Transcriptional Regulator Gene Research Articles

The IclR-family transcriptional regulator XyrR controls flotation, motility, antibiotic production and virulence in Serratia sp. ATCC 39006

The opportunistic pathogen Serratia sp. ATCC 39006 (S39006) is a rod-shaped, motile, Gram-negative bacterium that produces a 𝛽-lactam antibiotic (a carbapenem) and a bioactive red-pigmented tripyrrole antibiotic, prodigiosin. It is also the only known enterobacterium that naturally produces intracellular gas vesicles (GVs), enabling cells to float in static water columns. Regulation of GVs and secondary metabolites in S39006 can be coordinated but such pleiotropy is still poorly understood. To uncover novel inputs to this complex regulatory network, we used transposon mutagenesis to identify a mutant with an insertion in an IclR-type transcriptional regulator gene. The iclR mutant showed diminished production of carbapenem, prodigiosin, GVs and cellulase. Furthermore, the mutant also showed increased swimming and swarming motilities but exhibited attenuated virulence in planta and ability to kill the nematode C. elegans. Using differential expression analysis of the intracellular proteomes of the wild type and iclR mutant, we confirmed that the mutation negatively impacted expression of the corresponding GV, carbapenem and prodigiosin gene clusters. In contrast, flagellar and chemotaxis proteins were overexpressed, consistent with the increased motility of the mutant. We also found that the proteins encoded by a putative yagEF-yjhF operon, involved in xylonate catabolism and transport, showed a 5- to 7-fold increase in expression. Finally, we show that IclR is a repressor of xylonate catabolism in S39006 and suggest that xylonate is potentially involved in controlling carbapenem and prodigiosin biosynthesis. Our results indicate that IclR is a global regulator that controls antibiotic biosynthesis, flotation through modulating GV assembly, and has pleiotropic impacts on the physiology and virulence of S39006. Based on these findings, we propose the designation of this IclR-family transcriptional regulator as XyrR (Xylonate response Regulator).

ZnO-Rutin nanostructure as a potent antibiofilm agent against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common human pathogen that is resistant to multiple antibiotics due to its ability to form biofilms. Developing novel nanoformulations capable of inhibiting and removing biofilms offers a promising solution for controlling biofilm-related infections. In this study, we investigated the anti-biofilm activity of rutin-conjugated ZnO nanoparticles (ZnO-Rutin NPs) in pathogenic strains of P. aeruginosa. The synthesized ZnO-Rutin NPs had amorphous shapes with sizes ranging from 14 to 100 nm. The broth microdilution assay revealed that ZnO-Rutin NPs, with an MIC value of 2 mg/mL, exhibit greater antimicrobial activity than ZnO NPs and rutin alone. Based on crystal violet staining, the biofilm inhibition rate by ½ MIC of the conjugated nanoparticles was recorded at above 90%. The significant reduction in exopolysaccharide (62.75–66.37%) and alginate (38.3–57.61%) levels, as well as the formation of thin biofilms in the ZnO-Rutin NP-treated group, confirmed the anti-biofilm potential of these nanoparticles. Additionally, a significant decrease in the metabolic activity and viable cells of mature biofilms was observed after exposure to the conjugated nanoparticles. Furthermore, ZnO-Rutin NPs considerably attenuated the expression of the Las-Rhl quorum-sensing transcriptional regulator genes (lasR and rhlR) in P. aeruginosa by 0.39–0.40 and 0.25–0.42 folds, respectively. This work demonstrated that ZnO-Rutin NPs are remarkably capable of inhibiting the initial stage of biofilm formation and eradicating mature biofilms, suggesting they could be a useful agent for treating P. aeruginosa biofilm-related infections.

