Transplantation of human induced pluripotent stem cell derived keratinocytes accelerates deep second-degree burn wound healing.

BACKGROUND: Current evidence shows that human induced pluripotent stem cells (hiPSCs) can effectively differentiate into keratinocytes (KCs), but its effect on skin burn healing has not been reported. AIM: To observe the effects of hiPSCs-derived KCs transplantation on skin burn healing in mice and to preliminarily reveal the underlying mechanisms. METHODS: An analysis of differentially expressed genes in burn wounds based on GEO datasets GSE140926, and GSE27186 was established. A differentiation medium containing retinoic acid and bone morphogenetic protein 4 was applied to induce hiPSCs to differentiate into KCs. The expression of KCs marker proteins was detected using immunofluorescence staining. A model of a C57BL/6 mouse with deep cutaneous second-degree burn was created, and then phosphate buffered saline (PBS), hiPSCs-KCs, or hiPSCs-KCs with knockdown of COL7A1 were injected around the wound surface. The wound healing, re-epithelialization, engraftment of hiPSCs-KCs into wounds, proinflammatory factor level, and the NF-κB pathway proteins were assessed by hematoxylin-eosin staining, carboxifluorescein diacetate succinimidyl ester (CFSE) fluorescence staining, enzyme linked immunosorbent assay, and Western blotting on days 3, 7, and 14 after the injection, respectively. Moreover, the effects of COL7A1 knockdown on the proliferation and migration of hiPSCs-KCs were confirmed by immunohistochemistry, EdU, Transwell, and damage repair assays. RESULTS: HiPSCs-KCs could express the hallmark proteins of KCs. COL7A1 was down-regulated in burn wound tissues and highly expressed in hiPSCs-KCs. Transplantation of hiPSCs-KCs into mice with burn wounds resulted in a significant decrease in wound area, an increase in wound re-epithelialization, a decrease in proinflammatory factors content, and an inhibition of NF-κB pathway activation compared to the PBS group. The in vitro assay showed that COL7A1 knockdown could rescue the inhibition of hiPSCs-KCs proliferation and migration, providing further evidence that COL7A1 speeds up burn wound healing by limiting cell proliferation and migration. CONCLUSION: In deep, second-degree burn wounds, COL7A1 can promote KC proliferation and migration while also suppressing the inflammatory response.

Integrated analysis of the genomic and transcriptional profile of gliomas with isocitrate dehydrogenase-1 and tumor protein 53 mutations.

Background: The gene mutation of isocitrate dehydrogenase-1 (IDH1) is commonly found in LGG and some GBM patients and usually carries tumor protein 53 (TP53) mutations. However, the underlying mechanisms on both mutations of glioma patients in IDH1 and TP53 are still unclear. Aim: To find the potential target markers in GBM and LGG patients with IDH1 and TP53 mutation.Method: A total of 1122 glioma patients from The Cancer Genome Atlas were enrolled and divided as wild-type (without IDH1 and TP53 mutations) or both mutant (both IDH1 and TP53 mutations). The data of clinicopathological characteristics, mRNA, mutations, and copy number alteration were analyzed. Results: IDH1 and TP53 mutations, not gene expression, affect the survival probability of GBM and LGG patients, which might be related to neuron function, immune function, tumor invasion, and metastasis. The effects of the selected gene (EMILIN3, SAA1, VSTM2A, HAMP, IFT80, and CHIC2) on glioma patients could be regulated by IDH1 and TP53 mutations and had a higher survival possibility in these patients. Conclusions: The selected genes in GBM and LGG patients with IDH1 and TP53 mutations could be a potential prognosis marker in the future.

The role of anthraquinone-2-sulfonate on biohydrogen production by Klebsiella strain and mixed culture.

The role of anthraquinone-2-sulfonate (AQS) on biohydrogen production was explored in this study. The hydrogen molar yield (HMY) of the batch experiment with 0.2 mM AQS was not significantly improved comparing to that without AQS, but the acetate and ethanol yields were increased by 12.1% and 9.82%, respectively. According to the metabolites flux analysis, e- balance calculations and stoichiometry results, the biogenic anthrahydroquinone-2,6,-disulfonate (AH2QS) preferred to improve the H2 generation that catalyzed by hydrogenase, but not likely affect the H2 generation that through formate cleavage pathway (FCP). However, comparing to the mix culture without AQS, the hydrogen production rate and hydrogen yield were increased by 20.97% of 1.96%, respectively, in the presence of AQS. The results of this study provided better understanding of the role of AQS on biohydrogen production by the strain that follows FCP, and the synergistic biohydrogen production by the co-culture that follows FCP and butyrate/acetate pathway.

