Despite a long-suspected role in the development of human colorectal cancer (CRC), the composition of gut microbiota in CRC patients has not been adequately described. In this study, fecal bacterial diversity in CRC patients (n=46) and healthy volunteers (n=56) were profiled by 454 pyrosequencing of the V3 region of the 16S ribosomal RNA gene. Both principal component analysis and UniFrac analysis showed structural segregation between the two populations. Forty-eight operational taxonomic units (OTUs) were identified by redundancy analysis as key variables significantly associated with the structural difference. One OTU closely related to Bacteroides fragilis was enriched in the gut microbiota of CRC patients, whereas three OTUs related to Bacteroides vulgatus and Bacteroides uniformis were enriched in that of healthy volunteers. A total of 11 OTUs belonging to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus were significantly more abundant in the gut microbiota of CRC patients, and 5 OTUs belonging to the genus Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae were less abundant. Real-time quantitative PCR further validated the significant reduction of butyrate-producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-coenzyme A CoA transferase genes (Mann–Whitney test, P<0.01). Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.
colorectal cancer; gut microbiota; pyrosequencing
Nucleolin is implicated to play a role in angiogenesis, a vital process in tumor growth and metastasis. However, the presence and clinical relevance of nucleolin in human non small cell lung cancer (NSCLC) remains largely unknown. In this study, we explored the expression and prognostic implication of nucleolin in surgically resected NSCLC patients. A cohort of 146 NSCLC patients who underwent surgical resection was selected for tissue microarray. In this tissue microarray, nucleolin expression was measured by immunofluorescence. Staining for CD31, a marker of endothelial cells, was performed to mark blood vessels. A Cox proportional hazards model was used to assess the prognostic significance of nucleolin. Nucleolin expression was observed in 34.2% of all patients, and 64.1% in high CD31 expression patients. The disease-free survival (DFS) was significantly shorter in patients with high nucleolin (CD31hiNCLhi) compared to patients with low tumor blood vessels (CD31loNCLlo) (5 ys of DFS 24% vs 64%, p = 0.002). Such a difference was demonstrated in the following stratified analyses: stage I (p<0.001), squamous cell carcinoma and adenosquamous cell carcinoma (p = 0.028), small tumor (<5 cm, p = 0.008), and surgery alone (p = 0.015). Multivariate analysis further revealed that nucleolin expression independently predicted for worse survival (p = 0.003). This study demonstrates that nucleolin is associated with the clinical outcomes in postoperative NSCLC patients. Thus, the expression levels of nucleolin may provide a new prognostic marker to identify patients at higher risk for treatment failure, especially in some subgroups.
Staphylococcus aureus is an important pathogen that causes biofilm-associated infection in humans. Autoinducer 2 (AI-2), a quorum-sensing (QS) signal for interspecies communication, has a wide range of regulatory functions in both Gram-positive and Gram-negative bacteria, but its exact role in biofilm formation in S. aureus remains unclear.
Here we demonstrate that mutation of the AI-2 synthase gene luxS in S. aureus RN6390B results in increased biofilm formation compared with the wild-type (WT) strain under static, flowing and anaerobic conditions and in a mouse model. Addition of the chemically synthesized AI-2 precursor in the luxS mutation strain (ΔluxS) restored the WT phenotype. Real-time RT-PCR analysis showed that AI-2 activated the transcription of icaR, a repressor of the ica operon, and subsequently a decreased level of icaA transcription, which was presumably the main reason why luxS mutation influences biofilm formation. Furthermore, we compared the roles of the agr-mediated QS system and the LuxS/AI-2 QS system in the regulation of biofilm formation using the ΔluxS strain, RN6911 and the Δagr ΔluxS strain. Our data indicate a cumulative effect of the two QS systems on the regulation of biofilm formation in S. aureus.
These findings demonstrate that AI-2 can decrease biofilm formation in S. aureus via an icaR-activation pathway. This study may provide clues for therapy in S. aureus biofilm-associated infection.
The prevalence of obesity is higher in blacks than whites, especially in black women, and is known to be associated with major cardiovascular disease risk factors, which are also more prevalent in blacks than whites. Weight perception may contribute to these differences if blacks are more likely to underestimate their weight. We explored race and gender differences in underestimation of weight using body mass index (BMI) and waist circumference (WC), after adjusting for other cardiovascular risk factors.
