Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo

Adult peripheral blood-derived cells are able to differentiate into a variety of cell types, including nerve cells, liver-like cells and epithelial cells. However, their differentiation into retina-like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina-like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial-like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin-, MAP2 (microtubule-associated protein)- and GFAP (glial fibrillary acidic protein)-positive. Flow cytometry results suggested a significant increase in nestin-, MAP2- and CD16-positive cells in group I and nestin-, GFAP-, MAP2-, vimentin- and rhodopsin-positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate into the retina in vivo.     

Identification of fatty acid synthase from the Pacific white shrimp, Litopenaeus vannamei and its specific expression profiles during white spot syndrome virus infection

Fatty acid synthase (FAS) in animal tissues consists of two identical monomers and is known to be a complex multi-functional enzyme that plays an important role in energy homeostasis. However, there are few reports of studies focused on the relationship between FAS and virus infection in invertebrates. In the present study, we cloned the FAS gene from an economically important invertebrate, the Pacific white shrimp Litopenaeus vannamei. The full-length FAS cDNA is 8268 bp, including a 5'-terminal untranslated region of 137 bp, a 3'-terminal untranslated region of 601 bp and an open reading frame of 7530 bp. FAS cDNA encodes a polypeptide of 2509 amino acid residues that contains a typical β-ketoacyl synthase (KS) domain at the N-terminus, next to a malonyl/acetyltransferase (MAT) domain, a dehydrase domain, an enoyl reductase domain, a ketoacyl reductase domain, a phosphopantetheine attachment site domain and a thioesterase domain at the C-terminus. Quantitative real-time RT-PCR revealed the up-regulated expression of FAS in L. vannamei hepatopancreas and muscle after white spot syndrome virus (WSSV) infection. The expression of FAS in muscle was 13.03-fold greater than that in the control (p<0.05) and 2.93-fold greater in hepatopancreas (p>0.05). Meanwhile, expression of the fatty acid-binding protein (FABP), another important factor in lipid metabolism, was increased in muscle to 19.20-fold greater than that in the control (p<0.05) but only 0.76-fold in hepatopancreas (p>0.05). These results implied that WSSV infected body surface tissues, but there was very little infection of internal organs. We suggest that the increase of FAS expression is induced in WSSV-infected shrimps, and the virus changes the lipid metabolism of the host, which directly affects virus assembly or defense against virus infection.     

Heat shock protein 90-mediated inactivation of nuclear factor-κB switches autophagy to apoptosis through becn1 transcriptional inhibition in selenite-induced NB4 cells

Autophagy can protect cells while also contributing to cell damage, but the precise interplay between apoptosis and autophagy and the contribution of autophagy to cell death are still not clear. Previous studies have shown that supranutritional doses of sodium selenite promote apoptosis in human leukemia NB4 cells. Here, we report that selenite treatment triggers opposite patterns of autophagy in the NB4, HL60, and Jurkat leukemia cell lines during apoptosis and provide evidence that the suppressive effect of selenite on autophagy in NB4 cells is due to the decreased expression of the chaperone protein Hsp90 (heat shock protein 90), suggesting a novel regulatory function of Hsp90 in apoptosis and autophagy. Excessive or insufficient expression indicates that Hsp90 protects NB4 cells from selenite-induced apoptosis, and selenite-induced decreases in the expression of Hsp90, especially in NB4 cells, inhibit the activities of the IκB kinase/nuclear factor-κB (IKK/NF-κB) signaling pathway, leading to less nuclear translocation and inactivation of NF-κB and the subsequent weak binding of the becn1 promoter, which facilitates the transition from autophagy to apoptosis. Taken together, our observations provide novel insights into the mechanisms underlying the balance between apoptosis and autophagy, and we also identified Hsp90-NF-κB-Beclin1 as a potential biological pathway for signaling the switch from autophagy to apoptosis in selenite-treated NB4 cells.     

Three sorting nexins drive the degradation of apoptotic cells in response to PtdIns(3)P signaling

Apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Phagosome maturation requires phosphatidylinositol 3-phosphate [PtdIns(3)P], yet how PtdIns(3)P triggers phagosome maturation remains largely unknown. Through a genomewide PtdIns(3)P effector screen in the nematode Caenorhabditis elegans , we identified LST-4/SNX9, SNX-1, and SNX-6, three BAR domain-containing sorting nexins, that act in two parallel pathways to drive PtdIns(3)P-mediated degradation of apoptotic cells. We found that these proteins were enriched on phagosomal surfaces through association with PtdIns(3)P and through specific protein-protein interaction, and they promoted the fusion of early endosomes and lysosomes to phagosomes, events essential for phagosome maturation. Specifically, LST-4 interacts with DYN-1 (dynamin), an essential phagosome maturation initiator, to strengthen DYN-1's association to phagosomal surfaces, and facilitates the maintenance of the RAB-7 GTPase on phagosomal surfaces. Furthermore, both LST-4 and SNX-1 promote the extension of phagosomal tubules to facilitate the docking and fusion of intracellular vesicles. Our findings identify the critical and differential functions of two groups of sorting nexins in phagosome maturation and reveal a signaling cascade initiated by phagocytic receptor CED-1, mediated by PtdIns(3)P, and executed through these sorting nexins to degrade apoptotic cells.     

