The examination of some virulence factors in S. aureus isolates obtained from the healthy human population, sheep mastitis, and cheese.

Abstract. Background: Staphylococcus aureus is responsible for many infections in humans and animals from skin and soft tissue infections to life-threatening diseases. In this study to explore the origin of S. aureus infections in humans, the antibiotic resistance profile and the variety of virulence factors in S. aureus isolates were examined in three groups: a healthy human population, cheese, and the milk of sheep with mastitis. Aims: The examination of some virulence factors in S. aureus isolates obtained from the healthy human population, sheep mastitis, and cheese. Methods: A total of 400 nasal swab samples from healthy students, 30 cheese samples, and 122 sheep milk samples were collected for the detection of S. aureus isolates from January 1, 2018, to March 1, 2018. The frequency of hla, hlb, Acme/arcA, pvl, and tsst-1 virulence genes and mecA gene was determined in each group by PCR assay. Results: There was a direct relationship between the antibiotic susceptibility profile of the isolates from a healthy population and those from mastitis milk samples. Of 400 nasal samples, 15% (60/400) were positive for S. aureus, of which 60% (36/60) were positive for mecA. While 50% (15/30) of cheese samples were positive for S. aureus. of which 7 cases (46.66%, 7/15) were positive for mecA. The prevalence of S. aureus among students was dependent on gender (P=0.025). Also, 47.5% (58/122) of milk samples from sheep mastitis were positive for S. aureus, and 41.37% (24/58) were positive for the mecA gene. Based on PCR results, the highest rate of hla (68.33%, 41/60), hlb (53.33%, 32/60), and Acme/arcA (46.66%, 28/60) genes were related to a healthy population, and the highest frequency of pvl (41.38%, 24/58), and tsst-1 (27.59%, 16/58) was related to milk samples (P<0.05). A significant correlation was observed between the presence of the arginine catabolic mobile element (ACME)-arcA gene and resistance to methicillin (P<0.05). Conclusion: The high rate of virulence factors in the S. aureus isolates obtained from mastitis and dairy products is an alert point, because they could be source of the spreading of S. aureus to humans. There is an essential need for continuous monitoring to control staphylococcal food poisoning.

Spatiotemporal dynamics of self-organized branching in pancreas-derived organoids.

The development dynamics and self-organization of glandular branched epithelia is of utmost importance for our understanding of diverse processes ranging from normal tissue growth to the growth of cancerous tissues. Using single primary murine pancreatic ductal adenocarcinoma (PDAC) cells embedded in a collagen matrix and adapted media supplementation, we generate organoids that self-organize into highly branched structures displaying a seamless lumen connecting terminal end buds, replicating in vivo PDAC architecture. We identify distinct morphogenesis phases, each characterized by a unique pattern of cell invasion, matrix deformation, protein expression, and respective molecular dependencies. We propose a minimal theoretical model of a branching and proliferating tissue, capturing the dynamics of the first phases. Observing the interaction of morphogenesis, mechanical environment and gene expression in vitro sets a benchmark for the understanding of self-organization processes governing complex organoid structure formation processes and branching morphogenesis.

HDAC1 and HDAC2 integrate the expression of p53 mutants in pancreatic cancer.

Mutation of p53 is a frequent genetic lesion in pancreatic cancer being an unmet clinical challenge. Mutants of p53 have lost the tumour-suppressive functions of wild type p53. In addition, p53 mutants exert tumour-promoting functions, qualifying them as important therapeutic targets. Here, we show that the class I histone deacetylases HDAC1 and HDAC2 contribute to maintain the expression of p53 mutants in human and genetically defined murine pancreatic cancer cells. Our data reveal that the inhibition of these HDACs with small molecule HDAC inhibitors (HDACi), as well as the specific genetic elimination of HDAC1 and HDAC2, reduce the expression of mutant p53 mRNA and protein levels. We further show that HDAC1, HDAC2 and MYC directly bind to the TP53 gene and that MYC recruitment drops upon HDAC inhibitor treatment. Therefore, our results illustrate a previously unrecognized class I HDAC-dependent control of the TP53 gene and provide evidence for a contribution of MYC. A combined approach targeting HDAC1/HDAC2 and MYC may present a novel and molecularly defined strategy to target mutant p53 in pancreatic cancer.

