Hot Deformation Behavior and Processing Maps of ZnSnO3/Cu Composites.

In this work, we designed ternary ZnSnO3 particle-reinforced Cu matrix composites and evaluated the hot deformation behavior of ZnSnO3/Cu composites. The hot deformation characteristics of typical dynamic recrystallization were probed by the resulting true stress-strain curves of ZnSnO3/Cu composites. The influences of deformation conditions, including temperatures (650-850 °C) and strain rates (0.01-5 s-1), on the flow stress of the designed composites were investigated. This revealed that the peak stress increased with the increasing of strain rate and decreasing of temperature. Additionally, the activation energy was calculated to be 237.05 kJ/mol and followed by yielding a constitutive equation for low-stress ZnSnO3/Cu composites. The processing maps established by dynamic materials model theory indicated that the designed composites possessed excellent hot workability, and then the processing parameters (790-850 °C and 0.01-0.04 s-1) of the ZnSnO3/Cu composites were determined for practical industrial production. Our work discloses the deformation behavior of ZnSnO3/Cu matrix composites and extends the rational process design for ternary ceramic/metal materials with excellent hot workability.

Autophagy-based unconventional secretion of HMGB1 in glioblastoma promotes chemosensitivity to temozolomide through macrophage M1-like polarization.

BACKGROUND: Glioblastoma (GB) is the most common and highly malignant brain tumor characterized by aggressive growth and resistance to alkylating chemotherapy. Autophagy induction is one of the hallmark effects of anti-GB therapies with temozolomide (TMZ). However, the non-classical form of autophagy, autophagy-based unconventional secretion, also called secretory autophagy and its role in regulating the sensitivity of GB to TMZ remains unclear. There is an urgent need to illuminate the mechanism and to develop novel therapeutic targets for GB. METHODS: Cancer genome databases and paired-GB patient samples with or without TMZ treatment were used to assess the relationship between HMGB1 mRNA levels and overall patient survival. The relationship between HMGB1 protein level and TMZ sensitivity was measured by immunohistochemistry, ELISA, Western blot and qRT-PCR. GB cells were engineered to express a chimeric autophagic flux reporter protein consisting of mCherry, GFP and LC3B. The role of secretory autophagy in tumor microenvironment (TME) was analyzed by intracranial implantation of GL261 cells. Coimmunoprecipitation (Co-IP) and Western blotting were performed to test the RAGE-NFκB-NLRP3 inflammasome pathway. RESULTS: The exocytosis of HMGB1 induced by TMZ in GB is dependent on the secretory autophagy. HMGB1 contributed to M1-like polarization of tumor associated macrophages (TAMs) and enhanced the sensitivity of GB cells to TMZ. Mechanistically, RAGE acted as a receptor for HMGB1 in TAMs and through RAGE-NFκB-NLRP3 inflammasome pathway, HMGB1 enhanced M1-like polarization of TAMs. Clinically, the elevated level of HMGB1 in sera may serve as a beneficial therapeutic-predictor for GB patients under TMZ treatment. CONCLUSIONS: We demonstrated that enhanced secretory autophagy in GB facilitates M1-like polarization of TAMs to enhance TMZ sensitivity of GB cells. HMGB1 acts as a key regulator in the crosstalk between GB cells and tumor-suppressive M1-like TAMs in GB microenvironment and may be considered as an adjuvant for the chemotherapeutic agent TMZ.

Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis.

A large number of protease inhibitors have been found from leeches, which are essential in various physiological and biological processes. In the curret study, a novel elastase inhibitor was purified and characterized from the leech of Hirudinaria manillensis, which was named HMEI-A. Primary structure analysis showed that HMEI-A belonged to a new family of proteins. HMEI-A exerted inhibitory effects on elastase and showed potent abilities to inhibit elastase with an inhibition constant (Ki) of 1.69 × 10-8 mol·L-1. Further study showed that HMEI-A inhibited the formation of neutrophil extracellular trap (NET). These results suggested that HMEI-A from the leech of H. manillensis is a novel elastase inhibitor which can suppress NET formation. It may play a significant role in blood-sucking of leeches and is a potential candidate as an anti-inflammatory agent.

