Temperature-dependent development and oviposition models of Illeis koebelei (Coleoptera: Coccinellidae)

The yellow ladybeetle, Illeis koebelei Timberlake, is a potential biological agent for powdery mildew. The objective of this study was to construct development and oviposition models of I. koebelei. Development experiment was conducted at eight temperatures ranging from 15.4 to 39.5?°C. Development rates were well fitted with linear and nonlinear models. Lower developmental thresholds for egg, first instar, second instar, third instar, and fourth instar larva, pre-pupa, pupa, and total immature stage were estimated to be 3.6, 12.7, 12.1, 11.3, 11.3, 12.8, 14.7, and 14.2?°C, respectively. Their respective thermal requirements in degree days (DD) were 86.6, 16.0, 22.5, 30.2, 49.3, 14.5, 43.8 and 217.4, respectively. Survivorship was the highest at 25.1?°C for immatures. Oviposition experiment was conducted at nine temperatures, ranging from 15.4 to 35.3?°C. Mean fecundity ranged from 18.6 eggs at 29.3?°C to 205.3 eggs at 20.3?°C. It was well described by extreme value function. Adult survival and cumulative oviposition rates of I. koebelei were fitted to a sigmoid function and a two-parameter Weibull function, respectively. Findings of this study provide basic information for ecology of I. koebelei. They can be used to optimize environmental conditions for mass-rearing and shipping, comparing optimal occurrence conditions between I. koebelei and powdery mildew, and forecasting phenology and population dynamics of I. koebelei in the fields.     

A rule governing the FtsH-mediated proteolysis of the MgtC virulence protein from <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

A tightly controlled turnover of membrane proteins is required for lipid bilayer stability, cell metabolism, and cell viability. Among the energy-dependent AAA⁺ proteases in Salmonella, FtsH is the only membrane-bound protease that contributes to the quality control of membrane proteins. FtsH preferentially degrades the C-terminus or N-terminus of misfolded, misassembled, or damaged proteins to maintain physiological functions. We found that FtsH hydrolyzes the Salmonella MgtC virulence protein when we substitute the MgtC 226^th Trp, which is well conserved in other intracellular pathogens and normally protects MgtC from the FtsH-mediated proteolysis. Here we investigate a rule determining the FtsH-mediated proteolysis of the MgtC protein at Trp226 residue. Substitution of MgtC tryptophan 226^th residue to alanine, glycine, or tyrosine leads to MgtC proteolysis in a manner dependent on the FtsH protease whereas substitution to phenylalanine, methionine, isoleucine, leucine, or valine resists MgtC degradation by FtsH. These data indicate that a large and hydrophobic side chain at 226^th residue is required for protection from the FtsH-mediated MgtC proteolysis.     

Temperature-dependent development and oviposition models of Leptocorisa chinensis Dallas (Hemiptera: Alydidae)

Leptocorisa chinensis Dallas (Hemiptera: Alydidae) is known to cause pecky rice by sucking panicles of milk stage of rice. Based on its continuous spread and expanded damage area in Asian countries such as Japan with high reproduction potential, it is highly likely that L. chinensis will become an important rice pest in the near future. However, limited information is available to predict its distribution and occurrence. Thus, the objective of this study was to develop models for their development and oviposition. We investigated the development of L. chinensis immatures (from egg to adult) at 11 constant temperatures ranging from 16.2 to 35.3 °C and the oviposition of female adults at five constant temperatures ranging from 22.3 to 35.3 °C in this study. For L. chinensis immatures, the lower developmental threshold temperature, optimal developmental temperature, upper developmental threshold temperature were 12.7, 32.3, and 37.6 °C, respectively. The highest survival rate of immatures was observed at temperature of 25.2 °C and the highest mean total fecundity was 585.8 at 28.0 °C. This study provides basic information for the ecology of L. chinensis. It is applicable to forecast the phenology of its populations in the fields and to predict its future distribution under global warming.     

