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Mycobacterium indicus pranii (MIP), a benign saprophyte with potent immunomodulatory attributes, holds a pivotal position in mycobacterial evolution, potentially serving as the precursor to the pathogenic Mycobacterium avium complex (MAC). Despite its established immunotherapeutic efficacy against leprosy and notable outcomes in gram‐negative sepsis and COVID‐19 cases, the genomic and biochemical features of MIP remain largely elusive. This study explores the uncharted territory of toxin‐antitoxin (TA) systems within MIP, hypothesizing their role in mycobacterial pathogenicity regulation. Genome‐wide screening, employing diverse databases, unveils putative TA modules in MIP, setting the stage for a comparative analysis with known modules in Mycobacterium tuberculosis, Mycobacterium smegmatis, Escherichia coli, and Vibrio cholerae. The study further delves into the TA network of MAC and Mycobacterium intracellulare, unraveling interactive properties and family characteristics of identified TA modules in MIP. This comprehensive exploration seeks to illuminate the contribution of TA modules in regulating virulence, habitat diversification, and the evolutionary pathogenicity of mycobacteria. The insights garnered from this investigation not only enhance our understanding of MIP's potential as a vaccine candidate but also hold promise in optimizing tuberculosis drug regimens for expedited recovery. [ABSTRACT FROM AUTHOR]

Genome-wide screening is a potent approach for comprehensively understanding the molecular mechanisms of biological phenomena. However, despite its widespread use in the past decades across various biological targets, its application to biochemical reactions with temporal and reversible biological outputs remains a formidable challenge. To uncover the molecular machinery underlying various biochemical reactions, we have recently developed the revival screening method, which combines flow cytometry–based cell sorting with library reconstruction from collected cells. Our refinements to the traditional genome-wide screening technique have proven successful in revealing the molecular machinery of biochemical reactions of interest. In this article, we elucidate the technical basis of revival screening, focusing on its application to CRISPR-Cas9 single guide RNA (sgRNA) library screening. Finally, we also discuss the future of genome-wide screening while describing recent achievements from in vitro and in vivo screening. [ABSTRACT FROM AUTHOR]

In response to the global threat posed by bacterial pathogens, which are the second leading cause of death worldwide, vaccine development is challenged by the diversity of bacterial serotypes and the lack of immunoprotection across serotypes. To address this, we introduce BacScan, a novel genome-wide technology for the rapid discovery of conserved highly immunogenic proteins (HIPs) across serotypes. Using bacterial-specific serum, BacScan combines phage display, immunoprecipitation, and next-generation sequencing to comprehensively identify all the HIPs in a single assay, thereby paving the way for the development of universally protective vaccines. Our validation of this technique with Streptococcus suis, a major pathogenic threat, led to the identification of 19 HIPs, eight of which conferred 20-100% protection against S. suis challenge in animal models. Remarkably, HIP 8455 induced complete immunity, making it an exemplary vaccine target. BacScan's adaptability to any bacterial pathogen positions it as a revolutionary tool that can expedite the development of vaccines with broad efficacy, thus playing a critical role in curbing bacterial transmission and slowing the march of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Potassium (K+) transporters, particularly high-affinity K+ transporters (HKT), are crucial for plant growth and abiotic stress response. These transporters regulate Na+ and K+ balance in the cells to prevent sodium toxicity in plant tissues. Although HKT genes have been extensively studied in various crops, there is a deficiency in their characterization and understanding of their role in wheat, particularly regarding their involvement in adaptive response to environmental stresses like salinity. In this study, several bioinformatics tools including web-based platforms and software applications were employed to identify, characterize and localize HKTs genes within the wheat genome. A greenhouse trial was also conducted to investigate the mRNA expression levels of some of the significant salt-responsive genes in wheat seedlings grown under different concentrations of NaCl (0, 200, and 400 mM). Genome-wide analysis, resulted in identification of 35 TaHKTs that are unevenly distributed on ten chromosomes. The majority of these transporters are localized on plasma membrane. Analysis of promoter regions showed that most of the TaHKT genes have abundant cis-elements that involved in hormones and abiotic stress responses. Gene expression analysis indicated a significant upregulation of TaHKT6 and TaSOS1 genes were found in wheat seedlings treated with 400 mM NaCl. Expression levels of TaNHX1, TaNHX2 and TaTVP1 genes were also higher in 200 mM and 400 mM NaCl-treated plants when compared to non-stressed plants. Overall, the results provide further evidence for the role of the TaHKT in wheat's stress response to elevated salt levels and pave the way for further functional research in K+ transporters in plants.

The introduction of noninvasive prenatal testing has resulted in substantial reductions to previously accepted false-positive rates of prenatal screening. Despite this, the possibility of false-positive results remains a challenging consideration in clinical practice, particularly considering the increasing uptake of genome-wide noninvasive prenatal testing, and the subsequent increased proportion of high-risk results attributable to various biological events besides fetal aneuploidy. Confined placental mosaicism, whereby chromosome anomalies exclusively affect the placenta, is perhaps the most widely accepted cause of false-positive noninvasive prenatal testing. There remains, however, a substantial degree of ambiguity in the literature pertaining to the clinical ramifications of confined placental mosaicism and its potential association with placental insufficiency, and consequentially adverse pregnancy outcomes including fetal growth restriction. Other causes of false-positive noninvasive prenatal testing include vanishing twin syndrome, in which the cell-free DNA from a demised aneuploidy-affected twin triggers a high-risk result, technical failures, and maternal origins of abnormal cell-free DNA such as uterine fibroids or unrecognized mosaicisms. Most concerningly, maternal malignancies are also a documented cause of false-positive screening results. In this review, we compile what is currently known about the various causes of false-positive noninvasive prenatal testing. [Display omitted] [ABSTRACT FROM AUTHOR]

