Identification of cellular retinoic acid binding protein 2 (CRABP2) as downstream target of nuclear factor I/X (NFIX): implications for skeletal dysplasia syndromes.

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix+/+, Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice, compared with NfixDel2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in NfixDel2/Del2 MEFs with ≥2-fold-change (P <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in NfixDel2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%-100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.

Adding yeast extract to culture medium enhances replication of the avian coronavirus infectious bronchitis virus in chicken embryo kidney cells.

Infectious bronchitis virus (IBV), an avian coronavirus, can be isolated and cultured in tracheal organ cultures (TOCs), embryonated eggs and cell cultures, the first two of which are commonly used for viral isolation. Previous studies have suggested that foetal bovine serum (FBS) can inhibit coronavirus replication in cell cultures. In this study, the replication of IBV in chicken embryo kidney (CEK) cell cultures and the Leghorn hepatocellular carcinoma (LMH) cell line was assessed using two different cell culture media containing FBS or yeast extract (YE) and two different IBV strains. The highest concentrations of viral genomes were observed when the cell culture medium (CEK) contained YE. Similar results were observed in LMH cells. Examination of the infectivity by titration demonstrated that the cell lysate from CEK cell cultures in a medium including YE contained a higher median embryo infectious dose than that from CEK cell cultures in a medium containing FBS. These results indicate that improved replication of IBV in cell cultures can be achieved by replacing FBS with YE in the cell culture medium.

Invasive Oral Squamous Cell Carcinoma Mimicking a Dentoalveolar Abscess: Report of a Case in a 14-Year-Old Patient.

INTRODUCTION: A diagnosis of oral squamous cell carcinoma in adolescent patients is extremely rare. When an oral squamous cell carcinoma lesion arises near the teeth and/or periodontium, it can be easily misdiagnosed as an inflammatory condition of endodontic or periodontal origin. METHODS: This is a case report of an otherwise healthy 14-year-old patient who was referred for endodontic evaluation and treatment of a soft-tissue swelling in the anterior maxilla. RESULTS: The unexpected definitive diagnosis of invasive oral squamous cell carcinoma underscores the importance of proper diagnostic testing. CONCLUSIONS: Accurate interpretation of pulp testing results, periapical and cone beam computed tomography imaging, timely biopsy, and prompt definitive treatment are critical when a lesion of nonodontogenic origin is suspected.

GNAQ/GNA11 Mosaicism Causes Aberrant Calcium Signaling Susceptible to Targeted Therapeutics.

Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.

Clinical investigation and management of Brucella suis seropositive dogs: A longitudinal case series.

BACKGROUND: Brucellosis in dogs caused by Brucella suis is an emerging zoonotic disease. OBJECTIVES: To document clinical characteristics, serology, microbiology, and clinical response to treatment in B. suis-seropositive dogs. ANIMALS: Longitudinal study of 27 privately-owned dogs. Dogs that tested positive by serology, culture, or real-time polymerase chain reaction (qPCR) were included in the study. METHODS: Clinical (physical examination and imaging) and laboratory (serology, hematology, serum biochemistry, and qPCR or culture) assessments were made at baseline and after approximately 3, 6, 12, and 18 months. RESULTS: Dogs were followed for 10 895 dog days, with 17/27 dogs completing the 18-month follow-up. Ten dogs had signs consistent with brucellosis before enrollment (n = 4), at baseline (n = 2) or during follow-up (n = 6), with 2 dogs experiencing relapse of historical signs. Antibody titers persisted for the duration of follow-up in 15/17 dogs (88%). Radiographic (n = 5) and ultrasound (n = 11) findings, of variable clinical relevance, were observed. Brucella DNA and organisms were detected in 3 dogs, all of which had clinical signs, including in the milk of a bitch around the time of whelping. Brucella DNA was not detected in blood (n = 92 samples), urine (n = 80), saliva (n = 95) or preputial swabs (n = 78) at any time during follow-up. Six dogs underwent treatment, all of which achieved clinical remission although remission was not reflected by decreasing antibody titers. CONCLUSIONS AND CLINICAL IMPORTANCE: Most dogs with B. suis infections have subclinical infections. Serology is poorly associated with clinical disease. Excretion of organisms appears rare except in whelping bitches. Clinical management using antibiotics with or without surgery is recommended.

miR-3156-5p is downregulated in serum of MEN1 patients and regulates expression of MORF4L2.

