Restriction Site Research Articles

Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library’s breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

Changes in the users of the social‐ecological system around a reciprocal human‐dolphin relationship

Abstract In contrast to many contemporary negative human‐nature relationships, Indigenous Peoples and Local Communities have stewarded nature through cultural practices that include reciprocal contributions for both humans and nature. A rare example is the century‐old artisanal fishery in which net‐casting fishers and wild dolphins benefit by working together, but little is known about the persistence of the social‐ecological system formed around this cultural practice. Here, we frame the human‐dolphin cooperative fishery in southern Brazil as a social‐ecological system based on secondary data from the scientific and grey literature. To investigate the dynamics of this system, we survey the local and traditional ecological knowledge and examine potential changes in its main component—the artisanal fishers—over time and space. Over 16 years, we conducted four interview campaigns with 188 fishers in fishing sites that are more open (accessible) or closed (restricted) to external influence. We investigated their experience, engagement and economic dependence on dolphin‐assisted fishing, as well as the learning processes and transmission of the traditional knowledge required to cooperate with dolphins. Our qualitative data suggest that fishers using accessible and restricted fishing sites have equivalent fishing experience, but those in more restrictive sites tend to be more economically dependent on dolphins, relying on them for fishing year‐round. The traditional knowledge on how to cooperate with dolphins is mostly acquired via social learning, with a tendency for vertical learning to be frequent among fishers using sites more restrictive for outsiders. Experience, economic dependence and reliance on vertical learning seem to decrease recently, especially in the accessible site. Our quantitative analyses, however, suggest that some of these fluctuations were not significant. Our study outlines the key components of this social‐ecological system and identifies changes in the attributes of a main component, the users. These changes, when coupled with changes in other components such as governance and resource units (fish and dolphins), can have implications for the persistence of this cultural practice and the livelihoods of Local Communities. We suggest that continuous monitoring of this system can help to safeguard the reciprocal contributions of this human‐nature relationship in years to come. Read the free Plain Language Summary for this article on the Journal blog.

Structure of the human K 2P 13.1(THIK-1) channel reveals a novel hydrophilic pore restriction and lipid cofactor site.

The halothane-inhibited K 2P leak potassium channel K 2P 13.1 (THIK-1) 1-3 is found in diverse cells 1,4 including neurons 1,5 and microglia 6-8 where it affects surveillance 6 , synaptic pruning 7 , phagocytosis 7 , and inflammasome-mediated interleukin-1β release 6,8,9 . As with many K 2P s 1,5,10-14 and other voltage-gated ion channel (VGIC) superfamily members 3,15,16 , polyunsaturated fatty acid (PUFA) lipids modulate K 2P 13.1 (THIK-1) 1,5,14,17 via a poorly understood mechanism. Here, we present cryo-electronmicroscopy (cryo-EM) structures of human K 2P 13.1 (THIK-1) and mutants in lipid nanodiscs and detergent. These reveal that, unlike other K 2P s 13,18-24 , K 2P 13.1 (THIK-1) has a two-chamber aqueous inner cavity obstructed by a M4 transmembrane helix tyrosine (Tyr273, the flow restrictor). This hydrophilic barrier can be opened by an activatory mutation, S136P 25 , at natural break in the M2 transmembrane helix and by intrinsic channel dynamics. The structures also reveal a buried lipid in the P1/M4 intersubunit interface at a location, the PUFA site, that coincides with the TREK subfamily K 2P modulator pocket for small molecule agonists 18,26,27 . This overlap, together with the effects of mutation on K 2P 13.1 (THIK-1) PUFA responses, indicates that the PUFA site lipids are K 2P 13.1 (THIK-1) cofactors. Comparison with the PUFA-responsive VGIC Kv7.1 (KCNQ1) 28-31 reveals a shared role for the equivalent pore domain intersubunit interface in lipid modulation, providing a framework for dissecting the effects of PUFAs on the VGIC superfamily. Our findings reveal the unique architecture underlying K 2P 13.1 (THIK-1) function, highlight the importance of the P1/M4 interface in control of K 2P s by both natural and synthetic agents, and should aid development of THIK subfamily modulators for diseases such as neuroinflammation 6,32 and autism 6 .

