Cystatin C, a measure of renal function, as prognostic risk marker in acute heart failure : Studies on the cardiorenal syndrome

Acute heart failure (AHF) is a complex syndrome associated with exceptionally high mortality. Still, characteristics and prognostic factors of contemporary AHF patients have been inadequately studied. Kidney function has emerged as a very powerful prognostic risk factor in cardiovascular disease. This is believed to be the consequence of an interaction between the heart and kidneys, also termed the cardiorenal syndrome, the mechanisms of which are not fully understood. Renal insufficiency is common in heart failure and of particular interest for predicting outcome in AHF. Cystatin C (CysC) is a marker of glomerular filtration rate with properties making it a prospective alternative to the currently used measure creatinine for assessment of renal function. The aim of this thesis is to characterize a representative cohort of patients hospitalized for AHF and to identify risk factors for poor outcome in AHF. In particular, the role of CysC as a marker of renal function is evaluated, including examination of the value of CysC as a predictor of mortality in AHF. The FINN-AKVA (Finnish Acute Heart Failure) study is a national prospective multicenter study conducted to investigate the clinical presentation, aetiology and treatment of, as well as concomitant diseases and outcome in, AHF. Patients hospitalized for AHF were enrolled in the FINN-AKVA study, and mortality was followed for 12 months. The mean age of patients with AHF is 75 years and they frequently have both cardiovascular and non-cardiovascular co-morbidities. The mortality after hospitalization for AHF is high, rising to 27% by 12 months. The present study shows that renal dysfunction is very common in AHF. CysC detects impaired renal function in forty percent of patients. Renal function, measured by CysC, is one of the strongest predictors of mortality independently of other prognostic risk markers, such as age, gender, co-morbidities and systolic blood pressure on admission. Moreover, in patients with normal creatinine values, elevated CysC is associated with a marked increase in mortality. Acute kidney injury, defined as an increase in CysC within 48 hours of hospital admission, occurs in a significant proportion of patients and is associated with increased short- and mid-term mortality. The results suggest that CysC can be used for risk stratification in AHF. Markers of inflammation are elevated both in heart failure and in chronic kidney disease, and inflammation is one of the mechanisms thought to mediate heart-kidney interactions in the cardiorenal syndrome. Inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) correlate very differently to markers of cardiac stress and renal function. In particular, TNF-α showed a robust correlation to CysC, but was not associated with levels of NT-proBNP, a marker of hemodynamic cardiac stress. Compared to CysC, the inflammatory markers were not strongly related to mortality in AHF. In conclusion, patients with AHF are elderly with multiple co-morbidities, and renal dysfunction is very common. CysC demonstrates good diagnostic properties both in identifying impaired renal function and acute kidney injury in patients with AHF. CysC, as a measure of renal function, is also a powerful prognostic marker in AHF. CysC shows promise as a marker for assessment of kidney function and risk stratification in patients hospitalized for AHF.

Dynamic changes in mitogen-activated protein kinase (MAPK) activities in the corpus luteum of the bonnet monkey (Macaca radiata) during development, induced luteolysis, and simulated early pregnancy: A role for p38 MAPK in the regulation of luteal function

Changes in MAPK activities were examined in the corpus luteum (CL) during luteolysis and pregnancy, employing GnRH antagonist (Cetrorelix)-induced luteolysis, stages of CL, and hCG treatment to mimic early pregnancy as model systems in the bonnet monkey. We hypothesized that MAPKs could serve to phosphorylate critical phosphoproteins to regulate luteal function. Analysis of several indices for structural (caspase-3 activity and DNA fragmentation) and functional (progesterone and steroidogenic acute regulatory protein expression) changes in the CL revealed that the decreased luteal function observed during Cetrorelix treatment and late luteal phase was associated with increased caspase-3 activity and DNA fragmentation. As expected, human chorionic gonadotropin treatment dramatically increased luteal function, but the indices for structural changes were only partially attenuated. All three MAPKs appeared to be constitutively active in the mid-luteal-phase CL, and activities of ERK-1/2 and p38-MAPK (p38), but not Jun N-terminal kinase (JNK)-1/2, decreased significantly (P < 0.05) within 12 - 24 h after Cetrorelix treatment. During the late luteal phase, in contrast to decreased ERK-1/2 and p38 activities, JNK-1/2 activities increased significantly (P < 0.05). Although human chorionic gonadotropin treatment increased ERK-1/2 and p38 activities, it decreased JNK-1/2 activities. The activation status of p38 was correlated with the phosphorylation status of an upstream activator, MAPK kinase-3/6 and the expression of MAPK activated protein kinase-3, a downstream target. Intraluteal administration of p38 kinase inhibitor (SB203580), but not MAPK kinase-1/2 inhibitor (PD98059), decreased the luteal function. Together, these data suggest an important role for p38 in the regulation of CL function in primates.

