UPRmt and coordinated UPRER in type 2 diabetes.

The mitochondrial unfolded protein response (UPRmt) is a molecular mechanism that maintains mitochondrial proteostasis under stress and is closely related to various metabolic diseases, such as type 2 diabetes (T2D). Similarly, the unfolded protein response of the endoplasmic reticulum (UPRER) is responsible for maintaining proteomic stability in the endoplasmic reticulum (ER). Since the mitochondria and endoplasmic reticulum are the primary centers of energy metabolism and protein synthesis in cells, respectively, a synergistic mechanism must exist between UPRmt and UPRER to cooperatively resist stresses such as hyperglycemia in T2D. Increasing evidence suggests that the protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) signaling pathway is likely an important node for coordinating UPRmt and UPRER. The PERK pathway is activated in both UPRmt and UPRER, and its downstream molecules perform important functions. In this review, we discuss the mechanisms of UPRmt, UPRER and their crosstalk in T2D.

Characterization of Shallow Whole-Metagenome Shotgun Sequencing as a High-Accuracy and Low-Cost Method by Complicated Mock Microbiomes.

Characterization of the bacterial composition and functional repertoires of microbiome samples is the most common application of metagenomics. Although deep whole-metagenome shotgun sequencing (WMS) provides high taxonomic resolution, it is generally cost-prohibitive for large longitudinal investigations. Until now, 16S rRNA gene amplicon sequencing (16S) has been the most widely used approach and usually cooperates with WMS to achieve cost-efficiency. However, the accuracy of 16S results and its consistency with WMS data have not been fully elaborated, especially by complicated microbiomes with defined compositional information. Here, we constructed two complex artificial microbiomes, which comprised more than 60 human gut bacterial species with even or varied abundance. Utilizing real fecal samples and mock communities, we provided solid evidence demonstrating that 16S results were of poor consistency with WMS data, and its accuracy was not satisfactory. In contrast, shallow whole-metagenome shotgun sequencing (shallow WMS, S-WMS) with a sequencing depth of 1 Gb provided outputs that highly resembled WMS data at both genus and species levels and presented much higher accuracy taxonomic assignments and functional predictions than 16S, thereby representing a better and cost-efficient alternative to 16S for large-scale microbiome studies.

The maintenance of microbial community in human fecal samples by a cost effective preservation buffer.

In the burgeoning microbiome field, powerful sequencing approaches and accompanied bioanalytical methods have made tremendous contributions to the discoveries of breakthroughs, which favor to unravel the intimate interplay between gut microbiota and human health. The proper preservation of samples before being processed is essential to guarantee the authenticity and reliability of microbiome studies. Hence, the development of preservation methods is extremely important to hold samples eligible for the consequent analysis, especially population cohort-based investigations or those spanning species or geography, which frequently facing difficulties in suppling freezing conditions. Although there are several commercial products available, the exploration of cost-efficient and ready-to-use preservation methods are still in a large demand. Here, we performed shotgun metagenomic sequencing and demonstrated that microbial consortia in human fecal samples were substantially preserved within a temporary storage of 4 h, independent of the storage temperature. We also verified a previous reported self-made preservation buffer (PB buffer) could not only preserve fecal microbiota at room temperature up to 4 weeks but also enable samples to endure a high temperature condition which mimics temperature variations in summer logistics. Moreover, PB buffer exhibited suitability for human saliva as well. Collectively, PB buffer may be a valuable choice to stabilize samples if neither freezing facilities nor liquid nitrogen is available.

[Quantification of microbial DNA in laboratory environment during DNA extraction].

Metagenomic sequencing provides a powerful tool for microbial research. However, traditional experimental DNA extraction process will inevitably mix with environmental microorganisms which float in the air. It is still unclear whether the mixed environmental microbial DNA will heavily affect the metagenomic results of samples with extremely low microbial content. In this study, we first collected environmental bacteria in the laboratory and quantified the mixed environmental microbial DNA content during DNA extraction based on a qPCR-based quantification assay. We then extracted DNA from pure water in order to determine the mixed microbial taxons during extraction under open environment. At last, we extracted total DNA from a skin sample in a Biosafety cabinet or under open laboratory environment, to assess the impact of the mixed environmental microorganisms on the metagenomic results. Our results showed that DNA extraction under open laboratory environment in Beijing region resulted in 28.9 pg contaminant, which may accout for 30% of total DNA amount from skin samples. Metagenomic analysis revealed that the main incorporated environmental taxons were Cutibacterium acnes and Escherichia coli. Tens of environmental bacteria were foisted in the skin DNA samples, which largely decreased the relative abundance of dominant species and thus deteriorated the result accuracy. Therefore, analyzing microbial composition of samples with extremely low DNA content should better performed under aseptic environment.

