A review of pathological findings in impalas (Aepyceros melampus) in South Africa.

Impalas (Aepyceros melampus) are common African antelope. A retrospective study was conducted of 251 impala cases from game farms, national parks and zoos submitted by veterinarians and pathologists in South Africa (2003-2016). Histopathology slides as well as records of macroscopic lesions and additional diagnostic tests performed were examined. Non-infectious conditions, such as acute pulmonary congestion and oedema, cachexia, traumatic injury and anaesthetic-related mortality were the most common causes of morbidity and mortality. Bacterial sepsis was the most common infectious disease, whilst skeletal muscle and myocardial sarcocystosis and verminous cholangitis and pneumonia were the most common parasitic diseases. Although the retrospective nature of this study limits the significance of the relative prevalence of lesions in the three locations, management decisions and diagnostic plans may be informed by the results. Impala from game farms had significantly more cachexia cases than those from other locations. Impala from zoos had significantly more lymphoid depletion than those from other locations. These findings suggest that nutrition and pasture management, enclosure design, management of intra- and interspecies aggression and improved anaesthetic protocols could improve animal welfare and survival of impala on game farms and in zoos. This report presents a detailed survey of diseases and conditions found in impala that provides baseline data for veterinary pathologists.

Investigation of multiple mortality events in eastern box turtles (Terrapene carolina carolina).

Wildlife mortality investigations are important for conservation, food safety, and public health; but they are infrequently reported for cryptic chelonian species. Eastern box turtles (Terrapene carolina carolina) are declining due to anthropogenic factors and disease, and while mortality investigations have been reported for captive and translocated individuals, few descriptions exist for free-living populations. We report the results of four natural mortality event investigations conducted during routine health surveillance of three Illinois box turtle populations in 2011, 2013, 2014, and 2015. In April 2011, over 50 box turtles were found dead and a polymicrobial necrotizing bacterial infection was diagnosed in five survivors using histopathology and aerobic/anaerobic culture. This represents the first reported occurrence of necrotizing bacterial infection in box turtles. In August 2013, paired histopathology and qPCR ranavirus detection in nine turtles was significantly associated with occupation of moist microhabitats, identification of oral plaques and nasal discharge on physical exam, and increases in the heterophil count and heterophil to lymphocyte ratio (p < 0.05). In July 2014 and 2015, ranavirus outbreaks reoccurred within a 0.2km radius of highly-disturbed habitat containing ephemeral ponds used by amphibians for breeding. qPCR ranavirus detection in five individuals each year was significantly associated with use of moist microhabitats (p < 0.05). Detection of single and co-pathogens (Terrapene herpesvirus 1, adenovirus, and Mycoplasma sp.) was common before, during, and after mortality events, but improved sample size would be necessary to determine the impacts of these pathogens on the occurrence and outcome of mortality events. This study provides novel information about the causes and predictors of natural box turtle mortality events. Continued investigation of health, disease, and death in free-living box turtles will improve baseline knowledge of morbidity and mortality, identify threats to survival, and promote the formation of effective conservation strategies.

PHARMACOKINETICS, EFFICACY, AND SAFETY OF VORICONAZOLE AND ITRACONAZOLE IN HEALTHY COTTONMOUTHS (AGKISTRODON PISCIVORUS) AND MASSASAUGA RATTLESNAKES (SISTRURUS CATENATUS) WITH SNAKE FUNGAL DISEASE.

