Deletion of the major peroxiredoxin Tsa1 alters telomere length homeostasis

Reactive oxygen species (ROS) are proposed to play a major role in telomere length alterations during aging. The mechanisms by which ROS disrupt telomeres remain unclear. In Saccharomyces cerevisiae, telomere DNA consists of TG_(1–3) repeats, which are maintained primarily by telomerase. Telomere length maintenance can be modulated by the expression level of telomerase subunits and telomerase activity. Additionally, telomerase‐mediated telomere repeat addition is negatively modulated by the levels of telomere‐bound Rap1‐Rif1‐Rif2 protein complex. Using a yeast strain defective in the major peroxiredoxin Tsa1 that is involved in ROS neutralization, we have investigated the effect of defective ROS detoxification on telomere DNA, telomerase, telomere‐binding proteins, and telomere length. Surprisingly, the tsa1 mutant does not show significant increase in steady‐state levels of oxidative DNA lesions at telomeres. The tsa1 mutant displays abnormal telomere lengthening, and reduction in oxidative exposure alleviates this phenotype. The telomere lengthening in the tsa1 cells was abolished by disruption of Est2, subtelomeric DNA, Rap1 C‐terminus, or Rif2, but not by Rif1 deletion. Although telomerase expression and activity are not altered, telomere‐bound Est2 is increased, while telomere‐bound Rap1 is reduced in the tsa1 mutant. We propose that defective ROS scavenging can interfere with pathways that are critical in controlling telomere length homeostasis.     

Expression of the CMV‐CP Gene in Synechocystis 6803 Affects Cyanobacterial Photosynthesis

Previous work has shown that the presence of excess coat protein (CP) of cucumber mosaic virus (CMV) in the chloroplasts was related with mosaic symptoms. However, whether these mosaic symptoms are directly induced by the interaction between CP and chloroplasts is unknown. To directly demonstrate the interaction between CP and the chloroplast, Synechocystis sp. PCC 6803 was used as the chloroplast model. The cDNA encoding the CMV‐CP was cloned in a cyanobacterial shuttle vector (pKT‐CP) and transferred to Synechocystis sp. PCC 6803. The CP was expressed in the cyanobacterium with the psbA promoter. The expression of CMV‐CP hindered the growth of transgenic cyanobacterium cells and decreased its photosynthetic rate and the PS II activity. The transgenic cells showed increased fluorescence (F) from the phycobilisome terminal emitters and increased fluorescence (F) from PS II. The absorption spectra at room temperature showed the Chl and the phycocyanin absorption peak of the mutant strain significantly decreased. These results showed that CP may directly affect the cyanobacterium cells and decreased its photosynthesis, especially the PS II activity. These data might provide new evidence for mosaic symptoms being directly induced by the interaction between CP and chloroplasts.     

广西东部早泥盆世晚期盔甲鱼类一新属

描述了产自广西东部平乐下泥盆统贺县组的一脊椎动物群中的无颌类化石,建立了盔 甲鱼类的一新属、新种──平乐圆盘鱼（Ｄｉｓｃａｓｐｉｓ ｐｉｎｇｌｅｎｓｉｓ ｇｅｔ． ｅｔ ｓｐ． ｎｏｖ．）。圆盘鱼鳃囊 数目多达３２对。同层产出的尚有保存较完好的胴甲鱼目中华鱼科化石。该动物群的发现为 华南区早泥盆世晚期脊椎动物群的演化及其生物地层的对比提供了重要依据。     

广西下泥盆统大瑶山群盔甲鱼类的新发现

广西象州县大乐乡泥盆系剖面是华南象州型(底栖相)泥盆系的标准剖面(侯鸿飞、王士涛,1988),其下部大瑶山群为一套厚达上千米的石英砂岩夹粉砂岩沉积,底部以一层约2 m厚的砾岩与下伏的前泥盆系呈角度不整合接触(白顺良等,1982).     

