On the involvement of single-bond rotation in the primary photochemistry of photoactive yellow protein

Prior experimental observations, as well as theoretical considerations, have led to the proposal that C₄-C₇ single-bond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid chromophore, (5-hydroxy indan-(1E)-ylidene)acetic acid, in which rotation across the C₄-C₇ single bond has been locked with an ethane bridge, and we reconstituted the apo form of the wild-type protein and its R52A derivative with this chromophore analog. In PYP reconstituted with the rotation-locked chromophore, 1), absorption spectra of ground and intermediate states are slightly blue-shifted; 2), the quantum yield of photochemistry is ∼60% reduced; 3), the excited-state dynamics of the chromophore are accelerated; and 4), dynamics of the thermal recovery reaction of the protein are accelerated. A significant finding was that the yield of the transient ground-state intermediate in the early phase of the photocycle was considerably higher in the rotation-locked samples than in the corresponding samples reconstituted with p-coumaric acid. In contrast to theoretical predictions, the initial photocycle dynamics of PYP were observed to be not affected by the charge of the amino acid residue at position 52, which was varied by 1), varying the pH of the sample between 5 and 10; and 2), site-directed mutagenesis to construct R52A. These results imply that C₄-C₇ single-bond rotation in PYP is not an alternative to C₇=C₈ double-bond rotation, in case the nearby positive charge of R52 is absent, but rather facilitates, presumably with a compensatory movement, the physiological Z/E isomerization of the blue-light-absorbing chromophore.     

Persistent occurrence of meiotic abnormalities in a new hexaploid cytotype of <Emphasis Type="Italic">Thalictrum foetidum</Emphasis> from Indian cold deserts

Thalictrum foetidum L. (Ranunculaceae), a morphologically variable and widely distributed species of temperate and alpine Himalayas is worked out cytologically for the first time from India. Earlier studies from outside India were restricted to chromosome counts and karyotypic analysis. We studied the male meiosis, microsporogenesis and pollen viability in the wild accessions from the cold deserts of Lahaul-Spiti, Kinnaur and Pangi Valley of Himachal Pradesh. Present cytomorphological surveys in the species record the existence of two distinct morphotypes involving plant size; colour and size of leaf/leaflet; dentation of leaflet lobes; and degree of leaf pubescence. All the accessions in the two morphovariants share the same meiotic chromosome number (n = 21) and adds a new intraspecific hexaploid cytotype. The accessions show the phenomenon of cytomixis involving transfer of chromatin material among proximate pollen mother cells (PMCs) and associated meiotic abnormalities like, out of plate bivalents, interchromosomal connections, and laggards, bridges and micronuclei at anaphases/telophases. Microsporogenesis results into abnormal sporads (tetrads with micronuclei, dyads, triads and polyads). The products of such sporads resulted into some pollen sterility and pollen grains of heterogeneous sizes. The persistent occurrence of phenomenon of cytomixis and associated meiotic abnormalities and consequently pollen sterility and pollen grains of heterogeneous sizes in the hexaploid cytotype of T. foetidum seems to be under some genetic factors associated with the genome.     

Feed intake, milk production and composition of crossbred cows fed with insect-protected Bollgard II® cottonseed containing Cry1Ac and Cry2Ab proteins

Twenty crossbred lactating multiparous cows were used in a 28-day study to compare dry matter intake (DMI), milk yield, milk composition and Bacillus thuringiensis (Bt) protein concentrations in plasma when fed diets containing Bollgard II® cottonseed (BGII) or a control non-genetically modified isogenic cottonseed (CON). Bollgard II cottonseed contains the Cry1Ac and Cry2Ab insecticidal proteins that protect cotton plants from feeding damage caused by certain lepidopteran insects. Cows were assigned randomly to the BGII or CON treatments after a 2-week adjustment period. Cows consumed a concentrate containing 40% crushed cottonseed according to milk yield and green maize forage ad libitum. All cows received the same diet but with different crushed cottonseed sources. Cottonseed was included to provide approximately 2.9 kg per cow daily (dry matter basis). The ingredient composition of the concentrate was 40% crushed cottonseed, 15% groundnut cake, 20% corn, 22% wheat bran, 1% salt and 2% mineral mixture. Milk and blood plasma were analyzed for Cry1Ac and Cry2Ab proteins. DMI, BW, milk yield and milk components did not differ between cows on the BGII and CON treatments. Although milk yield and milk fat percentage were not affected by treatment, 4% fat-corrected milk (FCM) production and FCM/kg DMI for cows on the BGII treatment (14.0 kg/cow per day, 1.12 kg/kg) were significantly improved compared with cows on the CON treatment (12.1 kg/cow per day, 0.97 kg/kg). Gossypol contents in BGII cottonseed and conventional cottonseed were similar. Cry1Ac and Cry2Ab2 proteins in Bollgard II cottonseed were 5.53 and 150.8 μg/g, respectively, and were not detected in the milk or plasma samples. The findings suggested that Bollgard II cottonseed can replace conventional cottonseed in dairy cattle diets with no adverse effects on performance and milk composition.     

