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In this paper, we use a simple and cheap approach for the synthesis of herceptin-conjugated graphene biosensor to detect the HER2-positive breast cancer cells. The bifunctional graphene-herceptin nanosheets are prepared from graphite by a simple ultrasonic-mediated technique. The prepared protein-mediated graphene is fully characterized. The results show the exfoliation of graphene layers in herceptin solution. Moreover, herceptin is effectively conjugated into the surface of graphene nanosheets. The synthesized herceptin-conjugated graphene is applied for breast cancer detection. The linear range of this biosensor is 1–80 cells, which is significant. The biosensor shows an excellent selectivity performance for detection of HER2-positive cancer cells. Likewise, the stability and functionality of the biosensor is about 40 days. Based on the results, this device is a promising candidate for rapid and selective detection of cancer cells.

ObjectiveIn this large multi-institutional study, we aimed to analyze the prognostic power of computed tomography (CT)-based radiomics models in COVID-19 patients.MethodsCT images of 14,339 COVID-19 patients with overall survival outcome were collected from 19 medical centers. Whole lung segmentations were performed automatically using a previously validated deep learning-based model, and regions of interest were further evaluated and modified by a human observer. All images were resampled to an isotropic voxel size, intensities were discretized into 64-binning size, and 105 radiomics features, including shape, intensity, and texture features were extracted from the lung mask. Radiomics features were normalized using Z-score normalization. High-correlated features using Pearson (R2>0.99) were eliminated. We applied the Synthetic Minority Oversampling Technique (SMOT) algorithm in only the training set for different models to overcome unbalance classes. We used 4 feature selection algorithms, namely Analysis of Variance (ANOVA), Kruskal- Wallis (KW), Recursive Feature Elimination (RFE), and Relief. For the classification task, we used seven classifiers, including Logistic Regression (LR), Least Absolute Shrinkage and Selection Operator (LASSO), Linear Discriminant Analysis (LDA), Random Forest (RF), AdaBoost (AB), Naïve Bayes (NB), and Multilayer Perceptron (MLP). The models were built and evaluated using training and testing sets, respectively. Specifically, we evaluated the models using 10 different splitting and cross-validation strategies, including different types of test datasets (e.g. non-harmonized vs. ComBat-harmonized datasets). The sensitivity, specificity, and area under the receiver operating characteristic (ROC) curve (AUC) were reported for models evaluation.ResultsIn the test dataset (4301) consisting of CT and/or RT-PCR positive cases, AUC, sensitivity, and specificity of 0.83±0.01 (CI95%: 0.81-0.85), 0.81, and 0.72, respectively, were obtained by ANOVA feature selector + RF classifier. In RT-PCR-only positive test sets (3644), similar results were achieved, and there was no statistically significant difference. In ComBat harmonized dataset, Relief feature selector + RF classifier resulted in highest performance of AUC, reaching 0.83±0.01 (CI95%: 0.81-0.85), with sensitivity and specificity of 0.77 and 0.74, respectively. At the same time, ComBat harmonization did not depict statistically significant improvement relevant to non-harmonized dataset. In leave-one-center-out, the combination of ANOVA feature selector and LR classifier resulted in the highest performance of AUC (0.80±0.084) with sensitivity and specificity of 0.77 ± 0.11 and 0.76 ± 0.075, respectively.ConclusionLung CT radiomics features can be used towards robust prognostic modeling of COVID-19 in large heterogeneous datasets gathered from multiple centers. As such, CT radiomics-based model has significant potential for use in prospective clinical settings towards improved management of COVID-19 patients.

Introduction: Long non-coding RNAs (lncRNAs) have fundamental roles in cell migration, proliferation, invasion and metastasis. Methods: In the current study, we evaluated expression of a panel of lncRNAs in bladder cancer tissues, adjacent non-cancerous tissues (ANCTs) and normal bladder tissues to evaluate their diagnostic power. Results: PV1 was down-regulated in tumor tissues compared with both ANCTs and normal controls (Expression ratios of 0.48 and 0.14; P values of 0.4 and

Background: The outbreak of coronavirus disease 2019 (COVID-19) has become a major threat to all humans. Objectives: To assess the association between the patients’ clinical and laboratory records, computed tomography (CT) findings, and epidemiological features of COVID-19 with the severity of the disease. Patients and Methods: In this retrospective case-control study conducted on the medical records of confirmed COVID-19 pneumonia patients on admission, we investigated the CT manifestations and clinical and laboratory risk factors for progression to severe COVID-19 pneumonia. The medical records and radiological CT features of confirmed COVID-19 patients were reviewed in one public hospital and one respiratory clinic in Qom, Iran, from August 1 to September 30, 2020. Results: Of 236 confirmed COVID-19 cases, 62 were infected with moderate to severe COVID-19 and required hospital admission, and 174 were followed-up on an outpatient basis. A significant difference was found in the mean age of the outpatient and hospitalized groups. The incidence of bilateral lung involvement, consolidations, linear opacities, crazy-paving pattern, air bronchogram, and number of lobes involved were significantly higher in the hospitalized group compared to the outpatient group. However, the crazy-paving pattern was only significantly associated with an oxygen saturation (SpO2) level < 90% and, coughing. Our findings indicated that the crazy-paving pattern was significantly associated with the inflammatory phase. The presence of this pattern on admission, SpO2 < 90%, older age, and diabetes were independent risk factors for progression to severe COVID-19. Conclusion: The crazy-paving pattern can predict the severity of COVID-19, which is of great importance in the management and follow-up of COVID-19 pneumonia patients. Clinical factors, such as aging, male gender, and diabetes, may be risk factors for the crazy-paving pattern. Severe cough is the most important clinical sign related to this pattern, along with an SpO2 < 90%, which is an important sign of COVID-19 severity.

