Still trouble with bleeding: Risk factors for HCV transmission in men who have sex with men and behavioural trajectories from 2019 to 2021.

OBJECTIVES: To identify sexual/sex-associated risk factors for hepatitis C transmission among men who have sex with men (MSM) and visualise behavioural trajectories from 2019 to 2021. METHODS: We linked a behavioural survey to a hepatitis C cohort study (NoCo), established in 2019 across six German HIV/hepatitis C virus (HCV) treatment centres, and performed a case-control analysis. Cases were MSM with recent HCV infection, and controls were matched for HIV status (model 1) or proportions of sexual partners with HIV (model 2). We conducted conditional univariable and multivariable regression analyses. RESULTS: In all, 197 cases and 314 controls completed the baseline questionnaire and could be matched with clinical data. For regression models, we restricted cases to those with HCV diagnosed since 2018 (N = 100). Factors independently associated with case status included sex-associated rectal bleeding, shared fisting lubricant, anal douching, chemsex, intravenous and intracavernosal injections, with population-attributable fractions of 88% (model 1) and 85% (model 2). These factors remained stable over time among cases, while sexual partner numbers and group sex decreased during COVID-19 measures. CONCLUSIONS: Sexual/sex-associated practices leading to blood exposure are key factors in HCV transmission in MSM. Public health interventions should emphasize the importance of blood safety in sexual encounters. Micro-elimination efforts were temporarily aided by reduced opportunities for sexual encounters during the COVID-19 pandemic.

Recently acquired HCV infection in men who have sex with men in Germany in the direct-acting antivirals era and during the COVID-19 pandemic.

INTRODUCTION: Direct-acting antivirals (DAAs) are key to eliminating hepatitis C virus (HCV). In men who have sex with men (MSM) with HIV co-infection, recently acquired HCV infection is common. Sexual practices and reinfection rates may hamper micro-elimination despite high treatment rates. METHODS: The cohort included MSM with recently acquired HCV infection from 2014 to 2021. The patients' demographic, clinical, behavioural, and laboratory data and treatment and reinfection outcomes were documented. RESULTS: A total of 237 men with recently acquired HCV infection were included: 216 (91%) had HIV. The median age was 46 years (interquartile range [IQR] 39-52), and the median CD4 count was 660/mm3 (IQR 527-835). The annual incidence of recently acquired HCV remained between 0.28% and 0.43% but dropped to 0.02% in 2021 during the COVID pandemic, almost reaching micro-elimination. The reinfection incidence was 15.5 per 100 patient-years (95% confidence interval 12.6-18.8), and reinfection was associated with the use of crystal methamphetamine (p = 0.032) and ketamine (p = 0.042). In total, 31.3% had multiple reinfections, and four reinfections occurred in users of pre-exposure prophylaxis. CONCLUSIONS: High treatment and cure rates did not lead to HCV elimination. A change in sexual behaviour, potentially imposed by COVID-19 restrictions, led to micro-elimination in the NoCo cohort. As recently acquired HCV is prevalent in MSM with and without HIV, surveillance is necessary to consolidate elimination goals.

Weight loss in adult male Wistar rats by Roux-en-Y gastric bypass is primarily explained by caloric intake reduction and presurgery body weight.