Multiple endocrine neoplasia type 1 (MEN1), caused by mutations in the MEN1 gene encoding menin, is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic neuroendocrine tumours (NETs). Development of these tumours is associated with wide variations in their severity, order and ages (from <5 to >80 years), requiring life-long screening. To improve tumour surveillance and quality of life, better circulating biomarkers, particularly for pancreatic NETs that are associated with higher mortality, are required. We, therefore, examined the expression of circulating miRNA in the serum of MEN1 patients. Initial profiling analysis followed by qRT-PCR validation studies identified miR-3156-5p to be significantly downregulated (-1.3 to 5.8-fold, P < 0.05-0.0005) in nine MEN1 patients, compared to matched unaffected relatives. MEN1 knock-down experiments in BON-1 human pancreatic NET cells resulted in reduced MEN1 (49%, P < 0.05), menin (54%, P < 0.05) and miR-3156-5p expression (20%, P < 0.005), compared to control-treated cells, suggesting that miR-3156-5p downregulation is a consequence of loss of MEN1 expression. In silico analysis identified mortality factor 4-like 2 (MOR4FL2) as a potential target of miR-3156-5p, and in vitro functional studies in BON-1 cells transfected with either miR-3156-5p mimic or inhibitors showed that the miR-3156-5p mimic significantly reduced MORF4L2 protein expression (46%, P < 0.005), while miR-3156-5p inhibitor significantly increased MORF4L2 expression (1.5-fold, P < 0.05), compared to control-treated cells, thereby confirming that miR-3156-5p regulates MORF4L2 expression. Thus, the inverse relationship between miR-3156-5p and MORF4L2 expression represents a potential serum biomarker that could facilitate the detection of NET occurrence in MEN1 patients.

Field trial investigating the efficacy of a long-acting imidacloprid 10%/flumethrin 4.5% polymer matrix collar (Seresto®, Elanco) compared to monthly topical fipronil for the chemoprevention of canine tick-borne pathogens in Cambodia.

The tropical brown dog tick, Rhipicephalus linnaei, commonly infests canines in the tropics and is an important vector for disease-causing and sometimes lethal pathogens including Babesia spp., Ehrlichia canis, Hepatozoon canis and Anaplasma platys. In tropical climates ticks and their pathogens exert an extremely high infection pressure on unprotected dogs. To protect canines in such regions, effective acaricidal products possessing a speed of kill that blocks pathogen transmission is paramount. We conducted a 12-month community trial to compare the chemoprophylactic efficacy of two topical commercial acaricidal formulations: an imidacloprid 10% and flumethrin 4.5%, 8-month acting collar (Seresto®) against a monthly spot-on containing 12% w/v fipronil (Detick, Thailand). In a separate analysis, we used baseline data collected at the start of the trial to quantify tick-borne pathogen (TBP) infection status in dogs with a prior history of being administered a systemically-acting (isoxazoline) versus a topically-acting ectoparasiticide. We found that both topical products in the community trial demonstrated high efficacy at protecting dogs from ticks and TBP, with Seresto® demonstrating a moderate increase in protection at 3 (95% confidence interval (CI): 1-5) TBP-positive dogs per 100 dog-years at risk compared to 11 (95% CI: 4-26) TBP-positive dogs per 100 dog-years at risk for those on fipronil. Additionally, at baseline dogs treated with commercial systemic isoxazoline acaricides prior to the trial's commencement were 2.7 (95% CI: 0.5-15.0) times more likely to be TBP-positive compared to dogs that had been topically treated, highlighting such isoxazoline products as being less efficacious than topical products at preventing canine TBP acquisition in a tropical setting.

The Bartter-Gitelman Spectrum: 50-Year Follow-up With Revision of Diagnosis After Whole-Genome Sequencing.

