Loss of FMRP affects ovarian development and behaviour through multiple pathways in a zebrafish model of fragile X syndrome.

Fragile X syndrome (FXS) is an inherited neurodevelopmental disorder and the leading genetic cause of autism spectrum disorders. FXS is caused by loss of function mutations in Fragile X mental retardation protein (FMRP), an RNA binding protein that is known to regulate translation of its target mRNAs, predominantly in the brain and gonads. The molecular mechanisms connecting FMRP function to neurodevelopmental phenotypes are well understood. However, neither the full extent of reproductive phenotypes, nor the underlying molecular mechanisms have been as yet determined. Here, we developed new fmr1 knockout zebrafish lines and show that they mimic key aspects of FXS neuronal phenotypes across both larval and adult stages. Results from the fmr1 knockout females also showed that altered gene expression in the brain, via the neuroendocrine pathway contribute to distinct abnormal phenotypes during ovarian development and oocyte maturation. We identified at least three mechanisms underpinning these defects, including altered neuroendocrine signaling in sexually mature females resulting in accelerated ovarian development, altered expression of germ cell and meiosis promoting genes at various stages during oocyte maturation, and finally a strong mitochondrial impairment in late stage oocytes from knockout females. Our findings have implications beyond FXS in the study of reproductive function and female infertility. Dissection of the translation control pathways during ovarian development using models like the knockout lines reported here may reveal novel approaches and targets for fertility treatments.

An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites-a case study with Omicron Spike DNA.

Site-directed mutagenesis is an invaluable technique that enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations, is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost-effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis. We demonstrate this method using the Omicron Spike DNA construct as an example, and create a construct bearing 37 mutations (as compared to wild-type Spike DNA), as well as 4 other constructs bearing subsets of the full spectrum of mutations. We believe that this method can be an excellent alternative to gene synthesis, especially when three or more variants are required.

CRISPR-Cas9-induced gene knockout in zebrafish.

The application of CRISPR has greatly facilitated genotype-phenotype studies of human disease models. In this protocol, we describe CRISPR-Cas9-induced gene knockout in zebrafish, utilizing purified Cas9 protein and in vitro-transcribed sgRNA. This protocol targets the PHLPP1 gene in an Indian wild-caught strain, but is broadly applicable. Major factors influencing protocol success include zebrafish health and fecundity, sgRNA efficiency and specificity, germline transmission, and mutant viability. For complete details on the use and execution of this protocol, please refer to Balamurugan et al. (2022).

A Study of Head and Neck Cancer Patients with Reference to Tobacco Use, Gender, and Subsite Distribution.

Richa ChauhanContext Head and neck cancer (HNC) is very common in India, constituting 30% of all the cancers because of the widespread use of tobacco across India. The prevalence and pattern of tobacco use vary in different regions and states of the country. Although predominantly seen in males, studies have reported that the male-to-female ratio varies worldwide and also by anatomical subsite. Aims This study was done with an aim to determine the difference in pattern and prevalence of tobacco use in male and female patients with HNCs and compare them with different subsites' involvement in our region. Methods and Materials This is a retrospective analysis of 500 consecutive biopsy-proven HNC patients from a large comprehensive cancer hospital from Bihar during the period of January 2019 to June 2019. Data collected for the study included age, gender, site of the disease, and use of tobacco. The categorical data were analyzed by a chi-square test using SPSS (version 16). Results Our study showed a male-to-female ratio of 8.43:1 with tobacco addiction in 84.40% patients. Smokeless tobacco was used by 52.20%, combustible form by 12.80%, and both by 19.40% of the patients. Tobacco use was seen in 87.25% of male patients as compared with only 60.38% of female patients ( p -value = 0.0001). Oral cavity cancer was seen in 60.85% of male patients and 37.74% of female patients ( p -value = 0.0012), whereas oropharyngeal cancer was seen in only 11.63% of male patients as compared with 25.83% of female patients ( p -value = 0.0008). The subsite analysis showed that in patients with oral cavity cancers, no addiction was found in only 10.29% of male patients as compared with 30% of the female patients ( p -value = 0.008). Conclusions Our study confirms a high prevalence of tobacco use among HNC patients. So, we need to continue our efforts to create awareness against tobacco use. Besides, there is also a need for more studies to look into other etiological factors among nontobacco users.