Methods and Results
We studied 219 white and 240 black women and men as part of the META-Health Study. Perceived weight was assessed over the phone and categorized into three categories: underweight or normal weight, overweight, or obesity. Height, weight, and WC were measured at a subsequent visit, and BMI was calculated. Logistic regression was used to compare the likelihood of underestimating actual weight category by race, before and after adjusting for sociodemographic, lifestyle factors, and medical history. In multivariate analysis, the odds of underestimating BMI category was greater than threefold in blacks compared with whites (OR 3.1, 95% CI 1.9–4.8) and was larger for black women than for black men (p<0.01 for interaction). When abdominal adiposity was taken into account by utilizing WC as a measure of weight, the observed difference in weight underestimation remained.
Our data reveal a significant misperception of weight among blacks, particularly black women, who have the highest burden of obesity. A multifaceted approach with efficient identification of social, cultural, and environmental factors that give rise to obesity tolerance in blacks will provide potential targets for intervention, which may ameliorate weight misperception and the prevalence of excess weight in the black population.
Berberine, a major pharmacological component of the Chinese herb Coptis chinensis, which was originally used to treat bacterial diarrhea, has recently been demonstrated to be clinically effective in alleviating type 2 diabetes. In this study, we revealed that berberine effectively prevented the development of obesity and insulin resistance in high-fat diet (HFD)-fed rats, which showed decreased food intake. Increases in the levels of serum lipopolysaccharide-binding protein, monocyte chemoattractant protein-1, and leptin and decrease in the serum level of adiponectin corrected for body fat in HFD-fed rats were also significantly retarded by the co-administration of berberine at 100 mg/kg body weight. Bar-coded pyrosequencing of the V3 region of 16S rRNA genes revealed a significant reduction in the gut microbiota diversity of berberine-treated rats. UniFrac principal coordinates analysis revealed a marked shift of the gut microbiota structure in berberine-treated rats away from that of the controls. Redundancy analysis identified 268 berberine-responding operational taxonomic units (OTUs), most of which were essentially eliminated, whereas a few putative short-chain fatty acid (SCFA)-producing bacteria, including Blautia and Allobaculum, were selectively enriched, along with elevations of fecal SCFA concentrations. Partial least square regression models based on these 268 OTUs were established (Q2>0.6) for predicting the adiposity index, body weight, leptin and adiponectin corrected for body fat, indicating that these discrete phylotypes might have a close association with the host metabolic phenotypes. Taken together, our findings suggest that the prevention of obesity and insulin resistance by berberine in HFD-fed rats is at least partially mediated by structural modulation of the gut microbiota, which may help to alleviate inflammation by reducing the exogenous antigen load in the host and elevating SCFA levels in the intestine.
The development of CRS is believed to be the result of combined interactions between the genetic background of the affected subject and environmental factors.
To replicate and extend our recent findings from genetic association studies in chronic rhinosinusitis (CRS) performed in a Canadian Caucasian population in a Chinese population.
In a case-control replication study, DNA samples were obtained from CRS with (n = 306; CRSwNP) and without (n = 332; CRSsNP) nasal polyps, and controls (n = 315) in a Chinese population. A total of forty-nine single nucleotide polymorphisms (SNPs) selected from previous identified SNPs associated with CRS in Canadian population, and SNPs from the CHB HapMap dataset were individually genotyped.
We identified two SNPs respectively in RYBP (rs4532099, p = 2.15E–06, OR = 2.59) and AOAH (rs4504543, p = 0.0001152, OR = 0.58) significantly associated with whole CRS cohort. Subgroup analysis for the presence of nasal polyps (CRSwNP and CRSsNP) displayed significant association in CRSwNP cohorts regarding to one SNP in RYBP (P = 3.24E–006, OR = 2.76). Evidence of association in the CRSsNP groups in terms of 2 SNPs (AOAH_rs4504543 and RYBP_rs4532099) was detected as well. Stratifying analysis by gender demonstrated that none of the selected SNPs were associated with CRSwNP as well as CRSsNP. Meanwhile 3 SNPs (IL1A_rs17561, P = 0.005778; IL1A_rs1800587, P = 0.009561; IRAK4_rs4251513, P = 0.03837) were associated with serum total IgE level.
These genes are biologically plausible, with roles in regulation of transcription (RYBP) and inflammatory response (AOAH). The present data suggests the potential common genetic basis in the development of CRS in Chinese and Caucasian population.