Chronic suppression of testicular function by constant infusion of gonadotropin-releasing hormone agonist and testosterone supplementation in the bonnet monkey (Macaca radiata)

Objective: To study the efficacy of long-term buserelin acetate infusion to desensitize pituitary and block testicular function in adult male monkeys (Macaca radiata). Animals: Proven fertile male monkeys exhibiting normal testicular function. Protocol: Each of the control (n = 5) and experimental monkeys (n = 10) received a fresh miniosmotic pump every 21 days, whereas pumps in controls delivered vehicle of experimentals released 50-mu-g buserelin acetate every 24 hours. On day 170 (renewed every 60 days) a silastic capsule containing crystalline testosterone (T) was implanted in the experimental monkeys. At the end of 3 years, treatment was stopped, and recovery of testicular function and fertility monitored. Results: (1) Treatment resulted in marked reduction of nocturnal but not basal serum T; (2) the pituitary remained desensitized to buserelin acetate throughout the 3-year period; (3) animals were largely azoospermic with occasional oligospermia exhibited by two monkeys; and (4) withdrawal of treatment restored testicular function, with 70% of animals regaining fertility. Conclusion: Long-term infertility (but restorable) can be induced in male monkeys by constant infusion of buserelin acetate and T.

Role of lipid peroxidation in impairment of mitochondrial function at complex I by methyl isocyanate treatment of rats in vivo

The subcutaneous administration of methyl isocyanate (MIC) in 1.0 LD50 dose in rats caused a significant effect on hepatic mitochondrial function only at complex I region of the respiratory chain. MIC administration at 1.0 LD50 dose also resulted in significant increases in malondialdehyde and ferrous ion concentration in liver mitochondria. It is suggested that the augmented lipid peroxidation in hepatic mitochondria, catalyzed by iron, possibly mobilized from intracellular stores leads to the inhibition of enzymes of mitochondrial respiration at complex I region, in vivo, in rats receiving a lethal dose of MIC subcutaneously.

Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies

We report two antibodies, scFv 13B1 and MAb PD1.37, against the hinge regions of LHR and TSHR, respectively, which have similar epitopes but different effects on receptor function. While neither of them affected hormone binding, with marginal effects on hormone response, scFv 13B1 stimulated LHR in a dose-dependent manner, whereas MAb PD1.37 acted as an inverse agonist of TSHR. Moreover, PD1.37 could decrease the basal activity of hinge region CAMs, but had varied effects on those present in ECLs, whereas 13B1 was refractory to any CAMs in LHR. Using truncation mutants and peptide phage display, we compared the differential roles of the hinge region cysteine box-2/3 as well as the exoloops in the activation of these two homologus receptors. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Synthesis and application of weight function for edge cracked semicircular disk (ECSD)

We analyze the utility of edge cracked semicircular disk (ECSD) for rapid assessment of fracture toughness using compressive loading. Continuing our earlier work on ECSD, a theoretical examination here leads to a novel way for synthesizing weight functions using two distinct form factors. The efficacy of ECSD mode-I weight function synthesized using displacement and form factor methods is demonstrated by comparing with finite element results. Theory of elasticity in conjunction with finite element method is utilized to analyze crack opening potency of ECSD under eccentric compression to explore newer configurations of ECSD for fracture testing.