Targeting Transcriptional Regulators Affecting Acarbose Biosynthesis in Actinoplanes sp. SE50/110 Using CRISPRi Silencing

Acarbose, a pseudo-tetrasaccharide produced by Actinoplanes sp. SE50/110, is an α-glucosidase inhibitor and is used as a medication to treat type 2 diabetes. While the biosynthesis of acarbose has been elucidated, little is known about its regulation. Gene silencing using CRISPRi allows for the identification of potential regulators influencing acarbose formation. For this purpose, two types of CRISPRi vectors were established for application in Actinoplanes sp. SE50/110. The pCRISPomyces2i vector allows for reversible silencing, while the integrative pSETT4i vector provides a rapid screening approach for many targets due to its shorter conjugation time into Actinoplanes sp. These vectors were validated by silencing the known acarbose biosynthesis genes acbB and acbV, as well as their regulator, CadC. The reduction in product formation and the diminished relative transcript abundance of the respective genes served as evidence of successful silencing. The vectors were used to create a CRISPRi-based strain library, silencing 50 transcriptional regulators, to investigate their potential influence in acarbose biosynthesis. These transcriptional regulatory genes were selected from previous experiments involving protein–DNA interaction studies or due to their expression profiles. Eleven genes affecting the yield of acarbose were identified. The CRISPRi-mediated knockdown of seven of these genes significantly reduced acarbose biosynthesis, whereas the knockdown of four genes enhanced acarbose production.

Xylose utilization promotes Salmonella replication within macrophages and systemic infection in mice

ABSTRACT The intracellular pathogen Salmonella can cause systemic diseases via its survival and replication in host macrophages. Xylose is the second most abundant sugar in nature and Salmonella can use xylose as its sole carbon source for growth. However, whether xylose utilization contributes to the pathogenicity and intracellular growth of Salmonella has not yet been determined. In this study, we observed that the xylose concentration in macrophages increased during Salmonella infection. Moreover, there was an increase in expression of Salmonella xylose catabolic genes (xylA and xylB) and the transcriptional regulatory gene of xylose metabolism (xylR) in macrophages, revealing the possibility of using host-accumulated xylose by Salmonella for intracellular growth. Mutation of either xylAB or xylR reduced Salmonella replication in macrophages and attenuated the colonization of mouse systemic loci (e.g. the liver and spleen), indicating that xylose utilization promotes Salmonella replication within macrophages and systemic infection in mice. Moreover, we found that xylose utilization by intracellular Salmonella was activated by the cAMP-CRP complex upon detection of low glucose levels in the infected macrophages. Collectively, these findings reveal that although the available glucose decreases during infection, Salmonella can use xylose, which accumulates in infected macrophages, as an alternative carbon source to promote intracellular replication and virulence.

YeeE-like bacterial SoxT proteins mediate sulfur import for oxidation and signal transduction

Many sulfur-oxidizing prokaryotes oxidize sulfur compounds through a combination of initial extracytoplasmic and downstream cytoplasmic reactions. Facultative sulfur oxidizers adjust transcription to sulfur availability. While sulfur-oxidizing enzymes and transcriptional repressors have been extensively studied, sulfur import into the cytoplasm and how regulators sense external sulfur are poorly understood. Addressing this gap, we show that SoxT1A and SoxT1B, which resemble YeeE/YedE-family thiosulfate transporters and are encoded alongside sulfur oxidation and transcriptional regulation genes, fulfill these roles in the Alphaproteobacterium Hyphomicrobium denitrificans. SoxT1A mutants are sulfur oxidation-negative despite high transcription levels of sulfur oxidation genes, showing that SoxT1A delivers sulfur to the cytoplasm for its further oxidation. SoxT1B serves as a signal transduction unit for the transcriptional repressor SoxR, as SoxT1B mutants are sulfur oxidation-negative due to low transcription unless SoxR is also absent. Thus, SoxT1A and SoxT1B play essential but distinct roles in oxidative sulfur metabolism and its regulation.