Functional connective tissues have been developed using tissue engineering approach by seeding cells on biodegradable scaffolds such as polyglycolic acid (PGA). However, a major drawback of tissue engineering approaches that utilize synthetic polymers is the persistence of polymer remnants in engineered tissues at the end of culture. Such polymer fragments may significantly degrade tissue mechanics and stimulate local inflammatory responses in vivo. In this study, several polymeric materials with a range of degradation profiles were developed and evaluated for their potential applications in construction of collagen matrix-rich tissues, particularly tissue-engineered blood vessels. The degradation characteristics of these polymers were compared as were their characteristics vis-à-vis cell adhesion and proliferation, collagen synthesis, and ability to support growth of engineered vessels. Under aqueous conditions at 37°C, Polymer I (comprising 84% glycolide and 16% trimethylene carbonate [TMC]) had a similar degradation profile to PGA, Polymer II (comprising 84% glycolide, 14% TMC, and 2% polyethylene succinate) degradedly more slowly, but Polymer III (comprising 87% glycolide, 7% TMC, and 6% polyethylene glycol) had a more extensive degradation as compared to PGA. All polymers supported cell proliferation, but Polymer III improved collagen production and engineered vessel mechanics as compared with PGA. In addition, more slowly degrading polymers were associated with poorer final vessel mechanics. These results suggest that polymers that degrade more quickly during tissue culture actually result in improved engineered tissue mechanics, by virtue of decreased disruption of collagenous extracellular matrix.
With the expanding availability of sequencing technologies, research previously centered on the human genome can now afford to include the study of humans’ internal ecosystem (human microbiome). Given the scale of the data involved in this metagenomic research (two orders of magnitude larger than the human genome) and their importance in relation to human health, it is crucial to guarantee (along with the appropriate data collection and taxonomy) proper tools for data analysis. We propose to adapt the approaches defined for the analysis of gene-expression microarray in order to infer information in metagenomics. In particular, we applied SAM, a broadly used tool for the identification of differentially expressed genes among different samples classes, to a reported dataset on a research model with mice of two genotypes (a high density lipoprotein knockout mouse and its wild-type counterpart). The data contain two different diets (high-fat or normal-chow) to ensure the onset of obesity, prodrome of metabolic syndromes (MS). By using 16S rRNA gene as a genomic diversity marker, we illustrate how this approach can identify bacterial populations differentially enriched among different genetic and dietary conditions of the host. This approach faithfully reproduces highly-relevant results from phylogenetic and standard statistical analyses, used to explain the role of the gut microbiome in relation to obesity. This represents a promising proof-of-principle for using functional genomic approaches in the fast growing area of metagenomics, and warrants the availability of a large body of thoroughly tested and theoretically sound methodologies to this exciting new field.
human microbiome; functional genomic; metagenomics
Studies on the incidence and predictors of heart failure (HF) are often restricted to elderly persons or identify only inpatient cases.
Methods and Results
We determined the incidence and predictors of new HF diagnosed in either outpatient or inpatient settings, among 359 947 women and men (age ≥18 years) insured by Kaiser Permanente Georgia at any time during calendar years 2000 to 2005. Subjects were free of HF at baseline, and incident HF was identified with ICD-9 codes (1 inpatient or 2 outpatient HF visits). We developed multivariable Cox models to assess the association of antecedent factors (coronary heart disease, hypertension, diabetes mellitus, atrial fibrillation, and valvular heart disease) with incident HF. Separate models were created for each sex and for newly diagnosed HF in outpatient or inpatient settings. There were 4001 incident HF cases (50% women and 48% in subjects <65 years old), during 1 015 794 person-years of follow-up. The incidence rate of HF was greater in men than in women (4.24 versus 3.68 per 1000 person-years) but was stable across the study interval in both sexes. Two thirds of incident HF cases from this population occurred in outpatients. These 5 antecedent factors and age yielded excellent discrimination for incident HF in both outpatients and inpatients and in both sexes (C >0.85 in all models).
