Bayesian inference for a correlated 2 x 2 table with a structural zero

We develop a new Bayesian approach to interval estimation for both the risk difference and the risk ratio for a 2 x 2 table with a structural zero using Markov chain Monte Carlo (MCMC) methods. We also derive a normal approximation for the risk difference and a gamma approximation for the risk ratio. We then compare the coverage and interval width of our new intervals to the score-based intervals over various parameter and sample-size configurations. Finally, we consider a Bayesian method for sample-size determination.     

泛素化特异性酶（USP2a）与肿瘤发生的相关分子机制

去泛素化酶USP2a是去泛素化酶家族(DUBs)的一个成员,为半胱氨酸蛋白酶,是一种重要的特异性去泛素化水解酶。USP2a具有结构和功能多样性,其结构多样化使得这些酶具有一些特异性作用靶点,特别是在基因表达调控中靶向的生理底物种类繁多。特异性蛋白泛素化水平的动态变化涉及到基因表达活化和失活的多种机制以及信号通路转导的多个环节。越来越多的文献报道了去泛素化酶相互作用网络的组成及其重要性。USP2a调节多种重要的细胞生长和分化调节因子及信号转导因子的稳定性和功能,通过USP2a的去泛素化作用以及诱导它们之间相互反应对机体进行相应调控,特别是在调控转录因子、细胞周期和细胞凋亡自噬上发挥重要作用。USP2a的过表达在体内外都表现出致癌性,其靶蛋白通过各种途径影响肿瘤发生发展。通过对人类肿瘤发生发展的相关分子机制及信号通路影响的深入研究,USP2a有望成为肿瘤治疗的新靶点。现就去泛素化酶与人类肿瘤发生发展的相关分子机制及该领域的研究进展作一综述。     

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.     

Loss of the PLA2G2A gene in a sporadic colorectal tumor of a patient with a PLA2G2A germline mutation and absence of PLA2G2A germline alterations in patients with FAP

The Min (multiple intestinal neoplasia) mouse with a germline mutation in the adenomatous polyposis coli gene serves as an animal model for familial adenomatous polyposis coli (FAP). The number and age at onset of colorectal adenomas varies in the offspring of Min mice crossed with other strains. The murine gene for the secretory phospholipase A2 (PLA2G2A) was found to be the main candidate for these variations. To test the hypothesis of a correlation between PLA2G2A gene alterations and human tumor development, we screened 14 patients with FAP and 20 patients with sporadic colorectal cancer for germline and somatic PLA2G2A gene mutations. None of the individuals with FAP showed PLA2G2A germline alterations. However, a germline mutation was observed in one patient with an apparently sporadic colorectal tumor; the wildtype allele was somatically lost in the tumor of this patient. Received: 12 February 1997 / Accepted: 9 May 1997     

Visualization of the trimeric P2X2 receptor with a crown-capped extracellular domain

The P2X2 purinergic receptor permeates cationic ions in response to stimulation by ATP and mediates fast synaptic transmission. Here, we purified the P2X2 receptor using baculovirus-Sf9 cell expression system and observed its structure using electron microscopy. The FLAG-tagged P2X2 receptor, which has intact ion channel function, was purified to be a single peak by affinity purification and gel filtration chromatography. It was confirmed to be a trimer by introducing cross-linking. Negatively stained P2X2 protein images were homogeneous and picked up by automated pick-up programs, aligned, and classified using the modified growing neural gas network method. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. These images demonstrate an inverted three-sided pyramid with the dimensions of 215 A in height and 200 A in side length. It is composed of a high-density trunk and a stain-permeable swollen extracellular domain of a crown-shaped structure. The internal cavities and constituent segments were clearly demonstrated in both the raw images and the averaged images. The threefold symmetrical top view demonstrates the first visual evidence of the trimeric composition of the P2X receptor family.     

