Recombination Strategy Research Articles

Establishment of the auxin inducible degron system for Babesia duncani: a conditional knockdown tool to study precise protein regulation in Babesia spp.

BackgroundBabesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions.MethodsThe endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA).ResultsIn this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation.ConclusionsThis study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system.Graphical

Advances in recombinant protein production in microorganisms and functional peptide tags.

Recombinant protein production in prokaryotic and eukaryotic cells is a fundamental technology for both research and industry. Achieving efficient protein synthesis is key to accelerating the discovery, characterization, and practical application of proteins. This review focuses on recent advances in recombinant protein production and strategy for more efficient protein production especially using Escherichia coli and Saccharomyces cerevisiae. Additionally, this review summarizes the development of various functional peptide tags that can be employed for protein production, modification, and purification, including translation enhancing peptide tags developed by our research group.

Cascaded Cross-modal Alignment for Visible-Infrared Person Re-Identification

Visible-Infrared Person Re-Identification faces significant challenges due to cross-modal and intra-modal variations. Although existing methods explore semantic alignment from various angles, severe distribution shifts in heterogeneous data limit the effectiveness of single-level alignment approaches. To address this issue, we propose a Cascaded Cross-modal Alignment (CCA) framework that gradually eliminates distribution discrepancies and aligns semantic features from three complementary perspectives in a cascaded manner. First, at the input-level, we propose a Channel-Spatial Recombination (CSR) strategy that strategically reorganizes and preserves crucial details from channel and spatial dimensions to diminish visual discrepancies between modalities, thereby narrowing the modality gap in input images. Second, at the frequency-level, we introduce a Low Frequency Masking (LFM) module to emphasize global details that CSR might overlook by randomly masking low-frequency information, thus driving comprehensive alignment of identity semantics. Third, at the part-level, we design a Prototype-based Semantic Refinement (PSR) module to refine fine-grained features and mitigate the impact of irrelevant areas in LFM. It accurately aligns body parts and enhances semantic consistency guided by global discriminative clues from LFM and flipped views with pose variations. Comprehensive experimental results on the SYSU-MM01 and RegDB datasets demonstrate the superiority of our proposed CCA.

SsCyp86 modulates Sporisorium scitamineum mating/filamentation and pathogenicity through regulating fatty acid metabolism

ABSTRACT Background Fatty acid metabolism constitutes a significant and intricate biochemical process within microorganisms. Cytochrome P450 (CYP450) enzymes are found in most organisms and occupy a pivotal position in the metabolism of fatty acids. However, the role of CYP450 enzyme mediated fatty acid metabolism in the pathogenicity of pathogenic fungi remains unclear. Methods In this study, a CYP450 enzyme-encoding gene, SsCYP86, was identified in the sugarcane smut fungus Sporisorium scitamineum and its functions were characterized using a target gene homologous recombination strategy and metabonomics. Results We found that the expression of SsCYP86 was induced by or sugarcane wax or under the condition of mating/filamentation. Sexual reproduction assay demonstrated that the SsCYP86 deletion mutant was defective in mating/filamentation and significantly reduced its pathogenicity. Further fatty acid metabolomic analysis unravelled the levels of fatty acid metabolites were reduced in the SsCYP86 deletion mutant. Exogenous addition of fatty acid metabolites cis-11-eicosenoic acid (C20:1N9), pentadecanoic acid (C15:0), and linolenic acid (C18:3N3) partially restored the mating/filamentation ability of the SsCYP86 deletion mutant and restored the transcriptional level of the SsPRF1, a pheromone response transcription factor that is typically down-regulated in the absence of SsCYP86. Moreover, the constitutive expression of SsPRF1 in the SsCYP86 deletion mutant restored its mating/filamentation. Conclusion Our results indicated that SsCyp86 modulates the SsPRF1 transcription by fatty acid metabolism, and thereby regulate the sexual reproduction of S. scitamineum. These findings provide insights into how CYPs regulate sexual reproduction in S. scitamineum.