Examining the Effectiveness of Discriminant Function Analysis and Cluster Analysis in Species Identification of Male Field Crickets Based on Their Calling Songs

Traditional taxonomy based on morphology has often failed in accurate species identification owing to the occurrence of cryptic species, which are reproductively isolated but morphologically identical. Molecular data have thus been used to complement morphology in species identification. The sexual advertisement calls in several groups of acoustically communicating animals are species-specific and can thus complement molecular data as non-invasive tools for identification. Several statistical tools and automated identifier algorithms have been used to investigate the efficiency of acoustic signals in species identification. Despite a plethora of such methods, there is a general lack of knowledge regarding the appropriate usage of these methods in specific taxa. In this study, we investigated the performance of two commonly used statistical methods, discriminant function analysis (DFA) and cluster analysis, in identification and classification based on acoustic signals of field cricket species belonging to the subfamily Gryllinae. Using a comparative approach we evaluated the optimal number of species and calling song characteristics for both the methods that lead to most accurate classification and identification. The accuracy of classification using DFA was high and was not affected by the number of taxa used. However, a constraint in using discriminant function analysis is the need for a priori classification of songs. Accuracy of classification using cluster analysis, which does not require a priori knowledge, was maximum for 6-7 taxa and decreased significantly when more than ten taxa were analysed together. We also investigated the efficacy of two novel derived acoustic features in improving the accuracy of identification. Our results show that DFA is a reliable statistical tool for species identification using acoustic signals. Our results also show that cluster analysis of acoustic signals in crickets works effectively for species classification and identification.

Unraveling the Intricate Organization of Mammalian Mitochondrial Presequence Translocases: Existence of Multiple Translocases for Maintenance of Mitochondrial Function

Mitochondria are indispensable organelles implicated in multiple aspects of cellular processes, including tumorigenesis. Heat shock proteins play a critical regulatory role in accurately delivering the nucleus-encoded proteins through membrane-bound presequence translocase (Tim23 complex) machinery. Although altered expression of mammalian presequence translocase components had been previously associated with malignant phenotypes, the overall organization of Tim23 complexes is still unsolved. In this report, we show the existence of three distinct Tim23 complexes, namely, B1, B2, and A, involved in the maintenance of normal mitochondrial function. Our data highlight the importance of Magmas as a regulator of translocase function and in dynamically recruiting the J-proteins DnaJC19 and DnaJC15 to individual translocases. The basic housekeeping function involves translocases B1 and B2 composed of Tim17b isoforms along with DnaJC19, whereas translocase A is nonessential and has a central role in oncogenesis. Translocase B, having a normal import rate, is essential for constitutive mitochondrial functions such as maintenance of electron transport chain complex activity, organellar morphology, iron-sulfur cluster protein biogenesis, and mitochondrial DNA. In contrast, translocase A, though dispensable for housekeeping functions with a comparatively lower import rate, plays a specific role in translocating oncoproteins lacking presequence, leading to reprogrammed mitochondrial functions and hence establishing a possible link between the TIM23 complex and tumorigenicity.

De novo inference of protein function from coarse-grained dynamics

Inference of molecular function of proteins is the fundamental task in the quest for understanding cellular processes. The task is getting increasingly difficult with thousands of new proteins discovered each day. The difficulty arises primarily due to lack of high-throughput experimental technique for assessing protein molecular function, a lacunae that computational approaches are trying hard to fill. The latter too faces a major bottleneck in absence of clear evidence based on evolutionary information. Here we propose a de novo approach to annotate protein molecular function through structural dynamics match for a pair of segments from two dissimilar proteins, which may share even <10% sequence identity. To screen these matches, corresponding 1 mu s coarse-grained (CG) molecular dynamics trajectories were used to compute normalized root-mean-square-fluctuation graphs and select mobile segments, which were, thereafter, matched for all pairs using unweighted three-dimensional autocorrelation vectors. Our in-house custom-built forcefield (FF), extensively validated against dynamics information obtained from experimental nuclear magnetic resonance data, was specifically used to generate the CG dynamics trajectories. The test for correspondence of dynamics-signature of protein segments and function revealed 87% true positive rate and 93.5% true negative rate, on a dataset of 60 experimentally validated proteins, including moonlighting proteins and those with novel functional motifs. A random test against 315 unique fold/function proteins for a negative test gave >99% true recall. A blind prediction on a novel protein appears consistent with additional evidences retrieved therein. This is the first proof-of-principle of generalized use of structural dynamics for inferring protein molecular function leveraging our custom-made CG FF, useful to all. (C) 2014 Wiley Periodicals, Inc.