Diets varying in macronutrient composition, energy density, and/or palatability may cause differences in outcome of bariatric surgery. In the present study, rats feeding a healthy low-fat (LF) diet or an obesogenic high-fat/sucrose diet (HF/S) were either subjected to Roux-en-Y gastric bypass surgery (RYGB) or sham surgery, and weight loss trajectories and various energy balance parameters were assessed. Before RYGB, rats eating an HF/S (n = 14) diet increased body weight relative to rats eating an LF diet (n = 20; P < 0.01). After RYGB, absolute weight loss was larger in HF/S (n = 6) relative to LF feeding (n = 6) rats, and this was associated with reduced cumulative energy intake (EI; P < 0.05) and increased locomotor activity (LA; P < 0.05-0.001), finally leading to similar levels of reduced body fat content in HF/S and LF rats 3 wk after surgery. Regression analysis revealed that variation in RYGB-induced body weight loss was best explained by models including 1) postoperative cumulative EI and preoperative body weight (R2 = 0.87) and 2) postoperative cumulative EI and diet (R2 = 0.79), each without significant contribution of LA. Particularly rats on the LF diet became transiently more hypothermic and circadianally arrhythmic following RYGB (i.e., indicators of surgery-associated malaise) than HF/S feeding rats. Our data suggest that relative to feeding an LF diet, continued feeding an HF/S diet does not negatively impact recovery from RYGB surgery, yet it promotes RYGB-induced weight loss. The RYGB-induced weight loss is primarily explained by reduced cumulative EI and higher preoperative body weight, leading to comparably low levels of body fat content in HF/S and LF feeding rats.NEW & NOTEWORTHY Relative to feeding an LF diet, continued feeding an HF/S diet does not negatively impact recovery from RYGB surgery in rats. Relative to feeding an LF diet, continued feeding an HF/S diet promotes RYGB-induced weight loss. The RYGB-induced weight loss is primarily explained by reduced cumulative EI and higher preoperative body weight, leading to comparably low levels of body fat content in HF/S and LF feeding rats.

Laparoscopic Roux-Y-gastric bypass versus laparoscopic one-anastomosis gastric bypass for obesity: clinical & metabolic results of a prospective randomized controlled trial.

BACKGROUND: One anastomosis gastric bypass (OAGB) has been proposed as an effective alternative to the current standard procedure in Switzerland, Roux-en-Y gastric bypass (RYGB). Prospective data comparing both procedures are scarce. Therefore, we performed a non-inferiority randomized controlled trial assessing the effectiveness and safety of these 2 operative techniques. METHOD: Eighty patients were randomized 1:1. OAGB consisted of a very long gastric pouch with a 200 cm biliopancreatic limb, RYGB of a 150 cm ante-colic alimentary and a 60 cm biliopancreatic limb, respectively. Primary endpoint was the percent excess weight loss (%EWL) at 12 months after surgery. RESULTS: Mean %EWL at 12 months was 87.9% (SD24.4) in the RYGB group and 104.1% (SD24.6) in the OAGB group (p = 0.006). There was no mortality. The rate of marginal ulcers was higher in patients with OAGB compared to those with RYGB (p = 0.011), while the total number of late complications did not statistically differ between the two groups. Except for the remission of GERD, which was higher in the RYGB group compared to OAGB, there was no difference between the groups regarding the remission of comorbidities. OAGB showed improved glucose control compared to the RYGB after 1 year (p = 0.001). Furthermore, glucagon-like peptide-1 increase was significantly higher in OAGB at 6 weeks (p = 0.041) and 1 year after surgery (p = 0.029). Quality of life improved after both surgeries, without differences between the groups. CONCLUSIONS: %EWL 1 year after surgery was higher in OAGB than in RYGB. A better glycemic control with a higher increase in GLP-1 was observed after OAGB compared to RYGB. TRIAL REGISTRATION: This trial is registered on ClinicalTrials.gov under the identifier NCT02601092.

Efficacy and safety of switching to dolutegravir/lamivudine in virologically suppressed people with HIV-1 aged ≥ 50 years: week 48 pooled results from the TANGO and SALSA studies.

