Delayed Chronic Acidic Postconditioning Improves Poststroke Motor Functional Recovery and Brain Tissue Repair by Activating Proton-Sensing TDAG8.

Acidic postconditioning by transient CO2 inhalation applied within minutes after reperfusion has neuroprotective effects in the acute phase of stroke. However, the effects of delayed chronic acidic postconditioning (DCAPC) initiated during the subacute phase of stroke or other acute brain injuries are unknown. Mice received daily DCAPC by inhaling 5%/10%/20% CO2 for various durations (three cycles of 10- or 20-min CO2 inhalation/10-min break) at days 3-7, 7-21, or 3-21 after photothrombotic stroke. Grid-walk, cylinder, and gait tests were used to assess motor function. DCAPC with all CO2 concentrations significantly promoted motor functional recovery, even when DCAPC was delayed for 3-7 days. DCAPC enhanced the puncta density of GAP-43 (a marker of axon growth and regeneration) and synaptophysin (a marker of synaptogenesis) and reduced the amoeboid microglia number, glial scar thickness and mRNA expression of CD16 and CD32 (markers of proinflammatory M1 microglia) compared with those of the stroke group. Cerebral blood flow (CBF) increased in response to DCAPC. Furthermore, the mRNA expression of TDAG8 (a proton-activated G-protein-coupled receptor) was increased during the subacute phase of stroke, while DCAPC effects were blocked by systemic knockout of TDAG8, except for those on CBF. DCAPC reproduced the benefits by re-expressing TDAG8 in the peri-infarct cortex of TDAG8-/- mice infected with HBAAV2/9-CMV-TDAG8-3flag-ZsGreen. Taken together, we first showed that DCAPC promoted functional recovery and brain tissue repair after stroke with a wide therapeutic time window of at least 7 days after stroke. Brain-derived TDAG8 is a direct target of DCAPC that induces neuroreparative effects.

Whole-genome assembly and annotation for the little yellow croaker (Larimichthys polyactis) provide insights into the evolution of hermaphroditism and gonochorism.

The evolutionary direction of gonochorism and hermaphroditism is an intriguing mystery to be solved. The special transient hermaphroditic stage makes the little yellow croaker (Larimichthys polyactis) an appealing model for studying hermaphrodite formation. However, the origin and evolutionary relationship between of L. polyactis and Larimichthys crocea, the most famous commercial fish species in East Asia, remain unclear. Here, we report the sequence of the L. polyactis genome, which we found is ~706 Mb long (contig N50 = 1.21 Mb and scaffold N50 = 4.52 Mb) and contains 25,233 protein-coding genes. Phylogenomic analysis suggested that L. polyactis diverged from the common ancestor, L. crocea, approximately 25.4 million years ago. Our high-quality genome assembly enabled comparative genomic analysis, which revealed several within-chromosome rearrangements and translocations, without major chromosome fission or fusion events between the two species. The dmrt1 gene was identified as the male-specific gene in L. polyactis. Transcriptome analysis showed that the expression of dmrt1 and its upstream regulatory gene (rnf183) were both sexually dimorphic. Rnf183, unlike its two paralogues rnf223 and rnf225, is only present in Larimichthys and Lates but not in other teleost species, suggesting that it originated from lineage-specific duplication or was lost in other teleosts. Phylogenetic analysis shows that the hermaphrodite stage in male L. polyactis may be explained by the sequence evolution of dmrt1. Decoding the L. polyactis genome not only provides insight into the genetic underpinnings of hermaphrodite evolution, but also provides valuable information for enhancing fish aquaculture.

Delayed metformin treatment improves functional recovery following traumatic brain injury via central AMPK-dependent brain tissue repair.

