OBJECTIVE

High glucose content of peritoneal dialysis fluids (PDFs) has been shown to contribute to loss of peritoneal function during long-term peritoneal dialysis. However, hyperosmolality and hypertonicity of PDF are usually seen as similar stress events inducing osmotic stress-induced programmed cell death. In this study, we examined the impact of various osmotic agents on apoptosis induced by hyperosmolar PDFs, focusing on the mechanisms underlying the lethal effects of PDFs on peripheral blood mononuclear cells (PBMCs).

METHODS

We assessed apoptosis and necrosis by annexin V-propidium iodide (PI) labeling, and caspase-3 activity by fluorescence assay. F-actin remodeling was measured using fluorescent phalloidin labeling.

RESULTS

Hyperosmolality does not cause the cytotoxicity observed with PDF, but exposure to agents incapable of permeating cell membranes results in a significant increase in the percentage of apoptotic PBMCs by annexin V-PI labeling, which is confirmed by the increase in caspase-3 activity. Interestingly, inhibition of caspase-3 by Z-VAD-FMK did not suppress apoptosis. Extracellular hypertonicity produced polymerization of filamentous actin and cell shrinkage, which displayed similar time courses. Cell shrinkage was blocked by cytochalasin D, indicating an active role for actin cytoskeleton in hypertonicity-induced cell shrinkage. F-actin polymerization was related to an increase in intracellular ionic strength. Finally, we excluded a direct role for actin remodeling in osmotic stress-induced programmed cell death.

CONCLUSIONS

Exposure to osmolytes that cannot penetrate cell membranes results in a hypertonicity-induced apoptosis that cannot be blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. In addition, extracellular hypertonicity induced by impermeant solutes produces F-actin polymerization through an increase in intracellular ionic strength. The remodeling of the cytoskeleton does not modulate apoptosis but participates in cell shrinkage.

In continuous ambulatory peritoneal dialysis patients, treatment success is inextricably linked to the functional and morphological integrity of the peritoneal membrane. This membrane, however, is repeatedly exposed to peritoneal dialysis fluids (PDFs) with unphysiological composition (e.g., acidic pH, high glucose content, hyperosmolarity). More recently, attention of researchers and clinicians has been focused on the presence of glucose degradation products (GDPs) that are generated during heat sterilization of PDF. These GDPs were found to adversely affect peritoneal cell function both acutely and chronically. Recently, a new family of multi-chambered PDFs has been introduced into clinical practice. By keeping the glucose in a separate compartment at very low pH, the generation of GDPs during heat sterilization is markedly reduced. Initial clinical studies indicate that treatment with these novel PDFs may lead to improved clinical outcomes. The current article reviews recent experimental and clinical experience with both conventional and multi-chambered PDFs.

Long-term peritoneal dialysis (PD) is limited by morphological changes of the peritoneal membrane. Structural changes were promoted by toxicity of glucose degradation products (GDPs) which are generated during heat sterilization in peritoneal dialysis fluids (PDFs). Besides their direct toxicity GDPs promote formation of advanced glycation endproducts (AGEs). RAGE (receptor for AGE) is the best characterized signal transduction receptor for AGEs and is expressed on mesothelial cells. The effects of PDFs with different amounts of GDPs were compared on morphological changes in the peritoneal membrane in a RAGE -/- mouse model. It could be demonstrated that RAGE plays a pivotal role in structural damage (e.g. inflammation, neoangiogenesis and fibrosis) of the peritoneal membrane. Further investigations of this pathway with regard to preventing peritoneal fibrosis should be performed to maintain the integrity of the peritoneal membrane in peritoneal dialysis patients.

BACKGROUND

Peritoneal mesothelial cells lining the peritoneal cavity play a primary role in prevention of formation of peritoneal adhesions, which depends mainly on their fibrinolytic activity. During surgical procedures, the abdominal cavity is in most cases rinsed with normal saline solution, which may modify the fibrinolytic activity of the peritoneal mesothelium and predispose to formation of adhesions. The goal of our experiments was to evaluate how normal saline and other solutions affect the fibrinolytic properties of the peritoneal mesothelial cells.

METHODS

Experiments were performed on in vitro cultures of human peritoneal mesothelial cells. Mesothelial monolayers were exposed during 6 hours to the following solutions: culture medium (control), .9% NaCl, Hanks solution, Earles solution, new peritoneal dialysis fluid with low glucose degradation products (GDP) concentration (PDF), and peritoneal dialysis fluid with high concentration of GDP (PDF-GDP). Afterwards, morphology of the cells as well as leakage of lactate dehydrogenase (LDH) from their cytosol were evaluated. During the next 24 hours when the cells were cultured in standard medium synthesis of interleukin-6 (IL-6), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were studied.