BACKGROUND: As the population of people with HIV ages, concerns over managing age-related comorbidities, polypharmacy, immune recovery, and drug-drug interactions while maintaining viral suppression have arisen. We present pooled TANGO and SALSA efficacy and safety results dichotomized by age (< 50 and ≥ 50 years). METHODS: Week 48 data from the open-label phase 3 TANGO and SALSA trials evaluating switch to once-daily dolutegravir/lamivudine (DTG/3TC) fixed-dose combination vs continuing current antiretroviral regimen (CAR) were pooled. Proportions of participants with HIV-1 RNA ≥ 50 and < 50 copies/mL (Snapshot, intention-to-treat exposed) and safety were analyzed by age category. Adjusted mean change from baseline in CD4 + cell count was assessed using mixed-models repeated-measures analysis. RESULTS: Of 1234 participants, 80% of whom were male, 29% were aged ≥ 50 years. Among those aged ≥ 50 years, 1/177 (< 1%) DTG/3TC participant and 3/187 (2%) CAR participants had HIV-1 RNA ≥ 50 copies/mL at 48 weeks; proportions with HIV-1 RNA < 50 copies/mL were high in both treatment groups (≥ 92%), consistent with overall efficacy and similar to observations in participants aged < 50 years (≥ 93%). Regardless of age category, CD4 + cell count increased or was maintained from baseline with DTG/3TC. Change from baseline in CD4 + /CD8 + ratio was similar across age groups and between treatment groups. One CAR participant aged < 50 years had confirmed virologic withdrawal, but no resistance was detected. In the DTG/3TC group, incidence of adverse events (AEs) was similar across age groups. Proportions of AEs leading to withdrawal were low and comparable between age groups. Although drug-related AEs were generally low, across age groups, drug-related AEs were more frequent in participants who switched to DTG/3TC compared with those who continued CAR. While few serious AEs were observed in both treatment groups, more were reported in participants aged ≥ 50 years vs < 50 years. CONCLUSIONS: Among individuals with HIV-1, switching to DTG/3TC maintained high rates of virologic suppression and demonstrated a favorable safety profile, including in those aged ≥ 50 years despite higher prevalence of concomitant medication use and comorbidities. TRIAL REGISTRATION NUMBER: TANGO, NCT03446573 (February 27, 2018); SALSA, NCT04021290 (July 16, 2019).

Low Spontaneous Clearance Rates of Recently Acquired Hepatitis C Virus in Human Immunodeficiency Virus-Positive Men Who Have Sex With Men (PROBE-C Study).

BACKGROUND: Using direct-acting antivirals (DAAs) for recently acquired hepatitis C virus (RAHCV) infections, particularly in human immunodeficiency virus (HIV)-positive men who have sex with men (MSM), dramatically reduced the incidence of hepatitis C. However, implementation into clinical practice is challenging. The aim of this study was to analyze spontaneous clearance (SC) rates of RAHCV and to identify predictors of SC. METHODS: The PROBE-C study is an observational European cohort on RAHCV infections in HIV-positive MSM. Between 2007 and 2017, RAHCV infections were documented with ≥12 months of follow-up. Fisher exact, χ2, and Mann-Whitney U tests were used for statistical analysis. RESULTS: A total of 464 RAHCV infections were documented; 457 of 464 patients (98%) were male, and the median age (interquartile range [IQR]) was 41 (38-46) years. The main risk group for hepatitis C virus (HCV) transmission was MSM (98.9%). Most participants were infected with HCV genotype 1 (78.3%). The median baseline HCV RNA level (IQR) was 230 000 (135 000-474 432) IU/mL, and the median CD4+ T-cell count was 574/µL (547-604/µL. Of all cases, 92% received combination antiretroviral therapy, with 91% showing suppressed HIV RNA levels (<200 copies/mL). The median maximum alanine aminotransferase level (IQR) was 445 (402-522) U/L. SC of RAHCV infection occurred in 55 of 464 cases (11.9%). A >2-log decline in HCV RNA levels 4 weeks after diagnosis of RAHCV infection was the strongest predictor of SC (P < .001; sensitivity, 96.4%; specificity, 97.5%; positive predictive value, 84.1%; negative predictive value, 99.5%). CONCLUSIONS: SC of RAHCV in HIV-positive MSM is found in only 11.9% of cases and a <2-log drop in HCV RNA level at week 4 after diagnosis should prompt early DAA-based treatment. However, immediate DAA treatment for RAHCV infection may also be favored in patients with ongoing transmission risk behavior.

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA.

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.

Obesity-induced changes in gene expression in feline adipose and skeletal muscle tissue.

