Targeted whole-genome recovery of single viral species in a complex environmental sample.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. We present an experimental method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A unique gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. We demonstrate this method's efficacy by spiking two known viral genomes, Simian virus 40 (SV40, 5,243 bp) and Human Adenovirus 5 (HAd5, 35,938 bp), into a sewage sample with a final abundance in the droplets of around 0.1% and 0.015%, respectively. We achieve 100% recovery of the complete sequence of the spiked-in SV40 genome with uniform coverage distribution. For the larger HAd5 genome, we cover approximately 99.4% of its sequence. Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables single-genome whole-genome amplification and targeting characterizations of rare viral species and will facilitate our ability to access the mutational profile in single-virus genomes and contribute to an improved understanding of viral ecology.

Alternative polyadenylation factor CPSF6 regulates temperature compensation of the mammalian circadian clock.

A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3'-end cleavage and polyadenylation, significantly alters circadian temperature compensation in human U-2 OS cells. We apply a combination of 3'-end-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3' UTR length as well as gene and protein expression between wild-type and CPSF6 knockdown cells and their dependency on temperature. Since changes in temperature compensation behavior should be reflected in alterations of temperature responses within one or all of the 3 regulatory layers, we statistically assess differential responses upon changes in ambient temperature between wild-type and CPSF6 knockdown cells. By this means, we reveal candidate genes underlying circadian temperature compensation, including eukaryotic translation initiation factor 2 subunit 1 (EIF2S1).

Targeting the immunoproteasome in hypothalamic neurons as a novel therapeutic strategy for high-fat diet-induced obesity and metabolic dysregulation.

OBJECTIVE: Obesity represents a significant global health challenge characterized by chronic low-grade inflammation and metabolic dysregulation. The hypothalamus, a key regulator of energy homeostasis, is particularly susceptible to obesity's deleterious effects. This study investigated the role of the immunoproteasome, a specialized proteasomal complex implicated in inflammation and cellular homeostasis, during metabolic diseases. METHODS: The levels of the immunoproteasome ß5i subunit were analyzed by immunostaining, western blotting, and proteasome activity assay in mice fed with either a high-fat diet (HFD) or a regular diet (CHOW). We also characterized the impact of autophagy inhibition on the levels of the immunoproteasome ß5i subunit and the activation of the AKT pathway. Finally, through confocal microscopy, we analyzed the contribution of ß5i subunit inhibition on mitochondrial function by flow cytometry and mitophagy assay. RESULTS: Using an HFD-fed obese mouse model, we found increased immunoproteasome levels in hypothalamic POMC neurons. Furthermore, we observed that palmitic acid (PA), a major component of saturated fats found in HFD, increased the levels of the ß5i subunit of the immunoproteasome in hypothalamic neuronal cells. Notably, the increase in immunoproteasome expression was associated with decreased autophagy, a critical cellular process in maintaining homeostasis and suppressing inflammation. Functionally, PA disrupted the insulin-glucose axis, leading to reduced AKT phosphorylation and increased intracellular glucose levels in response to insulin due to the upregulation of the immunoproteasome. Mechanistically, we identified that the protein PTEN, a key regulator of insulin signaling, was reduced in an immunoproteasome-dependent manner. To further investigate the potential therapeutic implications of these findings, we used ONX-0914, a specific immunoproteasome inhibitor. We demonstrated that this inhibitor prevents PA-induced insulin-glucose axis imbalance. Given the interplay between mitochondrial dysfunction and metabolic disturbances, we explored the impact of ONX-0914 on mitochondrial function. Notably, ONX-0914 preserved mitochondrial membrane potential and attenuated mitochondrial ROS production in the presence of PA. Moreover, we found that ONX-0914 reduced mitophagy in the presence of PA. CONCLUSIONS: Our findings strongly support the pathogenic involvement of the immunoproteasome in hypothalamic neurons in the context of HFD-induced obesity and metabolic disturbances. Targeting the immunoproteasome highlights a promising therapeutic strategy to mitigate the detrimental effects of obesity on the insulin-glucose axis and cellular homeostasis. This study provides valuable insights into the mechanisms driving obesity-related metabolic diseases and offers potential avenues for developing novel therapeutic interventions.

Cultured Renal Proximal Tubular Epithelial Cells Resemble a Stressed/Damaged Kidney While Supporting BK Virus Infection.

