In vitro interaction and colocalization of HSV-1 ORF P with a cellular splicing factor (SC35) using pulldown assay

Herpes simplex virus type-1 (HSV-1) causes a variety of diseases in human. This virus is a neurotropic pathogen of human that establishes latent infection in the sensory ganglia innervating the site of primary infection. A number of genes including ICP34.5 control HSV-1 pathogenicity and ICP34.5 has been identified as HSV-1 virulence gene. Open reading frame P (ORF P) is also a HSV-1 gene that might have a role in latency. A complication in the analysis of the role of ICP34.5 and ORF P in the HSV-1 life cycle is that these two are overlapping antisense genes. ORF P is also deleted in ICP34.5 negative mutants and to date, no definite function is attributed to it. To attribute characteristics which were originally attributed solely to ICP34.5 to each of these two genes (ORF P or ICP34.5), an approach is to construct a number of HSV-1 recombinant viruses that express ICP34.5 and ORF P independently. An alternative way is to determine if ORF P interacts with any of the cellular and viral proteins both in vitro and in vivo. Using Glutathione-S-transferase (GST) pulldown assay and Western-blotting, we showed that ORF P interacts with a cellular splicing factor (SC35) in vitro. To investigate the colocalization of ORF P and SC35, nuclear and cytoplasmic fractionation of ORF P/SC35 was also carried out. Our results showed that both SC35 and ORF P are located in the nucleus of HSV-1 infected cells. Conclusively, because ORF P interacts and colocalizes with SC35, it might have a role in splicing

SIGNAL ABSTRACTION FOR ROOT CAUSE IDENTIFICATION OF CONTROL SYSTEMS MALFUNCTIONS IN CONNECTED VEHICLES

Today’s automotive control systems have gained huge advantage from using integrated software and hardware to reliably manage the performance of vehicles. The integration of largescale software with many hardware components, however, have increased the complexity of diagnosis and root cause analysis for a detected malfunction. High level of expertise and detailed knowledge of the underlying software and hardware are typically required to analyze a large list of variables and precisely identify the root cause of the malfunction. In this paper, an abstraction method is presented to identify the most important signals for a root cause analysis by leveraging data collected from a connected fleet of field vehicles. A novel label propagation methodology is proposed to select the most relevant signals for the root cause analysis by detecting linear and nonlinear correlations between an observed malfunction and candidate test signals of the control system. The proposed label propagation method eliminates the requirement for a priori known correlation kernel that is needed for a regression analysis. The signal abstraction method is applied and successfully tested for abstracting signals in the fuel control system, with high degree of interconnection between software and hardware, using data from more than 5000 connected vehicles

Detecting enteropathogenic Campylobacters in chicken feces by PCR and comparing with culture method

امروزه کامپیلوباکترها از شایع ترین علل اسهال های باکتریایی در سرتاسر جهان محسوب می شوند. در جنس کامپیلوباکتر 18 گونه و تحت گونه وجود دارد که گونه های Campylobacter jejuni و Campylobacter coli مسبب اکثر عفونت های انسانی ناشی از کامپیلوباکترها می باشند و مهم ترین بیماری آنها نیز اسهال می باشد. بیماری در انسان متعاقب مصرف آب و مواد غذایی بویژه گوشت ماکیان رخ می دهد. تشخیص باکتریولوژیک این باکتری با دشواری زیادی از قبیل طولانی بودن مدت انکوباسیون و محدودیت تست های افتراقی همراه است. بنابراین استفاده از روش های دیگر تشخیصی امروزه مورد توجه قرار گرفته است. تعداد 116 نمونه رکتال سواپ از مرغ های موجود در مرغداری های شهر اصفهان گرفته شده و پس از 18-12 ساعت غنی سازی در محیط Campy-Thio، در محیط اختصاصی Campylobacter selective agar کشت شدند. برای شناسایی و تعیین گونه ها از رنگ آمیزی گرم و تست های اکسیداز، کاتالاز، هیدرولیز هیپورات و سنجش حساسیت به نالیدیسیک اسید و سفالوتین استفاده شد. جهت استخراج DNA از کیت های اختصاصی استفاده شد و با استفاده از پرایمرهای ویژه گونه های ژژونی و کلی اقدام به انجام PCR (Palymeras Chain Reaction) گردید. نتیجه کشت در 11 مورد مثبت بود (5/9) که از این تعداد 8 مورد مربوط به گونه ژژونی و 3 مورد مربوط به گونه کلی بود. نتیجه PCR نمـونه هـا در 27 مـورد مثـبت بود (2/23) که از این تعداد 18 مورد مربوط به گونه ژژونی و 9 مورد مربوط به گونه کلی بود. حساسیت و ویژگی روش PCR در مقایسه با روش کشت بترتیب 100 و 7/84 درصد بود. روش PCR مورد استفاده در این تحقیق قادر به شناسایی تمام نمونه های کشت مثبت بود و نتایج تعیین گونه با این روش تطابق کاملی با روش بیوشیمیایی داشت. بنابراین با توجه به دقت بالا و سرعت زیاد به نظر می رسد روش PCR جایگزین مناسبی جهت کشت در تشخیص کامپیلوباکترها در نمونه های مرغ ها باشد

