Cloning Genes Research Articles

Engineering Bacterial Chitinases for Industrial Application: From Protein Engineering to Bacterial Strains Mutation! A Comprehensive Review of Physical, Molecular, and Computational Approaches.

Bacterial chitinases are integral in breaking down chitin, the natural polymer in crustacean and insect exoskeletons. Their increasing utilization across various sectors such as agriculture, waste management, biotechnology, food processing, and pharmaceutical industries highlights their significance as biocatalysts. The current review investigates various scientific strategies to maximize the efficiency and production of bacterial chitinases for industrial use. Our goal is to optimize the heterologous production process using physical, molecular, and computational tools. Physical methods focus on isolating, purifying, and characterizing chitinases from various sources to ensure optimal conditions for maximum enzyme activity. Molecular techniques involve gene cloning, site-directed mutation, and CRISPR-Cas9 gene editing as an approach for creating chitinases with improved catalytic activity, substrate specificity, and stability. Computational approaches use molecular modeling, docking, and simulation techniques to accurately predict enzyme-substrate interactions and enhance chitinase variants' design. Integrating multidisciplinary strategies enables the development of highly efficient chitinases tailored for specific industrial applications. This review summarizes current knowledge and advances in chitinase engineering to serve as an indispensable guideline for researchers and industrialists seeking to optimize chitinase production for various uses.

The RNA helicase LOS4 regulates pre-mRNA splicing of key genes (EIN2, ERS2, CTR1) in the ethylene signaling pathway.

The Arabidopsis RNA helicase LOS4 plays a key role in regulating pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. The plant hormone ethylene plays diverse roles in plant growth, development, and responses to stress. Ethylene is perceived by the membrane-bound ethylene receptors complex, and then triggers downstream components, such as EIN2, to initiate signal transduction into the nucleus, leading to the activation of ethylene-responsive genes. Over the past decades, substantial information has been accumulated regarding gene cloning, protein-protein interactions, and downstream gene expressions in the ethylene pathway. However, our understanding of mRNA post-transcriptional processing and modification of key genes in the ethylene signaling pathway remains limited. This study aims to provide evidence demonstrating the involvement of the Arabidopsis RNA helicase LOS4 in pre-mRNA splicing of the genes EIN2, CTR1, and ERS2 in ethylene signaling pathway. Various genetic approaches including RNAi gene silencing, CRISPR-Cas9 gene editing, and amino acid mutations were employed in this study. When LOS4 was silenced or knocked down, the ethylene sensitivity of etiolated seedlings was significantly enhanced. Further investigation revealed errors in the EIN2 pre-mRNA splicing when LOS4 was knocked down. In addition, aberrant pre-mRNA splicing was observed in the ERS2 and CTR1 genes in the pathway. Biochemical assays indicated that the los4-2 (E94K) mutant protein exhibited increased ATP binding and enhanced ATP hydrolytic activity. Conversely, the los4-1 (G364R) mutant had reduced substrate RNA binding and lower ATP binding activities. These findings significantly advanced our comprehension of the regulatory functions and molecular mechanisms of RNA helicase in ethylene signaling.

Cloning and expression of UbiA human gene using innovative methodologies for recombinant protein production in PUAST vector

Introduction: A systematic library of Gal4/UAS-regulated transgenes has proven to be a powerful genetic system for identifying genes and defining pathways in development. This system offers valuable insights that highlight the evolutionary conservation between animals and humans. Objectives: The objective of this study was to clone, express, and characterize the UbiA gene. The research presents a highly effective method for cloning genes, using the UbiA-pcDNA3 gene as a model for mammalian cloning. These genes were then integrated into the PUAST vector of Drosophila, an expression vector and eukaryotic cell system commonly used for producing recombinant proteins. Materials and Methods: UbiA was isolated from human cells, and complementary DNA was synthesized. An oligonucleotide primer pair was designed based on the UbiA gene sequence, incorporating XhoI and Xbal restriction sites at the 5´ end of the forward and reverse primers, respectively. The UbiA gene was then amplified by PCR, cloned into the pcDNA3 plasmid, and the resulting recombinant plasmid was sequenced. Subsequently, the gene was sub cloned into the PUAST vector and expressed in S2 cells as a eukaryotic cell system. Protein determination and verification were conducted through western blotting techniques. Results: Confirmation of UbiA gene cloning into the PUAST vector was achieved through colony-PCR and digestion by enzymes. Cloning and sub cloning techniques validated by enzymatic digestion, along with gene sequencing. The identity between cloned UbiA gene and the identical gene exhibited 99%. We revealed a singular band purified protein through western blotting with 60 kDa size. Conclusion: More protein synthesis of the UbiA gene can be achieved by using the eukaryotic expression system provided by the PUAST vector. This technique has been proven to be a suitable platform and can be instrumental in various applications such as therapeutics, pharmacology, and vaccine development.