BK virus (BKV; human polyomavirus 1) infections are asymptomatic in most individuals, and the virus persists throughout life without harm. However, BKV is a threat to transplant patients and those with immunosuppressive disorders. Under these circumstances, the virus can replicate robustly in proximal tubule epithelial cells (PT). Cultured renal proximal tubule epithelial cells (RPTE) are permissive to BKV and have been used extensively to characterize different aspects of BKV infection. Recently, lines of hTERT-immortalized RPTE have become available, and preliminary studies indicate they support BKV infection as well. Our results indicate that BKV infection leads to a similar response in primary and immortalized RPTE. In addition, we examined the patterns of global gene expression of primary and immortalized RPTE and compared them with uncultured PT freshly dissociated from human kidney. As expected, PT isolated from the healthy kidney express a number of differentiation-specific genes that are associated with kidney function. However, the expression of most of these genes is absent or repressed in cultured RPTE. Rather, cultured RPTE exhibit a gene expression profile indicative of a stressed or injured kidney. Inoculation of cultured RPTE with BKV results in the suppression of many genes associated with kidney stress. In summary, this study demonstrated similar global gene expression patterns and responses to BKV infection between primary and immortalized RPTE. Moreover, results from bulk transcriptome sequencing (RNA-seq) and SCT experiments revealed distinct transcriptomic signatures representing cell injury and stress in primary RPTE in contrast to the uncultured, freshly dissociated PT from human kidney. IMPORTANCE Cultured primary human cells provide powerful tools for the study of viral infectious cycles and host virus interactions. In the case of BKV-associated nephropathy, viral replication occurs primarily in the proximal tubule epithelia in the kidney. Consequently, cultured primary and immortalized renal proximal tubule epithelial cells (RPTE) are widely used to study BKV infection. In this work, using bulk and single-cell transcriptomics, we found that primary and immortalized RPTE responded similarly to BKV infection. However, both uninfected primary and immortalized RPTE have gene expression profiles that are markedly different from healthy proximal tubule epithelia isolated directly from human kidney without culture. Cultured RPTE are in a gene expression state indicative of an injured or stressed kidney. These results raise the possibility that BKV replicates preferentially in injured or stressed kidney epithelial cells during nephropathy.

Disease flares with baricitinib dose reductions and development of flare criteria in patients with CANDLE/PRAAS.

OBJECTIVES: Patients with chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature/proteasome-associated autoinflammatory syndrome (CANDLE/PRAAS) respond to the janus kinase inhibitor 1/2 inhibition with baricitinib at exposures higher than in rheumatoid arthritis. Baricitinib dose reductions to minimise exposure triggered disease flares which we used to develop 'flare criteria'. METHODS: Of 10 patients with CANDLE/PRAAS treated with baricitinib in an open-label expanded-access programme, baricitinib doses were reduced 14 times in 9 patients between April 2014 and December 2019. Retrospective data analysis of daily diary scores and laboratory markers collected before and after the dose reductions were used to develop 'clinical' and 'subclinical' flare criteria. Disease flare rates were compared among patients with <25% and >25% dose reductions and during study visits when patients received recommended 'optimized' baricitinib doses (high-dose visits) versus lower than recommended baricitinib doses (low-dose visits) using two-sided χ2 tests. RESULTS: In the 9/10 patients with CANDLE with dose reduction, 7/14 (50%) times the dose was reduced resulted in a disease flare. All four dose reductions of >25% triggered a disease flare (p <0.05). Assessment of clinical and laboratory changes during disease flares allowed the development of disease flare criteria that were assessed during visits when patients received high or low doses of baricitinib. Disease flare criteria were reached during 43.14% of low-dose visits compared with 12.75% of high-dose visits (p <0.0001). Addition of an interferon score as an additional flare criterion increased the sensitivity to detect disease flares. CONCLUSION: We observed disease flares and rebound inflammation with baricitinib dose reductions and proposed flare criteria that can assist in monitoring disease activity and in designing clinical studies in CANDLE/PRAAS.

Associations of current and childhood socioeconomic status and health outcomes amongst patients with knee or hip osteoarthritis in a Mexico City family-practice setting.

