Fluorescence characterization of clinically-important bacteria

Healthcare-associated infections (HCAI/HAI) represent a substantial threat to patient health during hospitalization and incur billions of dollars additional cost for subsequent treatment. One promising method for the detection of bacterial contamination in a clinical setting before an HAI outbreak occurs is to exploit native fluorescence of cellular molecules for a hand-held, rapid-sweep surveillance instrument. Previous studies have shown fluorescence-based detection to be sensitive and effective for food-borne and environmental microorganisms, and even to be able to distinguish between cell types, but this powerful technique has not yet been deployed on the macroscale for the primary surveillance of contamination in healthcare facilities to prevent HAI. Here we report experimental data for the specification and design of such a fluorescence-based detection instrument. We have characterized the complete fluorescence response of eleven clinically-relevant bacteria by generating excitation-emission matrices (EEMs) over broad wavelength ranges. Furthermore, a number of surfaces and items of equipment commonly present on a ward, and potentially responsible for pathogen transfer, have been analyzed for potential issues of background fluorescence masking the signal from contaminant bacteria. These include bedside handrails, nurse call button, blood pressure cuff and ward computer keyboard, as well as disinfectant cleaning products and microfiber cloth. All examined bacterial strains exhibited a distinctive double-peak fluorescence feature associated with tryptophan with no other cellular fluorophore detected. Thus, this fluorescence survey found that an emission peak of 340nm, from an excitation source at 280nm, was the cellular fluorescence signal to target for detection of bacterial contamination. The majority of materials analysed offer a spectral window through which bacterial contamination could indeed be detected. A few instances were found of potential problems of background fluorescence masking that of bacteria, but in the case of the microfiber cleaning cloth, imaging techniques could morphologically distinguish between stray strands and bacterial contamination

The Extrachromosomal EAST Protein of Drosophila Can Associate with Polytene Chromosomes and Regulate Gene Expression

The EAST protein of Drosophila is a component of an expandable extrachromosomal domain of the nucleus. To better understand its function, we studied the dynamics and localization of GFP-tagged EAST. In live larval salivary glands, EAST-GFP is highly mobile and localizes to the extrachromosomal nucleoplasm. When these cells are permeabilized, EAST-GFP rapidly associated with polytene chromosomes. The affinity to chromatin increases and mobility decreases with decreasing salt concentration. Deleting the C-terminal residues 1535 to 2301 of EAST strongly reduces the affinity to polytene chromosomes. The bulk of EAST-GFP co-localizes with heterochromatin and is absent from transcriptionally active chromosomal regions. The predominantly chromosomal localization of EAST-GFP can be detected in non-detergent treated salivary glands of pupae as they undergo apoptosis, however not in earlier stages of development. Consistent with this chromosomal pattern of localization, genetic evidence indicates a role for EAST in the repression of gene expression, since a lethal east mutation is allelic to the viable mutation suppressor of white-spotted. We propose that EAST acts as an ion sensor that modulates gene expression in response to changing intracellular ion concentrations

Formin 1-Isoform IV Deficient Cells Exhibit Defects in Cell Spreading and Focal Adhesion Formation

Background: Regulation of the cytoskeleton is a central feature of cell migration. The formin family of proteins controls the rate of actin nucleation at its barbed end. Thus, formins are predicted to contribute to several important cell processes such as locomotion, membrane ruffling, vesicle endocytosis, and stress fiber formation and disassociation. Methodology/Principal Findings: In this study we investigated the functional role of Formin1-isoform4 (Fmn1-IV) by using genetically null primary cells that displayed augmented protrusive behaviour during wound healing and delayed cell spreading. Cells deficient of Fmn1-IV also showed reduced efficiency of focal adhesion formation. Additionally, we generated an enhanced green fluorescence protein (EGFP)-fused Fmn1-IV knock-in mouse to monitor the endogenous subcellular localization of Fmn1-IV. Its localization was found within the cytoplasm and along microtubules, yet it was largely excluded from adherens junctions. Conclusions/Significance: It was determined that Fmn1-IV, as an actin nucleator, contributes to protrusion of the cell’s leading edge and focal adhesion formation, thus contributing to cell motility

