Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Binding of Cholesterol to the N-Terminal Domain of the NPC1L1 Transporter: Analysis of the Epimerization-Related Binding Selectivity and Loop Mutations.

Cholesterol is a fat-like substance with a pivotal physiological relevance in humans, and its homeostasis is tightly regulated by various cellular processes, including the import in the small intestine and the reabsorption in the biliary ducts by the Niemann-Pick C1 Like 1 (NPC1L1) importer. NPC1L1 can mediate the absorption of a variety of sterols but strikingly exhibits a large sensitivity to cholesterol epimerization. This study examines the molecular basis of the epimerization-related selective binding of cholesterol by combining extended unbiased molecular dynamics simulations of the apo and holo species of the N-terminal domain of wild-type NPC1L1, in conjunction with relative binding free energy, umbrella sampling, and well-tempered metadynamics calculations. The analysis of the results discloses the existence of two distinct binding modes for cholesterol and epi-cholesterol. The former binds deeper in the cavity, forming key hydrogen-bond interactions with Q95, S56, and a water molecule. In contrast, epi-cholesterol is shifted ca. 3 Å to the mouth of the cavity and the transition to the Q95 site is prevented by an energetic barrier of 4.1 kcal·mol-1. Thus, the configuration of the hydroxyl group of cholesterol, together with the presence of a structural water molecule, is a key feature for effective absorption. Finally, whereas these findings may seemingly be challenged by single-point mutations that impair cholesterol transport but have a mild impact on the binding of cholesterol to the Q95 binding site, our results reveal that they have a drastic influence on the conformational landscape of the α8/ß7 loop in the apo species compared to the wild-type protein. Overall, the results give support to the functional role played by the α8/ß7 loop in regulating the access of ligands to NPC1L1, and hence to interpreting the impact of these mutations on diseases related to disruption of sterol absorption, paving the way to understanding certain physiological dysfunctions.

Development of a functional outcome measure for riboflavin transporter deficiency.

BACKGROUND AND AIMS: Riboflavin transporter deficiency (RTD) is a progressive inherited neuropathy of childhood onset, characterised clinically by pontobulbar palsy, sensory ataxia, sensorineural deafness, muscle weakness, optic atrophy and respiratory failure. A robust and responsive functional outcome measure is essential for future clinical trials of disease-modifying therapies including genetic therapies. The Charcot-Marie-Tooth disease Pediatric Scale (CMTPedS) is a well-validated outcome measure for CMT and related neuropathies, and might have utility for measuring disease progression in individuals with RTD. However, the CMTPedS requires modifications to account for phenotypic differences between children with CMT and RTD. The aim of this study was to develop a functional outcome measure based on the CMTPedS for specific use in individuals with RTD. METHODS: The CMTPedS data collected over the last 10 years in individuals with RTD attending the Peripheral Neuropathy Management Clinic at the Children's Hospital at Westmead (Sydney, Australia) were reviewed to evaluate each item within the CMTPedS. A literature review of articles published until September 2021 for functional outcome measures generated an item pool for pilot testing. The results of this pilot testing, alongside analysis of existing CMTPedS item scores in the RTD cohort, informed the modification of the CMTPedS. RESULTS: CMTPedS data were reviewed for eight individuals over the past 10 years. Two items were identified as requiring modification or removal and additional items of proximal strength and function needed to be considered. Six studies were identified in the literature review, and five items were selected for pilot testing. 'Shoulder internal rotation' and the '30-s sit to stand test' were added as proximal measures of strength and function. The composite balance item comprising nine tasks in the CMTPedS showed a ceiling effect and was replaced with the single 'Feet apart on a line eyes open' balance item. 'Pinprick sensation' was removed due to a floor effect. INTERPRETATION: This study provides preliminary evidence that the Riboflavin Transporter Deficiency Pediatric Scale (RTDPedS) is a functional outcome measure covering strength, upper and lower limb function, balance and mobility for individuals with RTD to assess disease severity and progression in clinical trials and cohort studies.