Altered genomic methylation promotes Staphylococcus aureus persistence in hospital environment

Staphylococcus aureus can cause outbreaks and becomes multi-drug resistant through gene mutations and acquiring resistance genes. However, why S. aureus easily adapts to hospital environments, promoting resistance and recurrent infections, remains unknown. Here we show that a specific S. aureus lineage evolved from a clone that expresses the accessory gene regulator (Agr) system to subclones that reversibly suppressed Agr and caused an outbreak in the hospital setting. S. aureus with flexible Agr regulation shows increased ability to acquire antibiotic-resistant plasmids, escape host immunity, and colonize mice. Bacteria with flexible Agr regulation shows altered cytosine genomic methylation, including the decreased 5mC methylation in transcriptional regulator genes (pcrA and rpsD), compared to strains with normal Agr expression patterns. In this work, we discover how altered genomic methylation promotes flexible Agr regulation which is associated with persistent pathogen colonization in the hospital environment.

UBASH3B-mediated MRPL12 Y60 dephosphorylation inhibits LUAD development by driving mitochondrial metabolism reprogramming

BackgroundMetabolic reprogramming plays a pivotal role in tumorigenesis and development of lung adenocarcinoma (LUAD). However, the precise mechanisms and potential targets for metabolic reprogramming in LUAD remain elusive. Our prior investigations revealed that the mitochondrial ribosomal protein MRPL12, identified as a novel mitochondrial transcriptional regulatory gene, exerts a critical influence on mitochondrial metabolism. Despite this, the role and regulatory mechanisms underlying MRPL12’s transcriptional activity in cancers remain unexplored.MethodsHuman LUAD tissues, Tp53fl/fl;KrasG12D-driven LUAD mouse models, LUAD patient-derived organoids (PDO), and LUAD cell lines were used to explored the expression and function of MRPL12. The posttranslational modification of MRPL12 was analyzed by mass spectrometry, and the oncogenic role of key phosphorylation sites of MRPL12 in LUAD development was verified in vivo and in vitro.ResultsMRPL12 was upregulated in human LUAD tissues, Tp53fl/fl;KrasG12D-driven LUAD tissues in mice, LUAD PDO, and LUAD cell lines, correlating with poor patient survival. Overexpression of MRPL12 significantly promoted LUAD tumorigenesis, metastasis, and PDO formation, while MRPL12 knockdown elicited the opposite phenotype. Additionally, MRPL12 deletion in a Tp53fl/fl;KrasG12D-driven mouse LUAD model conferred a notable survival advantage, delaying tumor onset and reducing malignant progression. Mechanistically, we discovered that MRPL12 promotes tumor progression by upregulating mitochondrial oxidative phosphorylation. Furthermore, we identified UBASH3B as a specific binder of MRPL12, dephosphorylating tyrosine 60 in MRPL12 (MRPL12 Y60) and inhibiting its oncogenic functions. The decrease in MRPL12 Y60 phosphorylation impeded the binding of MRPL12 to POLRMT, downregulating mitochondrial metabolism in LUAD cells. In-depth in vivo, in vitro, and organoid models validated the inhibitory effect of MRPL12 Y60 mutation on LUAD.ConclusionThis study establishes MRPL12 as a novel oncogene in LUAD, contributing to LUAD pathogenesis by orchestrating mitochondrial metabolism reprogramming towards oxidative phosphorylation (OXPHOS). Furthermore, it confirms Y60 as a specific phosphorylation modification site regulating MRPL12’s oncogenic functions, offering insights for the development of LUAD-specific targeted drugs and clinical interventions.Graphical

Identification of Cold Tolerance Transcriptional Regulatory Genes in Seedlings of Medicago sativa L. and Medicago falcata L.

