CRB1-associated retinal degeneration is dependent on bacterial translocation from the gut.

The Crumbs homolog 1 (CRB1) gene is associated with retinal degeneration, most commonly Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Here, we demonstrate that murine retinas bearing the Rd8 mutation of Crb1 are characterized by the presence of intralesional bacteria. While normal CRB1 expression was enriched in the apical junctional complexes of retinal pigment epithelium and colonic enterocytes, Crb1 mutations dampened its expression at both sites. Consequent impairment of the outer blood retinal barrier and colonic intestinal epithelial barrier in Rd8 mice led to the translocation of intestinal bacteria from the lower gastrointestinal (GI) tract to the retina, resulting in secondary retinal degeneration. Either the depletion of bacteria systemically or the reintroduction of normal Crb1 expression colonically rescued Rd8-mutation-associated retinal degeneration without reversing the retinal barrier breach. Our data elucidate the pathogenesis of Crb1-mutation-associated retinal degenerations and suggest that antimicrobial agents have the potential to treat this devastating blinding disease.

Indole-3-carboxaldehyde ameliorates ionizing radiation-induced hematopoietic injury by enhancing hematopoietic stem and progenitor cell quiescence.

Indole-3-carboxaldehyde (I3A), one of tryptophan metabolites derived from gut microbiota, extends the lifespan of mice after high-dose ionizing radiation exposure. Persistent myelosuppression is the most common and fatal complication for victims of nuclear accidents and patients undergoing radiotherapy, with few therapeutic options available. However, whether and how I3A protects ionizing radiation-induced hematopoietic toxicity remain unknown. In this study, we demonstrated that I3A treatment effectively ameliorated radiation-induced hematopoietic injury through accelerating peripheral blood cells recovery, promoting bone marrow cellularity restoration and enhancing functional HSPC regeneration. Additionally, I3A also suppressed intracellular reactive oxygen species production and inhibited apoptosis in irradiated HSPCs. Mechanistically, I3A treatment significantly increased HSPC quiescence, thus conferring HSPCs with resistance against radiation injury. Finally, I3A treatment could improve survival of lethally irradiated mice. Taken together, our data suggest that I3A acts as a gut microbiota-derived paracrine factor that regulates HSPC regeneration and may serve as a promising therapeutic agent for ionizing radiation-induced myelosuppression.

Prediction model of axillary lymph node status using automated breast ultrasound (ABUS) and ki-67 status in early-stage breast cancer.

BACKGROUND: Automated breast ultrasound (ABUS) is a useful choice in breast disease diagnosis. The axillary lymph node (ALN) status is crucial for predicting the clinical classification and deciding on the treatment of early-stage breast cancer (EBC) and could be the primary indicator of locoregional recurrence. We aimed to establish a prediction model using ABUS features of primary breast cancer to predict ALN status. METHODS: A total of 469 lesions were divided into the axillary lymph node metastasis (ALNM) group and the no ALNM (NALNM) group. Univariate analysis and multivariate analysis were used to analyze the difference of clinical factors and ABUS features between the two groups, and a predictive model of ALNM was established. Pathological results were as the gold standard. RESULTS: Ki-67, maximum diameter (MD), posterior feature shadowing or enhancement and hyperechoic halo were significant risk factors for ALNM in multivariate logistic regression analysis (P < 0.05). The four risk factors were used to build the predictive model, and it achieved an area under the receiver operating characteristic (ROC) curve (AUC) of 0.791 (95% CI: 0.751, 0.831). The accuracy, sensitivity and specificity of the prediction model were 72.5%, 69.1% and 75.26%. The positive predictive value (PPV) and negative predictive value (NPV) were 66.08% and 79.93%, respectively. Distance to skin, MD, margin, shape, internal echo pattern, orientation, posterior features, and hyperechoic halo showed significant differences between stage I and stage II (P < 0.001). CONCLUSION: ABUS features and Ki-67 can meaningfully predict ALNM in EBC and the prediction model may facilitate a more effective therapeutic schedule.

Performance of novel deep learning network with the incorporation of the automatic segmentation network for diagnosis of breast cancer in automated breast ultrasound.