Background: The outbreak of coronavirus disease 2019 (COVID-19) becomes an enormous threat to all human beings. Via this retrospective study conducted on medical records of confirmed COVID-19 pneumonia patients on admission, we investigate the CT manifestation and clinical and laboratory risk factors associated with progression to severe COVID-19 pneumonia and assessed the association among clinical and laboratory records, CT findings, and epidemiological features. The medical records and radiological CT Features of 236 confirmed COVID-19 patients were reviewed at one public hospital and one respiratory clinic in Quom, from 1 August to 30 September 2020. Results: Among a total of 236 confirmed Covid-19 cases, 62 were infected with moderate to severe COVID-19 disease and required hospital admission, and 174 were followed up on outpatient bases. A significant difference was verified in the mean age between outpatients and hospitalized groups. The incidences of bilateral lung involvement, consolidation, linear opacities, crazy-paving pattern, air bronchogram sign, and the number of lobe involvement were significantly higher in hospitalized groups. However, only the crazy-paving pattern was significantly associated with an SpO2 level lower than 90%, with clinical sign of cough severity. Our data indicate that this pattern is also significantly associated with inflammatory levels and the presence of this pattern along with SpO2 level lower than 90%, older age, diabetes, on admission are independent risk factors for COVID-19 progression to severe level.Conclusions: The crazy-paving pattern can predict the severity of COVID-19, which is of great significance for the management and follow-up of COVID-19 pneumonia patients. The clinical factors of aging, male gender, and diabetes, may be risk factors for the crazy-paving pattern, whereas severe coughing is considered to be the most important clinical symptom related to this pattern, and SpO2 level lower than 90%, which is a matter of more severity.

Aim: miRNAs have been suggested as biomarkers for bladder cancer. We aimed to find a diagnostic panel of miRNAs based on differential expression of miRNAs in urine specimens of patient with bladder cancer compared with control group. Methods: miR-141, miR-10b, miR-34b and miR-103 were selected to assess their expression in urine samples of 66 bladder cancer patients and 53 matched controls using quantitative real time PCR. Results: miR-10b and miR-34b were upregulated in cases compared with controls. The combination of four miRNAs showed a sensitivity of 75% and specificity of 63.5% with a diagnostic power of 72%. Conclusion: Certain miRNAs can be used as biomarkers for early diagnosis of bladder cancer.

Background Bladder cancer (BC) is a frequent cancer with high morbidity and mortality. Method We assessed expression of NMP22, BAG3, BCL2 and BAX in BC samples, adjacent non-neoplastic tissues (ANNTs), urinary cell pellets (UCP) of the same patients and non-malignant conditions (NM). Results NMP22 was up-regulated in tumoral tissues compared with ANNTs and in UCP of BC patients compared with UCP of NM. BCL2 and BAX were down-regulated in BC tissues compared with ANNTs. BAG3 and BAX were down-regulated in UCP of BC compared with NM. The combination of all four genes had 100% sensitivity and specificity for detection of BC in urine. Conclusion Expression analysis of these genes in tumor or urine is a tool for detection of BC.

Aim: Cancer-testis antigens (CTAs) have specific expression in gametogenic tissues and aberrant expression in cancers. Materials & methods: We assessed expression of five testis-specific genes namely KIF2B, CST8, TMEM225, RBM46, OAZ3 in bladder cancer tissues, adjacent non-neoplastic tissues and urinary cell pellets (UCPs) of bladder cancer patients compared with nonmalignant conditions.Results: Expressions of all CTAs were higher in UCPs of bladder cancer patients compared with nonmalignant conditions. RBM46 expression in UCPs was higher in patients with recurrent tumors compared with primary tumors and in patients without hematuria compared with those having hematuria. TMEM225 expression in tumoral tissues was higher in high-grade tumors compared with low-grade tumors. Conclusion: Expression analysis of CTAs in UCP might provide diagnostic information about bladder cancer.

Fatemeh Yazarlou,1 Mohammad Hossein Modarressi,1 Seyed Javad Mowla,2 Vahid Kholghi Oskooei,3 Elahe Motevaseli,4 Leila Farhady Tooli,5 Leila Nekoohesh,6 Maryam Eghbali,1 Soudeh Ghafouri-Fard,3 Mandana Afsharpad7 1Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; 2Faculty of Biological Sciences, Department of Genetics, Tarbiat Modares University, Tehran, Iran; 3Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 4Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran; 5Department of Microbiology, School of Biology, College of Science, Tehran University, Tehran, Iran; 6Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran; 7Cancer Control Research Center, Cancer Control Foundation, Iran University of Medical Sciences, Tehran, Iran Background: Long non-coding RNAs (lncRNAs) and exosomes have been regarded as components of cell signal transmission that modulate indigenous cellular microenvironments. Exosomes also participate in relocation of functional lncRNAs between cells. Methods: In the present study, we evaluated expression of LINC00355, LINC00958, UCA1-201, UCA1-203, and MALAT1 lncRNAs in urinary exosomes isolated from transitional cell carcinoma (TCC) of bladder, non-malignant urinary disorders, and normal subjects. Results: LINC00355, UCA1-203, and MALAT1 expression was significantly higher in TCC patients compared to controls (non-malignant or normal samples). However, UCA1-201 expression was significantly decreased in TCC patients compared with controls. LINC00355 and MALAT1 expression was significantly lower in cigarette smokers and opium-addicted TCC patients, respectively. On the other hand, LINC00355 expression tended to be higher in opium-addicted TCC patients. The proposed panel of lncRNAs (composed of UCA1-201, UCA1-203, MALAT1, and LINC00355) had 92% sensitivity and 91.7% specificity for diagnosis of bladder cancer from normal samples. Conclusion: Transcript levels of lncRNAs in urinary exosomes are potential diagnostic biomarkers in bladder cancer. Keywords: lncRNA, exosome, bladder cancer

