The safety and short-term effect of mixed approach in laparoscopic right hemicolectomy for right colon cancer compared with middle approach: a retrospective study.

PURPOSE: To investigate whether the mixed approach is a safe and advantageous way to operate laparoscopic right hemicolectomy. METHODS: A retrospective study was performed on 316 patients who underwent laparoscopic right hemicolectomy in our center. They were assigned to the middle approach group (n = 158) and the mixed approach group (n = 158) according to the surgical approaches. The baseline data like gender、age and body mass index as well as the intraoperative and postoperative conditions including operation time, blood loss, postoperative hospital stay and complications were analyzed. RESULTS: There were no significant differences in age, sex, BMI, ASA grade and tumor characteristics between the two groups. Compared with the middle approach group, the mixed approach group was significantly lower in terms of operation time (217.61 min vs 154.31 min, p < 0.001), intraoperative blood loss (73.8 ml vs 37.97 ml, p < 0.001) and postoperative drainage volume. There was no significant difference in the postoperative complications like postoperative anastomotic leakage, postoperative infection and postoperative intestinal obstruction. CONCLUSIONS: Compared with the middle approach, the mixed approach is a safe and advantageous way that can significantly shorten the operation time, reduce intraoperative bleeding and postoperative drainage volume, and does not prolong the length of hospital stay or increase the morbidity postoperative complications.

Modular catalytic activity of nonribosomal peptide synthetases depends on the dynamic interaction between adenylation and condensation domains.

Nonribosomal peptide synthetases (NRPSs) are large multidomain enzymes for the synthesis of a variety of bioactive peptides in a modular and pipelined fashion. Here, we investigated how the condensation (C) domain and the adenylation (A) domain cooperate with each other for the efficient catalytic activity in microcystin NRPS modules. We solved two crystal structures of the microcystin NRPS modules, representing two different conformations in the NRPS catalytic cycle. Our data reveal that the dynamic interaction between the C and the A domains in these modules is mediated by the conserved "RXGR" motif, and this interaction is important for the adenylation activity. Furthermore, the "RXGR" motif-mediated dynamic interaction and its functional regulation are prevalent in different NRPSs modules possessing both the A and the C domains. This study provides new insights into the catalytic mechanism of NRPSs and their engineering strategy for synthetic peptides with different structures and properties.

A tRNA t6A modification system contributes to the sensitivity towards the toxin ß-N-methylamino-L-alanine (BMAA) in the cyanobacterium Anabaena sp. PCC 7120.

Cyanobacteria are oxygen-evolving photosynthetic autotrophs essential for nutrient cycling in the environment. They possess multiple control mechanisms for their cellular activities in order to adapt to the environment. While protein translation is essential for cell survival and adaptation, the regulation and the flexibility of this process are poorly understood in cyanobacteria. ß-N-methylamino-L-alanine (BMAA), an amino acid analog proposed as an environmental neurotoxin, is highly toxic to the filamentous diazotrophic cyanobacterium Anabaena PCC 7120. In this study, through genetic analysis of BMAA-resistant mutants, we demonstrate that the system responsible for modification of ANN-decoding tRNAs with N(6)-threonylcarbamoyl adenosine (t6A) is involved in BMAA sensitivity through the control of translation. Both BMAA and inactivation of the t6A biosynthesis pathway affect translational fidelity and ribosome assembly. However, the two factors display either additive effects on translational elongation, or attenuate each other over translational fidelity or the resistance/sensitivity to antibiotics that inhibit different steps of the translational process. BMAA has a broad effect on translation and transcription, and once BMAA enters the cells, the presence of the t6A biosynthesis pathway increases the sensitivity of the cells towards this toxin. BMAA-resistant mutants screening is an effective method for getting insight into the toxic mechanisms of BMAA. In addition, BMAA is a useful tool for probing translational flexibility of cyanobacteria, and the characterization of the corresponding resistant mutants should help us to reveal translational mechanism allowing cyanobacteria to adapt to changing environments.

