Multiplexed bacterial pathogen detection and clinical characteristics of orthopedic infection in hospitalized patients.

Introduction: Accurate identification of the etiology of orthopedic infection is very important for correct and timely clinical management, but it has been poorly studied. In the current study we explored the association of multiple bacterial pathogens with orthopedic infection. Methods: Hospitalized orthopedic patients were enrolled in a rural hospital in Qingdao, China. Wound or exudate swab samples were collected and tested for twelve bacterial pathogens with both culture and multiplex real time PCR. Results and discussion: A total of 349 hospitalized orthopedic patients were enrolled including 193 cases presenting infection manifestations upon admission and 156 with no sign of infection. Orthopedic infection patients were mainly male (72.5%) with more lengthy hospital stay (median 15 days). At least one pathogen was detected in 42.5% (82/193) of patients with infection while 7.1% (11/156) in the patients without infection (P < 0.001). S. aureus was the most prevalent causative pathogen (15.5%). Quantity dependent pathogen association with infection was observed, particularly for P. aeruginosa and K. pneumoniae, possibly indicating subclinical infection. Most of the patients with detected pathogens had a previous history of orthopedic surgery (odds ratio 2.8, P = 0.038). Pathogen specific clinical manifestations were characterized. Multiplex qPCR, because of its high sensitivity, superior specificity, and powerful quantification could be utilized in combination with culture to guide antimicrobial therapy and track the progression of orthopedic infection during treatment.

Multiple conformations of trimeric spikes visualized on a non-enveloped virus.

Many viruses utilize trimeric spikes to gain entry into host cells. However, without in situ structures of these trimeric spikes, a full understanding of this dynamic and essential process of viral infections is not possible. Here we present four in situ and one isolated cryoEM structures of the trimeric spike of the cytoplasmic polyhedrosis virus, a member of the non-enveloped Reoviridae family and a virus historically used as a model in the discoveries of RNA transcription and capping. These structures adopt two drastically different conformations, closed spike and opened spike, which respectively represent the penetration-inactive and penetration-active states. Each spike monomer has four domains: N-terminal, body, claw, and C-terminal. From closed to opened state, the RGD motif-containing C-terminal domain is freed to bind integrins, and the claw domain rotates to expose and project its membrane insertion loops into the cellular membrane. Comparison between turret vertices before and after detachment of the trimeric spike shows that the trimeric spike anchors its N-terminal domain in the iris of the pentameric RNA-capping turret. Sensing of cytosolic S-adenosylmethionine (SAM) and adenosine triphosphate (ATP) by the turret triggers a cascade of events: opening of the iris, detachment of the spike, and initiation of endogenous transcription.

MicroRNA-4732 is downregulated in non-small cell lung cancer and inhibits tumor cell proliferation, migration, and invasion.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death with increasing morbidity and mortality. MicroRNA-4732-5p (miR-4732-5p) has been reported to be dysregulated in various cancers and identified as a tumor suppressor. This study aims to explore the expression and role of miR-4732-5p in NSCLC. METHODS: Reverse transcription-quantitative polymerase chain reaction (qRT-PCR) assay was employed to detect the expression of miR-4732-5p in NSCLC. With the help of Kaplan-Meier survival and Cox regression, the prognostic significance of miR-4732-5p was investigated. Meanwhile, the effects of miR-4732-5p on cell proliferation, migration, and invasion were also studied. RESULTS: The expression of miR-4732-5p decreased in NSCLC tissues and cells. The downregulation of miR-4732-5p was closely associated with lymph node metastasis, TNM stage, and poor prognosis. Multivariate Cox regression analysis results showed that miR-4732-5p was an independent prognosis factor for NSCLC. In addition, the overexpression of miR-4732-5p inhibited the proliferation, migration, and invasion of NSCLC cells through modulating TSPAN13. CONCLUSIONS: These data showed that miR-4732-5p might be involved in the development of NSCLC, which can act as an independent prognostic biomarker and therapeutic target.

Structures of capsid and capsid-associated tegument complex inside the Epstein-Barr virus.

