Exogenous Hsp70 attenuates nitroglycerin-induced migraine-like symptoms in mice.

Although the connection between heat shock protein 70 (HSP70) and vestibular migraine is not clear, HSP70 is neuroprotective in other scenarios. This study aimed to investigate the potential of exogenous HSP70 for treating migraine-like symptoms in a mouse model of nitroglycerin (NTG)-induced migraine. HSP70 levels were assessed in patients with vestibular migraine and healthy individuals by ELISA. Migraine was induced in mice by NTG, and HSP70 expression was examined in the trigeminal nucleus caudalis (TNC) tissue of mice treated with NTG and NTG together with exogenous HSP70. The effects of exogenous HSP70 on migraine-like symptoms were assessed through behavioral assays. Finally, the impact of HSP70 on oxidative stress and NF-κB signaling in mice with migraine was investigated. Serum HSP70 in patients with vestibular migraine was significantly lower than that of healthy individuals. NTG administration significantly suppressed HSP70 expression in mouse TNC tissue, which was reversed by exogenous HSP70. HSP70 alleviated NTG-induced mechanical hypersensitivity, light aversion, and anxiety-like behavior. Finally, exogenous HSP70 suppressed NTG-induced oxidative stress and NF-κB signaling. Our study suggests that exogenous HSP70 may be a potential therapy for alleviating migraine symptoms and our promising finding warrants further investigation of HSP70 for clinical application.NEW & NOTEWORTHY The study suggests that exogenous HSP70 may be a potential therapy for alleviating migraine symptoms and our promising finding warrants further investigation of HSP70 for clinical application.

Establishment and validation of immune microenvironmental gene signatures for predicting prognosis in patients with head and neck squamous cell carcinoma.

Tumor cells influencing the microenvironment are essential for restrained immunity in head and neck squamous cell carcinoma (HNSCC). There has been considerable progress in the research on monoclonal antibodies for antigen-specific immunotherapy that overcome immunosuppressive checkpoint receptor/ligand signaling in patients with HNSCC. However, alteration of immunogenicity and formation of neoantigens that lead to dysregulation and immunosuppression in the HNSCC microenvironment is not well-defined. The aim of this study was to quantify the Immune, Stromal, and ESTIMATE scores based on the gene matrix of patients with HNSCC reported in The Cancer Genome Atlas (TCGA). We examined the association of the Immune, Stromal, and ESTIMATE scores with the pathologic characteristics of patients with HNSCC, using weighted gene co-expression network (WGCNA) and protein-protein interaction (PPI) analyses, and selected 17 hub gene signatures from the key gene module that was mostly correlated to immunocyte infiltration. Gene functional enrichment showed that this key gene module was closely related to the regulation of immune cell activation and its relevant pathways. In the prognostic analysis, high expression of CD3E, SASH3, CD2, SIRPG, UBASH3A, IKZF1, SPN, IL10RA, SLA, and CD3G was significantly associated with a good prognosis. Consequently, these prognosis-related genes were validated via analysis of mRNA expression in tumor-infiltrating lymphocytes (TILs) and matched peripheral blood lymphocytes (PBLs) in ten patients with HNSCC, and the expression of these genes was significantly higher in TILs compared to that in PBLs. These findings provide a novel understanding of the tumor immune targets for improved therapeutic regimes in patients with HNSCC.

Pattern extraction of structural responses of gut microbiota to rotavirus infection via multivariate statistical analysis of clone library data.

This study developed a new statistical strategy for analyzing clone library data to observe whether there is a defined pattern in structural responses of gut microbiota to environmental perturbations. A large clone library of genus Bacteroides was constructed with fecal samples for each subject in rotavirus-infected (Group R) and healthy children (Group H). In all, 665 clones of the 12 Group H subjects and 284 clones of the nine Group R subjects were sequenced and classified into 34 operational taxonomic units (OTUs) with a similarity cutoff at 98%. Partial least squares-discriminant analysis was used to observe the change of the Bacteroides spp. composition caused by rotavirus infection and to identify the most relevant species contributing to this shift. It was revealed that H subjects and R subjects were well separated. Bacteroides vulgatus, Bacteroides stercoris and Bacteroides fragilis were identified as the most important discriminating OTUs between two groups. The increased abundance of B. fragilis and the decreased populations of B. vulgatus and B. stercoris in infected guts observed in this study were in agreement with previous culture-based studies. The strategy developed in this work can be used to reveal patterns in structural responses of gut microbiota to environmental perturbations from large-scale 16S rRNA gene-based sequencing data.

