Comet Assay and Micronucleus Test in Circulating Erythrocytes of Ctenopharyngodon idella Exposed to Nickel Oxide Nanoparticles.

The number of pollutants released into freshwater and marine environments has increased due to the widespread use of nanoparticles. Nickel oxide nanoparticles (NiO-NPs) were tested for genotoxicity in fish fingerlings of the species Ctenopharyngodon idella. For 7, 14, and 21 days, fingerlings were exposed to NiO-NPs with each increasing concentrations of 2.25 mg/L, 4.50 mg/L, and 6.75 mg/L, respectively. The micronuclei assay and comet assay were used to evaluate the DNA damage. The experiment revealed that with the increase in nanoparticle concentration and exposure duration, the level of DNA damage also increased. The experiment resulted to be time and dose dependent, and the damage was found as follows: 6.75 mg/L > 4.50 mg/L > 2.25 mg/L against each exposure period. In terms of comet assay, the results showed that after 7 days, the level of DNA damage in all the concentrations was highly significant (P < 0.001). Increased DNA damage was calculated at the higher administered dose of 6.75 mg/L for 21 days of exposition, followed by 14 and 7 days, respectively. The second high toxic effect was observed in the fish blood at the exposure concentration of 4.50 mg/L for 21 days, followed by 14 and 7 days, respectively. The micronuclei induction in the nanoparticle's administered blood could be detected only for a 7-day exposition period. Whereas for the exposed duration of 14 and 21 days, the entire red blood cells of the grass carp were completely destroyed demonstrating the ability of the nanoparticles to cause anomalies in aquatic life.

Novel mutation in alpha-spectrin gene in Saudi patients with hereditary spherocytosis.

Hereditary spherocytosis (HS) is the most common hereditary hemolytic disorder induced by red blood cell (RBC) membrane defect. This study was undertaken to determine mutations in genes associated with RBC membrane defect in patients with HS such as α-spectrin gene (SPTA1), ß-spectrin gene (SPTB), ankyrin gene (ANK1), band 3 anion transport gene (SLC4A1) and erythrocyte membrane protein band 4.1 gene (EPB41). Blood samples were collected from 23 unrelated patients with HS. Patients were diagnosed according to the guidelines from the British Society for Hematology. All hematological examinations for the determination of RBC abnormalities and osmotic fragility tests were conducted. Genomic DNA were extracted from peripheral blood cells and coding exons of known genes for hereditary spherocytosis were enriched using Roche/KAPA sequence capture technology and sequenced on an Illumina system via next-generation sequencing (NGS). The data showed that most of the HS patients confirmed splenomegaly and showed elevated reticulocytes and abnormal bilirubin values. NGS analysis identified the heterozygous variant c.5501G > A in the exon 39 of SPTA1 gene, resulted in a Trp1834*, which leads to a premature stop codon and subsequent mRNA degradation (nonsense- mediated decay) or truncation in α spectrin. Moreover, our data also revealed conventional mutations in genes SPTB, ANK, SLC4A1 and EBP41 in severe patients of HS. In short, this is the first report that determined a novel mutation c.5501G > A in SPTA1 gene in the Saudi population. To the best of our knowledge, this variant c.5501G > A has not been described in global literature so far. This novel mutation in SPTA1 gene is unique in the Saudi population.

Exome sequencing identifies homozygous variants in MBOAT7 associated with neurodevelopmental disorder.

Intellectual disability (ID) is a large group of neurodevelopmental disorders characterized by a congenital limitation in intellectual functioning (reasoning, learning, and problem solving), adaptive behavior (conceptual, social, and practical skills), originated at birth and manifested before the age of 18. By whole exome sequencing of five consanguineous Pakistani families presenting hallmark features of ID, global developmental delay, aggressive and self-injurious behaviors, microcephaly, febrile seizures and facial dysmorphic features, we identified three novel homozygous missense variants (NM_024298.5: c.588G > T; p.Trp196Cys, c.736 T > C; p.Tyr246His and c.524A > C; p. Asp175Ala) and one rare homozygous in-frame deletion variant (c.758_778del;p.Glu253_Ala259del) in membrane-bound O-acyltransferase family member 7 (MBOAT7) gene previously associated with autosomal recessive neurodevelopmental disorder. The segregation of the variants was validated by Sanger sequencing in all family members. In silico homology modeling of wild-type and mutated proteins revealed substantial changes in the structure of both proteins, indicating a possible effect on function. The identification and validation of new pathogenic MBOAT7 variants in five cases of autosomal recessive ID further highlight the importance of this genes in proper brain function and development.

