Study of medicinal plants used in ethnoveterinary medical system in riverine areas of Punjab, Pakistan.

BACKGROUND: The use of medicinal plants to treat various veterinary illnesses has been practiced for millennia in many civilizations. Punjab is home to a diverse ethnic community, the majority of whom work in dairy farming, agriculture, and allied professions and have indigenous practices of treating animal illnesses using native flora. This study was designed to (1) document and preserve information about the applications of medicinal plant species in ethnoveterinary remedies among inhabitants of Punjab, Pakistan, and (2) identify popular plants for disease treatment by quantitative analysis of the obtained data and to assess the pharmacological relevance of these species. METHODS: To collect data from informants (N = 279), questionnaires and semi-structured interviews were used. The ethnoveterinary data were analyzed using principal component analysis, relative frequency citation, fidelity level, relative popularity level, and rank order priority. RESULTS: A total of 114 plant species utilized in the ethnoveterinary medicinal system were found, which were divided into 56 families and used to treat 16 different illnesses. The Poaceae family, with 16 species, was the most common in the region. The most commonly employed growth form in herbal preparation was herb (49%). The most used part in ethnoveterinary remedies was leaves (35%), while powder was the most commonly used way for preparing ethnoveterinary remedies (51 applications). According to principal component analysis, the most typically used species in the research region were grasses. Five grasses (Arundo donax, Desmostachya bipinnata, Eleusine indica, Hordeum vulgare, and Pennisetum glaucum) showed a 100% FL value when used to treat diuretics, helminthiasis, digestive problems, fever, cough, worm infestation, indigestion, galactagogue, oral infections, and genital prolapse. The maximum value of disease cured level (DCL%) was recorded at 87.6% for endo- and ecto-parasitic ailments in the study area. CONCLUSION: This study demonstrates that medicinal plants play an important part in satisfying farmers' animal healthcare demands, making it a feasible practice. The study also provides a wealth of knowledge regarding ethnoveterinary methods for further planning and application, providing an option for farmers who cannot afford allopathic therapy.

Piperine modulates IR/Akt/GLUT4 pathways to mitigate insulin resistance: Evidence from animal and computational studies.

The global prevalence of diabetes mellitus is rising, especially in India. Medicinal herbs, whether used alone or in combination with conventional medicines, have shown promise in managing diabetes and improving overall well-being. Piperine (PIP), a major bioactive compound found in pepper, is gaining attention for its beneficial properties. This study aimed to assess whether PIP could alleviate diabetes by targeting insulin pathway-related molecules in the adipose tissue of rats on a high-fat diet (HFD). After 60 days on the HFD, rats received PIP at a dose of 40 mg/kg body weight for one month. The results showed that PIP significantly improved metabolic indicators, antioxidant enzymes, and carbohydrate metabolic enzymes. It also regulated the mRNA and protein expression of insulin signaling, which had been disrupted by the diet and sucrose intake. Molecular docking analysis also revealed strong binding of PIP to key diabetes-related regulatory proteins, including Akt (-6.2 kcal/mol), IR (-7.02 kcal/mol), IRS-1 (-6.86 kcal/mol), GLUT4 (-6.24 kcal/mol), AS160 (-6.28 kcal/mol), and ß-arrestin (-6.01 kcal/mol). Hence, PIP may influence the regulation of glucose metabolism through effective interactions with these proteins, thereby controlling blood sugar levels due to its potent antilipidemic and antioxidant properties. In conclusion, our study provides in vivo experimental evidence against the HFD-induced T2DM model for the first time, making PIP a potential natural remedy to enhance the quality of life for diabetic patients and aid in their management.

Glyphosate potentiates insulin resistance in skeletal muscle through the modulation of IRS-1/PI3K/Akt mediated mechanisms: An in vivo and in silico analysis.

