Restriction Site Research Articles

Ce-UiO-66-F4-based composites decorated with green carbon dots for universal adsorption of organic pollutants containing hydrogen bond donors and its application exploration

Efficient and stable water purification is essential for the protection of water resources. Current adsorbents based on metal–organic frameworks face limitations of restricted active sites and single adsorption objects. In this study, we developed a cost-effective and environmentally friendly Tea-L-CDs@Ce-UiO-66-F4 composites by growing Tea and L-tryptophan carbon dots (Tea-L-CDs) over Ce-UiO-66-F4 MOF, and the interaction force was enhanced by hydrogen bonding (-F/O/N…H-O-, -F/O/N…H-N-, etc.) to achieve universal adsorption. In detail, the maximum adsorption capacity (qm) of the adsorbent for trypan blue (TB), congo red (CR), and CR (adjust pH) was 773.4, 1428.6, and 4761.9 mg/g. The universal experiment showed that the composites exhibited excellent qm for alizarin red S (ARS, 662.7 mg/g) and rhodamine B (RdB, 374.0 mg/g). Meanwhile, Tea-L-CDs@Ce-UiO-66-F4 composites maintained good anti-interference ability (salt solution, temperature, etc.). Remarkably, the adsorption capacity of the adsorbent for dyes exceeded 97 % of qm within 1 min of contact time. The adsorption performance of synthetic material for TB, CR, ARS, and RdB is better than most of the adsorbents, especially the qm for TB is higher than any adsorbent previously reported. Besides, this adsorbent had excellent storage stability in the air for 30 days. Based on this nanomaterial, we successfully developed an automated dye filtration and contamination assessment system for blocking dye-attached adsorbent and online detection of dye residues after water purification. Thus, the synthesized Tea-L-CDs@Ce-UiO-66-F4 composites, with universal adsorption, high efficiency, and good storage stability, hold great potential for wastewater purification.

Sequence analysis and secondary structure prediction of autosomal STR alleles using next generation sequencing (NGS) data

The inception of NGS technology in forensic DNA analysis explores the sequence-based alleles in STR markers. This allows the in-depth analysis of observed STR allelic sequences to understand their nature, stability, inheritance, and possible generation of artifacts during their analysis. In the current study, 100 allelic sequences at 20 STR markers generated using the NGS technique were analyzed for their sequence topographies and prediction of secondary structures. The G + C content of the alleles observed in 20 STR markers used in this study varied from 58.65 ± 1.367% (D2S1338) to 7.62 ± 0.844% (D12ATA63). The average exact mass of one stand and for the opposite stand mass was found to be highest in FGA (25,963.248 ± 1623.271; 27,501.720 ± 1712.691), whereas, D4S2408 generated the lowest exact mass of one stand and for the opposite stand mass (11,136.354 ± 1521.757; 11,582.021 ± 1585.486). As expected, none of the STR markers showed the presence of open reading frames, Codons, and CRISPR sequences. Three STR markers viz. D2S1338, TH01, and D5S2800 showed the presence of restriction sites for Cac8I, TspGWI, TspDTI, AccI, and Hpy8I enzymes. Phylogenetic analysis reveals the close association of alleles between the D12ATA63 and D19S433 markers. Stable pseudoknots were predicted at alleles of D2S1338 showing an average energy of −0.76 with the highest number of nucleotides present in the pseudoknots i.e., 21.33, suggesting this marker is more prone to generate amplification artifacts.

Mutagenic primer-based novel multiplex PCR-RFLP technique to genotype BECN1 SNPs rs10512488 and rs11552192.

