Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum.

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

Costs of TB services in India (No 1).

BACKGROUND: There is a dearth of economic analysis required to support increased investment in TB in India. This study estimates the costs of TB services from a health systems´ perspective to facilitate the efficient allocation of resources by India´s National Tuberculosis Elimination Programme.METHODS: Data were collected from a multi-stage, stratified random sample of 20 facilities delivering TB services in two purposively selected states in India as per Global Health Cost Consortium standards and using Value TB Data Collection Tool. Unit costs were estimated using the top-down (TD) and bottom-up (BU) methodology and are reported in 2018 US dollars.RESULTS: Cost of delivering 50 types of TB services and four interventions varied according to costing method. Key services included sputum smear microscopy, Xpert® MTB/RIF and X-ray with an average BU costs of respectively US$2.45, US$17.36 and US$2.85. Average BU cost for bacille Calmette-Guérin vaccination, passive case-finding, TB prevention in children under 5 years using isoniazid and first-line drug treatment in new pulmonary and extrapulmonary TB cases was respectively US$0.76, US$1.62, US$2.41, US$103 and US$98.CONCLUSION: The unit cost of TB services and outputs are now available to support investment decisions, as diagnosis algorithms are reviewed and prevention or treatment for TB are expanded or updated in India.

Nutritional status of Indian adolescents (15-19 years) from National Family Health Surveys 3 and 4: Revised estimates using WHO 2007 Growth reference.

BACKGROUND: The National Family Health Surveys (NFHS) in India apply adult cutoffs of nutritional status for the estimation of undernutrition/overweight in the 15-19 age group. The prevalence of thinness in boys and girls thus estimated is 58.1% and 46.8% in NFHS-3, and 45% and 42% in NFHS-4 respectively. But the WHO recommends using age and sex-specific reference for adolescents. We reanalyzed the nutritional status of the adolescents using the WHO 2007 Growth Reference to obtain revised estimates of thinness, overweight and stunting across states, rural-urban residence, and wealth quintiles. METHODS AND FINDINGS: Demographic information, anthropometric data, and wealth index were accessed from the Demographic and Health Survey (DHS) database. We re-analyzed the anthropometric data using WHO AnthroPlus software which uses the WHO 2007 Growth reference. The revised estimates of thinness assessed by BMI-for-age z-scores in boys and girls was 22.3% (95%CI: 21.6, 23.0) and 9.9% (95%CI: 9.5, 10.3) in NFHS-3 and 16.5% (95%CI: 16.0,17.0) and 9% (95%CI: 8.9, 9.2) in NFHS-4 respectively. Stunting was found to be 32.2% (95% CI: 31.6, 32.9) in boys and 34.4% (95% CI: 34.2, 34.7) in girls in NFHS-4. This was higher than that in NFHS-3; 25.2% (95% CI: 24.4, 26) in boys and 31.2 (95% CI: 30.6, 31.8) in girls. There was a clear socioeconomic gradient as there were higher thinness and stunting in rural areas. There was wide variation among the states with pockets of a double burden of malnutrition. CONCLUSION: Using the adult cutoffs significantly overestimates thinness in adolescents in the age group of 15-19 years old in India. Stunting, which is an indicator of long term nutrition is also widely prevalent in them. Future editions of DHS and NFHS should consider adolescents as a separate age group for nutritional assessment for a better understanding of nutritional transition in the population.

An assessment study of known pyrazolopyrimidines: Chemical methodology and cellular activity.

Heterocyclic compounds with nitrogen atom play a key role in the normal life cycle of a cell. Pyrazolopyrimidine is a privileged class of nitrogen containing fused heterocyclic compound contributing to a major portion of all lead molecules in medicinal chemistry. The thumbprint of pyrazolopyrimidine as a pharmacophore is always noticeable due to its analogy with the adenine base in DNA. Pyrazolopyrimidines are divided into five types [I, II, III, IV, V] based on the mechanism of action on the specific target conferring a wide scope of research which has accelerated the interest of researchers to investigate its biological profile. In 1956, the anti-cancer activity of pyrazolopyrimidine was evaluated for the first time with appreciable results. Since then, medicinal chemists centered their work on various methods of synthesis and evaluating the biological profile of pyrazolopyrimidine isomers. This report consists of novel methodologies followed to synthesize pyrazolopyrimidine isomers along with a note on their biological significance. To the best of our knowledge, this review article will be first of its kind to encompass different synthetic procedures along with anti-cancer, kinase inhibition, phosphodiesterase inhibition and receptor blocking activity of pyrazolopyrimidine moieties. IC50 values of potent compounds are added wherever necessary to understand the suitability of pyrazolopyrimidine skeletons for a specific biological activity.

