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Monkeypox virus (MPXV) resides in two forms; mature and enveloped, and depending on it, distinct proteins are displayed on the viral surface. Here, we expressed two MPXV antigens from the mature, and one from the enveloped form, and tested their reactivity to sera of 11 MPXV recoverees while comparing to sera from recently and past vaccinated individuals. 8 out of 11 recoverees exhibited detectable neutralization levels against Vaccinia Lister. Sera from all recoverees bound strongly to A35R and H3L antigens. Moreover, the responses to A35R were significantly higher within the recoverees compared to both recently and past vaccinated donors. Lastly, A35R- and H3L-specific IgG + B cells ranging from 0.03-0.46% and 0.11-0.36%, respectively, were detected in all recoverees (A35R), and in 9 out of 11 recoverees (H3L). Therefore, A35R and H3L represent MPXV immune targets and could be used in a heat-inactivated serological ELISA for the identification of recent MPXV infection., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s).)

Fundamental key processes in viral infection cycles generally occur in distinct cellular sites where both viral and host factors accumulate and interact. These sites are usually termed viral replication organelles, or viral factories (VF). The generation of VF is accompanied by the synthesis of viral proteins and genomes and involves the reorganization of cellular structure. Recently, rVSV-ΔG-spike (VSV-S), a recombinant VSV expressing the SARS-CoV-2 spike protein, was developed as a vaccine candidate against SARS-CoV-2. By combining transmission electron microscopy (TEM) tomography studies and immuno-labeling techniques, we investigated the infection cycle of VSV-S in Vero E6 cells. RT-real-time-PCR results show that viral RNA synthesis occurs 3-4 h post infection (PI), and accumulates as the infection proceeds. By 10-24 h PI, TEM electron tomography results show that VSV-S generates VF in multi-lamellar bodies located in the cytoplasm. The VF consists of virus particles with various morphologies. We demonstrate that VSV-S infection is associated with accumulation of cytoplasmatic viral proteins co-localized with dsRNA (marker for RNA replication) but not with ER membranes. Newly formed virus particles released from the multi-lamellar bodies containing VF, concentrate in a vacuole membrane, and the infection ends with the budding of particles after the fusion of the vacuole membrane with the plasma membrane. In summary, the current study describes detailed 3D imaging of key processes during the VSV-S infection cycle.

SARS-CoV-2 is evolving with increased transmission, host range, pathogenicity, and virulence. The original and mutant viruses escape host innate (Interferon) immunity and adaptive (Antibody) immunity, emphasizing unmet needs for high-yield, commercial-scale manufacturing to produce inexpensive vaccines/boosters for global/equitable distribution. We developed DYAI-100A85, a SARS-CoV-2 spike receptor binding domain (RBD) subunit antigen vaccine expressed in genetically modified thermophilic filamentous fungus, Thermothelomyces heterothallica C1, and secreted at high levels into fermentation medium. The RBD-C-tag antigen strongly binds ACE2 receptors in vitro. Alhydrogel ® '85'-adjuvanted RDB-C-tag-based vaccine candidate (DYAI-100A85) demonstrates strong immunogenicity, and antiviral efficacy, including in vivo protection against lethal intranasal SARS-CoV-2 (D614G) challenge in human ACE2-transgenic mice. No loss of body weight or adverse events occurred. DYAI-100A85 also demonstrates excellent safety profile in repeat-dose GLP toxicity study. In summary, subcutaneous prime/boost DYAI-100A85 inoculation induces high titers of RBD-specific neutralizing antibodies and protection of hACE2-transgenic mice against lethal challenge with SARS-CoV-2. Given its demonstrated safety, efficacy, and low production cost, vaccine candidate DYAI-100 received regulatory approval to initiate a Phase 1 clinical trial to demonstrate its safety and efficacy in humans.

Blocking the interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with its angiotensin-converting enzyme 2 (ACE2) receptor was proved to be an effective therapeutic option. Various protein binders as well as monoclonal antibodies that effectively target the receptor-binding domain (RBD) of SARS-CoV-2 to prevent interaction with ACE2 were developed. The emergence of SARS-CoV-2 variants that accumulate alterations in the RBD can severely affect the efficacy of such immunotherapeutic agents, as is indeed the case with Omicron that resists many of the previously isolated monoclonal antibodies. Here, we evaluate an ACE2-based immunoadhesin that we have developed early in the pandemic against some of the recent variants of concern (VoCs), including the Delta and the Omicron variants. We show that our ACE2-immunoadhesin remains effective in neutralizing these variants, suggesting that immunoadhesin-based immunotherapy is less prone to escape by the virus and has a potential to remain effective against future VoCs., Competing Interests: The Weizmann Institute has filed for a patent for the ACE2mod-Fc immunoadhesin., (© 2022 The Authors.)

The current monkeypox virus global spread and lack of data regarding clinical specimens' infectivity call for examining virus infectivity, and whether this correlates with results from PCR, the available diagnostic tool. We show strong correlation between viral DNA amount in clinical specimens and virus infectivity toward BSC-1 cell line. Moreover, we define a PCR threshold value (Cq ≥ 35, ≤ 4,300 DNA copies/mL), corresponding to negative viral cultures, which may assist risk-assessment and decision-making regarding protective-measures and guidelines for patients with monkeypox.

BriLife ® , a vector-based vaccine that utilizes the recombinant vesicular stomatitis virus (VSV) platform to express and present the spike antigen of SARS-CoV-2, is undergoing testing in a phase 2 clinical trial in Israel. A nonclinical repeated-dose (GLP) toxicity study in New Zealand white rabbits was performed to evaluate the potential toxicity, local tolerance, immunogenicity and biodistribution of the vaccine. rVSV-ΔG-SARS-CoV-2-S (or vehicle) was administered intramuscularly to two groups of animals (10 6 , 10 7 PFU/animal, n = 10/sex/group) on three occasions, at 2-week intervals, followed by a 3-week recovery period. Systemic clinical signs, local reactions, body weight, body temperature, food consumption, ophthalmology, urinalysis, clinical pathology, C-reactive protein, viremia and antibody levels were monitored. Gross pathology was performed, followed by organs/tissues collection for biodistribution and histopathological evaluation. Treatment-related changes were restricted to multifocal minimal myofiber necrosis at the injection sites, and increased lymphocytic cellularity in the iliac and mesenteric lymph nodes and in the spleen. These changes were considered related to the inflammatory reaction elicited, and correlated with a trend for recovery. Detection of rVSV-ΔG-SARS-CoV-2-S vaccine RNA was noted in the regional iliac lymph node in animals assigned to the high-dose group, at both termination time points. A significant increase in binding and neutralizing antibody titers was observed following vaccination at both vaccine doses. In view of the findings, it was concluded that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe. These results supported the initiation of clinical trials., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to shutoff of protein synthesis, and nsp1, a central shutoff factor in coronaviruses, inhibits cellular mRNA translation. However, the diverse molecular mechanisms employed by nsp1 as well as its functional importance are unresolved. By overexpressing various nsp1 mutants and generating a SARS-CoV-2 mutant, we show that nsp1, through inhibition of translation and induction of mRNA degradation, targets translated cellular mRNA and is the main driver of host shutoff during infection. The propagation of nsp1 mutant virus is inhibited exclusively in cells with intact interferon (IFN) pathway as well as in vivo, in hamsters, and this attenuation is associated with stronger induction of type I IFN response. Therefore, although nsp1's shutoff activity is broad, it plays an essential role, specifically in counteracting the IFN response. Overall, our results reveal the multifaceted approach nsp1 uses to shut off cellular protein synthesis and uncover nsp1's explicit role in blocking the IFN response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 10 8 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The emergence of rapidly spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major challenge to the ability of vaccines and therapeutic antibodies to provide immunity. These variants contain mutations of specific amino acids that might impede vaccine efficacy. BriLife ® (rVSV-ΔG-spike) is a newly developed SARS-CoV-2 vaccine candidate currently in phase II clinical trials. It is based on a replication-competent vesicular stomatitis virus (VSV) platform. The rVSV-ΔG-spike contains several spontaneously acquired spike mutations that correspond to SARS-CoV-2 variants' mutations. We show that human sera from BriLife ® vaccinees preserve comparable neutralization titers towards alpha, gamma, and delta variants and show less than a three-fold reduction in the neutralization capacity of beta and omicron compared to the original virus. Taken together, we show that human sera from BriLife ® vaccinees overall maintain a neutralizing antibody response against all tested variants. We suggest that BriLife ® -acquired mutations may prove advantageous against future SARS-CoV-2 VOCs.

SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The continued spread of SARS-CoV-2 increases the probability of influenza/SARS-CoV-2 coinfection, which may result in severe disease. In this study, we examine the disease outcome of influenza A virus (IAV) and SARS-CoV-2 coinfection in K18-hACE2 mice. Our data indicate enhance susceptibility of IAV-infected mice to developing severe disease upon coinfection with SARS-CoV-2 two days later. In contrast to nonfatal influenza and lower mortality rates due to SARS-CoV-2 alone, this coinfection results in severe morbidity and nearly complete mortality. Coinfection is associated with elevated influenza viral loads in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevents severe disease and mortality. This protection is antibody-dependent. These data experimentally support the necessity of seasonal influenza vaccination for reducing the risk of severe influenza/COVID-19 comorbidity during the COVID-19 pandemic., (© 2021. The Author(s).)

The COVID-19 pandemic initiated a worldwide race toward the development of treatments and vaccines. Small animal models included the Syrian golden hamster and the K18-hACE2 mice infected with SARS-CoV-2 to display a disease state with some aspects of human COVID-19. A group activity of animals in their home cage continuously monitored by the HCMS100 (Home cage Monitoring System 100) was used as a sensitive marker of disease, successfully detecting morbidity symptoms of SARS-CoV-2 infection in hamsters and in K18-hACE2 mice. COVID-19 convalescent hamsters rechallenged with SARS-CoV-2 exhibited minor reduction in group activity compared to naive hamsters. To evaluate the rVSV-ΔG-spike vaccination efficacy against SARS-CoV-2, we used the HCMS100 to monitor the group activity of hamsters in their home cage. A single-dose rVSV-ΔG-spike vaccination of the immunized group showed a faster recovery than the nonimmunized infected hamsters, substantiating the efficacy of rVSV-ΔG-spike vaccine. HCMS100 offers nonintrusive, hands-free monitoring of a number of home cages of hamsters or mice modeling COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Melamed, Politi, Grauer, Achdout, Aftalion, Gur, Tamir, Yahalom-Ronen, Maimon, Yitzhak, Weiss, Rosner, Erez, Yitzhaki, Shapira, Paran, Mamroud, Vagima and Israely.)

A wide range of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing monoclonal antibodies (mAbs) have been reported, most of which target the spike glycoprotein. Therapeutic implementation of these antibodies has been challenged by emerging SARS-CoV-2 variants harboring mutated spike versions. Consequently, re-assessment of previously identified mAbs is of high priority. Four previously selected mAbs targeting non-overlapping epitopes are now evaluated for binding potency to mutated RBD versions, reported to mediate escape from antibody neutralization. In vitro neutralization potencies of these mAbs, and two NTD-specific mAbs, are evaluated against two frequent SARS-CoV-2 variants of concern, the B.1.1.7 Alpha and the B.1.351 Beta. Furthermore, we demonstrate therapeutic potential of three selected mAbs by treatment of K18-human angiotensin-converting enzyme 2 (hACE2) transgenic mice 2 days post-infection with each virus variant. Thus, despite the accumulation of spike mutations, the highly potent MD65 and BL6 mAbs retain their ability to bind the prevalent viral mutants, effectively protecting against B.1.1.7 and B.1.351 variants., Competing Interests: Declaration of interests Patent application for the described antibodies was filed by the Israel Institute for Biological Research. None of the authors declared any additional competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causal agent of COVID-19 and stands at the center of the current global human pandemic, with death toll exceeding one million. The urgent need for a vaccine has led to the development of various immunization approaches. mRNA vaccines represent a cell-free, simple, and rapid platform for immunization, and therefore have been employed in recent studies toward the development of a SARS-CoV-2 vaccine. Herein, we present the design of an mRNA vaccine, based on lipid nanoparticles (LNPs)-encapsulated SARS-CoV-2 human Fc-conjugated receptor-binding domain (RBD-hFc). Several ionizable lipids have been evaluated in vivo in a luciferase (luc) mRNA reporter assay, and two leading LNPs formulations have been chosen for the subsequent RBD-hFc mRNA vaccine strategy. Intramuscular administration of LNP RBD-hFc mRNA elicited robust humoral response, a high level of neutralizing antibodies and a Th1-biased cellular response in BALB/c mice. The data in the current study demonstrate the potential of these lipids as promising candidates for LNP-based mRNA vaccines in general and for a COVID19 vaccine in particular.