Quinoxaline is a heterocyclic compound with a two-membered ring structure that undergoes redox cycling to produce toxic free radicals. It has antiviral, antibacterial, antifungal, and antitumor activities. However, the biological functions that are involved in mounting a response against the toxic effects of quinoxaline have not been investigated. Herein, we performed a genome-wide screen using the yeast haploid mutant collection and reported the identification of 12 mutants that displayed varying sensitivity towards quinoxaline. No mutant was recovered that showed resistance to quinoxaline. The quinoxaline-sensitive mutants were deleted for genes that encode cell cycle function, as well as genes that belong to other physiological pathways such as the vacuolar detoxification process. Three of the highly sensitive gene-deletion mutants lack the DDC1, DUN1, and MFT1 genes. While Ddc1 and Dun1 are known to perform roles in the cell cycle arrest pathway, the role of Mft1 remains unclear. We show that the mft1Δ mutant is as sensitive to quinoxaline as the ddc1Δ mutant. However, the double mutant ddc1Δ mft1Δ lacking the DDC1 and MFT1 genes, is extremely sensitive to quinoxaline, as compared to the ddc1Δ and mft1Δ single mutants. We further show that the mft1Δ mutant is unable to arrest in the G2/M phase in response to the drug. We conclude that Mft1 performs a unique function independent of Ddc1 in the cell cycle arrest pathway in response to quinoxaline exposure. This is the first demonstration that quinoxaline exerts its toxic effect likely by inducing oxidative DNA damage causing cell cycle arrest. We suggest that clinical applications of quinoxaline and its derivatives should entail targeting cancer cells with defective cell cycle arrest. [ABSTRACT FROM AUTHOR]

Quinoxaline is a heterocyclic compound with a two-membered ring structure that undergoes redox cycling to produce toxic free radicals. It has antiviral, antibacterial, antifungal, and antitumor activities. However, the biological functions that are involved in mounting a response against the toxic effects of quinoxaline have not been investigated. Herein, we performed a genome-wide screen using the yeast haploid mutant collection and reported the identification of 12 mutants that displayed varying sensitivity towards quinoxaline. No mutant was recovered that showed resistance to quinoxaline. The quinoxaline-sensitive mutants were deleted for genes that encode cell cycle function, as well as genes that belong to other physiological pathways such as the vacuolar detoxification process. Three of the highly sensitive gene-deletion mutants lack the DDC1, DUN1, and MFT1 genes. While Ddc1 and Dun1 are known to perform roles in the cell cycle arrest pathway, the role of Mft1 remains unclear. We show that the mft1Δ mutant is as sensitive to quinoxaline as the ddc1Δ mutant. However, the double mutant ddc1Δ mft1Δ lacking the DDC1 and MFT1 genes, is extremely sensitive to quinoxaline, as compared to the ddc1Δ and mft1Δ single mutants. We further show that the mft1Δ mutant is unable to arrest in the G2/M phase in response to the drug. We conclude that Mft1 performs a unique function independent of Ddc1 in the cell cycle arrest pathway in response to quinoxaline exposure. This is the first demonstration that quinoxaline exerts its toxic effect likely by inducing oxidative DNA damage causing cell cycle arrest. We suggest that clinical applications of quinoxaline and its derivatives should entail targeting cancer cells with defective cell cycle arrest.

ABSTRACTExtraintestinal pathogenic Escherichia coli (ExPEC) is an important zoonotic pathogen. Recently, ExPEC has been reported to be an emerging problem in pig farming. However, the mechanism of pathogenicity of porcine ExPEC remains to be revealed. In this study, we constructed a transposon (Tn) mutagenesis library covering Tn insertion in over 72% of the chromosome-encoded genes of a virulent and multi-drug resistant porcine ExPEC strain PCN033. By using a mouse infection model, a transposon-directed insertion site sequencing (TraDIS) assay was performed to identify in vivo fitness factors. By comparing the Tn insertion frequencies between the input Tn library and the recovered library from different organs, 64 genes were identified to be involved in fitness during systemic infection. 15 genes were selected and individual gene deletion mutants were constructed. The in vivo fitness was evaluated by using a competitive infection assay. Among them, ΔfimG was significantly outcompeted by the WT strain in vivo and showed defective adhesion to host cells. rfa which was involved in lipopolysaccharide biosynthesis was shown to be critical for in vivo fitness which may have resulted from its role in the resistance to serum killing. In addition, several metabolic genes including fepB, sdhC, fepG, gltS, dcuA, ccmH, ddpD, narU, glpD, malM, and yabL and two regulatory genes metJ and baeS were shown as important determinants of in vivo fitness of porcine ExPEC. Collectively, this study performed a genome-wide screening for in vivo fitness factors which will be important for understanding the pathogenicity of porcine ExPEC.

Insulin is essential in controlling blood glucose levels, and its synthesis and secretion have been well investigated. In contrast, how insulin secretory granules (ISGs) are degraded in pancreatic beta cells remains largely unknown. To clarify the mechanism, we constructed a fluorescent reporter detecting ISG degradation, where EGFP and mCherry are tandemly conjugated to a cytoplasmic region of ZnT8, an ISG membrane-localized protein. Depletion of serum and amino acid stimulated lysosomal ISG degradation detected with the reporter. Next, with MIN6 cells expressing Cas9 and the reporter, we investigated the involvement of conventional Atg5/7-dependent autophagy to show that it is dispensable for the ISG degradation process. Finally, we performed genome-wide screening by enriching the cells lacking the ISG degradation and showed that pathways regulating autophagy are not identified. These results suggest that alternative degradation in lysosomes, instead of conventional autophagy, may be involved in ISG degradation. • ・How intracellular insulin secretory granules are degraded remains largely unknown. • ・We constructed a ZnT8-mCherry-EGFP reporter to monitor the degradation. • ・The essential regulators of conventional autophagy are dispensable for the process. • ・Genome-wide screening neither identified pathways related to conventional autophagy. [ABSTRACT FROM AUTHOR]

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and ageing. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale. [ABSTRACT FROM AUTHOR]