A comparative analysis of the clinicopathological profile of early-onset versus late-onset rectal cancer patients.

Introduction: Colorectal cancer has been primarily considered a disease of the elderly, but recent data have shown an alarming rise among young people. It has been also suggested that young age is associated with aggressive histopathological characteristics and advanced stages of the disease at diagnosis. As there are few studies and none from our part of the country evaluating the clinicopathological profile of early-onset versus late-onset rectal cancer patients, this analysis was conducted to assess and compare the clinical and pathological characteristics of patients with rectal cancer diagnosed with ages over and below 50 years. Materials and method: The relevant details of all biopsy proven rectal cancer patients undergoing radiotherapy at a tertiary cancer hospital, from January 2017 to December 2019, were collected. All the data were categorised into two groups, an early-onset group (age <50 years) and a late-onset group (age ≥50 years), and comparison of the clinicopathological characteristics between the two groups was made. Results: A total of 224 patients with rectal cancer, 150 male and 74 female, were included in the study. About two-thirds of the patients were less than 50 years of age, with an average age of 42 years. The comparative analysis showed a significantly higher number of young patients presenting with bleeding and pain. Patients below 50 years also had a significantly higher number of adenocarcinoma grade III and clinical stage III than those in the late-onset group. Conclusion: Our study revealed a significant number of early-onset rectal cancer patients. There should be a high index of suspicion in any young patient presenting with symptoms suggestive of rectal malignancy and they should be evaluated promptly.

Arsenic exposure in Indo Gangetic plains of Bihar causing increased cancer risk.

Reportedly, 300 million people worldwide are affected by the consumption of arsenic contaminated groundwater. India prominently figures amongst them and the state of Bihar has shown an upsurge in cases affected by arsenic poisoning. Escalated arsenic content in blood, leaves 1 in every 100 human being highly vulnerable to being affected by the disease. Uncontrolled intake may lead to skin, kidney, liver, bladder, or lung related cancer but even indirect forms of cancer are showing up on a regular basis with abnormal arsenic levels as the probable cause. But despite the apparent relation, the etiology has not been understood clearly. Blood samples of 2000 confirmed cancer patients were collected from pathology department of our institute. For cross-sectional design, 200 blood samples of subjects free from cancer from arsenic free pockets of Patna urban agglomeration, were collected. Blood arsenic levels in carcinoma patients as compared to sarcomas, lymphomas and leukemia were found to be higher. The geospatial map correlates the blood arsenic with cancer types and the demographic area of Gangetic plains. Most of the cancer patients with high blood arsenic concentration were from the districts near the river Ganges. The raised blood arsenic concentration in the 2000 cancer patients strongly correlates the relationship of arsenic with cancer especially the carcinoma type which is more vulnerable. The average arsenic concentration in blood of the cancer patients in the Gangetic plains denotes the significant role of arsenic which is present in endemic proportions. Thus, the study significantly correlates and advocates a strong relation of the deleterious element with the disease. It also underlines the need to address the problem by deciphering the root cause of the elevated cancer incidences in the Gangetic basin of Bihar and its association with arsenic poisoning.

A Comparative Analysis of Body Mass Index with Estrogen Receptor, Progesterone Receptor and Human Epidermal Growth Factor Receptor 2 Status in Pre- and Postmenopausal Breast Cancer Patients.

