肠激酶在毕赤酵母中的分泌表达优化

肠激酶 (Enterokinase,EK) 是一类特异性识别切割DDDDK序列的丝氨酸蛋白酶,作为一种工具酶广泛应用于生物医药领域。目前,EK在毕赤酵母Pichia pastoris中的表达水平较低,难以应用。本研究比较了6种不同的信号肽SP1、SP2、SP3、SP4、SP7和SP8对毕赤酵母分泌表达EK的影响。在摇瓶水平上,与α-factor信号肽相比,SP1信号肽显著提高了EK的分泌表达 (从6.8 mg/L提高至14.3 mg/L),酶活从 (2 390±212) U/mL提高至 (4 995±378) U/mL。在此基础上,通过共表达毕赤酵母内源蛋白Kex2,EK酶活提高至 (7 219±489) U/mL。另外,N端融合WLR三个氨基酸进一步提高酶活至 (15 145±920) U/mL,比酶活为 (1 174 600±53 100) U/mg。EK在毕赤酵母中的高效分泌表达为未来应用奠定了基础。     

Phosphorus accumulates faster than nitrogen globally in freshwater ecosystems under anthropogenic impacts

Combined effects of cumulative nutrient inputs and biogeochemical processes that occur in freshwater under anthropogenic eutrophication could lead to myriad shifts in nitrogen (N):phosphorus (P) stoichiometry in global freshwater ecosystems, but this is not yet well‐assessed. Here we evaluated the characteristics of N and P stoichiometries in bodies of freshwater and their herbaceous macrophytes across human‐impact levels, regions and periods. Freshwater and its macrophytes had higher N and P concentrations and lower N : P ratios in heavily than lightly human‐impacted environments, further evidenced by spatiotemporal comparisons across eutrophication gradients. N and P concentrations in freshwater ecosystems were positively correlated and N : P was negatively correlated with population density in China. These results indicate a faster accumulation of P than N in human‐impacted freshwater ecosystems, which could have large effects on the trophic webs and biogeochemical cycles of estuaries and coastal areas by freshwater loadings, and reinforce the importance of rehabilitating these ecosystems.     

Identification of baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line.

A gene that promotes Autographa californica M nuclear polyhedrosis virus (AcMNPV) replication in IPLB-Ld652Y cells, a cell line that is nonpermissive for AcMNPV, was identified in Lymantria dispar M nuclear polyhedrosis virus (LdMNPV). Cotransfection of AcMNPV DNA and a plasmid carrying the LdMNPV gene into IPLB-Ld652Y cells results in AcMNPV replication. The gene maps between 43.3 and 43.8 map units on the 162-kbp genome of LdMNPV. It comprises a 218-codon open reading frame and encodes a polypeptide with a predicted molecular mass of 25.7 kDa. The predicted polypeptide is glutamic acid and valine rich and negatively charged, with a pI of 4.61. No protein sequence motifs were identified, and no matches with known nucleotide or peptide sequences were found in the AcMNPV genome or database searches that suggest how this gene might function. A recombinant AcMNPV bearing the LdMNPV gene overcomes a block in protein synthesis observed in AcMNPV-infected IPLB-Ld652Y cells. Using Southern blotting techniques, we were unable to identify a homolog in Orgyia pseudotsugata M nuclear polyhedrosis virus, a baculovirus that is routinely propagated in IPLB-Ld652Y cells. This suggests that the LdMNPV host range is unique among the baculoviruses studied to date. We named this gene hrf-1 (for host range factor 1).     

Blocking of the PD-1/PD-L1 interaction by a novel cyclic peptide inhibitor for cancer immunotherapy

The interaction of PD-1/PD-L1 allows tumor cells to escape from immune surveillance. Clinical success of the antibody drugs has proven that blockade of PD-1/PD-L1 pathway is a promising strategy for cancer immunotherapy. Here, we developed a cyclic peptide C8 by using Ph.D.-C7 C phage display technology. C8 showed high binding affinity with h PD-1 and could effectively interfere the interaction of PD-1/PD-L1. Furthermore, C8 could stimulate CD8+T cell activation in human peripheral blood mononuclear cells(PBMCs). We also observed that C8 could suppress tumor growth in CT26 and B16-OVA, as well as anti-PD-1 antibody resistant B16 mouse model. CD8+T cells infiltration significantly increased in tumor microenvironment, and IFN-γ secretion by CD8+T cells in draining lymph nodes also increased. Simultaneously, we exploited T cells depletion models and confirmed that C8 exerted anti-tumor effects via activating CD8+T cells dependent manner. The interaction model of C8 with h PD-1 was simulated and confirmed by alanine scanning. In conclusion, C8 shows anti-tumor capability by blockade of PD-1/PD-L1 interaction, and C8 may provide an alternative candidate for cancer immunotherapy.     