Can Volunteer Community Health Workers Decrease Child Morbidity and Mortality in Southwestern Uganda? An Impact Evaluation

Background

The potential for community health workers to improve child health in sub-Saharan Africa is not well understood. Healthy Child Uganda implemented a volunteer community health worker child health promotion model in rural Uganda. An impact evaluation was conducted to assess volunteer community health workers'' effect on child morbidity, mortality and to calculate volunteer retention.

Methodology/Principal Findings

Two volunteer community health workers were selected, trained and promoted child health in each of 116 villages (population ∼61,000) during 2006–2009. Evaluation included a household survey of mothers at baseline and post-intervention in intervention/control areas, retrospective reviews of community health worker birth/child death reports and post-intervention focus group discussions. Retention was calculated from administrative records. Main outcomes were prevalence of recent child illness/underweight status, community health worker reports of child deaths, focus group perception of effect, and community health worker retention. After 18–36 months, 86% of trained volunteers remained active. Post-intervention surveys in intervention households revealed absolute reductions of 10.2% [95%CI (−17.7%, −2.6%)] in diarrhea prevalence and 5.8% [95%CI (−11.5%, −0.003%)] in fever/malaria; comparative decreases in control households were not statistically significant. Underweight prevalence was reduced by 5.1% [95%CI (−10.7%, 0.4%)] in intervention households. Community health worker monthly reports revealed a relative decline of 53% in child deaths (<5 years old), during the first 18 months of intervention. Focus groups credited community health workers with decreasing child deaths, improved care-seeking practices, and new income-generating opportunities.

Conclusions/Significance

A low-cost child health promotion model using volunteer community health workers demonstrated decreased child morbidity, dramatic mortality trend declines and high volunteer retention. This sustainable model could be scaled-up to sub-Saharan African communities with limited resources and high child health needs.     

Enhancing Non-linear Response of Fullerene via Incorporation of Gold Nanoparticles

Plasmonics - Bi-functional nanocomposite thin films of fullerene C60 and C70 containing Au NPs were synthesized using thermal co-evaporation method. Different atomic concentrations of Au metal...     

Association between SPARC mRNA Expression,Prognosis and Response to Neoadjuvant Chemotherapy in Early Breast Cancer: A Pooled in-silico Analysis

Introduction

SPARC is an important regulator of the extracellular matrix and has been suggested to improve delivery of albumin-bound cytotoxics. However, little is known regarding its role in breast cancer (BC).

Methods

We conducted a pooled analysis of publically available datasets, in which BC patients who received no systemic therapy or received neoadjuvant chemotherapy were eligible. Patients were assigned to molecular subtypes using PAM-50. We computed a SPARC module (SPARC7), composed of genes with an absolute correlation with SPARC >0.7. In the systemically untreated cohort, we evaluated 1) expression of SPARC/SPARC7 according to breast cancer subtype, 2) association between SPARC/SPARC7 and biological processes related to proliferation, immune and stroma, and 3) association between SPARC/SPARC7 and relapse-free survival in a Cox model in all patients and in the different molecular subtypes adjusted for tumor size, nodal status, histological grade, and age. In the neoadjuvant cohort, we evaluated the association between SPARC and pCR in a logistic regression model, adjusted for the same clinicopathologic factors.