ADP-Ribosylation Factor Like GTPase 4C (ARL4C) augments stem-like traits of glioblastoma cells by upregulating ALDH1A3.

Glioma cells with stem cell-like properties are crucial for tumor initiation, progression and therapeutic resistance. Therefore, identifying specific factors in regulating stem-like traits is critical for the design of novel glioma therapeutics. Herein, we reported that ADP-Ribosylation Factor Like GTPase 4C (ARL4C) was highly expressed in glioma stem-like cells (GSLCs). GSLCs, determined by the efficiency of sphere formation in vitro and tumor growth in vivo, was increased by overexpression of ARL4C. ARL4C induced the tumorigenesis through ALDH1A3. Analyses of 325 patient specimens showed that ARL4C was highly expressed in glioblastoma (GBM) as compared with lower grade gliomas. In addition, higher level ARL4C expression in glioma was correlated with poorer progression-free survival and overall survival of patients. Therefore, ARL4C may act as a novel prognostic marker and a therapeutic target for GBM.

Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic worldwide. Currently, however, no effective drug or vaccine is available to treat or prevent the resulting coronavirus disease 2019 (COVID-19). Here, we report our discovery of a promising anti-COVID-19 drug candidate, the lipoglycopeptide antibiotic dalbavancin, based on virtual screening of the FDA-approved peptide drug library combined with in vitro and in vivo functional antiviral assays. Our results showed that dalbavancin directly binds to human angiotensin-converting enzyme 2 (ACE2) with high affinity, thereby blocking its interaction with the SARS-CoV-2 spike protein. Furthermore, dalbavancin effectively prevents SARS-CoV-2 replication in Vero E6 cells with an EC50 of ~12 nM. In both mouse and rhesus macaque models, viral replication and histopathological injuries caused by SARS-CoV-2 infection are significantly inhibited by dalbavancin administration. Given its high safety and long plasma half-life (8-10 days) shown in previous clinical trials, our data indicate that dalbavancin is a promising anti-COVID-19 drug candidate.

Purification and characterization of a novel anti-coagulant from the leech Hirudinaria manillensis.

Protease inhibitors have been reported rarely from the leech Hirudinaria manillensis. In this study, we purified a novel protease inhibitor (bdellin-HM-2) with anticoagulant properties from H. manillensis. With a molecular weight of 1.4x104, bdellin-HM-2 was also characterized with three intra-molecular disulfide bridges at the N-terminus and multiple HHXDD and HXDD motifs at the C-terminus. cDNA cloning revealed that the putative nucleotide-encoding protein of bdellin-HM-2 contained 132 amino acids and was encoded by a 399 bp open reading frame (ORF). Sequence alignment showed that bdellin-HM-2 shared similarity with the "non-classical" Kazal-type serine protease inhibitors, but had no inhibitory effect on trypsin, elastase, chymotrypsin, kallikrein, factor XIIa (FXIIa), factor XIa (FXIa), factor Xa (FXa), thrombin, or plasmin. Bdellin-HM-2 showed anticoagulant effects by prolonging the activated partial thromboplastin time (aPTT), indicating a role in enabling H. manillensis to obtain a blood meal from its host. Our results suggest that bdellin-HM-2 may play a crucial role in blood-sucking in this leech species and may be a potential candidate for the development of clinical anti-thrombotic drugs.

Mitochondrial pyruvate carrier 1 functions as a tumor suppressor and predicts the prognosis of human renal cell carcinoma.

Invasion and subsequent metastasis are major characteristics of malignant human renal cell carcinoma (RCC), though the mechanisms remain elusive. Mitochondrial pyruvate carrier (MPC), a key factor that controls pyruvate transportation in mitochondria, is frequently dysregulated in tumor cells and loss of MPC predicts poor prognosis in various types of cancer. However, the clinical relevance and functional significance of MPC in RCC remain to be elucidated. In this study, we investigated the expression of MPC1 and MPC2 in specimens from RCC patients and observed downregulation of MPC1, but not MPC2, in RCC tissues compared with adjacent non-cancerous tissue. Moreover, RCC patients with higher MPC1 expression exhibited longer overall survival rate than those with lower MPC1. Functionally, MPC1 suppressed the invasion of RCC cells in vitro and reduced the growth of RCC cells in vivo, possibly through inhibition of MMP7 and MMP9. Further studies revealed that loss of MPC1 was induced by hypoxia in RCC cells, and notably, MPC1 expression, was negatively correlated with HIF1α expression in RCC cells and patient samples. Taken together, our results identify anti-tumor function of MPC1 in RCC and revealed MPC1 as a novel prognostic biomarker to predict better patient survival.