Context: Red clover (Trifolium pratense L.) is an important legume forage in temperate agricultural zones. Evaluation of self- and cross-pollination fertility is important for setting up an effective breeding-program scale. However, the outcrossing rate of red clover under open-pollination conditions is not certain. Development of a reliable and time-saving marker system is needed to quantify and characterise outcrossing rates. Aim: We aimed to develop a duplex PCR-based protocol based on a genome-wide simple sequence repeat (SSR) screen, and to determine the outcrossing rate of red clover under open-pollination environments. Methods: We screened 209 SSR markers with pooled DNA samples of 60 plants from 20 red clover accessions, and selected 185 SSR markers that produced clear scorable bands for testing with 24 individual DNA samples to determine polymorphism. We selected 70 primer pairs, and then assembled a core set of 24 loci into 12 sets of duplex markers, which were used for outcrossing behaviour analysis of 60 maternal parents and their respective 22 half-sib progenies. Key results: Mean polymorphic information content (PIC) for the 70 markers was 0.490 (range 0.117–0.878). Minimum, mean and maximum PIC values for the 24 markers constituting the 12 duplexes were 0.226, 0.594 and 0.781, respectively. The outcrossing rate was identified as 99.4% for red clover in a natural environment. Conclusion: We successfully developed a duplex SSR-based PCR protocol consisting of 24 markers. This SSR system was applied to determine the outcrossing rate of red clover in a natural environment. Red clover is an important legume forage and green manure crop in temperate agricultural zones and has been considered an outcrossing species, although its outcrossing rate under open-pollination conditions is not certain. In this study, the outcrossing rate of diploid red clover in a natural environment was calculated for the first time, using microsatellite molecular markers. These results are valuable for future studies involving breeding research in red clover. [ABSTRACT FROM AUTHOR]

Context: Red clover (Trifolium pratense L.) is an important legume forage in temperate agricultural zones. Evaluation of self- and cross-pollination fertility is important for setting up an effective breeding-program scale. However, the outcrossing rate of red clover under open-pollination conditions is not certain. Development of a reliable and time-saving marker system is needed to quantify and characterise outcrossing rates. Aim: We aimed to develop a duplex PCR-based protocol based on a genome-wide simple sequence repeat (SSR) screen, and to determine the outcrossing rate of red clover under open-pollination environments. Methods: We screened 209 SSR markers with pooled DNA samples of 60 plants from 20 red clover accessions, and selected 185 SSR markers that produced clear scorable bands for testing with 24 individual DNA samples to determine polymorphism. We selected 70 primer pairs, and then assembled a core set of 24 loci into 12 sets of duplex markers, which were used for outcrossing behaviour analysis of 60 maternal parents and their respective 22 half-sib progenies. Key results: Mean polymorphic information content (PIC) for the 70 markers was 0.490 (range 0.117–0.878). Minimum, mean and maximum PIC values for the 24 markers constituting the 12 duplexes were 0.226, 0.594 and 0.781, respectively. The outcrossing rate was identified as 99.4% for red clover in a natural environment. Conclusion: We successfully developed a duplex SSR-based PCR protocol consisting of 24 markers. This SSR system was applied to determine the outcrossing rate of red clover in a natural environment. Red clover is an important legume forage and green manure crop in temperate agricultural zones and has been considered an outcrossing species, although its outcrossing rate under open-pollination conditions is not certain. In this study, the outcrossing rate of diploid red clover in a natural environment was calculated for the first time, using microsatellite molecular markers. These results are valuable for future studies involving breeding research in red clover. [ABSTRACT FROM AUTHOR]

Non-invasive prenatal testing (NIPT) using cell-free DNA can detect fetal chromosomal anomalies with high clinical sensitivity and specificity. In approximately 0.1% of clinical cases, the NIPT result and a subsequent diagnostic karyotype are discordant. Here we report a case of a 32-year-old pregnant patient with a 44.1 Mb duplication on the short arm of chromosome 4 detected by NIPT at 12 weeks' gestation. Amniocentesis was carried out at 18 weeks' gestation, followed by conventional and molecular cytogenetic analysis on cells from the amniotic fluid. SNP array analysis found a de novo deletion of 1.2 Mb at chromosome 4, and this deletion was found to be near the critical region of the Wolf-Hirschhorn syndrome. A normal 46,XY karyotype was identified by G-banding analysis. The patient underwent an elective termination and molecular investigations on tissues from the fetus, and the placenta confirmed the presence of type VI true fetal mosaicism. It is important that a patient receives counselling following a high-risk call on NIPT, with appropriate diagnostic analysis advised before any decisions regarding the pregnancy are taken. This case highlights the importance of genetic counselling following a high-risk call on NIPT, especially in light of the increasing capabilities of NIPT detection of sub-chromosomal deletions and duplications. [ABSTRACT FROM AUTHOR]

Objective To identify new human lung cancer suppressors in a genome-wide screening library during neoplastic transformation of normal human lung epithelial cells using CRISPR/Cas9-mediated genome editing, and preliminarily validate the Results in non-small cell lung cancer (NSCLC) sample databases. Methods Human normal lung epithelial BEAS-2B cell line with stable expression of Cas9 protein was constructed, and then infected with the Brunello lentiviral library encompassing 77 441 single-guide RNAs (sgRNAs) targeting 19 114 genes. Then the obtained cells were transplanted subcutaneously into nude mice. Genomic DNA of tumor cells (Tumor) and pre-transplantation cells (Input) were extracted. The extracted DNA and plasmid library were amplified for deep sequencing. By analyzing the proportion of sgRNA reads in Tumor cells compared to the Input cells and the Log Fold Change (LFC) of sgRNA reads, tumor suppressor genes that can induce oncogenic transformation of BEAS-2B cells after knockout were screened, and preliminary validation were performed using public data platforms. Results Library infections in BEAS-2B cells stably expressing Cas9 protein and subcutaneous tumorigenesis in nude mice were successfully established. Only cells infected with the library formed tumors at the inoculation sites. Sequencing Results showed that only a small part of sgRNAs were detected in tumor cells. Here, 38 genes with sgRNA reads accounting for more than 1% of the total reads or with 2 or more significantly enriched sgRNA were selected as effective candidate human lung tumor suppressor genes, including known tumor suppressor genes such as NF2 and PTEN etc, and some candidate tumor suppressor genes (AP2M1 and PSENEN) had not been previously reported in lung cancer. Enrichment analysis revealed that these candidate tumor suppressor genes were enriched in key biological pathways involved in cancer, such as Notch and Hippo signaling pathways. It is confirmed in the clinical NSCLC sample databases that 38 candidate tumor suppressor genes were mutated in NSCLC samples. Among the 17 genes with mutation rates greater than 2%, 14 genes were co-mutated with KRAS or TP53, and 3 ones were associated with the prognosis of NSCLC patients. There were 30 candidate genes with their expression in the samples of NSCLC different from those in para-tumor normal tissues, and the expression of 17 candidate genes were correlated with the prognosis of NSCLC patients. Conclusion By using an in vivo genome-wide CRISPR/Cas9 screening library, 38 human lung cancer candidate suppressor genes are identified and preliminarily verified in the clinical NSCLC sample databases.