Effects of obeticholic acid on lipoprotein metabolism in healthy volunteers.

The bile acid analogue obeticholic acid (OCA) is a selective farnesoid X receptor (FXR) agonist in development for treatment of several chronic liver diseases. FXR activation regulates lipoprotein homeostasis. The effects of OCA on cholesterol and lipoprotein metabolism in healthy individuals were assessed. Two phase I studies were conducted to evaluate the effects of repeated oral doses of 5, 10 or 25 mg OCA on lipid variables after 14 or 20 days of consecutive administration in 68 healthy adults. Changes in HDL and LDL cholesterol levels were examined, in addition to nuclear magnetic resonance analysis of particle sizes and sub-fraction concentrations. OCA elicited changes in circulating cholesterol and particle size of LDL and HDL. OCA decreased HDL cholesterol and increased LDL cholesterol, independently of dose. HDL particle concentrations declined as a result of a reduction in medium and small HDL. Total LDL particle concentrations increased because of an increase in large LDL particles. Changes in lipoprotein metabolism attributable to OCA in healthy individuals were found to be consistent with previously reported changes in patients receiving OCA with non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

Histochemical study of the oesophagus in the chukar partridge (Alectoris chukar) embryo.

Study of the histogenesis of different organs is a useful laboratory method which helps us achieve accurate basic information about organ development during the embryonic period. It also reveals histological differences of each organ in different species. This research was carried out to study the histogenesis of the oesophagus of chukar partridge. For this purpose, the embryonated eggs were placed in the incubator and the embryos were collected between the 5th to 24th days of incubation period. The specimens were fixed in Bouin's solution, and routine histotechnique processes were performed. The slides were finally stained with haematoxylin-eosin, Periodic Acid Schiff, Alcian Blue and Masson Trichrome, and the developmental changes of the oesophagus during the embryonic period were studied by light microscope. The four layers of oesophagus, including, the mucosa, submucosa, tunica muscularis and advantitia/serosa, both in cervical and thoracic oesophagus, were studied. During days 5 to 24 of incubation, developmental events in the oesophagus such as changes in the oesophageal epithelium, formation of muscularis mucosae and tunica muscularis, development of the mucous glands and the type of their secretion, were observed. Finally the results were compared with those of other studied avian species and the similarities and differences were discussed.

Kras(G12D) induces EGFR-MYC cross signaling in murine primary pancreatic ductal epithelial cells.

Epidermal growth factor receptor (EGFR) signaling has a critical role in oncogenic Kras-driven pancreatic carcinogenesis. However, the downstream targets of this signaling network are largely unknown. We developed a novel model system utilizing murine primary pancreatic ductal epithelial cells (PDECs), genetically engineered to allow time-specific expression of oncogenic Kras(G12D) from the endogenous promoter. We show that primary PDECs are susceptible to Kras(G12D)-driven transformation and form pancreatic ductal adenocarcinomas in vivo after Cdkn2a inactivation. In addition, we demonstrate that activation of Kras(G12D) induces an EGFR signaling loop to drive proliferation. Interestingly, pharmacological inhibition of EGFR fails to decrease Kras(G12D)-activated ERK or PI3K signaling. Instead our data provide novel evidence that EGFR signaling is needed to activate the oncogenic and pro-proliferative transcription factor c-MYC. EGFR and c-MYC have been shown to be essential for pancreatic carcinogenesis. Importantly, our data link both pathways and thereby explain the crucial role of EGFR for Kras(G12D)-driven carcinogenesis in the pancreas.

Transgenic expression of oncogenic BRAF induces loss of stem cells in the mouse intestine, which is antagonized by ß-catenin activity.