Phosphatidylinositol phosphates modulate interactions between the StarD4 sterol trafficking protein and lipid membranes.

There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain-containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P2 and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics (MD) simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP headgroup as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which PIPs modulate cholesterol transfer activity via StarD4.

Guard Cell Transcriptome Reveals Membrane Transport, Stomatal Development and Cell Wall Modifications as Key Traits Involved in Salinity Tolerance in Halophytic Chenopodium quinoa.

A comparative investigation was conducted to evaluate transcriptional changes in guard cells (GCs) of closely related halophytic (Chenopodium quinoa) and glycophytic (Spinacia oleracea) species. Plants were exposed to 3 weeks of 250 mM sodium chloride treatment, and GC-enriched epidermal fragments were mechanically prepared. In both species, salt-responsive genes were mainly related to categories of protein metabolism, secondary metabolites, signal transduction and transport systems. Genes related to abscisic acid (ABA) signaling and ABA biosynthesis were strongly induced in quinoa but not in spinach GCs. Also, expression of the genes encoding transporters of amino acids, proline, sugars, sucrose and potassium increased in quinoa GCs under salinity stress. Analysis of cell-wall-related genes suggests that genes involved in lignin synthesis (e.g. lignin biosynthesis LACCASE 4) were highly upregulated by salt in spinach GCs. In contrast, transcripts related to cell wall plasticity Pectin methylesterase3 (PME3) were highly induced in quinoa. Faster stomatal response to light and dark measured by observing kinetics of changes in stomatal conductance in quinoa might be associated with higher plasticity of the cell wall regulated by PME3 Furthermore, genes involved in the inhibition of stomatal development and differentiation were highly expressed by salt in quinoa, but not in spinach. These changes correlated with reduced stomatal density and index in quinoa, thus improving its water use efficiency. The fine modulation of transporters, cell wall modification and controlling stomatal development in GCs of quinoa may have resulted in high K+/Na+ ratio, lower stomatal conductance and higher stomatal speed for better adaptation to salinity stress in quinoa.

Polyethylene Degradation by a Rhodococcous Strain Isolated from Naturally Weathered Plastic Waste Enrichment.

Polyethylene (PE) is the most widely produced synthetic polymer and the most abundant plastic waste worldwide due to its recalcitrance to biodegradation and low recycle rate. Microbial degradation of PE has been reported, but the underlying mechanisms are poorly understood. Here, we isolated a Rhodococcus strain A34 from 609 day enriched cultures derived from naturally weathered plastic waste and identified the potential key PE degradation enzymes. After 30 days incubation with A34, 1% weight loss was achieved. Decreased PE molecular weight, appearance of C-O and C═O on PE, palmitic acid in the culture supernatant, and pits on the PE surface were observed. Proteomics analysis identified multiple key PE oxidation and depolymerization enzymes including one multicopper oxidase, one lipase, six esterase, and a few lipid transporters. Network analysis of proteomics data demonstrated the close relationships between PE degradation and metabolisms of phenylacetate, amino acids, secondary metabolites, and tricarboxylic acid cycles. The metabolic roadmap generated here provides critical insights for optimization of plastic degradation condition and assembly of artificial microbial communities for efficient plastic degradation.

Genotype-driven NPC1L1 and PCSK9 inhibition and reduced risk of periodontitis.

