BACKGROUND

The interest of clinical research in aging males increased in recent years and thereby the interest to measure health-related quality of life (HRQoL) and symptoms of aging men. The Aging Males' Symptoms scale (AMS) became the most commonly used scale to measure HRQoL and symptoms in aging males in many countries worldwide. The aim of this paper is to review the current state of the instrument particularly concerning versions of the scale in different languages in the light of the quality of the translation process.

UNASSIGNED

Most of the translations were performed following international methodological recommendations for linguistic & cultural adaptation of HRQoL instruments. Mainly the English version was used as source language for the translation into Dutch, Spanish, Portuguese, Italian, Swedish, and Japanese (attached as additional PDF-files). Preliminary versions that were derived only from forward translations are of secondary quality and available in Finnish, Flemish, and Russian. It is recommended to complete the translation process for the latter languages before using them in international studies.

UNASSIGNED

The AMS scale is in the process of consensus finding of two existing French versions, and the versions in the Korean, Thai, and Indonesian languages have not yet been completed in the translation process.

CONCLUSIONS

The AMS scale is obviously a valuable tool for assessing health related quality of life in aging men, because it is used worldwide. It is a standardized scale according to psychometric norms. Most of the currently available language versions were translated following international standards for linguistic and cultural translation of quality of life scales. Assistance is offered to help interested parties in the translation process.

Molecular oscillations that underlie the circadian clock are coupled to different output signals by which daily rhythms in downstream events are evoked and/or synchronized. Here the authors review the literature that describes circadian output mechanisms in Drosophila. They begin at the most proximal level, within oscillator cells themselves, by surveying studies of rhythmic gene expression within Drosophila heads. Next the authors describe the several neuron groups that compose the circadian pacemaker network underlying rhythmic locomotor activity, and they detail current models of how that network is organized and coordinated. The authors outline the body of evidence that describes a role for the neuropeptide pigment dispersing factor (PDF) as a circadian transmitter in the fly brain. Finally, in the context of PDF, they consider studies that address mechanisms of signaling from the circadian pacemaker network to downstream neurons and nonneuronal cells that directly control rhythmic outputs.

The human Ether-à-go-go related gene (hERG) potassium channel is responsible for the rapid delayed rectifier potassium current that plays a critical role in the repolarization of cardiomyocytes during the cardiac action potential. In humans, inhibition of hERG by drugs can prolong the electrocardiographic QT interval, which, in rare instance, leads to ventricular arrhythmia and sudden cardiac death. As such, several medications that block hERG channels in vitro have been withdrawn from the market due to QT prolongation and arrhythmias. The current FDA guidelines recommend that drug candidates destined for human use be evaluated for potential hERG activity ( www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm074963.pdf ). Here, we employed automated planar patch clamp (APPC), high-throughput fluorescent Tl(+) flux, and moderate-throughput [³H]dofetilide competition binding assays to characterize a panel of 49 drugs for their activities at the hERG channel. Notably, we used the same HEK293-hERG cell line for all assays, facilitating comparisons of hERG potencies across screening platforms. In general, hERG inhibitors were most potent in APPC assays, intermediate potent in [³H]dofetilide binding assays, and least potent in Tl(+) flux assays. Binding affinity constants (pK(i) values) and Tl(+) flux potencies (pEC₅₀ values) correlated well with APPC pEC₅₀ values. Further, the inhibitory potencies of many known hERG inhibitors in APPC matched literature values from manual and/or automated patch clamp systems. We also developed a novel fluorescent Tl(+) flux assays to measure the effects of drugs that modulate hERG trafficking and surface expression.