O-2 Substituted pyranosyl oxacarbenium ions are C-2-O-2 2-fold rotors with a strong syn preference

The substituent at O-2 of glycopyranosides is known to have a pronounced effect on both the formation and the cleavage of glycosides at C-1. This is primarily attributed to stereoelectronic effects on the formation and stability of the related glycopyranosyl oxacarbenium ions. Previous QM studies of 2-O-methyl substituted manno and gluco configured pyranosyl oxacarbenium ions found a preference for the methyl carbon to be syn to the CH-2 methine. This study examines the conformational preference of variously substituted O-2 tetrahydropyranosyl oxacarbenium ions and confirms this syn preference. Neutral analogues are shown to have the expected 3-fold rotation whereas the charged species exhibit 2-fold rotation about C-2-O-2. Natural bond order (NBO) calculations suggest that the dominant stabilizing interaction is a unimodal O-2 lone pair to C-1-O-5 pi-bond hyperconjugative interaction. This syn conformational preference has important implications for mimics of glycopyranosyl oxacarbenium ion transition states. It also suggests a conformational based mechanism that can be exploited to tune the reactivity of glycopyranosyl donors in the glycosylation reaction.     

Detection of a nucleolar 7-2 ribonucleoprotein and a cytoplasmic 8-2 ribonucleoprotein with autoantibodies from patients with scleroderma

In studies on antinucleolar antibodies in sera from 24 patients with scleroderma, an autoimmune disease, one serum, designated "anti-To", contained antibodies against a nucleolar 7-2 ribonucleoprotein and a novel cytoplasmic 8-2 ribonucleoprotein. The 7-2 and 8-2 RNAs are distinct RNAs with a pppG terminus. They are partially conserved between rat and human species and are present in distinct ribonucleoprotein particles. Eight sera contained antibodies that precipitated particles containing nucleolar U3 RNA; these antibodies appear to be directed against preribosomal particles containing U3 ribonucleoprotein, rather than the U3 ribonucleoprotein particles alone. All these ribonucleoproteins required proteins for antigenicity. These antibodies will be of use in studies on the structure and function of these novel small ribonucleoproteins.     

Immunosensitization with a Bcl-2 small molecule inhibitor

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x_L, was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.     

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H₂O₂ using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H₂O₂ produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H₂O₂. © 1997 John Wiley & Sons, Ltd.     

Acute modulation of PP2a and troponin I phosphorylation in ventricular myocytes: studies with a novel PP2a peptide inhibitor

The present study demonstrates that acute activation with either beta-adrenergic receptor agonists or H(2)O(2) treatment increases protein phosphatase 2a (PP2a) activity in ventricular myocytes. PP2a activation occurs concomitant with an increase in methylation of PP2a, changes in localization of a PP2a targeting subunit PP2aB56alpha, and a decrease in phosphorylation of PP2a substrates, such as troponin I (TnI) and ERK in ventricular myocytes. Okadaic acid, a well-established pharmacological inhibitor of PP2a, and the peptide Thr-Pro-Asp-Tyr-Phe-Leu (TPDYFL) were used to block PP2a methylation, localization, and phosphorylations. TPDYFL is a highly conserved sequence of the PP2a catalytic subunit COOH-terminus. Specifically, both okadaic acid and the peptide increased beta-adrenergic-cAMP-dependent phosphorylation of TnI and blocked the beta-adrenergic-cAMP-dependent translocation of PP2aB56alpha. TPDYFL, but not a scrambled version of this sequence, blocked H(2)O(2)-induced changes in PP2a methylation and TnI dephosphorylation. Okadaic acid produces similar inhibition of H(2)O(2) effects. Thus we propose that the novel peptide TPDYFL acts as an inhibitor of PP2a activity and may be a useful tool to increase our understanding of how PP2a is regulated and the role of PP2a in a variety of physiological and pathological processes. In addition, the present study is consistent with acute beta-adrenergic receptor activation and H(2)O(2) exposure, simultaneously activating kinases and PP2a to work on common substrates, such as TnI. We hypothesize that dual activation of opposing enzymes provides for a tighter regulation of substrate phosphorylations in ventricular myocytes.     