International rotations: A valuable source to supplement operative experience for acute-care surgery, trauma, and surgical critical care fellows.

BACKGROUND: Acute-care surgery (ACS), trauma, and surgical critical care (SCC) fellowships graduate fellows deemed qualified to perform complex cases immediately upon graduation. We hypothesize international fellow rotations can be a resource to supplement operative case exposure. METHODS: A survey was sent to all program directors (PDs) of ACS and SCC fellowships via e-mail. Data were captured and analyzed using the REDCap (Research Electronic Data Capture) tool. RESULTS: The survey was sent to 113 PDs, with a response rate of 42%. Most fellows performed less than 150 operative cases (59.5%). The majority of PDs thought the operative exposure either could be improved or was not enough to ensure expertise in trauma and emergent general surgery. Only a minority of the PDs found their case load exceptional (can be improved: 43%, not enough: 30% exceptional: 27%). Most PDs thought an international experience could supplement the breadth of cases, provide research opportunities, and improve understanding of trauma systems (70%). Ten sites offered international rotations (70%). Most fellowships would be willing to provide reciprocity to the host institution (90%). CONCLUSIONS: The majority of PDs for ACS, trauma, and SCC programs perceive a need for increased quality and quantity of operative cases. The majority recognize international fellow rotations as a valuable tool to supplement fellows' education.

DVT surveillance program in the ICU: analysis of cost-effectiveness.

BACKGROUND: Venous Thrombo-embolism (VTE--Deep venous thrombosis (DVT) and/or pulmonary embolism (PE)--in traumatized patients causes significant morbidity and mortality. The current study evaluates the effectiveness of DVT surveillance in reducing PE, and performs a cost-effectiveness analysis. METHODS: All traumatized patients admitted to the adult ICU underwent twice weekly DVT surveillance by bilateral lower extremity venous Duplex examination (48-month surveillance period--SP). The rates of DVT and PE were recorded and compared to the rates observed in the 36-month pre-surveillance period (PSP). All patients in both periods received mechanical and pharmacologic prophylaxis unless contraindicated. Total costs--diagnostic, therapeutic and surveillance--for both periods were recorded and the incremental cost for each Quality Adjusted Life Year (QALY) gained was calculated. RESULTS: 4234 patients were eligible (PSP--1422 and SP--2812). Rate of DVT in SP (2.8%) was significantly higher than in PSP (1.3%) - p<0.05, and rate of PE in SP (0.7%) was significantly lower than that in PSP (1.5%) - p<0.05. Logistic regression demonstrated that surveillance was an independent predictor of increased DVT detection (OR: 2.53 - CI: 1.462-4.378) and decreased PE incidence (OR: 0.487 - CI: 0.262-0.904). The incremental cost was $509,091/life saved in the base case, translating to $29,102/QALY gained. A sensitivity analysis over four of the parameters used in the model indicated that the incremental cost ranged from $18,661 to $48,821/QALY gained. CONCLUSIONS: Surveillance of traumatized ICU patients increases DVT detection and reduces PE incidence. Costs in terms of QALY gained compares favorably with other interventions accepted by society.

A multicenter prospective hospital-based surveillance to estimate the burden of rotavirus gastroenteritis in children less than five years of age in India.

BACKGROUND: Rotavirus is the leading cause of severe, dehydrating diarrhea in children aged <5 years globally, with an estimated 25 million outpatient visits and 2 million hospitalizations attributable to rotavirus infections each year. The aim of this hospital-based surveillance was to summarize the local epidemiological and virological features of rotavirus and to estimate the disease burden in the population under surveillance in India. METHODS: During the 16 months surveillance period from April 2011 through July 2012, a total of 4711 children under the age of 5 years were admitted with acute diarrhea at 12 medical centers attached to medical schools throughout India. Stool samples were randomly collected from 2051 (43.5%) subjects and were analyzed for rotavirus positivity using commercial enzyme immunoassay kit (Premier Rotaclone Qualitative Elisa) at the respective study centers. Rotavirus positive samples were genotyped for VP7 and VP4 by reverse-transcription polymerase chain reaction (RT-PCR) at a central laboratory. RESULTS: During the study period, maximum number of rotavirus related hospitalizations were reported from December 2011 through February 2012. Out of the 2051 stool samples tested for rotavirus, overall 541 (26.4%) samples were positive for rotavirus VP6 antigen in stool. The highest positivity was observed in the month of December, 2011 (52.5%) and lowest in the month of May, 2011 (10.3%). We found that majority of the rotavirus positive cases (69.7%) were in children <24 months of age. The most common genotypes reported were G1 (38%), G2 (18%), G9 (18%), G12 (9%) and mixed strains (17%). CONCLUSIONS: The results of this study confirm the significant burden of acute rotavirus gastroenteritis as a cause of hospitalizations in under five children in India.