Bioincompatibility of conventional glucose-based peritoneal dialysis fluids (PDF) has been partially attributed to the presence of glucose degradation products (GDP) generated during heat sterilization of PDF. Most previous studies on GDP toxicity were performed on animal and/or transformed cell lines, and the impact of GDP on peritoneal cells remains obscure. The short-term effects of six identified GDP on human peritoneal mesothelial cell (HPMC) functions were examined in comparison to murine L929 fibroblasts. Exposure of HPMC to acetaldehyde, formaldehyde, glyoxal, methylglyoxal, furaldehyde, but not to 5-hydroxymethyl-furfural, resulted in dose-dependent inhibition of cell growth, viability, and interleukin-1beta (IL-1beta)-stimulated IL-6 release; for several GDP, this suppression was significantly greater compared with L929 cells. Although the addition of GDP to culture medium at concentrations found in PDF had no major impact on HPMC function, the exposure of HPMC to filter-sterilized PDF led to a significantly smaller suppression of HPMC proliferation compared to that induced by heat-sterilized PDF. The growth inhibition mediated by filter-sterilized PDF could be increased after the addition of clinically relevant doses of GDP. These effects were equally evident in L929 cells. In conclusion, GDP reveal a significant cytotoxic potential toward HPMC that may be underestimated in test systems using L929 cells. GDP-related toxicity appears to be particularly evident in experimental systems using proliferating cells and the milieu of dialysis fluids. Thus, these observations may bear biologic relevance in vivo where HPMC are repeatedly exposed to GDP-containing PDF for extended periods of time.

The secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1,25D) has been shown to regulate the growth and differentiation of human prostate cancer (PCa) cells, although the precise molecular mechanisms mediating these effects have not been defined. Previous studies in our laboratory demonstrated that the antiproliferative effects of 1,25D on PCa cells are mediated through the nuclear vitamin D receptor (VDR). In the present study, we performed gene profiling of LNCaP human PCa cells following 1,25D treatment and identified the antitumorigenic gene, prostate derived factor (PDF), as being highly induced by 1,25D. PDF is a member of the TGF-beta superfamily and has been implicated in a variety of functions directly related totumorigenicity including antiproliferative and pro-apoptotic effects. Gene expression studies using 1,25D analogs and a VDR antagonist demonstrate that 1,25D-mediated induction of PDF message and protein in PCa cells is dependent on VDR action. PDF is a transcriptional target of the tumor suppressor, p53. Here we show that the expression of PDF in nine PCa cell lines is dependent on functional p53. Additionally, transfection of p53-null ALVA-31 PCa cells with a p53 expression plasmid, and expression of dominant negative p53 in LNCaP PCa cells, show that the ability of VDR to induce PDF requires functional p53. Importantly, forced PDF expression in PC-3 cells results in decreased cell proliferation, soft agar cloning, and xenograft tumor size. These data demonstrate that PDF exerts antitumorigenic properties on PCa cells and its regulation by 1,25D may provide insights into the action of 1,25D in PCa.

Substantial progress has been made in elucidating the molecular processes that impart a temporal control to physiology and behavior in most eukaryotes. In Drosophila, dorsal and ventral neuronal networks act in concert to convey rhythmicity. Recently, the hierarchical organization among the different circadian clusters has been addressed, but how molecular oscillations translate into rhythmic behavior remains unclear. The small ventral lateral neurons can synchronize certain dorsal oscillators likely through the release of pigment dispersing factor (PDF), a neuropeptide central to the control of rhythmic rest-activity cycles. In the present study, we have taken advantage of flies exhibiting a distinctive arrhythmic phenotype due to mutation of the potassium channel slowpoke (slo) to examine the relevance of specific neuronal populations involved in the circadian control of behavior. We show that altered neuronal function associated with the null mutation specifically impaired PDF accumulation in the dorsal protocerebrum and, in turn, desynchronized molecular oscillations in the dorsal clusters. However, molecular oscillations in the small ventral lateral neurons are properly running in the null mutant, indicating that slo is acting downstream of these core pacemaker cells, most likely in the output pathway. Surprisingly, disrupted PDF signaling by slo dysfunction directly affects the structure of the underlying circuit. Our observations demonstrate that subtle structural changes within the circadian network are responsible for behavioral arrhythmicity.

OBJECTIVE

The purpose of this paper is to provide a physical description of the amorphous state for pharmaceutical materials and to investigate the pharmaceutical implications. Techniques to elucidate structural differences in pharmaceutical solids exhibiting characteristic X-ray amorphous powder patterns are also presented.

METHODS

The X-ray amorphous powder diffraction patterns of microcrystalline cellulose, indomethacin, and piroxicam were measured with laboratory XRPD instrumentation. Analysis of the data were carried out using a combination of direct methods, such as pair distribution functions (PDF), and indirect material modeling techniques including Rietveld, total scattering, and amorphous packing.