Alzheimer's disease (AD) is known to be caused by amyloid β-peptide (Aβ) misfolded into β-sheets, but this knowledge has not yet led to treatments to prevent AD. To identify novel molecular players in Aβ toxicity, we carried out a genome-wide screen in Saccharomyces cerevisiae, using a library of 5154 gene knock-out strains expressing Aβ1–42. We identified 81 mammalian orthologue genes that enhance Aβ1–42 toxicity, while 157 were protective. Next, we performed interactome and text-mining studies to increase the number of genes and to identify the main cellular functions affected by Aβ oligomers (oAβ). We found that the most affected cellular functions were calcium regulation, protein translation and mitochondrial activity. We focused on SURF4, a protein that regulates the store-operated calcium channel (SOCE). An in vitro analysis using human neuroblastoma cells showed that SURF4 silencing induced higher intracellular calcium levels, while its overexpression decreased calcium entry. Furthermore, SURF4 silencing produced a significant reduction in cell death when cells were challenged with oAβ1–42, whereas SURF4 overexpression induced Aβ1–42 cytotoxicity. In summary, we identified new enhancer and protective activities for Aβ toxicity and showed that SURF4 contributes to oAβ1–42 neurotoxicity by decreasing SOCE activity. [ABSTRACT FROM AUTHOR]

High-throughput genetic screening serves as an indispensable approach for deciphering gene functions and the intricate relationships between phenotypes and genotypes. The CRISPR/Cas9 system, with its ability to precisely edit genomes on a large scale, has revolutionized the field by enabling the construction of comprehensive genomic libraries. This technology has become a cornerstone for genome-wide screenings in disease research. This review offers a comprehensive examination of how CRISPR/Cas9-based genetic screening has been leveraged to uncover genes that play a role in disease mechanisms, focusing on areas such as cancer development and viral replication processes. The insights presented in this review hold promise for the development of novel therapeutic strategies and precision medicine approaches., Competing Interests: Competing interests The authors declare no conflicts of interest relevant to this article., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mapping the essential pathways for neuronal differentiation can uncover new therapeutics and models for neurodevelopmental disorders. We thus utilized a genome-wide loss-of-function library in haploid human embryonic stem cells, differentiated into caudal neuronal cells. We show that essential genes for caudal neurogenesis are enriched for secreted and membrane proteins and that a large group of neurological conditions, including neurodegenerative disorders, manifest early neuronal phenotypes. Furthermore, essential transcription factors are enriched with homeobox (HOX) genes demonstrating synergistic regulation and surprising non-redundant functions between HOXA6 and HOXB6 paralogs. Moreover, we establish the essentialome of imprinted genes during neurogenesis, demonstrating that maternally expressed genes are non-essential in pluripotent cells and their differentiated germ layers, yet several are essential for neuronal development. These include Beckwith-Wiedemann syndrome- and Angelman syndrome-related genes, for which we suggest a novel regulatory pathway. Overall, our work identifies essential pathways for caudal neuronal differentiation and stage-specific phenotypes of neurological disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective Genetic polymorphisms might serve as useful prognostic markers for the timely diagnosis of RA. The purpose of this study was to identify genomic factors predictive of the occurrence of interstitial lung disease (ILD) in RA by performing a genome-wide association study of genetic variants, including single nucleotide polymorphisms (SNPs). Methods The study population included 306 RA patients. All patients were treated with conventional DMARDs, including 6–16 mg MTX per week. Clinical data and venous blood samples were collected from all patients before administration of DMARDs. A total of 278 347 SNPs were analysed to determine their association with ILD occurrence. Results Several SNPs were strongly associated with ILD occurrence (P  < 10−5). rs6578890, which is located on chromosome 11 in the intronic region of the gene encoding tyrosine phosphatase receptor type F polypeptide-interacting protein-binding protein 2 (PPFIBP2), showed the strongest association with ILD occurrence (odds ratio 4.32, P  = 10−7.93). Conclusion PPFIBP2 could be a useful genetic marker for occurrence of interstitial pneumonia in RA patients and might help to identify the risk of ILD occurrence before RA treatment, thereby improving patient outcomes. [ABSTRACT FROM AUTHOR]

Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identification of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact filtration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s flanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two flanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the field of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to efficiently localize genomic transposon integrations, which was confirmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

Abstract Background Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. Methods We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student’s t-test from at least four independent experiments. Results We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. Conclusions We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.

