The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-α and Trn-β, that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [ 13 C, 15 N] Trn-α and [ 13 C, 15 N]Trn-β were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the α-carbons of the modified residues, forming three sulfur to α-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S′-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to α-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora.two-component bacteriocin | posttranslational modifications | Clostridium difficile-associated disease | peptide | NMR

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Myocyte cell loss is a prominent and important pathogenic feature of cardiac ischemia. We have used cultured neonatal rat cardiac myocytes exposed to prolonged hypoxia as an experimental system to identify critical factors involved in cardiomyocyte death. Exposure of myocytes to hypoxia for 48 h resulted in intranucleosomal cleavage of genomic DNA characteristic of apoptosis and was accompanied by increased p53 transactivating activity and protein accumulation. Expression of p21/WAF-1/CIP-1, a well-characterized target of p53 transactivation, also increased in response to hypoxia. Hypoxia did not cause DNA laddering or cell loss in cardiac fibroblasts. To determine whether the increase in p53 expression in myocytes was sufficient to induce apoptosis, normoxic cultures were infected with a replication-defective adenovirus expressing wild-type human p53 (AdCMV.p53). Infected cells expressed high intracellular levels of p53 protein and exhibited the morphological changes and genomic DNA fragmentation characteristic of apoptosis. In contrast, no genomic DNA fragmentation was observed in myocytes infected with the control virus lacking an insert (AdCMV.null) or in cardiac fibroblasts infected with AdCMV.p53. These results suggest that the intracellular signaling pathways activated by p53 might play a critical role in the regulation of hypoxia-induced apoptosis of cardiomyocytes. ( J. Clin. Invest. 1997. 99:2635-2643.)

Background/Aims: Systemic inflammatory response (SIR) is widely considered as a preoperative risk factor for hepatocellular carcinoma (HCC) outcomes. The neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR), two of the prognostic indices, have been investigated in post-therapeutic recurrence and survival of HCC. Here, we quantify the prognostic value of these two biomarkers and evaluate their consistency in different HCC therapies. Methods: A systematic review of electronic database of the Web of Science, Embase, PubMed and the Cochrane Library was conducted to search for associations between the NLR and PLR in the blood and clinical outcomes of HCC. Overall survival (OS) and recurrence-free survival (RFS) were the primary outcomes, and hazard ratios (HRs) and 95% confidence intervals (95% CIs) were explored as effect measures. Subgroup analyses were performed to explore the heterogeneity of different therapies. Results: A total of 24 articles comprising 6318 patients were included in the meta-analysis. Overall, the pooled outcomes revealed that a high NLR before treatment predicted a poor OS (HR: 1.54, 95% CI: 1.34 to 1.76, p<0.001) and poor RFS (HR: 1.45, 95% CI: 1.16 to 1.82, p=0.001). Moreover, an increased PLR predicted a poor OS (HR: 1.63, 95% CI: 1.34 to 1.98, p<0.001) and earlier HCC recurrence (HR: 1.52, 95% CI: 1.21 to 1.91, p<0.001). In addition, both the NLR and PLR were identified as independent risk factors for predicting OS and RFS in HCC patients in a subgroup analysis of different treatment types, including curative or palliative therapy; however, these results were not found in the sorafenib subgroup due to limited clinical research. Conclusion: An increased NLR or PLR indicated poor outcomes for patients with HCC. The NLR and PLR may be considered as reliable and inexpensive biomarkers for making clinical decisions regarding HCC treatment.

Termination of wound healing requires a fine balance between collagen deposition and its hydrolysis. To dissect the underlying control mechanisms for this process, we established a keratinocyte/fibroblast co-culture system and subsequently demonstrated more than a 10-fold increase in collagenase expression in fibroblasts co-cultured with keratinocytes relative to that of control cells. This finding was further confirmed in fibroblasts grown in a keratinocyte/fibroblast collagen-GAG gel. The efficacy of keratinocyte-derived collagenase stimulatory factors on collagenase activity was evaluated, and the results showed that only conditioned medium derived from fibroblasts co-cultured with keratinocytes was able to break down markedly type I collagen to its one-quarter and three-quarter fragments of both alpha (alpha1 and alpha2) and beta (beta1.1 and beta1.2) chains. The results of a dose-response experiment showed that keratinocyte-conditioned medium (KCM) stimulates the expression of collagenase mRNA by dermal fibroblasts in a concentration-dependent fashion. In a similar experiment, the results of a time-response experiment revealed that KCM treatment increases the expression of collagenase mRNA in dermal fibroblasts as early as 6 h and reaches its maximum level within 24-48 h. Considering that this keratinocyte-releasable factor has a potent collagenase stimulatory effect on fibroblasts, which favors the resolution of accumulated type I and type III collagen found in fibrotic tissue, we referred to this protein as a keratinocyte-derived anti-fibrogenic factor (KDAF). In a series of chromatography experiments and a direct trypsin digestion of the proteins and subsequent peptide mapping, a keratinocyte-derived collagenase-stimulating factor turned out to be a releasable form of stratifin, also known as 14-3-3 sigma protein. To validate this finding, stratifin cDNA was cloned into a pGEX-6P-1 expressing vector and more than 50 mg of recombinant stratifin was generated and used to treat fibroblasts with various concentrations for 24 h. The results of northern analysis showed a remarkable dose-response increase in the expression of collagenase mRNA in stratifin-treated fibroblasts relative to that of the control. This finding was consistent with that obtained from collagenase activity assay. In conclusion, we identified a keratinocyte-releasable form of stratifin in KCM that mimics the collagenase stimulatory effect of KCM for dermal fibroblasts. This finding suggests that stratifin is likely to be, at least, one of the KDAFs found in KCM.

