The molecular mechanism of NELL2 movement and secretion in hippocampal progenitor HiB5 cells

Neural epidermal growth factor-like protein-like 2 (NELL2) is a secreted glycoprotein that is predominantly expressed in the nervous system, but little is known about the intracellular movement and secretion mechanism of this protein. By monitoring the localization and movements of enhanced green fluorescent protein (EGFP)-labeled NELL2 in living cultured hippocampal neuroprogenitor HiB5 cells, we determined the subcellular localization of NELL2 and its intracellular movement and secretion mechanism. Cterminal EGFP-fused NELL2 showed a typical expression pattern of secreted proteins, especially with respect to its localization in the endoplasmic reticulum, Golgi apparatus, and punctate structures. Vesicles containing NELL2 exhibited bidirectional movement in HiB5 cells. The majority of the vesicles (70.1%) moved in an anterograde direction with an average velocity of 0.454 μm/s, whereas some vesicles (28.7%) showed retrograde movement with an average velocity of 0.302 μm/s. The movement patterns of NELL2 vesicles were dependent upon the presence of microtubules in HiB5 cells. Anterograde movement of NELL2 did not lead to a detectable accumulation of NELL2 in the peripheral region of the cell, indicating that it was secreted into the culture medium. We also showed that the N-terminal 29 amino acids of NELL2 were important for secretion of this protein. Taken together, these results strongly suggest that the N-terminal region of NELL2 determines both the pattern of its intracellular expression and transport of NELL2 vesicles by high-velocity movement. Therefore, NELL2 may affect the cellular activity of cells in a paracrine or autocrine manner.     

ATPase‐driven oligomerization of RIG‐I on RNA allows optimal activation of type‐I interferon

The cytosolic pathogen sensor RIG‐I is activated by RNAs with exposed 5′‐triphosphate (5′‐ppp) and terminal double‐stranded structures, such as those that are generated during viral infection. RIG‐I has been shown to translocate on dsRNA in an ATP‐dependent manner. However, the precise role of the ATPase activity in RIG‐I activation remains unclear. Using in vitro‐transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG‐I oligomerizes on 5′‐ppp dsRNA in an ATP hydrolysis‐dependent and dsRNA length‐dependent manner, which correlates with the strength of type‐I interferon (IFN‐I) activation. These results establish a clear role for the ligand‐induced ATPase activity of RIG‐I in the stimulation of the IFN response.     

Lake restoration by biomanipulation using piscivore and Daphnia stocking; results of the biomanipulation in Japan

Biomanipulation has been employed in numerous locations throughout the world as a means for reducing phytoplankton biomass; however, it has not been employed very often in Japan. A common approach involves the introduction of piscivorous fish to reduce the abundance of planktivorous fish. In our study, to first apply biomanipulation, we stocked Lake Shirakaba (a high-altitude, protected area in a park) in central Japan with rainbow trout fingerlings and cladoceran Daphnia (Daphnia galeata) in 2000. A “pre-biomanipulation” data set (1997–1999) and “a post-biomanipulation” data set (2000–2006) allowed us to evaluate the lake's response to biomanipulation. After the biomanipulation, zoo-planktivorous pond smelt disappeared and a large population of Daphnia had been established, which substantially reduced the number of the previously dominant small cladocerans and rotifers. Water transparency increased from about 2 m (before biomanipulation) to more than 4 m (after biomanipulation). Reductions in algal biomass and increased transparency led to expansion of the submerged macrophyte Elodea nuttallii. Total phosphorus concentrations declined as well over this time period. Based on these results, we concluded that biomanipulation using piscivore and Daphnia stocking succeeded in improving lake water quality by reducing algal abundance and providing favorable conditions for the establishment of rooted plants.     

Loss of VHL promotes progerin expression,leading to impaired p14/ARF function and suppression of p53 activity

Renal cell carcinomas (RCCs) are frequently occurring genitourinary malignancies in the aged population. A morphological characteristic of RCCs is an irregular nuclear shape, which is used to index cancer grades. Other features of RCCs include the genetic inactivation of the von Hippel-Lindau gene, VHL, and p53 genetic-independent inactivation. An aberrant nuclear shape or p53 suppression has not yet been demonstrated. We examined the effect of progerin (an altered splicing product of the LMNA gene linked to Hutchinson Gilford progeria syndrome; HGPS) on the nuclear deformation of RCCs in comparison to that of HGPS cells. In this study, we showed that progerin was suppressed by pVHL and was responsible for nuclear irregularities as well as p53 inactivation. Thus, progerin suppression can ameliorate nuclear abnormalities and reactivate p53 in response to genotoxic addition. Furthermore, we found that progerin was a target of pVHL E3 ligase and suppressed p53 activity by p14/ARF inhibition. Our findings indicate that the elevated expression of progerin in RCCs results from the loss of pVHL and leads to p53 inactivation through p14/ARF suppression. Interestingly, we showed that progerin was expressed in human leukemia and primary cell lines, raising the possibility that the expression of this LMNA variant may be a common event in age-related cancer progression.     

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs.     