The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19 1 . Coronaviruses have developed a variety of mechanisms to repress host mRNA translation to allow the translation of viral mRNA, and concomitantly block the cellular innate immune response 2,3 . Although several different proteins of SARS-CoV-2 have previously been implicated in shutting off host expression 4-7 , a comprehensive picture of the effects of SARS-CoV-2 infection on cellular gene expression is lacking. Here we combine RNA sequencing, ribosome profiling and metabolic labelling of newly synthesized RNA to comprehensively define the mechanisms that are used by SARS-CoV-2 to shut off cellular protein synthesis. We show that infection leads to a global reduction in translation, but that viral transcripts are not preferentially translated. Instead, we find that infection leads to the accelerated degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. We reveal that the translation of transcripts that are induced in response to infection (including innate immune genes) is impaired. We demonstrate this impairment is probably mediated by inhibition of nuclear mRNA export, which prevents newly transcribed cellular mRNA from accessing ribosomes. Overall, our results uncover a multipronged strategy that is used by SARS-CoV-2 to take over the translation machinery and to suppress host defences.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic 1 . To understand the pathogenicity and antigenic potential of SARS-CoV-2 and to develop therapeutic tools, it is essential to profile the full repertoire of its expressed proteins. The current map of SARS-CoV-2 coding capacity is based on computational predictions and relies on homology with other coronaviruses. As the protein complement varies among coronaviruses, especially in regard to the variety of accessory proteins, it is crucial to characterize the specific range of SARS-CoV-2 proteins in an unbiased and open-ended manner. Here, using a suite of ribosome-profiling techniques 2-4 , we present a high-resolution map of coding regions in the SARS-CoV-2 genome, which enables us to accurately quantify the expression of canonical viral open reading frames (ORFs) and to identify 23 unannotated viral ORFs. These ORFs include upstream ORFs that are likely to have a regulatory role, several in-frame internal ORFs within existing ORFs, resulting in N-terminally truncated products, as well as internal out-of-frame ORFs, which generate novel polypeptides. We further show that viral mRNAs are not translated more efficiently than host mRNAs; instead, virus translation dominates host translation because of the high levels of viral transcripts. Our work provides a resource that will form the basis of future functional studies.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The COVID-19 pandemic caused by SARS-CoV-2 imposes an urgent need for rapid development of an efficient and cost-effective vaccine, suitable for mass immunization. Here, we show the development of a replication competent recombinant VSV-∆G-spike vaccine, in which the glycoprotein of VSV is replaced by the spike protein of SARS-CoV-2. In-vitro characterization of this vaccine indicates the expression and presentation of the spike protein on the viral membrane with antigenic similarity to SARS-CoV-2. A golden Syrian hamster in-vivo model for COVID-19 is implemented. We show that a single-dose vaccination results in a rapid and potent induction of SARS-CoV-2 neutralizing antibodies. Importantly, vaccination protects hamsters against SARS-CoV-2 challenge, as demonstrated by the abrogation of body weight loss, and  alleviation of the extensive tissue damage and viral loads in lungs and nasal turbinates. Taken together, we suggest the recombinant VSV-∆G-spike as a safe, efficacious and protective vaccine against SARS-CoV-2.

Objectives: Environmental surfaces have been suggested as likely contributors in the transmission of COVID-19. This study assessed the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contaminating surfaces and objects in two hospital isolation units and a quarantine hotel., Methods: SARS-CoV-2 virus stability and infectivity on non-porous surfaces was tested under controlled laboratory conditions. Surface and air sampling were conducted at two COVID-19 isolation units and in a quarantine hotel. Viral RNA was detected by RT-PCR and infectivity was assessed by VERO E6 CPE test., Results: In laboratory-controlled conditions, SARS-CoV-2 gradually lost its infectivity completely by day 4 at ambient temperature, and the decay rate of viral viability on surfaces directly correlated with increase in temperature. Viral RNA was detected in 29/55 surface samples (52.7%) and 16/42 surface samples (38%) from the surroundings of symptomatic COVID-19 patients in isolation units of two hospitals and in a quarantine hotel for asymptomatic and very mild COVID-19 patients. None of the surface and air samples from the three sites (0/97) were found to contain infectious titres of SARS-Cov-2 on tissue culture assay., Conclusions: Despite prolonged viability of SARS-CoV-2 under laboratory-controlled conditions, uncultivable viral contamination of inanimate surfaces might suggest low feasibility for indirect fomite transmission., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD. A subset of the antibodies exert their inhibitory activity by abrogating binding of the RBD to the human ACE2 receptor. The human monoclonal antibodies described here represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.

Objectives: Adolescent females in sub-Saharan Africa bear a disproportionate burden of new HIV infections but have been excluded from prognostic research, such as developed risk calculators. This study examines whether validated risk calculators, which calculate HIV risk among sub-Saharan African women, can be modified to assess HIV risk among adolescent girls. The performance of selected risk variables from validated calculators and the literature was evaluated among adolescent females using modern advanced statistical tools., Methods: Risk variables for the updated tool were selected from the CAPRISA 007 (CAP007) trial (2010-2012) questionnaires. An initially HIV-seronegative cohort of rural South African female high school students (n=1049) aged 14-25 years was selected. The number and characteristics of latent factors, or dimensions, underlying selected variables were assessed using exploratory factor analysis (EFA). The updated tool's effectiveness identifying trends in adolescent risk were assessed with latent class analysis (LCA)., Results: EFA identified two key latent factors: sexual behaviour and socioeconomic risk factors. Latent sexual behaviour risk influenced contraception use (0.883), perceived HIV risk (0.691) and pregnancy (-0.384). Latent socioeconomic risk influenced low HIV knowledge (0.371), financial dependence (0.532), prior HIV testing (-0.379) and alcohol use (-0.332). Using LCA, three underlying categories of adolescent females were identified: those with no, low and high risk of HIV (1.10%, 2.26% and 2.91% 1-year seroconversion rates, respectively). Herpes simplex virus serotype-2, condom contraception, alcohol use, pregnancy and age were significantly associated with higher risk class membership, while non-condom contraception was associated with lower risk class membership., Conclusions: Adolescent females are at unequal risk of acquiring HIV. Findings suggest the updated tool captures two main facets of adolescent characteristics and may identify differential risk. This work supports further investigation to inform development of targeted differentiated interventions and efficient prognostic tools for adolescents in high-risk settings., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To compare surgical site infections (SSI) rate after radical cystectomy (RC) over time and ascertain whether antibiotic prophylaxis should be enhanced., Methods: All medical records of RC patients in a single tertiary uro-oncology center between 2007 and 2017 were analyzed. SSI was defined using the criteria of the US Centers for Disease Control and Prevention. All bacterial culture results and antimicrobial resistance rates were recorded. Lastly, multivariable logistic regression analysis was performed to ascertain SSI predictors., Results: RC was performed in 405 patients, of which 96 (23.7%) developed SSI. No differences were demonstrated in the mean age, gender, NIDDM prevalence, neoadjuvant chemotherapy, positive preoperative urine culture, bowel preparation, and surgery time between both groups. However, statistically significant higher median BMI, age-adjusted Charlson Comorbidity score, usage of ceftriaxone preoperatively, and intensive care unit (ICU) hospitalization were noted in SSI patients. Overall, 62/96 (63.5%) SSI patients had a positive wound culture, with only 16.7% of the pathogens being sensitive to their perioperative antibiotics. Lastly, on multivariable analysis rising BMI, preoperative ceftriaxone and ICU hospitalization were associated with a higher SSI rate., Conclusions: Preoperative BMI reduction, and maximal preoperative medical optimization in an attempt to lower ICU admittance rates, should be part of the ideal strategy for lowering SSI rates. Additionally, preoperative antibiotics should be enhanced to harbor-wide spectrum coverage, based on local resistance rates.

We report a case of monkeypox in a man who returned from Nigeria to Israel in 2018. Virus was detected in pustule swabs by transmission electron microscopy and PCR and confirmed by immunofluorescence assay, tissue culture, and ELISA. The West Africa monkeypox outbreak calls for increased awareness by public health authorities worldwide.