Single Nucleotide Polymorphisms (SNPs) in candidate autophagy gene BECN1 could influence its functions thereby autophagy process. BECN1 noncoding SNPs were found to be significantly associated with neurodegenerative disease and type 2 diabetes mellitus. This study aimed to develop a simultaneous genotyping technique for two BECN1 SNPs (rs10512488 and rs11552192). A mutagenic primer-based approach was used to introduce a NdeI restriction site to genotype rs10512488 by Artificial-Restriction Fragment Length Polymorphism (A-RFLP) along with rs11552192 by Polymerase Chain Reaction (PCR)-RFLP. Multiplexing PCR and restriction digestion reactions were set up for simultaneous genotyping of both SNPs in 100 healthy individuals. Genotypic and allele frequencies were manually calculated, and the Hardy-Weinberg Equilibrium was assessed using the chi-square test. We successfully developed PCR and RFLP conditions for the amplification and restriction digestion of both SNPs within the same tube for genotyping. The results of genotyping by newly developed multiplexing PCR-RFLP technique were concordant with the genotypes obtained by Sanger sequencing of samples. Allelic frequencies of rs10512488 obtained were 0.15 (A) and 0.85 (G), whereas allelic frequencies of rs11552192 were 0.16 (T) and 0.84 (A). The newly developed technique is rapid, cost-effective and time-saving for large-scale applications compared to sequencing methods and would play an important role in low-income settings. For the first time, allelic frequencies of rs10512488 and rs11552192 were reported among the North Indian population.

Biological and Physical Controls on Multidecadal Acidification in a Eutrophic Estuary

AbstractEstuaries support ecologically and economically important resources that are vulnerable to ocean acidification from rising anthropogenic CO2. However, complex local processes in estuaries complicate and may disguise long‐term pH trends. For example, terrestrial nutrient runoff and coastal upwelling may exacerbate pH variability and declines. We investigated eutrophication impacts on acidification in a central California estuary, Elkhorn Slough, which receives high nutrient loads from intensive surrounding agriculture and upwelling of the California Current System. We examined drivers of acidification including nutrients, ecosystem metabolism, and upwelling by modeling pH trends over 20 years using a Generalized Additive Mixed Model at four sites from the National Estuarine Research Reserve Systemwide Monitoring Program and collected additional water samples to calculate aragonite saturation. Our models revealed acidification trends over two decades which were more pronounced near the marine inlet. Near the marine inlet, high nutrient levels and lower buffering were associated with the greatest rate of acidification in the estuary, which was four times greater than the trend from anthropogenic CO2 alone. Compared to fully tidal sites, a tidally restricted site showed diminished pH declines over time because of higher mean pH and aragonite saturation levels, but greater diel and seasonal variability associated with cycles of ecosystem metabolism and tidal range. Therefore, short‐term drops of saturation are threats to acidity in this location. The effects of enhanced seasonal cycles or long‐term trends in different zones of the estuary have implications for monitoring estuaries with a temporal frequency and scale to capture coastal acidification risks.

Weighted single step GWAS reveals genomic regions associated with economic traits in Murrah buffaloes

The objective of the present study was to identify genomic regions influencing economic traits in Murrah buffaloes using weighted single step Genome Wide Association Analysis (WssGWAS). Data on 2000 animals, out of which 120 were genotyped using a double digest Restriction site Associated DNA (ddRAD) sequencing approach. The phenotypic data were collected from NDRI, India, on growth traits, viz., body weight at 6M (month), 12M, 18M and 24M, production traits like 305D (day) milk yield, lactation length (LL) and dry period (DP) and reproduction traits like age at first calving (AFC), calving interval (CI) and first service period (FSP). The biallelic genotypic data consisted of 49353 markers post-quality check. The heritability estimates were moderate to high, low to moderate, low for growth, production, reproduction traits, respectively. Important genomic regions explaining more than 0.5% of the total additive genetic variance explained by 30 adjacent SNPs were selected for further analysis of candidate genes. In this study, 105 genomic regions were associated with growth, 35 genomic regions with production and 42 window regions with reproduction traits. Different candidate genes were identified in these genomic regions, of which important are OSBPL8, NAP1L1 for growth, CNTNAP2 for production and ILDR2, TADA1 and POGK for reproduction traits.

Phenotypic and genetic characterization of unexplored, potential cattle population of Madhya Pradesh.