Abstract LB227: Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution

Abstract Cancers are diseases of aging. Many cellular processes characteristic of cancer, e.g., epigenetic dysregulation, are similarly observed during aging. Acute myeloid leukemia (AML) is emblematic of this interplay. As a blood cancer stemming from hematopoietic stem and progenitors (HSPC), its median age of diagnosis is 65. AML is linked to age-related clonal hematopoiesis (CH), a condition resulting from the expansion of HSPC, marked by mutations in leukemia-associated genes. We aim to clarify the intrinsic role of aging in HSPC as it causes CH and leukemic transformation. Hypothesis: aging amplifies the heritable phenotypic variations in hematopoietic stem cells (HSC), enhancing epigenetic heterogeneity. This acts as a substrate for natural selection, driving leukemic transformation. Results: We found a significant increase in epigenetic and transcriptomic heterogeneity in long-term HSC (LT-HSC) from old mice compared to their younger counterparts via single-cell RNA-seq (scRNA-seq)s. This process occurs in an aged bone marrow with higher inflammation and altered HSPC support. Of relevance, we previously show that the rise of epigenetic and transcriptomic heterogeneity in HSC with Tet2 loss and Flt3 ITD precedes leukemic transformation. We further employed CellTag Indexing and scRNA-seq to track the fate of specific HSC subclones. We tracked HSPC cell culture (in vitro) after two-weeks and two-months following transplantation into recipient mice of matching ages (in vivo). Cells of the same clone manifest higher transcriptomic similarities than those from different clones, validating CellTag in mapping clonal evolution. We computed the progeny output activity (Ai) by the ratio of clone i abundance in the committed progenitors to its frequency in the HSC compartment. We found that young mice exhibit significantly higher variability and a higher mean of progeny output activity Ai, indicating the efficiency in self-renewal and cell differentiation. While both young and old mice have low-output (higher stemness) clones, only young mice had significant differential expression of key genes in low-output clones compared to high-output clones, e.g., Dpp4, Eps8, and Col5a1. Depite lower progeny output Ai variation, LT-HSCs from older mice exhibited greater clonal transcriptome diversification both in vitro and in vivo , evidenced by intra-clonal transcriptomic correlation. This pattern also existed in ST-HSC and MPP from older mice. Genes with higher expression variability in older mice highlighted pathways pivotal for HSPC fitness, e.g., ribosome synthesis (RS), inflammation response mechanisms (JAK-STAT), hematopoietic cell lineage, AML and cancer pathways (GSEA). The RS pathway were remarkably enriched in the megakaryocytic-erythroid progenitors, directly before differentiation where substantial protein translation is required. Conclusion: our results suggest that the synergy of aging and somatic mutations in epigenetic regulators may enhance cell-to-cell variations, offering additional heritable phenotypic variations. These, in turn, may facilitate clonal hematopoiesis and leukemic transformation. Future work will delineate specific epigenetic and transcriptomic configurations for mutant HSPC clonal expansion. This study provides a cohesive framework, linking the principles governing aging to cancer. Citation Format: Lamis Naddaf, Marco De Dominici, Xiaowen Chen, Travis Roeder, Hideyuki Oguro, Shilpita Karmakar, Eric Pietras, James DeGregori, Sheng Li. Accelerated intra-clonal transcriptomic diversification of hematopoietic stem cells during aging fuels leukemia evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB227.

Comparative transcriptomic analysis of rubber (Hevea brasiliensis) clones differing in disease tolerance provides novel insights into the disease response associated with abnormal leaf fall disease caused by Phytophthora meadii

