Mechanism of Photosignaling by Drosophila Cryptochrome: ROLE OF THE REDOX STATUS OF THE FLAVIN CHROMOPHORE*?

Cryptochrome (CRY) is the primary circadian photoreceptor in Drosophila. Upon light absorption, dCRY undergoes a conformational change that enables it to bind to Timeless (dTIM), as well as to two different E3 ligases that ubiquitylate dTIM and dCRY, respectively, resulting in their proteolysis and resetting the phase of the circadian rhythm. Purified dCRY contains oxidized flavin (FAD_ox), which is readily photoreduced to the anionic semiquinone through a set of 3 highly conserved Trp residues (Trp triad). The crystal structure of dCRY has revealed a fourth Trp (Trp-536) as a potential electron donor. Previously, we reported that the Trp triad played no role in photoinduced proteolysis of dCRY in Drosophila cells. Here we investigated the role of the Trp triad and Trp-536, and the redox status of the flavin on light-induced proteolysis of both dCRY and dTIM and resetting of the clock. We found that both oxidized (FAD_ox) and reduced (FAD^⨪) forms of dCRY undergo light-induced conformational change in vitro that enable dCRY to bind JET and that Trp triad and Trp-536 mutations that block known or presumed intraprotein electron transfer reactions do not affect dCRY phototransduction under bright or dim light in vivo as measured by light-induced proteolysis of dCRY and dTIM in Drosophila S2R+ cells. We conclude that both oxidized and reduced forms of dCRY are capable of photosignaling.     

Suspension trapping (STrap) sample preparation method for bottom‐up proteomics analysis

Despite recent developments in bottom‐up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS‐based protein extraction with rapid detergent removal, reactor‐type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack—this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow‐through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.     

Diversity of lactic acid bacteria of the bioethanol process

Background  

Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil.     

Alterations in Cytokines and Effects of Dexamethasone Immunosuppression during Subclinical Infections of Invasive Klebsiella pneumoniae with Hypermucoviscosity Phenotype in Rhesus (Macaca mulatta) and Cynomolgus (Macaca fascicularis) Macaques