Colon cancer cells frequently carry mutations that activate the ß-catenin and mitogen-activated protein kinase (MAPK) signaling cascades. Yet how oncogenic alterations interact to control cellular hierarchies during tumor initiation and progression is largely unknown. We found that oncogenic BRAF modulates gene expression associated with cell differentiation in colon cancer cells. We therefore engineered a mouse with an inducible oncogenic BRAF transgene, and analyzed BRAF effects on cellular hierarchies in the intestinal epithelium in vivo and in primary organotypic culture. We demonstrate that transgenic expression of oncogenic BRAF in the mouse strongly activated MAPK signal transduction, resulted in the rapid development of generalized serrated dysplasia, but unexpectedly also induced depletion of the intestinal stem cell (ISC) pool. Histological and gene expression analyses indicate that ISCs collectively converted to short-lived progenitor cells after BRAF activation. As Wnt/ß-catenin signals encourage ISC identity, we asked whether ß-catenin activity could counteract oncogenic BRAF. Indeed, we found that intestinal organoids could be partially protected from deleterious oncogenic BRAF effects by Wnt3a or by small-molecule inhibition of GSK3ß. Similarly, transgenic expression of stabilized ß-catenin in addition to oncogenic BRAF partially prevented loss of stem cells in the mouse intestine. We also used BRAF(V637E) knock-in mice to follow changes in the stem cell pool during serrated tumor progression and found ISC marker expression reduced in serrated hyperplasia forming after BRAF activation, but intensified in progressive dysplastic foci characterized by additional mutations that activate the Wnt/ß-catenin pathway. Our study suggests that oncogenic alterations activating the MAPK and Wnt/ß-catenin pathways must be consecutively and coordinately selected to assure stem cell maintenance during colon cancer initiation and progression. Notably, loss of stem cell identity upon induction of BRAF/MAPK activity may represent a novel fail-safe mechanism protecting intestinal tissue from oncogene activation.

Clinical and genetic characterization of Bardet-Biedl syndrome in Tunisia: defining a strategy for molecular diagnosis.

Bardet-Biedl syndrome (BBS, OMIM 209900) is a rare genetic disorder characterized by obesity, retinitis pigmentosa, post axial polydactyly, cognitive impairment, renal anomalies and hypogonadism. The aim of this study is to provide a comprehensive clinical and molecular analysis of a cohort of 11 Tunisian BBS consanguineous families in order to give insight into clinical and genetic spectrum and the genotype-phenotype correlations. Molecular analysis using combined sequence capture and high-throughput sequencing of 30 ciliopathies genes revealed 11 mutations in 11 studied families. Five mutations were novel and six were previously described. Novel mutations included c.1110G>A and c.39delA (p.G13fs*41) in BBS1, c.115+5G>A in BBS2, c.1272+1G>A in BBS6, c.1181_1182insGCATTTATACC in BBS10 (p.S396Lfs*6). Described mutations included c.436C>T (p.R146*) and c.1473+4A>G in BBS1, c.565C> (p.R189*) in BBS2, deletion of exons 4-6 in BBS4, c.149T>G (p.L50R) in BBS5, and c.459+1G>A in BBS8; most frequent mutations were described in BBS1 (4/11, 37%) and BBS2 (2/11, 18%) genes. No phenotype-genotype correlation was evidenced. This data expands the mutations profile of BBS genes in Tunisia and suggests a divergence of the genetic spectrum comparing Tunisian and other populations.

Detailed molecular characterisation of acute myeloid leukaemia with a normal karyotype using targeted DNA capture.

Advances in sequencing technologies are giving unprecedented insights into the spectrum of somatic mutations underlying acute myeloid leukaemia with a normal karyotype (AML-NK). It is clear that the prognosis of individual patients is strongly influenced by the combination of mutations in their leukaemia and that many leukaemias are composed of multiple subclones, with differential susceptibilities to treatment. Here, we describe a method, employing targeted capture coupled with next-generation sequencing and tailored bioinformatic analysis, for the simultaneous study of 24 genes recurrently mutated in AML-NK. Mutational analysis was performed using open source software and an in-house script (Mutation Identification and Analysis Software), which identified dominant clone mutations with 100% specificity. In each of seven cases of AML-NK studied, we identified and verified mutations in 2-4 genes in the main leukaemic clone. Additionally, high sequencing depth enabled us to identify putative subclonal mutations and detect leukaemia-specific mutations in DNA from remission marrow. Finally, we used normalised read depths to detect copy number changes and identified and subsequently verified a tandem duplication of exons 2-9 of MLL and at least one deletion involving PTEN. This methodology reliably detects sequence and copy number mutations, and can thus greatly facilitate the classification, clinical research, diagnosis and management of AML-NK.

