Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

Sweat gland density and response during high-intensity exercise in athletes with spinal cord injuries

Sweat production is crucial for thermoregulation. However, sweating can be problematic for individuals with spinal cord injuries (SCI), as they display a blunting of sudomotor and vasomotor responses below the level of the injury. Sweat gland density and eccrine gland metabolism in SCI are not well understood. Consequently, this study examined sweat lactate (S-LA) (reflective of sweat gland metabolism), active sweat gland density (SGD), and sweat output per gland (S/G) in 7 SCI athletes and 8 able-bodied (AB) controls matched for arm ergometry VO₂peak. A sweat collection device was positioned on the upper scapular and medial calf of each subject just prior to the beginning of the trial, with iodine sweat gland density patches positioned on the upper scapular and medial calf. Participants were tested on a ramp protocol (7 min per stage, 20 W increase per stage) in a common exercise environment (21±1°C, 45-65% relative humidity). An independent t-test revealed lower (p<0.05) SGD (upper scapular) for SCI (22.3 ±14.8 glands · cm⁻²) vs. AB. (41.0 ± 8.1 glands · cm⁻²). However, there was no significant difference for S/G between groups. S-LA was significantly greater (p<0.05) during the second exercise stage for SCI (11.5±10.9 mmol · l⁻¹) vs. AB (26.8±11.07 mmol · l⁻¹). These findings suggest that SCI athletes had less active sweat glands compared to the AB group, but the sweat response was similar (SLA, S/G) between AB and SCI athletes. The results suggest similar interglandular metabolic activity irrespective of overall sweat rate.     

Characterization of the genetic diversity of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> in São Paulo city,Brazil

Background

Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).

Findings

Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.

Conclusions

Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.

     

Dietary use and conservation concern of edible wetland plants at indo-burma hotspot: a case study from northeast India

Background

The wetlands of the North East India fall among the global hotspots of biodiversity. However, they have received very little attention with relation to their intrinsic values to human kind; therefore their conservation is hardly addressed. These wetlands are critical for the sustenance of the tribal communities.

Methods

Field research was conducted during 2003 to 2006 in seven major wetlands of four districts of Manipur state, Northeast India (viz. Imphal-East, Imphal-West, Thoubal, and Bishnupur). A total of 224 wetland-plant-collectors were interviewed for the use and economics of species using semi-structured questionnaires and interview schedules. Imphal, Bishenpur and Thoubal markets were investigated in detail for influx and consumption pattern of these plants. The collectors were also inquired for medicinal use of wetland species. Nutritive values of 21 species were analyzed in laboratory. The vouchers were collected for all the species and deposited in the CSIR-NEIST (Formerly Regional Research Laboratory), Substation, Lamphelpat, Imphal, Manipur, India.

Results

We recorded 51 edible wetland species used by indigenous people for food and medicinal purposes. Thirty eight species had high medicinal values and used in the traditional system to treat over 22 diseases. At least 27 species were traded in three markets studied (i.e. Imphal, Thoubal and Bishenpur), involving an annual turnover of 113 tons of wetland edible plants and a gross revenue of Rs. 907, 770/- (US$1 = Rs. 45/-). The Imphal market alone supplies 60% of the total business. Eighty per cent of the above mentioned species are very often used by the community. The community has a general opinion that the availability of 45% species has depleted in recent times, 15 species need consideration for conservation while another 7 species deserved immediate protection measures. The nutrient analysis showed that these species contribute to the dietary balance of tribal communities.

Conclusions

Considering the importance of wild wetland plants in local sustenance, it is suggested to protect their habitats, develop domestication protocols of selected species, and build programs for the long-term management of wetland areas by involving local people. Some medicinal plants may also be used to develop into modern medicines.

     

Inflammatory thresholds and the species-specific effects of colonising bacteria in stable chronic obstructive pulmonary disease

Background

There has been increasing interest in the use of newer, culture-independent techniques to study the airway microbiome of COPD patients. We investigated the relationships between the three common potentially pathogenic microorganisms (PPMs) Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis, as detected by quantitative PCR (qPCR), and inflammation and health status in stable patients in the London COPD cohort.