A novel RUNX2 mutation in exon 8, G462X,in a patient with Cleidocranial Dysplasia

To identify a novel mutation of Runx2 gene in Cleidocranial Dysplasia (CCD) patients and to characterize the functional consequences of this mutation. The subjects consisted of 12 Korean CCD patients. After oral epithelial cells were collected using a mouthwash technique, genomic DNA was extracted. Screening for Runx2 mutation was performed using direct sequencing of polymerase chain reaction (PCR) products for exons 1‐8. Restriction fragment length polymorphism (RFLP) analysis was performed to confirm the novel mutation. For functional studies, we performed luciferase assay for Runx2 transacting activity, cyclohexamide chase assay for Runx2 protein stability, real‐time PCR for mRNA level of Runx2 downstream bone marker genes, and alkaline phosphatase (ALP) staining assay in mesenchymal stem cells for osteoblast differentiation. Of the 12 patients, seven showed Runx2 mutations reported previously and four showed no mutation. A novel mutation, G462X in exon 8, which was located in the C‐terminus of proline/serine/threonine‐rich (PST) domain, was found in one patient. In the luciferase assay, Runx2 transacting activity was decreased in Runx2‐G462X transfected cells. In the cyclohexamide chase assay, Runx2‐G462X mutation reduced the stability of Runx2 protein. Expression of the bone marker genes (osteocalcin, ALP, Type I collagen αI, matrix metalloproteinase‐13, bone sialoprotein, and osteopontin) decreased in G462X‐transfected cells. In the ALP staining assay, osteoblast differentiation was reduced in Runx2‐G462X overexpressed cell. The G462X mutation might reduce the Runx2 transacting activity, lower the protein stability, downgrade the expression of bone marker genes, and eventually diminish osteoblast differentiation in CCD patients.     

Analysis of the free fatty acid metabolome in the plasma of patients with systemic lupus erythematosus and fever

Introduction

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease with heterogeneous clinical manifestations mediated by immune dysregulation.

Objectives

We aimed to analyze the metabolomic differences in free fatty acids (FFAs) between patients with SLE and healthy controls (HCs).

Methods

In this study, the levels of 24 FFAs, as their tert-butyldimethylsilyl derivatives, in the plasma of 41 patients with SLE and 41 HCs, were investigated using gas chromatography with mass spectrometry in selected-ion monitoring mode.

Results

The results showed that patients with SLE and HCs had significantly different levels of 13 of the 24 FFAs. The levels of myristic, palmitoleic, oleic, and eicosenoic acids were significantly higher, whereas the levels of caproic, caprylic, linoleic, stearic, arachidonic, eicosanoic, behenic, lignoceric, and hexacosanoic acids were significantly lower in patients with SLE, than in the HCs. In the partial-correlation analysis of the FFA profiles and markers of disease activity of SLE, several metabolic markers correlated with SLE disease activity.

Conclusions

Our results provide a comprehensive understanding of the relationship between FFAs and markers of SLE disease activity. Thus, this approach has promising potential for the discovery of metabolic biomarkers of SLE.

     

Association of Periodontitis with the Concentration Levels of Germanium and Tin in Hair

The purpose of this study was to investigate whether or not the concentration levels of certain kinds of trace elements in hair are associated with periodontitis. We studied a total of 109 participants, which are composed of 25 participants with periodontitis and 84 participants without periodontitis. Periodontal conditions were assessed by measuring the periodontal clinical attachment loss and pocket depth, which were determined at six sites of all teeth. Periodontitis was defined according to the criteria of periodontitis proposed by CDC-AAP. The hair samples were washed with acetone, water, and extran (1%v/v), and then aliquots of hair samples were wet-ashed. This sample solution was analyzed by Perkin-Elmer Mass Spectrometer. The odds ratios and 95% confidence intervals of the concentration levels of trace elements for periodontitis were calculated by multivariate logistic regression analysis. After adjusting all confounders, it was found that the higher concentration level of germanium in hair was significantly and positively associated with periodontitis (odds ratio [OR] 7.12; 95% confidential interval [CI] 2.03–25.00). The higher concentration level of tin in hair was significantly and negatively associated with periodontitis (OR 0.27; 95% CI 0.08–0.94). It was concluded that there was a significant relationship between periodontitis and the concentration level of germanium and tin in hair.     