LC-Q-TOF-MS/MS determination of darunavir and its metabolites in rat serum and urine: application to pharmacokinetics.

A simple, rapid and reliable liquid chromatography coupled with quadrupole time of flight mass spectrometry (LC-Q-TOF-MS/MS) method was developed and validated for simultaneous determination of darunavir and its metabolites in rat serum and urine. The separation was accomplished on an Agilent RP-18 (250×4.6mm, 5µm) column using 20mM ammonium acetate and methanol (40:60, v/v) as a mobile phase at a flow rate of 1.0mL/min in an isocratic mode. The [M+H](+) ions of darunavir (m/z 548) and metabolites-I (m/z 392) were monitored in positive mode of ionization, while [M-H](-) ion of metabolite-II (m/z 172) in negative mode selectively. The matrix effects of rat serum and urine were found to be negligible and the recoveries were 87-93% for all the analytes. The short and long term stability of darunavir and its metabolites was within acceptable limits and the lower limits of quantification were in the range of 3.63-5.24ng/mL with a linear range of 5-5000ng/mL in rat serum as well as urine. The method exhibited good intra- and inter-day performance in terms of 2.54-8.92% precision and 0-5% accuracy. The method was successfully applied to a single-dose pharmacokinetic study of darunavir boosted with ritonavir in Wistar rats.

Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins.

Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~100,281) in a large set of eukaryotic proteins (~22,995). Phosphorylation probability was found to be much higher in both the termini of protein sequences and this is much pronounced in transmembrane proteins. A large proportion (51.3%) of occupied sites had a nearby phosphorylation within a distance of 10 amino acids; however, this proportion is very high compared to the expected one (16.9%). The distribution of phosphorylated sites in proteins showed a strong deviation from the expected maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.

Brief violence interventions with community case management services are effective for high-risk trauma patients.

BACKGROUND: Currently there are few data that brief violence intervention (BVI) and community case management services (CCMS) are effective for trauma patients admitted for interpersonal violence in terms of recidivism, service utilization, or alcohol abuse. The objective of this study is to assess outcomes for a cohort of young trauma patients in a prospective, randomized trial comparing BVI with BVI + CCMS. METHODS: Intentionally injured patients, aged 10 years to 24 years, admitted to a Level I trauma center were randomized to receive a brief in-hospital psychoeducational violence intervention alone (Group I) or in combination with a 6 months wraparound CCMS (Group II) that included vocational, employment, educational, housing, mental health, and recreational assistance. Recidivism, alcohol use, and hospital and community service utilization were assessed at 6 weeks (6W) and 6 months (6M). RESULTS: Seventy-five of 376 eligible injured patients were randomized into Group I and II. The two groups had similar demographics, injuries, and clinical outcomes. After discharge, percent clinic visits maintained was 57% in both the groups. Group II showed better hospital service utilization, CMS, and risk factor reduction at 6W and 6M. One patient in each group sustained a reinjury at 6M. CONCLUSIONS: In-hospital BVI with community wraparound case management interventions can improve hospital and community service utilization both short- and long-term for high-risk injured patients. Longer follow-up is needed to show sustained reduction.

Biomarkers and personalised medicine in rheumatoid arthritis: a proposal for interactions between academia, industry and regulatory bodies.

Rheumatoid arthritis (RA) is one of the most appropriate conditions for the application of personalised medicine as a high degree of heterogeneity has been recognised, which remains to be explained. Such heterogeneity is also reflected in the large number of treatment targets and options. A growing number of biologics as well as small molecules are already in use and there are promising new drugs in development. In order to make the best use of treatment options, both targeted and non-targeted biomarkers have to be identified and validated. To this aim, new rules are needed for the interaction between academia and industry under regulatory control. Setting up multi-centre biosample collections with clear definition of access, organising early, possibly non-committing discussions with regulatory authorities, and defining a clear route for the validation, qualification and registration of the biomarker-drug combination are some of the more critical areas where effective collaboration between the drug industry, academia and regulators is needed.

A central venous line protocol decreases bloodstream infections and length of stay in a trauma intensive care unit population.