Unveiling genetics of non-syndromic albinism using whole exome sequencing: A comprehensive study of TYR, TYRP1, OCA2 and MC1R genes in 17 families.

BACKGROUND: Oculocutaneous albinism (OCA) is a group of skin depigmentation disorders. Clinical presentation of OCA includes defects in melanocyte differentiation, melanin biosynthesis, and melanosome maturation and transport. OBJECTIVES: A molecular diagnostics study of families presenting oculocutaneous albinism. METHODS: In this study, 17 consanguineous OCA families consisting of 93 patients were investigated. Whole Exome Sequencing (WES) of the index patient in each family were performed. Short listed variants of WES were Sanger validated for Mendelian segregation in obligate carriers and other available family members. Variant prioritization and pathogenicity were classified as per the criteria of American College Medical Genetics and Genomics (ACMG). Comparative computational modelling was performed to predict the potential damaging effect of the altered proteins. RESULTS: 15 pathogenic variations: c.132 T > A, c.346C > T, c.488C > G, c.1037G > A in TYR, c.1211C > T, c.1441G > A, c.1706_1707insT, c.2020C > G, c.2402G > C, c.2430del, in OCA2, c.1067G > A in TYRP1 and c.451C > T, c.515G > T, c.766C > T, c.917G > A in MC1R genes were identified. Three variants in OCA2 gene were characterized: c.1706_1707insT, c.2430del, and c.2402G > C, all of which were not reported before in OCA families. CONCLUSION: A few studies focusing on mutation screening of OCA patients have been reported before; however, this study has uniquely presents the Pakhtun ethnic population residing on the North-Western boarder. It explains that TYR, OCA2, TYRP1, and MC1R variations lead to non-syndromic OCA phenotype The overlapping phenotypes of OCA can precisely be diagnosed for its molecular pathogenicity using WES. This study recommends WES as a first-line molecular diagnostic tool, and provides a basis for developing customized genetic tests i.e. pre-marital screening to reduce the disease burden in the future generations.

Spatially explicit estimation of freshwater fish stock biomass with limited data: A case study of an endangered endemic fish on the Tibetan Plateau, China.

Accurate evaluation of fish stock biomass is essential for effective conservation management and targeted species enhancement efforts. However, this remains challenging owing to limited data availability. Therefore, we present an integrated modeling framework combining catch per unit effort with ensemble species distribution modeling called CPUESDM, which explicitly assesses and validates the spatial distribution of stock biomass for freshwater fish species with limited data, applied to Herzensteinia microcephalus. The core algorithm incorporates the Leslie regression model, ensemble species distribution modeling, and exploratory spatial interpolation techniques. We found that H. microcephalus biomass in the Yangtze River source area yielded an initial estimate of 113.52 tons. Our validation results demonstrate high accuracy with a Cohen's kappa coefficient of 0.78 and root mean square error of 0.05. Furthermore, our spatially-explicit, global, absolute biomass density map effectively identified areas with high and low concentrations of biomass distribution centers. Additionally, this study offers access to the source code, example raw data, and a step-by-step instruction manual for other researchers using field data to explore the application of this model. Our findings can help inform for future conservation efforts around fish stock biomass estimation, especially for endangered species.

Multifunctional and switchable metamaterial for terahertz polarization modulation in the reflection mode.

In this paper, a broadband multi-layered active metamaterial design is investigated, which can achieve a high polarization conversion efficiency over a wide band of frequencies in the terahertz regime. The design can be switched to an efficient metamaterial absorber using the phase transition property of vanadium dioxide (V O 2). Additionally, the designed structure can convert the linear polarization of the incoming wavefronts to its cross-polarization and linear polarization to circular polarization in the reflection mode. The broadband characteristic is achieved due to the strong anisotropic behavior of the metasurface. The structure is robust to a wide range of incident angles as well. The proposed switchable multifunctional design can contribute to the development of active plasmonic polarization devices and metamaterial absorbers.