Rubber is an important industrial commodity and Hevea brasiliensis is the commercial source of natural rubber (NR). Abnormal Leaf Fall (ALF) disease caused by Phytophthora meadii is one of the major diseases affecting yield of NR. Understanding Hevea-Phytophthora interaction mechanism is essential to manage this devastating disease. Transcriptome analysis was employed to gain an understanding on the molecular mechanisms that confer resistance to Phytophthora that would facilitate development of disease-tolerant Hevea clones. As our current understanding of Hevea-Phytophthora interaction is limited, an attempt was made to analyze and compare the transcriptomes of tolerant and susceptible clones of H. brasiliensis in response to P. meadii infection (in healthy and Phytophthora challenge-inoculated condition) during early, middle and late phases of infection by using the RNA-Seq technology. The transcriptome sequencing generated an average of 19,000 differentially expressed genes (DEGs) in both clones. When comparing the tolerant clone to the susceptible clone, there was an overall increase in the number of DEGs and a progression in the number of DEGs, indicating a more active engagement of transcriptional activity in the tolerant clone. Functional annotation, KEGG analysis and enrichment analysis revealed that the major pathways enriched for the DEGs were involved in plant hormone signal transduction and plant-pathogen interaction. An analysis of DEGs during each phase showed that the most active transcripts involved in defense mechanisms were activated during mid-phase and in the case of susceptible clone least transcripts were induced during mid-phase than in any other phase in both the clones. Many DEGs belonging to the MYB, NAC, WRKY and AP2/ERF transcription factor families were observed in both clones. Comparative studies revealed their differential involvement in tolerant and susceptible clones against Phytophthora infection. These findings may facilitate further exploration of Hevea-Phytophthora interaction and in cloning for defense related genes.

Unlocking the Power of Molecular Cloning: Revolutionizing Medical Microbiology Procedures

The revolutionary realm of molecular cloning, encompassing the creation of recombinant DNA molecules, has ignited a wave of progress within the life sciences. The advent of potent tools has facilitated the manipulation of DNA, resulting in an extraordinary surge in the versatility and breadth of applications in recombinant DNA technology. The once complex task of cloning genes has now been simplified, triggering a veritable explosion of insights into gene functionality. This has been achieved through the seamless fusion of multiple DNA fragments or the utilization of interchangeable gene cassettes, culminating in a state of unparalleled agility and expediency. In the 1970s, when restriction endonucleases, enzymes that cut DNA molecules selectively were discovered, molecular cloning technology has grown exponentially in application and intricacy, resulting in influential DNA manipulation tools. Recent decades have seen an explosion in our understanding of gene function due to the simplicity and efficiency of molecular cloning. It is expected that emerging technologies will offer superior potentials, such as stitching together multiple DNA fragments in under a few hours and transforming the resulting plasmids into bacteria, or the use of swappable genes, which can be easily moved between different paradigms, maximizing promptness and flexibility. It has been proved that cloning techniques provide a gold standard technique for polymicrobial infection, recombinant cytokines, antimicrobial peptides, epidemiology and gene therapy due to the limitations of culture-based methods. Due to molecular cloning technique, recombinant antigens are now being used to monitor patients against clinical infections. As a result of laboratory techniques that permit in vitro chemical synthesis of any DNA construct specified in silico, molecular cloning will likely undergo a paradigm shift in the coming future. As a result of these advances, DNA clones can be constructed faster and iteratively, which will speed up the growth of new vaccines, gene therapy vectors, and recombinant proteins. Here I present a detailed overview of the latest applications of molecular cloning techniques in medical microbiology.

Cloning and expression heterologous alanine dehydrogenase genes: Investigation of reductive amination potential of L-alanine dehydrogenases for green synthesis of alanine derivatives

Unnatural amino acids (UAAs) offer significant promise in a wide range of applications, including drug discovery, the custom design of peptides and proteins, and their utility and use as markers for monitoring molecular interactions in biological research. The synthesis of UAAs presents a formidable challenge and can be classified into two primary categories: enzymatic and chemical synthesis. Notably, the enzymatic route, specifically asymmetric synthesis, emerges as a an attractive method for procuring enantiopure UAAs with high efficiency, owing to its streamlined and concise reaction mechanism.The current study investigated the reductive amination activity mechanisms of alanine dehydrogenase (L-AlaDH), sourced from a combination of newly and previously characterized microorganisms. Our principal aim was to evaluate the catalytic efficiency of these L-AlaDH enzymes concerning a range of specific ketoacids and pyruvate to ascertain their capability for facilitating the production of both natural and unnatural amino acids. After the characterization processes, mutation points for TtAlaDH were determined and as a result of the mutations, mutants that could use ketocaproate and ketovalerate more effectively than the wild type were obtained.Among the enzymes studied, MetAlaDH exhibited the highest specific activity against pyruvate, 173 U/mg, and a KM value of 1.3 mM. VlAlaDH displayed the most favourable catalytic efficiency with a rate constant of 170 s−1mM−1. On the other hand, AfAlaDH demonstrated the highest catalytic efficiency against α-ketobutyrate (34.0 s−1mM−1) and α-ketovalerate (2.7 s−1mM−1). Of the enzymes investigated in the study, TtAlaDH exhibited the highest effectiveness among bacterial enzymes in catalyzing ketocaproate with a measured catalytic efficiency of about 0.6 s−1mM−1 and a KM value of approximately 0.3 mM. These findings provide valuable insights into the substrate specificity and catalytic performance of L-AlaDHs, enhancing our understanding of their potential applications in various biocatalytic processes.

