Genetic trans-complementation of L-protease fails to rescue the infectious foot-and-mouth disease virus from the Lbpro defective genome.

Outbreaks of the foot-and-mouth disease (FMD) have major economic impact on the global livestock industry by affecting the animal health and product safety. L-protease, a non-structural protein of FMDV, is a papain-like cysteine proteinase involved in viral protein processing as well as cleavage of host proteins for promoting the virus growth. FMDV synthesizes two forms of leader proteinase, Lpro (Labpro and Lbpro), where the deletion of Labpro is lethal and Lbpro deletion is reported to be attenuated. Defective replicons have been used by trans-complementing the deleted gene to produce one time replicating virus; thus, the bio-safety procedure can be compromised in the production units. Attempts are made to rescue of ΔLbproFMDV Asia1 virus by co-expressing the Lbpro protein carried in pcDNA plasmid. Mutant FMDV cDNA, pAsia-ΔLbpro, was constructed by PCR mediated mutagenesis using inverse primers. Transfection of BHK-21 cells with in-vitro transcribed RNA from the constructs failed to produce an infective mutant FMDV. Genetic trans-complementation of the Lbpro, which was done by co-transfecting the pcDNALbpro plasmid DNA along with the pAsia-ΔLbpro RNA in BHK-21 cells also failed to produce viable virus. Expression experiments of reporter genes and indirect immune-fluorescence confirmed the production of the viral proteins in wild type FMDV pAsiaWT; however, it was absent in the pAsia-ΔLbpro indicating that the leaderless virus was unable to produce infectious progeny and infect the cells. Failure to produce virus either by Lbpro deleted mutant clone or by genetic complementation suggests little chance of reversion of the disabled virus with large deletions of FMDV genome.

Repurposing endogenous Type I-D CRISPR-Cas system for genome editing in Synechococcus sp. PCC7002.

Synechococcus sp. PCC7002 has been considered as a photosynthetic chassis for the conversion of CO2 into biochemicals through genetic modification. However, conventional genetic manipulation techniques prove inadequate for comprehensive genetic modifications in this strain. Here, we present the development of a genome editing tool tailored for S. PCC7002, leveraging its endogenous type I-D CRISPR-Cas system. Utilizing this novel tool, we successfully deleted the glgA1 gene and iteratively edited the genome to obtain a double mutant of glgA1 and glgA2 genes. Additionally, large DNA fragments encompassing the entire type I-A (∼14 kb) or III-B CRISPR-Cas (∼21 kb) systems were completely knocked-out in S. PCC7002 using our tool. Furthermore, the endogenous pAQ5 plasmid, approximately 38 kb in length, was successfully cured from S. PCC7002. Our work demonstrates the feasibility of harnessing the endogenous CRISPR-Cas system for genome editing in S. PCC7002, thereby enriching the genetic toolkit for this species and providing a foundation for future enhancements in its biosynthetic efficiency.

Pleiotropic effects of PAB1 deletion: Extensive changes in the yeast proteome, transcriptome, and translatome.

Cytoplasmic poly(A)-binding protein (PABPC; Pab1 in yeast) is thought to be involved in multiple steps of post-transcriptional control, including translation initiation, translation termination, and mRNA decay. To understand both the direct and indirect roles of PABPC in more detail, we have employed mass spectrometry to assess the abundance of the components of the yeast proteome, as well as RNA-Seq and Ribo-Seq to analyze changes in the abundance and translation of the yeast transcriptome, in cells lacking the PAB1 gene. We find that pab1Δ cells manifest drastic changes in the proteome and transcriptome, as well as defects in translation initiation and termination. Defects in translation initiation and the stabilization of specific classes of mRNAs in pab1Δ cells appear to be partly indirect consequences of reduced levels of specific initiation factors, decapping activators, and components of the deadenylation complex in addition to the general loss of Pab1's direct role in these processes. Cells devoid of Pab1 also manifested a nonsense codon readthrough phenotype indicative of a defect in translation termination. Collectively, our results indicate that, unlike the loss of simpler regulatory proteins, elimination of cellular Pab1 is profoundly pleiotropic and disruptive to numerous aspects of post-transcriptional regulation.

