A human obesity-associated MC4R mutation with defective Gq/11α signaling leads to hyperphagia in mice.

Melanocortin 4 receptor (MC4R) mutations are the most common cause of human monogenic obesity and are associated with hyperphagia and increased linear growth. While MC4R is known to activate Gsα/cAMP signaling, a substantial proportion of obesity-associated MC4R mutations do not affect MC4R/Gsα signaling. To further explore the role of specific MC4R signaling pathways in the regulation of energy balance, we examined the signaling properties of one such mutant, MC4R (F51L), as well as the metabolic consequences of MC4RF51L mutation in mice. The MC4RF51L mutation produced a specific defect in MC4R/Gq/11α signaling and led to obesity, hyperphagia, and increased linear growth in mice. The ability of a melanocortin agonist to acutely inhibit food intake when delivered to the paraventricular nucleus (PVN) was lost in MC4RF51L mice, as well as in WT mice in which a specific Gq/11α inhibitor was delivered to the PVN; this provided evidence that a Gsα-independent signaling pathway, namely Gq/11α, significantly contributes to the actions of MC4R on food intake and linear growth. These results suggest that a biased MC4R agonist that primarily activates Gq/11α may be a potential agent to treat obesity with limited untoward cardiovascular and other side effects.

Intercellular communication within the virus microenvironment affects the susceptibility of cells to secondary viral infections.

Communication between infected cells and cells in the surrounding tissue is a determinant of viral spread. However, it remains unclear how cells in close or distant proximity to an infected cell respond to primary or secondary infections. We establish a cell-based system to characterize a virus microenvironment, distinguishing infected, neighboring, and distal cells. Cell sorting, microscopy, proteomics, and cell cycle assays allow resolving cellular features and functional consequences of proximity to infection. We show that human cytomegalovirus (HCMV) infection primes neighboring cells for both subsequent HCMV infections and secondary infections with herpes simplex virus 1 and influenza A. Neighboring cells exhibit mitotic arrest, dampened innate immunity, and altered extracellular matrix. Conversely, distal cells are poised to slow viral spread due to enhanced antiviral responses. These findings demonstrate how infection reshapes the microenvironment through intercellular signaling to facilitate spread and how spatial proximity to an infection guides cell fate.

Racial and Language Disparities in Pediatric Emergency Department Triage.

OBJECTIVE: The aim of this study was to assess the impact race and language have on emergency department (ED) triage scores while accounting for illness severity. We hypothesized that non-White and non-English-speaking patients were assigned lower-acuity triage scores compared with White and English-speaking patients, respectively. METHODS: We used a chart review-based retrospective cohort study design, examining patients aged 0 to 17 years at our pediatric ED from July 2015 through June 2016. Illness severity was measured using a truncated Modified Pediatric Early Warning Score calculated from patient vital signs. We used univariate and multivariate multinomial logistic regression to assess the association between race and language with Emergency Severity Index scores. RESULTS: Our final data set consisted of 10,815 visits from 8928 patients. Non-Hispanic (NH) White patients accounted for 34.6% of patients. In the adjusted analyses, non-White patients had significantly reduced odds of receiving a score of 2 (emergency) (odds ratio [OR], 0.4; 95% confidence interval [CI], 0.33-0.49) or 3 (urgent) (OR, 0.5; 95% CI, 0.45-0.56) and significantly higher odds of receiving a score of 5 (minor) (OR, 1.34; 95% CI, 1.07-1.69) versus a score of 4 (nonurgent). We did not find a consistent disparity in Emergency Severity Index scores when comparing English- and non-English-speaking patients. CONCLUSIONS: We confirm that non-White patients receive lower triage scores than White patients. A more robust tool is required to account for illness severity and will be critical to understanding whether the relationship we describe reflects bias within the triage system or differences in ED utilization by racial groups.

The Upper-Arm Basilic-Cephalic Loop: A Valueable Alternative for Below-Knee Arterial Reconstruction.