The Mitochondrial Distribution and Morphology Family 33 Gene FgMDM33 Is Involved in Autophagy and Pathogenesis in Fusarium graminearum.

The mitochondrial distribution and morphology family 33 gene (MDM33) regulates mitochondrial homeostasis by mediating the mitochondrial fission process in yeast. The wheat head blight Fusarium graminearum contains an FgMdm33 protein that is orthologous to Saccharomyces cerevisiae Mdm33, albeit its function remains unknown. We have reported here the roles of FgMdm33 in regulating fungal morphogenesis, mitochondrial morphology, autophagy, apoptosis, and fungal pathogenicity. The ΔFgmdm33 mutants generated through a homologous recombination strategy in this study exhibited defects in terms of mycelial growth, conidia production, and virulence. Hyphal cells lacking FgMDM33 displayed elongated mitochondria and a dispensable respiratory-deficient growth phenotype, indicating the possible involvement of FgMDM33 in mitochondrial fission. The ΔFgmdm33 mutants displayed a remarkable reduction in the proteolysis of GFP-FgAtg8, whereas the formation of autophagic bodies in the hyphal cells of mutants was recorded under the induction of mitophagy. In addition, the transcriptional expression of the apoptosis-inducing factor 1 gene (FgAIF1) was significantly upregulated in the ΔFgmdm33 mutants. Cumulatively, these results indicate that FgMDM33 is involved in mitochondrial fission, non-selective macroautophagy, and apoptosis and that it regulates fungal growth, conidiation, and pathogenicity of the head blight pathogen.

Soluble CD40 Ligand as a Promising Biomarker in Cancer Diagnosis.

Cancer remains a significant challenge in medicine due to its complexity and heterogeneity. Biomarkers have emerged as vital tools for cancer research and clinical practice, facilitating early detection, prognosis assessment, and treatment monitoring. Among these, CD40 ligand (CD40L) has gained attention for its role in immune response modulation. Soluble CD40 ligand (sCD40L) has shown promise as a potential biomarker in cancer diagnosis and progression, reflecting interactions between immune cells and the tumor microenvironment. This review explores the intricate relationship between sCD40L and cancer, highlighting its diagnostic and prognostic potential. It discusses biomarker discovery, emphasizing the need for reliable markers in oncology, and elucidates the roles of CD40L in inflammatory responses and interactions with tumor cells. Additionally, it examines sCD40L as a biomarker, detailing its significance across various cancer types and clinical applications. Moreover, the review focuses on therapeutic interventions targeting CD40L in malignancies, providing insights into cellular and gene therapy approaches and recombinant protein-based strategies. The clinical effectiveness of CD40L-targeted therapy is evaluated, underscoring the need for further research to unlock the full potential of this signaling pathway in cancer management.

Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

A potyvirus provides an efficient viral vector for gene expression and functional studies in Asteraceae plants.

Plant virus-derived vectors are rapid and cost-effective for protein expression and gene functional studies in plants, particularly for species that are difficult to genetically transform. However, few efficient viral vectors are available for functional studies in Asteraceae plants. Here, we identified a potyvirus named zinnia mild mottle virus (ZiMMV) from common zinnia (Zinnia elegans Jacq.) through next-generation sequencing. Using a yeast homologous recombination strategy, we established a full-length infectious cDNA clone of ZiMMV under the control of the cauliflower mosaic virus 35S promoter. Furthermore, we developed an efficient expression vector based on ZiMMV for the persistent and abundant expression of foreign proteins in the leaf, stem, root, and flower tissues with mild symptoms during viral infection in common zinnia. We showed that the ZiMMV-based vector can express ZeMYB9, which encodes a transcript factor inducing dark red speckles in leaves and flowers. Additionally, the expression of a gibberellic acid (GA) biosynthesis gene from the ZiMMV vector substantially accelerated plant height growth, offering a rapid and cost-effective method. In summary, our work provides a powerful tool for gene expression, functional studies, and genetic improvement of horticultural traits in Asteraceae plant hosts.