RESULTS

The observation of X-ray amorphous powder patterns may indicate the presence of amorphous, glassy or disordered nanocrystalline material in the sample. Rietveld modeling of microcrystalline cellulose (Avicel PH102) indicates that it is predominantly disordered crystalline cellulose Form Ibeta with some amorphous contribution. The average crystallite size of the disordered nanocrystalline cellulose was determined to be 10.9 nm. Total scattering modeling of ground samples of alpha, gamma, and delta crystal forms of indomethacin in combination with analysis of the PDFs provided a quantitative picture of the local structure during various stages of grinding. For all three polymorphs, with increased grinding time, a two-phase system, consisting of amorphous and crystalline material, continually transformed to a completely random close packed (RCP) amorphous structure. The same pattern of transformation was detected for the Form I polymorph of piroxicam. However, grinding of Form II of piroxicam initially produced a disordered phase that maintained the local packing of Form II but over a very short nanometer length scale. The initial disordered phase is consistent with continuous random network (CRN) glass material. This initial disordered phase was maintained to a critical point when a transition to a completely amorphous RCP structure occurred.

CONCLUSIONS

Treating X-ray amorphous powder patterns with different solid-state models, ranging from disordered nanocrystalline to glassy and amorphous, resulted in the assignment of structures in each of the systems examined. The pharmaceutical implications with respect to the stability of the solid are discussed.

Here, we report the identification, cloning, and functional characterization of three Caenorhabditis elegans G protein-coupled pigment dispersing factor (PDF) receptors, which we designated as Ce_PDFR-1a, -b, and -c. They represent three splice isoforms of the same gene (C13B9.4), which share a high degree of similarity with the Drosophila PDF receptor and are distantly related to the mammalian vasoactive intestinal peptide receptors (VPAC2) and calcitonin receptors. In a reverse pharmacological screen, three bioactive C. elegans neuropeptides, which were recently identified as the Drosophila PDF orthologues, were able to activate these receptors in a dose-dependent manner with nanomolar potency (isoforms a and b). Integrated green fluorescent protein reporter constructs reveal the expression of these PDF receptors in all body wall muscle cells and many head and tail neurons involved in the integration of environmental stimuli and the control of locomotion. Using a custom data analysis system, we demonstrate the involvement of this newly discovered neuropeptide signaling system in the regulation of locomotor behavior. Overexpression of PDF-2 phenocopies the locomotor defects of a PDF-1 null mutant, suggesting that they elicit opposite effects on locomotion through the identified PDF receptors. Our findings strengthen the hypothesis that the PDF signaling system, which imposes the circadian clock rhythm on behavior in Drosophila, has been functionally conserved throughout the protostomian evolutionary lineage.

Social cues contribute to the circadian entrainment of physiological and behavioral rhythms. These cues supplement the influence of daily and seasonal cycles in light and temperature. In Drosophila, the social environment modulates circadian mechanisms that regulate sex pheromone production and mating behavior. Here we demonstrate that a neuroendocrine pathway, defined by the neuropeptide Pigment-Dispersing Factor (PDF), couples the CNS to the physiological output of peripheral clock cells that produce pheromones, the oenocytes. PDF signaling from the CNS modulates the phase of the oenocyte clock. Despite its requirement for sustaining free-running locomoter activity rhythms, PDF is not necessary to sustain molecular rhythms in the oenocytes. Interestingly, disruption of the PDF signaling pathway reduces male sex pheromones and results in sex-specific differences in mating behavior. Our findings highlight the role of neuropeptide signaling and the circadian system in synchronizing the physiological and behavioral processes that govern social interactions.