Bawri or Garri, a non-descript cattle population managed under an extensive system in Madhya Pradesh state of India, was identified and characterized both genetically and phenotypically to check whether or not it can be recognised as a breed. The cattle have white and gray colour and are medium sized with 122.5 ± 7.5cm and 109.45 ± 0.39cm height at withers in male and female, respectively. Double-digest restriction site associated DNA (ddRAD) sequencing was employed to identify ascertainment bias free SNPs representing the entire genome cost effectively; resulting in calling 1,156,650 high quality SNPs. Observed homozygosity was 0.76, indicating Bawri as a quite unique population. However, the inbreeding coefficient was 0.025, indicating lack of selection. SNPs found here can be used in GWAS and genetic evaluation programs. Considering the uniqueness of Bawri cattle, it can be registered as a breed for its better genetic management.

A Smart DNA Hydrogel Enables Synergistic Immunotherapy and Photodynamic Therapy of Melanoma.

Immunotherapy faces insufficient immune activation and limited immune effectiveness. Herein, we report a smart DNA hydrogel that enables the release of multivalent functional units at the tumor site to enhance the efficacy of immunotherapy. The smart DNA hydrogel was assembled from two types of ultra-long DNA chains synthesized via rolling circle amplification. One DNA chain contained immune adjuvant CpG oligonucleotides and polyaptamers for loading natural killer cell-derived exosomes; the other chain contained multivalent G-quadruplex for loading photodynamic agents. DNA chains formed DNA hydrogel through base-pairing. HhaI restriction endonuclease sites were designed between functional units. Upon stimuli in the tumor sites, the hydrogel was effectively cleaved by the released HhaI and disassembled into functional units. Natural killer cell-derived exosomes played an anti-tumor role, and the CpG oligonucleotide activated antigen-presenting cells to enhance the immunotherapy. Besides the tumor-killing effect of photodynamic therapy, the generated cellular debris acted as an immune antigen to further enhance the immunotherapeutic effect. In a mouse melanoma orthotopic model, the smart DNA hydrogel as a localized therapeutic agent, achieved a remarkable tumor suppression rate of 91.2 %. The smart DNA hydrogel exhibited enhanced efficacy of synergistic immunotherapy and photodynamic therapy, expanding the application of DNA materials in biomedicine.

Designing a T-cell epitope-based vaccine using in silico approaches against the Sal k 1 allergen of Salsola kali plant

Allergens originated from Salsola kali (Russian thistle) pollen grains are one of the most important sources of aeroallergens causing pollinosis in desert and semi-desert regions. T-cell epitope-based vaccines (TEV) are more effective among different therapeutic approaches developed to alleviate allergic diseases. The physicochemical properties, and B as well as T cell epitopes of Sal k 1 (a major allergen of S. kali) were predicted using immunoinformatic tools. A TEV was constructed using the linkers EAAAK, GPGPG and the most suitable CD4+ T cell epitopes. RS04 adjuvant was added as a TLR4 agonist to the amino (N) and carboxyl (C) terminus of the TEV protein. The secondary and tertiary structures, solubility, allergenicity, toxicity, stability, physicochemical properties, docking with immune receptors, BLASTp against the human and microbiota proteomes, and in silico cloning of the designed TEV were assessed using immunoinformatic analyses. Two CD4+ T cell epitopes of Sal k1 that had high affinity with different alleles of MHC-II were selected and used in the TEV. The molecular docking of the TEV with HLADRB1, and TLR4 showed TEV strong interactions and stable binding pose to these receptors. Moreover, the codon optimized TEV sequence was cloned between NcoI and XhoI restriction sites of pET-28a(+) expression plasmid. The designed TEV can be used as a promising candidate in allergen-specific immunotherapy against S. kali. Nonetheless, effectiveness of this vaccine should be validated through immunological bioassays.