Background Long non-coding RNAs (lncRNAs) have several important functions in the regulation of cell homeostasis and cell fate. Consequently, abnormal transcription of lncRNAs has been correlated with malignant transformation of cells. These human transcripts have been shown to participate in the progression of gastric cancer. Methods In the current project, we evaluated expression of a panel of lncRNAs including HULC, MALAT1, FAS-AS1, GAS5, PVT1, OIP5-AS1 and THRIL in 30 gastric cancer tissues and paired adjacent non-cancerous tissues (ANCTs) using quantitative real-time PCR. Results HULC, OIP5-AS1 and THRIL transcription quantities were significantly lower in gastric tumors compared to ANCTs (P values = .02, 0.02 and 0.007, respectively). Relative transcription quantities of HULC, MALAT1, OIP5-AS1, PVT1, FAS-AS1 and THRIL were associated with the site of the primary tumor (P values = .002, 0.003, 0.002, 0.002, 0.002, and 0.001, respectively). Moreover, relative expression levels of PVT1 were associated with history of smoking (P value = .04). Correlations were identified between transcript quantities of these lncRNAs in both tumor samples and ANCTs. Receiver operating characteristic curve assessment demonstrated that THRIL had the highest diagnostic power among the mentioned lncRNAs (area under curve (AUC) = 0.72, P value = .001). HULC and OIP5-AS1 ranked afterwards (AUC values of 0.69 and 0.68; P values = .005 and 0.007, respectively). Conclusion The current investigation underscores the dysregulation of these transcripts in gastric cancer specimens and suggests a number of these transcripts for further assessments of their suitability as cancer biomarkers.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in thyroid tumors require accurate data normalization, however, there are no sufficient studies addressing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue specimens. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human papillary thyroid carcinoma tissue samples. The expression characteristics of 12 candidate reference genes (GAPDH, ACTB, HPRT1, TBP, B2M, PPIA, 18SrRNA, HMBS, GUSB, PGK1, RPLP0, and PGM1) were assessed by qRT-PCR in 45 thyroid tissue samples (15 papillary thyroid carcinoma, 15 paired normal tissues and 15 multinodular goiters). These twelve candidate reference genes were selected by a systematic literature search. GeNorm, NormFinder, and BestKeeper statistical algorithms were applied to determine the most stable reference genes. The three algorithms were in agreement in identifying GUSB and HPRT1 as the most stably expressed genes in all thyroid tumors investigated. According to the NormFinder software, the pair of genes including ‘GUSB and HPRT1’ or ‘GUSB and HMBS’ or ‘GUSB and PGM1’ were the best combinations for selection of pair reference genes. The optimal number of genes required for reliable normalization of qPCR data in thyroid tissues would be three according to calculations made by GeNorm algorithm. These results suggest that GUSB and HPRT1 are promising reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma.

With the growing rate of tumors, cancer has become one of the most important health concerns in Iran. The urgency with which Iranian researchers and health professionals address this challenge leads to a load of scientific materials.To reveal gaps in produced knowledge and suggest future research directions, applying well-validated scientometric tools, we assessed the trends of Iranian published scientific articles and citations in the field of oncology. The inclusion criteria consisted of all oncology-related articles that were data-based, and peer-reviewed; with at least an abstract published in English; and authored by at least one researcher affiliated with Iranian institutions.Amongst 5063525 oncology research records indexed in at least one of PubMed, Scopus, or Web of Science Core Collection (WoS) from the start to February 2019, Iranian researches accounted for about 24867 (0.49%). Published articles on all cancers by Iranian researchers had a sharp continuously ascending trend, with the same pattern for citations received. Some important topics such as complementary and alternative medicine (CAM) therapies have been missing and some such as diagnostic and pharmaceutical innovations have been less investigated. The most collaborative country was the United States, while no close collaboration was observed with China that was introduced as the most productive country in the field of oncology over the past decades.Despite the progressive trend in most oncology fields, some significant practical topics are still missing. Systematic reviews of produced theoretical innovations and translating them to functional knowledge can be of importance to fulfill the mentioned gaps.

The specific mRNA signature of urinary exfoliated cells (UECs) can be helpful for biomarker discovery and validation studies related to urological malignancies including cancers of bladder, kidney, prostate and testicles, as well as kidney allograft rejection, systemic autoimmune diseases such as lupus, and acquired proteinuric diseases. To the best of our knowledge, no systematic validation of reference genes has been reported for UECs Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) analysis up to now. To determine the stability of candidate housekeeping genes (HKGs) across UECs, they were isolated from first morning urine of patients diagnosed as high grade and low grade urothelial bladder cancer as cases and clinical controls including the patients suffering from bladder stone, benign prostatic hyperplasia, obstructive uropathy and healthy individuals. RT-qPCR was performed to determine 9 candidate HKGs stability. geNorm, Normfinder, and BestKeeper statistical algorithm were applied to determine the most stable reference genes. The non-parametric Mann-Whitney U test applied to compare HKGs expression levels between cases and controls samples. GAPDH and HSP90AB1 were selected as two most stable expressed HKGs by all three algorithms, while ACTB demonstrated to have the least stability. Although, GAPDH is a suitable reference gene for quantitative analysis of UECs in urological malignancies, based on normalization factor calculated by geNorm, we recommend using GAPDH, and HSP90AB1 together as normalizer to obtain more realistic expression analysis via relative qPCR.