Constitutive expression of Vitreoscilla haemoglobin in Sphingomonas elodea to improve gellan gum production

Aims: To improve a commercially used strain for gellan production by exogenous Vitreoscilla haemoglobin (VHb). Methods and Results: VHb gene was expressed in Sphingomonas elodea under the control of constitutive bla promoter. Biochemical activity of expressed VHb was confirmed by CO‐difference spectra analysis that exhibited a characteristic absorption maximum at 419 nm. During cultivation, not only enhanced cell growth was detected, but also 20% improvement in gellan production was observed after 48 h of incubation, with a maximum yield of 16·82 g l^?1. Moreover, maximum sucrose conversion efficiency (g gellan per g sucrose) was 57·8, 20% higher than that of the parental strain. We further examined the polysaccharide production of VHb‐expressing strain at different aeration levels in Erlenmeyer flasks. Again, in all cases, a significant enhancement of gellan production was observed, and the enhancement was more significant under oxygen‐limiting conditions (up to 26·8%). Conclusions: VHb exhibited positive effect on cell growth and gellan yield of S. elodea, especially under hypoxic conditions. Significance and Impact of the Study: This is the first application of VHb as an effective metabolic engineering strategy in S. elodea to regulate cell growth and optimize gellan yield.     

Knockdown of ribosomal protein S7 causes developmental abnormalities via p53 dependent and independent pathways in zebrafish

Ribosomal proteins (RPs), structural components of the ribosome involved in protein synthesis, are of significant importance in all organisms. Previous studies have suggested that some RPs may have other functions in addition to assembly of the ribosome. The small ribosomal subunits RPS7, has been reported to modulate the mdm2-p53 interaction. To further investigate the biological functions of RPS7, we used morpholino antisense oligonucleotides (MO) to specifically knockdown RPS7 in zebrafish. In RPS7-deficient embryos, p53 was activated, and its downstream target genes and biological events were induced, including apoptosis and cell cycle arrest. Hematopoiesis was also impaired seriously in RPS7-deficient embryos, which was confirmed by the hemoglobin O-dianisidine staining of blood cells, and the expression of scl, gata1 and α-E1 globin were abnormal. The matrix metalloproteinase (mmp) family genes were also activated in RPS7 morphants, indicating that improper cell migration might also cause development defects. Furthermore, simultaneously knockdown of the p53 protein by co-injecting a p53 MO could partially reverse the abnormal phenotype in the morphants. These results strengthen the hypothesis that specific ribosomal proteins regulate p53 and that their deficiency affects hematopoiesis. Moreover, our data implicate that RPS7 is a regulator of matrix metalloproteinase (mmp) family in zebrafish system. These specific functions of RPS7 may provide helpful clues to study the roles of RPs in human disease.     

ClC-3 chloride channel prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through mitochondria dependent pathway

ClC-3 Cl⁻ channel plays an important role in cell volume regulation and cell cycle. In vascular smooth muscle cells, we have found that ClC-3 was involved in ET-1 induced cell proliferation. The present study was designed to further investigate the role of ClC-3 Cl⁻ channel in H₂O₂-induced apoptosis and its underlying mechanisms in rat basilar arterial smooth muscle cell (BASMCs). By using ClC-3 cDNA and small interference RNA (siRNA) transfection strategy, it was found that overexpression of ClC-3 significantly decreased the apoptotic rate of H₂O₂-treated BASMCs and increased the cell viability, whereas silencing of ClC-3 with siRNA produced opposite effects and increased the apoptotic rate. ClC-3 overexpression decreased cytochrome C release and caspase-3 activation, and increased both the stability of mitochondrial membrane potential and the ratio of Bcl-2/Bax, whereas silencing of ClC-3 produced opposite effect. Furthermore, we demonstrated that overexpression of ClC-3 attenuated, whereas silencing of ClC-3 facilitated, the degradation of LaminA, one of the structural matrix proteins, in BASMCs. Our data suggest that ClC-3 Cl⁻ channel can modulate H₂O₂-induced apoptosis in BASMCs via the intrinsic, mitochondrial pathway.     

Prediction of novel pre-microRNAs with high accuracy through boosting and SVM

High-throughput deep-sequencing technology has generated an unprecedented number of expressed short sequence reads, presenting not only an opportunity but also a challenge for prediction of novel microRNAs. To verify the existence of candidate microRNAs, we have to show that these short sequences can be processed from candidate pre-microRNAs. However, it is laborious and time consuming to verify these using existing experimental techniques. Therefore, here, we describe a new method, miRD, which is constructed using two feature selection strategies based on support vector machines (SVMs) and boosting method. It is a high-efficiency tool for novel pre-microRNA prediction with accuracy up to 94.0% among different species. AVAILABILITY: miRD is implemented in PHP/PERL+MySQL+R and can be freely accessed at http://mcg.ustc.edu.cn/rpg/mird/mird.php.     

The coupling of epigenome replication with DNA replication

In multicellular organisms, each cell contains the same DNA sequence, but with different epigenetic information that determines the cell specificity. Semi-conservative DNA replication faithfully copies the parental nucleotide sequence into two DNA daughter strands during each cell cycle. At the same time, epigenetic marks such as DNA methylation and histone modifications are either precisely transmitted to the daughter cells or dynamically changed during S-phase. Recent studies indicate that in each cell cycle, many DNA replication related proteins are involved in not only genomic but also epigenomic replication. Histone modification proteins, chromatin remodeling proteins, histone variants, and RNAs participate in the epigenomic replication during S-phase. As a consequence, epigenome replication is closely linked with DNA replication during S-phase.