The microbial community in the feces of the giant panda (Ailuropoda melanoleuca) as determined by PCR-TGGE profiling and clone library analysis.

Despite having a typical carnivorous digestive tract, the giant panda has a diet consisting exclusively of bamboo, a low-efficiency food source. Given this paradox, we sought to investigate if the giant panda digestive tract is inhabited by organisms indicative of high cellulose diet or their gastrointestinal tract anatomy. The diversity and dynamics of the predominant bacteria in the fecal flora of two adult (male and female) and one young (male) giant panda reared in two different zoos over a 2-year period was studied using 16S rDNA-based approaches. The temperature gradient gel electrophoresis (TGGE) profiles of the 16S rDNA V3 region of the three individuals were highly similar. The structure of their fecal flora remained relatively stable over the 2-year period. Both the most predominant band in TGGE patterns shared by the three pandas and the biggest operational taxonomic unit (OTU) in the clone library were phylogenetically related to Escherichia coli. Gram-negative, facultative bacteria constituted almost 60% of the whole community in the clone library. All the OTUs were related to previously described phylotypes known to reside in the intestine or rumen. The results of our study indicate that the predominant bacterial populations in the intestine of the three pandas were markedly different from that of herbivores. The unbalanced intestinal community structure may play a role in the inefficient digestion of bamboo by the giant pandas.

Molecular profiling of the Clostridium leptum subgroup in human fecal microflora by PCR-denaturing gradient gel electrophoresis and clone library analysis.

A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, approximately 99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.

Microbial communities in the larval midgut of laboratory and field populations of cotton bollworm (Helicoverpa armigera).

We compared the bacterial communities in the larval midgut of field and laboratory populations of a polyphagous pest, the cotton bollworm (Helicoverpa armigera), using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences and 16S library sequence analysis. DGGE profiles and 16S rDNA library sequence analysis indicated similar patterns of midgut microbial community structure and diversity: specific bacterial types existed in both populations, and a more diverse microbial community was observed in caterpillars obtained from the field. The laboratory population harbored a rather simple gut microflora consisting mostly of phylotypes belonging to Enterococcus (84%). For the field population, phylotypes belonging to Enterococcus (28%) and Lactococcus (11%), as well as Flavobacterium (10%), Acinetobacter (19%), and Stenotrophomonas (10%) were dominant members. These results provided the first comprehensive description of the microbial diversity of the midgut of the important pest cotton bollworm and suggested that the environment and food supply might influence the diversity of the gut bacterial community.

[Analysis of Bifidobacteria spp. composition in human gut via temperature gradient gel electrophoresis (TGGE)].

Temperature Gradient Gel Electrophoresis (TGGE) method combined with 16S rDNA clone library profiling was used to analyze Bifidobacteria spp. composition in human gut in this study. Bifidobacteria group-specific TGGE patterns of 10 healthy human individuals showed that the Bifidobacteria population in humans was host-specific. The genomic diversity and species composition of Bifidobacteria in different individual was dissimilar. Through sequencing and TGGE comigration analysis of the Bifidobacteria group-specific amplicons for one healthy boy, it was revealed that the TGGE bands of this individual represented species Bifidobacterium bifidum, B. infantis, B. longum, B. adolescentis, B. pseudocatenulatum and one potentially new species, respectively. B. pseudocatenulatum was the most common species of tested samples. While as control, only two species- B. pseudocatenulatum and B. longum were isolated using traditional culture method. Bifidobacteria group-specific PCR based TGGE method combined with 16S rDNA clone library profiling is a sensitive and effective approach to resolve the population structure of Bifidobacteria in microflora of human intestinal tract.

