Glycosidic Bond Conformation Preference Plays a Pivotal Role in Catalysis of RNA Pseudouridylation: A Combined Simulation and Structural Study

The most abundant chemical modification on RNA is isomerization of uridine (or pseudouridylation) catalyzed by pseudouridine synthases. The catalytic mechanism of this essential process remains largely speculative, partly due to lack of knowledge of the pre-reactive state that is important to the identification of reactive chemical moieties. In the present study, we showed, using orthogonal space random-walk free-energy simulation, that the pre-reactive states of uridine and its reactive derivative 5-fluorouridine, bound to a ribonucleoprotein particle pseudouridine synthase, strongly prefer the syn glycosidic bond conformation, while that of the nonreactive 5-bromouridine-containing substrate is largely populated in the anti conformation state. A high-resolution crystal structure of the 5-bromouridine-containing substrate bound to the ribonucleoprotein particle pseudouridine synthase and enzyme activity assay confirmed the anti nonreactive conformation and provided the molecular basis for its confinement. The observed preference for the syn pre-reactive state by the enzyme-bound uridine may help to distinguish among currently proposed mechanisms.     

The regulatory mechanism of extracellular Hsp90{alpha} on matrix metalloproteinase-2 processing and tumor angiogenesis

Heat shock protein 90α (Hsp90α) is a ubiquitously expressed molecular chaperone that is essential for eukaryotic homeostasis. Hsp90α can also be secreted extracellularly, where it has been shown to be involved in tumor metastasis. Extracellular Hsp90α interacts with and promotes the proteolytic activity of matrix metalloproteinase-2 (MMP-2). However, the regulatory mechanism of Hsp90α on MMP-2 activity is still unknown. Here we show that Hsp90α stabilizes MMP-2 and protects it from degradation in tumor cells. Further investigation reveals that this stabilization effect is isoform-specific, ATP-independent, and mediated by the interaction between the Hsp90α middle domain and the MMP-2 C-terminal hemopexin domain. Moreover, this mechanism also applies to endothelial cells that secrete more Hsp90α in their proliferating status. Furthermore, endothelial cell transmigration, Matrigel plug, and tumor angiogenesis assays demonstrate that extracellular Hsp90α promotes angiogenesis in an MMP-2-dependent manner. In sum, this study provides new insights into the molecular mechanism of how Hsp90α regulates its extracellular client proteins and also reveals for the first time the function of extracellular Hsp90α in promoting tumor angiogenesis.     

Silkworm coatomers and their role in tube expansion of posterior silkgland

Background

Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation.

Methodology/Principal Findings

Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (α,β,β′, δ, ε, and ζ-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of α-, β′- and γ-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of α-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion.

Conclusions/Significance

The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and would facilitate the establishment of silkworm PSG as an efficient bioreactor.     

SCAR Makers and Multiplex PCR-Based Rapid Molecular Typing of Lentinula edodes Strains

Lentinula edodes is the second most important cultivated mushroom worldwide, the most commercial strains have been identified only through traditional phenotypic analysis. In this study, a simple rapid PCR-based molecular method was developed for distinguishing commercial strains of L. edodes by developing specific sequence characterized amplified region (SCAR) markers and establishing multiplex PCR assays with the SCAR primers. Derived from the randomly amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) techniques, 10 informative SCAR markers were generated from 10 polymorphic RAPD and SRAP bands. The differences in SCAR phenotypes among different strains made these SCAR markers potentially useful to characterize 6 strains and identify them from other studied strains. Moreover, different SCAR phenotypes also made the other 17 studied strains to be divided into four distinguishable groups. The multiplex PCR assays were further established for the joint use of some SCAR markers efficiently. Compared with some identification methods reported previously, the special feature of this new molecular method is technically rapid and convenient in the practical use and suitable for analyzing large numbers of samples. Thus, the simple rapid PCR-based molecular method can be used as a helpful assistant tool for the lentinula industry. To our knowledge, this study is the first to describe a development of a new SCAR maker-based multiplex PCR assay for rapid molecular typing of edible mushroom.     