Thermal recombination strategy designed R-CN as active shell to improve catalyst activity

A new recombinant CN material (R-CN) was prepared with g-C3N4 as precursor by hydrothermal method combined with thermal recombination method, which has 2D ultra-thin structure, nanoscale folds, graphite-like texture and some pores, and has richer N and O elements than g-C3N4. R-CN can reduce high temperature decomposition peak of ammonium perchlorate (AP) to 356.06 oC, and its promoting effect is obviously better than that of g-C3N4. Based on specific structural composition and physicochemical properties of R-CN, a novel R-CN@MWO composite with core-shell was designed, which was applied to catalytic decomposition of AP and pressure enlarging effect during combustion process of cyclotrimethylenetrinitramine (RDX)+nitrocellulose (NC). With the introduction of R-CN@MWO, decomposition peak temperature and activation energy of AP are reduced by 104.67oC and 59.24 kJ mol−1, and peak pressure and pressurization rate of RDX+NC constant volume combustion test are increased by 11.33 and 118.54 times, respectively. Moreover, the combustion rate of R-CN@MWO-AP/HTPB propellant is 3.23 times that of AP/HTPB propellant. To sum up, R-CN as a coating layer can significantly enhance catalytic activity of MgWO4 for thermal decomposition of energetic materials.

Dual auxotrophy coupled red labeling strategy for efficient genome editing in Saccharomyces cerevisiae

The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %–80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.

Crucial Involvement of Heme Biosynthesis in Vegetative Growth, Development, Stress Response, and Fungicide Sensitivity of Fusarium graminearum.

Heme biosynthesis is a highly conserved pathway from bacteria to higher animals. Heme, which serves as a prosthetic group for various enzymes involved in multiple biochemical processes, is essential in almost all species, making heme homeostasis vital for life. However, studies on the biological functions of heme in filamentous fungi are scarce. In this study, we investigated the role of heme in Fusarium graminearum. A mutant lacking the rate-limiting enzymes in heme synthesis, coproporphyrinogen III oxidase (Cpo) or ferrochelatase (Fc), was constructed using a homologous recombination strategy. The results showed that the absence of these enzymes was lethal to F. graminearum, but the growth defect could be rescued by the addition of hemin, so we carried out further studies with the help of hemin. The results demonstrated that heme was required for the activity of FgCyp51, and its absence increased the sensitivity to tebuconazole and led to the upregulation of FgCYP51 in F. graminearum. Additionally, heme plays an indispensable role in the life cycle of F. graminearum, which is essential for vegetative growth, conidiation, external stress response (especially oxidative stress), lipid accumulation, fatty acid β-oxidation, autophagy, and virulence.

Augmented antitumor immune responses of HER2-targeted pyroptotic induction by long-lasting recombinant immunopyroptotins

Pyroptosis is a well-documented form of programmed cell death caused by the gasdermin-driven perforation of cell membranes. Selective induction of pyroptosis in tumor cells represents a promising antitumor strategy to enhance the efficacy of immunotherapy. In this study, we established a recombinant protein-based immunopyroptotin strategy that led to the intratumoral induction of pyroptosis for HER2-directed therapy. Long-lasting immunopyroptotins were constructed by sequentially fusing the humanized anti-HER2 single-chain antibody P1h3, albumin-binding peptide (ABD035 or dAb7h8), cathepsin B-cleavable peptide B2, endosome-disruptive peptide E5C3, and active pyroptotic effector gasdermin D-N fragment (GN). After purification, we evaluated the cytotoxicity and antitumor immune responses primarily induced by the immunopyroptotins in HER2-overexpressing breast cancer cells. The resulting ABD035-immunoGN and dAb7h8-immunoGN showed improved in vitro cytotoxicity in HER2-overexpressing cancer cells compared with that in the immunotBid that we previously generated to induce tumor cell apoptosis. The binding of long-lasting immunopyroptotins to albumin increased the half-life by approximately 7-fold in nude mice. The enhanced antitumor efficacy of long-lasting immunopyroptotins was confirmed in both N87 tumor-bearing T cell-deficient mice and 4T1-hHER2 bilateral tumor-bearing immunocompetent mice. Immunopyroptotin treatment elicited systemic antitumor immune responses involving CD8+ T cells and mature dendritic cells and upregulated the expression of proinflammatory cytokines, leading to sustained remission of non-injected distant tumors. This study extends the repertoire of antibody-based therapeutics through the tumor-targeted delivery of a constitutively active pore-forming gasdermin-N fragment, which shows great potential for pyroptosis-based antitumor therapy.

