Production of the Streptomyces scabies coronafacoyl phytotoxins involves a novel biosynthetic pathway with an F420‐dependent oxidoreductase and a short‐chain dehydrogenase/reductase

Coronafacoyl phytotoxins are secondary metabolites that are produced by various phytopathogenic bacteria, including several pathovars of the Gram‐negative bacterium Pseudomonas syringae as well as the Gram‐positive potato scab pathogen Streptomyces scabies. The phytotoxins are composed of the polyketide coronafacic acid (CFA) linked via an amide bond to amino acids or amino acid derivatives, and their biosynthesis involves the cfa and cfa‐like gene clusters that are found in P. syringae and S. scabies, respectively. The S. scabies cfa‐like gene cluster was previously reported to contain several genes that are absent from the P. syringae cfa gene cluster, including one (oxr) encoding a putative F₄₂₀—dependent oxidoreductase, and another (sdr) encoding a predicted short‐chain dehydrogenase/reductase. Using gene deletion analysis, we demonstrated that both oxr and sdr are required for normal production of the S. scabies coronafacoyl phytotoxins, and structural analysis of metabolites that accumulated in the Δsdr mutant cultures revealed that Sdr is directly involved in the biosynthesis of the CFA moiety. Our results suggest that S. scabies and P. syringae use distinct biosynthetic pathways for producing coronafacoyl phytotoxins, which are important mediators of host‐pathogen interactions in various plant pathosystems.     

Proteolytic digestion of histones indicates that the nucleosomes of nuclei and of sheared chromatin are similar

Calf thymus nuclei were treated with trypsin, chymotrypsin or Pronase, and the rate of digestion of the various histone fractions was determined. The results differed from those obtained by digestion of DNA-free histones with the same set of enzymes but were identical to those obtained by digestion of calf thymus chromatin. Because these enzymes have such different specificities, the results of these digestions indicate that the histone fractions have similar locations in the chromosomal substructures of nuclei and chromatin, $\text{i.e.}$ that the structure of the nucleosomes which exist within nuclei is not changed markedly when chromatin is isolated from nuclei by a method which involves shearing.     

Measurement of 25-hydroxyvitamin D-1 alpha-hydroxylase activity in mammalian kidney

Until recently measurement of 25-OH-D3-1 alpha-hydroxylase activity in mammalian kidney has not been possible due to the presence of a protein which inhibits the enzyme by reducing available substrate. However, utilization of sufficient unlabeled 25-OH-D3 (80 nmol/ml renal homogenate) to overcome the effect of the inhibitor while maintaining optimal concentration for 1-hydroxylation has made quantitation of enzyme activity possible. We have modified this existing technique in order to increase the sensitivity and to permit detailed study of 1 alpha-hydroxylate regulation in mouse kidney. The modifications that we have incorporated include (i) simplifying the purification scheme for obtaining measurable 1,25-(OH)2D3 by reducing to one the necessary number of high-performance liquid chromatography steps and (ii) quantifying 1,25-(OH)2D3 by radioligand assay. The sensitivity of the assay is 10 pg, which, corrected for fractionation and recovery (50-60%), allows the measurement of 0.5 fmol 1,25-(OH)2D3 produced per milligram kidney per minute. Moreover, reliability and precision of the assay have been confirmed by demonstrating that samples from carefully matched, identically treated mice have reproducible enzyme activity (interassay coefficient of variation = 9.1%, n = 5) and show appropriate dilution characteristics. We have also demonstrated appropriate modulation of enzyme activity by known effectors of 1-hydroxylation. Kidneys from D-deficient mice exhibit significantly higher enzyme activity (15.28 +/- 1.17, n = 21) than do normal mouse kidneys (5.14 +/- 0.26, n = 33). In contrast, enzyme activity is suppressed significantly in kidneys obtained from calcium-loaded (1.20 +/- 0.04, n = 5) and parathyroidectomized animals (2.94 +/- 0.29, n = 5). Our assay now permits the indepth study of 1 alpha-hydroxylase regulation in mammalian (mouse) kidneys.     

A Systematic Review of Childhood Maltreatment Assessments in Population-Representative Surveys Since 1990