Rainwater harvesting for agriculture development using multi-influence factor and fuzzy overlay techniques.

Rainwater harvesting (RWH) is an essential technique for enhancing agricultural development, particularly in regions facing water scarcity or unreliable rainfall patterns. Water shortage, however, is one of the key causes of low crop production especially in mountainous regions like the Khyber Pakhtunkhwa province where most rainwater is lost by runoff. Therefore, rainwater harvesting could be a suitable to make better use of runoff and increase crop production. The study focuses on selecting suitable rainwater harvesting sites in District Karak to enhance agriculture by utilizing multi-influence factor (MIF) and fuzzy overlay techniques. We considered seven factors, i.e., land use land cover (LULC), slope, geology, soil, rainfall, lineament, drainage density, to create a ranking system to understand its application in site selection analysis. The results were combined into one overlay process to produce a rainwater harvesting suitability map. The weighted overlay analysis of the MIF model results reveals that 167.96 km2 area has a very high potential for rainwater harvesting, 874.17 km2 has a high potential, 1182.92 km2 has a moderate and 354.50 km2 has a poor potential for rainwater harvesting. The fuzzy overlay analysis revealed that 257.53 km2 has a very high potential for rainwater harvesting, 896.56 km2 area is classified as high, 1018.30 km2 moderate, and 407.7 km2 has poor potential for rainwater harvesting. The findings of this research work will help the policymakers and decision-makers construct various rainwater harvesting structures in the study area to overcome the water shortage problems.

Ambient Temperature and Cardiac Biomarkers: A Meta-Analysis.

This study quantified the effect of cold or heat exposure of ambient temperature on the alteration of well-known cardiac markers. A meta-analysis was performed using the PRISMA guidelines. Peer-reviewed studies on ambient temperature and cardiac biomarkers were retrieved from MEDLINE, ScienceDirect and Google Scholar from January 2000 to February 2022. The pooled effect sizes of ambient temperature on cardiac biomarkers c-reactive protein, soluble-cell adhesion-molecule-1, soluble-intercellular-adhesion-molecule-1, total cholesterol, low-densitylipoprotein, interleukin-6, B-type-Natriuretic-Peptide; systolic/diastolic blood pressure were quantified using a random-effects meta-analysis. A total of 26 articles were included in the metaanalysis after screening the titles, abstracts and full texts. The pooled results for a 1°C decrease of ambient temperature showed an increase of 0.31% (95% CI= 0.26 to 0.38) in cardiac biomarkers (p=0.00; I-squared=99.2%; Cochran's Q=5636.8). In contrast, the pooled results for a 1°C increase in ambient temperature showed an increase of 2.03% (95% CI= 1.08 to 3.82) in cardiac biomarkers (p=0.00; I-squared=95.7%; Cochran's Q=235.2). In the cardiovascular (CV) population, the percent increase in cardiac biomarkers levels due to a decrease/increase in ambient temperature was greater. This study showed the decrease/increase in ambient temperature has a direct correlation with the alterations in cardiac biomarkers. These findings are useful for managing temperatureassociated cardiovascular mortality.

Tunable metalensing based on plasmonic resonators embedded on thermosresponsive hydrogel.

Metalenses of adjustable power and ultrathin flat zoom lens system have emerged as a promising and key photonic device for integrated optics and advanced reconfigurable optical systems. Nevertheless, realizing an active metasurface retaining lensing functionality in the visible frequency regime has not been fully explored to design reconfigurable optical devices. Here, we present a focal tunable metalens and intensity tunable metalens in the visible frequency regime through the control of the hydrophilic and hydrophobic behavior of freestanding thermoresponsive hydrogel. The metasurface is comprised of plasmonic resonators embedded on the top of hydrogel which serves as dynamically reconfigurable metalens. It is shown that the focal length can be continuously tuned by adjusting the phase transition of hydrogel, the results reveal that the device is diffraction limited in different states of hydrogel. In addition, the versatility of hydrogel-based metasurfaces is further explored to design intensity tunable metalens, that can dynamically tailor the transmission intensity and confined it into the same focal spot under different states, i.e., swollen and collapsed. It is anticipated that the non-toxicity and biocompatibility make the hydrogel-based active metasurfaces suitable for active plasmonic devices with ubiquitous roles in biomedical imaging, sensing, and encryption systems.

