Monoclonal antibody-based serological methods for detection of Cucumber green mottle mosaic virus

<p>Abstract</p> <p>Background</p> <p>Cucumber green mottle mosaic virus (CGMMV), a member of the genus <it>Tobamovirus</it>, can be transmitted by seeds and infects many cucurbit species, causing serious yield losses in cucumber and watermelon plants. In this paper, five serological methods including antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), Dot-immunobinding assay (DBIA), direct tissue blot immunoassay (DTBIA) and immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) were described for detection and diagnosis of CGMMV.</p> <p>Results</p> <p>Using the purified CGMMV particles as immunogens, six murine monoclonal antibodies (MAbs) were produced. Five serological methods were established using the MAb 4H1 and detection sensitivity was compared using purified preparations and infected-plant tissue extracts. The detection sensitivity of ACP-ELISA was 0.16 ng of purified CGMMV, whereas TAS-ELISA was more sensitive than ACP-ELISA with a minimum detection of 0.04 ng of purified CGMMV. The sensitivities of TAS-ELISA and DBIA were similar for detecting CGMMV in infected-plant tissue extracts, and were four times higher than ACP-ELISA. The IC-RT-PCR was the most sensitive method, which could detect as little as 0.1 pg of purified virus. The detection sensitivity of IC-RT-PCR for CGMMV-infected plant tissues was about 400 times higher than that of TAS-ELISA and DBIA.</p> <p>Conclusions</p> <p>The established ACP-ELISA, TAS-ELISA, DBIA and DTBIA are suitable for routine CGMMV detection of large-scale samples in the field survey, while IC-RT-PCR is more sensitive and suitable for acquiring information about the viral genome.</p

A splicing isoform of TEAD4 attenuates the Hippo–YAP signalling to inhibit tumour proliferation

Aberrant splicing is frequently found in cancer, yet the biological consequences of such alterations are mostly undefined. Here we report that the Hippo–YAP signalling, a key pathway that regulates cell proliferation and organ size, is under control of a splicing switch. We show that TEAD4, the transcription factor that mediates Hippo–YAP signalling, undergoes alternative splicing facilitated by the tumour suppressor RBM4, producing a truncated isoform, TEAD4-S, which lacks an N-terminal DNA-binding domain, but maintains YAP interaction domain. TEAD4-S is located in both the nucleus and cytoplasm, acting as a dominant negative isoform to YAP activity. Consistently, TEAD4-S is reduced in cancer cells, and its re-expression suppresses cancer cell proliferation and migration, inhibiting tumour growth in xenograft mouse models. Furthermore, TEAD4-S is reduced in human cancers, and patients with elevated TEAD4-S levels have improved survival. Altogether, these data reveal a splicing switch that serves to fine tune the Hippo–YAP pathway

Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer

The transcription factor (TF) SOX2 is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition to its normal stem cell function, SOX2 over-expression is associated with cancer development. The ability to selectively target this and other oncogenic TFs in cells, however, remains a significant challenge due to the 'undruggable' characteristics of these molecules. Here, we employ a zinc finger (ZF)-based artificial TF (ATF) approach to selectively suppress SOX2 gene expression in cancer cells. We engineered four different proteins each composed of 6ZF arrays designed to bind 18 bp sites in the SOX2 promoter and enhancer region, which controls SOX2 methylation. The 6ZF domains were linked to the Kruppel Associated Box (SKD) repressor domain. Three engineered proteins were able to bind their endogenous target sites and effectively suppress SOX2 expression (up to 95% repression efficiencies) in breast cancer cells. Targeted down-regulation of SOX2 expression resulted in decreased tumor cell proliferation and colony formation in these cells. Furthermore, induced expression of an ATF in a mouse model inhibited breast cancer cell growth. Collectively, these findings demonstrate the effectiveness and therapeutic potential of engineered ATFs to mediate potent and long-lasting down-regulation of oncogenic TF expression in cancer cells

Breaking through an epigenetic wall: Re-activation ofOct4by KRAB-containing designer zinc finger transcription factors