AIM: Epidemiological and pre-clinical studies suggest a chemoprotective role of lipid-lowering agents in periodontitis. We tested the association of genetically proxied inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), Niemann-Pick C1-Like 1 (NPC1L1) and proprotein convertase subtilisin/kexin type 9 (PCSK9) with periodontitis. MATERIALS AND METHODS: Genetic variants in HMGCR, NCP1L1 and PCSK9 associated with low-density lipoprotein (LDL) cholesterol in a genome-wide association study (GWAS) meta-analysis (N = 188,578) were used to proxy therapeutic inhibition of HMGCR, NPC1L1 and PCSK9. For these genetic variants, associations with periodontitis were obtained from GWAS of 17,353 cases and 28,210 controls in the GeneLifestyle Interactions in Dental Endpoints consortium. Generalized weighted least squares analysis accounted for linkage disequilibrium of genotypes to derive pooled estimates. RESULTS: While genetically proxied HMGCR inhibition equivalent to 1 mmol/L reduction in LDL was not associated with odds of periodontitis (odds ratio [OR] = 0.92 [95% confidence interval [CI]: 0.73; 1.16]; p = .4905; false discovery rate [FDR] = 0.4905), genetically proxied NPC1L1 (OR = 0.53 [95% CI: 0.35; 0.81]; p = .0038; FDR = 0.0077) and PCSK9 (OR = 0.84 [95% CI: 0.74; 0.95]; p = .0051; FDR = 0.0077) inhibition lowered the odds of periodontitis. CONCLUSIONS: Genetically proxied inhibition of NCP1L1 and PCSK9 was associated with lower odds of periodontitis.

Immunoelectron Microscopic Analysis of Dipeptidyl-Peptidases and Dipeptide Transporter Involved in Nutrient Acquisition in Porphyromonas gingivalis.

Porphyromonas gingivalis is an asaccharolytic, Gram-negative, anaerobic bacterium representing a keystone pathogen in chronic periodontitis. The bacterium's energy production depends on the metabolism of amino acids, which are predominantly incorporated as dipeptides via the proton-dependent oligopeptide transporter (Pot). In this study, the localization of dipeptidyl-peptidases (DPPs) and Pot was investigated for the first time in P. gingivalis using immunoelectron microscopy with specific antibodies for the bacterial molecules and gold-conjugated secondary antibodies on ultrathin sections. High-temperature protein G and hemin-binding protein 35 were used as controls, and the cytoplasmic localization of the former and outer membrane localization of the latter were confirmed. P. gingivalis DPP4, DPP5, DPP7, and DPP11, which are considered sufficient for complete dipeptide production, were detected in the periplasmic space. In contrast, DPP3 was localized in the cytoplasmic space in accord with the absence of a signal sequence. The inner membrane localization of Pot was confirmed. Thus, spatial integration of the nutrient acquisition system exists in P. gingivalis, in which where dipeptides are produced in the periplasmic space by DPPs and readily transported across the inner membrane via Pot.

Genome-wide identification of Streptococcus sanguinis fitness genes in human serum and discovery of potential selective drug targets.

Streptococcus sanguinis is a primary colonizer of teeth and is associated with oral health. When it enters the bloodstream, however, this bacterium may cause the serious illness infective endocarditis. The genes required for survival and proliferation in blood have not been identified. The products of these genes could provide a rich source of targets for endocarditis-specific antibiotics possessing greater efficacy for endocarditis, and also little or no activity against those bacteria that remain in the mouth. We previously created a comprehensive library of S. sanguinis mutants lacking every nonessential gene. We have now screened each member of this library for growth in human serum and discovered 178 mutants with significant abundance changes. The main biological functions disrupted in these mutants, including purine metabolism, were highlighted via network analysis. The components of an ECF-family transporter were required for growth in serum and were shown for the first time in any bacterium to be essential for endocarditis virulence. We also identified two mutants whose growth was reduced in serum but not in saliva. This strategy promises to enable selective targeting of bacteria based on their location in the body, in this instance, treating or preventing endocarditis while leaving the oral microbiome intact.

Preassembled Cas9 Ribonucleoprotein-Mediated Gene Deletion Identifies the Carbon Catabolite Repressor and Its Target Genes in Coprinopsis cinerea.

