Molecular characterization, expression and immune functional analysis of cystatin 10 in turbot.

BACKGROUND: Cystatin is a protease inhibitor that also regulates genes expression linked to inflammation and plays a role in defense and regulation. METHODS AND RESULTS: Cystatin 10 (Smcys10) was cloned from Scophthalmus maximus and encodes a 145 amino acid polypeptide. The results of qRT-PCR showed that Smcys10 exhibited tissue-specific expression patterns, and its expression was significantly higher in the skin than in other tissues. The expression level of Smcys10 was significantly different in the skin, gill, head kidney, spleen and macrophages after Vibrio anguillarum infection, indicating that Smcys10 may play an important role in resistance to V. anguillarum infection. The recombinant Smcys10 protein showed binding and agglutinating activity in a Ca2+-dependent manner against bacteria. rSmcys10 treatment upregulated the expression of IL-10, TNF-α and TGF-ß in macrophages of turbot and hindered the release of lactate dehydrogenase (LDH) from macrophages after V. anguillarum infection, which confirmed that rSmcys10 reduced the damage to macrophages by V. anguillarum. The NF-κB pathway was suppressed by Smcys10, as demonstrated by dual-luciferase analysis. CONCLUSIONS: These results indicated that Smcys10 is involved in the host antibacterial immune response.

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.

Utilizing immobilized recombinant serine alkaline protease from Bacillus safensis lab418 in wound healing: Gene cloning, heterologous expression, optimization, and characterization.

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.

Functional Characterization of F3H Gene and Optimization of Dihydrokaempferol Biosynthesis in Saccharomyces cerevisiae.

The 1092 bp F3H gene from Trapa bispinosa Roxb., which was named TbF3H, was cloned and it encodes 363 amino acids. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbF3H with flavanone 3-hydroxylase from other plants. A functional analysis showed that TbF3H of Trapa bispinosa Roxb. encoded a functional flavanone 3-hydroxylase; it catalyzed the formation of dihydrokaempferol (DHK) from naringenin in S. cerevisiae. The promoter strengths were compared by fluorescence microscopy and flow cytometry detection of the fluorescence intensity of the reporter genes initiated by each constitutive promoter (FITC), and DHK production reached 216.7 mg/L by the promoter adjustment strategy and the optimization of fermentation conditions. The results presented in this study will contribute to elucidating DHK biosynthesis in Trapa bispinosa Roxb.

Expanding the Scope of Adenoviral Vectors by Utilizing Novel Tools for Recombination and Vector Rescue.

Recombinant adenoviruses are widely used in clinical and laboratory applications. Despite the wide variety of available sero- and genotypes, only a fraction is utilized in vivo. As adenoviruses are a large group of viruses, displaying many different tropisms, immune epitopes, and replication characteristics, the merits of translating these natural benefits into vector applications are apparent. This translation, however, proves difficult, since while research has investigated the application of these viruses, there are no universally applicable rules in vector design for non-classical adenovirus types. In this paper, we describe a generalized workflow that allows vectorization, rescue, and cloning of all adenoviral species to enable the rapid development of new vector variants. We show this using human and simian adenoviruses, further modifying a selection of them to investigate their gene transfer potential and build potential vector candidates for future applications.

Comparative genome analysis of three classical E. coli cloning strains designed for blue/white selection: JM83, JM109 and XL1-Blue.

The development of the Escherichia coli K-12 laboratory strains JM83, JM109 and XL1-Blue was instrumental in early gene technology. We report the comprehensive genome sequence analysis of JM83 and XL1-Blue using Illumina and Oxford Nanopore technologies and a comparison with both the wild-type sequence (MG1655) and the genome of JM109 deposited at GenBank. Our investigation provides insight into the way how the genomic background that allows blue/white colony selection-by complementing a functionally inactive ω-fragment of ß-galactosidase (LacZ) with its α-peptide encoded on the cloning vector-has been implemented independently in these three strains using classical bacterial genetics. In fact, their comparative analysis reveals recurrent motifs: (i) inactivation of the native enzyme via large deletions of chromosomal regions encompassing the lac locus, or a chemically induced frameshift deletion at the beginning of the lacZ cistron, and (ii) utilization of a defective prophage (ϕ80), or an F'-plasmid, to provide the lacZ∆M15 allele encoding its ω-fragment. While the genetic manipulations of the E. coli strains involved repeated use of mobile genetic elements as well as harsh chemical or physical mutagenesis, the individual modified traits appear remarkably stable as they can be found even in distantly related laboratory strains, beyond those investigated here. Our detailed characterization at the genome sequence level not only offers clues about the mechanisms of classical gene transduction and transposition but should also guide the future fine-tuning of E. coli strains for gene cloning and protein expression, including phage display techniques, utilizing advanced tools for site-specific genome engineering.

