MiRNAs related in signaling pathways of women's reproductive diseases: an overview.

BACKGROUND: One of the main health issues that can affect women's health is reproductive diseases, such as polycystic ovary syndrome (PCOS), endometriosis (EMs), uterine leiomyomas (ULs), and ovarian cancer (OC). Although these diseases are very common, we do not have a complete understanding of their underlying cellular and molecular mechanisms. It is important to mention that the majority of patients are diagnosed with these diseases at later stages because of the absence of early diagnostic techniques and dependable molecular indicators. Hence, it is crucial to discover novel and non-invasive biomarkers that have prognostic, diagnostic and therapeutic capabilities. MiRNAs, also known as microRNAs, are small non-coding RNAs that play a crucial role in regulating gene expression at the post-transcriptional level. They are short in length, typically consisting of around 22 nucleotides, and are highly conserved across species. Numerous studies have shown that miRNAs are expressed differently in various diseases and can act as either oncogenes or tumor suppressors. METHODS: The author conducted a comprehensive review of all the pertinent papers available in web of science, PubMed, Google Scholar, and Scopus databases. RESULTS: We achieved three goals: providing readers with better information, enhancing search results, and making peer review easier. CONCLUSIONS: This review focuses on the investigation of miRNAs and their involvement in various reproductive disorders in women, including their molecular targets. Additionally, it explores the role of miRNAs in the development and progression of these disorders.

Hsa-miR-15b-5p/miR-195-5p Controls Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells Through Regulating Indian Hedgehog Expression.

Osteoblastogenesis is regulated by several signaling pathways like hedgehog signaling. Of three types of mammalian Hedgehog genes, the Indian Hedgehog (Ihh) plays an important role in the formation of the skeleton. Mesenchymal stem cells (MSCs) isolated from adipose tissue have been considered a good source of osteoblast differentiation. Evidence also suggests that miRNAs play an important role in regulating key stages of osteoblast differentiation. In this study, two miRNAs targeting the Ihh were predicted by using bioinformatics analysis. ASCs were successfully derived, purified, and characterized from human adipose tissue. ASCs were chemically induced into osteoblast cells. Then, differentiation was confirmed by alkaline phosphatase (ALP) activity and Alizarin red staining. The relative expression of Ihh and related miRNAs was evaluated after 0, 7, 14, and 21 from the differentiation duration. The results of bioinformatics data showed that has-miR-195-5p and has-miR-15b-5p target the Ihh gene. The expression of Ihh significantly increased in a time-dependent manner in the differentiation process. In contrast, miR-195-5p and miR-15b-5p were significantly downregulated dependent on time duration (P < 0.01). Overall, the data indicate the antithetical regulation of Ihh versus has-miR-195-5p and has-miR-15b-5p during the differentiation process. These results support the hypothesis that these mi-RNAs could target the Ihh in the pathway of osteoblast differentiation derived from human ASCs.

A systems biology approach and in vitro experiment indicated Rapamycin targets key cancer and cell cycle-related genes and miRNAs in triple-negative breast cancer cells.

An anticancer drug known as Rapamycin acts by inhibiting the mammalian target of the Rapamycin pathway. This agent has recently been investigated for its potential therapeutic benefits in sensitizing drug-resistant breast cancer (BC) treatment. The molecular mechanism underlying these effects, however, is still a mystery. Using a systems biology method and in vitro experiment, this study sought to discover essential genes and microRNAs (miRNAs) targeted by Rapamycin in triple-negative BC (TNBC) cells to aid prospective new medications with less adverse effects in BC treatment. We developed the transcription factor-miRNA-gene and protein-protein interaction networks using the freely accessible microarray data sets. FANMOD and MCODE were utilized to identify critical regulatory motifs, clusters, and seeds. Then, functional enrichment analyses were conducted. Using topological analysis and motif detection, the most important genes and miRNAs were discovered. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the effect of Rapamycin on the expression of the selected genes and miRNAs to verify our findings. We performed flow cytometry to investigate Rapamycin's impact on cell cycle and apoptosis. Furthermore, wound healing and migration assays were done. Three downregulated (PTGS2, EGFR, VEGFA) and three upregulated (c-MYC, MAPK1, PIK3R1) genes were chosen as candidates for additional experimental verification. There were also three upregulated miRNAs (miR-92a, miR-16, miR-20a) and three downregulated miRNAs (miR-146a, miR-145, miR-27a) among the six selected miRNAs. The qRT-PCR findings in MDA-MB-231 cells indicated that c-MYC, MAPK1, PIK3R1, miR-92a, miR-16, and miR-20a expression levels were considerably elevated following Rapamycin treatment, whereas PTGS2, EGFR, VEGFA, miR-146a, and miR-145 expression levels were dramatically lowered (p < 0.05). These genes are engaged in cancer pathways, transcriptional dysregulation in cancer, and cell cycle, according to the top pathway enrichment findings. Migration and wound healing abilities of the cells declined after Rapamycin treatment, and the number of apoptotic cells increased. We demonstrated that Rapamycin suppresses cell migration and metastasis in the TNBC cell line. In addition, our data indicated that Rapamycin induces apoptosis in this cell line. The discovered vital genes and miRNAs affected by Rapamycin are anticipated to have crucial roles in the pathogenesis of TNBC and its therapeutic resistance.