The Transient Receptor Potential Channels TRPP2 and TRPC1 Form a Heterotetramer with a 2:2 Stoichiometry and an Alternating Subunit Arrangement

There is functional evidence that polycystin-2 (TRPP2) interacts with other members of the transient receptor potential family, including TRPC1 and TRPV4. Here we have used atomic force microscopy to study the structure of the TRPP2 homomer and the interaction between TRPP2 and TRPC1. The molecular volumes of both Myc-tagged TRPP2 and V5-tagged TRPC1 isolated from singly transfected tsA 201 cells indicated that they assembled as homotetramers. The molecular volume of the protein isolated from cells expressing both TRPP2 and TRPC1 was intermediate between the volumes of the two homomers, suggesting that a heteromer was being formed. The distribution of angles between pairs of anti-Myc antibodies bound to TRPP2 particles had a large peak close to 90° and a smaller peak close to 180°, consistent with the assembly of TRPP2 as a homotetramer. In contrast, the corresponding angle distributions for decoration of the TRPP2-TRPC1 heteromer by either anti-Myc or anti-V5 antibodies had predominant peaks close to 180°. This decoration pattern indicates a TRPP2:TRPC1 subunit stoichiometry of 2:2 and an alternating subunit arrangement.     

Thrombospondin 2, a matricellular protein with diverse functions.

Thrombospondin (TSP) 2 is a close relative of TSP1 but differs in its temporal and spatial distribution in the mouse. This difference in expression undoubtedly reflects the marked disparity in the DNA sequences of the promoters in the genes encoding the two proteins. The synthesis of TSP2 occurs primarily in connective tissues of the developing and growing mouse. In the adult animal the protein is again produced in response to tissue injury and in association with the growth of tumors. Despite the abnormalities in collagen fibrillogenesis, fragility of skin, and laxity of tendons and ligaments observed in the TSP2-null mouse, TSP2 does not appear to contribute directly to the structural integrity of connective tissue elements. Instead, emerging evidence supports a mode of action of TSP2 'at a distance', i.e. by modulating the activity and bioavailability of proteases and growth factors in the pericellular environment and, very likely, by interaction with cell-surface receptors. Thus, TSP2 qualifies as a matricellular protein, as defined in the introduction to this minireview series. The phenotype of TSP2-null mice has been very helpful in providing clues to the functions of TSP2. In addition to histological and functional abnormalities in connective tissues, these mice display an increased vascularity of the dermis and subdermal tissues, increased endosteal bone growth, a bleeding defect, and a marked adhesive defect of dermal fibroblasts. Our laboratory has established that TSP2 binds matrix metalloproteinase 2 (MMP2) and that the adhesive defect in TSP2-null fibroblasts results from increased MMP2 activity. The investigation of the basis for the other defects in the TSP2-null mouse is likely to yield equally interesting results.     

Enhancement of antibody-dependent cytotoxicity with a chimeric anti-GD2 antibody

A mouse/human chimeric antibody (ch14.18) was developed that reacts with the disialoganglioside GD2 on the surface of tumor cells of neuroectodermal origin. ch14.18 has the constant regions of a human IgG1 antibody and was expressed in a murine hybridoma. This antibody was produced in tissue culture at concentrations up to 180 mg/liter of spent culture fluid. ch14.18 was characterized and compared to 14.G2a, a murine mAb against GD2 of IgG2a isotype derived from the same parental hybridoma as ch14.18. Scatchard plot analysis of data from saturation binding studies on M21 melanoma cells showed identical binding for ch14.18 and 14.G2a. Indirect immunofluorescence revealed the same staining pattern for ch14.18 and 14.G2a on different melanoma cell lines. Both antibodies were equally capable of targeting M21 xenografts in athymic nude mice. ch14.18- and 14.G2a-activated human C-mediated cytolysis of melanoma cell; however, ch14.18-mediated antibody-dependent cytotoxicity of human effector cells against melanoma cells 50- to 100-fold more efficiently than 14.G2a.     