ARL4C stabilized by AKT/mTOR pathway promotes the invasion of PTEN-deficient primary human glioblastoma.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency in primary human glioblastoma (GBM) is associated with increased invasiveness and poor prognosis with unknown mechanisms. Therefore, how loss of PTEN promotes GBM progression remains to be elucidated. Herein, we identified that ADP-ribosylation factor like-4C (ARL4C) was highly expressed in PTEN-deficient human GBM cells and tissues. Mechanistically, loss of PTEN stabilized ARL4C protein due to AKT/mTOR pathway-mediated inhibition of ARL4C ubiquitination. Functionally, ARL4C enhanced the progression of GBM cells in vitro and in vivo. Moreover, microarray profiling and GST pull-down assay identified that ARL4C accelerated tumor progression via RAC1-mediated filopodium formation. Importantly, targeting PTEN potently inhibited GBM tumor progression in vitro and in vivo, whereas overexpression of ARL4C reversed the tumor progression impaired by PTEN overexpression. Clinically, analyses with patients' specimens validated a negative correlation between PTEN and ARL4C expression. Elevated ARL4C expression but PTEN deficiency in tumor was associated with poorer disease-free survival and overall survival of GBM patients. Taken together, ARL4C is critical for PTEN-deficient GBM progression and acts as a novel prognostic biomarker and a potential therapeutic candidate. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Effects of an immune stimulant, inactivated Mycobacterium phlei, on the growth performance as well as meat quality of fattening pigs.

Inactivated mycobacterium phlei (M. phlei) is well known for its immune-stimulatory functions in humans and livestock, but less information is available about the influence on meat quality of pigs when used as a feed additive. This study was designed to evaluate the effects of inactivated M. phlei on growth performance as well as meat quality of fattening pigs. A total of 240 cross-bred pigs ([Landrace × Yorkshire] × Duroc) with initial body weight of 80.14 ± 0.29 kg were randomly allocated to five treatments, each of which consisted of eight replicates with 6six pigs per replicate. The basal diet supplemented with five levels of inactivated M. phlei preparations (0, 3.5 × 109 [0.1% w/w], 7 × 109 [0.2%], 1.4 × 1010 [0.4%] or 2.1 × 1010 [0.6%] colony-forming units/kg) was respectively fed to the control group and four treatment groups for 30 days. Adding 0.4% of inactivated M. phlei to diet increased the average daily feed intake and average daily gain of pigs. Importantly, intramuscular fat percentage in the Longissimus dorsi (LD) was increased by feeding diet containing 0.2%, 0.4% and 0.6% of inactivated M. phlei, despite the pH value, drip loss, cooking loss and filter paper fluid uptake not being influenced. Analysis of the fatty acid components showed that some saturated fatty acids were decreased in LD after feeding inactivated M. phlei, but some monounsaturated fat acids (MUFAs) and polyunsaturated fatty acids were increased (PUFAs), which induced the total contents of MUFAs and PUFAs were improved. RT-PCR assay revealed that feeding inactivated M. phlei up-regulated genes implicated in fat metabolism in muscle, including ELOVL6, FASN, SCD1 and H-FABP. This study revealed that feeding inactivated M. phlei not only increased growth performance of fattening pigs, but also improved the meat quality by increasing intramuscular fat content, thus inactivated M. phlei probably has high utilization value in modern pig production.

Prospective evaluation of the diagnostic accuracy of hepatic copper content, as determined using the entire core of a liver biopsy sample.