Different subsets of Drosophila melanogaster's clock neurons are characterized by their specific functions in daily locomotor rhythms and the differences in their neurotransmitter composition. We investigated the function of the neuropeptide F (NPF) immunoreactive clock neurons in the rhythmic locomotor behavior of adult flies. We newly identified the fifth s-LN(v) and a subset of the l-LN(v)s as NPF-positive in addition to the three LN(d)s that have been described previously. We then selectively ablated different subsets of NPF-expressing neurons using npfGal4-targeted expression of the cell death gene head involution defective (hid) in combination with cryGal80 and pdfGal80. By analyzing daily locomotor rhythms in these flies, we show that the NPF-positive clock neurons--especially the fifth s-LN(v) and the LN(d)s--are involved in both the control of the free-running period in constant darkness (DD) and the phasing and amplitude of the evening activity in light-dark (LD) cycles. Furthermore, we show that the simultaneous ablation of NPF and pigment dispersing factor (PDF)-immunoreactive neurons has additive effects in LD, resulting in an evening peak phase that is even more advanced in comparison to PDF-ablated flies. We also found that this more advanced evening peak is additionally reduced in amplitude. To putatively assign the observed phenotypes to the action of NPF, we knocked it down in conjunction with PDF using RNA-interference (RNAi) and further suggest a possible role for NPF in the control of the flies' evening activity.

The role of the central neuropeptide pigment-dispersing factor (PDF) in circadian timekeeping in Drosophila is remarkably similar to that of vasoactive intestinal peptide (VIP) in mammals. Like VIP, PDF is expressed outside the circadian network by neurons innervating the gut, but the function and mode of action of this PDF have not been characterized. Here we investigate the visceral roles of PDF by adapting cellular and physiological methods to the study of visceral responses to PDF signaling in wild-type and mutant genetic backgrounds. We find that intestinal PDF acts at a distance on the renal system, where it regulates ureter contractions. We show that PdfR, PDF's established receptor, is expressed by the muscles of the excretory system, and present evidence that PdfR-induced cAMP increases underlie the myotropic effects of PDF. These findings extend the similarities between PDF and VIP beyond their shared central role as circadian regulators, and uncover an unexpected endocrine mode of myotropic action for an intestinal neuropeptide on the renal system.

Three metallo forms of peptide deformylase (PDF, EC 3.5.1.31) of Escherichia coli were prepared and crystallized (space group C2, diffraction limit 1.9 A) for initiating the X-ray structure determination of the metal center in correlation with the catalytic functionality of this enzyme. The native Fe2+ containing enzyme species was directly isolated from overproducing bacteria by using catalase as a buffer additive, which stabilizes the catalytic activity against oxidative destruction. The Ni2+ containing form, which is oxygen-insensitive, was obtained by metal exchange with free Ni2+ and found to be catalytically equally effective (kcat/KM = 10(5) M-1 s-1 for N-formyl-Met-Ala). The Zn2+ form, prepared from the apoenzyme or by displacement of bound Ni2+ by free Zn2+, proved virtually inactive.

Daily biological rhythms in both prokaryotes and eukaryotes are controlled by circadian clocks. In Drosophila, there is a good basic understanding of both the molecular and anatomical components of the clock. In this study we directly measure, for the first time, electrophysiological properties and anatomy of individual filled large lateral PDF-positive clock neurons, a cell group believed to be involved in synchronization of the clock in constant conditions. We find that the large PDF-positive neurons are morphologically homogeneous and that their resting membrane potential is modulated both by the clock and by light inputs. Expression of a leak channel, dORK-deltaC, which has been shown to disrupt circadian locomotor rhythms, hyperpolarizes these neurons, and blocks firing. These data imply that the firing properties of large PDF neurons are both regulated by and critical for clock function.

Low-molecular-weight beta-sulfonyl- and beta-sulfinylhydroxamic acid derivatives have been synthesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most of the compounds synthesized and tested displayed antibacterial activities that cover several pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these compounds as antibacterial agents is discussed with respect to selectivity, intracellular concentrations in bacteria, and potential for resistance development.

Analysis of experiments aimed at understanding the genetic mechanisms of differentiation and growth of the heart, calls for detailed insights into cardiac growth and proliferation rate of myocytes and their precursors. Such insights in mouse heart development are currently lacking. We quantitatively assessed the 3D patterns of proliferation in the forming mouse heart and in the adjacent splanchnic mesoderm, from the onset of heart formation till the developed heart at late gestation. These results are presented in an interactive portable document format (Suppl. PDF) to facilitate communication and understanding. We show that the mouse splanchnic mesoderm is highly proliferative, and that the proliferation rate drops upon recruitment of cells into the cardiac lineage. Concomitantly, the proliferation rate locally increases at the sites of chamber formation, generating a regionalized proliferation pattern. Quantitative analysis shows a gradual decrease in proliferation rate of the ventricular walls with progression of development, and a base-to-top decline in proliferation rate in the trabecules. Our data offers clear insights into the growth and morphogenesis of the mouse heart and shows that in early development the phases of tube formation and chamber formation overlap. The resulting interactive quantitative 3D atlas of cardiac growth and morphogenesis provides a resource for interpretation of mechanistic studies.

