BACKGROUND

The aim of this cohort study was to assess the cumulative effect of marginal donor criteria on initial graft function and patient survival after liver transplantation.

METHODS

We included 734 consecutive patients who underwent orthotopic liver transplantation at the Vienna General Hospital between January 1993 and December 2003. We employed the local registry of the Department of Transplant Surgery, where variables of all patients are routinely and prospectively recorded. Primary outcome was initial graft function, secondary outcome was patient survival.

RESULTS

Cumulative number of marginal donor criteria was significantly and linearly associated with an increased rate of primary dysfunction (PDF; p = 0.005). In patients with more than three cumulative marginal donor criteria the rate of PDF was 36%. Patient survival was not influenced by the cumulative number of donor criteria (log-rank test, p = 0.81). Independent marginal donor criteria to predict PDF were cold ischemia time >10 h [odds ratio (OR) 0.56; 95% CI 0.32-0.98] and donor peak serum sodium >155 mEq/L (OR 0.44; 95% CI 0.26-0.77), as assessed in a multivariate regression model.

CONCLUSIONS

The use of marginal liver donors with more than three marginal donor criteria shows deleterious effects on initial graft function. Noteworthy, patient survival was not associated with marginal donor criteria, which may be explained by early and successful retransplantation of liver recipients with primary non-function.

The neuropeptide pigment-dispersing factor (PDF) has been studied extensively in Drosophila, and its role in circadian time-keeping has been firmly established. The role of PDF outside of the clock circuit, however, is poorly understood. A recent study suggested that PDF may act on the ellipsoid body (EB) to link the clock and sleep/activity circuits. We performed whole brain optical imaging with the fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac1-camps expressed under control of the pdfR promoter to address how the clock and sleep deprivation affect the physiology of these cells. Basal cAMP levels in EB were regulated both by PDF and synaptic inputs that are controlled by the circadian clock. Acute application of PDF to the brain caused a significant, and PDF-receptor-dependent, increase in cAMP in EB cells. Application of TTX to block circuit-mediated effects of PDF increased the morning response but not the response at night, implying the existence of a temporally regulated, PDF-stimulated input that blocks cAMP generation. ACh produced both direct (TTX-insensitive) and indirect (TTX-sensitive) increases in cAMP during the day but was totally TTX-insensitive at night, indicating that ACh-stimulated inputs to the EB are suppressed at night. Sleep deprivation did not affect the cAMP responses of these cells to either PDF or ACh. These results suggest a novel role for PDF as a modulator of activity outside of the clock circuit. By elucidating the mechanisms by which the neuropeptide PDF act on its target cells, our work contributes to our understating of how the central clock coordinates activity and sleep.

BACKGROUND

Severe limitations in biocompatibility of conventional peritoneal dialysis fluids (PDF) can be partially attributed to the presence of glucose degradation products (GDP), which are generated during autoclaving of PDF. Formation of GDP can be significantly reduced by the use of multi-chamber bag systems. Recent clinical studies have revealed increased dialysate levels of pro-collagen I C-terminal peptide (PICP) in patients dialyzed with these solutions. Here, we briefly review the current knowledge on various aspects of GDP toxicity toward peritoneal cells and analyze the impact of GDP on PICP release by human peritoneal mesothelial cells (HPMC) in vitro.

METHODS

HPMC were exposed to a mixture of known GDP added to culture medium at clinically relevant doses. After 12 days, the amount of PICP released was measured using an immunoassay. Furthermore, the protein synthesis was assessed by 3H-proline incorporation in HPMC exposed to peritoneal effluent obtained from patients after three months of CAPD with either conventional PDF or low-GDP solution.

RESULTS

Exposure to GDP resulted in a significant decrease in PICP release by HPMC. In addition, the synthesis of new proteins secreted by HPMC was preserved significantly better in HPMC treated with effluent obtained when patients were dialyzed with low-GDP solutions rather than conventional PDF.

CONCLUSIONS

Exposure to GDP may impair protein synthesis and secretion by HPMC. Therefore, increased dialysate PICP levels in response to GDP-free PDF may be viewed as evidence of improved mesothelial cell function.