UNLABELLED: Hepatic copper determination is an important test for the diagnosis of Wilson's disease (WD). However, the method has not been standardized, the diagnostic accuracy has not been evaluated prospectively, and the optimal cut-off value remains controversial. Accordingly, we aimed to prospectively evaluate the diagnostic accuracy of hepatic copper content, as determined using the entire core of a liver biopsy sample. Patients for whom a liver biopsy was indicated were consecutively enrolled. Hepatic copper content was determined with atomic absorption spectroscopy. All assays were performed using careful quality control by a single technician. WD diagnosis was based on WD score or its combination with clinical follow-up results. A total of 3,350 consecutive patients underwent liver biopsy. Six hundred ninety-one patients, including 178 with WD, underwent two passes of liver biopsy with hepatic copper determination. Mean hepatic content in WD patients was 770.6 ± 393.2 µg/g dry weight (wt). Sensitivity, specificity, and positive and negative predictive values of hepatic copper content for WD diagnosis in the absence of primary biliary cirrhosis (PBC) or primary sclerosing cholangitis at the cut-off value of 250 µg/g dry wt. were 94.4%, 96.8%, 91.8%, and 97.8%, respectively. The most useful cut-off value was 209 µg/g dry wt, with a sensitivity and specificity of 99.4% and 96.1%, respectively. A total of 23.3% of patients without WD and PBC had hepatic copper content >75 µg/g dry wt. CONCLUSION: A liver biopsy sample of more than 1 mg dry wt may reliably reflect hepatic copper content and should be used for hepatic copper determination. Hepatic copper determination is a very valid procedure for the diagnosis of WD, and the most useful cut-off value is 209 µg/g dry wt.

Study on angle of immediate loading of immediate implant placement.

OBJECTIVE: To investigate the clinical immediate load at an angle after immediate placement of the implant. METHODS: Select 4 adult dogs; through establishing the angle loading animal experiment model, perform lateral loading on 32 implants respectively at vertical and 0°, 10°, and 20°, with which as a basis for grouping, determine the osseointegration index and new bone growth rate; and observe the peri-implant bone remodeling conditions. RESULTS: The 20° group is found with the most obvious bone absorption, and compared with other groups, its osseointegration index and new bone growth rate are statistically significant (P < 0.01); bone remodeling under 0° load stress is the best, with the formation of new bone and the highest bone contact ratio, which is the most reasonable under this the stress distribution compared with other angles. CONCLUSIONS: The implant stress distribution at 0° against the occlusal force direction is closer to physiologic optimum stress on the implant bone interface, and it is permitted for the long axis of the immediately implanted and immediately loaded implant to be tilted within about 10° against the load angle.

Adipose-derived stem cells transfected with pEGFP-OSX enhance bone formation during distraction osteogenesis.

This study was designed to investigate the effects of local delivery of adipose-derived stem cells (ADSCs) transfected with transcription factor osterix (OSX) on bone formation during distraction osteogenesis. New Zealand white rabbits (n=54) were randomly divided into three groups (18 rabbits per group). A directed cloning technique was used for the construction of recombinant plasmid pEGFP-OSX, where EGFP is the enhanced green fluorescence protein. After osteodistraction of the right mandible of all experimental rabbits, rabbits in group A were treated with ADSCs transfected with pEGFP-OSX, group B with ADSCs transfected with pEGFP-N1, and group C with physiological saline. Radiographic and histological examinations were processed after half of the animals within each group were humanely killed by injection of sodium pentothal at Week 2 or 6 after surgery. The distraction bone density was measured as its projectional bone mineral density (BMD). Three parameters were measured, namely, the thickness of new trabeculae (TNT), and the volumes of the newly generated cortical bone (NBV1) and the cancellous bone (NBV2) of the distracted regions. Good bone generation in the distraction areas was found in group A, which had the highest BMD, TNT, and NBV in the distraction zones among the groups. There was no significant difference in bone generation in the distraction areas between groups B and C. The results indicate that the transplantation of ADSCs transfected with pEGFP-OSX can effectively promote bone generation during distraction in vivo.

Transcription factor osterix modified bone marrow mesenchymal stem cells enhance callus formation during distraction osteogenesis.