Identification and Expression Profile Analysis of WOX Family Genes in the Formation of Eucalyptus Adventitious Root

The WUSCHEL-related homeobox (WOX) gene family are key players in the rooting process. Eucalyptus is an important plant species of artificial forests in China. It is mainly grown through tissue culture of many excellent clonal materials, in which rooting is a key step. In the present study, by using the genome data of Eucalyptus grandis, Corymbia citriodora, E. pellita, and E. urophylla × E. grandis, the members of the eucalyptus WOX gene family were identified and analyzed by bioinformatics techniques. The eucalyptus WOX gene family members are unstable proteins, with 7 acidic proteins and 24 basic proteins, and no signal peptide region was detected. Subcellular localization prediction indicated that all these proteins are localized in the nucleus. Motif analysis showed that eucalyptus WOX genes share the same motifs. Phylogenetic tree and gene expression analyses revealed that the eucalyptus WOX genes are highly conserved during the evolution process. Moreover, the WOX protein sequences are also highly conserved within the species, with higher similarity between woody plants. The EupWOX gene showed tissue-specific expression, with EupWOX1 and EupWOX11 specifically expressed in the roots of E. urophylla × E. pellita clonal tissue culture during the late-stage rooting. This finding suggests that EupWOX1 may be a key regulatory gene induced by the root primordium and is critically related to the rooting rate. EupWOX1, EupWOX5, and EupWOX13 could be the key regulatory genes for adventitious root formation. EupWOX1, EupWOX5, and EupWOX13 could be the key regulatory genes for the elongation of adventitious roots and the growth of adventitious lateral roots. EupWOX5 and EupWOX13 could play a critical role, not only in the formation of adventitious roots and adventitious lateral roots of E. urophylla clonal tissue culture but also in the root elongation process. These results will help us understand the complexity of rooting in different lines and provide valuable information for future functional characterization of specific genes in eucalyptus clones.

SF3B1 K700E Alters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS

Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novo methyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1 and DNMT3A mutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3A mutations have shorter progression-free survival than those with solely SF3B1 mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood. To investigate how Dnmt3a and Sf3b1 mutations disrupt HSC function in vivo we crossed two conditional knock-in mouse models of common point mutations in SF3B1 (K700E) and DNMT3A (R787H, murine equivalent to human DNMT3A R882H). We followed monthly blood counts and evaluated stem and progenitor cell composition in the bone marrow at 6 months. We found that Sf3b1/Dnmt3a double mutant mice display a similar phenotype to Sf3b1K700E mice, with a progressive anemia and an expansion in LT-HSCs. However, when assayed for HSC self-renewal potential, whole bone marrow and LT-HSCs isolated from Sf3b1/Dnmt3a mutant mice display increased serial colony formation capacities similar to Dnmt3a mutant hematopoietic stem and progenitor cells (HSPCs, 5 to 6 passages), but have a competitive disadvantage in secondary and tertiary transplant assays that is less severe than Sf3b1K700E HSPCs but distinct from the competitive advantage of Dnmt3a mutant HSPCs. Transcriptionally, double mutant HSCs clustered closer to Sf3b1 HSCs with shared expression of 74% of upregulated genes and 64% of downregulated genes and have decreased expression of stress response and metabolism genes. These results indicate Sf3b1 mutations may mitigate the preleukemic phenotype due to Dnmt3a mutations in MDS. To identify factors regulated by SF3B1 mutations that may mitigate the effect of DNMT3A loss on leukemic progression, we generated isogenic DNMT3A KO and SF3B1 K700E single and double mutant K562 cell lines using CRISPR/Cas9. Transcriptome analysis demonstrated that SF3B1K700E/DNMT3AKO double mutant clones clustered with and shared similar upregulated (69%) and downregulated (62%) genes as SF3B1 single mutant clones. Similar to our findings in murine HSPCs, pathway analysis of SF3B1 and SF3B1/DNMT3A mutant clones displayed enrichment of stress response and metabolic pathways. Alternative mRNA splicing analysis identified 13 events (3 alternative 3' splice site, 9 intron retention, and 1 skipped exon) in 8 genes that were upregulated in DNMT3A KO clones and downregulated in SF3B1 and SF3B1/DNMT3A mutant clones. We confirmed decreased protein expression of these mis-spliced genes in SF3B1 single and double mutant clones, but not DNMT3A KO clones. These genes include components of the integrated stress response pathway, which are also mis-spliced in SF3B1 and SF3B1/DNMT3A mutant MDS patient samples. Taken together our data suggest that aberrant splicing by mutant SF3B1 prevents metabolic reprogramming and decreases leukemic progression caused by DNMT3A mutation in MDS.