Whole exome sequencing identifies a novel compound heterozygous GFM1 variant underlying developmental delay, dystonia, polymicrogyria, and severe intellectual disability in a Pakhtun family.

Mitochondrial protein synthesis requires three elongation factors including EF-Tu (TUFM; OMIM 602389), EF-Ts (TSFM; OMIM 604723), and EF-G1 (GFM1; OMIM 606639). Pathogenic variants in any of these three members result in defective mitochondrial translation which can impart an oxidative phosphorylation (OXPHOS) deficiency. In this study, we investigated a consanguineous Pakhtun Pakistani family. There were four affected siblings at the time of this study and one affected girl had died in infancy. The index patient had severe intellectual disability, global developmental delay, dystonia, no speech development, feeding difficulties, and nystagmus. MRI brain presented thinning of corpus callosum and polymicrogyria. Whole exome sequencing revealed a novel compound heterozygous variant in GFM1 located on chromosome 3q25.32. Sanger sequencing confirmed recessive segregation of the maternal (NM_001308164.1:c.409G > A; p.Val137Met) and paternal (NM_001308164.1:c.1880G > A; p.Arg627Gln) variants in all the four affected siblings. These variants are classified as "likely-pathogenic" according to the recommendation of ACMG/AMP guideline. GFM1 alterations mostly lead to severe phenotypes and the patients may die in early neonatal life; however, four of the affected siblings had survived till the ages of 10-17 years, without developing any life-threatening conditions. Mostly, in cousin marriages, the pathogenic variants are identical-by-descent, and affected siblings born to such parents are homozygous. Three homozygous variants were shortlisted in the analysis of the WES data, but Sanger sequencing did not confirm their segregation with the disease phenotype. This is the first report from Pakistan expanding pathogenicity of GFM1 gene.

All-dielectric metasurface designs for spin-tunable beam splitting via simultaneous manipulation of propagation and geometric phases.

Metasurfaces offer diverse wavefront control by manipulating amplitude, phase, and polarization of light which is beneficial to design subwavelength scaled integrated photonic devices. Metasurfaces based tunable circular polarization (CP) beam splitting is one functionality of interest in polarization control. Here, we propose and numerically realize metasurface based spin tunable beam splitter which splits the incoming CP beam into two different directions and tune the splitting angles by switching the handedness of incident light polarization. The proposed design approach has potential in applications such as optical communication, multiplexing, and imaging.

Effect of Cypermethrin on Blood Hematology and Biochemical Parameters in Fresh Water Fish Ctenopharyngodon idella (Grass Carp).

The insecticide, cypermethrin, adversely affects biochemical parameters in blood and behavior in grass carp (Ctenopharyngodon idella). Fish were obtained from hatchery, reared in the laboratory. Different concentration of cypermethrin were applied. Blood was collected and hematological and biochemical parameters were measured. Biochemical parameters such as, protein levels, cholesterol, phosphorous and calcium in both acute and chronically cypermethrin treated groups decreased, with increasing exposure time from 24h to 15 days with more pronounced effects in the acute groups. Increased glucose, urea, serum glutamic pyruvic transaminase (SGPT), creatinine, and lactate dehydrogenase (LDH) levels were found in both acute and chronic groups with the increasing exposure time. Hematological parameters, such as red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MHCH), and red cell distribution width (RDW) were significantly reduced in both groups as the exposure time increases. However, the numbers of white blood cells (WBC) and platelets were increased. This study established both the acute and chronic toxicity of cypermethrin in grass carp, which likely occurs secondary to altered biochemical and blood parameters.