Snake fungal disease (SFD; Ophidiomyces ophiodiicola) is posing a significant threat to several free-ranging populations of pitvipers. Triazole antifungals have been proposed for the treatment of mycoses in reptiles; however, data are lacking about their safety and efficacy in snakes with SFD. Study 1 investigated in vitro susceptibility, and identified that plasma concentrations >250 ng/ml (voriconazole) and >1,000 ng/ml (itraconazole) may be effective in vivo for SFD. In Study 2, the pharmacokinetics after a single subcutaneous voriconazole injection were assessed in apparently healthy free-ranging cottonmouths (Agkistrodon piscivorus). Based on pilot-study results, four snakes were administered a single injection of voriconazole (5 mg/kg). One pilot snake and three full-study snakes died within 12 hr of voriconazole administration. All surviving snakes maintained plasma concentrations >250 ng/ml for 12-24 hr. In Study 3, two Eastern massasaugas (Sistrurus catenatus) and a timber rattlesnake (Crotalus horridus horridus) diagnosed with SFD were treated with voriconazole delivered by subcutaneous osmotic pumps. The timber rattlesnake (12.1-17.5 mg/kg/hr) reached therapeutic concentrations, whereas the massasaugas (1.02-1.6 mg/kg/hr) did not. In Study 4, the pharmacokinetics of a single 10-mg/kg per-cloaca dose of itraconazole (Sporanox®) was evaluated in seven apparently healthy free-ranging cottonmouths. Similarly, the plasma and tissue concentrations did not meet therapeutic concentrations based on in vitro data. The data presented in this report serve as an initial step toward understanding the pharmacokinetics, efficacy, and safety of triazole antifungals in pitviper species with SFD. Further study is needed to determine the appropriate dose and route of administration of triazole antifungals in pitviper species.

Pathway-Informed Discovery and Targeted Proteomic Workflows Using Mass Spectrometry.

Recent advancements in mass spectrometry (MS) and data analysis software have enabled new strategies for biological discovery using proteomics. Proteomics has evolved from routine discovery and identification of proteins to integrated multi-omics projects relating specific proteins to their genes and metabolites. Using additional information, such as that contained in biological pathways, has enabled the use of targeted protein quantitation for monitoring fold changes in expression as well as biomarker discovery. Here we discuss a full proteomic workflow from discovery proteomics on a quadrupole Time-of-Flight (Q-TOF) MS to targeted proteomics using a triple quadrupole (QQQ) MS. A discovery proteomics workflow encompassing acquisition of data-dependent proteomics data on a Q-TOF and protein database searching will be described which uses the protein abundances from identified proteins for subsequent statistical analysis and pathway visualization. From the active pathways, a protein target list is created for use in a peptide-based QQQ assay. These peptides are used as surrogates for target protein quantitation. Peptide-based QQQ assays provide sensitivity and selectivity allowing rapid and robust analysis of large batches of samples. These quantitative results are then statistically compared and visualized on the original biological pathways with a more complete coverage of proteins in the studied pathways.

CUTANEOUS SQUAMOUS CELL CARCINOMA IN A PANTHER CHAMELEON (FURCIFER PARDALIS) AND TREATMENT WITH CARBOPLATIN IMPLANTABLE BEADS.

A 3-yr-old male panther chameleon (Furcifer pardalis) presented with bilateral raised crusted skin lesions along the lateral body wall that were found to be carcinoma in situ and squamous cell carcinoma. Similar lesions later developed on the caudal body wall and tail. A subcutaneous implantable carboplatin bead was placed in the first squamous cell carcinoma lesion identified. Additional new lesions sampled were also found to be squamous cell carcinomas, and viral polymerase chain reaction was negative for papillomaviruses and herpesviruses. Significant skin loss would have resulted from excision of all the lesions, so treatment with only carboplatin beads was used. No adverse effects were observed. Lesions not excised that were treated with beads decreased in size. This is the first description of cutaneous squamous cell carcinoma and treatment with carboplatin implantable beads in a panther chameleon.

Human SRMAtlas: A Resource of Targeted Assays to Quantify the Complete Human Proteome.