Results

948 (10 datasets), and 791 (8 datasets) patients were included in the systemically untreated and neoadjuvant cohorts, respectively. High SPARC expression was associated with small tumor size, low histological grade and luminal-A tumors (all p<0.0001). There was a positive correlation between SPARC and stroma-related modules but negative correlation with proliferation modules. High SPARC expression was associated with poor prognosis in patients with basal and HER2+ breast cancer even after adjusting for clinicopathologic parameters. In the neoadjuvant cohort, a subgroup analysis suggested that high SPARC is associated with low rates of pCR in the HER2 subtype. Same results were observed on replacing SPARC by SPARC7.

Conclusion

This analysis suggests a potential role of SPARC in determining prognosis and response to primary chemotherapy in early BC. This information could guide further development of albumin-bound cytotoxics in BC.     

An FGF21-Adiponectin-Ceramide Axis Controls Energy Expenditure and Insulin Action in Mice

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Antioxidant components of naturally-occurring oils exhibit marked anti-inflammatory activity in epithelial cells of the human upper respiratory system

Background

Proteomic studies of respiratory disorders have the potential to identify protein biomarkers for diagnosis and disease monitoring. Utilisation of sensitive quantitative proteomic methods creates opportunities to determine individual patient proteomes. The aim of the current study was to determine if quantitative proteomics of bronchial biopsies from asthmatics can distinguish relevant biological functions and whether inhaled glucocorticoid treatment affects these functions.

Methods

Endobronchial biopsies were taken from untreated asthmatic patients (n = 12) and healthy controls (n = 3). Asthmatic patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months) and new biopsies were obtained. Proteins extracted from the biopsies were digested and analysed using isobaric tags for relative and absolute quantitation combined with a nanoLC-LTQ Orbitrap mass spectrometer. Spectra obtained were used to identify and quantify proteins. Pathways analysis was performed using Ingenuity Pathway Analysis to identify significant biological pathways in asthma and determine how the expression of these pathways was changed by treatment.

Results

More than 1800 proteins were identified and quantified in the bronchial biopsies of subjects. The pathway analysis revealed acute phase response signalling, cell-to-cell signalling and tissue development associations with proteins expressed in asthmatics compared to controls. The functions and pathways associated with placebo and budesonide treatment showed distinct differences, including the decreased association with acute phase proteins as a result of budesonide treatment compared to placebo.

Conclusions

Proteomic analysis of bronchial biopsy material can be used to identify and quantify proteins using highly sensitive technologies, without the need for pooling of samples from several patients. Distinct pathophysiological features of asthma can be identified using this approach and the expression of these features is changed by inhaled glucocorticoid treatment. Quantitative proteomics may be applied to identify mechanisms of disease that may assist in the accurate and timely diagnosis of asthma.

Trial registration

ClinicalTrials.gov registration NCT01378039     

Application of a validated ultra performance liquid chromatography-tandem mass spectrometry method for the quantification of darunavir in human plasma for a bioequivalence study in Indian subjects

A simple, precise and rapid ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the quantification of darunavir, a protease inhibitor, using darunavir-d9 as internal standard (IS). The method involved liquid-liquid extraction of darunavir and IS in methyl-tert-butyl ether from 50 μL human plasma. The chromatographic separation was achieved on an Acquity UPLC BEH C18 (50 mm × 2.1mm, 1.7 μm particle size) analytical column under gradient conditions, in a run time of 1.6 min. The precursor → product ion transitions for darunavir (m/z 548.1 → 392.0) and IS (m/z 557.1 → 401.0) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was extensively validated for its selectivity, sensitivity, carryover check, linearity, precision and accuracy, reinjection reproducibility, recovery, matrix effect, ion suppression/enhancement, stability and dilution integrity. The linearity of the method was established in the concentration range of 1.0-5000 ng/mL. The mean relative recovery for darunavir (100.8%) and IS (89.8%) from spiked plasma samples was consistent and reproducible. The application of this method for routine measurement of plasma darunavir concentration was demonstrated by a bioequivalence study conducted in 40 healthy Indian subjects for a 600 mg tablet formulation along with 100mg ritonavir as booster under fast and fed conditions. To demonstrate the reproducibility in the measurement of study data, an incurred sample reanalysis was done with 400 subject samples and the % change in concentration was within ± 12%.