The reviews on HIV-1/AIDS [1-8] highlighted the mechanism by which HIV-1 virions utilize dendritic cells (DCs) for transport from the genitals, the portal of virus infection, to the draining lymph nodes where DCs carry HIV-1 virions and present viral antigens by HLA class I and II to CD4(+) T cells. Interaction of the T cells with viral antigens presented by HLA class II molecules polarizes them to become Th2 cells, the targets of HIV-1 infection and producers of HIV-1 progeny virions. The T cells which interact with viral antigen presented by HLA class I polarize to become Th1 cells, which stimulate the CD8(+) T cell precursors to develop into antiviral cytotoxic T cells. In addition, HIV-1 virions shed gp120 glycoprotein molecules which bind to IgE immunoglobulin molecules bound to FCepsilonRI+ innate system cells (basophils, mast cells and monocytes) and induce them to release large amounts of Th2 cytokines (IL-4, IL-5, IL-10, IL-13), thereby creating an allergy-like condition. The present review attempts to define the role of chemokine receptors like CCR5 and CXCR4, and especially fractalkine receptor CX3CR1 in the trafficking of lymphocytes in healthy individuals and HIV-1/AIDS patients. The role of chemokine receptors as co-receptors for HIV-1 virion gp120 glycoprotein has been defined, but the role of fractalkine and fractalkine receptor has been clarified only recently [9-19]. In healthy individuals fractalkine is expressed by blood vessel endothelial cells and the CX3CR1 receptors are expressed on leukocytes that migrate in the peripheral blood in the direction of increased fractalkine concentration. In HIV-1/AIDS patients the virus-infected CD4(+) Th2 cells migrate to organs that harbor the adaptive immune system cells in the thymus, genitals, gastrointestinal tract, and to the brain. A most significant finding which revealed the importance of the human CX3CR1 gene expression to the progression of the infection to the stage of AIDS was recently reported by Faure and collaborators [20, 21] who showed that the delayed or rapid progression to AIDS was affected in HIV-1-infected individuals who had inherited a fractalkine receptor gene with the polymorphisms V249I or T280M, respectively, located in the sixth and seventh transmembrane domains of CX3CR1 protein. The T280M mutation in the CX3CR1 gene caused a rapid progression to AIDS, while in patients with the V249I mutation progression to AIDS was much slower. These studies led to the idea that it might be possible to slow or prevent HIV-1/AIDS progression in HIV-1 patients by treating them with fractalkine antagonists that will bind to and inhibit the activity of the fractalkine receptor. It is hypothesized that treatment of HIV-1/AIDS patients with a combination of fractalkine antagonists, IL-4 antagonist IL-4delta2 and the adjuvant CpG ODN induced release of type I IFN from PDF, and may inhibit HIV-1 infection, especially in HAART-treated patients infected with drug-resistant HIV-1 mutants due to prevention of the availability of immune cells needed for the viral evasion of the immune response. The hypothesis implies that the advantage of the suggested mode of treatment of HIV-1-infected people is prevention of cellular processes that are used by the viral protein to cause immunodeficiency, and prevention of HIV-1 replication without induction of resistant mutants.

BACKGROUND

The Japanese Society of Respiratory Care Medicine and the Japanese Society of Intensive Care Medicine provide here a clinical practice guideline for the management of adult patients with ARDS in the ICU.

METHODS

The guideline was developed applying the GRADE system for performing robust systematic reviews with plausible recommendations. The guideline consists of 13 clinical questions mainly regarding ventilator settings and drug therapies (the last question includes 11 medications that are not approved for clinical use in Japan).