Alfalfa species Medicago sativa L. (MS) and Medicago falcata L. (MF), globally prominent perennial leguminous forages, hold substantial economic value. However, our comprehension of the molecular mechanisms governing their resistance to cold stress remains limited. To address this knowledge gap, we scrutinized and compared MS and MF cold-stress responses at the molecular level following 24 h and 120 h low-temperature exposure (4 °C). Our study revealed that MF had superior physiological resilience to cold stress compared with MS, and its morphology was healthier under cold stress, and its malondialdehyde content and superoxide dismutase activity increased, first, and then decreased, while the soluble sugar content continued to accumulate. Transcriptome analysis showed that after 120 h of exposure, there were different gene-expression patterns between MS and MF, including 1274 and 2983 genes that were continuously up-regulated, respectively, and a total of 923 genes were included, including star cold-resistant genes such as ICE1 and SIP1. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed numerous inter-species differences in sustained cold-stress responses. Notably, MS-exclusive genes included a single transcription factor (TF) gene and several genes associated with a single DNA repair-related pathway, whereas MF-exclusive genes comprised nine TF genes and genes associated with 14 pathways. Both species exhibited high-level expression of genes encoding TFs belonging to AP2-EREBP, ARR-B, and bHLH TF families, indicating their potential roles in sustaining cold resistance in alfalfa-related species. These findings provide insights into the molecular mechanisms governing cold-stress responses in MS and MF, which could inform breeding programs aimed at enhancing cold-stress resistance in alfalfa cultivars.

Integrated Phenotypic Physiology and Transcriptome Analysis Revealed the Molecular Genetic Basis of Anthocyanin Accumulation in Purple Pak-Choi

Purple Pak-choi is rich in anthocyanins, which have both ornamental and edible health functions, and has been used more and more widely in facility cultivation. In order to further clarify the molecular mechanism of purple Pak-choi, two Pak-choi inbred lines (‘PQC’ and ‘HYYTC’) were selected for the determination of pigment content and transcriptome analysis, and the key genes controlling the formation of purple character in leaves of Pak-choi were discovered. The results of pigment determination showed that the anthocyanin content of ‘PQC’ was 0.29 mg/g, which was about 100 times than ‘HYYTC’; The chlorophyll content of ‘HYYTC’ was 2.25 mg/g, while ‘PQC’ only contained 1.05 mg/g. A total of 20 structural genes related to anthocyanin biosynthesis and 28 transcriptional regulatory genes were identified by transcriptome analysis. Weighted gene co-expression network analysis (WGCNA) was used to construct the weight network analysis map of 14 genes. The results showed that the cinnamate hydroxylase gene (BraA04002213, BrC4H3), flavanone-3- hydroxylase (BraA09004531, BrF3H1), and chalcone synthetase (BraA10002265, BrCHS1) were the core genes involved in the anthocyanin synthesis pathway of purple Pak-choi. The results identified the key genes controlling the formation of purple leaf traits, which laid a foundation for further analysis of the molecular mechanism of anthocyanin accumulation in purple Pak-choi and provided a theoretical basis for leaf color regulation.

The Biological Characteristics of Mycobacterium Phage Henu3 and the Fitness Cost Associated with Its Resistant Strains.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is an infectious disease that seriously affects human life and health. Despite centuries of efforts to control it, in recent years, the emergence of multidrug-resistant bacterial pathogens of M. tuberculosis due to various factors has exacerbated the disease, posing a serious threat to global health. Therefore, a new method to control M. tuberculosis is urgently needed. Phages, viruses that specifically infect bacteria, have emerged as potential biocontrol agents for bacterial pathogens due to their host specificity. In this study, a mycobacterium phage, Henu3, was isolated from soil around a hospital. The particle morphology, biological characteristics, genomics and phylogeny of Henu3 were characterized. Additionally, to explore the balance between phage resistance and stress response, phage Henu3-resistant strains 0G10 and 2E1 were screened by sequence passage and bidirectional validation methods, which significantly improved the sensitivity of phage to antibiotics (cefotaxime and kanamycin). By whole-genome re-sequencing of strains 0G10 and 2E1, 12 genes involved in cell-wall synthesis, transporter-encoded genes, two-component regulatory proteins and transcriptional regulatory factor-encoded genes were found to have mutations. These results suggest that phage Henu3 has the potential to control M. tuberculosis pathogens, and phage Henu3 has the potential to be a new potential solution for the treatment of M. tuberculosis infection.