Aph2, a protein with a zf-DHHC motif,interacts with c-Abl and has pro-apoptotic activity

c-Abl is a non-receptor tyrosine kinase implicated in DNA damage-induced cell death and in growth factor receptor signaling. To further understand the function and regulation of c-Abl, a yeast two-hybrid screen was performed to identify c-Abl-interacting proteins. Here we report the identification of Abl-philin 2 (Aph2), encoding a novel protein with a unique cysteine-rich motif (zf-DHHC) and a 53-amino acid stretch sharing homology with the creatine kinase family. The zf-DHHC domain is highly conserved from yeast to human. Two proteins containing this motif, Akr1p and Erf2p, have been characterized in Saccharomyces cerevisiae, both implicated in signaling pathways. Deletion analysis by two-hybrid assays revealed that the N-terminal portion of Aph2 interacts with the C terminus of c-Abl. Aph2 was demonstrated to interact with c-Abl by co-immunoprecipitation assays. Aph2 is expressed in most tissues tested and is localized in the cytoplasm, mainly in the endoplasmic reticulum (ER). The sequences required for ER location reside in the N terminus and the zf-DHHC motif of Aph2. It has been reported that a portion of c-Abl is localized in the ER. We demonstrate here that Aph2 and c-Abl are co-localized in the ER region. Overexpression of Aph2 leads to apoptosis as justified by TUNEL assays, and the induction of apoptosis requires the N terminus. Co-expression of c-Abl and Aph2 had a synergistic effect on apoptosis induction and led to a decreased expression of both proteins, suggesting either that these two proteins are mutually down-regulated or that cells expressing both c-Abl and Aph2 rapidly disappeared from the culture. These results suggest that Aph2 may be involved in ER stress-induced apoptosis in which c-Abl plays an important role.     

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1–10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

The outbreak of SARS-CoV-2 has caused great damage to human society, and early detection of this virus is important to contain its spread and save lives. Existing diagnostic methods are complex and require well-trained personnel, but this study describes a fast and accurate method which can be operated by lay-users and whose results can be visualized by the naked eye.     

The FT210 cell line is a mouse G2 phase mutant with a temperature-sensitive CDC2 gene product

The mouse cell FT210 was isolated as a G2 phase mutant with a possible defect in the histone H1 kinase. We determined that a temperature-sensitive lesion in this cell line lies in the CDC2 gene. DNA sequence analysis revealed two point mutations in highly conserved regions of the gene: an isoleucine to valine change in the PSTAIR region, and a proline to serine change at the C-terminal region of the protein p34. These mutations cause the p34 protein kinase to become inactivated and degraded in FT210 cells at the restrictive temperature, 39 degrees C. The consequence of this temperature-induced inactivation of the CDC2 gene product is cell cycle arrest at the mid to late G2 phase, and this arrest can be alleviated by the introduction of the human CDC2 homolog.     

Diabetes insipidus in mice with a mutation in aquaporin-2

Congenital nephrogenic diabetes insipidus (NDI) is a disease characterized by failure of the kidney to concentrate urine in response to vasopressin. Human kindreds with nephrogenic diabetes insipidus have been found to harbor mutations in the vasopressin receptor 2 (Avpr2) gene or the vasopressin-sensitive water channel aquaporin-2 (Aqp2) gene. Development of a treatment is rendered difficult due to the lack of a viable animal model. Through forward genetic screening of ethylnitrosourea-mutagenized mice, we report the identification and characterization of a mouse model of NDI, with an F204V mutation in the Aqp2 gene. Unlike previously attempted murine models of NDI, our mice survive to adulthood and more exactly recapitulate the human disorder. Previous in vitro experiments using renal cell lines suggest recessive Aqp2 mutations result in improper trafficking of the mutant water pore. Using these animals, we have directly proven this hypothesis of improper AQP2 translocation as the molecular defect in nephrogenic diabetes insipidus in the intact organism. Additionally, using a renal cell line we show that the mutated protein, AQP2-F204V, is retained in the endoplasmic reticulum and that this abnormal localization can be rescued by wild-type protein. This novel mouse model allows for further mechanistic studies as well as testing of pharmacological and gene therapies for NDI.