Assessment of efficacy of carboxytherapy in management of skin aging through evaluation of gene expression profile: a 2-split randomized clinical trial.

Skin aging is a continuous and irreversible process which results in impairment of the skin role as barrier against all aggressive exogenous factors. It mainly manifests by photoaging, laxity, sagging, wrinkling, and xerosis. Carboxytherapy is considered as a safe, minimally invasive modality used for rejuvenation, restoration, and recondition of the skin. In the current study, the efficacy of carboxytherapy in the treatment of skin aging was assessed through investigation of gene expression profile for Coll I, Coll III, Coll IV, elastin, FGF, TGF-ß1, and VEGF. Our study is a 2-split clinical trial in which carboxytherapy was performed on one side of the abdomen in 15 cases with intrinsically skin aging manifestations weekly for 10 sessions, while the other side of the abdomen was left without treatment. Two weeks after the last session, skin biopsies were taken from both the treated and control sides of the abdomen in order to assess gene expression profile by qRT-PCR. The analysis of gene expression levels for all of Coll I, Coll III, Coll IV, elastin, TGF-ß1, FGF and VEGF genes showed a statistically significant difference between the interventional and control groups. The findings for all of these seven genes showed increase in the interventional group, among which Coll IV, VEGF, FGF, and elastin showed the higher mean changes. Our study confirmed the effectiveness of carboxytherapy in treating and reversing the intrinsically aging skin.Clinical Trial Registration Code and Date of Registration: ChiCTR2200055185; 2022/1/2.

The role of miRNAs as a big master regulator of signaling pathways involved in lymphoblastic leukemia.

MicroRNAs (miRNAs) belong to small noncoding RNAs, which have long attracted researchers' attention because of their potency in acting either as oncogenes or tumor-suppressors in cancers. acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are two known types of leukemia with high mortality rates in adults and children. On a molecular basis, various signaling pathways are active in both types, making researchers consider the potential role of miRNAs in activating or suppressing these pathways to further hinder cancer development. In this review, we summarized the potential miRNAs, especially circulating ones, involved in essential signaling pathways in the ALL and CLL patients which serve as biomarkers and valuable targets in the treatment fields.

The role of microRNAs in diseases and related signaling pathways.

MicroRNAs (miRNAs) are epigenetic regulators of the gene expression and act through posttranslational modification. They bind to 3'-UTR of target mRNAs to inhibit translation or increase the degradation mRNA in many tissues. Any alteration in the level of miRNA expression in many human diseases indicates their involvement in the pathogenesis of many diseases. On the other hand, the regulation of the signaling pathways is necessary for the maintenance of natural and physiological characteristics of any cell. It is worth mentioning that dysfunction of the signaling pathways manifests itself as a disorder or disease. The significant evidence report that miRNAs regulate the several signaling pathways in many diseases. Base on previous studies, miRNAs can be used for therapeutic or diagnostic purposes. According to the important role of miRNAs on the cell signaling pathways, this article reviews miRNAs involvement in incidence of diseases by changing signaling pathways.