H3N2亚型猪流感病毒的拯救及HA与NA基因的替换

摘要：【目的】为研究流感病毒突破种间屏障分子机制,筛选流感基因工程疫苗株。【方法】本实验以猪流感病毒A/Swine/Henan/S4/01(H3N2)为亲本株,利用反向遗传学操作技术,采用RT-PCR技术对该病毒的8个基因片段分段进行扩增,通过与双向转录载体pHW2000连接, 重组质粒转染293T和MDCK共培养细胞,拯救出全部基因均来自于亲本株的猪流感病毒rgH3N2,并分别以人流感病毒A/ PR/8/34(H1N1)、禽流感病毒A/Duck/Nanchang /4-165/2000 (H4N6 )、马流感病毒A/Equine/Fuyun/2008/(H3N8)的HA和NA基因替换A/Swine/Henan/S4/01的相应基因,【结果】生物学实验结果表明rgH3N2在鸡胚半数感染量、组织培养半数感染量、稳定性试验等方面都与亲本株保持一致。rgH3N2经鸡胚多次传代后血凝价最高可达到1:256,接种MDCK细胞60 h后,血凝价可以达到1:64。基因替换后成功拯救出的重组病毒rgH1N1、rgH4N6和rgH3N8在鸡胚和细胞上均具有较高的增殖能力。【结论】病毒的成功拯救为流感病毒突破种间屏障分子机制,HA、NA基因在流感病毒跨种属传播中所扮演角色的研究和流感基因工程疫苗株的筛选奠定了基础。     

Association between S100B Levels and Long-Term Outcome after Aneurysmal Subarachnoid Hemorrhage: Systematic Review and Pooled Analysis

S100 calcium binding protein B (S100B), a well-studied marker for neurologic injury, has been suggested as a candidate for predicting outcome after subarachnoid hemorrhage. We performed a pooled analysis summarizing the associations between S100B protein in serum and cerebrospinal fluid (CSF) with radiographic vasospasm, delayed ischemic neurologic deficit (DIND), delayed cerebral infarction, and Glasgow Outcome Scale (GOS) outcome. A literature search using PubMed, the Cochrane Library, and the EMBASE databases was performed to identify relevant studies published up to May 2015. The weighted Stouffer’s Z method was used to perform a pooled analysis of outcome measures with greater than three studies. A total of 13 studies were included in this review. Higher serum S100B level was found to be associated with cerebral infarction as diagnosed by CT (p_adj = 3.1 x 10⁻⁴) and worse GOS outcome (p_adj = 5.5 x 10⁻¹¹). There was no association found between serum and CSF S100B with radiographic vasospasm or DIND. S100B is a potential prognostic marker for aSAH outcome.     

Linc00483 as ceRNA regulates proliferation and apoptosis through activating MAPKs in gastric cancer

Long non‐coding RNAs (lncRNAs) are important regulators of many cellular processes, and their aberrant expression and/or function is associated with many different diseases, including cancer. However, the identification of functional lncRNAs in gastric cancer is still a challenge. In this study, we describe a novel functional lncRNA, linc00483, that is upregulated and associated with tumorigenesis, tumour size, metastasis and poor prognosis in gastric cancer. In our study, linc00483 promoted gastric cancer cell proliferation, invasiveness and metastasis in vitro and in vivo. Mechanistically, upregulated expression of linc00483 in gastric cancer acts as a sponge to absorb endogenous tumour suppressor miR‐30a‐3p. Furthermore, it restores SPAG9 expression, which is negatively regulated by miR‐30a‐3p, and actives MAPK signaling pathway in gastric cancer cells. Thus, linc00483 is an oncogenic lncRNA in gastric cancer and targeting linc00483 or its pathway can potentially be useful in development of targeted therapies for patients with gastric cancer. Our results show that linc00483 is an important regulator in carcinogenesis and may be a useful biomarker to predict prognosis of gastric cancer patients. We believe our findings are novel and will be of interest to scientists working in many areas related to biomarkers in cancer.     

CD33‐targeting extracellular vesicles deliver antisense oligonucleotides against FLT3‐ITD and miR‐125b for specific treatment of acute myeloid leukaemia

IntroductionAcute Myeloid Leukaemia (AML) is the most common blood cancer in adults. Although 2 out of 3 AML patients go into total remission after chemotherapies and targeted therapies, the disease recurs in 60%–65% of younger adult patients within 3 years after diagnosis with a dramatically decreased survival rate. Therapeutic oligonucleotides are promising treatments under development for AML as they can be designed to silence oncogenes with high specificity and flexibility. However, there are not many well validated approaches for safely and efficiently delivering oligonucleotide drugs. This issue could be resolved by utilizing a new generation of delivery vehicles such as extracellular vesicles (EVs).MethodsIn this study, we harness red blood cell‐derived EVs (RBCEVs) and engineer them via exogenous drug loading and surface functionalization to develop an efficient drug delivery system for AML. Particularly, EVs are designed to target CD33, a common surface marker with elevated expression in AML cells via the conjugation of a CD33‐binding monoclonal antibody onto the EV surface.ResultsThe conjugation of RBCEVs with the CD33‐binding antibody significantly increases the uptake of RBCEVs by CD33‐positive AML cells, but not by CD33‐negative cells. We also load CD33‐targeting RBCEVs with antisense oligonucleotides (ASOs) targeting FLT3‐ITD or miR‐125b, 2 common oncogenes in AML, and demonstrate that the engineered EVs improve leukaemia suppression in in vitro and in vivo models of AML.ConclusionTargeted RBCEVs represent an innovative, efficient, and versatile delivery platform for therapeutic ASOs and can expedite the clinical translation of oligonucleotide drugs for AML treatments by overcoming current obstacles in oligonucleotide delivery.

In this study, we harness red blood cell‐derived EVs (RBCEVs) and engineer them with surface functionalization and exogenous drug loading to develop an efficient drug delivery system for AML. Anti‐CD33 antibody was conjugated to RBCEVs using an enzymatic method combined with the streptavidin‐biotin system. We load the antibody conjugated RBCEVs with ASOs targeting FLT3‐ITD or miR‐125b, 2 common oncogenes in AML, and demonstrate that the treatment with engineered EVs improve leukaemia suppression both in vitro and in vivo.