Transverse single spin asymmetry in e plus p up arrow -> e plus J/psi plus X and Q(2) evolution of Sivers function-II

We present estimates of single spin asymmetry in the electroproduction of J/psi taking into account the transverse momentum-dependent (TMD) evolution of the gluon Sivers function. We estimate single spin asymmetry for JLab, HERMES, COMPASS and eRHIC energies using the color evaporation model of J/psi. We have calculated the asymmetry using recent parameters extracted by Echevarria et al. using the Collins-Soper-Sterman approach to TMD evolution. These recent TMD evolution fits are based on the evolution kernel in which the perturbative part is resummed up to next-to-leading logarithmic accuracy. We have also estimated the asymmetry by using parameters which had been obtained by a fit by Anselmino et al., using both an exact numerical and an approximate analytical solution of the TMD evolution equations. We find that the variation among the different estimates obtained using TMD evolution is much smaller than between these on one hand and the estimates obtained using DGLAP evolution on the other. Even though the use of TMD evolution causes an overall reduction in asymmetries compared to the ones obtained without it, they remain sizable. Overall, upon use of TMD evolution, predictions for asymmetries stabilize.

De-DUFing the DUFs: Deciphering distant evolutionary relationships of Domains of Unknown Function using sensitive homology detection methods

Background: In the post-genomic era where sequences are being determined at a rapid rate, we are highly reliant on computational methods for their tentative biochemical characterization. The Pfam database currently contains 3,786 families corresponding to ``Domains of Unknown Function'' (DUF) or ``Uncharacterized Protein Family'' (UPF), of which 3,087 families have no reported three-dimensional structure, constituting almost one-fourth of the known protein families in search for both structure and function. Results: We applied a `computational structural genomics' approach using five state-of-the-art remote similarity detection methods to detect the relationship between uncharacterized DUFs and domain families of known structures. The association with a structural domain family could serve as a start point in elucidating the function of a DUF. Amongst these five methods, searches in SCOP-NrichD database have been applied for the first time. Predictions were classified into high, medium and low-confidence based on the consensus of results from various approaches and also annotated with enzyme and Gene ontology terms. 614 uncharacterized DUFs could be associated with a known structural domain, of which high confidence predictions, involving at least four methods, were made for 54 families. These structure-function relationships for the 614 DUF families can be accessed on-line at http://proline.biochem.iisc.ernet.in/RHD_DUFS/. For potential enzymes in this set, we assessed their compatibility with the associated fold and performed detailed structural and functional annotation by examining alignments and extent of conservation of functional residues. Detailed discussion is provided for interesting assignments for DUF3050, DUF1636, DUF1572, DUF2092 and DUF659. Conclusions: This study provides insights into the structure and potential function for nearly 20 % of the DUFs. Use of different computational approaches enables us to reliably recognize distant relationships, especially when they converge to a common assignment because the methods are often complementary. We observe that while pointers to the structural domain can offer the right clues to the function of a protein, recognition of its precise functional role is still `non-trivial' with many DUF domains conserving only some of the critical residues. It is not clear whether these are functional vestiges or instances involving alternate substrates and interacting partners. Reviewers: This article was reviewed by Drs Eugene Koonin, Frank Eisenhaber and Srikrishna Subramanian.

Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

BACKGROUND: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. PRINCIPAL FINDINGS: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SIGNIFICANCE: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.