Tumor microsatellite instability and clinicopathologic features in Iranian colorectal cancer patients at risk for Lynch syndrome

Background: Microsatellite instability (MSI) is a mutational signature that is the hallmark of Lynch syndrome, and MSI testing is a cost-effective method to screen the disease. Since there is no enough data about MSI status and associated clinicopathologic features of hereditary nonpolyposis colorectal cancer (HNPCC) in Iran, our study is a new trial to describe them in center of Iran (Isfahan). Materials and Methods: It is a descriptive retrospective study to screen HNPCC families using Amsterdam II criteria in Central Iran within 2000-2013. For MSI testing, we used a commercially available kit evaluating mononucleotide markers (BAT25, BAT-26, MON0-27, NR-21 and NR-24). After a fluorescent multiplex polymerase chain reaction amplification of the markers, samples were sequenced to fragment analysis. Data analysis was performed using SPSS 16 software (SPSS Inc., Chicago, IL, USA). Results: Overall, 31 of 45 screened HNPCC families were eventually included to MSI testing. Totally, 9/31 patients (29.0%) showed MSI in their tumor tissues. BAT-26 was the most instable marker with instability in 7/24 MSI tumors (29.2%). The mean age at diagnosis in microsatellite stable (MSS), MSI-Low (MSI-L), and MSI-High (MSI-H) probands was respectively 44.7 (standard deviation SD] = 11.83), 51.7 (SD = 16.17), and 36.0 (SD = 3.41) years. The most common tumor sites in MSS, MSI-L, and MSI-H probands were rectosigmoid (similar to 72.8%), rectum (66.7%) and right colon (50.0%), respectively. Of 186 cancer patients among 31 HNPCC families, 86 patients (46.2%) had colorectal cancer (CRC) and 100 patients (53.8%) had extracolonic cancers. The average of CRC affected members among MSS, MSI-L, and MSI-H groups of our HNPCC families was 2.2 (SD = 1.30), 3.3 (SD = 3.21), and 4.7 (SD = 2.42) patients per family, respectively. Stomach with 18.3% and 26.7% of all extracolonic cancers were most common involved organ in MSS and MSI-H families, respectively. Conclusion: Our different molecular results could be suggested to describe HNPCC families based on some new molecular mechanisms leading to MSS HNPCC phenotypes. Meanwhile, more evaluations within our population are recommended

Immunohistochemical analysis of mismatch repair proteins in Iranian Colorectal Cancer patients at risk for Lynch syndrome

Background: Hereditary non-polyposis colorectal cancer (HNPCC) is a common hereditary cancer predisposing syndrome has molecular and clinicopathological features still have remained ambiguous within Iranian populations. We discuss in this article some molecular and clinicopathological features of the condition. Methods: The study was a descriptive retrospective and designed on 1659 colorectal cancer (CRC) patients were screened based on early-onset disease and Amsterdam II criteria during 14 years (2000-2013). Immunohistochemistry (IHC) staining was set up to detect expression of mismatch repair (MMR) genes on paraffin-embedded tissue sections of 31 HNPCC-CRC tumors. SPSS 19 software was used to analyze the data. Results: IHC-MMR staining was absent in 7/31 individuals (22.6%) of which 4 cases showed IHC-Absent (IHC-A) in both MSH2 and MSH6 (57.1%), in 2 cases both MLH1 and PMS2 had negative staining (28.6%), and just in one case, MSH6 was defective (14.3%). The frequency of CRC among IHC-A and IHC-Present (IHC-P) families was 67.5% and 27.9%, respectively. Also the most frequent extracolonic cancers in IHC-A group were: stomach (10%), small bowel (5%), and prostate (5%); and in IHC-P group: stomach (18.4%), lung (10.9%), and breast (7.5%). Average age of IHC-A individuals at diagnosis was 38.0 versus 45.3 years in IHC-P individuals. Overall, 20.8% and 57.1% of our index CRCs were localized proximal to the splenic flexure in IHC-P and IHC-A groups, respectively. Conclusion: Given the lack of enough information about molecular aspects of hereditary cancer syndromes like HNPCC in Iran, more evaluations are necessary on larger samples using complementary techniques such as MSI-testing and mutation analyses

Familial colorectal cancertype X in central Iran: A new clinicopathologic description