Characterization of starvation‐induced dispersion in Pseudomonas putida biofilms: genetic elements and molecular mechanisms

Pseudomonas putida OUS82 biofilm dispersal was previously shown to be dependent on the gene PP0164 (here designated lapG). Sequence and structural analysis has suggested that the LapG geneproduct belongs to a family of cysteine proteinases that function in the modification of bacterial surface proteins. We provide evidence that LapG is involved in P. putida OUS82 biofilm dispersal through modification of the outer membrane‐associated protein LapA. While the P. putida lapG mutant formed more biofilm than the wild‐type, P. putida lapA and P. putida lapAG mutants displayed decreased surface adhesion and were deficient in subsequent biofilm formation, suggesting that LapG affects LapA, and that the LapA protein functions both as a surface adhesin and as a biofilm matrix component. Lowering of the intracellular c‐di‐GMP level via induction of an EAL domain protein led to dispersal of P. putida wild‐type biofilm but did not disperse P. putida lapG biofilm, indicating that LapG exerts its activity on LapA in response to a decrease in the intracellular c‐di‐GMP level. In addition, evidence is provided that associated to LapA a cellulase‐degradable exopolysaccharide is part of the P. putida biofilm matrix.     

Outer surface protein C is a dissemination-facilitating factor of Borrelia burgdorferi during mammalian infection

Background

The Lyme disease spirochete Borrelia burgdorferi dramatically upregulates outer surface protein C (OspC) in response to fresh bloodmeal during transmission from the tick vector to a mammal, and abundantly produces the antigen during early infection. As OspC is an effective immune target, to evade the immune system B. burgdorferi downregulates the antigen once the anti-OspC humoral response has developed, suggesting an important role for OspC during early infection.

Methodology/Principal Findings

In this study, a borrelial mutant producing an OspC antigen with a 5-amino-acid deletion was generated. The deletion didn''t significantly increase the 50% infectious dose or reduce the tissue bacterial burden during infection of the murine host, indicating that the truncated OspC can effectively protect B. burgdorferi against innate elimination. However, the deletion greatly impaired the ability of B. burgdorferi to disseminate to remote tissues after inoculation into mice.

Conclusions/Significance

The study indicates that OspC plays an important role in dissemination of B. burgdorferi during mammalian infection.     

Genome-wide screen identifies signaling pathways that regulate autophagy during Caenorhabditis elegans development

The mechanisms that coordinate the regulation of autophagy with developmental signaling during multicellular organism development remain largely unknown. Here, we show that impaired function of ribosomal protein RPL-43 causes an accumulation of SQST-1 aggregates in the larval intestine, which are removed upon autophagy induction. Using this model to screen for autophagy regulators, we identify 139 genes that promote autophagy activity upon inactivation. Various signaling pathways, including Sma/Mab TGF-β signaling, lin-35/Rb signaling, the XBP-1-mediated ER stress response, and the ATFS-1-mediated mitochondrial stress response, regulate the expression of autophagy genes independently of the TFEB homolog HLH-30. Our study thus provides a framework for understanding the role of signaling pathways in regulating autophagy under physiological conditions.Subject Categories: Autophagy & Cell Death; Signal Transduction; Membrane & Intracellular Transport     

Normal Thoracic Radiographic Appearance of the Cynomolgus Monkey (Macaca fascicularis)

Background

The cynomolgus monkey (Macaca fascicularis) has been increasingly used as a non-human primate model in biomedical research. As establishing baseline thoracic radiography for the cynomolgus monkey is essential, we tested the hypothesis that age and sex may affect the thoracic radiography parameters of this species.