INTRODUCTION: Despite advances of endovascular interventions, bypass surgery remains the gold standard for treatment of long and complex arterial occlusions in the lower limb. Autologous vein is regarded superior to other options. As the graft of first choice, the great saphenous vein (GSV) is often not available due to previous bypass, stripping or poor quality. Other options like arm veins (AV) are important alternatives. As forearm portions of AVs are often unusable, a graft created from the upper arm basilic and cephalic veins provides a valuable alternative. PATIENTS AND METHODS: We analyzed consecutive patients treated at an academic tertiary referral center between 01/1998 and 07/2018 using arm veins as the main peripheral bypass graft. Study endpoints were primary patency, secondary patency, limb salvage and survival. RESULTS: In the observed time period 2702 bypass procedures were performed at our institution for below-knee arterial reconstructions. Vein grafts used included the ipsilateral GSV (iGSV; n = 1937/71.7%), contralateral GSV (cGSV; 192/7.1%), small saphenous vein (SSV; 133/4.9%), prosthetic conduits (61/2.3%) and different configurations of AV (379/14%). In the majority of patients receiving AV grafts a complete continuous cephalic or basilic vein (CAV) was used (n = 292/77%). If it was not possible to use major parts of these 2 veins, either spliced arm vein grafts (SAV) (42/11%) or an upper arm basilic-cephalic loop graft (45/12%) were used. Median follow-up was 27 (interquartile range: 8-50) months. After 3 years secondary patency (CAV: 85%; SAV: 62%; loop: 66%; p = 0.125) and limb salvage rates (CAV: 79%, SAV: 68%; loop: 79%; p = 0.346) were similar between the 3 bypass options. CONCLUSION: The encouraging results of alternative AV configurations highlight their value in case the basilic or cephalic veins are not useable in continuity. Especially for infragenual redo-bypass procedures, these techniques should be considered to offer patients durable revascularization options.

Arm Vein versus Small Saphenous Vein for Lower Extremity Bypass in the Absence of Both Great Saphenous Veins.

BACKGROUND: Bypass surgery remains the gold standard for long and complex arterial occlusions in the lower limb. The vein is regarded superior to prosthetic conduits in peripheral arterial bypass surgery. However, this option is often limited because of previous bypass, stripping, or poor quality of the ipsilateral and/or contralateral great saphenous vein (GSV). Under these circumstances, the arm vein (AV) and small saphenous vein (SSV) are the only alternative autologous vein grafts. METHODS: We analyzed all consecutive patients treated at an academic tertiary referral center between January 1998 and July 2018 using either the AV or SSV as the main peripheral bypass graft. Study end points were primary patency, secondary patency, limb salvage, and survival. RESULTS: In total, 416 bypass procedures using exclusively AV (n = 327) or SSV (n = 89) were performed. There was a predominance of male gender. The majority of risk factors were evenly distributed between groups. The mean follow-up period was 2.3 years (0.9 to 13.3 years). Five-year primary and secondary patency rates were 39% (95% CI: 31-47%) and 67% (59-75%) for AV and 53% (41-66%) and 76% (67-86%) for SSV, respectively (P = 0.2 and 0.25). The five-year limb salvage and survival rates were 71% (68-81%) and 84% (77-90%) for AV and 78% (67-88%) and 90% (82-98%) for SSV, respectively (P = 0.52 and 0.11). CONCLUSIONS: Both AV and SSV are equally effective alternatives for peripheral bypass if no GSV is available. Although there was a trend toward better results with the SSV, there was no significant difference between the 2 options.

Gqα/G11α deficiency in dorsomedial hypothalamus leads to obesity resulting from decreased energy expenditure and impaired sympathetic nerve activity.