BACKGROUND: Breast cancer is now the most common cancer among Indian women. Recent studies have suggested a possible link between risk factors like high BMI and molecular subtypes of breast cancer. Studies from Western and Asian population have shown varying relationship between post- menopausal obesity and expression of ER, PR, Her2-neu receptors in breast cancer patients. AIM: This study was done with an aim to explore if overweight or obesity as defined by BMI and status of ER, PR and Her2-neu receptors differ in Indian pre-menopausal and post-menopausal breast cancer patients. METHODS AND MATERIAL: This is a retrospective analysis of 446 breast cancer patients treated at Mahavir Cancer Sansthan, Patna from July to December 2019. Their case records were evaluated and data regarding age, menopausal status, height, weight and ER, PR & HER2-neu receptor status were extracted for analyses. STATISTICAL ANALYSIS: Chi-square test was used to compare categorical variables between the pre-menopausal and post-menopausal group. RESULTS: The prevalence of obesity in the post-menopausal group was 2.3% more than the pre-menopausal group (P-value = 0.24). As compared to the pre-menopausal group, there was an increase in the ER/PR positivity in the postmenopausal group by 3.41% (P-value = 0.47) and in the Her2-neu positivity by 6.38% (P-value = 0.15). As compared to the pre-menopausal group, there was further increase in the ER/PR positivity in the post-menopausal group by 6.85% (P-value = 0.40) in sub-group of patients with BMI ≥ 25kg/m2. CONCLUSIONS: Our study showed slightly increased incidence of obesity in post-menopausal breast cancer patients. Overweight post-menopausal patients also had a higher percentage of ER/PR receptor positivity and lower percentage of Triple negative breast cancer. The percentage of Her2-neu receptor positivity was more in post-menopausal patients. A high BMI was found to be associated with a lower Her2neu positivity.

A DNAzyme based knockdown model for Fragile-X syndrome in zebrafish reveals a critical window for therapeutic intervention.

INTRODUCTION: FXS is the leading cause of intellectual disabilities in males and a major monogenic cause of ASD (Autism spectrum disorders). It occurs due to the loss of FMRP, whose role in early development is not well understood. In this study, we have used a novel DNAzyme based approach to create a larval model of FXS in zebrafish with specific focus on the early developmental window. METHODS: Fmr1specific DNAzymes were electroporated into embryos to create the knockdown. Changes in RNA and protein levels of FMRP and relevant biomarkers were measured in the 0-7dpf window. Behavioral tests to measure anxiety, cognitive impairments and irritability in the larvae were conducted at the 7dpf stage. Drug treatment was carried out at various time points in the 0-7dpf window to identify the critical window for pharmacological intervention. RESULTS: The DNAzyme based knockdown approach led to a significant knockdown of FMRP in the zebrafish embryos, accompanied by increased anxiety, irritability and cognitive impairments at 7dpf, thus creating a robust larval model of FXS. Treatment with the Mavoglurant was able to rescue the behavioral phenotypes in the FXS larvae, and found to be more efficacious in the 0-3dpf window. DISCUSSION: The results from this study have revealed that a) a DNAzyme based knockdown approach can be used to create robust larval zebrafish model of disease, in a high-throughput manner and b) optimal window for therapeutic intervention for FXS as well as other pediatric diseases with a monogenic cause can be identified using such a model.

An RNA aptamer to HP1/Swi6 facilitates heterochromatin formation at an ectopic locus in S.pombe.

In the fission yeast Schizosaccharomyces pombe (S.pombe), heterochromatin domains are established and maintained by protein complexes that contain numerous RNA binding domains among their components. The fission yeast HP1 protein Swi6 is one such component and contains an unstructured RNA-binding hinge, which is important for the integrity and silencing of heterochromatin. In this study, we have used an RNA aptamer that likely binds to the Swi6 hinge with high affinity, as a tool to perturb the natural interactions mediated by this domain. When the hinge is blocked by the aptamer RNA, Swi6 appears to become less restricted to the pericentromeres and is enriched at specific euchromatic loci. This suggests a role for the Swi6 hinge, along with the chromoshadow domain (previously shown) in controlling the spread of heterochromatin in S.pombe. The study also highlights the potential of using a synthetic aptamer RNA as a tool to perturb nucleic acid - protein interaction in vivo with the objective of understanding the functional relevance of such an interaction.

Larval zebrafish model for studying the effects of valproic acid on neurodevelopment: An approach towards modeling autism.