Genetic optimization of the human gut bacterium Phocaeicola vulgatus for enhanced succinate production.

The demand for sustainably produced bulk chemicals is constantly rising. Succinate serves as a fundamental component in various food, chemical, and pharmaceutical products. Succinate can be produced from sustainable raw materials using microbial fermentation and enzyme-based technologies. Bacteroides and Phocaeicola species, widely distributed and prevalent gut commensals, possess enzyme sets for the metabolization of complex plant polysaccharides and synthesize succinate as a fermentative end product. This study employed novel molecular techniques to enhance succinate yields in the natural succinate producer Phocaeicola vulgatus by directing the metabolic carbon flow toward succinate formation. The deletion of the gene encoding the methylmalonyl-CoA mutase (Δmcm, bvu_0309-0310) resulted in a 95% increase in succinate production, as metabolization to propionate was effectively blocked. Furthermore, deletion of genes encoding the lactate dehydrogenase (Δldh, bvu_2499) and the pyruvate:formate lyase (Δpfl, bvu_2880) eliminated the formation of fermentative end products lactate and formate. By overproducing the transketolase (TKT, BVU_2318) in the triple deletion mutant, succinate production increased from 3.9 mmol/g dry weight in the wild type to 10.9 mmol/g dry weight. Overall, succinate yield increased by 180% in the new mutant strain P. vulgatus Δmcm Δldh Δpfl pG106_tkt relative to the parent strain. This approach is a proof of concept, verifying the genetic accessibility of P. vulgatus, and forms the basis for targeted genetic optimization. The increase of efficiency highlights the huge potential of P. vulgatus as a succinate producer with applications in sustainable bioproduction processes. KEY POINTS: • Deleting methylmalonyl-CoA mutase gene in P. vulgatus doubled succinate production • Triple deletion mutant with transketolase overexpression increased succinate yield by 180% • P. vulgatus shows high potential for sustainable bulk chemical production via genetic optimization.

The role of aroA and ppk1 in Aeromonas veronii pathogenicity and the efficacy evaluation of mutant strain AV-ΔaroA/ppk1 as a live attenuated vaccine.

Aeromonas veronii is an opportunistic pathogen that poses great threat to aquaculture and human health, so there is an urgent need for green and efficient methods to deal with its infection. In this study, single and double gene deletion strains (AV-ΔaroA, AV-Δppk1 and AV-ΔaroA/ppk1) that can be stably inherited were constructed. Pathogenicity test showed that the toxicity of AV-ΔaroA and AV-ΔaroA/ppk1 was significantly lower compared to wild-type A. veronii. Biological characterization analysis revealed that the decrease in pathogenicity might be due to the declined growth, motility, biofilm formation abilities and the expression of virulence-related genes in mutants. Subsequently, we evaluated the efficacy of AV-ΔaroA/ppk1 as a live attenuated vaccine (LAV). Safety assessment experiments showed that AV-ΔaroA/ppk1 injected at a concentration of 3 × 107 CFU/mL was safe for C. carassius. The relative percentage survival of AV-ΔaroA/ppk1 was 67.85 %, significantly higher than that of the inactivated A. veronii, which had an RPS of 54.84 %. This improved protective effect was mainly attributed to the increased levels of A. veronii specific IgM antibody, enhanced alkaline phosphatase, lysozyme and superoxide dismutase activities, as well as higher expression levels of several immune related genes. Together, these findings deepen our understanding of the functional roles of aroA and ppk1 in A. veronii pathogenicity, provide a good candidate of LAV for A. veronii.

Effects of AI-2 quorum sensing related luxS gene on Streptococcus suis formatting monosaccharide metabolism-dependent biofilm.