Tear proteome profiling may generate useful information for the understanding of the interaction between an eye and its contacting objects, such as a contact lens or a lens implant. This is important for designing improved eye-care devices and maintaining the health of an eye. Proteome profiles of tear fluids may also be used for disease diagnosis and prognosis. However, only a small volume of tear fluid (<5 microL) can be collected in a clinical laboratory under normal operational conditions, which makes proteome profiling a challenge. In this work we apply several proteomic analysis techniques, including gel-based and solution-based approaches with LC-ESI and LC-MALDI MS and MS/MS to gauge the relative merits of producing proteome profiles and to generate as broad a coverage of the tear proteome as possible from this small amount of sample. It is shown that a total of 54 proteins can be confidently identified using less than 5 microL of tear fluid. Of these, 44 proteins can be detected by LC-MALDI MS alone with a consumption of 2 microL of tear fluid. Furthermore, LC-MALDI can be used to determine post-translational modifications (PTMs), such as glycosylation and phosphorylation, without any sample enrichment or treatment. This work represents one of the most extensive proteome profiles (i.e., proteins identified and PTMs characterized) generated from tear fluids using clinically relevant amounts of sample.

The 70-kD S6 kinase (p70S6K) has been implicated in the regulation of protein synthesis in many cell types and in the angiotensin II-stimulated hypertrophy of cardiac myocytes. Our purpose was to determine whether p70S6K plays a role in cardiomyocyte hypertrophy induced by the alpha 1-adrenergic receptor (alpha 1-AR) agonist phenylephrine (PE). PE stimulated the activity of p70S6K > 3-fold, and this increase was blocked by rapamycin, an immunosuppressant macrolide that selectively inhibits p70S6K. When administered for 3 days, PE stimulated a 30% increase in total protein content, a 2-fold increase in the incorporation of [14C]phenylalanine (14C-Phe) into protein, and a 50% increase in two-dimensional myocyte area. Rapamycin pretreatment (> or = 500 pg/mL) significantly inhibited each of these PE-stimulated changes. Two days of PE treatment resulted in a 1.6-fold increase in total RNA yield per dish, a 2-fold increase in incorporation of [14C]uridine into myocyte RNA, and increases in relative mRNA levels of the hypertrophy-associated atrial natriuretic factor (ANF, 2.1-fold) and skeletal alpha-actin (SK, 2.2-fold) genes. Although rapamycin abolished the PE-stimulated increases in total RNA and incorporation of [14C]uridine, it had no effect on the induction of the ANF and SK genes. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited PE-stimulated increases in p70S6K activity and the incorporation of labeled precursors into myocyte protein and RNA. These results demonstrate that p70S6K is activated by the hypertrophic agent PE and that a PI3-K or PI3-K-like activity is required for p70S6K activation and myocyte hypertrophy. The data suggest that p70S6K activation may be required for PE-stimulated hypertrophy of cardiac myocytes. Our results demonstrate that intracellular signaling pathways responsible for transcriptional and translational responses diverge early after alpha 1-AR stimulation in cardiac myocytes.

Background Colon cancer (CC) is a common malignant cancer. Recently, circFNDC3B was found to exert biological function in multiple cancers. However, it was unclear whether the potential protein encoded by circFNDC3B is involved in carcinogenesis of CC. Methods We used Sanger sequence and RNase R digestion assay to confirm the existence of circFNDC3B, and quantitative real-time PCR was used to evaluate the circRNA’s expression. Then fluorescence in situ hybridization (FISH) was performed to study location of circFNDC3B. The identification of protein encoded by circFNDC3B was performed using LC-MS/MS. The function of circFNDC3B-218aa on proliferation, invasion and migration were assessed by CCK8 assays, colony formation assays, transwell assays, wound-healing assays and animal experiments. RNA-sequencing and western blot were used to identify the gene regulated by circFNDC3B-218aa. Finally, glucose metabolism-related assays were performed to further investigate function of circFNDC3B-218aa. Results CircFNDC3B was localized mostly in the cytoplasm, and was decreased in CC cell lines and tissues. The patients with low circFNDC3B expression had a shorter OS (P = 0.0014) than patients with high expression. Moreover, circFNDC3B inhibited the proliferation, invasion and migration of CC cells. Next, we identified that circFNDC3B could encode a novel protein circFNDC3B-218aa. Furthermore, circFNDC3B-218aa, not circFNDC3B, inhibited the proliferation, invasion and migration of CC. Additionally, the in vivo experiments implied that up-regulated circFNDC3B-218aa exhibited an inhibitory effect on CC progression. By RNA-sequencing, western blot and glucose metabolism-related assays, we found that circFNDC3B-218aa inhibited the expression of Snail, and subsequently promoted the tumor-suppressive effect of FBP1 in CC. Conclusions The novel circFNDC3B-218aa may serve as a tumor suppressive factor and potential biomarker which may supply the potential therapeutic target for CC.