Molecular Marker Profiles Research Articles

Profile of molecular markers of Sulfadoxine-Pyrimethamine-resistant Plasmodium falciparum in individuals living in southern area of Brazzaville, Republic of Congo

BackgroundAlthough the seasonal and perennial malaria chemopreventions are not implemented in the Republic of Congo, resistance to Sulfadoxine-pyrimethamine (SP) threatens the intermittent preventive treatment during pregnancy (IPTp-SP) and others treatments using the drug. The objective of this study was to determine the prevalence of molecular markers of P.falciparum resistance to SP in individuals with microscopic malaria infection in the south of Brazzaville. MethodsTwo parallel surveys (health facilities and community-based cross sectional studies) were carried out in urban and rural areas in southern Brazzaville. Between March and October 2021, blood samples were collected from 328 P. falciparum microscopic positive individuals (1- 83 years old, and sex ratio female/male of 1.1) to characterize dhfr and dhps genes involved in the P.falciparum resistance to SP. Restriction Fragment Length Polymorphism PCR was used for the detection of mutations within these parasite genes. ResultsHigh prevalence of mutations was reported within Pfdhfr gene: N51I; 328/328 (100%) ratio (prevalence) [95 CI uncertainty], C59R; 317/328 (96.6 %) [94.1-98.1%], S108N; 326/326 (100%), N164L; 3/326 (0.9%) [0.3-2.7%], and Pfdhps gene: A437G; 292/327 (89.3%) [85.5-92.2%], K540E; 140/327(42.8 %) [37.6-48.2%], A581G; 136/325 (41.8%) [36.6-42.3%]. The quintuple mutant (N51I+ C59R + S108N + A437G + K540E) and sextuple mutant haplotypes (N51I+ C59R + S108N + A437G + K540E + A581G) were reported for 11/144 (7.6%) [4.3-13.2%] and 5/144 (3.4% ) [1.5-7.9%]) of the participants respectively. The K540E and A437G mutants were more prevalent in the rural community; 81/139 (58.3%) [50.0-66.1%] and 135/139 (97.1%) [92.8-98.9%] respectively) than in the urban community; 21/50 (46.3%) [33.7-59.4%] and 47/54(87.0%) [75.6-93.6%] (p= 0.004 and p˂0.0001 respectively) ConclusionThese results indicate high prevalence of SP resistance mutations within the dhfr and dhps genes of P. falciparum isolates circulating in study sites, which may limit the efficacy of treatments using SP in these settings.

Analysis of molecular marker expression in cutaneous lesions and cervical carcinoma associated with HPV infection.

The study sought to systematically compare the expression of molecular markers in benign cutaneous lesions and squamous cell cervical carcinoma associated with HPV infection to better understand the pathophysiological mechanisms involved in HPV-related lesions and their progression to malignancy. We included 200 patients recruited from a gynecological clinic divided into two groups: 100 patients with positive HPV tests presenting with cutaneous lesions and 100 patients diagnosed with squamous cell cervical carcinoma and testing positive for HPV. The participants were selected to ensure diverse ethnic and demographic representation. The study utilized different statistical analyses, including Chi-square tests to assess associations between categorical variables and logistic regression to evaluate factors influencing lesion progression and compare marker expressions across different lesion types. The results indicated significant differences in the expression of specific molecular markers between cutaneous lesions and cervical carcinomas, highlighting distinct molecular pathways involved in HPV-related lesion development. Notably, markers such as p16, p53, and E-cadherin showed varying expression, suggesting their potential role in distinguishing between benign and malignant lesions. The findings emphasize the significance of molecular marker profiling in improving diagnostic and therapeutic strategies for HPV-related lesions. The differential expression of molecular markers can offer valuable insights into the pathogenesis of HPV-induced lesions and help develop targeted interventions to prevent malignant transformation. Further research is necessary to validate these markers in larger cohorts and diverse populations.