Invasive Klebsiella pneumoniae with the hypermucoviscosity phenotype (HMV K. pneumoniae) is an emerging human pathogen that also has been attributed to fatal multisystemic disease in African green monkeys at our institution. Combining a cluster of subclinically infected macaques identified in March and April 2008 and the animals documented during a subsequent survey of more than 300 colony nonhuman primates yielded a total of 9 rhesus macaques and 6 cynomolgus macaques that were subclinically infected. In an attempt to propagate the responsible HMV K. pneumoniae strain, a subset of these animals was immunosuppressed with dexamethasone. None of the treated animals developed clinical disease consistent with the multisystemic disease that affected colony African green monkeys. However, cytokine analysis revealed significant alterations of secreted cytokines in macaques subclinically infected with HMV K. pneumoniae when compared with noninfected macaques, thereby calling into question the suitability of animals subclinically infected with HMV K. pneumoniae for use in immunologic or infectious disease research.Abbreviations: HMV, hypermucoviscosity phenotype; rmpA, regulator of the mucoid phenotype gene; magA, mucoviscosity-associated geneKlebsiella pneumoniae is a gram-negative member of the Enterobacteriaceae family that comprises part of the normal fecal and oral flora of many nonhuman primates¹⁹ but also has been implicated in cases of peritonitis, septicemia, pneumonia, and meningitis in both Old and New World primates.^17,20,37 Over the past 20 y, strains of invasive K. pneumoniae with a unique hypermucoviscosity phenotype (HMV K. pneumoniae) have been reported to cause community-acquired primary liver abscesses, meningitis, and endophthalmitis in humans in Taiwan and other Asian countries,^{10, 26–31,33,44,48,51} mostly in people with diabetes mellitus.^7,8,44 In addition, HMV K. pneumoniae has caused clinical disease in the United States and other nonAsian countries.^18,30,33 The HMV phenotype is determined based on a positive string test, which is performed by touching a colony with a bacterial loop and gently lifting. If a mucoid ‘string’ of at least 5 mm forms, the string test is considered positive.^3,14,45,51Capsular serotypes K1 and K2 have been reported as the major virulence determinants for human HMV K. pneumoniae liver abscesses.^9,15,49,50 The products of the mucoviscosity-associated gene (magA), which encodes a structural outer membrane protein of the K1 serotype, and the regulator of the mucoid phenotype gene (rmpA) have also been proposed as virulence factors.^{16,34,42,52,53}HMV K. pneumoniae has been reported to cause multisystemic abscesses in African green monkeys (Chlorocebus aethiops).⁴⁵ In late 2005 and early 2006, 7 African green monkeys in the research colony at our institution, the US Army Medical Research Institute for Infectious Diseases, were found to have abscesses in multiple locations; all 7 animals either succumbed or were euthanized because of poor prognosis due to surgically nonresectable abdominal abscesses.⁴⁵ The etiology of the final case was determined to be HMV K. pneumoniae with the K2 serotype and rmpA, and all 6 other cases had similar clinical, microbiologic, and pathologic characteristics. Prior to the current study, we believe these 7 cases were the only documented natural infections attributed specifically to HMV K. pneumoniae in nonhuman primates.⁴⁵As a result of those findings, our institution instituted a policy to report K. pneumoniae positive cultures in nonhuman primates during quarantine periods and on routine semiannual examination. Over several months in spring and summer 2008, a group of 19 macaques tested positive on oropharyngeal or rectal culture for HMV K. pneumoniae; 15 of those 19 animals were isolated in a single room for 2 to 4 mo to better characterize the infection.³ None of the animals showed clinical signs of disease during the isolation period, and abdominal palpation failed to suggest the presence of abdominal abscesses like those seen in African green monkeys. Testing of isolates suggested that the macaques harbored subclinical infections and that multiple genotypes of HMV K. pneumoniae were present.³In July 2008, a cynomolgus macaque from the colony that was experimentally challenged with monkeypox virus survived beyond the normal time-to-death window (12 to 16 d after infection). However, on day 22 after infection (6 to 10 d beyond this window), this macaque died unexpectedly. Histopathologic analysis of tissues from this NHP revealed a concurrent gram-negative bacterial infection, based on Gram stains and immunohistochemistry. Although cultures were not available, PCR analysis of DNA extracted from formalin-fixed, paraffin-embedded tissues revealed the presence of K. pneumoniae through the amplification of rmpA,³⁹ which is consistent with the HMV phenotype. This animal was considered to have survived infection with monkeypox based on time to death after infection. Monkeypox is reported to target the mononuclear phagocyte system and associated dendritic cells,⁵⁴ and we theorized that the monkeypox infection in this macaque led to suppression of the immune system, which then allowed development of a fatal HMV K. pneumoniae septicemia.The present project sought to explore the pathophysiology of HMV K. pneumoniae in macaques. We hypothesized that immunosuppression of subclinically infected macaques would produce lesions similar to those observed in the coinfected macaque. In addition, we hypothesized that subclinically infected macaques would have a different immune profile from that of noninfected primates. We measured and analyzed cytokine levels as an indication of altered immune status because such a state potentially could confound research into immunologic responses and infectious disease.     

Sample size determination in clinical proteomic profiling experiments using mass spectrometry for class comparison

Mass spectrometric profiling approaches such as MALDI‐TOF and SELDI‐TOF are increasingly being used in disease marker discovery, particularly in the lower molecular weight proteome. However, little consideration has been given to the issue of sample size in experimental design. The aim of this study was to develop a protocol for the use of sample size calculations in proteomic profiling studies using MS. These sample size calculations can be based on a simple linear mixed model which allows the inclusion of estimates of biological and technical variation inherent in the experiment. The use of a pilot experiment to estimate these components of variance is investigated and is shown to work well when compared with larger studies. Examination of data from a number of studies using different sample types and different chromatographic surfaces shows the need for sample‐ and preparation‐specific sample size calculations.     