OBJECTIVE: To develop novel deep learning network (DLN) with the incorporation of the automatic segmentation network (ASN) for morphological analysis and determined the performance for diagnosis breast cancer in automated breast ultrasound (ABUS). METHODS: A total of 769 breast tumors were enrolled in this study and were randomly divided into training set and test set at 600 vs. 169. The novel DLNs (Resent v2, ResNet50 v2, ResNet101 v2) added a new ASN to the traditional ResNet networks and extracted morphological information of breast tumors. The accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), area under the receiver operating characteristic (ROC) curve (AUC), and average precision (AP) were calculated. The diagnostic performances of novel DLNs were compared with those of two radiologists with different experience. RESULTS: The ResNet34 v2 model had higher specificity (76.81%) and PPV (82.22%) than the other two, the ResNet50 v2 model had higher accuracy (78.11%) and NPV (72.86%), and the ResNet101 v2 model had higher sensitivity (85.00%). According to the AUCs and APs, the novel ResNet101 v2 model produced the best result (AUC 0.85 and AP 0.90) compared with the remaining five DLNs. Compared with the novice radiologist, the novel DLNs performed better. The F1 score was increased from 0.77 to 0.78, 0.81, and 0.82 by three novel DLNs. However, their diagnostic performance was worse than that of the experienced radiologist. CONCLUSIONS: The novel DLNs performed better than traditional DLNs and may be helpful for novice radiologists to improve their diagnostic performance of breast cancer in ABUS. KEY POINTS: • A novel automatic segmentation network to extract morphological information was successfully developed and implemented with ResNet deep learning networks. • The novel deep learning networks in our research performed better than the traditional deep learning networks in the diagnosis of breast cancer using ABUS images. • The novel deep learning networks in our research may be useful for novice radiologists to improve diagnostic performance.

Structure of LINC00511-siRNA-conjugated nanobubbles and improvement of cisplatin sensitivity on triple negative breast cancer.

The drug resistance of triple negative breast cancer (TNBC) is considered as a major obstacle for the curative effect of chemotherapy. Long intergenic noncoding RNA 00511 (LINC00511) has been considered as a target gene of drug resistance. A novel theranostic agent loaded with LINC00511-siRNA to deliver siRNA was structured, and the responses of drug sensitivity in TNBC were detected. Next-generation high-throughput RNA sequencing (RNA-Seq) was performed to accurately analyze the differential expression of mRNAs and lncRNA targets after LINC00511-siRNA transfection with low-frequency ultrasound (LFUS). The LINC00511-siRNA conjugated nanobubble complexes showed appropriate characterization, with a mean diameter of 516.1 ± 24.7 nm and a zeta potential of -38.05 ± 0.24 mV. The transfection efficiency of nanobubble complexes was approximately 50% with LFUS. By RNA-Seq, the differential expressions of lncRNA transcripts and mRNA transcripts were identified, and then analyzed. The GO and KEGG enrichment analyses revealed the TNBC drug resistance related target genes and pathways. The combination of LFUS irradiation and nanobubble complexes is regarded as an efficient and safe method for siRNA transfection. The TNBC drug resistance occurs as a result of synergistic reactions between a variety of genes and a variety of pathways.

Diarrhea and altered inflammatory cytokine pattern in severe coronavirus disease 2019: Impact on disease course and in-hospital mortality.