Biofilm is the primary cause of persistent infections caused by Streptococcus suis (S. suis). Metabolism and AI-2 quorum sensing are intricately linked to S. suis biofilm formation. Although the role of the AI-2 quorum sensing luxS gene in S. suis biofilm has been reported, its specific regulatory mechanism remains unclear. This study explored the differences in biofilm formation and monosaccharide metabolism among the wild type (WT), luxS mutant (ΔluxS) and complement strain (CΔluxS), and Galleria mellonella larvae were used to access the effect of luxS gene deletion on the virulence of S. suis in different monosaccharide medias. The results indicated that deletion of the luxS gene further compromised the monosaccharide metabolism of S. suis, impacting its growth in media with fructose, galactose, rhamnose, and mannose as the sole carbon sources. However, no significant impact was observed in media with glucose and N-acetylglucosamine. This deletion also weakened EPS synthesis, thereby diminishing the biofilm formation capacity of S. suis. Additionally, the downregulation of adhesion gene expression due to luxS gene deletion was found to be independent of the monosaccharide medias of S. suis.

EbsA is essential for both motility and biofilm formation in the filamentous cyanobacterium Nostoc punctiforme.

Many cyanobacteria, both unicellular and filamentous, exhibit surface motility driven by type IV pili (T4P). While the component parts of the T4P machinery described in other prokaryotes are largely conserved in cyanobacteria, there are also several T4P proteins that appear to be unique to this phylum. One recently discovered component is EbsA, which has been characterized in two unicellular cyanobacteria. EbsA was found to form a complex with other T4P proteins and is essential for motility. Additionally, deletion of ebsA in one of these strains promoted the formation of biofilms. To expand the understanding of ebsA in cyanobacteria, its role in motility and biofilm formation were investigated in the model filamentous cyanobacterium Nostoc punctiforme. Expression of ebsA was strictly limited to hormogonia, the motile filaments of N. punctiforme. Deletion of ebsA did not affect hormogonium development but resulted in the loss of motility and the failure to accumulate surface pili or produce hormogonium polysaccharide (HPS), consistent with pervious observations in unicellular cyanobacteria. Protein-protein interaction studies indicated that EbsA directly interacts with PilB, and the localization of EbsA-GFP resembled that previously shown for both PilB and Hfq. Collectively, these results support the hypothesis that EbsA forms a complex along with PilB and Hfq that is essential for T4P extension. In contrast, rather than enhancing biofilm formation, deletion of both ebsA and pilB abolish biofilm formation in N. punctiforme, implying that distinct modalities for the relationship between motility, T4P function and biofilm formation may exist in different cyanobacteria.

Isolation and characterization of Saccharomyces cerevisiae mutants with increased cell wall chitin using fluorescence-activated cell sorting.

Yeast cell wall chitin has been shown to bind grape pathogenesis-related chitinases that are the primary cause of protein haze in wines, suggesting that yeast cell walls may be applied for haze protection. Here, we present a high-throughput screen to identify yeast strains with high cell wall chitin using a reiterative enrichment strategy and fluorescence-activated cell sorting of cells labelled with either GFP-tagged chitinase or Calcofluor white. To assess the validity of the strategy, we first used a pooled deletion strain library of Saccharomyces cerevisiae. The strategy enriched for deletion mutants with genes that had previously been described as having an impact on chitin levels. Genes that had not previously been linked to chitin biosynthesis or deposition were also identified. These genes are involved in cell wall maintenance and/or membrane trafficking functions. The strategy was then applied to a mutagenized population of a commercial wine yeast strain, S. cerevisiae EC1118. Enriched mutant strains showed significantly higher cell wall chitin than the wild type and significantly reduced the activity of chitinases in synthetic model wine, suggesting that these strains may be able to reduce haze formation in wine.

Ccn2 Deletion Reduces Cardiac Dysfunction, Oxidative Markers, and Fibrosis Induced by Doxorubicin Administration in Mice.

