The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Evaluation of the lncRNA-miRNA-mRNA ceRNA network in lungs of miR-147 -/- mice.

Background: Previous studies have documented important roles for microRNA-147 (miR-147) in inflammation, radiation-induced injury, cancer, and a range of other diseases. Murine lungs exhibit high levels of miRNA, mRNA, and lncRNA expression. However, very little research to date has focused on the lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) networks associated with miR-147, and the regulation of lncRNAs and miRNAs in this setting remains poorly understood. Methods: After establishing a miR-147-/- model mouse, samples of lung tissue were harvested for RNA-sequencing, and differentially expressed lncRNAs, miRNAs, and mRNAs were identified. The miRNA targets of these lncRNAs and the identified miRNAs were first overlapped to facilitate the prediction of target mRNAs, with analyses then examining the overlap between these targets and mRNAs that were differentially expressed. Then, these target mRNAs were subjected to pathway enrichment analyses. These results were ultimately used to establish a miR-147-related ceRNA network. Results: Relative to wild-type mice, the lungs of miR-147-/- mice exhibited 91, 43, and 71 significantly upregulated lncRNAs, miRNAs, and mRNAs, respectively, together with 114, 31, and 156 that were significantly downregulated. The lncRNA-miRNA-mRNA network established based on these results led to the identification of Kcnh6 as a differentially expressed hub gene candidate and enabled the identification of a range of regulatory relationships. KEGG pathway enrichment showed that the mRNA targets of differentially expressed lncRNAs and miRNAs in the mice were associated with tumor-related signaling, endometrial cancer, bladder cancer, and ErbB signaling. Conclusion: These results suggest that the identified ceRNA network in miR-147-/- mice shapes tumor-associated signaling activity, with miR-147 potentially regulating various lncRNAs and miRNAs through Kcnh6, ultimately influencing tumorigenesis. Future studies of the lncRNA, miRNA, and mRNA regulatory targets shown to be associated with miR-147 in the present study may ultimately lead to the identification of novel clinically relevant targets through which miR-147 shapes the pathogenesis of cancer and other diseases.

The structural and molecular mechanisms of type II PETases: a mini review.

The advent of plastics has led to significant advances for humans, although the accompanying pollution has also been a source of concern for countries globally. Consequently, a biological method to effectively degrade polyethylene terephthalate (PET) has been an area of significant scientific interest. Following the report of the highly efficient PET hydrolase from the bacterium Ideonella sakaiensis strain 201-F6 (i.e., IsPETase) in 2016, its structure has been extensively studied, showing that it belongs to the type II PETase group. Unlike type I PETases that include most known cutinases, structural investigations of type II PETases have only been conducted since 2017. Type II PETases are further divided into type IIa and IIb enzymes. Moreover, even less research has been conducted on type IIa plastic-degrading enzymes. Here, we present a review of recent studies of the structure and mechanism of type II PETases, using the known structure of the type IIa PETase PE-H from the marine bacterium Pseudomonas aestusnigri in addition to the type IIb enzyme IsPETase as representatives. These studies have provided new insights into the structural features of type II PETases that exhibit PET catalytic activity. In addition, recent studies investigating the rational design of IsPETases are reviewed and summarized alongside a discussion of controversies surrounding PETase investigations.

Understanding calcium functionality by examining growth characteristics and structural aspects in calcium-deficient grapevine.

This study characterized growth characteristics and cellular details employing microscopy techniques in hydroponically-grown Ca2+-sufficient and Ca2+-deficient grapevines (Vitis vinifera) in a glasshouse. The Ca2+-deficient vines exhibited significant reductions in shoot length, shoot and trunk fresh weights, leaf area, chlorophyll, which eventually led to drooping, yellowing, and chlorosis of leaves. Roots were less dense and primarily dark and necrotic. Furthermore, their xylem vessels were small, polygonal, and appeared to be collapsed yet increased in number and developed lateral roots. Despite such alterations, the anatomical organization of leaves was not affected, yet they developed with more xylem vessels with thick walls and lignin in their mesophyll and vascular tissues. The chloroplasts in internodes' chlorenchyma, phloem, and cambium underwent significant ultrastructural modifications. The concentrations of macro and micronutrients varied significantly among the roots, trunk, canes, and leaves, including the growth characteristics. These structural and growth modifications of calcium deficiency enable us to understand better the link between the symptoms and functions and for a holistic understanding of Ca2+ functionalities.

Regulatory effect of root restriction on aroma quality of Red Alexandria grape.