We evaluated the benefit of a central venous line (CVL) protocol on bloodstream infections (BSIs) and outcome in a trauma intensive care unit (ICU) population. We prospectively compared three groups: Group 1 (January 2003 to June 2004) preprotocol; Group 2 (July 2004 to June 2005) after the start of the protocol that included minimizing CVL use and strict universal precautions; and Group 3 (July 2005 to December 2006) after the addition of a line supply cart and nursing checklist. There were 1622 trauma patients admitted to the trauma ICU during the study period of whom 542 had a CVL. Group 3 had a higher Injury Severity Score (ISS) compared with both Groups 2 and 1 (28.3 +/- 13.0 vs 23.5 +/- 11.7 vs 22.8 +/- 12.0, P = 0.0002) but had a lower BSI rate/1000 line days (Group 1: 16.5; Group 2: 15.0; Group 3: 7.7). Adjusting for ISS group, three had shorter ICU length of stay (LOS) compared with Group 1 (12.11 +/- 1.46 vs 18.16 +/- 1.51, P = 0.01). Logistic regression showed ISS (P = 0.04; OR, 1.025; CI, 1.001-1.050) and a lack of CVL protocol (P = 0.01; OR, 0.31; CI, 0.13-0.76) to be independent predictors of BSI. CVL protocols decrease both BSI and LOS in trauma patients. Strict enforcement by a nurse preserves the integrity of the protocol.

Enantioselective separation and determination of adrafinil and modafinil on Chiralcel OJ-H column in rat serum and urine using solid-phase extraction followed by HPLC.

A simple and rapid normal-phase HPLC method for enantiospecific separation of a psychostimulant, adrafinil (ADL), and its metabolite modafinil (MDL) in rat serum and urine was developed. The separation was accomplished on a normal-phase polysaccharide stationary phase Chiralcel OJ-H using n-hexane-ethanol (62:38 v/v) as a mobile phase at a flow rate of 1.0 mL/min. Detection was carried out at 225 nm using a photo diode array (PDA) detector. The elution order of the enantiomers was determined by a polarimeter connected in series with the PDA. ADL and its metabolite were recovered from rat serum and urine by solid phase extraction using Oasis HLB cartridges and the mean recoveries were >or=80%. The enantiomers were eluted within 15 min without any interference from endogenous substances. The calibration curves were linear (r(2) > 0.998) in the concentration range of 1.20-500 microg/mL for ADL and MDL. The assay was specific, accurate, precise and reproducible (intra- and inter-day precisions RSDs <7.2%). ADL in rat serum was stable over three freeze-thaw cycles at ambient temperature for 4 h. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.

Rapid determination of rifaximin in rat serum and urine by direct injection on to a shielded hydrophobic stationary phase by HPLC.

A simple and rapid reversed-phase HPLC method for determination of rifaximin in rat serum and urine was developed. Separation of rifaximin from biological matrix was achieved by direct injection of rat serum and urine onto a restricted-access medium, Supelco LC-Hisep, a shielded hydrophobic stationary phase, using acetonitrile:water:acetic acid (18:82:0.1 v/v/v) as a mobile phase. The linear range was 0.10-20 microg/mL (r(2 )> 0.999, n = 6), intraday and interday variation was <6.10%. The limits of detection and quantification were 0.03 (signal-to-noise ratio >3) and 0.10 microg/mL (signal-to-noise ratio >10), respectively. The method was successfully applied to pharmacokinetic studies of rifaximin after an oral administration to rats.

Is the lateral cervical spine plain film obsolete?

BACKGROUND: The objective of this study was to determine the utility of a lateral cervical spine plain film in the evaluation of blunt trauma patients. METHODS: We prospectively evaluated blunt trauma patients from February 2004 to September 2006 who had both a lateral cervical spine (LCS) film and a computed tomography of the cervical spine (CTC), comparing the diagnostic accuracy of the LCS to the CTC. RESULTS: There were 1004 patients who met inclusion criteria. Eighty-four patients had a cervical spine fracture while 920 patients had no fracture on CTC. Of the 84 patients with fractures by CTC, 68 had a negative or incomplete LCS. Of the 920 negative CTC, there were 7 false positive LCSs. LCS compared with CTC showed a sensitivity of 19% (16/84) and positive predictive value of 69.6% (16/23). Of the 981 negative or incomplete LCS films, 96.9% were incomplete (951/981). Of the seven patients with a false positive LCS (negative CTC), none was subsequently found to have a cervical spine fracture on further evaluation. Elimination of the LCS would result in charge savings of $265,056.00 (LCS charges with interpretation, $264 each) and increase patient safety by eliminating error. CONCLUSIONS: LCS has no value as a screening tool in the blunt trauma patient since most are either inaccurate or incomplete. It should be eliminated from the Advanced Trauma Life Support algorithm, and CTC should receive emphasis as the diagnostic gold standard.

