A Novel CaM Kinase II Pathway Controls the Location of Neuropeptide Release from Caenorhabditis elegans Motor Neurons

Neurons release neuropeptides via the regulated exocytosis of dense core vesicles (DCVs) to evoke or modulate behaviors. We found that Caenorhabditis elegans motor neurons send most of their DCVs to axons, leaving very few in the cell somas. How neurons maintain this skewed distribution and the extent to which it can be altered to control DCV numbers in axons or to drive release from somas for different behavioral impacts is unknown. Using a forward genetic screen, we identified loss-of-function mutations in UNC-43 (CaM kinase II) that reduce axonal DCV levels by ∼90% and cell soma/dendrite DCV levels by ∼80%, leaving small synaptic vesicles largely unaffected. Blocking regulated secretion in unc-43 mutants restored near wild-type axonal levels of DCVs. Time-lapse video microscopy showed no role for CaM kinase II in the transport of DCVs from cell somas to axons. In vivo secretion assays revealed that much of the missing neuropeptide in unc-43 mutants is secreted via a regulated secretory pathway requiring UNC-31 (CAPS) and UNC-18 (nSec1). DCV cargo levels in unc-43 mutants are similarly low in cell somas and the axon initial segment, indicating that the secretion occurs prior to axonal transport. Genetic pathway analysis suggests that abnormal neuropeptide function contributes to the sluggish basal locomotion rate of unc-43 mutants. These results reveal a novel pathway controlling the location of DCV exocytosis and describe a major new function for CaM kinase II.     

A thiocyanate-forming protein generates multiple products upon allylglucosinolate breakdown in Thlaspi arvense

Glucosinolates, amino acid-derived thioglycosides found in plants of the Brassicales order, are one of the best studied classes of plant secondary metabolites. Together with myrosinases and supplementary proteins known as specifier proteins, they form the glucosinolate–myrosinase system that upon tissue damage gives rise to a number of biologically active glucosinolate breakdown products such as isothiocyanates, epithionitriles and organic thiocyanates involved in plant defense. While isothiocyanates are products of the spontaneous rearrangement of the glucosinolate aglycones released by myrosinase, the formation of epithionitriles and organic thiocyanates depends on both myrosinases and specifier proteins. Hydrolysis product profiles of many glucosinolate-containing plant species indicate the presence of specifier proteins, but only few have been identified and characterized biochemically. Here, we report on cDNA cloning, heterologous expression and characterization of TaTFP, a thiocyanate-forming protein (TFP) from Thlaspi arvense L. (Brassicaceae), that is expressed in all plant organs and can be purified in active form after heterologous expression in Escherichia coli. As a special feature, this protein promotes the formation of allylthiocyanate as well as the corresponding epithionitrile upon myrosinase-catalyzed hydrolysis of allylglucosinolate, the major glucosinolate of T. arvense. All other glucosinolates tested are converted to their simple nitriles when hydrolyzed in the presence of TaTFP. Despite its ability to promote allylthiocyanate formation, TaTFP has a higher amino acid sequence similarity to known epithiospecifier proteins (ESPs) than to Lepidium sativum TFP. However, unlike Arabidopsis thaliana ESP, its activity in vitro is not strictly dependent on Fe²⁺ addition to the assay mixtures. The availability of TaTFP in purified form enables future studies to be aimed at elucidating the structural bases of specifier protein specificities and mechanisms. Furthermore, identification of TaTFP shows that product specificities of specifier proteins can not be predicted based on amino acid sequence similarity and raises interesting questions about specifier protein evolution.     

Recommendations for the Empirical Treatment of Complicated Urinary Tract Infections Using Surveillance Data on Antimicrobial Resistance in the Netherlands

Background

Complicated urinary tract infections (c-UTIs) are among the most common nosocomial infections and a substantial part of the antimicrobial agents used in hospitals is for the treatment of c-UTIs. Data from surveillance can be used to guide the empirical treatment choices of clinicians when treating c-UTIs. We therefore used nation-wide surveillance data to evaluate antimicrobial coverage of agents for the treatment of c-UTI in the Netherlands.

