Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve.

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.

Delirium in psychiatric settings: risk factors and assessment tools in patients with psychiatric illness: a scoping review.

BACKGROUND: Delirium is a common disorder affecting patients' psychiatric illness, characterized by a high rate of underdiagnosis, misdiagnosis, and high risks. However, previous studies frequently excluded patients with psychiatric illness, leading to limited knowledge about risk factors and optimal assessment tools for delirium in psychiatric settings. OBJECTIVES: The scoping review was carried out to (1) identify the risk factors associated with delirium in patients with psychiatric illness; (2) synthesize the performance of assessment tools for detecting delirium in patients with psychiatric illness in psychiatric settings. DESIGN: Scoping review. DATA SOURCES: PubMed, Web of Science, and Embase were searched to identify primary studies on delirium in psychiatric settings from inception to Dec 2023 inclusive. Two independent reviewers screened eligible studies against inclusion criteria. A narrative synthesis of the included studies was conducted. RESULTS: A final set of 36 articles meeting the inclusion criteria, two main themes were extracted: risk factors associated with delirium in patients with psychiatric illness and assessment tools for detecting delirium in psychiatric settings. The risk factors associated with delirium primarily included advanced age, physical comorbid, types of psychiatric illness, antipsychotics, anticholinergic drug, Electroconvulsive therapy, and the combination of lithium and Electroconvulsive therapy. Delirium Rating Scale-Revised-98, Memorial Delirium Assessment Scale, and Delirium Diagnostic Tool-Provisional might be valuable for delirium assessment in patients with psychiatric illness in psychiatric settings. CONCLUSIONS: Delirium diagnosis in psychiatric settings is complex due to the overlapping clinical manifestations between psychiatric illness and delirium, as well as their potential co-occurrence. It is imperative to understand the risk factors and assessment methods related to delirium in this population to address diagnostic delays, establish effective prevention and screening strategies. Future research should focus on designing, implementing, and evaluating interventions that target modifiable risk factors, to prevent and manage delirium in patients with psychiatric illness.

Functions and substrate selectivity of diacylglycerol acyltransferases from Mortierella alpina.

Mortierella alpina produces various polyunsaturated fatty acids in the form of triacylglycerols (TAG). Diacylglycerol acyltransferase (DGAT) catalyzes the binding of acyl-CoA to diacylglycerol to form TAG and is the key enzyme involved in TAG synthesis. A variety of DGATs are present in M. alpina; however, comparative analysis of the functional properties and substrate selectivity of these DGATs is insufficient. In this study, DGAT1 (MaDGAT1A/1B/1C) and DGAT2 (MaDGAT2A/2B) isoforms from M. alpina were analyzed and heterologously expressed in S. cerevisiae H1246. The results showed that MaDGAT1A/1B/2A/2B were able to restore TAG synthesis, and the corresponding TAG content in recombinant yeasts was 2.92 ± 0.42%, 3.62 ± 0.22%, 0.86 ± 0.34%, and 0.18 ± 0.09%, respectively. In S. cerevisiae H1246, MaDGAT1A preferred C16:1 among monounsaturated fatty acids, MaDGAT1B preferred C16:0 among saturated fatty acids (SFAs), and MaDGAT2A/2B preferred C18:0 among SFAs. Under exogenous addition of polyunsaturated fatty acids (PUFAs), MaDGAT1A and 2A preferentially assembled linoleic acid into TAG, and MaDGAT2B had substrate selectivity for eicosapentaenoic and linoleic acids in ω-6 PUFAs. In vitro, MaDGAT1A showed no obvious acyl-CoA selectivity and MaDGAT1B preferred C20:5-CoA. MaDGAT1A/1B preferred C18:1/C18:1-DAG compared with C20:4/C20:4-DAG. This study indicates that MaDGATs have the potential to be used in the production of LA/EPA-rich TAG and provide a reference for improving the production of TAGs in oleaginous fungi. KEY POINTS: • MaDGAT1A preferred C16:1 among MUFAs, MaDGAT1B and MaDGAT2A/2B preferred C16:0 and C18:0 among SFAs, respectively • MaDGAT1A/2A preferentially assembled linoleic acid into TAG, and MaDGAT2B has substrate selectivity for eicosapentaenoic acid and linoleic acid in ω-6 PUFAs • MaDGAT1A showed no obvious acyl-CoA selectivity, and MaDGAT1B preferred C20:5-CoA. MaDGAT1A/1B preferred to select C18:1/C18:1-DAG compared with C20:4/C20:4-DAG.