RESULTS

The recommendations for adult patients with ARDS include: we suggest against early tracheostomy (GRADE 2C), we suggest using NPPV for early respiratory management (GRADE 2C), we recommend the use of low tidal volumes at 6-8 mL/kg (GRADE 1B), we suggest setting the plateau pressure at 30cmH20 or less (GRADE2B), we suggest using PEEP within the range of plateau pressures less than or equal to 30cmH2O, without compromising hemodynamics (Grade 2B), and using higher PEEP levels in patients with moderate to severe ARDS (Grade 2B), we suggest using protocolized methods for liberation from mechanical ventilation (Grade 2D), we suggest prone positioning especially in patients with moderate to severe respiratory dysfunction (GRADE 2C), we suggest against the use of high frequency oscillation (GRADE 2C), we suggest the use of neuromuscular blocking agents in patients requiring mechanical ventilation under certain circumstances (GRADE 2B), we suggest fluid restriction in the management of ARDS (GRADE 2A), we do not suggest the use of neutrophil elastase inhibitors (GRADE 2D), we suggest the administration of steroids, equivalent to methylprednisolone 1-2mg/kg/ day (GRADE 2A), and we do not recommend other medications for the treatment of adult patients with ARDS (GRADE1B; inhaled/intravenous β2 stimulants, prostaglandin E1, activated protein C, ketoconazole, and lisofylline, GRADE 1C; inhaled nitric oxide, GRADE 1D; surfactant, GRADE 2B; granulocyte macrophage colony-stimulating factor, N-acetylcysteine, GRADE 2C; Statin.).

CONCLUSIONS

This article was translated from the Japanese version originally published as the ARDS clinical practice guidelines 2016 by the committee of ARDS clinical practice guideline (Tokyo, 2016, 293p, available from http://www.jsicm.org/ARDSGL/ARDSGL2016.pdf). The original article, written for Japanese healthcare providers, provides points of view that are different from those in other countries.

The National Institute for Occupational Safety and Health conducted an initial, task-based comparative assessment to determine the potential for release of carbon nanofibers (CNFs) during dry material handling, wet cutting, grinding, and sanding (by machine and hand) of plastic composite material containing CNFs. Using a combination of direct-reading instruments and filter-based air sampling methods for airborne mass and transmission electron microscopy (TEM), concentrations were measured and characterized near sources of particle generation, in the breathing zone of the workers, and in the general work area. Tasks such as surface grinding of composite material and manually transferring dry CNFs produced substantial increases in particle number concentration (range = 20,000-490,000 1-cm(-3)). Concomitant increases in mass concentration were also associated with most tasks. Nearly 90% of all samples examined via TEM indicated that releases of CNFs do occur and that the potential for exposure exists. These findings also indicate that improperly designed, maintained, or installed engineering controls may not be completely effective in controlling releases. Unprotected skin exposure to CNFs was noted in two instances and indicated the need for educating workers on the need for personal protective equipment. [Supplementary materials are available for this article. Go to the publisher's online edition of Journal of Occupational and Environmental Hygiene for the following free supplemental resource: a PDF file containing information on materials, evaluated processes, personal protective equipment, and existing ventilation/engineering controls.].

We demonstrate the robust scale-invariance in the probability density function (PDF) of detrended healthy human heart rate increments, which is preserved not only in a quiescent condition, but also in a dynamic state where the mean level of the heart rate is dramatically changing. This scale-independent and fractal structure is markedly different from the scale-dependent PDF evolution observed in a turbulentlike, cascade heart rate model. These results strongly support the view that a healthy human heart rate is controlled to converge continually to a critical state.

Clinical heterogeneity within t(12;21) or TEL/AML1-positive ALL (25% of childhood common/preB ALL) indicates that additional genetic changes might contribute to outcome. We studied the relation between additional genetic changes in TEL(ETV6) and AML1(RUNX1) (FISH), drug sensitivity (MTT assay) and clinical outcome in 143 DCOG and COALL-treated t(12;21)-positive ALL patients. Additional genetic changes in TEL and AML1 were present in 83% of the patients, and consisted of (partial) deletion of the second TEL gene (70%), an extra AML1 gene (23%) or an extra der(21)t(12;21) (10%). More than one additional change was observed in 20%. Disease-free survival (pDFS) of DCOG patients without additional genetic changes (4 years pDFS +/- s.e. 53 +/- 17%) and of those with an extra der(21)t(12;21) (60 +/- 22%) is poorer than that of compared to patients with other additional genetic changes in TEL or AML1 (79 +/- 6%; P-trend = 0.02). This was mainly due to the occurrence of early relapses within 2.5 years after the first diagnosis. Similar observations were found in the COALL cohort, albeit not significant owing to limited follow-up. Multivariate analysis including age, WBC and genetic abnormalities in TEL and/or AML1 showed that especially, in vitro resistance to prednisolone (hazard ratio 5.78, 95% CI 1.45-23.0; P=0.01) is an independent prognostic factor in DCOG- and COALL-treated t(12;21)-positive ALL.