To systematically study the impact of root restriction (RR) on the aroma quality of grape berry, in this study, free and bound compounds were investigated in 'Red Alexandria' grape skin and pulp produced with and without RR during development and ripening. Compared with the control, RR advanced the initiation of free-terpene synthesis and increased their concentrations at 14-18 weeks post-flowering (wpf) by promoting the conversion of bound terpenes to free terpenes. In addition, RR significantly regulated the aromatic series at 14-18 wpf and advanced the date of aroma maturation. Network analyses indicated that the correlations among bound compounds were more conserved than those among free compounds, and the skin network displayed tight coordination compared with the pulp network. Terpenes were highly intercorrelated and played a core role in these networks. Finally, 10 bound compounds in pulp were screened out as indicators of the developmental timing of grape.

Comparative Metabolic Profiling of Grape Pulp during the Growth Process Reveals Systematic Influences under Root Restriction.

The compositions and contents of metabolites in the pulp tissue play critical roles in the fruit quality for table grape. In this study, the effects of root restriction (RR) on the primary and secondary metabolites of pulp tissue at five developmental stages were studied at the metabolomics level, using "Red Alexandria" grape berry (Vitis vinifera L.) as materials. The main results were as follows: 283 metabolites were annotated by using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS); 28 and 16 primary metabolites contents were increased and decreased, and 11 and 19 secondary metabolites contents were increased and decreased, respectively, along the berry development; RR significantly decreased 12 metabolites (four amino acids and derivatives, three organic acids, four flavonoids and one other compound) contents, and improved 40 metabolites (22 amino acids and derivatives, six nucleotides, four carbohydrates, four cofactors, three cinnamic acids and one other compound) accumulation at the different developmental stages. Altogether, our study would be helpful to increase our understanding of grape berry's responses to RR stress.

A novel antitumor dithiocarbamate compound inhibits the EGFR/AKT signaling pathway and induces apoptosis in esophageal cancer cells.

Dithiocarbamate has been reported to possess a potent antitumor efficacy against several types of cancer, such as ovarian cancer, breast cancer and hepatocellular carcinoma; however, only a few studies have investigated its inhibitory effect on esophageal cancer. Dipyridylhydrazone dithiocarbamate (DpdtC) is a novel dithiocarbamate derivative that was recently designed, synthesized and evaluated in our previous study. In the present study, the cell growth inhibition and apoptosis induced by DpdtC were measured using the CCK-8 and Annexin V-FITC/propidium iodide staining assays, respectively. Epidermal growth factor receptor (EGFR) signaling pathway and apoptosis related protein levels were examined by western blotting. In vivo effect of DpdtC was evaluated in nude mice bearing KYSE-450 ×enograft tumors. The aims of the present study were to further evaluate the antitumor effects of DpdtC on esophageal cancer cells (KYSE-150 and KYSE-450 cells), and to investigate its potential mechanism of action in vitro and in vivo. It was found that DpdtC significantly inhibited KYSE-150 and KYSE-450 cell proliferation by regulating the EGFR/AKT signaling pathway and inducing apoptosis. In addition, this effect was further identified in vivo; DpdtC inhibited the growth of the KYSE-450 esophageal cancer xenografts by regulating the EGFR/AKT signaling pathway. Furthermore, DpdtC did not affect the body weight in mice. Collectively, the present results suggested that DpdtC may be a promising antitumor drug candidate for the treatment of esophageal cancer.

Crystal structure of human archease, a key cofactor of tRNA splicing ligase complex.

The human archease, hereafter named HArch, is identified as a key cofactor of the tRNA-splicing ligase complex, and a potential therapeutic target for treating nervous system injuries. However, little is known about the structural basis of HArch in tRNA maturation, mRNA splicing, and RNA repair. Here we report the crystal structures of HArch and its two mutants D51A and D178A with resolutions ranging from 1.96 Å to 3.4 Å. HArch is composed of an extended N-terminal protrusion domain (NTD) and one compacted C-terminal domain (CTD). Unlike previously reported homologous proteins, the NTD of the first subunit interacts with the CTD of the second one, and this interaction might be important for maintaining protein stability. Moreover, HArch interacts and colocalizes with RNA ligase RTCB in cells. Our current study reveals the atomic structure of HArch and may help us understand its function in mRNA splicing.

Purification and enzymatic characterization of the RNA ligase RTCB from Thermus thermophilus.

OBJECTIVE: To identify the key residues of Thermus thermophilus (T. thermophilus) RTCB in RNA ligation and DNA activation. RESULTS: The biochemical activities of RTCB from T. thermophilus were purified, characterized, and compared. Structure and sequence alignment between T. thermophilus RTCB and Pyrococcus horikoshii (P. horikoshii) RTCB identified six conserved residues (D64, D95, N203, H204, E207, H399) that were essential for RNA ligation. Mutation analysis showed that the expression levels of mutants D95A, N203A, H204A, E207A and H399A were relatively low. Compared to wide-type RTCB, variant D64A protein had no RNA ligation and DNA activation activity. In addition, T. thermophilus RTCB showed acceptable catalytic activity of 3'-phosphate DNA activation at 37 °C. CONCLUSION: D64 was proved to be essential for RTCB-catalyzed RNA ligation and DNA activation (from 37 to 70 °C) in T. thermophilus.

