A modified version of the digestion–ligation cloning method for more efficient molecular cloning

Here we describe a modified version of the digestion–ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and blunt-end cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.     

Chemical composition of Enterococcus faecalis in biofilm cells initiated from different physiologic states

Enterococcus faecalis is a ubiquitous bacterium of the gut that is observed in persistent periradicular infections. Its pathogenicity is associated with biofilm formation and the ability to survive under nutrient-poor (starvation) conditions. However, characteristics of chemical composition of biofilm cells developed by starved E. faecalis cells remain poorly understood. In this study, E. faecalis cells in exponential, stationary, and starvation phases were prepared and separately cultured to form biofilms. Confocal laser scanning microscopy was performed to verify biofilm formation. Raman microscopy was used to investigate the chemical composition of cells within the biofilms. Compared to cells in exponential or stationary phase, starved cells developed biofilms with fewer culturable cells (P?E. faecalis.     

Analysis of mitochondrial DNA mutations in D-loop region in thyroid lesions

Background

Mitochondrial defects have been associated with various human conditions including cancers.

Methods

We analyzed the mutations at the mitochondrial DNA (mtDNA) in patients with different thyroid lesions. In particular, in order to investigate if the accumulation of mtDNA mutations play a role in tumor progression, we studied the highly variable main control region of mtDNA, the displacement-loop (D-loop) in patients with non-tumor nodular goiters, with benign thyroid adenomas, and with malignant thyroid carcinomas. Total thyroid tumor or goiter samples were obtained from 101 patients, matched with nearby normal tissue and blood from the same subject.

Results

Noticeably, mitochondrial microsatellite instability (mtMSI) was detected in 2 of 19 nodular goiters (10.53%), and 8 of 77 (10.39%) malignant thyroid carcinomas. In addition, 6 patients, including 5 (6.49%) with malignant thyroid carcinomas and 1 (5.26%) with nodular goiter, were found to harbor point mutations. The majority of the mutations detected were heteroplasmic.

General significance

Our results indicate that mtDNA alterations in the D-loop region could happen before tumorigenesis in thyroid, and they might also accumulate during tumorigenesis.     

Complexity and Specificity of Precursor microRNAs Driven by Transposable Elements in Rice

In eukaryotes, small noncoding RNA molecules of 16–29 nucleotides in length play crucial roles in the regulation of gene expression. Some 377 sequences representing rice pseudo-microRNAs (miRNAs) are available in release 13.0 of the miRBase sequence database () and are grouped into 143 families. Most newly deposited miRNA sequences are likely to be species-specific. To understand the relationship between miRNAs and transposable elements (TEs) in rice, the RepeatMasker application () was used to screen single-stranded precursor miRNA (pre-miRNA) sequences. This analysis revealed that 33.1% of miRNAs and 36.4% of miRNA families are associated with interspersed repeats, and most of them are species-specific. Furthermore, multiple miRNA families can be encoded by the same TE class. Alignment analysis revealed that miR439 originated from an MuDR4-OS TE, which amplified and diversified in the genome as an inverted repeat of the core sequence followed by multiple repeats. Multiple copies of miR445 and its complexity originate from and are driven by the DNA/Tourist TE class. These results provide an important contribution to the elucidation of TE-driven mechanisms that regulate the species specificity and complexity of rice miRNAs.     

Urinary tract abnormalities in Chinese rural children who consumed melamine-contaminated dairy products: a population-based screening and follow-up study

Background

Kidney damage related to consumption of melamine-contaminated dairy products by young children in China has been described. However, no studies have reported on the population-based prevalence of kidney damage among exposed children or on the condition of affected children after follow-up.

Methods

We conducted an ultrasound-based screening in September 2008 of 7933 children younger than 36 months of age who lived in a rural area in China where the dairy products most highly contaminated with melamine were sold. We monitored children who had evidence of nephrolithiasis or hydronephrosis at screening using renal ultrasonography after one, three and six months. We also collected information from the mothers of affected children about consumption of melamine-contaminated products between June and August 2008.

Results

The overall prevalence of urinary tract abnormalities among screened children was 0.61% (95% confidence interval [CI] 0.45%–0.80%). The mean exposure dose of melamine was estimated to be 116 (range 36–220) mg per day. Of the 48 affected children, 43 (89.6%) were asymptomatic, 2 had symptoms and were hospitalized, and 3 had symptoms but treatment had been not sought for them. Of the 46 children for whom six-month follow-up information was available, renal abnormalities persisted in 5 children and resolved in the remaining 41.

