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Human adenoviruses (HAdVs) are prevalent worldwide and are a common cause of respiratory tract infection in people of all ages. However, little is known about HAdV infection among children with severe acute respiratory infection (SARI). The present study retrospectively analysed the molecular typing and epidemiological characteristics of HAdV-positive samples from children with SARI from January 2017 to December 2021 in Huzhou. The results showed that 89 (8·27%) of 1078 SARI paediatric patients were positive for HAdVs. Children <5 years of age accounted for 87·64% of the positive cases. The peak seasons for HAdV infection were the first quarter and the fourth quarter. In addition, HAdV-B and HAdV-C were circulating among paediatric patients with SARI, of which the B3 genotype (n = 30, 51·72%) was the most prevalent and was detected every year, indicating that B3 is the main epidemic strain in the Huzhou area, followed by C1 (n = 9, 15·52%), C2 (n = 7, 12·07%) and B7 (n = 5, 8·62%). These findings provide a benchmark for future epidemiology and prevention strategies for HAdVs., (© 2022 Society for Applied Microbiology.)

A new type of nitrogen and chloride co-doped carbon dots (N/Cl-CDs) based on choline chloride-urea-glycine ternary deep eutectic solvents (DESs) was synthesized using a one-step hydrothermal method. The prepared N/Cl-CDs exhibited oxidase-like activity and excellent antibacterial activity against Escherichia coli, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA). The addition of silver nanoparticles (Ag NPs) (i.e. N/Cl-CDs + Ag NPs) to the N/Cl-CDs also significantly enhanced the oxidase and antibacterial activities. The nanocomposite (1·8 mg ml -1 ) completely inactivated 10 5  CFU per ml of MRSA in 90 min. E. coli and S. aureus were labelled with the N/Cl-CDs, enabling multicolour fluorescence imaging at different excitation wavelengths. The nanocomposites have high antibacterial efficiency as a new bactericidal agent, as well as application potential with good biocompatibility and low toxicity., (© 2022 The Society for Applied Microbiology.)

Aims: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is now the leading cause of death from infectious disease, thus rapid diagnostic and screening techniques for TB are urgently needed., Methods and Results: Here, a detection of MTB using multiple cross displacement amplification coupling with nanoparticles-based lateral flow device (MCDA-LFD) was developed and validated, targeting the specific sdaA gene. The whole detection procedure, including rapid genomic DNA extraction (15 min), amplification (30 min) and result reporting (2 min), was completed within 50 min. No cross-reaction with non-mycobacteria and non-tuberculous mycobacteria (NTM) strains was observed. The sensitivity of sdaA-MCDA-LFD, Xpert MTB/RIF assay and culture results was 81·6, 48·3 and 37·9%, respectively, in TB patients. Among positive culture samples, the sensitivity of sdaA-MCDA-LFD and Xpert MTB/RIF assay was 93·9% (31/33) and 81·8% (27/33), respectively. Among culture-negative samples, the sensitivity of sdaA-MCDA-LFD and Xpert MTB/RIF assay was 74·1% (40/54) and 27·8% (15/54), respectively. The specificity of sdaA-MCDA-LFD and Xpert MTB/RIF was 95·4% (62/65) and 100% (65/65) in clinical samples from non-TB patients., Conclusion: The sdaA-MCDA-LFD assay was a rapid, simple, specific and sensitive TB diagnostic test., Significance and Impact of the Study: The sdaA-MCDA-LFD assay holds promise for application as a useful point-of-care test to detect MTB, and will play an important role in controlling and preventing TB., (© 2020 The Society for Applied Microbiology.)

Microbial diversity of pit mud (PM) plays a significant role in Baijiu's flavour. Here we explored the microbial community structures and aroma substances of Wenwang Winery with high-throughput sequencing coupling with headspace solid-phase microextraction-gas chromatography-mass spectrometry. We discovered that the odorant was mainly derived from 14 aroma compounds because of their OAVs ≥ 1 (OAV, the ratio of substance concentration to aroma threshold; s, on behalf of the plural), such as ethyl hexanoate (2438), ethyl octanoate (975), caproic acid (52) and etc. Moreover we also revealed that Lactobacillaceae (97·08%) was the mainly bacterial microbial community in 2-year-old PM, companied by the primarily fungi including Aspergillaceae (55·45%), Unclassified Ascomycota (11·13%) and Dipodascaceae (5·72%). Compared with the 2-year-old PM, bacterial floras in 20-year-old PM and 30-year-old PM were more abundant (i.e. Dysgonomonadaceae, Clostridium and Synerggstaceas), while no fungi were detected. Besides, the physicochemical analysis showed that the content of Lactobacillaceae was inversely associated with moisture, pH and ammonia nitrogen. By further Spearman's correlation coefficient analysis, we verified that the content of Lactobacillaceae was positively correlated with ethyl hexanoate, while negatively correlated with ethyl octanoate and caproic acid. Meanwhile, ethyl octanoate and caproic acid were positively correlated with most flora including Ruminococcaceae, Dysgonomonadaceae and Clostridiacea, which were related to physicochemical indexes. This work demonstrates promise for adjusting the physicochemical indexes of PM to affect the micro-organisms and aroma, which may provide a reference for the production of high-quality Baijiu., (© 2020 The Society for Applied Microbiology.)

Aim: Quinoline is a recalcitrant pollutant in coking wastewater which has been broadly investigated with many isolates possessing aerobic quinoline-degrading ability. However, studies on anaerobic degradation and the corresponding bacteria are very scarce. This study attempted to investigate the role of diverse functional members and the redundancy of quinoline degradation in a lab-scale quinoline denitrifying bioreactor., Methods and Results: Antibiotics were added to the batch culture under denitrifying conditions to disturb the microbial community of the quinoline-degrading bioreactor. According to the results, the nitrate removal rate remained stable, and the quinoline removal rate increased by 9·7% after treatment with streptomycin. However, PCoA analysis of 16S rRNA gene sequencing data of these samples indicated a significant shift in microbial community structures. Specifically, 12 operational taxonomic units (OTUs), including OTU1 (Pseudomonas) and OTU2 (Achromobacter), were significantly enriched. OTU1 replaced OTU8 (Thauera) as the most predominant denitrifying quinoline-degrading member. However, OTU8 and other predominant OTUs (Comamonas and Pseudoxanthomonas), which were hypothesized to contribute essentially to quinoline degradation in the origin bioreactor, became almost undetectable., Conclusion: Functional redundancy due to high biological diversity allowed the role reversal of predominant quinoline-degrading bacteria and other rare bacteria when disturbed by antibiotic stress. Although the abundance of OTU1 was much lower initially, it replaced the essential role of the predominant member OTU8 in the bioreactor community for quinoline degradation once the environmental condition changed., Significance and Impact of the Study: This study indicated that the high biological diversity in a wastewater treatment bacterial community is crucial for maintaining the degrading function of organic pollutants, especially in a changing environment due to external disturbance or stress., (© 2020 The Society for Applied Microbiology.)

Congenital self-healing reticulohistiocytosis (CSHR) is a rare disorder characterized by benign skin lesions with a tendency to self-heal. Multiple skin lesions are usually present in CSHR. It is very difficult to distinguish between CSHR and an invasive Langerhans cell histiocytosis. We present a case of a 5-month-old infant girl who had hypopigmented skin lesions distributed over her neck, thorax and torso. The skin lesions regressed spontaneously 2 months after the diagnosis of CSHR and the child has remained in complete remission without any sign of recurrence over a 2-year follow-up. BRAF V600E mutation was detected in lesional cells along with a low Ki-67 proliferative activity of about 6%. BRAF oncogene-induced senescence might contribute to a mechanism of self-regression in CSHR; however, the exact role of the somatic BRAF V600E mutation in CSHR remains to be determined., (© 2019 British Association of Dermatologists.)