Biallelic inheritance in a single Pakistani family with intellectual disability implicates new candidate gene RDH14.

In a multi-branch family from Pakistan, individuals presenting with palmoplantar keratoderma segregate in autosomal dominant fashion, and individuals with intellectual disability (ID) segregate in apparent autosomal recessive fashion. Initial attempts to identify the ID locus using homozygosity-by-descent (HBD) mapping were unsuccessful. However, following an assumption of locus heterogeneity, a reiterative HBD approach in concert with whole exome sequencing (WES) was employed. We identified a known disease-linked mutation in the polymicrogyria gene, ADGRG1, in two affected members. In the remaining two (living) affected members, HBD mapping cross-referenced with WES data identified a single biallelic frameshifting variant in the gene encoding retinol dehydrogenase 14 (RDH14). Transcription data indicate that RDH14 is expressed in brain, but not in retina. Magnetic resonance imaging for the individuals with this RDH14 mutation show no signs of polymicrogyria, however cerebellar atrophy was a notable feature. RDH14 in HEK293 cells localized mainly in the nucleoplasm. Co-immunoprecipitation studies confirmed binding to the proton-activated chloride channel 1 (PACC1/TMEM206), which is greatly diminished by the mutation. Our studies suggest RDH14 as a candidate for autosomal recessive ID and cerebellar atrophy, implicating either disrupted retinoic acid signaling, or, through PACC1, disrupted chloride ion homeostasis in the brain as a putative disease mechanism.

Whole exome sequencing identified a novel missense alteration in CC2D2A causing Joubert syndrome 9 in a Pakhtun family.

BACKGROUND: Joubert syndrome (JBTS) is a heterogenous disorder characterized by intellectual disability, developmental delays, molar tooth sign in brain imaging, hypotonia, ocular motor apraxia and overlapping features of ciliopathies. There are 36 clinical subtypes of JBTS, with an equal number of genes known so far for this phenotype. METHODS: Whole exome sequencing (WES) and Sanger sequencing were performed for the molecular diagnosis of a Pakhtun family affected with Joubert syndrome type 9 (JBTS9). RESULTS: A novel homozygous missense variant (c.4417C>G; Pro1473Ala) in exon 34 was identified in coiled-coil and C2 domains-containing the protein 2A (CC2D2A; NM_001080522) gene. The variant co-segregated in autosomal recessive fashion within the family and was not found in 200 ethnically matched unaffected individuals. In silico analyses supported the pathogenic effect of the altered CC2D2A protein. CONCLUSIONS: To the best of our knowledge, this is the first report of CC2D2A alteration co-segragating with a JBTS9 phenotype in a Pakhtun family from Pakistan. Our findings broaden the pathogenic spectrum of JBTS9, adding a novel variant to CC2D2A variation pool. WES analysis is a successful molecular diagnostic tool for rare genetic disorders, especially in those populations where the marriage of cousins is more frequent. Efficient and accurate genetic testing and counselling of the affected families are helpful for patient management and for reducing the disease burden in future generations.

Absence of CD74 Isoform at 41kDa Prevents the Heterotypic Associations between CD74 and CD44 in Human Lung Adenocarcinoma-derived Cells.

Lung cancer is a leading cause of cancer-associated death in all over the globe. This study was undertaken to determine the expression and interaction of membrane-bound receptors CD74 and CD44 in human lung adenocarcinoma cells and their associated signaling was also attempted. Levels of CD74 and CD44 were studied in human lung adenocarcinoma-evolved cells A549 and H460. CD74-mediated downstream signaling was studied by the nuclear-transcription-factor NF-κB and prostaglandin E2 (PGE2) production. Flow-cytometric analysis showed that both CD74 and CD44 were perfectly expressed in A549 cells. Importantly, Western immunoblotting showed that A549 cells expressed only two isoforms of CD74 at 33 and 35 kDa but isoform at 41 kDa was absent. These results were verified in H460 cells. Confocal microscopy showed CD74 and CD44 was colocalized but heterotypic interaction between them was missing in both A549 and H460 cells. Activation of NF-κB and production of PGE2 in human lung cancer cells were comparable with other cancer cells. In conclusion, this is the first study that shows A549 and H460 cells expressed two distinctive isoforms of CD74 but isoform at 41 kDa was absent. Due to the absence of this isoform, the direct physical interaction between them CD74 and CD44 was lacking. Furthermore, the data also demonstrated that lacking of direct physical interaction between CD74 and CD44 had no effect on NF-κB activation and PGE2 production indicating that CD74-mediated downstream signaling occurs either through coreceptors or indirect interaction with CD44 in human lung cancer cells.Abbreviation: CD: cluster of differentiation; SCLC: small cell lung cancer; NSCLC: nonsmall cell lung cancer; SCC: squamous cell carcinoma; ADC: adenocarcinoma; LCC: large cell carcinoma.