Assessment of serum macro and trace element homeostasis and the complexity of inter-element relations in bipolar mood disorders.

BACKGROUND: Bipolar disorders are complex neuropsychiatric in nature and are clinically classified as Type I, Type II, and Type V. The etiological factors include environmental-genetic inter-relations. Trace metals play a significant role in neurological disorders. There is very limited information on the role of macro and trace elements in bipolar disorders. METHODS: Trace elements namely Na, K, S, Ca, Mg, P, Cu, Fe, Zn, Mn and Al were analyzed in serum samples of 3 bipolar types: bipolar I, bipolar II and bipolar V with a control group using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The patients were assessed as per the standard diagnostic criteria and classified into the bipolar type I, II hypomanic, II depressives and V. RESULTS: In bipolar I (mania), Na, K, P, Cu, Al and Mn were increased significantly (p<0.001). In bipolar II hypomania, Na, S, Al and Mn were increased significantly (p<0.02), while in bipolar II depression, Na, K, Cu and Al were increased (p<0.001). In bipolar V, Na, Mg, P, Cu, and Al were increased significantly (p<0.002), though S (p<0.00001), Fe (p<0.002) and Zn (p<0.004) were decreased in all 3 bipolar groups. CONCLUSIONS: There is a disturbance in the charge distribution and element-element interdependency in bipolar serum when compared to controls. These results suggest that there is a definite imbalance in macro and trace element homeostasis as evidenced by element inter-relationships in serum samples of bipolar groups when compared to controls.

Determination of antibiotics in aquatic environment by solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometry.

A robust and sensitive solid-phase extraction followed by liquid chromatography-electrospray ionization mass spectrometric (LC-ESI-MS) method for determination of antibiotics viz., fluoroquinolones, sulfamethoxazole, trimethoprim and cephalosporines in surface waters was developed. The sample recoveries on Oasis HLB cartridges were found to be >80%. Identification was carried out by LC-ESI-MS/MS. The positive ion ESI mass spectra containing the peaks of quasimolecular ions [M+H](+) allowed the determination of molecular masses whereas the fragment ions obtained by MS/MS of [M+H](+) ions permitted the structural assignment. Quantification was carried out by selective ion monitoring (SIM) using the quasimolecular ions [M+H](+) of the parent compounds. The detection and quantification limits were found to be in the range of 0.6-8.1 and 2.0-24.0 microg/L. The surface waters of different lakes and tanks of Hyderabad, India were found to contain a few antibiotics.

Development of a validated RP-LC/ESI-MS-MS method for separation, identification and determination of related substances of tamsulosin in bulk drugs and formulations.

A reversed-phase high performance liquid chromatographic (RP-HPLC) method for evaluation of purity of tamsulosin in bulk drugs and pharmaceuticals was developed. The separation was accomplished on an Inertsil C(18) column using 10 mM ammonium acetate: acetonitrile as a mobile phase in a gradient elution mode. A photodiode array detector set at 280 nm was used for detection. The impurities were identified by ESI-MS-MS. The detection limits were 0.06-0.11 microg/ml. The method was validated with respect to accuracy, precision, linearity, ruggedness and limits of detection and quantification. It finds application not only for monitoring the reactions during the process development but also on quality assurance of tamsulosin.

Xylitol production from corn fiber and sugarcane bagasse hydrolysates by Candida tropicalis.

A natural isolate, Candida tropicalis was tested for xylitol production from corn fiber and sugarcane bagasse hydrolysates. Fermentation of corn fiber and sugarcane bagasse hydrolysate showed xylose uptake and xylitol production, though these were very low, even after hydrolysate neutralization and treatments with activated charcoal and ion exchange resins. Initial xylitol production was found to be 0.43 g/g and 0.45 g/g of xylose utilised with corn fiber and sugarcane bagasse hydrolysate respectively. One of the critical factors for low xylitol production was the presence of inhibitors in these hydrolysates. To simulate influence of hemicellulosic sugar composition on xylitol yield, three different combinations of mixed sugar control experiments, without the presence of any inhibitors, have been performed and the strain produced 0.63 g/g, 0.68 g/g and 0.72 g/g of xylose respectively. To improve yeast growth and xylitol production with these hydrolysates, which contain inhibitors, the cells were adapted by sub culturing in the hydrolysate containing medium for 25 cycles. After adaptation the organism produced more xylitol 0.58 g/g and 0.65 g/g of xylose with corn fiber hydrolysate and sugarcane bagasse hydrolysate respectively.