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The rumen harbors a complex microbial mixture of archaea, bacteria, protozoa and fungi that efficiently breakdown plant biomass and its complex dietary carbohydrates into soluble sugars that can be fermented and subsequently converted into metabolites and nutrients utilized by the host animal. While rumen bacterial populations have been well documented, only a fraction of the rumen eukarya are taxonomically and functionally characterized, despite the recognition that they contribute to the cellulolytic phenotype of the rumen microbiota. To investigate how anaerobic fungi actively engage in digestion of recalcitrant fiber that is resistant to degradation, we resolved genome-centric metaproteome and metatranscriptome datasets generated from switchgrass samples incubated for 48 hours in nylon bags within the rumen of cannulated dairy cows. Across a gene catalogue covering anaerobic rumen bacteria, fungi and viruses, a significant portion of the detected proteins originated from fungal populations. Intriguingly, the carbohydrate-active enzyme (CAZyme) profile suggested a domain-specific functional specialization, with bacterial populations primarily engaged in the degradation of polysaccharides such as hemicellulose, whereas fungi were inferred to target recalcitrant cellulose structures via the detection of a number of endo- and exo-acting enzymes belonging to the glycoside hydrolase (GH) family 5, 6, 8 and 48. Notably, members of the GH48 family were amongst the highest abundant CAZymes and detected representatives from this family also included dockerin domains that are associated with fungal cellulosomes. A eukaryote-selected metatranscriptome further reinforced the contribution of uncultured fungi in the ruminal degradation of recalcitrant fibers. These findings elucidate the intricate networks ofin siturecalcitrant fiber deconstruction, and importantly, suggests that the anaerobic rumen fungi contribute a specific set of CAZymes that complement the enzyme repertoire provided by the specialized plant cell wall degrading rumen bacteria.

This paper presents an image enhancement technique based on super-resolution approach. The method uses fractional filters and reconstructs the output image by projection on convex sets (POCS) method. First, we generate a reference frame by using low-resolution frames and enhanced it by an adaptive fractional mask. Then the speed-up robust feature (SURF) is used to find the matching between low-resolution frames and the reference frame. Finally, the residuals between matching are reduced by the POCS reconstruction approach. To recover the high-frequency components, we have used a fractional integral mask in the POCS reconstruction process. We have compared the experimental results with some other existing methods from literature. Simulation results show that the proposed approach is efficient and returns a good quality image.

This paper presents a new image denoising algorithm based on fractional filters. The fractional filters are derived using a newly introduced fractional operator. The proposed algorithm identifies the noisy pixels based on pixel-density and upgrades them by an adaptive fractional integral mask. To maintain the correlation and recover the lost information, the noise-free pixels are also processed by an adaptive fractional differential mask. We formulate the order function for the fractional mask with the help of gradient features and variance of the image. The algorithm is applied to standard images of different characteristics. The experimental results are compared with some other existing techniques. Evaluation parameters and visual perceptions show that the proposed method performs better than most of the discussed methods. The proposed approach is applicable for image denoising due to its applicability over different types of noises and denoising performance.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2–180 μmol/kg/d) of benzo[a]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site. In hepatic microsomes from exposed mice, we measured amounts of active CYPs using activity-based protein profiling and total CYP expression using global proteomics. We observed rapid Cyp1a1 induction after 6 hr at the lowest PAH exposures and broad induction of many CYPs after 3 daily PAH doses at 72 hr following the first dose. Using samples displaying Cyp1a1 induction, we observed significantly higher metabolic affinity for BaP metabolism (K(m) reduced 3-fold), 3-fold higher intrinsic clearance, but no changes to the V(max). Mice dosed with the highest PAH exposures exhibited 1.7 to 5-fold higher intrinsic clearance rates for BaP compared to controls and higher V(max) values indicating greater amounts of enzymes capable of metabolizing BaP. This study demonstrates exposure to PAHs found at Superfund Sites induces enzymes in dose- and time-dependent patterns in mice. Accounting for specific changes in enzyme profiles, and relative rates of PAH bioactivation and detoxification, and resulting risk will help translate internal dosimetry of animal models to humans and improve risk assessments of PAHs at Superfund sites.