Biological Roles of Lipids in Rice.

Lipids are organic nonpolar molecules with essential biological and economic importance. While the genetic pathways and regulatory networks of lipid biosynthesis and metabolism have been extensively studied and thoroughly reviewed in oil crops such as soybeans, less attention has been paid to the biological roles of lipids in rice, a staple food for the global population and a model species for plant molecular biology research, leaving a considerable knowledge gap in the biological roles of lipids. In this review, we endeavor to furnish a current overview of the advancements in understanding the genetic foundations and physiological functions of lipids, including triacylglycerol, fatty acids, and very-long-chain fatty acids. We aim to summarize the key genes in lipid biosynthesis, metabolism, and transcriptional regulation underpinning rice's developmental and growth processes, biotic stress responses, abiotic stress responses, fertility, seed longevity, and recent efforts in rice oil genetic improvement.

Transcriptional regulators SP110 and SP140 modulate inflammatory response genes in Mycobacterium tuberculosis-infected human macrophages.

Tuberculosis (TB) is one of the most serious infectious diseases, with high morbidity and mortality worldwide. C3HeB/FeJ mice are widely utilized for evaluating anti-TB drugs because their drug sensitivity and pathology during M. tuberculosis infection resemble those of human TB, including the development of necrotizing granulomas. Downregulation of the transcriptional regulatory genes Sp110 and Sp140 in C3HeB/FeJ mice has been demonstrated to activate gene expression associated with inflammatory responses during M. tuberculosis infection, resulting in susceptibility to the infection. Here, we examined the regulatory manner of SP110 and SP140 using transcriptomic analysis in M. tuberculosis-infected human macrophages. Depletion of SP110 and/or SP140 in M. tuberculosis-infected THP-1 macrophages impaired the induction of gene expression associated with inflammatory responses, including interferon response genes, compared with that in control macrophages. These results suggest that human SP110 and SP140 act as positive regulators for genes associated with inflammatory responses upon M. tuberculosis infection.

Methylome-proteome integration after late-life voluntary exercise training reveals regulation and target information for improved skeletal muscle health.