Fatemeh Yazarlou,1 Seyed Javad Mowla,2 Vahid Kholghi Oskooei,3 Elahe Motevaseli,4 Leila Farhady Tooli,5 Mandana Afsharpad,6 Leila Nekoohesh,7 Nafiseh Sadat Sanikhani,4 Soudeh Ghafouri-Fard,3 Mohammad Hossein Modarressi1 1Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran; 2Faculty of Biological Sciences, Department of Genetics, Tarbiat Modares University, Tehran, Iran; 3Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 4Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran; 5Department of Microbiology, School of Biology, College of Science, Tehran University, Tehran, Iran; 6Cancer Control Research Center, Cancer Control Foundation, Iran University of Medical Sciences, Tehran, Iran; 7Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Background: Exosomes have been regarded as emerging tools for cancer diagnosis. Tumor-derived exosomes contain molecules that enhance cancer progression and affect immune responses. Material and methods: In the present study, we evaluated expression of seven cancer-testis antigens (CTAs) that are regarded as putative biomarkers and immunotherapeutic targets along with NMP22 in urinary exosomes of bladder cancer patients, healthy subjects and patients affected with nonmalignant urinary disorders. Results: Exosomal expression of MAGE-B4 was significantly higher in bladder cancer patients compared with normal samples (expression ratio=2.68, P=0.01). However, its expression was lower in bladder cancer patients compared with benign prostate hyperplasia (BPH) patients (expression ratio=0.17, P=0.01). Exosomal expression of NMP22 was significantly higher in bladder cancer patients compared with BPH patients (expression ratio=9.22, P=0.02). Expressions of other genes were not significantly different between bladder cancer patients and normal/nonmalignant samples. We found significant correlation between MAGE-A3 and MAGE-B4 expressions in exosomes obtained from controls. In addition, TSGA10 expression was correlated with expression of NMP22 in both cancer patients and controls. Conclusion: The present study provides evidences for differential expression of CTAs in urinary exosomes of bladder cancer patients and urogenital disorders and warrants further studies for assessment of their significance in cancer diagnosis and immunotherapeutic approaches. Keywords: cancer-testis antigen, bladder cancer, exosome

This study aims to investigate the expression level of mir-let7b-3p and mir-548, which are involved in PTEN expression in tissue samples of prostate cancer patients versus benign prostate hyperplasia (BPH) and normal adjacent tissue.Prostate cancer tissues were obtained from patients after receiving informed consent. Total RNA extraction and cDNA synthesis were performed for determining gene expression.Ten patients were determined to have high Gleason scores (7), 36 and seven samples had intermediate Gleason scores (7?) and BPH, respectively, and 40 samples were derived from normal adjacent tissue. Downreg-ulation of mir-let7b and upregulation of mir-548 expression significantly correlated with high-risk Gleason scores.The present study showed that miR-let7b and/or mir-548 can be considered as potential targets in prostate cancer therapy.

Deregulation of key signaling pathways is one of the primary phenomena in carcinogenesis. DAB2IP and SPRY2 are regulatory elements, which act as feedback inhibitors of receptor tyrosine kinases signaling in mitogen-activated protein kinase pathway. These elements have also been implicated in the pathophysiology of cancer. Therefore, this study is aimed to investigate the expression of all known splice variants of DAB2IP and SPRY2 in prostate tissue. Fresh Prostate tissue samples (50 prostate cancer/ matched normal tissue and 30 BPH) were collected and total RNA was extracted followed by cDNA synthesis. The expression of DAB2IP and SPRY2 transcript variants were evaluated using RT-PCR and quantitative Real-time PCR. The results indicated significant down-regulation of DAB2IP transcript variant 1 in cancerous tissues compared to paired normal tissues (P = 0.001) as well as SPRY2 transcript variant 2 in cancerous tissues in comparison with the normal counterparts and BPH (P = 0.008 and P = 0.025, respectively). In addition, there was a significant negative correlation between DAB2IP.1 and SPRY2.2 expression with PSA levels in prostate cancer (P = 0.039 ρ =−0.24 and P = 0.045 ρ =−0.3, respectively). Interestingly, the down-regulation of DAB2IP.1 mRNA and SPRY2.2 mRNA was positively correlated in tumor samples (P = 0.002 ρ = 0.434). For the first time, this experiment highlights the deregulation of DAB2IP and SPRY2 transcript variants in human prostate cancer. The present study confirms and extends the previous reports through indicating transcript-specific down-regulation and significant association of DAB2IP and SPRY2 in prostate tumorigenesis.

Breast cancer is the most frequent cancer with second mortality rate in women worldwide. Lack of validated biomarkers for early detection of breast cancer to warranty the diagnosis and effective treatments in early stages has directed to the new therapeutic approach. Cancer/testis antigens which have restricted normal expression in testis and aberrant expression in different cancers are promising targets for generating cancer vaccines, monoclonal antibodies, or dendritic cell-based immunotherapy. In this context, we investigated the expression of two known cancer testis genes, Aurora kinase C (AURKC) and testis expressed 101 (TEX101), and one new candidate, deleted in azoospermia 1 (DAZ1), in six breast cancer cell lines including two ductal carcinomas, T47D and BT-474, and four adenocarcinomas, MDA-MB-231, MDA-MB-468, MCF7, and SKBR3 as well as 50 breast cancer tumors in comparison to normal mammary epithelial cells using quantitative real-time reverse transcription PCR (RT-PCR). Results showed significant overexpression (p = 0.000) of all three genes in BT474, DAZ1 in MDA-MB-231, and AURKC and DAZ1 in SKBR3 and significant downregulation (p = 0.000) of AURKC in MCF7 cell line relative to normal breast epithelial cells. Breast tumors showed significant overexpression of AURKC in comparison to normal breast tissues (p = 0.016). The results are noticeable especially in the case of AURKC; however, there is a little knowledge about the nature, causes, consequences, and effects of cancer/testis antigens activation in different cancers. It is suggested that AURKC has effects on cell division via its serin/threonin kinases activity and organizing microtubules in relation to centrosome/spindle function during mitosis.