Cellular Communication Network Factor 2 (CCN2) is a matricellular protein implicated in cell communication and microenvironmental signaling. Overexpression of CCN2 has been documented in various cardiovascular pathologies, wherein it may exert either deleterious or protective effects depending on the pathological context, thereby suggesting that its role in the cardiovascular system is not yet fully elucidated. In this study, we aimed to investigate the effects of Ccn2 gene deletion on the progression of acute cardiac injury induced by doxorubicin (DOX), a widely utilized chemotherapeutic agent. To this end, we employed conditional knockout (KO) mice for the Ccn2 gene (CCN2-KO), which were administered DOX and compared to DOX-treated wild-type (WT) control mice. Our findings demonstrated that the ablation of CCN2 ameliorated DOX-induced cardiac dysfunction, as evidenced by improvements in ejection fraction (EF) and fractional shortening (FS) of the left ventricle. Furthermore, DOX-treated CCN2-KO mice exhibited a significant reduction in the gene expression and activation of oxidative stress markers (Hmox1 and Nfe2l2/NRF2) relative to DOX-treated WT controls. Additionally, the deletion of Ccn2 markedly attenuated DOX-induced cardiac fibrosis. Collectively, these results suggest that CCN2 plays a pivotal role in the pathogenesis of DOX-mediated cardiotoxicity by modulating oxidative stress and fibrotic pathways. These findings provide a novel avenue for future investigations to explore the therapeutic potential of targeting CCN2 in the prevention of DOX-induced cardiac dysfunction.

AB095. Methylation class infant-type hemispheric glioma with CDKN2A/B deletion: a rare case report.

BACKGROUND: Gliomas are the most common central nervous system (CNS) tumors in infant but with incidence rate only 1.38 per 100,000. Due to distinctive clinical, histologic, and molecular features, the current World Health Organization (WHO) CNS5 separate gliomas in children from adult as pediatric-type diffuse high-grade and low-grade gliomas. Infant hemispheric gliomas constitute a biologically and clinically distinct subgroup of pediatric-type diffuse high-grade. In this case we present clinical, radiographic, intraoperative, and methylation profiling of the first infant-type hemispheric glioma diagnosed in Indonesia. CASE DESCRIPTION: This is a case report of infant operated at Dr. Cipto Mangunkusumo National General Hospital, Jakarta, Indonesia in February 2024. A 6-month-old male infant brought to regional hospital due to head enlargement compared to infant of the same age, head circumference was 50 cm [>2 standard deviation (SD)] with frontal bossing. Brain MRI showed large multi-loculated cystic lesion at left parietooccipital region, which appeared hypointense on T1-weithgted (T1W), hyperintense on T2-weighted (T2W) and fluid-attenuated inversion recovery (FLAIR), with irregular contrast enhancing border. There was isointense lesion on T1W with inhomogeneous contrast enhancement. The largest volume of cystic lesion was 216 cm. Intraoperatively, parietal bone was thinner than usual. The brain was tense, purplish, and non-pulsating, giving the impression of a tumor with indistinct borders with the normal cortex. Dark clear yellowish fluid was spurt after the cortex was incised. Histopathological findings revealed moderate to high cellularity tumor tissue with mitosis, microvascular proliferation, palisading necrosis. In collaboration with German Cancer Research Center (DKFZ), DNA methylation array analysis showed the tumor to match the Infant-type Hemispheric Glioma methylation class (calibrated score 0.94) with deletion of cyclin dependent kinase inhibitor 2A/B (CDKN2A/B). CONCLUSIONS: Methylation class (MC) infant-type hemispheric glioma may present with macrocephaly. On magnetic resonance imaging (MRI) it may appear as large multi-loculated cystic lesion and irregular contrast enhancing border. The key diagnostic criteria for infant-type hemispheric glioma involve combination of clinical, pathological, and molecular feature.

AB003. Clinical analysis of central nervous system (CNS) World Health Organization (WHO) grade 4 gliomas with IDH-mutant versus IDH-wildtype focused on CDKN2A homozygous deletion.