Novel homozygous variant in the TPO gene associated with congenital hypothyroidism and mild-intellectual disability.

Congenital hypothyroidism (CH) is one of the most common hereditary disorders affecting neonates worldwide. CH is a multifactorial complex disorder and can be caused by either environmental factors or genetic factors. We studied one Pakistani family with segregating mutations in CH inherited in an autosomal recessive manner. Using whole-exome sequencing (WES), we found a novel homozygous missense variant (c.2315A>G; p.Tyr772Cys) in the thyroid peroxidase (TPO) gene. Different bioinformatics prediction tools and Sanger sequencing were performed to verify the identified variant. Our findings highlight the importance of this gene in causing CH and mild-intellectual disability (ID) in two affected brothers. WES is a convenient and useful tool for the clinical diagnosis of CH and other associated disorders.

Highly Recurrent Copy Number Variations in GABRB2 Associated With Schizophrenia and Premenstrual Dysphoric Disorder.

OBJECTIVE: Although single-nucleotide polymorphisms in GABRB2, the gene encoding for GABAA receptors ß2 subunit, have been associated with schizophrenia (SCZ), it is unknown whether there is any association of copy number variations (CNVs) in this gene with either SCZ or premenstrual dysphoric disorder (PMDD). METHODS: In this study, the occurrences of the recurrent CNVs esv2730987 in Intron 6 and nsv1177513 in Exon 11 of GABRB2 in Chinese and German SCZ, and Chinese PMDD patients were compared to controls of same ethnicity and gender by quantitative PCR (qPCR). RESULTS: The results demonstrated that copy-number-gains were enriched in both SCZ and PMDD patients with significant odds ratios (OR). For combined-gender SCZ patients versus controls, about two-fold increases were observed in both ethnic groups at both esv2730987 (OR = 2.15, p = 5.32E-4 in Chinese group; OR = 2.79, p = 8.84E-3 in German group) and nsv1177513 (OR = 3.29, p = 1.28E-11 in Chinese group; OR = 2.44, p = 6.17E-5 in German group). The most significant copy-number-gains were observed in Chinese females at nsv1177513 (OR = 3.41), and German females at esv2730987 (OR=3.96). Copy-number-gains were also enriched in Chinese PMDD patients versus controls at esv2730987 (OR = 10.53, p = 4.34E-26) and nsv1177513 (OR = 2.39, p = 3.19E-5). CONCLUSION: These findings established for the first time the association of highly recurrent CNVs with SCZ and PMDD, suggesting the presence of an overlapping genetic basis with shared biomarkers for these two common psychiatric disorders.

Interleukin-6 polymorphisms in HCC patients chronically infected with HCV.