To evaluate the diagnostic potential of 23 candidate genes, belonging to a category of tumor-specific antigens known as cancer-testis antigens (CTAs), in transitional cell carcinoma (TCC) patients. The expression of 16 known candidate CTAs and seven testis restricted/selective genes, predominantly expressed in the testis, was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Urinary exfoliated cells (UECs) and cancerous tissues of 73 TCC patients were used as cases, while 25 tumor-free adjacent bladder tissue specimens along with bladder tissue specimens and UECs of five non-TCC individuals were analyzed as controls. Among the known CTAs only MAGEA3, MAGEB4, TSGA10, PIWIL2, OIP5, and ODF4 were expressed specifically in TCC tissues and UEC samples. ACTL7A, AURKC, and CGB2 were testis-restricted/selective genes that indicated specific expression in cases in comparison to controls. MAGEA3, MAGEB4, and ODF4 mRNA was detectable in more than 50% of both TCC tissues, and UEC samples. Slight differences were detected in the mRNA expression pattern of candidate genes between the UEC samples and tumor tissues. Different panels formed by combinations of these genes can show up to 95.9% and 94.5% of positivity in TCC tissues and UEC samples, respectively, suggesting their diagnostic and surveillance potential. Meanwhile the RT-PCR assay of at least MAGEA3, MAGEB4, and ODF4 may be particularly useful for diagnostic and surveillance of TCC in the form of a multi-biomarker panel.

Testis specific gene antigen 10 (TSGA10) is a cancer testis antigen involved in the process of spermatogenesis. TSGA10 could also play an important role in the inhibition of angiogenesis by preventing nuclear localization of HIF-1α. Although it has been shown that TSGA10 messenger RNA (mRNA) is mainly expressed in testis and some tumors, the transcription pattern and regulatory mechanisms of this gene remain largely unknown. Here, we report that human TSGA10 comprises at least 22 exons and generates four different transcript variants. It was identified that using two distinct promoters and splicing of exons 4 and 7 produced these transcript variants, which have the same coding sequence, but the sequence of 5'untanslated region (5'UTR) is different between them. This is significant because conserved regulatory RNA elements like upstream open reading frame (uORF) and putative internal ribosome entry site (IRES) were found in this region which have different combinations in each transcript variant and it may influence translational efficiency of them in normal or unusual environmental conditions like hypoxia. To indicate the transcription pattern of TSGA10 in breast cancer, expression of identified transcript variants was analyzed in 62 breast cancer samples. We found that TSGA10 tends to express variants with shorter 5'UTR and fewer uORF elements in breast cancer tissues. Our study demonstrates for the first time the expression of different TSGA10 transcript variants in testis and breast cancer tissues and provides a first clue to a role of TSGA10 5'UTR in regulation of translation in unusual environmental conditions like hypoxia.

In this study, the nature and extent of genetic diversity of Plasmodium vivax populations circulating in Afghanistan have been investigated by analyzing three genetic markers: csp, msp-1, and msp-3 alpha. Blood samples (n=202) were collected from patients presenting with vivax malaria from south-western (Herat) and south-eastern (Nangarhar) parts of Afghanistan, and analysed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 revealed type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant in parasites in both study areas. The sequence analysis of 57 P. vivax isolates identified a total of 26 distinct alleles. Genotyping pvcsp gene showed that VK210 type (86.6%) is predominant in Afghanistan. Moreover, three major types of the pvmsp-3 alpha locus: type A, type B and type C were distinguished among Afghani isolates. The predominant fragments among Nangarhar and Herat parasites were type A (70.8% and 67.9%, respectively). PCR/RFLP products with Hha I and Alu I were detected 52 and 38 distinct variants among Nangarhar and Herat isolates, respectively. These results strongly indicate that the P. vivax populations in Afghanistan are highly diverse.

Introduction Basically chronic graft rejection is a type-IV hypersensitivity process, also known as delayed-type hypersensitivity (DTH). The role of cytokines secreted from T-cell lymphocytes is known in this process. Among these cytokines, the biggest role belongs to the transforming growth factor beta (TGF-β) and interleukin-2 (IL-2). Many studies have been published on the effect of low level lasers (LLL) on DTH, but not many have focused on the mechanism of LLL effects on DTH, which is also the mechanism of chronic graft rejection. If LLL are able to decrease the level of cytokines effectively in this process, then it would be possible to use it to prevent chronic graft rejection, either alone or combined with routine treatments, to give fewer treatment side effects. This study was designed to evaluate the effect of LLL on the amount of cytokines secreted by T-cells after stimulation in vitro by foreign antigens. Method and materials Human peripheral blood mononuclear cells were cultured and the cells were divided into 4 groups (control #1, control #2, case #1, and case #2). Control group #1 was the base group and the cells received neither stimulation nor irradiation. Control group #2 was only stimulated by phytohaemagglutinin (PHA); no laser irradiation was carried out. Case group #1 received daily laser irradiation for a total of 9 days, starting one day prior to stimulation with PHA. Case group #2 was given daily laser irradiation for a total of 7 days, starting one day after the stimulation with PHA. Both case groups were irradiated with the same helium-neon (HeNe) laser device (parameters: 632.8 nm, 4 J/cm2). On days 3, 5 and 8 of the experiment, the level of the cytokines interleukins IL-2 and IL-12, TGF-β and interferon gamma (INT-γ) were checked by the ELISA method in all the groups and the data were analyzed using SPSS 11.5. Results Our results showed that low-level irradiation can decrease the level of cytokines that are known to be stimulators for chronic graft rejection (IL-2, IL-12, TGF-β and INF-γ). However, we did not find any significant differences between our two case groups. Discussion Although this study suggests the successful role of LLL in preventing chronic graft rejection by affecting the cytokines known to have significant role in this process, further experimental studies and dosage determination experiments are required before using LLL in clinical treatment procedures.