Examining the impact of green technological specialization and the integration of AI technologies on green innovation performance: evidence from China.

China's commitment to achieving carbon neutrality by 2060 has sparked scholars' interest in examining the environmental ramifications of green technologies in the digital era. While plenty of them provide eco-efficiency policy such as increasing R&D investment or stimulating green exports, little attention has been paid to the firm-level technological management and recombination strategies such as differentiation/specialization of green portfolios along with AI integration, which can significantly impact the pace of net-zero transitions. To address these gaps, this study investigates the moderating effect of technological specialization on levels of AI integration into green technologies estimated by green-AI technological distance and enterprises' innovation performance in Chinese contemporary contexts. Regression results of fixed-effect model in Chinese patent data (2011-2020) indicate that enterprises' green innovation performance is significantly improved as AI integrates more into the green technologies due to the legitimacy and the inability to appropriate more green values. Interestingly, specialized green-technological enterprises demonstrate superior performance in integrating distant AI technologies. This occurrence could potentially be driven by the governments' incentives and the organization's risk attitudes, shaping green innovation outcomes. Hence, the study underscores the importance of considering both the AI integration and green specialization in shaping innovation outcomes amidst green transitions.

Predicting dust pollution from dry bulk ports in coastal cities: A hybrid approach based on data decomposition and deep learning

Dust pollution from storage and handling of materials in dry bulk ports seriously affects air quality and public health in coastal cities. Accurate prediction of dust pollution helps identify risks early and take preventive measures. However, there remain challenges in solving non-stationary time series and selecting relevant features. Besides, existing studies rarely consider impacts of port operations on dust pollution. Therefore, a hybrid approach based on data decomposition and deep learning is proposed to predict dust pollution from dry bulk ports. Port operational data is specially integrated into input features. A secondary decomposition and recombination (SDR) strategy is presented to reduce data non-stationarity. A dual-stage attention-based sequence-to-sequence (DA-Seq2Seq) model is employed to adaptively select the most relevant features at each time step, as well as capture long-term temporal dependencies. This approach is compared with baseline models on a dataset from a dry bulk port in northern China. The results reveal the advantages of SDR strategy and integrating operational data and show that this approach has higher accuracy than baseline models. The proposed approach can mitigate adverse effects of dust pollution from dry bulk ports on urban residents and help port authorities control dust pollution.

Abstract 5633: Unravelling the pleiotropic role of HNF4α in invasive mucinous adenocarcinoma of the lung (IMA)