An Autonomous Log Storage Management Protocol with Blockchain Mechanism and Access Control for the Internet of Things.

As the Internet of Things (IoT) has become prevalent, a massive number of logs produced by IoT devices are transmitted and processed every day. The logs should contain important contents and private information. Moreover, these logs may be used as evidences for forensic investigations when cyber security incidents occur. However, evidence legality and internal security issues in existing works were not properly addressed. This paper proposes an autonomous log storage management protocol with blockchain mechanism and access control for the IoT. Autonomous model allows sensors to encrypt their logs before sending it to gateway and server, so that the logs are not revealed to the public during communication process. Along with blockchain, we introduce the concept "signature chain". The integration of blockchain and signature chain provides efficient management functions with valuable security properties for the logs, including robust identity verification, data integrity, non-repudiation, data tamper resistance, and the legality. Our work also employs attribute-based encryption to achieve fine-grained access control and data confidentiality. The results of security analysis using AVSIPA toolset, GNY logic and semantic proof indicate that the proposed protocol meets various security requirements. Providing good performance with elliptic curve small key size, short BLS signature, efficient signcryption method, and single sign-on solution, our work is suitable for the IoT.

From discovery to spread: The evolution and phylogeny of Getah virus.

Getah virus (GETV) was first isolated in Malaysia in 1955. Since then, epidemics in horses and pigs caused by GETV have resulted in huge economic losses. At present, GETV has spread across Eurasia and Southeast Asia, including mainland China, Korea, Japan, Mongolia, and Russia. Data show that the Most Recent Common Ancestor (MRCA) of GETV existed about 145years ago (95% HPD: 75-244) and gradually evolved into four distinct evolutionary populations: Groups I-IV. The MRCA of GETVs in Group III, which includes all GETVs isolated from mosquitoes, pigs, horses, and other animals since the 1960s (from latitude 19°N to 60°N), existed about 51years ago (95% HPD: 51-72). Group III is responsible for most viral epidemics among domestic animals. An analysis of the GETV E2 protein sequence and structure revealed seven common amino acid mutation sites. These sites are responsible for the structural and electrostatic differences detected between widespread Group III isolates and the prototype strain MM2021. These differences may account for the recent geographical radiation of the virus. Considering the economic significance of GETV infection in pigs and horses, we recommend the implementation of strict viral screening and monitoring programs.

The Papaya Transcription Factor CpNAC1 Modulates Carotenoid Biosynthesis through Activating Phytoene Desaturase Genes CpPDS2/4 during Fruit Ripening.

Papaya fruits accumulate carotenoids during fruit ripening. Although many papaya carotenoid biosynthesis pathway genes have been identified, the transcriptional regulators of these genes have not been characterized. In this study, a NAC transcription factor, designated as CpNAC1, was characterized from papaya fruit. CpNAC1 was localized exclusively in nucleus and possessed transcriptional activation activity. Expression of carotenoid biosynthesis genes phytoene desaturases (CpPDSs) and CpNAC1 was increased during fruit ripening and by propylene treatment, which correlates well with the elevated carotenoid content in papaya. The gel mobility shift assays and transient expression analyses demonstrated that CpNAC1 directly binds to the NAC binding site (NACBS) motifs in CpPDS2/4 promoters and activates them. Collectively, these data suggest that CpNAC1 may act as a positive regulator of carotenoid biosynthesis during papaya fruit ripening possibly via transcriptional activation of CpPDSs such as CpPDS2/4.

Molecular characterization of full-length genome of Japanese encephalitis virus genotype V isolated from Tibet, China.

OBJECTIVE: To determine the molecular characterization of full-length genome of Japanese encephalitis virus (JEV) genotype V. METHODS: The full-length nucleotide sequences of JEV strains isolated from different locations and sources were used in sequence and phylogenetic analysis. RESULTS: The full-length genome of genotypes V JEV, XZ0934, and Muar strain were composed of 10 983 and 10 988 nucleotides respectively and shared a lower level of identity with JEV genotypes I-IV, ranging from 78.4% (G I, KV1899) to 79.7% (G III, JaGAr01), for the nucleotide sequences, and from 90.0% (G I, KV1899) to 91.8% (G III, JaGAr01) for the amino acid sequences. The open reading frame (ORF) of JEV genotype V spanned nucleotides 96 to 10 397 and encoded 3 433 amino acids. Interestingly, a comparison with JEV genotype I-IV revealed that 3 nucleotides (encoded with a serine residue) were inserted in the NS4A gene of JEV genotype V, and the insertion of nucleotides was also found in downstream of the ORF stop codon in 3'-untranslated region. Moreover, numerous amino acid mutations were observed in 3 functional domains of the E gene of JEV genotype V. CONCLUSION: The molecular characterization of JEV genotype V is significantly different from that of the known genotypes I-IV. The mutations located in the coding region and the non-coding region may be molecular markers of JEV genotype V and warrant further studies to determine their effects on biology and immunogenicity of genotype V strains.