The gene Oct4 encodes a transcription factor critical for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition, improper re-activation of Oct4 contributes to oncogenic processes. Herein, we describe a novel designer zinc finger protein (ZFP) capable of upregulating the endogenous Oct4 promoter in a panel of breast and ovarian cell lines carrying a silenced gene. In some ovarian tumor lines, the ZFP triggered a strong reactivation of Oct4, with levels of expression comparable with exogenous Oct4 cDNA delivery. Surprisingly, the reactivation of Oct4 required a KRAB domain for effective upregulation of the endogenous gene. While KRAB-containing ZFPs are traditionally described as transcriptional repressors, our results suggest that these proteins could, in certain genomic contexts, function as potent activators and, thus, outline an emerging novel function of KRAB-ZFPs. In addition, we document a novel ZFP that could be used for the epigenetic reprograming of cancer cells

Identification of floR Variants Associated With a Novel Tn4371-Like Integrative and Conjugative Element in Clinical Pseudomonas aeruginosa Isolates

Florfenicol is widely used to control respiratory diseases and intestinal infections in food animals. However, there are increasing reports about florfenicol resistance of various clinical pathogens. floR is a key resistance gene that mediates resistance to florfenicol and could spread among different bacteria. Here, we investigated the prevalence of floR in 430 Pseudomonas aeruginosa isolates from human clinical samples and identified three types of floR genes (designated floR, floR-T1 and floR-T2) in these isolates, with floR-T1 the most prevalent (5.3%, 23/430). FloR-T2 was a novel floR variant identified in this study, and exhibited less identity with other FloR proteins than FloRv. Moreover, floR-T1 and floR-T2 identified in P. aeruginosa strain TL1285 were functionally active and located on multi-drug resistance region of a novel incomplete Tn4371-like integrative and conjugative elements (ICE) in the chromosome. The expression of the two floR variants could be induced by florfenicol or chloramphenicol. These results indicated that the two floR variants played an essential role in the host’s resistance to amphenicol and the spreading of these floR variants might be related with the Tn4371 family ICE

The Splicing Factor RBM4 Controls Apoptosis, Proliferation, and Migration to Suppress Tumor Progression

Splicing dysregulation is one of the molecular hallmarks of cancer. However, the underlying molecular mechanisms remain poorly defined. Here we report the splicing factor RBM4 suppresses proliferation and migration of various cancer cells by specifically controlling cancer-related splicing. Particularly, RBM4 regulates Bcl-x splicing to induce apoptosis, and co-expression of Bcl-xL partially reverses the RBM4-mediated tumor suppression. Moreover, RBM4 antagonizes an oncogenic splicing factor, SRSF1, to inhibit mTOR activation. Strikingly, RBM4 expression is dramatically decreased in cancer patients, and RBM4 level is positively correlated with improved survival. In addition to providing mechanistic insights of cancer-related splicing dysregulation, this study establishes RBM4 as a tumor suppressor with therapeutic potentials and clinical values as a prognostic factor

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Mesoscience in cell biology and cancer research

Abstract Mesoscale characteristics and their interdimensional correlation are the focus of contemporary interdisciplinary research. Mesoscience is a discipline that has the potential to radically update the existing knowledge structure, which differs from the conventional unit‐scale and system‐scale research models, revealing a previously untouchable area for scientific research. Integrative biology research aims to dissect the complex problems of life systems by conducting comprehensive research and integrating various disciplines from all biological levels of the living organism. However, the mesoscientific issues between different research units are neglected and challenging. Mesoscale research in biology requires the integration of research theories and methods from other disciplines (mathematics, physics, engineering, and even visual imaging) to investigate theoretical and frontier questions of biological processes through experiments, computations, and modeling. We reviewed  integrative paradigms and methods for the biological mesoscale problems (focusing on oncology research) and prospected the potential of their multiple dimensions and upcoming challenges. We expect to establish an interactive and collaborative theoretical platform for further expanding the depth and width of our understanding on the nature of biology