Restriction Site Research Articles

A computational approach to developing a multi-epitope vaccine for combating Pseudomonas aeruginosa-induced pneumonia and sepsis.

Pseudomonas aeruginosa is a complex nosocomial infectious agent responsible for numerous illnesses, with its growing resistance variations complicating treatment development. Studies have emphasized the importance of virulence factors OprE and OprF in pathogenesis, highlighting their potential as vaccine candidates. In this study, B-cell, MHC-I, and MHC-II epitopes were identified, and molecular linkers were active to join these epitopes with an appropriate adjuvant to construct a vaccine. Computational tools were employed to forecast the tertiary framework, characteristics, and also to confirm the vaccine's composition. The potency was weighed through population coverage analysis and immune simulation. This project aims to create a multi-epitope vaccine to reduce P. aeruginosa-related illness and mortality using immunoinformatics resources. The ultimate complex has been determined to be stable, soluble, antigenic, and non-allergenic upon inspection of its physicochemical and immunological properties. Additionally, the protein exhibited acidic and hydrophilic characteristics. The Ramachandran plot, ProSA-web, ERRAT, and Verify3D were employed to ensure the final model's authenticity once the protein's three-dimensional structure had been established and refined. The vaccine model showed a significant binding score and stability when interacting with MHC receptors. Population coverage analysis indicated a global coverage rate of 83.40%, with the USA having the highest coverage rate, exceeding 90%. Moreover, the vaccine sequence underwent codon optimization before being cloned into the Escherichia coli plasmid vector pET-28a (+) at the EcoRI and EcoRV restriction sites. Our research has developed a vaccine against P. aeruginosa that has strong binding affinity and worldwide coverage, offering an acceptable way to mitigate nosocomial infections.

Methodological approaches to gene identification of tea raw materials and raw material composition of tea-based soft drinks

Tea or tea shrub is a plant of the Camellia sinensis species, the leaves of which, previously prepared in a special way, are the traditional raw material for the production of tea products. Varietal gene identification of tea allows us to increase the level of assessment of the authenticity of tea raw materials and tea products. It is predominantly based on DNA technologies for the detection and interpretation of SNP markers (Single Nucleotide Polymorphism), represented by a wide arsenal of both expensive high-tech methods and publicly available laboratory approaches. Species gene identification of the raw material composition of tea-based soft drinks is an equally important area of research due to the risk of falsification of this type of product. The purpose of this study was to find methodological approaches to the varietal gene identification of tea raw materials and to the species gene identification of the raw material composition of tea-based soft drinks. As a result of a bioinformatics study to identify polymorphic restriction sites in the nucleotide sequences of Camellia sinensis genome loci, diagnostically significant restriction enzymes were selected that were capable of detecting SNPs and identifying tea genotypes using the analyzed markers. At the same time, 16 loci had potential for practical application, of which 11 belonged to the group of the most informative SNP markers. A post-analytical assessment of tea varieties was carried out with them regarding their genotypic affiliation and identifiability as part of solving the first task of the study. To achieve the second task, a molecular genetic approach to the species identification of the raw composition of soft drinks based on green tea was tested. The study included the analysis of experimental drinks (with natural flavoring “Lemon” and synthetic flavoring “Peach 716”), as well as commercial concentrates “TIAKVA” (based on extracts from the coarse stems of green or black tea). The methods used in the work were PCR (Polymerase Chain Reaction), RFLP (Restriction Fragment Length Polymorphism) and direct sequencing of the amplified chloroplast DNA locus. The combination of two methods (PCR and sequencing) showed its effectiveness in establishing the belonging of the analyzed nucleic acid samples to the Camellia sinensis species, the raw material base of the studied drinks and concentrates. However, to unlock the authentication potential of PCR with primers #1 and #2 combined with RFLP analysis, it will be necessary to select diagnostically significant restriction enzymes suitable for generating species-specific combinations of PCR-RFLP profiles of marker sequence.

Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method.

Seamless site-directed mutagenesis is an important technique for studying protein functions, tuning enzyme catalytic activities and modifying genetic elements in multiple rounds because it can insert, delete or substitute nucleotides, DNA segments or even entire genes at the target site without introducing any unwanted change. To facilitate seamless site-directed mutagenesis in large plasmids and bacterial artificial chromosomes (BACs) with repetitive sequences, we recently developed the RedEx strategy. Compared with previous methods, our approach achieves the recovery of correct recombinants with high accuracy by circumventing unwanted recombination between repetitive sequences. RedEx readily yields more than 80% accuracy in seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters, which are among the most difficult targets due to the large number of repetitive DNA sequences in modules encoding almost identical enzymes. Here we present the RedEx method by describing in detail the seamless site-directed mutagenesis in a BAC vector. Overall, the process includes three parts: (1) insertion of the RedEx cassette containing the desired mutation together with selection-counterselection markers flanked by unique restriction sites and 20-bp overlapping sequences into the target site by recombineering, (2) removal of the selection-counterselection markers in the BAC by restriction digestion and (3) circularization of the linear BAC by exonuclease-mediated in vitro DNA annealing. This protocol can be performed within 3 weeks and will enable researchers with DNA cloning experience to master seamless site-directed mutagenesis to accelerate their research.