Indoor-confined cats are prone to developing obesity due to a sedentary life and an energy intake exceeding energy requirements. As in humans, feline obesity decreases insulin sensitivity and increases the risk of developing feline diabetes mellitus, but the pathophysiological mechanisms are currently poorly understood. Human obesity-related metabolic alterations seem to relate to changes in the expression of genes involved in glucose metabolism, insulin action and inflammation. The objective of the current study was to investigate changes in the expression of genes relating to obesity, glucose metabolism and inflammation in cats with non-experimentally induced obesity. Biopsies from the sartorius muscle and subcutaneous adipose tissue were obtained from 73 healthy, neutered, indoor-confined domestic shorthaired cats ranging from lean to obese. Quantification of obesity-related gene expression levels relative to glyceraldehyde-3-phosphate dehydrogenase was performed by quantitative real-time polymerase chain reaction. A negative association between obesity and adiponectin expression was observed in the adipose tissue (mean ± SD; normal weight, 27.30 × 10-3 ± 77.14 × 10-3 ; overweight, 2.89 × 10-3 ± 0.38 × 10-3 and obese, 2.93 × 10-3 ± 4.20 × 10-3 , p < 0.05). In muscle, the expression of peroxisome proliferative activated receptor-γ2 and plasminogen activator inhibitor-1 was increased in the obese compared to the normal-weight cats, and resistin was increased in the normal-weight compared to the overweight cats. There were no detectable obesity-related changes in the messenger RNA levels of inflammatory cytokines. In conclusion, a possible obesity-related low-grade inflammation caused by increased expression of key proinflammatory regulators was not observed. This could imply that the development of feline obesity and ensuing insulin resistance may not be based on tissue-derived inflammation, but caused by several determining factors, many of which still need further investigation.

Hyperleptinemia as a contributing factor for the impairment of glucose intolerance in obesity.

Obesity has emerged as a major risk factor for insulin resistance leading to the development of type 2 diabetes (T2D). The condition is characterized by high circulating levels of the adipose-derived hormone leptin and a state of chronic low-grade inflammation. Pro-inflammatory signaling in the hypothalamus is associated with a decrease of central leptin- and insulin action leading to impaired systemic glucose tolerance. Intriguingly, leptin not only regulates body weight and glucose homeostasis but also acts as a pro-inflammatory cytokine. Here we demonstrate that increasing leptin levels (62,5 µg/kg/d, PEGylated leptin) in mice fed a high-fat diet (HFD) exacerbated body weight gain and aggravated hypothalamic micro- as well as astrogliosis. In contrast, administration of a predetermined dose of a long-acting leptin antagonist (100 µg/kg/d, PESLAN) chosen to block excessive leptin signaling during diet-induced obesity (DIO) showed the opposite effect and significantly improved glucose tolerance as well as decreased the total number of microglia and astrocytes in the hypothalamus of mice fed HFD. These results suggest that high levels of leptin, such as in obesity, worsen HFD-induced micro-and astrogliosis, whereas the partial reduction of hyperleptinemia in DIO mice may have beneficial metabolic effects and improves hypothalamic gliosis.

Calcitonin receptor antibody validation and expression in the rodent brain.

BACKGROUND AND AIM: Therapeutics that reduce calcitonin gene-related peptide activity are effective migraine treatments. However, gaps remain in our understanding of the molecular mechanisms that link calcitonin gene-related peptide to migraine. The amylin 1 receptor responds potently to calcitonin gene-related peptide, and to the related peptide amylin, but its role in relation to either peptide or to migraine is unclear. We sought to better understand the expression of the amylin 1 receptor protein subunit, the calcitonin receptor, in the rodent brain. METHODS: We profiled three antibodies for immunodetection of calcitonin receptor, using immunocytochemistry, western blotting, and calcitonin receptor conditional knockout mouse tissue. Selected migraine-relevant rat brain regions were then examined for calcitonin receptor-like immunoreactivity. RESULTS: All three antibodies detected calcitonin receptor protein but only one (188/10) produced robust immunostaining in rodent brain, under the conditions used. Calcitonin receptor-like immunoreactivity was apparent in the rat brainstem and midbrain including the locus coeruleus, periaqueductal grey and spinal trigeminal nucleus. CONCLUSIONS: Anti-calcitonin receptor antibodies require comprehensive profiling to ensure confidence in the detection of calcitonin receptor. Using a validated antibody, calcitonin receptor-like immunoreactivity was detected in several brain regions relevant to migraine. Further research is needed to understand the functional consequences of calcitonin receptor expression for calcitonin gene-related peptide or amylin physiology and pathophysiology.