RESULTS

Mesothelial monolayers exposed to .9% NaCl or to PDF-GDP showed destruction of their morphology after 6 hours incubation and in case of PDF-GDP release of LDH from the cytosol was increased by 275% versus the control (P<.05). During subsequent culture of all cells in standard medium, the release of IL-6 was decreased in cases of cells pretreated with .9% NaCl (-58%, P<.05) or PDF-GDP (-93%, P<.001). The release of t-PA was also reduced from cells pretreated with .9% NaCl (-71%, P<.01) or with PDF-GDP (-74%, P<.01) but increased after exposure of these cells to PDF (+35%, P<.05). Statistically significant decrease of PAI-1 synthesis was observed in cells preexposed to .9% NaCl (-69%, P<.01) or to PDF-GDP (-82%, P<.05). When changes in the PAI-1/t-PA ratio were calculated, a strong tendency for increase of that value was seen in cells pretreated with .9% NaCl or Earles salts solution but not with PDF. However, in cases of Hanks solution, a significant increase in the PAI-1/t-PA ratio was observed (+104%, P<.01).

CONCLUSIONS

Exposure of the peritoneal mesothelial cells to .9% NaCl, Hanks, Earles salts solution, or PDF-GDP results either in reduction of their viability or in loss of their fibrinolytic activity. Peritoneal dialysis fluid with a low content of glucose degradation products appears to be the optimal solution causing the least damage to mesothelial cells and therefore may be the ideal solution for rinsing the abdominal cavity with low risk of inducing deterioration of the mesothelial cells fibrinolytic activity and formation of adhesions. We postulate therefore that such hypertonic peritoneal dialysis fluids should be used not only during peritoneal dialysis but also for rinsing the abdominal cavity during any surgical procedures.

Differential expression of pneumococcal virulence proteins has been demonstrated. We previously demonstrated challenge route-dependent differences in pneumococcal surface protein C (PspC) expression during bacteremia. In this study, we investigated differences in PspC expression during the transition of pneumococci from the peritoneum to the blood. Time course analysis of PspC expression using flow cytometry demonstrated that Streptococcus pneumoniae D39 collected from blood expressed significantly more PspC than did D39 collected from the peritoneum of intraperitoneally (i.p.)-infected mice. Various challenge models were then used to determine whether host responses originating from the peritoneum can influence PspC expressed by pneumococci in the blood. Using heat-inactivated D39 (HI-D39) and sterile peritoneal dialysis fluid (PDF), we investigated whether stimulation of peritoneal responses can influence PspC expression. Injection of mice i.p. with HI-D39 or PDF immediately prior to intravenous (i.v.) infection with D39 caused a significant increase in PspC expressed by D39 in the blood. Finally, we used cytokine array analysis to investigate specific inflammatory mediators that may result in differential PspC expression. Of the 96 inflammatory cytokines assayed, D39 i.p. challenge led to increased expression of 33 cytokines in serum; whereas D39 i.v. challenge led to increased expression of 15 and decreased expression of 11 cytokines relative to serum of the uninfected control. These results indicate that PspC is differentially regulated during growth in vivo and that the level of expression varies depending on the host environment.

BACKGROUND

Acute exposure of mesothelial cells to peritoneal dialysis fluid (PDF) has been shown not only to result in injury but also to induce cytoprotective heat shock proteins (HSP). The aim of the present study was to evaluate the expression of HSP in a more chronic in vitro PDF exposure system, searching for a role of glucose degradation products (GDP).

METHODS

Human peritoneal mesothelial cells (HPMC) were chronically incubated in filter- or heat-sterilized PDF (mixed 1:1 with cell culture medium), or in control cell culture medium. After incubation periods of 1, 3 and 10 days, cell extract was assessed for Ezrin, Hsp27 and Hsp72, and supernatant for IL-6 and IL-8. After 24-h exposure to the GDP 3.4-di-deoxyglucosone-3-ene (3.4-DGE), HPMC were assessed for expression of Hsp27 and Hsp72, and for release of LDH, IL-6 and IL-8.

RESULTS

In vitro PDF exposure for more than 1 day resulted in reduced cell mass, lower expression of the epithelial marker Ezrin and depressed cellular levels of both HSP, associated with increased IL-6 and IL-8 release. These effects occurred earlier and stronger with heat-sterilized than with filter-sterilized PDF. Exposure of HPMC to 3.4-DGE resulted in suppression of HSP, and increased release of LDH, IL-6 and IL-8.

