Tumor microenvironment activation amplify oxidative stress promoting tumor energy remodeling for mild photothermal therapy and cuproptosis.

Tumor metabolic reprogramming requires high levels of adenosine triphosphate (ATP) to maintain treatment resistance, which poses major challenges to chemotherapy and photothermal therapy. Especially, high levels of ATP promote copper ion efflux for limiting the curative effect of cuproptosis. Here, an H2S-responsive mesoporous Cu2Cl(OH)3-loading chemotherapeutic cisplatin (CDDP) was synthesized, and the final nanoparticle, CDDP@Cu2Cl(OH)3-CDs (CDCuCDs), was encapsulated by electrostatic action with carbon dots (CDs). CDCuCDs reacted with overproduction H2S in colon tumor to produce photothermic copper sulfide for photothermal therapy. CDDP was released by lysis to achieve chemotherapeutic effects. Importantly, CDDP elevated H2O2 levels in cells through a cascade reaction and continuously transforms H2O2 into highly cytotoxic •OH through chemodynamic therapy between H2O2 and Cu+, which enables nanoparticles to generate •OH and improve the chemotherapeutic efficacy. Highly toxic •OH disrupts mitochondrial homeostasis, prohibiting it from performing normal energy-supplying functions. Down-regulated ATP inhibits heat shock protein expression, which promotes the therapeutic effect of mild photothermal therapy and reduces the efflux of intracellular copper ions, thus improving the therapeutic effect of cuproptosis. Our research provides a potential therapeutic strategy using overproduction H2S responses in tumors, allowing tumor microenvironment-activated •OH nanogenerators to promote tumor energy remodeling for cancer treatment.

H2S-driven chemotherapy and mild photothermal therapy induced mitochondrial reprogramming to promote cuproptosis.

The elevated level of hydrogen sulfide (H2S) in colon cancer hinders complete cure with a single therapy. However, excessive H2S also offers a treatment target. A multifunctional cascade bioreactor based on the H2S-responsive mesoporous Cu2Cl(OH)3-loaded hypoxic prodrug tirapazamine (TPZ), in which the outer layer was coated with hyaluronic acid (HA) to form TPZ@Cu2Cl(OH)3-HA (TCuH) nanoparticles (NPs), demonstrated a synergistic antitumor effect through combining the H2S-driven cuproptosis and mild photothermal therapy. The HA coating endowed the NPs with targeting delivery to enhance drug accumulation in the tumor tissue. The presence of both the high level of H2S and the near-infrared II (NIR II) irradiation achieved the in situ generation of photothermic agent copper sulfide (Cu9S8) from the TCuH, followed with the release of TPZ. The depletion of H2S stimulated consumption of oxygen, resulting in hypoxic state and mitochondrial reprogramming. The hypoxic state activated prodrug TPZ to activated TPZ (TPZ-ed) for chemotherapy in turn. Furthermore, the exacerbated hypoxia inhibited the synthesis of adenosine triphosphate, decreasing expression of heat shock proteins and subsequently improving the photothermal therapy. The enriched Cu2+ induced not only cuproptosis by promoting lipoacylated dihydrolipoamide S-acetyltransferase (DLAT) heteromerization but also performed chemodynamic therapy though catalyzing H2O2 to produce highly toxic hydroxyl radicals ·OH. Therefore, the nanoparticles TCuH offer a versatile platform to exert copper-related synergistic antitumor therapy.

PORCN promotes hepatocellular carcinogenesis via Wnt/ß-catenin-dependent manner.