Creating the amylin story.

This paper is based on a presentation given at the Annual Meeting of the Society for the Study of Ingestive Behavior in July 2021 and provides a personal view on some of the milestones in the discovery of amylin as a constituent of pancreatic islet amyloid deposits, as a pancreatic beta-cell hormone, and on its role in physiology and pathophysiology. Only selected effects of amylin are discussed here because we recently published extensive reviews on the physiology and pathophysiology of amylin. Amylin was discovered as the main constituent of islet amyloid that is predominantly found in pancreatic islets in type 2 diabetics. These deposits, and in particular small oligomer aggregates of amylin seem to contribute to the progressive beta-cell damage seen in type 2 diabetics. Amylin is also a physiologically relevant circulating hormone with diverse metabolic functions, e.g. inhibition of eating, of pancreatic glucagon secretion and of gastric emptying. Knowledge of these types of functions and amylin's mechanisms of action lead to the development of amylin analogues that are now among the most promising anti-obesity targets in clinical testing. With this review, I want to give a short overview of 35 exciting years of amylin research.

The Deubiquitinase OTUB1 Is a Key Regulator of Energy Metabolism.

Dysregulated energy metabolism is a major contributor to a multitude of pathologies, including obesity and diabetes. Understanding the regulation of metabolic homeostasis is of utmost importance for the identification of therapeutic targets for the treatment of metabolically driven diseases. We previously identified the deubiquitinase OTUB1 as substrate for the cellular oxygen sensor factor-inhibiting HIF (FIH) with regulatory effects on cellular energy metabolism, but the physiological relevance of OTUB1 is unclear. Here, we report that the induced global deletion of OTUB1 in adult mice (Otub1 iKO) elevated energy expenditure, reduced age-dependent body weight gain, facilitated blood glucose clearance and lowered basal plasma insulin levels. The respiratory exchange ratio was maintained, indicating an unaltered nutrient oxidation. In addition, Otub1 deletion in cells enhanced AKT activity, leading to a larger cell size, higher ATP levels and reduced AMPK phosphorylation. AKT is an integral part of insulin-mediated signaling and Otub1 iKO mice presented with increased AKT phosphorylation following acute insulin administration combined with insulin hypersensitivity. We conclude that OTUB1 is an important regulator of metabolic homeostasis.

Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale.

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.

A selective role for receptor activity-modifying proteins in subchronic action of the amylin selective receptor agonist NN1213 compared with salmon calcitonin on body weight and food intake in male mice.

The role of receptor activity-modifying proteins (RAMPs) in modulating the pharmacological effects of an amylin receptor selective agonist (NN1213) or the dual amylin-calcitonin receptor agonist (DACRA), salmon calcitonin (sCT), was tested in three RAMP KO mouse models, RAMP1, RAMP3 and RAMP1/3 KO. Male wild-type (WT) and knockout (KO) littermate mice were fed a 45% high-fat diet for 20 weeks prior to the 3-week treatment period. A decrease in body weight after NN1213 was observed in all WT mice, whereas sCT had no effect. The absence of RAMP1 had no significant effect on NN1213 efficacy, and sCT was still inactive. However, the absence of RAMP3 impeded NN1213 efficacy but improved sCT efficacy. Similar results were observed in RAMP1/3 KO suggesting that the amylin receptor 3 (AMY3 = CTR + RAMP3) is necessary for NN1213's maximal action on body weight and food intake and that the lack of AMY3 allowed sCT to be active. These results suggest that the chronic use of DACRA such as sCT can have unfavourable effect on body weight loss in mice (which differs from the situation in rats), whereas the use of the amylin receptor selective agonist does not. AMY3 seems to play a crucial role in modulating the action of these two compounds, but in opposite directions. The assessment of a long-term effect of amylin and DACRA in different rodent models is necessary to understand potential physiological beneficial and unfavourable effects on weight loss before its transition to clinical trials.