Herbicides have been linked to a higher risk of developing diabetes. Certain herbicides also operate as environmental toxins. Glyphosate is a popular and extremely effective herbicide for weed control in grain crops that inhibits the shikimate pathway. It has been shown to negatively influence endocrine function. Few studies have demonstrated that glyphosate exposure results in hyperglycemic and insulin resistance; but the molecular mechanism underlying the diabetogenic potential of glyphosate on skeletal muscle, a primary organ that includes insulin-mediated glucose disposal, is unknown. In this study, we aimed to evaluate the impact of glyphosate on the detrimental changes in the insulin metabolic signaling in the gastrocnemius muscle. In vivo results showed that glyphosate exposure caused hyperglycemia, dyslipidemia, increased glycosylated hemoglobin (HbA1c), liver function, kidney function profile, and oxidative stress markers in a dose-dependent fashion. Conversely, hemoglobin and antioxidant enzymes were significantly reduced in glyphosate-induced animals indicating its toxicity is linked to induce insulin resistance. The histopathology of the gastrocnemius muscle and RT-PCR analysis of insulin signaling molecules revealed glyphosate-induced alteration in the expression of IR, IRS-1, PI3K, Akt, ß-arrestin-2, and GLUT4 mRNA. Lastly, molecular docking and dynamics simulations confirmed that glyphosate showed a high binding affinity with target molecules such as Akt, IRS-1, c-Src, ß-arrestin-2, PI3K, and GLUT4. The current work provides experimental proof that glyphosate exposure has a deleterious effect on the IRS-1/PI3K/Akt signaling pathways, which in turn causes the skeletal muscle to become insulin resistant and eventually develop type 2 diabetes mellitus.

Novel Associations between BRCA1 Variants C.181 T>G (Rs28897672) and Ovarian Crisk in Saudi Females.

BACKGROUND: Mutations in BRCA1 gene have been implicated in ovarian cancers, and BRCA testing may be conducted in high-risk women. This study was designed to determine the frequency of three single nucleotide polymorphisms (SNPs) variants in BRCA1 gene and BRCA1 expression in Saudi females with ovarian cancer. METHODS: Expression levels of mRNA of BRCA1 gene were studied in 10 ovarian cancer and 10 normal ovarian tissues, by quantitative real time polymerase chain reaction (qPCR). The study also included 28 females who had suffered from ovarian cancer and had been successfully operated upon and 90 healthy females with no history of cancer. Blood was drawn in EDTA tubes and used for extraction of DNA. The genotyping was carried out using Taqman® SNP Genotyping kit by RT-PCR. The variants investigated included c.871 T>C (rs799917), c.1040 G>A (rs4986852), c.181 T>G (rs28897672) in BRCA1 gene. RESULTS: The c.181 T>G (rs28897672) showed significantly different genotype and allele frequencies between the patients and the control subjects (p value = 0.002 and 0.02, respectively). The genotype TG was significantly protective (OR = 0.36, p value = 0.024). The mRNA expression of BRCA1 gene was found to be low in the ovarian cancer tissues. CONCLUSIONS: This study showed that c.181 T>G in BRCA1 genes is associated with the development of ovarian cancer in Saudis. More studies are needed to unveil other SNPs that may be associated with ovarian cancer and to understand the mechanism(s) involved in reducing the expression of BRCA1 gene in ovarian cancer tissues.

Genetic polymorphism and expression of HSF1 gene is significantly associated with breast cancer in Saudi females.

The transcription factor, heat shock factor 1 (HSF1), influences the expression of heat shock proteins as well as other activities like the induction of tumor suppressor genes, signal transduction pathway, and glucose metabolism. We hypothesized that single nucleotide polymorphisms (SNPs) in HSF1 gene might affect its expression or function which might have an influence on the development of breast cancer. The study group included 242 individuals (146 breast cancer patients and 96 healthy controls). From the cancer patients, genomic DNA was extracted from 96 blood samples and 50 Formalin-Fixed Paraffin Embedded (FFPE) tissues, while from the controls DNA were extracted from blood only. Genotype was carried out for four SNPs in the HSF1 gene (rs78202224, rs35253356, rs4977219 and rs34404564) using Taqman genotyping assay method. The HSF1 expression was investigated using immunohistochemistry on FFPE tissues (cancer tissue and adjacent normal tissue). The SNP rs78202224 (G>T) was significantly associated with increased risk of breast cancer. The combined TT + GT genotype (OR: 6.91; p: 0.035) and the T allele showed high risk (OR: 5.81; p:0.0085) for breast cancer development. The SNP rs34404564 (A>G) had a protective effect against the development of breast cancer. The genotype AG (OR: 0.41; p = 0.0059) and GG+AG (OR: 0.52; p: 0.026) occurred at a significantly lower frequency in the breast cancer patients compared to the frequency in healthy controls. No significant relationship was identified between either rs35253356 (A>G) or rs4977219 (A>C) and breast cancer in Saudi. The HSF1 protein expression was higher in all invasive and in situ breast carcinoma compared to the normal tissue. A stronger positive staining for HSF1 was found in the nucleus compared to the cytoplasm. Our results show that HSF1 gene expression is elevated in breast cancer tissue and two of the studied SNPs correlate significantly with cancer development.