CONCLUSIONS

Our data show that GDP (dys)regulate the mesothelial cell stress response. This was associated with reduced cell mass, loss of the epithelial phenotype and sterile cellular inflammation following extended exposure to heat-sterilized PDF. Toxic effects of PDF might thus be extended to reduced mesothelial cell stress responses.

Peritoneal dialysis (PD) is now an established and successful alternative to hemodialysis. Multiple studies have confirmed its equivalent dialysis adequacy, mortality and fluid balance status, at least for the first 4-5 years. Peritoneal membrane failure is now one of the leading cause of technique failure. This review describes the role of glucose, glucose degradation product, pH, lactate, advanced glycosylation end product (AGE) in causing this membrane damage, and gives insight how the use of newer peritoneal dialysis fluids (PDFs) containing icodextrin, amino acids and bicarbonate buffer can prevent peritoneal membrane damage.

1. In tuberculosis meningitis there is a tuberculosis endarteritis characterized by the formation of intimal tubercles and a diffuse subendothelial, intimal proliferation due to implantation of tubercle bacili from the blood. From the endarteritis the infiltration may spread into the muscular coat and the adventitia, and the whole wall may undergo caseous and hyaline degeneration. 2. Tuberculous proliferation in the adventitia may invade the media and the intima, and the whole wall of the arterial segment may undergo degeneration. 3. The veins are constantly the seat of more or less extensive infiltration, which always results from adjacent extravascular or arterial foci. 4. The epithelioid cells of the subendothelial, tuberculous intimal See PDF for Structure proliferation are most likely derived from the subendothelial layer of connective tissue and not from the endothelial lining.

BACKGROUND

Residual renal function (RRF) contributes to better patient survival in peritoneal dialysis (PD). It is known that glucose degradation products (GDP) and advanced glycation end-products (AGE) resorbed from the peritoneal cavity do not only cause local peritoneal toxicity but also systemic damage resulting in a loss of RRF. We hypothesize that GDP and AGE affect the structure and function of podocytes and investigate whether these effects can be rescued by human RAGE antibody (hRAGEab) to prevent AGE/RAGE interaction and podocyte damage.

METHODS

Differentiated human podocytes were pre-incubated with ±hRAGEab to block the AGE/RAGE interaction and incubated afterwards either with control solution (control), PD fluid (PDF) or a GDP mixture (GDP) for 48 h. We analysed podocyte damage and rescue by hRAGEab using immunofluorescence, western blot, enzyme-linked immunosorbent assay and a functional migration assay. For quantitation, a semiquantitative score was used.

RESULTS

When pre-incubating podocytes with hRAGEab, damage mediated by PDF and GDP was reduced. We observed lower expression of AGE in PDF and GDP as well as decreased levels of inflammation as shown by activation of nuclear factor kappa B and interleukin-6 release. The reorganization of the podocyte actin cytoskeleton was reduced in the presence of hRAGEab as well as ameliorated synaptopodin expression could be observed, both functionally associated with normal podocyte motility. Finally, podocytes underwent less apoptosis.

CONCLUSIONS

It has been investigated that GDP-containing PDF causes a loss of RRF in PD. Our findings suggest that hRAGEab confers protection against PDF- and GDP-induced podocyte dysfunction. Blocking the AGE/RAGE interaction provides specific protective effects against PDF- and GDP-induced cytoskeletal reorganization, dynamics and stabilizes podocyte survival; this might be an implication for the preservation of RRF in PD.