MRI significantly improves the accuracy and reliability of target delineation in radiation therapy for certain tumors due to its superior soft tissue contrast compared to CT. A treatment planning process with MRI as the sole imaging modality will eliminate systematic CT/MRI co-registration errors, reduce cost and radiation exposure, and simplify clinical workflow. However, MRI lacks the key electron density information necessary for accurate dose calculation and generating reference images for patient setup. The purpose of this work is to develop a unifying method to derive electron density from standard T1-weighted MRI. We propose to combine both intensity and geometry information into a unifying probabilistic Bayesian framework for electron density mapping. For each voxel, we compute two conditional probability density functions (PDFs) of electron density given its: (1) T1-weighted MRI intensity, and (2) geometry in a reference anatomy, obtained by deformable image registration between the MRI of the atlas and test patient. The two conditional PDFs containing intensity and geometry information are combined into a unifying posterior PDF, whose mean value corresponds to the optimal electron density value under the mean-square error criterion. We evaluated the algorithm's accuracy of electron density mapping and its ability to detect bone in the head for eight patients, using an additional patient as the atlas or template. Mean absolute HU error between the estimated and true CT, as well as receiver operating characteristics for bone detection (HU>> 200) were calculated. The performance was compared with a global intensity approach based on T1 and no density correction (set whole head to water). The proposed technique significantly reduced the errors in electron density estimation, with a mean absolute HU error of 126, compared with 139 for deformable registration (p = 2 × 10(-4)), 283 for the intensity approach (p = 2 × 10(-6)) and 282 without density correction (p = 5 × 10(-6)). For 90% sensitivity in bone detection, the proposed method achieved a specificity of 86%, compared with 80, 11 and 10% using deformable registration, intensity and without density correction, respectively. Notably, the Bayesian approach was more robust against anatomical differences between patients, with a specificity of 62% in the worst case (patient), compared to 30% specificity in registration-based approach. In conclusion, the proposed unifying Bayesian method provides accurate electron density estimation and bone detection from MRI of the head with highly heterogeneous anatomy.

Classification of a given observation to one of three classes is an important task in many decision processes or pattern recognition applications. A general analysis of the performance of three-class classifiers results in a complex 6-D receiver operating characteristic (ROC) space, for which no simple analytical tool exists at present. We investigate the performance of an ideal observer under a specific set of assumptions that reduces the 6-D ROC space to 3-D by constraining the utilities of some of the decisions in the classification task. These assumptions lead to a 3-D ROC space in which the true-positive fraction (TPF) can be expressed in terms of the two types of false-positive fractions (FPFs). We demonstrate that the TPF is uniquely determined by, and therefore is a function of, the two FPFs. The domain of this function is shown to be related to the decision boundaries in the likelihood ratio plane. Based on these properties of the 3-D ROC space, we can define a summary measure, referred to as the normalized volume under the surface (NVUS), that is analogous to the area under the ROC curve (AUC) for a two-class classifier. We further investigate the properties of the 3-D ROC surface and the NVUS for the ideal observer under the condition that the three class distributions are multivariate normal with equal covariance matrices. The probability density functions (pdfs) of the decision variables are shown to follow a bivariate log-normal distribution. By considering these pdfs, we express the TPF in terms of the FPFs, and integrate the TPF over its domain numerically to obtain the NVUS. In addition, we performed a Monte Carlo simulation study, in which the 3-D ROC surface was generated by empirical "optimal" classification of case samples in the multidimensional feature space following the assumed distributions, to obtain an independent estimate of NVUS. The NVUS value obtained by using the analytical pdfs was found to be in good agreemen- t with that obtained from the Monte Carlo simulation study. We also found that, under all conditions studied, the NVUS increased when the difficulty of the classification task was reduced by changing the parameters of the class distributions, thereby exhibiting the properties of a performance metric in analogous to AUC. Our results indicate that, under the conditions that lead to our 3-D ROC analysis, the performance of a three-class classifier may be analyzed by considering the ROC surface, and its accuracy characterized by the NVUS.