Expression, Purification, and Determination of Sensitivity to Calcium Ions of Ctenophore Photoproteins.

Light-sensitive Ca2+-regulated photoproteins of ctenophores are single-chain polypeptide proteins of 206-208 amino acids in length comprising three canonical EF-hand Ca2+-binding sites, each of 12 contiguous residues. These photoproteins are a stable complex of apoprotein and 2-hydroperoxy adduct of coelenterazine. Addition of calcium ions to photoprotein is only required to trigger bright bioluminescence. However, in contrast to the related Ca2+-regulated photoproteins of jellyfish their capacity to bioluminescence disappears on exposure to light over the entire absorption spectral range of ctenophore photoproteins. Here, we describe protocols for expression of gene encoding ctenophore photoprotein in Escherichia coli cells, obtaining of the recombinant apoprotein of high purity and its conversion into active photoprotein with synthetic coelenterazine as well as determination of its sensitivity to calcium ions using light-sensitive Ca2+-regulated photoprotein berovin from ctenophore Beroe abyssicola as an illustrative case.

Functional Screening of cDNA Expression Library for Novel Ctenophore Photoproteins.

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.

Fully in vitro iterative construction of a 24 kb-long artificial DNA sequence to store digital information.

In the absence of a DNA template, the ab initio production of long double-stranded DNA molecules of predefined sequences is particularly challenging. The DNA synthesis step remains a bottleneck for many applications such as functional assessment of ancestral genes, analysis of alternative splicing or DNA-based data storage. In this report we propose a fully in vitro protocol to generate very long double-stranded DNA molecules starting from commercially available short DNA blocks in less than 3 days using Golden Gate assembly. This innovative application allowed us to streamline the process to produce a 24 kb-long DNA molecule storing part of the Declaration of the Rights of Man and of the Citizen of 1789 . The DNA molecule produced can be readily cloned into a suitable host/vector system for amplification and selection.

CRISPR/Cas9 Vector Construction for Gene Knockout.

This protocol outlines the construction of a plant transformation plasmid to express both the Cas9 nuclease and individual guide RNA (gRNA), facilitating the induction of double-stranded breaks (DSBs) in DNA and subsequent imprecise repair via the non-homologous end-joining (NHEJ) pathway. The gRNA expression cassettes are assembled from three components. First, the Medicago truncatula U6.6 (MtU6) promoter (352 bp) and scaffold (83 bp) sequences are amplified from a pUC-based plasmid. Additionally, a third fragment, corresponding to the target sequence, is synthesized as an oligonucleotide. The three gRNA expression fragments are then loosely assembled in a ligation-free cloning reaction and used as a template for an additional PCR step to amplify a single gRNA expression construct, ready for assembly into the transformation vector. The benefits of this design include cost efficiency, as subsequent cloning reactions only require 59 oligonucleotides and standard cloning reagents. Researchers engaged in CRISPR/Cas9-mediated genome editing in plants will find this protocol a clear and resource-efficient approach to create transformation plasmids for their experiments.

Rapid human genomic DNA cloning into mouse artificial chromosome via direct chromosome transfer from human iPSC and CRISPR/Cas9-mediated translocation.

A 'genomically' humanized animal stably maintains and functionally expresses the genes on human chromosome fragment (hCF; <24 Mb) loaded onto mouse artificial chromosome (MAC); however, cloning of hCF onto the MAC (hCF-MAC) requires a complex process that involves multiple steps of chromosome engineering through various cells via chromosome transfer and Cre-loxP chromosome translocation. Here, we aimed to develop a strategy to rapidly construct the hCF-MAC by employing three alternative techniques: (i) application of human induced pluripotent stem cells (hiPSCs) as chromosome donors for microcell-mediated chromosome transfer (MMCT), (ii) combination of paclitaxel (PTX) and reversine (Rev) as micronucleation inducers and (iii) CRISPR/Cas9 genome editing for site-specific translocations. We achieved a direct transfer of human chromosome 6 or 21 as a model from hiPSCs as alternative human chromosome donors into CHO cells containing MAC. MMCT was performed with less toxicity through induction of micronucleation by PTX and Rev. Furthermore, chromosome translocation was induced by simultaneous cleavage between human chromosome and MAC by using CRISPR/Cas9, resulting in the generation of hCF-MAC containing CHO clones without Cre-loxP recombination and drug selection. Our strategy facilitates rapid chromosome cloning and also contributes to the functional genomic analyses of human chromosomes.

A Modular Cloning Toolkit Including CRISPRi for the Engineering of the Human Fungal Pathogen and Biotechnology Host Candida glabrata.