[Analysis of differential genes related to dormancy release of Paris polyphylla var. chinensis seeds by transcriptome sequencing].

The study aims at exploring the expression of differential genes and related metabolic pathways in the process of seed dormancy release. The dormant embryo and the dormant released embryo of Paris polyphylla var. chinensis were used as the test materials, a new generation high-throughput sequencing methods to sequence the transcriptome of the samples was used to carry out systematic bioinformatics analysis. We obtained 62 882 650 and 62 263 366 clean reads from the DNA libraries of the samples before and after dormancy breaking. A total of 69 248 differentially expressed genes(DEGs) were obtained, 56 426 up-regulated genes and 12 822 down-regulated genes. There are 138 267 differentially expressed genes in the process of embryo dormancy release, which were annotated by GO function to 58 subclasses of biological processes, molecular functions and cell components. The annotated differentially expressed genes were closely related to metabolic processes, biological regulation, cell component synthesis and enzyme catalytic activity. We found 139 metabolic pathways through pathway analysis of 58 722 differentially expressed genes. Before and after dormancy, DEGs were mainly enriched in carbon metabolism, secondary metabolite biosynthesis and polysaccharide metabolism. Based on the annotation results in KEGG database, we found 16 metabolic pathways related to the dormancy release of P. polyhoylla var. chinensis. A large number of differentially expressed genes were involved in embryo morphogenesis, polysaccharide decomposition and protein synthesis during seed development and dormancy release. It involves the interaction of multiple metabolic pathways and constitutes a complex regulation network for dormancy relief.

[Identification and differential expression of miRNA related to seed dormancy of Paris polyphylla var. chinensis].

The purpose of this study was to explore the expression pattern of miRNA in the process of embryo dormancy and provide a reference for the mechanism of regulating seed dormancy and germination by miRNA. We used high-throughput sequencing technology, bioinformatics analysis and real-time fluorescent quantitative PCR(qPCR) technology to sequence, screen and identify miRNAs of dormant and dormant embryos. The results showed that there were 23 811 977, 24 276 695, 20 611 876 and 20 601 811 unique sequences in the four sample libraries during the period of dormancy and dormancy release. MiRNAs are mainly distributed between 21 and 24 nt, among which the length of 24 nt occurred most frequently. A total of 31 known miRNAs were identified, belonging to 13 different families. 93 new miRNAs were predicted by bioinformatics software. Ten miRNAs(mir156 a-5 p, mir160 a-5 p, mir160 h-1, mir169 a-5 p, mir157 d, mir159 a-1, mir395-3, mir156 f-5 p, mir156-2 and mir171 a-3 p) were screened out. In this study, 10 miRNAs related to seed dormancy release were identified. The target genes mainly involved carbohydrate metabolism, plant hormone signal transduction, cell division and growth. The results of qRT-PCR showed that the sequencing results were consistent with the actual results.

Three Substrains of the Cyanobacterium Anabaena sp. Strain PCC 7120 Display Divergence in Genomic Sequences and hetC Function.