Induction of jasmonate signalling regulators MaMYC2s and their physical interactions with MaICE1 in methyl jasmonate-induced chilling tolerance in banana fruit.

MYC2, a basic helix-loop-helix (bHLH) transcription factor, is a key regulator in the activation of jasmonate (JA) response. However, the molecular details of MYC2 involving in methyl jasmonate (MeJA)-induced chilling tolerance of fruit remain largely unclear. In the present work, two MYC2 genes, MaMYC2a and MaMYC2b, and one homolog of the inducer of the C-repeat-binding factor (CBF) gene, MaICE1 were isolated and characterized from banana fruit. MaMYC2s and MaICE1 were found to be all localized in the nucleus. In addition, the proline-rich domain (PRD) and the acidic domain (AD) in the N-terminus were important for the transcriptional activation of MaMYC2 in yeast cells. Unlike MaICE1's constitutive expression, MaMYC2a and MaMYC2b were induced rapidly following MeJA treatment during cold storage. Moreover, protein-protein interaction analysis confirmed that MaMYC2s interacted with MaICE1. The expression of ICE-CBF cold-responsive pathway genes including MaCBF1, MaCBF2, MaCOR1, MaKIN2, MaRD2 and MaRD5 was also significantly induced by MeJA. Taken together, our work provides strong evidence that MaMYC2 is involved in MeJA-induced chilling tolerance in banana fruit through physically interacting and likely functionally coordinating with MaICE1, revealing a novel mechanism for ICE1 in response to cold stress as well as during development of induced chilling tolerance.

[Study on the Fast Testing Strategy for identifying the wild poliovirus I].

OBJECTIVE: To explore the Fast Testing Sstrategy (FTS) for wild poliovirus I (WP1). METHODS: Epidemiological investigations were carried out on 671 students from WP1 epidemic areas in China. A set of real time RT-PCR assays, including panenterovirus testings (PE) assay, poliovirus serotypings (PS) assay and the assay distinguishing wild strain from vaccine strain of poliovirus I (DWV) were introduced into the screening program for WPV1 to replace the conventional RT-PCR, recommended by the China National Polio Laboratory (GNPL). Additionally, sensitivities of all the assays were assessed by poliovirus type I to III (Sabin stain) and the isolated WPV1. RESULTS: (1) 33 non-poliovirus enterovirus (NPEV) cases were detected, with 16 polio vaccine-related cases including 5 polio I, 1 polio II, 3 polio III, 1 polio I + II, 4 polio I + III and 2 polio I + II + III. Three WPV1 cases were also detected in this study and confirmed by CNPL. (2) For polio virus vaccine strain, sensitivities of the set of real time RT-PCR assays ranged from 1 to 100 times than that of the in-house RT-PCR assay. The sensitivities of PE and PS assays for the detection of polio II were 100 times than that of the RT-PCR assay and the sensitivity of DWV assay used for the detection of polio I were 10 times than that of the RT-PCR assay. For WPV1, the sensitivity of three real time RT-PCR was 10 times hight than that of the RT-PCR assay. CONCLUSION: The novel FTS for WPV1 suggested by this study would include PE, PS and DWV. It not only could greatly shorten the testing time but also more sensitive than the RT-PCR and suited for emergency detection for WPV1.

[Genotype I Japanese encephalitis virus is the main genotype in mosquito in Fujian province].

OBJECTIVE: To grasp the infection rate and genotypes of Japanese encephalitis virus (JEV) in mosquito in Fujian province. METHODS: Mosquito specimens in Sanming city, Jianyang city and Fuzhou city in Fujian province were collected in 2010. RT-PCR was used to detect the JEV sequence from the mosquitoes by specific primers. The sequence splicing and the differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree were performed by the software of ATGC, Clustal X (1.83), MegAlign, GeneDoc 3.2 and Mega (4.0). RESULTS: Totally 6987 mosquitoes were collected and main species was Culex tritaeniorhynchus and Anopheles sinensis. The infection rate of JEV in mosquitoes in Sanming, Jianyang and Fuzhou were 1.25%, 1.76% and 0.65%, respectively. One full genome in the positive specimens was sequenced. And further study showed that the positive JEV sequences belonged to genotype I. CONCLUSION: Genotype I Japanese encephalitis virus is the main genotype in mosquitos in Fujian province.