Purpose This paper aims to present a high-order scheme to approximate generalized derivative of Caputo type for μ ∈ (0,1). The scheme is used to find the numerical solution of generalized fractional advection-diffusion equation define in terms of the generalized derivative. Design/methodology/approach The Taylor expansion and the finite difference method are used for achieving the high order of convergence which is numerically demonstrated. The stability of the scheme is proved with the help of Von Neumann analysis. Findings Generalization of fractional derivatives using scale function and weight function is useful in modeling of many complex phenomena occurring in particle transportation. The numerical scheme provided in this paper enlarges the possibility of solving such problems. Originality/value The Taylor expansion has not been used before for the approximation of generalized derivative. The order of convergence obtained in solving generalized fractional advection-diffusion equation using the proposed scheme is higher than that of the schemes introduced earlier.

This paper presents a new algorithm for image denoising using a fractional integral mask of the K-operator. K-operator is the generalized fractional operator, and it reduces to Riemann–Liouville and Caputo fractional derivatives in a special case. The proposed algorithm is applied to digital images of different nature to demonstrate the performance of image denoising. Experimental results are compared with other existing filters together with block matching and 3-D filtering, and weighted nuclear norm minimization-based approaches. The obtained experimental results show that the proposed algorithm is computationally efficient and its average performance is comparatively better than other discussed methods.

Protein S-glutathionylation is an important reversible post-translational modification implicated in redox signaling. Oxidative modifications to protein thiols can alter the activity of metabolic enzymes, transcription factors, kinases, phosphatases, and the function of contractile proteins. However, the extent to which muscle contraction induces oxidative modifications in redox sensitive thiols is not known. The purpose of this study was to determine the targets of S-glutathionylation redox signaling following fatiguing contractions. Anesthetized adult male CB6F1 (BALB/cBy × C57BL/6) mice were subjected to acute fatiguing contractions for 15 min using in vivo stimulations. The right (stimulated) and left (unstimulated) gastrocnemius muscleswere collected 60 min after the last stimulation and processed for redox proteomics assay of S-glutathionylation. Using selective reduction with a glutaredoxin enzyme cocktail and resin-assisted enrichment technique, we quantified the levels of site-specific protein S-glutathionylation at rest and following fatiguing contractions. Redox proteomics revealed over 2200 sites of S-glutathionylation modifications, of which 1290 were significantly increased after fatiguing contractions. Muscle contraction leads to the greatest increase in S-glutathionylation in the mitochondria (1.03%) and the smallest increase in the nucleus (0.47%). Regulatory cysteines were significantly S-glutathionylated on mitochondrial complex I and II, GAPDH, MDH1, ACO2, and mitochondrial complex V among others. Similarly, S-glutathionylation of RYR1, SERCA1, titin, and troponin I2 are known to regulate muscle contractility and were significantly S-glutathionylated after just 15 min of fatiguing contractions. The largest fold changes (> 1.6) in the S-glutathionylated proteome after fatigue occurred on signaling proteins such as 14-3-3 protein gamma and MAP2K4, as well as proteins like SERCA1, and NDUV2 of mitochondrial complex I, at previously unknown glutathionylation sites. These findings highlight the important role of redox control over muscle physiology, metabolism, and the exercise adaptive response. This study lays the groundwork for future investigation into the altered exercise adaptation associated with chronic conditions, such as sarcopenia. Keywords: Muscle contraction, Post-translational modification, S-glutathionylation, Redox signaling, Skeletal muscle, Thiol redox proteomics

Biochemical networks mediating normal lung morphogenesis and function have important implications for ameliorating morbidity and mortality in premature infants. Although several transcript-level studies have examined normal lung development, corresponding protein-level analyses are lacking. Here we performed proteomics analysis of murine lungs from embryonic to early adult ages to identify the molecular networks mediating normal lung development. We identified 8,932 proteins, providing a deep and comprehensive view of the lung proteome. Analysis of the proteomics data revealed discrete modules and the underlying regulatory and signaling network modulating their expression during development. Our data support the cell proliferation that characterizes early lung development and highlight responses of the lung to exposure to a nonsterile oxygen-rich ambient environment and the important role of lipid (surfactant) metabolism in lung development. Comparison of dynamic regulation of proteomic and recent transcriptomic analyses identified biological processes under posttranscriptional control. Our study provides a unique proteomic resource for understanding normal lung formation and function and can be freely accessed at Lungmap.net.

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nano-droplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

Given the wide-ranging potential applications of metal organic frameworks (MOFs), an emerging imperative is to understand their formation with atomic scale precision. This will aid in designing syntheses for next-generation MOFs with enhanced properties and functionalities. Major challenges are to characterize the early-stage seeds, and the pathways to framework growth, which require synthesis coupled with in situ structural characterization sensitive to nanoscale structures in solution. Here we report measurements of an in situ synthesis of a prototypical MOF, ZIF-8, utilizing synchrotron X-ray atomic pair distribution function (PDF) analysis optimized for sensitivity to dilute species, complemented by mass spectrometry, electron microscopy, and density functional theory calculations. We observe that despite rapid formation of the crystalline product, a high concentration of Zn(2-MeIm)4 (2-MeIm = 2-methylimidazolate) initially forms and persists as stable clusters over long times. A secondary, amorphous phase also pervades during the synthesis, which has a structural similarity to the final ZIF-8 and may act as an intermediate to the final product.

