p62 is a key regulator of nutrient sensing in the mTORC1 pathway

The signaling adaptor p62 is a critical mediator of important cellular functions, owing to its ability to establish interactions with various signaling intermediaries. Here, we identify raptor as an interacting partner of p62. Thus, p62 is an integral part of the mTORC1 complex and is necessary to mediate amino acid signaling for the activation of S6K1 and 4EBP1. p62 interacts in an amino acid-dependent manner with mTOR and raptor. In addition, p62 binds the Rags proteins and favors formation of the active Rag heterodimer that is further stabilized by raptor. Interestingly, p62 colocalizes with Rags at the lysosomal compartment and is required for the interaction of mTOR with Rag GTPases in vivo and for translocation of the mTORC1 complex to the lysosome, a crucial step for mTOR activation.     

Pyrosequencing for mini-barcoding of fresh and old museum specimens

DNA barcoding is an effective approach for species identification and for discovery of new and/or cryptic species. Sanger sequencing technology is the method of choice for obtaining standard 650 bp cytochrome c oxidase subunit I (COI) barcodes. However, DNA degradation/fragmentation makes it difficult to obtain a full-length barcode from old specimens. Mini-barcodes of 130 bp from the standard barcode region have been shown to be effective for accurate identification in many animal groups and may be readily obtained from museum samples. Here we demonstrate the application of an alternative sequencing technology, the four-enzymes single-specimen pyrosequencing, in rapid, cost-effective mini-barcode analysis. We were able to generate sequences of up to 100 bp from mini-barcode fragments of COI in 135 fresh and 50 old Lepidoptera specimens (ranging from 53-97 year-old). The sequences obtained using pyrosequencing were of high quality and we were able to robustly match all the tested pyro-sequenced samples to their respective Sanger-sequenced standard barcode sequences, where available. Simplicity of the protocol and instrumentation coupled with higher speed and lower cost per sequence than Sanger sequencing makes this approach potentially useful in efforts to link standard barcode sequences from unidentified specimens to known museum specimens with only short DNA fragments.     

嗜热厌氧菌Caldicellulosiruptor sp. F32中降解β-1,3-1,4葡聚糖水解酶的协同性分析及糖基化对F32EG5热稳定性影响

【目的】通过研究来自极端嗜热厌氧菌Caldicellulosiruptor sp. F32中3个可降解β-1,3-1,4葡聚糖(β-葡聚糖)的糖苷水解酶,解析其在降解β-葡聚糖过程中协同作用,以及异源表达的糖基化修饰对β-葡聚糖酶F32EG5热稳定性的影响,为该系列水解酶的应用提供考据。【方法】通过大肠杆菌异源表达β-葡聚糖酶F32EG5和Lam16A-GH,以及β-葡萄糖苷酶BlgA,利用DNS、TLC等方法检测其在β-葡聚糖降解过程中的协同性及底物耐受能力。随后,利用毕赤酵母对F32EG5进行异源表达,以及对糖基化修饰的p-F32EG5进行酶学对比。【结果】β-葡聚糖酶F32EG5和Lam16A-GH单独作用于底物时,水解产物不同。但混合使用时,低聚合度寡糖的比例增加。β-葡萄糖苷酶BlgA分别与F32EG5和Lam16A-GH复配时,均展示出良好的协同效应和底物耐受能力。此外,利用毕赤酵母异源表达的p-F32EG5,没有明显改变其最适pH和最适温度,但在超高温下(80–90°C)的热稳定性和催化效率相对于未被糖基化的F32EG5提高2倍以上。【结论】葡萄糖糖苷水解酶BlgA分别与β-葡聚糖酶F32EG5、Lam16A-GH复配,在水解β-葡聚糖过程中表现出良好的协同性和底物耐受能力,同时毕赤酵母异源表达的糖基化修饰能提高在超高温环境下的热稳定性能,有利于酶制剂生产造粒过程中的酶活保留,从而使F32EG5具备应用化潜力。     

Th1- and Th2-dependent endothelial progenitor cell recruitment and angiogenic switch in asthma

Increased numbers of submucosal vessels are a consistent pathologic component of asthmatic airway remodeling. However, the relationship between new vessel formation and asthmatic inflammatory response is unknown. We hypothesized that angiogenesis is a primary event during the initiation of airway inflammation and is linked to the recruitment of bone marrow-derived endothelial progenitor cells (EPC). To test this hypothesis, circulating EPC and EPC-derived endothelial cell colony formation of individuals with asthma or allergic rhinitis and health controls was evaluated. Circulating EPC were increased in asthma, highly proliferative, and exhibited enhanced incorporation into endothelial cell tubes as compared with controls. In an acute allergen challenge murine asthma model, EPC mobilization occurred within hours of challenge and mobilized EPC were selectively recruited into the challenged lungs of sensitized animals, but not into other organs. EPC recruitment was Th1 and Th2 dependent and was temporally associated with an increased microvessel density that was noted within 48 h of allergen challenge, indicating an early switch to an angiogenic lung environment. A chronic allergen challenge model provided evidence that EPC recruitment to the lung persisted and was associated with increasing microvessel density over time. Thus, a Th1- and Th2-dependent angiogenic switch with EPC mobilization, recruitment, and increased lung vessel formation occurs early but becomes a sustained and cumulative component of the allergen-induced asthmatic response.     

A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing

Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward–reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n² independent libraries for each set of ‘n’ forward and ‘n’ reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.     

The interaction of high and low-risk human papillomavirus genotypes increases the risk of developing genital warts: A population-based cohort study

Cervical cancer is among the most common type of cancers in women and is associated with human papillomavirus (HPV) infection. Genital warts are also reported to be linked with HPV infection types 11 and 6. In turn, clinical characteristics and morphological features of warts may be useful in the prediction of prognosis and in making treatment decisions. Thus, we have investigated the association of high and low-risk HPVs genotype with genital wart risk, as well as pathological and cytological information in cases recruited from a population-based cohort study of 1380 patients. Patients infected with HPV genotype 6 or 11 had an increased risk of having warts, with OR of 2.34 (95% CI: 0.955-5.737, P = 0.06). Also, this association was enhanced in the presence of high plus low-risk HPV for having genital wart (OR: 2.814; 95%: 1.208-6.55, P = 0.017) and cases having high-risk HPV (OR: 2.329; 95% CI: 1.029-5.269, P = 0.042). Moreover, we observed patients with genital warts having CIN2/3, indicating the importance of informing the physician to the patient to prevent more severe lesions. Our data demonstrated that patients with both low/high-risk HPV types had an increased risk of developing genital warts and persistent infection with HPV was a necessary precursor for the increase in cervical lesions.     

Quinol type compound in cytochrome c preparations leads to non-enzymatic reduction of cytochrome c during the measurement of complex III activity

Measurement of complex III activity is critical to the diagnosis of human mitochondrial disease and the study of mitochondrial pathobiology. Activity is measured as the maximal rate of antimycin A-sensitive reduction of exogenous cytochrome c by detergent-solubilized mitochondria. Complex III activity exhibited an unexpected variation based upon the commercial source of cytochrome c owing to an increase in the antimycin A-insensitive background reduction of cytochrome c and variable increases in total activity. Analysis of cytochrome c (producing a high-background) by fast protein liquid chromatography yielded a contaminant peak containing a lipid extractable component with redox spectra and mass spectroscopy fragmentation suggestive of a quinol. Measurement of inhibitor-sensitive rates are critical for the accurate and reproducible measurement of complex III activity and serve as a key quality control to screen for non-enzymatic reactions that obscure complex III activity.