Molecular characterization of banana NAC transcription factors and their interactions with ethylene signalling component EIL during fruit ripening.

The plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factors (TFs) play important roles in plant growth, development, and stress responses. However, the precise role of NAC TFs in relation to fruit ripening is poorly understood. In this study, six NAC genes, designated MaNAC1-MaNAC6, were isolated and characterized from banana fruit. Subcellular localization showed that MaNAC1-MaNAC5 proteins localized preferentially to the nucleus, while MaNAC6 was distributed throughout the entire cell. A transactivation assay in yeast demonstrated that MaNAC4 and MaNAC6, as well as their C-terminal regions, possessed trans-activation activity. Gene expression profiles in fruit with four different ripening characteristics, including natural, ethylene-induced, 1-methylcyclopropene (1-MCP)-delayed, and a combination of 1-MCP with ethylene treatment, revealed that the MaNAC genes were differentially expressed in peel and pulp during post-harvest ripening. MaNAC1 and MaNAC2 were apparently upregulated by ethylene in peel and pulp, consistent with the increase in ethylene production. In contrast, MaNAC3 in peel and pulp and MaNAC5 in peel were constitutively expressed, and transcripts of MaNAC4 in peel and pulp and MaNAC6 in peel decreased, while MaNAC5 or MaNAC6 in pulp increased slightly during fruit ripening. Furthermore, the MaNAC2 promoter was activated after ethylene application, further enhancing the involvement of MaNAC2 in fruit ripening. More importantly, yeast two-hybrid and bimolecular fluorescence complementation analyses confirmed that MaNAC1/2 physically interacted with a downstream component of ethylene signalling, ethylene insensitive 3 (EIN3)-like protein, termed MaEIL5, which was downregulated during ripening. Taken together, these results suggest that MaNACs such as MaNAC1/MaNAC2, may be involved in banana fruit ripening via interaction with ethylene signalling components.

[Rapid identification of Beijing measles vaccine virus and wild virus by multiplex real-time fluorescent PCR].

OBJECTIVE: To identify wild measles virus and vaccine virus by detection nucleic acid of clinical samples from measles patients with immunization history circulating in Beijing through multiplex real-time fluorescent PCR technology. METHODS: From July 2011 to February 2012, 10 throat swabs and 15 urine specimens were collected from 16 suspected measles patients who were 8 - 9 months old infants with immunization history in Beijing. The specificity of multiplex real-time fluorescent PCR was firstly tested by measles vaccine virus, wild virus and other respiratory virus. Then the vaccine virus and wild virus were titrated and diluted to test the sensitivity of the PCR method. The result was then compared with it analyzed by PCR-RFLP method. Meanwhile, the clinical sample of the measles patients were tested and confirmed by sequencing method. RESULTS: The primer-probe sets of Fam, Joe and Cy5 showed great specificity of measles virus, and could distinguish the measles vaccine virus and wild virus well. The sensitivity of this method to detect measles vaccine virus reached 0.1 CCID(50)/0.1 ml; and the sensitivity to wild virus reached 0.006 CCID(50)/0.1 ml; which were both higher than the sensitivity of PCR-RFLP method. Out of the 16 measles patients with vaccination history, 3 were negative and the other 13 all belonged to measles virus genotype A, and were confirmed to be vaccine virus by sequencing method. CONCLUSION: Multiplex real-time PCR method is accurate, rapid and sensitive to identify measles vaccine virus and wild virus. The method could greatly help us to find measles patients and to identify the vaccine-related cases.

Genotype v Japanese encephalitis virus is emerging.

Japanese encephalitis (JE) is a global public health issue that has spread widely to more than 20 countries in Asia and has extended its geographic range to the south Pacific region including Australia. JE has become the most important cause of viral encephalitis in the world. Japanese encephalitis viruses (JEV) are divided into five genotypes, based on the nucleotide sequence of the envelope (E) gene. The Muar strain, isolated from patient in Malaya in 1952, is the sole example of genotype V JEV. Here, the XZ0934 strain of JEV was isolated from Culex tritaeniorhynchus, collected in China. The complete nucleotide and amino acid sequence of XZ0934 strain have been determined. The nucleotide divergence ranged from 20.3% to 21.4% and amino acid divergence ranged from 8.4% to 10.0% when compared with the 62 known JEV isolates that belong to genotype I-IV. It reveals low similarity between XZ0934 and genotype I-IV JEVs. Phylogenetic analysis using both complete genome and structural gene nucleotide sequences demonstrates that XZ0934 belongs to genotype V. This, in turn, suggests that genotype V JEV is emerging in JEV endemic areas. Thus, increased surveillance and diagnosis of viral encephalitis caused by genotype V JEV is an issue of great concern to nations in which JEV is endemic.