This study was designed to analyze the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) mutations as markers of chloroquine (CQ) resistance in 200 blood samples collected from malaria patients in south-eastern Iran during 2002-2005. Among these, 25 (post-treatment) fulfilled the 28-day follow-up study. A high number of Iranian P. falciparum (97%) strains harbored quadruple mutations at codons 76T, 220S, 326D, and 356L. All post-treatment isolates harbored the mutant allele 76T, but low rates of the mutant allele 86Y (44%) of the pfmdr1 gene were detected. No wild haplotype of pfcrt (72-CVMNKAQNIR-371) was found in post-treatment samples; however, 56% of clinical "failure" samples carried the wild type of pfmdr1 (NYSND). The present results suggest a strong association between pfcrt 76T, but not pfmdr1 86Y mutation and in vivo CQ resistance. Furthermore, we found the CQ resistance-associated SVMNT haplotype, which previously had been seen in South American isolates. Although Iran is located more proximally to Southeast Asia than to South America, no CQ resistance-associated CVIET haplotye has been observed in this region. Therefore, these results were not consistent with the earlier presumed spread of CQR parasites from Southeast Asia to Africa via the Indian subcontinent. In conclusion, P. falciparum mutations associated with resistance to CQ are abundant in south-eastern Iran and this finding strongly supports that CQ as the first line drug is inadequate for treatment of uncomplicated falciparum malaria in Iran.

Background Artemisinin-based combination therapy (ACT) is the mainstay of global efforts for treatment of Plasmodium falciparum malaria, but decline in its efficacy is the most important obstacle towards malaria control and elimination. Therefore, the present molecular analysis provides information on putative mutations associated with artemisinin resistance in P. falciparum clinical population unexposed and exposed to artesunate 4 years after adoption of ACT as the first-line anti-malarial therapy in Iran. Methods In this study, blood samples (n = 226) were collected from uncomplicated P. falciparum-infected patients from different health centers of Chabahar district in Sistan and Baluchistan province in the south-eastern part of Iran, during 2003 to 2010. All collected isolates were analysed for putative candidate mutations (TTA) L263E (GAA), (GAA) E431K (AAA), (GCA) A623E (GAA) and (AGT) S769N (AAT) of pfatpase6 gene using nested PCR/RFLP, followed by sequencing. Furthermore, the gene copy number was assessed by real-time quantitative PCR (RT-qPCR) in the presence of SYBR green. Results Neither the pfatpase6 L263E nor the A623E mutation was detected among all examined isolates. The E431K mutation was found in 23% of the analysed samples unexposed to ACT; however, it was detected in 17.8% (34/191) of P. falciparum isolates exposed to artesunate after 2007. High frequency of this single nucleotide polymorphisms (SNP) (overall 18.6%) among both examined groups (X2 test, P>0.05) indicated that this SNP should be considered as an unrelated mutation to artemisinin resistance. In contrast, S769N mutation was not detected in unexposed isolates; however, it was found in 2.6% (5/191), four years after introduction of ACT in this malaria setting. Also, detected SNPs were not significantly frequent in both unexposed and exposed examined isolates (X2 test, P> 0.05). Investigation in the copy number of pfatpase6 gene revealed a similar number of copy (n = 1) as in an isolate sensitive to artemisinin. Conclusion Taken together, the results suggest, in particular, that pfatpase6 S769N gene needs more consideration for its possible association with artesunate resistance among P. falciparum isolates.

International audience; The main objective of this investigation was whether the combination therapy of sulfadoxine pyrimethamine (SP) plus artesunate (AS) protects against the spread of resistance to SP in malaria-endemic south-eastern Iran. Infected blood samples of Plasmodium falciparum (n=170) were collected during 2008-2010 after the adoption of SP-AS as the first line treatment in Iran. Four different genes of P. falciparum [dihydropteroate synthetase (pfdhps), dihydrofolate reductase (pfdhfr), chloroquine (CQ) resistance transporter (pfcrt K76T) and multidrug resistance1 (pfmdr1 N86Y)], associated with SP and CQ resistance were analyzed using PCR-RFLP methods. The result showed 4.1, 95.9 and 100% prevalence of pfdhfr 51I, 59R and 108N, respectively and the majority of patients (95.9%) were found to carry both 59R and 108N. The prevalence of single mutation at pfdhps 437G gene was 26.9% before the adoption of SP-AS, but as SP was used as the first line treatment; this mutation started to increase and reached a high level of 55.5% in 2008 (χ(2) test, P