BACKGROUND AND AIM: Dynamic changes of immunocyte subsets and inflammatory profiles in coronavirus disease 2019 (COVID-19) patients with gastrointestinal symptoms were undetermined. METHODS: A single-center retrospective analysis of 409 severe, hospitalized COVID-19 patients from 20 January to 29 February 2020 was performed. The longitudinal characteristics of immune inflammatory cytokines in patients with/without diarrhea were analyzed. The relations of diarrhea and immuno-inflammatory factors with illness course and clinical outcomes were further explored. RESULTS: Diarrhea was more common and more serious with longer duration (4.9 ± 1.5 vs 4.2 ± 1.5 days, P = 0.039) and higher frequency (5.5 ± 2.1 vs 4.0 ± 2.0 times/day, P = 0.001) in deceased patients than in the survivors. Also, diarrhea patients were more inclined to develop multi-organ damage: survivors have longer illness course (media 41.0 vs 36.0 days, P = 0.052) and hospital stays (media 27.0 vs 23.0 days, P = 0.041), and the deceased patients had higher mortality (33.0% vs 22.6%, P = 0.045) and earlier death (media 20.0 vs 25.0 days, P = 0.038). Progressively, neutrophilia and lymphopenia, especially the declined CD8+ T cells, were demonstrated in diarrhea patients relative to the non-diarrhea cases. The inflammatory cytokines including IL-6, IL-10, and TNF-α were intensively increased in patients with diarrhea. The multivariable logistic analysis showed longer duration of diarrhea (P = 0.036), higher neutrophil counts (P = 0.011), and lower lymphocyte counts (P < 0.001) were independent risk factors of in-hospital death. The proportional hazards model indicated that longer duration of diarrhea (P = 0.002), higher frequency of diarrhea (P = 0.058), higher neutrophil counts (P = 0.001), lower lymphocyte counts (P = 0.035), and decreased proportion of CD8+ T cells (P < 0.001) were independently associated with longer illness course of the survivors. CONCLUSIONS: Diarrhea patients were more likely to present with neutrophilia, lymphopenia, and cytokine storm and to develop multi-organ damage. The inflammatory patterns were independent factors associated with illness course of the survivors and in-hospital death of severe COVID-19.

Single-cell RNA sequencing reveals the mechanism of sonodynamic therapy combined with a RAS inhibitor in the setting of hepatocellular carcinoma.

BACKGROUND: Ras activation is a frequent event in hepatocellular carcinoma (HCC). Combining a RAS inhibitor with traditional clinical therapeutics might be hampered by a variety of side effects, thus hindering further clinical translation. Herein, we report on integrating an IR820 nanocapsule-augmented sonodynamic therapy (SDT) with the RAS inhibitor farnesyl-thiosalicylic acid (FTS). Using cellular and tumor models, we demonstrate that combined nanocapsule-augmented SDT with FTS induces an anti-tumor effect, which not only inhibits tumor progression, and enables fluorescence imaging. To dissect the mechanism of a combined tumoricidal therapeutic strategy, we investigated the scRNA-seq transcriptional profiles of an HCC xenograft following treatment. RESULTS: Integrative single-cell analysis identified several clusters that defined many corresponding differentially expressed genes, which provided a global view of cellular heterogeneity in HCC after combined SDT/FTS treatment. We conclude that the combination treatment suppressed HCC, and did so by inhibiting endothelial cells and a modulated immunity. Moreover, hepatic stellate secretes hepatocyte growth factor, which plays a key role in treating SDT combined FTS. By contrast, enrichment analysis estimated the functional roles of differentially expressed genes. The Gene Ontology terms "cadherin binding" and "cell adhesion molecule binding" and KEGG pathway "pathway in cancer" were significantly enriched by differentially expressed genes after combined SDT/FTS therapy. CONCLUSIONS: Thus, some undefined mechanisms were revealed by scRNA-seq analysis. This report provides a novel proof-of-concept for combinatorial HCC-targeted therapeutics that is based on a non-invasive anti-tumor therapeutic strategy and a RAS inhibitor.

Preparation of novel targeting nanobubbles conjugated with small interfering RNA for concurrent molecular imaging and gene therapy in vivo.

The aim of this study was to establish a novel targeting nanobubble (TNB) conjugated with small interfering RNA (siRNA)-cyanine 5 (Cy5) and to validate its theranostic ability in vivo. The TNB conjugated with neuroepithelial transforming gene 1 (NET-1) siRNA-Cy5 was prepared by thin-film hydration and mechanical sonication method. A hepatocellular carcinoma (HCC) xenograft model was established by subcutaneously injecting SMMC-7721 cells in BALB/c nude mice. The NET-1 siRNA-conjugated TNB was utilized for accurate contrast-enhanced ultrasound in vivo imaging, which was enabled by the target ligand GPC-3 antibody and specific gene transfection with the aid of low-frequency ultrasound (LFUS) irradiation. BALB/c nude mice bearing tumors were randomized into 5 groups and irradiated with LFUS for 5 min after TNB administration; mice were treated twice a week for a total of 60 d. The mean particle size of TNB was <500 nm. Mice treated with NET-1 siRNA-conjugated TNB showed a significant decrease in tumor growth and the highest survival rate. Our findings offer an effective and safe gene vehicle and probe for molecular imaging in vivo. It may improve the early diagnosis and treatment effects of HCC.-Wu, B., Shang, H., Liang, X., Sun, Y., Jing, H., Han, X., Cheng, W. Preparation of novel targeting nanobubbles conjugated with small interfering RNA for concurrent molecular imaging and gene therapy in vivo.