Coronavirus disease 2019 (COVID-19) is a novel respiratory illness caused by SARS-CoV-2. Viral entry is mediated through viral spike protein and host ACE2 enzyme interaction. Most cases are mild; severe disease often involves cytokine storm and organ failure. Therapeutics including antivirals, immunomodulators, and vaccines are in development. To view this SnapShot, open or download the PDF.

OBJECTIVE

The aims of this study was to investigate the effects of visual and auditory cues on the freezing of gait in Parkinson disease patients (PDF) compared with Parkinson disease patients without freezing of gait (PDNF).

METHODS

Fifteen PDF, 10 PDNF, and 10 age-matched healthy volunteers were recruited. Subjects walked back and forth on a 7-m walkway under three different conditions: baseline condition without cues, with visual cues, and with auditory cues. Visual cues consisted of white stripes located along the walkway. For auditory cues, a metronome was used. Gait was analyzed using three-dimensional computerized analysis.

RESULTS

In the PDF group, both visual and auditory cues significantly affected visuospatial and kinematic gait parameters. PDF group benefited more from visual cues than auditory cues. In the PDNF and healthy volunteer groups, visual cues significantly decreased patient velocity. Auditory cues affected some kinematic parameters on PDNF group. Compared among three groups, visual cues more positively affected the PDF group, and auditory cues more positively affected kinematic parameters in the PDNF group.

CONCLUSIONS

This study suggests that gait training using visual and auditory cues can improve PDF patient gait and that auditory cues enhance gait in PDNF patients with hypokinetic gait patterns.

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. Here we studied the rhythmic behavior of neural mutants with modified arborizations of the large PDF neurons. In sine oculis(1) (so(1)) mutants we found a higher density of PDF fibers in the fly's pacemaker center, the accessory medulla. These flies exhibited a significantly longer period (24.6 h) than control flies. When PDF levels were elevated to very high levels in the dorsal brain as true for so(mda) mutants and small optic lobes;so(1) double mutants (sol(1);so( 1)), a short-period component split off the long period in behavioral rhythmicity. The short period became shorter the higher the amount of PDF in this brain region and reached a value of approximately 21 h. The period alterations were clearly dependent on PDF, because so(1);Pdf 01 and so(mda);Pdf 01 double mutants showed a single free-running component with a period similar to Pdf 01 mutants (approximately 22.5 h) and significantly longer than the short period of so(mda) mutants. These observations indicate that PDF feeds back on the clock neurons and changes their period. Obviously, PDF lengthens the period of some clock neurons and shortens that of others.

BACKGROUND

In recent years, laboratory and clinical research has suggested the need for peritoneal dialysis fluids (PDFs) that are more biocompatible than the conventional PDFs commonly used today. Bioincompatibility of PDF has been attributed to low pH, lactate, glucose, glucose degradation products (GDPs), and osmolality. PDFs with neutral pH and low GDPs are now available commercially. In vitro and early clinical studies suggest that these solutions are indeed more biocompatible but, as of now, there is no evidence that their use improves patient outcome.

METHODS

Using a dedicated database of over 2000 patients treated with PD in Korea, we were able to conduct a retrospective observational study comparing outcomes for incident continuous ambulatory PD patients treated with a standard, conventional, heat-sterilized PDF to the outcomes for patients treated with a novel, low GDP, neutral-pH PDF prepared in a dual-compartment, double-bag PD system (Balance; Fresenius Medical Care, St. Wendel, Germany). In an intention-to-treat analysis, patient and technique survival, peritonitis-free survival, and peritonitis rates were compared in 611 patients treated with Balance for up to 30 months and 551 patients with a standard PDF (stay . safe; Fresenius Medical Care) treated in the same era and with equivalent follow-up.

