Lipases are differentially regulated by hormones to maintain free fatty acid homeostasis for insect brain development.

BACKGROUND: Free fatty acids (FFAs) play vital roles as energy sources and substrates in organisms; however, the molecular mechanism regulating the homeostasis of FFA levels in various circumstances, such as feeding and nonfeeding stages, is not fully clarified. Holometabolous insects digest dietary triglycerides (TAGs) during larval feeding stages and degrade stored TAGs in the fat body during metamorphosis after feeding cessation, which presents a suitable model for this study. RESULTS: This study reported that two lipases are differentially regulated by hormones to maintain the homeostasis of FFA levels during the feeding and nonfeeding stages using the lepidopteran insect cotton bollworm Helicoverpa armigera as a model. Lipase member H-A-like (Lha-like), related to human pancreatic lipase (PTL), was abundantly expressed in the midgut during the feeding stage, while the monoacylglycerol lipase ABHD12-like (Abhd12-like), related to human monoacylglycerol lipase (MGL), was abundantly expressed in the fat body during the nonfeeding stage. Lha-like was upregulated by juvenile hormone (JH) via the JH intracellular receptor methoprene-tolerant 1 (MET1), and Abhd12-like was upregulated by 20-hydroxyecdysone (20E) via forkhead box O (FOXO) transcription factor. Knockdown of Lha-like decreased FFA levels in the hemolymph and reduced TAG levels in the fat body. Moreover, lipid droplets (LDs) were small, the brain morphology was abnormal, the size of the brain was small, and the larvae showed the phenotype of delayed pupation, small pupae, and delayed tissue remodeling. Knockdown of Abhd12-like decreased FFA levels in the hemolymph; however, TAG levels increased in the fat body, and LDs remained large. The development of the brain was arrested at the larval stage, and the larvae showed a delayed pupation phenotype and delayed tissue remodeling. CONCLUSIONS: The differential regulation of lipases expression by different hormones determines FFAs homeostasis and different TAG levels in the fat body during the feeding larval growth and nonfeeding stages of metamorphosis in the insect. The homeostasis of FFAs supports insect growth, brain development, and metamorphosis.

Biodiesel Production from Waste Cooking Oil Using Recombinant Escherichia coli Cells Immobilized into Fe3O4-Chitosan Magnetic Microspheres.

Developing reusable and easy-to-operate biocatalysts is of significant interest in biodiesel production. Here, magnetic whole-cell catalysts constructed through immobilizing recombinant Escherichia coli cells (containing MAS1 lipase) into Fe3O4-chitosan magnetic microspheres (termed MWCC@MAS1) were used for fatty acid methyl ester (FAME) production from waste cooking oil (WCO). During the preparation process of immobilized cells, the effects of chitosan concentration and cell concentration on their activity and activity recovery were investigated. Optimal immobilization was achieved with 3% (w/v) chitosan solution and 10 mg wet cell/mL cell suspension. Magnetic immobilization endowed the whole-cell catalysts with superparamagnetism and improved their methanol tolerance, enhancing the recyclability of the biocatalysts. Additionally, we studied the effects of catalyst loading, water content, methanol content, and reaction temperature on FAME yield, optimizing these parameters using response surface methodology and Box-Behnken design. An experimental FAME yield of 89.19% was gained under the optimized conditions (3.9 wt% catalyst loading, 22.3% (v/w) water content, 23.0% (v/w) methanol content, and 32 °C) for 48 h. MWCC@MAS1 demonstrated superior recyclability compared to its whole-cell form, maintaining about 86% of its initial productivity after 10 cycles, whereas the whole-cell form lost nearly half after just five cycles. These results suggest that MWCC@MAS1 has great potential for the industrial production of biodiesel.

Efficient Expression and Application of a Modified Rhizomucor miehei Lipase for Simultaneous Production of Biodiesel and Eicosapentaenoic Acid Ethyl Ester from Nannochloropsis Oil.