Commercially available peritoneal dialysis fluids (PDFs) are known to impair peritoneal cellular defense mechanisms. We have investigated the influence of glucose polymer-containing PDFs on phagocytic function in vitro. Polymorphonuclear neutrophils (PMNLs) and monocytes (MNs) from 10 continuous ambulatory peritoneal dialysis patients and 10 healthy donors were incubated in PDFs containing either 7.5% icodextrin (glucose polymer) or 1.5% glucose at original pH and pH 7.4. Chemiluminescence response and H202 production were measured following stimulation with preopsonized Staphylococcus epidermidis or phorbol myristate acetate. Phagocytosis of radiolabeled bacteria and killing capacity of the cells were determined. A comparison of the impact of glucose polymer versus glucose-containing solutions at their original pH on the oxidative metabolism of the cells showed a highly significant difference (P < 0.0001) in favor of glucose polymers for H202 production of PMNLs (7.78 +/- 4.5 nmol cytochrome C reduction/10(6) cells/min v 1.11 +/- 0.67 nmol cytochrome C reduction/10(6) cells/min) and MNs (7.66 +/- 3.6 nmol cytochrome C reduction/10(6) cells/min v 1.29 +/- 0.86 nmol cytochrome C reduction/10(6)cells/min). Correspondingly, PMNLs and MNs incubated in glucose polymers showed a significantly higher chemiluminescence response irrespective of the stimulant used (P < 0.0001). Applying the killing assay on PMNLs also revealed a significantly higher percentage of inactivated bacteria (45.5% +/- 11.0% v 29.2% +/- 15.5%; P < 0.05). After adjustment of pH to 7.4, a significant difference could only be found for H202 production of PMNLs in favor of glucose polymers (16.73 +/- 6.98 nmol cytochrome C reduction/10(6) cells/min v 11.65 +/- 5.37 nmol cytochrome C reduction/10(6) cells/min; P < 0.05). In addition, we compared the glucose-polymer solution to an otherwise equally composed equiosmolar solution that contained 0.274% glucose instead of glucose polymers. No significant differences were detected with any of the tests applied. Our data suggest that glucose polymer solutions are comparatively less suppressive to phagocytic function than currently used glucose-containing PDFs. This effect may be attributed to the low osmolarity of these solutions.

Conventional peritoneal dialysis fluids (PDFs) lead to formation of advanced glycation end-products (AGE) in the peritoneal membrane. In this study, we investigated in vitro the dependence of AGE formation on regular changes of PDFs, as performed during continuous ambulatory peritoneal dialysis (CAPD), and on the contribution of high glucose concentration versus glucose degradation products (GDPs). Under conditions similar to CAPD, protein glycating activity of a conventional single chamber bag PDF (CAPD 4.25%), two double chamber bag PDFs (CAPD Balance 4.25% and CAPD Bicarbonate 4.25%) and a sterile filtered control was measured in vitro by N(epsilon)-(carboxymethyl)lysine (CML) and imidazolones, two well characterized, physiologically relevant AGE structures. Regular changes of PDFs increased AGE formation (CML 3.3-fold and imidazolone 2.6-fold) compared to incubation without changes. AGE formation by CAPD 4.25% was increased compared to control (imidazolones 7.9-fold and CML 3.3-fold) and the use of double chamber bag PDFs led to a decrease of imidazolones by 79% (CAPD Bicarbonate 4.25%) and by 66% (CAPD Balance 4.25%) and to CML contents similar to the control. These results indicate that a major part of AGEs were formed by GDPs in PDFs, whereas only a minor part was due to high glucose concentration. The use of double chamber bag fluids can reduce AGE formation considerably.

BACKGROUND

The peritoneum is impaired by exposure to biocompatible dialysis solutions. Because icodextrin peritoneal dialysis fluid (PDF) is made from cornstarch, a possibility that it induces intraperitoneal inflammation has been reported. In the present study, patients on glucose PDF were switched to icodextrin PDF and then switched back to glucose PDF. Icodextrin PDF-induced intraperitoneal inflammation was investigated based on changes in peritoneal permeability and inflammatory reactions.

METHODS

The subjects were 7 stable peritoneal dialysis patients (4 men, 3 women), with a mean age of 59.1 +/- 3.8 years (range: 55.2 - 64.6 years). The mean duration of peritoneal dialysis was 58.3 +/- 27.4 months (range: 34.3 - 97.7 months), and the cause of end-stage renal disease was chronic glomerulonephritis in all patients. For the overnight dwell, glucose PDF was changed to icodextrin PDF, and the patients returned to glucose PDF 30 months later. To evaluate peritoneal permeability, a peritoneal equilibrium test (PET) was performed, and dialysate-to-plasma (D/P) ratios of creatinine (Cr), beta(2)-microglobulin (beta2M), albumin, immunoglobulin G (IgG), and alpha(2)-macroglobulin (alpha2M) were measured in the overnight dialysate and serum. As markers of inflammation and fibrinolysis or coagulation, interleukin-6 (IL-6) and fibrinogen degradation products (FDPs) were measured in overnight effluent. The evaluations were made every 6 months for 36 months.