Introduction: Breast cancer (BC) is the second most common cause of cancer death among women in the USA. Recent proteogenomic studies of human BC have identified many potential therapeutic biomarkers for the most common types of BC. Here, we strive to comprehensively understand the proteogenomic landscape of difficult-to-treat breast cancer (DTBC) tumors. Methods: 118 primary breast tumors, with a focus on DTBC patients, were selected from the IRB-approved protocol. The study cohort included 30 triple negative, 16 HER2+, 16 Luminal A, 39 Luminal B1, and 17 Luminal B2 selected by immunohistochemistry. Breast tumors were embedded in OCT (Optimum Cutting Temperature) and processed by laser microdissection (LMD) to remove non-tumorous tissue. DNA, RNA, and protein were simultaneously extracted from each tumor using the illustra triplePrep kit. Paired-end RNA sequencing and whole genome sequencing (WGS) were performed for 118 and 100 tumors, respectively, using the Illumina HiSeq platform. Quantitative global proteomics and phosphoproteomic analyses were performed on 113 and 50 tumors, respectively, using isobaric TMT 6-plex labeling with the “universal reference” strategy. Results: Unlike other BC cohorts, our WGS cohort had an almost equal number of Estrogen(ER)+ (55.6%) and ER- (44.4%) cases, which gave us a nominally higher percentage TP53 (57%) and lower PIK3CA (32%) non-silent somatic mutation. Similar to previous reports, GATA3 was mutated in 13% of only ER+ cases. Unlike a recent report on proteomics clustering of bulk-processed tumors, our data show that a stromal-enriched cluster was not mixed with all subtypes but a majority of Luminal A (LumA) subtype, probably because LMD excluded stromal components in other tumor subtypes. Consensus clustering of proteomics data separated intrinsic Her2 patients with outcome differences. Her2 patients associated with the Basal-enriched proteome cluster had better survival than those associated with the LumA-enriched proteome cluster. Previously reported marker PGK1, a predictor for poor survival in BC, was upregulated in Her2 tumors associated with the LumA-enriched proteome cluster. The clustering of phosphoproteomics data separated intrinsic Basal patients into groups with and without relapse. We identified 3 and 18 genes with upregulated kinase activities in the relapse-free and relapsed Basal patients, respectively. In conclusion, this study provides a high-quality proteogenomic resource for DTBC investigation and identifies potential molecular markers for predicting outcomes for patients with less responsive tumors. The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, DoD, USUHS, HJF. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government. Citation Format: Praveen-Kumar Raj-Kumar, Tao Liu, Lori A. Sturtz, Albert J. Kovatich, Marina A. Gritsenko, Vladislav A. Petyuk, Brenda Deyarmin, Jianfang Liu, Anupama Praveen-Kumar, Jason E. McDermott, Anil K. Shukla, Ronald J. Moore, Matthew E. Monroe, Bobbie-Jo M. Webb-Robertson, Jeffrey A. Hooke, Leigh Fantacone-Campbell, Leonid Kvecher, Jennifer Kane, Jennifer Melley, Stella Somiari, Stephen C. Benz, Justin Golovato, Shahrooz Rabizadeh, Patrick Soon-Shiong, Richard D. Smith, Richard J. Mural, Karin D. Rodland, Craig D. Shriver, Zhaoyu Li, Hai Hu. Proteogenomics of laser microdissected difficult-to-treat breast cancers identifies Basal and Her2 patients associated with outcome differences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2172.

Dissociation of methanol molecular cations, CH 3 OH + , to CH 2 OH + on collision with a self-assembled monolayer surface of fluorinated alkyl thiol on gold 111 crystal has been studied at 12.5 eV collision energy. Two energetically and spatially distinct processes contribute to the dissociation process: one involving loss of very large amount of energy approaching the initial kinetic energy of the primary ions with scattering of fragment ions over a broad angular range between surface normal and surface parallel while the second process results from small amount of energy loss with fragment ions scattered over a narrow angular range close to the surface parallel. There is a third process with relatively small contribution to total dissociation whose characteristics are very similar to the low energy loss process. These results demonstrate that surface-induced dissociation of methanol cations via hydrogen loss is non-ergodic.