Young drivers are over-represented in road injury statistics, partly because they engage in more risky driving than older people. Although it is assumed that younger people have greater risk-propensity, defined as a positive attitude to risk, relevant theory is imprecise and relevant research is clouded by inappropriate measures. 89 participants aged 16-25 and 110 participants aged over 35 were recruited outside motor registries. Participants completed a battery of questionnaires including Rohrmann's [Rohrmann, B. 2004. Risk attitude scales: concepts and questionnaires. Project report. Available at http://www.rohrmannresearch.net/pdfs/rohrmann-racreport.pdf (last accessed 12th February 2008)] measures of risk-aversion, risk-propensity, and risk-related motives for risky driving, as well as measures of risk-perception and risky driving. Compared to older drivers, younger drivers demonstrated lower risk-aversion, and higher propensity for taking accident risks, as well as stronger motives for risky driving in relation to experience-seeking, excitement, sensation-seeking, social influence, prestige-seeking, confidence/familiarity, underestimation of risk, irrelevance of risk, "letting off steam", and "getting there quicker". Further, these variables were associated with risky driving. Some evidence was observed for the possibility that risk propensity moderates the relationship between perceived risk and risky behaviour. These results suggest approaches to targeting the "young driver problem".

BACKGROUND

Previous studies have documented strategies to promote off-label use of drugs using journal publications and other means. Few studies have presented internal company communications that discussed financial reasons for manipulating the scholarly record related to off-label indications. The objective of this study was to build on previous studies to illustrate implementation of a publication strategy by the drug manufacturer for four off-label uses of gabapentin (Neurontin, Pfizer, Inc.): migraine prophylaxis, treatment of bipolar disorders, neuropathic pain, and nociceptive pain.

METHODS

We included in this study internal company documents, email correspondence, memoranda, study protocols and reports that were made publicly available in 2008 as part of litigation brought by consumers and health insurers against Pfizer for fraudulent sales practices in its marketing of gabapentin (see http://pacer.mad.uscourts.gov/dc/cgi-bin/recentops.pl?filename=saris/pdf/ucl%20opinion.pdf for the Court's findings).We reviewed documents pertaining to 20 clinical trials, 12 of which were published. We categorized our observations related to reporting biases and linked them with topics covered in internal documents, that is, deciding what should and should not be published and how to spin the study findings (re-framing study results to explain away unfavorable findings or to emphasize favorable findings); and where and when findings should be published and by whom.

RESULTS

We present extracts from internal company marketing assessments recommending that Pfizer and Parke-Davis (Pfizer acquired Parke-Davis in 2000) adopt a publication strategy to conduct trials and disseminate trial findings for unapproved uses rather than an indication strategy to obtain regulatory approval. We show internal company email correspondence and documents revealing how publication content was influenced and spin was applied; how the company selected where trial findings would be presented or published; how publication of study results was delayed; and the role of ghost authorship.

CONCLUSIONS

Taken together, the extracts we present from internal company documents illustrate implementation of a strategy at odds with unbiased study conduct and dissemination. Our findings suggest that Pfizer and Parke-Davis's publication strategy had the potential to distort the scientific literature, and thus misinform healthcare decision-makers.

Primary dysfunction (PDF) still occurs after orthotopic liver transplantation (OLT). Celsior solution (CS) might offer some advantages over the conventional University of Wisconsin (UW) solution for organ preservation, but to date, this has not been prospectively evaluated in the context of OLT. In this prospective, randomized, multicenter, pilot study, 215 potential liver donors were enrolled and randomized. In 42 cases, the livers were unsuitable for transplantation; therefore, 173 randomized livers ultimately were implanted after perfusion and cold preservation with CS (n = 83) or UW solution (n = 90). In accord with the indications of the CS manufacturing company, total CS infusion volume was 90 mL/kg, greater than that of UW solution (60 mL/kg). The main aim of the study is to compare the prevalence of PDF between the two groups. Donor and recipient variables were similar in the two groups. Episodes of PDF were numerically lower in the CS (2.4%) than UW group (7.8%), but the difference was not statistically significant. There was a trend toward a lesser need for early re-OLT (<30 days) in the CS group (P =.0507), but again, no statistically significant difference emerged. Overall and time-differentiated postoperative deaths also were similar. One-year actuarial patient (UW, 89% v CS, 87%) and graft (UW, 83% v CS, 85%) survival rates were similar. In conclusion, CS was similar to UW solution as a preservation solution in the clinical setting of OLT at the infusion volumes described, although some theoretical advantages of CS composition suggest that CS might prove a valid alternative to UW preservation solution in multiorgan harvesting, including the liver. A study on a larger patient basis is needed.