RESULTS

A significant elevation in FDP levels was detected in overnight effluent 6 months after the switch to icodextrin PDF, and IL-6 levels tended to increase. The D/P ratios of Cr, beta2M, and albumin were also significantly increased, and the D/P ratios of IgG and alpha2M tended to increase. The D/P ratio of Cr as measured by PET was slightly increased, but the elevation was not significant. In 5 patients, after icodextrin PDF was switched back to glucose PDF at 30 months, the D/P ratios of Cr, beta2M, albumin, IgG, and alpha2M in overnight effluent were significantly reduced. The FDP levels decreased slightly in those patients. In the remaining 2 patients, the D/P ratios of Cr on PET and of Cr, beta2M, albumin, IgG, and alpha2M in overnight effluent, and the FDP and IL-6 levels in overnight effluent were markedly elevated after the switching from glucose to icodextrin PDF, and they remained high after the switch back to glucose PDF. One of these 2 patients developed pre-EPS and was treated with prednisolone and concomitant hemodialysis. The other was switched from peritoneal dialysis to hemodialysis.

CONCLUSIONS

Icodextrin dialysis solution may induce an inflammatory reaction in the peritoneum. Further investigation is necessary for the long-term use of icodextrin PDF.

Peritonitis remains the most important factor in patient morbidity and technical failure associated with continuous ambulatory peritoneal dialysis (CAPD). In vitro examination of bacterial infection of cultured human peritoneal mesothelial cells (HPMC) is an attractive approach to the study of peritonitis in CAPD, yet there are few reports on this subject. Previous studies have shown two limitations: (i) cell cultures of HPMC lasted for days only when incubated in culture medium and (ii) short-term studies of <30 min were done in HPMC when incubated with peritoneal dialysis fluid (PDF). Human peritoneal mesothelial cells, maintained in a conventional single chamber culture system with PDF alone, were unable to survive more than 40 min. The present study was designed to prolong the viability of HPMC cultured in PDF, with the object of using cells under different conditions, such as that of simulating CAPD. HPMC were cultured using plastic microtiter plates, where they were grown to confluence and growth was arrested. PDF containing different concentrations of NaHCO3and human serum albumin was added. Cell viability after exposure for up to 24 h was measured by trypan blue, Cell Death Detection ELISA and Annex-V flow cytometry. The data confirmed the 'toxic' effect of PDF, with cell viability being <40% after 2 h incubation in 4.25% glucose in PDF. However, the survival time of HPMC increased significantly in 4.25% glucose PDF at a physiological pH and even further after the addition of human albumin. These experimental conditions simulating CAPD may allow future in vitro studies of mesothelial physiology and peritonitis related to CAPD treatment.

BACKGROUND

The Portable Document Format (PDF) is the most commonly used file format for online scientific publications. The absence of effective means to extract text from these PDF files in a layout-aware manner presents a significant challenge for developers of biomedical text mining or biocuration informatics systems that use published literature as an information source. In this paper we introduce the 'Layout-Aware PDF Text Extraction' (LA-PDFText) system to facilitate accurate extraction of text from PDF files of research articles for use in text mining applications.

RESULTS

Our paper describes the construction and performance of an open source system that extracts text blocks from PDF-formatted full-text research articles and classifies them into logical units based on rules that characterize specific sections. The LA-PDFText system focuses only on the textual content of the research articles and is meant as a baseline for further experiments into more advanced extraction methods that handle multi-modal content, such as images and graphs. The system works in a three-stage process: (1) Detecting contiguous text blocks using spatial layout processing to locate and identify blocks of contiguous text, (2) Classifying text blocks into rhetorical categories using a rule-based method and (3) Stitching classified text blocks together in the correct order resulting in the extraction of text from section-wise grouped blocks. We show that our system can identify text blocks and classify them into rhetorical categories with Precision1 = 0.96% Recall = 0.89% and F1 = 0.91%. We also present an evaluation of the accuracy of the block detection algorithm used in step 2. Additionally, we have compared the accuracy of the text extracted by LA-PDFText to the text from the Open Access subset of PubMed Central. We then compared this accuracy with that of the text extracted by the PDFPDF. Finally, we discuss preliminary error analysis for our system and identify further areas of improvement.

CONCLUSIONS

LA-PDFText is an open-source tool for accurately extracting text from full-text scientific articles. The release of the system is available at http://code.google.com/p/lapdftext/.

This article has been corrected. To see what has changed, please read the Letter to the Editor and the authors' response. The original version (PDF) is appended to this article as a Supplement.

UNASSIGNED

The Severe Sepsis and Septic Shock Early Management Bundle (SEP-1), the sepsis performance measure introduced in 2015 by the Centers for Medicare & Medicaid Services (CMS), requires the reporting of up to 5 hemodynamic interventions, as many as 141 tasks, and 3 hours to document for a single patient.

