Phosphoinositide 3-OH kinases (PI3Ks) are a group of major intracellular signaling molecules. In our previous study, we found that inhibition of PI3K activity suppressed the androgen receptor (AR)-mediated gene expression in prostate cancer cells. The AR has been considered as a critical determinant for the development and progression of human prostate cancers. In this study, we sought to identify the PI3K isoforms involved in AR transactivation. Using a gene-specific small interference RNA (siRNA) approach, we determined that the regulatory isoform p85a and the catalytic isoform p110b, but not p110a, were required for androgen-stimulated AR transactivation and cell proliferation in prostate cancer cells. Consistently, overexpression of wild-type p110b but not p110a gene led to androgen-independent AR transactivation. Silencing p110b gene in prostate cancer cells abolished tumor growth in nude mice. Of the dual (lipid and protein) kinase activities, p110b's lipid kinase activity was required for AR transactivation. Further analysis by a chromatin immunoprecipitation assay showed that p110b is indispensable for androgen-induced AR-DNA interaction. Finally, gene expression analysis of clinical specimens showed that both p85a and p110b were highly expressed in malignant prostate tissues compared to the nonmalignant compartments, and their expression levels correlated significantly with disease progression. Taken together, our data demonstrated that p85a and p110b are essential for androgen-stimulated AR transactivation, and their aberrant expression or activation might play an important role in prostate cancer progression.

Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides a new potential therapeutic target for HO prevention.

It is well known that high rates of fusion failure and pseudoarthrosis development (5~35%) are concomitant in spinal fusion surgery, which was ascribed to the shortage of suitable materials for bone regeneration. Citrate was recently recognized to play an indispensable role in enhancing osteconductivity and osteoinductivity, and promoting bone formation. To address the material challenges in spinal fusion surgery, we have synthesized mechanically robust and fast degrading citrate-based polymers by incorporating N-methyldiethanolamine (MDEA) into clickable poly(1, 8-octanediol citrates) (POC-click), referred to as POC-M-click. The obtained POC-M-click were fabricated into POC-M-click-HA matchstick scaffolds by compositing with hydroxyapatite (HA) for interbody spinal fusion in a rabbit model. Spinal fusion was analyzed by radiography, manual palpation, biomechanical testing, and histological evaluation. At 4 and 8 weeks post surgery, POC-M-click-HA scaffolds presented optimal degradation rates that facilitated faster new bone formation and higher spinal fusion rates (11.2±3.7, 80±4.5 at week 4 and 8, respectively) than the poly(L-lactic acid)-HA (PLLA-HA) control group (9.3±2.4 and 71.1±4.4) (p<0.05). The POC-M-click-HA scaffold-fused vertebrates possessed a maximum load and stiffness of 880.8±14.5 N and 843.2±22.4 N/mm, respectively, which were also much higher than those of the PLLA-HA group (maximum: 712.0±37.5 N, stiffness: 622.5±28.4 N/mm, p<0.05). Overall, the results suggest that POC-M-click-HA scaffolds could potentially serve as promising bone grafts for spinal fusion applications.

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the roles of GEF-H1 (guanine-nucleotide exchange factor-H1)-RhoA signaling in LPS-induced interleukin-8 (IL-8, CXCL8) production in endothelial cells. First, we observed that GEF-H1 expression was upregulated in a dose- and time-dependent manner as consistent with TLR4 (Toll-like receptor 4) expression after LPS stimulation. Afterwards, Clostridium difficile toxin B-10463 (TcdB-10463), an inhibitor of Rho activities, reduced LPS-induced NF-κB phosphorylation. Inhibition of GEF-H1 and RhoA expression reduced LPS-induced NF-κB and p38 phosphorylation. TLR4 knockout blocked LPS-induced activity of RhoA, however, MyD88 knockout did not impair the LPS-induced activity of RhoA. Nevertheless, TLR4 and MyD88 knockout both significantly inhibited transactivation of NF-κB. GEF-H1-RhoA and MyD88 both induced significant changes in NF-κB transactivation and IL-8 synthesis. Co-inhibition of GEF-H1-RhoA and p38 expression produced similar inhibitory effects on LPS-induced NF-κB transactivation and IL-8 synthesis as inhibition of p38 expression alone, thus confirming that activation of p38 was essential for the GEF-H1-RhoA signaling pathway to induce NF-κB transactivation and IL-8 synthesis. Taken together, these results demonstrate that LPS-induced NF-κB activation and IL-8 synthesis in endothelial cells are regulated by the MyD88 pathway and GEF-H1-RhoA pathway.