Few reports exist on one-step enzymatic methods for the simultaneous production of biodiesel and eicosapentaenoic acid ethyl ester (EPA-EE), a high-value pharmaceutical compound. This study aimed to efficiently express Rhizomucor miehei lipase (pRML) in Pichia pastoris X-33 via propeptide mutation and high-copy strain screening. The mutated enzyme was then used to simultaneously catalyze the production of both biodiesel and EPA-EE. The P46N mutation in the propeptide (P46N-pRML) significantly boosted its production, with the four-copy strain increasing enzyme yield by 3.7-fold, reaching 3425 U/mL. Meanwhile, its optimal temperature increased to 45-50 °C, pH expanded to 7.0-8.0, specific activity doubled, Km reduced to one-third, and kcat/Km increased 7-fold. Notably, P46N-pRML efficiently converts Nannochloropsis gaditana oil's eicosapentaenoic acid (EPA). Under optimal conditions, it achieves up to 93% biodiesel and 92% EPA-EE yields in 9 h. Our study introduces a novel, efficient one-step green method to produce both biodiesel and EPA-EE using this advanced enzyme.

A PNPLA3-Deficient iPSC-Derived Hepatocyte Screen Identifies Pathways to Potentially Reduce Steatosis in Metabolic Dysfunction-Associated Fatty Liver Disease.

The incidence of nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is increasing in adults and children. Unfortunately, effective pharmacological treatments remain unavailable. Single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain-containing protein (PNPLA3 I148M) have the most significant genetic association with the disease at all stages of its progression. A roadblock to identifying potential treatments for PNPLA3-induced NAFLD is the lack of a human cell platform that recapitulates the PNPLA3 I148M-mediated onset of lipid accumulation. Hepatocyte-like cells were generated from PNPLA3-/- and PNPLA3I148M/M-induced pluripotent stem cells (iPSCs). Lipid levels were measured by staining with BODIPY 493/503 and were found to increase in PNPLA3 variant iPSC-derived hepatocytes. A small-molecule screen identified multiple compounds that target Src/PI3K/Akt signaling and could eradicate lipid accumulation in these cells. We found that drugs currently in clinical trials for cancer treatment that target the same pathways also reduced lipid accumulation in PNPLA3 variant cells.

Association of copy number variation in X chromosome-linked PNPLA4 with heterotaxy and congenital heart disease.

BACKGROUND: Heterotaxy (HTX) is a thoracoabdominal organ anomaly syndrome and commonly accompanied by congenital heart disease (CHD). The aim of this study was to analyze rare copy number variations (CNVs) in a HTX/CHD cohort and to examine the potential mechanisms contributing to HTX/CHD. METHODS: Chromosome microarray analysis was used to identify rare CNVs in a cohort of 120 unrelated HTX/CHD patients, and available samples from parents were used to confirm the inheritance pattern. Potential candidate genes in CNVs region were prioritized via the DECIPHER database, and PNPLA4 was identified as the leading candidate gene. To validate, we generated PNPLA4 -overexpressing human induced pluripotent stem cell lines as well as pnpla4 -overexpressing zebrafish model, followed by a series of transcriptomic, biochemical and cellular analyses. RESULTS: Seventeen rare CNVs were identified in 15 of the 120 HTX/CHD patients (12.5%). Xp22.31 duplication was one of the inherited CNVs identified in this HTX/CHD cohort, and PNPLA4 in the Xp22.31 was a candidate gene associated with HTX/CHD. PNPLA4 is expressed in the lateral plate mesoderm, which is known to be critical for left/right embryonic patterning as well as cardiomyocyte differentiation, and in the neural crest cell lineage. Through a series of in vivo and in vitro analyses at the molecular and cellular levels, we revealed that the biological function of PNPLA4 is importantly involved in the primary cilia formation and function via its regulation of energy metabolism and mitochondria-mediated ATP production. CONCLUSIONS: Our findings demonstrated a significant association between CNVs and HTX/CHD. Our data strongly suggested that an increased genetic dose of PNPLA4 due to Xp22.31 duplication is a disease-causing risk factor for HTX/CHD.

Genotypic variation in CYP2E1, GCKR, and PNPLA3 among nonalcoholic steatohepatitis patients of Turkish origin.