Background: Memory T cells, a highly effective subset of T lymphocytes, have been reported to be involved in many inflammatory skin disorders. However, the potential role of memory T cells in keloid disease (KD) remains unclear., Objectives: Due to their important role in regulating inflammation, we investigated the characteristics of CD45RO + memory T cells in KD., Methods: Primary cutaneous cells were isolated from keloid scars and normal skin by enzymic digestion. Peripheral blood mononuclear cells were isolated from a related blood sample, and flow cytometry was applied to identify the phenotypic and functional abnormalities of memory T cells in KD., Results: We observed that the majority of T lymphocytes in keloid scars had the memory phenotype, and a greater number of the CD8 + memory T cells in keloid scars produced lower levels of tumour necrosis factor (TNF)-α. This abnormal cytokine production was even more distinct in Forkhead box (FOX)P3 -  CD8 - memory T cells, with lower TNF-α production and enhanced interferon-γ production. Furthermore, FOXP3 +  CD8 - memory T cells in keloid scars were abnormal, including showing reduced CD25 and cytotoxic T-lymphocyte-associated antigen 4 expression and interleukin-10 production. In addition, a significant decrease in the number of CD4 +  CD25 high  FOXP3 + regulatory T cells was identified in patients with multiple keloid scars. We also found that there was significantly increased infiltration of CD103 +  CD8 + memory T cells in keloid scars., Conclusions: Our findings preliminarily elucidate the abnormalities of CD45RO + memory T cells in keloid scars and provide early evidence that a disrupted T-cell response contributes to the progression of KD., (© 2017 British Association of Dermatologists.)

Bacteria of Dechloromonas were recognized as potential functional important denitrifiers in a long-term shell sand-amended peat soil. Different microcosms in a solid matrix and slurry systems with the addition of carbon and nitrogen sources, for example, clover leaves, glutamate and nitrate, were established. The bacterial community structures were analysed by pyrosequencing of the 16S rRNA gene to select the conditions for enriching bacteria of Dechloromonas. The results showed that a relatively even bacterial community in the initial soil shifted to communities dominated by a few types of nitrate-reducing bacteria after the incubation, which strongly responded to the carbon substrates addition and consumption. The bacteria of several genera including Dechloromonas, Pseudomonas, Clostridium, Aeromonas and Ferribacterium were significantly enriched after a certain period of time. The bacteria of Dechloromonas became one of the most predominant bacteria in the incubated community. Especially when added the mixed carbon substrates into the solid soil matrix, as high as 34% of abundance was detected. This study proved that the functional important bacteria from the genus of Dechloromonas could be enriched to an extremely high abundance by using proper culture condition which will benefit to the isolation or direct metagenomics study for Dechloromonas., Significance and Impact of the Study: The study of key players in a microbial community is always of important. In this study, the functional important denitrifiers in a shell sand-amended peat soil were investigated. Using different carbon sources in the incubation, we found the bacteria from the genus of Dechloromonas were enriched to an abundance of higher than 34% with several other denitrifiers together. This work provides us helpful insights not only for knowing the diversity of denitrifiers in the studied peat soil, but also for understanding their response to the carbon sources and the culture conditions., (© 2017 The Society for Applied Microbiology.)

Esophageal cancer is the eighth most common cancer worldwide. It is the fourth most common cause of cancer death in China and esophageal squamous cell carcinoma (ESCC) is the most prevalent histologic type. Many clinical trials have explored the value of neoadjuvant or adjuvant chemoradiation therapy in potentially resectable ESCC; however, these studies have produced conflicting results. This retrospective study was performed to investigate whether patients with resectable stage II/III ESCC should receive neoadjuvant or adjuvant therapy in addition to surgery. A review of stage II/III thoracic ESCC patients who underwent esophagectomy and either neoadjuvant or adjuvant chemoradiation was performed. Chemotherapy regimen consisted of cisplatin 75 mg/m2 divided into 3 days and fluorouracil 500 mg/m2 on days 1 to 5. The patients who underwent neoadjuvant therapy were treated with one cycle of chemotherapy concurrently with radiotherapy (40 Gy in 20 fractions, 5 days/week), and those receiving adjuvant therapy were treated with two cycles of chemotherapy concurrently with radiotherapy (46-50 Gy in 23-25 fractions, 5 days/week). A total of 122 patients met inclusion criteria, of which 49 underwent neoadjuvant chemoradiation and 73 underwent adjuvant chemoradiation. Median follow up was 36.5 months. The median survival times and 3, 5-year overall survival (OS) rates for the neoadjuvant and adjuvant groups were 39.3 versus 31.5 months, and 53.0%, 45.7% versus 42.9%, 29.7%, respectively (P = 0.091). For the patients with stage III ESCC, the median survival times and 5-year OS rates for the neoadjuvant and adjuvant groups were 39.3 versus 21.3 months, and 43.4% versus 21.0%, respectively (P = 0.021). Among lymph node-positive patients, the median survival times and 5-year OS rates for the neoadjuvant and adjuvant groups were 55.6 versus 23.7 months, and 43.0% versus 25.7%, respectively (P = 0.085). The incidence of perioperative and postoperative complications was comparable between the two groups (P > 0.05). For patients with resectable stage II/III ESCC, neoadjuvant chemoradiation does not increase postoperative complications and is associated with a trend toward better OS when compared to adjuvant chemoradiation., (© The Authors 2017. Published by Oxford University Press on behalf of International Society for Diseases of the Esophagus. All rights reserved. For permissions, please e-mail:journals.permissions@oup.com.)

Aims: To explore the distribution disciplinarian of alginate on the chalcopyrite concentrate surface during bioleaching., Methods and Results: The evolution of Sulfobacillus thermosulfidooxidans secreting alginate during bioleaching of chalcopyrite concentrate was investigated through gas chromatography coupled with mass spectrometry (GC-MS) and confocal laser scanning microscope (CLSM), and the critical synthetic genes (algA, algC, algD) of alginate were analysed by real-time polymerase chain reaction (RT-PCR). The GC-MS analysis results indicated that there was a little amount of alginate formed on the mineral surface at the early stage, while increasing largely to the maximum value at the intermediate stage, and then kept a stable value at the end stage. The CLSM analysis of chalcopyrite slice showed the same variation trend of alginate content on the mineral surface. Furthermore, the RT-PCR results showed that during the early stage of bioleaching, the expressions of the algA, algC and the algD genes were all overexpressed. However, at the final stage, the algD gene expression decreased in a large scale, and the algA and algC decreased slightly. This expression pattern was attributed to the fact that algA and algC genes were involved in several biosynthesis reactions, but the algD gene only participated in the alginate biosynthesis and this was considered as the key gene to control alginate synthesis., Conclusions: The content of alginate on the mineral surface increased largely at the beginning of bioleaching, and remained stable at the end of bioleaching due to the restriction of algD gene expression., Significance and Impact of the Study: Our findings provide valuable information to explore the relationship between alginate formation and bioleaching of chalcopyrite., (© 2017 The Society for Applied Microbiology.)

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by extensive immune response, including over-activation of T and B cell development of pathogenic autoantibodies, organ damage induced by the formation and deposition of immune complex and the abnormal elevation of type I interferon. Semaphorin5A (Sema5A) is involved essentially in immune cell regulation and is also implicated in the pathogenesis of autoimmune disorders. We aimed to evaluate the role of Sema5A in patients with SLE. Serum levels of Sema5A were tested by enzyme-linked immunosorbent assay (ELISA) in 152 SLE patients and 48 healthy controls. The message ribonucleic acid (mRNA) expression levels of Sema5A and ADAM metallopeptidase domain 17 (ADAM17) in the peripheral blood mononuclear cells (PBMC) from 43 patients with SLE and 19 healthy controls were detected by the real-time-quantitative polymerase chain reaction (qPCR). Serum Sema5A levels were increased significantly in SLE patients compared with healthy controls (P < 0·001). Elevated levels of Sema5A were correlated positively with 24-h proteinuria excretion (r = 0·558, P < 0·0001), SLE disease activity index (SLEDAI) (r = 0·278, P = 0·0006) and C-reactive protein (CRP) (r = 0·266, P = 0·002), but negatively with planet (PLT) (r = -0·294, P = 0·0003) and complement 3 (C3) (r = -0·287, P = 0·0004) in SLE patients. Patients with elevated Sema5A levels showed higher incidence of rash, serositis and nephritis (P < 0·05 or P < 0·001). Patients with decreased PLT, C3 or positive for proteinuria also showed elevated Sema5A (P < 0·001 or P < 0·05). The mRNA ADAM17 was increased in SLE patients and correlated positively with serum Sema5A levels. Our data demonstrated that elevated serum Sema5A in SLE patients correlated with disease activity and are involved in kidney and blood system damage; ADAM17 might be involved in the release of secreted Sema5A., (© 2017 British Society for Immunology.)