A phase II study of LFP therapy (5-FU (5-fluorourasil) continuous infusion (CVI) and Low-dose consecutive (Cisplatin) CDDP) in advanced biliary tract carcinoma

BACKGROUND: Unresectable biliary tract carcinoma is known to demonstrate a poor prognosis. We conducted a single arm phase II study of LFP therapy (5-FU (5-fluorourasil) continuous infusion (CVI) and Low-dose consecutive (Cisplatin) CDDP) for advanced biliary tract malignancies basically on an outpatient basis. METHODS: Between February 1996 and September 2003, 42 patients were enrolled in this trial. LFP THERAPY: By using a total implanted CV-catheter system, 5-FU (160 mg/m(2)/day) was continuously infused over 24 hours for 7 consecutive days and CDDP (6 mg/m(2)/day) was infused for 30 minutes twice a week as one cycle. The administration schedule consisted of 4 cycles as one course. RESIST criteria (Response evaluation criteria for solid tumors) and NCI-CTC (National Cancer Institute-Common Toxicity Criteria) (ver.3.0) were used for evaluation of this therapy. The median survival time (MST) and median time to treatment failure (TTF) were calculated by the Kaplan-Meier method. RESULTS: Patients characteristics were: mean age 66.5(47–79): male 24 (54%): BDca (bile duct carcinoma) 27 GBca (Gallbladder carcinoma) 15: locally advanced 26, postoperative recurrence 16. The most common toxicity was anemia (26.2%). Neither any treatment related death nor grade 4 toxicity occurred. The median number of courses of LFP Therapy which patients could receive was two (1–14). All the patients are evaluable for effects with an over all response rates of 42.9% (95% confidence interval C.I.: 27.7–59.0) (0 CR, 18 PR, 13 NC, 11 PD). There was no significant difference regarding the anti tumor effects against both malignant neoplasms. Figure 2 Shows the BDca a longer MST and TTF than did GBca (234 vs 150, 117 vs 85, respectively), but neither difference was statistically significant. The estimated MST and median TTF were 225 and 107 days, respectively. The BDca had a longer MST and TTF than GBca (234 vs 150, 117 vs 85, respectively), but neither difference was statistically significant. CONCLUSION: LFP therapy appears to be useful modality for the clinical management of advanced biliary tract malignancy

Outpatient chemotherapy with gemcitabine and oxaliplatin in patients with biliary tract cancer

This phase II study was conducted to determine the efficacy and toxicity of a gemcitabine (GEM) and oxaliplatin (OX) chemotherapy protocol in patients with unresectable biliary tract cancer (BTC). Patients were treated with GEM 1000 mg m−2 (30 min infusion) on days 1, 8, 15, and OX 100 mg m−2 (2 h infusion) on days 1 and 15 (gemcitabine and oxaliplatin (GEMOX-3 protocol), repeated every 28 days. The data were collected according to the Simon 2-stage design for a single centre phase II study (α=0.05; β=0.2). Primary end point was response rate; secondary end points were time-to-progression (TTP), median survival, and safety profile. Thirty-one patients were enrolled in the study between July 2002 and April 2005. Therapeutic responses were as follows: partial response in eight patients (26%, 95% confidence interval (CI) 14–44), stable disease in 14 patients (45%, 95%CI 29–62), resulting in a disease control rate of 71%. Nine patients (29%, 95%CI 16–47) had progressive disease. Median TTP was 6.5 months. Median overall survival was 11 months. Common Toxicity Criteria (CTC) Grade 3–4 toxicities were transient thrombocytopenia (23%), peripheral sensory neuropathy (19%), leucopenia (16%), and anaemia (10%). In conclusion the GEMOX-3 protocol is active and well tolerated in patients with advanced BTC. It can be applied in an outpatient setting with three visits per month only

Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy

Background A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets. Methods Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis. Results A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001). Conclusion We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty

Systemic Treatments for Mesothelioma: Standard and Novel

Systemic therapy is the only treatment option for the majority of mesothelioma patients, for whom age, co-morbid medical illnesses, non-epithelial histology, and locally advanced disease often preclude surgery. For many years, chemotherapy had a minimal impact on the natural history of this cancer, engendering considerable nihilism. Countless drugs were evaluated, most of which achieved response rates below 20% and median survival of <1 year. Several factors have hampered the evaluation of systemic regimens in patients with mesothelioma. The disease is uncommon, affecting only about 2500 Americans annually. Thus, most clinical trials are small, and randomized studies are challenging to accrue. There is significant heterogeneity within the patient populations of these small trials, for several reasons. Since all of the staging systems for mesothelioma are surgically based, it is almost impossible to accurately determine the stage of a patient who has not been resected. Patients with very early stage disease may be lumped together with far more advanced patients in the same study. The disease itself is heterogenous, with many different prognostic factors, most notably three pathologic subtypes—epithelial, sarcomatoid, and biphasic—that have different natural histories, and varying responses to treatment. Finally, response assessment is problematic, since pleural-based lesions are difficult to measure accurately and reproducibly. Assessment criteria often vary between trials, making some cross-trial comparisons difficult to interpret. Despite these limitations, in recent years, there has been a surge of optimism regarding systemic treatment of this disease. Several cytotoxic agents have been shown to generate reproducible responses, improve quality of life, or prolong survival in mesothelioma. Drugs with single-agent activity include pemetrexed, raltitrexed, vinorelbine, and vinflunine. The addition of pemetrexed or raltitrexed to cisplatin prolongs survival. The addition of cisplatin to pemetrexed, raltitrexed, gemcitabine, irinotecan, or vinorelbine improves response rate. The combination of pemetrexed plus cisplatin is considered the benchmark front-line regimen for this disease, based on a phase III trial in 456 patients that yielded a response rate of 41% and a median survival of 12.1 months. Vitamin supplementation with folic acid is essential to decrease toxicity, though recent data suggests that there may be an optimum dose of folic acid that should be administered; higher doses may diminish the effectiveness of pemetrexed. There are also several unresolved questions about the duration and timing of treatment with pemetrexed that are the subject of planned clinical trials. It is essential to recognize that the improvements observed with the pemetrexed/cisplatin combination, though real, are still modest. Other active drugs or drug combinations may be more appropriate for specific individuals, and further research is still needed to improve upon these results. Since the majority of mesotheliomas in the United States occur in the elderly, non-cisplatin-containing pemetrexed combinations may be more appropriate for some patients. Now that effective agents have been developed for initial treatment, several classical cytotoxic drugs and many novel agents are being evaluated in the second-line setting. These include drugs targeted against the epidermal growth factor, platelet-derived growth factor, vascular endothelial growth factor, src kinase, histone deacetylase, the proteasome, and mesothelin. Given the progress made in recent years, there is reason to believe that more effective treatments will continue to be developed

Fibroblasts Express Immune Relevant Genes and Are Important Sentinel Cells during Tissue Damage in Rainbow Trout (Oncorhynchus mykiss)