Tcf4 Controls Neuronal Migration of the Cerebral Cortex through Regulation of Bmp7.

Background: Transcription factor 4 (TCF4) is found to be associated with schizophrenia. TCF4 mutations also cause Pitt-Hopkins Syndrome, a neurodevelopmental disorder associated with severe mental retardation. However, the function of TCF4 during brain development remains unclear. Results: Here, we report that Tcf4 is expressed in the developing cerebral cortex. In utero suppression of Tcf4 arrested neuronal migration, leading to accumulation of ectopic neurons in the intermediate zone. Knockdown of Tcf4 impaired leading process formation. Furthermore, Bone Morphogenetic Protein 7 (Bmp7) is upregulated in Tcf4-deficient neurons. In vivo gain of function and rescue experiments demonstrated that Bmp7 is the major downstream effector of Tcf4 required for neuronal migration. Conclusion: Thus, we have uncovered a new Tcf4/Bmp7-dependent mechanism underlying neuronal migration, and provide insights into the pathogenesis of neurodevelopmental disorders.

RAB18, a protein associated with Warburg Micro syndrome, controls neuronal migration in the developing cerebral cortex.

BACKGROUND: Loss of function mutations in RAB18, has been identified in patients with the human neurological and developmental disorder Warburg Micro syndrome. However, the function of RAB18 in brain remains unknown. RESULTS: In this study, we report that RAB18 is a critical regulator of neuronal migration and morphogenesis. Using in utero electroporation suppression of RAB18 in the mouse brain impairs radial migration. Overexpression of dominant negative RAB18 or disruption of RAB3GAP (RAB18GEF) also results in delayed neuronal migration in the developing mouse cortex and inhibition of neurite growth in vitro. Moreover, loss of RAB18 induces an acceleration of N-cadherin degradation by lysosomal pathway resulting in the decrease of surface level of N-cadherin on neurons. CONCLUSIONS: RAB18 regulates neuronal migration and morphogenesis during development. Our findings highlight the critical role of RAB3GAP-RAB18 pathway in the developing cerebral cortex and might explain some of clinical features observed in patients with Warburg Micro syndrome.

Ribosomal L1 domain and lysine-rich region are essential for CSIG/ RSL1D1 to regulate proliferation and senescence.

The expression change of cellular senescence-associated genes is underlying the genetic foundation of cellular senescence. Using a suppressive subtractive hybridization system, we identified CSIG (cellular senescence-inhibited gene protein; RSL1D1) as a novel senescence-associated gene. CSIG is implicated in various process including cell cycle regulation, apoptosis, and tumor metastasis. We previously showed that CSIG plays an important role in regulating cell proliferation and cellular senescence progression through inhibiting PTEN, however, which domain or region of CSIG contributes to this function? To clarify this question, we investigated the functional importance of ribosomal L1 domain and lysine (Lys) -rich region of CSIG. The data showed that expression of CSIG potently reduced PTEN expression, increased cell proliferation rates, and reduced the senescent phenotype (lower SA-ß-gal activity). By contrast, neither the expression of CSIG N- terminal (NT) fragment containing the ribosomal L1 domain nor C-terminal (CT) fragment containing Lys-rich region could significantly altered the levels of PTEN; instead of promoting cell proliferation and delaying cellular senescence, expression of CSIG-NT or CSIG-CT inhibited cell proliferation and accelerated cell senescence (increased SA-ß-gal activity) compared to either CSIG over-expressing or control (empty vector transfected) cells. The further immunofluorescence analysis showed that CSIG-CT and CSIG-NT truncated proteins exhibited different subcellular distribution with that of wild-type CSIG. Conclusively, both ribosomal L1 domain and Lys-rich region of CSIG are critical for CSIG to act as a regulator of cell proliferation and cellular senescence.

NDUFV2 regulates neuronal migration in the developing cerebral cortex through modulation of the multipolar-bipolar transition.

Abnormalities during brain development are tightly linked several psychiatric disorders. Mutations in NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) are responsible for schizophrenia, bipolar disorder and Parkinson׳s disease. However, the function of NDUFV2 during brain development remains unclear. Here we reported that ndufv2 is expressed in the developing cerebral cortex. In utero suppression of ndufv2 arrested neuronal migration, leading to accumulation of ectopic neurons in the intermediate zone. ndufv2 inhibition did not affect radial glia scaffold, progenitor cells or neurons survival. However, the loss of ndufv2 impairs neuronal multipolar-bipolar transition in vivo and polarization in vitro. Moreover, ndufv2 affected actin cytoskeleton and tubulin stabilization in cortical neurons. Overall, our findings establish a new NDUFV2 dependent mechanism underlying neuronal migration and psychiatric disorders.