Methods

We included the first isolate per patient of urine samples of hospitalised patients collected by the Infectious Disease Surveillance Information System for Antibiotic Resistance (ISIS-AR) in 2012, and determined the probability of inadequate coverage for antimicrobial agents based on species distribution and susceptibility. Analyses were repeated for various patient groups and hospital settings.

Results

The most prevalent bacteria in 27,922 isolates of 23,357 patients were Escherichia coli (47%), Enterococcus spp. (14%), Proteus mirabilis (8%), and Klebsiella pneumoniae (7%). For all species combined, the probability of inadequate coverage was <5% for amoxicillin or amoxicillin-clavulanic acid combined with gentamicin and the carbapenems. When including gram-negative bacteria only, the probability of inadequate coverage was 4.0%, 2.7%, 2.3% and 1.7%, respectively, for amoxicillin, amoxicillin-clavulanic acid, a second or a third generation cephalosporin in combination with gentamicin, and the carbapenems (0.4%). There were only small variations in results among different patient groups and hospital settings.

Conclusions

When excluding Enterococcus spp., considered as less virulent, and the carbapenems, considered as last-resort drugs, empirical treatment for c-UTI with the best chance of adequate coverage are one of the studied beta-lactam-gentamicin combinations. This study demonstrates the applicability of routine surveillance data for up-to-date clinical practice guidelines on empirical antimicrobial therapy, essential in patient care given the evolving bacterial susceptibility.     

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

Bone morphogenetic protein 4 (BMP4) signaling in retinoblastoma cells

Bone morphogenetic proteins (BMPs) - expressed in the developing retina - are known to be involved in the regulation of cell proliferation and apoptosis in several tumor entities. The objective of this study was to determine the role of the BMP4 pathway in retinoblastoma cells, which are absent in a functional retinoblastoma (RB1) gene. BMP receptors were detected in all retinoblastoma cell lines investigated. A correct transmission of BMP signaling via the Smad1/5/8 pathway could be demonstrated in WERI-Rb1 retinoblastoma cells and application of recombinant human BMP4 resulted in an increase in apoptosis, which to a large extend is caspase independent. Cell proliferation was not affected by BMP4 signaling, although the pRb-related proteins p107 and p130, contributing to the regulation of the same genes, are still expressed. WERI-Rb1 cells exhibit elevated endogenous levels of p21(CIP1) and p53, but we did not detect any increase in p53, p21(CIP1)or p27(KIP1) expression levels. Id proteins became, however, strongly up-regulated upon exogenous BMP4 treatment. Thus, RB1 loss in WERI-Rb1 cells is obviously not compensated for by pRb-independent (e.g. p53-dependent) cell cycle control mechanisms, preventing an anti-proliferative response to BMP4, which normally induces cell cycle arrest.     

Frequent genes in rare diseases: panel‐based next generation sequencing to disclose causal mutations in hereditary neuropathies

Hereditary neuropathies comprise a wide variety of chronic diseases associated to more than 80 genes identified to date. We herein examined 612 index patients with either a Charcot‐Marie‐Tooth phenotype, hereditary sensory neuropathy, familial amyloid neuropathy, or small fiber neuropathy using a customized multigene panel based on the next generation sequencing technique. In 121 cases (19.8%), we identified at least one putative pathogenic mutation. Of these, 54.4% showed an autosomal dominant, 33.9% an autosomal recessive, and 11.6% an X‐linked inheritance. The most frequently affected genes were PMP22 (16.4%), GJB1 (10.7%), MPZ, and SH3TC2 (both 9.9%), and MFN2 (8.3%). We further detected likely or known pathogenic variants in HINT1, HSPB1, NEFL, PRX, IGHMBP2, NDRG1, TTR, EGR2, FIG4, GDAP1, LMNA, LRSAM1, POLG, TRPV4, AARS, BIC2, DHTKD1, FGD4, HK1, INF2, KIF5A, PDK3, REEP1, SBF1, SBF2, SCN9A, and SPTLC2 with a declining frequency. Thirty‐four novel variants were considered likely pathogenic not having previously been described in association with any disorder in the literature. In one patient, two homozygous mutations in HK1 were detected in the multigene panel, but not by whole exome sequencing. A novel missense mutation in KIF5A was considered pathogenic because of the highly compatible phenotype. In one patient, the plasma sphingolipid profile could functionally prove the pathogenicity of a mutation in SPTLC2. One pathogenic mutation in MPZ was identified after being previously missed by Sanger sequencing. We conclude that panel based next generation sequencing is a useful, time‐ and cost‐effective approach to assist clinicians in identifying the correct diagnosis and enable causative treatment considerations.