The T-lineage phenotype in children with acute lymphoblastic leukaemia (ALL) is associated with in vitro drug resistance and a higher relapse-risk compared to a precursor B phenotype. Our study was aimed to investigate whether mutations in the ATM gene occur in childhood T-lineage acute lymphoblastic leukaemia (T-ALL) that are linked to drug resistance and clinical outcome. In all, 20 different single nucleotide substitutions were found in 16 exons of ATM in 62/103 (60%) T-ALL children and 51/99 (52%, P = 0.21) controls. Besides the well-known polymorphism D1853N, five other alterations (S707P, F858L, P1054R, L1472W, Y1475C) in the coding part of ATM were found. These five coding alterations seem to occur more frequently in T-ALL (13%) than controls (5%, P = 0.06), but did not associate with altered expression levels of ATM or in vitro resistance to daunorubicin. However, T-ALL patients carrying these five coding alterations presented with a higher white blood cell count at diagnosis (P = 0.05) and show an increased relapse-risk (5-year probability of disease-free survival (pDFS) = 48%) compared to patients with other alterations or wild-type ATM (5-year pDFS = 76%, P = 0.05). The association between five coding ATM alterations in T-ALL, their germline presence, white blood cell count and unfavourable outcome may point to a role for ATM in the development of T-ALL in these children.

The brain of larval Rhodnius prolixus releases neurohormones with a circadian rhythm, indicating that a clock system exists in the larval brain. Larvae also possess a circadian locomotor rhythm. The present paper is a detailed analysis of the distribution and axonal projections of circadian clock cells in the brain of the fifth larval instar. Clock cells are identified as neurons that exhibit circadian cycling of both PER and TIM proteins. A group of eight lateral clock neurons (LNs) in the proximal optic lobe also contain pigment-dispersing factor (PDF) throughout their axons, enabling their detailed projections to be traced. LNs project to the accessory medulla and thence laterally toward the compound eye and medially into a massive area of arborizations in the anterior protocerebrum. Fine branches radiate from this area to most of the protocerebrum. A second group of clock cells (dorsal neurons [DNs]), situated in the posterior dorsal protocerebrum, are devoid of PDF. The DNs receive two fine axons from the LNs, indicating that clock cells throughout the brain are integrated into a timing network. Two axons of the LNs cross the midline, presumably coordinating the clock networks of left and right sides. The neuroarchitecture of this timing system is much more elaborate than any previously described for a larval insect and is very similar to those described in adult insects. This is the first report that an insect timing system regulates rhythmicity in both the endocrine system and behavior, implying extensive functional parallels with the mammalian suprachiasmatic nucleus.

We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the β-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA). The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment. Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively. The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose. According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 μm when the bacterial growth is inhibited by the CEZ treatment. In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly. There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria. Thus, the drug resistance of GNB to β-lactam antibiotics can be rapidly assessed. Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method. Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria.

Past studies have implicated the Drosophila LARK protein in the circadian control of adult eclosion behavior. LARK has a broad tissue pattern of distribution, and is pan-neuronal in the differentiated brain. In certain peptidergic neurons, LARK abundance changes in a circadian manner. However, the precise cellular requirement for LARK, with respect to circadian behavior, is still not known. To explore this issue, we employed the GAL4/UAS binary expression system to increase LARK abundance in defined neuronal cell types. Interestingly, LARK expression in Crustacean Cardioactive Peptide (CCAP) neurons caused an early-eclosion phenotype, whereas a similar perturbation in the Eclosion Hormone (EH) cells resulted in abnormally late peaks of eclosion. Surprisingly, LARK expression in Pigment Dispersing Factor (PDF)- or TIMELESS (TIM)-containing clock neurons caused behavioral arrhythmicity, even though clock protein cycling was found to be normal in these flies. Although the observed effects of LARK expression mirrored those seen with genetic ablation of the relevant peptidergic populations, there was no evidence of defective cell development or morphology. This suggests that an alteration of cell function rather than cell death is the cause of the aberrant phenotypes. Diminished PDF immunoreactivity in flies expressing LARK in the PDF neurons suggests that an effect on neuropeptide synthesis, transport, or release may contribute to the observed arrhythmicity. Importantly, the expression of LARK in several other cell populations did not have detectable effects on development, viability or behavior, indicating a specificity of action within certain cell types.

