BRCA2 Polymorphisms and Breast Cancer Susceptibility: a Multi-Tools Bioinformatics Approach.

BACKGROUND/AIMS: The main focus of this investigation is to identify deleterious single nucleotide polymorphisms (SNPs) located in the BRCA2 gene through in silico approach, thereby,providing an understanding of potential consequences regarding the susceptibility to breast cancer. METHODS: The GenomAD database was used to identify SNPs. To determine the potential adverse consequences, our study employed various prediction tools, including SIFT, PolyPhen, PredictSNP, SNAP2, PhD-SNP, and ClinVar. The pathogenicity associated with the deleterious snSNPs was evaluated bu MutPred and Fathmm. Additionally, I-Mutant and MuPro were used to assess the stability, followed by conservation and protein-protein interaction analysis using robust computational tools. The 3D structure of BRCA2 protein was generated by SwissModel, followed by validation using PROCHECK and Errat. RESULTS: The GenomAD database was used to identify a total of 7, 921 SNPs, including 1940 missense SNPs. A set of 69 SNPs predicted by consensus to be damaging across all platforms was identified. Mutpred and Fathmm identified 48 and 38 SNPs, respectively to be associated with cancer. While I- Mutant and MuPro assays suggested 22 SNPs to decrease protein stability. Additionally, these 22 SNPs reside within highly conserved regions of the BRCA2 protein. Domain analysis, utilizing InterPro, pinpointed 18 deleterious mutations within crucial DNA binding domains and one in the BRC repeat region. CONCLUSION: This study establishes a foundation for future experimental validations and the creation of breast cancer-targeted treatment approaches.

Significant Association of Candidate Genes (AGTR1 and TGF-Β1) Polymorphism with Diabetic Nephropathy in Diabetes Mellitus Type 2 Patients.

BACKGROUND/AIMS: Diabetic nephropathy (DN) is one of the complications of diabetes mellitus (DM). This study aimed to investigate the association between genetic polymorphisms, specifically AGTR1 (rs5186) and TGF-ß1 (rs1800470), and the risk of developing Diabetic nephropathy (DN) in type 2 diabetes mellitus patients, compared to those without DN and healthy controls. METHODS: A case-control study was conducted on 165 diabetic patients (59 with diabetic nephropathy (DN) and 54 without DN (DM)), and 52 healthy controls (HC). The genotyping was done using amplification refractory mutation system method (ARMS-PCR). Age, gender, and duration of diabetes were matched across groups. Clinical parameters including FBS, RBS, HbA1C, creatinine, urea, SBP, DBP, total cholesterol, triglycerides, LDL, and BMI were assessed. RESULTS: Diabetic patients with nephropathy exhibited significantly higher levels of clinical parameters compared to those without nephropathy and healthy controls. The risk allele of AGTR1 , C (p <0.0001), and risk allele containing genotypes AC (p <0.0001) and CC (p - 0.0010) were significantly higher in DN patients compared to DM and HC groups. Similarly, the TGF-ß1 risk allele C (p - 0.0001), and corresponding genotypes TC (p - 0.0038) and CC (p - 0.0027) were significantly associated with increased risk of diabetic nephropathy compared to DM and HC groups. CONCLUSION: The data showed significant association of AGTR1 (rs5186) and TGF-ß1 (rs1800470) polymorphism with an increased risk of diabetic nephropathy in type 2 diabetes mellitus patients. More investigation will be required to disseminate the results, while increasing the samples size and using whole genome sequencing.

Phosphate solubilizing Aspergillus Niger PH1 ameliorates growth and alleviates lead stress in maize through improved photosynthetic and antioxidant response.