Viral infections of the central nervous system (CNS) lead to a broad range of pathologies. CNS infections with Orthopox viruses have been mainly documented as an adverse reaction to smallpox vaccination with vaccinia virus. To date, there is insufficient data regarding the mechanisms underlying pathological viral replication or viral clearance. Therefore, informed risk assessment of vaccine adverse reactions or outcome prediction is limited. This work applied a model of viral infection of the CNS, comparing neurovirulent with attenuated strains. We followed various parameters along the disease and correlated viral load, morbidity, and mortality with tissue integrity, innate and adaptive immune response and functionality of the blood⁻brain barrier. Combining these data with whole brain RNA-seq analysis performed at different time points indicated that neurovirulence is associated with host immune silencing followed by induction of tissue damage-specific pathways. In contrast, brain infection with attenuated strains resulted in rapid and robust induction of innate and adaptive protective immunity, followed by viral clearance and recovery. This study significantly improves our understanding of the mechanisms and processes determining the consequence of viral CNS infection and highlights potential biomarkers associated with such outcomes., Competing Interests: The authors declare no conflict of interest.

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-κB (NF-kB) and inhibitor κB (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Many types of crops are severely affected by at least one important bacterial disease. Chemical control of bacterial plant diseases in the field vastly relies on copper-based bactericides, yet with limited efficacy. In this study, we explored the potential of two random peptide mixture (RPM) models as novel crop protection agents. These unique peptide mixtures consist of random combination of l-phenylalanine and l- or d-lysine (FK-20 and FdK-20, respectively) along the 20 mer chain length of the peptides. Both RPMs displayed powerful bacteriostatic and bactericidal activities towards strains belonging to several plant pathogenic bacterial genera, for example, Xanthomonas, Clavibacter and Pseudomonas. In planta studies in the glasshouse revealed that RPMs significantly reduced disease severity of tomato and kohlrabi plants infected with Xanthomonas perforans and Xanthomonas campestris pv. campestris respectively. Moreover, RPM effects on reduction in disease severity were similar to those exerted by the commercial copper-based bactericide Kocide 2000 that was applied at a 12-fold higher concentration of the active compound relative to the RPM treatments. Importantly, the two tested RPM compounds had no toxic effect on survival of bees and Caco-2 mammalian cells. This study demonstrates the potential of these innovative RPMs to serve as crop protection agents against crop diseases caused by phytopathogenic bacteria., (© 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Objectives: Ultrasound (US) is an important modality for the detection of acute appendicitis in children but has limited sensitivity and specificity. Therefore, additional US findings may contribute to the diagnosis. In our experience, children with acute appendicitis often have increased renal cortical echogenicity on US imaging. The purpose of this study was to examine the association of increased renal cortical echogenicity with appendicitis., Methods: This study included 240 consecutive pediatric patients with no renal or liver disease who underwent US examinations for suspected appendicitis between February 2014 and January 2016. Ultrasound images of the liver and right kidney were retrospectively reviewed, and the echogenicity of the renal cortex was classified as less than the liver, equal to the liver, or greater than the liver., Results: The renal cortex was abnormally hyperechoic in 38 (50%) of all of the patients who had appendicitis according to US (P < .001) and in 47% of patients who underwent appendectomy (P = .002). Overall, 36% of patients with increased renal cortical echogenicity had a diagnosis of appendicitis. After correction for variables, patients with renal hyperechogenicity had a 2.5 times chance of appendicitis (odds ratio, 2.5)., Conclusions: There is a statistically significant association between increased renal cortical echogenicity and appendicitis. In the absence of hepatic or renal disease, this finding may increase the accuracy of the US diagnosis of appendicitis. Increased renal cortical echogenicity may be added to the list of US findings accompanying acute appendicitis in children., (© 2017 by the American Institute of Ultrasound in Medicine.)

With nearly one million HIV-infected women in India, prevention of mother-to-child transmission (PMTCT) programs serve an important role. While PMTCT programs focus on mothers living with HIV infection, offering them to uninfected pregnant women may prevent maternal HIV infections. To inform future efforts to offer PMTCT programs to uninfected women, we conducted focus groups with 24 uninfected women in the South Indian state of Karnataka who had given birth within the previous 2 years to explore their perceptions and experiences about HIV education and screening during pregnancy. Although all the participants had undergone HIV testing at a public health facility during pregnancy, they reported little knowledge about HIV transmission or prevention. Revisions are needed in existing PMTCT program curricula and instruction methods before they can be offered to uninfected women as an HIV prevention strategy., (Copyright © 2017 Association of Nurses in AIDS Care. Published by Elsevier Inc. All rights reserved.)

Eczema vaccinatum is a severe and occasionally lethal complication of smallpox vaccine, characterized by systemic viral dissemination, distant from the initial inoculation site of the vaccine. A major risk factor for eczema vaccinatum is a background of atopic dermatitis, a chronic, common allergic, relapsing disorder, manifested by dry and inflamed skin, itchy rash, Th2 biased immune response and hypersensitivity to various antigens. Unlike the severe manifestations of eczema vaccinatum in humans, current models present only mild symptoms that limits examination of potential therapeutics for eczema vaccinatum. The atopic dermatitis and eczema vaccinatum models we present here, are the first to simulate the severity of the diseases in humans. Indeed, dermatitic mice display persistent severe dermatitis, characterized by dry and inflamed skin with barrier dysfunction, epidermal hyperplasia and significant elevation of serum IgE. By exposing atopic dermatitis mice to ectromelia virus, we generated eczema vaccinatum that mimic the human disease better than known eczema vaccinatum models. Similarly to humans, eczematous mice displayed enlarged and disseminated skin lesions, which correlated with elevated viral load. Cidofovir and antiviral antibodies conferred protection even when treatment started at a late eczematous stage. Moreover, we are the first to demonstrate that despite a severe background of atopic dermatitis, modified vaccinia Ankara virus (MVA) vaccination protects against lethal ectromelia virus exposure. We finally show that protection by MVA vaccination is dependent on CD4 + T cells and is associated with significant activation of CD8 + cytotoxic T cells and induction of humoral immunity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Little is known about the role of oxytocin (OT) in colostrum during early gut colonization. We previously showed that transient OT receptor (OTR) expression on newborn rat enterocytes coincides with the milk-suckling period, and that OT activates endoplasmic reticulum stress sensors in cultured enterocytes. Here, we explored whether colostrum-OT attenuates stress in newborn villi primed and unprimed by colostrum by measuring levels of stress markers including BiP (an ER chaperone), eIF2a (translation initiation factor), and pPKR (eIF2a kinase). We also measured two inflammation-signaling proteins NF-κB and its inhibitor IκB. To test the impact of colostrum on autophagy, we measured a marker of autophagy initiation, LC3A. Colostrum increased inactive p-eIF2a, p-PKR and IκB and reduced p-IκB, BiP and LC3A. LPS increased and OT decreased p-IkB. BiP (GRP78) was higher in unprimed than primed villi. Together, these data suggest that colostrum OT attenuates the impact of inflammation on postnatal gut villi and that OT enhances autophagy to protect against amino acid insufficiency-induced stress during the interval between birth and the first feeding., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Introduction: To identify factors influencing human papillomavirus (HPV) vaccination up taking decision making among vaccinated and nonvaccinated Hispanic college women., Participants: Hispanic young women between the ages of 18 and 24 years (N = 49). In total, 26 had not received the HPV vaccine, and 23 had started/completed the vaccine series., Method: Participants registered for the study via a psychology research pool at a large public university in the southeast United States after institutional review board approval. After completing a demographic information and HPV knowledge Web-based survey, participants were individually interviewed., Results: Differences in HPV vaccine knowledge emerged between vaccinated and nonvaccinated women. Fear of side effects, perceptions of risk, and sources of encouragement influenced willingness to be vaccinated against HPV. Health care providers played a central role in addressing concerns and promoting vaccination., Conclusions: Health care providers must address and integrate unique decision-making processes influencing Hispanic young adult women's perceptions of HPV vaccination., (© The Author(s) 2016.)