BACKGROUND: We are primarily investigating the prognostic role of cell-cycle-dependent kinase inhibitor (CDKN)-2A homozygous deletion in central nervous system (CNS) World Health Organization (WHO) grade 4 gliomas. Additionally, traditional prognostic factors for grade 4 gliomas will be examined, and our results will be validated. METHODS: We conducted a retrospective analysis of glioma cohorts in our institute. Medical records were reviewed for 142 glioblastoma patients for 15 years, and pathological slides were examined again for the updated diagnosis according to the 2021 WHO classification of CNS tumors. The isocitrate dehydrase (IDH) mutation and CDKN2A deletion were examined by next generation sequencing (NGS) analysis using ONCO accuPanel®. Traditional prognostic factors including age, WHO performance status, extent of resection, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation were examined. RESULTS: After the exclusion of 6 patients with poor status of pathologic samples, 136 glioblastoma that were diagnosed by previous WHO criteria were changed into 29 (21.3%) astrocytoma, IDH-mutant, CNS WHO grade 4 and 107 (78.7%) glioblastoma, IDH-wildtype, CNS WHO grade 4. Among them, 61 patients (56.0%) had CDKN2A deletion. Group A with IDH-wildtype and CDKN2A deletion had a mean overall survival (OS) of 15.70 months [95% confident interval (CI): 13.86-17.54], group B with IDH-mutant and CDKN2A deletion had a mean OS of 19.37 months (95% CI: 13.43-25.30), group C with IDH-wildtype and intact CDKN2A had a mean OS of 22.63 months (95% CI: 20.10-25.17), and group D with IDH-mutant and intact CDKN2A had a mean OS of 33.38 months (95% CI: 29.35-37.40). Multifactor analysis showed following factors were independently associated with OS: age [≥50 vs. <50 years; hazard ratio (HR) 4.642], extent of resection (gross total resection vs. others; HR 5.523), WHO performance (0, 1 vs. 2; HR 5.007), MGMT promoter methylation, (methylated vs. unmethylated; HR 5.075), IDH mutation (mutant vs. wildtype; HR 6.358), and CDKN2A deletion (absence vs. presence; HR 13.452). CONCLUSIONS: The presenting study suggests that CDKN2A deletion should play a powerful prognostic role in CNS WHO grade 4 gliomas as well as low-grade glioma. Even if CNS WHO grade 4 gliomas had mutant IDH, they can have poor clinical outcomes due to CDKN2A deletion.

The Rate of NAD+ Breakdown Is Maintained Constant against Deletion or Overexpression of NAD+-Degrading Enzymes in Mammalian Cells.

Cellular NAD+ is continuously degraded and synthesized under resting conditions. In mammals, NAD+ synthesis is primarily initiated from nicotinamide (Nam) by Nam phosphoribosyltransferase, whereas poly(ADP-ribose) polymerase 1 (PARP1) and 2 (PARP2), sirtuin1 (SIRT1), CD38, and sterile alpha and TIR motif containing 1 (SARM1) are involved in NAD+ breakdown. Using flux analysis with 2H-labeled Nam, we found that when mammalian cells were cultured in the absence of Nam, cellular NAD+ levels were maintained and NAD+ breakdown was completely suppressed. In the presence of Nam, the rate of NAD+ breakdown (RB) did not significantly change upon PARP1, PARP2, SIRT1, or SARM1 deletion, whereas stable expression of CD38 did not increase RB. However, RB in PARP1-deleted cells was much higher compared with that in wild-type cells, in which PARP1 activity was blocked with a selective inhibitor. In contrast, RB in CD38-overexpressing cells in the presence of a specific CD38 inhibitor was much lower compared with that in control cells. The results indicate that PARP1 deletion upregulates the activity of other NADases, whereas CD38 expression downregulates the activity of endogenous NADases, including PARP1 and PARP2. The rate of cellular NAD+ breakdown and the resulting NAD+ concentration may be maintained at a constant level, despite changes in the NAD+-degrading enzyme expression, through the compensatory regulation of NADase activity.

AcuM and AcuK: The global regulators controlling multiple cellular metabolisms in a dimorphic fungus Talaromyces marneffei.