Among the several threats to humanity by anthropogenic activities, contamination of the environment by heavy metals is of great concern. Upon entry into the food chain, these metals cause serious hazards to plants and other organisms including humans. Use of microbes for bioremediation of the soil and stress mitigation in plants are among the preferred strategies to provide an efficient, cost-effective, eco-friendly solution of the problem. The current investigation is an attempt in this direction where fungal strain PH1 was isolated from the rhizosphere of Parthenium hysterophorus which was identified as Aspergillus niger by sequence homology of the ITS 1 and ITS 4 regions of the rRNA. The strain was tested for its effect on growth and biochemical parameters as reflection of its potential to mitigate Pb stress in Zea mays exposed to 100, 200 and 500 µg of Pb/g of soil. In the initial screening, it was revealed that the strain has the ability to tolerate lead stress, solubilize insoluble phosphate and produce plant growth promoting hormones (IAA and SA) and other metabolites like phenolics, flavonoids, sugar, protein and lipids. Under 500 µg of Pb/g of soil, Z. mays exhibited significant growth retardation with a reduction of 31% in root length, 30.5% in shoot length, 57.5% in fresh weight and 45.2% in dry weight as compared to control plants. Inoculation of A. niger to Pb treated plants not only restored root and shoot length, rather promoted it to a level significantly higher than the control plants. Association of the strain modulated the physio-hormonal attributes of maize plants that resulted in their better growth which indicated a state of low stress. Additionally, the strain boosted the antioxidant defence system of the maize there by causing a significant reduction in the ascorbic acid peroxidase (1.5%), catalase (19%) and 1,1-diphenyl-2 picrylhydrazyl (DPPH) radical scavenging activity (33.3%), indicating a lower stress condition as compared to their non-inoculated stressed plants. Based on current evidence, this strain can potentially be used as a biofertilizer for Pb-contaminated sites where it will improve overall plant health with the hope of achieving better biological and agricultural yields.

IAA is more effective than EDTA in enhancing phytoremediation potential for cadmium and copper contaminated soils.

Enhanced phytoremediation offers a rapid and eco-friendly approach for cleaning agricultural soil contaminated with copper and cadmium which pose a direct threat to food scarcity and security. The current study aimed to compare the effectiveness of the two commonly used additives, IAA and EDTA, for the remediation of copper (Cu) and cadmium (Cd) contaminated soils using sunflower and maize. The plants were cultivated in pots under controlled conditions with four sets of treatments: control (0), Cu50/Cd50, Cu50/Cd50 + EDTA, and Cu50/Cd50 + IAA. The results showed that Cu50/Cd50 mg/kg drastically compromised the phytoremediation potential of both plants, as evident by reduced shoot and root length, and lower biomass. However, the augmentation of Cu50/Cd50 with EDTA or IAA improved the tested parameters. In sunflower, EDTA enhanced the accumulation of Cu and Cd by 58% and 21%, respectively, and improved plant biomass by 41%, compared to control treatment. However, IAA exhibited higher accumulation of Cu and Cd by 64% and 25%, respectively, and enhanced plant biomass by 43%. In case of maize, IAA was superior to EDTA which enhanced the accumulation of Cu and Cd by 87% and 32% respectively, and increased the plant biomass by 57%, compared to control treatment. Our findings demonstrate that foliar IAA is more effective than EDTA in enhancing the phytoremediation potential of sunflower and maize for Cu and Cd.

Xanthan gum-based edible coating effectively preserve postharvest quality of 'Gola' guava fruits by regulating physiological and biochemical processes.

BACKGROUND: Guava is a fruit prone to rapid spoilage following harvest, attributed to continuous and swift physicochemical transformations, leading to substantial postharvest losses. This study explored the efficacy of xanthan gum (XG) coatings applied at various concentrations (0.25, 0.5, and 0.75%) on guava fruits (Gola cultivar) over a 15-day storage period. RESULTS: The results indicated that XG coatings, particularly at 0.75%, substantially mitigated moisture loss and decay, presenting an optimal concentration. The coated fruits exhibited a modified total soluble soluble solids, an increased total titratable acidity, and an enhanced sugar-acid ratio, collectively enhancing overall quality. Furthermore, the XG coatings demonstrated the remarkable ability to preserve bioactive compounds, such as total phenolics, flavonoids, and antioxidants, while minimizing the levels of oxidative stress markers, such as electrolyte leakage, malondialdehyde, and H2O2. The coatings also influenced cell wall components, maintaining levels of hemicellulose, cellulose, and protopectin while reducing water-soluble pectin. Quantitative analysis of ROS-scavenging enzymes, including superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase, revealed significant increases in their activities in the XG-coated fruits compared to those in the control fruits. Specifically, on day 15, the 0.75% XG coating demonstrated the highest SOD and CAT activities while minimizing the reduction in APX activity. Moreover, XG coatings mitigated the activities of fruit-softening enzymes, including pectin methylesterase, polygalacturonase, and cellulase. CONCLUSIONS: This study concludes that XG coatings play a crucial role in preserving postharvest quality of guava fruits by regulating various physiological and biochemical processes. These findings offer valuable insights into the potential application of XG as a natural coating to extend the shelf life and maintain the quality of guava fruits during storage.

Microbiome analysis reveals the differences in gut fungal community between Dutch Warmblood and Mongolian horses.