Outcomes and implications of diarrhea in patients with SARS-CoV-2 infection.

BACKGROUND AND AIMS: Diarrhea was not uncommon in patients with coronavirus disease 2019 (COVID-19), but the significance remains undetermined. METHODS: This retrospective study included 157 diarrhea cases form 564 hospitalized COVID-19 patients who were admitted to Wuhan Union Hospital from January 20 to February 29, 2020. Clinical characteristics, the course and the outcome of patients with diarrhea were analyzed. The correlation between diarrhea and fecal presence of coronavirus was also determined. RESULTS: The overall morbidity of diarrhea was 27.8% (157/564) in COVID-19 patients. Among them, 38 cases presented only with diarrhea, and 119 cases in both diarrhea and respiratory symptoms. Patients with diarrhea and respiratory symptoms had higher levels of inflammatory activity, longer hospital stay (27.5 vs. 23.0 vs. 22.0 days, p = .029) and higher odds ratio of mortality (3.2 times and 2.2 times, respectively) than those with diarrhea only or respiratory symptoms only. However, patients with diarrhea had longer time from onset to admission (14.5 days vs. 11.0 days, p = .04), higher positive viral RNA in stool (80.0% vs. 52.4%, p = .016) than those with both diarrhea and respiratory symptoms. CONCLUSIONS: Diarrhea caused by high enteric viral burden may lead to long course and poor outcome in COVID-19 patients. The patients with diarrhea and respiratory symptoms were prone to serious condition, and had worse outcomes. However, the patients with diarrhea alone showed mild illness but delayed health-seeking.

Formation of Zerovalent Iron in Iron-Reducing Cultures of Methanosarcina barkeri.

Methanogenic archaea have been shown to reduce iron from ferric [Fe(III)] to ferrous [Fe(II)] state, but minerals that form during iron reduction by different methanogens remain to be characterized. Here, we show that zerovalent iron (ZVI) minerals, ferrite [α-Fe(0)] and austenite [γ-Fe(0)], appear in the X-ray diffraction spectra minutes after the addition of ferrihydrite to the cultures of a methanogenic archaeon, Methanosarcina barkeri (M. barkeri). M. barkeri cells and redox-active, nonenzymatic soluble organic compounds in organic-rich spent culture supernatants can promote the formation of ZVI; the latter compounds also likely stabilize ZVI. Methanogenic microbes that inhabit organic- and Fe(III)-rich anaerobic environments may similarly reduce Fe(III) to Fe(II) and ZVI, with implications for the preservation of paleomagnetic signals during sediment diagenesis and potential applications in the protection of iron metals against corrosion and in the green synthesis of ZVI.

Study on the Diversity and Function of Gut Microbiota in Pigs Following Long-Term Antibiotic and Antibiotic-Free Breeding.

In-feed antibiotics can influence intestinal microbial structures in born and early-life within a period. However, the impact of antibiotics on gut microbiota during long-term antibiotic-free and antibiotic breeding at porcine-fattening phase have not been studied extensively so far. Here, we conducted a systematic 16S rRNA gene sequencing-based study combined with metagenomic analysis to reveal the variation of diversity and function of gut microbiota between antibiotic-free (treatment group, TG) and antibiotic (a mixture of flavomycin and enramycin, control group, CG) breeding at various stages of fattening pigs. In the present study, Bacteroidetes, Firmicutes, and Proteobacteria phyla were the core microbiomes in fattening pig gut microbiota. The ratio between Firmicutes and Bacteroidetes significantly increased with age (P = 0.03). TG showed significantly higher relative abundance of Proteobacteria and Fibrobacteres phyla than CG. The microbial community can be divided into several notably clustered blocks based on cooperative and competitive correlations. These blocks centered on numerous special genera, which play essential roles in body development and disease prevention. TG showed obviously higher proportions of metabolic pathways related to metabolism, endocrine system, nervous system and excretory system, but pathways included carbohydrate metabolism and immune system diseases in CG. Collectively, this study has comprehensively demonstrated microbial diversities, differences and correlations among gut microbiota, microbial metabolism and gene functions during long-term antibiotic-free breeding. This work provides a novel resource and information with positive implications for pig husbandry production and disease prevention.