Accumulating evidence suggests that chronic metformin posttreatment offers potent neuroreparative effects against acute brain injury. However, in previous studies, metformin was not initially administered beyond 24 h postinjury, and the effects of delayed metformin treatment in traumatic brain injury (TBI) and other types of acute brain injury and the related mechanisms are unclear. To test this, male C57BL/6 mice received once daily metformin treatment (20, 50 or 100 mg/kg/d, i.p.) at day 1-14, day 1-2, day 1-10, day 3-10, day 5-12 or day 5-28 after cryogenic TBI (cTBI). The results showed that 100 mg/kg/d metformin administered at day 1-14 postinjury significantly promoted motor functional recovery in the beam walking and gait tests and reduced the infarct volume. Metformin (100 mg/kg/d) administered at day 1-10 or day 3-10 but not day 1-2 or day 5-12 after cTBI significantly improved motor functional outcomes at day 7 and 14, and reduced the infarct volume at day 14. Interestingly, the therapeutic time window was further expanded when the duration of metformin treatment starting at day 5 postinjury was extended to 2 weeks. Furthermore, compared with cTBI, the administration of metformin at day 3-10 or day 5-28 after cTBI significantly elevated the expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and growth associated protein 43 (an axonal regeneration marker) and the number of vascular branch points and decreased the area of glial scar and the number of amoeboid microglia in the peri-infarct area at day 14 or 28 postinjury. The above beneficial effects of metformin were blocked by the intracerebroventricular injection of the AMPK inhibitor compound C (40 µg/mouse/d). Our data provide the first evidence that metformin has a wide therapeutic time window for at least 5 days after cTBI, during which it can improve functional recovery by promoting tissue repair and inhibiting glial scar formation and microglial activation in a central AMPK-dependent manner.

A determining factor for insect feeding preference in the silkworm, Bombyx mori.

Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.

Effects of long-term rapamycin treatment on glial scar formation after cryogenic traumatic brain injury in mice.

Glial scar impedes axon regeneration and functional recovery following traumatic brain injury (TBI). Although it has been shown that rapamycin (a specific inhibitor of mammalian target of rapamycin) can reduce astrocyte reactivation in the early stage of TBI, its effect on glial scar formation has not been characterized in TBI and other acute brain injury models. To test this, ICR mice received daily administration of rapamycin (0.5 or 1.5 mg/kg, i.p.) beginning at 1 h after cryogenic TBI (cTBI). The results showed that at 3 d post-injury, 1.5 mg/kg rapamycin increased cTBI-induced motor functional deficits and infarct size, and attenuated astrocyte reactivation in the ipsilateral cortex, while 0.5 mg/kg rapamycin did not worsen brain damage and only slightly attenuated astrocyte reactivation. Furthermore, at 7 and 14 d after cTBI, 0.5 mg/kg rapamycin group showed a better motor functional performance than cTBI group. At 14 d post-injury, 0.5 mg/kg rapamycin significantly reduced the area and thickness of glial scar and chondroitin sulfate proteoglycan expression, accompanied by decreased expression of p-S6 and enhanced expression of growth associated protein 43 (an axon regeneration marker) in the region of glial scar. Our data suggest that long-term treatment with rapamycin can inhibit glial scar formation after cTBI, which may be involved in the mechanisms of increased axon regeneration and improved neurological functional recovery, and low-dose rapamycin may be more beneficial for such a therapy.

Interleukin-6 as a potential molecular target in esophageal squamous cell carcinoma.

Knowledge of potential tumor markers may improve chemotherapeutic efficacy. Interleukin-6 (IL-6) expression in local tumor tissues is associated with cancer progression and poor prognosis in variety of cancer types. The aim of the present study was to investigate the role and potential application of IL-6 in determining the prognosis of esophageal carcinoma. KYSE170 and TE13 esophageal cancer cell lines were used to conduct cell- and animal-based experiments investigating biological changes and tumor behavior. Immunohistochemical analysis revealed that 70-80% of cancer cells exhibited positive staining for IL-6, compared with <15% of non-malignant epithelial cells. These immunohistochemical results were consistent with the mRNA expression levels detetced. The IL-6 silencing vector significantly reduced invasion and proliferation of the two cell lines and attenuated tumor growth in xenograft mouse models (P<0.05). The IL-6 silencing vector markedly reduced the presence of Ki-67 (a typical proliferation marker) and microvessel density, indicating that downregulation of IL-6 levels may greatly affect tumor growth and inhibition. The IL-6 silencing vector increased E-cadherin and matrix metalloproteinase (MMP)-9 expression levels in the two esophageal carcinoma cell lines. This vector also regulated the release of IL-6 in cell supernatant and serum in KYSE170- and TE13-tumor-bearing mice. The secretion of vascular endothelial growth factor and cluster of differentiation 31 (a nuclear protein) immunoreactive molecules were also reduced by the IL-6 silencing vector. Therefore, IL-6 may be an important trigger in the progression of angiogenesis and endothelial tube formation within the tumor, and targeting IL-6 may be a promising strategy for the treatment of esophageal cancer.