Background: Porcupine O-acyltransferase (PORCN), a membrane-bound O-acyltransferase, is crucial in Wnt ligand palmitoylation. However, the roles of PORCN in the development of hepatocellular carcinoma (HCC) remain unknown. Methods: Western blot, real-time quantitative polymerase chain reaction (RT-qPCR) assays, and The Cancer Genome Atlas (TCGA) database were used to study the expression and prognostic values of PORCN in patients with HCC. Following this, Cell Counting Kit-8 (CCK-8), wound-healing tests, Transwell assay, and a xenograft mouse model were employed to examine the effect of PORCN on HCC cells. Finally, the underlying molecular mechanisms involved in cell proliferation and migration caused by PORCN were identified. Results: The protein and messenger RNA (mRNA) levels of PORCN in HCC tissues were higher than those of adjacent normal tissues. The analysis of TCGA database indicated that patients with higher PORCN expression had a lower overall survival (OS) rate. Overexpression of PORCN could promote the proliferation and migration abilities of HCC cells both in vitro and in vivo. Gene set enrichment analysis (GSEA) showed that the effect of PORCN on the biological characteristics of HCC cells mainly centered on the Wnt-ß-catenin signaling pathway. Mechanically, immunofluorescence staining and subcellular protein fraction assays showed that PORCN could induce epithelial-mesenchymal transition (EMT) by promoting the translocation of ß-catenin from the cytoplasm to nucleus, ultimately promoting the progression of HCC. Conclusions: The findings of this study suggest that PORCN can promote HCC cell proliferation and migration by stimulating the Wnt-ß-catenin signaling pathway. Therefore, PORCN may be a promising therapeutic target for HCC.

KRT17 Promotes the Activation of HSCs via EMT in Liver Fibrosis.

Background and Aims: Although activation of hepatic stellate cells (HSCs) plays a central role in the development of liver fibrosis, the mechanism underlying the activation of HSCs remains unclear. Keratin 17 (KRT17), a member of the intermediate filament family, can regulate tumor cell proliferation and migration. The current study aimed to elucidate the role of KRT17 in the activation of HSCs and the mechanisms underlying liver fibrosis. Methods: The expression of KRT17 was determined using immunohistochemistry in tissue microarray. Western blotting and qRT-PCR assays were used to determine the KRT17 expression in fibrotic liver tissues obtained from human subjects and mice. LX-2 cells were treated with TGF-ß1 recombinant protein and adipocyte differentiation mixture (MDI) mix to induce and reverse LX-2 cell activation, respectively, in order to explore the correlation between KRT17 and HSC activation. Additionally, cell proliferation and migration abilities of LX-2 cells transfected with KRT17-overexpressing plasmid or small interfering RNA were determined using CCK-8, flow cytometry, Transwell, and wound healing assays. Finally, rescue assay was used to explore the role of KRT17 in HSC activation and epithelial-mesenchymal transition (EMT). Results: The expression of KRT17 was higher in the human and mouse fibrotic liver tissues than in healthy liver tissues, and it was positively correlated with HSC activation. Upregulated KRT17 enhanced proliferation, migration, HSC activation and EMT in LX-2 cells, while knockdown of KRT17 reversed these effects. TGF-ß1 recombinant protein accelerated KRT17-mediated EMT, HSC activation and proliferation, while TGF-ß1 inhibitor counteracted the effect of KRT17 in vitro. Conclusions: KRT17 promoted HSC activation, proliferation and EMT in hepatic fibrosis probably via TGF-ß1 signaling, and KRT17 might serve as a therapeutic target for the treatment of liver fibrosis.

PSME4 Activates mTOR Signaling and Promotes the Malignant Progression of Hepatocellular Carcinoma.