Interpretation

Among children who underwent screening, 0.61% showed ultrasonographic evidence of nephrolithiasis or hydronephrosis. Most of the affected children were asymptomatic. The majority of the affected children recovered from the toxic effects of melamine over time without specific treatment. Renal abnormalities remained in 12% of the affected children.Contamination of dairy products with melamine in China has resulted in a widespread outbreak of serious kidney damage in children.^1–4 On Sept. 12, 2008, the Chinese government announced to the public that the outbreak had occurred and initiated various emergency responses, including the set-up of a high-level national coordinating group, free screening and treatment of affected children, thorough inspection of all dairy products and producers, timely release of information to the public, recalls of contaminated products, suspension of production of the contaminated milk and compensation to the families of affected children. More than 50 000 children have been hospitalized and six have died because of kidney damage.⁵Melamine is a nitrogen-containing compound commonly used in chemical industry. Because its addition to milk elevates apparent protein content, raw milk was intentionally adulterated with melamine in the production-chain, leading to contamination of dairy products and high-level exposure of thousands of children.⁶Melamine is known to cause formation of calculi in weanling rats and has led to acute renal failure in cats and dogs consuming melamine-contaminated pet foods.^7,8 In humans, melamine-related disease has been recognized only recently, and the full adverse effects of exposure remain unknown.^4,9 A clinicopathologic study suggests that the size of urinary stones is related to melamine concentration.⁹ A recent hospital-based study in Beijing reported the prevalence of nephrolithiasis was 8.5% among children who were exposed and referred by other hospitals.¹⁰However, no studies have reported on the population-based prevalence of kidney disease among exposed children, nor have follow-up reports been released about affected children. We performed a population-based screening and follow-up study involving residents of a rural area situated close to the manufacturer of Sanlu dairy products, which was the source of the most severely melamine-contaminated products in the mainland of China.¹¹ The study was approved by the Institutional Review Board of Peking University.     

An h-type thioredoxin functions in tobacco defense responses to two species of viruses and an abiotic oxidative stress

Various thioredoxin (Trx) proteins have been identified in plants. However, many of the physiological roles played by these proteins remain to be elucidated. We cloned a TRXh-like gene predicted to encode an h-type Trx in tobacco (Nicotiana tabacum) and designated it NtTRXh3, based on the biochemical activity of the NtTRXh3 protein. Overexpression of NtTRXh3 conferred resistance to Tobacco mosaic virus and Cucumber mosaic virus, both of which showed reduced multiplication and pathogenicity in NtTRXh3-overexpressing plants compared with controls. NtTRXh3 overexpression also enhanced tobacco resistance to oxidative stress induced by paraquat, an herbicide that inhibits the production of reducing equivalents by chloroplasts. The NtTRXh3 protein localized exclusively to chloroplasts in coordination with the maintenance of cellular reducing conditions, which accompanied an elevation in the glutathione/glutathione disulfide couple ratio. NtTRXh3 gene expression and NtTRXh3 protein production were necessary for these defensive responses, because they were all arrested when NtTRXh3 was silenced and the production of NtTRXh3 protein was abrogated. These results suggest that NtTRXh3 is involved in the resistance of tobacco to virus infection and abiotic oxidative stress.     

云南稻籼粳亚种间功能性成分含量差异

对111份云南改良稻种和77份元阳地方稻种采用程氏指数法进行籼粳分类,比色法测定抗性淀粉、γ-氨基丁酸和总黄酮含量,并用SPSS软件对数据进行统计分析,结果表明:(1)总体抗性淀粉含量籼型偏籼偏粳粳型,γ-氨基丁酸含量则相反,黄酮含量与籼粳分化的关系不明显;(2)抗性淀粉、总黄酮含量有色米无色米,γ-氨基丁酸含量无色米有色米。总体功能性成分含量有色米无色米,籼稻粳稻,糙米精米,地方种改良种,有色、籼型糙米功能性成分最高,揭示了籼粳分化和米色与抗性淀粉、γ-氨基丁酸与总黄酮含量的关系。