Similar to gut bacterial community, gut fungal community are also an important part of the gut microbiota and play crucial roles in host immune regulation and metabolism. However, most studies have focused on the gut bacterial community, and research on the gut fungal community has been limited. Dutch Warmblood (DWH) and Mongolian horses (MGH) are important equine breeds, but little research has been done on their gut fungal community. Here, we assessed differences in gut fungal community between two horse species. Results showed that a total of 2159 OTUs were found in the Dutch Warmblood and Mongolian horses, of which 308 were common. Between-group analyzes of microbial diversity showed no differences in the alpha and beta diversity of gut fungal community between the two horse species. Microbiological taxonomic surveys showed that the dominant fungal phyla (Neocallimastigomycota and Ascomycota) and genera (unclassified_Neocallimastigaceae and Anaeromyces) were the same without being affected by species. Although the types of dominant fungal phyla did not change, the abundances of some fungal genera changed significantly. Results of Metastats analysis showed that there were a total of 206 fungal genera that were significantly different between the two horses, among which 78 genera showed an increase and 127 genera significantly decreased in Dutch Warmblood horses compared with Mongolian horses. In conclusion, this study investigated the composition and structure of the gut fungal community of Dutch Warmblood and Mongolian horses and found significant differences in gut fungal community between both breeds. Notably, this is the first exploration of the differences in the gut fungal community of both breeds, which may help to understand the distribution characteristics of the gut fungal community of different breeds of horses and reveal the differences in the traits of different horses.

Developing new Hydrazine Carbothioamide Molecules for Selective Colorimetric/Fluorometric Detection of Environmentally Essential Hg2+ and Ag+ Metal Ions in Mixed Aqueous Media.

A novel colorimetric and fluorogenic probe L based on hydrazine carbothioamide and 1,8-naphthalimide moieties has been designed and synthesized for the hypersensitive detection of Hg2+ or Ag+ ions. The observed probe L showed colorimetric and fluorometric responses for these studies when Hg2+ or Ag+ was added to the DMSO - HEPES buffer solution (pH = 7). An interference test with other metal ions was determined, and the high selectivity of Hg2+ and Ag+ did not interfere with other metal ions in colorimetric and fluorogenic methods. The possible mechanism of binding of these metal ions and the probe L 1:1 complex was determined by H1 NMR. Additionally, the reversibility of the affinity of probe L with mercury (Hg2+) and silver (Ag+) ions was investigated by adding Na2EDTA. The naked eye detected the "Off-On" type fluorescence sensor in the presence of Hg2+ and EDTA. The tested test strip kits provided a strong probability of probe L with high response and rapid, sensitive detection with Hg2+ ion, which may be suitable for practical use.

BRAF and RET polymorphism association with thyroid cancer risk, a preliminary study from Khyber Pakhtunkhwa population.

BACKGROUND: Thyroid cancer, originating in the neck's thyroid gland, encompasses various types. Genetic mutations, particularly in BRAF and RET genes are crucial in its development. This study investigates the association between BRAF (rs113488022) and RET (rs77709286) polymorphisms and thyroid cancer risk in the Khyber Pakhtunkhwa (KP) population. METHODS: Blood samples from 100 thyroid cancer patients and 100 healthy controls were genotyped using ARMS-PCR followed by gel electrophoresis and statistical analysis. RESULTS: Analysis revealed a significant association between the minor allele T of BRAF (rs113488022) and thyroid cancer risk (P = 0.0001). Both genotypes of BRAF (rs113488022) showed significant associations with thyroid cancer risk (AT; P = 0.0012 and TT; P = 0.045). Conversely, the minor allele G of RET (rs77709286) exhibited a non-significant association with thyroid cancer risk (P = 0.2614), and neither genotype showed significant associations (CG; P = 0.317, GG; P = 0.651). Demographic and clinical parameters analysis using SPSS showed a non-significant association between BRAF and RET variants and age group (P = 0.878 and P = 0.536), gender (P = 0.587 and P = 0.21), tumor size (P = 0.796 and P = 0.765), or tumor localization (P = 0.689 and P = 0.727). CONCLUSION: In conclusion, this study emphasizes the significant association between BRAF polymorphism and thyroid cancer risk, while RET polymorphism showed a less pronounced impact. Further validation using larger and specific datasets is essential to establish conclusive results.

Anticancer effects of rutin from Fagopyrum tataricum (tartary buckwheat) against osteosarcoma cell line.