OBJECTIVES: To examine the association of current and childhood socioeconomic status (SES) with patient-reported functional status, quality of life and disability in patients with knee or hip osteoarthritis (OA). METHODS: Cross-sectional study amongst individuals seeking care for any medical reason in a primary care family-practice clinic in Mexico City. We included individuals with self-reported doctor-diagnosed arthritis, recruited through waiting-room posters and invitations by treating family physicians. We administered a survey using validated Spanish language versions of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), the Osteoarthritis of Lower Limbs and Quality of Life (AMICAL), and the Stanford Health Assessment Questionnaire-Disability Index (HAQ-DI). To estimate current and childhood SES, we collected data on education level and occupation type for both the patient and their parents, as well as using a validated tool to estimate income quintile. RESULTS: We recruited 154 patients and excluded 8 patients. There was a high correlation between outcome scores. Estimated income and education levels were correlated with WOMAC, AMICAL and HAQ-DI scores, and significant differences were found in all scores by occupation type. The associations for current SES variables and outcome scores remained significant independently of age, sex, BMI, and presence of diabetes or hypertension, and were largely explained by current income in mutually adjusted models. Childhood SES - in particular as measured through maternal education - was best correlated with AMICAL scores, though its effect seemed largely mediated by its association with current SES. CONCLUSIONS: Current Socioeconomic Status impacts functional status, quality of life and disability amongst OA patients in Mexico City. The WOMAC, AMICAL and HAQ-DI scores correlate with each other and are all potentially useful markers of disease severity. More research is needed to elucidate the relationships between childhood SES and OA outcomes. Awareness of life-course SES may be useful in identifying patients at risk for worse outcomes.

Increased femoral anteversion in females with anterior knee pain relates to both the neck and the shaft of the femur.

INTRODUCTION: Increased femoral anteversion (FAV) can have many clinical manifestations, including anterior knee pain (AKP). To our knowledge, no studies have measured the location of FAV in a cohort of female AKP patients. The objective of this research is to determine whether the increased FAV in AKP females originates above the lesser trochanter, below the lesser trochanter or at both levels. MATERIALS AND METHODS: Thrity-seven consecutive AKP female patients (n = 66 femurs) were recruited prospectively. There were 17 patients (n = 26 femurs; mean age of 28 years) in whom the suspicion for the increased FAV of the femur was based on the clinical examination (pathological group-PG). The control group (CG) consisted of 20 patients (n = 40 femurs; mean age of 29 years) in whom there was no increased FAV from the clinical standpoint. All of them underwent a torsional computed tomography of the lower limbs. FAV was measured according to Murphy´s method. A segmental analysis of FAV was performed using the lesser trochanter as a landmark. RESULTS: Significant differences in the total FAV (18.7 ± 5.52 vs. 42.46 ± 6.33; p < 0.001), the neck version (54.88 ± 9.64 vs. 64.27 ± 11.25; p = 0.0006) and the diaphysis version (- 36.17 ± 8.93 vs. - 21.81 ± 11.73; p < 0.001) were observed between the CG and the PG. The difference in the diaphyseal angle between CG and PG accounts for 60% of the total difference between healthy and pathological groups, while the difference between both groups in the angle of the neck accounts for 40%. CONCLUSION: In chronic AKP female patients with increased FAV, the two segments of the femur contribute to the total FAV, with a different pattern among patients and controls, being the compensation mechanism of the diaphysis much lower in the pathological femurs than in the controls.

Malnutrition in COVID-19 survivors: prevalence and risk factors.