Bartter syndrome (BS) and Gitelman syndrome (GS) are renal tubular disorders affecting sodium, potassium, and chloride reabsorption. Clinical features include muscle cramps and weakness, in association with hypokalemia, hypochloremic metabolic alkalosis, and hyperreninemic hyperaldosteronism. Hypomagnesemia and hypocalciuria are typical of GS, while juxtaglomerular hyperplasia is characteristic of BS. GS is due to SLC12A3 variants, whereas BS is due to variants in SLC12A1, KCNJ1, CLCNKA, CLCNKB, BSND, MAGED2, or CASR. We had the opportunity to follow up one of the first reported cases of a salt-wasting tubulopathy, who based on clinical features was diagnosed with GS. The patient had presented at age 10 years with tetany precipitated by vomiting or diarrhea. She had hypokalemia, a hypochloremic metabolic alkalosis, hyponatremia, mild hypercalcemia, and normomagnesemia, and subsequently developed hypocalciuria and hypomagnesemia. A renal biopsy showed no evidence for juxtaglomerular hyperplasia. She developed chronic kidney failure at age 55 years, and ocular sclerochoroidal calcification, associated with BS and GS, at older than 65 years. Our aim was therefore to establish the genetic diagnosis in this patient using whole-genome sequencing (WGS). Leukocyte DNA was used for WGS analysis, and this revealed a homozygous c.226C > T (p.Arg76Ter) nonsense CLCNKB mutation, thereby establishing a diagnosis of BS type-3. WGS also identified 2 greater than 5-Mb regions of homozygosity that suggested likely mutational heterozygosity in her parents, who originated from a Greek island with fewer than 1500 inhabitants and may therefore have shared a common ancestor. Our results demonstrate the utility of WGS in establishing the correct diagnosis in renal tubular disorders with overlapping phenotypes.

Ap2s1 mutation causes hypercalcaemia in mice and impairs interaction between calcium-sensing receptor and adaptor protein-2.

Adaptor protein 2 (AP2), a heterotetrameric complex comprising AP2α, AP2ß2, AP2µ2 and AP2σ2 subunits, is ubiquitously expressed and involved in endocytosis and trafficking of membrane proteins, such as the calcium-sensing receptor (CaSR), a G-protein coupled receptor that signals via Gα11. Mutations of CaSR, Gα11 and AP2σ2, encoded by AP2S1, cause familial hypocalciuric hypercalcaemia types 1-3 (FHH1-3), respectively. FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with hypercalcaemia, which may be marked and symptomatic, and occasional hypophosphataemia and osteomalacia. To further characterize the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcaemia, hypermagnesaemia, hypophosphataemia, and increases in alkaline phosphatase activity and fibroblast growth factor-23. Plasma 1,25-dihydroxyvitamin D was normal, and no alterations in bone mineral density or bone turnover were noted. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. Co-immunoprecipitation studies showed that the AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2σ2 and the other AP2 subunits, and also with the CaSR. Cinacalcet, a CaSR positive allosteric modulator, decreased plasma calcium and parathyroid hormone concentrations in Ap2s1+/L15 mice, but had no effect on the diminished AP2σ2-CaSR interaction in vitro. Thus, our studies have established a mouse model that is representative for FHH3 in humans, and demonstrated that the AP2S1 p.Arg15Leu mutation causes a predominantly calcitropic phenotype, which can be ameliorated by treatment with cinacalcet.

Multiple Endocrine Neoplasia Type 1 (MEN1) 5'UTR Deletion, in MEN1 Family, Decreases Menin Expression.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of parathyroid, pancreatic and pituitary tumors, and is due to mutations in the coding region of the MEN1 gene, which encodes menin. We investigated a family with identical twins that had MEN1, with different MEN1 tumors. DNA sequence analysis of the MEN1 coding region had not identified any abnormalities and we hypothesized that deletions and mutations involving the untranslated regions may be involved. Informed consent and venous blood samples were obtained from five family members. Sanger DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses were performed using leukocyte DNA. This revealed a heterozygous 596bp deletion (Δ596bp) between nucleotides -1087 and -492 upstream of the translation start site, located within the MEN1 5' untranslated region (UTR), and includes the core promoter and multiple cis-regulatory regions. To investigate the effects of this 5'UTR deletion on MEN1 promoter activity, we generated luciferase reporter constructs, containing either wild-type 842bp or mutant 246bp MEN1 promoter, and transfected them into human embryonic kidney HEK293 and pancreatic neuroendocrine tumor BON-1 cells. This revealed the Δ596bp mutation to result in significant reductions by 37-fold (p < 0.0001) and 16-fold (p < 0.0001) in luciferase expression in HEK293 and BON-1 cells, respectively, compared to wild-type. The effects of this 5'UTR deletion on MEN1 transcription and translation were assessed using qRT-PCR and Western blot analyses, respectively, of mRNA and protein lysates obtained from Epstein-Barr-virus transformed lymphoblastoid cells derived from affected and unaffected individuals. This demonstrated the Δ596bp mutation to result in significant reductions of 84% (p < 0.05) and 88% (p < 0.05) in MEN1 mRNA and menin protein, respectively, compared to unaffected individuals. Thus, our results report the first germline MEN1 5'UTR mutation and highlight the importance of investigating UTRs in MEN1 patients who do not have coding region mutations. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

The Effect of Pet Insurance on Presurgical Euthanasia of Dogs With Gastric Dilatation-Volvulus: A Novel Approach to Quantifying Economic Euthanasia in Veterinary Emergency Medicine.