BACKGROUND: The present study is analysisof the seeds of buckwheat (Fagopyrum sp.),member of the Polygonaceae family for isolation of rutin and its anticancer property againstOsteosarcoma celllines (SAOS2). The selected plant is traditionally used for diabetes and cancer. It has several biological properties such as antibacterial, antioxidant and anti-aging. PURPOSE: Thirty-five buckwheat cultivars were obtained from Nepal Agriculture Genetic Resources Centre (NAGRC) Khumaltar, Kathmandu, Nepal, and Kumrek Sikkim. These plant varieties are scientifically evaluated their biological properties. METHODS: Rutin wasfractionated from buckwheat seeds using methanol fraction and analysed for quality by HPLC method. The rutin fraction of the cultivar NGRC03731 a tartary buck wheat and standard rutin was used against Osteosarcoma cell lines (SAOS2) and human gingival fibroblast cells (hGFs) for anticancer activity. The cell viability using rutin fraction and standard rutin treated with SAOS2 cells were assessed by MTT assay. For further research, the best doses (IC-50: 20 g/ml) were applied. By using AO/EtBr dual staining, the effects of Rutin fraction on SAOS2 cell death were analysed. The scratch wound healing assay was used to analyse cell migration. Real-time PCR was used to analyse the pro-/anti-apoptotic gene expression. RESULTS: The seeds with the highest rutin content, NGRC03731 seeds, had 433 mg/100 g of rutin.The rutin fraction treatment and standard rutin significantly reduced cell viability in the MTT assay, and osteosarcoma cells were observed on sensitive to the IC-50 dose at a concentration of 20 g/ml after 24 h.The SAOS2 cells exposed to rutin fraction at 20 g/ml and standard rutin at 10 g/ml exhibited significant morphological alterations, cell shrinkage and decreased cell density, which indicate apoptotic cells.Rutin-fraction treated cells stained with acridine orange/ethidium bromide (AO/EtBr) dual staining cells turned yellow, orange, and red which indicatesto measure apoptosis.The anti-migration potential of rutin fraction, results prevented the migration of SAOS2 cancer cells.Rutin-fraction significantly increased the expression of pro-apoptotic proteinsBad, using real-time PCR analysis (mRNA for Bcl-2 family proteins) resulted Bcl-2's expression is negatively regulated. CONCLUSION: Osteosarcoma (SAOS2) cell lines' proliferation, migration, and ability to proliferate were reduced markedly by rutin fraction and it also causes apoptosis of Osteosarcoma cell lines (SAOS2).

Neurobehavioral and bioaccumulative toxicity in adult in-vivo zebrafish model due to prolonged cadmium exposure in the presence of ketoprofen.

Increasing industrial activity causes the release of chemical compounds into aquatic habitats, including toxic heavy metals like cadmium and medications like ketoprofen, posing considerable ecological concerns. Although previous studies have shown that cadmium and ketoprofen individually cause cognitive impairment, there is a lack of information on the combined neurological effects of the two substances. We investigated the neurological consequences of persistent cadmium exposure in the presence of ketoprofen on adult zebrafish, providing an essential model for understanding cumulative impacts on vertebrate organisms. Behavioral assessments, bioaccumulation rates, biochemical studies, and histopathological exams were conducted over 42 days in authentic environmental settings. The results of our study show that cadmium (10 µg/L) and ketoprofen (10 and 100 µg/L) at environmentally relevant concentrations had a significant impact on locomotor activity, social interactions, and cognitive responses, indicating cumulative neurotoxicity in co-exposure groups compared to single pollutant groups. Biochemical tests show disturbances in antioxidant defense systems, while histological examinations reveal structural changes in zebrafish brain regions. Ketoprofen influences cadmium accumulation in the brain, underscoring the importance of conducting complete evaluations to understand the intricate interactions between environmental pollutants. This study improves our understanding of the complex interactions between heavy metals and medications, stressing the need to consider combined exposure when assessing the neurological effects on vertebrate models.

Cadmium and ketoprofen accumulation influences aquatic ecosystem demonstrated using in-vivo zebrafish model.