Background: The neuropeptide neuromodulator and hormone oxytocin (OT) activates signaling pathways involved in mRNA translation in response to endoplasmic reticulum stress and reduces inflammation associated with experimental colitis in rats. The anti-inflammatory effects of OT may serve a vital role in the development, survival and function of newborn-type enterocytes during microbial gut colonization, which coincides with the milk suckling period when OT receptor expression peaks in the gut. Furthermore, mice deficient in the OT receptor have abnormal gut structure and function, underscoring OT's developmental importance., Methods: We tested the effect of OT upon lipopolysaccharide (LPS)-induced markers of the inflammatory response in Caco2BB gut cells in vitro using automated immunocapillary electrophoresis., Results: We demonstrate that OT suppresses NF-κB signaling and presumably inflammatory transcriptional programs, which are unleashed by LPS through the modulation of IκB. We show that OT counteracts LPS-elicited silencing of the unfolded protein response, a pathway limiting endoplasmic reticulum stress by suppressing protein translation. OT selectively activates dsRNA-activated kinase (PKR), X-box binding protein 1 (XBP1), immunoglobulin binding protein (BiP), A20 (TNFα-induced protein 3) and inositol requiring enzyme 1a (IRE1a). OT inactivates eukaryotic translation initiation factor 2a (eIF2a) without significant activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK)., Conclusions: Mild, preemptive stimulation of endoplasmic reticulum stress sensors by OT may precondition newborn enterocytes to resist apoptosis associated with inflammation and may support their differentiation and development by modulating cellular metabolism., General Significance: OT may protect enterocytes and other cell types, such as neurons, from stress-related complications during postnatal development., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Modest antidepressant response rates of mood disorders (MD) encourage benzodiazepine (BZD) co-medication with debatable benefit. Adult hippocampal neurogenesis may underlie antidepressant responses, but diazepam co-administration impairs murine neuron maturation and survival in response to fluoxetine. We counted neural progenitor cells (NPCs), mitotic cells, and mature granule neurons post-mortem in dentate gyrus (DG) from subjects with: untreated Diagnostic and Statistical Manual of Mental Disorders (DSM) IV MD (n = 17); antidepressant-treated MD (MD*ADT, n = 10); benzodiazepine-antidepressant-treated MD (MD*ADT*BZD, n = 7); no psychopathology or treatment (controls, n = 18). MD*ADT*BZD had fewer granule neurons vs. MD*ADT in anterior DG and vs. controls in mid DG, and did not differ from untreated-MD in any DG subregion. MD*ADT had more granule neurons than untreated-MD in anterior and mid DG and comparable granule neuron number to controls in all dentate subregions. Untreated-MD had fewer granule neurons than controls in anterior and mid DG, and did not differ from any other group in posterior DG. MD*ADT*BZD had fewer NPCs vs. MD*ADT in mid DG. MD*ADT had more NPCs vs. untreated-MD and controls in anterior and mid DG. MD*ADT*BZD and MD*ADT had more mitotic cells in anterior DG vs. controls and untreated-MD. There were no between-group differences in mid DG in mitotic cells or in posterior DG for any cell type. Our results in mid-dentate, and to some degree anterior dentate, gyrus are consistent with murine findings that benzodiazepines counteract antidepressant-induced increases in neurogenesis by interfering with progenitor proliferation. We also confirmed, in this expanded sample, our previous finding of granule neuron deficit in untreated MD.

We have shown that oxytocin receptor (OTR) expression in neonatal rat enterocytes is robust from birth to weaning, but OTR function during this period is unknown. We previously reported that oxytocin (OT) stimulation of Caco2BB cells (enterocytes in vitro) inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling. The unfolded protein response (UPR) is known to protectively reduce translation during endoplasmic reticulum (ER) stress. Because the mTORC1 pathway is linked to cellular stress, we investigated markers of UPR in OT-stimulated Caco2BB cells. We report that OT modulates several factors involved in sensing and translation of ER stress. High OT (62.5 nM) reduced translation initiation factor 4E-BP1 phosphorylation (Ser65), which is known to inhibit cap-dependent translation via its rate-limiting eukaryotic translation initiation factor 4E (eIF4E). Importantly, high OT increased phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) phospho-Ser51, which inhibits eIF2a. High OT also increased protein kinase RNA-like endoplasmic reticulum kinase phosphorylation, a sensor of ER stress and a kinase of eIF2a. Both high and low OT activated inositol requiring enzyme1 (IRE1), which generates the transcription factor X-box binding protein 1 (XBP1) and induces the UPR. We also show that OT modulates XBP1 splicing and induces tribbles 3 (TRIB3; a negative regulator of Akt and protein involved in autophagy) and immunoglobulin binding protein (BiP; ER-chaperone). Taken together, these results indicate that OT modulates sensors of ER stress and autophagy. These findings support our hypothesis that transiently elevated OTR expression in neonatal gut may serve a protective function during a critical postnatal developmental period.

Smaller hippocampal volume is reported in major depressive disorder (MDD). We hypothesize that it may be related to fewer granule neurons (GN) in the dentate gyrus (DG), a defect possibly reversible with antidepressants. We studied age-, sex-, and postmortem interval-matched groups: no major psychopathology (controls); unmedicated-MDD; and MDD treated with serotonin reuptake inhibitors (MDD*SSRI) or tricyclics (MDD*TCA). Frozen right hippocampi were fixed, sectioned (50 μm), immunostained with neuronal nuclear marker (NeuN), and counterstained with hematoxylin. GN and glial number, and DG and granule cell layer (GCL) volumes were stereologically estimated. Fewer GNs in the anterior DG were present in unmedicated-MDDs compared with controls (p=0.013). Younger age of MDD onset correlated with fewer GNs (p=0.021). Unmedicated-MDDs had fewer mid-DG GNs than MDD*SSRIs (p=0.028) and controls (p=0.032). Anterior GCL glial number did not differ between groups. Anterior/mid GCL volume was smaller in unmedicated-MDDs vs controls (p=0.008) and larger in MDD*SSRIs vs unmedicated-MDDs (p<0.001), MDD*TCAs (p<0.001), and controls (p<0.001). Anterior GCL volume and GN number (r=0.594, p=0.001), and mid DG volume and GN number (r=0.398, p=0.044) were correlated. Anterior DG capillary density correlated with GN number (p=0.027), and with GCL (p=0.024) and DG (r=0.400, p=0.047) volumes. Posterior DG volume and GN number did not differ between groups. Fewer GNs in unmedicated-MDD without fewer neuronal progenitor cells, as previously reported, suggests a cell maturation or survival defect, perhaps related to MDD duration. This may contribute to a smaller hippocampus and is potentially reversed by SSRIs. Postmortem studies are correlative and animal studies are needed to test implied causal relationships.