A metabolism targeting three-pronged attack significantly attenuates breast cancer stem cell related markers toward therapeutic application

Tumor metabolism has provided researchers with a promising window to cancer therapy. The metabolic pathways adopted by cancer cells are different from those of normal cells. Thus, metabolism can be considered a linchpin in targeted cancer therapy. Glycolysis, pentose phosphate pathway, and mitochondria represent three critical metabolic spots with important roles in cancer cell survival and proliferation. In the present study, we aimed to target these pathways using three different inhibitors: 2-deoxyglucose, 6-aminonicotinamide, and doxycycline, separately and in combination. Accordingly, cell viability, lactate production, cell cycle profile, apoptotic profile, and expression of surface and molecular markers of MCF-7 and MDA-MB-231 breast cancer cell lines were investigated under adherent and sphere conditions. Our results from our set conditions indicated various inhibitory effects of these compounds on the breast cancer cell lines. Based on this all-around attack, the combination of drugs demonstrated the most effective inhibitory action compared to separate usage. This study suggests the combined application of these drugs in future investigations and more experimental settings in order to introduce this therapeutic strategy as an efficient anti-cancer treatment.

Morphology of the esophagus of ferrets and expression profile of molecular markers

To examine the morphological characteristics and the expression profile of molecular markers of ferret esophagus and assess the feasibility of using ferrets as animal models for studying human esophageal diseases. Frozen sections and paraffin- embedded specimens of the esophageal tissues were obtained from adult ferrets (aged 6 to 8 months) and ferrets aged 1 day, 3 days, 5 days, 1 week and 2 weeks. HE staining and periodic acid-Schiff (PAS) staining were used for morphological analysis of the esophageal submucosal glands (SMGs) of adult ferrets, and the expressions of MUC5B and MUC5AC were tested using Mucin staining; The expressions of cytokeratins (CK4, CK5, CK7, CK8, CK14, CK17, CK18, CK19, and CK20) in adult ferret esophagus were examined using HE staining and immunofluorescence assay. The expressions of LEF1 in the esophageal epithelium and SMGs were detected with immunofluorescence assay. In adult ferrets, the esophageal SMGs were connective tissues below the muscularis mucosa of the esophagus with secretory functions. Cytokeratins were expressed differentially in different esophageal cells: CK4, CK8 and CK20 were expressed mainly in the mucous cells, ductal cells and epithelial cells, respectively, while the mucous cells expressed the largest variety of cytokeratins. Mucin staining showed positive MUC5B and MUC5AC expression in the cytoplasm and lumen of adult ferret esophageal glands. Lectin from DBA, ECL, GSLI, GSL Ⅱ, SBA, Tacalin bioylated, ULEX, WGA, GSL Ⅰ and GSL Ⅱ were expressed on ductal cell membrane, and ECL, PNA and WGA were detected on epithelial cell membrane. Lectin with ConA, PHA-E and PHA-L were expressed on serous cell membrane. Immunofluorescence assay showed that LEF1 in the developing glands were visible from 3 days to 1 week of age and then disappeared as the glands matured. The intensity of LEF1 expression in the esophageal glands differed significantly between ferrets aged 1 to 7 days and those aged two weeks. Ferrets and human share similar esophageal tissue structures and some common molecular markers, suggesting the possibility of using ferrets as animal models of human esophageal diseases.

Profile of Molecular Markers of Cardiac Fibrosis in Rats Exposed to Different Doxorubicin Doses

Profile of Molecular Markers of Cardiac Fibrosis in Rats Exposed to Different Doxorubicin Doses

Immunophenotypic Analysis of T Lymphocytes and Cytokine Production in Elderly Practicing Physical Activities and Its Relationship with Quality of Life and Depression.

