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Rho-associated coiled-coil containing protein kinases (ROCKs) are members of the cyclic adenosine monophosphate-dependent protein kinase/protein kinase G/protein kinase C family that participates in a variety of important physiological functions, including smooth muscle contraction, cell proliferation, cell adhesion, migration, and inflammatory responses. In this study, we focused on ROCK1 and ROCK2, which are targets of the Food and Drug Administration-approved inhibitor 2-(3-(4-((1[Formula: see text]-Indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-[Formula: see text]-isopropylacetamide (belumosudil). We constructed four representative belumosudil/ROCK complex structures by molecular docking. The interactions between belumosudil and ROCK were then investigated via molecular dynamics simulations and binding free energy calculations. It was found that belumosudil showed a stronger binding affinity toward ROCK2 than toward ROCK1. Binding free energy calculations and free energy decompositions suggested that the modification of various regions of the belumosudil structure may enhance its binding affinity with ROCK, in addition to improving the selectivity between ROCK1 and ROCK2. This work therefore provides useful information to aid future drug design.

Here, we report a novel mechanism to selectively degrade target proteins. 3-(3-Phenoxybenzyl)amino-β-carboline (PAC), a tubulin inhibitor, promotes selective degradation of αβ-tubulin heterodimers. Biochemical studies have revealed that PAC specifically denatures tubulin, making it prone to aggregation that predisposes it to ubiquitinylation and then degradation. The degradation is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and βGlu198, which is identified as a low-barrier hydrogen bond (LBHB). In contrast, another two tubulin inhibitors that only form normal hydrogen bonds with βGlu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. We then screened for compounds capable of forming an LBHB with βGlu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimer degradation through the LBHB. Our study provided a unique example of LBHB function and identified a novel approach to obtain tubulin degraders.

Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin-α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.—Wei, H., Sundararaman, A., Dickson, E., Rennie-Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome.

Low barrier hydrogen bond (LBHB) is a special type of hydrogen bond which occurs where two heteroatoms with similar pKa values share a single proton resulting in an unusually strong and short hydrogen bond. LBHBs in protein play important roles in enzyme catalysis and maintaining protein structural integrity but its other biochemical roles are unknown. Here we report a novel function of LBHB in selectively inducing tubulin protein degradation. A tubulin inhibitor, 3-(3-Phenoxybenzyl) amino-β-carboline (PAC), promotes selective degradation of αβ-tubulin heterodimers by binding to the colchicine site of β-tubulin. Biochemical studies have revealed that PAC specifically destabilizes tubulin, making it prone to aggregation that then predisposes it to ubiquitinylation and then degradation. Structural activity analyses have indicated that the destabilization is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and βGlu198, which is identified as a LBHB. In contrast, another two tubulin inhibitors only forming normal hydrogen bonds with βGlu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. Most importantly, we screened for compounds capable of forming LBHB with βGlu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimers degradation in a PAC-like manner as expected. Our study has identified a novel approach for designing tubulin degraders, providing a unique example of LBHB function and suggests that designing small molecules to form LBHBs with protein residues resulting in the highly specific degradation of a target protein could be a new strategy for drug development.

A novel binding site on α-tubulin shows promise as a target for a new generation of anticancer drugs., Microtubules, composed of αβ-tubulin heterodimers, have remained popular anticancer targets for decades. Six known binding sites on tubulin dimers have been identified thus far, with five sites on β-tubulin and only one site on α-tubulin, hinting that compounds binding to α-tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our x-ray crystallography study reveals that, in addition to binding to the vinblastine site, cevipabulin also binds to a new site on α-tubulin. We find that cevipabulin at this site pushes the αT5 loop outward, making the nonexchangeable GTP exchangeable, which reduces the stability of tubulin, leading to its destabilization and degradation. Our results confirm the existence of a new agent binding site on α-tubulin and shed light on the development of tubulin degraders as a new generation of antimicrotubule drugs targeting this novel site.