Molecular study on virulence and resistance genes of ST131 clone (uropathogenic/enteropathogenic Escherichia coli) hybrids in children.

Aim: To analyze ST131 clones and other characteristics in uropathogenic and atypical enteropathogenic Escherichia coli hybrids. Methods: Samples were collected from children with urinary tract infections and underwent testing for antimicrobial susceptibility, multidrug resistance and extended-spectrum β-lactamases, in vitro biofilm formation and virulence, resistance genes, hybrid pathotypes and ST131 clones. Results: E. coli isolates showed high levels of antibiotic resistance, extended-spectrum β-lactamase production, virulence genes, multidrug resistance and biofilm formation. Four (5.0%) isolates were identified as uropathogenic/atypical enteropathogenic E. coli hybrids, all of which belonged to the high-risk ST131 clone. Conclusion: Our results provide promising insights about hybrid isolates and should be addressed to improve prevention measures for hybrid pathotypes.

Nosocomial dissemination of blaIMP-4 among Klebsiella pneumoniae by horizontal gene transfer and clonal spread: the epidemic IncN plasmids and the emerging high-risk IMP-4-producing ST101 clone.

To determine the genomic features of IMP-4-producing Klebsiella pneumoniae isolates recovered from paediatric patients and the transmission dynamics of blaIMP-4. IMP-producing K. pneumoniae isolates were collected from paediatric patients in Shanghai Children's Medical Center from 2013 to 2020. WGS was performed for all isolates, and the complete genomes of three IMP-4-producing isolates were generated. The distribution of blaIMP-4-harbouring plasmids was determined, and a conjugation assay was employed to investigate the horizontal transfer of blaIMP-4-harbouring plasmids. We collected 21 blaIMP-carrying K. pneumoniae isolates, with IMP-4 (16/21, 76.2%) as the predominant subtype, followed by IMP-8 (n = 3) and IMP-26 (n = 2). IMP-4-producing isolates displayed a diverse population structure and all blaIMP-4 genes were located on plasmids, including IncN (n = 9), IncHI5 (n = 5), IncFII(K) (n = 1) and IncFII(pKP91) (n = 1), although only IncN plasmids were conjugative. Clonal transmission of ST101 strains carrying IncHI5 blaIMP-4-harbouring plasmids was observed, and the acquisition of blaIMP-4 by the international high-risk ST101 clone constituted a novel combination of ST101 clone and carbapenemase genes. Plasmid analysis demonstrated that the conjugal transfer of the IncHI5 blaIMP-4-harbouring plasmid might be blocked by the ST101 bacterial host. The horizontal transfer of IncN plasmids and clonal spread of the international high-risk ST101 clone facilitated the nosocomial dissemination of blaIMP-4 among K. pneumoniae. The emerging IMP-4-producing ST101 clone displays diverse combinations of carbapenemase genes, and this clone could be a continually evolving threat and warrants prospective monitoring.

Physiological Analysis and Genetic Mapping of Short Hypocotyl Trait in Brassica napus L.

Hypocotyl length is a botanical trait that affects the cold tolerance of Brassica napus L. (B. napus). In this study, we constructed an F2 segregating population using the cold-resistant short hypocotyl variety '16VHNTS158' and the cold-sensitive long hypocotyl variety 'Tianyou 2288' as the parents, and BSA-seq was employed to identify candidate genes for hypocotyl length in B. napus. The results of parental differences showed that the average hypocotyl lengths of '16VHNTS158' and 'Tianyou 2288' were 0.41 cm and 0.77 cm at the 5~6 leaf stage, respectively, after different low-temperature treatments, and '16VHNTS158' exhibited lower relative ion leakage rates compared to 'Tianyou 2288'. The contents of indole acetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) in hypocotyls of '16VHNTS158' and 'Tianyou 2288' increased with decreasing temperatures, but the IAA and GA contents were significantly higher than those of 'Tianyou 2288', and the BR content was lower than that of 'Tianyou 2288'. The genetic analysis results indicate that the genetic model for hypocotyl length follows the 2MG-A model. By using SSR molecular markers, a QTL locus associated with hypocotyl length was identified on chromosome C04. The additive effect value of this locus was 0.025, and it accounted for 2.5% of the phenotypic variation. BSA-Seq further localized the major effect QTL locus on chromosome C04, associating it with 41 genomic regions. The total length of this region was 1.06 Mb. Within this region, a total of 20 non-synonymous mutation genes were identified between the parents, and 26 non-synonymous mutation genes were found within the pooled samples. In the reference genome of B. napus, this region was annotated with 24 candidate genes. These annotated genes are predominantly enriched in four pathways: DNA replication, nucleotide excision repair, plant hormone signal transduction, and mismatch repair. The findings of this study provide a theoretical basis for cloning genes related to hypocotyl length in winter rapeseed and their utilization in breeding.