Small is beautiful? Making sense of ‘shrinking’ homes

Current land pressures in the world cities of the global North are encouraging a move towards denser urban living and the development of smaller homes than has been the case for many decades. While this appears environmentally beneficial when compared with the alternative of suburban sprawl, it comes at a cost: the number of extremely small homes appears to be increasing particularly rapidly, with less communal and public space available to those living in compact homes which offer little room for socialising, storing possessions or working from home. Drawing specifically on the experience of England and Wales, with a focus on the overheated property market in London, this commentary sets out an international agenda for the study of small homes, noting the growing evidence of the negative impact of dense urban living on mental and physical health, home-working and familial and intimate relations, as well as its failure to solve the crisis of affordability. The article suggests that rather than being a reasoned response to the housing and environmental crises, the phenomenon of ‘shrinking homes’ indicates the growing role of finance in the development of cities, suggestive of the way that developers are extracting maximum value from restricted urban sites in an era of planning deregulation. In conclusion, the commentary argues that urban scholarship needs to compile more evidence of space inequality in cities, pushing for policies designed to enforce minimal space standards while reducing the ability of the wealthy to construct very large homes.

Development and clinical evaluation of a real-time multiple cross displacement amplification assay for rapid and sensitive detection of Mycobacterium tuberculosis

Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the Mycobacterium tuberculosis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 min) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.

Molecular Identification of Morus ssp. in Duhok Using Nuclear ITS Region and Chloroplast Matk Gene

Since Mulberries (Morus)is a tree species with a considerable plant variety. Molecular techniques are methods used to distinguish between species accurately, easily and quickly. This study examines a Molecular method for distinguishing different Morus species in the Duhok - Kurdistan region/ Iraq. The method is based on the use of four techniques: matK gene, the ITS region, PCR-RFLP, and SRAP markers. Twelve Morus species have been selected for this study from different region of Duhok. The ITS region's PCR result was 700 bp, but the matK gene's PCR produce was 900 bp. The same restriction site was found for all utilized cultivars when the 700bp of ITS fragment was used for PCR-RFLP with two restriction enzymes, RsaI GT/AC and HaeIII GG/CC. This study also used six combinations of SRAP markers to aid in grouping and identifying genetic similarities. The results of PCR-RFLP demonstrated an insufficient link between Morus physical appearance and genetic traits, but differences across studied cultivars could be identified using SRAP markers. Furthermore, this study demonstrated the possibility of DNA barcoding Morus cultivars, as well as additional sequence analysis and the identification of probable SNP between cultivars.

A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction.

The construction of gene expression vectors is an important component of laboratory work in experimental biology. With technical advancements like Gibson Assembly, vector construction becomes relatively simple and efficient. However, when the full-length genome of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) cannot be easily amplified by a single polymerase chain reaction (PCR) from cDNA, or it is difficult to acquire a full-length gene expression vector by homologous recombination of multiple inserts in vitro, the current Gibson Assembly technique fails to achieve this goal. Consequently, we aimed to divide the PRRSV genome into several fragments and introduce appropriate restriction sites into the reverse primer for obtaining PCR-amplified fragments. After joining the previous DNA fragment into the vector by homologous recombination technology, the new vector acquired the restriction enzyme cleavage site. Thus, we can linearize the vector by using the newly added enzyme cleavage site and introduce the next DNA fragment downstream of the upstream DNA fragment. The introduced restriction enzyme cleavage site at the 3' end of the upstream DNA fragment will be eliminated, and a new cleavage site will be introduced into the 3' end of the downstream DNA fragment. In this way, we can join DNA fragments to the vector one by one. This method is applicable to successfully construct the PRRSV expression vector and is an effective method for assembling a large number of fragments into the expression vector.

Genomic evaluation of mud sunfish in the atlantic slope drainage

Mud Sunfish (Acantharchus pomotis, Baird), a cryptic species native to the Atlantic and Gulf slope drainages of North America, have experienced a documented reduction in their geographic range. Atlantic Slope populations of Mud Sunfish were evaluated to determine the population structure, assess genetic diversity, and to identify presence of evolutionary significant units (ESU) in order to develop fundamental understandings of its occurrence. Tissue samples were collected from across the Atlantic Slope for genomic comparison. Restriction site Associated DNA Sequencing (RAD-Seq) was performed on 95 individuals from 14 populations. Mantel test provided evidence of a correlation between genetic and geographical distances, suggesting a pattern of isolation-by-distance. Differences among populations of Mud Sunfish largely occur along a north-south latitudinal gradient. Bayesian clustering of putatively outlier SNP loci, suggests that regional adaptive pressure is occurring. Despite a large geographic distance and significant differences between the majority of the populations of Mud Sunfish, Bayesian clustering analysis suggests that two unresolved clades are persisting within the Atlantic Slope Drainage: a northern clade of Mud Sunfish ranging from New Jersey to North Carolina and a southern clade ranging from South Carolina to Florida. A population of Mud Sunfish in Florida appears distinct from all others in the Atlantic Coastal plain and likely represents a distinct management unit.

