggressive subtypes Luminal B, HER2 and TN. Likewise, we observed positive correlation between MIF and IL-17A (p < 0.001). In addition, IL-17E was lower in BC versus HW (p <0.001). Likewise, we observed a positive correlation between MIF and IL-17A (p < 0.001). In conclusion, both MIF and IL-17A were associated with high risk for breast cancer and aggressive molecular subtypes.

The autographa californica multiple nucleopolyhedrovirus Ac12: A non-essential F box-like protein that interacts with cellular SKP1 component of the E3 ubiquitin ligase complex.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.

Gut microbial features can predict host phenotype response to protein deficiency.

Malnutrition remains a major health problem in low- and middle-income countries. During low protein intake, <0.67 g/kg/day, there is a loss of nitrogen (N2 ) balance, due to the unavailability of amino acid for metabolism and unbalanced protein catabolism results. However, there are individuals, who consume the same low protein intake, and preserve N2 balance for unknown reasons. A novel factor, the gut microbiota, may account for these N2 balance differences. To investigate this, we correlated gut microbial profiles with the growth of four murine strains (C57Bl6/J, CD-1, FVB, and NIH-Swiss) on protein deficient (PD) diet. Results show that a PD diet exerts a strain-dependent impact on growth and N2 balance as determined through analysis of urinary urea, ammonia and creatinine excretion. Bacterial alpha diversity was significantly (P < 0.05, FDR) lower across all strains on a PD diet compared to normal chow (NC). Multi-group analyses of the composition of microbiomes (ANCOM) revealed significantly differential microbial signatures between the four strains independent of diet. However, mice on a PD diet demonstrated differential enrichment of bacterial genera including, Allobaculum (C57Bl6/J), Parabacteroides (CD-1), Turicibacter (FVB), and Mucispirillum (NIH-Swiss) relative to NC. For instance, selective comparison of the CD-1 (gained weight) and C57Bl6/J (did not gain weight) strains on PD diet also demonstrated significant pathway enrichment of dihydroorodate dehydrogenase, rRNA methyltransferases, and RNA splicing ligase in the CD-1 strains compared to C57Bl6/J strains; which might account in their ability to retain growth despite a protein deficient diet. Taken together, these results suggest a potential relationship between the specific gut microbiota, N2 balance and animal response to malnutrition.

Androgen excess in pancreatic ß cells and neurons predisposes female mice to type 2 diabetes.

Androgen excess predisposes women to type 2 diabetes (T2D), but the mechanism of this is poorly understood. We report that female mice fed a Western diet and exposed to chronic androgen excess using dihydrotestosterone (DHT) exhibit hyperinsulinemia and insulin resistance associated with secondary pancreatic ß cell failure, leading to hyperglycemia. These abnormalities are not observed in mice lacking the androgen receptor (AR) in ß cells and partially in neurons of the mediobasal hypothalamus (MBH) as well as in mice lacking AR selectively in neurons. Accordingly, i.c.v. infusion of DHT produces hyperinsulinemia and insulin resistance in female WT mice. We observe that acute DHT produces insulin hypersecretion in response to glucose in cultured female mouse and human pancreatic islets in an AR-dependent manner via a cAMP- and mTOR-dependent pathway. Acute DHT exposure increases mitochondrial respiration and oxygen consumption in female cultured islets. As a result, chronic DHT exposure in vivo promotes islet oxidative damage and susceptibility to additional stress induced by streptozotocin via AR in ß cells. This study suggests that excess androgen predisposes female mice to T2D following AR activation in neurons, producing peripheral insulin resistance, and in pancreatic ß cells, promoting insulin hypersecretion, oxidative injury, and secondary ß cell failure.

Gut Microbiota: FFAR Reaching Effects on Islets.

The G protein-coupled receptors, free fatty acid (FFA) receptors 2 and 3 (FFA2 and FFA3), belonging to the free fatty acid receptor (FFAR) class, sense a distinct class of nutrients, short chain fatty acids (SCFAs). These receptors participate in both immune and metabolic regulation. The latter includes a role in regulating secretion of metabolic hormones. It was only recently that their role in pancreatic ß cells was recognized; these receptors are known now to affect not only insulin secretion but also ß-cell survival and proliferation. These observations make them excellent potential therapeutic targets in type 2 diabetes. Moreover, expression on both immune and ß cells makes these receptors possible targets in type 1 diabetes. Furthermore, SCFAs are generated by gut microbial fermentative activity; therefore, signaling by FFA2 and FFA3 represents an exciting novel link between the gut microbiota and the ß cells. This review enumerates the role of these receptors in ß cells revealed so far and discusses possible roles in clinical translation.

Hyaluronan content governs tissue stiffness in pancreatic islet inflammation.

We have identified a novel role for hyaluronan (HA), an extracellular matrix polymer, in governing the mechanical properties of inflamed tissues. We recently reported that insulitis in type 1 diabetes of mice and humans is preceded by intraislet accumulation of HA, a highly hygroscopic polymer. Using the double transgenic DO11.10 × RIPmOVA (DORmO) mouse model of type 1 diabetes, we asked whether autoimmune insulitis was associated with changes in the stiffness of islets. To measure islet stiffness, we used atomic force microscopy (AFM) and developed a novel "bed of nails"-like approach that uses quartz glass nanopillars to anchor islets, solving a long-standing problem of keeping tissue-scale objects immobilized while performing AFM. We measured stiffness via AFM nanoindentation with a spherical indenter and found that insulitis made islets mechanically soft compared with controls. Conversely, treatment with 4-methylumbelliferone, a small-molecule inhibitor of HA synthesis, reduced HA accumulation, diminished swelling, and restored basal tissue stiffness. These results indicate that HA content governs the mechanical properties of islets. In hydrogels with variable HA content, we confirmed that increased HA leads to mechanically softer hydrogels, consistent with our model. In light of recent reports that the insulin production of islets is mechanosensitive, these findings open up an exciting new avenue of research into the fundamental mechanisms by which inflammation impacts local cellular responses.