Carboxypeptidase A4 promotes proliferation and stem cell characteristics of hepatocellular carcinoma.

Carboxypeptidase A4 (CPA4), a member of the metallo-carboxypeptidase family, is overexpressed in liver cancer and is associated with cancer progression. The role of CPA4 in hepatocellular carcinoma (HCC) remains unclear. In this study, we aimed to evaluate the relevance of CPA4 to the proliferation and expression of stem cell characteristics of hepatocellular carcinoma cells. Western blot analysis showed high CPA4 expression in the liver cancer cell line Bel7402 and low expression in HepG2 cells. Knock-down of CPA4 decreased cancer cell proliferation as detected by MTT and clone formation assays. The serum-free culture system revealed that downregulated CPA4 suppressed the sphere formation capacities of tumour cells. However, upregulated CPA4 increased the proliferation and sphere formation capacity. In addition, the protein expression of CD133, ALDH1 and CD44 also increased in cells with upregulated CPA4. In vivo, the overexpression of CPA4 in tumour cells that were subcutaneously injected into nude mice markedly increased the growth of the tumours. These data suggest that CPA4 expression leads to poor prognoses by regulating tumour proliferation and the expression of stem cell characteristics and may therefore serve as a potential therapeutic target of HCC.

Sugiol (127horbar;hydroxyabieta-8,11,13-trien-7-one) targets human pancreatic carcinoma cells (Mia-PaCa2) by inducing apoptosis, G2/M cell cycle arrest, ROS production and inhibition of cancer cell migration.

PURPOSE: Plants produce a diversity of molecular scaffolds with tremendous pharmacological potential. In the present study we evaluated the anticancer activity of the plant-derived natural product sugiol. We also evaluated its effects on apoptosis-related key proteins, cell cycle phase distribution, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). METHODS: Cell viability was evaluated by MTT assay while clonogenic assay was done to determine the effects of sugiol on the cancer cell colony formation. Flow cytometric measurements were carried out in order to assess the effects of sugiol on cell cycle progression, apoptosis, MMP and ROS generation. RESULTS: Sugiol reduced the cell viability of Mia-PaCa2 human pancreatic cancer cells in a concentration-dependent manner. The IC50 of sugiol on the cell line was 15 µM. The anticancer activity of sugiol was found to be ROS-mediated alterations in MMP, ultimately favoring apoptosis as determined by the annexin V/propidium iodide (PI). Additionally, sugiol caused cell cycle arrest in G2/M phase of the cell cycle and upregulated the expression of Bax, with concomitant downregulation of Bcl-2 expression in comparison to the untreated cells. It also inhibited the migratory capacity of Mia-PaCa2 cells at the IC50 concentration. CONCLUSION: In conclusion our results indicate that sugiol is a potent anticancer molecule and may prove essential in pancreatic cancer therapy.

Creation of miniature pig model of human Waardenburg syndrome type 2A by ENU mutagenesis.

Human Waardenburg syndrome 2A (WS2A) is a dominant hearing loss (HL) syndrome caused by mutations in the microphthalmia-associated transcription factor (MITF) gene. In mouse models with MITF mutations, WS2A is transmitted in a recessive pattern, which limits the study of hearing loss (HL) pathology. In the current study, we performed ENU (ethylnitrosourea) mutagenesis that resulted in substituting a conserved lysine with a serine (p. L247S) in the DNA-binding domain of the MITF gene to generate a novel miniature pig model of WS2A. The heterozygous mutant pig (MITF +/L247S) exhibits a dominant form of profound HL and hypopigmentation in skin, hair, and iris, accompanied by degeneration of stria vascularis (SV), fused hair cells, and the absence of endocochlear potential, which indicate the pathology of human WS2A. Besides hypopigmentation and bilateral HL, the homozygous mutant pig (MITF L247S/L247S) and CRISPR/Cas9-mediated MITF bi-allelic knockout pigs both exhibited anophthalmia. Three WS2 patients carrying MITF mutations adjacent to the corresponding region were also identified. The pig models resemble the clinical symptom and molecular pathology of human WS2A patients perfectly, which will provide new clues for better understanding the etiology and development of novel treatment strategies for human HL.