Euthanasia of companion animals in veterinary emergency medicine is a common cause of death. Euthanasia is economic when it is the consequence of the pet owner's inability to afford essential treatment while a viable medical alternative to euthanasia exists. Gastric dilatation-volvulus (GDV) is an acute life-threatening emergency condition of dogs; if left untreated, rapid death is highly likely. Surgical treatment leads to survival of around 80-90% of dogs; however, such treatment is costly. Therefore, pre-surgical euthanasia may be largely economically motivated. Having pet insurance, a financial instrument to reduce the burden of unforeseen veterinary medical costs on pet owners, would be expected to abolish the risk for pre-surgical economic euthanasia. We therefore aimed to determine whether pet insurance attenuates the risk of pre-surgical economic euthanasia in dogs with GDV. Non-referred dogs (n = 260) with GDV and known insurance status seen at 24 emergency clinics over a 2-year period were included. Relevant data (e.g., insurance status, age, comorbidities, outcome) were retrospectively extracted from a pet insurer's claim records (insured animals) or from electronic medical records of participating hospitals (non-insured animals). Forty-one percent of dogs (106 of 260 dogs) did not survive to hospital discharge; 82 (77%) of non-survivors died before surgery, all through euthanasia. The pre-surgical euthanasia rate was 10% in insured and 37% in non-insured dogs (p < 0.001). When adjusted for the effect of age, deposit size, comorbidities, and blood lactate concentration, the absence of insurance increased the odds of pre-surgical euthanasia by a factor of 7.4 (95% CI 2.0 to 37; p = 0.002). Of dogs undergoing surgery, 86% survived to hospital discharge. Overall, 80% of insured animals and 53% of non-insured animals survived to hospital discharge (p < 0.001). Thus, insurance was associated with a marked decrease in risk of pre-surgical euthanasia indicating that the cause of pre-surgical euthanasia of dogs with GDV is predominantly economic in nature. The rate of pre-surgical euthanasia in dogs with GDV may emerge as a suitable marker to quantify economic decision making of pet owners and to measure the impact of financial interventions aimed at mitigating economic duress associated with cost of veterinary emergency care.

Multiple Endocrine Neoplasia Type 1 (MEN1) Phenocopy Due to a Cell Cycle Division 73 (CDC73) Variant.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined occurrence of parathyroid tumors, pituitary adenomas, and pancreatic neuroendocrine neoplasms (PNENs). MEN1 is caused by germline MEN1 mutations in > 75% of patients, and the remaining 25% of patients may have mutations in unidentified genes or represent phenocopies with mutations in genes such as cell cycle division 73 (CDC73), the calcium sensing receptor (CASR), and cyclin-dependent kinase inhibitor 1B (CDKN1B), which are associated with the hyperparathyroidism-jaw tumor syndrome, familial hypocalciuric hypercalcemia type 1, and MEN4, respectively. Here, we report a heterozygous c.1138C>T (p.Leu380Phe) CDC73 germline variant in a clinically diagnosed MEN1 patient, based on combined occurrence of primary hyperparathyroidism, acromegaly, and a PNEN. Characterization of the PNEN confirmed it was a neuroendocrine neoplasm as it immuno-stained positively for chromogranin and glucagon. The rare variant p.Leu380Phe occurred in a highly conserved residue, and further analysis using RNA-Scope indicated that it was associated with a significant reduction in CDC73 expression in the PNEN. Previously, CDC73 mutations have been reported to be associated with tumors of the parathyroids, kidneys, uterus, and exocrine pancreas. Thus, our report of a patient with PNEN and somatotrophinoma who had a CDC73 variant, provides further evidence that CDC73 variants may result in a MEN1 phenocopy.

Aberrant methylation underlies insulin gene expression in human insulinoma.