The G-protein subunits Gqα and G11α (Gq/11α) couple receptors to phospholipase C, leading to increased intracellular calcium. In this study we investigated the consequences of Gq/11α deficiency in the dorsomedial hypothalamus (DMH), a critical site for the control of energy homeostasis. Mice with DMH-specific deletion of Gq/11α (DMHGq/11KO) were generated by stereotaxic injection of adeno-associated virus (AAV)-Cre-green fluorescent protein (GFP) into the DMH of Gqαflox/flox:G11α-/- mice. Compared with control mice that received DMH injection of AAV-GFP, DMHGq/11KO mice developed obesity associated with reduced energy expenditure without significant changes in food intake or physical activity. DMHGq/11KO mice showed no defects in the ability of the melanocortin agonist melanotan II to acutely stimulate energy expenditure or to inhibit food intake. At room temperature (22°C), DMHGq/11KO mice showed reduced sympathetic nervous system activity in brown adipose tissue (BAT) and heart, accompanied with decreased basal BAT uncoupling protein 1 (Ucp1) gene expression and lower heart rates. These mice were cold intolerant when acutely exposed to cold (6°C for 5 h) and had decreased cold-stimulated BAT Ucp1 gene expression. DMHGq/11KO mice also failed to adapt to gradually declining ambient temperatures and to develop adipocyte browning in inguinal white adipose tissue although their BAT Ucp1 was proportionally stimulated. Consistent with impaired cold-induced thermogenesis, the onset of obesity in DMHGq/11KO mice was significantly delayed when housed under thermoneutral conditions (30°C). Thus our results show that Gqα and G11α in the DMH are required for the control of energy homeostasis by stimulating energy expenditure and thermoregulation.NEW & NOTEWORTHY This paper demonstrates that signaling within the dorsomedial hypothalamus via the G proteins Gqα and G11α, which couple cell surface receptors to the stimulation of phospholipase C, is critical for regulation of energy expenditure, thermoregulation by brown adipose tissue and the induction of white adipose tissue browning.

Alternative Venous Conduits for Below Knee Bypass in the Absence of Ipsilateral Great Saphenous Vein.

OBJECTIVE: Vein is regarded superior to artificial graft in peripheral arterial bypass surgery. However, this option is often limited owing to previous use or removal of the ipsilateral greater saphenous vein (iGSV). In this case, the contralateral great saphenous vein (cGSV), the small saphenous vein (SSV), or arm veins (AV) are possible alternatives. Experience with all three grafts for below knee vein bypass is reported. METHODS: Consecutive patients treated at an academic tertiary referral centre between January 1998 and July 2018 using the cGSV, SSV, or AV as the main peripheral bypass graft were analysed. Study end points were primary patency, secondary patency, limb salvage, and survival. RESULTS: Over the observed time period, 2642 bypass operations for treatment of peripheral artery disease with below knee target arteries were performed at the authors' institution: 1937 procedures using the iGSV; 644 bypass procedures using the cGSV (n = 186; 28.9%), SSV (n = 101; 15.7%), or AV (n = 357; 55.4%); and 61 procedures using a prosthetic graft. The median follow up period was 2.3 years (range 9 days-18.5 years). Thirty day mortality was 1.9% for the whole group and similar between the three groups. After five years, primary and secondary patency rates were comparable between the three groups. Secondary patency was 75% (95% confidence interval [CI] 66-83) in the cGSV and SSV groups, and 65% (95% CI 57-73) in the AV group (p = .47). Limb salvage and survival after five years were, respectively, 73% (95% CI 65-81) and 89% (95% CI 82-95) in the cGSV group, 79% (95% CI 69-89) and 87% (95% CI 79-95) in the SSV group, and 74% (95% CI 68-80) and 83% (77-89) in the AV group (p = .46). CONCLUSION: All three types of alternative autologous vein graft are equal regarding outcome parameters. Vascular surgeons should consider all autologous options if their preferred choice is not available.