INTRODUCTION: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder of early onset, characterized by impaired sociability, cognitive function and stereotypies. The etiology of ASD involves a multidimensional link between genetic, neurobiological and environmental factors. Since existing, comprehensive animal models for ASD are time consuming and laborious, the need for simple, quick approaches to study subsets of ASD-associated characteristics has always been in demand for better understanding of disease. The aim of the present study was to develop a cost and time effective zebrafish model with quantifiable parameters to facilitate mechanistic studies as well as high-throughput screening of new molecules for autism. METHODS: Zebrafish embryos were treated with valproic acid (75 µM) beginning at 4-h post fertilization to 5-days post fertilization. A series of behavioral tests (anxiety, inattentive behavior and circling behavior) and molecular studies were performed as surrogate parameters of ASD-like characteristic on the larvae at 7-dayspost fertilization for a quick screen. The study was followed by validation of model by screening positive control and negative control drugs. The social interaction test was performed on 21-days post fertilization to confirm that the surrogate phenotypes were indicative of social deficit (a core symptom of ASD). RESULTS: The model showed a significant behavioral impairment (2-4fold difference) in valproic acid treated larvae compared to control larvae, which was further supported by alterations in select high-risk genes and proteins, implicated in human ASD. Reversal of behavioral impairments using standard drugs marketed for symptomatic treatment in ASD and no effect on behaviors when treated with paracetamol (negative control) signifies the role of model in preliminary drug screening. CONCLUSION: The model shows robust parameters to study behavior, molecular mechanism and drug screening approach in a single frame. Thus, we postulate that our 7-day larval model could be a useful preliminary screening tool to identify novel targets as well as potential drugs for autism and also can be applied to develop a high-throughput screening approach.

Leiomyosarcoma of Penis: An Aggressive and Exceptionally Rare Entity.

Penile leiomyosarcoma is a very rare disease of penile mesenchymal tissue, most of them are of vascular origin and pathologically classified into the superficial and deep type. Because of the small number of cases reported so far, the conclusions about treatment and prognosis are equivocal. Here we report a case of 40-year old patient who presented with leiomyosracoma of penis; despite adequate surgery patient developed local recurrence and distant metastasis indicating aggressive nature of leiomyosarcoma entity of penis.

Localization of angiotensin-II type 1(AT1) receptors on buffalo spermatozoa: AT1 receptor activation during capacitation triggers rise in cyclic AMP and calcium.

The purpose of this study was to determine the role of Ang-II in buffalo spermatozoa; localize angiotensin type 1 (AT1) receptors on the sperm surface and understand the signaling mechanisms involved therein. Immunoblotting and immunocytochemistry using polyclonal Rabbit anti-AT1 (N-10) IgG were performed to confirm the presence of AT1 receptors. Intracellular levels of cyclic adenosine monophosphate (cAMP) were determined by non-radioactive enzyme immunoassay, while that of Calcium [Ca(2+)] were estimated by fluorimetry using Fura2AM dye. The results obtained showed that AT1 receptors were found on the post-acrosomal region, neck and tail regions. Immunoblotting revealed a single protein band with molecular weight of 40 kDa. Ang-II treated cells produced significantly higher level of cAMP compared to untreated cells (22.66 ± 2.4 vs. 10.8 ± 0.98 pmol/10(8) cells, p < 0.01). The mean levels of Ca(2+) were also higher in Ang-II treated cells compared to control (117.4 ± 6.1 vs. 61.15 ± 4.2 nmol/10(8) cells; p < 0.01). The stimulatory effect of Ang-II in both the cases was significantly inhibited in the presence of Losartan (AT1 antagonist; p < 0.05) indicating the involvement of AT1 receptors. Further, presence of neomycin (protein kinase C inhibitor) inhibited significantly the Ang-II mediated rise in Ca(2+) indicating the involvement of PKC pathway. These findings confirm the presence of AT1 receptors in buffalo spermatozoa and that Ang-II mediates its actions via the activation of these receptors. Ang-II stimulates the rise in intracellular levels of cAMP and Ca(2+) during capacitation.

Effects of cow ghee (clarified butter oil) & soybean oil on carcinogen-metabolizing enzymes in rats.