Construction of an Elastin-like Polypeptide Gene in a High Copy Number Plasmid Using a Modified Method of Recursive Directional Ligation

Elastin-like polypeptides (ELPs) are popular biomaterials due to their reversible, temperature-dependent phase separation and their tunability, which is achievable by evolving procedures in recombinant technology. In particular, recursive direction ligation by plasmid reconstruction (PRe-RDL) is the predominant cloning technique used to generate ELPs of varying lengths. Pre-RDL provides precise control over the number of (VPGXG)n repeat units in an ELP due to the selection of type IIS restriction enzyme (REs) sites in the reconstructed pET expression plasmid, which is a low-to-medium copy number plasmid. While Pre-RDL can be used to seamlessly repeat essentially any gene sequence and overcome limitations of previous cloning practices, we modified the Pre-RDL technique, where a high copy number plasmid (pBluescript II SK(+)—using a new library of type IIS REs) was used instead of a pET plasmid. The modified technique successfully produced a diblock ELP gene of 240 pentapeptide repeats from 30 pentapeptide “monomers” composed of alanine, tyrosine, and leucine X residues. This study found that the large, GC-rich ELP gene compromised plasmid yields in pBluescript II SK(+) and favored higher plasmid yields in the pET19b expression plasmid. Additionally, the BL21 E. coli strain expression consistently provided a higher transformation efficiency and higher plasmid yield than the high cloning efficiency strain TOP10 E. coli. We hypothesize that the plasmid/high GC gene ratio may play a significant role in these observations, and not the total plasmid size or the total plasmid GC content. While expression of the final gene resulted in a diblock ELP with a phase separation temperature of 34.5 °C, future work will need to investigate RDL techniques in additional plasmids to understand the primary driving factors for improving yields of plasmids with large ELP-encoding genes.

Analyzing genetic diversity in luffa and developing a Fusarium wilt-susceptible linked SNP marker through a single plant genome-wide association (sp-GWAS) study

BackgroundLuffa (Luffa spp.) is an economically important crop of the Cucurbitaceae family, commonly known as sponge gourd or vegetable gourd. It is an annual cross-pollinated crop primarily found in the subtropical and tropical regions of Asia, Australia, Africa, and the Americas. Luffa serves not only as a vegetable but also exhibits medicinal properties, including anti-inflammatory, antidiabetic, and anticancer effects. Moreover, the fiber derived from luffa finds extensive applications in various fields such as biotechnology and construction. However, luffa Fusarium wilt poses a severe threat to its production, and existing control methods have proven ineffective in terms of cost-effectiveness and environmental considerations. Therefore, there is an urgent need to develop luffa varieties resistant to Fusarium wilt. Single-plant GWAS (sp-GWAS) has been demonstrated as a promising tool for the rapid and efficient identification of quantitative trait loci (QTLs) associated with target traits, as well as closely linked molecular markers.ResultsIn this study, a collection of 97 individuals from 73 luffa accessions including two major luffa species underwent single-plant GWAS to investigate luffa Fusarium wilt resistance. Utilizing the double digest restriction site associated DNA (ddRAD) method, a total of 8,919 high-quality single nucleotide polymorphisms (SNPs) were identified. The analysis revealed the potential for Fusarium wilt resistance in accessions from both luffa species. There are 6 QTLs identified from 3 traits, including the area under the disease progress curve (AUDPC), a putative disease-resistant QTL, was identified on the second chromosome of luffa. Within the region of linkage disequilibrium, a candidate gene homologous to LOC111009722, which encodes peroxidase 40 and is associated with disease resistance in Cucumis melo, was identified. Furthermore, to validate the applicability of the marker associated with resistance from sp-GWAS, an additional set of 21 individual luffa plants were tested, exhibiting 93.75% accuracy in detecting susceptible of luffa species L. aegyptiaca Mill.ConclusionIn summary, these findings give a hint of genome position that may contribute to luffa wild resistance to Fusarium and can be utilized in the future luffa wilt resistant breeding programs aimed at developing wilt-resistant varieties by using the susceptible-linked SNP marker.