BACKGROUND: As hepatocellular carcinoma (HCC) having the second-highest mortality rate globally, the early diagnosis and prognosis of HCC have always been the focus of various studies. Although PSME4 has been reported to be closely related to several malignancies, its role in HCC remains unclear. MATERIALS AND METHODS: The TCGA-LIHC database and HCC tissues were used to explore the expression of PSME4 in HCC. Gene set enrichment analysis (GSEA) was used to forecast the biological behavior of HCC cells that PSME4 might be involved in regulation. In addition, CCK-8, colony formation and flow cytometry assays were used to explore the effect of PSME4 on HCC cells. Furthermore, the underlying PSME4-related signaling pathways in HCC were further confirmed using GSEA. RESULTS: We found that the expression of PSME4 in HCC tissues was significantly higher than that in adjacent normal tissues, and patients with high PSME4 expression have a poor prognosis. CCK-8, colony formation and flow cytometry assays shown that knockdown of PSME4 inhibits HCC cell proliferation of HCC cells, promotes cell apoptosis and moves the cell cycle away from the S phase. Mechanistically, PSME4 may promote the development of HCC through mTOR signaling pathway. CONCLUSION: The high expression of PSME4 in HCC promotes the proliferation of HCC cells via the mTOR signalling pathway. Therefore, PSME4 is an emerging tumour marker for the early diagnosis and prognosis of HCC.

Keratin 17 upregulation promotes cell metastasis and angiogenesis in colon adenocarcinoma.

Colon adenocarcinoma (COAD), having high malignancy and poor prognosis, is the main pathological type of colon cancer. Previous studies show that Keratin 17 (KRT17) plays an important role in the development of many malignant tumors. However, its role and the molecular mechanism underlying COAD remain unclear. Using TCGA and ONCOMINE databases, as well as immunohistochemistry, we found that the expression of KRT17 was higher in COAD tissues as compared to that in the adjacent normal tissues. Cell- and animal-based experiments showed that overexpression of KRT17 promoted the invasion and metastasis of colon cancer cells while knocking down KRT17 reversed these processes both in vitro and in vivo. In addition, we also showed that KRT17 promoted the formation of new blood vessels. Mechanistically, KRT17 could regulate the WNT/ß-catenin signaling pathway, and APC may be involved in this process by interacting with KRT17. In summary, these findings suggested that high expression of KRT17 could promote cell metastasis and angiogenesis of colon cancer cells by regulating the WNT/ß-catenin signaling pathway. Thus, KRT17 could be a potential therapeutic target for COAD treatment.

Maternal and neonatal outcomes in transverse and vertical skin incision for placenta previa : Skin incision for placenta previa.

BACKGROUND: Placenta previa, a serious obstetric issue, should be managed by experienced teams. The safe and appropriate mode of delivery for placenta previa is by cesarean delivery. However, no studies were found comparing either maternal or neonatal outcomes for different skin incision in women with placenta previa. The aim of this study was to compare maternal and neonatal outcomes by skin incision types (transverse compared with vertical) in a large cohort of women with placenta previa who were undergoing cesarean delivery. METHODS: This was a retrospective cohort study carried out between January 2014 and June 2019. All pregnant women with placenta previa had confirmed by ultrasonologist before delivery and obstetrician at delivery. The primary outcome was the estimated blood loss during the surgery and within the first 24 hours postoperatively. Mean (standard deviation), median (interquartile range) or frequency (percentage) was reported to variables. Appropriate parametric and nonparametric tests were used to analyses. RESULTS: The study included 1098 complete records, 332 (30.24%) cases in the vertical skin incision group and 766 (69.76%) cases in the transverse skin incision group. Those with vertical incision showed a higher percentage of preterm delivery, anterior placenta, abnormally invasive placenta, and history of previous cesarean delivery, and a lower percentage of first pregnancy, in vitro fertilization, and emergency cesarean delivery. After controlling for confounding factors, higher incidence of post-partum hemorrhage (OR 5.47, 95% CI 3.84-7.79), maternal intensive care unit (OR 4.30, 95% CI 2.86-6.45), transfusion (OR 5.97, 95% CI 4.15-8.58), and 5-min APGAR< 7 (OR 9.03, 95% CI 1.83-44.49), a more estimated blood loss (ß 601.85, 95%CI 458.78-744.91), and a longer length of hospital stay after delivery (ß 0.54, 95%CI 0.23-0.86) were found in the vertical skin incision group. CONCLUSIONS: Our data demonstrated that transverse skin incision group showed the better perinatal outcomes in women with placenta previa. Future collaborative studies are needed to be done by centers for placenta previa to have a better understanding of the characteristics and the outcomes of the disease in the choosing skin incision.