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding “G site” and a substrate binding “H site”. Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the “G site”, and (2) an ABP designed to mimic a substrate molecule and have “H site” activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.

Electrospray ionization of alkali halide solutions in the negative ion mode results in the formation of cluster ions of the general formula, (MX) n X − . However, alkali iodides form triiodide anion, I 3 − , in high abundance in addition to cluster ions. Br 3 − ions are observed in low abundance. Also, mixed tri-halide anions, I 2 Y − , are observed in high abundance when a small amount (

Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. Herein, we report use of long (≥1M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5MPa or 14Kpsi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5-5μm particles were used as packings and long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50kDa. Larger proteoforms (50-110kDa) were chromatographed on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for10kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200-400Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ∼900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC-MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Our initial data indicate that MS detection and fragmentation inefficiencies provided by current high-resolution mass spectrometers are key challenges for characterization of larger proteoforms.

ObjectiveThe aim of this study is to identifyin vivothe main blood supply to the bony chin and to determine the efficacy of ultrasound Doppler in determining this aspect and then compare it with radiograph.Materials and methodsThe study was carried on 50 subjects divided into two equal groups. In the first group, the lingual foramen was identitifed on intraoral periapical (IOPA) radiograph of the mandibular incisor region. Ultrasound Doppler identification of blood supply to the anterior part of the mandible was carried out. In the second group of 25 subjects, the procedure was reversed. This was done to avoid procedural bias.DiscussionIn our patients, the midline artery entering the lingual foramen was seen by ultrasound and radiograph. Their average diameters and velocities were measured. It was identified in 46 cases by the ultrasound Doppler examination. The IOPA radiographic examination was successful in identifying the foramen in 30 cases. The average diameter of the midline artery entering the lingual foramen was found to be 0.14 cm. The average velocity of blood flow in this artery was found to be 25.5 cm/second.ConclusionThe ultrasound Doppler is a reliable tool to visualize and measure the blood supply to the anterior part of the mandible.How to cite this articleKiran MS, Manjunath M, Shukla AK, Vidya S. Reliability of Ultrasound in the Identification and Measurement of Blood Supply to the Anterior Part of the Mandible. J Med Sci 2017;3(1):20-24.

This abstract was not presented at the symposium.

Phycobilisomes (PBSs) are large (3&ndash, 5 megadalton) pigment-protein complexes in cyanobacteria that associate with thylakoid membranes and harvest light primarily for photosystem II. PBSs consist of highly ordered assemblies of pigmented phycobiliproteins (PBPs) and linker proteins that can account for up to half of the soluble protein in cells. Cyanobacteria adjust to changing environmental conditions by modulating PBS size and number. In response to nutrient depletion such as nitrogen (N) deprivation, PBSs are degraded in an extensive, tightly controlled, and reversible process. In Synechococcus elongatus UTEX 2973, a fast-growing cyanobacterium with a doubling time of two hours, the process of PBS degradation is very rapid, with 80% of PBSs per cell degraded in six hours under optimal light and CO2 conditions. Proteomic analysis during PBS degradation and re-synthesis revealed multiple proteoforms of PBPs with partially degraded phycocyanobilin (PCB) pigments. NblA, a small proteolysis adaptor essential for PBS degradation, was characterized and validated with targeted mass spectrometry. NblA levels rose from essentially 0 to 25,000 copies per cell within 30 min of N depletion, and correlated with the rate of decrease in phycocyanin (PC). Implications of this correlation on the overall mechanism of PBS degradation during N deprivation are discussed.

ER-positive breast tumors represent ∼70#x0025; of all breast cancer cases. Although their treatment with endocrine therapies is effective in the adjuvant or recurrent settings, the development of resistance compromises their effectiveness. The binding of estrogen to ERα, a transcription factor, triggers the regulation of the target genes (genomic pathway). Additionally, a cytoplasmic fraction of estrogen-bound ERα activates oncogenic signaling pathways such as PI3K/AKT/mTOR (nongenomic pathway). The upregulation of the estrogenic and the PI3K/AKT/mTOR signaling pathways are frequently associated with a poor outcome. To better characterize the connection between these two pathways, we performed a phosphoproteome analysis of ER-positive MCF7 breast cancer cells treated with estrogen or estrogen and the mTORC1 inhibitor rapamycin. Many proteins were identified as estrogen-regulated mTORC1 targets and among them, DEPTOR was selected for further characterization. DEPTOR binds to mTOR and inhibits the kinase activity of both mTOR complexes mTORC1 and mTORC2, but mitogen-activated mTOR promotes phosphorylation-mediated DEPTOR degradation. Although estrogen enhances the phosphorylation of DEPTOR by mTORC1, DEPTOR levels increase in estrogen-stimulated cells. We demonstrated that DEPTOR accumulation is the result of estrogen-ERα-mediated transcriptional upregulation of DEPTOR expression. Consequently, the elevated levels of DEPTOR partially counterbalance the estrogen-induced activation of mTORC1 and mTORC2. These results underscore the critical role of estrogen-ERα as a modulator of the PI3K/AKT/mTOR signaling pathway in ER-positive breast cancer cells. Additionally, these studies provide evidence supporting the use of dual PI3K/mTOR or dual mTORC1/2 inhibitors in combination with endocrine therapies as a first-line treatment option for the patients with ER-positive advanced breast cancer.

