Cloning and functional characterization of the HRASLS2 gene

The HRAS-like suppressor 2 (HRASLS2) gene belongs to the H-REV107 gene family involved in the regulation of cell growth and differentiation. HRASLS2 is expressed at high levels in normal tissues of the small intestine, kidney, and trachea. We cloned HRASLS2 cDNA from human SW480 colon cancer cells. Most wild-type, and some N- and C-terminal truncated HRASLS2 (HRASLS2DeltaNDeltaC) were expressed as a granular pattern located at perinuclear region in HtTA cervical cancer cells, while truncation at the C-terminus only (HRASLS2DeltaC) resulted in a diffuse pattern. Wild-type HRASLS2 significantly suppressed colony formation of HeLa and HCT116 cells. HRASLS2DeltaNDeltaC significantly inhibited colony formation of HCT116 cells, but HRASLS2DeltaC did not affect cell growth. HRASLS2 suppressed the RAS-GTP levels and total RAS protein by 44% and 25%, respectively in HtTA cells; however, the suppression was not observed in truncated HRASLS2 variants. In conclusion, the HRASLS2 protein suppressed growth and RAS activities of cancer cells, and the C-terminal hydrophobic domain appeared to be indispensable for both activities.     

High-performance liquid chromatographic–electrochemical assay for the quantitation of BMS-181885 in monkey plasma

A high-performance liquid chromatographic–electrochemical assay was developed and validated for the quantitation of BMS-181885 (I), an anti-migraine agent, in monkey plasma. The assay involved a solid-phase extraction of I and BMY-46317 (internal standard; I.S.) on a 1-ml cyano cartridge using the automatic solid-phase extraction cartridge (ASPEC) system. Immediately following the conditioning of the cyano column (3 ml of methanol and 2 ml of 1% glacial acetic acid), plasma (0.25 ml) was loaded on to the column. The column was then washed with a 3 ml of 0.1 M ammonium acetate buffer (pH 6). The final elution of the analytes was performed using 2 ml of methanol. The eluate was then evaporated to dryness (gentle stream of nitrogen at 40°C) and the residue was dissolved in the mobile phase and injected on to a YMC basic column (15 cm×4.6 mm; 5 μm particle size) at a flow-rate of 1 ml/min. A mixture of 0.1 M ammonium acetate at pH 6–acetonitrile–methanol (70:20:10, v/v) was used as the mobile phase. Standard curves, with a lower limit of quantitation of 2 ng/ml of I were linear (r²≥0.998; range: 2–50 ng/ml). Based on the analysis of the quality control (QC) samples, the assay was both accurate and precise. The stability of I was established following freeze–thaw cycles and storage at or below −20°C. The extraction recovery of I from monkey plasma was about 82%. The validated assay method was applied to determine the pharmacokinetics of I in monkeys following a single 1 mg/kg intravenous dose.     

A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides

An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant.     

Mating,births, and transitions: a flexible two-sex matrix model for evolutionary demography

Models of sexually-reproducing populations that consider only a single sex cannot capture the effects of sex-specific demographic differences and mate availability. We present a new framework for two-sex demographic models that implements and extends the birth-matrix mating-rule approach of Pollak. The model is a continuous-time matrix model that explicitly includes the processes of mating (which is nonlinear but homogeneous), offspring production, and demographic transitions and survival. The resulting nonlinear model converges to exponential growth with an equilibrium population composition. The model can incorporate age- or stage-structured life histories and flexible mating functions. As an example, we apply the model to analyze the effects of mating strategies (polygamy or monogamy, and mated unions composed of males and females, of variable duration) on the response to sex-biased harvesting. The combination of demographic complexity with the interaction of the sexes can have major population dynamic effects and can change the outcome of evolution on sex-related characters.     

Interplay of two functionally and structurally distinct domains of the c-fos AU-rich element specifies its mRNA-destabilizing function.

AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs.     

A demographic model for sex ratio evolution and the effects of sex‐biased offspring costs

The evolution of the primary sex ratio, the proportion of male births in an individual's offspring production strategy, is a frequency‐dependent process that selects against the more common sex. Because reproduction is shaped by the entire life cycle, sex ratio theory would benefit from explicitly two‐sex models that include some form of life cycle structure. We present a demographic approach to sex ratio evolution that combines adaptive dynamics with nonlinear matrix population models. We also determine the evolutionary and convergence stability of singular strategies using matrix calculus. These methods allow the incorporation of any population structure, including multiple sexes and stages, into evolutionary projections. Using this framework, we compare how four different interpretations of sex‐biased offspring costs affect sex ratio evolution. We find that demographic differences affect evolutionary outcomes and that, contrary to prior belief, sex‐biased mortality after parental investment can bias the primary sex ratio (but not the corresponding reproductive value ratio). These results differ qualitatively from the widely held conclusions of previous models that neglect demographic structure.     