Determination of ITS1 haplotypes of Fritillariae Cirrhosae Bulbus by amplicon sequencing

BackgroundFritillariae Cirrhosae Bulbus is an antitussive and expectorant Chinese medicinal material derived from the dried bulbs of six Fritillaria species. In the 2015 edition of the Chinese Pharmacopoeia, the polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) is the officially listed method for their authenfication. Specifically, the ~ 300-bp ITS1 amplicon of only Fritillariae Cirrhosae Bulbus but not other Fritillaria species can be cleaved into two smaller fragments with restriction enzyme SmaI. Considering repeated reported cases of incomplete digestion of ITS1 amplicon, this study aims to investigate the possibility of heterogeneous ITS1 sequences contained in the Fritillariae Cirrhosae Bulbus.MethodsIn this study, ITS1 amplicons of Fritillaria Cirrhosae Bulbus and four other Fritillaria species were sequenced on Illumina platform. We utilised high-throughout amplicon sequencing to determine ITS1 haplotypes and their frequencies in Fritillaria genomes.ResultsOur results showed that all six botanical sources of Fritillariae Cirrhosae Bulbus indeed possess ITS1 haplotypes with no SmaI restriction site, and the average percentages of ITS1 reads containing SmaI restriction site ranged from 63.60% to 91.81%.ConclusionOur findings suggest that the incomplete digestion in PCR–RFLP analysis of Fritillariae Cirrhosae Bulbus is caused by the presence of ITS1 haplotypes without SmaI restriction site due to intragenomic heterogeneity.

A rapid and scalable approach to build synthetic repetitive hormone-responsive promoters.

Advancement of DNA-synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high-complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single-tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single-tube digestion-ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone-responsive, synthetic, repetitive proximal promoters were generated and tested in planta in the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene-responsive promoter 10x2EBS-S10 fused to the GUS reporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution.

Modeling of PCR-RFLP genotyping of cattle by polymorphic markers of iNOS gene

Relevance. The study of the Bos taurus iNOS gene polymorphism and its association with resistance to bovine leukemia, as well as with breeding value in terms of milk productivity, is a topical subject of genetic selection research.The purpose of this study was to identify and map polymorphic restriction sites in 4 SNP markers (AH13-1, AH13-2, AH13-3, AH13-4) of the Bos taurus iNOS gene, followed by PCR-RFLP profiling of the encountered genotypes and modeling of the method of gene testing of cattle by the listed polymorphic markers of the analyzed locus.Methods. An effective tool for visualizing the result of computer simulation of the experiment, which contributed to predicting the applicability of the developed method of gene testing, was in silico modeling of the generated PCR-RFLP profiles of the encountered genotypes of polymorphic markers of the Bos taurus iNOS gene. At the same time, the very result of mapping the identified polymorphic restriction sites and subsequent profiling of the encountered genotypes indicated the technical feasibility of detecting polymorphic positions of 4 SNP markers of the Bos taurus iNOS gene by PCR-RFLP analysis. This, in turn, initiated the modeling of the method of gene testing of cattle by the listed polymorphic markers with the provision of successive stages of DNA isolation from whole preserved blood of cattle, PCR with oligonucleotide primers iNOS-F and iNOS-R, RFLP with selected restriction endonucleases (HinfI, Sau96I (AspS9I isoschizomer), HpyAV and MluCI (Sse9I isoschizomer), as well as performing gel electrophoresis detection with visualization of the obtained electrophoregrams in a UV transilluminator.

Identification of Chromoblastomycosis and Phaeohyphomycosis Agents through ITS-RFLP.

Chromoblastomycosis (CBM) and phaeohyphomycosis (FEO) are infections caused by melanized filamentous fungal agents, primarily found in tropical and subtropical regions. Both infections pose significant challenges for the correct identification of the causative agent due to their morphological similarity, making conventional methods of morphological analysis highly subjective. Therefore, molecular techniques are necessary for the precise determination of these species. In this regard, this study aimed to contribute to a new methodology based on PCR-RFLP for the identification of agents causing CBM and FEO. Sequences from the Internal Transcribed Spacer (ITS) region were used to identify potential restriction enzyme sites in silico, followed by in vitro validation using the selected restriction enzymes. The obtained results were compared with species identification through morphological analyses and sequencing. The results demonstrated that the PCR-RFLP applied in this study accurately identified two major agents of chromoblastomycosis, Fonsecaea pedrosoi and Fonsecaea monophora, as well as Cladophialophora bantiana and Exophiala dermatitidis, both causative agents of phaeohyphomycosis. In this context, the proposed assay can complement current methods for identifying these species, aiding in diagnosis, and contributing to the proper management of these infections.