This study was designed to investigate the effects of local delivery of bone marrow mesenchymal stem cells (BMMSCs) with or without osterix (OSX) gene transfected on bone regeneration in the distracted zone using a rabbit model of mandibular lengthening. Fifty-four New Zealand white rabbits underwent osteodistraction of the left mandible and were then randomly divided into group A, group B, and group C (n = 18 for each group). At the end of distraction BMMSCs transfected with OSX, autologous BMMSCs and physiological saline were injected into the distraction gaps in groups A, B, and C, respectively. Nine animals from each group were humanely killed at 2 and 6 weeks after completion of distraction. The distracted mandibles were harvested and processed for radiographic, histological, and immunohistochemical examination. Excellent bone formation in the distracted callus was observed in group A and group B; the former showed better bone formation and highest bone mineral density (BMD), thickness of new trabeculae (TNT, mm) and volumes of the newly formed bone area (NBV) in the distraction zones. Group C animals showed poor bone formation in the distracted callus when compared with groups A and B. Positive immunostaining of bone sialoprotein (BSP) was observed in the distracted callus in all groups; however, BSP expression was much stronger in group A than in groups B and C. The results of this study suggest transplantation of BMMSCs can promote bone formation in DO; OSX-mediated ex vivo gene therapy was more effective during bone deposition and callus formation in distraction osteogenesis.

Expression of augmenter of liver regeneration in rats with gentamicin-induced acute renal failure and its protective effect on kidney.

Augmenter of liver regeneration (ALR) enhances the proliferation of hepatocytes and accelerates recovery from acute liver failure in animal models. ALR is expressed in both the liver and kidney; however, the specific location of ALR expression and its biological effects in the kidney remain unknown. We aimed to investigate the efficacy of ALR in rats with gentamicin (GM)-induced acute renal failure (ARF). Rats were randomized into the normal group, GM+saline group, GM+vehicle group, and GM+rrALR group. Blood urea nitrogen, serum creatinine, and urine beta-N-acetyl-D-glucosaminidase were measured, and histological analyses of the kidneys were performed. The expression of ALR protein was determined by immunohistochemistry and Western blotting. In vitro incorporation of tritiated thymidine was used to measure the proliferation of renal tubular epithelial cells. In normal rats, the expression of ALR protein was faint in the medulla and absent in the cortex. However, in ARF rats, ALR expression increased significantly in both the renal cortex and medulla. Histological analyses revealed that treatment with recombinant rat ALR (rrALR) reduced the extent of injury of tubular cells in the renal cortex. Serum/urine biochemical parameters also showed that renal dysfunction was improved by the administration of rrALR. Intraperitoneal injection of rrALR enhanced the proliferation of tubular cells in vivo. We also confirmed that rrALR could promote the proliferation of renal tubular cells in vitro. These results indicate that rrALR effectively accelerates kidney recovery after ARF induced by gentamicin, and that the protective effect is associated with enhanced proliferation of renal tubular cells.

Differentiation of human umbilical cord blood stem cells into hepatocytes in vivo and in vitro.

AIM: To study the condition and potentiality of human umbilical cord blood stem cells (HUCBSC) to differentiate into hepatocytes in vivo or in vitro. METHODS: In a cell culture study of human umbilical cord blood stem cell (HUCBSC) differentiation, human umbilical cord blood mononuclear cells (HUCBMNC) were separated by density gradient centrifugation. Fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) and the supernatant of fetal liver were added in the inducing groups. Only FGF was added in the control group. The expansion and differentiation of HUCBMNC in each group were observed. Human alpha fetoprotein (AFP) and albumin (ALB) were detected by immunohistochemistry. In the animal experiments, the survival SD rats with acute hepatic injury after carbon tetrachloride (CCL4) injection 48 h were randomly divided into three groups. The rats in group A were treated with human umbilical cord blood serum. The rats in group B were treated with HUCBMNC transplantation. The rats in group C were treated with HUCBMNC transplantation followed by intraperitoneal cyclophosphamide for 7 d. The rats were killed at different time points after the treatment and the liver tissue was histopathologically studied and human AFP and ALB detected by immunohistochemistry. The human X inactive-specific transcript gene fragment in the liver tissue was amplified by PCR to find human DNA. RESULTS: The results of cell culture showed that adherent cells were stained negative for AFP or ALB in control group. However, the adherent cells in the inducing groups stained positive for AFP or ALB. The result of animal experiment showed that no human AFP or ALB positive cells present in the liver tissue of group A (control group). However, many human AFP or ALB positive cells were scattered around sinus hepaticus and the central veins of hepatic lobules and in the portal area in group B and group C after one month. The fragment of human X chromagene could be detected in the liver tissue of groups B and C, but not in group A. CONCLUSION: Under certain conditions HUCBSC can differentiate into liver cells in vivo and in vitro.

