Gut microbiome in gastrointestinal cancer: a friend or foe?

The impact of the gut microbiome on host health is becoming increasingly recognized. To date, there is growing evidence that the complex characteristics of the microbial community play key roles as potential biomarkers and predictors of responses in cancer therapy. Many studies have shown that altered commensal bacteria lead to cancer susceptibility and progression in diverse pathways. In this review, we critically assess the data for gut microbiota related to gastrointestinal cancer, including esophageal, gastric, pancreatic, colorectal cancer, hepatocellular carcinoma and cholangiocarcinoma. Importantly, the underlying mechanisms of gut microbiota involved in cancer occurrence, prevention and treatment are elucidated. The purpose of this review is to provide novel insights for applying this understanding to the development of new therapeutic strategies in gastrointestinal cancer by targeting the microbial community.     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

民勤绿洲边缘积沙带植物群落健康分析

甘肃河西走廊经过60多年的防沙治沙,在绿洲边缘形成了一条积沙带。其中,民勤绿洲边缘积沙带相对最典型、最完整。积沙带上的植被不仅是积沙带的形成因素,更是维持积沙带稳定的重要因素。从一定意义上说,积沙带上植被的健康状况,决定着积沙带的稳定性。为了分析积沙带上的植被状况,在民勤绿洲边缘积沙带上等间距设定了11条样线,通过60多个样方的植被调查,对民勤绿洲边缘积沙带上植物群落进行了健康序列分析。结果表明:民勤绿洲边缘积沙带上的植被普遍是不健康的,民勤绿洲边缘人工固沙林积沙带上的植被健康状况相对好于天然灌丛积沙带上的植被健康状况,乔灌混交林积沙带上的植被健康状况最差。民勤绿洲下游积沙带上的优势种植物柽柳严重枯死,健康状况最差,积沙带潜在风蚀活化的危险。农田弃耕后,沙区农田边缘防护林及其积沙带的保护是一个新的值得考虑的问题。     

The small nonstructural protein NP1 of human bocavirus 1 directly interacts with Ku70 and RPA70 and facilitates viral DNA replication

Human bocavirus 1 (HBoV1), a member of the genus Bocaparvovirus of the family Parvoviridae, causes acute respiratory tract infections in young children. Well-differentiated pseudostratified human airway epithelium cultured at an air-liquid interface (HAE-ALI) is an ideal in vitro culture model to study HBoV1 infection. Unique to other parvoviruses, bocaparvoviruses express a small nonstructured protein NP1 of ~25 kDa from an open reading frame (ORF) in the center of the viral genome. NP1 plays an important role in viral DNA replication and pre-mRNA processing. In this study, we performed an affinity purification assay to identify HBoV1 NP1-inteacting proteins. We identified that Ku70 and RPA70 directly interact with the NP1 at a high binding affinity, characterized with an equilibrium dissociation constant (K_D) of 95 nM and 122 nM, respectively. Furthermore, we mapped the key NP1-interacting domains of Ku70 at aa266-439 and of RPA70 at aa181-422. Following a dominant negative strategy, we revealed that the interactions of Ku70 and RPA70 with NP1 play a significant role in HBoV1 DNA replication not only in an in vitro viral DNA replication assay but also in HBoV1-infected HAE-ALI cultures. Collectively, our study revealed a novel mechanism by which HBoV1 NP1 enhances viral DNA replication through its direct interactions with Ku70 and RPA70.     

Quantitative regulation of the thermal stability of enveloped virus vaccines by surface charge engineering to prevent the self-aggregation of attachment glycoproteins

The development of thermostable vaccines can relieve the bottleneck of existing vaccines caused by thermal instability and subsequent poor efficacy, which is one of the predominant reasons for the millions of deaths caused by vaccine-preventable diseases. Research into the mechanism of viral thermostability may provide strategies for developing thermostable vaccines. Using Newcastle disease virus (NDV) as model, we identified the negative surface charge of attachment glycoprotein as a novel determinant of viral thermostability. It prevented the temperature-induced aggregation of glycoprotein and subsequent detachment from virion surface. Then structural stability of virion surface was improved and virus could bind to and infect cells efficiently after heat-treatment. Employing the approach of surface charge engineering, thermal stability of NDV and influenza A virus (IAV) vaccines was successfully improved. The increase in the level of vaccine thermal stability was determined by the value-added in the negative surface charge of the attachment glycoprotein. The engineered live and inactivated vaccines could be used efficiently after storage at 37°C for at least 10 and 60 days, respectively. Thus, our results revealed a novel surface-charge-mediated link between HN protein and NDV thermostability, which could be used to design thermal stable NDV and IAV vaccines rationally.     

Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR

CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy ‘Ligation-Assisted Homologous Recombination’ (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.     

基于NGS的染色质测序在肿瘤研究中的应用

染色质是真核生物细胞核内由核酸和蛋白质组成的复合结构,有着精密且复杂的三维结构。染色质除基本的DNA序列外,内部还存在着不同化学修饰,DNA-蛋白质相互作用,DNA-DNA相互作用和DNA-RNA相互作用,以上这些若发生改变都可能在肿瘤发生发展过程中起到至关重要的作用。通过不同的染色质测序方法,可以解析出这些改变,并进一步加深研究者对肿瘤形成机制的理解,最终应用于肿瘤的治疗。本文对常见的染色质测序技术部分原理和应用进行综述。     

ER-lysosome lipid transfer protein VPS13C/PARK23 prevents aberrant mtDNA-dependent STING signaling

Mutations in VPS13C cause early-onset, autosomal recessive Parkinson’s disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the ER and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS-STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation in a model human cell line and place VPS13C in pathways relevant to PD pathogenesis.