Aging is a complex process often associated with a chronic inflammatory profile that alters several biological functions, including the immune system and cognitive and physical capacity. The practice of physical activity is increasingly gaining popularity as a method of preventing infections, depression, and other disorders that affect the quality of life of the elderly. Thus, this work analyzes the profile of cytokines and molecular markers expressed in immune cells of elderly people who practice physical activities or not, evaluating their impacts on the immune system and quality of life. For this, 48 individuals were recruited, and peripheral blood samples were collected for hemogram analysis, cytokine determination, and immunophenotyping. Elderly people were separated into two groups: practitioners with low-intensity physical activity and non-practitioners. Quality of life was assessed using the Whoqol-Old instrument, and depression was assessed using the Beck II Depression Inventory. When comparing the scores of the Whoqol-Old and Beck questionnaires, we observed a significant negative correlation between these two factors. The perception of a higher quality of life was present in the elderly who exercised and was related to greater autonomy and sensory abilities, whereas the presence of depression was lower. In the hemogram, we observed higher basophil and segmented counts in the sedentary elderly, whereas lymphocytes and monocytes had lower counts. Elderly practitioners of physical activities had higher levels of IFN-γ, IL-4, and IL-10; increased expression of CD69, PD1, and TIM-3 in CD4+ T lymphocytes and increased CD14+CD80+ and CD14+CD86+ monocytes. Elderly people with an increased perception of quality of life had higher levels of IFN-γ, higher expression of CD14+CD80+CD86+, and decreased levels of TRAIL. An increase in TRAIL was observed in individuals with depression, in addition to an increased expression of CD14+CD86+. These results show a clear correlation between the quality of life, level of depression, physical activity, and immune system function. Although some cytokines with a typical proinflammatory profile (IFN-γ) were observed, the results point to a protective state with benefits reflected in the general well-being of the elderly who exercise.

Utility of mutational analysis for risk stratification of indeterminate thyroid nodules in a real-world setting.

American Thyroid Association (ATAn) 2015 guidelines recommend repeat fine-needle aspiration with molecular marker profiling (MMP) or diagnostic lobectomy in thyroid nodules yielding atypia of unknown significance/follicular lesion of unknown significance (AUS/FLUS) or follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) cytology. Our objective is to describe the molecular profiles and histological correlates of these cytologically indeterminate nodules (CIN) to aid risk stratification. Retrospective chart review. Adults with CIN that underwent MMP from 2017 to 2020. Pearsons' χ2 , Fisher's exact test, nonparametric testing and multiple regression analysis were performed. A total of 89 CIN underwent mutational analysis. Of 55% (n = 49) were Bethesda class III AUS/FLUS and 45% (n = 40) were Bethesda class IV FN/SFN. The US phenotype of a CIN was isoechoic (53%) or hypoechoic (32%) with well-defined margins (98%), absence of calcifications (75%) and mildly increased internal vascularity (70%). A total of 84% and 87% of nodules were classified as mild/moderate or low/intermediate risk per the Thyroid Imaging Reporting and Data System and ATA classifications, respectively. Based on the Thyroseq patient management resource, 6.7% (n = 6) of nodules had a high predicted probability of cancer (≥95%), 41.6% (n = 37) were intermediate probability (40%-94%) and 51.7% (n = 46) were low probability (<40%). MMP revealed positive mutations in 45% (n = 40) of nodules, with 71% demonstrating RAS mutations. Of the nodules that underwent resection (n = 38), 39% (n = 15) had malignant pathology. Increasing the threshold to recommend surgical resection to a Thyroseq predicted probability of cancer to ≥50%, had a 100% sensitivity and 65% specificity for detecting malignant nodules (area under the ROC curve: 0.86). The positive predictive value was 37% and the negative predictive value was 100%. US phenotypes of CIN nodules were variable and did not aid in differentiating malignant from benign nodules. Of the CIN nodules with a positive MMP, most were RAS and had a benign pathology. With the exception of high-risk genetic markers for malignancy, the threshold to recommend surgical resection should be raised for CIN. Further studies to improve risk stratification in these nodules are required.

Immunohistochemical Profile of Molecular Markers of Mammary Carcinomas in Libreville