Cre1 is an important transcription factor that regulates carbon catabolite repression (CCR) and is widely conserved across fungi. The cre1 gene has been extensively studied in several Ascomycota species, whereas its role in gene expression regulation in the Basidiomycota species remains poorly understood. Here, we identified and investigated the role of cre1 in Coprinopsis cinerea, a basidiomycete model mushroom that can efficiently degrade lignocellulosic plant wastes. We used a rapid and efficient gene deletion approach based on PCR-amplified split-marker DNA cassettes together with in vitro assembled Cas9-guide RNA ribonucleoproteins (Cas9 RNPs) to generate C. cinerea cre1 gene deletion strains. Gene expression profiling of two independent C. cinerea cre1 mutants showed significant deregulation of carbohydrate metabolism, plant cell wall degrading enzymes (PCWDEs), plasma membrane transporter-related and several transcription factor-encoding genes, among others. Our results support the notion that, like reports in the ascomycetes, Cre1 of C. cinerea orchestrates CCR through a combined regulation of diverse genes, including PCWDEs, transcription factors that positively regulate PCWDEs, and membrane transporters which could import simple sugars that can induce the expression of PWCDEs. Somewhat paradoxically, though in accordance with other Agaricomycetes, genes related to lignin degradation were mostly downregulated in cre1 mutants, indicating they fall under different regulation than other PCWDEs. The gene deletion approach and the data presented here will expand our knowledge of CCR in the Basidiomycota and provide functional hypotheses on genes related to plant biomass degradation. IMPORTANCE Mushroom-forming fungi include some of the most efficient lignocellulosic plant biomass degraders. They degrade dead plant materials by a battery of lignin-, cellulose-, hemicellulose-, and pectin-degrading enzymes, the encoding genes of which are under tight transcriptional control. One of the highest-level regulations of these metabolic enzymes is known as carbon catabolite repression, which is orchestrated by the transcription factor Cre1, and ensures that costly lignocellulose-degrading enzyme genes are expressed only when simple carbon sources (e.g., glucose) are not available. Here, we identified the Cre1 ortholog in a litter decomposer Agaricomycete, Coprinopsis cinerea, knocked it out, and characterized transcriptional changes in the mutants. We identified several dozen lignocellulolytic enzyme genes as well as membrane transporters and other transcription factors as putative target genes of C. cinerea cre1. These results extend knowledge on carbon catabolite repression to litter decomposer Basidiomycota.

Hitchhiking into the Deep: How Microplastic Particles are Exported through the Biological Carbon Pump in the North Atlantic Ocean.

Understanding residence times of plastic in the ocean is a major knowledge gap in plastic pollution studies. Observations report a large mismatch between plastic load estimates from worldwide production and disposal and actual plastics floating at the sea surface. Surveys of the water column, from the surface to the deep sea, are rare. Most recent work, therefore, addressed the "missing plastic" question using modeling or laboratory approaches proposing biofouling and degradation as the main removal processes in the ocean. Through organic matrices, plastic can affect the biogeochemical and microbial cycling of carbon and nutrients. For the first time, we provide in situ measured vertical fluxes of microplastics deploying drifting sediment traps in the North Atlantic Gyre from 50 m down to 600 m depth, showing that through biogenic polymers plastic can be embedded into rapidly sinking particles also known as marine snow. We furthermore show that the carbon contained in plastic can represent up to 3.8% of the total downward flux of particulate organic carbon. Our results shed light on important pathways regulating the transport of microplastics in marine systems and on potential interactions with the marine carbon cycle, suggesting microplastic removal through the "biological plastic pump".

Genetic screening in patients of Meige syndrome and blepharospasm.

OBJECTIVE: Meige syndrome (MS) is cranial dystonia, including bilateral eyelid spasms (blepharospasm; BSP) and involuntary movements of the jaw muscles (oromandibular dystonia; OMD). Up to now, the pathogenic genes of MS and BSP are still unclear. METHODS: We performed Sanger sequencing of GNAL, TOR1A, TOR2A, THAP1, and REEP4 exons on 78 patients, including 53 BSP and 25 MS and 96 healthy controls. RESULTS: c.845G > C[R282P] of TOR1A, c.629delC[p.Gly210AlafsTer60] of TOR2A, c.1322A > G[N441S] of GNAL, c.446G > A[R149Q], and c.649C > T[R217C] of REEP4 were identified and predicated as deleterious probably damaging variants. Three potential alterations of splicing variants of TOR1A and TOR2A were identified in patients. The frequencies of TOR1A rs1435566780 and THAP1 rs545930392 were higher in patients than in controls. CONCLUSIONS: TOR1A rs1435566780 (c.*16G > C(G > A)) and THAP1 rs545930392 (c.192G > A[K64K]) may contribute to the etiology of MS and BSP. Other identified rare mutations predicted as deleterious probably damaging need further confirmation. Larger MS and BSP cohorts and functional studies will need to be performed further to elucidate the association between these genes and the diseases.