Human insulinomas are rare, benign, slowly proliferating, insulin-producing beta cell tumors that provide a molecular "recipe" or "roadmap" for pathways that control human beta cell regeneration. An earlier study revealed abnormal methylation in the imprinted p15.5-p15.4 region of chromosome 11, known to be abnormally methylated in another disorder of expanded beta cell mass and function: the focal variant of congenital hyperinsulinism. Here, we compare deep DNA methylome sequencing on 19 human insulinomas, and five sets of normal beta cells. We find a remarkably consistent, abnormal methylation pattern in insulinomas. The findings suggest that abnormal insulin (INS) promoter methylation and altered transcription factor expression create alternative drivers of INS expression, replacing canonical PDX1-driven beta cell specification with a pathological, looping, distal enhancer-based form of transcriptional regulation. Finally, NFaT transcription factors, rather than the canonical PDX1 enhancer complex, are predicted to drive INS transactivation.

Genetic background influences tumour development in heterozygous Men1 knockout mice.

Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder caused by MEN1 germline mutations, is characterised by parathyroid, pancreatic and pituitary tumours. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP), a milder condition causing hyperparathyroidism only. Identical mutations can cause either MEN1 or FIHP in different families, thereby implicating a role for genetic modifiers in altering phenotypic expression of tumours. We therefore investigated the effects of genetic background and potential for genetic modifiers on tumour development in adult Men1+/- mice, which develop tumours of the parathyroids, pancreatic islets, anterior pituitary, adrenal cortex and gonads, that had been backcrossed to generate C57BL/6 and 129S6/SvEv congenic strains. A total of 275 Men1+/- mice, aged 5-26 months were macroscopically studied, and this revealed that genetic background significantly influenced the development of pituitary, adrenal and ovarian tumours, which occurred in mice over 12 months of age and more frequently in C57BL/6 females, 129S6/SvEv males and 129S6/SvEv females, respectively. Moreover, pituitary and adrenal tumours developed earlier, in C57BL/6 males and 129S6/SvEv females, respectively, and pancreatic and testicular tumours developed earlier in 129S6/SvEv males. Furthermore, glucagon-positive staining pancreatic tumours occurred more frequently in 129S6/SvEv Men1+/- mice. Whole genome sequence analysis of 129S6/SvEv and C57BL/6 Men1+/- mice revealed >54,000 different variants in >300 genes. These included, Coq7, Dmpk, Ccne2, Kras, Wnt2b, Il3ra and Tnfrsf10a, and qRT-PCR analysis revealed that Kras was significantly higher in pituitaries of male 129S6/SvEv mice. Thus, our results demonstrate that Kras and other genes could represent possible genetic modifiers of Men1.

NADPH Oxidase 4 Regulates Inflammation in Ischemic Heart Failure: Role of Soluble Epoxide Hydrolase.

Aims: Oxidative stress is implicated in cardiomyocyte cell death and cardiac remodeling in the failing heart. The role of NADPH oxidase 4 (NOX4) in cardiac adaptation to pressure overload is controversial, but its function in myocardial ischemic stress has not been thoroughly elucidated. This study examined the function of NOX4 in the pathogenesis of ischemic heart failure, utilizing mouse models, cell culture, and human heart samples. Results:Nox4-/- mice showed a protective phenotype in response to permanent left anterior descending coronary artery ligation with smaller infarction area, lower cardiomyocyte cross-sectional area, higher capillary density, and less cell death versus wild-type (WT) mice. Nox4-/- mice had lower activity of soluble epoxide hydrolase (sEH), a potent regulator of inflammation. Nox4-/- mice also showed a 50% reduction in the number of infiltrating CD68+ macrophages in the peri-infarct zone versus WT mice. Adenoviral overexpression of NOX4 in cardiomyoblast cells increased sEH expression and activity and CCL4 and CCL5 levels; inhibition of sEH activity in NOX4 overexpressing cells attenuated the cytokine levels. Human hearts with ischemic cardiomyopathy showed adverse cardiac remodeling, increased NOX4 and sEH protein expression and CCL4 and CCL5 levels compared with control nonfailing hearts. Innovation and Conclusion: These data from the Nox4-/- mouse model and human heart tissues show for the first time that oxidative stress from increased NOX4 expression has a functional role in ischemic heart failure. One mechanism by which NOX4 contributes to ischemic heart failure is by increasing inflammatory cytokine production via enhanced sEH activity.

MiR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs.