Ultrasound evaluation of deep fascia thickness: Reliability and association with clinically evaluated changes

BackgroundThere is growing evidence of fascial involvement in musculoskeletal pathologies; thus, measuring the fascia's properties is clinically essential. Objectives1) to evaluate intra- and inter-tester reliability of sonographic measurements of deep fascia thickness in healthy individuals; 2) to assess whether fascial thickness in areas of fascial movement restriction (fascial densification in Stecco's Fascial Manipulation method), differs from contralateral areas with normal fascia. MethodsPart 1 (reliability study): Fascial thickness was sonographically measured in 10 sites (5 on each side), twice by a single examiner to assess intra-rater reliability and once by a second examiner to evaluate inter-rater reliability.Part 2 (cross-sectional study): Unilateral fascial movement restrictions were detected in 5 bilateral sites by palpation. The fascial thickness on the ultrasound image was measured at the restriction site and its normal contralateral side by a blinded assessor. Results21 healthy individuals were evaluated in part 1, 15 healthy individuals in part 2. Intra-tester reliability (Interclass correlation – ICC) values ranged from 0.677 to 0.975; inter-tester ICC values ranged from 0.473 to 0.966. No significant differences in fascia thickness between the right and left body sides were observed in most sites. Significant differences were found between the fascial thickness of the site of fascial movement restrictions vs. normal counterpart. ConclusionsIntra- and inter-rater reliability of sonographic measuring fascial thickness was high, and fascial thickness of the site of fascial movement restrictions was greater than in the normal counterpart. Ultrasonography may be used as a reliable method to evaluate fascial alterations.

An artificial-restriction fragment length polymorphism (A-RFLP) method for genotyping intronic SNP rs7587633 C/T of ATG16L1

SNPs could either cause a disorder or directly alter the efficacy of a particular treatment and act as biological markers. The SNP rs7587633 C/T present in the intronic region of the ATG16L1 gene has been studied for its role in psoriasis vulgaris and Palmoplantar pustulosis. To genotype rs7587633 C/T using PCR-RFLP no restriction site is present for any of the restriction enzymes at the SNP position. To develop an artificial-RFLP method for genotyping rs7587633 C/T, the forward primer was designed in such a way that it resulted in the creation of an EcoRI restriction site in the amplified product which could further be digested with EcoRI to find the genotype of the individual. The newly developed A-RFLP method was applied to genotype the SNP rs7587633 C/T in DNA samples of 100 healthy control individuals. The allelic and genotypic frequencies of the SNPs were 0.80(C), 0.20(T) and 65%(CC), 31%(CT) and 4%(TT), respectively. In conclusion, we developed an A-RFLP method to genotype the SNP rs7587633 C/T which is not present in any of the natural restriction sites and this method could be applied to genotype this SNP in various populations/diseases to find its role.

Cloning Sheep Cytokin Genes and Preliminary Transfect to HT29 Cell Line

The study was carried out to clone sheep cytokine genes for protein expression in mammalian cell culture. Once expressed it is hypothesized that these proteins when added to a primary mast cell culture will allow the growth of these cells. Mast cells are used by sheep to combat gastro-intestinal nematode (GIN) by helping to establish the host immune response and to eliminate of GIN. Kit ligand and IL3 proteins are growth factors of mast cells that were derived from extraction of total RNA from lung and thymus tissues, respectively. The IL3 and kit ligand genes were cloned into bacterial expression pGEM – T easy vector to determine correct sequences before being sub-cloned into the mammalian expression pWPI vector. The Pac 1 enzyme was used to cut restriction sites of pGEM – T easy vector, pWPI vector, IL3 and kit ligand genes in cloning strategies. The study also established 24 different conditions for transient transfection IL3 and Kit ligand genes into HT29 cells and screen positive cells on the flow cytometry. Results showed that clones of IL3 and kit ligand with the correct sequences were created for using transient transfection. The transfection efficiency of IL3 and kit ligand DNA/ pWPI plasmid into HT29 cells were extremely low with approximately 9% for the best condition. This low transfection efficiency leads to shortage of IL3 and kit ligand for growing mast cells. Therefore, stable transfection will be required to have highest efficiencies that produce the large quantities of IL3 and kit ligand proteins required for culture of sheep mast cell lines.