Occurrence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) across the Gulf Corporation Council countries: Four years update.

The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infections has become a global issue of dire concerns. MERS-CoV infections have been identified in many countries all over the world whereas high level occurrences have been documented in the Middle East and Korea. MERS-CoV is mainly spreading across the geographical region of the Middle East, especially in the Arabian Peninsula, while some imported sporadic cases were reported from the Europe, North America, Africa, and lately Asia. The prevalence of MERS-CoV infections across the Gulf Corporation Council (GCC) countries still remains unclear. Therefore, the objective of the current study was to report the prevalence of MERS-CoV in the GCC countries and to also elucidate on its demographics in the Arabian Peninsula. To date, the World Health Organization (WHO) has reported 1,797 laboratory-confirmed cases of MERS-CoV infection since June 2012, involving 687 deaths in 27 different countries worldwide. Within a time span of 4 years from June 2012 to July 2016, we collect samples form MERS-CoV infected individuals from National Guard Hospital, Riyadh, and Ministry of health Saudi Arabia and other GCC countries. Our data comprise a total of 1550 cases (67.1% male and 32.9% female). The age-specific prevalence and distribution of MERS-CoV was as follow: <20 yrs (36 cases: 3.28%), 20-39 yrs (331 cases: 30.15%), 40-59 yrs (314 cases: 28.60%), and the highest-risk elderly group aged ≥60 yrs (417 cases: 37.98%). The case distribution among GCC countries was as follows: Saudi Arabia (1441 cases: 93%), Kuwait (4 cases: 0.3%), Bahrain (1 case: 0.1%), Oman (8 cases: 0.5%), Qatar (16 cases: 1.0%), and United Arab Emirates (80 cases: 5.2%). Thus, MERS-CoV was found to be more prevalent in Saudi Arabia especially in Riyadh, where 756 cases (52.4%) were the worst hit area of the country identified, followed by the western region Makkah where 298 cases (20.6%) were recorded. This prevalence update indicates that the Arabian Peninsula, particularly Saudi Arabia, is the hardest hit region regarding the emerging MERS-CoV infections worldwide. GCC countries including Saudi Arabia now have the infrastructure in place that allows physicians and scientific community to identify and immediately respond to the potential risks posed by new outbreaks of MERS-CoV infections in the region. Given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of MERS-CoV in our region.

Optimization of expression and purification of HSPA6 protein from Camelus dromedarius in E. coli.

The HSPA6, one of the members of large family of HSP70, is significantly up-regulated and has been targeted as a biomarker of cellular stress in several studies. Herein, conditions were optimized to increase the yield of recombinant camel HSPA6 protein in its native state, primarily focusing on the optimization of upstream processing parameters that lead to an increase in the specific as well as volumetric yield of the protein. The results showed that the production of cHSPA6 was increased proportionally with increased incubation temperature up to 37 °C. Induction with 10 µM IPTG was sufficient to induce the expression of cHSPA6 which was 100 times less than normally used IPTG concentration. Furthermore, the results indicate that induction during early to late exponential phase produced relatively high levels of cHSPA6 in soluble form. In addition, 5 h of post-induction incubation was found to be optimal to produce folded cHSPA6 with higher specific and volumetric yield. Subsequently, highly pure and homogenous cHSPA6 preparation was obtained using metal affinity and size exclusion chromatography. Taken together, the results showed successful production of electrophoretically pure recombinant HSPA6 protein from Camelus dromedarius in Escherichia coli in milligram quantities from shake flask liquid culture.

Spectroscopic and thermodynamic properties of recombinant heat shock protein A6 from Camelus dromedarius.

Heat shock protein A6, also known as HSP70B', is a member of the Hsp70 family of molecular chaperones. Under stressed conditions, the level of HSPA6 increases substantially, and the protein has been targeted as a biomarker of cellular stress in several studies. We report the spectroscopic and thermodynamic properties of Arabian camel species cHSPA6, determined by measurement of intrinsic and extrinsic fluorescence emission, and use of far-UV circular dichroism and dynamic multimode spectroscopy. Our results showed that cHSPA6 has similar binding affinity for both ATP and ADP (K D = ~50 nM). Binding of ATP and ADP reduced the surface hydrophobicity of the protein, and slightly altered its secondary structure, suggesting localized conformational rearrangement after ATP or ADP binding. Dynamic multimode spectroscopy revealed that cHSPA6 unfolds through three transitions with melting points (T m) of 42.3 ± 0.2, 61.3 ± 0.1, and 81.2 ± 0.2 °C. To the best of the author's knowledge, and literature search, this is the first report of the spectroscopic and thermodynamic properties of the Arabian camel heat shock protein.