Talaromycosis is a fungal infection caused by an opportunistic dimorphic fungus Talaromyces marneffei. During infection, T. marneffei resides inside phagosomes of human host macrophages where the fungus encounters nutrient scarcities and host-derived oxidative stressors. Previously, we showed that the deletion of acuK, a gene encoding Zn(2)Cys(6) transcription factor, caused a decreased ability for T. marneffei to defend against macrophages, as well as a growth impairment in T. marneffei on both low iron-containing medium and gluconeogenic substrate-containing medium. In this study, a paralogous gene acuM was deleted and characterized. The ΔacuM mutant showed similar defects with the ΔacuK mutant, suggesting their common role in gluconeogenesis and iron homeostasis. Unlike the pathogenic mold Aspergillus fumigatus, the ΔacuK and ΔacuM mutants unexpectedly exhibited normal siderophore production and did not show lower expression levels of genes involved in iron uptake and siderophore synthesis. To identify additional target genes of AcuK and AcuM, RNA-sequencing analysis was performed in the ΔacuK and ΔacuM strains growing in a synthetic dextrose medium with 1% glucose at 25 °C for 36 hours. Downregulated genes in both mutants participated in iron-consuming processes, especially in mitochondrial metabolism and anti-oxidative stress. Importantly, the ΔacuM mutant was sensitive to the oxidative stressors menadione and hydrogen peroxide while the ΔacuK mutant was sensitive to only hydrogen peroxide. The yeast form of both mutants demonstrated a more severe defect in antioxidant properties than the mold form. Moreover, ribosomal and ribosomal biogenesis genes were expressed at significantly lower levels in both mutants, suggesting that AcuK and AcuM could affect the protein translation process in T. marneffei. Our study highlighted the role of AcuK and AcuM as global regulators that control multiple cellular adaptations under various harsh environmental conditions during host infection. These transcription factors could be potentially exploited as therapeutic targets for the treatment of this neglected infectious disease.

Genetic Manipulation of Candida glabrata.

Candida glabrata (Nakaseomyces glabratus) is an opportunistic fungal pathogen that has become a significant concern in clinical settings due to its increasing resistance to antifungal treatments. Understanding the genetic basis of its pathogenicity and resistance mechanisms is crucial for developing new therapeutic strategies. One powerful method of studying gene function is through targeted gene deletion. This paper outlines a comprehensive protocol for the deletion of genes in C. glabrata, encompassing primer design, preparation of electrocompetent cells, transformation, and finally confirmation of the gene deletion. The protocol begins with the identification and design of primers necessary for generating deletion constructs, involving the precise targeting of up- and downstream regions flanking the gene of interest to ensure high specificity and efficiency of homologous recombination. Followed is the preparation of electrocompetent cells, a critical step for successful transformation. Transformation of the competent cells is achieved through electroporation, facilitating the introduction of exogenous DNA into the cells. This is followed by the selection and confirmation of successfully transformed colonies. Confirmation involves the use of colony PCR to verify the correct integration of the NAT resistance cassette and deletion of the target gene. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Primer design for gene deletion in C. glabrata Basic Protocol 2: Preparing competent C. glabrata cells Basic Protocol 3: Transforming C. glabrata using electroporation Basic Protocol 4: Confirming deletion strains with colony PCR.

DUF1127-containing protein and ProQ had opposite effects on biofilm formation in Vibrio alginolyticus.

The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus.

Enhanced accumulation of γ-aminobutyric acid by deletion of aminotransferase genes involved in γ-aminobutyric acid catabolism in engineered Halomonas elongata.