As the first discovered human cancer virus, Epstein-Barr virus (EBV) causes Burkitt's lymphoma and nasopharyngeal carcinoma. Isolating virions for determining high-resolution structures has been hindered by latency-a hallmark of EBV infection-and atomic structures are thus available only for recombinantly expressed EBV proteins. In the present study, by symmetry relaxation and subparticle reconstruction, we have determined near-atomic-resolution structures of the EBV capsid with an asymmetrically attached DNA-translocating portal and capsid-associated tegument complexes from cryogenic electron microscopy images of just 2,048 EBV virions obtained by chemical induction. The resulting atomic models reveal structural plasticity among the 20 conformers of the major capsid protein, 2 conformers of the small capsid protein (SCP), 4 conformers of the triplex monomer proteins and 2 conformers of the triplex dimer proteins. Plasticity reaches the greatest level at the capsid-tegument interfaces involving SCP and capsid-associated tegument complexes (CATC): SCPs crown pentons/hexons and mediate tegument protein binding, and CATCs bind and rotate all five periportal triplexes, but notably only about one peri-penton triplex. These results offer insights into the EBV capsid assembly and a mechanism for recruiting cell-regulating factors into the tegument compartment as 'cargoes', and should inform future anti-EBV strategies.

Structure of the human ClC-1 chloride channel.

ClC-1 protein channels facilitate rapid passage of chloride ions across cellular membranes, thereby orchestrating skeletal muscle excitability. Malfunction of ClC-1 is associated with myotonia congenita, a disease impairing muscle relaxation. Here, we present the cryo-electron microscopy (cryo-EM) structure of human ClC-1, uncovering an architecture reminiscent of that of bovine ClC-K and CLC transporters. The chloride conducting pathway exhibits distinct features, including a central glutamate residue ("fast gate") known to confer voltage-dependence (a mechanistic feature not present in ClC-K), linked to a somewhat rearranged central tyrosine and a narrower aperture of the pore toward the extracellular vestibule. These characteristics agree with the lower chloride flux of ClC-1 compared with ClC-K and enable us to propose a model for chloride passage in voltage-dependent CLC channels. Comparison of structures derived from protein studied in different experimental conditions supports the notion that pH and adenine nucleotides regulate ClC-1 through interactions between the so-called cystathionine-ß-synthase (CBS) domains and the intracellular vestibule ("slow gating"). The structure also provides a framework for analysis of mutations causing myotonia congenita and reveals a striking correlation between mutated residues and the phenotypic effect on voltage gating, opening avenues for rational design of therapies against ClC-1-related diseases.

pH-dependent gating mechanism of the Helicobacter pylori urea channel revealed by cryo-EM.

The urea channel of Helicobacter pylori (HpUreI) is an ideal drug target for preventing gastric cancer but incomplete understanding of its gating mechanism has hampered development of inhibitors for the eradication of H. pylori. Here, we present the cryo-EM structures of HpUreI in closed and open conformations, both at a resolution of 2.7 Å. Our hexameric structures of this small membrane protein (~21 kDa/protomer) resolve its periplasmic loops and carboxyl terminus that close and open the channel, and define a gating mechanism that is pH dependent and requires cooperativity between protomers in the hexamer. Gating is further associated with well-resolved changes in the channel-lining residues that modify the shape and length of the urea pore. Site-specific mutations in the periplasmic domain and urea pore identified key residues important for channel function. Drugs blocking the urea pore based on our structures should lead to a new strategy for H. pylori eradication.

Investigation of multiple polymers in a denitrifying sulfur conversion-EBPR system: The structural dynamics and storage states.

Polyhydroxyalkanoates (PHAs), polyphosphate (poly-P) and polysulfide or elemental sulfur (poly-S) are the key functionally relevant polymers involved in the recently reported Denitrifying Sulfur conversion-associated Enhanced Biological Phosphorus Removal (DS-EBPR) process. However, little is known about the structural dynamics and storage states of these polymers. In particular, investigating the poly-S generated in this process is quite a superior challenge. This study was thus aimed at simultaneously qualitative-quantitative investigating poly-S and associated poly-P and PHAs through the integrated chemical analysis and Raman micro-spectroscopy coupled with multiple microscopic methods (i.e. optical microscopy, confocal laser scanning microscopy, and differential interference contrast microscopy). The chemical analytical results displayed a stable DS-EBPR phenotype in terms of sulfur conversion, P release/uptake and the dynamics of relevant polymers. The multiple microscopic images and Raman spectrum profiles further clearly demonstrated the existence of the polymers and their dynamic changes under alternating anaerobic-anoxic conditions, consistent with the chemical analytical results. In particular, Raman analysis for the first time unraveled the co-existence of S0/Sn2- species stored either intracellularly or extracellularly; and the dynamic conversions between S0/Sn2- and other sulfur species suggest that there might be a universal pool of bioavailable sulfur. The results reveal the mechanisms underlying the structural dynamics and changes in storage states of the relevant polymers that are functionally relevant to the carbon/phosphorus/sulfur-cycles during different metabolic phases. These mechanisms would otherwise not be obtained only using a traditional chemical analysis-based approach.