Multiple Endocrine Neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic islet tumours, and is due to mutations of the MEN1 gene, which encodes the tumour suppressor protein menin. Menin has multiple roles in genome stability, transcription, cell division and proliferation, but its mechanistic roles in tumourigenesis remain to be fully elucidated. MicroRNAs (miRNA) are non-coding single stranded RNAs that post-transcriptionally regulate gene expression and have been associated with tumour development, although the contribution of miRNAs to MEN1-associated tumourigenesis and their relationship with menin expression are not fully understood. Alterations in miRNA expression, including downregulation of three putative 'tumour suppressor' miRNAs, miR-15a, miR-16-1 and let-7a, have been reported in several tumour types including non-MEN1 pituitary adenomas. We have therefore investigated the expression of miR-15a, miR-16-1 and let-7a in pituitary tumours that developed after 12 months of age in female mice with heterozygous knock out of the Men1 gene (Men1+/- mice). The miRNAs miR-15a, miR-16-1 and let-7a were significantly downregulated in pituitary tumours (by 2.3-fold, p<0.05; 2.1-fold p<0.01 and 1.6-fold p<0.05, respectively) of Men1+/- mice, compared to normal wild type pituitaries. MiR-15a and miR-16-1 expression inversely correlated with expression of cyclin D1, a known pro-tumourigenic target of these miRNAs, and knock down of menin in a human cancer cell line (HeLa), and AtT20 mouse pituitary cell line resulted in significantly decreased expression of miR-15a (p<0.05), indicating that the decrease in miR-15a may be a direct result of lost menin expression.

The epidemiology of Taenia spp. infection and Taenia solium cysticerci exposure in humans in the Central Highlands of Vietnam.

BACKGROUND: Vietnam is endemic for taeniasis and T. solium cysticercosis. Despite this, information on the epidemiological characteristics of the diseases in the Central Highlands of Vietnam are poorly described. The aims of this study were to determine the epidemiological characteristics of taeniasis (Taenia spp.) and T. solium cysticerci exposure in humans in Dak Lak province in the Central Highlands, Vietnam. METHODS: This cross-sectional study was carried out in six villages in three districts of Dak Lak. A total of 190 households were visited. From each household, between one and five individuals were asked to donate a single faecal and blood sample and respond to a questionnaire. Serum samples were subjected to lentil lectin purified glycoprotein enzyme-linked immunoelectrotransfer blot assay to detect antibodies against T. solium cysticerci. Multiplex real-time PCR was used to detect Taenia spp. infection in faecal samples. A fixed-effects logistic regression model was developed to identify factors associated with the probability of Taenia spp. infection or T. solium cysticerci exposure risk. The contribution of each of identified factor was quantified using population attributable fractions. RESULTS: The prevalence of seroexposure to T. solium in Dak Lak was 5% (95% CI 3% to 8%). Consumption of raw vegetables, sourcing drinking water from lakes, streams or ponds and the practice of outdoor defaecation were identified as primary risk factors for the prevalence of T. solium cysticerci exposure, while consuming undercooked pork and beef, pork tongue and observing Taenia proglottids in stool were associated with Taenia spp. infection. Consumption of raw vegetables attributed to 74% of T. solium cysticerci exposure-positive cases and consumption of undercooked beef attributed to 77% of taeniasis cases in these communities. CONCLUSIONS: The prevalence of T. solium seroexposure in Dak Lak is consistent with those reported in other regions of Vietnam. The identified risk factors associated with the prevalence of T. solium seroexposure and taeniasis infection in Dak Lak are modifiable and thus advocate for targeted community intervention programs to mitigating these risks.

Spatial distribution of Taenia solium exposure in humans and pigs in the Central Highlands of Vietnam.

