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Abstract Frequent RNA virus mutations raise concerns about evolving virulent variants. The purpose of this study was to investigate genetic variation in salmonid alphavirus-3 (SAV3) over the course of an experimental infection in Atlantic salmon and brown trout. Atlantic salmon and brown trout parr were infected using a cohabitation challenge, and heart samples were collected for analysis of the SAV3 genome at 2-, 4- and 8-weeks post-challenge. PCR was used to amplify eight overlapping amplicons covering 98.8% of the SAV3 genome. The amplicons were subsequently sequenced using the Nanopore platform. Nanopore sequencing identified a multitude of single nucleotide variants (SNVs) and deletions. The variation was widespread across the SAV3 genome in samples from both species. Mostly, specific SNVs were observed in single fish at some sampling time points, but two relatively frequent (i.e., major) SNVs were observed in two out of four fish within the same experimental group. Two other, less frequent (i.e., minor) SNVs only showed an increase in frequency in brown trout. Nanopore reads were de novo clustered using a 99% sequence identity threshold. For each amplicon, a number of variant clusters were observed that were defined by relatively large deletions. Nonmetric multidimensional scaling analysis integrating the cluster data for eight amplicons indicated that late in infection, SAV3 genomes isolated from brown trout had greater variation than those from Atlantic salmon. The sequencing methods and bioinformatics pipeline presented in this study provide an approach to investigate the composition of genetic diversity during viral infections.

Abstract Delousing strategies, including mechanical delousing, are typically used to treat Atlantic salmon (Salmo salar) sea lice infestations. In this study, we evaluate the impact of mechanical delousing (Hydrolicer) on the skin bacterial microbiome of broodstock female and male Atlantic salmon. 16S rDNA sequencing of salmon skin microbial communities was performed immediately before delousing, right after delousing and 2 and 13 days post-delousing (dpd). The skin bacterial community of female salmon was more diverse than that of males at the start of the experiment. Overall, hydrolycer caused losses in alpha diversity in females and increases in alpha diversity in males. Hydrolicer also caused rapid shifts in the skin microbial community composition immediately after delicing in a sex-specific manner. There was a decrease in abundance of Proteobacteria and Bacteriodetes in both female and male salmon, whereas Firmicutes and Tenericutes abundances increased. Interestingly, the female community recovered faster, while the male community remained dysbiotic 13 dpd due to expansions in Bacteroidetes (Pseudomonadaceae) and Firmicutes. Our data suggest that female broodstock are more resilient to Hydrolicer treatment due to their more diverse skin microbiota community, and that sex influences the skin microbial community and therefore host health outcomes during common farming manipulations.

Background: Neutropenic fever management decisions are complex and result in prolonged duration of broad-spectrum antibiotics. Strategies for antibiotic stewardship in this context have been studied, including de-escalation of antibiotics prior to resolution of neutropenia, with unclear implementation. Here, we present the first survey study to describe real-world neutropenic fever management practices in US healthcare institutions, with particular emphasis on de-escalation strategies after initiation of broad-spectrum antibiotics. Methods: Using REDCap, we conducted a survey of US healthcare institutions through the SHEA Research Network (SRN). Questions pertained to antimicrobial prophylaxis and supportive care in the management of oncology patients and neutropenic fever management (including specific antimicrobial choices and clinical scenarios). Hematologic malignancy hospitalization (2020) and bone-marrow transplantation (2016–2020) volumes were obtained from CMS and Health Resources & Services Administration databases, respectively. Results: Overall, 23 complete responses were recorded (response rate, 35.4%). Collectively, these entities account for ~11.0% of hematologic malignancy hospitalizations and 13.3% bone marrow transplantations nationwide. Of 23 facilities, 19 had institutional guidelines for neutropenic fever management and 18 had institutional guidelines for prophylaxis, with similar definitions for neutropenic fever. Firstline treatment universally utilized antipseudomonal broad-spectrum IV antibiotics (20 of 23 use cephalosporin, 3 of 23 use penicillin agent, and no respondents use carbapenem). Fluoroquinolone prophylaxis was common for leukemia induction patients (18 of 23) but was mixed for bone-marrow transplantation (10 of 23). We observed significant heterogeneity in treatment decisions. For stable neutropenic fever patients with no clinical source of infection identified, 13 of 23 respondents continued IV antibiotics until ANC (absolute neutrophil count) recovery. The remainder had criteria for de-escalation back to prophylaxis prior to this (eg, a fever-free period). Respondents were more willing to de-escalate prior to ANC recovery in patients with identified clinical sources (14 of 23 de-escalations in patients with pneumonia) or microbiological sources (15 of 23 de-escalations in patients with bacteremia) after dedicated treatment courses. In free-text responses, several respondents described opportunities for more systemic de-escalation for antimicrobial stewardship in these scenarios. Conclusions: Our results illustrate the real-world management of neutropenic fever in US hospitals, including initiation of therapy, prophylaxis, and treatment duration. We found significant heterogeneity in de-escalation of empiric antibiotics relative to ANC recovery, highlighting a need for more robust evidence for and adoption of this practice.