Evidence of trem2 variant associated with triple risk of Alzheimer's disease.

Alzheimer's disease is one of the main causes of dementia among elderly individuals and leads to the neurodegeneration of different areas of the brain, resulting in memory impairments and loss of cognitive functions. Recently, a rare variant that is associated with 3-fold higher risk of Alzheimer's disease onset has been found. The rare variant discovered is a missense mutation in the loop region of exon 2 of Trem2 (rs75932628-T, Arg47His). The aim of this study was to investigate the evidence for potential structural and functional significance of Trem2 gene variant (Arg47His) through molecular dynamics simulations. Our results showed the alteration caused due to the variant in TREM2 protein has significant effect on the ligand binding affinity as well as structural configuration. Based on molecular dynamics (MD) simulation under salvation, the results confirmed that native form of the variant (Arg47His) might be responsible for improved compactness, hence thereby improved protein folding. Protein simulation was carried out at different temperatures. At 300K, the deviation of the theoretical model of TREM2 protein increased from 2.0 Šat 10 ns. In contrast, the deviation of the Arg47His mutation was maintained at 1.2 Šuntil the end of the simulation (t = 10 ns), which indicated that Arg47His had reached its folded state. The mutant residue was a highly conserved region and was similar to "immunoglobulin V-set" and "immunoglobulin-like folds". Taken together, the result from this study provides a biophysical insight on how the studied variant could contribute to the genetic susceptibility to Alzheimer's disease.

DNA mismatch repair MSH2 gene-based SNP associated with different populations.

We screened for the major essential single-nucleotide polymorphism (SNP) variant that might be associated with the MSH2 gene based on the data available from three types of human tissue samples [156 lymphoblastoid cell variations (LCL), 160 epidermis, 166 fat]. An association analysis confirmed that the KCNK12 SNP variant (rs748780) was highly associated (p value 9 × 10(-4)) with the MSH2 gene for all three samples. Using SNP identification, we further found that the recognized SNP was also relevant among Hapmap populations. Techniques that display specific SNPs associated with the gene of interest or nearby genes provide more reliable genetic associations than techniques that rely on data from individual SNPs. We investigated the MSH2 gene regional linkage association with the determined SNP (rs748780), KCNK12 variant (Allele T>C) in the intronic region, in HapMap3 full dataset populations, Yoruba in Ibadan, Nigeria (YRI), Utah residents with ancestry from northern Europe (CEU), Han Chinese in Beijing, China (CHB), and a population of Mexican ancestry in Los Angeles, California (MEX). A gene-based SNP association analysis analyzes the combined impact of every variant within the gene while creating referrals to linkage disequilibrium or connections between markers. Our results indicated that among the four populations studied, this association was highest in the MEX population based on the r(2) value; a similar pattern was also observed in the other three populations. The relevant SNP rs748780 in KCNK12 is related to a superfamily of potassium channel pore-forming P-domain proteins as well as to other non-pore-forming proteins and has been shown to be relevant to neurological disorder predisposition in MEX as well as in other populations.

Evidence of colorectal cancer risk associated variant Lys25Ser in the proximity of human bone morphogenetic protein 2.

Colorectal cancer (CRC) is the third most prevalent cancer and fourth leading cause of cancer-related deaths globally. It has been shown that the nsSNP variants play an important role in diseases, however it remained unclear how these variants are associated with the disease. Recently, several CRC risk associated SNPs have been discovered, however rs961253 (Lys25Arg at 20p12.3) located in the proximity of bone morphogenetic protein 2 (Bmp2) and fermitin family homolog 1 Fermt1 genes have been reported to be highly associated with the CRC risk. Here we provide evidence for the first time in silico biological functional and structural implications of non-synonymous (nsSNPs) CRC disease-associated variant Lys25Arg via molecular dynamic (MD) simulation. Protein structural analysis was performed with a particular variant allele (A/C, Lys25Arg) and compared with the predicted native protein structure. Our results showed that this nsSNP will cause changes in the protein structure and as a result is associated with the disease. In addition to the native and mutant 3D structures of CRC associated risk allele protein domain (CRAPD), they were also analyzed using solvent accessibility models for further protein stability confirmation. Taken together, this study confirmed that this variant has functional effect and structural impact on the CRAPD and may play an important role in CRC disease progression; hence it could be a reasonable approach for studying the effect of other deleterious variants in future studies.