Fibroblasts have shown to be an immune competent cell type in mammals. However, little is known about the immunological functions of this cell-type in lower vertebrates. A rainbow trout hypodermal fibroblast cell-line (RTHDF) was shown to be responsive to PAMPs and DAMPs after stimulation with LPS from E. coli, supernatant and debris from sonicated RTHDF cells. LPS was overall the strongest inducer of IL-1β, IL-8, IL-10, TLR-3 and TLR-9. IL-1β and IL-8 were already highly up regulated after 1 hour of LPS stimulation. Supernatant stimuli significantly increased the expression of IL-1β, TLR-3 and TLR-9, whereas the debris stimuli only increased expression of IL-1β. Consequently, an in vivo experiment was further set up. By mechanically damaging the muscle tissue of rainbow trout, it was shown that fibroblasts in the muscle tissue of rainbow trout contribute to electing a highly local inflammatory response following tissue injury. The damaged muscle tissue showed a strong increase in the expression of the immune genes IL-1β, IL-8 and TGF-β already 4 hours post injury at the site of injury while the expression in non-damaged muscle tissue was not influenced. A weaker, but significant response was also seen for TLR-9 and TLR-22. Rainbow trout fibroblasts were found to be highly immune competent with a significant ability to express cytokines and immune receptors. Thus fish fibroblasts are believed to contribute significantly to local inflammatory reactions in concert with the traditional immune cells

Comparative evaluation of the treatment efficacy of suberoylanilide hydroxamic acid (SAHA) and paclitaxel in ovarian cancer cell lines and primary ovarian cancer cells from patients

BACKGROUND: In most patients with ovarian cancer, diagnosis occurs after the tumour has disseminated beyond the ovaries. In these cases, post-surgical taxane/platinum combination chemotherapy is the "gold standard". However, most of the patients experience disease relapse and eventually die due to the emergence of chemotherapy resistance. Histone deacetylase inhibitors are novel anticancer agents that hold promise to improve patient outcome. METHODS: We compared a prototypic histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), and paclitaxel for their treatment efficacy in ovarian cancer cell lines and in primary patient-derived ovarian cancer cells. The primary cancer cells were isolated from malignant ascites collected from five patients with stage III ovarian carcinomas. Cytotoxic activities were evaluated by Alamar Blue assay and by caspase-3 activation. The ability of SAHA to kill drug-resistant 2780AD cells was also assessed. RESULTS: By employing the cell lines OVCAR-3, SK-OV-3, and A2780, we established SAHA at concentrations of 1 to 20 μM to be as efficient in inducing cell death as paclitaxel at concentrations of 3 to 300 nM. Consequently, we treated the patient-derived cancer cells with these doses of the drugs. All five isolates were sensitive to SAHA, with cell killing ranging from 21% to 63% after a 72-h exposure to 20 μM SAHA, while four of them were resistant to paclitaxel (i.e., <10% cell death at 300 nM paclitaxel for 72 hours). Likewise, treatment with SAHA led to an increase in caspase-3 activity in all five isolates, whereas treatment with paclitaxel had no effect on caspase-3 activity in three of them. 2780AD cells were responsive to SAHA but resistant to paclitaxel. CONCLUSION: These ex vivo findings raise the possibility that SAHA may prove effective in the treatment of paclitaxel-resistant ovarian cancer in vivo

Mining and state-space modeling and verification of sub-networks from large-scale biomolecular networks

<p>Abstract</p> <p>Background</p> <p>Biomolecular networks dynamically respond to stimuli and implement cellular function. Understanding these dynamic changes is the key challenge for cell biologists. As biomolecular networks grow in size and complexity, the model of a biomolecular network must become more rigorous to keep track of all the components and their interactions. In general this presents the need for computer simulation to manipulate and understand the biomolecular network model.</p> <p>Results</p> <p>In this paper, we present a novel method to model the regulatory system which executes a cellular function and can be represented as a biomolecular network. Our method consists of two steps. First, a novel scale-free network clustering approach is applied to the large-scale biomolecular network to obtain various sub-networks. Second, a state-space model is generated for the sub-networks and simulated to predict their behavior in the cellular context. The modeling results represent <it>hypotheses </it>that are tested against high-throughput data sets (microarrays and/or genetic screens) for both the natural system and perturbations. Notably, the dynamic modeling component of this method depends on the automated network structure generation of the first component and the sub-network clustering, which are both essential to make the solution tractable.</p> <p>Conclusion</p> <p>Experimental results on time series gene expression data for the human cell cycle indicate our approach is promising for sub-network mining and simulation from large-scale biomolecular network.</p