Abstract IMA is a distinct subtype of lung adenocarcinoma (LUAD), marked by a cellular identity shift from a pulmonary to a gastric lineage with the loss of the pulmonary lineage specifier NKX2-1. This distinctive transformation underscores the complexity of lung cancer heterogeneity and highlights the critical role of lineage-specifying transcription factors in dictating tumor behavior. Notably, both human and murine models of IMA overexpress HNF4α, a known lineage specifier for gastric differentiation in the adult mouse mucosa. Also, concomitant deletion of Hnf4a and Nkx2-1 at tumor initiation impairs tumor growth and the extent of HNF4α in regulating IMA tumor growth, cellular identity and drug response remains unknown at large. Here, we utilized genetically engineered mouse models (GEMM) that relies on a sequential recombination strategy to delete Hnf4a in established tumors. We induce tumor formation through intratracheal delivery of Flp-O virus, activating the expression of mutant KRASG12D and Cre in lung epithelial cells. Tamoxifen administration recombines either Nkx2-1 alone (KN) or in combination with Hnf4a (KNH). We have extensively studied the phenotypic role of HNF4α in GEMM and organoids and identified gene sets regulated by HNF4α in IMA at the bulk level. To complement our findings, we profiled both the tumor and stromal compartments via scRNAseq analysis on our GEMM to gain insights into the cellular diversity within the tumor microenvironment (TME) and the role HNF4α plays. Additionally, we performed ChIPSeq in organoids to elucidate the genome occupancy of HNF4α and determine the mechanisms of gene regulation in IMA. We find that deletion of Hnf4a impairs tumor growth in GEMM and this is phenocopied in organoids. We identified numerous differentially expressed genes (DEGs) in KNH compared to KN and validated several DEGs in vivo. GSEA analysis reveals a significant involvement in regulating cellular identity and suggests a potential impact on anti-tumor immune responses. To this, our scRNAseq data revealed an enrichment of N2-TAN (tumor associated neutrophils) associated genes in KNH compared to KN, suggesting a nuanced interplay between HNF4α and the TME. Additionally, HNF4α exhibited dual functionality, acting as an activator and indirectly as a repressor by modulating the activity of other transcription factors like FOXA1. FOXA1 was found to bind de novo target genes following the loss of HNF4α. These findings so far collectively illuminate the multifaceted role of HNF4α in IMA, primarily driving tumorigenesis in a cell autonomous manner and secondarily skewing the TME towards a protumor via N2-TAN following loss of HNF4α in tumors. Citation Format: Headtlove Essel Dadzie, Gabriela Fort, Katherine Gillis, Pengshu Fang, Walter Orellana, Eric L. Snyder. Unravelling the pleiotropic role of HNF4α in invasive mucinous adenocarcinoma of the lung (IMA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5633.

Design, Synthesis, and Fungicidal Activities of Novel N-(Pyrazol-5-yl)benzamide Derivatives Containing a Diphenylamine Moiety.

To accelerate the development of novel fungicides, a variety of N-(pyrazol-5-yl)benzamide derivatives with a diphenylamine moiety were designed and synthesized using a pharmacophore recombination strategy based on the structure of pyrazol-5-yl-aminophenyl-benzamides. The bioassay results demonstrated that most of the target compounds had excellent in vitro antifungal activities against Sclerotinia sclerotiorum, Valsa mali, and Botrytis cinerea. In particular, compound 5IIIh exhibited remarkable activity against S. sclerotiorum (EC50 = 0.37 mg/L), which was similar to that of fluxapyroxad (EC50 = 0.27 mg/L). In addition, compound 5IIIc (EC50 = 1.32 mg/L) was observed to be more effective against V. mali than fluxapyroxad (EC50 = 12.8 mg/L) and comparable to trifloxystrobin (EC50 = 1.62 mg/L). Furthermore, compound 5IIIh demonstrated remarkable in vivo protective antifungal properties against S. sclerotiorum, with an inhibition rate of 96.8% at 100 mg/L, which was close to that of fluxapyroxad (99.6%). Compounds 5IIIc (66.7%) and 5IIIh (62.9%) exhibited good in vivo antifungal effects against V. mali at 100 mg/L, which were superior to that of fluxapyroxad (11.1%) but lower than that of trifloxystrobin (88.9%). The succinate dehydrogenase (SDH) enzymatic inhibition assay was conducted to confirm the mechanism of action. Molecular docking analysis further revealed that compound 5IIIh has significant hydrogen-bonding, π-π, and p-π conjugation interactions with ARG 43, SER 39, TRP 173, and TYR 58 in the binding site of SDH, and the binding mode was similar to that of the commercial fungicide fluxapyroxad. All of the results suggest that compound 5IIIh could be a potential SDH inhibitor, offering a valuable reference for future studies.

Hybrid whale algorithm with evolutionary strategies and filtering for high-dimensional optimization: Application to microarray cancer data.