Efficacy and safety of umbilical cord mesenchymal stem cells in treatment of cesarean section skin scars: a randomized clinical trial.

BACKGROUND: Pathological skin scars, caused by cesarean section, affected younger mothers esthetically and psychosocially and to some extent frustrated obstetricians and dermatologists. Umbilical cord mesenchymal stem cells (UC-MSCs), as a population of multipotent cells, are abundant in human tissues, providing several possibilities for their effects on skin scar tissues. Herein, we performed a randomized, double-blind, placebo-controlled, three-arm clinical trial, aiming to assess the efficacy and safety of UC-MSCs in the treatment of cesarean section skin scars among primiparous singleton pregnant women. METHODS: Ninety primiparous singleton pregnant women undergoing elective cesarean section were randomly allocated to receive placebo, low-dose (3 × 106 cells), or high-dose (6 × 106 cells) transdermal hydrogel UC-MSCs on the surface of the skin incision. The primary outcome was cesarean section skin scars followed after the sixth month, assessed by the Vancouver Scar Scale (VSS). RESULTS: All the participants completed their trial of the primary outcome according to the protocol. The mean score of estimated total VSS was 5.52 in all participants at the sixth-month follow-up, with 6.43 in the placebo group, 5.18 in the low-dose group, and 4.71 in the high-dose group, respectively. No significant difference was found between-group in the mean scores for VSS at the sixth month. Additional prespecified secondary outcomes were not found with significant differences among groups either. No obvious side effects or adverse effects were reported in any of the three arms. CONCLUSION: This randomized clinical trial showed that UC-MSCs did not demonstrate the effects of improvement of cesarean section skin scars. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02772289. Registered on 13 May 2016.

Random placenta margin incision for control hemorrhage during cesarean delivery complicated by complete placenta previa: a prospective cohort study.

Introduction: Complete placenta previa (CPP) is one of the most problematic types of abnormal placenta, which is further complicated by placenta accreta or percreta that can unexpectedly lead to catastrophic blood loss, infection, multiple complications, emergency hysterectomy, and even death. The present study aimed to assess the efficacy of random placenta margin incision in controlling intraoperative and total blood loss during cesarean section for CPP women. Methods: A prospective cohort study, including a total of 100 consecutive pregnant women with CPP, was performed at a tertiary university-affiliated medical center between March 2016 and July 2017. All of them underwent random placenta margin incision, and intraoperative and total blood loss were analyzed. Through antenatal diagnosis using color Doppler, women were further divided into abnormally invasive placenta (AIP) and non-AIP groups, and anterior and posterior placenta groups. The protocol was registered with the Clinical Trial Registry under registration number NCT02695069. Results: Mean maternal age and gestational age at delivery were 32.26 ± 5.03 years old and 36.21 ± 2.07 weeks, respectively. Total duration of the surgical procedure time was 52.50 (42.43-64.00) min. Median estimated intraoperation blood loss was 746.43 (544.44-1092.86) ml. Total blood loss was 875.00 (604.50-1196.67) ml, and 38 (38.0%) had post-partum hemorrhage. The change from baseline in the median hemoglobin level was -0.33 (6.00-13.20). No women underwent hysterectomy due to massive hemorrhage during the study period. No women had an intraoperative urinary bladder injury, postoperative wound infection, and required relaparotomy, owing to intra-abdominal bleeding. The median hospitalization time was 5.41 (4.18-7.58) d. Conclusion: The random placenta margin incision may be a potentially valuable surgical procedure to control the volumes of intraoperative and postoperative blood loss and reduce the incidence of postpartum hemorrhage among women with complete placenta previa.

Diagnostic accuracy of cystoscopy and ultrasonography in the prenatal diagnosis of abnormally invasive placenta.