Consensus mutagenesis and computational simulation provide insight into the desaturation catalytic mechanism for delta 6 fatty acid desaturase.

Fatty acid desaturase (FADS) generates double bond at a certain position of the corresponding polyunsaturated fatty acids (PUFAs) with high selectivity, the enzyme activity and PUFAs products of which are essential to biological systems and are associated with a variety of physiological diseases. Little is known about the structure of FADSs and their amino acid residues related to catalytic activities. Identifying key residues of Micromonas pusilla delta 6 desaturase (MpFADS6) provides a point of departure for a better understanding of desaturation. In this study, conserved amino acids were anchored through gene consensus analysis, thereby generating corresponding variants by site-directed mutagenesis. To achieve stable and high-efficiency expression of MpFADS6 and its variants in Saccharomyces cerevisiae, the key points of induced expression were optimized. The contribution of conserved residues to the function of enzyme was determined by analyzing enzyme activity of the variants. Molecular modeling indicated that these residues are essential to catalytic activities, or substrate binding. Mutants MpFADS6[Q409R] and MpFADS6[M242P] abolished desaturation, while MpFADS6[F419V] and MpFADS6[A374Q] significantly reduced catalytic activities. Given that certain residues have been identified to have a significant impact on MpFADS6 activities, it is put forward that histidine-conserved region III of FADS6 is related to electronic transfer during desaturation, while histidine-conserved regions I and II are related to desaturation. These findings provide new insights and methods to determine the structure, mechanism and directed transformation of membrane-bound desaturases.

The relationship between amino acid and lipid metabolism in oleaginous eukaryotic microorganism.

Amino acids are the building blocks of protein, promoting the balance between growth and lipid synthesis. However, the accumulation of microbial lipids involves multiple pathways, which requires the analysis of the global cellular metabolic network in which amino acid metabolism is involved. This review illustrates the dependence patterns of intracellular amino acids and lipids of oleaginous eukaryotic microorganisms in different environments and points out the contribution of amino acid metabolic precursors to the de novo synthesis of fatty acids. We emphasized the key role of amino acid metabolism in lipid remodeling and autophagy behavior and highlighted the regulatory effects of amino acids and their secondary metabolites as signal factors for microbial lipid synthesis. The application prospects of omics technology and genetic engineering technology in the field of microbial lipids are described. KEY POINTS: • Overview of microbial lipid synthesis mediated by amino acid metabolism • Insight into metabolic mechanisms founding multiple regulatory networks is provided • Description of microbial lipid homeostasis mediated by amino acid excitation signal.

Shared and Non-Shared sIgA-Coated and -Uncoated Bacteria in Intestine of Mother-Infant Pairs.

The infant gut microbiota is critical for promoting and maintaining early-life health. The study aimed to analyze the composition of sIgA-coated and sIgA-uncoated bacterial communities at genus level and lactobacilli and bifidobacterial communities at species level in human breast milk (HBM) and infant and maternal feces. Eleven pregnant women were recruited successfully. HBM; infant feces during colostrum, transition, and mature stages; and maternal feces within the mature stage were collected. sIgA-coated and sIgA-uncoated bacteria were separated with magnetic-activated cell sorting. Then, 16S rRNA sequencing, bifidobacterial groEL gene sequencing, and lactobacilli groEL gene sequencing were performed to analyze the bacterial community. PCoA revealed that the compositions of sIgA-coated and sIgA-uncoated bacteria were different among HBM and infant and maternal feces. Higher relative abundance of sIgA-uncoated Bifidobacterium was found in the three lactation stages in infant feces compared to the corresponding HBM, and a higher relative abundance of sIgA-uncoated Faecalibacterium was found in maternal feces compared to HBM and infant feces. For bifidobacterial community, sIgA-coated and sIgA-uncoated B. longum subsp. infantis and B. pseudocatenulatum was dominant in infant feces and maternal feces, respectively. The relative abundance of sIgA-uncoated B. longum subsp. infantis was significantly higher in infant feces compared to that in maternal feces. For the Lactobacillus community, L. paragasseri and L. mucosae were dominant in infant and maternal feces, respectively. HBM and infant and maternal feces showed distinct diversity and composition of both sIgA-coated and sIgA-uncoated bacteria at genus level. Infant and maternal feces showed similar composition of Bifidobacterium at species level. The same Bifidobacterium species could be detected both in sIgA-coated and -uncoated form. This article provided deeper understanding on the microbiota profile in HBM and infant and maternal feces.