Comparative Metabolic Profiling of Grape Skin Tissue along Grapevine Berry Developmental Stages Reveals Systematic Influences of Root Restriction on Skin Metabolome.

This research aimed to comparatively evaluate the influences of root restriction (RR) cultivation and traditional cultivation (RC) on grape berry skin metabolomics using a non-targeted metabolomics method. Two-hundred-and-ninety-one metabolites were annotated and the kinetics analyses showed that berry skin metabolome is stage- and cultivation-dependent. Our results showed that RR influences significantly the metabolomes of berry skin tissues, particularly on secondary metabolism, and that this effect is more obvious at pre-veraison stage, which was evidenced by the early and fast metabolic shift from primary to secondary metabolism. Altogether, this study provided an insight into metabolic adaptation of berry skin to RR stress and expanded general understanding of berry development.

In-Depth Aroma and Sensory Profiling of Unfamiliar Table-Grape Cultivars.

We present an in-depth analysis of aroma profiles and sensory attributes, employing solid-phase microextraction gas chromatography/mass spectrometry (SPME-GC-MS) to identify the key compounds driving consumer preference in 19 unfamiliar cultivars. In combination with popular cultivars, we identified a total of 100 compounds in all table grapes, of which 26 key volatiles were correlated with consumer liking. Based on this relationship, five aroma combinations (AC) were formulated, wherein 33 compounds contributed to aroma intensity, and thus, were viewed as active volatiles. The fruity, floral, and sweet aromas were further divided into secondary aromatic series, of which the apple, citrus, orange, rose, geranium, violet, and honey aromas constituted the predominant series in unfamiliar cultivars. Xiangyue and Heikuixiang emerged as the preferred table grapes according to our analysis. By comparison, the popular cultivars showed relatively fewer volatiles, but their contents were much greater than the large number of volatiles identified in the unfamiliar cultivars.

Characterization of the ABA Receptor VlPYL1 That Regulates Anthocyanin Accumulation in Grape Berry Skin.

ABA plays a crucial role in controlling several ripening-associated processes in grape berries. The soluble proteins named as PYR (pyrabactin resistant)/PYL (PYR-like)/RCAR (regulatory component of ABA receptor) family have been characterized as ABA receptors. Here, the function of a grape PYL1 encoding gene involved in the response to ABA was verified through heterologous expression. The expression level of VlPYL1 was highest in grape leaf and fruit tissues of the cultivar Kyoho, and the expression of VlPYL1 was increased during fruit development and showed a reduction in ripe berries. Over-expression of VlPYL1 enhances ABA sensitivity in Arabidopsis. Using the transient overexpression technique, the VlPYL1 gene was over-expressed in grape berries. Up-regulation of the VlPYL1 gene not only promoted anthocyanin accumulation but also induced a set of ABA-responsive gene transcripts, including ABF2 and BG3. Although tobacco rattle virus (TRV)-induced gene silencing (VIGS) was not successfully applied in the "Kyoho" grape, the application of the transient overexpression technique in grape fruit could be used as a novel tool for studying grape fruit development.

Structure insight of GSDMD reveals the basis of GSDMD autoinhibition in cell pyroptosis.

Recent findings have revealed that the protein gasdermin D (GSDMD) plays key roles in cell pyroptosis. GSDMD binds lipids and forms pore structures to induce pyroptosis upon microbial infection and associated danger signals. However, detailed structural information for GSDMD remains unknown. Here, we report the crystal structure of the C-terminal domain of human GSDMD (GSDMD-C) at 2.64-Å resolution. The first loop on GSDMD-C inserts into the N-terminal domain (GSDMD-N), which helps stabilize the conformation of the full-length GSDMD. Substitution of this region by a short linker sequence increased levels of cell death. Mutants F283A and F283R can increase protein heterogeneity in vitro and are capable of undergoing cell pyroptosis in 293T cells. The small-angle X-ray-scattering envelope of human GSDMD is consistent with the modeled GSDMD structure and mouse GSDMA3 structure, which suggests that GSDMD adopts an autoinhibited conformation in solution. The positive potential surface of GSDMD-N covered by GSDMD-C is exposed after being released from the autoinhibition state and can form high-order oligomers via a charge-charge interaction. Furthermore, by mapping different regions of GSDMD, we determined that one short segment is sufficient to kill bacteria in vitro and can efficiently inhibit cell growth in Escherichia coli and Mycobacterium Smegmatis These findings reveal that GSDMD-C acts as an auto-inhibition executor and GSDMD-N could form pore structures via a charge-charge interaction upon cleavage by caspases during cell pyroptosis.