The aim of this study was to compare the accuracy of cystoscopy and ultrasonography for the prenatal diagnosis of abnormally invasive placenta (AIP), including its subgroups: placenta accreta (PA), placenta increta (PI), and placenta percreta (PP).A retrospective observational study including a total of 85 pregnant women at high risk for AIP underwent prenatal cystoscopy and ultrasonography evaluations. The sensitivity (Se), specificity (Sp), positive predictive value, negative predictive value, and exact diagnosed were calculated and compared for both cystoscopy and ultrasonography. Se and Sp values of cystoscopy and ultrasonography were compared by means of the McNemar test.Of the 85 patients, there were 24 (28.2%) PA, 35 (41.2%) PI, 4 (4.7%) PP, and 22 (25.9%) nonadherent placenta. The mean maternal age and gestational age of delivery were 31.88 ±â€Š4.42 years and 36.14 ±â€Š1.84 weeks, respectively. No one was found to develop any complications with cystoscopy like urinary tract infection, or ureteral injury or perforations. Se in the diagnosis of AIP was 50.8% with ultrasonography and 61.9% for cystoscopy. Sp was 86.4% with cystoscopy and 72.7% for ultrasonography. In subgroups, Se with cystoscopy was 25.0%, 62.9%, and 100.0% in PA, PI, and PP, respectively, and 37.5%, 74.3%, and 100.0%, respectively, for ultrasonography; Sp remained unchanged with 86.4% for cystoscopy and 72.7% for ultrasonography. After McNemar test, no difference was found in either Se or Sp between cystoscopy and ultrasonography in AIP and its subgroups.According to the depth of invasion, the diagnostic value of cystoscopy and ultrasonography is all conspicuous increased and they have similar test validity for prenatal diagnosis of AIP and its subgroups.

Mesenchymal stem cells in the treatment of Cesarean section skin scars: study protocol for a randomized, controlled trial.

BACKGROUND: Cesarean delivery has already become a very common method of delivery around the world, especially in low-income countries. Hypertrophic scars and wound infections have affected younger mothers and frustrated obstetricians for a long time. Mesenchymal stem cells (MSCs) have strong potential for self-renewal and differentiation to multilineage cells. Previous studies have demonstrated that MSCs are involved in enhancing diabetic wound healing. Therefore, this study is designed to investigate the safety and efficacy of using MSCs in the treatment of Cesarean section skin scars. METHODS: This trial is a prospective, randomized, double-blind, placebo-controlled, single-center trial with three parallel groups. Ninety eligible participants will be randomly allocated to placebo, low-dose (transdermal hydrogel MSCs; 3 × 106 cells) or high-dose (transdermal hydrogel MSCs; 6 × 106 cells) groups at a 1:1:1 allocation ratio according to a randomization list, once a day for six consecutive days. Study duration will last for 6 months, comprising a 1 week run-in period and 24 weeks of follow-up. The primary aim of this trial is to compare the difference in Vancouver Scar Scale rating among the three groups at the 6th month. Adverse events, including severe and slight signs or symptoms, will be documented in case report forms. The study will be conducted at the Department of Obstetric of Southern Medical University Affiliated Maternal & Child Health Hospital of Foshan. DISCUSSION: This trial is the first investigation of the potential for therapeutic use of MSCs for the management of women's skin scar after Cesarean delivery. The results will give us an effective therapeutic strategy to combat Cesarean section skin scars, even with uterine scarring. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02772289 . Registered on 10 May 2016.

Umbilical cord mesenchyme stem cell local intramuscular injection for treatment of uterine niche: Protocol for a prospective, randomized, double-blinded, placebo-controlled clinical trial.