BackgroundPopulation-representative surveys that assess childhood maltreatment and health are a valuable resource to explore the implications of child maltreatment for population health. Systematic identification and evaluation of such surveys is needed to facilitate optimal use of their data and to inform future research.ObjectivesTo inform researchers of the existence and nature of population-representative surveys relevant to understanding links between childhood maltreatment and health; to evaluate the assessment of childhood maltreatment in this body of work.MethodsWe included surveys that: 1) were representative of the non-institutionalized population of any size nation or of any geopolitical region ≥ 10 million people; 2) included a broad age range (≥ 40 years); 3) measured health; 4) assessed childhood maltreatment retrospectively; and 5) were conducted since 1990. We used Internet and database searching (including CINAHL, Embase, ERIC, Global Health, MEDLINE, PsycINFO, Scopus, Social Policy and Practice: January 1990 to March 2014), expert consultation, and other means to identify surveys and associated documentation. Translations of non-English survey content were verified by fluent readers of survey languages. We developed checklists to abstract and evaluate childhood maltreatment content.ResultsFifty-four surveys from 39 countries met inclusion criteria. Sample sizes ranged from 1,287-51,945 and response rates from 15%-96%. Thirteen surveys assessed neglect, 15 emotional abuse; 18 exposure to family violence; 26 physical abuse; 48 sexual abuse. Fourteen surveys assessed more than three types; six of these were conducted since 2010. In nine surveys childhood maltreatment assessments were detailed (+10 items for at least one type of maltreatment). Seven surveys’ assessments had known reliability and/or validity.

Conclusions and Implications

Data from 54 surveys can be used to explore the population health relevance of child maltreatment. Assessment of childhood maltreatment is not comprehensive but there is evidence of recent improvement.     

Phospholipid Ether Analogs for the Detection of Colorectal Tumors

The treatment of localized colorectal cancer (CRC) depends on resection of the primary tumor with adequate margins and sufficient lymph node sampling. A novel imaging agent that accumulates in CRCs and the associated lymph nodes is needed. Cellectar Biosciences has developed a phospholipid ether analog platform that is both diagnostic and therapeutic. CLR1502 is a near-infrared fluorescent molecule, whereas ^124/131I-CLR1404 is under clinical investigation as a PET tracer/therapeutic agent imaged by SPECT. We investigated the use of CLR1502 for the detection of intestinal cancers in a murine model and ¹³¹I-CLR1404 in a patient with metastatic CRC. Mice that develop multiple intestinal tumors ranging from adenomas to locally advanced adenocarcinomas were utilized. After 96 hours post CLR1502 injection, the intestinal tumors were analyzed using a Spectrum IVIS (Perkin Elmer) and a Fluobeam (Fluoptics). The intensity of the fluorescent signal was correlated with the histological characteristics for each tumor. Colon adenocarcinomas demonstrated increased accumulation of CLR1502 compared to non-invasive lesions (total radiant efficiency: 1.76×10¹⁰ vs 3.27×10⁹ respectively, p = 0.006). Metastatic mesenteric tumors and uninvolved lymph nodes were detected with CLR1502. In addition, SPECT imaging with ¹³¹I-CLR1404 was performed as part of a clinical trial in patients with advanced solid tumors. ¹³¹I-CLR1404 was shown to accumulate in metastatic tumors in a patient with colorectal adenocarcinoma. Together, these compounds might enhance our ability to properly resect CRCs through better localization of the primary tumor and improved lymph node identification as well as detect distant disease.     

Calpastatin is upregulated in non-immune neuronal cells via toll-like receptor 2 (TLR2) pathways by lipid-containing agonists

Calpain (intracellular Ca^2 +-dependent protease) and calpastatin (calpain specific endogenous inhibitor) are widely distributed in biological systems, and have been implicated in many cellular physiological and pathological processes. Calpastatin level is of central importance to the control of calpain activity. We demonstrated for the first time that calpastatin is overexpressed in mycoplasma-contaminated cultured cells (SH-SY5Y cells that are infected by a strain of Mycoplasma hyorhinis (NDMh)). We have found that the calpastatin-upregulating activity resides in the mycoplasmal membrane lipoproteins, and is associated with NF-κB activation. Calpain-promoted proteolysis is attenuated in the NDMh lipoprotein-treated cells. Here we show that the NDMh lipoproteins promoted an increase in calpastatin in SH-SY5Y cells via the TLR2/TAK1/NF-κB pathway. The synthetic mycoplasmal lipopeptide MALP-2 and the bacterial lipopeptide PAM3CSK4 (TLR2 agonists) also promoted calpastatin upregulation. LPS (TLR4 agonist) activated NF-κB without calpastatin increase in the cell. In contrast, lipoteichoic acid (TLR2 agonist) upregulated calpastatin not via NF-κB activation, but via the MEK1/ELK1 pathway. Zymosan and peptidoglycan, TLR2 agonists that lack lipids, did not induce calpastatin upregulation. Cell treatment with a calpastatin-upregulating agonist (lipoteichoic acid) led to the attenuation of Ca^2 +-promoted calpain activity, whereas agonists that do not upregulate calpastatin (LPS, Zymosan) were ineffective. Overall, the results indicate that in these non-immune cells, calpastatin is upregulated by TLR2-agonists containing lipids, with more than one downstream pathway involved. Such agonists may be useful for studying mechanisms and factors involved in calpastatin regulation. In addition, suitable TLR2 agonists may be of interest in devising treatments for pathological processes involving excessive calpain activation.