Influential Factors for and Outcomes of Hospitalized Patients with Suicide-Related Behaviors: A National Record Study in Taiwan from 1997–2010

Background

Investigating the factors related to suicide is crucial for suicide prevention. Psychiatric disorders, gender, socioeconomic status, and catastrophic illnesses are associated with increased risk of suicide. Most studies have typically focused on the separate influences of physiological or psychological factors on suicide-related behaviors, and have rarely used national data records to examine and compare the effects of major physical illnesses, psychiatric disorders, and socioeconomic status on the risk of suicide-related behaviors.

Objectives

To identify the characteristics of people who exhibited suicide-related behaviors and the multiple factors associated with repeated suicide-related behaviors and deaths by suicide by examining national data records.

Design

This is a cohort study of Taiwan’s national data records of hospitalized patients with suicide-related behaviors from January 1, 1997, to December 31, 2010.

Participants

The study population included all people in Taiwan who were hospitalized with a code indicating suicide or self-inflicted injury (E950–E959) according to the International Classification of Disease, Ninth Revision, Clinical Modification.

Results

Self-poisoning was the most common method of self-inflicted injury among hospitalized patients with suicide-related behaviors who used a single method. Those who were female, had been hospitalized for suicide-related behaviors at a younger age, had a low income, had a psychiatric disorder (i.e., personality disorder, major depressive disorder, bipolar disorder, schizophrenia, alcohol-related disorder, or adjustment disorder), had a catastrophic illness, or had been hospitalized for suicide-related behaviors that involved two methods of self-inflicted injury had a higher risk of hospitalization for repeated suicide-related behaviors. Those who were male, had been hospitalized for suicide-related behaviors at an older age, had low income, had schizophrenia, showed repeated suicide-related behaviors, had a catastrophic illness, or had adopted a single lethal method had an increased risk of death by suicide.

Conclusions

High-risk factors should be considered when devising suicide-prevention strategies.     

Enhancing antimicrobial activity of mastoparan-B by amino acid substitutions

This study evaluated antimicrobial and hemolytic activities of mastoparan-B (MP-B) isolated from the venom of the hornet, Vespa basalis, and its analogs after substituting certain amino acid (aa) residues. MP-B exhibited significantly different antimicrobial activities against bacteria species/strains tested, especially two Escherichia coli strains, Staphylococcus xylosus and Citrobacter koseri at low dosages, and was non-specific against certain Gram-positive and -negative bacteria. Our results indicated that hydrophobicity modification by single aa substitution may enhance their antimicrobial activities. An aa substituted MP-B, viz., MP-B-1, in which Trp substituted for Leu³, became more effective than the original peptide at inhibiting or killing the bacterial species tested, especially Klebsiella pneumonia, Salmonella typhimurium, and Salmonella Cholerasuis, and even up to 8 times more effective in some cases. However, MP-B-2 was virtually similar to MP-B against each bacterial species assayed, while MP-B-3 reduced its effectiveness greatly compared to others. On the other hand, MP-B and its analogs were not effective against the beneficial probiotics and they were not hemolytic to erythrocytes at the dosages tested. Our results suggested that MP-B becomes more potent against specific pathogenic bacteria and safe to the probiotics after undergoing appropriate amino acid substitutions.     

MicroRNA-208a Increases Myocardial Fibrosis via Endoglin in Volume Overloading Heart

MicroRNA-208a (mir-208a) is essential for cardiac hypertrophy and fibrosis. Endoglin, a co-receptor of transforming growth factor-β is also essential for cardiac fibrosis. Endoglin has been shown to be a target of mir-208a in the in vitro mechanical stress model. Volume overload can lead to heart failure and cardiac fibrosis. The role of mir-208a and endoglin in volume overload heart failure is well known. We sought to investigate the mechanism of regulation of mir-208a and endoglin in volume overload-induced heart failure. Aorta-caval (AV) shunt was performed in adult Sprague-Dawley rats to induce volume overload. Heart weight and heart weight/body weight ratio significantly increased in AV shunt animals. AV shunt significantly increased left ventricular end-diastolic dimension as compared to sham group. Mir-208a was significantly induced by AV shunt from 3 to 14 days. Endoglin, myosin heavy chain-β and brain natriuretic peptide were significantly induced by AV shunt from 3 to 14 days. Overexpression of mir-208a in the sham group without AV shunt significantly increased endoglin expression similar to the AV shunt group. Antagomir-208a attenuated the endoglin expression induced by AV shunt. Pretreatment with atorvastatin also attenuated the endoglin expression induced by AV shunt. AV shunt significantly increased myocardial fibrosis as compared to sham group. Overexpression of mir-208a in the sham group significantly increased myocardial fibrosis. Antagomir-208a and atorvastatin significantly attenuated the myocardial fibrosis induced by AV shunt. In conclusion, mir-208a increased endoglin expression to induce myocardial fibrosis in volume overloaded heart failure. Treatment with atorvastatin can attenuate the myocardial fibrosis induced by volume overload through inhibition of endoglin expression.