The role of phenylalanine hydroxylase in lipogenesis in the oleaginous fungus Mortierella alpina.

Phenylalanine hydroxylase (PAH) catalyses the irreversible hydroxylation of phenylalanine to tyrosine, which is the rate-limiting reaction in phenylalanine metabolism in animals. A variety of polyunsaturated fatty acids can be synthesized by the lipid-producing fungus Mortierella alpina, which has a wide range of industrial applications in the production of arachidonic acid. In this study, RNA interference (RNAi) with the gene PAH was used to explore the role of phenylalanine hydroxylation in lipid biosynthesis in M. alpina. Our results indicated that PAH knockdown decreased the PAH transcript level by approximately 55% and attenuated cellular fatty acid biosynthesis. Furthermore, the level of NADPH, which is a critical reducing agent and the limiting factor in lipogenesis, was decreased in response to PAH RNAi, in addition to the downregulated transcription of other genes involved in NADPH production. Our study indicates that PAH is part of an overall enzymatic and regulatory mechanism supplying NADPH required for lipogenesis in M. alpina.

Role of beta-isopropylmalate dehydrogenase in lipid biosynthesis of the oleaginous fungus Mortierella alpina.

Branched-chain amino acids (BCAAs) play an important role in lipid metabolism by serving as signal molecules as well as a potential acetyl-CoA source. Our previous study found that in the oleaginous fungus Mucor circinelloides, beta-isopropylmalate dehydrogenase (IPMDH), an important enzyme participating in the key BCAA leucine biosynthesis, was differentially expressed during lipid accumulation phase and has a positive role on lipogenesis. To further analyze its effects on lipogenesis in another oleaginous fungus Mortierella alpina, the IPMDH-encoding gene MaLeuB was homologously expressed. It was found that the total fatty acid content in the recombinant strain was increased by 20.2% compared with the control strain, which correlated with a 4-fold increase in the MaLeuB transcriptional level. Intracellular metabolites analysis revealed significant changes in amino acid biosynthesis and metabolism, tricarboxylic acid cycle and butanoate metabolism; specifically, leucine and isoleucine levels were upregulated by 6.4-fold and 2.2-fold, respectively. Our genetic engineering approach and metabolomics study demonstrated that MaLeuB is involved in fatty acid metabolism in M. alpina by affecting BCAAs metabolism, and this newly discovered role of IPMDH provides a potential bypass route to increase lipogenesis in oleaginous fungi.

Advances in improving the biotechnological application of oleaginous fungus Mortierella alpina.

Mortierella alpina is an oleaginous filamentous fungus with considerable lipid productivity, and it has been widely used for industrial production of arachidonic acid. The fermentation process of M. alpina is complicated and can be affected by various factors; therefore, a comprehensive knowledge of its metabolic characteristics and key factors governing lipid biosynthesis is required to further improve its industrial performance. In this review, we discuss the metabolic features and extracellular factors that affect lipid biosynthesis in M. alpina. The current progress in fermentation optimisation and metabolic engineering to improve lipid yield are also summarised. Moreover, we review the applications of M. alpina in the food industry and propose fermentation strategies for better utilisation of this genus in the future. In our opinion, the economic performance of M. alpina should be enhanced from multiple levels, including strains with ideal traits, efficient fermentation strategies, controllable fermentation costs, and competitive products of both high value and productivity. By reviewing the peculiarities of M. alpina and current progress to improve its suitability for biotechnological production, we wish to provide more efficient strategies for future development of M. alpina as a high-value lipid cell factory. KEY POINTS: • Understanding M. alpina metabolism is helpful for rational design of its fermentation processes. • Nitrogen source is a key point that affects PUFA's component and fermentation cost in M. alpina. • Dynamic fermentation strategy combined with breeding is needed to increase lipid yield in M. alpina.

Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC-ELSD to reveal accumulation differences of sugar and lipid.

OBJECTIVES: To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes between carbohydrate and lipid in fermentation process. RESULTS: The extraction of mycelia with HCl following a photometric phenol-sulphuric acid reaction was identified as an optimal method for total carbohydrate analysis in Mortierella alpina, which the extraction efficiency performed 1.1-3.6 fold than other five methods. The total carbohydrate content increased from initial 19.26 to 25.86% during early fermentation process and declined gradually thereafter, while the fatty acid was increasing from 8.47 to 31.03%. For separation and qualitative estimation of intracellular saccharides, the acetonitrile/water freeze-thaw method for extraction and Sugar-Pak I column for separation proved to be possible. With the glucose rapidly decreasing at the beginning of growth, the trehalose accumulated rapidly from 1.63 to 5.04% and then decreased slightly but maintain above 4% of dry biomass. CONCLUSIONS: This work established comprehensive carbohydrate extraction and analysis methods of Mortierella alpina and identified the main saccharide in fermentation process which indicated that the accumulation of fatty acids was related to the change of intracellular carbohydrate content.