Anabaena sp. strain PCC 7120 is a model strain for molecular studies of cell differentiation and patterning in heterocyst-forming cyanobacteria. Subtle differences in heterocyst development have been noticed in different laboratories working on the same organism. In this study, 360 mutations, including single nucleotide polymorphisms (SNPs), small insertion/deletions (indels; 1 to 3 bp), fragment deletions, and transpositions, were identified in the genomes of three substrains. Heterogeneous/heterozygous bases were also identified due to the polyploidy nature of the genome and the multicellular morphology but could be completely segregated when plated after filament fragmentation by sonication. hetC is a gene upregulated in developing cells during heterocyst formation in Anabaena sp. strain PCC 7120 and found in approximately half of other heterocyst-forming cyanobacteria. Inactivation of hetC in 3 substrains of Anabaena sp. PCC 7120 led to different phenotypes: the formation of heterocysts, differentiating cells that keep dividing, or the presence of both heterocysts and dividing differentiating cells. The expression of P hetZ -gfp in these hetC mutants also showed different patterns of green fluorescent protein (GFP) fluorescence. Thus, the function of hetC is influenced by the genomic background and epistasis and constitutes an example of evolution under way.IMPORTANCE Our knowledge about the molecular genetics of heterocyst formation, an important cell differentiation process for global N2 fixation, is mostly based on studies with Anabaena sp. strain PCC 7120. Here, we show that rapid microevolution is under way in this strain, leading to phenotypic variations for certain genes related to heterocyst development, such as hetC This study provides an example for ongoing microevolution, marked by multiple heterogeneous/heterozygous single nucleotide polymorphisms (SNPs), in a multicellular multicopy-genome microorganism.

Transcriptome analysis reveals self-incompatibility in the tea plant (Camellia sinensis) might be under gametophytic control.

BACKGROUND: Self-incompatibility (SI) is under genetic control and prevents inbreeding depression in angiosperms. SI mechanisms are quite complicated and still poorly understood in many plants. Tea (Camellia sinensis L.) belonging to the family of Theaceae, exhibits high levels of SI and high heterozygosity. Uncovering the molecular basis of SI of the tea plant may enhance breeding and simplify genomics research for the whole family. RESULTS: The growth of pollen tubes following selfing and crossing was observed using fluorescence microscopy. Self-pollen tubes grew slower than cross treatments from 24 h to 72 h after pollination. RNA-seq was employed to explore the molecular mechanisms of SI and to identify SI-related genes in C. sinensis. Self and cross-pollinated styles were collected at 24 h, 48 h and 72 h after pollination. Six RNA-seq libraries (SP24, SP48, SP72, CP24 CP48 and CP72; SP = self-pollinated, CP = cross-pollinated) were constructed and separately sequenced. In total, 299.327 million raw reads were generated. Following assembly, 63,762 unigenes were identified, and 27,264 (42.76 %) unigenes were annotated in five public databases: NR, KOG, KEGG, Swiss-Port and GO. To identify SI-related genes, the fragments per kb per million mapped reads (FPKM) values of each unigene were evaluated. Comparisons of CP24 vs. SP24, CP48 vs. SP48 and CP72 vs. SP72 revealed differential expression of 3,182, 3,575 and 3,709 genes, respectively. Consequently, several ubiquitin-mediated proteolysis, Ca(2+) signaling, apoptosis and defense-associated genes were obtained. The temporal expression pattern of genes following CP and SP was analyzed; 6 peroxidase, 1 polyphenol oxidase and 7 salicylic acid biosynthetic process-related genes were identified. The RNA-seq data were validated by qRT-PCR of 15 unigenes. Finally, a unigene (CL25983Contig1) with strong homology to the S-RNase was analyzed. It was mainly expressed in styles, with dramatically higher expression in self-pollinated versus cross-pollinated tissues at 24 h post-pollination. CONCLUSIONS: The present study reports the transcriptome of styles after cross- and self-pollination in tea and offers novel insights into the molecular mechanism behind SI in C. sinensis. We believe that this RNA-seq dataset will be useful for improvement in C. sinensis as well as other plants in the Theaceae family.

Accumulation of catechins and expression of catechin synthetic genes in Camellia sinensis at different developmental stages.