The growing concern about pollution and toxicity in aquatic as well as terrestrial organisms is predominantly caused due to waterborne exposure and poses a risk to environmental systems and human health. This study addresses the co-toxic effects of cadmium (Cd) and ketoprofen (KPF), representing heavy metal and pharmaceutical discharge pollutants, respectively, in aquatic ecosystems. A 96-h acute toxicity assessment was conducted using zebrafish embryos. The results indicated that high dosages of KPF (10, 15, and 100 µg/mL) and Cd (10 and 15 µg/mL) reduced survivability and caused concentration-dependent deformities such as scoliosis and yolk sac edema. These findings highlight the potential defects in development and metabolism, as evidenced by hemolysis tests demonstrating dose-dependent effects on blood cell integrity. Furthermore, this study employs adult zebrafish for a 42-day chronic exposure to Cd and KPF (10 and 100 µg/L) alone or combined (10 + 10 and 100 + 100 µg/L) to assess organ-specific Cd and KPF accumulation in tissue samples. Organ-specific accumulation patterns underscore complex interactions impacting respiratory, metabolic, and detoxification functions. Prolonged exposure induces reactive oxygen species formation, compromising antioxidant defense systems. Histological examinations reveal structural changes in gills, gastrointestinal, kidney, and liver tissues, suggesting impairments in respiratory, osmoregulatory, nutritional, and immune functions. This study emphasizes the importance of conducting extensive research on co-toxic effects to assist with environmental risk assessments and safeguard human health and aquatic ecosystems.

Effects of Cryptosporidium parvum infection on intestinal fungal microbiota in yaks (Bos grunniens).

During the last decade, researchers had started to focus on the relationship between intestinal parasitic infection and variation of intestinal microflora. Cryptosporidium is a widely known opportunistic and zoonotic pathogen. Several studies have shown that Cryptosporidium infection has impact to alter the gut microflora. However, there are only few studies referring to the fungal microflora changes in response to Cryptosporidium infection in highland ruminants. Therefore, the present study was performed for exploring the alternations of intestinal fungal microbiota in yaks after exposure to Cryptosporidium infection. In present study, Amplicon sequencing of ITS regions was used to study the variations of fungal microflora in yaks. After filtering the raw data, over 45 000 and 62 000 clean data were obtained in uninfected and infected yaks, respectively. By using alpha diversity analysis, it was found that there is no significant difference in the richness and evenness when positive samples were compared with negative ones, whereas intestinal fungal communities in different taxa in yaks were changed. The results of present study depicted that 2-phyla and 21-genera in the infected animals had significantly (P < 0.05) changed. These genera were Septoria, Coniothyrium, Cleistothelebolus, Bensingtonia, Cystobasidium, Filobasidium, Coprotus, Carex, Blumeria, Coprinellus, Leucosporidium, Phialophora, Isolepis, Ascobolus, Thecaphora, Mortierella, Urocystis, Symmetrospora and Lasiobolus. In addition, we found variations in 28 enzymes suggesting that the function of microbiota was also affected. It is concluded that there are drastic changes in the fungal microflora and microbiota functions after exposure to Cryptosporidium infection in yak. Our results help to focus on the prompt way for the development of new therapies to control Cryptosporidiosis.

Assessment of HER1 (rs11543848) and HER2 (rs1136201) polymorphism and their association with colorectal cancer susceptibility in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Colorectal cancer (CRC) is a widespread malignancy characterized by uncontrolled growth in the colon or rectum and remains a leading cause of cancer-related mortality globally. Various genes polymorphisms have been linked with the risk of CRC, but our study aimed to investigate the association between HER1 (rs11543848) and HER2 (rs1136201) polymorphisms with the risk of CRC in the Khyber Pakhtunkhwa (KPK) population of Pakistan. The association of the selected polymorphisms (rs11543848 and rs1136201) with CRC risk has been investigated in various ethnic groups, but their impact remains unexplored in Pakistan, particularly within the KPK population, highlighting the need of the study in this region. METHODS: In this study 120 CRC patients and 120 healthy controls were enrolled. The DNA was extracted from the blood by salting-out method and genotyping was done using ARMS-PCR. RESULTS: Our investigations provided convincing evidence of a strong association between HER1 (rs11543848) and the risk of CRC. Both the genotypes heterozygous GA (OR = 2.07, CI = 1.18 to 3.64, P = 0.01) and homozygous AA (OR = 6.22, CI = 2.56 to 15.08, P = 0.0001) showed higher risk and significant association with the CRC risk. Similarly, heterozygous genotype AG of HER2 (rs1136201) was significantly associated (OR = 3.16, 95% CI = 1.78 to 5.58, P = 0.0001) while mutant genotype GG showed higher risk but non-significant association (OR = 3.23, 95% CI = 0.84 to 12.43, P = 0.08) with CRC patients. HER1 (rs11543848) demonstrated a significant association (P = 0.003) with the age at diagnosis in CRC patients, while HER2 (rs1136201) showed a non-significant association (P = 0.434). Both the SNPs were non-significantly associated with gender (P = 0.793 and 0.117), metastasis (P = 0.582 and 0.129), location of the tumor (P = 0.555 and 0.993), tumor grade (P = 0.290 and 0.920), tumor size (P = 0.535 and 0.289) and stages of cancer (P = 0.892 and 0.352). CONCLUSION: In conclusion, both the polymorphisms rs11543848 and rs1136201 displayed susceptibility with CRC in the KPK population. However, further investigations are recommended while using whole exome sequencing on a larger sample size for more precise results.