Information theory-based algorithm for in silico prediction of PCR products with whole genomic sequences as templates.

BACKGROUND: A new algorithm for assessing similarity between primer and template has been developed based on the hypothesis that annealing of primer to template is an information transfer process. RESULTS: Primer sequence is converted to a vector of the full potential hydrogen numbers (3 for G or C, 2 for A or T), while template sequence is converted to a vector of the actual hydrogen bond numbers formed after primer annealing. The former is considered as source information and the latter destination information. An information coefficient is calculated as a measure for fidelity of this information transfer process and thus a measure of similarity between primer and potential annealing site on template. CONCLUSION: Successful prediction of PCR products from whole genomic sequences with a computer program based on the algorithm demonstrated the potential of this new algorithm in areas like in silico PCR and gene finding.

Molecular profiling of Bacteroides spp. in human feces by PCR-temperature gradient gel electrophoresis.

A group-specific PCR-based temperature gradient gel electrophoresis (TGGE) method was developed to study the population composition of genus Bacteroides in human gut. Highly reproducible and well-separated bands in TGGE fingerprints of ten unrelated human fecal samples showed complex and host-specific Bacteroides species composition. Dynamic monitoring over 22 months of samples from one healthy 10-year-old boy indicated a relatively stable population profile of Bacteroides. The species identity of each band in TGGE gel of this boy was also resolved via comigration analysis of sequenced inserts in a Bacteroides group-specific clone library. This work provides a rapid and effective technique for analyzing the species composition of Bacteroides in human gut.

ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts.

Bacterial populations common to healthy human guts may play important roles in human health. A new strategy for discovering genomic sequences as markers for these bacteria was developed using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting. Structural features within microbial communities are compared with ERIC-PCR followed by DNA hybridization to identify genomic fragments shared by samples from healthy human individuals. ERIC-PCR profiles of fecal samples from 12 diseased or healthy human and piglet subjects demonstrated stable, unique banding patterns for each individual tested. Sequence homology of DNA fragments in bands of identical size was examined between samples by hybridization under high stringency conditions with DIG-labeled ERIC-PCR products derived from the fecal sample of one healthy child. Comparative analysis of the hybridization profiles with the original agarose fingerprints identified three predominant bands as signatures for populations associated with healthy human guts with sizes of 500, 800 and 1000 bp. Clone library profiling of the three bands produced 17 genome fragments, three of which showed high similarity only with regions of the Bacteroides thetaiotaomicron genome, while the remainder were orphan sequences. Association of these sequences with healthy guts was validated by sequence-selective PCR experiments, which showed that a single fragment was present in all 32 healthy humans and 13 healthy piglets tested. Two fragments were present in the healthy human group and in 18 children with non-infectious diarrhea but not in eight children with infectious diarrhea. Genome fragments identified with this novel strategy may be used as genome-specific markers for dynamic monitoring and sequence-guided isolation of functionally important bacterial populations in complex communities such as human gut microflora.

[Type III secretion system in Se9R can recognize and secret harpin in Erwinia amylovora].

Bacterial pathogens of both animals and plants use type III secretion machines to secret virulence proteins. The signal that leads to tye type III secretion is encoded as stem loop structure in the messenger RNA. Harpin, produced by Erwinia anylovora is secreted via type III secretion. Erwinia carotovora Se9R and Erwinia amylovora belong to the same genus and they use type III secretion machines to secret virulence proteins. We used PCR to amplify hrpN from pCPP430 which harbors hrpN gene cluster, and cloned it to pGEM-T vector to get pWGF1, which was then chemically transformed into DH5 alpha (pCPP430hrpN-) and electrotransformed into Se9R. DH5 alpha (pCPP430hrpN-/pWGF1) induced hypersensitive response on tomato leaf and Se9R(pWGF1) showed significantly reduced virulence than Se9R on Chinese cabbage leaf. Western-blotting analysis of the CFEP of Se9R(pWGF1) showed production of harpin protein. These results suggest the type III secretion machine in Se9R can recognize secretion singal in the gene of harpin in Erwinia amylovora and secret it as a biologically native form.