BACKGROUND: Catechins are the main polyphenol compounds in tea (Camellia sinensis). To understand the relationship between gene expression and product accumulation, the levels of catechins and relative expressions of key genes in tea leaves of different developmental stages were analyzed. RESULTS: The amounts of catechins differed significantly in leaves of different stages, except for gallocatechin gallate. Close correlations between the expression of synthesis genes and the accumulation of catechins were identified. Correlation analysis showed that the expressions of chalcone synthase 1, chalcone synthase 3, anthocyanidin reductase 1, anthocyanidin reductase 2 and leucoanthocyanidin reductase genes were significantly and positively correlated with total catechin contents, suggesting their expression may largely affect total catechin accumulation. Anthocyanidin synthase was significantly correlated with catechin. While both ANRs and LAR were significantly and positively correlated with the contents of (-)-epigallocatechin gallate and (-)-epicatechin gallate. CONCLUSION: Our results suggest synergistic changes between the expression of synthetic genes and the accumulation of catechins. Based on our findings, anthocyanidin synthase may regulate earlier steps in the conversion of catechin, while the anthocyanidin reductase and leucoanthocyanidin reductase genes may both play important roles in the biosynthesis of galloylated catechins.

Unravelling the cross-talk between iron starvation and oxidative stress responses highlights the key role of PerR (alr0957) in peroxide signalling in the cyanobacterium Nostoc PCC 7120.

The cyanobacterial phylum includes oxygenic photosynthetic prokaryotes of a wide variety of morphologies, metabolisms and ecologies. Their adaptation to their various ecological niches is mainly achieved by sophisticated regulatory mechanisms and depends on a fine cross-talk between them. We assessed the global transcriptomic response of the filamentous cyanobacterium Nostoc PCC 7120 to iron starvation and oxidative stress. More than 20% of the differentially expressed genes in response to iron stress were also responsive to oxidative stress. These transcripts include antioxidant proteins-encoding genes that confirms that iron depletion leads to reactive oxygen accumulation. The activity of the Fe-superoxide dismutase was not significantly decreased under iron starvation, indicating that the oxidative stress generated under iron deficiency is not a consequence of (SOD) deficiency. The transcriptional data indicate that the adaptation of Nostoc to iron-depleted conditions displays important differences with what has been shown in unicellular cyanobacteria. While the FurA protein that regulates the response to iron deprivation has been well characterized in Nostoc, the regulators in charge of the oxidative stress response are unknown. Our study indicates that the alr0957 (perR) gene encodes the master regulator of the peroxide stress. PerR is a peroxide-sensor repressor that senses peroxide by metal-catalysed oxidation.

The alr2505 (osiS) gene from Anabaena sp. strain PCC7120 encodes a cysteine desulfurase induced by oxidative stress.

NifS-like cysteine desulfurases are widespread enzymes involved in the mobilization of sulfur from cysteine. The genome of the filamentous diazotrophic cyanobacterium Anabaena PCC 7120 contains four open reading frames potentially encoding NifS-like proteins. One of them, alr2505, belongs to the pkn22 operon, which enables Anabaena to cope with oxidative stress. The Alr2505 protein was purified and found to share all the features characteristic of cysteine desufurases. This is the first NifS-like enzyme to be functionally characterized in this bacterium. On the basis of the transcriptional profiling of all nifS-like genes in Anabaena, it is concluded that alr2505 is the only cysteine desulfurase-encoding gene induced by oxidative stress. The function of Alr2505, which was termed OsiS, is discussed.

PrxQ-A, a member of the peroxiredoxin Q family, plays a major role in defense against oxidative stress in the cyanobacterium Anabaena sp. strain PCC7120.

The genome of the cyanobacterium Anabaena PCC 7120 encodes seven polypeptides showing sequence similarities with peroxiredoxins (Prx-s). One of them, prxQ-A (alr2503), which encodes a Prx Q homologue, is located in the same gene cluster as pkn22, which encodes a Ser/Thr kinase. Here we report that the pkn22-knockout mutant (Mp22) is sensitive to oxidative stress because it fails to synthesize PrxQ-A; the expression of prxQ-A is significantly induced under oxidative stress conditions. The hypersensitivity of the Mp22 mutant to oxidative stress was restored by inducing the expression of the prxQ-A gene in trans. The recombinant PrxQ-A protein shows antioxidant activity protecting the DNA from being degraded by reactive oxygen species, catalyzes the reduction of H2O2 in the presence of DTT, and shows thioredoxin-dependent peroxidase activity in vitro. The conserved Cys47 residue is the peroxide oxidation site, since the replacement of Cys47 by a Ser residue completely abolished the peroxidase activity. All these data suggest that PrxQ-A may efficiently protect this organism from oxidative stress.