Boosting Aluminum Storage in Highly Stable Covalent Organic Frameworks with Abundant Accessible Carbonyl Groups

AbstractAluminum batteries employing organic electrode materials present an appealing avenue for sustainable and large‐scale energy storage. Nevertheless, conventional organic materials encounter limitations due to their restricted active sites, known instability, and sluggish redox kinetics. In this study, a redox‐active covalent organic framework supported by CNT is reported, enriched with substantial C═O groups, as an advanced cathode material for Al‐organic batteries. Theoretical simulation and ex situ analysis unveil the pivotal roles of C═O groups in effectively storing AlCl2+. As a result, Al batteries with the organic cathode exhibit a specific capacity of 290 mAh g−1 at 0.2 A g−1 and outstanding rate performance. Furthermore, it retains a reversible capacity of 170 mAh g−1 even after 32 000 cycles at 10 A g−1 and attains an energy density of 389 Wh kg−1. The remarkable performance stems not only from the abundant C═O and C─N groups enabling the storage of multiple AlCl2+ by the favorable pseudocapacitive process, but also from the synergistic interplay between the robust COF network and the conductive CNT channels that significantly enhances structural stability and accelerates ion/electron diffusion. This work stands to inspire further research in the pursuit of stable organic cathodes, fostering designs with plentiful accessible redox‐active sites to boost energy storage capabilities.

دور تعدد الأشكال لجين APE1 Asp148Glu على خطر الإصابة بسرطان الدم النخاعي الحاد في المرضى العراقيين

ابيضاض الدم النخاعي الحاد هو أكثر أنواع امراض الدم الخبيثة شيوعًا عند البالغين. يعد انتشار الخلايا الطافرة الغير مسيطر عليها للأسلاف النخاعية الغير متمايزة سمة مميزة لهذا المرض. يمكن إصلاح تلف الحمض النووي عن طريق مجموعة متنوعة من الأنظمة الأنزيمية الموجودة في الخلايا حقيقية النواة. تتشكل الخلايا الطافرة عندما لا يمكن إصلاح تلف الحمض النووي بواسطة آليات إصلاح الخلية. إحدى آليات إصلاح الحمض النووي هي مسار إصلاح استئصال القاعدة ، التي تكون المسئولة عن تصحيح الأضرار الصغيرة الناتجة عن التلف ألتأكسدي أو والقلوية أو المثيلة. اشتملت الدراسة على 70 مريضًا مصابًا بسرطان الدم النخاعي الحاد - 37 أنثى و 33 ذكرًا - بالإضافة إلى 30 فردًا سليمًا كمجموعة سيطرة. تم استخدام مجموعة استخلاص الحمض النووي gSYNCTM لاستخراج الحمض النووي من عينات الدم الكاملة من مجموعات الدراسة. تم استخدام طريقة PCR-RFLP لتحديد تعدد الأشكال Asp148 Glu للجين APE1 في التحليل الجيني ، تبين أن الزيادة في النمط الجيني T/T وأليل T في تعدد الأشكال في كودون APE1 148 يلعب دورًا وقائيًا في مرض ابيضاض الدم النخاعي الحادAML ، وأن الزيادة في النمط الجيني G / G يمكن أن تترافق مع خطر الإصابة بسرطان الدم الحاد. . أظهرت النتائج أن النمط الجيني المتغاير لجين APE1 ، Asp 148 Glu (T / G) وأليل G كانت أعلى بشكل ملحوظ (P <0.05) في مرضى AML مقارنة بمجموعة السيطرة . في تسلسل المنطقة التي تم دراستها ، وجد أن هناك موقعًا للتغاير يقع بين GATC و GAGC ، حيث يمثل التغيير في النيوكليوتيدات T إلى G موقع تقييد الإنزيم القاطع. حيث ارتبط النمط الجيني متعدد الأشكال 148 Glu> Asp من جين APE1 مع تطور AML في المرضى العراقيين. أثبتت الدراسة أن حاملي النمط الجيني T / T لديهم مخاطر أقل لتطور مرض ابيضاض الدم النخاعي الحادAML ، في حين أن حاملي الأليلات G لديهم مخاطر متزايدة.