BACKGROUND: This study examines genetic variations in CYP2E1 (rs6413432, rs3813867), GCKR (rs780094, rs1260326), and PNPLA3 (rs738409) among Turkish patients to assess their influence on nonalcoholic steatohepatitis. METHODS: Allele and genotype frequencies were compared between 245 NASH patients and 120 healthy controls using SNP genotyping via polymerase chain reaction-restriction fragment length polymorphism. Additionally, the deviation of the observed genotype frequencies from Hardy-Weinberg proportion was examined. RESULTS: No significant differences were found in the allelic and genotypic distributions of rs6413432, rs3813867, and rs780094 between NASH patients and healthy controls. However, significant disparities were noted for rs1260326 and rs738409. Gender and age-specific distributions showed no notable differences. The only observed deviation from Hardy-Weinberg proportion was in the genotype frequency of rs738409. CONCLUSIONS: Variants in GCKR (rs1260326) and PNPLA3 (rs738409) are significantly associated with increased NASH risk in the Turkish population, with the rs738409 variant potentially playing a more prominent role in NASH development.

Recombinant Expression and Characterization of a Novel Thermo-Alkaline Lipase with Increased Solvent Stability from the Antarctic Thermophilic Bacterium Geobacillus sp. ID17.

Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg-1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency (kcat/Km) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v/v ethanol and 70% v/v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.

Repurposing promethazine hydrochloride to inhibit biofilm formation against Burkholderia thailandensis.

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, an intracellular pathogen with a high mortality rate and significant antibiotic resistance. The high mortality rate and resistance to antibiotics have drawn considerable attention from researchers studying melioidosis. This study evaluated the effects of various concentrations (75, 50, and 25 µg/mL) of promethazine hydrochloride (PTZ), a potent antihistamine, on biofilm formation and lipase activity after 24 h of exposure to B. thailandensis E264. A concentration-dependent decrease in both biofilm biomass and lipase activity was observed. RT-PCR analysis revealed that PTZ treatment not only made the biofilm structure loose but also reduced the expression of btaR1, btaR2, btaR3, and scmR. Single gene knockouts of quorum sensing (QS) receptor proteins (∆btaR1, ∆btaR2, and ∆btaR3) were successfully constructed. Deletion of btaR1 affected biofilm formation in B. thailandensis, while deletion of btaR2 and btaR3 led to reduced lipase activity. Molecular docking and biological performance results demonstrated that PTZ inhibits biofilm formation and lipase activity by suppressing the expression of QS-regulated genes. This study found that repositioning PTZ reduced biofilm formation in B. thailandensis E264, suggesting a potential new approach for combating melioidosis.

[Clinical characteristics and genetic analysis of a child with Neutral lipid storage disease with myopathy].

OBJECTIVE: To explore the clinical phenotype and genetic basis of a child with Neutral lipid storage disease with myopathy (NLSDM). METHODS: A child who was admitted to the First Affiliated Hospital of Zhengzhou University in February 2021 for a history of elevated creatine kinase (CK) for over 2 months was selected as the study subject. Clinical and laboratory examinations were carried out, and the child was subjected to whole exome sequencing. Candidate variants were validated by Sanger sequencing of her family members. RESULTS: The patient, a 9-year-old female, had exhibited weakness in the lower limbs, elevated CK level, and refractory cardiomyotrophy. Genetic testing revealed that she has harbored c.32C>G (p.S11W) and c.516C>G (p.N172K) compound heterozygous variants of the PNPLA2 gene, which were respectively inherited from her mother and father. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were rated as likely pathogenic (PM1+PM2_Supporting+PP3+PP4). CONCLUSION: The c.32C>G (p.S11W) and c.516C>G (p.N172K) compound heterozygous variants of the PNPLA2 gene probably underlay the myasthenia gravis and elevated creatine kinase in this child.

Genome-wide identification of the GDSL-type esterase/lipase protein (GELP) gene family in Ricinus communis and its transcriptional regulation during germination and seedling establishment under different abiotic stresses.

GDSL-type esterase/lipase protein (GELP) genes are crucial in the specialized lipid metabolism, in the responses to abiotic stresses, and in the regulation of plant homeostasis. R. communis is an important oilseed crop species that can sustain growth and productivity when exposed to harsh environmental conditions. Herein, we raised the question of whether the GELP gene family could be involved in the acquisition of R. communis tolerance to abiotic stresses during seed germination and seedling establishment. Thus, we used bioinformatics and transcriptomics to characterize the R. communis GELP gene family. R. communis genome possesses 96 GELP genes that were characterized by extensive bioinformatics, including phylogenetic analysis, subcellular localization, exon-intron distribution, the analysis of regulatory cis-elements, tandem duplication, and physicochemical properties. Transcriptomics indicated that numerous RcGELP genes are readily responsive to high-temperature and salt stresses and might be potential candidates for genome editing techniques to develop abiotic stress-tolerant crops.