Methods

We prospectively collected sputum, serum and plasma samples for analysis of airway bacterial presence and load, and airway and systemic inflammation from 99 stable COPD patients between January 2011 and October 2012. Health status was measured with St George’s Respiratory Questionnaire and COPD Assessment Test.

Results

Airway inflammation and plasma fibrinogen, but not C-reactive protein, were greater in samples with PPM detection (p < 0.001, p = 0.049 and p = 0.261, respectively). Increasing total bacterial load was associated with increasing airway (p < 0.01) but not systemic inflammation (p > 0.05). Samples with high total bacterial loads had significantly higher airway inflammation than both samples without PPM detection and those with lower loads. Haemophilus influenzae presence was associated with significantly higher levels of airway but not systemic inflammation for all given pathogen loads (p < 0.05), and was significantly greater than with other PPMs. No association was observed between inflammation and health status (p > 0.05).

Conclusions

Airway and systemic inflammation, as measured by fibrinogen, is greater in stable COPD patients with PPMs detected using the culture-independent qPCR technique. The airway, but not systemic inflammatory response, appears to have a total pathogen-load threshold and appears attributable to Haemophilus influenzae. However, discordance between inflammation and health status was observed.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0114-1) contains supplementary material, which is available to authorized users.     

PILOT_PROTEIN: Identification of Unmodified and Modified Proteins via High-Resolution Mass Spectrometry and Mixed-Integer Linear Optimization

A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized. A sequence tag procedure is then incorporated along with the untargeted PTM identification algorithm, PILOT_PTM, to determine a list of all modification types and sites for each protein. The unmodified protein identification algorithm, PILOT_PROTEIN, is compared to the methods SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both prepared protein samples and a more complex chromatin digest. The algorithm demonstrates superior protein identification accuracy with a lower false positive rate. All materials are freely available to the scientific community at http://pumpd.princeton.edu .     

A Mixed Integer Linear Optimization Framework for the Identification and Quantification of Targeted Post-translational Modifications of Highly Modified Proteins Using Multiplexed Electron Transfer Dissociation Tandem Mass Spectrometry