To establish the immunohistochemical profile of the molecular markers of free-ranging breast carcinomas. Descriptive retrospective study over 3 years from July 2014 to September 2017. The tumour samples came from the Anatomical Pathology Laboratories of the Omar Bongo Ondimba Army Instruction Hospital and the University of Health Sciences. In total, the records of 60 patients with histologically proven breast carcinoma with the immunohistochemical study were included in the study. The following molecular markers, hormone receptors (estrogen receptors, progesterone receptors), the HER2/Neu oncogene, and the Ki67 cell proliferation marker, was identified using the Immunohistochemistry technique.The average age was 47.6 years, with extremes of 15 and 69 years. Depending on the location, the right breast was most commonly affected (50%). Histologically, there was a predominance of infiltrating ductal carcinomas (66.6%) and a majority SBR III grade (50%). This study revealed an immunohistochemical profile of positive hormone receptors: ERs + (13.3%); PRs + (8.3%) and a Ki67 profile positive in 10% of tumors. The molecular classification into 4 subtypes (Luminal A, Luminal B, HER2 and Triple Negative) places the Luminal A group in the first rank (33.3%) followed by the Luminal B (15%), Triple Negative (11.6%) and HER2 (8.3%). The present work is the first study reporting the immunohistochemical profile of molecular markers of mammary carcinomas in Gabon. It would be necessary to continue this study on a larger and wider cohort throughout Gabon because the knowledge of the immunohistochemical profile gives an indication of the origin of breast cancers and allows to consider better management of patients by a targeted therapy to avoid unnecessary toxic effects resulting from ineffective treatment.

Genetic diversity and population structure of Seriphidium Sub-genus of Artemisia from different terrains of Balochistan, Pakistan

Abstract. Rehman A, Saeed S, Ahmed A. 2021. Genetic diversity and population structure of Seriphidium Sub-genus of Artemisia from different terrains of Balochistan, Pakistan. Biodiversitas 22: 2658-2664. Seriphidium is one of the largest sub-genus of Artemisia growing in different geographical populations and terrains of Balochistan Pakistan. This genus is economically important and used as folk medicine. The genus is highly variable, besides morphological characterization, molecular authentication is also needed for novel drug discovery. Three significant species of the genus viz., S. quettense, S. oliverianum, and S. maritimum were selected for morphological and molecular characterization and phytochemical investigation. Genetic variability assessment was carried out by polymorphic bands analysis by using molecular markers in 18 accessions collected from two sites, namely, Ziarat and Hazarganji-Chiltan National Park, Balochistan, Pakistan. A combined molecular marker system by using 17 Randomly Amplified Polymorphic DNA (RAPD) and 9 Inter-Simple Sequence Repeats (ISSR) amplified 231 loci, of which 111 were polymorphic (48% polymorphism). Phenolic contents and Flavonoids were estimated for phytochemical assessment. Results revealed that the highest phenolic compounds were in S. quettense when compared with other investigated taxa. In conclusion, molecular marker profiling together with phytochemical variation of all three species exhibited genetic diversity and chemical variation. The findings of the current investigation can further be used as a baseline study for the implementation of conservation strategies. In addition, phytochemical assessment can be utilized for modern drug discovery

Clinical Features and Molecular Markers on Diffuse Midline Gliomas With H3K27M Mutations: A 43 Cases Retrospective Cohort Study.

PurposeDiffuse midline gliomas (DMG) with H3K27M mutations have been identified as a rare distinctive entity with unique genetic features, varied molecular alterations, and poor prognosis. The current study aimed to evaluate the clinical characteristics and profile of molecular markers on patients with a DMG harboring H3K27M mutations, and explore the impact of this genetic makeup on overall survival.MethodsWe retrospectively analyzed 43 consecutive patients diagnosed with a DMG harboring H3K27M mutations (age range 3 to 75 years) and treated in a tertiary institution within China between January 2017 to December 2019. Various clinical and molecular factors were evaluated to assess their prognostic value in this unique patient cohort.ResultsThe median overall survival (OS) was 12.83 months. Preoperative Karnofsky Performance Score (KPS) and adjuvant radiotherapy were found to be independent clinical parameters influencing the OS by multivariate analysis (p = 0.027 and p < 0.001 respectively). Whereas extent of tumor resection failed to demonstrate statistical significance. For molecular markers, P53 overexpression was identified as a negative prognostic factor for overall survival by multivariate analysis (p = 0.030).ConclusionLow preoperative KPS, absence of radiotherapy and P53 overexpression were identified as predictors of a dismal overall survival in patients with DMG and H3K27M mutations.

Transcriptome Analysis Reveals High Similarities between Adult Human Cardiac Stem Cells and Neural Crest-Derived Stem Cells.