Acute and chronic exposure to organophosphates (OPs), including agricultural pesticides, industrial chemicals, and chemical warfare agents, remain a significant worldwide health risk. The mechanisms by which OPs alter development and cognition in exposed individuals remain poorly understood, in part due to the large number of structurally diverse OPs and the wide range of affected proteins and signaling pathways. To investigate the influence of structure on OP targets in mammalian systems, we have developed a series of probes for activity-based protein profiling (ABPP) featuring two distinct reactive groups that mimic OP chemical reactivity. FOP features a fluorophosphonate moiety, and PODA and CODA utilize a dialkynyl phosphate ester; both reactive group types target serine hydrolase activity. As the oxon represents the highly reactive and toxic functional group of many OPs, the new probes described herein enhance our understanding of tissue-specific reactivity of OPs. Chemoproteomic analysis of mouse tissues treated with the probes revealed divergent protein profiles, demonstrating the influence of probe structure on protein targeting. These targets also vary in sensitivity towards different OPs. The simultaneous use of multiple probes in ABPP experiments may therefore offer more comprehensive coverage of OP targets; FOP consistently labeled more targets in both brain and liver than PODA or CODA, suggesting the dialkyne warhead is more selective for enzymes in major signaling pathways than the more reactive fluorophosphonate warhead. Additionally, the probes can be used to assess reactivation of OP-inhibited enzymes by N-oximes and may serve as diagnostic tools for screening of therapeutic candidates in a panel of protein targets. These applications will help clarify the short- and long-term effects of OP toxicity beyond acetylcholinesterase inhibition, investigate potential points of convergence for broad spectrum therapeutic development, and support future efforts to screen candidate molecules for efficacy in various model systems.

Commensal microorganisms in the mammalian gut play important roles in host health and physiology, but a central challenge remains in achieving a detailed mechanistic understanding of specific microbial contributions to host biochemistry. New function-based approaches are needed that analyze gut microbial function at the molecular level by coupling detection and measurements of in situ biochemical activity with identification of the responsible microbes and enzymes. We developed a platform employing β-glucuronidase selective activity-based probes to detect, isolate, and identify microbial subpopulations in the gut responsible for this xenobiotic metabolism. We find that metabolic activity of gut microbiota can be plastic and that between individuals and during perturbation, phylogenetically disparate populations can provide β-glucuronidase activity. Our work links biochemical activity with molecular-scale resolution without relying on genomic inference.

RATIONALE Electrospray ionization (ESI) results in the formation of multiply charged cluster ions which then fragment via two competing processes: fission (charge separation) via Coulomb repulsion, and evaporation of one or more neutrals from the fragmenting ion, as described by Rayleigh's liquid drop model. Unimolecular and collision-induced dissociation (CID) of doubly charged lithium acetate clusters, (CH3 COOLi)n Li2 (2+) , demonstrated that Coulomb fission via charge separation is the dominant dissociation process with no contribution from the neutral evaporation processes for all such ions from the critical limit to larger cluster ions, although the latter process was normally observed in most earlier studies of multiply charged clusters. METHODS Doubly and higher charged lithium acetate cluster ions were generated by ESI. Unimolecular and CID of mass selected doubly charged cluster ions above the critical limit were recorded at high resolution using an LTQ Velos Orbitrap mass spectrometer. RESULTS Unimolecular and collision-induced dissociation of doubly charged lithium acetate clusters proceed via symmetric fission processes and the intensity of the fragment ions decreases as the cluster size increases. Dissociations via neutral evaporation of a monomer or larger clusters are not observed. CONCLUSIONS Doubly charged clusters of lithium acetate above the critical limit dissociate via symmetrical fission processes and not neutral evaporation. These results are clearly in disagreement with the Rayleigh liquid drop model that has been used previously to predict the critical size and explain the fragmentation behavior of multiply charged clusters. Copyright © 2016 John Wiley & Sons, Ltd.