BACKGROUND: Uterine niche is defined as a triangular anechoic structure at the site of the scar or a gap in the myometrium at the site of a previous caesarean section. The main clinical manifestations are postmenstrual spotting and intrauterine infection, which may seriously affect the daily life of nonpregnant women. Trials have shown an excellent safety and efficacy for the potential of mesenchymal stem cells (MSCs) as a therapeutic option for scar reconstruction. Therefore, this study is designed to investigate the safety and efficacy of using MSCs in the treatment for the uterine niche. METHODS/DESIGN: This phase II clinical trial is a single-center, prospective, randomized, double-blind, placebo-controlled with 2 arms. One hundred twenty primiparous participants will be randomly (1:1 ratio) assigned to receive direct intramuscular injection of MSCs (a dose of 1*10 cells in 1 mL of 0.9% saline) (MSCs group) or an identical-appearing 1 mL of 0.9% saline (placebo-controlled group) near the uterine incision. The primary outcome of this trial is to evaluate the proportion of participants at 6 months who is found uterine niche in the uterus by transvaginal utrasonography. Adverse events will be documented in a case report form. The study will be conducted at the Department of Obstetric of Southern Medical University Affiliated Maternal & Child Health Hospital of Foshan. DISCUSSION: This trial is the first investigation of the potential for therapeutic use of MSCs for the management of uterine niche after cesarean delivery. CONCLUSION: This protocol will help to determine the efficacy and safety of MSCs treatment in uterine niche and bridge the gap with regards to the current preclinical and clinical evidence. TRIAL REGISTRATION NUMBER: NCT02968459 (Clinical Trials.gov: http://clinicaltrials.gov/).

Prevalence of prenatally diagnosed congenital cystic adenomatoid malformation among fetuses in China.

The prevalence of congenital cystic adenomatoid malformation among fetuses still varies in different studies in China. The present meta-analysis was intended to evaluate the pooled prevalence of fetuses in China. Four English (Pubmed, Elsevier Science Direct, Web of Science and the Cochrane Library) and four Chinese (the Chinese Biological Medical Literature database, the Chinese National Knowledge Infrastructure database, VIP database and Wanfang Data) databases were searched from inception to July 2016. Meta-analyses were performed using Stata (version 12.0), with prevalence and corresponding 95% confidence intervals using the random effect model. Five studies with 393496 fetuses were chosen for this meta-analysis. The overall pooled prevalence was 4.01/10000 (2.03/10000 - 6.00/10000) fetuses. Sensitivity analysis revealed that the results were stable, and Begg's test and Egg's test showed no potential risk of publication bias. This is, to our knowledge, the first study to systematically evaluate the literature of the prevalence of congenital cystic adenomatoid malformation among fetuses in China. Results showed that the prevalence among fetuses should be considerable. A large-scale multicenter study on the epidemiology across different areas in China is required.

Successfully treated congenital cystic adenomatoid malformation by open fetal surgery: A care-compliant case report of a 5-year follow-up and review of the literature.

BACKGROUND: Congenital cystic adenomatoid malformation (CCAM) is a rare hamartomatous cystic lesion. Open fetal surgery currently provides a potential therapeutic option for management of a fetus with CCAM diagnosis. CASE SUMMARY: A 22-year-old G2P0 woman presented at (Equation is included in full-text article.)weeks' gestation for evaluation of a fetus with a left lung lesion and diagnosed as CCAM at (Equation is included in full-text article.)weeks' gestation. Open fetal surgery was performed to resection the lesion at (Equation is included in full-text article.)weeks' gestation under deep maternal general anesthesia. The mother presented at (Equation is included in full-text article.)weeks after open fetal surgery with preterm premature rupture of membranes (PPROM) and underwent cesarean delivery at (Equation is included in full-text article.)weeks' gestation. A vigorous woman infant of 1955 g, with good Apgar score, was delivered. At 1 month, 4 years, and present, 5 years after birth, she has continued to do well without any obvious deficit and both respiration and circulation were well maintained. CONCLUSION: We present one case of CCAM which was cured by open fetal surgery and continued to do well at follow-up of 5 years. The success of treatment provided preliminary experience for further carrying out such interventions in China.

Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines.

Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.

The time-dependent expression of α7nAChR during skeletal muscle wound healing in rats.

The study on time-dependent expression of α7 nicotine acetylcholine receptor (α7nAChR) was performed by immunohistochemistry, Western blotting, and real-time PCR during skeletal muscle wound healing in rats. Furthermore, co-localization of α7nAChR with macrophage or myofibroblast marker was detected by double immunofluorescence. A total of 50 Sprague-Dawley male rats were divided into control and contusion groups (3 h, 6 h, 12 h, 1 day, 3 days, 5 days, 7 days, 10 days, and 14 days post-injury). In the uninjured controls, α7nAChR positive staining was observed in the sarcolemma and sarcoplasm of normal myofibers. In wounded specimens, a small number of polymorphonuclear cells, a number of macrophages and myofibroblasts showed positive reaction for α7nAChR in contused zones. Morphometrically, the average ratios of α7nAChR-positive cells were over 50 % from 3 to 10 days after contusion, and exceeded 60 % at 5 and 7 days post-injury. Besides, the positive ratios of α7nAChR were <50 % at the other posttraumatic intervals. By Western blotting analysis, the average ratio of α7nAChR protein expression maximized at 7 days after injury, which was >2.13. Similarly, the relative quantity of α7nAChR mRNA expression peaked at 7 days post-wounding as compared with control by real-time PCR detection, showing a relative quantity of >2.65. In conclusion, the expression of α7nAChR is upregulated and temporally distributed in macrophages and myofibroblasts during skeletal muscle wound healing, which might be closely involved in inflammatory response and fibrotic repair after injury. Moreover, α7nAChR is promising as a useful marker for wound age determination of skeletal muscle.

Differential protein expression in perfusates from metastasized rat livers.

BACKGROUND: Liver perfusates exhibit theoretical advantages regarding the discovery of disease biomarkers because they contain proteins that readily enter the blood-stream, and perfusion preserves the disease state in its natural context. The purpose of the study is to explore the value of liver perfusate proteome in the biomarker discovery of liver diseases. RESULTS: In this study, 86 differentially expressed proteins were identified in perfusates from isolated rat livers metastasized by Walker-256 tumor cells. Among these proteins, 27 were predicted to be secreted, and 59 were intracellular or membrane proteins. Most of the secretory proteins (70.4%) were decreased in metastasized liver perfusates. The main canonical ingenuity pathway to which these secretory proteins belonged was acute phase response, which indicated that the liver-associated immune reaction was damaged by the metastasis. In contrast, most of the intracellular or membrane proteins (86.4%) exhibited higher relative abundances in the metastasized liver perfusates. Some of these proteins, including Rpl21, Atic, Eif3s2, Echs1, Eps15 and Ywhab, have previously been reported to be involved in cancer genesis and progression. As a member of the 14-3-3 protein family, Ywhab plays a key role in cellular proliferation and oncogenic transformation and has been reported to be involved in the development of breast cancer. Its abundance was elevated by 3.5-fold in the metastasized perfusates. Validation by Western blotting revealed a 3.7-fold increase in the abundance of this protein in metastasized plasma. CONCLUSIONS: These results show that perfusate proteome can be used as an alternative initial resource for biomarker identification, which ultimately requires validation in serum.

Ii-Key/HPV16 E7 hybrid peptide immunotherapy for HPV16+ cancers.