Simple SummaryThe regeneration of nearly all organs of the human body mainly depends on the functionality of adult stem cell populations that reside in their respective niches and can be activated upon injuries or other damages. These stem cell populations greatly differ in their expression profile of molecular markers, which greatly influences their potential use in regenerative medicine. Neural crest-derived stem cells are a prominent subpopulation of adult stem cells and are known for their high regenerative potential. Within this study, we compared two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells from the nasal cavity and human cardiac stem cells from the heart, using global gene expression profiling. Here, we found differences that correspond to the tissue sources of origin but also similarities in the expression of markers that are associated with the neural crest. Further classifying nasal stem cells and cardiac stem cells in a broader context, we identified clear similarities between both populations and other adherent stem cell populations compared to non-adherent progenitor cells of the blood system. The analyses provided here might help to understand the differences and similarities between different adult human stem cell populations.For the identification of a stem cell population, the comparison of transcriptome data enables the simultaneous analysis of tens of thousands of molecular markers and thus enables the precise distinction of even closely related populations. Here, we utilized global gene expression profiling to compare two adult human stem cell populations, namely neural crest-derived inferior turbinate stem cells (ITSCs) of the nasal cavity and human cardiac stem cells (hCSCs) from the heart auricle. We detected high similarities between the transcriptomes of both stem cell populations, particularly including a range of neural crest-associated genes. However, global gene expression likewise reflected differences between the stem cell populations with regard to their niches of origin. In a broader analysis, we further identified clear similarities between ITSCs, hCSCs and other adherent stem cell populations compared to non-adherent hematopoietic progenitor cells. In summary, our observations reveal high similarities between adult human cardiac stem cells and neural crest-derived stem cells from the nasal cavity, which include a shared relation to the neural crest. The analyses provided here may help to understand underlying molecular regulators determining differences between adult human stem cell populations.

Analysis of genetic aberrations in pediatric high-grade gliomas

Background. High-grade gliomas are characterized by a wide range of genetic abnormalities. The heterogeneous genomic landscape of pediatric high-grade gliomas allows identifying distinct subgroups of the disease in children and young adults. Most importantly, these subgroups differ by clinical course and prognosis, as well as treatment response to standard therapy.Objective: to assess the profile of molecular genetic markers of high-grade gliomas in children.Materials and methods. In the current study, we examine the frequency of H3F3A, Hist1H3B, BRAF, IDH1 / 2 mutations, the copy number alterations of CDKN2A / 2B genes and the expression of ETV6‑NTRK3 fusion gene in a cohort of 53 pediatric high-grade gliomas.Results. Driver mutations and CDKN2A / 2B deletions were observed in 24 (45 %) and 15 (28 %) of 53 tumors, respectively. Overall, the studied high-grade gliomas harbored 41 genetic aberrations including 24 (58.5 %) somatic missense mutations, 1 (2.4 %) genetic variant of unknown clinical significance, 1 (2.4 %) oncogenic fusion gene and 15 (36.6 %) deletions of the tumor suppressor genes.Conclusion. These findings point to the importance of molecular profiling of tumors for the optimal clinical care and development of new approaches to treatment aimed at molecular targets for personalized anticancer therapies.

Detailed Molecular and Immune Marker Profiling of Archival Prostate Cancer Samples Reveals an Inverse Association between TMPRSS2:ERG Fusion Status and Immune Cell Infiltration

Prostate cancer is a significant global health issue, and limitations to current patient management pathways often result in overtreatment or undertreatment. New ways to stratify patients are urgently needed. We conducted a feasibility study of such novel assessments, looking for associations between genomic changes and lymphocyte infiltration. An innovative workflow using an in-house targeted sequencing panel, immune cell profiling using an image analysis pipeline, RNA sequencing, and exome sequencing in select cases was tested. Gene fusions were profiled by RNA sequencing in 27 of 27 cases, and a significantly higher tumor-infiltrating lymphocyte (TIL) count was noted in tumors without a TMPRSS2:ERG fusion compared with those with the fusion (P=0.01). Although this finding was not replicated in a larger validation set (n=436) of The Cancer Genome Atlas images, there was a trend in the same direction. Differential expression analysis of TIL-high and TIL-low tumors revealed the enrichment of both innate and adaptive immune response pathways. Mutations in mismatch repair genes (MLH1 and MSH6 mutations in 1 of 27 cases) were identified. We describe a potential immune escape mechanism in TMPRSS2:ERG fusion-positive tumors. Detailed profiling, as shown herein, can provide novel insights into tumor biology. Likely differences with findings with other cohorts are related to methods used to define region of interest, but this warrants further study in a larger cohort.