Molecular Modelling and Atomistic Insights into the Binding Mechanism of MmpL3 Mtb.

Mycobacterial membrane proteins Large (MmpLs), which belong to the resistance, nodulation, and division (RND) protein superfamily, play critical roles in transporting polymers, lipids, and immunomodulators. MmpLs have become one of the important therapeutic drug targets to emerge in recent times. In this study, two homology modelling techniques, Modeller and SWISS-MODEL, were used in modelling the three-dimensional protein structure of the MmpL3 of Mycobacterium tuberculosis using that of M. smegmatis as template. MmpL3 inhibitors, namely BM212, NITD304, SPIRO, and NITD349, in addition to the co-crystalized ligands AU1235, ICA38, SQ109 and rimonabant, were screened against the modelled structure and the Mmpl3 of M. smegmatis using molecular docking techniques. Protein-ligand interactions were analysed using molecular dynamics simulations and Molecular Mechanics Poisson-Boltzmann surface area computations. Novel residues Gln32, Leu165, Ile414, and Phe35 were identified as critical for binding to M. tuberculosis MmpL3, and conformational dynamics upon inhibitor binding were discussed.

The membrane transporter lactose permease increases lipid bilayer bending rigidity.

Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers. In contrast, much less is known about the influence of integral membrane proteins on bending rigidity under physiological conditions. We focus on an exemplar member of the ubiquitous major facilitator superfamily of transporters and assess the influence of lactose permease on the bending rigidity of lipid bilayers. Fluctuation analysis of giant unilamellar vesicles (GUVs) is a useful means to measure bending rigidity. We find that using a hydrogel substrate produces GUVs that are well suited to fluctuation analysis. Moreover, the hydrogel method is amenable to both physiological salt concentrations and anionic lipids, which are important to mimic key aspects of the native lactose permease membrane. Varying the fraction of the anionic lipid in the lipid mixture DOPC/DOPE/DOPG allows us to assess the dependence of membrane bending rigidity on the topology and concentration of an integral membrane protein in the lipid bilayer of GUVs. The bending rigidity gradually increases with the incorporation of lactose permease, but there is no further increase with greater amounts of the protein in the membrane.

Messenger RNA Expression of Albumin, Transferrin, Transthyretin, Asialoglycoprotein Receptor, Cytochrome P450 Isoform, Uptake Transporter, and Efflux Transporter Genes as a Function of Culture Duration in Prolonged Cultured Cryopreserved Human Hepatocytes as Collagen-Matrigel Sandwich Cultures: Evidence for Redifferentiation upon Prolonged Culturing.

Hepatic gene expression as a function of culture duration was evaluated in prolonged cultured human hepatocytes. Human hepatocytes from seven donors were maintained as near-confluent collagen-Matrigelsandwich cultures, with messenger RNA expression for genes responsible for key hepatic functions quantified by real-time polymerase chain reaction at culture durations of 0 (day of plating), 2, 7, 9, 16, 23, 26, 29, 36, and 43 days. Key hepatocyte genes were evaluated, including the differentiation markers albumin, transferrin, and transthyretin; the hepatocyte-specific asialoglycoprotein receptor 1 cytochrome P450 isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A7; uptake transporter isoforms SLC10A1, SLC22A1, SLC22A7, SLCO1B1, SLCO1B3, and SLCO2B1; efflux transporter isoforms ATP binding cassette (ABC)B1, ABCB11, ABCC2, ABCC3, ABCC4, and ABCG2; and the nonspecific housekeeping gene hypoxanthine ribosyl transferase 1 (HPRT1). The well established dedifferentiation phenomenon was observed on day 2, with substantial (>80%) decreases in gene expression in day 2 cultures observed for all genes evaluated except HPRT1 and efflux transporters ABCB1, ABCC2, ABCC3 (<50% decrease in expression), ABCC4 (>400% increase in expression), and ABCG2 (no decrease in expression). All genes with a >80% decrease in expression were found to have increased levels of expression on day 7, with peak expression observed on either day 7 or day 9, followed by a gradual decrease in expression up to the longest duration evaluated of 43 days. Our results provide evidence that cultured human hepatocytes undergo redifferentiation upon prolonged culturing. SIGNIFICANCE STATEMENT: This study reports that although human hepatocytes underwent dedifferentiation upon 2 days of culture, prolonged culturing resulted in redifferentiation based on gene expression of differentiation markers, uptake and efflux transporters, and cytochrome P450 isoforms. The observed redifferentiation suggests that prolonged (>7 days) culturing of human hepatocyte cultures may represent an experimental approach to overcome the initial dedifferentiation process, resulting in "stabilized" hepatocytes that can be applied toward the evaluation of drug properties requiring an extended period of treatment and evaluation.

Sec62 Suppresses Foot-and-Mouth Disease Virus Proliferation by Promotion of IRE1α-RIG-I Antiviral Signaling.

Foot-and-mouth disease virus (FMDV) is highly infectious and causes a major plague in animal farming. Unfolded protein response is one of the major cellular responses to pathogenic infections, which performs a crucial role in cell survival, apoptosis, and antiviral innate immune response. In this study, we showed that FMDV infection activated two unfolded protein response branches (PERK-eIF2α and ATF6 signaling) in both baby hamster kidney cells (BHK-21) and porcine kidney (PK-15) cells, whereas it suppressed the IRE1α-XBP1 signaling by decreasing IRE1α level. Further study revealed IRE1α signaling as an important antiviral innate immune mechanism against FMDV. Sec62, the transport protein, was greatly decreased at the late stages of FMDV infection. By overexpression and knockdown study, we also found that the expression of Sec62 was positively involved in the levels of IRE1α and RIG-I and subsequent activation of downstream antiviral signaling pathways in FMDV-infected PK-15 cells. Taken together, our study demonstrates that Sec62 is an important antiviral factor that upregulates IRE1α-RIG-I-dependent antiviral innate immune responses, and FMDV evades antiviral host defense mechanism by downregulating Sec62-IRE1α/RIG-I.

Downregulation of Prolactin-Induced Protein Promotes Osteogenic Differentiation of Periodontal Ligament Stem Cells.

BACKGROUND Periodontal ligament stem cells (PDLSCs) are promising seed cells for bone tissue engineering and periodontal regeneration applications. However, the mechanism underlying the osteogenic differentiation process remains largely unknown. Previous reports showed that prolactin-induced protein (PIP) was upregulated after PDLSCs osteogenic induction. However, few studies have reported on the function of PIP in osteogenic differentiation. The purpose of the present study was to investigate the effect of PIP on osteogenic differentiation of PDLSCs. MATERIAL AND METHODS The expression pattern of PIP during PDLSCs osteogenic differentiation was detected and the effect of each component in the osteogenic induction medium on PIP was also tested by qRT-PCR. Then, the PIP knockdown cells were established using lentivirus. The knockdown efficiency was measured and the proliferation, apoptosis, and osteogenic differentiation ability were examined to determine the functional role of PIP on PDLSCs. RESULTS QRT-PCR showed that PIP was sustainedly upregulated during the osteogenic induction process and the phenomenon was mainly caused by the stimulation of dexamethasone in the induction medium. CCK-8 and flow cytometer showed that knocking down PIP had no influence on proliferation and apoptosis of PDLSCs. ALP staining and activity, Alizarin Red staining, and western blot analysis demonstrated PIP knockdown enhanced the osteogenic differentiation and mineralization of PDLSCs. CONCLUSIONS PIP was upregulated after osteogenic induction; however, PIP knockdown promoted PDLSCs osteogenic differentiation. PIP might be a by-product of osteogenic induction, and downregulating of PIP might be a new target in bone tissue engineering applications.

Genome and secretome analysis of jute endophyte Grammothele lineata strain SDL-CO-2015-1: Insights into its lignocellulolytic structure and secondary metabolite profile.

Grammothele lineata strain SDL-CO-2015-1, jute (Corchorus olitorius) endophyte has been reported to produce anti-cancer drug paclitaxel in culture condition. Here we investigated the genome using different bioinformatic tools to find its association with the production of commercially important compounds including taxol. Carbohydrate-active enzymes, proteases, and secretory proteins were annotated revealing a complex endophytic relationship with its plant host. The presences of a diverse range of CAZymes including numerous lignocellulolytic enzymes support its potentiality in biomass degradation. Genome annotation led to the identification of 28 clusters for secondary metabolite biosynthesis. Several biosynthesis gene clusters were identified for terpene biosynthesis from antiSMASH analysis but none could be specifically pinned to taxol synthesis. This study will direct us to understand the genomic organization of endophytic basidiomycetes with a potential for producing numerous commercially important enzymes and secondary metabolites taking G. lineata as a model.

The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins.

Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from Escherichia coli cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into E. coli membrane vesicles. MPIase-depleted membrane vesicles lacked both 3L-Pf3 coat and MtlA integration, indicating that MPIase is involved in the integration of both proteins. We developed a reconstitution system in which disordered spontaneous integration was precluded, which revealed that SecYEG, YidC, or both, are not sufficient for Sec-dependent and -independent integration. Although YidC had no effect on MPIase-dependent integration of Sec-independent substrates in the conventional assay system, YidC significantly accelerated the integration when the substrate amounts were increased in our PURE system-based assay. Similar acceleration by YidC was observed for MtlA integration. YidC mutants with amino acid substitutions in the hydrophilic cavity inside the membrane were defective in the acceleration of the Sec-independent integration. Of note, MPIase was up-regulated upon YidC depletion. These results indicate that YidC accelerates the MPIase-dependent integration of membrane proteins, suggesting that MPIase and YidC function sequentially and cooperatively during the catalytic cycle of membrane protein integration.

Chronic arsenic exposure impairs adaptive thermogenesis in male C57BL/6J mice.

The global prevalence of type 2 diabetes (T2D) has doubled since 1980. Human epidemiological studies support arsenic exposure as a risk factor for T2D, although the precise mechanism is unclear. We hypothesized that chronic arsenic ingestion alters glucose homeostasis by impairing adaptive thermogenesis, i.e., body heat production in cold environments. Arsenic is a pervasive environmental contaminant, with more than 200 million people worldwide currently exposed to arsenic-contaminated drinking water. Male C57BL/6J mice exposed to sodium arsenite in drinking water at 300 µg/L for 9 wk experienced significantly decreased metabolic heat production when acclimated to chronic cold tolerance testing, as evidenced by indirect calorimetry, despite no change in physical activity. Arsenic exposure increased total fat mass and subcutaneous inguinal white adipose tissue (iWAT) mass. RNA sequencing analysis of iWAT indicated that arsenic dysregulated mitochondrial processes, including fatty acid metabolism. Western blotting in WAT confirmed that arsenic significantly decreased TOMM20, a correlate of mitochondrial abundance; PGC1A, a master regulator of mitochondrial biogenesis; and, CPT1B, the rate-limiting step of fatty acid oxidation (FAO). Our findings show that chronic arsenic exposure impacts the mitochondrial proteins of thermogenic tissues involved in energy expenditure and substrate regulation, providing novel mechanistic evidence for arsenic's role in T2D development.