Gluteal hernias are extremely uncommon and occur as a result of deficiency or defect in the gluteal musculature. Our case reports one such incidence in a one year old girl with review of available literature. The clinical presentation and imaging findings are discussed comprehensively. We thus lay emphasis on the importance of real-time ultrasonography as the method of choice to identify hernia contents, peristalsis and obstruction if any.

Primary tumors of the diaphragm are quite rare. About 150 cases have been reported in the literature. Fibrosarcomas are the most common malignant neoplasms of the diaphragm; however, only a few (less than 20) cases have been reported to date. We present a case of an extremely rare tumor of the diaphragm. A 65-year-old woman presented with history of vague upper abdominal pain since 2 months and distension for 2 weeks. Ultrasonography features were in favor of a mass arising from left dome of diaphragm with evidence of vascularity on Doppler; lesion was displacing spleen inferiorly. Contrast-enhanced computed tomography scan of the abdomen revealed a mass located in the region of the left dome of diaphragm and deriving blood supply from the branches of abdominal aorta. Surgical excision was planned, keeping in mind the diagnosis of a left diaphragmatic tumor. Laparotomy revealed a left diaphragmatic tumor growing caudally into the upper abdomen.How to cite this articleAnand S, Suresh N, Reddy AK, Shukla AK. Imaging of a Rare Case of Diaphragmatic Tumor. J Med Sci 2017;3(1):25-27.

This paper presents a new fractional filter and an algorithm for retinal blood vessel segmentation. The proposed fractional filter is designed with the help of a weighted fractional derivative and an exponential weight factor. We have utilized the fractional filter and the eigenvalue maps of a local covariance matrix to develop the algorithm for retinal vessel segmentation. The local covariance matrix is formed by a second-order image moment. Experiments are performed on two well-studied evaluation databases named STARE and DRIVE. Experimental results show that the proposed method is computationally efficient, and the average accuracy of the vessel segmentation on STARE and DRIVE databases are 95.73% and 94.76%, respectively. The performance of the proposed method is compared with other existing methods. Simulation results show that the average performance of the proposed method is comparatively better than most of the discussed methods.

Each year, thousands of patients are at risk of cerebral ischemic injury, due to iatrogenic responses to surgical procedures. Prophylactic treatment of these patients as standard care could minimize potential neurological complications. We have shown that protection of brain tissue, in a non-human primate model of cerebral ischemic injury, is possible through pharmacological preconditioning using the immune activator D192935. We postulate that preconditioning with D192935 results in neuroprotective reprogramming that is evident in the brain following experimentally induced cerebral ischemia. We performed quantitative proteomic analysis of cerebral spinal fluid (CSF) collected post-stroke from our previously published efficacy study to determine whether CSF protein profiles correlated with induced protection. Four groups of animals were examined: naive animals (no treatment or stroke); animals treated with vehicle prior to stroke; D192935 treated and stroked animals, further delineated into two groups, ones that were protected (small infarcts) and those that were not protected (large infarcts). We found that distinct protein clusters defined the protected and non-protected animal groups, with a 16-member cluster of proteins induced exclusively in D192935 protected animals. Seventy percent of the proteins induced in the protected animals have functions that would enhance neuroprotection and tissue repair, including several members associated with M2 macrophages, a macrophage phenotype shown to contribute to neuroprotection and repair during ischemic injury. These studies highlight the translational importance of CSF biomarkers in defining mechanism and monitoring responses to treatment in development of stroke therapeutics.

Proteome profiling of circulating tumor cells (CTCs) can provide crucial insight into disease progression and the role of CTCs in tumor metastasis. We describe an integrated workflow to measure global protein expression in 1-5 spiked CTCs enriched from whole blood by immunodensity gradient centrifugation. Enriched CTCs were purified and collected by laser capture microdissection, prepared using a recently developed nanodroplet-based processing platform (nanoPOTS), and finally analyzed by ultrasensitive nanoLC-MS/MS. The workflow was capable of identifying an average of 164 and 607 protein groups from samples comprising 1 and 5 LNCaP cells, respectively, that were isolated from human whole blood. A panel of prostate cancer-specific proteins were identified and quantified, which was used to differentiate between spiked CTCs and white blood cells.