The emergence and spread of Plasmodium falciparum resistant to the commonly used anti-malarial drugs is a major challenge in the control and elimination of malaria. The present study provides information on genetic analysis in multidrug resistance 1 (pfmdr1) (N86Y/Y184F/S1034C/N1042D/F1226Y/D1246Y) and multidrug resistance protein 1 (pfmrp1) (H191Y/S437A/I876V/F1390I/K1466R) genes that are probably associated with artemisinin as well as chloroquine resistance transporter (pfcrt) 76T in P. falciparum clinical isolates (N=200) exposed to artemisinin-based combination therapy (ACT) 4years after its adoption in Iran. Also, the copy number of pfmdr1 gene was screened for its association with pfmdr1 mutations to incriminate artemisinin resistance. By using nested PCR-RFLP and sequencing analysis, none of the samples had any mutation at codons 1034, 1042, 1226 and 1246 of pfmdr1, while 86Y and 184F mutations were detected in 46% and 2% of the examined samples, respectively. Also, no significant difference was identified among analyzed samples collected before (baseline, 2002-2005) and after adoption of ACT (2007-2010) (P>0.05). As with pfmrp1 gene, the mutations at positions 191Y (76.5%), 437A (69.5%), 876V (64.5%) and 1390I (17%) were detected and no samples displayed mutation at codon 1466R. In total, 42.5% of the examined isolates carried both pfmdr1 86Y and pfcrt 76T and none of the parasites simultaneously harbored pfcrt 76T, pfmdr1 86Y, 184F and pfmrp1 191Y, 437A, 876V, 1390I mutations. In addition, the copy number of pfmdr1 gene (N = 1) was similar as a sensitive isolate, 3D7, to artemisinin. In summary, none of the potential mutations associated with artemisinin and its derivatives resistance was significantly changed 4years after adoption of ACT in Iran.

International audience; The increasing use of sulfadoxine/pyrimethamine (SP) for treatment of chloroquine-resistant Plasmodium falciparum has resulted in increased exposure of Plasmodium vivax parasites in areas where both species co-exist. In this study, the extent of mutations/haplotypes in pvdhfr and pvdhps was examined using PCR-RFLP methods in 427 P. vivax isolates in Iran after 4 years of introducing SP as the first-line anti-malarial drug in Iran. Mutations were detected in three codons of pvdhfr (F57L, S58R and S117N) and in one of pvdhps (A383G) and the majority of isolates had double mutations (58R/117N, 45.4%). In addition, the frequency of 57L mutation was detected in 8.2% of P. vivax isolates. This frequency was significantly increased when compared with a similar study on P. vivax isolates in 2005 (X(2) test, P

Species identification and information on transmission pattern of malaria parasite in any malaria endemic area is key to success for a malaria control programme. In this investigation, malaria diagnosis using molecular method was used to assess the transmission pattern of malaria parasite in three malaria endemic regions: Afghanistan, Iran and Pakistan.Blood samples were collected from the patients presenting with vivax malaria from Afghanistan (n=108), Iran (n=200) and Pakistan (n=199). Malaria parasite detection was made by the gold standard (microscopy) and also nested-PCR assay, using 18S small sub-unit ribosomal RNA (ssrRNA) gene.Based on microscopy method, the level of mixed infection was zero to 2.5 per cent; however, nested-PCR assay detected 6.5, 22 and 23.5 per cent mixed infections in samples collected from Afghanistan, Iran and Pakistan, respectively. The present results showed that the co-infection of P. vivax with P. falciparum was frequent in malaria endemic regions of Iran and Pakistan.The present data suggest the need for improving microscopy diagnosis method and the clinician should also have careful clinical observation, along with the reports on Giemsa- stained thick blood films, particularly in summer time when P. vivax is predominant. Also sharing information on transmission pattern of mixed infection among these countries may help in designing better control strategies for malaria.

The main aim of the present study was to investigate the frequency of SNPs-haplotypes of dhfr and dhps genes associated to sulfadoxine-pyrimethamine (SP) resistance in Plasmodium vivax clinical isolates circulating in a malaria endemic area, Pakistan. All 164 collected isolates were analyzed for SNPs-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of pvdhfr and 383 and 553 of pvdhps genes using PCR-RFLP methods. All examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 15.2% and 53.6% of isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B (79.3%) was the most prevalent variant. The combination of pvdhfr and pvdhps haplotypes demonstrated nine distinct haplotypes. The three most prevalent haplotypes were I(13)P(33)F(57)S(58)T(61)S(117)I(173)/A(383)A(553) (43.9%), I(13)P(33)F(57)S(58)T(61)N(117)I(173)/A(383)A(553) (33.6%) and I(13)P(33)F(57)R(58)T(61)N(117)I(173)/A(383)A(553) (12.2%). The presence of mutant haplotypes is worrying and indicates the emergence of drug tolerant/resistant P. vivax isolates in Pakistan in near future.

Background Analysis of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations in Plasmodium vivax wild isolates has been considered to be a valuable molecular approach for mapping resistance to sulphadoxine-pyrimethamine (SP). The present study investigates the frequency of SNPs-haplotypes in the dhfr and dhps genes in P. vivax clinical isolates circulating in two malaria endemic areas in Afghanistan. Methods P. vivax clinical isolates (n = 171) were collected in two different malaria endemic regions in north-west (Herat) and east (Nangarhar) Afghanistan in 2008. All collected isolates were analysed for SNP-haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the pvdhfr and 383 and 553 of the pvdhps genes using PCR-RFLP methods. Results All 171 examined isolates were found to carry wild-type amino acids at positions 13, 33, 57, 61 and 173, while 58R and 117N mutations were detected among 4.1% and 12.3% of Afghan isolates, respectively. Based on the size polymorphism of pvdhfr genes at repeat region, type B was the most prevalent variant among Herat (86%) and Nangarhar (88.4%) isolates. Mixed genotype infections (type A/B and A/B/C) were detected in only 2.3% (2/86) of Herat and 1.2% (1/86) of Nangarhar isolates, respectively. The combination of pvdhfr and pvdhps haplotypes among all 171 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were wild-type (86%) and single mutant haplotype I13P33F57S58T61N 117I173/A383A553 (6.4%). Double (I13P33S57R58T61N117I173/A383A553) and triple mutant haplotypes (I13P33S57R 58T61N117I173/G383A553) were found in 1.7% and 1.2% of Afghan isolates, respectively. This triple mutant haplotype was only detected in isolates from Herat, but in none of the Nangarhar isolates. Conclusion The present study shows a limited polymorphism in pvdhfr from Afghan isolates and provides important basic information to establish an epidemiological map of drug-resistant vivax malaria, and updating guidelines for anti-malarial policy in Afghanistan. The continuous usage of SP as first-line anti-malarial drug in Afghanistan might increase the risk of mutations in the dhfr and dhps genes in both P. vivax and Plasmodium falciparum isolates, which may lead to a complete SP resistance in the near future in this region. Therefore, continuous surveillance of P. vivax and P. falciparum molecular markers are needed to monitor the development of resistance to SP in the region.