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 K2P13.1(THIK-1) channel reveals a novel hydrophilic pore restriction and lipid cofactor site.

The halothane-inhibited K2P leak potassium channel K2P13.1 (THIK-1)1-3 is found in diverse cells1,4 including neurons1,5 and microglia6-8 where it affects surveillance6, synaptic pruning7, phagocytosis7, and inflammasome-mediated interleukin-1β release6,8,9. As with many K2Ps1,5,10-14 and other voltage-gated ion channel (VGIC) superfamily members3,15,16, polyunsaturated fatty acid (PUFA) lipids modulate K2P13.1 (THIK-1)1,5,14,17 via a poorly understood mechanism. Here, we present cryo-electronmicroscopy (cryo-EM) structures of human K2P13.1 (THIK-1) and mutants in lipid nanodiscs and detergent. These reveal that, unlike other K2Ps13,18-24, K2P13.1 (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, S136P25, 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 K2P modulator pocket for small molecule agonists18,26,27. This overlap, together with the effects of mutation on K2P13.1 (THIK-1) PUFA responses, indicates that the PUFA site lipids are K2P13.1 (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 K2P13.1 (THIK-1) function, highlight the importance of the P1/M4 interface in control of K2Ps by both natural and synthetic agents, and should aid development of THIK subfamily modulators for diseases such as neuroinflammation6,32 and autism6.

Designing and development of efficient multi-epitope-based peptide vaccine candidate against emerging avian rotavirus strains: A vaccinomic approach

BackgroundEnteric avian rotavirus (ARV) is the etiological agent of several health problems that pose a global threat to commercial chickens. Therefore, to avoid these widespread epidemics and high mortality rates, only vaccine and strict biosecurity are required. MethodThe present study employs computational techniques to design a unique multi-epitope-based vaccine candidate that successfully activates immune cells against the ARV by combining adjuvant, linker, and B and T-cell epitopes. Starting, homologous sequences in the various ARV serotypes were revealed in the NCBI BLAST database, and then the two surface proteins (VP4 and VP7) of the ARV were retrieved from the UniprotKB database. The Clustal Omega server was then used to identify the conserved regions among the homologous sequences, and the B and T-cell epitopes were predicted using IEDB servers. Then, superior epitopes—2 MHC-1 epitopes, 2 MHC-2 epitopes, and 3B-cell epitopes—were combined with various adjuvants to create a total of four unique vaccine candidates. Afterward, the designed vaccine candidates underwent computational validation to assess their antigenicity, allergenicity, and stability. The vaccine candidate (V2) that demonstrated non-antigenicity, a high VaxiJen score, and non-allergenicity was ultimately chosen for molecular docking and dynamic simulation. ResultsAlthough the V2 and V4 vaccine candidates were highly immunogenic, V2 had a higher solubility rate. The predicted values of the aliphatic index and GRAVY value were 30.4 and 0.417, respectively. In terms of binding energy, V2 outperformed V4. Being successfully docked with TLRs, V2 was praised as the finest. After adaptation, the sequence’s 50.73 % GC content outside of the BglII or ApaI restriction sites indicated that it was equivalently safe to clone. The chosen sequence was then inserted into the pET28a(+) vector within the BglII and ApaI restriction sites. This resulted in a final clone that was 4914 base pairs long, with the inserted sequence accounting for 478 bp and the vector accounting for the remainder. ConclusionsThe immune-mediated simulation results for the selected vaccine construct showed significant response; thus, the study confirmed that the selected V2 vaccine candidate could enhance the immune response against ARV.

Analysis of CYP1B1 Polymorphisms in Lung Cancer Patients Using Novel, Quick and Easy Methods Based on CAPS and ACRS-PCR Techniques.

Within the sequence of the CYP1B1 gene, more than 50 polymorphisms, resulting from single-nucleotide polymorphisms (SNPs), have been described. Some of them play an important role as specific genetic markers in the process of carcinogenesis and for therapeutic purposes. In this publication, we present methods we have developed that enable the specific and unambiguous identification of four polymorphisms that result in amino acid changes: c. 142C > G, c. 355G > T, c. 1294C > G, and c. 1358A > G. Our studies are based on cleaved amplified polymorphic sequences (CAPSs) and artificially created restriction site (ACRS) PCR techniques; therefore, they require only basic laboratory equipment and low financial outlays. Utilizing the described methods allows for the reduction of research time and cost, and the minimization of errors. Their effectiveness and efficiency depend on the careful design of appropriate primers and the precise selection of suitable restriction enzymes. As a result, further confirmation by sequencing is not necessary. Using the developed method, we examined 63 patients diagnosed with lung cancer and observed a 1.5 to 2.1 times higher frequency of the analyzed single-nucleotide polymorphisms compared to the frequency in the European population.

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.

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.