CSIG promotes hepatocellular carcinoma proliferation by activating c-MYC expression.

Cellular senescence-inhibited gene (CSIG) protein significantly prolongs the progression of replicative senescence, but its role in tumorigenesis is unclear. To reveal the role of CSIG in HCC, we determined its expression in HCC tissues and surrounding tissues and its functions in tumor cell proliferation in vitro and in vivo. CSIG protein was overexpressed in 86.4% of the human HCC cancerous tissues as compared with matched surrounding tissues, and its protein expression was greater in HCC cells than the non-transformed hepatic cell line L02. Furthermore, upregulation of CSIG significantly increased the colony formation of SMMC7721 and HepG2 cells, and silencing CSIG could induce cell cycle arrest and cell apoptosis. The tumorigenic ability of CSIG was confirmed in vivo in a mouse xenograft model. Our results showed that CSIG promoted the proliferation of HepG2 and SMMC7721 cells in vivo. Finally, CSIG protein directly interacted with c-MYC protein and increased c-MYC protein levels; the ubiquitination and degradation of c-MYC protein was increased with knockdown of CSIG. CSIG could also increase the expression of c-MYC protein in SMMC7721 cells in vivo, and it was noted that the level of c-MYC protein was also elevated in most human cancerous tissues with high level of CSIG.

The retinoblastoma protein selectively represses E2F1 targets via a TAAC DNA element during cellular senescence.

The retinoblastoma (Rb) protein mediates heterochromatin formation at the promoters of E2 transcription factor 1 (E2F1) target genes, such as proliferating cell nuclear antigen and cyclin A2 (CCNA2), and represses these genes during cellular senescence. However, the selectivity of Rb recruitment is still not well understood. Here, we demonstrate that a senescence-associated gene is a direct target of E2F1 and is also repressed by heterochromatin in senescent cells. In contrast, ARF and p27(KIP1), which are also E2F1 targets, are not repressed by Rb and heterochromatin formation. By comparing the promoter sequences of these genes, we found a novel TAAC element that is present in the cellular senescence-inhibited gene, proliferating cell nuclear antigen, and CCNA2 promoters but absent from the ARF and p27(KIP1) promoters. This TAAC element associates with Rb and is required for Rb recruitment. We further determined that TAAC element-mediated Rb association requires the E2F1 binding site, but not E2F1 protein. These results provide a novel molecular mechanism for the different expression patterns of E2F1 targets and afford new mechanistic insight regarding the selectivity of Rb-mediated heterochromatin formation and gene repression during cellular senescence.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) delays cellular senescence by promoting p27(Kip1) degradation in human diploid fibroblasts.

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) plays an important role in the proliferation of tumor cells and the lifespan of Caenorhabditis elegans. However, the role of WWP1 in cellular senescence is still unknown. Here, we show that the expression patterns of p27(Kip1) and WWP1 are inversely correlated during cellular senescence. Moreover, the overexpression of WWP1 delayed senescence, whereas the knockdown of WWP1 led to premature senescence in human fibroblasts. Furthermore, we demonstrate that WWP1 repressed endogenous p27(Kip1) expression through ubiquitin-proteasome-mediated degradation. Additionally, WWP1 had a strong preference for catalyzing the Lys-48-linked polyubiquitination of p27(Kip1) in vitro. Finally, we demonstrate that WWP1 markedly inhibited the replicative senescence induced by p27(Kip1) by promoting p27(Kip1) degradation. Therefore, our study provides a new molecular mechanism for the regulation of cellular senescence.

Transcriptional activation of the senescence regulator Lsh by E2F1.

Lsh, a protein related to the SNF2 family of chromatin-remodeling ATPases, is a major epigenetic regulator that is essential for DNA methylation and histone acetylation at repetitive elements. Lsh represses endogenous p16(INK4a) expression by recruiting HDAC to the p16(INK4a) promoter, which in turn delays cell senescence. However, the molecular mechanisms that govern loss of Lsh expression during cellular senescence have yet to be elucidated. Here we investigate the transcriptional regulation of the human Lsh promoter. We find that the minimal Lsh promoter is located between positions -216 and -119 relative to the transcription start site, and contains two putative E2F binding sites. Ectopic E2F1 increases expression of Lsh at both transcriptional and translational levels. E2F1 physically interacts with the Lsh promoter by binding to each of the two putative binding sites and transactivates the Lsh promoter. E2F1 also induces Lsh protein expression and transactivates the Lsh promoter in 2BS cells. At the same time, E2F1-induced Lsh promoter activity is reduced in senescent cells compared to young cells. These results indicate that E2F1 plays a crucial role in transcriptional control of the human Lsh gene and the decrease of Lsh expression in senescent cells is related to the repression of E2F1.