Only two mutations detected in 15 Tunisian patients with 11ß-hydroxylase deficiency: the p.Q356X and the novel p.G379V.

Steroid 11ß-hydroxylase deficiency is the second most common cause of congenital adrenal hyperplasia, resulting in virilization, glucocorticoid deficiency and hypertension. The 11ß-hydroxylase enzyme is encoded by the CYP11B1 gene and mutations in this gene are responsible for this disease. The aim of this study was to characterize mutations in the CYP11B1 gene and to determine their frequencies in a cohort of Tunisian patients. The molecular genetic analysis was performed by direct nucleotide sequencing of the CYP11B1 gene in 15 unrelated Tunisian patients suffering from classical 11ß-hydroxylase deficiency. Only two mutations were detected in homozygous state in the CYP11B1 gene of all patients, the p.Q356X in exon 6 (26.6%) and the novel p.G379V in exon 7 with large prevalence (73.3%). This is the first report of screening for mutations of CYP11B1 gene in the Tunisian population and even in the Arab population.

A novel UBE3A truncating mutation in large Tunisian Angelman syndrome pedigree.

We identified in a large Tunisian pedigree a novel UBE3A frameshift mutation in exon 16 coding region, and we expect that the resulting UBE3A truncated protein in our patients is non-functional since the mutation implies the catalytic region of the enzyme. The family includes 14 affected patients born from four sisters. This mutation was found in all surviving affected individuals and their mothers pointing out the importance of genetic counseling possibility in Angelman syndrome (AS). All patients had severe mental retardation with epilepsy and microcephaly. Minor clinical expression variation was observed among the investigated patients. The severity of clinical expression is related to the detected molecular variation: deletion of 15 bp and insertion of 7 bp. These results are concordant with the gene expression observed in previously reported individuals with AS and truncated UBE3A protein.

[Genetic analysis of Turner syndrome: 89 cases in Tunisia]. / Analyse génétique du syndrome de Turner : étude tunisienne de 89 cas.

Turner's syndrome (TS) affects about 1/2500 female infants born alive. The syndrome results from total or partial absence of one of the two X chromosomes normally present in females. We report the results of a retrospective analysis of 89 cases of TS observed during a six-year period (2000-2005). The patients' age ranged from two days to 51 years at the time of this analysis. Most patients were adults (48%). The aim of this study is to ascertain the principal clinical features leading to a request for a karyotype, searching for a possible relationship between chromosomal anomalies and clinical expression of TS. Pediatric patients were referred for statural retardation or dysmorphic features, while reproduction anomalies were the main indication for karyotyping in patients aged over 20 years. Mosaicism was prevalent (47%), whereas the homogeneous karyotype 45,X was found in only 32% of the patients; structural anomalies were found in 21%. Regarding the advanced age of our patients, we established a relationship between chromosome anomalies and the clinical expression of TS, based on an analysis of stature and reproduction disorders. Short stature and primary amenorrhea were correlated with total deletion of one chromosome X or imbalanced gene dosage due to structural X anomalies. Whereas cases of infertility, recurrent miscarriages and secondary amenorrhea were associated with a mosaic karyotype pattern (45,X/46,XX or 45,X/46,XX/47,XXX ...), with a slight mosaicism in most cases. Thus, chromosome investigations should be performed in cases of reproduction failure even for women with normal stature.

Y chromosome microdeletions in Tunisian infertile males.