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

Pelvic organ prolapse (POP) is a serious health issue that affects many adult women. Surgical treatments for POP patients comprise a common strategy in which scaffold materials are used to reconstruct the prolapsed pelvic. However, the existing materials for pelvic reconstruction cannot meet clinical requirements in terms of biocompatibility, mechanics and immunological rejection. To address these concerns, polypropylene (PP) mesh was selected because of its strong mechanical properties. Small intestinal submucosa (SIS) was used to modify the PP mesh via a mussel-inspired polydopamine coating to enhance its biocompatibility. The scanning electron microscopy (SEM) and atomic force microscopy (AFM) results demonstrated that SIS was successfully conjugated on the surface of the PP mesh. Moreover, the cytotoxicity results indicated that the PP mesh and SIS-modified PP mesh were safe to use. Furthermore, in vivo tests demonstrated that the fibroplasia around the implanted site in the SIS-modified PP mesh group was significantly less than the fibroplasia around the PP mesh group. In addition, the immunohistochemistry staining results indicated that the expression of pro-inflammatory macrophages (M1) was substantially lower and that the expression of pro-healing macrophages (M2) was higher in the SIS-modified PP mesh group. Furthermore, ELISA detection indicated that the expression of IL-1β and IL-6 in the SIS-modified PP mesh group was reduced compared with the PP mesh group. These findings suggest that a SIS-modified polypropylene hybrid mesh via a mussel-inspired polydopamine coating is a promising approach in pelvic reconstruction.

Table S1. The diagnosis of pre-diabetes (n =â 1514). (DOCX 16 kb)

Table S2. Multiple logistic regression odds ratio (OR) and 95% confidence interval (CI) for the association of tertiles of serum 25(OH)D (ng/ml) with pre-diabetes1 (DOCX 18Â kb)

Figure S1. Length distribution of total sRNAs. Figure S2. Pie charts of sRNA percentages. Figure S3. Top 10 miRNA reads. Figure S4. GO functional enrichment annotations for the target genes of DE miRNAs. Table S1. Overview of miRNA high-throughput sequencing data. Table S2. Expression profiles of miRNAs. Table S3. Differentially expressed miRNAs. Table S4. Target prediction for DE miRNAs. Table S5. Integrated expression analysis of DE miRNAs and their target mRNA. Table S6. DE miRNAs in vivo and vitro under viral infection. Table S7. Primers used for stem-loop qRT-PCR. (DOCX 2983 kb)

Background Porcine torovirus (PToV) is a member of the genus Torovirus which is responsible for gastrointestinal disease in both human beings and animals with particular prevalence in youth. Torovirus infections are generally asymptomatic, however, their presence may worsen disease consequences in concurrent infections with other enteric pathogens. Methods A total of 872 diarrheic fecal samples from pigs of different ages were collected from 12 districts of Sichuan Province in the southwest of China. RT-PCR was done with PToV S gene specific primers to detect the presence of PToV positive samples. M gene specific primers were used with the PToV positive samples and the genes were sequenced. A phylogenetic tree was constructed based on the M gene nucleotide sequences from the 19 selected novel Sichuan strains and 21 PToV and BToV M gene sequences from GenBank. Results A total of 331 (37.96%, 331/872) samples were found to be positive for PToV and the highest prevalence was observed in piglets aged from 1 to 3 weeks old. Through phylogenetic inference the 40 PToV M gene containing sequences were placed into two genotypes (I & II). The 19 novel Sichuan strains of genotype I showed strong correlations to two Korean gene sequences (GU-07-56-11 and GU-07-56-22). Amino-acid sequence analysis of the 40 PToV M gene strains revealed that the M gene protein was highly conserved. Conclusions This study uncovered the presence of PToV in Sichuan Province, and demonstrated the need for continuous surveillance PToV of epidemiology.

Background Torque teno sus virus (TTSuV), infecting domestic swine and wild boar, is a non-enveloped virus with a circular, single-stranded DNA genome. which has been classified into the genera Iotatorquevirus (TTSuV1) and Kappatorquevirus (TTSuV2) of the family Anelloviridae. A molecular study was conducted to detect evidence of a phylogenic relationship between these two porcine TTSuV genogroups from the sera of 244 infected pigs located in 21 subordinate prefectures and/or cities of Sichuan. Results Both genogroups of TTSuV were detected in pig sera collected from all 21 regions examined. Of the 244 samples, virus from either genogroup was detected in 203 (83.2%), while 44 animals (18.0%) were co-infected with viruses of both genogroups. Moreover, TTSuV2 (186/244, 76.2%) was more prevalent than TTSuV1 (61/244, 25%). There was statistically significant difference between the prevalence of genogroups 1 infection alone (9.4%, 23/244) and 2 alone (64.8%, 158/244), and between the prevalence of genogroups 2 (76.2%, 186/244) and both genogroups co-infection (18.0%, 44/244). The untranslated region of the swine TTSuV genome was found to be an adequate molecular marker of the virus for detection and surveillance. Phylogenetic analysis indicated that both genogroups 1 and 2 could be further divided into two subtypes, subtype a and b. TTSuV1 subtype b and the two TTSuV2 subtypes are more prevalent in Sichuan Province. Conclusions Our study presents detailed geographical evidence of TTSuV infection in China.