The yeast Candida glabrata is an emerging, often drug-resistant opportunistic human pathogen that can cause severe systemic infections in immunocompromised individuals. At the same time, it is a valuable biotechnology host that naturally accumulates high levels of pyruvate─a valuable chemical precursor. Tools for the facile engineering of this yeast could greatly accelerate studies on its pathogenicity and its optimization for biotechnology. While a few tools for plasmid-based expression and genome engineering have been developed, there is no well-characterized cloning toolkit that would allow the modular assembly of pathways or genetic circuits. Here, by characterizing the Saccharomyces cerevisiae-based yeast molecular cloning toolkit (YTK) in C. glabrata and by adding missing components, we build a well-characterized CgTK (C. glabrata toolkit). We used the CgTK to build a CRISPR interference system for C. glabrata that can be used to generate selectable phenotypes via single-gRNA targeting such as is required for genome-wide library screens.

Phage N15-Based Vectors for Gene Cloning and Expression in Bacteria and Mammalian Cells.

Bacteriophage N15 is the first virus known to deliver linear prophage into Escherichia coli. During its lysogenic cycle, N15 protelomerase (TelN) resolves its telomerase occupancy site (tos) into hairpin telomeres. This protects the N15 prophage from bacterial exonuclease degradation, enabling it to stably replicate as a linear plasmid in E. coli. Interestingly, purely proteinaceous TelN can retain phage DNA linearization and hairpin formation without involving host- or phage-derived intermediates or cofactors in the heterologous environment. This unique feature has led to the advent of synthetic linear DNA vector systems derived from the TelN-tos module for the genetic engineering of bacterial and mammalian cells. This review will focus on the development and advantages of N15-based novel cloning and expression vectors in the bacterial and mammalian environments. To date, N15 is the most widely exploited molecular tool for the development of linear vector systems, especially the production of therapeutically useful miniDNA vectors without a bacterial backbone. Compared to typical circular plasmids, linear N15-based plasmids display remarkable cloning fidelity in propagating unstable repetitive DNA sequences and large genomic fragments. Additionally, TelN-linearized vectors with the relevant origin of replication can replicate extrachromosomally and retain transgenes functionality in bacterial and mammalian cells without compromising host cell viability. Currently, this DNA linearization system has shown robust results in the development of gene delivery vehicles, DNA vaccines and engineering mammalian cells against infectious diseases or cancers, highlighting its multifaceted importance in genetic studies and gene medicine.

Are there any good experiments that should not be done?

Is there any research that should not be done? Could you think of an experiment and then decide not to do it? These questions get to the heart of the power of modern genetics to mix up and alter genes.

Molecular characterization and functional analysis of Bxy-octr-1 in Bursaphelenchus xylophilus.

Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus.

Characterization of silicon transporter gene family in Saccharum and functional analysis of the ShLsi6 gene in biotic stress.

Silicon, one of the most prevalent elements in the soil, is beneficial for plant growth and defense against different stresses. The silicon transporter gene (Lsi) plays an important role in the uptake and transport of silicon in higher plants. In this study, a total of 32 Lsi genes, including 20 SsLsi in sugarcane wild species Saccharum spontaneum, 5 ShLsi in Saccharum hybrid cultivar R570 and 7 SbLsi in sugarcane related species Sorghum bicolor, were identified and classified into three groups. Bioinformatics analysis showed that instability, hydrophobicity, localization of cell membranes and vacuoles were the main features of the Lsi proteins. Whole genome and segmental duplication contributed to the main expansion of Lsi gene family. Collinearity analysis of the Lsi genes showed that S. spontanum and R570 had a collinear relationship with monocotyledonous plants S. bicolor and Oryza sativa, but not with dicotyledonous plants Arabidopsis thaliana and Vitis vinifera. The replicated Lsi genes were mainly subjected to strong selection pressure for purification. The diverse cis-regulatory elements in the promoter of SsLsi, ShLsi and SbLsi genes suggested that they were widely involved in the response of plants to various stresses and the regulation of the growth and development. Transcriptome data and real time quantitative PCR analysis showed that the Lsi genes exhibited different expression profiles in sugarcane tissues and under Sporisorium scitamineum, drought and cold stresses. In addition, the cDNA and genomic DNA sequences of ShLsi6 that was homologous to SsLsi1b gene was cloned from Saccharum hybrid cultivar ROC22. Transient expression analysis showed that, compared with the control, Nicotiana benthamiana leaves which overexpressed the ShLsi6 gene showed a high sensitivity after inoculation with tobacco pathogens Ralstonia solanacearum and Fusarium solani var. coeruleum. This study provides important information for further functional analysis of Lsi genes and resistant breeding in sugarcane.

Molecular cloning and functional characterization of KCNQ1 in shell biomineralisation of pearl oyster Pinctada fucata martensii.