Activation of antigen-specific CD4+ T cells is critical for vaccine design. We have advanced a novel technology for enhancing activation of antigen-specific CD4+ T helper cells whereby a fragment of the MHC class II-associated invariant chain (Ii-Key) is linked to an MHC class II epitope. An HLA-DR4-restricted HPV16 E7 epitope, HPV16 E7(8-22), was used to create a homologous series of Ii-Key/HPV16 E7 hybrids testing the influence of spacer length on in vivo enhancement of HPV16 E7(8-22)-specific CD4+ T lymphocyte responses. HLA-DR4-tg mice were immunized with Ii-Key/HPV16 E7(8-22) hybrids or the epitope-only peptide HPV16 E7(8-22). As measured by IFN-gamma ELISPOT assay of splenocytes from immunized mice, one of the Ii-Key/HPV16 E7(8-22) hybrids enhanced epitope-specific CD4+ T cell activation 5-fold compared to the HPV16 E7(8-22) epitope-only peptide. We further demonstrated that enhanced CD4+ T cell activation augments the CTL activity of a H-2D(b)-restricted HPV16 E7(49-57) epitope in HLA-DR4+ mice using an in vivo CTL assay. Binding assays indicated that the Ii-Key/HPV16 hybrid has increased affinity to HLA-DR4+ cells relative to the epitope-only peptide, which may explain its increased potency. In summary, Ii-Key hybrid modification of the HLA-DR4-restricted HPV16 E7(8-22) MHC class II epitope generates a potent immunotherapeutic peptide vaccine that may have potential for treating HPV16+ cancers in HLA-DR4+ patients.

Using enrichment index for quality control of secretory protein sample and identification of secretory proteins.

Analysis of secretory proteins is an important area in proteomic research. We propose that a good secretory protein sample should be enriched with known secretory proteins, and a secretory protein should be enriched in the secretory protein sample compared with its corresponding soluble cell lysate. Positive identifications of proteins were subjected to quantitation of spectral counts, which reflect relative protein abundance. Enrichment index of the sample (EIS) and the enrichment index for protein (EIP) were obtained by comparing proteins identified in the secretory protein sample and those in the soluble cell lysate sample. The quality of the secretory protein sample can be represented by EIS. EIP was used to identify the secretory proteins.The secretory proteins from mouse dendritic cell sarcoma (DCS) were analyzed by MS. The EISs of two samples were 75.4 and 84.65, respectively. 72 proteins were significantly enriched in secretory protein samples, of which 42 proteins were either annotated in Swiss-Prot and/or predicted by signal peptides to be secretory. In the remaining 30 proteins, 12 and 15 proteins were positively predicted by SecretomeP and ProP, respectively, and 5 proteins were positive by both methods. Furthermore, 11 proteins were found to be present in exosome in other studies that involved mice dendritic cell lines. We suggest that this assessment method is helpful for systemic research of secretory proteins and biomarker discovery for diseases such as cancer.

[Comparative proteomics analysis of human adenomyosis].

OBJECTIVE: To set up the proteomic protein profiling of adenomyotic tissue and normal uterine muscle and identify the abnormally expressed proteins in adenomyotic tissue. METHODS: Samples of adenomyotic tissue (adenomyosis group) and age-matched healthy uterine muscle (control group) were collected from totally 10 patients undergoing transabdominal hysterectomy for adenomyosis and cervical diseases at Peking Union Medical College Hospital from January 2007 to October 2007. The proteomics profiling of adenomyotic tissue and normal uterine tissue were established using two dimensional gel electrophoresis (2-DE) and gel staining method. The differently expressed protein spots were detected by gel comparison using image analysis software and identified using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: In Coomassie blue stained gels there were on average (512 +/- 36) spots and compared with the reference gel the matching rate was 83.7%. In silver stained gels there were (762 +/- 54) spots and compared with the reference gel the matching rate was 81.1%. Compared with normal uterine muscle, there were 15 protein spots disregulated in adenomyotic tissue. Among them 10 protein spots were successfully identified by mass spectrometry. The functions of these disregulated proteins included cell skeleton, oxidation, apoptosis and immune reaction. CONCLUSIONS: Comparative proteomics analysis is a useful approach for the study of adenomyosis. Compared with normal uterine muscle there are abnormalities in cell skeleton, oxidation, apoptosis and immune reaction. These life processes may participate in pathophysiology of adenomyosis.