Piscine orthoreovirus -1 (PRV-1) causes the disease heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon, and the virus has been detected in wild anadromous Atlantic salmon and brown trout. However, the infection prevalence, viral kinetics, and disease severity in different life stages of Atlantic salmon and brown trout are unknown. The current study aimed to evaluate and compare susceptibility to PRV-1 infection and development of HSMI in different life stages of anadromous Atlantic salmon (Salmo salar) and brown trout (Salmo trutta). We challenged Atlantic salmon and brown trout fry, parr, and post-smolts with PRV-1 by bath, cohabitation, or IP injection. The kinetics of viral infection and disease development were evaluated by RT-qPCR, in situ hybridization, and histology. Our results indicated that PRV-1 infection prevalence and viral kinetics depend on the developmental stage and challenge method in both Atlantic salmon and brown trout. All developmental stages of Atlantic salmon and brown trout can be infected with PRV-1. However, brown trout showed a lower infection prevalence, with positive cases exhibiting only mild infections without any pathological changes in the target organs, while all life stages of Atlantic salmon developed heart lesions characteristic of HSMI. These results strongly suggest that brown trout are less susceptible to PRV-1 infection than Atlantic salmon and further confirm the species-specific susceptibility and disease development for PRV-1 infection.

Objective: Obesity-related adipose tissue dysfunction has been linked to the development of insulin resistance, type 2 diabetes, and cardiovascular disease. Impaired calcium homeostasis is associated with altered adipose tissue metabolism; however, the molecular mechanisms that link disrupted calcium signaling to metabolic regulation are largely unknown. Here, we investigated the contribution of a calcium-sensing enzyme, calcium/calmodulin-dependent protein kinase II (CAMK2), to adipocyte function, obesity-associated insulin resistance, and glucose intolerance. Methods: To determine the impact of adipocyte CAMK2 deficiency on metabolic regulation, we generated a conditional knockout mouse model and acutely deleted CAMK2 in mature adipocytes. We further used in vitro differentiated adipocytes to dissect the mechanisms by which CAMK2 regulates adipocyte function. Results: CAMK2 activity was increased in obese adipose tissue, and depletion of adipocyte CAMK2 in adult mice improved glucose intolerance and insulin resistance without an effect on body weight. Mechanistically, we found that activation of CAMK2 disrupted adipocyte insulin signaling and lowered the amount of insulin receptor. Further, our results revealed that CAMK2 contributed to adipocyte lipolysis, tumor necrosis factor alpha (TNFα)–induced inflammation, and insulin resistance. Conclusions: These results identify a new link between adipocyte CAMK2 activity, metabolic regulation, and whole-body glucose homeostasis.

Down-regulation or loss of MHC class I expression is a major mechanism used by cancer cells to evade immunosurveillance and increase their oncogenic potential. MHC I mediated antigen presentation is a complex regulatory process, controlled by antigen processing machinery (APM) dictating immune response. Transcriptional regulation of the APM that can modulate gene expression profile and their correlation to MHC I mediated antigen presentation in cancer cells remain enigmatic. Here, we reveal that Scaffold/Matrix-Associated Region 1- binding protein (SMAR1), positively regulates MHC I surface expression by down-regulating calnexin, an important component of antigen processing machinery (APM) in cancer cells. SMAR1, a bonafide MAR binding protein acts as a transcriptional repressor of several oncogenes. It is down-regulated in higher grades of cancers either through proteasomal degradation or through loss of heterozygosity (LOH) at the Chr.16q24.3 locus where the human homolog of SMAR1 (BANP) has been mapped. It binds to a short MAR region of the calnexin promoter forming a repressor complex in association with GATA2 and HDAC1. A reverse correlation between SMAR1 and calnexin was thus observed in SMAR1-LOH cells and also in tissues from breast cancer patients. To further extrapolate our findings, influenza A (H1N1) virus infection assay was performed. Upon viral infection, the levels of SMAR1 significantly increased resulting in reduced calnexin expression and increased MHC I presentation. Taken together, our observations establish that increased expression of SMAR1 in cancers can positively regulate MHC I surface expression thereby leading to higher chances of tumor regression and elimination of cancer cells.