HSPB6 Is Depleted in Colon Cancer Patients and Its Expression Is Induced by 5-aza-2'-Deoxycytidine In Vitro.

Background and Objectives: Colon cancer (CC) is the second most common cancer in Saudi Arabia, and the number of new cases is expected to increase by 40% by 2040. Sixty percent of patients with CC are diagnosed in the late stage, causing a reduced survival rate. Thus, identifying a new biomarker could contribute to diagnosing CC in the early stages, leading to delivering better therapy and increasing the survival rate. Materials and Methods: HSPB6 expression was investigated in extracted RNA taken from 10 patients with CC and their adjacent normal tissues, as well as in DMH-induced CC and a colon treated with saline taken from a male Wistar rat. Additionally, the DNA of the LoVo and Caco-2 cell lines was collected, and bisulfite was converted to measure the DNA methylation level. This was followed by applying 5-aza-2'-deoxycytidine (AZA) to the LoVo and Caco-2 cell lines for 72 h to see the effect of DNA methylation on HSPB6 expression. Finally, the GeneMANIA database was used to find the interacted genes at transcriptional and translational levels with HSPB6. Results: We found that the expression of HSPB6 was downregulated in 10 CC tissues compared to their adjacent normal colon tissues, as well as in the in vivo study, where its expression was lower in the colon treated with the DMH agent compared to the colon treated with saline. This suggests the possible role of HSPB6 in tumor progression. Moreover, HSPB6 was methylated in two CC cell lines (LoVo and Caco-2), and demethylation with AZA elevated its expression, implying a mechanistic association between DNA methylation and HSPB6 expression. Conclusions: Our findings indicate that HSPB6 is adversely expressed with tumor progression, and its expression may be controlled by DNA methylation. Thus, HSPB6 could be a good biomarker employed in the CC diagnostic process.

Higher Expression Levels of SSX1 and SSX2 in Patients with Colon Cancer: Regulated In Vitro by the Inhibition of Methylation and Histone Deacetylation.

Background and Objectives: Colon cancer (CC) has a high mortality rate and is often diagnosed at an advanced stage in Saudi Arabia. Thus, the identification and characterization of potential new cancer-specific biomarkers are imperative for improving the diagnosis of CC by detecting it at an early stage. Cancer-testis (CT) genes have been identified as potential biomarkers for the early diagnosis of various cancers. Among the CT genes are those belonging to the SSX family. In order to assess the usefulness of SSX family genes as cancer biomarkers for the detection of early-stage CC, the goal of this research was to validate the expressions of these genes in patients with CC and in matched patients with normal colons (NCs). Materials and Methods: RT-PCR assays were used to analyze the SSX1, SSX2, and SSX3 family gene expression levels in 30 neighboring NC and CC tissue samples from male Saudi patients. Epigenetic alterations were also tested in vitro using qRT-PCR analysis to determine whether reduced DNA methyltransferase or histone deacetylation could stimulate SSX gene expression via 5-aza-2'-deoxycytidine and trichostatin treatments, respectively. Results: The RT-PCR results showed SSX1 and SSX2 gene expression in 10% and 20% of the CC tissue specimens, respectively, but not in any of the NC tissue specimens. However, no SSX3 expression was detected in any of the examined CC or NC tissue samples. In addition, the qRT-PCR results showed significantly higher SSX1 and SSX2 expression levels in the CC tissue samples than in the NC tissue samples. The 5-aza-2'-deoxycytidine and trichostatin treatments significantly induced the mRNA expression levels of the SSX1, SSX2, and SSX3 genes in the CC cells in vitro. Conclusions: These findings suggest that SSX1 and SSX2 are potentially suitable candidate biomarkers for CC. Their expressions can be regulated via hypomethylating and histone deacetylase treatments, subsequently providing a potential therapeutic target for CC.