Here we present a novel methodology for the identification of the targeted post-translational modifications present in highly modified proteins using mixed integer linear optimization and electron transfer dissociation (ETD) tandem mass spectrometry. For a given ETD tandem mass spectrum, the rigorous set of modified forms that satisfy the mass of the precursor ion, within some tolerance error, are enumerated by solving a feasibility problem via mixed integer linear optimization. The enumeration of the entire superset of modified forms enables the method to normalize the relative contributions of the individual modification sites. Given the entire set of modified forms, a superposition problem is then formulated using mixed integer linear optimization to determine the relative fractions of the modified forms that are present in the multiplexed ETD tandem mass spectrum. Chromatographic information in the mass and time dimension is utilized to assess the likelihood of the assigned modification states, to average several tandem mass spectra for confident identification of lower level forms, and to infer modification states of partially assigned spectra. The utility of the proposed computational framework is demonstrated on an entire LC-MS/MS ETD experiment corresponding to a mixture of highly modified histone peptides. This new computational method will facilitate the unprecedented LC-MS/MS ETD analysis of many hypermodified proteins and offer novel biological insight into these previously understudied systems.Accurate identification of post-translational modifications (PTMs)¹ is a critical and often difficult task in proteomics. Most standard mass spectrometry-based techniques for the identification of protein modifications utilize a “bottom up” approach where the proteins are enzymatically digested into smaller peptides that are subsequently ionized and fragmented via CID to derive their sequence information (1–9). The identification of all the modifications present in a protein hinges on the successful identification of the PTM modifications of its corresponding peptides. This protocol can be limited by (a) insufficient elution and detection of all the peptides that cover the entire sequence of the protein, (b) false or incomplete identifications at the peptide level, and (c) the existence of different modification states of the same protein. Additional complications arise when using CID to study labile PTMs such as phosphorylation, glycosylation, or sulfonation. In these instances, the preferred reaction is often the cleavage of the PTM as opposed to the backbone of the peptide, resulting in a high intensity peak corresponding to the difference of the parent mass and the cleaved modification. The advent of electron capture dissociation (ECD) (10, 11) and electron transfer dissociation (ETD) (12–15) has enabled researchers to address the aforementioned issues associated with bottom up approaches using CID by adopting a complementary top down or middle down analysis strategy.ECD and ETD both involve the reaction of an electron with a highly protonated cation to form an odd electron peptide. This process induces large amounts of backbone cleavage to yield c and z· ions that are analogous to the b and y ion series typically encountered in CID tandem mass spectra. Unlike CID, ECD/ETD cleavage is weakly affected by the composition and number of amino acids in the peptide and for certain systems can provide more fragmentation coverage than CID alone, especially for bigger peptides with higher charge states. Both ECD and ETD also prevent the cleavage of labile modifications, and thus PTMs are retained on the corresponding c and z· ions. The aforementioned benefits make ECD/ETD particularly well suited for the LC-MS/MS top down and middle down analysis of post-translationally modified proteins. These top down and middle down approaches also enable the approximate inference of protein abundance from the chromatogram and MS¹ information because the full protein sequence elutes from the column (16).In recent years, there has been significant interest in the identification of highly modified proteins, such as histones. Histone proteins are key regulators of many important DNA processes in eukaryotes, and recent studies have elucidated complex relationships between histone modifications and many nuclear events. It has also been shown that differences in global histone modifications in tissues can be used to predict the clinical outcome of cancer patients (17). Early MS or immunoassay studies were only able to analyze these modifications on a site-by-site basis and as a result lost important connectivity information on the molecular level because several modified forms of the same protein exist concurrently. In MS-based applications, the use of traditional reversed phase HPLC for the separation of a highly modified protein results in poor chromatographic resolution because all the modified forms are physically similar. Successful off-line techniques for the separation of highly modified histone forms have been achieved using cation exchange hydrophilic interaction chromatography (HILIC) (18), which separates the modified species primarily by the number of acetyl groups and secondly by the degree of methylation. The separation must be conducted off line because the mobile phase additives used are non-volatile components, and subsequent fractionation is necessary for mass spectrometric analysis. This protocol has made it possible to analyze the first 50 amino acids of the N-terminal tail of histone H3 and provided important insight regarding connectivity information between the modification sites. A major disadvantage of this approach is that the off-line nature of the experimental protocol is extremely time-consuming (on the order of months) and thus prohibits the ability to conduct multiple runs for high throughput studies and statistical validation. Additionally, other off-line techniques have been successful in the extraction and purification of modified histone proteins using acid-urea gel electrophoresis (19) but suffer from similar throughput constraints.We have recently developed chromatography that is particularly suited for LC-MS ETD analysis of highly modified polypeptides with successful applications to histone proteins (20). The protocol uses a “saltless” pH gradient to elute the various modified forms in a weak cation exchange HILIC. Unprecedented separation of the modified histone forms is achieved within a single LC-MS/MS ETD experiment, thereby introducing important chromatographic information that can be utilized in the subsequent identification and quantification of these post-translational modifications. Although the achieved separation is exceptional in comparison with previous attempts, the complexity and relative similarity of the modified forms still results in minor species co-eluting with similar mass and retention times, thus resulting in multiplexed tandem mass spectra. The term “multiplexed” as used here refers to the fact that several species are dissociated and measured in a single tandem mass spectrum (21) and should not be confused with the multiplex experimental protocols. Computational methodologies that utilize the extensive and complementary information contained within these LC-MS/MS data sets are nonexistent as the technology has only recently been developed.In this work, we present a novel mixed integer linear optimization (MILP) computational framework for the identification and quantification of highly modified proteins using LC-MS and ETD tandem mass spectrometry. Key concepts of the proposed framework are illustrated using histone H3.2 as an example system. For a given primary sequence, the entire set of post-translational modifications that satisfy a precursor mass are enumerated by solving an MILP feasibility problem. Given this set of PTM forms, an MILP superposition problem is then solved to determine the relative fractions of the modified forms that are present in the multiplexed ETD tandem mass spectrum. An important aspect of the proposed framework is that chromatographic information is used to correlate the modification states as a function of modification position, mass, and time. The proposed computational framework is applied to an entire LC-MS/MS ETD experiment corresponding to a mixture of highly modified histone peptides to demonstrate its utility.