The effects of superchilling with modified atmosphere packaging on the physicochemical properties and shelf life of swimming crab.

The objective of the present study was to investigate the effects of superchilling with modified atmosphere packaging on the physicochemical properties and shelf life of swimming crab. As the storage time increased, the rates at which the total aerobic plate count, total volatile basic nitrogen, pH, peroxide value and thiobarbituric acid-reactive substances value increase were significantly lower for the superchilling with modified atmosphere packaging (SCS + MAP) treatment compared to superchilling storage (SCS) and chilling storage (CS). With increasing storage time, the carbonyl content of the proteins increased from 1.21 nmol/mg of protein (0 day) to 2.03, 1.87, 1.66 nmol carbonyl/mg protein on the 6th day for CS, SCS and SCS + MAP, respectively. The disulfide bonds increased in a similar manner, and the total sulfhydryl content, salt extractable protein and Ca-ATPase stability decreased. Sodium dodecyl sulphate polyacrylamide gel elcetrophoresis (SDS-PAGE) and microstructure analysis also indicated that SCS + MAP could reduce the degree of protein degradation. These results suggested that superchilling with modified atmosphere packaging offers an effective approach to slowdown protein and lipid oxidation, and extends the shelf life of swimming crab. However, superchilling with high-CO2 packaging had a negative effect on the surface hydrophobicity and drip loss of swimming crab.

Expression of Bama Minipig and Human CYP3A Enzymes: Comparison of the Catalytic Characteristics with Each Other and Their Liver Microsomes.

Minipigs represent a good animal model because of the physiologic and anatomic similarities they share with humans. Three cytochrome P450 (CYP) 3A isozymes, CYP3A22, CYP3A29, and CYP3A46, have recently been reported to be expressed in Bama minipigs, which have limited data relating to their metabolic characteristics. In the present study, Bama minipig CYP3A22, CYP3A29, and CYP3A46 were recombinantly expressed and their metabolic manners were compared with those of human CYP3A4 and CYP3A5 and also human and Bama minipig liver microsomes. The results indicated Bama minipigs and human CYP3A enzymes showed similar metabolic kinetics and metabolite profiles using testosterone, midazolam, and nifedipine as substrates. However, the differences in amino acid sequences change the elimination velocity and metabolic preference of CYP3A enzymes to their substrates. It was demonstrated that CYP3A29, CYP3A4, and CYP3A5 were the most active enzymes for all reactions, whereas CYP3A46 was the least active enzyme. Substrate-dependent metabolism characteristics between human and Bama minipig CYP3A isoenzymes exist.

CP-31398 prevents the growth of p53-mutated colorectal cancer cells in vitro and in vivo.

Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Small molecule CP-31398 was shown to restore mutant p53 tumor suppressor functions in cancer cells. Here, we determined the effects of CP-31398 on the growth of p53-mutated colorectal cancer (CRC) cells in vitro and in vivo. CRC cells which carry p53 mutation in codon 273 were treated with CP-31398 and the control, and the effects of CP-31398 on cell cycle, cell apoptosis, and proliferation were determined. The expression of p53-responsive downstream genes was evaluated by quantitative reverse transcriptase PCR (RT-PCR) and Western blot. CP-31398 was administrated into xenograft tumors created by the inoculation of HT-29 cells, and then the effect of CP-31398 on the growth of xenograft tumors was examined. CP-31398 induced p53 downstream target molecules in cultured HT-29 cells, which resulted in the inhibition of CRC cell growth assessed by the determination of cell cycle, apoptosis, and cell proliferation. In xenograft tumors, CP-31398 modulated the expression of Bax, Bcl-2, caspase 3, cyclin D, and Mdm2 and then blocked the growth of xenograft tumors. CP-31398 would be developed as a therapeutic candidate for p53-mutated CRC due to the restoration of mutant p53 tumor suppressor functions.