Alterations in The Plasma Expression of mir-15b, mir-195 and the Tumor-Suppressor Gene DLEU7 in Patients with B-Cell Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is one of the most prevalent forms of leukemia in adults. Inactivation of the DLEU7 gene is frequently observed in patients with CLL. Furthermore, microRNAs (miRNAs) have been observed to have a critical role in the pathogenesis of several cancers, including leukemia. Considering the tumor-suppressive role of DLEU7, as well as the tumor suppressor or oncogenic role of microRNAs (miRNAs), the aim of the present study was to evaluate the potential miRNAs targeting the DLEU7 gene in B-cells and explore expression changes these genes in the plasma of B-CLL patients. METHODS: The miRNAs interacting with the DLEU7 gene were predicted and selected using bioinformatics tools. A total of 80 plasma samples were collected from 40 patients with B-cells and 40 healthy individuals, then subjected to RNA extraction and cDNA synthesis. The expression profiles of the predicted miRNAs and the DLEU7 gene in the plasma of B-CLL patients and healthy individuals were determined by RT-qPCR analysis. RESULTS: The bioinformatics prediction indicated that miR-15b and miR-195 target the DLEU7 gene. The expression levels of miR-15b and miR-195 were significantly higher in the plasma of patients with B-CLL compared to the healthy individuals (91.6, p= 0.001) (169, p= 0.001). However, the expression level of the DLEU7 gene was found to be significantly lower in the patient group compared to healthy controls (0.304, p= 0.001). CONCLUSION: Both miR-15b and miR-195, have the potential to function as novel and non-invasive biomarkers in the diagnosis and prognosis of patients with B-CLL.

Pap Smear miR-92a-5p and miR-155-5p as potential diagnostic biomarkers of squamous intraepithelial cervical cancer.

BACKGROUND: one of the female-specific diseases with a high incidence and mortality is cervical cancer. The main cause of cervical cancer is infection with Human papilloma virus (HPV). Low-grade squamous intraepithelial lesions (LSIL) and High-grade squamous intraepithelial lesions (HSIL) usually is caused by an HPV infection. Considering the role of microRNAs (miRNAs) as diagnostic biomarkers for a variety of cancers, the aim of this study was to determine miR-92a-5p and miR-155-5p expression levels in LSIL and HSIL Pap Smear samples. METHODS: After initial bioinformatic studies, A total of 75 samples (25 samples of patients with LSIL, 25 patients with HSIL and 25 healthy individuals) were subjected to RNA extraction and cDNA synthesis. The expressions levels of confirmed miRNAs in samples of patients with LSIL, HSIL and healthy individuals were evaluated by Real time PCR analysis. To demonstration the role of predicted miRNAs as novel biomarkers in diagnosis of LSIL and HSIL, ROC curve analysis was done. RESULTS: Bioinformatics results showed that miR-92a-5p and miR-155-5p target the HPV E6 and E7 genes. The expression levels of these miRNAs were strikingly higher in Pap smear of patients with LSIL than in the healthy individuals (35.36, P = 0.001) (62.23, P = 0.001). Similarity, expression levels of miR-92a-5p and miR-155-5p were amazingly higher in patients with HSIL than in the healthy individuals (33.62, P= 0.001) (69.07, P= 0.001). Although, the levels of miR-92a-5p (0.95, P = 0. 85) and miR-155-5p (1.11, P = 0.84) exhibited no statistical differences between patients with LSIL and HSIL. Also, ROC curve analyses verified that miR-92a-5p and miR-155-5p are specific and sensitive and may serve as new biomarkers for the early detection of cervical cancer. CONCLUSION: These data suggest miR-92a-5p and miR-155-5p, which are upregulated in LSIL and HSIL, can be consider as predictive biomarkers for the prognosis of cervical cancer patients.
.