Sodium bicarbonate (NaHCO3) is an important inorganic salt. It is not only widely used in industrial production and daily life, but is also the main stress in alkaline saline soil. NaHCO3 has a strong ability to inhibit the growth of fungi in both natural environment and daily application. However, the mechanism by which fungi respond to NaHCO3 stress is not fully understood. To further clarify the toxic mechanisms of NaHCO3 stress and identify the specific cellular genes and pathways involved in NaHCO3 resistance, we performed genome-wide screening with NaHCO3 using a Saccharomyces cerevisiae deletion mutant library. A total of 33 deletion mutants with NaHCO3 sensitivity were identified. Compared with wild-type strains, these mutants had significant growth defects in the medium containing NaHCO3. Bioinformatics analysis found that the corresponding genes of these mutants are mainly enriched in the cell cycle, mitophagy, cell wall integrity, and signaling pathways. Further study using transcriptomic analysis showed that 309 upregulated and 233 downregulated genes were only responded to NaHCO3 stress, when compared with yeast transcriptomic data under alkaline and saline stress. Upregulated genes were mainly concentrated in amino acid metabolism, steroid biosynthesis, and cell wall, while downregulated genes were enriched in various cellular metabolisms. In summary, we have identified the cellular pathways and key genes that respond to NaHCO3 stress in the whole genome, providing resource and direction for understanding NaHCO3 toxicity and cellular resistance mechanisms. [ABSTRACT FROM AUTHOR]

Sodium bicarbonate (NaHCO3) is an important inorganic salt. It is not only widely used in industrial production and daily life, but is also the main stress in alkaline saline soil. NaHCO3 has a strong ability to inhibit the growth of fungi in both natural environment and daily application. However, the mechanism by which fungi respond to NaHCO3 stress is not fully understood. To further clarify the toxic mechanisms of NaHCO3 stress and identify the specific cellular genes and pathways involved in NaHCO3 resistance, we performed genome-wide screening with NaHCO3 using a Saccharomyces cerevisiae deletion mutant library. A total of 33 deletion mutants with NaHCO3 sensitivity were identified. Compared with wild-type strains, these mutants had significant growth defects in the medium containing NaHCO3. Bioinformatics analysis found that the corresponding genes of these mutants are mainly enriched in the cell cycle, mitophagy, cell wall integrity, and signaling pathways. Further study using transcriptomic analysis showed that 309 upregulated and 233 downregulated genes were only responded to NaHCO3 stress, when compared with yeast transcriptomic data under alkaline and saline stress. Upregulated genes were mainly concentrated in amino acid metabolism, steroid biosynthesis, and cell wall, while downregulated genes were enriched in various cellular metabolisms. In summary, we have identified the cellular pathways and key genes that respond to NaHCO3 stress in the whole genome, providing resource and direction for understanding NaHCO3 toxicity and cellular resistance mechanisms.

Reverse genetics offers precise functional insights into genes through the targeted manipulation of gene expression followed by phenotypic assessment. While these approaches have proven effective in model organisms such as Saccharomyces cerevisiae, large-scale genetic manipulations in human cells were historically unfeasible due to methodological limitations. However, recent advancements in functional genomics, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-based screening technologies and next-generation sequencing platforms, have enabled pooled screening technologies that allow massively parallel, unbiased assessments of biological phenomena in human cells. This review provides a comprehensive overview of cutting-edge functional genomic screening technologies applicable to human cells, ranging from short hairpin RNA screens to modern CRISPR screens. Additionally, we explore the integration of CRISPR platforms with single-cell approaches to monitor gene expression, chromatin accessibility, epigenetic regulation, and chromatin architecture following genetic perturbations at the omics level. By offering an in-depth understanding of these genomic screening methods, this review aims to provide insights into more targeted and effective strategies for genomic research and personalized medicine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Conjugal transfer is a major driving force of genetic exchange in eubacteria, and the system in IncP1-type broad-host-range plasmids transfers DNA even to eukaryotes and archaea in a process known as trans-kingdom conjugation (TKC). Although conjugation factors encoded on plasmids have been extensively analyzed, those on the donor chromosome have not. To identify the potential conjugation factor(s), a genome-wide survey on a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection) as donors to Saccharomyces cerevisiae recipients was performed using a conjugal transfer system mediated by the type IV secretion system (T4SS) of the IncP1α plasmid. Out of 3,884 mutants, three mutants (ΔfrmR, ΔsufA, and ΔiscA) were isolated, which showed an increase by one order of magnitude in both E. coli–E. coli and E. coli–yeast conjugations without an increase in the mRNA accumulation level for the conjugation related genes examined. The double-knockout mutants for these genes (ΔfrmRΔsufA and ΔiscAΔfrmR) did not show synergistic effects on the conjugation efficiency, suggesting that these factors affect a common step in the conjugation machinery. The three mutants demonstrated increased conjugation efficiency in IncP1β-type but not in IncN- and IncW-type broad-host-range plasmid transfers, and the homologous gene knockout mutants against the three genes in Agrobacterium tumefaciens also showed increased TKC efficiency. These results suggest the existence of a specific regulatory system in IncP1 plasmids that enables the control of conjugation efficiency in different hosts, which could be utilized for the development of donor strains as gene introduction tools into bacteria, eukaryotes, and archaea.

Background: Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages.Methods: We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student's t-test from at least four independent experiments.Results: We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production.Conclusions: We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy. [ABSTRACT FROM AUTHOR]

Sensory systems allow for the transfer of environmental stimuli into internal cues that can alter physiology and behavior. Many studies of visual systems focus on opsins to compare spectral sensitivity among individuals, populations, and species living in different lighting environments. This requires an understanding of the cone opsins, which can be numerous. The bluefin killifish is a good model for studying the interaction between environments and visual systems as they are found in both clear springs and tannin-stained swamps. We conducted a genome-wide screening and demonstrated that the bluefin killifish has 9 cone opsins: 1 SWS1 (354 nm), 2 SWS2 (SWS2B : 359 nm, SWS2A : 448 nm), 2 RH2 (RH2-2 : 476 nm, RH2-1 : 537 nm), and 4 LWS (LWS-1 : 569 nm, LWS-2 : 524 nm, LWS-3 : 569 nm, LWS-R : 560 or 569 nm). These 9 cone opsins were located on 4 scaffolds. One scaffold contained the 2 SWS2 and 3 of the 4 LWS opsins in the same syntenic order as found in other cyprinodontoid fishes. We also compared opsin expression in larval and adult killifish under clear water conditions, which mimic springs. Two of the newly discovered opsins (LWS-2 and LWS-3) were expressed at low levels (<0.2%). Whether these opsins make meaningful contributions to visual perception in other contexts (i.e. swamp conditions) is unclear. In contrast, there was an ontogenetic change from using LWS-R to LWS-1 opsin. Bluefin killifish adults may be slightly more sensitive to longer wavelengths, which might be related to sexual selection and/or foraging preferences. [ABSTRACT FROM AUTHOR]