Structures and operating principles of the replisome.

Visualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo-electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A ß hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.

T2DM inhibition of endothelial miR-342-3p facilitates angiogenic dysfunction via repression of FGF11 signaling.

Understanding the function and molecular relevance of distinct miRNAs in endothelial cells (ECs) paves avenues for possible therapeutic intervention by targeting epigenetic mechanisms in vascular endothelial dysfunction, one of the major complications of type 2 diabetes mellitus (T2DM). MiR-342-3p, an obesity-associated miRNA, has recently been shown to be significantly upregulated in human angiosarcoma compared to benign hemangioma, indicating its potential involvement as a proangiogenic factor. Herein, we show that endothelial miR-342-3p expression was significantly compromised in T2DM organisms and this inhibition powerfully blocked vasculogenesis in vivo by repressing endothelial proliferation and migration. From a mechanistic standpoint, miR-342-3p promoted the transactivation of fibroblast growth factor 11 (FGF11) by directly targeting its 3' untranslated regions (3'UTRs). Functionally, overexpression of exogenous FGF11 successfully rescued miR-342-3p deficiency-impaired endothelial proliferation and migration. Thus, perturbation of miR-342-3p/FGF11 cascade by hyperinsulinemia plays a causative role in the induction of vascular dysfunction in T2DM. Overall, the current study underscore an endothelial facet of miR-342-3p, which may operate as a novel epigenetic integrator linking adipogenic homeostasis and angiogenesis.

Increased neutrophil-lymphocyte ratio independently predicts poor survival in non-metastatic triple-negative breast cancer patients.

Inflammation plays an important role in tumor initiation, progression, and metastasis. The neutrophil-lymphocyte ratio (NLR) is widely used to evaluate global inflammation in various tumor types. However, the prognostic role of NLR in non-metastatic triple-negative breast cancer (TNBC) patients was poorly known. The aim of this study was to explore the association between pre-treatment NLR and survival in TNBC patients. Data were collected for patients with stages I-III TNBC from 2006 to 2013 at Linyi Central Hospital to analyze pre-treatment NLR and survival. Overall survival (OS) and disease-free survival (DFS) were estimated by the Kaplan-Meier method, and Cox analysis was performed to determine clinicopathological parameters for their prognostic relevance. A total of 406 patients were eligible. Patients with NLR lower than 2.85 exhibited significantly higher OS (P < 0.001) and DFS (P < 0.001) than patients with higher NLR. Higher pre-treatment NLR was independently correlated with poor OS and DFS, with hazard ratios of 2.69 (95% confidence interval [CI]: 1.94-3.72, P = 0.001) and 2.13 (95% CI: 1.68-2.65, P = 0.008), respectively, in the Cox proportional multivariate hazard model. In conclusion, our results indicate that pre-treatment NLR may be correlated with OS and DFS in early-stage TNBC patients, and that it may have considerable clinical applications. © 2018 IUBMB Life, 70(6):529-535, 2018.

Down-regulation of FOXR2 inhibits non-small cell lung cancer cell proliferation and invasion through the Wnt/ß-catenin signaling pathway.

Forkhead box R2 (FOXR2), a new member of the FOX family, is an important player in a wide range of cellular processes such as proliferation, migration, differentiation and apoptosis. Recently, FOXR2 has been reported to be implicated in cancer development. However, the biological functions of FOXR2 in non-small cell lung cancer (NSCLC) remain unclear. In this study, we investigated the specific role of FOXR2 in NSCLC. The results showed that down-regulation of FOXR2 significantly inhibited NSCLC cell proliferation and invasion in vitro and suppressed NSCLC cell growth and metastasis in vivo. In addition, the decrease in FOXR2 expression markedly reduced the protein levels of ß-catenin, cyclinD1 and c-Myc and hence inactivated the Wnt/ß-catenin pathway in NSCLC cells. Taken together, we concluded that FOXR2 might be considered as a promising therapeutic target for NSCLC treatment.