Diagnostic Value of Plasma miR-145 and miR-185 as Targeting of the APRIL Oncogene in the B-cell Chronic Lymphocytic Leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is one of the most common hematologic malignancy in adults worldwide. This cancer has a poor prognosis at different stages. So, the identification of new biomarkers is important for diagnosis of B-CLL. Considering the oncogenic role of APRIL molecule in this leukemia as well as the regulatory role of miRNAs in different signaling pathways, the present study evaluated the miRNAs targeting APRIL gene in B-CLL. METHODS: The miRNAs were predicted and selected using bioinformatics algorithms. A total of 80 plasma samples were subjected to RNA extraction and synthesis of cDNA. The expressions levels of predicted miRNAs and APRIL gene in plasma of B-CLL patients and healthy individuals were assessed by Real time PCR analysis. ROC analysis was performed to investigate the role predicted miRNAs as novel biomarkers in diagnosis of B-CLL. RESULTS: The results of the prediction showed that miR-145-5p and miR-185-5p target the APRIL gene. The expression level of APRIL gene was strikingly higher in plasma of B-CLL patients than in the healthy individuals (102, P= 0.001). On the other hand, expression levels of miR-145-5p and miR-185-5p were strikingly lower in B-CLL patients than in the healthy individuals (0.07, P= 0.001) (0.29, P= 0.001). Also, ROC curve analyses demonstrated that miR-145-5p and miR-185-5p are specific and sensitive and may serve as new biomarkers for the detection of B-CLL. (AUC; 0.95, sensitivity; %90) (AUC; 0.87, sensitivity; %63). CONCLUSION: These data suggest that miR-145-5p and miR-185-5p target the APRIL gene and might have a role in diagnosis of B-CLL. Therefore, these two miRNAs can be served as a novel and potential biomarker for detection of B-CLL.

Multi-Drug-Resistant Diarrheagenic Escherichia coli Pathotypes in Pediatric Patients with Gastroenteritis from Central Iran.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) is a significant cause of gastroenteritis and a major public health problem. This study investigates the prevalence and the antibiotic resistance patterns of DEC that were isolated from infectious diarrhea samples of pediatric patients from central Iran. PATIENTS AND METHODS: Pediatric diarrhea samples were collected from 230 pediatric patients visiting the hospital. E. coli pathotypes were diagnosed by using conventional culture methods and PCR. Antibiotic resistance profiles, the frequency of multi-drug resistance (MDR), and the phenotypic and genotypic characteristics of extended spectrum-ß-lactamase (ESBL), AmpC and integron-associated genes were analyzed. RESULTS: Of the 230 samples of infectious diarrhea, 91 (39.5%) produced E. coli isolates. Of these, 32 cases (35.1%) were identified as DEC by culture and PCR. The frequency of the E. coli pathotypes obtained was as follows: EAEC 11/32 (34.3%), EPEC 9/32 (28.1%), ETEC 6/32 (18.7%), EIEC 3/32 (9.3%), and EHEC 3/32 (9.3%). The antibiotic resistance rates were greater for nalidixic acid (30/32; 93.7%), ampicillin (29/32; 90.6%), and tetracycline (25/32; 78.1%) than for any of the other tested antibiotics. High levels of MDR (25/32; 78.1%) and the presence of ESBL (18/32; 56.2%) and AmpC (9/32; 28.1%) were observed in the DEC isolates. The isolates showed a higher frequency of the ESBL genes [blaTEM (18/18; 100%), blaCTX-M15 (17/18; 94.4%)], and AmpC [bla CIT (4/9; 44.4%) and blaDHA (4/9; 44.4%)] than of the other ESBL and AmpC genes. CONCLUSION: Compared to the previous study, DEC appeared to be the second-most abundant agent of diarrhea in pediatric patients after Campylobacter jejuni, with frequent MDR and ESBL presence.

Alteration in Expression of miR-32 and FBXW7 Tumor Suppressor in Plasma Samples of Patients with T-cell Acute Lymphoblastic Leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and malignant neoplasm that arises from the hematopoietic T-cell precursors. Inactivation of FBXW7 gene is frequently observed in T-cell acute lymphoblastic leukemia, suggesting a significant tumor-suppressive role for FBXW7 in the pathobiology of this leukemia. Considering the role of microRNAs in cell proliferation and regulation of apoptosis, the aim of this study was to identify novel oncogenic microRNAs that suppress FBXW7 in patients with T-ALL. PATIENTS AND METHODS: The expression levels of two bioinformatically predicted microRNAs - miR-32 and miR-107 were compared in patients with T-ALL and a control group. A total of 80 plasma samples were subjected to RNA extraction, and the microRNA expression profiles were assessed by the RT-qPCR. The expression level of miR-103 was used as the endogenous reference for normalization of quantitative data. RESULTS: The plasma levels of miR-32 and miR-107 in patients with T-ALL were significantly higher (5.65, P < 0.001) and lower (0.432, P = 0.002), respectively. On the other hand, the expression levels of FBXW7 gene were significantly downregulated by -76.9 fold in T-ALL patients (P < 0.001). The results of the ROC curve analysis indicated that overexpression of miR-32 might be used to distinguish T-ALL patients with reasonable sensitivity and specificity. CONCLUSION: miR-32 is considered as a novel oncomir that targets FBXW7 and might have a role in the etiology or progression of T-ALL. Furthermore, miR-32 can potentially serve as a non-invasive biomarker for detection of T-ALL.