The ability to reliably and reproducibly measure any protein of the human proteome in any tissue or cell type would be transformative for understanding systems-level properties as well as specific pathways in physiology and disease. Here, we describe the generation and verification of a compendium of highly specific assays that enable quantification of 99.7% of the 20,277 annotated human proteins by the widely accessible, sensitive, and robust targeted mass spectrometric method selected reaction monitoring, SRM. This human SRMAtlas provides definitive coordinates that conclusively identify the respective peptide in biological samples. We report data on 166,174 proteotypic peptides providing multiple, independent assays to quantify any human protein and numerous spliced variants, non-synonymous mutations, and post-translational modifications. The data are freely accessible as a resource at http://www.srmatlas.org/, and we demonstrate its utility by examining the network response to inhibition of cholesterol synthesis in liver cells and to docetaxel in prostate cancer lines.

State of the Human Proteome in 2014/2015 As Viewed through PeptideAtlas: Enhancing Accuracy and Coverage through the AtlasProphet.

The Human PeptideAtlas is a compendium of the highest quality peptide identifications from over 1000 shotgun mass spectrometry proteomics experiments collected from many different laboratories, all reanalyzed through a uniform processing pipeline. The latest 2015-03 build contains substantially more input data than past releases, is mapped to a recent version of our merged reference proteome, and uses improved informatics processing and the development of the AtlasProphet to provide the highest quality results. Within the set of ∼20,000 neXtProt primary entries, 14,070 (70%) are confidently detected in the latest build, 5% are ambiguous, 9% are redundant, leaving the total percentage of proteins for which there are no mapping detections at just 16% (3166), all derived from over 133 million peptide-spectrum matches identifying more than 1 million distinct peptides using AtlasProphet to characterize and classify the protein matches. Improved handling for detection and presentation of single amino-acid variants (SAAVs) reveals the detection of 5326 uniquely mapping SAAVs across 2794 proteins. With such a large amount of data, the control of false positives is a challenge. We present the methodology and results for maintaining rigorous quality along with a discussion of the implications of the remaining sources of errors in the build.

Pancreatic carcinoma in an African clawed frog (Xenopus laevis).

This report describes the histologic features of a pancreatic carcinoma in an adult female African clawed frog (Xenopus laevis). The animal was found to be in poor body condition and subsequently euthanized for a complete necropsy. Histologically, the pancreas was effaced by packets of polyhedral cells consistent with a pancreatic islet cell carcinoma. Metastatic disease was not identified. Pancreatic tumors are uncommon in amphibians, and this report is the first to describe a pancreatic carcinoma in an African clawed frog.

Novel high-molecular weight fucosylated milk oligosaccharides identified in dairy streams.

Oligosaccharides are the third largest component in human milk. This abundance is remarkable because oligosaccharides are not digestible by the newborn, and yet they have been conserved and amplified during evolution. In addition to encouraging the growth of a protective microbiota dominated by bifidobacteria, oligosaccharides have anti-infective activity, preventing pathogens from binding to intestinal cells. Although it would be advantageous adding these valuable molecules to infant milk formula, the technologies to reproduce the variety and complexity of human milk oligosaccharides by enzymatic/organic synthesis are not yet mature. Consequently, there is an enormous interest in alternative sources of these valuable oligosaccharides. Recent research has demonstrated that bovine milk and whey permeate also contain oligosaccharides. Thus, a thorough characterization of oligosaccharides in bovine dairy streams is an important step towards fully assessing their specific functionalities. In this study, bovine milk oligosaccharides (BMOs) were concentrated by membrane filtration from a readily available dairy stream called "mother liquor", and analyzed by high accuracy MALDI FT-ICR mass spectrometry. The combination of HPLC and accurate mass spectrometry allowed the identification of ideal processing conditions leading to the production of Kg amount of BMO enriched powders. Among the BMOs identified, 18 have high-molecular weight and corresponded in size to the most abundant oligosaccharides present in human milk. Notably 6 oligosaccharides contained fucose, a sugar monomer that is highly abundant in human milk, but is rarely observed in bovine milk. This work shows that dairy streams represent a potential source of complex milk oligosaccharides for commercial development of unique dairy ingredients in functional foods that reproduce the benefits of human milk.