The microbial communities that live in symbiosis with the mucosal surfaces of animals provide the host with defense strategies against pathogens. These microbial communities are largely shaped by the environment and the host genetics. Triploid Atlantic salmon (Salmo salar) are being considered for aquaculture as they are reproductively sterile and thus cannot contaminate the natural gene pool. It has not been previously investigated how the microbiome of triploid salmon compares to that of their diploid counterparts. In this study, we compare the steady-state skin and gill microbiome of both diploid and triploid salmon, and determine the effects of salmonid alphavirus 3 experimental infection on their microbial composition. Our results show limited differences in the skin-associated microbiome between triploid and diploid salmon, irrespective of infection. In the gills, we observed a high incidence of the bacterial pathogen Candidatus Branchiomonas, with higher abundance in diploid compared to triploid control fish. Diploid salmon infected with SAV3 showed greater histopathological signs of epitheliocystis compared to controls, a phenomenon not observed in triploid fish. Our results indicate that ploidy can affect the alpha diversity of the gills but not the skin-associated microbial community. Importantly, during a natural outbreak of Branchiomonas sp. the gill microbiome of diploid Atlantic salmon became significantly more dominated by this pathogen than in triploid animals. Thus, our results suggest that ploidy may play a role on Atlantic salmon gill health and provide insights into co-infection with SAV3 and C. Branchiomonas in Atlantic salmon.

Amyand's hernia is an exceedingly rare condition in which the appendix is found in an inguinal hernia. The appendix can be normal, inflamed, infected, or perforated. Here, we present a case of perforated appendicitis in an Amyand's hernia in an extremely preterm infant with subsequent post-operative complications including necrotic bowel and venous thrombosis of the mesentery. This article stresses the importance of complications that can arise in Amyand's hernia, especially in an extremely preterm infant.

Introduction. Hepatopulmonary fusion is a very rare finding associated with right-sided congenital diaphragmatic hernia. With less than 50 reported cases, management and outcomes of hepatopulmonary fusion are poorly understood. This report highlights that clinical presentation is not a reliable indicator of outcomes in this rare disease. Case Presentation. A term neonate admitted for tachypnea and complete opacification of the right hemithorax was diagnosed with right-sided congenital diaphragmatic hernia. Preoperative respiratory support was minimal, and the only symptom exhibited was tachypnea. During surgical repair, fusion of the lung and liver were noted, consistent with a diagnosis of hepatopulmonary fusion. Postoperatively, the patient’s pulmonary hypertension worsened and required extracorporeal membrane oxygenation. Conclusions. Many patients with hepatopulmonary fusion and only mild symptoms die postoperatively from severe pulmonary hypertension and progressive respiratory failure. Preoperative clinical status is not indicative of postoperative outcomes, and literature suggests that patients who require less support preoperatively have high mortality rates. The availability of ECMO for postoperative complications may be necessary in patients requiring repair of hepatopulmonary fusion.

Wnt signaling is one of the central mechanisms regulating tissue morphogenesis during embryogenesis and repair. The pivot of this signaling cascade is the Wnt ligand, which binds to receptors belonging to the Frizzled family or the ROR1/ROR2 and RYK family. This interaction governs the downstream signaling cascade (canonical/non-canonical), ultimately extending its effect on the cellular cytoskeleton, transcriptional control of proliferation and differentiation, and organelle dynamics. Anomalous Wnt signaling has been associated with several cancers, the most prominent ones being colorectal, breast, lung, oral, cervical, and hematopoietic malignancies. It extends its effect on tumorigenesis by modulating the tumor microenvironment via fine crosstalk between transformed cells and infiltrating immune cells, such as leukocytes. This review is an attempt to highlight the latest developments in the understanding of Wnt signaling in the context of tumors and their microenvironment. A dynamic process known as immunoediting governs the fate of tumor progression based on the correlation of various signaling pathways in the tumor microenvironment and immune cells. Cancer cells also undergo a series of mutations in the tumor suppressor gene, which favors tumorigenesis. Wnt signaling, and its crosstalk with various immune cells, has both negative as well as positive effects on tumor progression. On one hand, it helps in the maintenance and renewal of the leucocytes. On the other hand, it promotes immune tolerance, limiting the antitumor response. Wnt signaling also plays a role in epithelial-mesenchymal transition (EMT), thereby promoting the maintenance of Cancer Stem Cells (CSCs). Furthermore, we have summarized the ongoing strategies used to target aberrant Wnt signaling as a novel therapeutic intervention to combat various cancers and their limitations.