Spermidine/spermine N(1)-acetyltransferase-1 binds to hypoxia-inducible factor-1alpha (HIF-1alpha) and RACK1 and promotes ubiquitination and degradation of HIF-1alpha

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis that controls the expression of genes encoding proteins that play key roles in angiogenesis, erythropoiesis, and glucose/energy metabolism. The stability of the HIF-1alpha subunit is regulated by ubiquitination and proteasomal degradation. In aerobic cells, O(2)-dependent prolyl hydroxylation of HIF-1alpha is required for binding of the von Hippel-Lindau tumor suppressor protein VHL, which then recruits the Elongin C ubiquitin-ligase complex. SSAT2 (spermidine/spermine N-acetyltransferase-2) binds to HIF-1alpha and promotes its ubiquitination/degradation by stabilizing the interaction of VHL and Elongin C. Treatment of cells with heat shock protein HSP90 inhibitors induces the degradation of HIF-1alpha even under hypoxic conditions. HSP90 competes with RACK1 for binding to HIF-1alpha, and HSP90 inhibition leads to increased binding of RACK1, which recruits the Elongin C ubiquitin-ligase complex to HIF-1alpha in an O(2)-independent manner. In this work, we demonstrate that SSAT1, which shares 46% amino acid identity with SSAT2, also binds to HIF-1alpha and promotes its ubiquitination/degradation. However, in contrast to SSAT2, SSAT1 acts by stabilizing the interaction of HIF-1alpha with RACK1. Thus, the paralogs SSAT1 and SSAT2 play complementary roles in promoting O(2)-independent and O(2)-dependent degradation of HIF-1alpha.     

Structure-activity relationship study of thiazolyl-hydroxamate derivatives as selective histone deacetylase 6 inhibitors

Several human diseases are associated with aberrant epigenetic pathways mediated by histone deacetylases (HDACs), especially HDAC6, a class IIb HDACs, which has emerged as an attractive target for neurodegenerative and autoimmune disease therapeutics. In a previous study, we developed the novel HDAC6-selective inhibitor 9a ((E)-N-hydroxy-4-(2-styrylthiazol-4-yl)butanamide) and showed that it has anti-sepsis activity in vivo. In this study, we conducted structure-activity relationship (SAR) studies to optimize the activity and selectivity of HDAC6, synthesizing its derivatives with various aliphatic linker sizes and cap structures. We identified 6u ((E)-N-hydroxy-3-(2-(4-fluorostyryl)thiazol-4-yl)propanamide), which has nanomolar inhibition activity and a 126-fold selectivity for HDAC6 over HDAC1. Through the docking analyses of 6u against HDAC subtypes, we revealed the importance of the optimal aliphatic linker size, as well as the electronic substituent effect and rigidity of the aryl cap group. Thus, we suggest a new rationale for the design of HDAC6-selective inhibitors.     

Reducing proteolytic liability of a MMP‐14 inhibitory antibody by site‐saturation mutagenesis

Playing pivotal roles in tumor growth and metastasis, matrix metalloproteinase‐14 (MMP‐14) is an important cancer target. Potent inhibitory Fab 3A2 with therapy‐desired high selectivity has been isolated from a synthetic antibody library carrying long CDR‐H3s. However, like many standard mechanism protease inhibitors, Fab 3A2 can be cleaved by high concentrations of MMP‐14 after extended incubation at acidic pH. Edman sequencing of generated 3A2 fragments indicated that cleavage occurred within its CDR‐H3 between residues N100h (P1) and L100i (P1’). To improve proteolytic stability of 3A2, three positions adjacent to its cleavage site (P1, P1’, and P3’) were subjected to site‐saturation mutagenesis (SSM). Mutations at P1’ (L100i) resulted in loss of inhibition function, while screening of 3A2 Fab mutants at P1 (N100h) or P3’ (A100k) positions identified four clones exhibiting improvements in both stability and inhibition potency. The majority of these mutants with improved stability were substitutions to either hydrophobic (Lue, Trp) or basic residues (Arg, Lys, His). Combinations of these beneficial mutations resulted in a double mutant N100hR/A100kR, which prolonged half‐life twofold with an inhibition potency K_I of 6.6 nM. Enzyme kinetics and competitive ELISA suggested that N100hR/A100kR was a competitive inhibitor overlapping its binding epitope with that of nTIMP‐2. This study demonstrated that site‐directed mutagenesis at or near the cleavage position reduced proteolytic liability of standard mechanism protease inhibitors especially inhibitory antibodies.