The primary objective of this study was to evaluate the safety and clinical efficacy of autologous cytokine-induced killer (CIK) cells combined with cyclophosphamide in the treatment of polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin changes (POEMS) syndrome patients. We evaluated five POEMS syndrome patients treated with autologous CIK cell immunotherapy combined with cyclophosphamide from 1 May 2012 to 30 November 2014. The Overall Neuropathy Limitation Scale (ONLS), computed tomography of the chest and abdomen, ultrasound of the abdomen, serum vascular endothelial growth factor (VEGF) level and lymphocyte count findings in the five patients were recorded. The median age of the patients was 40 years (range: 25-62), and all the patients were male. CIK cells were generated routinely from peripheral blood mononuclear cells (PBMCs) of all five patients, and the numbers of CIK cells increased by approximately 105-fold after 14 days of culture. All five patients (100%) responded to their neuropathy treatment, the ONLS scores were reduced by at least 1 and a paired-sample t-test revealed a significant difference (t = 5·715, P = 0·003 < 0·01). The extravascular volume overload responses indicated partial remission (PR = 60%) or stable disease (SD = 40%), and no cases of progressive disease (PD) or complete remission (CR) were observed. During clinical treatment, the serum VEGF of patient 5 decreased after one cycle of transfusion within 1 month. The lymphocyte counts of all the patients increased significantly after CIK transfusion, and a paired-sample t-test revealed a significant difference (t = 5·101, P = 0·004 < 0·01). Autologous CIK cell infusion combined with cyclophosphamide was found to be highly safe and elicited no adverse reactions. CIK cells can improve both the symptoms and quality of life, decrease serum VEGF levels and increase lymphocyte counts in patients with POEMS syndrome., (© 2016 British Society for Immunology.)

The role of NOD-like receptor family (NLRP3) has been confirmed in various inflammatory diseases. The association between NLRP3 and hyper-bileacidaemia during the sepsis remains unclear. We aimed to investigate whether NLRP3 silencing protects against the sepsis-induced hyper-bileacidaemia. Sepsis was induced by caecum ligation and puncture (CLP). Gene silencing of NLRP3 was performed by injecting rats with NLRP3 short hairpin RNA plasmids (NLRP3 shRNA) 48 h before surgery. Rats were divided into four groups: group 1: sham; group 2: sepsis; group 3: NLRP3 shRNA + sepsis (called the 'NLRP3 shRNA' group); and group 4: scrambled shRNA + sepsis (called the 'scrambled shRNA' group). The serum levels of bile acids, hepatic expression of hepatocyte membrane transporters, hepatic cytokine levels and behaviours of immune cells were compared among the groups. Hepatic NLRP3 expression was increased dramatically during the sepsis, but was suppressed by pretreatment with NLRP3 shRNA. Compared with rats in the sepsis and the scrambled shRNA groups, rats in the NLRP3 shRNA group exhibited significantly decreased serum levels of glycine and taurine conjugated-bile acids, with rehabilitated expression of hepatocyte transporters, suppressed hepatic cytokine levels, decreased hepatic neutrophils infiltration and attenuated macrophages pyroptosis. Gene silencing of NLRP3 ameliorates sepsis-induced hyper-bileacidaemia by rehabilitating hepatocyte transporter expression, reducing hepatic cytokine levels, neutrophil infiltration and macrophages pyroptosis. NLRP3 may be a pivotal target for sepsis management., (© 2014 British Society for Immunology.)

Background: BRAF (v-raf murine sarcoma viral oncogene homologue B) V600E mutations have been detected with high frequency in melanocytic naevi. Few studies have stratified analyses by naevus dermoscopic pattern., Objectives: To determine the frequency of BRAF V600E expression and histopathological pattern in acquired melanocytic naevi distinguished by a globular vs. reticular dermoscopic pattern., Methods: We retrospectively identified histologically proven melanocytic naevi with banal reticular or globular dermoscopic patterns and evaluated BRAF V600E expression using immunohistochemistry., Results: BRAF V600E expression was detected in 11 of 12 globular naevi vs. four of 13 reticular naevi (91·7% vs. 30·1%, P = 0·004). A predominantly dermal growth pattern (P < 0·001) and the presence of large junctional nests (P = 0·017) were each associated with a globular dermoscopic pattern. The presence of either a predominantly dermal growth pattern or large junctional nests was found in 13 of 15 naevi positive for BRAF V600E and in two of 10 naevi negative for BRAF V600E (86·7% vs. 20%, P = 0·002)., Conclusions: The frequency of BRAF V600E mutations differs in naevi distinguished by unique dermoscopic structures and microanatomical growth patterns. Globular naevi, which most often histologically correspond to a predominantly dermal growth pattern and/or the presence of large junctional nests, are significantly more likely to express BRAF V600E than reticular naevi. These preliminary results require validation, but may directly inform future studies of naevogenesis and melanoma genesis., (© 2014 British Association of Dermatologists.)

Eosinophilic esophagitis is characterized by eosinophil-predominant inflammation in the esophagus. How eosinophils migrate and infiltrate into the esophagus, however, is less clear. Our previous study demonstrated that mast cell activation led to eosinophil infiltration in the esophagus. Prostaglandin D2 (PGD2) is an important mediator released from activated mast cells. The present study aims to determine whether PGD2 induces eosinophil infiltration into the esophagus via a d-type prostanoid receptor 2 (DP2) receptor-dependent mechanism. Using an in vivo guinea pig model, PGD2, d-type prostanoid receptor 1 (DP1) agonist, or DP2 agonist were injected into the esophagus. Esophageal tissues were removed 2 hours after injections and proceeded to either hematoxylin-eosin (HE) staining or immunofluorescent staining of eosinophil major basic protein (MBP) to compare each treatment-induced eosinophil infiltration in the esophagus. In a separate study, ovalbumin (OVA)-sensitized guinea pigs were pretreated with either DP2 or DP1 antagonists, followed by inhalation of OVA to induce mast cell activation. Esophageal tissues were then processed for immunofluorescent staining of MBP. PGD2 injection in the esophagus led to an increase of eosinophil infiltration in esophageal epithelium at the injection site as revealed by HE staining. Increased infiltration of eosinophils was further confirmed by the increased presence of MBP-labeled immunopositive (MBP-LI) cells in esophageal epithelium. Injection with DP2 agonist 15(R)-PGD2, but not DP1 agonist BW 245C, mimicked the PGD2-induced response. In OVA-sensitized animals, antigen inhalation increased MBP-LI cells in esophageal epithelium. Pretreatment with DP2 antagonist BAY-u3405, but not DP1 antagonist BW 868C, inhibited the antigen inhalation-induced increase of MBP-LI cells in esophageal epithelium. These data support the hypothesis that PGD2 induces eosinophil trafficking into the esophageal epithelium via a DP2-mediated pathway, suggesting a role of DP2 antagonist in the prevention of eosinophilic esophagitis., (© 2013 Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.)

Aims: There is a need for novel treatments for Clostridium difficile infection(CDI). Antibacterial flavonoids are part of a large family of polyphenol phytochemicals with a long history of use in ethnomedicine, but are unexamined against Cl. difficile. We explored their anti-difficile properties., Methods and Results: Anti-difficile activities were determined for several naturally occurring flavonoids, olympicin A and synthetic 4-chromanone and chalcone analogues. With the exception of olympicin A, most naturally occurring phytochemicals tested were poorly active. Diversified synthetic flavonoids resembling olympicin A retained anti-difficile activity, suggesting olympicin A could act as a pharmacophore to obtain novel agents. They also demonstrated concentration-dependent killing of logarithmic and stationary phase cultures and reduced sporulation and toxin production. Olympicin A and some synthetic flavonoids dissipated the bacterial transmembrane potential. Interestingly, mutants could only be selected with the analogue 207 at a frequency of 10(-9)., Conclusions: Based on the potent anti-difficile properties of olympicin A and modified flavonoids, further exploration of this class of phytochemicals is warranted., Significance and Impact of the Study: Clostridium difficile infection is a major problem in developed countries. These studies point to there being an avenue for optimizing plant-derived flavonoids, and related antibacterial phytochemicals, as nature-inspired approaches to treat CDI.

The ataxia-telangiectasia group D complementing gene (ATDC) plays significant roles in various human cancers. However, the clinical significance of ATDC in esophageal squamous cell carcinoma (ESCC) has not been investigated. The ATDC messenger RNA level of 40 paired ESCC and nonneoplastic tissues were evaluated using quantitative real-time polymerase chain reaction, 10 pairs of which were also used for Western blot analysis. In addition, immunohistochemical staining was performed to detect the ATDC expression in 118 paraffin-embedded cancerous and matched nonneoplastic tissues, and the correlation of ATDC expression with the clinicopathological parameters and prognosis of the ESCC patients was analyzed. The quantitative real-time polymerase chain reaction, immunohistochemical staining, and Western blot results demonstrated that the expression level of ATDC was significantly higher in ESCC tissue than in matched noncancerous tissues. Both ATDC messenger RNA and protein expression in the ESCC tissue were significantly correlated with tumor differentiation, stage, and lymph node metastasis. However, there was no significant difference in ATDC expression based on patient age or gender. Moreover, the results of both univariate and multivariate analyses showed that increased ATDC expression was correlated with a shorter 5-year survival time for ESCC patients after surgery. We concluded that increased ATDC expression is associated with poor clinical outcomes and that this marker might be a useful indicator for prognosis and a promising target for therapy in ESCC patients., (© 2012 Copyright the Authors. Journal compilation © 2012, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.)

Aims:   To investigate the antimicrobial efficacy of an alkaloid, harmaline alone and in combination with chlorhexidine digluconate (CHG) against clinical isolates of Staphylococcus aureus (S. aureus) grown in planktonic and biofilm cultures., Methods:   Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for each micro-organism grown in suspension and in biofilm using microbroth dilution method. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between harmaline and CHG, and the some of results were verified by confocal laser scanning microscopy., Results:   Harmaline and CHG showed effective antimicrobial activity against suspensions and biofilm cultures of S. aureus, respectively. As determined by fractional inhibitory concentration index (FICI), synergistic antimicrobial effects between harmaline and CHG were observed in nine and 11 of the 13 S. aureus strains when in suspension and in biofilm, respectively. FICI values were from 0·375 to 1·25 when in suspension and from 0·25 to 1·25 when in biofilm., Conclusions:   Synergistic activity of harmaline and CHG against clinical isolates of S. aureus (in suspension and in biofilm) was observed in vitro., Significance and Impact of the Study:   This study might provide alternative methods to overcome the problem of drug-resistance of S. aureus both in suspension and in biofilm., (© 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.)

Background: Alopecia areata (AA) is a hair loss disease caused by T-cell-mediated autoimmune reactions against anagen-stage hair follicles. Although the exact aetiology is poorly understood, there is evidence to suggest that both genetic and environmental factors are involved in AA pathogenesis., Objectives: To analyse DNA methylation and histone modification patterns in peripheral blood mononuclear cells (PBMCs) of patients with AA., Methods: PBMC samples were obtained from 25 patients with AA and 20 healthy controls. Global DNA methylcytosine levels, as well as histone acetylation and methylation levels, were measured by enzyme-linked immunosorbent assay. mRNA expression levels were determined using real-time quantitative reverse transcription-polymerase chain reaction., Results: Genomic DNA methylation in PBMCs of patients with AA was increased relative to controls. DNMT1, MBD1 and MBD4 expression levels were significantly higher in AA PBMCs than in controls, and DNMT1 transcription levels positively correlated with global DNA methylation levels in patient samples. Histone H3 acetylation was significantly increased and histone H3 lysine 4 methylation was significantly decreased in patient PBMCs compared with healthy controls. Histone H3 acetylation levels were positively correlated with AA disease severity, and with RANTES (CCL5) mRNA expression in PBMCs of patients with AA. These changes were accompanied by increased p300 (EP300), histone deacetylase 1 (HDAC1), myeloid/lymphoid or mixed lineage leukemia (MLL), SET7/9 (SETD7), G9A (EHMT2), JMJD2C (KDM4C) and JARID1A (KDM5A) expression, as well as reduced HDAC2, HDAC7, LSD1 (KDM1A), JMJD2A (KDM4A) and JMJD2B (KDM4B) expression., Conclusions: DNA methylation and histone modification status are altered in PBMCs of patients with AA, possibly due to the deregulation of epigenetic regulatory genes. These changes may contribute to the activation of pathological immune responses in AA., (© 2011 The Authors. BJD © 2011 British Association of Dermatologists.)

Aims: To evaluate the interaction of 1-(1-naphthylmethyl)-piperazine (NMP) and ciprofloxacin (CPFX) in vitro against fluoroquinolone (FQ)-resistant clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA)., Methods and Results: The in vitro interaction of NMP and CPFX in 12 FQ-resistant clinical isolates of MRSA was assessed using a checkerboard microdilution method. In the study, a synergistic antimicrobial effect between NMP and CPFX was observed in all 12 FQ-resistant strains tested, as determined by the fractional inhibitory concentration index (FICI), and in 10 strains using ΔE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed using the time-killing test and agar diffusion assay for the selected strain, MRSA 1862; synergistic activity was observed when NMP was combined with the first-line antimicrobial agent CPFX against Staph. aureus., Conclusions: Synergistic activity between NMP and CPFX against clinical isolates of FQ-resistant Staph. aureus was observed in vitro., Significance and Impact of the Study: This report might provide alternative methods to reduce the resistance of Staph. aureus to CPFX., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Background: Vitiligo is a skin disorder characterized by the destruction of melanocytes by autoreactive lymphocytes. The genetic and environmental factors that trigger the autoimmune response are poorly understood. However, alterations to epigenetic DNA methylation patterns contribute to many other autoimmune diseases., Objectives: To investigate genomic and gene-specific DNA methylation levels in peripheral blood mononuclear cells (PBMCs) of patients with vitiligo and to relate any changes to the expression of genes that regulate methylation, as well as the autoimmune-related gene IL10., Methods: We quantified global methylcytosine levels in PBMCs from 20 patients with vitiligo and 20 healthy controls. mRNA levels of DNA methyltransferases (DNMTs), methyl-DNA binding domain proteins (MBDs) and interleukin (IL)-10 were measured by real-time reverse transcriptase-polymerase chain reaction. Methylation of an IL10 regulatory element domain was determined by bisulphite genomic sequencing., Results: Genomic DNA methylation in PBMCs of patients with vitiligo was increased relative to healthy controls (P = 0·012). DNMT1, MBD1, MBD3, MBD4 and MeCP2 expression was significantly higher than in control PBMCs (P = 0·013, 0·001, 0·005, 0·001 and 0·001, respectively). MBD1 and MBD3 expression correlated positively with global DNA methylation in vitiligo PBMCs (MBD1: r = 0·519, P = 0·019; MBD3: r = 0·529, P = 0·016). IL10 expression was significantly decreased (P = 0·030), and an IL-10 enhancer region was hypermethylated in vitiligo PBMCs compared with controls (P = 0·014)., Conclusions: These data show that levels of DNA methylation are altered in PBMCs of patients with vitiligo, and this may contribute to disease activity by affecting the expression of autoimmunity-related genes., (© 2010 The Authors. BJD © 2010 British Association of Dermatologists.)

Aims: To isolate a fucoidan-utilizing strain from seawater for sea cucumber fucoidan degradation., Methods and Results: The utilization of sea cucumber fucoidan was monitored by H(2)SO(4)-phenol assay for neutral sugar. The bacterium CZ1127 was isolated from seawater and shown to have a relatively large maximum fucoidan-utilizing rate of 81.5%. CZ1127 was confirmed to belong to the family Flavobacteriaceae by 16S rDNA and physiological analyses. This strain has an ability to utilize fucoidans extracted from various sea cucumbers to different degrees. Both extracellular and intracellular enzymes of CZ1127 could degrade sea cucumber fucoidan, as confirmed by high-performance size exclusion chromatography. The M(r) of sea cucumber fucoidan could be reduced from 792.6 kDa to at least 3.7 kDa by the crude intracellular enzyme of this strain., Conclusions: The marine bacterial strain CZ1127, which belongs to the family Flavobacteriaceae, was found to utilize various sea cucumber fucoidans and furthermore showed promise in sea cucumber fucoidan enzymatic degradation and oligosaccharide preparation., Significance and Impact of the Study: The finding of a novel source can be applied in sea cucumber fucoidan enzymatic degradation. Furthermore, it is the first definite report of a bacterial strain that can utilize the fucoidans from various sea cucumbers.

Aim: To determine the transcriptional responses of model fungus Saccharomyces cerevisiae (S. cerevisiae) cells upon exposure to thymol (THY)., Methods and Results: Commercial oligonucleotide microarrays and quantitative real-time RT-PCR were used to study the transcriptional responses of S. cerevisiae to THY. Compared with the transcriptional profiles of untreated cultures, 305 genes were significantly upregulated, and 212 genes were significantly downregulated in THY-treated cells. We interpreted the microarray data using a hierarchical clustering tool, T-profiler. Glucose-dependent efflux of rhodamine 6G (R6G) from S. cerevisiae was performed to assay a phenotypic correlation between ATP-binding cassette transporter overexpression induced by THY and efflux of R6G. The addition of THY resulted in an increase in the concentration of released fluorescence, following the addition of glucose. Additional phenotype analysis showed that the intracellular concentration of thiamine was decreased by THY, and THY led to lesions in the membranes; these were consistent with the results of microarray., Conclusion: The transcriptional response of S. cerevisiae to THY was determined, and several changes in genetic regulation were verified in physiological effects on the cell., Significance and Impact of the Study: Taken a whole-genome view to elucidate the mechanism of THY as a potential antifungal agent.

Background: Gonadal damage is a consequence of therapy for pediatric malignancies. Prepubertal males have no semen or mature spermatozoa, posing a challenge for fertility preservation. Testicular tissue cryopreservation is a potential option but is still experimental. We report on a pilot protocol that offered testicular biopsy cryopreservation to families of prepubertal boys with newly diagnosed malignancy. The aims were to determine the acceptability and safety of this procedure., Methods: Parents of prepubertal boys with diagnoses at highest risk for treatment-related gonadal damage were offered the option of testicular cryopreservation. Half of the biopsy was frozen for the subject's potential future use and the remainder used for research. Data on negative intraoperative and/or 7 day post-operative sequelae of testicular biopsies were assessed. Two to four weeks later, parents were asked to complete a questionnaire on factors influencing their decision to have the biopsy or not., Results: Since January 2008, 24 boys have met the eligibility criteria but three required immediate treatment and were excluded. Sixteen of 21 families (76%) consented to testicular biopsy, indicating the prospective acceptability of this option to parents of boys aged 3 months to 14 years; 14 underwent the procedure without any negative intra- or post-operative sequelae. Although the time at diagnosis is stressful, families can give thoughtful consideration to this option. Factors such as religion, finance, ethics and the experimental nature of cryopreservation did not play a major role in decision-making., Conclusions: Parents of prepubertal boys with cancer are willing to pursue testicular tissue cryopreservation at diagnosis, and testicular biopsy caused no acute adverse effects.

Tissue integrity relies on barriers formed between epithelial cells. In the testis, the barrier is formed at the initiation of puberty by a tight junction complex between adjacent Sertoli cells, thereby defining an adluminal compartment where meiosis and spermiogenesis occur. Claudin 11 is an obligatory protein for tight junction formation and barrier integrity in the testis. It is expressed by Sertoli cells, and spermatogenesis does not proceed beyond meiosis in its absence, resulting in male sterility. Sertoli cell maturation--arrest of proliferation and expression of proteins to support germ cell development--parallels tight junction assembly; however, the pathophysiology underlying the loss of tight junctions in the mature testis remains largely undefined. Here, using immunohistochemistry and microarrays we demonstrate that adult Cldn11(-/-) mouse Sertoli cells can proliferate while maintaining expression of mature markers. Sertoli cells detach from the basement membrane, acquire a fibroblast cell shape, are eliminated through the lumen together with apoptotic germ cells, and are found in epididymis. These changes are associated with tight junction regulation as well as actin-related and cell cycle gene expression. Thus, Cldn11(-/-) Sertoli cells exhibit a unique phenotype whereby loss of tight junction integrity results in loss of the epithelial phenotype.

Motivation: With the increasing use of post-genomics techniques to examine a wide variety of biological systems in laboratories throughout the world, scientists are often presented with lists of genes that they must make sense of. A consistently challenging problem is that of defining co-regulated genes within those gene lists. In recent years, microRNAs have emerged as a mechanism for regulating several cellular processes. In this article, we report on how gene lists and microRNA targets data may be integrated to test for significant associations between gene lists and microRNAs., Results: We discuss CORNA, a package written in R and released under the GNU GPL, which allows users to test gene lists for significant microRNA-target associations using one of three separate statistical tests, to link microRNA targets to functional annotation and to visualize quantitative data associated with those data., Availability: CORNA is available as an R package from http://corna.sf.net.

Background: Genetic defects in the oocyte can lead to female infertility due to failures in early embryogenesis. NLRP5 (NLR family, Pyrin domain containing 5) is a maternal effect factor required for early embryo development beyond the 2-cell stage in mice. The NLRP5 gene is conserved in mammals. Applying RNA interference (RNAi) technique in the macaque oocyte, we studied the in vitro effect of NLRP5 in preimplantation embryo development in rhesus macaques., Methods: Macaque MII oocytes were collected and subjected to NLRP5 long double-stranded (ds) RNA microinjection, followed by intracytoplasmic sperm injection and in vitro embryo culture. NLRP5 mRNA and protein expression were examined, and the progress of preimplantation embryo development was monitored until the blastocyst stage., Result: While 46.7 and 41.4% of embryos derived from uninjected and EGFP (Enhanced Green Fluorescence Protein) dsRNA-injected oocytes (control) reached the blastocyst stage within 10 days of culture, respectively, only 7.0% of the NLRP5 knockdown embryos developed to term. The majority of NLRP5 knockdown embryos were arrested between the 8-cell and the 16-cell stages., Conclusions: The work provides the first evidence that long dsRNA-mediated RNAi can be applied to specific gene silencing in rhesus macaque oocytes, and demonstrates that maternal NLRP5 plays an important role in preimplantation embryogenesis in non-human primates.

Activated nuclear factor-kappa B (NF-kappaB) in the pretreatment cancer tissue of patients with localized esophageal adenocarcinoma (LEA) undergoing preoperative chemoradiation is associated with poor prognosis. It is known that constitutively activated NF-kappaB prior to any therapy portends poor prognosis, and it is also known that activated NF-kappaB in the treated specimen is associated with poor prognosis. However, the prognosis of patients who have treatment-induced activation of NF-kappaB (meaning their cancers activate NF-kappaB during or after therapy) is not been reported. We hypothesized that the treatment-induced activation of NF-kappaB would impart poor prognosis similar to that imparted by constitutively activated NF-kappaB cancer. Patients with LEA who had undergone preoperative chemoradiation plus surgery and had pre- and post-therapy cancer tissue available were selected. Pre- and post-therapy cancer tissues were stained by immunohistochemistry for nuclear staining of NF-kappaB. The overall survival (OS) and disease-free survival were assessed and compared for patients who had intrinsic constitutively activated NF-kappaB cancer with those who had induced activation of NF-kappaB only post-therapy. A total of 41 patients with LEA were investigated. Twenty-five patients had NF-kappaB positive cancer at baseline, and 16 had NF-kappaB negative cancer at baseline but became positive post-therapy. There was no difference in the location, histology grade, clinical stage, or the curative resection (RO) resection rate in the two populations. OS (P = 0.71), disease-free survival (P = 0.86), and median survivals (Converters: 24 months [95% confidence intervals: 7.78 to 40.22]vs. Nonconverters: 34.13 months [95% confidence intervals: 3.54 to 64.27]) were not different between the two groups. Our data suggest that activation of NF-kappaB in response to stress/injury of therapy leads to poor OS. These results need to be confirmed in a larger number of patients. It may be that only pre-therapy evaluation of NF-kappaB is insufficient to assess prognosis of patients with LEA. Additional implications include that when effective anti-NF-kappaB therapies become available, they may have to be considered in patients whose cancers do not have constitutively activated NF-kappaB or cancer may have to be monitored during therapy with biomarker assessments.

Aims: To determine whether resuscitation-promoting factor (RPF) from Mycobacterium tuberculosis can promote mycobacterial growth and shorten culture time., Method and Results: We cloned, expressed and purified an RPF from M. tuberculosis, Rv1009 protein and subsequently studied the biological activity of the recombinant Rv1009 (rRv1009) in liquid and on solid media. Our results indicate that the molecular weight of rRv1009 protein expressed in Escherichia coli BL21 was approximately 39 kDa. At picomolar and micromolar concentrations, rRv1009 protein could increase the optical density of freeze-dried Mycobacterium bovis BCG three to fivefold in Middlebrook 7H9 medium, stimulate the growth of viable mycobacteria on solid medium, and shorten positive growth detection time of a small number of M. tuberculosis in BACTEC 960 medium., Conclusions: The rRv1009 could promote proliferation of mycobacteria. It may be useful for culture of mycobacteria presented in clinical samples., Significance and Impact of the Study: rRv1009 protein can be used as a growth-promoting reagent of mycobacteria in the medium to shorten the time of culture.

Sorghum is a major cereal crop in the USA. However, sorghum has been underutilized as a renewable feedstock for bioenergy. The goal of this research was to improve the bioconversion efficiency for biofuels and biobased products from processed sorghum. The main focus was to understand the relationship among "genetics-structure-function-conversion" and the key factors impacting ethanol production, as well as to develop an energy life cycle analysis model (ELCAM) to quantify and prioritize the saving potential from factors identified in this research. Genetic lines with extremely high and low ethanol fermentation efficiency and some specific attributes that may be manipulated to improve the bioconversion rate of sorghum were identified. In general, ethanol yield increased as starch content increased. However, no linear relationship between starch content and fermentation efficiency was found. Key factors affecting the ethanol fermentation efficiency of sorghum include protein digestibility, level of extractable proteins, protein and starch interaction, mash viscosity, amount of phenolic compounds, ratio of amylose to amylopectin, and formation of amylose-lipid complexes in the mash. A platform ELCAM with a base case showed a positive net energy value (NEV) = 25,500 Btu/gal EtOH. ELCAM cases were used to identify factors that most impact sorghum use. For example, a yield increase of 40 bu/ac resulted in NEV increasing from 7 million to 12 million Btu/ac. An 8% increase in starch provided an incremental 1.2 million Btu/ac.

Although the delayed-type hypersensitivity skin test reaction to tuberculin purified protein derivative (PPD) is used worldwide for tuberculosis (TB) detection, it is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacille Calmette-Guérin (BCG) vaccination or infection with non-tuberculous Mycobacteria. As a result, there is an urgent need for a more specific diagnostic tool for TB. This study reports the skin reactions of guinea pigs and human volunteers to recombinant early secreted antigen target 6 (rESAT6), a secretory protein found only in MTB, M. bovis and few other mycobacterial species. These volunteers had varying histories of BCG vaccination and exposure to MTB, allowing us to determine the specificity of their response to TB exposure. Our results show that 1.0 microg of the purified MTB rESAT6 antigen elicited a positive skin response in both animals and humans exposed to MTB, as well as in animals exposed to M. bovis and M. marinum, all species of Mycobacteria that contain the gene for early secreted antigen target 6 (ESAT6). ESAT6 appears to be more specific to MTB infection than PPD, as demonstrated by the fact that we saw no skin responses in the BCG-vaccinated volunteers, nor in the guinea pigs sensitized with BCG vaccine, or with Mycobacteria that do not contain the gene encoding ESAT6. We believe that this is the first report of the use of a rESAT6 protein in a skin test in human volunteers, and that these data support its use in the specific detection of MTB infection.

Aims: To discover if human colorectal cancer expresses granulocyte colony stimulating factor receptor (G-CSFR) and if granulocyte colony stimulating factor (G-CSF) plays an important part in the development and progression of human colorectal cancer., Methods: Forty two specimens of colorectal cancer and normal colorectal mucosa were investigated, taken from the colon or rectum in group of colorectal cancer patients. Immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) technique were used to show G-CSFR expression. The relation between expression of G-CSFR and clinical or pathological factors was analysed., Results: Immucohistochemical analyses showed that G-CSFR was expressed in the human colorectal cancer (25 of 42, 59.52%) and seemed to be up-regulated compared with the normal mucosa (14 of 42, 33.33%, p<0.001). In pronounced contrast with mostly strongly positive tumours, corresponding normal colorectal mucosa was negative or weakly positive. A significant correlation was found between G-CSFR expression and tumour stage (p = 0.001), tumour differentiation (p<0.001), but there was no significant relation between the expression of G-CSFR and the age, sex, and tumour size (p = 0.346, p = 0.686, p = 0.459). In RT-PCR, 21 of all 42 tumours had G-CSFR mRNA expression, while only 11 of 42 normal colorectal mucosa had such expression., Conclusions: These data show that G-CSFR is commonly expressed in human colorectal cancers, thus supporting a possible role for G-CSF in colorectal cancer physiology.

Compared with the sc depot, omental (om) adipose tissue is relatively resistant to the metabolic actions of insulin. Protein-tyrosine phosphatases (PTPases) modulate receptor kinase activation and signal transduction in insulin-sensitive tissues, and their activity is dependent on the reduced state of the cysteine thiol required for catalysis. Using a novel anaerobic technique to avoid air oxidation, we found that the mean endogenous PTPase activity was 2.1-fold higher in om compared with paired samples of sc adipose tissue (P < 0.003). The specific activity of PTP1B isolated under anaerobic conditions was also 41% higher in om adipose tissue (P < 0.001). Interestingly, the total PTPase activity from both adipose depots and the specific activity of PTP1B was increased by 42-71% after reduction in vitro with dithiothreitol, indicating that a major fraction of the cellular PTPase activity can be reactivated by sulfhydryl reduction. The mass of the insulin receptor beta-subunit and the PTPases PTP1B and leukocyte antigen related was not significantly different between the two adipose depots. These studies provide the first demonstration that endogenous PTPase activity, including PTP1B, is increased in om adipose tissue and may contribute to the relative insulin resistance of this fat depot. The finding that a substantial fraction of PTPase activity in human adipose tissue is present in a latent, oxidized form also suggests a potential means of in vivo regulation of these important cellular enzymes that modulate the insulin signaling cascade.

Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) beta, zeta or eta. Base excision repair was also proficient without Polbeta. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polalpha (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Poldelta (pol3-1) or Polvarepsilon (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Poldelta and Polvarepsilon, respectively. Deleting the polymerase catalytic domain of Polvarepsilon (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Polvarepsilon, but not by purified yeast Poleta. These results suggest that efficient repair synthesis of yeast NER requires both Poldelta and Polvarepsilon in vitro, and that the low fidelity Poleta is not accessible to repair synthesis during NER.

In the yeast Saccharomyces cerevisiae, DNA polymerase zeta (Polzeta) is required in a major lesion bypass pathway. To help understand the role of Polzeta in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions. Purified yeast Polzeta performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3' T, and predominantly A opposite the 5' T. Purified yeast Polzeta predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine. The lesion, however, significantly inhibited subsequent extension. Furthermore, yeast Polzeta catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3' T of the TT (6-4) photoproduct with varying efficiencies. Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3' T of the TT (6-4) photoproduct. In contrast, the 3' T of a cis-syn TT dimer completely blocked purified yeast Polzeta, whereas the 5' T was readily bypassed. These results support the following dual-function model of Polzeta. First, Polzeta catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency. Secondly, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Polzeta.

The RecQ helicases constitute a small but highly conserved helicase family. Proteins in this family are of particular interest because they are critical to maintenance of genomic stability in prokaryotes and eukaryotes. Eukaryotic RecQ helicase family members have been shown to unwind not only DNA duplexes but also DNAs with alternative structures, including structures stabilized by G quartets (G4 DNAs). We report that Escherichia coli RecQ can also unwind G4 DNAs, and that unwinding requires ATP and divalent cation. RecQ helicase is comparably active on duplex and G4 DNA substrates, as measured by direct comparison of protein activity and by competition assays. The porphyrin derivative, N-methyl mesoporphyrin IX (NMM), is a highly specific inhibitor of RecQ unwinding activity on G4 DNA but not duplex DNA: the inhibition constant (K(i)) for NMM inhibition of G4 DNA unwinding is 1.7 microM, approximately two orders of magnitude below the K(i) for inhibition of duplex DNA unwinding (>100 microM). NMM may therefore prove to be a valuable compound for substrate-specific inhibition of other RecQ family helicases in vitro and in vivo.

Lesion bypass is an important mechanism to overcome replication blockage by DNA damage. Translesion synthesis requires a DNA polymerase (Pol). Human Pol iota encoded by the RAD30B gene is a recently identified DNA polymerase that shares sequence similarity to Pol eta. To investigate whether human Pol iota plays a role in lesion bypass we examined the response of this polymerase to several types of DNA damage in vitro. Surprisingly, 8-oxoguanine significantly blocked human Pol iota. Nevertheless, translesion DNA synthesis opposite 8-oxoguanine was observed with increasing concentrations of purified human Pol iota, resulting in predominant C and less frequent A incorporation opposite the lesion. Opposite a template abasic site human Pol iota efficiently incorporated a G, less frequently a T and even less frequently an A. Opposite an AAF-adducted guanine, human Pol iota was able to incorporate predominantly a C. In both cases, however, further DNA synthesis was not observed. Purified human Pol iota responded to a template TT (6-4) photoproduct by inserting predominantly an A opposite the 3' T of the lesion before aborting DNA synthesis. In contrast, human Pol iota was largely unresponsive to a template TT cis-syn cyclobutane dimer. These results suggest a role for human Pol iota in DNA lesion bypass.

Vascular endothelial growth factor (VEGF) is a major inducer of tumor angiogenesis and edema in human astrocytomas by its interaction with cognate endothelial-specific receptors (VEGFR1/R2). Tie1 and Tie2/Tek are more recently identified endothelial-specific receptors, with angiopoietins being ligands for the latter. These angiogenic factors and receptors are crucial for the maturation of the vascular system, but their role in tumor angiogenesis, particularly in astrocytomas, is unknown. In this study, we demonstrate that the angiopoietin family member Ang1 is expressed by some of the astrocytoma cell lines. In contrast to VEGF, Ang1 is down regulated by hypoxia. Ang2 was not overexpressed. Expression profiles of low-grade astrocytoma specimens were similar to those of normal brain, with low levels of Ang1, Ang2, and VEGF expression. Glioblastoma multiforme expressed higher levels of Angl, but not to the same degree as pseudopalisading astrocytoma cells around necrotic and hypoxic zones expressed VEGF, as shown in previous studies. Ang2 expression in the highly proliferative tumor vascular endothelium was also increased, as was phosphorylated Tie2/Tek. The expression profile of these angiogenic factors and their endothelial cell receptors in human glioblastomas multiforme was similar to that in a transgenic mouse model of glioblastoma multiforme. These data suggest that both VEGF and angiopoietins are involved in regulating tumor angiogenesis in human astrocytomas.

In an earlier study of 37 candidate genes for polycystic ovary syndrome (PCOS), the strongest evidence for genetic linkage was found with the region of the follistatin gene. We have now carried out studies to detect variation in the follistatin gene and assess its relevance to PCOS. By sequencing the gene in 85 members of 19 families of PCOS patients, we found sequence variants at 17 sites. Of these, 16 sites have variants that are too rare to make a major contribution to susceptibility; the only common variant is a single base pair change in the last exon at a site that is not translated. In our sample of 249 families, the evidence for linkage between PCOS and this variant is weak. We also examined the expression of the follistatin gene; messenger RNA levels in cultured fibroblasts from PCOS and control women did not differ appreciably. We conclude that contributions to the etiology of PCOS from the follistatin gene, if any, are likely to be small.

DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase eta (Pol(eta)), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Pol(eta), we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Pol(eta) efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Pol(eta) effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant -1 deletion was also observed when the template base 5' to the abasic site is a T. Human Pol(eta) partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N:(2)-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in mammalian cells. These results show that human Pol(eta) is capable of error-prone translesion DNA syntheses in vitro and suggest that Pol(eta) may bypass certain lesions with a mutagenic consequence in humans.

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

Escherichia coli DNA polymerase IV encoded by the dinB gene is involved in untargeted mutagenesis. Its human homologue is DNA polymerase kappa (Polkappa) encoded by the DINB1 gene. Our recent studies have indicated that human Polkappa is capable of both error-free and error-prone translesion DNA synthesis in vitro. However, it is not known whether human Polkappa also plays a role in untargeted mutagenesis. To examine this possibility, we have measured the fidelity of human Polkappa during DNA synthesis from undamaged templates. Using kinetic measurements of nucleotide incorporations and a fidelity assay with gapped M13mp2 DNA, we show that human Polkappa synthesizes DNA with extraordinarily low fidelity. At the lacZalpha target gene, human Polkappa made on average one error for every 200 nucleotides synthesized, with a predominant T-->G transversion mutation at a rate of 1/147. The overall error rate of human Polkappa is 1.7-fold lower than human Poleta, but 33-fold higher than human Polbeta, a DNA polymerase with very low fidelity. Thus, human Polkappa is one of the most inaccurate DNA polymerases known. These results support a role for human Polkappa in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage.

Brassinosteroids (BRs) are plant growth-promoting natural products required for plant growth and development. Physiological studies have demonstrated that exogenous BR, alone or in combination with auxin, enhance bending of the lamina joint of rice. However, little is known about the function of endogenous BR in rice or other grass species. We report here the phenotypical and molecular characterization of a rice dwarf mutant, d61, that is less sensitive to BR compared to the wild type. We cloned a rice gene, OsBRI1, with extensive sequence similarity to that of the Arabidopsis BRI gene, which encodes a putative BR receptor kinase. Linkage analysis showed that the OsBRI1 gene is closely linked to the d61 locus. Single nucleotide substitutions found at different sites of the d61 alleles would give rise to amino acid changes in the corresponding polypeptides. Furthermore, introduction of the entire OsBRI1 coding region, including the 5' and 3' flanking sequences, into d61 plants complemented the mutation to display the wild-type phenotype. Transgenic plants carrying the antisense strand of the OsBRI1 transcript showed similar or even more severe phenotypes than those of the d61 mutants. Our results show that OsBRI1 functions in various growth and developmental processes in rice, including (1) internode elongation, by inducing the formation of the intercalary meristem and the longitudinal elongation of internode cells; (2) bending of the lamina joint; and (3) skotomorphogenesis.

During DNA replication, lesion bypass is an important cellular response to unrepaired damage in the genome. In the yeast Saccharomyces cerevisiae, Rad6 and Rad18 are required for both the error-free and error-prone lesion bypass mechanisms. Furthermore, Rad6-Rad18 interaction is thought to be critical at an early step during lesion bypass in yeast. Two closely related human homologs of yeast Rad6 have been identified as HHR6A and HHR6B. Here, we report a full-length cDNA coding for the human homolog of yeast Rad18. The human RAD18 gene codes for a protein of 484 amino acid residues with a calculated molecular weight of 54 804 Da, and the gene is localized to chromosome 3 between reference intervals D3S3591 and D3S1283. Human RAD18 protein (hRAD18) was found to interact with HHR6A and HHR6B. When co-expressed in yeast cells, stable hRAD18-HHR6A and hRAD18-HHR6B protein complexes were identified and purified to near homogeneity. Thus, through interaction and complex formation with HHR6A and HHR6B, RAD18 protein may play an important role in lesion bypass mechanisms in humans. Consistent with its role as a fundamental lesion bypass protein, the RAD18 gene is ubiquitously expressed in various human tissues.

Background: We hypothesize that accumulation of genetic damage is dependent on an individual's intrinsic carcinogen sensitivity and on various humoral factors (e.g., insulin-like growth factors [IGFs]) that enhance proliferation, resistance to apoptotic cell death, and clonal outgrowth of genetically damaged cells. We tested this hypothesis by determining whether proliferation potential and genetic instability are associated with the risk of lung cancer., Methods: In a study of 183 lung cancer patients and 227 matched control subjects, we examined the joint effects of latent genetic instability (measured as mutagen sensitivity) and elevated proliferation potential (assessed by measuring IGFs) in lung cancer risk. Levels of IGF-I, IGF-II, and IGF-binding protein-3 (IGFBP-3) in plasma were measured by use of immunoassay kits. Mutagen sensitivity was assessed by quantitating bleomycin- and benzo[a]pyrene diol epoxide (BPDE)-induced chromatid breaks in peripheral blood lymphocyte cultures., Results: Although not statistically significant, the mean levels of IGF-I and the molar ratio of IGF-I/IGFBP-3 were higher in patients with advanced or poorly differentiated disease than in patients with early or well-differentiated disease. Variation in IGFs was not associated with any specific histologic type or tumor stage. High levels of IGF-I and enhanced mutagen sensitivity were individually associated with increased risk of lung cancer: odds ratio (OR) of 2.13 (95% confidence interval [CI] = 1.20-3.78) for IGF-I, 2.50 (95% CI = 1. 49-4.20) for bleomycin sensitivity, and 2.95 (95% CI = 1.72-5.06) for BPDE sensitivity. The OR was statistically significantly elevated to 8.88 for both higher IGF-I and bleomycin sensitivity (95% CI = 3.67-21.50) and to 13.53 for higher IGF-I and BPDE sensitivity combined (95% CI = 4.48-40.89). With all three risk factors considered together, the OR was 17.09 (95% CI = 4.16-70.27). High levels of IGFBP-3 alone were associated with reduced lung cancer risk: OR = 0.59 (95% CI = 0.33-1.05)., Conclusions: Our data suggest that individuals with genetic instability and higher proliferation potential are at enhanced risk for lung cancer.

Background: Cardiovascular disease is rare in China, but there are few data on the prevalence of electrocardiographic (ECG) abnormalities in Chinese populations., Methods: The ECG surveys were carried out in four Chinese population samples, in a total of 9,666 adults aged 35-54 in Beijing and Guangzhou, China from 1981 to 1984. Twelve-lead resting ECG tracings were coded by the Minnesota Code., Results: Prevalence per 1,000 of abnormal ECG ranged from 77.4 to 209.8, and was higher for men than women and higher for Guangzhou than Beijing. Prevalence per 1,000 of major abnormalities in Guangzhou was 29.8 for men and 78.4 for women, higher than the 18.4 and 29.6 for counterparts in Beijing. The ECG changes attributed in 'Western' populations to coronary heart disease (CHD), such as large Q waves (Minnesota Code 1-1, 1-2) and ST-T abnormalities, were similar between Beijing and Guangzhou men, but Guangzhou women had much higher prevalence of ST-T abnormalities than Beijing women. Other ECG abnormalities such as A-V block, left branch bundle block, and left ventricular hypertrophy were rare in people of both sites., Conclusions: Compared with similar data from the US, these Chinese populations had a relatively low prevalence of ECG abnormalities putatively related to CHD. This corresponds with the low incidence of CHD in the Chinese population. However, within the Chinese populations of this study, a high abnormality rate appeared in a population with low incidence of CHD and hypertension (Guangzhou women). Reasons why ECG abnormalities do not parallel prevalence levels of CHD and hypertension remain to be elucidated.

Lentiviral vectors efficiently transduce human CD34(+) cells that mediate long-term engraftment of nonobese diabetic/severe combined immunodeficient mice. However, hematopoiesis in these animals is abnormal. Typically, 95% of the human cells in peripheral blood are B lymphocytes. To determine whether lentiviral vectors efficiently transduce stem cells that maintain normal hematopoiesis in vivo, we isolated Sca-1(+)c-Kit(+)Lin(-) bone marrow cells from mice without 5-fluorouracil treatment, and transduced these cells in the absence of cytokine stimulation with a novel lentiviral vector containing a GFP (green flourescent protein) reporter gene. These cells were transplanted into lethally irradiated C57Bl/6 mice. In fully reconstituted animals, GFP expression was observed in 8.0% of peripheral blood mononuclear cells for 20 weeks posttransplantation. Lineage analysis demonstrated that a similar percentage (approximately 8.0%) of GFP-positive cells was detected in peripheral blood B cells, T cells, granulocytes and monocytes, bone marrow erythroid precursor cells, splenic B cells, and thymic T cells. In secondary transplant recipients, up to 20% of some lineages expressed GFP. Our results suggest that quiescent, hematopoietic stem cells are efficiently transduced by lentiviral vectors without impairing self-renewal and normal lineage specification in vivo. Efficient gene delivery into murine stem cells with lentiviral vectors will allow direct tests of genetic therapies in mouse models of hematopoietic diseases such as sickle cell anemia and thalassemia, in which corrected cells may have a selective survival advantage.

DNA is frequently damaged by various physical and chemical agents. DNA damage can lead to mutations during replication. In the yeast Saccharomyces cerevisiae, the damage-induced mutagenesis pathway requires the Rev1 protein. We have isolated a human cDNA homologous to the yeast REV1 gene. The human REV1 cDNA consists of 4255 bp and codes for a protein of 1251 amino acid residues with a calculated molecular weight of 138 248 Da. The human REV1 gene is localized between 2q11.1 and 2q11.2. We show that the human REV1 protein is a dCMP transferase that specifically inserts a dCMP residue opposite a DNA template G. In addition, the human REV1 transferase is able to efficiently and specifically insert a dCMP opposite a DNA template apurinic/apyrimidinic (AP) site or a uracil residue. These results suggest that the REV1 transferase may play a critical role during mutagenic translesion DNA synthesis bypassing a template AP site in human cells. Consistent with its role as a fundamental mutagenic protein, the REV1 gene is ubiquitously expressed in various human tissues.

We have generated rat monoclonal antibodies specific for the mouse eotaxin receptor, C-C chemokine receptor 3 (CCR3). Several anti-CCR3 mAbs proved to be useful for in vivo depletion of CCR3-expressing cells and immunofluorescent staining. In vivo CCR3 mAbs of the IgG2b isotype substantially depleted blood eosinophil levels in Nippostrongyus brasiliensis-infected mice. Repeated anti-CCR3 mAb treatment in these mice significantly reduced tissue eosinophilia in the lung tissue and bronchoalveolar lavage fluid. Flow cytometry revealed that mCCR3 was expressed on eosinophils but not on stem cells, dendritic cells, or cells from the thymus, lymph node, or spleen of normal mice. Unlike human Th2 cells, mouse Th2 cells did not express detectable levels of CCR3 nor did they give a measurable response to eotaxin. None of the mAbs were antagonists or agonists of CCR3 calcium mobilization. To our knowledge, the antibodies described here are the first mAbs reported to be specific for mouse eosinophils and to be readily applicable for the detection, isolation, and in vivo depletion of eosinophils.