AIM: To determine frequency of Y microdeletions in azoospermic and oligospermic Tunisian infertile males. METHODS: A Sample of 146 Tunisian infertile males with a low sperm count (<5 x 10(6) sperms per mililiter) and normal karyotype was screened for Y chromosome microdeletions. 76 men were azoospermic and 70 men were oligospermic. Genomic DNA was isolated from blood and multiplex PCR was carried out with a set of 20 AZFa, AZFb and AZFc STS markers to detect the microdeletions as recommended by the European Academy of Andrology. RESULTS: In 10/146 (6.85%) subjects AZF deletions were observed. Of these ten males with microdeletions, 9/10 subjects were azoospermic (90%), 1/10 was oligospermic (10%). Frequency of microdeletions in azoospermic men was 9/76 (11.84%). None of the patients showed isolated microdeletion in the AZFa region, but one azoospermic man had deletion in the AZFb region. Eight azoospermic patients and one oligospremic man have AZFc microdeletions. AZFc and AZFb were deleted in three azoospermic patients. AZFc, AZFb and AZFa were deleted in three azoospermic patients We estimate the sensitivity of the test comprising six STS in our sample to be 90%. CONCLUSION: The incidence of Yq microdeletions in the study population of infertile Tunisian men falls within the range published in other countries. We suggest to analyze 9STS in the first step to detect efficiently Y microdeletions in our population.

[Myalgia in familial Mediterranean fever]. / Myalgies au cours d'une fièvre méditerranéenne familiale.

INTRODUCTION: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of fever and painful episodes of sterile peritonitis, pleuritis and arthritis. Among rare symptoms of the disease, muscular manifestations, first described in 1945, sometimes as one of the main clinical manifestations or as its sole feature should be recognized. We present a patient with FMF in whom severe myalgia were predominant. CASE REPORT: An 18 year-old Tunisian boy treated with corticosteroids for an "inflammatory myopathy" in another institution was admitted for abdominal pain. FMF was suspected because of a history of paroxysmal abdominal pain with fever from the age of 5 leading two times to laparotomy and one attack of left knee arthritis at the age of 14. FMF diagnosis was confirmed genetically, corticosteroids were tapered and a treatment with colchicine was started. Two years and a half later, he was admitted for severe and incapacitating myalgia of the upper and lower limbs without fever nor abdominal pain that responded well to rest and colchicine. Myalgia was then definitively attached to FMF. CONCLUSION: Three clinical patterns of myalgia are now well identified in FMF: the spontaneous pattern as observed in our patient, the exercise-induced pattern and the protracted febrile myalgia syndrome. The three patterns differ in the severity of pain, grade of fever and duration of the episode.

Lack of RUNX3 regulation in human gastric cancer.

It has been proposed that the transcription factor RUNX3 is the product of a gastric tumour suppressor gene. We examined RUNX3 expression in gastric biopsies from 105 patients with different histological presentations. Surprisingly, immunohistochemical staining detected RUNX3 protein expression only in infiltrating leukocytes but not in the gastric epithelium. Using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we confirmed that the level of RUNX3 mRNA expression in the gastric epithelium was very low and was influenced neither by H. pylori infection nor by neoplastic transformation. Instead, RUNX3 was highly expressed in the gastric stroma and the level of expression correlated with the magnitude of H. pylori-induced gastric inflammation. The low level of RUNX3 expression in gastric epithelium and the absence of downregulation in gastric cancer do not support the hypothesis that RUNX3 functions as a gastric tumour suppressor gene.

Outcome of Kaposi's sarcoma and graft following discontinuation of immunosuppressive drugs in renal transplant recipients.

PURPOSE: Owing to the use of immunosuppressive drugs, renal transplant recipients are at risk for malignancies including Kaposi's sarcoma (KS). Following the diagnosis, physicians tend to decrease the doses of immunosuppressive drugs to lower tumor progression rate. On the other hand, those who receive lower doses of immunosuppressive drugs are at a higher risk for acute rejection. In this study, we evaluated the outcome of KS on renal allografts following discontinuation or decrease in the doses of drugs. METHODS: Since 1984, 14 (nine men and five women) among 2000 cases of renal transplantation have been diagnosed as KS. In 11 patients, cyclosporine was completely discontinued, the dosage was decreased to half of the initial dose in other cases. Except one case, we discontinued either azathioprine or mycophenolate mofetil. RESULTS: During 57 months of follow-up on average, the serum creatinine level remained normal in 10 but increased in four cases. Kidney function deteriorated in two of these four patients at the beginning of study. Three patients died with normal serum creatinine levels. Discontinuation of immunosuppressive drugs caused complete remission of KS in all patients except one who received chemotherapy. CONCLUSION: Discontinuation of immunosuppressants following the diagnosis of KS caused complete remission of this cancer in almost all patients and seemed to be relatively safe for kidney graft function.