BACKGROUND: Nutritional status is a critical factor throughout COVID-19 disease course. Malnutrition is associated with poor outcomes in hospitalized COVID-19 patients. AIM: To assess the prevalence of malnutrition and identify its associated factors in COVID-19 survivors. METHODS: Study cohort included 1230 COVID-19 survivors aged 18-86 attending a post-COVID-19 outpatient service. Data on clinical parameters, anthropometry, acute COVID-19 symptoms, lifestyle habits were collected through a comprehensive medical assessment. Malnutrition was assessed according to Global Leadership Initiative on Malnutrition (GLIM) criteria. RESULTS: Prevalence of malnutrition was 22% at 4-5 months after acute disease. Participants who were not hospitalized during acute COVID-19 showed a higher frequency of malnutrition compared to those who needed hospitalization (26% versus 19%, p < 0.01). Malnutrition was found in 25% COVID-19 survivors over 65 years of age compared to 21% younger participants (p < 0.01). After multivariable adjustment, the likelihood of being malnourished increased progressively and independently with advancing age (Odds ratio [OR] 1.02; 95% CI 1.01-1.03) and in male participants (OR 5.56; 95% CI 3.53-8.74). Malnutrition was associated with loss of appetite (OR 2.50; 95% CI 1.73-3.62), and dysgeusia (OR 4.05; 95% CI 2.30-7.21) during acute COVID-19. DISCUSSION: In the present investigation we showed that malnutrition was highly prevalent in a large cohort of COVID-19 survivors at 4-5 months from acute illness. CONCLUSIONS: Our findings highlight the need to implement comprehensive nutritional assessment and therapy as an integral part of care for COVID-19 patients.

BMAL1 Regulates Glucokinase Expression Through E-Box Elements In Vitro.

The organization of a circadian system includes an endogenous pacemaker system, input pathways for environmental synchronizing (entraining) stimuli, and output pathways through which the clock regulates physiological and behavioral processes, for example, the glucose-sensing mechanism in the liver. The liver is the central regulator of metabolism and one of our peripherals clocks. In mammals, central to this pacemaker are the transcription factors Circadian Locomotor Output Cycles Kaput (CLOCK) and BMAL1 (Brain and Muscle ARNT-Like 1). BMAL1 dimerizes with CLOCK, and this heterodimer then binds to the E-box promoter elements (CACGTG) present in clock and clock-controlled genes (CCGs). However, we are just beginning to understand how output pathways and regulatory mechanisms of CCGs are involved in rhythmic physiological processes. Glucokinase (GCK) is a fundamental enzyme in glucose homeostasis, catalyzing the high Km phosphorylation of glucose and allowing its storage. Moreover, gck is a dependent circadian gene. This study aims to determine the contribution of clock genes to hepatic gck expression and to define the specific role of E-box sequences on the circadian regulation of hepatic gck. Results showed that gck expression follows a circadian rhythm in rat hepatocytes in vitro. Accordingly, bmal1 expression induces the glucokinase circadian rhythmic expression in hepatocytes and the analysis of human and rat gck promoters, indicating the presence of E-box regions. Moreover, the basal activity of gck promoter was increased by clock/bmal1 co-transfection but inhibited by Period1/Period2 (per1/per2) co-transfection. Thus, the data suggest that the clock proteins tightly regulate the transcriptional activity of the gck promoter.

Ultrasound-Based Recovery of Anti-Inflammatory and Antimicrobial Extracts of the Acidophilic Microalga Coccomyxa onubensis.

In the present study, the recovery of valuable molecules of proven anti-inflammatory and antimicrobial activity of the acidophilic microalga Coccomyxa onubensis (C. onubensis) were evaluated using green technologies based on ultrasound-assisted extraction (UAE). Using a factorial design (3 × 2) based on response surface methodology and Pareto charts, two types of ultrasonic equipment (bath and probe) were evaluated to recover valuable compounds, including the major terpenoid of C. onubensis, lutein, and the antimicrobial activity of the microalgal extracts obtained under optimal ultrasound conditions (desirability function) was evaluated versus conventional extraction. Significant differences in lutein recovery were observed between ultrasonic bath and ultrasonic probe and conventional extraction. Furthermore, the antimicrobial activity displayed by C. onubensis UAE-based extracts was greater than that obtained in solvent-based extracts, highlighting the effects of the extracts against pathogens such as Enterococcus hirae and Bacillus subtilis, followed by Staphylococcus aureus and Escherichia coli. In addition, gas chromatography-mass spectrometry was performed to detect valuable anti-inflammatory and antimicrobial biomolecules present in the optimal C. onubensis extracts, which revealed that phytol, sterol-like, terpenoid, and even fatty acid structures could also be responsible for the antibacterial activities of the extracts. Moreover, UAE displayed a positive effect on the recovery of valuable molecules, improving biocidal effects. Our study results facilitate the use of green technology as a good tool in algal bioprocess engineering, improving energy consumption and minimizing environmental impacts and process costs, as well as provide a valuable product for applications in the field of biotechnology.

Central sensitization negatively influences the level of disability in female patients with anterior knee pain.