Our recent findings of a weaning-related pattern of oxytocin (OT) and OT receptor (OTR) expression in the rat enteric nervous system and in villus-crypt enterocytes, together with the known high level and stability of OT in breast milk support that OT may play a role in gut function and development. We previously described a biphasic dose-response of the PI3K/Akt pathway in gut cells treated with OT. Activation peaked at 62.5 nM OT (30 min) and coincided with OTR internalization. Here we use automated Western blotting to further explore OT-elicited changes in Akt and pAkt(T308), as well as in downstream substrates p70 S6 kinase-1 (S6K1) and eIF-4E binding protein 1 (4E-BP1). Relative to fresh growth medium (FGM) alone, our results showed OT in FGM reduced the abundance and phosphorylation of S6K1 and the phosphorylation of 4E-BP1, both substrates of mammalian target of rapamycin complex 1 (mTORC1). Phosphorylation of mTORC1 regulator, Raptor(S792), was increased by high and low OT concentrations, with predicted inhibitory effects on mTORC1. OT thus downregulates anabolic effects induced by FGM activity catalyzed by mTORC1. OT is a regulator of the PI3K/Akt/mTORC1 pathway in Caco2BB cells and may modulate translation in gut cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Striated muscle fibers are characterized by their tightly organized cytoplasm. Here, we show that the Drosophila melanogaster KASH proteins Klarsicht (Klar) and MSP-300 cooperate in promoting even myonuclear spacing by mediating a tight link between a newly discovered MSP-300 nuclear ring and a polarized network of astral microtubules (aMTs). In either klar or msp-300(ΔKASH), or in klar and msp-300 double heterozygous mutants, the MSP-300 nuclear ring and the aMTs retracted from the nuclear envelope, abrogating this even nuclear spacing. Anchoring of the myonuclei to the core acto-myosin fibrillar compartment was mediated exclusively by MSP-300. This protein was also essential for promoting even distribution of the mitochondria and ER within the muscle fiber. Larval locomotion is impaired in both msp-300 and klar mutants, and the klar mutants were rescued by muscle-specific expression of Klar. Thus, our results describe a novel mechanism of nuclear spacing in striated muscles controlled by the cooperative activity of MSP-300, Klar, and astral MTs, and demonstrate its physiological significance.

Zinc enhances epithelial proliferation, protects the digestive epithelial layer and has profound antiulcerative and antidiarrheal roles in the colon. Despite the clinical significance of this ion, the mechanisms linking zinc to these cellular processes are poorly understood. We have previously identified an extracellular Zn(2+) sensing G-protein coupled receptor (ZnR) that activates Ca(2+) signaling in colonocytes, but its molecular identity as well as its effects on colonocytes' survival remained elusive. Here, we show that Zn(2+), by activation of the ZnR, protects HT29 colonocytes from butyrate induced cell death. Silencing of the G-protein coupled receptor GPR39 expression abolished ZnR-dependent Ca(2+) release and Zn(2+)-dependent survival of butyrate-treated colonocytes. Importantly, GPR39 also mediated ZnR-dependent upregulation of Na(+)/H(+) exchange activity as this activity was found in native colon tissue but not in tissue obtained from GPR39 knock-out mice. Although ZnR-dependent upregulation of Na(+)/H(+) exchange reduced the cellular acid load induced by butyrate, it did not rescue HT29 cells from butyrate induced cell death. ZnR/GPR39 activation however, increased the expression of the anti-apoptotic protein clusterin in butyrate-treated cells. Furthermore, silencing of clusterin abolished the Zn(2+)-dependent survival of HT29 cells. Altogether, our results demonstrate that extracellular Zn(2+), acting through ZnR, regulates intracellular pH and clusterin expression thereby enhancing survival of HT29 colonocytes. Moreover, we identify GPR39 as the molecular moiety of ZnR in HT29 and native colonocytes.

Recently, we discovered oxytocin receptor (OTR) expression in the developing gut villus epithelium that emerges in villus-crypt junctions after weaning. Oxytocin (OT) and OTR regulate many physiological functions in various tissues; however, their function in gut epithelium is unknown. We explored responses of PI3K and Akt phosphoisoforms to OT stimuli in the Caco2BB human gut cell line. In Caco2BB cells, PI3K and pAkt levels peaked at 62.5  nM OT. At higher concentrations, PI3K decreased more gradually than pAkt(S473) suggesting that the pAkt(S473) response is separate from PI3K. At ≤7.8  nM OT, pAkt(T308) increased while pAkt(S473) decreased. Using a specific OTR antagonist, we demonstrated that responses of pAkt(T308) to OT depend on OTR in contrast to the partial OTR-dependence of the pAkt(S473) response. Differential pAkt phosphoisoform responses included pAkt phosphoserine 473 persistently free of phosphothreonine 308. The reduction in PI3K after 62.5  nM OT for 30  min coincided with OTR internalization. The PI3K/Akt activation profile was somewhat different in other cell lines (MCF-7 breast cancer cells, HT29 gut cells), which have PI3K activating mutations, that were examined to establish experimental parameters. In Caco2BB cells, the divergent effects of OT upon pAkt phosphoisoforms suggests separate sub-pathways; pAkt (T308) activation depends on OTR via the PI3K pathway and pAkt(S473) presumably results from its specific kinase mTORC2 (mammalian target of rapamycin complex 2). Thus, OT may modulate gut cell functions downstream of mTOR complexes (e.g., translation control as suggested by others in uterine cells). We will next explore OT-stimulated kinase activities downstream of mTOR related to pAkt phosphoisoforms., (Copyright © 2011 Wiley Periodicals, Inc.)

Chiliadenus iphionoides (Asteraceae), a shrub endemic to the Mediterranean region and widespread throughout Israel, is used in the traditional eastern Mediterranean medicine. Although recent research confirmed its pharmacological potential, C. iphionoides essential oil has not been adequately characterized chemically. Essential-oil samples were collected from representative wild populations throughout Israel and characterized by GC/MS analysis. Considerable interpopulation variation was found for the composition of the essential oils. Multivariate analysis showed a significant correlation between the chemical composition and the geographic location, with three main chemotypes identified., (Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.)