[Clinical and experimental study of the therapeutic effect of umbilical cord blood stem cell transplantation on liver failure and heart damage in severe viral hepatitis patients].

OBJECTIVE: To investigate and compare the therapeutic effect of umbilical cord blood stem cell transplantation (UCBSCT) or adult fresh plasma in severe viral hepatitis liver failure with/without heart damage, and to study the effect of UCBSCT on liver lesions in rats. METHODS: 83 severe hepatitis patients with/without heart damage were included in the study between January 1994 and June 2003. The patients were treated with UCBSCT or given adult plasma transfusions. The therapeutic effect was evaluated by serial determination of liver function and myocardium enzymes in all patients before and after the treatment. The model of experimental hepatic failure was constructed in SD rats by injecting carbon tetrachloride. Then, the rats were given normal saline, neonate cord blood serum or neonate cord blood stem cells respectively. The expression of human AFP and Alb in SD rat livers was detected by immunohistochemistry; and human special DNA was detected by PCR. RESULTS: The UCBSCT group had much better effects in the improvement of liver function than the adult plasma group had, no matter whether the patients had heart damage or not. Moreover, UCBSCT can decrease heart impairment of the patients. The animal experiment demonstrated that AFP and Alb positive cells were present in the neonate cord blood stem cell group after 21 days and 1 month; human special DNA was detected by PCR in these SD rat livers. CONCLUSION: UCBSCT displayed good therapeutic effects on severe viral hepatitis and improvement of heart injury of the patients. The rat liver immunohistochemistry indicated that neonate cord blood stem cell application can decrease the liver damage and increase hepatocellular regeneration. Human umbilical cord blood stem cells can differentiate into liver cells in acute damaged SD rat livers.

Clinical and experimental study on therapeutic effect of umbilical cord blood transplantation on severe viral hepatitis.

AIM: To investigate the therapeutic effect of umbilical cord blood transplantation (UCBT) on patients with severe viral hepatitis and on liver lesions in rats. METHODS: One hundred and fifty three patients with severe viral hepatitis were included in the study between April 1990 and July 2002. The patients were treated with adult plasma transfusion (control), UCBT, plasma exchange (PE) and UCBT combined with PE (UCBT+PE) respectively. The therapeutic effectiveness was evaluated by serial determinations of liver function, lipids and immune function in all patients before and after the treatment. The model of experimental hepatic failure was constructed in SD rats by injecting carbon tetrachloride. Then, the rats were given normal saline, adult plasma or neonate cord blood intraperitoneally. After detection of liver function, the rats were killed and morphological changes of the liver were microscopically observed. RESULTS: UCBT group and UCBT+PE group had much better improvement in liver and immune functions than control group and PE group. The patients in UCBT+PE group had the best clinical efficacy. UCBT was safe and had no side effects. The animal experiment showed significant improvements in liver function and survival rate in neonate cord blood group as compared with adult plasma group. The histopathology of rat's liver indicated that neonate cord blood application could decrease the liver injury and increase hepatocellular regeneration. CONCLUSION: UCBT demonstrated a good therapeutic effect on severe viral hepatitis and no obvious side effects. Umbilical cord blood can attenuate the liver lesions and reproduce hepatocyte. The treatment of UCBT combined with PE was much better than that of single plasma exchange, thus UCBT can enhance the therapeutic effect of plasma exchange on severe viral hepatitis.