Exercise is a potent stimulus for combatting skeletal muscle ageing. To study the effects of exercise on muscle in a preclinical setting, we developed a combined endurance-resistance training stimulus for mice called progressive weighted wheel running (PoWeR). PoWeR improves molecular, biochemical, cellular and functional characteristics of skeletal muscle and promotes aspects of partial epigenetic reprogramming when performed late in life (22-24months of age). In this investigation, we leveraged pan-mammalian DNA methylome arrays and tandem mass-spectrometry proteomics in skeletal muscle to provide detailed information on late-life PoWeR adaptations in female mice relative to age-matched sedentary controls (n=7-10 per group). Differential CpG methylation at conserved promoter sites was related to transcriptional regulation genes as well as Nr4a3, Hes1 and Hox genes after PoWeR. Using a holistic method of -omics integration called binding and expression target analysis (BETA), methylome changes were associated with upregulated proteins related to global and mitochondrial translation after PoWeR (P=0.03). Specifically, BETA implicated methylation control of ribosomal, mitoribosomal, and mitochondrial complex I protein abundance after training. DNA methylation may also influence LACTB, MIB1 and UBR4 protein induction with exercise - all are mechanistically linked to muscle health. Computational cistrome analysis predicted several transcription factors including MYC as regulators of the exercise trained methylome-proteome landscape, corroborating prior late-life PoWeR transcriptome data. Correlating the proteome to muscle mass and fatigue resistance revealed positive relationships with VPS13A and NPL levels, respectively. Our findings expose differential epigenetic and proteomic adaptations associated with translational regulation after PoWeR that could influence skeletal muscle mass and function in aged mice. KEY POINTS: Late-life combined endurance-resistance exercise training from 22-24months of age in mice is shown to improve molecular, biochemical, cellular and in vivo functional characteristics of skeletal muscle and promote aspects of partial epigenetic reprogramming and epigenetic age mitigation. Integration of DNA CpG 36k methylation arrays using conserved sites (which also contain methylation ageing clock sites) with exploratory proteomics in skeletal muscle extends our prior work and reveals coordinated and widespread regulation of ribosomal, translation initiation, mitochondrial ribosomal (mitoribosomal) and complex I proteins after combined voluntary exercise training in a sizeable cohort of female mice (n=7-10 per group and analysis). Multi-omics integration predicted epigenetic regulation of serine β-lactamase-like protein (LACTB - linked to tumour resistance in muscle), mind bomb 1 (MIB1 - linked to satellite cell and type 2 fibre maintenance) and ubiquitin protein ligase E3 component N-recognin 4 (UBR4 - linked to muscle protein quality control) after training. Computational cistrome analysis identified MYC as a regulator of the late-life training proteome, in agreement with prior transcriptional analyses. Vacuolar protein sorting 13homolog A (VPS13A) was positively correlated to muscle mass, and the glycoprotein/glycolipid associated sialylation enzyme N-acetylneuraminate pyruvate lyase (NPL) was associated to in vivo muscle fatigue resistance.

Exploring essential oil-based bio-composites: molecular docking and in vitro analysis for oral bacterial biofilm inhibition.

Oral bacterial biofilms are the main reason for the progression of resistance to antimicrobial agents that may lead to severe conditions, including periodontitis and gingivitis. Essential oil-based nanocomposites can be a promising treatment option. We investigated cardamom, cinnamon, and clove essential oils for their potential in the treatment of oral bacterial infections using in vitro and computational tools. A detailed analysis of the drug-likeness and physicochemical properties of all constituents was performed. Molecular docking studies revealed that the binding free energy of a Carbopol 940 and eugenol complex was -2.0kcal/mol, of a Carbopol 940-anisaldehyde complex was -1.9kcal/mol, and a Carbapol 940-eugenol-anisaldehyde complex was -3.4kcal/mol. Molecular docking was performed against transcriptional regulator genes 2XCT, 1JIJ, 2Q0P, 4M81, and 3QPI. Eugenol cinnamaldehyde and cineol presented strong interaction with targets. The essential oils were analyzed against Staphylococcus aureus and Staphylococcus epidermidis isolated from the oral cavity of diabetic patients. The cinnamon and clove essential oil combination presented significant minimum inhibitory concentrations (MICs) (0.0625/0.0312mg/mL) against S. epidermidis and S. aureus (0.0156/0.0078mg/mL). In the anti-quorum sensing activity, the cinnamon and clove oil combination presented moderate inhibition (8mm) against Chromobacterium voilaceum with substantial violacein inhibition (58% ± 1.2%). Likewise, a significant biofilm inhibition was recorded in the case of S. aureus (82.1% ± 0.21%) and S. epidermidis (84.2% ± 1.3%) in combination. It was concluded that a clove and cinnamon essential oil-based formulation could be employed to prepare a stable nanocomposite, and Carbapol 940 could be used as a compatible biopolymer.

Histiocytosis and adult-onset orbital xanthogranuloma in 2023: a review of the literature and mini case series.