The standard whale algorithm is prone to suboptimal results and inefficiencies in high-dimensional search spaces. Therefore, examining the whale optimization algorithm components is critical. The computer-generated initial populations often exhibit an uneven distribution in the solution space, leading to low diversity. We propose a fusion of this algorithm with a discrete recombinant evolutionary strategy to enhance initialization diversity. We conduct simulation experiments and compare the proposed algorithm with the original WOA on thirteen benchmark test functions. Simulation experiments on unimodal or multimodal benchmarks verified the better performance of the proposed RESHWOA, such as accuracy, minimum mean, and low standard deviation rate. Furthermore, we performed two data reduction techniques, Bhattacharya distance and signal-to-noise ratio. Support Vector Machine (SVM) excels in dealing with high-dimensional datasets and numerical features. When users optimize the parameters, they can significantly improve the SVM's performance, even though it already works well with its default settings. We applied RESHWOA and WOA methods on six microarray cancer datasets to optimize the SVM parameters. The exhaustive examination and detailed results demonstrate that the new structure has addressed WOA's main shortcomings. We conclude that the proposed RESHWOA performed significantly better than the WOA.

Empowering Protein Engineering through Recombination of Beneficial Substitutions.

Directed evolution stands as a seminal technology for generating novel protein functionalities, a cornerstone in biocatalysis, metabolic engineering, and synthetic biology. Today, with the development of various mutagenesis methods and advanced analytical machines, the challenge of diversity generation and high-throughput screening platforms is largely solved, and one of the remaining challenges is: how to empower the potential of single beneficial substitutions with recombination to achieve the epistatic effect. This review overviews experimental and computer-assisted recombination methods in protein engineering campaigns. In addition, integrated and machine learning-guided strategies were highlighted to discuss how these recombination approaches contribute to generating the screening library with better diversity, coverage, and size. A decision tree was finally summarized to guide the further selection of proper recombination strategies in practice, which was beneficial for accelerating protein engineering.

Genomic insights into virulence, antimicrobial resistance, and adaptation acumen of Escherichia coli isolated from an urban environment.

Evolutionary patterns of E. coli bacteria convey that they evolve into highly pathogenic forms by acquiring fitness advantages, such as AMR, and various virulence factors through the horizontal gene transfer (HGT)-mediated acquisition of MGEs. However, limited research on the genetic profiles of environmental E. coli, particularly from India, hinders our understanding of their transition to pathogenic forms and impedes the adoption of a comprehensive approach to address the connection between environmentally dwelling E. coli populations and human and veterinary public health. This study focuses on high-resolution genomic analysis of the environmental E. coli isolates aiming to understand the genetic similarities and differences among isolates from different environmental niches and uncover the survival strategies employed by these bacteria to thrive in their surroundings. Our approach involved molecular characterization of environmental samples using PCR-based DNA fingerprinting and subsequent WGS analysis. This multidisciplinary approach is likely to provide valuable insights into the understanding of any potential spill-over to human and animal populations and locales. Investigating these environmental isolates has significant potential for developing epidemiological strategies against transmission and understanding niche-specific evolutionary patterns.

Analysis of protein-protein and protein-membrane interactions by isotope-edited infrared spectroscopy.

The objective of this work is to highlight the power of isotope-edited Fourier transform infrared (FTIR) spectroscopy in resolving important problems encountered in biochemistry, biophysics, and biomedical research, focusing on protein-protein and protein membrane interactions that play key roles in practically all life processes. An overview of the effects of isotope substitutions in (bio)molecules on spectral frequencies and intensities is given. Data are presented demonstrating how isotope-labeled proteins and/or lipids can be used to elucidate enzymatic mechanisms, the mode of membrane binding of peripheral proteins, regulation of membrane protein function, protein aggregation, and local and global structural changes in proteins during functional transitions. The use of polarized attenuated total reflection FTIR spectroscopy to identify the spatial orientation and the secondary structure of a membrane-bound interfacial enzyme and the mode of lipid hydrolysis is described. Methods of production of site-directed, segmental, and domain-specific labeling of proteins by the synthetic, semisynthetic, and recombinant strategies, including advanced protein engineering technologies such as nonsense suppression and frameshift quadruplet codons are overviewed.