Plasma Level of MicroRNAs, MiR-107, MiR-194 and MiR-210 as Potential Biomarkers for Diagnosis Intestinal-Type Gastric Cancer in Human

Background: Timely and sensitive diagnosis of gastric cancer is crucial for efficient treatment and survival of the patients. microRNAs have been considered as diagnostic biomarkers in different type of cancers including gastric cancer. In the present study, the expression profile of four microRNAs, miR-103, miR-107, miR-194 and miR-210 were evaluated in patients with intestinal-type of gastric cancer (IGC) in order to assess their diagnosis utility as noninvasive biomarkers. Methods: A total number of 100 plasma samples from patients with gastric cancer and healthy controls were obtained and total RNA was extracted using a commercial monophasic solution of phenol and guanidium thiocyanate. Reverse transcription (RT) reactions were performed by specific stem-loop RT primers and M-MuLV RT-enzyme. The expression patterns of microRNAs were assessed using reverse transcription quantitative real-time PCR (RT-qPCR) method and the expression of SNORD47 RNA was used as the reference for normalization. Results: The results indicate that the plasma levels of miR-107, miR-194, and miR-210 were significantly lower in patients. Receiver operating characteristic (ROC) curve analysis showed that the patients could be distinguished from healthy individuals at the cutoff levels of 0.504, 0.266, and 0.394 of miR-107, miR-194, and miR-210, respectively. On the other hand, the expression levels of these miRNAs were not significantly different in different clinicopathological stages of the disease. Conclusion: These findings suggest that the plasma levels of miR-107, miR-194 and miR-210 were downregulated in patients with ICG and propose these molecules as potential non-invasive biomarkers for detection of IGC.

Transgenerational consequences of prepregnancy chronic morphine use on spatial learning and hippocampal Mecp2 and Hdac2 expression.

There has recently been increasing interest in the transgenerational effects of opioids. Herein, transgenerational consequences of maternal chronic morphine consumption before gestation were investigated at the behavioral and molecular levels of next two generations. Twelve female Wistar rats were randomly divided into two groups: pregestation morphine-consuming and control mothers. Morphine-consuming mothers had access to morphine solution ad libitum for 2 months, whereas the control mothers received only tap water. One month after stopping morphine consumption, rats were mated. After parturition, male and female offspring and later grandoffspring of morphine-consuming and control mothers were divided and used as the study groups. Behavioral testing comprised spatial memory assessment using Morris water maze. Hippocampal expressions of Mecp2 and Hdac2 were investigated through real-time PCR. Spatial memory was significantly diminished in male but not female offspring and grandoffspring of morphine-consuming mothers versus control (P<0.01). Hippocampal Mecp2 and Hdac2 were significantly upregulated in male but not in female offspring and grandoffspring of morphine-consuming mothers compared with control (P<0.05). Consequences of prepregnancy morphine consumption have sex differences and are detectable at the behavioral and molecular level over at least the next two generations.

Two Triacylglycerol Pathway Genes, CTDNEP1 and LPIN1, are Down-Regulated by hsa-miR-122-5p in Hepatocytes.

BACKGROUND: Expression of miR-122 is highly specific to hepatocytes of the liver.  This miRNA is involved in lipid hemostasis of the tissue; however, there is no comprehensive understanding of its function in lipid hemostasis. MATERIALS AND METHODS: Since hepatocytes are responsible for part of Triacylglycerol (TAG) synthesis in the body, we hypothesized that miR-122, as the most abundant miRNA in the tissue, might regulate TAG metabolism by targeting key enzymes that are involved in its production pathway. A systematic computational analysis of putative targets of miR-122 identified CTDNEP1 and LPIN1 genes in the TAG pathway. We used dual-luciferase reporter assay, quantitative RT-PCR as well as western blot to confirm the repressive effect of miR-122 on CTDNEP1 and LPIN1 in TAG pathway. RESULTS: Real time PCR on liver needle biopsies with hepatosteatosis showed that miR-122 is up-regulated in hepatosteatosis. Surprisingly, the protein and RNA level of identified targets of miR-122 are also up-regulated in clinical samples, probably as a disproportionate feedback response to the high level of miR-122. CONCLUSION: Our findings suggest that up-regulation of miR-122 can trigger the compensatory response of LPIN1 and CTDNEP1 in hepatosteatosis.