Multiple precursor ion scanning of gangliosides and sulfatides with a reversed-phase microfluidic chip and quadrupole time-of-flight mass spectrometry.

Precise profiling of polar lipids including gangliosides and sulfatides is a necessary step in understanding the diverse physiological role of these lipids. We have established an efficient method for the profiling of polar lipids using reversed-phase nano high-performance liquid chromatography microfluidic chip quadrupole time-of-flight mass spectrometry (nano-HPLC-chip Q-TOF/MS). A microfluidic chip design provides improved chromatographic performance, efficient separation, and stable nanospray while the advanced high-resolution mass spectrometer allowed for the identification of complex isobaric polar lipids such as NeuAc- and NeuGc-containing gangliosides. Lipid classes were identified based on the characteristic fragmentation product ions generated during data-dependent tandem mass spectrometry (MS/MS) experiments. Each class was monitored by a postprocessing precursor ion scan. Relatively simple quantitation and identification of intact ions was possible due to the reproducible retention times provided by the nano-HPLC chip. The method described in this paper was used to profile polar lipids from mouse brain, which was found to contain 17 gangliosides and 13 sulfatides. Types and linkages of the monosaccharides and their acetyl modifications were identified by low-energy collision-induced dissociation (CID) (40 V), and the type of sphingosine base was identified by higher energy CID (80 V). Accurate mass measurements and chromatography unveiled the degree of unsaturation and hydroxylation in the ceramide lipid tails.

Functional analysis of conserved motifs in influenza virus PB1 protein.

The influenza virus RNA polymerase complex is a heterotrimer composed of the PB1, PB2, and PA subunits. PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. A previous study demonstrated the importance of several of these conserved amino acids in PB1 for influenza polymerase activity through mutational analysis. However, a small number of viruses isolated in nature possesses non-consensus amino acids in one of the four motifs, most of which have not been tested for their replicative ability. Here, we assessed the transcription/replication activities of 25 selected PB1 mutations found in natural isolates by using minireplicon assays in human and avian cells. Most of the mutations tested significantly reduced polymerase activity. One exception was mutation K480R, observed in several pandemic (H1N1) 2009 viruses, which slightly increased polymerase activity relative to wild-type. However, in the background of the pandemic A/California/04/2009 (H1N1) virus, this mutation did not affect virus titers in cell culture. Our results further demonstrate the functional importance of the four conserved PB1 motifs in influenza virus transcription/replication. The finding of natural isolates with non-consensus PB1 motifs that are nonfunctional in minireplicon assays suggests compensatory mutations and/or mixed infections which may have 'rescued' the inactive PB1 protein.

An assessment of current bioinformatic solutions for analyzing LC-MS data acquired by selected reaction monitoring technology.

Selected reaction monitoring (SRM) is an accurate quantitative technique, typically used for small-molecule mass spectrometry (MS). SRM has emerged as an important technique for targeted and hypothesis-driven proteomic research, and is becoming the reference method for protein quantification in complex biological samples. SRM offers high selectivity, a lower limit of detection and improved reproducibility, compared to conventional shot-gun-based tandem MS (LC-MS/MS) methods. Unlike LC-MS/MS, which requires computationally intensive informatic postanalysis, SRM requires preacquisition bioinformatic analysis to determine proteotypic peptides and optimal transitions to uniquely identify and to accurately quantitate proteins of interest. Extensive arrays of bioinformatics software tools, both web-based and stand-alone, have been published to assist researchers to determine optimal peptides and transition sets. The transitions are oftentimes selected based on preferred precursor charge state, peptide molecular weight, hydrophobicity, fragmentation pattern at a given collision energy (CE), and instrumentation chosen. Validation of the selected transitions for each peptide is critical since peptide performance varies depending on the mass spectrometer used. In this review, we provide an overview of open source and commercial bioinformatic tools for analyzing LC-MS data acquired by SRM.

Nano-LC-MS/MS of glycopeptides produced by nonspecific proteolysis enables rapid and extensive site-specific glycosylation determination.