Discovery and characterization of a novel PKD-Fn3 domains containing GH44 endoglucanase from a Tibetan metagenomic library.

To explore novel microbial endoglucanases with unique properties derived from extreme environments by using metagenomics approach. A Tibetan soil metagenomic library was applied for screening cellulase-active clones by function-based metagenomics. The candidate genes in the active clones were identified through bioinformatic analyses and heterologously expressed using an Escherichia coli system. The recombinant endoglucanases were purified and characterized using enzyme assays to determine their bioactivities, stabilities, substrate specificities, and other enzymatic properties. A novel endoglucanase gene Zfeg1907 was identified, which consisted of a glycoside hydrolase family 44 (GH44) catalytic domain along with a polycystic kidney disease (PKD) domain and a fibronectin type Ⅲ (Fn3) domain at the C terminal. Recombinant enzyme ZFEG1907 and its truncated mutant ZFEG1907t (ΔPKDΔFn3) were successfully expressed and purified. The two recombinants exhibited catalytic activities towards carboxymethyl cellulose, konjac glucomannan and lichenan. Both enzymes had an optimal temperature of 50°C and an optimal pH value of 5.0. The catalytic activities of both recombinant enzymes were promoted by adding Zn2+ and Ca2+ at the final concentration of 10mM. The Km value of ZFEG1907 were lower while the kcat/Km value of ZFEG1907 was higher than those of of ZFEG1907t when using carboxymethyl cellulose, konjac glucomannan and lichenan as substrates. Structure prediction of two recombinants revealed that PKD-Fn3 domains consisted of a flexible linker and formed a β-sandwich structure. A novel endoglucanase ZFEG1907 contained a GH44 catalytic domain and a PKD-Fn3 domain was characterized. The PKD-Fn3 domains were not indispensable for the activity but contributed to the enzyme binding of the polysaccharide substrates as a carbohydrate-binding module (CBM).

Mechanical Characterization and Corrosion Behavior of Magnesium/Boron Carbide Composites

“Product Design & Injection Mold Design Development of Car Chassis Upper Cover Part” This project fully concentrated on how to design of double impression injection Mold for wheel hub for the car sing the least economic processing and tooling method and it also helps to get the very deep knowledge about the “Split Activation Method”. Finally, the aim of the project is to estimation of the mold which should most economic and also detail about mention above things. It is all the transcendence of human race that people today reach the height of maximum, luxury upgrading from the days of their very ancient stone and cave age. What human being has achieved today was once only a dream out of reach. But man’s curiosity perhaps the most wonderful gift given to man by God was behind his quest leading to wholehearted efforts to convert all impossible dreams of yesteryears into reality. Today in the 21st century, men have reason enough to be proud for their unique achievements from stepping into the moon to cloning genes; there has been long list of tremendous success. Designing of product and mold for plastics is one of the important achievements in this century. This project is our sincere effort for the design of Bottom inlet valve components and their molds. This component has good aesthetics and optimum production cost which helps in withstanding the market competition.

Plastics Product & Injection Mold Design Development of Car Chassis Upper Cover

“Product Design & Injection Mold Design Development of Car Chassis Upper Cover Part” This project fully concentrated on how to design of double impression injection Mold for wheel hub for the car sing the least economic processing and tooling method and it also helps to get the very deep knowledge about the “Split Activation Method”. Finally, the aim of the project is to estimation of the mold which should most economic and also detail about mention above things. It is all the transcendence of human race that people today reach the height of maximum, luxury upgrading from the days of their very ancient stone and cave age. What human being has achieved today was once only a dream out of reach. But man’s curiosity perhaps the most wonderful gift given to man by God was behind his quest leading to wholehearted efforts to convert all impossible dreams of yesteryears into reality. Today in the 21st century, men have reason enough to be proud for their unique achievements from stepping into the moon to cloning genes; there has been long list of tremendous success. Designing of product and mold for plastics is one of the important achievements in this century. This project is our sincere effort for the design of Bottom inlet valve components and their molds. This component has good aesthetics and optimum production cost which helps in withstanding the market competition

Abstract 3526: The transcription factor ZEB1 shapes osteosarcoma aggressiveness by affecting tumor cell differentiation and stemness features