An autologous humanized patient-derived-xenograft platform to evaluate immunotherapy in ovarian cancer

ObjectiveThe aim of this study was to “humanize” ovarian cancer patient-derived xenograft (PDX) models by autologous transfer of patient-matched tumor infiltrating lymphocytes (TILs) to evaluate immunotherapies. MethodsOrthotopic high-grade serous ovarian cancer (HGSOC) PDX models were established from three patient donors. Models were molecularly and histologically validated by immunohistochemistry. TILs were expanded from donor tumors using a rapid expansion protocol. Ex vivo TIL and tumor co-cultures were performed to validate TIL reactivity against patient-matched autologous tumor cells. Expression of TIL activation markers and cytokine secretion was quantitated by flow cytometry and ELISA. As proof of concept, the efficacy of anti-PD-1 monotherapy was tested in autologous TIL/tumor HGSOC PDX models. ResultsEvaluation of T-cell activation in autologous TIL/tumor co-cultures resulted in an increase in HLA-dependent IFNγ production and T-cell activation. In response to increased IFNγ production, tumor cell expression of PD-L1 was increased. Addition of anti-PD-1 antibody to TIL/tumor co-cultures increased autologous tumor lysis in a CCNE1 amplified model. Orthotopic HGSOC PDX models from parallel patient-matched tumors maintained their original morphology and molecular marker profile. Autologous tumor-reactive TIL administration in patient-matched PDX models resulted in reduced tumor burden and increased survival, in groups that also received anti-PD-1 therapy. ConclusionsThis study validates a novel, clinically relevant model system for in vivo testing of immunomodulating therapeutic strategies for ovarian cancer, and provides a unique platform for assessing patient-specific T-cell response to immunotherapy.

H3K27M, IDH1, and ATRX expression in pediatric GBM and their clinical and prognostic significance

Pediatric glioblastoma (pGBM) tumors have been identified as an entity distinct and different from the adult variety of GBM not only with respect to pathogenesis, genetics, and molecular alterations but also in clinical outcomes and overall survival. This study aims to evaluate the immunohistochemical profile of molecular markers in pediatric GBM and correlate them with clinical features and prognosis. We retrospectively analyzed 29 pGBMs (age range 3 to 18years), operated at our institute between 2009 and 2014, and evaluated their clinical and histopathological features along with the immunohistochemical expression of clinically relevant molecular markers: H3K27M, p53, ATRX, and IDH1 (R132H), and correlated their expression with clinical features. We further assessed the prognostic value of these markers in our cohort of patients. The median overall survival (OS) of the cohort was 6.00 ± 0.882months. The mean overall survival was 7.571 ± 1.118months which was lower than in most studies. Preoperative Karnofsky Performance Score (KPS), extent of surgical resection, and adjuvant radiotherapy were found to be the clinical factors strongly influencing median survival (p < 0.05). Loss of ATRX expression was predominantly noted in hemispheric tumors (84%), while p53 staining was maximum in thalamic tumors (8 out of 9 cases). H3K27M mutant protein expression was noted in 8/9 thalamic tumors and 5/7 tumors in the brain stem-cerebellar-peduncular region. Patients with tumors showing H3K27M immunopositivity had the worst prognosis with a mean OS of 5months ± 0.832months, as against patients with H3K27M-immunonegative tumors, which was 10.143 ± 1.866months(p = 0.006). Other markers like p53, ATRX, and IDH1 did not influence the prognosis in this patient cohort. ATRX loss of expression was associated with a better OS, with a trend to significance, and such an association has not been reported earlier. Ours is one among the few studies from India describing the clinical parameters and evaluating the key immunohistochemical markers in pGBM and deriving their prognostic significance. The study reiterates the poor prognostic significance of H3K27M immunopositivity.