Halomonas elongata OUT30018 is a moderately halophilic bacterium that synthesizes and accumulates ectoine as an osmolyte by activities of the enzymes encoded by the high salinity-inducible ectABC operon. Previously, we engineered a γ-aminobutyric acid (GABA)-producing H. elongata GOP-Gad (ΔectABC::mCherry-HopGadmut) from an ectoine-deficient mutant of this strain due to its ability to use high-salinity biomass waste as substrate. Here, to further increase GABA accumulation, we deleted gabT, which encodes GABA aminotransferase (GABA-AT) that catalyzes the first step of the GABA catabolic pathway, from the H. elongata GOP-Gad genome. The resulting strain H. elongata ZN3 (ΔectABC::mCherry-HopGadmut ΔgabT) accumulated 291 µmol/g cell dry weight (CDW) of GABA in the cells, which is a 1.5-fold increase from H. elongata GOP-Gad's 190 µmol/g CDW. This result has confirmed the role of GABA-AT in the GABA catabolic pathway. However, redundancy in endogenous GABA-AT activity was detected in a growth test, where a gabT-deletion mutant of H. elongata OUT30018 was cultured in a medium containing GABA as the sole carbon and nitrogen sources. Because L-2,4-diaminobutyric acid aminotransferase (DABA-AT), encoded by an ectB gene of the ectABC operon, shares sequence similarity with GABA-AT, a complementation analysis of the gabT and the ectB genes was performed in the H. elongata ZN3 genetic background to test the involvement of DABA-AT in the redundancy of GABA-AT activity. Our results indicate that the expression of DABA-AT can restore GABA-AT activity in H. elongata ZN3 and establish DABA-AT's aminotransferase activity toward GABA in vivo. IMPORTANCE: In this study, we were able to increase the yield of GABA by 1.5 times in the GABA-producing H. elongata ZN3 strain by deleting the gabT gene, which encodes GABA-AT, the initial enzyme of the GABA catabolic pathway. We also report the first in vivo evidence for GABA aminotransferase activity of an ectB-encoded DABA-AT, confirming a longstanding speculation based on the reported in vitro GABA-AT activity of DABA-AT. According to our findings, the DABA-AT enzyme can catalyze the initial step of GABA catabolism, in addition to its known function in ectoine biosynthesis. This creates a cycle that promotes adequate substrate flow between the two pathways, particularly during the early stages of high-salinity stress response when the expression of the ectB gene is upregulated.

A rare case of compound heterozygous Southeast Asian double α-globin gene deletion and Haemoglobin Quong Sze in a Malay proband.

INTRODUCTION: Haemoglobin (Hb) Quong Sze is a non-deletional α-thalassaemia subtype that occurs due to missense mutation at codon 125 of the HBA2 gene. Interaction between Hb QS with Southeast Asian double α-globin gene deletion results in non-deletional HbH disease, which is more severe than deletional HbH. CASE REPORT: A 3-month-old baby boy was presented with neonatal anaemia and mild hepatomegaly. Full blood count revealed severe hypochromic microcytic anaemia. There was an abundance of HbH inclusion bodies in his red blood cells. High-performance liquid chromatography showed a reduced HbA2 level with the presence of pre-run peak. Capillary electrophoresis showed the presence of HbH and Hb Barts. Molecular analysis found a common α0-thalassaemia (--SEA) in one allele and mutation in codon 125 in the other allele. DISCUSSION: Non-deletional HbH disease due to a combination of deletional and non-deletional mutations may present with severe clinical manifestations than those with deletion mutations, which warrants accurate diagnosis using molecular techniques.

VmSom1 is essential for growth, development, maintenance of cell wall integrity and virulence in Valsa mali.