Factors Associated with Dengue Shock Syndrome: A Systematic Review and Meta-Analysis

Background

The pathogenesis of dengue shock syndrome (DSS, grade 3 and 4) is not yet completely understood. Several factors are reportedly associated with DSS, a more severe form of dengue infection that reportedly causes 50 times higher mortality compared to that of dengue patients without DSS. However, the results from these reports remain inconclusive. To better understand the epidemiology, clinical manifestation, and pathogenesis of DSS for development of new therapy, we systematically reviewed and performed a meta-analysis of relevant studies that reported factors in both DSS and dengue hemorrhagic fever (DHF, grade 1 and 2) patients.

Methods and Findings

PubMed, EMBASE, Scopus, Google Scholar, Dengue Bulletin, Cochrane Library, Virtual Health Library, and a manual search of reference lists of articles published before September 2010 were used to retrieve relevant studies. A meta-analysis using fixed- or random-effects models was used to calculate pooled odds ratios (OR) or event rate with corresponding 95% confidence intervals. Assessment of heterogeneity and publication bias, meta-regression analysis, subgroup analysis, sensitivity analysis, and analysis of factor-specific relationships were further performed. There were 198 studies constituting 203 data sets that met our eligibility criteria. Our meta-regression analysis showed a sustained reduction of DSS/dengue hemorrhagic fever (DHF) ratio over a period of 40 years in Southeast Asia, especially in Thailand. The meta-analysis revealed that age, female sex, neurological signs, nausea/vomiting, abdominal pain, gastrointestinal bleeding, hemoconcentration, ascites, pleural effusion, hypoalbuminemia, hypoproteinemia, hepatomegaly, levels of alanine transaminase and aspartate transaminase, thrombocytopenia, prothrombin time, activated partial thromboplastin time, fibrinogen level, primary/secondary infection, and dengue virus serotype-2 were significantly associated with DSS when pooling all original relevant studies.

Conclusions

The results improve our knowledge of the pathogenesis of DSS by identifying the association between the epidemiology, clinical signs, and biomarkers involved in DSS.     

The Signal Sequence Influences Post-Translational ER Translocation at Distinct Stages

The metazoan Sec61 translocon transports polypeptides into and across the membrane of the endoplasmic reticulum via two major routes, a well-established co-translational pathway and a post-translational alternative. We have used two model substrates to explore the elements of a secretory protein precursor that preferentially direct it towards a co- or post-translational pathway for ER translocation. Having first determined the capacity of precursors to enter ER derived microsomes post-translationally, we then exploited semi-permeabilized mammalian cells specifically depleted of key membrane components using siRNA to address their contribution to the membrane translocation process. These studies suggest precursor chain length is a key factor in the post-translational translocation at the mammalian ER, and identify Sec62 and Sec63 as important components acting on this route. This role for Sec62 and Sec63 is independent of the signal sequence that delivers the precursor to the ER. However, the signal sequence can influence the subsequent membrane translocation process, conferring sensitivity to a small molecule inhibitor and dictating reliance on the molecular chaperone BiP. Our data support a model where secretory protein precursors that fail to engage the signal recognition particle, for example because they are short, are delivered to the ER membrane via a distinct route that is dependent upon both Sec62 and Sec63. Although this requirement for Sec62 and Sec63 is unaffected by the specific signal sequence that delivers a precursor to the ER, this region can influence subsequent events, including both Sec61 mediated transport and the importance of BiP for membrane translocation. Taken together, our data suggest that an ER signal sequence can regulate specific aspects of Sec61 mediated membrane translocation at a stage following Sec62/Sec63 dependent ER delivery.     

Peripheral Afferent Mechanisms Underlying Acupuncture Inhibition of Cocaine Behavioral Effects in Rats

Administration of cocaine increases locomotor activity by enhancing dopamine transmission. To explore the peripheral mechanisms underlying acupuncture treatment for drug addiction, we developed a novel mechanical acupuncture instrument (MAI) for objective mechanical stimulation. The aim of this study was to evaluate whether acupuncture inhibition of cocaine-induced locomotor activity is mediated through specific peripheral nerves, the afferents from superficial or deep tissues, or specific groups of nerve fibers. Mechanical stimulation of acupuncture point HT7 with MAI suppressed cocaine-induced locomotor activity in a stimulus time-dependent manner, which was blocked by severing the ulnar nerve or by local anesthesia. Suppression of cocaine-induced locomotor activity was elicited after HT7 stimulation at frequencies of either 50 (for Meissner corpuscles) or 200 (for Pacinian corpuscles) Hz and was not affected by block of C/Aδ-fibers in the ulnar nerve with resiniferatoxin, nor generated by direct stimulation of C/Aδ-fiber afferents with capsaicin. These findings suggest that HT7 inhibition of cocaine-induced locomotor activity is mediated by A-fiber activation of ulnar nerve that originates in superficial and deep tissue.     

Theoretical study on the adsorption of phenol on activated carbon using density functional theory

Density functional theory (DFT) calculations performed at the PBE/DZP level using the DFT-D2 method were utilized to investigate the adsorption of phenol on pristine activated carbon (AC) and on activated carbon functionalized with OH, CHO, or COOH groups. Over the pristine AC, the phenol molecule undergoes weak physical adsorption due to van der Waals interactions between the aromatic part of the phenol and the basal planes of the AC. Among the three functional groups used to functionalize the AC, the carboxylic group was found to interact most strongly with the hydroxyl group of phenol. These results suggest that functionalized AC-COOH has great potential for use in environmental applications as an adsorbent of phenol molecules in aqueous phases.