Holocene mangrove dynamics and environmental change in the Rufiji Delta, Tanzania

Holocene mangrove dynamics are reconstructed from pollen, sediment and radiocarbon analyses of three cores (ANR, BNR, CNR) located across a 20 km transect in the Rufiji Delta, Tanzania. At the base of the sediment sequence, dated to about 5600 cal. year b.p., the mangroves which are present suggest a low intertidal ecosystem in response to wet conditions and a higher sea level than at the present day. After around 5600 cal. year b.p. in core BNR, mangroves retreated seaward probably due to a lower sea level and drier environmental conditions. At around 4640 cal. year b.p., mangroves shifted landward suggesting a phase of sea level rise. In the late Holocene, mangroves became established at higher elevations of the Rufiji Delta, which is now a paddy field. Mangrove taxa decreased after 1170 cal. year b.p., suggesting drier conditions and less inundation frequency, possibly due to a lower sea level. Marked vegetation changes from mangroves to terrestrial vegetation occurred after around 750 cal. year b.p., possibly related to sea level regression and/or a desiccation phase recorded during the late Holocene. Paddy fields replaced mangroves in the landward part of the transect, reflecting an increase in human settlement in this area, a trend that continues to the present day. The recent decrease of mangrove species, particularly Rhizophora mucronata, could suggest less inundation by saline water and a lower sea level, although these changes may also be due to human activities during the last millennia as indicated by charcoal analysis.     

Inhalation by design: dual pharmacology β-2 agonists/M3 antagonists for the treatment of COPD

This paper describes the successful design and development of dual pharmacology β-2 agonists-M3 antagonists, for the treatment of chronic obstructive pulmonary disorder using the principles of ‘inhalation by design’. A key feature of this work is the combination of balanced potency and pharmacodynamic duration with desirable pharmacokinetic and material properties, whilst keeping synthetic complexity to a minimum.     

Increasing skeletal muscle carnitine content in older individuals increases whole‐body fat oxidation during moderate‐intensity exercise

Intramyocellular lipid (IMCL) utilization is impaired in older individuals, and IMCL accumulation is associated with insulin resistance. We hypothesized that increasing muscle total carnitine content in older men would increase fat oxidation and IMCL utilization during exercise, and improve insulin sensitivity. Fourteen healthy older men (69 ± 1 year, BMI 26.5 ± 0.8 kg/m²) performed 1 h of cycling at 50% VO₂max and, on a separate occasion, underwent a 60 mU/m²/min euglycaemic hyperinsulinaemic clamp before and after 25 weeks of daily ingestion of a 220 ml insulinogenic beverage (44.4 g carbohydrate, 13.8 g protein) containing 4.5 g placebo (n = 7) or L‐carnitine L‐tartrate (n = 7). During supplementation, participants performed twice‐weekly cycling for 1 h at 50% VO₂max. Placebo ingestion had no effect on muscle carnitine content or total fat oxidation during exercise at 50% VO₂max. L‐carnitine supplementation resulted in a 20% increase in muscle total carnitine content (20.1 ± 1.2 to 23.9 ± 1.7 mmol/kg/dm; p < 0.01) and a 20% increase in total fat oxidation (181.1 ± 15.0 to 220.4 ± 19.6 J/kg lbm/min; p < 0.01), predominantly due to increased IMCL utilization. These changes were associated with increased expression of genes involved in fat metabolism (ACAT1, DGKD & PLIN2; p < 0.05). There was no change in resting insulin‐stimulated whole‐body or skeletal muscle glucose disposal after supplementation. This is the first study to demonstrate that a carnitine‐mediated increase in fat oxidation is achievable in older individuals. This warrants further investigation given reduced lipid turnover is associated with poor metabolic health in older adults.