BRCA mutations: screening for germ-line founder mutations among early-onset Syrian breast cancer patients

BackgroundBreast cancer (BC) is the most common female cancers in many countries including Syria. Familial breast cancer or previous family cancer history are considered significant risk factors. Therefore, detecting the prevalence and founder mutations in the population facilitates genetic counselling, risk assessment and the development of a cost-effective screening strategy. In this study, we investigated the three germ-line founder mutations in the BRCA1/2 genes: [NM_007294.4 (BRCA1):c.68_69del (p.Glu23fs), NM_007294.4 (BRCA1):c.5266dup (p.Gln1756fs) and NM_000059.4 (BRCA2):c.5946del (p.Ser1982fs)], to examine their incidence and frequency in early-onset breast cancer cases and determine if they are connected to familial breast cancer. One hundred early diagnosed BC females (≤ 40 years old) with no other type of cancer were recruited. Genomic DNA was isolated from peripheral blood samples, and mutations were investigated using the Amplification-Created Restriction Site (ACRS) method.ResultsThe family history of cancer was observed in 61% of the cases, of which 35% were breast cancer; however, none of the screened mutations were detected among BC patients.ConclusionsThe investigated germ-line mutations were not common among Syrian female patients with early-onset BC and were not associated with familial BC. Other mutations in the BRCA1/2 genes or other genes may have a contributing role. Future studies and the need to launch nationwide mutation screening tests for BRCA 1/BRCA2 in the Syrian population are recommended.

PUdOs: Concise Plasmids for Bacterial and Mammalian Cells.

The plasmids from the Université d'Ottawa (pUdOs) are 28 small plasmids each comprising one of four origins of replication and one of seven selection markers, which together afford flexible use in Escherichia coli and several related gram-negative bacteria. The promoterless multicloning site is insulated from upstream spurious promoters by strong transcription terminators and contains type IIP or IIS restriction sites for conventional or Golden Gate cloning. pUdOs can be converted into efficient expression vectors through the insertion of a promoter at the user's discretion. For example, we demonstrate the utility of pUdOs as the backbone for an improved version of a Type III Secretion System reporter in Shigella. In addition, we derive a series of pUdO-based mammalian expression vectors, affording distinct levels of expression and transfection efficiency comparable to commonly used mammalian expression plasmids. Thus, pUdOs could advantageously replace traditional plasmids in a wide variety of cell types and applications.

Thermodynamic Allosteric Switch-Actuated 3D DNA Nanomachine for Ultrasensitive Electrochemical/Fluorescent Dual-Mode Biosensing of a Transcription Factor.

Herein, we reported an innovative thermodynamic allosteric switch-actuated 3D DNA nanomachine for selective, sensitive, and accurate electrochemical (EC)/fluorescent (FL) dual-mode biosensing of a microphthalmia-associated transcription factor (MITF). The thermodynamic allosteric switch was ingeniously customized as a hairpin probe (HP) that was in dynamic equilibrium but rapidly interconverting conformations. At the "inactive state", the MITF-binding region and the switch part were "sequestered". Upon the introduction of MITF, an MITF-HP complex promptly formed, and the equilibrium of HP thermodynamically inclined from the "inactive state" toward the "active state" conformation. Immediately, the exposed switch on HP effectively actuated the 3D DNA nanomachine and synchronously produced the restriction site for Nb.BbvCI nicking endonuclease. After the autonomous conveying of the 3D DNA nanomachine by means of the high-efficiency circularly nicking endonuclease signal amplification (NESA), not only was MB-S1 in the supernatant used for FL measurements but also MB-SP/MNs/S2 in the precipitate was adapted for EC analysis, significantly improving the utilization of output products derived from the 3D DNA nanomachine. Accordingly, benefiting from the efficient DNA nanomachine signal amplification manner and the self-calibration function of a dual-mode bioassay, the constructed biosensor exhibits superior sensitivity and accuracy for MITF determination.