Expression of genes associated with ethylene-signalling pathway in harvested banana fruit in response to temperature and 1-MCP treatment.

BACKGROUND: Little attention has been paid to characterising the ethylene-signalling pathway genes in relation to abnormal ripening of harvested banana fruit during storage at high temperature. The aim of the present study was to investigate banana fruit abnormal ripening and the expression of ten genes associated with the ethylene-signalling pathway, namely MaACS1, MaACO1, MaERS1-4 and MaEIL1-4, at high temperature. Changes in these parameters of banana fruit at high temperature in response to 1-MCP pretreatment were also investigated. RESULTS: High temperature accelerated the decline in fruit firmness, increased ethylene production and inhibited degreening in banana fruit, resulting in fruit abnormal ripening. In addition, the expression of MaACS1, MaACO1, MaERS2, MaERS3, MaERS4, MaEIL1, MaEIL3 and MaEIL4 was enhanced in banana fruit stored at high temperature. However, application of 1-MCP prior to high temperature storage delayed fruit abnormal ripening and simultaneously suppressed the expression of MaACS1, MaERS2, MaERS3, MaEIL1, MaEIL3 and MaEIL4. CONCLUSION: The findings of this study suggested that the expression of genes associated with the ethylene-signalling pathway might be involved in banana fruit abnormal ripening at high temperature. Application of 1-MCP suppressed the expression of genes associated with the ethylene-signalling pathway, which may be attributed at least partially to 1-MCP delaying fruit abnormal ripening at high temperature.

Changes in alpha-L-arabinofuranosidase activity in peel and pulp of banana (Musa sp.) fruits during ripening and softening.

Arabinose is one of the most dynamic cell wall glycosyl residues released during fruit ripening, alpha-L-arabinofuranosidase (alpha-Arab) are major glycosidases that may remove arabinose units from fruit cell wall polysaccharides. To find out whether alpha-Arab plays important roles in banana fruit softening, the enzyme activities in peel and pulp, fruit firmness, respiration rate and ethylene release rate were assayed during banana softening. The results showed that alpha-Arab activities in banana pulp and peel increased slightly at the beginning of storage and reached their maxima when the fruit firmness decreased drastically, alpha-Arab activity increased by more than ten folds in both pulp and peel during ripening and alpha-Arab activities were higher in pulp than in peel. Treatment of banana fruits with ethylene absorbent postponed the time of reaching of its maxima of respiration and ethylene, enhanced the firmness of pup and decreased alpha-Arab activity in the peel and pulp. These results suggest that alpha-Arab induced the decrease of fruit firmness and played an important role in banana fruit softening, and its activity was regulated by ethylene.

Cloning and expression analysis of beta-galactosidase gene related to softening of banana (Musa sp.) fruit.

Beta-galactosidase is thought to be involved in fruit softening through cleaving beta(1-->4) galactan bonds in cell wall hemicellulose. To study the relationship between fruit softening and beta-Gal during banana (Musa sp.) fruit ripening, a beta-Gal cDNA fragment, named MA-Gal, has been cloned from banana fruit pulp using RT-PCR in this study. The results of sequence analysis showed that MA-Gal contained 927 bp, encoding a polypeptide of 309 amino acids, the deduced protein was highly homologous to plant beta-galactosidase expressed in fruit ripening. The MA-Gal putative amino acids have five homologous domains (typical eukaryotic beta-Gals have seven domains), contain the GGPIILSQIENEY(F) motif, which is a presumptive catalytic active site conserved in all beta-Gals. Phylogenetic analysis showed that MA-Gal was in plant beta-Gal group I, which was expressed in fruit tissue. The changes in beta-Gal activity and firmness in pulp during three different stages were also assayed. The Northern analysis showed that the MA-Gal transcripts in pulp were detected at low level at preclimacteric stage, while an increasing progression was observed during fruit ripening, and the highest transcript amount was found at the postclimacteric stage. These results suggest that MA-Gal may play a role during banana fruit ripening and softening.