Molecular authentication of Trichosanthes species traded as “Patola:” An ayurvedic drug resource

Background: Different interpretations of ayurvedic texts have recognized/recommended taxonomically distinct species/varieties of Trichosanthes as “Patola,” which is used extensively in the Indian traditional system of medicine for curing a variety of disorders. Sources of “Patola” also vary depending on region-specific availability and diverse traditions in different parts of India, which creates ambiguity in correct identity of “Patola.” Since “Patola” is one of the medicinal plant species traded in high volumes, its exact taxonomic identity and authentication is very essential. Objectives: Molecular profiling of different taxa recognized as “Patola” was carried out for correct identification and authentication of commercial crude drug samples. Materials and Methods: Molecular marker profiles of different taxa recognized as “Patola” were generated using inter-simple sequence repeat (ISSR) markers. Attempt was also made to authenticate commercial crude drug samples, using ISSR markers. Results: Selected 10 polymorphic ISSR primers generated distinct amplification profiles for the taxa traded as “Patola.” Moreover, two varieties of Trichosanthes cucumerina (var. cucumerina and var. anguina) could be clearly distinguished based on the specific bands produced by primers UBC 812, 822, 887, 888, and 889. Molecular profiling of market samples along with the genuine plant samples confirmed the trade of different Trichosanthes species as well as Momordica charantia under the name of “Patola.” Conclusion: To our knowledge, this is a first report on the application of ISSR markers for identification and authentication of “Patola.” Developed DNA profiles should be useful in investigations of adulteration in market samples and establishment of correct identity. Abbreviations used: AFLP: Amplified fragment length polymorphism; AHMA: Agharkar Herbarium at Maharashtra Association; DNA: Deoxyribonucleic acid; ISSR: Inter-simple sequence repeat; MC: Momordica charantia; PCR: Polymerase chain reaction; RAPD: Random amplified polymorphic DNA; TAE: Tris acetate-EDTA buffer; TC: Trichosanthes cucumerina; TCA: Trichosanthes cucumerina var. anguina; TCC: Trichosanthes cucumerina var. cucumerina; TD: Trichosanthes dioica; TT: Trichosanthes tricuspidata; TE: Tris-EDTA buffer; UBC: University of british Columbia, Vancouver, Canada.

Indicators of Marine Pollution in the North Pacific Ocean.

The complex nature of ocean pollution underscores the utility in identifying and characterizing a limited number of "indicators" that enables scientists and managers to track trends over space and time. This paper introduces a special issue on indicators of marine pollution in the North Pacific Ocean and builds on a scientific session that was held at the North Pacific Marine Science Organization. The special issue highlights studies using a variety of indicators to provide insight into the identification of legacy and emerging contaminants, the ranking of priority pollutants from various sources, and the effects of contaminants on ecosystem health in the North Pacific Ocean. Examples include the use of mussels to illustrate spatial and temporal trends of a number of contaminants following the 2011 tsunami in Japan, the use of molecular marker (linear alkylbenzenes, hopanes, and polycyclic aromatic hydrocarbons) profiles to identify pollution sources, and the use of plastic resin pellets to illustrate spatial trends of petroleum pollution around the world. Stable isotopes were used to strengthen the utility of the Glaucous-winged gull (Larus glaucescens) as an indicator of marine pollution. Examples also demonstrate the development and application of biomarker approaches, including gene transcripts, oxidative stress, estradiol, hatchability, and respiration and swimming behavior abnormalities, as a function of exposure to polychlorinated biphenyls, sulfur-diesel, Pinghu crude oil, galaxolide and antifouling biocides. We provide a brief review of indicators of marine pollution, identify research gaps, and summarize key findings from the articles published within the issue. This special issue represents the first compilation of research pertaining to marine pollution indicators in the North Pacific Ocean and provides guidance to inform mitigation and monitoring efforts of contaminants in the region.

A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress

BackgroundWe present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms.MethodsFive Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward’s Agglomeration method whereas MSAP characteristics for ANOVA.ResultsRelative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines.ConclusionsOur relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII–MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control and stressed Al-tolerant and non-tolerant triticale inbred lines. The method could also be used to analyze methylation events affecting CG and CHG contexts, which were differentially methylated under Al stress. We cannot exclude that the methylation changes revealed among lines as well as between Al-tolerant and non-tolerant groups of lines were due to some experimental errors or that the number of lines was too small for ANOVA to prove the influence of Al stress. Nevertheless, we suspect that Al tolerance in triticale could be partly regulated by epigenetic factors acting at the level of DNA methylation. This method provides a valuable tool for studies of abiotic stresses in plants.