Whole-brain mapping of amylin-induced neuronal activity in receptor activity-modifying protein 1/3 knockout mice.

The pancreatic hormone amylin plays a central role in regulating energy homeostasis and glycaemic control by stimulating satiation and reducing food reward, making amylin receptor agonists attractive for the treatment of metabolic diseases. Amylin receptors consist of heterodimerized complexes of the calcitonin receptor and receptor-activity modifying proteins subtype 1-3 (RAMP1-3). Neuronal activation in response to amylin dosing has been well characterized, but only in selected regions expressing high levels of RAMPs. The current study identifies global brain-wide changes in response to amylin and by comparing wild type and RAMP1/3 knockout mice reveals the importance of RAMP1/3 in mediating this response. Amylin dosing resulted in neuronal activation as measured by an increase in c-Fos labelled cells in 20 brain regions, altogether making up the circuitry of neuronal appetite regulation (e.g., area postrema (AP), nucleus of the solitary tract (NTS), parabrachial nucleus (PB), and central amygdala (CEA)). c-Fos response was also detected in distinct nuclei across the brain that typically have not been linked with amylin signalling. In RAMP1/3 knockout amylin induced low-level neuronal activation in seven regions, including the AP, NTS and PB, indicating the existence of RAMP1/3-independent mechanisms of amylin response. Under basal conditions RAMP1/3 knockout mice show reduced neuronal activity in the hippocampal formation as well as reduced hippocampal volume, suggesting a role for RAMP1/3 in hippocampal physiology and maintenance. Altogether these data provide a global map of amylin response in the mouse brain and establishes the significance of RAMP1/3 receptors in relaying this response.

Noradrenaline signaling in the LPBN mediates amylin's and salmon calcitonin's hypophagic effect in male rats.

The LPBN (lateral parabrachial nucleus) plays an important role in feeding control. CGRP (calcitonin gene-related peptide) LPBN neurons activation mediates the anorectic effects of different gut-derived peptides, including amylin. Amylin and its long acting analog sCT (salmon calcitonin) exert their anorectic actions primarily by directly activating neurons located in the area postrema (AP). A large proportion of projections from the AP and the adjacent nucleus of the solitary tractNTS to the LPBN, are noradrenergic (NA), and amylin-activated NAAP neurons are critical in mediating amylin's hypophagic effects. Here, we determine the functional role of NAAP amylin activated neurons to activate CGRP and non-CGRP LPBN neurons. To this end, NA was specifically depleted in the rat LPBN through a stereotaxic microinfusion of 6-OHDA, a neurotoxic agent that destroys NA terminals. While amylin (50 µg/kg) and sCT (5 µg/kg) reduced eating in sham-lesioned rats, no reduction in feeding occurred in NA-depleted animals. Further, the amylin-induced c-Fos response in the LPBN and c-Fos/CGRP colocalization were reduced in NA-depleted animals compared to controls. We conclude that AP â†’ LPBN NA signaling, through the activation of LPBN CGRP neurons, mediates part of amylin's hypophagic effect.

A protein architecture guided screen for modification dependent restriction endonucleases.

Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

Reinfection With the Hepatitis C Virus in Men Who Have Sex With Men After Successful Treatment With Direct-acting Antivirals in Germany: Current Incidence Rates, Compared With Rates During the Interferon Era.