Abstract Osteosarcoma (OS) is a mesenchymal bone tumor affecting mainly children and young adults, characterized by a particularly aggressive behavior, with 20% of patients showing lung micro metastasis already at diagnosis. To date, treatment options for OS are still based on multi-drug chemotherapy and prognosis for metastatic patients is dismal, making urgent the identification of new therapies. OS is considered a differentiation disease, because of the incapability of mesenchymal stem cells or osteoblastic progenitors to proceed toward terminal differentiation. We tested the effects on OS cells of targeting ZEB1, a transcription factor known to be critical for the maintenance of mesenchymal and stemness features in cancer. We inhibited ZEB1 expression in a murine OS cell line by lentiviral vectors based on CRISPR-Cas9 technology. We firstly tested the effects of ZEB1 deficiency in vitro, evaluating OS cell stemness potential by sarcosphere forming assay and osteogenic differentiation by alkaline phosphatase (Alp) staining. Gene expression profile (GEP) comparing ZEB1 KO clones and control ones was also performed to investigate the transcriptional changes induced by ZEB1 deletion and potentially identify alternative, more easily druggable, therapeutic targets. We also investigated in vivo the effect of ZEB1 inhibition. ZEB1 KO clones showed decreased stemness potential and increased Alp staining, both features suggestive of a more differentiated phenotype. Preliminary GEP analysis showed 849 down-regulated genes and 1093 up-regulated genes in ZEB1 KO clones versus ZEB1-competent controls. Gene set enrichment analysis indicated a down-modulation of pathways related to cellular proliferation and survival such as MTORC1 signaling, MYC targets, GM2 checkpoint, E2F targets and oxidative phosphorylation in ZEB1 KO clones. In vivo we observed a reduced tumor growth in ZEB1 KO clones that were also characterized by a more differentiated morphology in comparison to controls. Interestingly, the absence of ZEB1 in tumor cells affected also the immune infiltrate, as tumors derived from ZEB1 KO clones were significantly less infiltrated by pro-tumoral CD206+ M2-like macrophages. Moreover, we observed a reduced metastatic potential in ZEB1 KO clones than controls when we inject the cells intravenously. Taken together, these results support the hypothesis of ZEB1 as a key factor in OS aggressiveness, affecting tumor cell differentiation, stemness ability and in vivo growth capability. Citation Format: Caterina Cascini, Daniele Lecis, Laura Botti, Chiara Ratti, Valeria Cancila, Katia Scotlandi, Claudio Tripodo, Mario Paolo Colombo, Claudia Chiodoni. The transcription factor ZEB1 shapes osteosarcoma aggressiveness by affecting tumor cell differentiation and stemness features. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3526.

Cloning and Functional Characterization of 2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase (LiMCT) Gene in Oriental Lily (Lilium 'Sorbonne').

2-C-methyl-D-erythritol-phosphate cytidylyltransferase (MCT) is a key enzyme in the MEP pathway of monoterpene synthesis, catalyzing the generation of 4- (5'-pyrophosphate cytidine)-2-C-methyl-D-erythritol from 2-C-methyl-D-erythritol-4-phosphate. We used homologous cloning strategy to clone gene, LiMCT, in the MEP pathway that may be involved in the regulation of floral fragrance synthesis in the Lilium oriental hybrid 'Sorbonne.' The full-length ORF sequence was 837bp, encoding 278 amino acids. Bioinformatics analysis showed that the relative molecular weight of LiMCT protein is 68.56 kD and the isoelectric point (pI) is 5.12. The expression pattern of LiMCT gene was found to be consistent with the accumulation sites and emission patterns of floral fragrance monoterpenes in transcriptome data (unpublished). Subcellular localization indicated that the LiMCT protein is located in chloroplasts, which is consistent with the location of MEP pathway genes functioning in plastids to produce isoprene precursors. Overexpression of LiMCT in Arabidopsis thaliana affected the expression levels of MEP and MVA pathway genes, suggesting that overexpression of the LiMCT in A. thaliana affected the metabolic flow of C5 precursors of two different terpene synthesis pathways. The expression of the monoterpene synthase AtTPS14 was elevated nearly fourfold in transgenic A. thaliana compared with the control, and the levels of carotenoids and chlorophylls, the end products of the MEP pathway, were significantly increased in the leaves at full bloom, indicating that LiMCT plays an important role in regulating monoterpene synthesis and in the synthesis of other isoprene-like precursors in transgenic A. thaliana flowers. However, the specific mechanism of LiMCT in promoting the accumulation of isoprene products of the MEP pathway and the biosynthesis of floral monoterpene volatile components needs further investigation.

Identification and validation of quantitative trait loci for fertile spikelet number per spike and grain number per fertile spikelet in bread wheat (Triticum aestivum L.)