Novel photoresponsive molecularly imprinted polymers based on etched silicon core with enabling enhanced selectivity and sensitivity for the detection of sulfamethazine

AbstractSulfamethymidine, a commonly employed sulfonamide, is introduced into the human body through the food chain, posing a threat to human health. Consequently, the development of a rapid and efficient detection technique is imperative. In this investigation, a photoresponsive sulfamethyridiazine‐imprinted polymer was synthesized to etch a silicon core, thereby effectively addressing the limitations encountered with conventional molecularly imprinted polymers including diminished binding capacity, restricted site accessibility and sluggish binding kinetics. The photosensitive monomer utilized in the molecularly imprinted polymers is 5[(4 (methylacryloxy) phenyl) diazene] isophthalic acid, which exhibits a stimulating reaction mechanism. The N=N bond undergoes photoisomerization, transitioning between trans and cis configurations. The adsorption experiment provides additional evidence that the hollow molecularly imprinted polymers exhibit a higher adsorption capacity, achieving a value of 0.192 mmol L−1. The experiments conducted to assess selectivity and repeatability confirm that the photoresponsive molecularly imprinted polymers exhibit a high level of selectivity and favorable repeatability. Following four cycles, the adsorption rate remains consistently at 64.3%. Additionally, the recoveries of the actual samples ranged from 95.6% to 99.7%. This finding presents a novel approach to detecting the concentration of trace pollutants in intricate substrates. © 2024 Society of Industrial Chemistry.

Construction of SARS-CoV-2 full - length S gene with point mutations relevant for COVID-19 vaccine strain candidate

To overcome the pandemic of COVID-19, prevent the disease or its post-conditions effectively, a vaccine with the updates of viral strains is needed. The full S gene encoding the glycoprotein spike of SARS-CoV-2 was recommended for vaccine production by the World Health Organization. To create a vaccine strain candidate for a COVID-19, we have cloned the full-length S gene of the SARS-CoV 2 containing mutations that improve thermal stability of the protein (HexaPro) and three strain-specific mutations of D614G, L452R and E484Q that are present in rapidly and widely circulating variants. The fragments of the full-length S gene were first amplified from the two commercial plasmids, in which one plasmid contains the HexaPro mutations and the other was used to amplify the 3’ fragment of the gene, these fragments were then assembled. Strain-specific mutations were generated using primers with mutations. The measles (MeV) overlaping sequences and AscI (5’- GCGCGC-3’) restriction sites were also added to the two ends of the S gene. The full length of the cloned S gene with mutations and restriction sites was confirmed by sequencing and is ready to be inserted into the measles backbone for creating a chimeric genome of MeV_SARS CoV-2.

Sequencing vs. amplification for the estimation of allele dosages in sugarcane (Saccharum spp.)

AbstractPremiseDetecting single‐nucleotide polymorphisms (SNPs) in a cost‐effective way is fundamental in any plant breeding pipeline. Here, we compare three genotyping techniques for their ability to reproduce the allele dosage of SNPs of interest in sugarcane (Saccharum spp.).MethodsTo identify a reproducible technique to estimate allele dosage for the validation of SNP markers, the correlation between Flex‐Seq, kompetitive allele‐specific PCR (KASP), and genotyping‐by‐sequencing and restriction site–associated DNA sequencing (GBS+RADseq) was determined for a set of 76 SNPs. To find alternative methodologies for allele dosage estimation, the KASP and Flex‐Seq techniques were compared for the same set of SNPs. For the three techniques, a population of 53 genotypes from the diverse sugarcane panel of the Centro de Investigación de la Caña de Azúcar (Cenicaña), Colombia, was selected.ResultsThe average Pearson correlation coefficients between GBS+RADseq and Flex‐Seq, GBS+RADseq and KASP, and Flex‐Seq and KASP were 0.62 ± 0.27, 0.38 ± 0.27, and 0.38 ± 0.30, respectively.DiscussionFlex‐Seq reproduced the allele dosages determined using GBS+RADseq with good levels of precision because of its depth of sequencing and ability to target specific positions in the genome. Additionally, Flex‐Seq outperformed KASP by allowing the conversion of a higher number of SNPs and a more accurate estimation of the allele dosage. Flex‐Seq has therefore become the genotyping methodology of choice for marker validation at Cenicaña.

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.