Modification 1T-MoS2 with surface ions towards electrocatalytic hydrogen evolution reaction

Molybdenum disulfide (MoS2) is considered as a thriving and cost-efficient electrocatalyst for hydrogen evolution reaction (HER). Unfortunately, the semiconductor phase of 2H-MoS2 itself has a conservative electronic intrinsic transport rate and the restricted active sites to edge regions, leading to unsatisfactory electrocatalytic performance of HER. Here, we have fabricated highly conductive metal phase 1T-MoS2 nanosheets by a hydrothermal method and introduced a series of transition metal ions on their surfaces by an in-situ reduction strategy to achieve MoS2 surface modification. It was found that the metals Ni and Co not only enhanced the intrinsic conductivity of 1T-MoS2, but also significantly increased the number of active sites in the catalyst, which resulted in the catalyst exhibiting excellent HER activity. Ni@1T-MoS2 exhibits fast reaction kinetics (86 mV·dec-1), good mechanical stability (~28 h) and low energy consumption in 1.0 M KOH medium, as well as an over-potential of only 187 mV at 50 mA·cm-2.

Assessing Environmental Management Plan Implementation in Water Supply Construction Projects: Key Performance Indicators

Assessing the implementation of environmental management plans (EMPs) in construction projects is crucial for meeting environmental sustainability goals and reducing potential adverse impacts. By using performance indicators (PIs), stakeholders can objectively measure the performance of EMP implementation, identifying areas of success and areas that may require improvement. Therefore, this study aims to examine the PIs for assessing EMP implementation in water supply construction projects, using Saudi Arabia as a case study. Data from semi-structured interviews and a systematic literature review were used to develop a potential list of PIs. Then, the PIs were used to create a survey and distributed to industry professionals. Data from 112 respondents were analyzed using mean ranking analysis, the normalization method, exploratory factor analysis (EFA), and fuzzy synthetic evaluation (FSE). Eighteen critical PIs for assessing EMP implementation in water supply construction projects were identified, including public safety, road safety hazards, construction waste, clogged drainage, irregular flooding, the spilling of chemical substances, slope failures, soil erosion, landslide occurrence, increased schedule waste, changes in the color of bodies of water, oil/fuel spills, restricted site accessibility, the smell of run-off water, traffic accidents on construction sites, the spread of disease, changes in the color of run-off water, and overflowing silt traps. The EFA revealed that PIs can be grouped into three underlying constructs: fluid-related indicators, health and safety-related indicators, and site environment-related indicators. The FSE results confirmed that all PIs are between moderately critical to critical. This study’s significance lies in its examination of PIs that aim to improve the environmental performance of water supply construction projects. Understanding which indicators are most effective allows for targeted improvements, helping to minimize negative environmental impacts and ensuring sustainable practices. Finally, this study is a pioneer in examining the critical PIs for assessing EMP implementation in water supply construction projects.

Analysis of ddRAD-seq data provides new insights into the genomic structure and patterns of diversity in Italian donkey populations.

With more than 150 recognized breeds, donkeys assume relevant economic importance, especially in developing countries. Even if the estimated number of heads worldwide is 53M, this species received less attention than other livestock species. Italy has traditionally been considered one of the cradles of European donkey breeding, and despite a considerable loss of biodiversity, today still counts nine autochthonous populations. A total of 220 animals belonging to 9 different populations were genotyped using the double-digest restriction site associated DNA (ddRAD) sequencing to investigate the pattern of diversity using a multi-technique approach. A total of 418,602,730 reads were generated and successfully demultiplexed to obtain a medium-density SNP genotypes panel with about 27K markers. The diversity indices showed moderate levels of variability. The genetic distances and relationships, largely agree with the breeding history of the donkey populations under investigation. The results highlighted the separation of populations based on their genetic origin or geographical proximity between breeding areas, showed low to moderate levels of admixture, and indicated a clear genetic difference in some cases. For some breeds, the results also validate the success of proper management conservation plans. Identified ROH islands, mapped within genomic regions related to immune response and local adaptation, are consistent with the characteristics of the species known for its rusticity and adaptability. This study is the first exhaustive genome-wide analysis of the diversity of Italian donkey populations. The results emphasized the high informativeness of genome-wide markers retrieved through the ddRAD approach. The findings take on great significance in designing and implementing conservation strategies. Standardized genotype arrays for donkey species would make it possible to combine worldwide datasets to provide further insights on the evolution of the genomic structure and origin of this important genetic resource.