Small antibody mimetics comprising two complementarity-determining regions and a framework region for tumor targeting

Here we show that fusion of two complementarity-determining regions (CDRs), VHCDR1 and VLCDR3, through a cognate framework region (VHFR2) yields mimetics that retain the antigen recognition of their parent molecules, but have a superior capacity to penetrate tumors. The antigen-recognition abilities of these approximately 3 kDa mimetics surpass those of comparable fragments lacking the framework region. In vivo activities of the mimetics suggests that the structural orientation of their CDRs approximates the conformation of the CDRs in the complex of the parent antibody with antigen. We linked the antibody mimetics to the bacterial toxin colicin Ia to create fusion proteins called "pheromonicins," which enable targeted inhibition of tumor growth. In mice bearing human malignant tumors, pheromonicins directed against tumor-specific surface markers show greater capacity to target and penetrate tumors than their parent antibodies. Rational recombination of selected VH/VL binding sites and their framework regions might provide useful targeting moieties for cytotoxic cancer therapies.     

Defective Repair of Uracil Causes Telomere Defects in Mouse Hematopoietic Cells

Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity.     

Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.     

Deglycosylation to obtain stable and homogeneous Pichia pastoris-expressed N-A1 domains of carcinoembryonic antigen

Carcinoembryonic antigen (CEA) is a seven domain membrane glycoprotein widely used as a tumour marker for adenocarcinomas and as a target for antibody-directed therapies. Structural models have proposed that the first two domains of CEA (the N terminal and adjoining A1 domains) bind MFE-23, a single chain Fv antibody in experimental clinical use. We aimed to produce recombinant N-A1 to test this hypothesis. The N-A1 domains were expressed as soluble protein with a C-terminal hexahistidine tag (His6-tag) in the yeast Pichia pastoris. His6-tagged N-A1 was captured from the supernatant by batch purification with copper-loaded Streamline Chelating, an immobilised metal affinity chromatography (IMAC) matrix usually utilised in expanded bed techniques. Purified N-A1 was heterogeneous with a molecular weight range from 38 to 188 kDa. Deglycosylation with endoglycosidase H (Endo H) resulted in three discrete molecular weight forms of N-A1, one partially mannosylated, one fully Endo H-digested and one fully Endo H-digested but lacking the His6-tag. These were separated by concanavalin A chromatography followed by HiTrap IMAC. The procedure resulted in single-band-purity, mannose-free N-A1. The binding interaction of MFE-23 to N-A1 was analysed by surface plasmon resonance. The affinity constants retrieved were KD = 4.49 x 10(-9)M for the P. pastoris expressed, native N-A1, and 5.33 x 10(-9) M for the Endo H-treated N-A1. To our knowledge this is the first time that two consecutive domains of CEA have been stably expressed and purified from P. pastoris. This work confirms that the CEA epitope recognised by MFE-23 resides in N-A1.     

The efficacy and feasibility of total reconstruction versus nontotal reconstruction of the pelvic floor on short-term and long-term urinary continence rates after radical prostatectomy: a meta-analysis

Background

Recently, total pelvic floor reconstruction (TR) has been the treatment of choice for improving urinary incontinence (UI) after radical prostatectomy (RP). However, the superiority of TR with respect to urinary continence recovery following RP remains controversial. This study identified the effect of TR versus nonTR of the pelvic floor on short-term and long-term continence rates after RP.

Methods

A literature search was performed in November 2017 using the PubMed, Embase, and Web of Science databases. Only comparative research or clinical studies reporting urinary continence outcomes was included in the meta-analysis, and the quality of evidence was evaluated using the 2011 Level of Evidence for therapy research.

Results

We analyzed ten studies reporting urinary continence rates after RP at one or more postoperative time points (1, 2, 4, 12, 24, and 52 weeks). TR was associated with significantly better urinary continence outcomes at 1 week (OR 2.76, 95% CI 1.58–4.84, P?<?0.001), 2 weeks (OR 2.57, 95% CI 1.74–3.80, P?<?0.001), 4 weeks (OR 2.61, 95% CI 1.56–4.38, P?<?0.001), 12 weeks (OR 4.33, 95% CI 2.01–9.33, P?<?0.001), 24 weeks (OR 3.83, 95% CI 1.54–9.55, P?=?0.004), 52 weeks (OR 4.10, 95% CI 1.80–9.38, P?<?0.001) after RP. There was no difference in the rate of complications between the two arms (OR 0.54, 95% CI 0.19–1.54, P?=?0.25).

Conclusions

Compared with nonTR, TR is significantly and positively associated with a return to continence but not with complication rate in men following RP, suggesting that TR may be useful for decreasing the urinary incontinence rate after surgery.