Common modifiable risk factors accurately discriminate women and men at risk for HF diagnosed in either outpatient or inpatient settings. Approximately two thirds of new HF cases in our insured population were diagnosed in outpatients; more research is needed to characterize these subjects and their prognosis.
heart failure; epidemiology; risk factors
Enterotoxigenic Escherichia coli (ETEC) strains with K88 fimbriae are often associated with the outbreaks of diarrhea in newborn and weaned piglets worldwide. In the present study, we observed that 108 CFU/ml of K88+ ETEC strain JG280 caused more death of pig intestinal IPEC-J2 cells than did 109 CFU/ml, suggesting that ETEC-induced cell death was cell density dependent and that quorum sensing (QS) may play a role in pathogenesis. Subsequent investigations demonstrated a positive correlation between autoinducer 2 (AI-2) activity of JG280 and death of IPEC-J2 cells during the infection for up to 3 h. However, there was a negative correlation between AI-2 activity and expression of the JG280 enterotoxin genes estA and estB when IPEC-J2 cells were exposed to the pathogen at 108 CFU/ml. We therefore cloned the luxS gene (responsible for AI-2 production) from JG280 and overexpressed it in E. coli DH5α, because deletion of the luxS gene was retarded by the lack of suitable antibiotic selection markers and the resistance of this pathogen to a wide range of antibiotics. The addition of culture fluid from E. coli DH5α with the overexpressed luxS reduced cell death of IPEC-J2 cells by 108 CFU/ml JG280. The addition also reduced the estA expression by JG280. Nonpathogenic K88+ strain JFF4, which lacks the enterotoxin genes, caused no death of IPEC-J2 cells, although it produced AI-2 activity comparable to that produced by JG280. These results suggest the involvement of AI-2-mediated quorum sensing in K88+ ETEC pathogenesis, possibly through a negative regulation of STa production.
The objectives of this study were to monitor the stability of rifampin (RIF) in Löwenstein-Jensen medium (L-J medium) and 7H9 broth, which are the media commonly used for drug susceptibility testing (DST) of Mycobacterium tuberculosis. Rifampin degradation in stock solution, 7H9 broth, and L-J medium and during the inspissation process for L-J medium preparation was serially monitored by high-performance liquid chromatography (HPLC). L-J medium-based DST was conducted to examine the effect of L-J medium storage on the DST outcome. The RIF stock solution was stable for at least 3 months when kept at either 4°C or −20°C; RIF in 7H9 broth and L-J medium was almost 50% decayed after 1 week of storage at 37°C, and rifampin could not be detected in 7H9 or L-J medium after 3 weeks or 6 weeks of storage at 37°C. Approximately half of the drug was decomposed after 4 months of storage at 4°C for both media, and after 6 months of storage at 4°C, RIF in L-J medium was undetectable, while 38% of RIF remained in 7H9 medium. Approximately 21, 24, 29, and 35% RIF degradations were detected when the L-J medium was coagulated at 75°C, 80°C, 85°C, and 90°C, respectively. The DST outcomes when using L-J medium stored for different periods of time were consistent with the RIF concentration monitoring data. Rifampin in stock solution is stable for at least 3 months at a reduced storage temperature. Media containing RIF should be prepared strictly according to validated standard operating procedures. RIF degradation is a possible reason for false resistance categorizations of M. tuberculosis isolates in the clinical laboratory.
Interleukin-1 receptor-associated kinase-4 (IRAK-4) encodes a kinase that is essential for NF-kB activation in Toll-like receptor and T-cell receptor signaling pathways, indicating a possible crosstalk between innate and acquired immunities. We attempted to determine whether the polymorphisms in the Interleukin-1 receptor-associated kinase-4 (IRAK-4) gene are associated with allergic rhinitis (AR) in the Han Chinese population.
A population of 379 patients with AR and 333 healthy controls was studied. Blood was drawn for DNA extraction and total serum immunoglobulin E (IgE). A total of 11 single nucleotide polymorphisms (SNPs) in IRAK-4 were selected and individually genotyped.
Significant allelic differences between cases and controls were obtained for the SNP of rs3794262 in the IRAK-4 gene. In the stratified analysis for gender, two SNPs (rs4251431 and rs6582484) in males appeared as significant associations. Subgroup analysis for the presence of different allergen sensitivities displayed associations only in the house dust mite-allergic cohorts (rs3794262, rs4251481). None of the selected SNPs in IRAK-4 was associated with total IgE level. The haplotype analyisis indicated GCCTGCGA was significantly associated with AR. The SNP-SNP interaction information analysis indicated that the selected sets of polymorphisms had no synergistic effect.
Our findings did not support the potential contribution of the IRAK-4 gene to serum IgE levels. However, the results demonstrated a gender- and allergen-dependant association pattern between polymorphisms in IRAK-4 and AR in Chinese population.
Persons with serious mental illnesses (SMI) have elevated rates of comorbid medical conditions, but may also face challenges in effectively managing those conditions.
The study team developed and pilot-tested the Health and Recovery Program (HARP), an adaptation of the Chronic Disease Self-Management Program (CDSMP) for mental health consumers. A manualized, six-session intervention, delivered by mental health peer leaders, helps participants become more effective managers of their chronic illnesses. A pilot trial randomized 80 consumers with one or more chronic medical illness to either the HARP program or usual care.
At six month follow-up, participants in the HARP program had a significantly greater improvement in patient activation than those in usual care (7.7% relative improvement vs. 5.7% decline, p=0.03 for group*time interaction), and in rates of having one or more primary care visit (68.4% vs. 51.9% with one or more visit, p=0.046 for group*time interaction). Intervention advantages were observed for physical health related quality of life (HRQOL), physical activity, medication adherence, and, and though not statistically significant, had similar effect sizes as those seen for the CDSMP in general medical populations. Improvements in HRQOL were largest among medically and socially vulnerable subpopulations.
This peer-led, medical self-management program was feasible and showed promise for improving a range of health outcomes among mental health consumers with chronic medical comorbidities. The HARP intervention may provide a vehicle for the mental health peer workforce to actively engage in efforts to reduce morbidity and mortality among mental health consumers.
serious mental illness; chronic disease; wellness; recovery; self management
While advances in regenerative medicine and vascular tissue engineering have been substantial in recent years, important stumbling blocks remain. In particular, the limited lifespan of differentiated cells that are harvested from elderly human donors is an important limitation in many areas of regenerative medicine. Recently, a mutant of the human telomerase reverse transcriptase enzyme (TERT) was described, which is highly processive and elongates telomeres more rapidly than conventional telomerase. This mutant, called pot1-TERT, is a chimeric fusion between the DNA binding protein pot1 and TERT. Because pot1-TERT is highly processive, it is possible that transient delivery of this transgene to cells that are utilized in regenerative medicine applications may elongate telomeres and extend cellular lifespan while avoiding risks that are associated with retroviral or lentiviral vectors. In the present study, adenoviral delivery of pot1-TERT resulted in transient reconstitution of telomerase activity in human smooth muscle cells, as demonstrated by telomeric repeat amplification protocol (TRAP). In addition, human engineered vessels that were cultured using pot1-TERT expressing cells had greater collagen content and somewhat better performance in vivo than control grafts. Hence, transient delivery of pot1-TERT to elderly human cells may be useful for increasing cellular lifespan and improving the functional characteristics of resultant tissue engineered constructs.
telomerase; senescence; tissue engineering; vascular grafts; smooth muscle cells
Immunoglobulin E (IgE) is a central player in the allergic response, and raised total IgE levels are considered as an indicator of atopy or potential development of atopy. A recent genome-wide scan in a German population-based cohort of adults identified the gene encoding the alpha chain of the high affinity receptor for IgE (FCER1A) as a susceptibility locus influencing total serum IgE levels. The aim of this study was to investigate whether the polymorphisms in the FCER1A gene are associated with allergic rhinitis (AR) in a Han Chinese population.
A population of 378 patients with AR and 288 healthy controls was studied. Precise phenotyping of patients was accomplished by means of a questionnaire and clinical examination. Blood was drawn for DNA extraction and total serum immunoglobulin E (IgE) measurement. A total of 16 single nucleotide polymorphisms (SNPs) in FCER1A were selected and individually genotyped. None of the SNPs in the FCER1A showed an association with AR. Similarly, the lack of association was also evident in subgroup analysis for the presence of different allergen sensitivities. None of the selected SNPs in FCER1A was associated with total IgE level.
Although FCER1A presents itself as a good candidate for contributing to total serum IgE, this study failed to find an association between SNPs in the FCER1A gene region and IgE level or AR susceptibility.
Autoinducer 2 (AI-2) is widely recognized as a signal molecule for intra- and interspecies communication in Gram-negative bacteria, but its signaling function in Gram-positive bacteria, especially in Staphylococcus aureus, remains obscure. Here we reveal the role of LuxS in the regulation of capsular polysaccharide synthesis in S. aureus NCTC8325 and show that AI-2 can regulate gene expression and is involved in some physiological activities in S. aureus as a signaling molecule. Inactivation of luxS in S. aureus NCTC8325 resulted in higher levels of transcription of capsular polysaccharide synthesis genes. The survival rate of the luxS mutant was higher than that of the wild type in both human blood and U937 macrophages. In comparison to the luxS mutant, a culture supplemented with chemically synthesized 4,5-dihydroxy-2,3-pentanedione (DPD), the AI-2 precursor molecule, restored all the parental phenotypes, suggesting that AI-2 has a signaling function in S. aureus. Furthermore, we demonstrated that the LuxS/AI-2 signaling system regulates capsular polysaccharide production via a two-component system, KdpDE, whose function has not yet been clarified in S. aureus. This regulation occurred via the phosphorylation of KdpE binding to the cap promoter.
Paper pulp wastewater resulting from alkaline extraction of wheat straw, known as black liquor, is very difficult to be treated and causes serious environmental problems due to its high pH value and chemical oxygen demand (COD) pollution load. Lignin, semicellulose and cellulose are the main contributors to the high COD values in black liquor. Very few microorganisms can survive in such harsh environments of the alkaline wheat straw black liquor. A naturally developed microbial community was found accidentally in a black liquor storing pool in a paper pulp mill of China. The community was effective in pH decreasing, color and COD removing from the high alkaline and high COD black liquor.
Thirty-eight strains of bacteria were isolated from the black liquor storing pool, and were grouped as eleven operational taxonomy units (OTUs) using random amplified polymorphic DNA-PCR profiles (RAPD). Eleven representative strains of each OTU, which were identified as genera of Halomonas and Bacillus, were used to construct a consortium to treat black liquor with a high pH value of 11.0 and very high COD pollution load of 142,600 mg l−1. After treatment by the constructed consortium, about 35.4% of color and 39,000 mg l−1 (27.3%) CODcr were removed and the pH decreased to 7.8. 16S rRNA gene polymerase chain reaction denaturant gradient gel electrophoresis (PCR-DGGE) and gas chromatography/mass spectrometry (GC/MS) analysis suggested a two-stage treatment mechanism to elucidate the interspecies collaboration: Halomonas isolates were important in the first stage to produce organic acids that contributed to the pH decline, while Bacillus isolates were involved in the degradation of lignin derivatives in the second stage under lower pH conditions.
Tolerance to the high alkaline environment and good controllability of the simple consortium suggested that the constructed consortium has good potential for black liquor treatment. Facilitating the treatment process by the constructed consortium would provide a promising opportunity to reduce the pollution, as well as to save forest resources and add value to a waste product.
The impact of long-term organic and inorganic amendments on the actinobacterial community in soils was studied. Denaturing gradient gel electrophoresis patterns based on the V3 region of 16S rRNA suggested that there was no significant difference between the communities occurring in the different amendments. However, analysis of the clone libraries of the actinobacterial communities by the use of multiple statistical approaches showed that these communities were significantly different from each other. Results showed that long-term organic and inorganic soil amendments did not significantly alter the overall phylogenetic diversity of the actinobacterial communities but did significantly change the community structure.
To understand the role of mucosa-associated microbiota in the pathogenicity of ulcerative colitis (UC), paired biopsies were obtained during colonoscopy from the ulcerated and nonulcerated gut mucosa of 24 patients with UC. Denaturing gradient gel electrophoresis analysis was employed to profile the composition of the dominant bacteria (16S rRNA gene V3 region) and three important groups: lactobacilli, the Clostridium leptum subgroup, and Bacteroides spp. The Pearson coefficient was used to estimate similarities between the bacterial communities of the paired biopsies for each patient. The average similarity values of bacterial composition between the paired samples were 94.8 ± 3.8% for dominant bacteria, 59.9 ± 26.1% for lactobacilli, 79.2 ± 22.6% for the Clostridium leptum subgroup, and 88.7 ± 16.4% for Bacteroides spp. The data revealed that lactobacilli and the Clostridium leptum subgroup were significantly different between the ulcerated and the nonulcerated regions. It also was noted that for lactobacilli, the composition varied significantly between biopsy sites irrespective of the location of UC in the gut but that the composition of the Clostridium leptum subgroup showed significant differences between paired samples from UC in the rectum and not in the left colon. Localized dysbiosis of the mucosa-associated intestinal microflora, especially for lactobacilli and the Clostridium leptum subgroup, may be closely related to UC.
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, ∼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.
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.
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.
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.