Trimethyl Chitosan Improves Anti-HIV Effects of Atripla as a New Nanoformulated Drug.

BACKGROUND: Highly active antiretroviral therapy (HAART) has been commonly used for HIV treatment. Its main drawbacks like drug resistance and side effects raised researcher's interest to find new approaches for its treatment. Trimethyl chitosan is one of the drug carriers which has been introduced recently. MATERIALS AND METHODS: the conjugated atripla-trimethyl chitosan was designed and characterized by zetasizer, AFM and FTIR techniques. The drug conjugation with trimethyl chitosan and cellular uptake of nano-conjugate were determined by spectrophotometry. XTT test was used to measure the cytotoxicity. Anti-retroviral efficiency was studied by ELISA test. RESULTS: Zetasizer Results proved that the average size of nano-conjugate particles agglomeration was 493.4±24.6 nm but the size of the majority of the particles was 177.2±7.8 nm with the intensity of 87.9%. AFM technique revealed that the sizes of nano-conjugate and trimethyl chitosan were 129 nm and 59.78 nm, respectively. Zeta potential was -1.35±0.04 mv for nano-conjugate and -7.69±0.3 mv for drug. Conjugation efficiency of atripla with trimethyl chitosan was 5.27%. Measured cellular uptake with spectrophotometry for nano-conjugate was about twice of the free drug in examined concentrations (P=0.007). Compared to atripla, the nano-conjugate showed a higher inhibitory effect on HIV replication (P=0.0001). CONCLUSION: The result showed that atripla-TMC conjugate does not have a significant cytotoxicity effect. Due to the higher inhibitory effect of nano-conjugate on viral replication, it can be used in lower concentration for antiviral treatment, which resulted in reduction of drug resistance and other side effects.

Synergistic effect of programmed cell death protein 1 blockade and secondary lymphoid tissue chemokine in the induction of anti-tumor immunity by a therapeutic cancer vaccine.

The use of DNA vaccines has become an attractive approach for generating antigen-specific cytotoxic CD8+ T lymphocytes (CTLs), which can mediate protective antitumor immunity. The potency of DNA vaccines encoding weakly immunogenic tumor-associated antigens (TAAs) can be improved by using an adjuvant injected together with checkpoint antibodies. In the current study, we evaluated whether the therapeutic effects of a DNA vaccine encoding human papilloma virus type 16 (HPV-16) E7 can be enhanced by combined application of an immune checkpoint blockade directed against the programmed death-1 (PD-1) pathway and secondary lymphoid tissue chemokine (SLC) also known as CCL21 adjuvant, in a mouse cervical cancer model. The therapeutic effects of the DNA vaccine in combination with CCL21 adjuvant plus PD-1 blockade was evaluated using a tumor growth curve. To further investigate the mechanism underlying the antitumor response, cytolytic and lymphocyte proliferation responses in splenocytes were measured using non-radioactive cytotoxicity and MTT assays, respectively. Vascular endothelial growth factor (VEGF) and IL-10 expression in the tumor and the levels of IFN-γ and IL-4 in supernatants of spleno-lymphocyte cultures were measured using ELISA. The immune efficacy was evaluated by in vivo tumor regression assay. The results showed that vaccination with a DNA vaccine in combination with the CCL21 adjuvant plus PD-1 blockade greatly enhanced cytotoxic T lymphocyte production and lymphocyte proliferation rates and greatly inhibited tumor progression. Moreover, the vaccine in combination with adjuvant and blockade significantly reduced intratumoral VEGF, IL-10 and splenic IL-4 but induced the expression of splenic IFN-γ. This formulation could be an effective candidate for a vaccine against cervical cancers and merits further investigation.