CTRP9 Promotes Brown Adipose Tissue Lipolysis in Mice Fed a High-Fat Diet.

BACKGROUND: This study aimed to elucidate the molecular mechanism through which C1q/tumor necrosis factor (TNF)-related protein 9 (CTRP9) acts in the formation and differentiation of brown adipose tissue (BAT). METHODS: Adenovirus particles encoding CTRP9 and green fluorescent protein were inoculated into the scapula of C57BL/6J mice and fed a high-fat diet for 8 weeks; the body weight, lipid droplet morphology, glucose tolerance, insulin tolerance, and protein expression levels were analyzed. In addition, CTRP9 adenovirus was transfected into brown preadipocytes, and differentiation was induced to identify the effect of CTRP9 overexpression on adipocyte differentiation. RESULTS: CTRP9 overexpression significantly increased the weight gain of mice. Additionally, the CTRP9 overexpression group exhibited significantly increased adipose tissue weight and glucose clearance rates and decreased insulin sensitivity and serum triglyceride levels compared to the control group. Furthermore, CTRP9 overexpression significantly upregulated the adipose triglyceride lipase (ATGL) and perilipin 1 protein expression levels in BAT. The cell experiment results confirmed that CTRP9 overexpression significantly inhibited the adipogenesis of brown adipocytes as evidenced by the downregulation of uncoupling protein 1, beta-3 adrenergic receptor, ATGL, and hormone-sensitive lipase mRNA levels and the significant suppression of uncoupling protein 1, ATGL, and perilipin 1 protein levels in brown adipocytes. CONCLUSIONS: The finding of this study demonstrated that CTRP9 promotes lipolysis by upregulating ATGL expression in vivo and inhibits the differentiation of brown preadipocytes in vitro.

The impact of genetic risk on the prevalence of advanced fibrosis and cirrhosis in prospectively assessed patients with type 2 diabetes.

BACKGROUND: Genetic factors contribute to the risk and severity of metabolic dysfunction-associated steatotic liver disease (MASLD). However, the utility of genetic testing in risk stratification remains poorly characterised. AIMS: To examine the influence of genetic risk on advanced fibrosis and cirrhosis in patients with type 2 diabetes mellitus (T2DM) and the utility of a polygenic risk score (PRS) in screening guidelines. METHODS: We prospectively enrolled adults aged ≥50 years with T2DM recruited from clinics. PRS was the sum of risk alleles in PNPLA3, TM6SF2 and SERPINA1 minus the protective variant in HSD17B13. We performed magnetic resonance elastography and vibration-controlled transient elastography to assess for advanced fibrosis and cirrhosis. RESULTS: Of 382 included patients, the mean age and BMI were 64.8 (±8.4) years and 31.7 (±6.2) kg/m2 respectively. The prevalence of advanced fibrosis and cirrhosis were 12.3% and 5.2% respectively; higher PRS was associated with increased risk of cirrhosis (2.7% vs. 7.5%, p = 0.037). High PRS was associated with increased risk of advanced fibrosis among those with fibrosis-4 index (FIB-4) index <1.3 (9.6% vs. 2.3%, p = 0.036) but was not significantly different in other FIB-4 categories. Incorporating PRS determination into the American Gastroenterological Association and American Association for the Study of Liver Diseases screening guidelines prevented approximately 20% of all participants with advanced fibrosis from being inappropriately classified as low risk. CONCLUSIONS: Utilising a well-phenotyped, prospective cohort of adults with T2DM, we found that adding an assessment of genetic risk to recommendations to screen at-risk populations may improve risk prediction.

Use of PNPLA3, TM6SF2, and HSD17B13 for detection of fibrosis in MASLD in the general population.