ABSTRACT: Objective: To describe an unusual presentation of a follicle-stimulating hormone-secreting pituitary adenoma leading to ovarian hyperstimulation syndrome. We also discuss the pathophysiology and subsequent management of these tumors.Methods: This is a case report and review of the literature. A 37-year-old female with menorrhagia was found to have bilateral multilocular adnexal cysts with concern for ovarian hyperstimulation syndrome.Results: Labs revealed elevated follicle-stimulating hormone, and an eminent estradiol level. Pituitary magnetic resonance imaging revealed a macroadenoma with cavernous sinus invasion. The patient went on to have a successful transsphenoidal resection with normalization of hormones and subsequent resumption of menstrual cycles.Conclusion: With the help of this case report, we illustrate the pathogenesis of functioning gonadotroph adenomas as well as the management challenges associated with these tumors. Our case is unique in its presentation with severe hyperestrogenemia and cavernous sinus invasion pointing towards a clinically aggressive adenoma. It is important to increase awareness of these tumors to ensure timely and effective management of their complications.Abbreviations: FGA = functioning gonadotroph adenoma FSH = follicle-stimulating hormone LH = luteinizing hormone MRI = magnetic resonance imaging OHSS = ovarian hyperstimulation syndrome

Large pyogenic liver abscess is a rare and difficult to treat entity in pediatric patients. Percutaneous drainage rather than open surgical drainage has become more common in recent years, even for large abscesses. Percutaneous drainage can be complicated by catheter obstruction. We present the case of a 16-year-old male presenting with abdominal pain, fever, and chills. He was diagnosed with a 9-centimeter liver abscess. A CT-guided percutaneous drainage was placed. The catheter initially drained well, but then became occluded. Tissue plasminogen activator was instilled into the catheter every 6 hours for a total of five doses, resulting in increased drainage and improved clinical state of the patient. To our knowledge, this is the first reported use of tissue plasminogen activator in pyogenic liver abscess in the pediatric population.

Mechanical stimulation is known to influence intervertebral disc (IVD) cell behavior and function, but the effect on disc cells is routinely considered in isolation from other microenvironmental factors. Acidic pH has been shown to be a prominent and detrimental microenvironmental factor present in degenerate IVDs, but its influence on the human disc cell mechanoresponse has never been studied. We investigated the response of agarose‐encapsulated human nucleus pulposus (NP) cells to 0.004 MPa, 1.0 Hz and 1 hour of compression (Flexcell FX4000 Compression System) under pH conditions representative of nondegenerate (pH 7.1) and degenerate (pH 6.5) IVDs. Cell viability, extracellular matrix production, and expression of anabolic/anti‐catabolic and catabolic genes were assessed. We report that preculture of NP cells in agarose gels was required in order for cells to be mechanoresponsive, and this correlated with increased type VI collagen, α5β1 integrin, and fibronectin expression. Furthermore, the matrix homeostatic response observed at pH 7.1 (representative of nondegenerate IVDs; increased aggrecan [AGC], tissue inhibitor of metalloproteinases‐1 [TIMP1], matrix metalloproteinase‐3 [MMP3], a disintegrin and metalloproteinase with thrombospondin motif‐5 [ADAMTS5] gene expression) was RGD‐integrin dependent, whereas only MMP3 remained mechanoresponsive at pH 6.5, and this was independent of RGD‐integrins. Our findings suggest differential mechanotransduction pathways operating for specific genes, with RGD‐integrin dependent AGC expression, but not RGD‐independent MMP3 expression, inhibited at pH representative of degenerate IVDs (pH 6.5), which could contribute to the catabolic phenotype observed during IVD degeneration. Clinical significance Characterizing the influence of the mechanical and chemical intervertebral disc microenvironment on disc cells, particularly in disc degeneration, could help develop future therapeutic strategies for the treatment of discogenic back pain.