UNASSIGNED

To evaluate whether moderate- or high-level evidence shows that use of the 2015 SEP-1 or its hemodynamic interventions improves survival in adults with sepsis.

UNASSIGNED

PubMed, Embase, Scopus, Web of Science, and ClinicalTrials.gov from inception to 28 November 2017 with no language restrictions.

UNASSIGNED

Randomized and observational studies of death among adults with sepsis who received versus those who did not receive either the entire SEP-1 bundle or 1 or more SEP-1 hemodynamic interventions, including serial lactate measurements; a fluid infusion of 30 mL/kg of body weight; and assessment of volume status and tissue perfusion with a focused examination, bedside cardiovascular ultrasonography, or fluid responsiveness testing.

UNASSIGNED

Two investigators independently extracted study data and assessed each study's risk of bias; 4 authors rated level of evidence by consensus using CMS criteria published in 2013. High- or moderate-level evidence required studies to have no confounders and low risk of bias.

UNASSIGNED

Of 56 563 references, 20 studies (18 reports) met inclusion criteria. One single-center observational study reported lower in-hospital mortality after implementation of the SEP-1 bundle. Sixteen studies (2 randomized and 14 observational) reported increased survival with serial lactate measurements or 30-mL/kg fluid infusions. None of the 17 studies were free of confounders or at low risk of bias. In 3 randomized trials, fluid responsiveness testing did not alter survival.

UNASSIGNED

Few trials, poor-quality and confounded studies, and no studies (with survival outcomes) of the focused examination or bedside cardiovascular ultrasonography. Use of the 2015 version of SEP-1 and 2013 version of CMS evidence criteria, both of which were updated in 2017.

UNASSIGNED

No high- or moderate-level evidence shows that SEP-1 or its hemodynamic interventions improve survival in adults with sepsis.

UNASSIGNED

National Institutes of Health. (PROSPERO: CRD42016052716).

Circadian clocks generate 24-hr rhythms in physiology and behavior. Despite numerous studies, it is still uncertain how circadian rhythms emerge from their molecular and neural constituents. Here, we demonstrate a tight connection between the molecular and neuronal circadian networks. Using fluorescent transcriptional reporters in a Drosophila ex vivo brain culture system, we identified a reciprocal negative regulation between the master circadian regulator CLK and expression of pdf, the main circadian neuropeptide. We show that PDF feedback is required for maintaining normal oscillation pattern in CLK-driven transcription. Interestingly, we found that CLK and neuronal firing suppresses pdf transcription, likely through a common pathway involving the transcription factors DHR38 and SR, establishing a direct link between electric activity and the circadian system. In sum, our work provides evidence for the existence of an uncharacterized CLK-PDF feedback loop that tightly wraps together the molecular oscillator with the circadian neuronal network in Drosophila.

Most current microarray oligonucleotide probe design strategies are based on probe design factors (PDFs), which include probe hybridization free energy (PHFE), probe minimum folding energy (PMFE), dimer score, hairpin score, homology score and complexity score. The impact of these PDFs on probe performance was evaluated using four sets of microarray comparative genome hybridization (aCGH) data, which included two array manufacturing methods and the genomes of two species. Since most of the hybridizing DNA is equimolar in CGH data, such data are ideal for testing the general hybridization properties of almost all candidate oligonucleotides. In all our data sets, PDFs related to probe secondary structure (PMFE, hairpin score and dimer score) are the most significant factors linearly correlated with probe hybridization intensities. PHFE, homology and complexity score are correlating significantly with probe specificities, but in a non-linear fashion. We developed a new PDF, pseudo probe binding energy (PPBE), by iteratively fitting dinucleotide positional weights and dinucleotide stacking energies until the average residue sum of squares for the model was minimized. PPBE showed a better correlation with probe sensitivity and a better specificity than all other PDFs, although training data are required to construct a PPBE model prior to designing new oligonucleotide probes. The physical properties that are measured by PPBE are as yet unknown but include a platform-dependent component. A practical way to use these PDFs for probe design is to set cutoff thresholds to filter out bad quality probes. Programs and correlation parameters from this study are freely available to facilitate the design of DNA microarray oligonucleotide probes.

OBJECTIVE

Acute liver failure (ALF) is a critical illness with high mortality. Plasma diafiltration (PDF) is a blood purification therapy that is useful for ALF patients, but it is difficult to use when those patients have multiple organ failure or unstable hemodynamics. In these patients, symptoms are also likely to exacerbate immediately after PDF therapy. We developed continuous PDF (CPDF) as a new concept in PDF therapy, and assessed its efficacy and safety in ALF patients.