PURPOSE: To define the prevalence of Central Sensitization (CS) in patients with Anterior Knee Pain (AKP) and determine whether there is an association between CS and the magnitude of pain, disability, quality-of-life and psychological impairment. METHODS: The data of a total of 44 AKP female patients with a mean age of 27.7 years (15-50) recruited consecutively from hospital outpatient knee clinics were prospectively included in this study. The patients had no antecedents of knee trauma or surgery and no history of injury or disease of the nervous system. There were also 50 healthy female controls with a mean age of 26.1 years (16-46). CS was evaluated using the Central Sensitization Inventory (CSI). Quality-of-life was evaluated using the EuroQoL-5D questionnaire. Self-reporting of clinical pain intensity was obtained using the Visual Analogue Scale. The Kujala Knee Scale and IKDC form were used to evaluate disability. Anxiety and depression were evaluated using the Hospital Anxiety and Depression Subscale (HAD). Kinesiophobia was measured with the Tampa Scale for Kinesiophobia (TSK-11) and catastrophizing by means of the Pain Catastrophizing Scale (PCS). RESULTS: Sixteen AKP patients (36%), and 2 (4%) of the healthy controls presented with central sensitization (p < 0.01). AKP patients with CS have a greater degree of disability based on the Kujala Scale and higher levels of anxiety and depression than AKP patients without CS. The score of AKP patients in the CSI correlated weakly with disability and quality of life and moderately with anxiety and depression. However, no association was seen between CSI score and pain intensity, nor with catastrophizing and kinesiophobia. A multivariate logistic regression analysis showed that only depression was statistically significant in the prediction of the presence of CS (odds ratio 1.45; 95% CI 1.07 to 1.96). CONCLUSIONS: AKP patients have a significantly higher prevalence of CS in comparison with what has been reported for the general population. This finding suggests the presence of altered pain modulation in a subgroup of AKP patients. LEVEL OF EVIDENCE: Level III.

Spent coffee grounds as feedstock for the production of biosurfactants and the improved recovery of melanoidins.

Spent coffee grounds (SCG) are wastes generated in high amounts worldwide. Their composition makes them a promising feedstock for biotechnological processes. Here we show that the production of the biosurfactant surfactin by submerged culture of a Bacillus subtilis strain growing on SCG is possible, reaching concentrations up to 8.8 mg/L when using SCG at 8.3 g/L in the medium. In addition, we report a synergy between the production of surfactin and the recovery of melanoidins, an added-value compound already present in SCG. More specifically, the concentration of melanoidins in the culture medium increased between 2.1 and 2.5 times thanks to the presence of the B. subtilis in the culture. Furthermore, we have observed a strong interaction between surfactin and melanoidin aggregates through dynamic light scattering measurements, and that both of them can be co-purified with an acid precipitation. We have also characterized the interfacial and antioxidant properties of the cell-free supernatant and surfactin extract, as well as the distribution of the congeners of the biosurfactant. Altogether, this work describes a promising approach to obtain biosurfactants and antioxidant molecules in a single operation, which can be used to design several new formulations of interest for bioremediation, amendment of soils, food and cosmetics.

Single-Cell Transcriptomics Reveals a Heterogeneous Cellular Response to BK Virus Infection.