Two DNA sites for MelR in the same orientation are sufficient for optimal MelR-dependent repression at the Escherichia coli melR promoter.

The Escherichia coli melR gene encodes the MelR transcription factor that controls melibiose utilization. Expression of melR is autoregulated by MelR, which represses the melR promoter by binding to a target that overlaps the transcript start. Here, we show that MelR-dependent repression of the melR promoter can be enhanced by the presence of a second single DNA site for MelR located up to 250 base pairs upstream. Parallels with AraC-dependent repression at the araC-araBAD regulatory region and the possibility of the MelR-dependent repression loop formation are discussed. The results show that MelR bound at two distal loci can cooperate together in transcriptional repression.

In silico analysis of single nucleotide polymorphism (SNPs) in human ß-globin gene.

Single amino acid substitutions in the globin chain are the most common forms of genetic variations that produce hemoglobinopathies--the most widespread inherited disorders worldwide. Several hemoglobinopathies result from homozygosity or compound heterozygosity to beta-globin (HBB) gene mutations, such as that producing sickle cell hemoglobin (HbS), HbC, HbD and HbE. Several of these mutations are deleterious and result in moderate to severe hemolytic anemia, with associated complications, requiring lifelong care and management. Even though many hemoglobinopathies result from single amino acid changes producing similar structural abnormalities, there are functional differences in the generated variants. Using in silico methods, we examined the genetic variations that can alter the expression and function of the HBB gene. Using a sequence homology-based Sorting Intolerant from Tolerant (SIFT) server we have searched for the SNPs, which showed that 200 (80%) non-synonymous polymorphism were found to be deleterious. The structure-based method via PolyPhen server indicated that 135 (40%) non-synonymous polymorphism may modify protein function and structure. The Pupa Suite software showed that the SNPs will have a phenotypic consequence on the structure and function of the altered protein. Structure analysis was performed on the key mutations that occur in the native protein coded by the HBB gene that causes hemoglobinopathies such as: HbC (E→K), HbD (E→Q), HbE (E→K) and HbS (E→V). Atomic Non-Local Environment Assessment (ANOLEA), Yet Another Scientific Artificial Reality Application (YASARA), CHARMM-GUI webserver for macromolecular dynamics and mechanics, and Normal Mode Analysis, Deformation and Refinement (NOMAD-Ref) of Gromacs server were used to perform molecular dynamics simulations and energy minimization calculations on ß-Chain residue of the HBB gene before and after mutation. Furthermore, in the native and altered protein models, amino acid residues were determined and secondary structures were observed for solvent accessibility to confirm the protein stability. The functional study in this investigation may be a good model for additional future studies.

Molecular cloning and characterization of cDNA encoding a putative stress-induced heat-shock protein from Camelus dromedarius.

Heat shock proteins are ubiquitous, induced under a number of environmental and metabolic stresses, with highly conserved DNA sequences among mammalian species. Camelus dromedaries (the Arabian camel) domesticated under semi-desert environments, is well adapted to tolerate and survive against severe drought and high temperatures for extended periods. This is the first report of molecular cloning and characterization of full length cDNA of encoding a putative stress-induced heat shock HSPA6 protein (also called HSP70B') from Arabian camel. A full-length cDNA (2417 bp) was obtained by rapid amplification of cDNA ends (RACE) and cloned in pET-b expression vector. The sequence analysis of HSPA6 gene showed 1932 bp-long open reading frame encoding 643 amino acids. The complete cDNA sequence of the Arabian camel HSPA6 gene was submitted to NCBI GeneBank (accession number HQ214118.1). The BLAST analysis indicated that C. dromedaries HSPA6 gene nucleotides shared high similarity (77-91%) with heat shock gene nucleotide of other mammals. The deduced 643 amino acid sequences (accession number ADO12067.1) showed that the predicted protein has an estimated molecular weight of 70.5 kDa with a predicted isoelectric point (pI) of 6.0. The comparative analyses of camel HSPA6 protein sequences with other mammalian heat shock proteins (HSPs) showed high identity (80-94%). Predicted camel HSPA6 protein structure using Protein 3D structural analysis high similarities with human and mouse HSPs. Taken together, this study indicates that the cDNA sequences of HSPA6 gene and its amino acid and protein structure from the Arabian camel are highly conserved and have similarities with other mammalian species.