Reverse pharmacology of Nimbin-N2 attenuates alcoholic liver injury and promotes the hepatoprotective dual role of improving lipid metabolism and downregulating the levels of inflammatory cytokines in zebrafish larval model.

Alcoholic liver disease is one of the most prominent liver diseases in the world. Lipid accumulation accompanied by oxidative stress and inflammation in the liver is the most important pathogenesis of ALD. This study was designed to investigate the anti-oxidative, fat metabolism-regulating, and anti-inflammatory potential of N2, a seminatural analog of Nimbin. The ethanol exposure was found to induce liver injury on zebrafish larvae, such as liver inflammation, lipid accumulation, oxidative stress, and hepatocytes apoptosis. N2 was subjected to ADMET screening in-silico, and it was observed N2's co-exposure decreased the ROS, apoptosis, lipid peroxidation, and macrophage accumulation in the liver of larval zebrafish. To further study the mechanism behind ethanol hepatotoxicity and the hepatoprotective behavior of N2, gene expression changes were determined in zebrafish. The results of this study revealed that ethanol exposure upregulated mRNA expressions of SREBP1, C/EBP-α, FAS and provoked more severe oxidative stress and hepatitis via upregulation of inflammatory cytokines TNF-α, IL-10, IL-1ß, iNOS, COX-2. However, the N2 co-exposure protected the hepatocyte damage and almost reversed the condition by downregulating the mRNA levels. The study suggested that N2 could be an effective therapeutic agent for the treatment of ALD and other inflammatory conditions.

ß-cells regeneration by WL15 of cysteine and glycine-rich protein 2 which reduces alloxan induced ß-cell dysfunction and oxidative stress through phosphoenolpyruvate carboxykinase and insulin pathway in zebrafish in-vivo larval model.

BACKGROUND: Pancreatic ß-cells are susceptible to oxidative stress, leading to ß-cell death and dysfunction due to enhanced ROS levels and type 2 diabetes. To inhibit the ß-cells damages induced by the oxidative stress, the present study investigates the beneficial effect of various peptides (WL15, RF13, RW20, IW13 and MF18) of immune related proteins (cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively). Also, the molecular mechanism of WL15 from cysteine and glycine-rich protein 2 on ß-cell regeneration was identified through PEPCK and insulin pathway. MATERIALS AND METHODS: In this study, a total of five peptides including WL15, RF13, RW20, IW13, and MF18 were derived from immune-related proteins such as cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively. These protein sequences were obtained from an earlier constructed transcriptome database of a teleost Channa striatus. The identified peptides were evaluated for their antioxidant as well as antidiabetic activity. Based on the in silico analysis and in-vitro screening experiments, WL15 was predicted to have better antioxidant and antidiabetic activity among the five different peptides. Therefore, WL15 alone was further analyzed for apoptosis, antioxidant capacity, glucose metabolism, and gene expression performance, which was investigated on the alloxan (500 µM) induced zebrafish in vivo larval model. RESULTS: The results showed alloxan exposure to zebrafish larvae for a day, the ROS was generated in the ß-cells. Interestingly, WL15 treatment showed a protective effect by reducing the toxicity of alloxan exposed zebrafish larvae by increasing their survival and heart rate. Moreover, WL15 reduced the intracellular ROS level and apoptosis in alloxan-induced larvae. The superoxide anion and lipid peroxidation levels are also reduced by improving the glutathione content after the WL15 treatment. Besides, WL15 treatment increased the proliferation rate of ß-cells and decreased the glucose level. Further, the gene expression studies revealed that WL15 treatment normalized the PEPCK expression while upregulating the insulin expression in alloxan exposed larvae. CONCLUSION: Overall, the findings indicate that WL15 of cysteine and glycine-rich protein 2 can act as a potential antioxidant for type 2 diabetes patients in respect of improving ß-cell regeneration.

Pro-inflammatory cytokine molecules from Boswellia serrate suppresses lipopolysaccharides induced inflammation demonstrated in an in-vivo zebrafish larval model.