The TOTEM RRMS (Testosterone Treatment on neuroprotection and Myelin Repair in Relapsing Remitting Multiple Sclerosis) trial: study protocol for a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Central nervous system damage in multiple sclerosis (MS) is responsible for serious deficiencies. Current therapies are focused on the treatment of inflammation; however, there is an urgent need for innovative therapies promoting neuroregeneration, particularly myelin repair. It is demonstrated that testosterone can act through neural androgen receptors and several clinical observations stimulated an interest in the potential protective effects of testosterone treatment for MS. Here, we sought to demonstrate the effects of a testosterone supplementation in testosterone-deficient men with relapsing-remitting MS. METHODS/DESIGN: This report presents the rationale and methodology of TOTEM RRMS, a French, phase 2, multicenter, randomized, placebo-controlled, and double-blind trial, which aims to prevent the progression of MS in men with low testosterone levels by administration of testosterone undecanoate, who were kept under natalizumab (Tysabri®) to overcome the anti-inflammatory effect of testosterone. Forty patients will be randomized into two groups receiving either a testosterone treatment (Nebido®) or a matching placebo. The intervention period for each group will last 66 weeks (treatment will be injected at baseline, week 6, and then every 12 weeks). The main objective is to determine the neuroprotective and remyelinating effects of testosterone using tensor diffusion imaging techniques and thalamic atrophy analyses. As secondary objectives, impacts of the testosterone supplementation will be studied using other conventional and unconventional MRI parameters and with clinical outcomes. DISCUSSION: The action of testosterone is observed in different experimental autoimmune encephalomyelitis models and epidemiological studies in humans. However, despite several preclinical data and some small clinical trials in MS, clear evidence for a therapeutic effect of hormone therapy is still missing. Therefore, our goal is to demonstrate the effects of testosterone therapies in MS. As there is no effective treatment currently available on fatigue in MS, careful attention should also be paid to secondary endpoints: fatigue, cognitive functions, and other symptoms that may improve life quality. Assuming a positive outcome of the trial, this treatment could be considered as a new neuroprotective and remyelinating therapy in relapsing-remitting MS and could be applicable to other demyelinating diseases. TRIAL REGISTRATION: ClinicalTrials.gov NCT03910738. Registered on 10 April 2019.

The Small Saphenous Vein: An Underestimated Source for Autologous Distal Vein Bypass.

OBJECTIVES: The small saphenous vein (SSV) is a potential vein source for bypass if neither greater saphenous vein nor arm vein is available. This study reports experience using SSV as part of an all autologous vein bypass policy. METHODS: This study comprised single centre retrospective data analysis of all consecutive patients treated at an academic tertiary referral centre from January 1998 to February 2017 using the SSV as the main peripheral bypass graft. Study endpoints were primary patency, secondary patency, limb salvage, and survival. RESULTS: One hundred and twenty operations were performed in 118 patients using SSV. Indications were peripheral arterial occlusive disease (n = 91; Rutherford classification 3: n = 11; 4: n = 21; 5,6: n = 59), acute ischaemia (n = 14), popliteal artery aneurysm (n = 12), and bypass revisions (n = 3). Median follow up was 30.5 months (10 months-13.7 years). Primary patency after one, three and five years was 68% (CI: 59-77%), 58% (49-68%), and 54% (45-64%). Secondary patency was 83% (76-89%) after one year and 77% (69-85%) after three and five years. Limb salvage after one year was 88% (82-94%) and 78% (70-86%) after five years. Survival was 96% (92-99%) after one year and 91% (85-97%) at five years. Multivariable analysis identified redo surgery as an independent risk factor. Patients receiving a primary (n = 59) vs. a redo bypass (n = 61) were compared. Primary patency and secondary patency were both significantly better in the primary bypass group than in the redo group (p = .0036 and p = .0003, respectively). Limb salvage was also significantly better in primary bypass patients than in the redo group (p = .0007), whereas overall survival did not differ significantly (p = .48). CONCLUSION: The SSV is a valuable alternative vein graft in peripheral bypass surgery. It achieves excellent long-term results, particularly in patients with primary procedures but also acceptable results in patients with redo surgery.

Grf10 and Bas1 Regulate Transcription of Adenylate and One-Carbon Biosynthesis Genes and Affect Virulence in the Human Fungal Pathogen Candida albicans.

Candida albicans is an opportunistic human fungal pathogen that causes superficial fungal infections and lethal systemic infections. To colonize and establish infections, C. albicans coordinates the expression of virulence and metabolic genes. Previous work showed that the homeodomain transcription factor Grf10 is required for formation of hyphae, a virulence factor. Here we report global gene expression analysis of a grf10Δ strain using a DNA microarray and identify genes for de novo adenylate biosynthesis (ADE genes), one-carbon metabolism, and a nucleoside permease (NUP). Upregulation of these genes in response to adenine limitation required both Grf10 and the myb protein Bas1, as shown by quantitative real-time PCR (qRT-PCR). Phenotypic analysis showed that both mutants exhibited growth defects when grown in the absence of adenine, and the doubling time was slower for the bas1Δ mutant. Bas1 is required for basal expression of these genes, whereas NUP expression is more dependent upon Grf10. Disruption of BAS1 led to only modest defects in hypha formation and weak attenuation of virulence in a systemic mouse model of infection, as opposed to the previously reported strong effects found in the grf10Δ mutant. Our data are consistent with a model in which Grf10 coordinates metabolic effects on nucleotide metabolism by interaction with Bas1 and indicate that AMP biosynthesis and its regulation are important for C. albicans growth and virulence. IMPORTANCECandida albicans is a commensal and a common constituent of the human microbiota; however, it can become pathogenic and cause infections in both immunocompetent and immunocompromised people. C. albicans exhibits remarkable metabolic versatility as it can colonize multiple body sites as a commensal or pathogen. Understanding how C. albicans adapts metabolically to each ecological niche is essential for developing novel therapeutic approaches. Purine metabolism has been targeted pharmaceutically in several diseases; however, the regulation of this pathway has not been fully elucidated in C. albicans. Here, we report how C. albicans controls the AMP de novo biosynthesis pathway in response to purine availability. We show that the lack of the transcription factors Grf10 and Bas1 leads to purine metabolic dysfunction, and this dysfunction affects the ability of C. albicans to establish infections.