[Mapping of the lethal genes in the sex-linkaged balanced lethal silkworm Bombyx mori using SSR markers].

The males of sex-linkaged balanced lethal silkworm strain (S-14) has two non-allelic recessive genes (lethal gene 1, l1 and lethal gene 2, l2). The two genes are located on two different Z chromosomes and cause death of embryos at body pigmentation stage and end reversal embryo stage, respectively. We firstly hybridized the males of S-14 strain with the females having wild-type genes of P50 strain and then backcrossed the males of F1 with females of P50 strain. A total of 1660 female moths of BC1 generation were divided into two groups, 1100 in BC1-l1 and 560 in BC1-l2 according to the lethal gene carried by these female moths' fathers-fame moths of F1, respectively. Based on the nucleotide sequence information from the published physical map of Bombyx mori, we developed 16 polymorphic SSR markers in l1 gene region and 18 polymorphic SSR makers in l2 gene region compared to the allelic region of P50 strain and used these SSR markers and groups of BC1-l1 and BC1-12 to map the two lethal genes, respectively. Gene l1 was mapped on the region of Z chromosome, covering a physical distance of 2.60 Mb. Gene l2 was fine mapped on the region of Z chromosome, covering a physical distance of 0.69 Mb.

[Agrobacterium tumefaciens-mediated transformation of the pathogen of Botrytis cinerea].

Employing gfp as a reporter gene and hygromycin gene (hph) as a selection marker, the recombinant vector pKPG was constructed and transformed into fresh conidia of Botrytis cinerea via Agrobacterium tumefaciens. Transformants were identified by PCR analysis of gfp and hph cassette, green fluorescence observation with microscope and Southern hybridization. Results confirmed that target genes were successfully integrated into the genome of Botrytis cinerea.

[Cloning and analysis of adenine nucleotide translocase gene in Helicoverpa armigera].

A cDNA clone encoding the ADP/ATP translocase in Helicoverpa armigera has been identified by RT-PCR, 5'and 3'RACE methods. Sequence analysis shows that it is 1,190 bp long and contains a single open reading frame (ORF, 133-1,033 bp) encoding a protein of 300 amino acids (GenBank submission number, AY253868). The protein has a 22 aa signal peptide on its N-terminal, which leads the protein locating onto the inner membrane of the mitochondria. It also has three conserved domains of the mitochondrial carrier protein forming a channel to exchange ATP and ADP energy molecule through the inner membrane of the mitochondria. It shows extensive similarities to the known ADP/ATP translocase poly-peptides. The ADP/ATP translocase similarity was up to 90% in the Lepidoptera.

[Cloning and structural analysis of MLP in the silkworm, Bombyx mori].

The LIM domain is found in a wide variety of eukaryotic proteins that regulate gene expression and cell differentiation during development. Muscle LIM protein (MLP) gene in Bombyx mori has been cloned by blasting its EST database and PCR test in present report. The resulting sequence covers 2 327 bp of cDNA (GenBank accession No. DQ311195). It has a complete open reading fragment and encodes a 494 amino acid protein. Genomic DNA sequence contains 11 exons and 10 introns, with intron splicing following the GT-AG rule. M.W. and PI of the predicted MLP in Bombyx mori are 53.03 kDa and 8.29 respectively. A single LIM domain linked to a glyscine-rich region is found in a previously deposited LIM protein (AAR23823) in Bombyx mori. MLP identified in this report encodes a protein with five tandem LIM-glycine modules. The two LIM proteins could be produced by alternative splicing and both are probably involved in muscle cell differentiation. This work provides foundation for further research on the in vivo function of MLP.

[Cloning and mapping of DpKettin gene on Z chromosome of the pine caterpillar by real-time quantitative PCR].