     

Acid Sphingomyelinase Serum Activity Predicts Mortality in Intensive Care Unit Patients after Systemic Inflammation: A Prospective Cohort Study

Introduction

Acid sphingomyelinase is involved in lipid signalling pathways and regulation of apoptosis by the generation of ceramide and plays an important role during the host response to infectious stimuli. It thus has the potential to be used as a novel diagnostic marker in the management of critically ill patients. The objective of our study was to evaluate acid sphingomyelinase serum activity (ASM) as a diagnostic and prognostic marker in a mixed intensive care unit population before, during, and after systemic inflammation.

Methods

40 patients admitted to the intensive care unit at risk for developing systemic inflammation (defined as systemic inflammatory response syndrome plus a significant procalcitonin [PCT] increase) were included. ASM was analysed on ICU admission, before (PCT_before), during (PCT_peak) and after (PCT_low) onset of SIRS. Patients undergoing elective surgery served as control (N = 8). Receiver-operating characteristics curves were computed.

Results

ASM significantly increased after surgery in the eight control patients. Patients from the intensive care unit had significantly higher ASM on admission than control patients after surgery. 19 out of 40 patients admitted to the intensive care unit developed systemic inflammation and 21 did not, with no differences in ASM between these two groups on admission. In patients with SIRS and PCT peak, ASM between admission and PCT_before was not different, but further increased at PCT_peak in non-survivors and was significantly higher at PCT_low compared to survivors. Survivors exhibited decreased ASM at PCT_peak and PCT_low. Receiver operating curve analysis on discrimination of ICU mortality showed an area under the curve of 0.79 for ASM at PCT_low.

Conclusions

In summary, ASM was generally higher in patients admitted to the intensive care unit compared to patients undergoing uncomplicated surgery. ASM did not indicate onset of systemic inflammation. In contrast to PCT however, it remained high in non-surviving ICU patients after systemic inflammation.     

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Molecular Biology Reports - Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic...     

Assessing maximum depth distribution,vegetated area,and production of submerged macrophytes in shallow,turbid coastal lagoons of the southern Baltic Sea

Submerged macrophytes improve water quality in shallow coastal lagoons but eutrophication often resulted in a degradation of macrophytes. Management measures that protect and restore macrophyte stands require knowledge on what limits macrophyte distribution. Information on macrophyte production and distribution in the Darss-Zingst Bodden Chain (southern Baltic Sea) is lacking since an almost complete loss of submerged vegetation in the 1980s. Nutrient input was reduced in the 1990s and macrophytes seem to recover, although turbidity is high and light conditions are still poor. However, this recovery raised hope that returning macrophytes could stabilize sediments and improve water clarity. In this study, seasonal changes in photosynthesis–irradiance curves of selected macrophyte species were used to calculate potential primary production in different depths and turbidity situations. Bathymetry of the area is then used to assess depth distribution and vegetated area. Since the so-calculated depth limits correspond well with the actual depth distribution in the field, macrophyte depth distribution is concluded to be mostly determined by light conditions. Most macrophytes grow in very shallow areas up to 50 cm depth where also 70% of potential primary production takes place. Present light conditions do not support a further expansion of macrophyte distribution in the DZBC.