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

Prognostic significance and treatment strategies for IKZF1 deletion in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: The predictive importance of IKZF1del in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has shown variability across different studies. Thus, the optimal treatment approach for children with IKZF1del BCP-ALL remains contentious, with the ongoing debate surrounding the use of IKZF1del-based high-risk stratification versus a minimal residual disease (MRD)-guided protocol. METHODS: IKZF1 status was reliably determined in 804 patients using multiplex ligation-dependent probe amplification (MLPA) data obtained from four hospitals in Fujian, a province of China. In the Chinese Children Leukemia Group (CCLG)-ALL 2008 cohort, IKZF1 status was included in the risk assignment, with all IKZF1del patients receiving a high-risk regimen. Conversely, in the Chinese Children's Cancer Group (CCCG)-ALL 2015 cohort, IKZF1del was not incorporated into the risk assignment, and patients were treated based on an MRD-guided risk stratification protocol. RESULTS: IKZF1del was found in 86 patients (86/804, 10.7%) overall and in 30 (30/46, 65.2%) BCR::ABL1-positive patients. Overall, IKZF1del was a poor prognostic predictor for patients, though the significance diminished upon age adjustment, white blood cell (WBC) count at diagnosis, treatment group, and MRD status. In the CCLG-ALL 2008 cohort, IKZF1del conferred a notably lower 5-year overall survival (OS) and event-free survival (EFS) and a significantly higher 5-year cumulative incidence of relapse (CIR) than IKZF1wt. In the CCLG-ALL 2015 cohort, IKZF1del conferred a lower 5-year OS and EFS and a higher 5-year CIR than IKZF1wt, but the differences were insignificant. The IKZF1del patients treated with higher intensity chemotherapy (CCLG-ALL 2008 high-risk regimen) had a markedly lower 5-year OS and EFS compared with those treated with the MRD-guided protocol (CCCG-ALL 2015 protocol). Furthermore, patients treated with the CCLG-ALL 2008 high-risk regimen experienced a higher frequency of serious adverse events (SAEs), especially infection-related SAEs, compared with those treated with the CCCG-ALL 2015 MRD-guided protocol. CONCLUSIONS: The prognostic effect of IKZF1del may vary in different protocols. Compared with higher intensity chemotherapy, the MRD-guided protocol may be a more effective approach to treating BCP-ALL with IKZF1del in children.

The robustness of porin-cytochrome gene clusters from Geobacter metallireducens in extracellular electron transfer.

To investigate their roles in extracellular electron transfer (EET), the porin-cytochrome (pcc) gene clusters Gmet0825-0828, Gmet0908-0910, and Gmet0911-0913 of the Gram-negative bacterium Geobacter metallireducens were deleted. Failure to delete all pcc gene clusters at the same time suggested their essential roles in extracellular reduction of Fe(III)-citrate by G. metallireducens. Deletion of Gmet0825-0828 had no impact on bacterial reduction of Fe(III)-citrate but diminished bacterial reduction of ferrihydrite and abolished anode reduction and direct interspecies electron transfer (DIET) to Methanosarcina barkeri and Geobacter sulfurreducens. Although it had no impact on the bacterial reduction of Fe(III)-citrate, deletion of Gmet0908-0910 delayed ferrihydrite reduction, abolished anode reduction, and diminished DIET. Deletion of Gmet0911-0913 had little impact on DIET but diminished bacterial reductions of Fe(III)-citrate, ferrihydrite, and anodes. Most importantly, deletions of both Gmet0825-0828 and Gmet0908-0910 restored bacterial reduction of ferrihydrite and anodes and DIET. Enhanced expression of Gmet0911-0913 in this double mutant when grown in coculture with G. sulfurreducens ΔhybLΔfdnG suggested that this cluster might compensate for impaired EET functions of deleting Gmet0825-0828 and Gmet0908-0910. Thus, these pcc gene clusters played essential, distinct, overlapping, and compensatory roles in EET of G. metallireducens that are difficult to characterize as deletion of some clusters affected expression of others. The robustness of these pcc gene clusters enabled G. metallireducens to mediate EET to different acceptors for anaerobic growth even when two of its three pcc gene clusters were inactivated by mutation. The results from this investigation provide new insights into the roles of pcc gene clusters in bacterial EET. IMPORTANCE: The Gram-negative bacterium Geobacter metallireducens is of environmental and biotechnological significance. Crucial to the unique physiology of G. metallireducens is its extracellular electron transfer (EET) capability. This investigation sheds new light on the robust roles of the three porin-cytochrome (pcc) gene clusters, which are directly involved in EET across the bacterial outer membrane, in the EET of G. metallireducens. In addition to their essential roles, these gene clusters also play distinct, overlapping, and compensatory roles in the EET of G. metallireducens. The distinct roles of the pcc gene clusters enable G. metallireducens to mediate EET to a diverse group of electron acceptors for anaerobic respirations. The overlapping and compensatory roles of the pcc gene clusters enable G. metallireducens to maintain and restore its EET capability for anaerobic growth when one or two of its three pcc gene clusters are deleted from the genome.