BK virus (BKV) is a human polyomavirus that is generally harmless but can cause devastating disease in immunosuppressed individuals. BKV infection of renal cells is a common problem for kidney transplant patients undergoing immunosuppressive therapy. In cultured primary human renal proximal tubule epithelial (RPTE) cells, BKV undergoes a productive infection. The BKV-encoded large T antigen (LT) induces cell cycle entry, resulting in the upregulation of numerous genes associated with cell proliferation. Consistently, microarray and transcriptome sequencing (RNA-seq) experiments performed on bulk infected cell populations identified several proliferation-related pathways that are upregulated by BKV. These studies revealed few genes that are downregulated. In this study, we analyzed viral and cellular transcripts in single mock- or BKV-infected cells. We found that the levels of viral mRNAs vary widely among infected cells, resulting in different levels of LT and viral capsid protein expression. Cells expressing the highest levels of viral transcripts account for approximately 20% of the culture and have a gene expression pattern that is distinct from that of cells expressing lower levels of viral mRNAs. Surprisingly, cells expressing low levels of viral mRNA do not progress with time to high expression, suggesting that the two cellular responses are determined prior to or shortly following infection. Finally, comparison of cellular gene expression patterns of cells expressing high levels of viral mRNA with those of mock-infected cells or cells expressing low levels of viral mRNA revealed previously unidentified pathways that are downregulated by BKV. Among these are pathways associated with drug metabolism and detoxification, tumor necrosis factor (TNF) signaling, energy metabolism, and translation.IMPORTANCE The outcome of viral infection is determined by the ability of the virus to redirect cellular systems toward progeny production countered by the ability of the cell to block these viral actions. Thus, an infected culture consists of thousands of cells, each fighting its own individual battle. Bulk measurements, such as PCR or RNA-seq, measure the average of these individual responses to infection. Single-cell transcriptomics provides a window to the one-on-one battle between BKV and each cell. Our studies reveal that only a minority of infected cells are overwhelmed by the virus and produce large amounts of BKV mRNAs and proteins, while the infection appears to be restricted in the remaining cells. Correlation of viral transcript levels with cellular gene expression patterns reveals pathways manipulated by BKV that may play a role in limiting infection.

Update of Dicrocoeliidae (Digenea) parasitizing rodents from the Americas with a description of a new Platynosomoides species from Argentina.

Digeneans are common parasites of small mammals. Dicrocoeliidae is a family with a cosmopolitan distribution, with 18 genera previously recorded from mammals in the Americas, six of them parasitizing rodents in Argentina. In this study, an updated compilation is provided of Dicrocoeliidae from rodents in the Americas. Also, a new Platynosomoides species is described parasitizing the cricetid rodent Akodon montensis of the Atlantic Forest in Argentina. Digital repositories were used to search for Dicrocoeliidae from rodents in the Americas. Rodents were collected in four localities of the Atlantic Forest, Argentina. Digeneans were removed from the rodent's bile duct, and conventional studies were used for the morphological description. A total of 15 Dicrocoeliidae species were found parasitizing 18 rodent species from eight countries in the Americas. The new species of Platynosomoides from Akodon montensis differs from the other two species of genus by the size of body, testes, ovary, cecum length and position and length of the vitelline bands. Dicrocoeliidae show growing diversity, and the compilation of species in a rodent host base allows a clearer comparison and identification of new taxa in the future.

How a Medication for Opioid Use Disorder Curriculum Translates into Experiences and Internal Medicine Residents' Understanding of Patients with Opioid Use Disorder.

PROBLEM: The number of people with an Opioid Use Disorder (OUD) continues to outpace access to associated medication. Ninety-six percent of states report higher rates of OUD than access to medications, and, despite being the standard of care, only 3% of physicians currently prescribe medication for opioid use disorder (MOUD). Prior studies have shown that decreasing barriers, such as a lack of knowledge about MOUD, increased physicians' willingness to prescribe. However, most internal medicine residency programs do not have a required addiction curriculum. As a result, we created a curriculum and conducted qualitative interviews with residents to better understand experiences with the curriculum. INTERVENTION: In an effort to overcome physician-centered barriers associated with prescribing MOUD, we developed and implemented a week-long curriculum, Addiction Week, for second and third year Internal Medicine Residents at Indiana University School of Medicine in a safety-net clinic. The curriculum included the following: didactics on substance use disorder (SUD), including OUD and alcohol use disorder, and MOUD (mostly buprenorphine), and mostly web-based, peer-reviewed and guideline based readings about addiction, direct observation of addiction counselors, direct discussion with people receiving MOUD, observation of a group therapy session, informal discussion with providers who prescribe MOUD, and, for some residents, observation of a physician prescribing MOUD. After completing the curriculum, the residents participated in an hour long audio-recorded interview to better understand their experiences with the curriculum. CONTEXT: This study was completed at a residency program where residents were not previously exposed to outpatient MOUD prescribing. Due to limited availability of faculty treating patients with MOUD, residents spent the majority of their time shadowing a social worker. IMPACT: Residents described gaining a deeper understanding of OUD by having the opportunity to interact with patients in a stable outpatient setting, which for many led to increased confidence and willingness to prescribe MOUD for people with OUD. LESSONS LEARNED: The greater understanding of addiction and willingness to prescribe MOUD described by residents in this study indicate that this type of curriculum may be a promising way to increase MOUD prescribing. Further studies are needed to evaluate whether this intervention can change prescribing behaviors.Supplemental data for this article is available online at https://doi.org/10.1080/10401334.2021.1897597.