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3alpha (msp-1 and msp-3alpha) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n=187) and Iran (n=150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3alpha, three major types: type A (1800bp), type B (1500bp) and type C (1200bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3alpha with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.

Background In Iran, co-infections of Plasmodium vivax and Plasmodium falciparum are common and P. vivax infections are often exposed to sulphadoxine-pyrimethamine (SP). In the present study, the frequency distribution of mutations associated to SP resistance was investigated in pvdhfr and pvdhps genes from field isolates. Methods Clinical isolates of P. vivax were collected in two different malaria endemic regions in northern and south-eastern Iran, between 2001 and 2006. All 189 collected isolates were analysed for SNP/haplotypes at positions 13, 33, 57, 58, 61, 117 and 173 of the pvdhfr and 383 and 553 of pvdhps genes using nested PCR-RFLP methods Results All 189 examined isolates were found to carry wild-type amino acids at positions 13, 33, 61 and 173, while 57L and 58R and 117N mutations in pure form was detected among 1.1%, 17.5% and 26% examined samples, respectively, with no polymorphisms in different loci of dhps genes. Based on size polymorphism of pvdhfr genes at repeat region, among northern isolates, the frequency distribution for type A and B were 2.2% and 97.8% respectively. However, in southern samples the prevalence of type A, B and C were 7%, 89.5% and 7.7%, respectively. Mixed genotype infections (type B and C) were detected in only 4.2% (6/143) of southern, but in none of the northern isolates. The combination of pvdhfr and pvdhps haplotypes among all 189 samples demonstrated six distinct haplotypes. The two most prevalent haplotypes among all examined samples were I13P33F57S58T61S117I173/A383A553 (65.6%) and I13P33F57S58T61N117I173 (16.4%). Two other alleles with one point mutation I13P33F57R58T61S117I173/A383A553 and two mutations I13P33F57R58T61N117I173/A383A553 accounted for 7.4% and 9.5% of the total isolates. Conclusion The present molecular data provide important information for making decisions on population based drug use in Iran. In addition, since October 2005, with more availability of SP as first-line treatment, P. vivax isolates are more exposed to SP and the selection or spread of resistant pvdhfr and pvdhps alleles might increase in the near future in this region.

Background This work was carried out to assess the patterns and prevalence of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in Iran. Methods The prevalence of pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, C59R, S108N/T and I164L and codons S436F/A, A437G, K540E, A581E, and A613S/T in pfdhps genes were genotyped by PCR/RFLP methods in 206 Plasmodium falciparum isolates from Chabahar and Sarbaz districts in Sistan and Baluchistan province, Iran, during 2003–2005. Results All P. falciparum isolates carried the 108N, while 98.5% parasite isolates carried the 59R mutation. 98.5% of patients carried both 108N and 59R. The prevalence of pfdhps 437G mutation was 17% (Chabahar) and 33% (Sarbaz) isolates. 20.4% of samples presented the pfdhfr 108N, 59R with pfdhps 437G mutations. The frequency of allele pfcrt 76T was 98%, while 41.4% (Chabahar) and 27.7% (Sarbaz) isolates carried pfmdr1 86Y allele. Eight distinct haplotypes were identified in all 206 samples, while the most prevalent haplotype was T76/N86/N51R59N108/A437 among both study areas. Conclusion Finding the fixed level of CQ resistance polymorphisms (pfcrt 76T) suggests that CQ must be withdrawn from the current treatment strategy in Iran, while SP may remain the treatment of choice for uncomplicated malaria.

Summary Objective The objective of this study was to determine the frequency of dhfr and dhps resistance-associated haplotypes in Plasmodium falciparum isolates, three years after the introduction of sulfadoxine–pyrimethamine (SP) as the first-line antimalarial treatment in Iran. Methods Blood samples ( N =182) were collected from patients presenting with falciparum malaria from southeastern Iran, and analyzed by nested-PCR/restriction fragment length polymorphism, followed by sequencing analysis. Results In pfdhfr , double mutation at positions 59R and 108N was a predominant allele with a prevalence of 95.7%. The pure double mutations of pfdhfr ( I 51 N 108 ) were detected, and showed an increase from 0.7% to 4.3% after the introduction of SP as first-line drug. Furthermore, a significant decrease in double mutations/wild-type of pfdhfr/pfdhps ( R 59 N 108 /A 437 ) was observed from 2004 (83.5%) to 2008 (44%) after changes in treatment policy. With regards to pfdhps , the results showed a rapid increase in frequency of the single pure form of pfdhps at position 437G (54.4%) and that of triple pfdhfr/pfdhps ( R 59 N 108 / G 437 ) mutant haplotype (51.7%) after three years. Conclusions The absence of quintuple mutations in the examined isolates supports the continued use of SP as the treatment of choice for uncomplicated malaria as a partner drug to artemisinin combination therapy in Iran. However, the increase in the triple pfdhfr/pfdhps ( R 59 N 108 / G 437 ) mutant haplotypes indicates that the P. falciparum parasite populations have the potential to evolve into dhfr/dhps quintuple mutants in the near future. Therefore, monitoring the status of dhps alleles as a predictor of the development of clinical resistance to sulfadoxine should be a high priority in this region.