The ratio of Z chromosome number to autosomal chromosome (A) is different between male and female lepidoptera insects. In males: 2Z : 2A=1, and in females: Z : 2A=0.5. The ratio of the copies of gene (such as K) on Z chromosome number to the copies of gene (such as N) on autosomal chromosome (K/N) is also different between male and female. In males: 2K : 2N =1, and in females: K : 2N=0.5. The DpKettin gene of the pine caterpillar Dendrolimus punctatus was cloned with the primers designed according to the sequences of the BmKettin of the silkworm and the HaKettin of the cotton bollworm. The ratio of copies between DpKettin and ANT (adenine nucleotide translocator) in male and female was determined by the real-time quantitative PCR technique. They were 1.0 in males and 0.5 in females. These ratios were equal to the ratios of the copies of gene on Z chromosome number to the copies of gene on autosomal chromosome. It indicates that DpKettin is located on Z chromosome in the genome of the pine caterpillar.

[The antibacterial effect of cecropin B on pseudomonas aeruginosa infection of wounds in mice].

OBJECTIVE: To investigate the antibacterial effect of a particular antimicrobial peptide Cecropin B(CB) on Pseudomonas aeruginosa infection of wound in mice. METHODS: Thirty ICR mice were enrolled in the study, and the Pseudomonas aeruginosa infection model was reproduced by excision of the full layer of dorsal skin with an area of 1 cm x 1 cm. Then they were randomly divided into C ( control, n = 10, with wet compress of isotonic saline at 3 postinjury hour( PIH) ) , M (with hydropathic compress of 100 g/L mafenide at 3 PIH), A (with wet compress of 1 000 mg/L Cecropin B at 3 PIH) groups. The changes in body temperature and hemogram in each group were determined before and 4 days after injury. Quantitative examination were used to detect the quantity of bacteria in muscular tissue of the wounds, and the survival of the mice were observed on 4 post-injury day( PID). RESULTS: The wounds were moist with more exudation in C group,while that in other groups were dry without obvious exudation. The body temperature of the majority of the mice in each group were elevated, but the number of leucocytes in each group was lowered after operation. The quantity of bacteria in muscle in A group[ (42 +/- 50) CFU/g] was obviously lower than that in M group [(886+/-804) CFU/g, P <0.05] , and it was all obviously lower than that in C group[ (41 +/-28) x 10(5) CFU/g, P <0.01]. The number of surviving mice after 4 PID in C group was evidently smaller than that in A and M groups( P <0. 05). CONCLUSION: The cecropin B possesses obvious anti-bacterial effect on the Pseudomonas Aeruginosa infected wounds of ICR mice, and it can reduce the mortality.

Cloning and alternative splicing analysis of Bombyx mori transformer-2 gene using silkworm EST database.

We have identified Bombyx mori transformer-2 gene (Bmtra-2) cDNA by blasting the EST database of B. mori. It was expressed in the whole life of the male and female silkworm and was observed as a band of 1.3 kb by Northern blot analysis. By comparing corresponding ESTs to the Bmtra-2 DNA sequence, it was revealed that there were eight exons and seven introns, and all splice sites of exons/introns conformed to the GT/AG rule. Bmtra-2 pre-mRNA can produce multiple mRNAs encoding six distinct isoforms of BmTRA-2 protein using an alternative splicing pathway during processing. Six types of Bmtra-2 cDNA clones were identified by reverse transcription-polymerase chain reaction. All isoforms of BmTRA-2 protein contain two arginine/serine-rich domains and one RNA recognition motif, showing striking organizational similarity to Drosophila TRA-2 proteins.

[Cloning and expression of the cecropin B-thanatin hybrid antimicrobial peptide in Escherichia coli].

A 44-residue hybrid peptide (CB (1-24)-Arg-Ser-Tyr-Tan (4-21)) incorporating 1-24 residues of cecropin B (CB) and 4-21 residues of thanatin (Tan) was designed and constructed. The CB-Tan gene was cloned into expression plasmid pGEX-3X and expressed in E. coli BL21. The fusion protein was purified by affinity chromatography. After digested with enterokinase the gene product released with antibacterial activity and gave one band in Tricine-SDS-PAGE.