In the Drosophila brain, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is expressed in the small and large Lateral ventral neurons (LNvs) and regulates circadian locomotor behavior. Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections. Despite the relevance of this phenomenon to circuit plasticity and behavior, the underlying mechanisms remain poorly understood. In this work we provide evidence that PDF along with matrix metalloproteinases (Mmp1 and 2) are key in the control of circadian structural remodeling. Adult-specific downregulation of PDF levels per se hampers circadian axonal remodeling, as it does altering Mmp1 or Mmp2 levels within PDF neurons post-developmentally. However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior. In vitro analysis demonstrated that PDF is hydrolyzed by Mmp1, thereby suggesting that Mmp1 could directly terminate its biological activity. These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.

The first article of this series showed that it was possible to express mathematically the phenomenon of cicatrization. The principal point consisted in determining by means of an equation, a constant, or index, characterizing each wound. The calculation had to be made for each patient for each wound, and required two observations, 4 days apart. The index having proved to be a continuous function of the size of the wound and of the age of the patient, of the form See PDF for Equation where S is the area, i the index, a a decimal exponent, and K a constant, it was then possible to draw a chart by means of which this index i could be obtained without calculation. The advantage of the new way of determining the index is, above all, that this index is a general, average, normal index, and no longer an individual index. Hence, the differences between the observed rate of cicatrization of man and the normal rate may give some indication of the general state of the patient. Another advantage is that the determination of the index is no longer controlled by the temporary accidents which may happen between the two observations of S and S'.

Reptation theory is a highly successful approach for describing polymer dynamics in entangled systems. In turn, this molecular process is the basis of viscoelasticity. We apply a modified version of reptation dynamics to develop an actual physical model of ion channel gating. We show that at times longer than microseconds these dynamics predict an alpha-helix-screw motion for the amphipathic protein segment that partially lines the channel pore. Such motion has been implicated in several molecular mechanics studies of both voltage-gated and transmitter-gated channels. The experimental probability density function (pdf) for this process follows t-3/2 which has been observed in several experimental systems. Reptation theory predicts that channel gating will occur on the millisecond time scale and this is consistent with experimental results from single-channel recording. We examine the consequences of reptation over random barriers and we show that, to first order, the pdf remains unchanged. In the case of a charged helix undergoing reptation in the presence of a transmembrane potential we show that the tail of the pdf will be exponential. We provide a list of practical experimental predictions to test the validity of this physical theory.

With approximately 7000 species, snakes and lizards, collectively known as squamates, are by far the most species-rich group of reptiles. It was from reptile-like ancestors that mammals and birds evolved and squamates can be viewed as phylogenetically positioned between them and fishes. Hence, their hearts have been studied for more than a century yielding insights into the group itself and into the independent evolution of the fully divided four-chambered hearts of mammals and birds. Structurally the heart is complex and debates persist on rudimentary issues such as identifying structures critical to understanding ventricle function. In seeking to resolve these controversies we have generated three-dimensional (3D) models in portable digital format (pdf) of the anaconda and anole lizard hearts ('typical' squamate hearts) and the uniquely specialized python heart with comprehensive annotations of structures and cavities. We review the anatomy and physiology of squamate hearts in general and emphasize the unique features of pythonid and varanid lizard hearts that endow them with mammal-like blood pressures. Excluding pythons and varanid lizards it is concluded that the squamate heart has a highly consistent design including a disproportionately large right side (systemic venous) probably due to prevailing pulmonary bypass (intraventricular shunting). Unfortunately, investigations on rudimentary features are sparse. We therefore point out gaps in our knowledge, such as the size and functional importance of the coronary vasculature and of the first cardiac chamber, the sinus venosus, and highlight areas with implications for vertebrate cardiac evolution.