METHODS

Ten ALF patients (gender: M/F 6/4, Age: 47 ± 14) were employed CPDF therapy. The primary outcomes were altered liver function, measured by the model for end-stage liver disease (MELD) score, and total bilirubin and prothrombin time international normalized ratios (PT-INR), 5 days after CPDF therapy. Secondary outcomes included sequential organ failure assessment (SOFA) scores, 5 days after CPDF therapy, and the survival rate 14 days after this therapy.

RESULTS

The MELD score (34.5-28.0; P = 0.005), total bilirubin (10.9-7.25 mg/dL; P = 0.048), PT-INR (1.89-1.31; P = 0.084), and SOFA score (10.0-7.5; P < 0.039) were improved 5 days after CPDF therapy. Nine patients were alive, and one patient died because of acute pancreatitis, complicated by ALF. There were no major adverse events related to this therapy under hemodynamic stability.

CONCLUSIONS

In the present study, CPDF therapy safely supported liver function and generally improved the condition of critically ill patients with ALF.

In Drosophila, opsin visual photopigments as well as blue-light-sensitive cryptochrome (CRY) contribute to the synchronization of circadian clocks. We focused on the relatively simple larval brain, with nine clock neurons per hemisphere: five lateral neurons (LNs), four of which express the pigment-dispersing factor (PDF) neuropeptide, and two pairs of dorsal neurons (DN1s and DN2s). CRY is present only in the PDF-expressing LNs and the DN1s. The larval visual organ expresses only two rhodopsins (RH5 and RH6) and projects onto the LNs. We recently showed that PDF signaling is required for light to synchronize the CRY(-) larval DN2s. We now show that, in the absence of functional CRY, synchronization of the DN1s also requires PDF, suggesting that these neurons have no direct connection with the visual system. In contrast, the fifth (PDF(-)) LN does not require the PDF-expressing cells to receive visual system inputs. All clock neurons are light-entrained by light-dark cycles in the rh5(2);cry(b), rh6(1) cry(b), and rh5(2);rh6(1) double mutants, whereas the triple mutant is circadianly blind. Thus, any one of the three photosensitive molecules is sufficient, and there is no other light input for the larval clock. Finally, we show that constant activation of the visual system can suppress molecular oscillations in the four PDF-expressing LNs, whereas, in the adult, this effect of constant light requires CRY. A surprising diversity and specificity of light input combinations thus exists even for this simple clock network.

BACKGROUND

Melatonin has been suggested to have antiproliferative effects on cancer cells. These effects can be attributed to immunomodulation, growth factor inhibition, induction of apoptosis and prooxidant properties. Melatonin is considered as a safe drug with minimal adverse effects.

OBJECTIVE

We planned to investigate the effects of melatonin in hepatoma (Hep G2) cell line. In this study, different concentrations of melatonin were studied to assess its effects on human hepatoma (Hep G2) cell line in vitro.

METHODS

In this study, different doses (5 x 10(-5) M, 5 x 10(-4) M, 10(-3) M) of melatonin were administered into hepatocellular carcinoma cell line in vitro. After an incubation period of 72 hours, the studied and control groups were evaluated for cell cycle, morphology, proliferating index and apoptosis percentage.

RESULTS

A significant decrease in percentage of phase G0/G1 cells was found in high-dose melatonin group (10(-3) M) compared to control group. Melatonin increased the cell counts in S phase of cell cycle at high doses as well. However, phase G2/M cell percentage did not change with the administration of melatonin. Cell proliferation was increased in all melatonin groups, but the only statistically significant difference was found between the high-dose and control groups. There was a significant increase in proliferative index between the control group and high-dose melatonin group.

CONCLUSIONS

High dose of melatonin increases the cell count in S phase and shows an antiproliferative effect on hepatoma cells. This indicates that melatonin can be considered a promising drug when used along with other antineoplastic agents for the treatment of hepatoma.However, it has no effect on apoptosis and colony counts (Tab. 1, Fig. 2, Ref. 19). Full Text (Free, PDF) www.bmj.sk.