Conjugal transfer is a major driving force of genetic exchange in eubacteria, and the system in IncP1-type broad-host-range plasmids transfers DNA even to eukaryotes and archaea in a process known as trans -kingdom conjugation (TKC). Although conjugation factors encoded on plasmids have been extensively analyzed, those on the donor chromosome have not. To identify the potential conjugation factor(s), a genome-wide survey on a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection) as donors to Saccharomyces cerevisiae recipients was performed using a conjugal transfer system mediated by the type IV secretion system (T4SS) of the IncP1α plasmid. Out of 3,884 mutants, three mutants (Δ frmR , Δ sufA , and Δ iscA) were isolated, which showed an increase by one order of magnitude in both E. coli – E. coli and E. coli –yeast conjugations without an increase in the mRNA accumulation level for the conjugation related genes examined. The double-knockout mutants for these genes (Δ frmR Δ sufA and Δ iscA Δ frmR) did not show synergistic effects on the conjugation efficiency, suggesting that these factors affect a common step in the conjugation machinery. The three mutants demonstrated increased conjugation efficiency in IncP1β-type but not in IncN- and IncW-type broad-host-range plasmid transfers, and the homologous gene knockout mutants against the three genes in Agrobacterium tumefaciens also showed increased TKC efficiency. These results suggest the existence of a specific regulatory system in IncP1 plasmids that enables the control of conjugation efficiency in different hosts, which could be utilized for the development of donor strains as gene introduction tools into bacteria, eukaryotes, and archaea. [ABSTRACT FROM AUTHOR]

In response to the global threat posed by bacterial pathogens, which are the second leading cause of death worldwide, vaccine development is challenged by the diversity of bacterial serotypes and the lack of immunoprotection across serotypes. To address this, we introduce BacScan, a novel genome-wide technology for the rapid discovery of conserved highly immunogenic proteins (HIPs) across serotypes. Using bacterial-specific serum, BacScan combines phage display, immunoprecipitation, and next-generation sequencing to comprehensively identify all the HIPs in a single assay, thereby paving the way for the development of universally protective vaccines. Our validation of this technique with Streptococcus suis , a major pathogenic threat, led to the identification of 19 HIPs, eight of which conferred 20-100% protection against S. suis challenge in animal models. Remarkably, HIP 8455 induced complete immunity, making it an exemplary vaccine target. BacScan's adaptability to any bacterial pathogen positions it as a revolutionary tool that can expedite the development of vaccines with broad efficacy, thus playing a critical role in curbing bacterial transmission and slowing the march of antimicrobial resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dong, Zhang, Yang, Zhao, Miao, Pei, Qin, Jing, Wen, Zhang and Tao.)

Abstract Extracellular interactions between cell surface receptors are necessary for signaling and adhesion but identifying them remains technically challenging. We describe a cell-based genome-wide approach employing CRISPR activation to identify receptors for a defined ligand. We show receptors for high-affinity antibodies and low-affinity ligands can be unambiguously identified when used in pools or as individual binding probes. We apply this technique to identify ligands for the adhesion G-protein-coupled receptors and show that the Nogo myelin-associated inhibitory proteins are ligands for ADGRB1. This method will enable extracellular receptor-ligand identification on a genome-wide scale.

The latent HIV‐1 reservoir is a major barrier to viral eradication. However, our understanding of how HIV‐1 establishes latency is incomplete. Here, by performing a genome‐wide CRISPR‐Cas9 knockout library screen, we identify phosphatidylethanolamine‐binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), as a novel gene inducing HIV latency. Depletion of PEBP1 leads to the reactivation of HIV‐1 in multiple models of latency. Mechanistically, PEBP1 de‐phosphorylates Raf1/ERK/IκB and IKK/IκB signaling pathways to sequestrate NF‐κB in the cytoplasm, which transcriptionally inactivates HIV‐1 to induce latency. Importantly, the induction of PEBP1 expression by the green tea compound epigallocatechin‐3‐gallate (EGCG) prevents latency reversal by inhibiting nuclear translocation of NF‐κB, thereby suppressing HIV‐1 transcription in primary CD4+ T cells isolated from patients receiving antiretroviral therapy (ART). These results suggest a critical role for PEBP1 in the regulation of upstream NF‐κB signaling pathways governing HIV transcription. Targeting of this pathway could be an option to control HIV reservoirs in patients in the future. Synopsis: The latent HIV‐1 reservoir prevents efficient viral eradication. The phosphatase PEBP1 plays a critical role in the regulation of NF‐κB‐dependent pathways governing HIV‐1 transcription, and its targeting could control HIV reservoirs in patients. Depletion of PEBP1 leads to the reactivation of HIV‐1 in multiple HIV latency models.EGCG induces PEBP1 protein expression and prevents latency reversal in patients receiving antiretroviral therapy.PEBP1 is induced by IFN signaling during HIV‐1 infection and promotes HIV latency in primary CD4+ T cells. [ABSTRACT FROM AUTHOR]