BACKGROUND: Boswellia serrate is an ancient and highly valued ayurvedic herb. Its extracts have been used in medicine for centuries to treat a wide variety of chronic inflammatory diseases. However, the mechanism by which B. serrata hydro alcoholic extract inhibited pro-inflammatory cytokines in zebrafish (Danio rerio) larvae with LPS-induced inflammation remained unknown. METHODS: LC-MS analysis was used to investigate the extract's phytochemical components. To determine the toxicity of B. serrata extract, cytotoxicity and embryo toxicity tests were performed. The in-vivo zebrafish larvae model was used to evaluate the antioxidant and anti-inflammatory activity of B. serrata extract. RESULTS: According to an in silico study using molecular docking and ADMET, the compounds acetyl-11-keto-boswellic and 11-keto-beta-boswellic acid present in the extract had higher binding affinity for the inflammatory specific receptor, and it is predicted to be an orally active molecule. In both in-vitro L6 cells and in-vivo zebrafish larvae, 160 µg/mL concentration of extract caused a high rate of lethality. The extract was found to have a protective effect against LPS-induced inflammation at concentrations ranged between 10 and 80 µg/mL. In zebrafish larvae, 80 µg/mL of treatment significantly lowered the level of intracellular ROS, apoptosis, lipid peroxidation, and nitric oxide. Similarly, zebrafish larvae treated with B. serrata extract (80 µg/mL) showed an increased anti-inflammatory activity by lowering inflammatory specific gene expression (iNOS, TNF-α, COX-2, and IL-1). CONCLUSIONS: Overall, our findings suggest that B. serrata can act as a potent redox scavenger against LPS-induced inflammation in zebrafish larvae and an inhibitor of specific inflammatory genes.

Azadirachta indica-wrapped copper oxide nanoparticles as a novel functional material in cardiomyocyte cells: An ecotoxicity assessment on the embryonic development of Danio rerio.

This research reports on the production of copper oxide nanoparticles (CuO NPs) through the green synthesis method using Azadirachta indica (Ai) flower extract. Synthesized Ai-CuO NPs are characterized by Zeta Potential, TGA, SEM and TEM analysis. The Ai-CuO NPs gave a maximum peak at 270 nm. As per XRD studies, the Ai-CuO NPs obtained were crystalline. FTIR spectrum Ai-CuO NPs showed the presence of functional groups like the O-H group, aromatic group, etc. TEM and SEM assist in investigating the size and morphology of the Ai-CuO NPs, which were spherical and varied in size between 10.11 nm and 17.54 nm. EDAX showed that Ai-CuO NPs were pure with no impurities. The synthesized Ai-CuO NPs were then analyzed for their cytotoxicity at various concentrations (5, 10, 20, 30, 40 and 50 µg/mL) against H9c2 cardiomyocyte cells using MTT assay. DOX-induced H9c2 cell damage of apoptosis and ROS. The nanoparticle formed by Ai-CuO was cured with different concentrations (5, 10 and 20 µg/mL). In zebrafish, 48 hpf and 72 hpf were measured at 75 µM to reduce dysfunction and mortality during organ development. These results can have a beneficial impact on eco-toxicological effects.

Deacetylepoxyazadiradione Derived from Epoxyazadiradione of Neem (Azadirachta indica A. Juss) Fruits Mitigates LPS-Induced Oxidative Stress and Inflammation in Zebrafish Larvae.

Reactive oxygen species (ROS) produced by cell metabolism have a duplex role in oxidation and inflammation reactions which involve cell damage or repair responses. Excess ROS production has detrimental effects on the survival of cells. We examined the protective effect of a semi-natural compound NF2 (deacetylepoxyazadiradione), for its protective activity against free radical-mediated stress and inflammatory response to lipopolysaccharide (LPS) using zebrafish larvae. Preliminary antioxidant assays indicated an increase in scavenging of free radicals from NF2 than NF1 (Epoxyazadiradione) in a concentration-dependent manner. Cell cytotoxicity was determined using rat myoblast cell lines (L6), and more than 95 % of cell viability was obtained. Zebrafish developmental toxicity test indicated that NF2 is not toxic even at 150 µM. The percentage of ROS, lipid peroxidation, nitric oxide and apoptosis were reduced significantly in NF2 treated LPS-stressed zebrafish larvae. The reduced number of employed macrophages on NF2 treatment was observed in neutral red dye-marked macrophage localization images. Relative expression of antioxidant genes in zebrafish larvae after treatment with NF2 is significantly increased. The RT-PCR quantification of antioxidant and anti-inflammatory gene expression indicated decreased relative folds of pro-inflammatory cytokines, iNOS and increased relative folds of mitochondrial antioxidant genes (GR, GST and GPx) in LPS stressed zebrafish larvae after treatment with NF2. From the overall obtained results, it can be concluded that NF2 reduced the oxidative stress and inflammatory response by scavenging free radicals caused by LPS.