Within the large umbrella of histiocytosis are a few similar yet heterogenous entities involving the orbit and periocular tissues with or without systemic infiltration, termed adult onset xanthogranuloma or orbital xanthogranuloma. Due to rarity of these conditions, different classifications in use, diverse clinical presentations and still unknown etiology, the aim of this paper was to provide an up-to-date literature review of the actual understanding of histiocytosis and its subgroups involving the orbit and periocular area, diagnostic strategies and therapeutic modalities. We present a review of literature and small case series comprising four patients diagnosed and treated in the period from 2001 until 2023 in our hospital. Clinical files of 4 patients with adult-onset xanthogranulomatous disease of the orbit and ocular adnexa (AOXGD) were reviewed retrospectively. Clinical, laboratory, radiological, histopathological, and immunohistochemical findings were reexamined. Reviewing medical records of our patients with AOXGD, we found significant overlap between histiocytosis and different immune disorders. A broad workup should be considered in these patients as they can harbour severe immune disfunctions and hematologic disorders. Preferred treatment modality depends on a histopathologic type of AOXGD, clinical presentation and systemic involvement and should be conducted multidisciplinary. The diagnosis is often delayed because of its rarity and diverse clinical findings. Development of molecular genetic tests, detection of BRAF V600E mutation and different types of kinase mutations, mutations in transcriptional regulatory genes as well as tyrosine kinase receptors have shed a new light on the etiopathogenesis and potential targeted treatment of histiocytosis.

The neuron-specific IIS/FOXO transcriptome in aged animals reveals regulatory mechanisms of cognitive aging

Cognitive decline is a significant health concern in our aging society. Here, we used the model organism C. elegans to investigate the impact of the IIS/FOXO pathway on age-related cognitive decline. The daf-2 Insulin/IGF-1 receptor mutant exhibits a significant extension of learning and memory span with age compared to wild-type worms, an effect that is dependent on the DAF-16 transcription factor. To identify possible mechanisms by which aging daf-2 mutants maintain learning and memory with age while wild-type worms lose neuronal function, we carried out neuron-specific transcriptomic analysis in aged animals. We observed downregulation of neuronal genes and upregulation of transcriptional regulation genes in aging wild-type neurons. By contrast, IIS/FOXO pathway mutants exhibit distinct neuronal transcriptomic alterations in response to cognitive aging, including upregulation of stress response genes and downregulation of specific insulin signaling genes. We tested the roles of significantly transcriptionally-changed genes in regulating cognitive functions, identifying novel regulators of learning and memory. In addition to other mechanistic insights, a comparison of the aged vs young daf-2 neuronal transcriptome revealed that a new set of potentially neuroprotective genes is upregulated; instead of simply mimicking a young state, daf-2 may enhance neuronal resilience to accumulation of harm and take a more active approach to combat aging. These findings suggest a potential mechanism for regulating cognitive function with age and offer insights into novel therapeutic targets for age-related cognitive decline.

The neuron-specific IIS/FOXO transcriptome in aged animals reveals regulatory mechanisms of cognitive aging.

Cognitive decline is a significant health concern in our aging society. Here, we used the model organism C. elegans to investigate the impact of the IIS/FOXO pathway on age-related cognitive decline. The daf-2 Insulin/IGF-1 receptor mutant exhibits a significant extension of learning and memory span with age compared to wild-type worms, an effect that is dependent on the DAF-16 transcription factor. To identify possible mechanisms by which aging daf-2 mutants maintain learning and memory with age while wild-type worms lose neuronal function, we carried out neuron-specific transcriptomic analysis in aged animals. We observed downregulation of neuronal genes and upregulation of transcriptional regulation genes in aging wild-type neurons. By contrast, IIS/FOXO pathway mutants exhibit distinct neuronal transcriptomic alterations in response to cognitive aging, including upregulation of stress response genes and downregulation of specific insulin signaling genes. We tested the roles of significantly transcriptionally-changed genes in regulating cognitive functions, identifying novel regulators of learning and memory. In addition to other mechanistic insights, a comparison of the aged vs young daf-2 neuronal transcriptome revealed that a new set of potentially neuroprotective genes is upregulated; instead of simply mimicking a young state, daf-2 may enhance neuronal resilience to accumulation of harm and take a more active approach to combat aging. These findings suggest a potential mechanism for regulating cognitive function with age and offer insights into novel therapeutic targets for age-related cognitive decline.