Flow Cytometry-Based Assessment of Antibody Function Against Malaria Pre-erythrocytic Infection.

The development of new interventional strategies against pre-erythrocytic malaria is hampered by the lack of standardized approaches to assess inhibition of sporozoite infection of hepatocytes. The following methodology, based on flow cytometry, can be used to quantitatively assess P. falciparum sporozoite infection in vitro in medium throughput. In addition to assessing the efficacy of antibodies, this assay has a wide variety of applications for investigating basic science questions about the malaria liver stage. This approach is easily applied in a variety of laboratory settings, assesses the functionality of antibody responses against malaria sporozoites, and can be adapted for the limited quantities of sample which are typically available from clinical investigations.

[Trocar site herniation (TSH) following laparoscopic cholecystectomy: incidence, pathogenesis, and prevention -- animal study]. / A trokárok helyén kialakult hasfali sérvekrol (Trocar Site Hernia) laparoscopos cholecystectomia után: incidencia és megelozés -- állatkísérletes tanulmány.

INTRODUCTION: In 1968 R. E. Fear first reported a trocar site hernia (TSH) in his large series on laparoscopy. Currently, the incidence of TSH is estimated to be 0.65-2.80%. Ports ≥10-mm are usually closed, but ports of the 5-mm trocars are always left open, which may lead to herniation. MATERIAL AND METHODS: Authors guided teaching courses for hands-on animal laparoscopic cholecystectomy (LC) operations, where trainees performed LC-s on 60 animals. Two and four weeks following the operations the animals underwent second look laparoscopy to detect adhesion formation. RESULTS: Trocar site herniation was observed, and in 20% of the animals herniation was found. 70% of the hernias were situated in the 5-mm ports and 30% in the 10-mm ports. CONCLUSION: Port sites should be closed to prevent the formation of TSH. Attention should be payed on the closure of 5-mm trocar sites as well.

Suppression of host p53 is critical for Plasmodium liver-stage infection.

Plasmodium parasites infect the liver and replicate inside hepatocytes before they invade erythrocytes and trigger clinical malaria. Analysis of host signaling pathways affected by liver-stage infection could provide critical insights into host-pathogen interactions and reveal targets for intervention. Using protein lysate microarrays, we found that Plasmodium yoelii rodent malaria parasites perturb hepatocyte regulatory pathways involved in cell survival, proliferation, and autophagy. Notably, the prodeath protein p53 was substantially decreased in infected hepatocytes, suggesting that it could be targeted by the parasite to foster survival. Indeed, mice that express increased levels of p53 showed reduced liver-stage parasite burden, whereas p53 knockout mice suffered increased liver-stage burden. Furthermore, boosting p53 levels with the use of the small molecule Nutlin-3 dramatically reduced liver-stage burden in vitro and in vivo. We conclude that perturbation of the hepatocyte p53 pathway critically impacts parasite survival. Thus, host pathways might constitute potential targets for host-based antimalarial prophylaxis.

A rapid and scalable density gradient purification method for Plasmodium sporozoites.

BACKGROUND: Malaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes. METHODS: Here is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants. RESULTS: This method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax) and rodent (Plasmodium yoelii) infective species with excellent recovery rates. CONCLUSIONS: This novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.