Methods

Here, 697 healthy cynomolgus monkeys were segregated by sex and age (three age groups: 25–36 months, 37–48 months, 49–60 months). The lung length (LL), maximal interior thoracic depth (TD), maximal interior thoracic breadth (TBr), cardiac silhouette breadth (CBr), cardiothoracic ratio (CR), right and left costophrenic angles (RCA and LCA), and right hilar height ratio (R-HHR) were assessed by chest film. Statistical analysis was applied to examine the effect of age, sex, and age × sex interactions.

Results

Significant effects by age were shown for LL, TD, TBr, CBr, and CR. Significant effects by sex were found for TD, TBr, CBr, CR, and R-HHR. Significant effects by age × sex were observed for TD, TBr, CBr, and CR. Both TD and TBr increased with age in both sexes, and both were significantly higher in males than in females in the group aged 49–60 months. CBr increased with age and was significantly higher in males than in females across all age groups. CR declined with age and was significantly higher in males than females across all age groups, and CR was similar or slightly higher relative to those previously found in other non-human primate species. As to the other parameters with no significant sex nor age-related differences, the R-HHR was greater than 1.00, and the angulation of bilateral costophrenic angles were sharp.

Conclusions

The thoracic radiographic parameters for the healthy cynomolgus monkey presented here should prove useful in veterinary practice, research involving non-human primate models of respiratory or cardiovascular disorders, and morphological studies on cynomolgus monkeys.     

Body Height,Estimated Cerebrospinal Fluid Pressure and Open-Angle Glaucoma. The Beijing Eye Study 2011

Purpose

To examine potential associations between body height, cerebrospinal fluid pressure (CSFP), trans-lamina cribrosa pressure difference (TLCPD) and prevalence of open-angle glaucoma (OAG) in a population-based setting.

Methods

The population-based Beijing Eye Study 2011 included 3468 individuals with a mean age of 64.6±9.8 years (range:50–93 years). A detailed ophthalmic examination was performed. Based on a previous study with lumbar cerebrospinal fluid pressure (CSFP) measurements, CSFP was calculated as CSFP[mmHg] = 0.44×Body Mass Index[kg/m²]+0.16×Diastolic Blood Pressure[mmHg]-0.18×Age[Years]-1.91

Results

Data of IOP and CSFP were available for 3353 (96.7%) subjects. Taller body height was associated with higher CSFP (P<0.001; standardized correlation coefficient beta:0.13; regression coefficient B:0.29; 95% confidence interval (CI):0.25,0.33) after adjusting for male gender, urban region of habitation, higher educational level, and pulse rate. If TLCPD instead of CSFP was added, taller body height was associated with lower TLCPD (P<0.001;beta:−0.10;B:−0.20;95%CI:−0.25,−0.15). Correspondingly, higher CSFP was associated with taller body height (P = 0.003;beta:0.02;B:0.01;95%CI:0.00,0.02), after adjusting for age, gender, body mass index, pulse, systolic blood pressure, and blood concentration of cholesterol. If IOP was added to the model, higher CSFP was associated with higher IOP (P<0.001;beta:0.02;B:0.02;95%CI:0.01,0.03). TLCPD was associated with lower body height (P = 0.003;beta:−0.04;B −0.02,95%CI:−0.04,−0.01) after adjusting for age, body mass index, systolic blood pressure, pulse, blood concentrations of triglycerides, axial length, central corneal thickness, corneal curvature radius, and anterior chamber depth. Adding the prevalence of OAG to the multivariate analysis revealed, that taller body height was associated with a lower OAG prevalence (P = 0.03;beta:−0.03;B:−1.20;95%CI:−2.28,−0.12) after adjusting for educational level and gender.

Conclusions

Taller body height was associated with higher CSFP and lower TLCPD (and vice versa), after adjusting for systemic and ocular parameters. Parallel to the associations between a higher prevalence of glaucoma with a lower CSFP or higher TLCPD, taller body height was associated with a lower prevalence of OAG.