Role of the mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina.

OBJECTIVES: The transport of citrate from the mitochondria to the cytoplasm is essential during lipid accumulation. This study aimed to explore the role of mitochondrial citrate-oxoglutarate carrier in lipid accumulation in the oleaginous fungus Mortierella alpina. RESULTS: Homologous MaYHM (the gene encoding the mitochondrial citrate-oxoglutarate carrier) was overexpressed in M. alpina. The fatty acid content of MaYHM-overexpressing recombinant strains was increased by up to 30% compared with the control. Moreover, the intracellular α-ketoglutarate level in recombinant strains was increased by 2.2 fold, together with a 23-35% decrease in NAD+-isocitrate dehydrogenase activity compared with the control. The overexpression of MaYHM altered the metabolic flux in the glutamate dehydrogenase shunt and 4-aminobutyric acid shunt during metabolic reprogramming, supplying more carbon to synthesize fatty acids. CONCLUSIONS: Overexpression of MaYHM resulted in more efflux of citrate from mitochondria to the cytoplasm and enhanced lipid accumulation. These findings provide new perspectives for the improvement of industrial lipid production in M. alpina.

The role of MTHFDL in mediating intracellular lipogenesis in oleaginous Mortierella alpina.

The oleaginous fungus Mortierella alpina can synthesize a variety of polyunsaturated fatty acids, which are used extensively in industry for the production of arachidonic acid (AA). NADPH is the limiting factor and critical reducing agent in lipid biosynthesis. In the folate cycle, methylenetetrahydrofolate dehydrogenase (MTHFDL) catalyzes the conversion of methylene tetrahydrofolate into 10-formyl-tetrahydrofolate with the reduction of NADP+ to NADPH. MTHFDL RNAi was used to investigate the role of the folate cycle in lipogenesis. Gene knockdown decreased the transcript levels of MTHFDL by about 50 % and attenuated cell fatty acid synthesis. The observation of decreased NADPH levels and downregulated NADPH-producing genes in response to MTHFDL RNAi indicates a novel aspect of the NADPH regulatory mechanism. Thus, our study demonstrates that MTHFDL plays key role in the mediation of NADPH in lipogenesis in M. alpina.

Δ6 fatty acid desaturases in polyunsaturated fatty acid biosynthesis: insights into the evolution, function with substrate specificities and biotechnological use.

Δ6 fatty acid desaturases (FADS6) have different substrate specificities that impact the ratio of omega-6/omega-3 polyunsaturated fatty acids, which are involved in regulating multiple signalling pathways associated with various diseases. For decades, FADS6 with different substrate specificities have been characterized and the functions of these crucial enzymes have been investigated, while it remains enigmatic that the substrate specificities of FADS6 from various species have a huge difference. This review summarizes the substrate specificities of FADS6 in different species and reveals the underlying relationship. Further evaluation of biochemical properties has revealed that the FADS6 prefer linoleic acid that is more hydrophilic and stable. Domain-swapping and site-directed mutagenesis have been employed to delineate the regions and sites that affect the substrate specificities of FADS6. These analyses improve our understanding of the functions of FADS6 and offer information for the discovery of novel biological resources. KEY POINTS: • Outline of the excavation and identification of Δ6 fatty acid desaturases. • Overview of methods used to determine the pivotal resides of desaturases. • Application of substrate properties to generate specific fatty acids.

Two-stage pH control combined with oxygen-enriched air strategies for the highly efficient production of EPA by Mortierella alpina CCFM698 with fed-batch fermentation.

Dissolved oxygen and pH are critical factors influencing cell growth and metabolism. In our previous work, we constructed the recombinant strain Mortierella alpina CCFM698, which has the ability to produce EPA at room temperature. However, our experiments showed that the dissolved oxygen produced by the aeration and agitation of the fermenter was insufficient for cell growth and EPA synthesis by this recombinant strain. Moreover, the optimum pH for cell growth was incompatible with that of EPA accumulation. This study introduced a combined strategy of two-stage pH control with oxygen-enriched air in fed-batch fermentation to facilitate both cell growth and EPA production in M. alpina CCFM698. After 10 days of fermentation in a 7.5 L tank, the biomass production reached 41.2 g/L, with a lipid content of 31.5%, and EPA accounting for 26.7% of total lipids. The final EPA production reached 3.47 g/L, which is the highest yet achieved by M. alpina. This study reveals the critical role of dissolved oxygen and pH control for EPA production of M. alpina, and provides an easy and efficient strategy for industrial production of EPA.