Potential Role of Lauric Acid in Milk Fat Synthesis in Chinese Holstein Cows Based on Integrated Analysis of Ruminal Microbiome and Metabolome.

The composition and metabolic profile of the ruminal microbiome have an impact on milk composition. To unravel the ruminal microbiome and metabolome affecting milk fat synthesis in dairy cows, 16S rRNA and internal transcribed spacer (ITS) gene sequencing, as well as ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) methods were used to investigate the significant differences in ruminal bacterial and fungal communities as well as metabolome among Chinese Holstein cows with contrasting milk fat contents under the same diet (H-MF 5.82 ± 0.41% vs. L-MF 3.60 ± 0.12%). Another objective was to culture bovine mammary epithelial cells (BMECs) to assess the effect of metabolites on lipid metabolism. Results showed that the acetate-to-propionate ratio and xylanase activity in ruminal fluid were both higher in H-MF. Microbiome sequencing identified 10 types of bacteria and four types of fungi differently abundant at the genus level. Metabolomics analysis indicated 11 different ruminal metabolites between the two groups, the majority of which were lipids and organic acids. Among these, lauric acid (LA) was enriched in fatty acid biosynthesis with its concentration in milk fat of H-MF cows being greater (217 vs. 156 mg per 100 g milk), thus, it was selected for an in vitro study with BMECs. Exogenous LA led to a marked increase in intracellular triglyceride (TG) content and lipid droplet formation, and it upregulated the mRNA abundance of fatty acid uptake and activation (CD36 and ACSL1), TG synthesis (DGAT1, DGAT2 and GPAM), and transcriptional regulation (SREBP1) genes. Taken together, the greater relative abundance of xylan-fermenting bacteria and fungi, and lower abundance of bacteria suppressing short-chain fatty acid-producing bacteria or participating in fatty acid hydrogenation altered lipids and organic acids in the rumen of dairy cows. In BMECs, LA altered the expression of genes involved in lipid metabolism in mammary cells, ultimately promoting milk fat synthesis. Thus, it appears that this fatty acid plays a key role in milk fat synthesis.

Improving Surfactin Production in Bacillus subtilis 168 by Metabolic Engineering.

Surfactin is widely used in the petroleum extraction, cosmetics, biopharmaceuticals and agriculture industries. It possesses antibacterial and antiviral activities and can reduce interfacial tension. Bacillus are commonly used as production chassis, but wild-type Bacillus subtilis 168 cannot synthesise surfactin. In this study, the phosphopantetheinyl transferase (PPTase) gene sfp* (with a T base removed) was overexpressed and enzyme activity was restored, enabling B. subtilis 168 to synthesise surfactin with a yield of 747.5 ± 6.5 mg/L. Knocking out ppsD and yvkC did not enhance surfactin synthesis. Overexpression of predicted surfactin transporter gene yfiS increased its titre to 1060.7 ± 89.4 mg/L, while overexpression of yerP, ycxA and ycxA-efp had little or negative effects on surfactin synthesis, suggesting YfiS is involved in surfactin efflux. By replacing the native promoter of the srfA operon encoding surfactin synthase with three promoters, surfactin synthesis was significantly reduced. However, knockout of the global transcriptional regulator gene codY enhanced the surfactin titre to 1601.8 ± 91.9 mg/L. The highest surfactin titre reached 3.89 ± 0.07 g/L, with the yield of 0.63 ± 0.02 g/g DCW, after 36 h of fed-batch fermentation in 5 L fermenter. This study provides a reference for further understanding surfactin synthesis and constructing microbial cell factories.

Expression of a unique M. tuberculosis DNA MTase Rv1509 in M. smegmatis alters the gene expression pattern and enhances virulence.

Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis.