Regulation of bacteriocin production in Lactobacillus plantarum depends on a conserved promoter arrangement with consensus binding sequence

Bacteriocin production in Lactobacillus plantarum C11 is regulated by a three-component signal transduction system comprising a peptide pheromone (PlnA), a histidine protein kinase (PlnB), and two homologous response regulators (RRs; PlnC and PlnD). Both RRs are DNA-binding proteins that bind to promoter-proximal elements in the pln regulon. The binding site for the two regulators consists of two 9-bp direct repeats, that conform to the consensus sequence 5'-TACGTTAAT-3', and the repeats are separated by an intervening 12-bp AT-rich spacer region. In the present work, the plhA promoter was used as a model to evaluate the significance of the binding sequence and conserved promoter arrangement. Point substitutions in the consensus sequence, particularly those in invariant positions, either abolished or significantly reduced binding of PlnC and PlnD. Both regulators bind as homodimers to DNA fragments containing a complete set of regulatory elements, while removal of either repeat, or alterations in the length of the spacer region, significantly weakened binding of both protein dimers. DNase I footprinting demonstrated that PlnC and PlnD both bind to, and protect, the direct repeats. By fusing the plnA promoter region to the beta-glucuronidase (GUS) gene, it was shown that promoter activity is dependent on an intact set of accurately organized repeats. The in vitro and in vivo results presented here confirm the involvement of the repeats as regulatory elements in the regulation of bacteriocin production.     

N-acylation of phosphatidylethanolamine and its biological functions in mammals

N-acylphosphatidylethanolamine (NAPE) and N-acylplasmenylethanolamine (pNAPE) are widely found phospholipids, and they are precursors for N-acylethanolamines, a group of compounds that has a variety of biological effects and encompasses the endocannabinoid anandamide. NAPE and pNAPE are synthesized by the transfer of an acyl chain from a donor phospholipid, to the amine in phosphatidylethanolamine or plasmenylethanolamine. NAPE has been reported to stabilize model membranes during brain ischemia, and to modulate food intake in rodents, thus having bioactive effects besides its precursor role. This paper reviews the metabolism, occurrence and assay of NAPE and pNAPE, and discusses the putative biological functions in mammals of these phospholipids. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

肠内营养对乳腺癌术后化疗患者肠道菌群及生活质量的影响

目的研究肠内营养对乳腺癌术后化疗患者肠道菌群、生活质量、营养指标水平的影响。方法选取2012年7月至2018年1月于我院进行乳腺癌术后化疗的91例女性乳腺癌患者为研究对象,根据是否进行肠内营养将患者分为肠内营养组和对照组,观察化疗前后2组患者肠道菌群、生活质量、营养指标水平的变化及不良反应发生情况。结果治疗前2组患者肠道菌群、生活质量、营养指标水平差异无统计学意义(均P0.05)。治疗后,肠内营养组患者肠道双歧杆菌数量为(6.7±0.5)lg CFU/g、乳杆菌数量为(8.5±0.4)lg CFU/g,均显著高于治疗前,同时显著高于对照组(均P0.05)。治疗后肠内营养组患者出现反酸、腹胀、便秘等胃肠道不良反应的比例与对照组比较差异无统计学意义(均P0.05)。治疗后两组患者总体健康、认知功能、情绪功能、角色功能、社会功能、活力、躯体疼痛、躯体功能评分显著高于治疗前(均P0.05),且肠内营养组患者生活质量各指标的评分显著高于对照组(均P0.05)。治疗后肠内营养组患者血清总蛋白(TB)、血清清蛋白(ALB)、血清前清蛋白(PA)、转铁蛋白(TF)水平均显著高于对照组(均P0.05)。结论肠内营养可预防乳腺癌术后化疗患者肠道菌群的紊乱及营养不良的发生,安全有效,有助于提高乳腺癌患者的生活质量。     

Rapid Diagnosis of Diarrhea Caused by Shigella sonnei Using Dipsticks; Comparison of Rectal Swabs,Direct Stool and Stool Culture

Background

We evaluated a dipstick test for rapid detection of Shigella sonnei on bacterial colonies, directly on stools and from rectal swabs because in actual field situations, most pathologic specimens for diagnosis correspond to stool samples or rectal swabs.

Methodology/Principal Findings

The test is based on the detection of S. sonnei lipopolysaccharide (LPS) O-side chains using phase I-specific monoclonal antibodies coupled to gold particles, and displayed on a one-step immunochromatographic dipstick. A concentration as low as 5 ng/ml of LPS was detected in distilled water and in reconstituted stools in 6 minutes. This is the optimal time for lecture to avoid errors of interpretation. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 4 x 10⁶ CFU/ml of S. sonnei. The specificity was 100% when tested with a battery of Shigella and different unrelated strains. When tested on 342 rectal swabs in Chile, specificity (281/295) was 95.3% (95% CI: 92.9% - 97.7%) and sensitivity (47/47) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 95.5 % of cases (328/342) in comparative studies. Positive and negative predictive values were 77% (95% CI: 65% - 86.5%) and 100% respectively. When tested on 219 stools in Chile, Vietnam, India and France, specificity (190/198) was 96% (95% CI 92%–98%) and sensitivity (21/21) was 100%. Stool cultures and the immunochromatographic test showed concordant results in 96.3 % of cases (211/219) in comparative studies. Positive and negative predictive values were 72.4% (95% CI 56.1%–88.6%) and 100 %, respectively.

Conclusion

This one-step dipstick test performed well for diagnosis of S. sonnei both on stools and on rectal swabs. These data confirm a preliminary study done in Chile.     

Hybridization between diploid Centaurea pseudophrygia and tetraploid C. jacea (Asteraceae): the role of mixed pollination,unreduced gametes,and mentor effects

We studied hybridization between the diploid Centaurea pseudophrygia and the tetraploid C. jacea by performing crossing experiments and screening natural populations using flow cytometry. The experiments confirm that the studied species exhibit strong reproductive isolation. Interspecific hybrids were formed at a low frequency, including triploids (originating from reduced gametes) and tetraploids (involving unreduced gametes of the diploids). In contrast, hybrids were almost absent among seeds and adult plants of natural mixed populations and among the offspring from experimental pollinations with a mixture of pollen of both ploidy levels. We found that mixed pollination is an important mechanism for preventing hybridization between plants of different ploidy levels and sustaining the reproduction of the tetraploids. A mentor effect (induced selfing in the presence of pollen of different ploidy levels) was observed in both diploids and tetraploids, reinforcing the reproductive isolation between cytotypes. Higher ploidy levels (pentaploid, hexaploid) involving unreduced gametes of the tetraploid species were identified. Notably, pentaploids were discovered for the first time in Centaurea sect. Jacea. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 93–106.     

马卵母细胞体外成熟的初步研究

采用抽吸法和切割法两种采卵方法收集马卵母细胞,显示采用切割法的卵母细胞回收率为83%,高于抽吸法的回收率46.5%(P＜0.05),在卵母细胞的成熟上,使用M199和DMEM/F12两种培养液为基础液的成熟体系,紧凑型(Cp)COCs和扩展型(Ex)COCs在以M199为基础液和以DMEM/F12为基础液的培养液中的卵母细胞成熟率分别为41.7%和64.7%、46.7%和66.7%,差异不显著(P＞0.05).两种培养体系中成熟的Cp COCs采用Ionomycin与6-DMAP和CHX联合激活,在以M199和以DMEM/F12为基础液的培养液中成熟的卵母细胞的卵裂率分别为45%和57.1%,差异不明显(P＞0.05).     

The folding pathway of T4 lysozyme: an on-pathway hidden folding intermediate

T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable submillisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N and C-terminal regions. However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we re-examined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate as the wild-type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state.     

Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome

Mutations in the rpoB gene of Escherichia coli result in resistance to the antibiotic rifampicin (Rif(r)) by altering the beta subunit of RNA polymerase. Previous studies have identified 39 single base substitutions in the rpoB gene that lead to Rif(r) at 37 degrees C and an additional two mutations that result in temperature sensitive cells. We have extended this work and identified an additional 30 single base substitutions that result in the Rif(r) phenotype. With these mutations the rpoB/Rif(r) system now allows the monitoring of 69 base substitutions at 37 degrees at 37 sites (base pairs) distributed among 24 coding positions. Each of the six possible base substitutions is represented by 8-17 mutations. More than 90% of the mutations are within a small enough region of the rpoB gene to allow PCR amplification with a single pair of oligonucleotide primers, followed by sequencing with a single primer, leading to rapid analysis of numerous mutations. The remaining mutations can be monitored using an additional primer pair. To calibrate this system we sequenced over 500 mutations in rpoB occurring spontaneously or generated by different mutagens and mutators with known specificity. These results show that rpoB/Rif(r) is an accurate and easy to employ detection system, and offers the advantage of allowing analysis of mutations occurring on the chromosome rather than on an extrachromosomal element. The mutS, mutT, mutY, M mutators, as well as the mutagenic agents ethyl methanesulfonate (EMS), ultraviolet (UV) irradiation, 2-aminopurine (2AP), 5-azacytidine (5AZ), and cisplatin (CPT) gave results predicted by their characterized specificities. The number of different sequence contexts is sufficient to reveal significant hotspots among the spontaneous mutS, 2-aminopurine, ultraviolet light, 5-azacytidine, and cisplatin mutational spectra. The cisplatin distribution is particularly striking, with 68% of the mutations resulting from an A:T-->T:A transversion at a single site. Because of the conservation of key regions of RNA polymerase among many microorganisms, using the Rif(r)/rpoB system may be a general method for studying mutational processes in microorganisms without well developed genetic systems.     

Paratransgenesis: an approach to improve colony health and molecular insight in honey bees (Apis mellifera)?

The honey bee (Apis mellifera) is highly valued as a commercial crop pollinator and a model animal in research. Over the past several years, governments, beekeepers, and the general public in the United States and Europe have become concerned by increased losses of honey bee colonies, calling for more research on how to keep colonies healthy while still employing them extensively in agriculture. The honey bee, like virtually all multicellular organisms, has a mutually beneficial relationship with specific microbes. The microbiota of the gut can contribute essential nutrients and vitamins and prevent colonization by non-indigenous and potentially harmful species. The gut microbiota is also of interest as a resource for paratransgenesis; a Trojan horse strategy based on genetically modified symbiotic microbes that express effector molecules antagonizing development or transmission of pathogens. Paratransgenesis was originally engineered to combat human diseases and agricultural pests that are vectored by insects. We suggest an alternative use, as a method to promote health of honey bees and to expand the molecular toolbox for research on this beneficial social insect. The honey bees' gut microbiota contains lactic acid bacteria including the genus Lactobacillus that has paratransgenic potential. We present a strategy for transforming one Lactobacillus species, L. kunkeei, for use as a vector to promote health of honey bees and functional genetic research.