Primer design and restriction site analysis targeting POU domain class 1 transcription factor 1 gene of Friesian Holstein in silico

This study was conducted to design primer set and to map restriction site targeting synonymous mutation c.297A>G of the POU domain class 1 transcription factor 1 gene (POU1F1) of Friesian Holstein in silico. Primer set was designed to target exon 3 of the POU1F1 based on nucleotide sequence at GenBank database with accession number NC_037328.1 using primer3. Simulations of amplification targeting POU1F1 fragment and restriction fragment length polymorphism (RFLP) were conducted in silico using serial cloner version 2.6 and NEBcutter version 3.0, respectively. This study used a primer pair (5′-CCT TTT AGA ACT GAG ACT GGC TG-3′ and 5′-CCC ACA GCT GTT AAC AAG CA-3′) that produce 356 bp of estimated product size. Moreover, a synonymous mutation, c.297A>G of the POU1F1, could be detected using BsII restriction enzyme in silico. The BsII did not have restriction site for AA genotype. On the other hand, it could cut the PCR product size into two fragments (167 and 189 bp) for GG genotype. It can be concluded that in silico analysis successfully amplified target region using primer designed in this study and RFLP simulation using BsII could detect synonymous mutation c.297A>G of the POU1F1. Further in vitro study should be conducted to identify c.297A>G in Friesian Holstein population.

Synonymous variant of TLR7 at restriction site rs864058 identified in Covid 19 Pakistani patients.

TLR7, the receptor accountable for immune response to RNA viruses, has been studied extensively to identify its variants related to the severity of Covid-19 in different populations worldwide. However, the genotype of Pakistani population is still unknown. This study aimed to determine the TLR7 genotypes and their relation with severity in our population. This cross sectional study collected data on 151 Covid-19 positive patients (aged 18-80 years), from June 2022 to May 2023, after an informed consent, from Ziauddin University and Hospital. Prior to that approval from ethics review committee was taken. The demographic variables and comorbidities were recorded along with health status till LAMA (Leave Against Medical Advise), recovery or death. The DNA was extracted from collected blood samples, PCR and Sanger sequencing was done for identification of TLR7 variants. SPSS was used for data analyses and Chi-Square for categorical variables. P-values of <0.05 was considered significant. Out of 151 patients' sequencing was done for 59 samples. The restriction site, rs864058 of TLR7 gene, identified G/A and G/G variants. This missense variant of TLR7 identified at rs864058 of TLR7 gene, has not been previously reported in population control databases. The genotype G/G was main variant of 49 (83%) patients, whereas, G/A was found in 10 (17%). Majority, 25 (51%) of patients with mild covid-19 had GG genotype but results were not significant (P=0.684). Among female patients the main genotype was GA 8 (80%) while male had G/G 29 (59.2%) with significant results (P=0.024). Since G/G genotype was the major genotype, high percentage was found in hypertensives [20 (40.8%)], Diabetics [13 (26.5%)], depression [24 (49%)] and pneumonia patients [20 (40.8%)]. However, significant association (P=0.023) was only found with pneumonia. Males, in majority had severe [17 (68%)] infection and death [40 (26.4%)], whereas, females had mild [14 (25%)] with [12 (7.9%)] deaths. A variant rs864058 "G/A" of TLR7, in relation to covid-19 were found in our population. Males were found more at risk of morbidity and mortality due to covid-19. Larger studies are required to further confirm these results.