Specific detection of common pathogens of acute bacterial meningitis using an internally controlled tetraplex-PCR assay.

Accurate and timely diagnosis of acute bacterial meningitis is critical for antimicrobial treatment of patients. Although PCR-based methods have been widely used for the diagnosis of acute meningitis caused by bacterial pathogens, the main disadvantage of these methods is their high cost. This disadvantage has hampered the widespread use of molecular assays in many developing countries. The application of multiplex assays and "in-house" protocols are two main approaches that can reduce the overall cost of a molecular test. In the present study, an internally controlled tetraplex-PCR was developed and validated for the specific detection of Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in cerebrospinal fluid (CSF) samples. The analysis of a panel of other human pathogens showed no cross-reactivity in the assay. The analytical sensitivity of the in-house assay was 792.3 copies/ml, when all three bacteria were presentin the specimens. This value was calculated as 444.5, 283.7, 127.8 copies/ml when only S. pneumoniae, N. meningitidis and H. influenzae, respectively, were present. To demonstrate the diagnostic performance of the assay, a total of 150 archival CSF samples were tested and compared with a commercial multiplex real-time PCR kit. A diagnostic sensitivity of 92.8% and a specificity of 95.1% were determined for the present tetraplex-PCR assay. The results indicate that the established method is sensitive, specific and cost-effective, and can be used particularly in situations where the high cost of commercial kits prevents the use of molecular methods for the diagnosis of bacterial meningitis.

Monitoring human cytomegalovirus infection in pediatric hematopoietic stem cell transplant recipients: using an affordable in-house qPCR assay for management of HCMV infection under limited resources.

Quantitative real-time PCR (qPCR) assay is accepted as the method of choice for monitoring human cytomegalovirus (HCMV) infection in hematopoietic stem cell transplant recipients, but the high cost of commercial kits has hampered its use in many developing countries. In this study, an affordable in-house qPCR was used to manage HCMV infection in pediatric patients and the diagnostic value of this method was compared with the conventional pp65 antigenemia assay. A total number of 1179 samples from 82 recipients were used in this study, and the effect of some potential risk factors on HCMV reactivation was evaluated. The qPCR was able to detect HCMV reactivation earlier and with higher sensitivity than antigenemia assay. Forty-six episodes of reactivation were detected in 39 patients, of which all were detected by the qPCR assay, while only 21 episodes were diagnosed by antigenemia. The DNAemia level of 1284 IU/ml plasma was defined as the optimal cutoff value for starting pre-emptive therapy. It was shown that the acute GVHD severity and the relationship of donor and recipient are the most significant risk factors for HCMV reactivation. The data suggest that the antigenemia method for monitoring HCMV reactivation could be substituted by the qPCR assay.

MicroRNA-124 regulates neuronal differentiation of mesenchymal stem cells by targeting Sp1 mRNA.

MicroRNAs play an important role in neuronal development and function. miR-124 is the most abundantly expressed miRNA in the nervous system. Several different mRNA targets have been proposed for miR-124, but the precise function of endogenous miR-124 and its mRNA targets remain to be further elucidated. Specificity protein 1 (Sp1) is a transcription factor that plays key roles in many cell processes including cell cycle. However, this transcription factor is nearly absent in differentiated neurons and a remarkable suppression of Sp1 expression was shown after neurogenesis. Since miR-124 is expressed abundantly in neurons and because Sp1 levels decrease during neurogenesis, it is possible that miR-124 could regulate the expression of Sp1 during neuronal development. Therefore, the aim of the present study was to evaluate the putative targeting of Sp1 by miR-124. Overexpression of miR-124 using a plasmid coding for pri-miR-124 in HEK293 cells decreased the expression of Sp1 mRNA. The results of dual-luciferase reporter assay demonstrated that miR-124 directly targeted the 3'-untranslated regions of Sp1 mRNA. To evaluate whether Sp1 expression was regulated by miR-124 during the process of neuronal differentiation, Adipose-derived mesenchymal stem cells (A-MSCs) were differentiated into neuron-like cells. The results of qPCR analysis showed that with the gradual increase of miR-124 expression during neurogenesis, the expression of Sp1 mRNA decreased accordingly. In summary, this study demonstrated for the first time that miR-124 is able to suppress Sp1 expression, which in turn affected the neuronal differentiation of mesenchymal stem cells.