BACKGROUND: Taenia solium, a pork-borne parasitic zoonosis, is the cause of taeniasis and cysticercosis in humans. In Vietnam, poor sanitation, the practice of outdoor defecation and consumption of raw/undercooked pork have been associated with infection/exposure to T. solium in both humans and pigs. The broad-scale geographic distribution of the prevalence of T. solium varies throughout the country with infection restricted to isolated foci in the north and a more sporadic geographic distribution in the Central Highlands and the south. While cross-sectional studies have allowed the broad-scale geographic distribution of T. solium to be described, details of the geographic distribution of T. solium at finer spatial scales have not been described in detail. This study provides a descriptive spatial analysis of T. solium exposure in humans and pigs and T. solium taeniasis in humans within individual households in village communities of Dak Lak in the Central Highlands of Vietnam. METHODOLOGY/PRINCIPAL FINDINGS: We used Ripley's K-function to describe spatial dependence in T. solium exposure positive and negative human and pig households and T. solium taeniasis exposure positive and negative households in villages within the districts of Buon Don, Krong Nang and M'Drak of Dak Lak province in the Central Highlands of Vietnam. The prevalence of exposure to T. solium in pigs in Dak Lak province was 9 (95% CI 5 to 17) cases per 1000 pigs at risk. The prevalence of exposure to the parasite in humans was somewhat higher at 5 (95% CI 3 to 8) cases per 100 individuals at risk. Spatial aggregations of T. solium exposure-positive pig and human households occurred in some, but not all of the villages in the three study districts. Human exposure-positive households were found to be aggregated within a distance of 200 to 300 m in villages in Krong Nang district compared with distances of up to 1500 m for pig exposure-positive households in villages in M'Drak district. Although this study demonstrated the aggregation of households in which either T. solium exposure- or taeniasis-positive individuals were present, we were unable to identify an association between the two due to the very low number of T. solium taeniasis-positive households. CONCLUSIONS: Spatial aggregations of T. solium exposure-positive pig and human households occurred in some, but not all of the villages in the three study districts. We were unable to definitively identify reasons for these findings but speculate that they were due to a combination of demographic, anthropological and micro-environmental factors. To more definitively identify characteristics that increase cysticercosis risk we propose that cross-sectional studies similar in design to that described in this paper should be applied in other provinces of Vietnam.

Molecular Genetic Studies of Pancreatic Neuroendocrine Tumors: New Therapeutic Approaches.

Pancreatic neuroendocrine tumors (PNETs) arise sporadically or as part of familial syndromes. Genetic studies of hereditary syndromes and whole exome sequencing analysis of sporadic NETs have revealed the roles of some genes involved in PNET tumorigenesis. The multiple endocrine neoplasia type 1 (MEN1) gene is most commonly mutated. Its encoded protein, menin, has roles in transcriptional regulation, genome stability, DNA repair, protein degradation, cell motility and adhesion, microRNA biogenesis, cell division, cell cycle control, and epigenetic regulation. Therapies targeting epigenetic regulation and MEN1 gene replacement have been reported to be effective in preclinical models.

The epidemiology of porcine Taenia solium cysticercosis in communities of the Central Highlands in Vietnam.

BACKGROUND: Taenia solium cysticercosis, recognized as a neglected tropical disease by the WHO, is distributed mostly in developing countries of Latin America, sub-Saharan Africa and Asia. Pigs and humans act as intermediate hosts, acquiring T. solium cysticerci (larval stage) in their tissue, through the ingestion of T. solium eggs shed in the faeces of humans infected with adult tapeworms. The disease has a negative impact on rural economies due to losses in productivity arising from human disease, pork carcass condemnations and loss of market access. The aim of this study was to estimate the prevalence of T. solium cysticercosis in pigs in Dak Lak Province in the Central Highlands of Vietnam and to identify household level characteristics associated with T. solium porcine cysticercosis. METHODS: This was a cross-sectional study of household pigs in three districts of Dak Lak Province. A total of 408 households in six villages in three districts were visited between June and October 2015. A questionnaire was administered to the head of each household, and within each household, serum samples were collected from three pigs. Serum samples were analyzed using the recombinant T24H antigen in enzyme-linked immunoelectrotransfer blot assay and lentil lectin purified glycoprotein in EITB assay. A Bayesian, mixed-effects logistic regression model was developed to identify management factors associated with the probability of a household having at least one cysticercosis-positive pig. RESULTS: The prevalence of porcine T. solium cysticercosis in this study was low at 0.94 [95% confidence interval (CI) 0.51-1.68] cases per 100 pigs at risk, in agreement with other studies conducted throughout Vietnam. Scavenging of food and coprophagy were associated with T. solium cysticercosis [odds ratios 1.98 (95% CrI: 0.55-4.74) and 2.57 (95% CrI: 1.22-4.66), respectively]. CONCLUSIONS: This study proves that the seroprevalence of porcine cysticercosis in Dak Lak Province was as low as that of other studies conducted throughout Vietnam. Scavenging of food and coprophagy are modifiable factors, providing the opportunity to decrease the prevalence of porcine cysticercosis further in the province.