Triploid Atlantic salmon (Salmo salar L.) may play an important role in the sustainable expansion of the Norwegian aquaculture industry. Therefore, the susceptibility of triploid salmon to common infections such as salmonid alphavirus (SAV), the causative agent of pancreas disease (PD), requires investigation. In this study, shortly after seawater transfer, diploid and triploid post-smolts were exposed to SAV type 3 (SAV3) using a bath challenge model where the infectious dose was 48 TCID50 ml-1 of tank water. Copy number analysis of SAV3 RNA in heart tissue showed that there was no difference in viral loads between the diploids and triploids. Prevalence reached 100% by the end of the 35-day experimental period in both infected groups. However, prevalence accumulated more slowly in the triploid group reaching 19% and 56% at 14 and 21 days post exposure (dpe) respectively. Whereas prevalence in the diploid group was 82% and 100% at the same time points indicating some differences between diploid and triploid fish. Both heart and pancreas from infected groups at 14 dpe showed typical histopathological changes associated with pancreas disease. Observation of this slower accumulation of prevalence following a natural infection route was possible due to the early sampling points and the exposure to a relatively low dose of virus. The triploid salmon in this study were not more susceptible to SAV3 than diploid salmon indicating that they could be used commercially to reduce the environmental impact of escaped farmed fish interbreeding with wild salmon. This is important information regarding the future use of triploid fish in large scale aquaculture where SAV3 is a financial threat to increased production.

[This corrects the article DOI: 10.1371/journal.pone.0175468.].

Interactions among host, microbiota and viral pathogens are complex and poorly understood. The goal of the present study is to assess the changes in the skin microbial community of Atlantic salmon (Salmo salar L.) in response to experimental infection with salmonid alphavirus (SAV). The salmon skin microbial community was determined using 16S rDNA pyrosequencing in five different experimental groups: control, 7 days after infection with low-dose SAV, 14 days after infection with low-dose SAV, 7 days after infection with high-dose SAV, and 14 days after infection with high-dose SAV. Both infection treatment and time after infection were strong predictors of the skin microbial community composition. Skin samples from SAV3 infected fish showed an unbalanced microbiota characterized by a decreased abundance of Proteobacteria such as Oleispira sp. and increased abundances of opportunistic taxa including Flavobacteriaceae, Streptococcaceae and Tenacibaculum sp. These results demonstrate that viral infections can result in skin dysbiosis likely rendering the host more susceptible to secondary bacterial infections.

Background : Perforation peritonitis is a common surgical emergency. Geriatric patient presenting with perforation peritonitis is a challenge for an anaesthetist. Risk and outcome in such patients depend upon age, the patient′s physiological status, co-existing disease and whether the surgery is elective or emergency and type of procedure. Preoperative optimization, hemodynamic stability can reduce intraoperative and postoperative morbidity and mortality. Considering decreased physiological reserve of all body organs and systems intensive care in critical care setting is essential.

Viruses are threatening pathogens for fish aquaculture. Some of them are transmitted through gonad fluids or gametes as occurs with nervous necrosis virus (NNV). In order to be transmitted through the gonad, the virus should colonize and replicate inside some cell types of this tissue and avoid the subsequent immune response locally. However, whether NNV colonizes the gonad, the cell types that are infected, and how the immune response in the gonad is regulated has never been studied. We have demonstrated for the first time the presence and localization of NNV into the testis after an experimental infection in the European sea bass (Dicentrarchus labrax), and in the gilthead seabream (Sparus aurata), a very susceptible and an asymptomatic host fish species, respectively. Thus, we localized in the testis viral RNA in both species using in situ PCR and viral proteins in gilthead seabream by immunohistochemistry, suggesting that males might also transmit the virus. In addition, we were able to isolate infective particles from the testis of both species demonstrating that NNV colonizes and replicates into the testis of both species. Blood contamination of the tissues sampled was discarded by completely fish bleeding, furthermore the in situ PCR and immunocytochemistry techniques never showed staining in blood vessels or cells. Moreover, we also determined how the immune and reproductive functions are affected comparing the effects in the testis with those found in the brain, the main target tissue of the virus. Interestingly, NNV triggered the immune response in the European sea bass but not in the gilthead seabream testis. Regarding reproductive functions, NNV infection alters 17β-estradiol and 11-ketotestosterone production and the potential sensitivity of brain and testis to these hormones, whereas there is no disruption of testicular functions according to several reproductive parameters. Moreover, we have also studied the NNV infection of the testis in vitro to assess local responses. Our in vitro results show that the changes observed on the expression of immune and reproductive genes in the testis of both species are different to those observed upon in vivo infections in most of the cases.