A major and stable QTL for fertile spikelet number per spike and grain number per fertile spikelet identified in a 4.96-Mb interval on chromosome 2A was validated in different genetic backgrounds. Fertile spikelet number per spike (FSN) and grain number per fertile spikelet (GNFS) contribute greatly to wheat yield improvement. To detect quantitative trait loci (QTL) associated with FSN and GNFS, we used a recombinant inbred line population crossed by Zhongkemai 13F10 and Chuanmai 42 in eight environments. Two Genomic regions associated with FSN were detected on chromosomes 2A and 6A using bulked segregant exome sequencing analysis. After the genetic linkage maps were constructed, four QTL QFsn.cib-2A, QFsn.cib-6A, QGnfs.cib-2A and QGnfs.cib-6A were identified in three or more environments. Among them, two major QTL QFsn.cib-2A (LOD = 4.67-9.34, PVE = 6.66-13.05%) and QGnfs.cib-2A (LOD = 5.27-11.68, PVE = 7.95-16.71%) were detected in seven and six environments, respectively. They were co-located in the same region, namely QFsn/Gnfs.cib-2A. The developed linked Kompetitive Allele Specific PCR (KASP) markers further validated this QTL in a different genetic background. QFsn/Gnfs.cib-2A showed pleiotropic effects on grain number per spike (GNS) and spike compactness (SC), and had no effect on grain weight. Since QFsn/Gnfs.cib-2A might be a new locus, it and the developed KASP markers can be used in wheat breeding. According to haplotype analysis, QFsn/Gnfs.cib-2A was identified as a target of artificial selection during wheat improvement. Based on haplotype analysis, sequence differences, spatiotemporal expression patterns, and gene annotation, the potential candidate genes for QFsn/Gnfs.cib-2A were predicted. These results provide valuable information for fine mapping and cloning gene(s) underlying QFsn/Gnfs.cib-2A.

A molecular cell biology toolkit for the study of meiosis in the silkworm Bombyx mori.

Meiosis is usually described as 4 essential and sequential processes: (1) homolog pairing; (2) synapsis, mediated by the synaptonemal complex; (3) crossing over; and (4) segregation. In this canonical model, the maturation of crossovers into chiasmata plays a vital role in holding homologs together and ensuring their segregation at the first meiotic division. However, Lepidoptera (moths and butterflies) undergo 3 distinct meiotic processes, only one of which is canonical. Lepidoptera males utilize 2 meiotic processes: canonical meiosis that produces nucleated fertile sperm, and a noncanonical meiosis that produces anucleated nonfertile sperm which are nonetheless essential for reproduction. Lepidoptera females, which carry heteromorphic sex chromosomes, undergo a completely achiasmate (lacking crossovers) meiosis, thereby requiring an alternative mechanism to ensure proper homolog segregation. Here, we report that the development of a molecular cell biology toolkit designed to properly analyze features of meiosis, including the synaptonemal complex structure and function, in the silkworm Bombyx mori. In addition to standard homology searches to identify Bombyx orthologs of known synaptonemal complex encoding genes, we developed an ortholog discovery app (Shinyapp) to identify Bombyx orthologs of proteins involved in several meiotic processes. We used this information to clone genes expressed in the testes and then created antibodies against their protein products. We used the antibodies to confirm the localization of these proteins in normal male spermatocytes, as well as using in vitro assays to confirm orthologous interactions. The development of this toolkit will facilitate further study of the unique meiotic processes that characterize meiosis in Lepidoptera.

Characterization of somatic mutations in the pathogenesis of lipedema.

Lipedema, a complex and enigmatic adipose tissue disorder, remains poorly understood despite its significant impact on the patients' quality of life. Genetic investigations have uncovered potential contributors to its pathogenesis, including somatic mutations, which are nonheritable genetic alterations that can play a pivotal role in the development of this disease. This review aims to elucidate the role of somatic mutations in the etiology of lipedema by examining their implications in adipose tissue biology, inflammation, and metabolic dysfunction. Studies focusing on leukocyte clones, genetic alterations like TET2 and DNMT3A, and the intricate interplay between adipose tissue and other organs have shed light on the underlying mechanisms driving lipedema. From the study of the scientific literature, mutations to genes correlated to three main pathways could be involved in the somatic development of lipedema: genes related to mitochondrial activity, genes related to localized disorders of subcutaneous adipose tissue, and genes of leukocyte clones. The insights gained from these diverse studies converge to highlight the complex genetic underpinnings of lipedema and offer potential avenues for therapeutic interventions targeting somatic mutations to alleviate the burden of this condition on affected individuals.