BACKGROUND & OBJECTIVES: Our previous study showed that cow ghee relative to soybean oil had a protective effect against carcinogen induced mammary cancer in rats. The objective of this study was to elucidate its biochemical mechanism. METHODS: Two groups of 21 day old rats (20 each) were fed for 44 wk diet containing cow ghee or soybean oil (10%). Five animals from each group were sacrificed at 0 day and at 5, 21 and 44 wk for analysis of phase I and phase II pathways enzymes of carcinogen metabolism. RESULTS: Dietary cow ghee relative to soybean oil decreased the activities of cytochrome P450 (CYP) enzymes, CYP1A1, CYP1A2, CYP1B1 and CYP2B1, responsible for activation of carcinogen in liver. Carcinogen detoxification activities of uridinediphospho-glucuronosyl transferase (UDPGT) and quinone reductase (QR) in liver, and γ-glutamyltranspeptidase (GGTP) and QR in mammary tissue were significantly higher in cow ghee fed rats than in soybean oil fed rats. The hepatic GGTP activity decreased on soybean oil diet; while in cow ghee group it remained unaffected. INTERPRETATION & CONCLUSIONS: Our findings show that dietary cow ghee compared to soybean oil downregulates the enzyme activities responsible for carcinogen activation in liver and upregulates carcinogen detoxification activities in liver and mammary tissues.

Study on cow ghee versus soybean oil on 7,12-dimethylbenz(a)-anthracene induced mammary carcinogenesis & expression of cyclooxygenase-2 & peroxisome proliferators activated receptor-γ in rats.

BACKGROUND & OBJECTIVES: Breast cancer is a leading cause of cancer death in women; dietary fat is the one of the factors that influences its incidence. In the present study we investigated the effect of feeding cow ghee versus soybean oil on 7,12-dimethylbenz(a)anthracene (DMBA) induced mammary cancer in rat and expression of cyclooxygenase-2 and peroxisome proliferators activated receptor-γ (PPAR-γ) in mammary gland. METHODS: Two groups of 21 day old female rats (30 each) were fed for 44 wk diet containing cow ghee or soybean oil (10%). The animals were given DMBA (30 mg/kg body weight) through oral intubation after 5 wk feeding. Another two groups (8 each) fed similarly but not given DMBA served as control for the gene expression study. RESULTS: In DMBA treated groups, the animal fed soybean oil had higher tumour incidence (65.4%), tumour weight (6.18 g) and tumour volume (6285 mm3) compared to those fed cow ghee (26.6%, 1.67 g, 1925 mm3, respectively). Tumour latency period was 23 wk on soybean oil compared to 27 wk on cow ghee. Histological analysis of tumours showed that the progression of carcinogenesis was more rapid on soybean oil than on cow ghee. The expression of cyclooxygenase-2 was observed only in DMBA treated rats and it was significantly less on cow ghee than on soybean oil. The expression of PPAR-γ was significantly more on cow ghee than on soybean oil. INTERPRETATION & CONCLUSIONS: Our results show that dietary cow ghee opposed to soybean oil attenuates mammary carcinogenesis induced by DMBA; and the effect is mediated by decreased expression of cyclooxygenase-2 and increased expression of PPAR-γ in the former group.

Dietary intervention of cow ghee and soybean oil on expression of cell cycle and apoptosis related genes in normal and carcinogen treated rat mammary gland.

The present study investigated the effect of cow ghee (clarified butter fat) versus soybean oil on the expression of cyclins A and D1, and apoptosis regulating Bax, Bcl-2 and PKC-α genes in mammary gland of normal and 7,12-dimethylbenz(a)anthracene (DMBA) treated rats. Two groups of 21 days old female rats were fed for 44 weeks diet containing cow ghee or soybean oil (10%). The animals were given DMBA (30 mg/kg body weight) through oral intubation after 5 weeks feeding. Another two groups fed similarly but not given DMBA served as respective controls. In control groups, the expression of cyclin A was similar on both cow ghee and soybean oil, but that of cyclin D1 was more on soybean oil diet. However, in DMBA treated groups, the expression levels of cyclins A and D1 were significantly greater on soybean oil than on cow ghee. The expression levels of Bax, Bcl-2 and PKC-α were similar in two control groups. However, in tumor tissue expression levels of Bcl-2 and PKC-α were significantly lower in cow ghee fed rats than in soybean oil fed ones, but Bax was similarly expressed in both DMBA treated groups. The pro-apoptotic ratio Bax/Bcl-2 increased and the anti-apoptotic ratio PKC-α(Bcl-2/Bax) decreased in cow ghee group compared to soybean oil group in DMBA treated rats. Hence, the decreased expressions of cyclins A and D1, Bcl-2 and PKC-α mediate the mechanism by which cow ghee protects from mammary carcinogenesis.