Cell type-resolved proteome analyses of the brain, heart and liver have been reported, however a similar effort on the lipidome is currently lacking. Here we applied liquid chromatography-tandem mass spectrometry to characterize the lipidome of major lung cell types isolated from human donors, representing the first lipidome map of any organ. We coupled this with cell type-resolved proteomics of the same samples (available at Lungmap.net). Complementary proteomics analyses substantiated the functional identity of the isolated cells. Lipidomics analyses showed significant variations in the lipidome across major human lung cell types, with differences most evident at the subclass and intra-subclass (i.e. total carbon length of the fatty acid chains) level. Further, lipidomic signatures revealed an overarching posture of high cellular cooperation within the human lung to support critical functions. Our complementary cell type-resolved lipid and protein datasets serve as a rich resource for analyses of human lung function.

Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs). We detected P450 activity primarily in the postnatal lung; using our ATP-ABP, we characterized a wide range of ATPases and other active nucleotide- and nucleic acid-binding enzymes involved in multiple facets of cellular metabolism throughout development. ATP-ABP targets include kinases, phosphatases, NAD- and FAD-dependent enzymes, RNA/DNA helicases, and others. The serine hydrolase-targeting probe detected changes in the activities of several proteases during the course of lung development, yielding insights into protein turnover at different stages of development. Select activity-based probe targets were then correlated with RNA in situ hybridization analyses of lung tissue sections.

Transcriptional and translational feedback loops in fungi and animals drive circadian rhythms in transcript levels that provide output from the clock, but post-transcriptional mechanisms also contribute. To determine the extent and underlying source of this regulation, we applied newly developed analytical tools to a long-duration, deeply sampled, circadian proteomics time course comprising half of the proteome. We found a quarter of expressed proteins are clock regulated, but >40% of these do not arise from clock-regulated transcripts, and our analysis predicts that these protein rhythms arise from oscillations in translational rates. Our data highlighted the impact of the clock on metabolic regulation, with central carbon metabolism reflecting both transcriptional and post-transcriptional control and opposing metabolic pathways showing peak activities at different times of day. The transcription factor CSP-1 plays a role in this metabolic regulation, contributing to the rhythmicity and phase of clock-regulated proteins.

Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to 3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues., There is a great need of developing highly sensitive mass spectrometry-based proteomics analysis for small cell populations. Here, the authors establish a robotically controlled chip-based nanodroplet processing platform and demonstrate its ability to profile the proteome from 10–100 mammalian cells.

Current mass spectrometry (MS)-based proteomics approaches are ineffective for mapping protein expression in tissue sections with high spatial resolution because of the limited overall sensitivity of conventional workflows. Here we report an integrated and automated method to advance spatially resolved proteomics by seamlessly coupling laser capture microdissection (LCM) with a recently developed nanoliter-scale sample preparation system termed nanoPOTS (Nanodroplet Processing in One pot for Trace Samples). The workflow is enabled by prepopulating nanowells with DMSO, which serves as a sacrificial capture liquid for microdissected tissues. The DMSO droplets efficiently collect laser-pressure catapulted LCM tissues as small as 20 μm in diameter with success rates >87%. We also demonstrate that tissue treatment with DMSO can significantly improve proteome coverage, likely due to its ability to dissolve lipids from tissue and enhance protein extraction efficiency. The LCM-nanoPOTS platform was able to identify 180, 695, and 1827 protein groups on average from 12-μm-thick rat brain cortex tissue sections having diameters of 50, 100, and 200 μm, respectively. We also analyzed 100-μm-diameter sections corresponding to 10–18 cells from three different regions of rat brain and comparatively quantified ∼1000 proteins, demonstrating the potential utility for high-resolution spatially resolved mapping of protein expression in tissues.

Although they are classified as Gram-positive bacteria, Corynebacterineae possess an asymmetric outer membrane that imparts structural and thereby physiological similarity to more distantly related Gram-negative bacteria. Like lipopolysaccharide in Gram-negative bacteria, lipids in the outer membrane of Corynebacterineae have been associated with the virulence of pathogenic species such as Mycobacterium tuberculosis (Mtb). For example, Mtb strains that lack long, branched-chain alkyl esters known as dimycocerosates (DIMs) are significantly attenuated in model infections. The resultant interest in the biosynthetic pathway of these unusual virulence factors has led to the elucidation of many of the steps leading to the final esterification of the alkyl β-diol, phthiocerol, with branched-chain fatty acids known as mycocerosates. PapA5 is an acyltransferase implicated in these final reactions. Here, we show that PapA5 is indeed the terminal enzyme in DIM biosynthesis by demonstrating its dual esterification activity and chain-length preference using synthetic alkyl β-diol substrate analogues. By applying these analogues to a series of PapA5 mutants, we also revise a model for the substrate binding within PapA5. Finally, we demonstrate that the Mtb Ser/Thr kinases PknB and PknE modify PapA5 on three overlapping Thr residues and that a fourth Thr is unique to PknE phosphorylation. These results clarify the DIM biosynthetic pathway and indicate post-translational modifications that warrant further elucidation for their roles in the regulation of DIM biosynthesis.