A pair of iron-responsive genes encoding protein kinases with a Ser/Thr kinase domain and a His kinase domain are regulated by NtcA in the Cyanobacterium Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 can fix N(2) when combined nitrogen is not available in the growth medium. It has a family of 13 genes encoding proteins with both a Ser/Thr kinase domain and a His kinase domain. The function of these enzymes is unknown. Two of them are encoded by pkn41 (alr0709) and pkn42 (alr0710). These two genes are separated by only 72 bp on the chromosome, and our results indicate that they are cotranscribed. The expression of pkn41 and pkn42 is induced by iron deprivation irrespective of the nature of the nitrogen source. Mutants inactivating either pkn41, pkn42, or both grow similarly to the wild type under normal conditions, but their growth is impaired either in the presence of an iron chelator or under conditions of nitrogen fixation and iron limitation, two situations where the demand for iron is particularly strong. Consistent with these results, these mutants display lower iron content than the wild type and a higher level of expression for nifJ1 and nifJ2, which encode pyruvate:ferredoxin oxidoreductases. Both nifJ1 and nifJ2 are known to be induced by iron limitation. NtcA, a global regulatory factor for different metabolic pathways, binds to the putative promoter region of pkn41, and the induction of pkn41 in response to iron limitation no longer occurs in an ntcA mutant. Our results suggest that ntcA not only regulates the expression of genes involved in nitrogen and carbon metabolism but also coordinates iron acquisition and nitrogen metabolism by activating the expression of pkn41 and pkn42.

A lithium-sensitive and sodium-tolerant 3'-phosphoadenosine-5'-phosphatase encoded by halA from the cyanobacterium Arthrospira platensis is closely related to its counterparts from yeasts and plants.

3'-Phosphoadenosine-5'-phosphatase (PAPase) is required for the removal of toxic 3'-phosphoadenosine-5'-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg2+ for its activity and could use PAP or 3'-phosphoadenosine-5'-phosphosulfate as a substrate. HalA is sensitive to Li+ (50% inhibitory concentration [IC50] = 3.6 mM) but only slightly sensitive to Na+ (IC50 = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li+ and Na+, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li+ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.

Iron starvation leads to oxidative stress in Anabaena sp. strain PCC 7120.

We establish here that iron deficiency causes oxidative stress in the cyanobacterium Anabaena sp. strain PCC 7120. Iron starvation leads to a significant increase in reactive oxygen species, whose effect can be abolished by treatment with the antioxidant tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl). Oxidative stress induced by iron starvation could be a common feature of photosynthetic bacteria.

pkn22 (alr2502) encoding a putative Ser/Thr kinase in the cyanobacterium Anabaena sp. PCC 7120 is induced by both iron starvation and oxidative stress and regulates the expression of isiA.

In cyanobacteria, the isiA gene is required for cell adaptation to oxidative damage caused by the absence of iron. We show here that a putative Ser/Thr kinase gene, pkn22 (alr2052), is activated by iron deficiency and oxidative damage in Anabaena sp. PCC 7120. A pkn22 insertion mutant is unable to grow when iron is limiting. pkn22 regulates the expression of isiA (encoding CP43'), but not of isiB (encoding flavodoxin) and psbC (CP43). Fluorescence measurement at 77 K reveals the absence of the typical signature of CP43' associated with photosystem I in the mutant under iron-limiting conditions. We propose that Pkn22 is required for the function of isiA/CP43' and constitutes a regulatory element necessary for stress response.