Antiproliferation Activity and Mechanism of c9, t11, c15-CLNA and t9, t11, c15-CLNA from Lactobacillus plantarum ZS2058 on Colon Cancer Cells.

Conjugated linolenic acid (CLNA) is a type of ω-3 fatty acid which has been proven to have a series of benefits. However, there is no study about the function of Lactobacillus-derived CLNA isomer. Lactobacillus plantarum ZS2058 has been proven to manifest comprehensive functions and can produce CLNA. To investigate the specific functions of CLNA produced by this probiotic bacterium, two different conjugated α-linolenic acid (CLNA) isomers were successfully isolated. These isoforms, CLNA1 (c9, t11, c15-CLNA, purity 97.48%) and CLNA2 (c9, t11, t15-CLNA, purity 99.00%), both showed the ability to inhibit the growth of three types of colon cancer cells in a time- and concentration-dependent manner. In addition, the expression of MDA in Caco-2 cells was increased by CLNA1 or CLNA2, which indicated that lipid peroxidation was related to the antiproliferation activity of CLNAs. An examination of the key protein of pyroptosis showed that CLNA1 induced the cleavage of caspase-1 and gasdermin-D, while CLNA2 induced the cleavage of caspase-4, 5 and gasdermin-D. The addition of relative inhibitors could alleviate the pyroptosis by CLNAs. CLNA1 and CLNA2 showed no effect on caspase-3, 7, 9 and PARP-1, which were key proteins associated with apoptosis. No sub-diploid apoptotic peak appeared in the result of PI single staining test. In conclusion, CLNA1 activated caspase-1 and induced Caco-2 cell pyroptosis, whereas CLNA2 induced pyroptosis through the caspase-4/5-mediated pathway. The inhibition of Caco-2 cells by the two isomers was not related to apoptosis. This is the first study on the function of Lactobacillus-derived CLNA isomer. The inhibition pathway of Lactobacillus-derived CLNA isomer on colon cancer cells were proved.

Evaluation of metabolome sample preparation and extraction methodologies for oleaginous filamentous fungi Mortierella alpina.

INTRODUCTION: Metabolomics has been successfully applied to guide the rational engineering of industrial strains and improve the performance of bioprocesses. Mortierella alpina has traditionally been one of the most popular industrial strains for the production of polyunsaturated fatty acids. However, a systematic comparison and optimisation of the metabolomic analysis methods of M. alpina has not yet been reported. OBJECTIVE: We sought to identify potential weaknesses that are important for accurate metabolomic analysis. We also aimed to determine an efficient sample preparation protocol for metabolomics studies in the oleaginous filamentous fungus M. alpina. METHODS: In this study, using GC-MS, we evaluated three sample preparation protocols and five solvent mixtures by assessment of the metabolite profile differences, the sum of peak intensities and the reproducibility of metabolite quantification. RESULTS: The freeze-dried biomass had better reproducibility and recovery than fresh biomass for metabolite extraction and data normalisation that is part of a metabolomics analysis of filamentous fungi M. alpina. Methanol:water (1:1) was superior for the profiling of metabolites in oleaginous fungi M. alpina. The unbiased metabolite profiling difference between the growth phase and lipids synthesis phase revealed that the degradation of amino acids were critical nodes for the efficient synthesis of lipids in M. alpina. CONCLUSION: The use of freeze-dried biomass for metabolite extraction and data normalisation was more efficient at measuring the active state of the intracellular metabolites in M. alpina. We recommend extracting the intracellular metabolites with methanol:water (1:1). An important role of amino acid oxidation in the nitrogen limitation-mediated lipid accumulation was found.

Role of 10-hydroxy-cis-12-octadecenic acid in transforming linoleic acid into conjugated linoleic acid by bifidobacteria.