BACKGROUND: Micro-elimination of the hepatitis C virus (HCV) includes treatment in populations at risk of ongoing HCV transmission, such as men who have sex with men (MSM) or people who inject drugs (PWID). We analyzed the HCV reinfection incidence rates of participants in the German hepatitis C cohort (GECCO) and compared our data to previous findings from the interferon era. METHODS: Patients with HCV reinfections in the multi-centric GECCO cohort were compared to patients in whom no reinfection occurred. The HCV reinfection incidence rate in MSM was also compared to the incidence rate in the interferon era (using data from the European Acquired Immunodeficiency Syndrome Treatment Network [NEAT]). RESULTS: Between January 2014 and April 2018, 48 HCV reinfections occurred in 2298 individuals (2%), with 2346 cured HCV episodes. The median time to reinfection was 500 days (range 16-1160) and the overall HCV reinfection incidence rate was 1.89 per 100 person-years (py; 95% confidence interval [CI], 1.41-2.48). In a multivariate analysis, the transmission risk in MSM was the only independent risk factor of HCV reinfection (odds ratio, 39.3; 95% CI, 4.57-334.40; P = .001). The incidence rate in MSM was 9.02 (95% CI, 6.48-12.26) per 100 py, compared to 1.14 per 100 py in PWID (95% CI, .56-2.09). The incidence rate for a first HCV reinfection in MSM was similar in the direct-acting antiviral era, compared to the interferon era, with a hazard ratio of 1.05 (95% CI, .64-1.74; P = .831). CONCLUSIONS: HCV reinfection remains a frequent finding among MSM in Germany. In addition to behavioral interventions, early HCV treatment and retreatment should be implemented for this subgroup to prevent HCV transmission.

Evaluation of Acute Mountain Sickness by Unsedated Transnasal Esophagogastroduodenoscopy at High Altitude.

BACKGROUND & AIMS: It is not clear how rapid ascent to a high altitude causes the gastrointestinal symptoms of acute mountain sickness (AMS). We assessed the incidence of endoscopic lesions in the upper gastrointestinal tract in healthy mountaineers after a rapid ascent to high altitude, their association with symptoms, and their pathogenic mechanisms. METHODS: In a prospective study, 25 mountaineers (10 women; mean age, 43.8 ± 9.5 y) underwent unsedated, transnasal esophagogastroduodenoscopy in Zurich (490 m) and then on 2 test days (days 2 and 4) at a high altitude laboratory in the Alps (Capanna Regina Margherita, 4559 m). Symptoms were assessed using validated instruments for AMS (the acute mountain sickness score and the Lake Louise scoring system) and visual analogue scales (scale, 0-100). Levels of messenger RNAs (mRNAs) in duodenal biopsy specimens were measured by quantitative polymerase chain rection. RESULTS: The follow-up endoscopy at high altitude was performed in 19 of 25 patients on day 2 and in 23 of 25 patients on day 4. The frequency of endoscopic lesions increased from 12% at baseline to 26.3% on day 2 and to 60.9% on day 4 (P < .001). The incidence of ulcer disease increased from 0 at baseline to 10.5% on day 2 and to 21.7% on day 4 (P = .014). Mucosal lesions were associated with lower hunger scores (37.3 vs 67.4 in patients without lesions; P = .012). Subjects with peptic lesions had higher levels of HIF2A mRNA, which encodes a hypoxia-induced transcription factor, and ICAM1 mRNA, which encodes an adhesion molecule, compared with subjects without lesions (fold changes, 1.38 vs 0.63; P = .001; and 1.37 vs 0.66; P = .011, respectively). CONCLUSIONS: In a prospective study of 25 mountaineers, fast ascent to a high altitude resulted in rapid onset of clinically meaningful mucosal lesions and ulcer disease. Duodenal biopsy specimens from these subjects had increased levels of HIF2A mRNA and ICAM1 mRNA, which might contribute to the formation of hypoxia-induced peptic lesions. Further studies are needed of the mechanisms of this process.

Modular Conjugation of a Potent Anti-HER2 Immunotoxin Using Coassociating Peptides.

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.