Hepatocellular carcinoma is a primary liver malignancy in which the risk of development is always multifunctional. Interleukin-6 is a proinflammatory and multifunctional cytokine, which plays an important role in the immune response, haematopoiesis and defence against viral infection. We aimed to evaluate the frequency of Interleukin-6 mutations (rs2069837 and rs17147230) associated with genetic risk of hepatocellular carcinoma in Khyber Pakthunkhwa population. A total of 72 hepatocellular carcinoma cases and 38 controls were included in this study. The genomic DNA was extracted from the peripheral blood cells and Interleukin-6 genotyping was performed using T-ARMS-PCR technique. Our results show a significant increase risk of developing hepatocellular carcinoma with the mutation within Interleukin-6 gene with heterozygous G allele (rs2069837) (OR = 10.667, 95%CI = 3.923-29.001, p = < 0.0001) and heterozygous T allele (rs17147230) (OR = 75.385, 95%CI = 9.797-580.065, p = < 0.0001). However, under recessive gene model the results were insignificant in case of Interleukin-6 rs2069837 (OR = 0.605, 95%CI = 0.217-1.689, p = 0.337), while significant in case of Interleukin-6 rs17147230 (OR = 0.298, 95%CI = 0.121-0.734, p = 0.0085). In conclusion, Interleukin-6 mutation is associated with hepatocellular carcinoma susceptibility. More related studies with other associated interleukins and their whole gene sequencing will be required.

A novel approach for high-level expression and purification of GST-fused highly thermostable Taq DNA polymerase in Escherichia coli.

Polymerases are enzymes that synthesize long chains or polymers of nucleic acids including DNA or RNA from nucleotides. They assemble nucleic acids by copying a DNA or RNA template strand using base-pairing interactions. One of the polymerase enzymes, Taq DNA polymerase, originally isolated from Thermus aquaticus (Taq) is a widely used enzyme in molecular biology so far. The thermostable properties of this enzyme have contributed majorly to the specificity, automation, and efficacy of the polymerase chain reaction (PCR), making it a powerful tool for today's molecular biology researches across the globe. The purification of Taq DNA polymerase from the native host results in low yield, more labor and time consumption. Therefore, many studies have been previously conducted to obtain this enzyme using alternative hosts. So far, all the existing methodologies are more laborious, time-consuming and require heavy expense. We used a novel approach to purify the enzyme with relatively high efficiency, yield and minimum time consumption using Escherichia coli (E. coli) as an alternative host. We cloned a 2500 base pair Taq DNA polymerase gene into pGEX-4T-1 vector, containing a GST-tag, downstream of tac promoter and overexpressed it using isopropyl ß-d-1-thiogalactopyranoside (IPTG) as an inducer. The enzyme was efficiently purified using novel chromatography approaches and was used in routine PCR assays in our laboratory. Our findings suggest a novel approach to facilitate the availability of polymerases for molecular and diagnostic studies. In the future, it may be used for the purification of other recombinant peptides or proteins used in structural biology and proteomics-based researches.

Clinical Characteristics, Mortality and Associated risk factors in COVID-19 patients reported in ten major hospitals of Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: COVID-19 is an ongoing public health issue across the world. Several risk factors associated with mortality in COVID-19 have been reported. The present study aims to describe clinical and epidemiological characteristics and predictors of mortality in hospitalized patients from Khyber Pakhtunkhwa, a province in Pakistan with highest COVID-19 associated case fatality rate. METHODS: This multicentre, retrospective study was conducted in hospitalized COVID-19 patients who died or discharged alive until 1st May 2020. Data about sociodemographic characteristics, clinical and laboratory findings, treatment and outcome were obtained from hospital records and compared between survivors and non-survivors. Statistical tests were applied to determine the risk factors associated with mortality in hospitalized patients. RESULTS: Of the total 179 patients from the 10 designated hospitals, 127 (70.9%) were discharged alive while 52 (29.1%) died in the hospital. Overall, 109 (60.9%) patients had an underlying comorbidity with hypertension being the commonest. Multivariate logistics regression analysis showed significantly higher odds of in-hospital death from COVID-19 in patients with multiple morbidities (OR 3.2, 95% CI 1.1, 9.1, p-value=0.03), length of hospital stay (OR 0.8, 95% CI 0.7, 0.9, p-value <0.001), those presenting with dyspnoea (OR 4.0, 95% CI 1.1, 14.0, p-value=0.03) and oxygen saturation below 90 (OR 9.6, 95% CI: 3.1, 29.2, p-value <0.001). CONCLUSION: Comorbidity, oxygen saturation and dyspnoea on arrival and length of stay in hospital (late admission) are associated with COVID-19 mortality. The demographic, clinical and lab characteristics could potentially help clinician and policy makers before potential second wave in the country.