As promising biomarkers and therapy targets, microRNAs (miRNAs) are involved in various physiological and tumorigenic processes. Genetic variants in miRNA‐binding sites can lead to dysfunction of miRNAs and contribute to disease. However, systematic investigation of the miRNA‐related single nucleotide polymorphisms (SNPs) for pancreatic cancer (PC) risk remains elusive. We performed integrative bioinformatics analyses to select 31 SNPs located in miRNA‐target binding sites using the miRNASNP v2.0, a solid database providing miRNA‐related SNPs for genetic research, and investigated their associations with risk of PC in two large case‐control studies totally including 1847 cases and 5713 controls. We observed that the SNP rs3802266 is significantly associated with increased risk of PC (odds ratio (OR) = 1.21, 95% confidence intervals (CI) = 1.11‐1.31, P = 1.29E‐05). Following luciferase reporter gene assays show that rs3802266‐G creates a stronger binding site for miR‐181a‐2‐3p in 3′ untranslated region (3′UTR) of the gene ZHX2. Expression quantitative trait loci (eQTL) analysis suggests that ZHX2 expression is lower in individuals carrying rs3802266‐G with increased PC risk. In conclusion, our findings highlight the involvement of miRNA‐binding SNPs in PC susceptibility and provide new clues for PC carcinogenesis. [ABSTRACT FROM AUTHOR]

Wheat sharp eyespot is a soil-borne disease caused by Rhizoctonia cerealis , which occurs in many countries worldwide and significantly reduces the yield. Thaumatin-like protein (TLP), also known as PR5, is a member of the pathogen response protein family and plays an essential role in plant resistance to pathogen infection. In this study, 131 TaTLP genes were identified from the wheat genome, of which 38 TaTLPs were newly discovered. The TaTLP gene family contains many tandem duplications and fragment duplications, which is a major pathway for gene amplification. Besides, we also analyzed the physicochemical properties, gene structure and promoter cis-acting regulatory elements of all the TaTLP genes. In addition, the expression patterns of nine TaTLPs in response to R. cerealis were analyzed by RT-qPCR. Six TaTLP proteins expressed in vitro had no significant inhibitory effect on R. cerealis , suggesting that these TaTLP proteins may function in other ways. Finally, we performed gene silencing of TaTLP44 in wheat, which increased the expression of some defense-associated genes and improved resistance to R. cerealis. In summary, we systematically analyzed TaTLP family members and demonstrated that TaTLP44 negatively regulates the resistance to R. cerealis by controlling expression of defense-associated genes. These results provide new insights into the functional mechanism of TaTLP proteins. • 131 TaTLP genes in wheat were identified and systematically analyzed. • The R. cerealis responsive TaTLP genes were identified from transcriptomic data and their expression patterns were verified. • Subcellular localization indicated that the 6 TaTLPs may function in cell membrane, cell wall or extracellular region. • TaTLP44 negatively regulates wheat resistance to R. cerealis by affecting the expression of defensive genes. [ABSTRACT FROM AUTHOR]

The conjugal transfer is a major driving force in the spread of antibiotic resistance genes. Nevertheless, an effective approach has not yet been developed to target conjugal transfer to prevent the acquisition of antibiotic resistance by this mechanism. This study aimed to identify potential targets for plasmid transfer blockade by isolating mutants defective in the completion of the acquisition of antibiotic resistance via conjugal transfer. We performed genome-wide screening by combining an IncP1α-type broad host range plasmid conjugation system with a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection; 3884 mutants). We followed a six-step screening procedure to identify the mutants showing conjugation deficiency precisely. No mutants defective in the conjugal transfer were isolated, strongly suggesting that E. coli cannot escape from being a recipient organism for P1α plasmid transfer. However, several mutants with low viability were identified, as well as mutants defective in establishing resistance to chloramphenicol, which was used for transconjugant selection. These results suggest that developing drugs capable of inhibiting the establishment of antibiotic resistance is a better approach than attempting to prevent the conjugal transfer to block the spread of antibiotic resistance genes. Our screening system based on the IncP1α-type plasmid transfer can be extended to isolation of target genes for other drugs. This study could be the foundation for further research to understand its underlying molecular mechanism through functional analysis of the identified genes.

ABSTRACT A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp. Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-Kp. IMPORTANCE Klebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae. This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine.

The use of oxidizing agents is one of the most favorable approaches to kill bacteria in daily life. However, bacteria have been evolving to survive in the presence of different oxidizing agents. In this study, we aimed to obtain a comprehensive list of genes whose expression can make Escherichiacoli cells resistant to different oxidizing agents. For this purpose, we utilized the ASKA library and performed a genome-wide screening of ~4200 E. coli genes. Hydrogen peroxide (H2O2) and hypochlorite (HOCl) were tested as representative oxidizing agents in this study. To further validate our screening results, we used different E. coli strains as host cells to express or inactivate selected resistance genes individually. More than 100 genes obtained in this screening were not known to associate with oxidative stress responses before. Thus, this study is expected to facilitate both basic studies on oxidative stress and the development of antibacterial agents.

The conjugal transfer is a major driving force in the spread of antibiotic resistance genes. Nevertheless, an effective approach has not yet been developed to target conjugal transfer to prevent the acquisition of antibiotic resistance by this mechanism. This study aimed to identify potential targets for plasmid transfer blockade by isolating mutants defective in the completion of the acquisition of antibiotic resistance via conjugal transfer. We performed genome-wide screening by combining an IncP1α-type broad host range plasmid conjugation system with a comprehensive collection of Escherichia coli gene knockout mutants (Keio collection; 3884 mutants). We followed a six-step screening procedure to identify the mutants showing conjugation deficiency precisely. No mutants defective in the conjugal transfer were isolated, strongly suggesting that E. coli cannot escape from being a recipient organism for P1α plasmid transfer. However, several mutants with low viability were identified, as well as mutants defective in establishing resistance to chloramphenicol, which was used for transconjugant selection. These results suggest that developing drugs capable of inhibiting the establishment of antibiotic resistance is a better approach than attempting to prevent the conjugal transfer to block the spread of antibiotic resistance genes. Our screening system based on the IncP1α-type plasmid transfer can be extended to isolation of target genes for other drugs. This study could be the foundation for further research to understand its underlying molecular mechanism through functional analysis of the identified genes. [ABSTRACT FROM AUTHOR]