Proteomic analysis of pear (Pyrus pyrifolia) ripening process provides new evidence for the sugar/acid metabolism difference between core and mesocarp.

Pears are one of the most popular nutrient-rich fruits in the world. The pear core and mesocarp have significantly different metabolism, although they display similar profiles. Most strikingly, the core is more acidic in taste. Our results showed that there is more titrated acid but lower total soluble solids in the core compared to the mesocarp, and the content of citric acid was more than 17-fold higher in the core compared to the mesocarp at the ripening stage. Proteomics was used to investigate the difference between core and mesocarp tissues during "Cuiguan" pear ripening. Fifty-four different protein expression patterns were identified in the core and mesocarp. In general, common variably expressed proteins between the core and mesocarp were associated with important physiological processes, such as glycolysis, pyruvate metabolic processes, and oxidative stress. Further, protein level associated qRT-PCR verification revealed a higher abundance of fructose-bisphosphate aldolase and NADP-dependent malic enzymes, which may play a role in the low acid content in the mesocarp, whereas a higher abundance of disulfide isomerase-like 2-2 and calcium-dependent lipid-binding in the core may explain why it is less prone to accumulate sugar. The different levels of a few typical ROS scavenger enzymes suggested that oxidative stress is higher in the core than in the mesocarp. This study provides the first characterization of the pear core proteome and a description of its variation compared to the mesocarp during ripening.

Aroma characterization based on aromatic series analysis in table grapes.

Aroma is an important part of quality in table grape, but the key aroma compounds and the aroma series of table grapes remains unknown. In this paper, we identified 67 aroma compounds in 20 table grape cultivars; 20 in pulp and 23 in skin were active compounds. C6 compounds were the basic background volatiles, but the aroma contents of pulp juice and skin depended mainly on the levels of esters and terpenes, respectively. Most obviously, 'Kyoho' grapevine series showed high contents of esters in pulp, while Muscat/floral cultivars showed abundant monoterpenes in skin. For the aroma series, table grapes were characterized mainly by herbaceous, floral, balsamic, sweet and fruity series. The simple and visualizable aroma profiles were established using aroma fingerprints based on the aromatic series. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) showed that the aroma profiles of pulp juice, skin and whole berries could be classified into 5, 3, and 5 groups, respectively. Combined with sensory evaluation, we could conclude that fatty and balsamic series were the preferred aromatic series, and the contents of their contributors (ß-ionone and octanal) may be useful as indicators for the improvement of breeding and cultivation measures for table grapes.

Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20-243 CrossMab.

Although rituximab has revolutionized the treatment of hematological malignancies, the acquired resistance is one of the prime obstacles for cancer treatment, and development of novel CD20-targeting antibodies with potent anti-tumor activities and specificities is urgently needed. Emerging evidence has indicated that lysosomes can be considered as an "Achilles heel" for cancer cells, and might serve as an effective way to kill resistant cancer cells. HLA-DR antibody L243 has been recently reported to elicit potent lysosome-mediated cell death in lymphoma and leukemia cells, suggesting that HLA-DR could be used as a potential target against lymphoma. In this study, we generated a bispecific immunoglobulin G-like antibody targeting both CD20 and HLA-DR (CD20-243 CrossMab) through CrossMab technology. We found that the CrossMab could induce remarkably high levels of complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and anti-proliferative activity. Notably, although HLA-DR is expressed on normal and malignant cells, the CrossMab exhibited highly anti-tumor specificity, showing efficient eradication of hematological malignancies both in vitro and in vivo. Our data indicated that combined targeting of CD20 and HLA-DR could be an effective approach against malignancies, suggesting that CD20-243 CrossMab would be a promising therapeutic agent against lymphoma.

Eradication of non-Hodgkin lymphoma through the induction of tumor-specific T-cell immunity by CD20-Flex BiFP.

Although monoclonal antibodies, including CD20 antibody rituximab, are standard therapeutics for several cancers, their efficacy remains variable and often modest. There is an urgent need to enhance the efficacy of the current generation of anticancer antibodies. Flt3 ligand, a soluble protein, has the ability to induce substantial expansion of dendritic cells (DCs). In this study, we constructed a bispecific immunoglobulin G-like bispecific fusion protein (BiFP) targeting both CD20 and Flt3 (CD20-Flex) by using CrossMab technology. We found that the BiFP exhibited stabilities that were comparable with the parental antibody rituximab and were able to bind to both targets with unaltered binding affinity. Notably, our data indicated that CD20-Flex BiFP could not only eliminate lymphoma temporarily but also potentiate tumor-specific T-cell immunity, which affords a long-lasting protection from tumor recurrence. The results showed that the expansion and infiltration of DCs into tumor tissues by CD20-Flex BiFP could be an effective way to generate protective immune responses against cancer, suggesting that the CD20-Flex BiFP could be a promising therapeutic agent against B-cell lymphomas.