Cracking the DNA Code for V(D)J Recombination.

To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the "beyond 12/23" restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.

Conformation-Directed Formation of Self-Healing Diblock Copolypeptide Hydrogels via Polyion Complexation.

Synthetic diblock copolypeptides were designed to incorporate oppositely charged ionic segments that form ß-sheet-structured hydrogel assemblies via polyion complexation when mixed in aqueous media. The observed chain conformation directed assembly was found to be required for efficient hydrogel formation and provided distinct and useful properties to these hydrogels, including self-healing after deformation, microporous architecture, and stability against dilution in aqueous media. While many promising self-assembled materials have been prepared using disordered or liquid coacervate polyion complex (PIC) assemblies, the use of ordered chain conformations in PIC assemblies to direct formation of new supramolecular morphologies is unprecedented. The promising attributes and unique features of the ß-sheet-structured PIC hydrogels described here highlight the potential of harnessing conformational order derived from PIC assembly to create new supramolecular materials.

miR-24 suppression of POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1) protects endothelial cell from diabetic damage.

The regulatory transcriptional factor PATZ1 is abnormally up-regulated in diabetic endothelial cells (ECs) where it acts as an anti-angiogenic factor via modulation of fatty acid-binding protein 4 (FABP4) signaling. The aim of the present work was to elucidate the upstream molecular events regulating PATZ1 expression in diabetic angiogenesis. The bioinformatics search for microRNAs (miRNAs) able to potentially target PATZ1 led to the identification of several miRNAs. Among them we focused on the miR-24 since the multiple targets of miR-24, which have so far been identified in beta cells, cardiomyocytes and macrophages, are all involved in diabetic complications. miR-24 expression was significantly impaired in the ECs isolated from diabetic hearts. Functionally, endothelial migration was profoundly inhibited by miR-24 suppression in Ctrl ECs, whereas miR-24 overexpression by mimics treatment effectively restored the migration rate in diabetic ECs. Mechanistically, miR-24 directly targeted the 3'untranslated region (3'UTR) of PATZ1, and miR-24 accumulation potentiated endothelial migration by reducing the mRNA stability of PATZ1. Together, these results suggest a novel mechanism regulating endothelial PATZ1 expression based on the down-regulation of miR-24 expression caused by hyperglycemia. Interfering with PATZ1 expression via miRNAs or miRNA mimics could potentially represent a new way to target endothelial PATZ1-dependent signaling of vascular dysfunction in diabetes.

[Clinical Significance of Detecting CyclinD1 and BCL-2 in Patients with B-Cell Lymphoma by Using Flow Cytometry].

OBJECTIVE: To explore the feasibility and value of detecting CyclinD1 and BCL-2 in patients with B-cell lymphoma by using flow cytometry. METHODS: Fifty-three patients with lymphoma were selected, and 50 healthy persons in the same period were selected as control. The expression levels of CyclinD1 and BCL-2 in patients with various subtypes of lymphoma were detected by using flow cytometry (FCM). RESULTS: When the dilution time was 1 min and the dilution proportion was 1:20, the cell morphology was the best complete, at the 4 min the cell morphology was best status. The mean fluorescence intensity of CyclinD1 and BCL-2 between persons of control group and patients with B-cell lymphoma showed significant difference, the CyclinD1 level (1.824 ± 0.315) and BCL-2 levels (4.257 ± 0.528) of patients with B-cell lymphoma were obviously higher than the CyclinD1 level (0.634 ± 0.153) and BCL-2 level (1.926 ± 0.328) of persons in control group, the CyclinD1 and BCL-2 expression levels of patients with HL were significantly lower than CyclinD1 and BCL-2 levels of patients with NHL (P < 0.01). After treatment, the expression levels of CyclinD1 and BCL-2 in patients with B lymphoma were significantly lower than these befor treatment. CONCLUSION: Using the method of flow cytometry for detecting CyclinD1 and BCL-2 expression levels in lymphoma cells of patients is feasible, and it can be applied clinically to evaluate the treatment efficacy.