BACKGROUND: Genetic testing can be used to evaluate disease risk. We evaluated if the use of three Single Nucleotide Polymorphisms (SNPs), alone or combined into a genetic risk score (GRS), can aid identify significant fibrosis in subjects with metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: We assessed three known risk variants: PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs72613567. The study included 414 adult individuals invited from the Danish population, who were defined as at-risk of MASLD due to elevated ALT and body mass index (BMI) >25 kg/m2. Participants were assessed clinically and by the Fibrosis-4 (FIB-4) index and Fibroscan. RESULTS: In total, 17 participants (4.1 %) had alcohol-related liver disease, 79 (19.1 %) had no evidence of liver disease, and four (1.0 %) were diagnosed with other liver diseases, including malignant disease. The remaining 314 participants (75.8 %) were diagnosed with MASLD. Of the 27 who underwent a liver biopsy for suspected fibrosis, 15 had significant fibrosis (≥F2) and 12 had no/mild fibrosis (F0/F1). The GRS was not associated with significant fibrosis (p = 0.09) but PNPLA3 was with an odds ratio of 6.75 (95 % CI 1.29 - 50.7; p = 0.039) risk allele CG/GG versus CC. The diagnostic accuracy of PNPLA3 combined with an increased Fib-4 (>1.3) was excellent for detecting significant fibrosis with a sensitivity of 1.00 (95 % CI 0.72-1.00), but the specificity was no better than for FIB-4 alone. CONCLUSIONS: This study found no evidence to support the use of GRS for diagnosing significant fibrosis in MASLD. However, the combination of PNPLA3 and Fib-4 increased sensitivity considerably. In addition, ALT remains a useful tool for screening diagnosing other liver diseases than MASLD.

Does Genetic Variation in PNPLA3, TM6SF2 and HSD17B13 have a Role in the Development or Prognosis of Hepatocellular Carcinoma in Turkish Patients with Hepatitis B?

BACKGROUND AND AIMS: Progression to hepatocellular carcinoma (HCC) is restricted by viral suppression in chronic hepatitis B (CHB); however, some patients still progress despite antiviral therapy. Presence of single nucleotide polymorphisms (SNPs) such as PNPLA3 rs738409 and TM6SF2 rs58542926 are associated with the development and progression of steatotic liver disease to HCC, whereas a splice variant in HSD17B13 rs72613567:TA has been shown to be protective. We investigated the role of these SNPs in the development or prognosis of HCC in pure CHB etiology, in the absence of hepatic steatosis, remains unknown. MATERIALS: We analysed PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs72613567 SNPs in a prospectively recruited cohort (n=323) consisting of healthy controls, CHB and CHB-HCC patients without hepatic steatosis. SNPs were determined by PCR analysis and associations for the alleles and genotypes were investigated using adjusted-logistic regression analyses. The overall survival (OS) data were collected from CHB-HCC patients for survival analysis. RESULTS: The genotype and allelic distribution of PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs72613567 were similar between healthy controls, CHB, and CHB-HCC groups. No genotype, allele or haplotype analysis was found to be associated with increased risk for CHB-HCC. Survival analysis revealed no genotype or allele to be associated with OS in patients with CHB-HCC. CONCLUSIONS: We could not demonstrate any association of PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs72613567 with the development or prognosis of CHB-HCC, supporting the initial hypothesis that they should be considered specific hotspots for liver diseases characterized with hepatic steatosis.

PNPLA3 is a triglyceride lipase that mobilizes polyunsaturated fatty acids to facilitate hepatic secretion of large-sized very low-density lipoprotein.

The I148M variant of PNPLA3 is closely associated with hepatic steatosis. Recent evidence indicates that the I148M mutant functions as an inhibitor of PNPLA2/ATGL-mediated lipolysis, leaving the role of wild-type PNPLA3 undefined. Despite showing a triglyceride hydrolase activity in vitro, PNPLA3 has yet to be established as a lipase in vivo. Here, we show that PNPLA3 preferentially hydrolyzes polyunsaturated triglycerides, mobilizing polyunsaturated fatty acids for phospholipid desaturation and enhancing hepatic secretion of triglyceride-rich lipoproteins. Under lipogenic conditions, mice with liver-specific knockout or acute knockdown of PNPLA3 exhibit aggravated liver steatosis and reduced plasma VLDL-triglyceride levels. Similarly, I148M-knockin mice show decreased hepatic triglyceride secretion during lipogenic stimulation. Our results highlight a specific context whereby the wild-type PNPLA3 facilitates the balance between hepatic triglyceride storage and secretion, and suggest the potential contribution of a loss-of-function by the I148M variant to the development of fatty liver disease in humans.