Background: Familial colorectal cancer type X (FCCX) is a subtype of mismatchrepair (MMR)-proficient colorectal cancerin whichthe patients are clinicallyat risk for Lynch syndrome (LS), a common hereditary cancer predisposing syndrome.In this study, we describeda new clinicopathological feature of the condition in central Iran. Subjects and Methods: We designed a descriptive, retrospective study to screenat-riskcolorectal cancer (CRC) patients,usingAmsterdam II criteria and Molecular analysis in Isfahan (central Iran) throughout 2000-2013 period. Results: 219 early-onset (≤ 50 years) CRC patients of 1659 were selected for the evaluation. Amsterdam II criteria were positive in 45 families; of whom 31 were finally analyzed by molecular testing. MMR deficiency was detected in 7/31 probands (22.6%) as affected to LS, so 24 families (77.4%) were identified as FCCX. The mean age of the probands at diagnosis among FCCX families was 45.3 years (range 24-69) versus 38.0 years (range 31-50) in LS families.The frequency of CRC among FCCX and LS families was calculated 27.9% and 67.5%, respectively. Also, the most frequent extracolonic cancer among both FCCX and LS families was stomach by 25.5% and 30.8%, respectively. Tumor site was proximal to the splenic flexure in 20.8% and 57.1% of index CRC patients in FCCX and LS families, respectively. Conclusion: Given the relative high frequency of FCCXand its different phenotype among Iranian populations, we need to set up more advanced molecular studies for exploration of unknown molecular pathways leading to tumorigenesis in this class of CRC patients

Evaluation of Soluble Human Leukocyte Antigen-G (sHLA-G) Isoforms and Regulatory T Cells in Relapsing-Remitting Multiple Sclerosis.

Soluble forms of nonclassical human leukocyte antigen (HLA)-G have recently been suggested as immunomodulatory factors in multiple sclerosis (MS). HLA-G inhibits the effecter function of T cells and natural killer (NK) cells. Also regulatory T cells (Treg) are considered as pivotal players in MS pathogenesis. Thus, we aimed to evaluate the presence of HLA-G molecules and Treg cells in Relapsing-Remitting Multiple Sclerosis (RRMS) patients and compare it to healthy controls. Patients with RRMS (n=205, mean age=31.32±8.53) and healthy subjects (n=205, mean age=32.2±7.48) were studied. The patients subgrouped to untreated and treated with Interferon beta. Then sHLA-G levels (sHLA-G1 and sHLA-G5) were measured using ELISA method. Treg (CD4+ CD25+ Foxp3+) cells in patients who had sHLA-G>10 U/ml were characterized by using flow cytometry. Our data showed that there was no significant differences between RRMS patients and healthy controls in sHLA-G concentration (p>0.05). Treg cell frequencies were higher in the patients who had sHLA-G >10 U/ml compared to healthy subjects (p<0.05). Collectively, there was significant correlation between sHLA-G and frequency of Treg cells in treated RRMS patients and healthy individuals. It seems that high level sHLA-G has been instrumental in raising frequency of Treg cells in treated patients and could be associated with remission of MS disease

Simplified microsatellite instability detection protocol provides equivalent sensitivity to robust detection strategies in Lynch syndrome patients

Objective: Germline mutations in mismatch repair (MMR) genes cause Lynch syndrome (LS). LS is an inherited disease, and an important consequence of MMR deficiency is microsatellite instability (MSI) phenotype. MSI phenotype influences the efficacy of 5 fluorouracil (5-FU) chemotherapy. Reproducible, cost effective, and easy to perform laboratory tests are required to include MSI detection in routine laboratory practice. Evaluation of CAT25 as monomorphic short tandem repeat sequence enables CAT25 to be an efficient screening tool among hereditary nonpolyposis colorectal cancer (HNPCC) patients compared with other methods used currently. Methods: Based on Amsterdam II criteria, 31 patients in 31 families were shortlisted from a total number of 1,659 colorectal cancer patients. MSI status was examined in these patients using CAT25 and a commercially available Promega MSI five-marker-based detection system as well as immunohistochemical (IHC) staining of four important MMR proteins. Patients were scored as high microsatellite instable (MSI-H), low (MSI-L), or stable (MSS). MSI status determined by CAT25 single mononucleotide marker was compared with that of five mononucleotide markers, Promega commercial kit, and IHC method. Results: MMR protein deficiency was observed on 7/31 probands using IHC methodology and 6/31 categorized as MSI-H using commercial kit or CAT25 single marker. The sensitivity and specificity of the CAT25 single marker were the same as those detected by five-marker Promega commercial kit in our patients. Conclusions: Based on our results, the performance of the CAT25 single mononucleotide marker for MSI status determination in our HNPCC patients is the same as that of the five-marker-based commercial kit