New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate.

Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea. This enzyme has several analogies with agmatinase, which catalyzes the hydrolysis of agmatine into putrescine and urea. However, this contrasts with the highlighted specificity that each one presents for their respective substrate. A comparison of available crystal structures for arginases reveals an important difference in the extension of two loops located in the entrance of the active site. The first, denominated loop A (I129-L140) contains the residues that interact with the alpha carboxyl group or arginine of arginase, and the loop B (D181-P184) contains the residues that interact with the alpha amino group of arginine. In this work, to determine the importance of these loops in the specificity of arginase, single, double, and triple arginase mutants in these loops were constructed, as well as chimeras between type I human arginase and E. coli agmatinase. In previous studies, the substitution of N130D in arginase (in loop A) generated a species capable of hydrolyzing arginine and agmatine. Now, the specificity of arginase is completely altered, generating a chimeric species that is only active with agmatine as a substrate, by substituting I129T, N130Y, and T131A together with the elimination of residues P132, L133, and T134. In addition, Quantum Mechanic/Molecular Mechanic (QM/MM) calculations were carried out to study the accommodation of the substrates in in the active site of this chimera. With these results it is concluded that this loop is decisive to discriminate the type of substrate susceptible to be hydrolyzed by arginase. Evidence was also obtained to define the loop B as a structural determinant for substrate affinity. Concretely, the double mutation D181T and V182E generate an enzyme with an essentially unaltered kcat value, but with a significantly increased Km value for arginine and a significant decrease in affinity for its product ornithine.

Association of the dopamine D2 receptor rs1800497 polymorphism with food addiction, food reinforcement, and eating behavior in Chilean adults.

PURPOSE: The regulation of food intake and body weight involves two interacting systems: (a) The homeostatic system (including biological regulators of hunger and satiety) and (b) the non-homeostatic system, (involving concepts of food reinforcement and food addiction). Studies have established a strong genetic component in eating behavior and obesity. The TaqI A1 polymorphism (rs1800497) has previously been associated with eating behavior, diminished dopamine D2 receptor (DRD2) density, higher body mass, and food reinforcement, but relations to food addiction remain unclear. AIM: To evaluate the association between the polymorphism rs1800497 with eating behavior, food reinforcement and food addiction in Chilean adults. METHODS: This cross-sectional study recruited a convenience sample of 97 obese, 25 overweight and 99 normal-weight adults (18-35 years). Anthropometric measurements were performed by standard procedures. Eating behavior was assessed using the: Yale Food Addiction Scale (YFAS), the Three Factor Eating Behavior Questionnaire and the Food Reinforcement Value Questionnaire (FRVQ). The DRD2 genotype (rs1800497) was determined by taqman assays. RESULTS: Twenty-two percentage of the participants met the criteria for food addiction. Food addiction was higher in women than men (26% vs 10.7%) and in obese compared to non-obese (40% vs 6%). There was no relationship between food addiction and DRD2 genotype. However when stratified by sex and nutritional status, obese female carriers of the A1 allele reported greater scores on emotional eating and snack food reinforcement compared to non-carriers. CONCLUSIONS: The DRD2 polymorphism is associated with some hedonic aspects of eating behavior, namely food reinforcement and emotional eating but not food addiction, and this association may be moderated by sex and obesity status, with obese women who are carriers of this genetic variant at higher risk. LEVEL OF EVIDENCE: Level V: evidence obtained from a cross-sectional descriptive study.

The FGF2-induced tanycyte proliferation involves a connexin 43 hemichannel/purinergic-dependent pathway.