RESULTS

The patients were well matched for most relevant characteristics except older age distribution for the patients treated with the standard PDF. Patients treated with Balance had significantly superior survival compared to those treated with the standard PDF (74% vs 62% at 28 months, p = 0.0032). In a multivariate Cox regression model including age, diabetes, and gender, the survival advantage persisted (relative risk of death for Balance 0.75, 95% confidence interval 0.56 - 0.99, p = 0.0465). Modality technique survival was similarin Kaplan-Meieranalysis for both PDFs. No differences were detected in peritonitis-free survival or in peritonitis rates between the two solutions.

CONCLUSIONS

This study, for the first time, suggests that treatment with a novel biocompatible PDF with low GDP concentration and neutral pH confers a significant survival advantage. The exact mechanisms for such a survival advantage cannot be determined from this study. The usual criticisms of observational studies apply and the results reported here strongly warrant the undertaking of appropriately designed, randomized, controlled clinical trials.

To gain insight into regulatory mechanisms of tissue-specific Corazonin (Crz) gene expression and its functions in Drosophila, we cloned the Crz genes from four Drosophila species (D. melanogaster, D. simulans, D. erecta, and D. virilis) and performed comparative analyses of Crz gene sequences and expression patterns using in situ hybridization and immunohistochemistry. Although Crz gene sequences showed a great deal of diversity, its expression patterns in the CNS were highly conserved in the Drosophila species examined here. In D. melanogaster larva, Crz expression was found in four pairs of neurons per cerebral lobe and in eight pairs of bilateral neurons in the ventral nerve cord; in adult, the number of Crz-producing neurons increased to 6-8 in the pars lateralis of each brain lobe, whereas neurons in the ventral nerve cord were no longer detectable. Crz transcripts were also found in the optic lobes; however, these mRNAs do not seem to be translated. Such adult-like Crz expression patterns were established within 48 hours after pupation. Somata of Crz-neurons in the pars lateralis are located in the vicinity of terminals emanating from PDF-containing pacemaking neurons, indicating a functional connection between the two peptidergic nervous systems. A subset of Crz neurons coexpressed the period clock gene; however, normal Crz transcription was unaffected by central clockworks. Two pairs of ectopic Crz cells were detected in the adult brains of behaviorally arrhythmic Clock(Jrk) or cycle(02) mutants, suggesting that CLOCK and CYCLE proteins negatively regulate Crz transcription in a cell-specific manner.

A two-step solid-state reaction for preparing cobalt molybdenum nitride with a nanoscale morphology has been used to produce a highly active and stable electrocatalyst for the hydrogen evolution reaction (HER) under acidic conditions that achieves an iR-corrected current density of 10 mA cm(-2) at -0.20 V vs RHE at low catalyst loadings of 0.24 mg/cm(2) in rotating disk experiments under a H2 atmosphere. Neutron powder diffraction and pair distribution function (PDF) studies have been used to overcome the insensitivity of X-ray diffraction data to different transition-metal nitride structural polytypes and show that this cobalt molybdenum nitride crystallizes in space group P63/mmc with lattice parameters of a = 2.85176(2) Å and c = 10.9862(3) Å and a formula of Co0.6Mo1.4N2. This space group results from the four-layered stacking sequence of a mixed close-packed structure with alternating layers of transition metals in octahedral and trigonal prismatic coordination and is a structure type for which HER activity has not previously been reported. Based on the accurate bond distances obtained from time-of-flight neutron diffraction data, it is determined that the octahedral sites contain a mixture of divalent Co and trivalent Mo, while the trigonal prismatic sites contain Mo in a higher oxidation state. X-ray photoelectron spectroscopy (XPS) studies confirm that at the sample surface nitrogen is present and N-H moieties are abundant.