Given the biological importance of glycosylation on proteins, the identification of protein glycosylation sites is integral to understanding broader biological structure and function. Unfortunately, the determination of the microheterogeneity at the site of glycosylation still remains a significant challenge. Nanoflow liquid chromatography with tandem mass spectrometry provides both separation of glycopeptides and the ability to determine glycan composition and site-specific glycosylation. However, because of the size of glycopeptides, they are not often amenable to tandem MS. In this work, proteins are digested with multiple proteases to produce glycopeptides that are of suitable size for tandem MS analysis. The conditions for collision-induced dissociation are optimized to obtain diagnostic ions that maximize glycan and peptide information. The method is applied to glycoproteins with contrasting glycans and multiple sites of glycosylation and identifies multiple glycan compositions at each individual glycosylation site. This method provides an important improvement in the routine determination of glycan microheterogeneity by mass spectrometry.

Label-free liquid chromatography-tandem mass spectrometry analysis with automated phosphopeptide enrichment reveals dynamic human milk protein phosphorylation during lactation.

Protein phosphorylation is a critical posttranslational modification that affects cell-cell signaling and protein function. However, quantifying the relative site-specific changes of phosphorylation occupancies remains a major issue. An online enrichment of phosphopeptides using titanium dioxide incorporated in a microchip liquid chromatography device was used to analyze trypsin-digested human milk proteins with mass spectrometry. The method was validated with standards and used to determine the dynamic behavior of protein phosphorylation in human milk from the first month of lactation. α-Casein, ß-casein, osteopontin, and chordin-like protein 2 phosphoproteins were shown to vary during this lactation time in an independent manner. In addition, changes in specific regions of these phosphoproteins were found to vary independently. Novel phosphorylation sites were discovered for chordin-like protein 2, α-lactalbumin, ß-1,4-galactosyl transferase, and poly-Ig (immunoglobulin) receptor. Coefficients of variation for the quantitation were comparable to those in other contemporary approaches using isotopically labeled peptides, with a median value of 11% for all phosphopeptide occupancies quantified.

Profile of native N-linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time-of-flight mass spectrometry.

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N-linked oligosaccharides released from human serum without derivatization has been developed using on-line nanoLC and high resolution TOF MS. The N-linked oligosaccharides were analyzed with MALDI FT-ICR MS and microchip LC MS (HPLC-Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43 x 0.075 mm(2) i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140 x 0.075 mm(2) i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N-linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for approximately 96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining approximately 4%.

Agreement between glucose trends derived from three simultaneously worn continuous glucose sensors.

BACKGROUND: Sensors detect the rate and direction of glucose trend. They need to be accurate and reproducible as could be evidenced by strong agreement between multiple sensors. We evaluated this relationship through simultaneously worn glucose sensors using several methods of slope analysis. METHODS: Ten type 1 diabetic, insulin pump-treated subjects were studied while simultaneously wearing three CGMS Gold sensors each. Sensors were placed in the right abdomen (reference), left abdomen, and left upper arm. Sensors were calibrated and chronologically aligned. Data were only interpreted and included if there were 24 hours of data simultaneously obtained from all three sensors. RESULTS: Using a two-point derived slope, increasing the duration of the trend from 5 to 60 minutes improved agreement between sensors. Using a 20-minute rolling average trend (using every 5-minute glucose value during the 20 minutes) improved the agreement to 94.3%. Finally, using whichever of the two comparator sensor rolling average trends was closest to the reference (better of two), the agreement improved to 98.2%. However, for these trend analysis methods, when the absolute reference rate of change was more than 1 mg/dl/min, the agreement decreased. Even with the best analysis approach, at an absolute reference sensor rate of change of >2 mg/dl/min, the agreement between sensors was only 40.0%. CONCLUSION: Despite several methods of analysis, trend agreement from multiple sensors diminishes as the absolute rate of change of reference glucose increases.