Fragile X syndrome (FXS), caused by loss of fragile X mental retardation 1 (FMR1) gene function, is the most common heritable cause of intellectual disability and autism spectrum disorders. The FMR1 product (FMRP) is an RNA-binding protein best established to function in activity-dependent modulation of synaptic connections. In the Drosophila FXS disease model, loss of functionally-conserved dFMRP causes synaptic overgrowth and overelaboration in pigment dispersing factor (PDF) peptidergic neurons in the adult brain. Here, we identify a very different component of PDF neuron misregulation in dfmr1 mutants: the aberrant retention of normally developmentally-transient PDF tritocerebral (PDF-TRI) neurons. In wild-type animals, PDF-TRI neurons in the central brain undergo programmed cell death and complete, processive clearance within days of eclosion. In the absence of dFMRP, a defective apoptotic program leads to constitutive maintenance of these peptidergic neurons. We tested whether this apoptotic defect is circuit-specific by examining crustacean cardioactive peptide (CCAP) and bursicon circuits, which are similarly developmentally-transient and normally eliminated immediately post-eclosion. In dfmr1 null mutants, CCAP/bursicon neurons also exhibit significantly delayed clearance dynamics, but are subsequently eliminated from the nervous system, in contrast to the fully persistent PDF-TRI neurons. Thus, the requirement of dFMRP for the retention of transitory peptidergic neurons shows evident circuit specificity. The novel defect of impaired apoptosis and aberrant neuron persistence in the Drosophila FXS model suggests an entirely new level of "pruning" dysfunction may contribute to the FXS disease state.

In the fruit fly Drosophila melanogaster, social interactions especially among heterosexual couples have been shown to have significant impact on the circadian timing system. Olfaction plays a major role in such interactions; however, we do not know yet specifically which receptor(s) are involved. Further, the role of circadian clock neurons in the rhythmic regulation of such sociosexual interactions (SSIs) is not fully understood. Here, we report the results of our study in which we assayed the locomotor activity and sleep-wake behaviors of male-male (MM), female-female (FF), and male-female (MF) couples from several wild-type and mutant strains of Drosophila with an aim to identify specific olfactory receptor(s) and circadian clock neurons involved in the rhythmic regulation of SSI. The results indicate that Or47b receptor neurons are necessary for SSI, as ablation or silencing of these neurons has a severe impact on SSI. Further, the neuropeptide pigment dispersing factor (PDF) and PDF-positive ventral lateral (LN(v)) clock neurons appear to be dispensable for the regulation of SSI; however, dorsal neurons may be involved.

Peptide deformylase (PDF), a metallohydrolase essential for bacterial growth, is an attractive target for use in the discovery of novel antibiotics. Focused chelator-based chemical libraries were constructed and screened for inhibition of enzymatic activity, inhibition of Staphylococcus aureus growth, and cytotoxicity. Positive compounds were selected based on the results of all three assays. VRC3375 [N-hydroxy-3-R-butyl-3-(2-S-(tert-butoxycarbonyl)-pyrrolidin-1-ylcarbonyl)propionamide] was identified as having the most favorable properties through an integrated combinatorial and medicinal chemistry effort. This compound is a potent PDF inhibitor with a K(i) of 0.24 nM against the Escherichia coli Ni(2+) enzyme, possesses activity against gram-positive and gram-negative bacterial pathogens, and has a low cytotoxicity. Mechanistic experiments demonstrate that the compound inhibits bacterial growth through PDF inhibition. Pharmacokinetic studies of this drug in mice indicate that VRC3375 is orally bioavailable and rapidly distributed among various tissues. VRC3375 has in vivo activity against S. aureus in a murine septicemia model, with 50% effective doses of 32, 17, and 21 mg/kg of body weight after dosing by intravenous (i.v.), subcutaneous (s.c.), and oral (p.o.) administration, respectively. In murine single-dose toxicity studies, no adverse effects were observed after dosing with more than 400 mg of VRC3375 per kg by i.v., p.o., or s.c. administration. The in vivo efficacy and low toxicity of VRC3375 suggest the potential for developing this class of compounds to be used in future antibacterial drugs.