The purpose of this note is to evaluate the relationship between the stochastic errors in CT numbers and the standard deviation of the computed proton beam range in radiotherapy planning. The stochastic voxel-to-voxel variation in CT numbers called 'noise,' may be due to signal registration, processing and numerical image reconstruction technique. Noise in CT images may cause a deviation in the computed proton range from the physical proton range, even assuming that the error due to CT number-stopping power calibration is removed. To obtain the probability density function (PDF) of the computed proton range, we have used the continuing slowing down approximation (CSDA) and the uncorrelated white Gaussian noise along the proton path. The model of white noise was accepted because for the slice-based fan-beam CT scanner; the power-spectrum properties apply only to the axial (x, y) domain and the noise is uncorrelated in the z domain. However, the possible influence of the noise power spectrum on the standard deviation of the range should be investigated in the future. A random number generator was utilized for noise simulation and this procedure was iteratively repeated to obtain convergence of range PDF, which approached a Gaussian distribution. We showed that the standard deviation of the range, sigma, increases linearly with the initial proton energy, computational grid size and standard deviation of the voxel values. The 95% confidence interval width of the range PDF, which is defined as 4sigma, may reach 0.6 cm for the initial proton energy of 200 MeV, computational grid 0.25 cm and 5% standard deviation of CT voxel values. Our results show that the range uncertainty due to random errors in CT numbers may be significant and comparable to the uncertainties due to calibration of CT numbers.

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neuromodulator functioning downstream of the insect brain's circadian clock, modulating daily rhythms of locomotor activity. Recently, we found that PDF precursors of the cricket Gryllus bimaculatus comprise a nuclear localization signal (NLS). Moreover, the nuclear localization of PDF immunoreactivity and the translocation of GFP-fused PDF precursor into the nucleus have both been demonstrated. These suggest a fundamental role for PDF peptide in the circadian clock system within the nucleus, in addition to its role in downstream neural events. In the present study, we carried out the cDNA cloning of PDF from adult brains of the last-summer cicada Meimuna opalifera, and found that an isolated clone (545 bp) encodes an ordinary PDF precursor protein. PDF peptide itself shows a high sequence identity (78-94%) and similarity (89-100%) to insect PDFs and also to the crustacean beta-PDH peptides. The computer-assisted sequence analysis of PDF precursor revealed a possible translocation into the nucleus, despite the lack of a definite NLS-like sequence. Using immunocytochemistry, the optic lobes of M. opalifera revealed PDF-immunoreactive neurons in both the medulla and lamina neuropiles. All these PDF cells exhibited prominent immunolabeling of both their perikarya and axons, but not their nuclei. Our results provide the first structural and immunocytochemical identification of PDF neurons in Hemiptera.

The German cockroach, Blattella germanica, and the double-striped cockroach, B. bisignata, are sibling species with a similar period sequence but a distinctive circadian rhythm in locomotion. The cell distribution of immunoreactivity (ir) against three clock-related proteins, Period (PER), Pigment Dispersing Factor (PDF), and Corazonin (CRZ), was compared between the species. The PER-ir cells tend to form clusters and are sprayed out in the central nervous system. Three major PER-ir cells are located in the optic lobes, which are the sites of the major circadian clock. They are interconnected with PER-ir axon bundles. Interestingly, the potential output signal of the circadian clock, PDF, is co-localized with PER in all three groups of cells. However, only two CRZ-ir cells and their axons are found in the optic lobes and they are not co-localized with PER-ir or PDF-ir cells and axons. Since only one circadian rhythm is expressed in locomotion, the time signals from both major clocks in optic lobes are coupled by connection with PDF-ir axons. A group of 3-4 PER-ir cells in the protocerebrum display typical characteristics of neurosecretary cells. In addition, there are numerous, small PER-ir and PDF-ir co-localized cells in the pars intercerebralis (PI), which have direct connections with the neurohemoorgan, corpora cardiaca, through PER-ir and PDF-ir axons. Based on these findings, the cellular connection shows a circadian control through the endocrine route. For the rest of central nervous system, only a few PER-ir and PDF-ir cells or axons are detected. This finding implies the circadian clock for locomotion is not located in subesophageal ganglion, thoracic or abdominal ganglia, but may use other neural messengers to pass on circadian signals. Since the overall distribution pattern of the clock cells are the same for B. germanica and B. bisignata, the possible explanation for the different expressions of locomotion between the species depends on genes downstream of per, pdf, and crz.

Colloidally synthesized quantum dots of CsPbBr3 are highly promising for light-emitting applications. Previous reports based on benchtop diffraction conflict as to the crystal structure of CsPbBr3 quantum dots. We present X-ray diffraction and PDF analysis of X-ray total scattering data that indicate that the crystal structure is unequivocally orthorhombic (Pnma).