Congenital diaphragmatic hernia is a well-described condition that occurs in about 1 in 5,000 live births. A majority of the patients are diagnosed either antenatal or will present in the first few hours of life with respiratory distress. Presentation in adults is extremely rare and accounts for about 5 to 25% of diaphragmatic hernias. Patients, who present with late diaphragmatic hernias, complain of a wide variety of symptoms and diagnosis can be difficult. It consists of herniation of bowel, and occasionally solid organs, into the chest. It is more common on the left side (seen in 80% of cases), as the liver provides a relative barrier on the right side. The major clinical problem is pulmonary hypoplasia, a result of the lung having failed to developin uteroas the thoracic cavity is filled with abdominal contents. These congenital diaphragmatic defects have also been described in the adult population, and the widespread use of computed tomography has led to the recognition that these hernias are not uncommon and are often asymptomatic.How to cite this articleSanjay S, Pramila VVS, Shukla AK, Nagesh R. A Rare Case of Late Presentation of Congenital Diaphragmatic Hernia. J Med Sci 2016;2(4):65-67.

IntroductionSuperior vena cava syndrome results from the blockage of blood flow through the superior vena cava. It can be a medical emergency if it causes laryngeal or cerebral edema. Hence, prompt diagnosis is of utmost importance.Case reportA 65-year-old male patient came with complaints of progressive dyspnea and cough since 3 weeks. A contrast- enhanced multidetector computed tomography (MDCT) scan was done for the patient, which showed thrombus in the superior vena cava with a rich network of collaterals. The three-dimensional reconstruction was also done to demonstrate the collaterals.ConclusionThe MDCT provides an excellent tool to diagnose and assess the cause of superior vena cava obstruction.How to cite this articleBacker PH, VVP Seetha, Shukla AK, Nagesh R. Multidetector Computed Tomography Evaluation of Malignant Superior Vena Cava Syndrome. J Med Sci 2016;2(3):53-55.

There is an unmet technical challenge for mass spectrometry (MS)-based proteomic analysis of single mammalian cells. Quantitative proteomic analysis of single cells has been previously achieved by antibody-based immunoassays but is limited by the availability of high-quality antibodies. Herein we report a facile targeted MS-based proteomics method, termed cPRISM-SRM (carrier-assisted high-pressure, high-resolution separations with intelligent selection and multiplexing coupled to selected reaction monitoring), for reliable analysis of low numbers of mammalian cells. The method capitalizes on using “carrier protein” to assist processing of low numbers of cells with minimal loss, high-resolution PRISM separation for target peptide enrichment, and sensitive SRM for protein quantification. We have demonstrated that cPRISM-SRM has sufficient sensitivity to quantify proteins expressed at ≥200,000 copies per cell at the single-cell level and ≥3000 copies per cell in 100 mammalian cells. We envision that with further improvement cPRISM-SRM has the potential to move toward targeted MS-based single-cell proteomics.

Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n = 5) were fed 13C6-labeled lysine ("heavy") feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed ("light"), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins and quantify heavy/light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ∼70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing turnover measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein biomarkers through better understanding of processes governing biomarker kinetics.

Lung immaturity is a major cause of morbidity and mortality in premature infants. Understanding the molecular mechanisms driving normal lung development could provide insights on how to ameliorate disrupted development. While transcriptomic and proteomic analyses of normal lung development have been previously reported, characterization of changes in the lipidome is lacking. Lipids play significant roles in the lung, such as dipalmitoylphosphatidylcholine in pulmonary surfactant; however, many of the roles of specific lipid species in normal lung development, as well as in disease states, are not well defined. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the murine lipidome during normal postnatal lung development. Lipidomics analysis of lungs from post-natal day 7, day 14 and 6–8 week mice (adult) identified 924 unique lipids across 21 lipid subclasses, with dramatic alterations in the lipidome across developmental stages. Our data confirmed previously recognized aspects of post-natal lung development and revealed several insights, including in sphingolipid-mediated apoptosis, inflammation and energy storage/usage. Complementary proteomics, metabolomics and chemical imaging corroborated these observations. This multi-omic view provides a unique resource and deeper insight into normal pulmonary development.