Background: The pathogenesis of inflammatory bowel disease is unknown; however, the disorder is aggravated by psychological stress and is itself psychologically stressful. Chronic intestinal inflammation, moreover, has been reported to activate forebrain neurons. We tested the hypotheses that the chronically inflamed bowel signals to the brain through the vagi and that administration of a combination of secretin (S) and oxytocin (OT) inhibits this signaling., Methods: Three daily enemas containing 2,4,6-trinitrobenzene sulfonic acid (TNBS), which were given to rats produced chronic colitis and ongoing activation of Fos in brain neurons., Key Results: Fos was induced in neurons in the paraventricular nucleus of the hypothalamus, basolateral amygdala, central amygdala, and piriform cortex. Subdiaphragmatic vagotomy failed to inhibit this activation of Fos, suggesting that colitis activates forebrain neurons independently of the vagi. When administered intravenously, but not when given intracerebroventricularly, in doses that were individually ineffective, combined S/OT prevented colitis-associated activation of central neurons. Strikingly, S/OT decreased inflammatory infiltrates into the colon and colonic expression of tumor necrosis factor-alpha and interferon-gamma., Conclusions & Inferences: These observations suggest that chronic colonic inflammation is ameliorated by the systemic administration of S/OT, which probably explains the parallel ability of systemic S/OT to inhibit the colitis-associated activation of forebrain neurons. It is possible that S and OT, which are endogenous to the colon, might normally combine to restrict the severity of colonic inflammatory responses and that advantage might be taken of this system to develop novel means of treating inflammation-associated intestinal disorders.

Although oxytocin (OT) and oxytocin receptor (OTR) are known for roles in parturition and milk let-down, they are not hypothalamus-restricted. OT is important in nurturing and opposition to stress. Transcripts encoding OT and OTR have been reported in adult human gut, and OT affects intestinal motility. We tested the hypotheses that OT is endogenous to the enteric nervous system (ENS) and that OTR signaling may participate in enteric neurophysiology. Reverse transcriptase polymerase chain reaction confirmed OT and OTR transcripts in adult mouse and rat gut and in precursors of enteric neurons immunoselected from fetal rats. Enteric OT and OTR expression continued through adulthood but was developmentally regulated, peaking at postnatal day 7. Coincidence of the immunoreactivities of OTR and the neural marker Hu was 100% in the P3 and 71% in the adult myenteric plexus, when submucosal neurons were also OTR-immunoreactive. Co-localization with NeuN established that intrinsic primary afferent neurons are OTR-expressing. Because OTR transcripts and protein were detected in the nodose ganglia, OT signaling might also affect extrinsic primary afferent neurons. Although OT immunoreactivity was found only in approximately 1% of myenteric neurons, extensive OT-immunoreactive varicosities surrounded many others. Villus enterocytes were OTR-immunoreactive through postnatal day 17; however, by postnatal day 19, immunoreactivity waned to become restricted to crypts and concentrated at crypt-villus junctions. Immunoelectron microscopy revealed plasmalemmal OTR at enterocyte adherens junctions. We suggest that OT and OTR signaling might be important in ENS development and function and might play roles in visceral sensory perception and neural modulation of epithelial biology.

Serotonergic 5-HT(1A) receptor signaling leading to nuclear factor-kappaB (NF-kappaB) activation appears to be critical for cell survival. Adenylyl cyclase and protein kinase A (AC/PKA) are effectors of the 5-HT(1A) receptor that are inhibited by Galpha(i) subunits. Conversely, Gbetagamma(i) subunits downstream from the 5-HT(1A) receptor participate in the activation of extracellular signal-regulated kinases (ERK1/2), phosphatidylinositol 3-kinase (PI3K), Akt, and NF-kappaB. To model the contribution of pro- and antiapoptotic signaling cascades downstream of activated 5-HT(1A) receptor in cell survival, Chinese hamster ovarian (CHO) cells were employed that exogenously overexpress 5-HT(1A) receptors. Stimulation with the 5-HT(1A) receptor agonist 8-OH-DPAT and pharmacological agonists of AC induced PKA and protein phosphatase 2A (PP2A) activity, which in turn inhibited: Akt activity, IkappaBalpha degradation, nuclear translocation of NF-kappaB, and expression of X-linked inhibitor of apoptosis protein (XIAP/BIRC4). Pharmacological inhibition of PP2A with calyculin A potentiated Akt activity while attenuating ERK1/2 signaling via increased inhibitory phosphorylation of Raf (pSer259). In contrast, increased cAMP levels enhanced Bax translocation to the mitochondria, resulting in the release of cytochrome c, caspase-3 activation, and apoptosis induction. Our data suggest a central role of cAMP/PKA-dependent PP2A in shifting the homeostasis of intracellular signaling downstream of activated 5-HT(1A) receptor toward cell death in biological systems linked to neuropsychiatric disorders.

In the vertebrate head, mesoderm cells fuse together to form a myofiber, which is attached to specific cranial neural crest (CNC)-derived skeletal elements in a highly coordinated manner. Although it has long been recognized that CNC plays a role in the formation of the head musculature, the precise molecular underpinnings of this process remain elusive. In the present study we explored the nature of the crosstalk between CNC and mesoderm cells during head muscle development, employing three models for genetic perturbations of CNC development in mice, as well as experimental ablation of CNC in chick embryos. We demonstrate that although early myogenesis is CNC-independent, the migration, patterning and differentiation of muscle precursors are regulated by CNC. In the absence of CNC cells, accumulated myoblasts are kept in a proliferative state, presumably because of an increase of Fgf8 in adjacent tissues, which leads to abnormalities in both differentiation and subsequent myofiber organization in the head. These results have uncovered a surprising degree of complexity and multiple distinct roles for CNC in the patterning and differentiation of muscles during craniofacial development. We suggest that CNC cells control craniofacial development by regulating positional interactions with mesoderm-derived muscle progenitors that together shape the cranial musculoskeletal architecture in vertebrate embryos.

Purpose: Serotonin1A (5-HT1A) receptors exist in high- and low-affinity states, and agonist ligands bind preferentially to the high-affinity state of the receptor and provide a measure of functional 5-HT1A receptors. Although the antagonist tracers are established PET ligands in clinical studies, a successful 5-HT1A receptor agonist radiotracer in living brain has not been reported. [11C]MPT, our first-generation agonist radiotracer, shows in vivo specificity in baboons; however, its utility is limited owing to slow washout and immeasurable plasma free fraction. Hence we performed structure-activity relationship studies of MPT to optimize a radiotracer that will permit valid quantification of 5-HT1A receptor binding. We now report the synthesis and evaluation of [11C]MMP as an agonist PET tracer for 5-HT1A receptors in baboons., Methods: In vitro binding assays were performed in bovine hippocampal membranes and membranes of CHO cells expressing 5-HT1A receptors. [11C] labeling of MMP was performed by reacting desmethyl-MMP with [11C]CH(3)OTf. In vivo studies were performed in baboons, and blocking studies were conducted by pretreatment with 5-HT1A receptor ligands WAY-100635 and (+/-)-8-OH-DPAT., Results: MMP is a selective 5-HT1A receptor agonist (Ki 0.15 nM). Radiosynthesis of [11C]MMP was achieved in 30 +/- 5% (n = 15) yield at EOS with a specific activity of 2,600 +/- 500 Ci/mmol (n = 12). PET studies in baboons demonstrated specific binding of [11C]MMP to 5-HT1A receptor-enriched brain regions, as confirmed by blockade with WAY-100635 and (+/-)-8-OH-DPAT., Conclusion: We identified [11C]MMP as an optimal agonist PET tracer that shows quantifiable, specific binding in vivo to 5-HT1A receptors in baboons.