Complex human diseases such as metabolic disorders, cancer, neurodegenerative diseases, and mitochondrial dysfunctions arise from the biochemical or genetic defects in various cellular processes. Therefore, it is important to understand which metabolic processes are affected by which cellular impairment. Because genome-wide screening of mutant collections (haploid/diploid deletion library) provides important clues for the understanding of conserved biological processes and for finding potential target genes, we screened the haploid mutant collection of Schizosaccharomyces pombe with wortmannin that inhibits phosphatidylinositol-3-kinase signaling. Using genome-wide screening, we determined that 52 mutants were resistant to this chemical. When 52 genes that are deleted in these mutants were grouped in 41 different biological processes, we found that 37 of them have human orthologues and 4 genes were associated with human metabolic disorders. In addition, when we examined the pathways in which these 52 genes function, we determined that 9 genes were related to phosphorylation process. These results might provide new insights for better understanding of certain human diseases. [ABSTRACT FROM AUTHOR]

During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311–342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311–347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

The emergence of the technology-critical-element yttrium as a contaminant in the environment raises concern regarding its toxicological impact on living organisms. The molecular mechanisms underlying yttrium toxicity must be delineated. We considered the genomic phenotyping of a mutant collection of Saccharomyces cerevisiae to be of particular interest to decipher key cellular pathways involved either in yttrium toxicity or detoxification mechanisms. Among the 4733 mutants exposed to yttrium, 333 exhibited modified growth, of which 56 were sensitive and 277 were resistant. Several functions involved in yttrium toxicity mitigation emerged, primarily vacuolar acidification and retrograde transport. Conversely, functional categories overrepresented in the yttrium toxicity response included cytoskeleton organization and endocytosis, protein transport and vesicle trafficking, lipid metabolism, as well as signaling pathways. Comparison with similar studies carried out using other metals and stressors showed a response pattern similar to nickel stress. One third of the identified mutants highlighted peculiar cellular effects triggered by yttrium, specifically those affecting the pheromone-dependent signaling pathway or sphingolipid metabolic processes. Taken together, these data emphasize the role of the plasma membrane as a hotspot for yttrium toxicity. The up-to-now lack of data concerning yttrium toxicity at the cellular and molecular levels makes this pioneer study using the model S. cerevisiae an excellent first basis for the assessment of yttrium toxicity toward eukaryotes.

Drugs called bisphosphonates are used to treat a range of bone diseases, but how do they reach the enzymes that are their target?

Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here, we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

The increasing use of lithium (Li) in new technologies raises the question of its impact on living microorganisms. In the present study, we aimed to identify putative Li targets and resistance mechanisms in the yeast model Saccharomyces cerevisiae using a deletomic approach based on the screening of a collection of 4733 knockout mutants under Li exposure. This screening highlighted 60 mutants resistant to Li and 124 mutants sensitive to Li. Through functional enrichment analyses, transport systems were identified as playing a central role in cell resistance to toxic concentrations of Li. In contrast, the AKT/protein kinase B family, signal transduction or cell communication were identified as potential toxic targets of Li. The majority of the mutants with a Li-sensitive phenotype were also sensitive to other alkali and alkaline earth metals, whereas the Li-resistance phenotype was mostly resistant to Na but poorly resistant to other metals. A comparison with the results of deletomics studies carried out in the presence of other metals highlighted Li-specific phenotypes. Three genes (NAM7, NMD2, UPF3) of the nonsense-mediated decay pathway were specifically involved in resistance to Li. In contrast, mutants with the NCA2, SPT20, GCN5, YOR376W, YPK3, and DCW1 genes deleted were specifically resistant to Li. These genes encode various functions from putative mannosidase to constitution of the Spt-Ada-Gcn5 acetyltransferase complex. This work provides a better understanding of potential specific resistance mechanisms and cellular targets of Li in yeast., (© The Author(s) 2023. Published by Oxford University Press.)

Autophagy is a highly conserved physiological process that maintains cellular homeostasis by recycling cellular contents. Selective autophagy is based on the specificity of cargo recognition and has been implicated in various human diseases, including neurodegenerative diseases and cancer. Selective autophagy receptors and modulators play key roles in this process. Identifying these receptors and modulators and their roles is critical for understanding the machinery and physiological function of selective autophagy and providing therapeutic value for diseases. Using modern researching tools and novel screening technologies, an increasing number of selective autophagy receptors and modulators have been identified. A variety of Strategies and approaches, including protein-protein interactions (PPIs)-based identification and genome-wide screening, have been used to identify selective autophagy receptors and modulators. Understanding the strengths and challenges of these approaches not only promotes the discovery of even more such receptors and modulators but also provides a useful reference for the identification of regulatory proteins or genes involved in other cellular mechanisms. In this review, we summarize the functions, disease association, and identification strategies of selective autophagy receptors and modulators., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Abstract Background The parasite Leishmania panamensis is the main cause of leishmaniasis in Panama. The disease is largely uncontrolled, with a rising incidence and no appropriate control measures. While microsatellites are considered some of the best genetic markers to study population genetics and molecular epidemiology in these and other parasites, none has been developed for L. panamensis. Findings Here we have developed and tested a new panel of microsatellites for this species, based on high-throughput genome-wide screening. The new set of microsatellites is composed of seventeen loci, mainly spanning trinucleotide or longer motifs. We have evaluated the sensitivity and specificity of the panel based on a sample of 27 isolates obtained from cutaneous leishmaniasis patients from central Panama and also several reference species from both L. (Leishmania) and L. (Viannia) subgenera. The genetic equilibrium was assessed both intra- and inter-loci, while the reproductive mode was evaluated using several tests. The new SSR panel shows high polymorphism and sensitivity, as well as good specificity. The preliminary data described here for L. panamensis suggest extensive departure from Hardy-Weinberg proportions, significant linkage disequilibrium and strong deficit of heterozygotes. Several recombination tests involving multilocus linkage disequilibrium and a phylogenetic approach allowed rejection of frequent recombination in our dataset. Conclusions The genome-wide strategy described here proved to be useful to identify and test new polymorphic SSR loci in Leishmania. The new panel of polymorphic microsatellites is a valuable contribution to the existing molecular markers for the study of genetic structure and other aspects of this important species.