KCNQ1, a voltage-gated potassium ion channel, plays an important role in various physiological processes, including osteoblast differentiation in higher animals. However, its function in lower invertebrates such as marine shellfish remains poorly understood. Pearl oysters, such as P. fucata martensii, are ideal for studying biomineralisation. In this study, a full-length cDNA of KCNQ1 from P. fucata martensii (PfKCNQ1) was obtained, and its function in shell formation was investigated. The full-length 3945 bp cDNA of PfKCNQ1 included an open reading frame (ORF) of 1944 bp encoding a polypeptide of 647 amino acids. Multiple sequence alignment revealed high homology with KCNQ1 from other species, with six transmembrane domains (S1 - S6) and a pore (P) region. Expression pattern analysis showed that PfKCNQ1 was expressed in all tested tissues, with highest expression in mantle and heart, and shell notching induced PfKCNQ1 expression. Silencing PfKCNQ1 expression inhibited PfKCNQ1 expression and downregulated four biomineralisation-related genes (Shematrin, Pif80, N16 and MSI60). Disordered crystals or "hollows" were visible in the shell ultrastructure by scanning electron microscopy following PfKCNQ1 knockdown. The results suggested that PfKCNQ1 may participate in or regulate biomineralisation and shell formation in pearl oyster.

Effect of pH on the structure and function of cyclin-dependent kinase 6.

Cyclin-dependent kinase 6 (CDK6) is an important protein kinase that regulates cell growth, development, cell metabolism, inflammation, and apoptosis. Its overexpression is associated with reprogramming glucose metabolism through alternative pathways and apoptosis, which ultimately plays a significant role in cancer development. In the present study, we have investigated the structural and conformational changes in CDK6 at varying pH employing a multi-spectroscopic approach. Circular dichroism (CD) spectroscopy revealed at extremely acidic conditions (pH 2.0-4.0), the secondary structure of CDK6 got significantly disrupted, leading to aggregates formation. These aggregates were further characterized by employing Thioflavin T (ThT) fluorescence. No significant secondary structural changes were observed over the alkaline pH range (pH 7.0-11.0). Further, fluorescence and UV spectroscopy revealed that the tertiary structure of CDK6 was disrupted under extremely acidic conditions, with slight alteration occurring in mild acidic conditions. The tertiary structure remains intact over the entire alkaline range. Additionally, enzyme assay provided an insight into the functional aspect of CDK at varying pH; CDK6 activity was optimal in the pH range of 7.0-8.0. This study will provide a platform that provides newer insights into the pH-dependent dynamics and functional behavior of CDK6 in different CDK6 directed diseased conditions, viz. different types of cancers where changes in pH contribute to cancer development.

The Ubiquitin E3 Ligase Nedd4 Regulates the Expression and Amyloid-ß Peptide Export Activity of P-Glycoprotein.

The ATP-binding cassette transporter, P-glycoprotein (P-gp), has been demonstrated to facilitate the clearance of amyloid-beta (Aß) peptides, exporting the neurotoxic entity out of neurons and out of the brain via the blood-brain barrier. However, its expression and function diminish with age and in Alzheimer's disease. P-gp is known to undergo ubiquitination, a post-translational modification that results in internalisation and/or degradation of the protein. NEDD4-1 is a ubiquitin E3 ligase that has previously been shown to ubiquitinate P-gp and reduce its cell surface expression. However, whether this effect translates into altered P-gp activity remains to be determined. siRNA was used to knockdown the expression of Nedd4 in CHO-APP cells. Western blot analysis confirmed that absence of Nedd4 was associated with increased P-gp protein expression. This was accompanied by increased transport activity, as shown by export of the P-gp substrate calcein-AM, as well as enhanced secretion of Aß peptides, as shown by ELISA. These results implicate Nedd4 in the regulation of P-gp, and highlight a potential approach for restoring or augmenting P-gp expression and function to facilitate Aß clearance from the brain.

In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components.

Rapid assessment of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based genome editing (GE) tools and their components is a critical aspect for successful GE applications in different organisms. In many bacteria, double-strand breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient nonhomologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide (nt) editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs, cytidine base editor (CBE) and adenine base editor (ABE), capable of converting C to T and A to G, respectively, have been recently developed but yet to be tested for editing characteristics in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising a combination of promoters and terminators enabling the expression of nCas9-based BE and sgRNA to nontoxic levels, eventually leading to successful base editing. We demonstrate the use of IRI-CCE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e) for bacteria, exhibiting that each independent BE has its specific editing pattern for a given target site depending on protospacer length. In summary, CRISPR-BE components expressed without lethal effects on cell survival in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and sgRNAs.