Synthesis and in vivo evaluation of 2-{4-[4-(3-methoxyphenyl)piperazin-1-yl]-butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (5 or MMT), a high affinity and selective serotonin 5-HT1AR agonist PET tracer, are described. GTPgammaS assay shows that MMT is an agonist with an EC50 comparable to 5-HT. Radiolabeling of 5 was achieved in 30% yield (EOS) from desmethyl-MMT (4) with >99% chemical and radiochemical purities and a specific activity >1000 Ci/mmol. PET studies in baboon show that [11C]5 penetrates the blood-brain barrier but, because of low specific binding and fast clearance of radioactivity it is not a suitable PET tracer for the in vivo quantification of 5-HT1AR.

Major depressive disorder (MDD) and suicide are associated with deficient serotonergic neurotransmission. Tryptophan hydroxylase (TPH) is the rate-limiting biosynthetic enzyme for serotonin. Previously, we reported elevated levels of TPH protein in the dorsal raphe nucleus (DRN) of depressed suicides and now examine expression of neuronal TPH2 mRNA in a cohort of matched controls and depressed suicides (n = 11 pairs). DRN TPH2 mRNA was measured by densitometric analysis of autoradiograms from in situ hybridization histochemistry experiments. TPH2 mRNA is confirmed as the raphe-specific isoform of TPH in human brain, and is expressed in neurons throughout the anteroposterior extent of the DRN and median raphe nucleus (MRN). TPH2 mRNA expression correlates with TPH protein distribution in the DRN, and has a negative correlation with age. In drug-free suicides, TPH2 expression is 33% higher in the DRN and 17% higher in the MRN as compared to matched nonpsychiatric controls. Higher levels of TPH2 mRNA were found throughout the entire extent of the rostrocaudal axis of the DRN, and were not specific to any single subnucleus. Higher TPH2 mRNA expression may explain more TPH protein observed in depressed suicides and reflect a homeostatic response to deficient brain serotonergic transmission.

Antagonist 5-HT(1A) PET ligands are available, but an agonist ligand would give more information about signal transduction capacity. Synthesis and in vivo evaluation of [O-methyl-(11)C]2-{4-[4-(7-methoxynaphthalen-1-yl)piperazin-1-yl]butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (10), a potential high affinity (K(i) = 1.36 nM) 5-HT(1A) agonist PET tracer is described. Piperazine 10 is a 5-HT(1A) agonist with an EC(50) comparable to serotonin, based on cAMP formation and GTP(gamma)S binding assays. Radiosynthesis of [(11)C]10 has been achieved by reacting 2-{4-[4-(7-hydroxynaphthalen-1-yl)piperazin-1-yl]butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (9) and [(11)C]CH(3)OTf in 25 +/- 5% (n = 15) yield at the end of synthesis (EOS). The chemical and radiochemical purities of [(11)C]10 were >99% with a specific activity 1500 +/- 300 Ci/mmol (n =15). PET studies in anesthetized baboon demonstrate [(11)C]10 specific binding in brain regions rich in 5-HT(1A) receptors. Binding of [(11)C]10 was blocked by WAY100635 and 8-OH-DPAT. The regional brain volumes of distribution (V(T)) of [(11)C]10 in baboon correlate with [(11)C]WAY100635 V(T) in baboons. These data provide evidence that [(11)C]10 is the first promising agonist PET tracer for the 5-HT(1A) receptors.

Purpose: The management of giant omphalocele presents a major challenge to pediatric surgeons. Current treatment modalities often result in wound infection, fascial separation, and abdominal domain loss. Human acellular dermis (AlloDerm), as a primary abdominal fascial substitute, may prevent these complications. We present our experience with its application in neonates with giant omphalocele., Methods: Charts of patients with giant omphalocele from January 2003 to September 2004 were reviewed and data collected regarding wound healing, rate of infection, ventilatory support, and outcome., Results: Three neonates underwent abdominal wall closure with AlloDerm (gestational ages: 38, 37, and 28 weeks; birthweights 2880, 2640, and 1160 g, respectively). All had cardiac anomalies; 1 required cardiac surgery and 1 was ventilator-dependent, secondary to pulmonary hypoplasia. Omphalocele repair was performed on day-of-life 9, 2, and 87. No fascial dehiscence or infection was encountered. Neovascularization was noted by day 7. Two died of cardiopulmonary disease (6 months and 1 year). The third exhibited normal growth and development without complication., Conclusions: AlloDerm provides visceral coverage without compromising cardiopulmonary function, diminishing abdominal domain, or requiring multiple operations, allowing prompt treatment of associated anomalies. AlloDerm represents an exciting alternative in the treatment of giant omphalocele. Further study is required to determine long-term benefits.

To investigate the functional consequences of cross-talk between multiple effectors of serotonin (5-HT) 1A receptor, we employed transfected Chinese hamster ovary cells. Activation of 5-HT 1A receptor stimulated extracellular signal-regulated kinase (ERK)1/2, Akt and nuclear transcription factor-kappaB (NF-kappaB). Stimulation of cells with 5-HT 1A receptor agonist induced a rapid but transient ERK1/2 phosphorylation followed by increased phosphorylation of Akt. Elevated Akt activity in turn suppressed Raf activity and induced a decline in ERK activation. The activation of ERK and Akt downstream of 5-HT 1A receptor was sensitive to inhibitors of Ras, Raf and phosphatidylinositol 3-kinase (PI3K). Stimulation of 5-HT 1A receptor also resulted in activation of NF-kappaB through a decrease in inhibitor of nuclear transcription factor-kappaB. In support of the importance of 5-HT 1A receptor signaling for cell survival, inhibition of NF-kappaB facilitated caspase 3 activation and cleavage of poly (ADP-ribose) polymerase, while treatment of cells with agonist inhibited caspase 3, DNA fragmentation and cell death. Both agonist-dependent NF-kappaB activation and cell survival were decreased by Akt Inhibitor II or by overexpression of dominant-negative Akt. These findings suggest a role for 5-HT 1A receptor signaling in the Ras/Raf-dependent regulation of multiple intracellular signaling pathways that include ERK and PI3K/Akt. Of these, only PI3K/Akt and NF-kappaB activation were required for 5-HT 1A receptor-dependent cell survival, implying that the relative distribution of signals between competing transduction pathways determines the functional outcome of 5-HT 1A receptor activation.