In the adult hypothalamus, the neuronal precursor role is attributed to the radial glia-like cells that line the third-ventricle (3V) wall called tanycytes. Under nutritional cues, including hypercaloric diets, tanycytes proliferate and differentiate into mature neurons that moderate body weight, suggesting that hypothalamic neurogenesis is an adaptive mechanism in response to metabolic changes. Previous studies have shown that the tanycyte glucosensing mechanism depends on connexin-43 hemichannels (Cx43 HCs), purine release, and increased intracellular free calcium ion concentration [(Ca2+ )i ] mediated by purinergic P2Y receptors. Since, Fibroblast Growth Factor 2 (FGF2) causes similar purinergic events in other cell types, we hypothesize that this pathway can be also activated by FGF2 in tanycytes to promote their proliferation. Here, we used bromodeoxyuridine (BrdU) incorporation to evaluate if FGF2-induced tanycyte cell division is sensitive to Cx43 HC inhibition in vitro and in vivo. Immunocytochemical analyses showed that cultured tanycytes maintain the expression of in situ markers. After FGF2 exposure, tanycytic Cx43 HCs opened, enabling release of ATP to the extracellular milieu. Moreover, application of external ATP was enough to induce their cell division, which could be suppressed by Cx43 HC or P2Y1-receptor inhibitors. Similarly, in vivo experiments performed on rats by continuous infusion of FGF2 and a Cx43 HC inhibitor into the 3V, demonstrated that FGF2-induced ß-tanycyte proliferation is sensitive to Cx43 HC blockade. Thus, FGF2 induced Cx43 HC opening, triggered purinergic signaling, and increased ß-tanycytes proliferation, highlighting some of the molecular mechanisms involved in the cell division response of tanycyte. This article has an Editorial Highlight see https://doi.org/10.1111/jnc.15218.

Human polyomavirus BKV infection of endothelial cells results in interferon pathway induction and persistence.

Polyomavirus BKV is highly prevalent among humans. The virus establishes an asymptomatic persistent infection in the urinary system in healthy people, but uncontrolled productive infection of the virus in immunocompromised patients can lead to serious diseases. In spite of its high prevalence, our knowledge regarding key aspects of BKV polyomavirus infection remains incomplete. To determine tissue and cell type tropism of the virus, primary human epithelial cells, endothelial cells and fibroblasts isolated from the respiratory and urinary systems were tested. Results from this study demonstrated that all 9 different types of human cells were infectable by BKV polyomavirus but showed differential cellular responses. In microvascular endothelial cells from the lung and the bladder, BKV persistent infection led to prolonged viral protein expression, low yield of infectious progeny and delayed cell death, in contrast with infection in renal proximal tubular epithelial cells, a widely used cell culture model for studying productive infection of this virus. Transcriptomic profiling revealed the activation of interferon signaling and induction of multiple interferon stimulated genes in infected microvascular endothelial cells. Further investigation demonstrated production of IFNß and secretion of chemokine CXCL10 by infected endothelial cells. Activation of IRF3 and STAT1 in infected endothelial cells was also confirmed. In contrast, renal proximal tubular epithelial cells failed to mount an interferon response and underwent progressive cell death. These results demonstrated that microvascular endothelial cells are able to activate interferon signaling in response to polyomavirus BKV infection. This raises the possibility that endothelial cells might provide initial immune defense against BKV infection. Our results shed light on the persistence of and immunity against infection by BKV polyomavirus.

HIF1α-dependent metabolic reprogramming governs mesenchymal stem/stromal cell immunoregulatory functions.

Hypoxia-inducible factor 1 α (HIF1α), a regulator of metabolic change, is required for the survival and differentiation potential of mesenchymal stem/stromal cells (MSC). Its role in MSC immunoregulatory activity, however, has not been completely elucidated. In the present study, we evaluate the role of HIF1α on MSC immunosuppressive potential. We show that HIF1α silencing in MSC decreases their inhibitory potential on Th1 and Th17 cell generation and limits their capacity to generate regulatory T cells. This reduced immunosuppressive potential of MSC is associated with a metabolic switch from glycolysis to OXPHOS and a reduced capacity to express or produce some immunosuppressive mediators including Intercellular Adhesion Molecule (ICAM), IL-6, and nitric oxide (NO). Moreover, using the Delayed-Type Hypersensitivity murine model (DTH), we confirm, in vivo, the critical role of HIF1α on MSC immunosuppressive effect. Indeed, we show that HIF1α silencing impairs MSC capacity to reduce inflammation and inhibit the generation of pro-inflammatory T cells. This study reveals the pivotal role of HIF1α on MSC immunosuppressive activity through the regulation of their metabolic status and identifies HIF1α as a novel mediator of MSC immunotherapeutic potential.