TRAP and SRAP molecular marker based profiling of radiation induced mutants of sugarcane (Saccharum officinarum L.)

Genetic variability is the mainstay for crop improvement programmes which includes generation of mutational repository, their selection and study of associated genetic diversity. Molecular marker profiling using 57 markers, comprising of 27 TRAP and 30 SRAP markers was carried out to validate the extent of genetic variability in the gamma ray-induced sugarcane mutants. Collectively these markers produced 260 PCR amplicons among which 147 were polymorphic (56.54%). The TRAP marker based analysis showed that the mutants AKTS-01 and AKTS-16 were more diverse (GS=94 and 92%, respectively) than rest of the mutants. The polymorphic information content (PIC) based on TRAP marker analysis ranged from 0.0 to 0.22 (average 0.11), while 11 combinations showed a PIC≥0.17. The resolving power (Rp) of TRAP analysis ranged from 0.0 to 6.18 (average 1.70). However, the SRAP marker based analysis showed minimum diversity between parent cultivar CoC-671 and high sugar mutant AKTS-02 (GS>99%), followed by fairly good divergence (GS=96%) among the mutants AKTS-01, AKTS-08, AKTS-11, AKTS-19 and AKTS-20; along with maximum divergence (GS=91%) was shown by AKTS-06, AKTS-13 and AKTS-16. The PIC values for SRAP marker based analysis ranged from 0.0 to 0.39 (average 0.10), while 9 combinations showed a PIC≥0.17 with Rp values in the range of 0.0 to 3.45 (average 0.82). The low sugar check genotype had the least similarity with high sugar cultivar CoC-671 and its induced mutants (GS in range of 40 to 60%). Nucleotide sequencing and in silico analysis of two polymorphic amplicons generated using SRAP markers showed homology with chloroplastic ATP synthase CF1 alpha chain and gypsy-like retrotransposon element. In silico analysis revealed homology of the retrotransposon sequence with flanking regions of mobile elements in close vicinity to the serine hydroxy methyltransferase (SHMT) coding region. In summary, our results suggest, for the first time, the application of TRAP and SRAP based markers for the characterization of the mutant germplasm developed through radiation induced mutagenesis in sugarcane.

Immunophenotypic and Molecular Analysis of Human Dental Pulp Stem Cells Potential for Neurogenic Differentiation.

Background:Growing evidence shows that dental pulp (DP) tissues could be a potential source of adult stem cells for the treatment of devastating neurological diseases and several other conditions.Aims:Exploration of the expression profile of several key molecular markers to evaluate the molecular dynamics in undifferentiated and differentiated DP-derived stem cells (DPSCs) in vitro.Settings and Design:The characteristics and multilineage differentiation ability of DPSCs were determined by cellular and molecular kinetics. DPSCs were further induced to form adherent (ADH) and non-ADH (NADH) neurospheres under serum-free condition which was further induced into neurogenic lineage cells and characterized for their molecular and cellular diversity at each stage.Statistical Analysis Used:Statistical analysis used one-way analysis of variance, Student's t-test, Livak method for relative quantification, and R programming.Results:Immunophenotypic analysis of DPSCs revealed >80% cells positive for mesenchymal markers CD90 and CD105, >70% positive for transferring receptor (CD71), and >30% for chemotactic factor (CXCR3). These cells showed mesodermal differentiation also and confirmed by specific staining and molecular analysis. Activation of neuronal lineage markers and neurogenic growth factors was observed during lineage differentiation of cells derived from NADH and ADH spheroids. Greater than 80% of cells were found to express β-tubulin III in both differentiation conditions.Conclusions:The present study reported a cascade of immunophenotypic and molecular markers to characterize neurogenic differentiation of DPSCs under serum-free condition. These findings trigger the future analyses for clinical applicability of DP-derived cells in regenerative applications.