Transgenic expression of human cytoxic T-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) by porcine skin for xenogeneic skin grafting.

Porcine skin is frequently used as a substitute of human skin to cover large wounds in clinic practice of wound care. In our previous work, we found that transgenic expression of human cytoxicT-lymphocyte associated antigen4-immunoglobulin (hCTLA4Ig) in murine skin graft remarkably prolonged its survival in xenogeneic wounds without extensive immunosuppression in recipients, suggesting that transgenic hCTLA4Ig expression in skin graft may be an effective and safe method to prolong xenogeneic skin graft survival. In this work, using a transgene construct containing hCTLA4Ig coding sequence under the drive of human Keratine 14 (k14) promoter, hCTLA4Ig transgenic pigs were generated by somatic nuclear transfer. The derived transgenic pigs were healthy and exhibited no signs of susceptibility to infection. The hCTLA4Ig transgene was stably transmitted through germline over generations, and thereby a transgenic pig colony was established. In the derived transgenic pigs, hCTLA4Ig expression in skin was shown to be genetically stable over generations, and detected in heart, kidney and corneal as well as in skin. Transgenic hCTLA4Ig protein in pigs exhibited expected biological activity as it suppressed human lymphocyte proliferation in human mixed lymphocyte culture to extents comparable to those of commercially purchased purified hCTLA4Ig protein. In skin grafting from pigs to rats, transgenic porcine skin grafts exhibited remarkably prolonged survival compared to the wild-type skin grafts derived from the same pig strain (13.33 ± 3.64 vs. 6.25 ± 2.49 days, P < 0.01), further indicating that the transgenic hCTLA4Ig protein was biologically active and capable of extending porcine skin graft survival in xenogeneic wounds. The transgenic pigs generated in this work can be used as a reproducible resource to provide porcine skin grafts with extended survival for wound coverage, and also as donors to investigate the impacts of hCTLA4Ig on xenotransplantation of other organs (heart, kidney and corneal) due to the ectopic transgenic hCTLA4Ig expression.

Evaluation of tumor markers for the differential diagnosis of benign and malignant ascites.

INTRODUCTION: The diagnosis of malignant ascites is a challenging problem in clinical practice, non-invasive techniques should be developed to improve diagnostic accuracy. The diagnostic performances of tumor markers in malignant ascites remained unsettled. Our aim was to evaluate diagnostic performance of tumor markers in differential diagnosis of benign and malignant ascites. MATERIAL AND METHODS: A total of 437 patients were enrolled, and the relevant parameters of the patients were analyzed for the differentiation of benign ascites from malignant ascites. RESULTS: At the predetermined cutoff values of tumor makers, tumor markers in ascitic fluid showed better diagnostic performance than those in serum. Combined use of tumor markers and the cytology increased the diagnostic yield of the latter by 37%. In cytologically negative malignant ascites, tumor markers provided assistance in differentiating malignant ascites from benign ascites, and the combination of ascitic tumor markers yielded 86% sensitivity, 97% specificity. CONCLUSION: Use of a panel of tumor markers exhibited excellent diagnostic performance in diagnosing malignant ascites, which indicated the detection of tumor markers may represent a beneficial adjunct to cytology, thus guiding the selection of patients who might benefit from further invasive procedures.

Probing long-lived radioactive isotopes on the double-logarithmic Segrè chart.

Isotopes have been widely applied in a variety of scientific subjects; many aspects of isotopes, however, remain not well understood. In this study, I investigate the relation between the number of neutrons (N) and the number of protons (Z) in stable isotopes of non-radioactive elements and long-lived isotopes of radioactive elements at the double-linear scale (conventional Segrè chart) and the double-logarithmic scale. Statistical analyses show that N is a power-law function of Z for these isotopes: N = 0.73 × Z 1.16. This power-law relation provides better predictions for the numbers of neutrons in stable isotopes of non-radioactive elements and long-lived isotopes of radioactive elements than the linear relation on the conventional Segrè chart. The power-law pattern reveled here offers empirical guidance for probing long-lived isotopes of unknown radioactive elements.