Chlamydia trachomatis is an obligate intracellular bacterium responsible for a number of human diseases. The mechanism underlying the intracellular parasitology of Chlamydiae remains poorly understood. In searching for host factors required for chlamydial infection, we discovered that C. trachomatis growth was effectively inhibited with GM6001 and TAPI-0, two compounds known as specific inhibitors of matrix metalloproteases. The inhibition was independent of chlamydial entry of the cell, suggesting that the loss of extracellular metalloprotease activities of the host cell is unlikely to be the mechanism for the growth suppression. Nucleotide sequences of candidate metalloprotease genes remained unchanged in a chlamydial variant designated GR10, which had been selected for resistance to the inhibitors. Nevertheless, GR10 displayed a single base mutation in the presumable promoter region of the gene for peptide deformylase (PDF), a metal-dependent enzyme that removes the N-formyl group from newly synthesized bacterial proteins. The mutation correlated with an increased PDF expression level and resistance to actinonin, a known PDF inhibitor with antibacterial activity, as compared with the parental strain. Recombinant chlamydial PDF was covalently labeled with a hydroxamate-based molecular probe designated AspR1, which was developed for the detection of metalloproteases. The AspR1 labeling of the chlamydial PDF became significantly less efficient in the presence of excessive amounts of GM6001 and TAPI-0. Finally, the PDF enzyme activity was efficiently inhibited with GM6001 and TAPI-0. Taken together, our results suggest that the metalloprotease inhibitors suppress chlamydial growth by targeting the bacterial PDF. These findings have important biochemical and medical implications.

BACKGROUND

In early postpartum haemorrhage (PPH), a low concentration of fibrinogen is associated with excessive subsequent bleeding and blood transfusion. We hypothesized that pre-emptive treatment with fibrinogen concentrate reduces the need for red blood cell (RBC) transfusion in patients with PPH.

METHODS

In this investigator-initiated, multicentre, double-blinded, parallel randomized controlled trial, we assigned subjects with severe PPH to a single dose of fibrinogen concentrate or placebo (saline). A dose of 2 g or equivalent was given to all subjects independent of body weight and the fibrinogen concentration at inclusion. The primary outcome was RBC transfusion up to 6 weeks postpartum. Secondary outcomes were total blood loss, total amount of blood transfused, occurrence of rebleeding, haemoglobin <58 g litre(-1), RBC transfusion within 4 h, 24 h, and 7 days, and as a composite outcome of 'severe PPH', defined as a decrease in haemoglobin of >40 g litre(-1), transfusion of at least 4 units of RBCs, haemostatic intervention (angiographic embolization, surgical arterial ligation, or hysterectomy), or maternal death.

RESULTS

Of the 249 randomized subjects, 123 of 124 in the fibrinogen group and 121 of 125 in the placebo group were included in the intention-to-treat analysis. At inclusion the subjects had severe PPH, with a mean blood loss of 1459 (sd 476) ml and a mean fibrinogen concentration of 4.5 (sd 1.2) g litre(-1). The intervention group received a mean dose of 26 mg kg(-1) fibrinogen concentrate, thereby significantly increasing fibrinogen concentration compared with placebo by 0.40 g litre(-1) (95% confidence interval, 0.15-0.65; P=0.002). Postpartum blood transfusion occurred in 25 (20%) of the fibrinogen group and 26 (22%) of the placebo group (relative risk, 0.95; 95% confidence interval, 0.58-1.54; P=0.88). We found no difference in any predefined secondary outcomes, per-protocol analyses, or adjusted analyses. No thromboembolic events were detected.

CONCLUSIONS

We found no evidence for the use of 2 g fibrinogen concentrate as pre-emptive treatment for severe PPH in patients with normofibrinogenaemia.

BACKGROUND

ClinicalTrials.gov: http://clinicaltrials.gov/show/NCT01359878. Published protocol: http://www.trialsjournal.com/content/pdf/1745-6215-13-110.pdf.

1. The variation of the experimental values (R (omega)), (C (omega)) of the resistance and capacity of blood for increasing frequencies is approximately represented by the equation: See PDF for Equation in which R(o) and C(o) are the resistance and capacity of the blood at low frequency and See PDF for Equation is the resistance of the blood at infinite frequency. Formulae (1) and (2) are derived by considering the blood as equivalent to the system shown in the diagram (a) of Fig. 1. 2. By the application of formula (1) to our experimental data the value of R(infinity) can be extrapolated with high accuracy. R(infinity) represents the resistance) which would have been obtained at low frequency, if the membranes around the corpuscles could have been removed. 3. The specific resistance of the corpuscle interior can be calculated by equation (5), using experimental values for R(infinity), for the volume concentration of the blood and for the specific resistance of the serum. 4. The specific resistance of the interior of the red corpuscle of the calf is found to be 3.5 +/- 10 per cent times the specific resistance of the serum.