Background: To determine the distribution and antimicrobial susceptibility pattern of pathogenic bacteria in patients with chronic cutaneous wounds on a national scale.Methods: A retrospective study was conducted using the data recorded between January 1, 2018 and January1, 2020 in 195 hospitals across China. After screening the data, 815 patients with chronic wounds were finally analyzed. The data collected included information about the patients' general condition and local cutaneous wound assessments, especially microbial culture and antibiotic susceptibility tests. The analyses were performed using SPSS Version 26.Results: The study included 815 patients (290 [35.6%] females; 63 [50–74] years). The most common causes of chronic cutaneous wounds were diabetes (183, 22.5%), infection (178, 21.8%), and pressure (140, 17.2%). Among these, 521(63.9%) samples tested yielded microbial growth, including 70 (13.4%) polymicrobial infection and 451 (86.6%) monomicrobial infection. The positive rate of microbial culture was highest in wound tissue of ulcers caused by infection (87.6%), followed by pressure (77.1%), diabetes (68.3%), and venous diseases (67.7%). Bates-Jensen wound assessment tool (BWAT) scores &gt;25 and wounds that lasted for more than 3 months had a higher positive rate of microbial culture. BWAT scores &gt;25 and wounds in the rump, perineum, and feet were more likely to exhibit polymicrobial infection. A total of 600 strains were isolated, of which 46.2% (277 strains) were Gram-positive bacteria, 51.3% (308 strains) were Gram-negative bacteria, and 2.5% (15 strains) were fungi. The most common bacterial isolates were Staphylococcus aureus (29.2%), Escherichia coli (11.5%), Pseudomonas aeruginosa (11.0%), Proteus mirabilis (8.0%), and Klebsiella pneumoniae (5.8%). The susceptibility tests showed that 116 cultured bacteria were Multidrug resistant (MDR) strains. The resistance rates of S. aureus were 92.0% (161/175) to penicillin, 58.3% (102/175) to erythromycin, and 50.9% (89/175) to clindamycin. Vancomycin was the most effective antibiotic (0% resistance rate) against all Gram-positive bacteria. Besides, the resistance rates of E. coli were 68.1% (47/69) to ampicillin, 68.1% (47/69) to ciprofloxacin, 60.9% (42/69) to levofloxacin. However, all the isolated Gram-negative bacteria showed low resistance rates to tigecycline (3.9%) and amikacin (3.6%).Conclusions: The distribution of bacteria isolated from chronic cutaneous wounds varies with the BWAT scores, causes, duration, and the location of wounds. Multidrug resistance is a serious health issue, and therefore antibiotics used in chronic wounds must be under strict regulation. Our findings may help clinicians in making informed decisions regarding antibiotic therapy.

Attempts to replicate native tissue architecture have lead to the design of biomimetic scaffolds focused on improving functionality. In this study, biomimetic citrate-based poly (octanediol citrate) – click hydroxyapatite (POC-Click-HA) scaffolds were developed to simultaneously replicate the compositional and architectural properties of native bone tissue while providing immediate structural support for large segmental defects following implantation. Biphasic scaffolds were fabricated with 70% internal phase porosity and various external phase porosities (between 5–50%) to mimic the bimodal distribution of cancellous and cortical bone, respectively. Biphasic POC-Click-HA scaffolds displayed compressive strengths up to 37.45 ± 3.83 MPa, which could be controlled through the external phase porosity. The biphasic scaffolds were also evaluated in vivo for the repair of 10-mm long segmental radial defects in rabbits and compared to scaffolds of uniform porosity as well as autologous bone grafts after 5, 10, and 15 weeks of implantation. The results showed that all POC-Click-HA scaffolds exhibited good biocompatibility and extensive osteointegration with host bone tissue. Biphasic scaffolds significantly enhanced new bone formation with higher bone densities in the initial stages after implantation. Biomechanical and histomorphometric analysis supported a similar outcome with biphasic scaffolds providing increased compression strength, interfacial bone ingrowth, and periosteal remodeling in early time points, but were comparable to all experimental groups after 15 weeks. These results confirm the ability of biphasic scaffold architectures to restore bone tissue and physiological functions in the early stages of recovery, and the potential of citrate-based biomaterials in orthopedic applications.

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The androgen receptor (AR) is the most critical factor in prostate cancer progression. We previously demonstrated that silencing the AR using 2 unique small interfering RNAs (no. 8 and no. 31 AR siRNA) induces apoptotic cell death in AR-positive prostate cancer cells. To develop this AR siRNA technique into a therapy for prostate cancers, we generated an adeno-associated virus (AAV) vector to stably express a short hairpin-structured RNA (shRNA) against the AR gene in vivo. In addition to the no. 8 AR shRNA (ARHP8), we also screened a group of AR shRNAs with different sequences and identified a less effective AR shRNA (ARHP4) that was used as an shRNA control. An empty AAV vector (AAV-GFP) was used as a negative control. Intratumoral injection of AAV-ARHP8 viruses significantly suppressed tumor growth of xenografts derived from either androgen-responsive or castration-resistant prostate cancer cells. Most interestingly, systemic delivery of the AAV-ARHP8 but not AAV-ARHP4 or AAV-GFP viruses via tail vein injection eliminated xenografts within 10 days. Further analysis revealed that AAV-ARHP8 viruses dramatically reduced the expression of AR-regulated cellular survival genes and caused a dramatic apoptotic response. Taken together, our data strongly suggest that AAV-ARHP8 viruses induced a strong AR gene silencing in vivo and that systemic delivery of ARHP8 siRNA via an AAV vector or any other means might be considered as novel gene therapy for prostate cancers.Clinically, nearly all prostate cancers including castration-resistant diseases retain a functional androgen receptor (AR) signaling pathway, which is the most critical factor in prostate cancer progression. 1 Current evidence favors a mechanism by which aberrant activation of intracellular signal transduction pathways stimulates the AR in the presence of residue ligand after chemical or surgical castration or even in case of androgen antagonist. 2-5 Thus, it is being considered that targeting the AR itself is more relevant compared to ablating its ligand that often results in clinical failure. 3,4 RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing, which can be activated by introducing small double-stranded interfering RNA molecule (siRNA) corresponding to any endogenous gene of interest, resulting in mRNA degradation of the targeted gene. 6 Therefore, siRNA is referred as an extremely powerful and simple