Microbial cell factory for butyl butyrate production: Knowledges and perspectives.

Butyl butyrate is a short-chain fatty acid ester (C8) with a fruity aroma. It has broad prospects in the fields of foods, cosmetics and biofuels. At present, butyl butyrate is produced by chemical synthesis in the industry, but it is highly dependent on petroleum-based products. The growing concerns regarding the future scarcity of fossil fuels have been strongly promoted the transition from traditional fossil fuels and products to renewable bioenergy and biochemicals. Therefore, it is necessary to develop a green biochemical technology to replace traditional petroleum-based materials. In recent years, microorganisms such as Escherichia coli and Clostridium have been engineered to serve as cell factories for the sustainable one-pot production of short-chain fatty acid esters, including butyl butyrate. This opinion highlights the recent development in the use of lipases and alcohol acyltransferases (AATs) for butyl butyrate production in microbial fermentation, as well as future perspectives.

Distinct Gut Microbial Signature and Host Genetic Variants in Association with Liver Fibrosis Severity in Patients with MASLD.

Gut microbiota might affect the severity and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). We aimed to characterize gut dysbiosis and clinical parameters regarding fibrosis stages assessed by magnetic resonance elastography. This study included 156 patients with MASLD, stratified into no/mild fibrosis (F0-F1) and moderate/severe fibrosis (F2-F4). Fecal specimens were sequenced targeting the V4 region of the 16S rRNA gene and analyzed using bioinformatics. The genotyping of PNPLA3, TM6SF2, and HSD17B13 was assessed by allelic discrimination assays. Our data showed that gut microbial profiles between groups significantly differed in beta-diversity but not in alpha-diversity indices. Enriched Fusobacterium and Escherichia_Shigella, and depleted Lachnospira were found in the F2-F4 group versus the F0-F1 group. Compared to F0-F1, the F2-F4 group had elevated plasma surrogate markers of gut epithelial permeability and bacterial translocation. The bacterial genera, PNPLA3 polymorphisms, old age, and diabetes were independently associated with advanced fibrosis in multivariable analyses. Using the Random Forest classifier, the gut microbial signature of three genera could differentiate the groups with high diagnostic accuracy (AUC of 0.93). These results indicated that the imbalance of enriched pathogenic genera and decreased beneficial bacteria, in association with several clinical and genetic factors, were potential contributors to the pathogenesis and progression of MASLD.

A lipase from Lacticaseibacillus rhamnosus IDCC 3201 with thermostability and pH resistance for use as a detergent additive.

Lipases are important biocatalysts and ubiquitous in plants, animals, and microorganisms. The high growth rates of microorganisms with low production costs have enabled the wide application of microbial lipases in detergent, food, and cosmetic industries. Herein, a novel lipase from Lacticaseibacillus rhamnosus IDCC 3201 (Lac-Rh) was isolated and its activity analyzed under a range of reaction conditions to evaluate its potential industrial application. The isolated Lac-Rh showed a molecular weight of 24 kDa and a maximum activity of 3438.5 ± 1.8 U/mg protein at 60 °C and pH 8. Additionally, Lac-Rh retained activity in alkaline conditions and in 10% v/v concentrations of organic solvents, including glycerol and acetone. Interestingly, after pre-incubation in the presence of multiple commercial detergents, Lac-Rh maintained over 80% of its activity and the stains from cotton were successfully removed under a simulated laundry  setting. Overall, the purified lipase from L. rhamnosus IDCC 3201 has potential for use as a detergent in industrial applications. KEY POINTS: • A novel lipase (Lac-Rh) was isolated from Lacticaseibacillus rhamnosus IDCC 3201 • Purified Lac-Rh exhibited its highest activity at a temperature of 60 °C and a pH of 8, respectively • Lac-Rh remains stable in commercial laundry detergent and enhances washing performance.

The proteome of Nicotiana benthamiana is shaped by extensive protein processing.

Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed processing events in vivo. This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana.