A large-scale, targeted metabolomics method for the analysis and quantification of metabolites in human plasma via liquid chromatography-mass spectrometry.

Metabolomics is the study of small molecules, primarily metabolites, that are produced during metabolic processes. Analysis of the composition of an organism's metabolome can yield useful information about an individual's health status at any given time. In recent years, the development of large-scale, targeted metabolomic methods has allowed for the analysis of biological samples using analytical techniques such as LC-MS/MS. This paper presents a large-scale metabolomics method for analysis of biological samples, with a focus on quantification of metabolites found in blood plasma. The method comprises a 10-min chromatographic separation using HILIC and RP stationary phases combined with positive and negative electrospray ionization in order to maximize metabolome coverage. Complete analysis of a single sample can be achieved in as little as 40 min using the two columns and dual modes of ionization. With 540 metabolites and the inclusion of over 200 analytical standards, this method is comprehensive and quantitatively robust when compared to current targeted metabolomics methods. This study uses a large-scale evaluation of metabolite recovery from plasma that enables absolute quantification of metabolites by correcting for analyte loss throughout processes such as extraction, handling, or storage. In addition, the method was applied to plasma collected from adjuvant breast cancer patients to confirm the suitability of the method to clinical samples.

Selection and Validation of Reference Genes for Gene Expression Studies in an Equine Adipose-Derived Mesenchymal Stem Cell Differentiation Model by Proteome Analysis and Reverse-Transcriptase Quantitative Real-Time PCR.

Adipose-derived stem cells (ADSCs) are used in tissue regeneration therapies. The objective of this study is to identify stable reference genes (RGs) for use in gene expression studies in a characterized equine adipose-derived mesenchymal stem cell (EADMSC) differentiation model. ADSCs were differentiated into adipocytes (ADs) or osteoblasts (OBs), and the proteomes from these cells were analyzed by liquid chromatography tandem mass spectrometry. Proteins that were stably expressed in all three cells types were identified, and the mRNA expression stabilities for their corresponding genes were validated by RT-qPCR. PPP6R1, CCDC97, and then either ACTB or EPHA2 demonstrated the most stable mRNA levels. Normalizing target gene Cq data with at least three of these RGs simultaneously, as per MIQE guidelines (PPP6R1 and CCDC97 with either ACTB or EPHA2), resulted in congruent conclusions. FABP5 expression was increased in ADs (5.99 and 8.00 fold, p = 0.00002 and p = 0.0003) and in OBs (5.18 and 5.91 fold, p = 0.0011 and p = 0.0023) relative to ADSCs. RUNX2 expression was slightly higher in ADs relative to ADSCs (1.97 and 2.65 fold, p = 0.04 and p = 0.01), but not in OBs (0.9 and 1.03 fold, p = 0.58 and p = 0.91).

KIT D816V is dimerization-independent and activates downstream pathways frequently perturbed in mastocytosis.

KIT, a type III tyrosine kinase receptor, plays a crucial role in haematopoietic development. The KIT receptor forms a dimer after ligand binding; this activates tyrosine kinase activity leading to downstream signal transduction. The D816V KIT mutation is extensively implicated in haematological malignancies, including mastocytosis and leukaemia. KIT D816V is constitutively active, but the molecular nuances that lead to constitutive tyrosine kinase activity are unclear. For the first time, we present experimental evidence that the KIT D816V mutant does not dimerize like KIT wild type. We further show evidence of decreased stabilization of the tyrosine kinase domain in the KIT D816V mutant, a phenomenon that might contribute to its constitutive activity. Since the mechanism of KIT D816V activation varies from that of the wild type, we explored downstream signal transduction events and found that even though KIT D816V targets similar signalling moieties, the signalling is amplified in the mutant compared to stem cell factor-activated wild type receptor. Uniquely, KIT D816V induces infection-related pathways and the spliceosome pathway, providing alternate options for selective as well as combinatorial therapeutic targeting.

Liquid Biopsy Versus Tissue Biopsy to Determine Front Line Therapy in Metastatic Non-Small Cell Lung Cancer (NSCLC).

In the last decade, non-small-cell lung cancer (NSCLC) treatment has improved with the approval of multiple therapies to target specific genetic alterations. Though, next generation sequencing (NGS) has traditionally been conducted from tissue biopsy samples, developing data supports the use of plasma-based circulating tumor DNA (ctDNA), also known as "liquid biopsy," to complement tissue biopsy approaches in guiding front-line therapy. This study is a retrospective analysis of 170 new NSCLC patients treated at 2 cancer centers within a 5-year period who received both tissue and liquid biopsy NGS as standard of care. Based on a treatment schema defined by testing sufficiency, biomarker detection, and turnaround time (TAT), physicians based the majority of their treatments on liquid biopsy results (73.5%) versus tissue biopsy (25.9%). Liquid biopsy NGS returned results on average 26.8 days faster than tissue and reported higher testing success. For guideline-recommended biomarkers, liquid biopsy was 94.8% to 100% concordant with tissue. In comparing testing modalities, a liquid-first approach identified guideline-recommended biomarkers in 76.5% of patients versus 54.9% in a tissue-first approach. There was no significant difference in time-to-treatment, or survival outcomes (overall survival and progression free survival) based on liquid versus tissue biopsy findings. This research demonstrates that liquid biopsy NGS is an effective tool to capture actionable genetic alterations in NSCLC. Due to its high concordance to tissue, faster TAT, and similarity in outcomes and time-to-treatment, liquid biopsy can be used either as a first-line test or concordantly with tissue biopsy to guide treatment decisions in NSCLC.

Successful Dietary Therapy in Paediatric Crohn's Disease is Associated with Shifts in Bacterial Dysbiosis and Inflammatory Metabotype Towards Healthy Controls.

BACKGROUND AND AIMS: Nutritional therapy with the Crohn's Disease Exclusion Diet + Partial Enteral Nutrition [CDED+PEN] or Exclusive Enteral Nutrition [EEN] induces remission and reduces inflammation in mild-to-moderate paediatric Crohn's disease [CD]. We aimed to assess if reaching remission with nutritional therapy is mediated by correcting compositional or functional dysbiosis. METHODS: We assessed metagenome sequences, short chain fatty acids [SCFA] and bile acids [BA] in 54 paediatric CD patients reaching remission after nutritional therapy [with CDED + PEN or EEN] [NCT01728870], compared to 26 paediatric healthy controls. RESULTS: Successful dietary therapy decreased the relative abundance of Proteobacteria and increased Firmicutes towards healthy controls. CD patients possessed a mixture of two metabotypes [M1 and M2], whereas all healthy controls had metabotype M1. M1 was characterised by high Bacteroidetes and Firmicutes, low Proteobacteria, and higher SCFA synthesis pathways, and M2 was associated with high Proteobacteria and genes involved in SCFA degradation. M1 contribution increased during diet: 48%, 63%, up to 74% [Weeks 0, 6, 12, respectively.]. By Week 12, genera from Proteobacteria reached relative abundance levels of healthy controls with the exception of E. coli. Despite an increase in SCFA synthesis pathways, remission was not associated with increased SCFAs. Primary BA decreased with EEN but not with CDED+PEN, and secondary BA did not change during diet. CONCLUSION: Successful dietary therapy induced correction of both compositional and functional dysbiosis. However, 12 weeks of diet was not enough to achieve complete correction of dysbiosis. Our data suggests that composition and metabotype are important and change quickly during the early clinical response to dietary intervention. Correction of dysbiosis may therefore be an important future treatment goal for CD.

Heat Shock Alters the Proteomic Profile of Equine Mesenchymal Stem Cells.

The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was observed, and many of the specific differentially expressed proteins were involved in cell-signaling pathways such as Notch and Wnt signaling, which are known to regulate cellular development. Furthermore, cytoskeletal proteins profilin, DSTN, SPECC1, and DAAM2 showed increased protein levels in osteoblasts differentiated at 42 °C as compared with 37 °C, and these cells, while they appeared to accumulate calcium, did not organize into a whorl agglomerate as is typically seen at physiological temperatures. This altered proteome composition observed suggests that heat stress could have long-term impacts on cellular development. We propose that this in vitro stem cell culture model of cell differentiation is useful for investigating molecular mechanisms that impact cell development in response to stressors.

Metabolome Changes With Diet-Induced Remission in Pediatric Crohn's Disease.

BACKGROUND & AIMS: The Crohn's disease (CD) exclusion diet (CDED) plus partial enteral nutrition (PEN) and exclusive enteral nutrition (EEN) both induce remission in pediatric CD. CDED+PEN is better tolerated and able to sustain remission. We characterized the changes in fecal metabolites induced by CDED+PEN and EEN and their relationship with remission. METHODS: A total of 216 fecal metabolites were measured in 80 fecal samples at week (W) 0, W6, and W12, of children with mild to moderate CD in a prospective randomized trial comparing CDED+PEN vs EEN. The metabolites were measured using liquid chromatography coupled to mass spectrometry. Metagenome Kyoto Encyclopedia of Genes and Genomes Orthology analysis was performed to investigate the differential functional gene abundance involved in specific metabolic pathways. Data were analyzed according to clinical outcome of remission (W6_rem), no remission (W6_nr), sustained remission (W12_sr), and nonsustained (W12_nsr) remission. RESULTS: A decrease in kynurenine and succinate synthesis and an increase in N-α-acetyl-arginine characterized CDED+PEN W6_rem, whereas changes in lipid metabolism characterized EEN W6_rem, especially reflected by lower levels in ceramides. In contrast, fecal metabolites in EEN W6_nr were comparable to baseline/W0 samples. CDED+PEN W6_rem children maintained metabolome changes through W12. In contrast, W12_nsr children in the EEN group, who resumed a free diet after week 6, did not. The metabolome of CDED+PEN differed from EEN in the purine, pyrimidine, and sphingolipid pathways. A significant differential abundance in several genes involved in these pathways was detected. CONCLUSION: CDED+PEN- and EEN-induced remission are associated with significant changes in inflammatory bowel disease-associated metabolites such as kynurenine, ceramides, amino acids, and others. Sustained remission with CDED+PEN, but not EEN, was associated with persistent changes in metabolites. CLINICALTRIALS: gov, Number NCT01728870.

Analysis of human serum and urine for tentative identification of potentially carcinogenic pesticide-associated N-nitroso compounds using high-resolution mass spectrometry.

Human serum and urine samples were analyzed for a suite of nitrosatable pesticides and potentially carcinogenic pesticide-associated N-nitroso (PANN) compounds. Formation of PANN compounds may occur in vivo after consumption of food or water containing trace amounts of nitrosatable pesticide residues and nitrate. Using a modified version of the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method, nine nitrosatable pesticides and byproducts were extracted from serum and urine from 64 individuals from two different sample populations in Atlantic Canada: (i) Prince Edward Island, a region where nitrate and trace amounts of nitrosatable pesticides have been detected in groundwater; and (ii) Halifax, Nova Scotia, a non-agricultural urban area. Samples were then analyzed using ultra-high pressure liquid chromatography (UHPLC) coupled with high-resolution accurate mass (HRAM) single-stage orbitrap mass spectrometry (MS), which allows for semi-targeted analysis and tentative identification of a virtually limitless number of exposure biomarkers. Two nitrosatable target analytes, ethylenethiourea (ETU) and 3,5,6-trichloro-2-pyridinol (TCPy) were found in serum, while atrazine (ATR) and ETU were detected in urine. Five and six PANN compounds were tentatively identified in serum and urine, respectively. The two PANN compounds that were most frequently tentatively identified in serum were N-nitroso dimethoate (N-DIM) and N-nitroso omethoate (N-OME) with detection frequencies of 78% and 95%, respectively. This is the first biomonitoring study of its kind to investigate PANN compounds in human serum and urine.

Gut bacterial gene changes following pegaspargase treatment in pediatric patients with acute lymphoblastic leukemia.

Treatment of pediatric acute lymphoblastic leukemia (ALL) with pegaspargase exploits ALL cells dependency on asparagine. Pegaspargase depletes asparagine, consequentially affecting aspartate, glutamine and glutamate. The gut as a confounding source of these amino acids (AAs) and the role of gut microbiome metabolism of AAs has not been examined. We examined asparagine, aspartate, glutamine and glutamate in stool samples from patients over pegaspargase treatment. Microbial gene-products, which interact with these AAs were identified. Stool asparagine declined significantly, and 31 microbial genes changed over treatment. Changes were complex, and included genes involved in AA metabolism, nutrient sensing, and pathways increased in cancers. While we identified changes in a gene (iaaA) with limited asparaginase activity, it lacked significance after correction leaving open other mechanisms for asparagine decline, possibly including loss from gut to blood. Understanding pathways that change AA availability, including by microbes in the gut, could be useful in optimizing pegaspargase therapy.

N-linked glycosylation enzymes in the diatom Thalassiosira oceanica exhibit a diel cycle in transcript abundance and favor for NXT-type sites.

N-linked glycosylation is a posttranslational modification affecting protein folding and function. The N-linked glycosylation pathway in algae is poorly characterized, and further knowledge is needed to understand the cell biology of algae and the evolution of N-linked glycosylation. This study investigated the N-linked glycosylation pathway in Thalassiosira oceanica, an open ocean diatom adapted to survive at growth-limiting iron concentrations. Here we identified and annotated the genes coding for the essential enzymes involved in the N-linked glycosylation pathway of T. oceanica. Transcript levels for genes coding for calreticulin, oligosaccharyltransferase (OST), N-acetylglucosaminyltransferase (GnT1), and UDP-glucose glucosyltransferase (UGGT) under high- and low-iron growth conditions revealed diel transcription patterns with a significant decrease of calreticulin and OST transcripts under iron-limitation. Solid-phase extraction of N-linked glycosylated peptides (SPEG) revealed 118 N-linked glycosylated peptides from cells grown in high- and low-iron growth conditions. The identified peptides had 81% NXT-type motifs, with X being any amino acids except proline. The presence of N-linked glycosylation sites in the iron starvation-induced protein 1a (ISIP1a) confirmed its predicted topology, contributing to the biochemical characterization of ISIP1 proteins. Analysis of extensive oceanic gene databases showed a global distribution of calreticulin, OST, and UGGT, reinforcing the importance of glycosylation in microalgae.

TRPM8 as the rapid testosterone signaling receptor: Implications in the regulation of dimorphic sexual and social behaviors.

Testosterone regulates dimorphic sexual behaviors in all vertebrates. However, the molecular mechanism underlying these behaviors remains unclear. Here, we report that a newly identified rapid testosterone signaling receptor, Transient Receptor Potential Melastatin 8 (TRPM8), regulates dimorphic sexual and social behaviors in mice. We found that, along with higher steroid levels in the circulation, TRPM8-/- male mice exhibit increased mounting frequency indiscriminate of sex, delayed sexual satiety, and increased aggression compared to wild-type controls, while TRPM8-/- females display an increased olfaction-exploratory behavior. Furthermore, neuronal responses to acute testosterone application onto the amygdala were attenuated in TRPM8-/- males but remained unchanged in females. Moreover, activation of dopaminergic neurons in the ventral tegmental area following mating was impaired in TRPM8-/- males. Together, these results demonstrate that TRPM8 regulates dimorphic sexual and social behaviors, and potentially constitutes a signalosome for mediation of sex-reward mechanism in males. Thus, deficiency of TRPM8 might lead to a delayed sexual satiety phenomenon.

Impact of Nasal Swabs on Empiric Treatment of Respiratory Tract Infections (INSERT-RTI).

Methicillin-resistant Staphylococcus aureus (MRSA) polymerase-chain-reaction nasal swabs (PCRNS) are a rapid diagnostic tool with a high negative predictive value. A PCRNS plus education "bundle" was implemented to inform clinicians on the utility of PCRNS for anti-MRSA therapy de-escalation in respiratory tract infections (RTI). The study included patients started on vancomycin with a PCRNS order three months before and after bundle implementation. The primary objective was the difference in duration of anti-MRSA therapy (DOT) for RTI. Secondary objectives included hospital length of stay (LOS), anti-MRSA therapy reinitiation, 30-day readmission, in-hospital mortality, and cost. We analyzed 62 of 110 patients screened, 20 in the preintervention and 42 in the postintervention arms. Mean DOT decreased after bundle implementation by 30.3 h (p = 0.039); mean DOT for patients with a negative PCRNS decreased by 39.7 h (p = 0.014). Median cost was lower after intervention [USD$51.69 versus USD$75.30 (p < 0.01)]. No significant difference in LOS, mortality, or readmission existed. The bundle implementation decreased vancomycin therapy and cost without negatively impacting patient outcomes.

Protection of the transplant kidney during cold perfusion with doxycycline: proteomic analysis in a rat model.

BACKGROUND: It has been previously shown that doxycycline (Doxy) protects the kidney from preservation injury by inhibition of matrix metalloproteinase. However, the precise molecular mechanism involved in this protection from injury is not known. We used a pharmaco-proteomics approach to identify potential molecular targets associated with kidney preservation injury. METHODS: Rat kidneys were cold perfused with or without doxycycline (Doxy) for 22 h. Kidneys perfusates were analyzed for the presence of injury markers such as lactate dehydrogenase (LDH), and neutrophil-gelatinase associated lipocalin (NGAL). Proteins extracted from kidney tissue were analyzed by 2-dimensional gel electrophoresis. Proteins of interest were identified by mass spectrometry. RESULTS: Triosephosphate isomerase, PGM, dihydropteridine reductase-2, pyridine nucleotide-disulfide oxidoreductase, phosphotriesterase-related protein, and aminoacylase-1A were not affected by cold perfusion. Perfusion with Doxy increased their levels. N(G),N(G)-dimethylarginine dimethylaminohydrolase and phosphoglycerate kinase 1 were decreased after cold perfusion. Perfusion with Doxy led to an increase in their levels. CONCLUSIONS: This study revealed specific metabolic enzymes involved in preservation injury and in the mechanism whereby Doxy protects the kidney against injury during cold perfusion.

Demonstration of beta-tropomyosin (Tpm2) and duplication of the alpha-slow tropomyosin gene (TPM3) in Atlantic salmon Salmo salar.

Beta tropomyosin (Tpm2) is demonstrated for the first time at the protein level in a fish species, using a combination of electrophoresis, mass spectrometric peptide mapping and end-group analysis. Tpm2 accounts for 50% of the total tropomyosin in slow trunk muscle of the adult Atlantic salmon as determined by quantitative carboxypeptidase digestion and is also present in the head and pectoral fin. It is absent in the fast skeletal (lighter-toned) trunk muscle, the most abundant muscle, which is composed solely of an alpha-fast (Tpm1) isoform. In contrast to the mammalian homologues, salmon Tpm2 migrates faster than salmon Tpm1 in the presence of anionic detergent. Other distinguishing characteristics are a reduced content of cysteine (one per chain) and tyrosine (five per chain) and a unique carboxyl-terminal region (residues 276-284). Two isoforms (paralogs) of alpha-slow tropomyosin (Tpm3) having different contents of methionine and histidine exist in slow trunk muscle indicating duplication of the TPM3 gene. