Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-ß-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.

Superadsorbent aerogel based on sunflower stem pith cellulose and layered double hydroxides modified montmorillonite for methylene blue removal from water solution.

Sunflower stem pith, an agricultural residue, was used as a starting material for the preparation of bio-based products. Sunflower stem pith nanocellulose (SSP-C) was prepared by sodium hydroxide/urea from the SSP cellulose. The prepared SSP-C was typical of cellulose II. To improve the adsorption capacity of the SSP-C, a bio-based aerogel (SSP-MH) with adsorbed methylene blue (MB) was prepared by compounding layered double hydroxides modified montmorillonite (MH) with SSP-C-based adsorbent, and the chemical characteristics and topology of the adsorbent were determined. The removal performance of SSP-MH in different MB concentrations was examined. Adsorption tests showed that hydrogels containing the same content of MH had higher removal efficiency. The removal rate of MB by SSP-MH was >87.5 % in MB solution (1 g/L), and its maximum adsorption capacity was 263.3 mg/g. The kinetics studies of MB removal were well by quasi-secondary adsorption kinetic model and Langmuir isotherm model. Moreover, the standard free Gibbs energy change of adsorption (ΔG0) was <0, which was favorable for adsorption of MB. The adsorption efficiency of SSP-MH on MB was still above 95 % by the five cycles of the adsorption/desorption experiment. The prepared samples were conducive to the high-value utilization of SSP.

Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Metformin Reduces Bone Resorption in Apical Periodontitis Through Regulation of Osteoblast and Osteoclast Differentiation.

INTRODUCTION: We have previously demonstrated that auxiliary metformin therapy promotes healing of apical periodontitis. Here we aimed to investigate the effects of metformin on osteoblast differentiation and osteoclast formation in cultured cells and rat apical periodontitis. METHODS: Murine pre-osteoblasts MC3T3-E1 and macrophages RAW264.7 were cultured under hypoxia (2% oxygen) or normoxia (21% oxygen) and stimulated with receptor activator of nuclear factor-κB ligand (RANKL) when indicated. Metformin was added to the cultures to evaluate its anti-hypoxic effects. Expressions of osteoblast differentiation regulator runt-related transcription factor 2 (RUNX2), RANKL, and osteoclast marker tartrate-resistant acid phosphatase (TRAP) were assessed by Western blot. Apical periodontitis was induced in mandibular first molars of 10 Sprague-Dawley rats. Root canal therapy with or without metformin supplement was performed. Periapical bone resorption was measured by micro-computed tomography. Immunohistochemistry was used to examine RUNX2, RANKL, and TRAP expressions. RESULTS: Hypoxia suppressed RUNX2 expression and enhanced RANKL synthesis in pre-osteoblasts. TRAP production increased in macrophages after hypoxia and/or RANKL stimulation. Metformin reversed hypoxia-induced RUNX2 suppression and RANKL synthesis in pre-osteoblasts. Metformin also inhibited hypoxia and RANKL-enhanced TRAP synthesis in macrophages. Intracanal metformin diminished bone loss in rat apical periodontitis. Comparing with vehicle control, cells lining bone surfaces in metformin-treated lesions had significantly stronger expression of RUNX2 and decreased synthesis of RANKL and TRAP. CONCLUSIONS: Alleviation of bone resorption by intracanal metformin was associated with enhanced osteoblast differentiation and diminished osteoclast formation in rat apical periodontitis. Our results endorsed the role of metformin as an effective medicament for inflammatory bone diseases.

Co-production of biohydrogen and biomethane utilizing halophytic biomass Atriplexcrassifolia by two-stage anaerobic fermentation process.

The excessive use of fossil has resulted in the drastic exhaustion of natural energy sources, leading to environmental challenges and energy crises. Owing to rising energy demand there is a dire need to shift towards renewable energies from lignocellulosic biomass. The present study assessed the co-production of biohydrogen (H2) and biomethane (CH4) by utilizing a less explored halophyte Atriplexcrassifolia. Various reaction parameters were evaluated for their effect on biohydrogen and biomethane production in batch experiments. One parameter at a time experimental strategy was chosen for production optimization. Hydrogen and methane yields along with their production rates were assessed at different incubation times, temperatures, pH, substrate concentrations, and inoculum sizes in acidogenesis and methanogenesis stages, respectively. In the first stage, maximum cumulative hydrogen production of 66 ± 0.02 mL, with hydrogen yield of 13.2 ± 0.03 mL/g, and hydrogen production rate (HPR) of 1.37 ± 0.05 mL/h was attained when the reaction mixture (5 g Atriplexcrassifolia and 10 mL pretreated sewage sludge) was processed at 37°C and pH 5.5 after 48 h of incubation. While in the second stage, maximum cumulative methane production, i.e., 343 ± 0.12 mL, methane yield (MY) of 8.5 ± 0.07 mL/mL, and methane production rate (MPR) of 0.8 ± 0.05 mL/h was achieved after 18 days of incubation of reaction mixture (40 mL of hydrogenic slurry with 80 mL inoculum) at 45°C and pH 8. Furthermore, a 51% and 24% rise in biohydrogen and biomethane production respectively were recorded when the gases were produced at these optimized reaction conditions. The results ensure halophyte Atriplexcrassifolia as an imperative renewable energy resource and proposed that effective optimization of the process further increased the coproduction of biohydrogen and biomethane.

Isolated Soy Protein Supplementation Combined With Resistance Training Improves Muscle Strength, Mass, and Physical Performance of Aging Female Mice.

Background/Purpose: In recent years, the aging population has gradually increased, and the aging process is accompanied by health-associated problems, such as loss of muscle mass and weakness. Therefore, it is important to explore alternative strategies for improving the health status and physical fitness of the aged population. In this study, we investigated the effect of soy protein supplementation combined with resistance training on changes in the muscle mass, muscle strength, and functional activity performance of aging mice. Methods: Female Institute of Cancer Research (ICR) mice were divided into four groups (n = 8 per group): sedentary control (SC), isolated soy protein (ISP) supplementation, resistance training (RT), and a combination of ISP and RT (ISP + RT). The mice in designated groups received oral ISP supplementation (0.123 g/kg/day), RT (5 days/week for a period of 4 weeks), or a combination of both ISP plus RT for 4 weeks. Afterward, we assessed muscle strength, endurance, and anaerobic endurance performance and analyzed blood biochemical and pathological tissue sections to investigate whether there were adverse effects or not in mice. Results: ISP supplementation effectively improved the muscle mass, muscle endurance, and endurance performance of aging female mice. The RT group not only showed similar results with ISP but also increased muscle strength and glycogen content. Nevertheless, the combination of ISP supplementation and RT had greater beneficial effects on muscle strength, physical performance, and glycogen levels (p < 0.05). In addition, the combination of ISP supplementation and RT had significantly increased type II muscle percentage and cross-sectional area (p < 0.05). Conclusion: Although ISP or RT alone improved muscle mass and performance, the combination of ISP with RT showed greater beneficial effects in aging mice. Our findings suggest that regular exercise along with protein supplementation could be an effective strategy to improve overall health and physical fitness among the elderly.

Small GTPase Rab40C is upregulated by 20-hydroxyecdysone and insulin pathways to regulate ovarian development and fecundity.

The insulin and 20-hydroxyecdysone (20E) pathways coordinately regulate insect vitellogenesis and ovarian development. However, the detailed molecular mechanisms such as the genes mediating the cooperation of the interaction of these 2 pathways in regulating insect reproductive development are not well understood. In the present study, a small GTPase, Rab40C, was identified from the notorious agricultural pest Bactrocera dorsalis. In addition to the well-known RAB domain, it also has a unique SOCS-box domain, which is different from other Rab-GTPases. Moreover, we found that Rab40C was enriched in the ovaries of sexually mature females. RNA interference (RNAi)-mediated knockdown of BdRab40C resulted in a decrease in vitellogenin synthesis, underdeveloped ovaries, and low fertility. Furthermore, depletion of insulin receptor InR or the heterodimer receptor of 20E (EcR or USP) by RNAi significantly decreased the transcription of BdRab40C and resulted in lower fecundity. Further studies revealed that the transcription of BdRab40C could be upregulated by the injection of insulin or 20E. These results indicate that Rab40C participates in the insulin and 20E pathways to coordinately regulate reproduction in B. dorsalis. Our results not only provide new insights into the insulin- and 20E-stimulated regulatory pathways controlling female reproduction in insects but also contribute to the development of potential eco-friendly strategies for pest control.

A Novel Green Diluent for the Preparation of Poly(4-methyl-1-pentene) Membranes via a Thermally-Induced Phase Separation Method.

The use of green solvents satisfies safer chemical engineering practices and environmental security. Herein, myristic acid (MA)-a green diluent-was selected to prepare poly- (4-methyl-1-pentene) (PMP) membranes with bicontinuous porous structure via a thermally induced phase separation (TIPS) process to maintain a high gas permeability. Firstly, based on the Hansen solubility parameter 'distance', Ra, the effect of four natural fatty acids on the PMP membrane structure was compared and studied to determine the optimal green diluent, MA. The thermodynamic phase diagram of the PMP-MA system was calculated and presented to show that a liquid-liquid phase separation region could be found during the TIPS process and the monotectic point was around 34.89 wt%. Then, the effect of the PMP concentration on the morphologies and crystallization behavior was systematically investigated to determine a proper PMP concentration for the membrane preparation. Finally, PMP hollow fiber (HF) membranes were fabricated with a PMP concentration of 30 wt% for the membrane performance characterization. The resultant PMP HF membranes possessed good performances that the porosity was 70%, the tensile strength was 96 cN, and the nitrogen flux was 8.20 ± 0.10 mL·(bar·cm2·min)-1. We believe that this work can be a beneficial reference for people interested in the preparation of PMP membranes for medical applications.

Differences between the temporal and mandibular components of the temporomandibular joint in topographic distribution of osseous degenerative features on cone-beam computerized tomography.

BACKGROUND/PURPOSE: Temporomandibular joint osteoarthritis (TMJOA) pathology is characterized by degenerative changes of the subchondral bone. The topographic distribution of osseous degenerative changes in TMJ is not clear. This study aimed to evaluate the topographic distribution of osseous degenerative features in the TMJ by using cone-beam computerized tomography (CBCT). MATERIALS AND METHODS: The CBCT images of 26 female patients diagnosed to have TMJOA were retrieved from the database of the National Taiwan University Hospital. The images of left and right TMJs were evaluated independently by 2 examiners. The evaluated degenerative features included surface erosion, subcortical cysts, subcortical sclerosis, and osteophytes in the mandibular condyle and temporal component of the TMJ. The topographic distribution at different portions in the mandibular condyle and temporal component of the TMJ was statistically analyzed. RESULTS: Significant differences in the topographic distribution of the osseous degenerative features were observed (a) between the mandibular condyle and the temporal component and (b) between the anterior/central portion and posterior portion of the temporal component. No significant differences were observed in the topographic distribution of the TMJOA features in the condyle, except for surface erosion between the central and lateral portion of the condyle. CONCLUSION: The results suggest that the mandibular condyle and temporal component react differently in TMJ osseous degeneration, with the condyle being more vulnerable than the temporal component. Mandibular activities that require the mandibular condyle to function outside the fossa may be more destructive to the health and integrity of the TMJ.

The advantage of cone-beam computerized tomography over panoramic radiography and temporomandibular joint quadruple radiography in assessing temporomandibular joint osseous degenerative changes.

BACKGROUND/PURPOSE: The clinical diagnosis of temporomandibular joint (TMJ) degenerative joint disease (DJD) is based primarily on radiographic features of the condyle and articular eminence. The purpose of this study was to compare the reliability, sensitivity, and specificity of using plain radiography to that of cone-beam computerized tomography (CBCT) in identifying different types of osseous degenerative features in the TMJ condyle. MATERIALS AND METHODS: The panoramic radiography (PANO), TMJ quadruple radiography (TMJQR) and CBCT images of 29 patients' TMJs were retrieved from a computer database and independently evaluated by a young oral surgeon and a senior TMD specialist. The examiners diagnosed osseous degenerative features on the radiographic images. The radiologist-assisted CBCT diagnoses were used as a reference standard and the reliability, sensitivity, and specificity of using the three radiographic modalities were statistically analyzed. RESULTS: There were cases of indeterminate diagnoses using the PANO and TMJQR due to superimposition from surrounding structures, but none using CBCT. Reliability was generally poor when using PANO and TMJQR for detecting osseous degenerative features of the TMJ condyle but good to excellent when using CBCT. The sensitivity and specificity in the use of PANO and TMJQR were typically below acceptable, but the levels were generally satisfactory when using CBCT. CONCLUSION: CBCT is superior to plain radiographic modalities for diagnosing osseous degenerative features of TMJs with regard to indeterminate cases, reliability, sensitivity, and specificity. It is recommended that CBCT can be used as an effective tool in identifying TMJ osteoarthritis.

Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol.

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo-electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.

An insertion/deletion polymorphism within 3'UTR of RYR2 modulates sudden unexplained death risk in Chinese populations.

Sudden unexplained death (SUD) constitutes a part of the overall sudden death that can not be underestimated. Over the last years, genetic testing on SUD has revealed that inherited channelopathies might play important roles in the pathophysiology of this disease. Ryanodine receptor type-2 (RYR2) is a kind of ion channel extensively distributed in the sarcoplasmic reticulum (SR) of myocardium. Studies on RYR2 have suggested that either dysfunction or abnormal expression of it could lead to arrhythmia, which may cause cardiac arrest. In this study, we conducted a case-control study to evaluate the association of a 4-base pair (4-bp) Indel polymorphism (rs10692285) in the 3'UTR of RYR2 with the risk of SUD and sudden cardiac death induced by coronary heart disease (SCD-AS) in a Chinese population. Logistic regression analysis showed that the insertion allele of rs10692285 had significantly increased the risk of SUD [OR=2.03; 95% confidence interval (CI)=1.08-3.77; P=0.0161; statistical power=0.743]. No relevance was observed between rs10692285 and SCD-AS. Further genotype-phenotype association analysis suggested that the expression level of RYR2 in human myocardium tissues with the insertion allele was higher than that with the deletion allele at both mRNA and protein levels. Dual-Luciferase activity assay system was used to detect the effect of rs10692285 on the transcription activity of RYR2. As expected, the result indicated that the transcription activity of RYR2 with the ins/ins genotype was higher than that with the del/del genotype. Finally, in-silico prediction revealed that different alleles of rs10692285 could alter the local structure of RYR2 mRNA and microRNA (miRNA) binding. In summary, our findings provided evidence that rs10692285 might contribute to SUD susceptibility through affecting the expression of RYR2, which suggest that abnormal ion channel activity is very likely to be the underlying mechanism of SUD, but not for SCD-AS. Thus, rs10692285 may become a potential marker for molecular diagnosis and genetic counseling of SUD.

RNA sequencing to characterize transcriptional changes of sexual maturation and mating in the female oriental fruit fly Bactrocera dorsalis.

BACKGROUND: Female reproductive potential plays a significant role in the survival and stability of species, and sexual maturation and mating processes are crucial. However, our knowledge of the reproductive genes involved in sexual maturation and mating has been largely limited to model organisms. The oriental fruit fly Bactrocera dorsalis is a highly invasive agricultural pest, known to cause major economic losses; thus, it is of great value to understand the transcriptional changes involved in sexual maturation and mating processes as well as the related genes. Here, we used a high-throughput sequencing method to identify multiple genes potentially involved in sexual maturation and mating in female B. dorsalis. RESULTS: We sequenced 39,999 unique genes with an average length of 883 bp. In total, 3264 differentially expressed genes (DEGs) were detected between mature virgin and immature Bactrocera dorsalis libraries, whereas only 83 DEGs were identified between flies that had mated or were mature virgins. These DEGs were functionally annotated using the GO and KEGG pathway annotation tools. Results showed that the main GO terms associated with the DEGs from the mature virgin vs. immature groups were primarily assigned to the metabolic and developmental processes, which we focused on, whereas those from the mated vs. mature virgin group largely belonged to the response to stimulus and immune system processes. Additionally, we identified multiple DEGs during sexual maturation that are involved in reproduction, and expression pattern analysis revealed that the majority DEGs detected were highly enriched in those linked to the ovaries or fat bodies. Several mating responsive genes differentially expressed after mating were also identified, and all antimicrobial peptides detected were highly enriched in fat body and significantly up-regulated approximately 2- to 10-fold at 24 h after mating. CONCLUSION: This study supplied female reproductive genes involved in sexual maturation and the post-mating response in B. dorsalis, based on RNA-seq. Our data will facilitate molecular research related to reproduction and provide abundant target genes for effective control of this agricultural pest.

[Expression of EV71-VP1, PSGL-1 and SCARB2 in Tissues of Infants with Brain Stem Encephalitis].

OBJECTIVE: To understand the correlation of enterovirus 71 (EV71), P-selectin glycoprotein ligand-1 (PSGL-1), and scavenger receptor B2 (SCARB2) and to explore the possible pathway and mechanism of EV71 infection by observing the expression of EV71, PSGL-1 and SCARB2 in tissues of infants with brain stem encephalitis. METHODS: The organs and tissues of infants with EV71-VP1 positivity in their brain stems were chosen. Expression and distribution of EV71-VP1, PSGL-1, and SCARB2 were detected and compared by immunohistochemistry. RESULTS: Strong staining of EV71 -VP1 was observed in the neuron, glial cells, the inflammatory cells of perivascular cuffing, parietal cells of the gastric fundus gland while alveolar macrophages, intestinal gland epithelium cells, mucosa lymphoid nodule and lymphocyte of palatine tonsil showed moderate staining and weak staining were displayed in mesenteric lymph nodes and lymphocyte of spleen. PSGL-1 expression was detected in parietal cells of the gastric fundus gland, tonsillar crypt squamous epithelium, alveolar macrophages and leukocytes in each tissue. SCARB2 expression was observed in all the above tissues except the intestines and spleen. CONCLUSION: The distribution of EV71 correlates with SCARB2 expression. SCARB2 plays an important role in virus infection and replication. Stomach may be an important site for EV71 replication.