10-hydroxy-cis-12 octadecenoic acid (10-HOE) is a type of octadecenoic acid with a hydroxyl on the C10 carbon. It is generated from linoleic acid (LA) catalyzed by linoleate hydratase in lactobacilli, which was initially named as myosin-cross-reactive antigen (MCRA). In lactobacilli, 10-HOE is the first intermediate in the production of conjugated LA (CLA). Although MCRA from bifidobacteria can generate 10-HOE, the precise role of 10-HOE in CLA production in bifidobacteria remains unknown. In the current work, 10-HOE and LA were added to the medium as the substrate both separately and synchronously to analyze their influence on CLA production. Using 10-HOE as the substrate, bifidobacteria were able to generate CLA by first converting it to LA, followed by CLA accumulation. Recombinant MCRA catalyzed the conversion of 10-HOE to LA, indicating that bifidobacterial MCRA can account for the reversible conversion between LA and 10-HOE. This is the first report to demonstrate the precise role of 10-HOE in the process of CLA production among bifidobacteria.

Role of B-Cell Lymphoma 2 Ovarian Killer (BOK) in Acute Toxicity of Human Lung Epithelial Cells Caused by Cadmium Chloride.

BACKGROUND B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) is a Bcl-2 family member with sequence homology to pro-apoptotic BAX and BAK, but its physiological and pathological roles remain largely unclear. Exposure of cells to cadmium may cause DNA damage, decrease DNA repair capacity, and increase genomic instability. MATERIAL AND METHODS The present study investigated the effects of BOK on the toxicity of cadmium chloride (CdCl2) to human bronchial epithelial (16HBE) cells. We constructed BOK over-expressing (16HBE-BOK) cells and BOK knockdown (16HBE-shBOK) cells using the BOK-ORF plasmid and BOK-siRNA. qRT-PCR for BOK mRNA expression. We used Trypan blue exclusion assay for cell growth, MTT colorimetric assays for cells inhibition rate, and Comet assays for detecting damaged DNA. RESULTS CdCl2, at various concentrations and exposure times, increased BOK mRNA. 16HBE-BOK cells (BOK over-expressing) proliferated more than 16HBE cells after 72 h; 16HBE-shBOK (BOK knockdown) cells proliferated less. In addition, BOK deficiency enhanced cell death induced by CdCl2. Similarly, CdCl2- and H2O2-induced DNA damage was greater in BOK-deficient cells. CONCLUSIONS These findings support a role for BOK in CdCl2-induced DNA damage and cell death.

Potential Functions of the Gastrointestinal Microbiome Inhabiting the Length of the Rat Digest Tract.

The rat is an important model animal used frequently in biological researches exploring the correlations between gut microbiome and a wide array of diseases. In this study, we used an extended ancestral-state reconstruction algorithm to predict the functional capabilities of the rat gastrointestinal microbiome. Our results indicate an apparent tendency toward metabolic heterogeneity along the longitudinal and transverse axes of the rat gastrointestinal tract (GIT). This heterogeneity was suggested by the enriched small-molecule transport activity and amino acid metabolism in the upper GIT, the aerobic energy metabolism in the stomach and the mucolysis-related metabolism in the lower GIT mucus layer. In contrast to prior results, many functional overlaps were observed when the gastrointestinal microbiomes of different hosts were compared. These overlaps implied that although both the biogeographic location and host genotype were prominent driving forces in shaping the gastrointestinal microbiota, the microbiome functions were similar across hosts when observed under similar physicochemical conditions at identical anatomical sites. Our work effectively complements the rat microbial biogeography dataset we released in 2017 and, thus, contributes to a better understanding and prediction of disease-related alterations in microbial community function.

Structural Determinants of Substrate Specificity of Omega-3 Desaturases from Mortierella alpina and Rhizophagus irregularis by Domain-Swapping and Molecular Docking.

Although various ω-3 fatty acid desaturases (ω3Des) have been identified and well-studied regarding substrate preference and regiospecificity, the molecular mechanism of their substrate specificities remains to be investigated. Here we compared two ω3Des, FADS15 from Mortierella alpina and oRiFADS17 from Rhizophagus irregularis, which possessed a substrate preference for linoleic acid and arachidonic acid, respectively. Their sequences were divided into six sections and a domain-swapping strategy was used to test the role of each section in catalytic activity. Heterologous expression and fatty acid experiments of hybrid enzymes in Saccharomyces cerevisiae INVSc1 indicated that the sequences between his-boxes I and II played critical roles in influencing substrate preference. Based on site-directed mutagenesis and molecular docking, the amino acid substitutions W129T and T144W, located in the upper part of the hydrocarbon chain, were found to be involved in substrate specificity, while V137T and V152T were confirmed to interfere with substrate recognition. This study provides significant insight into the structure-function relationship of ω3Des.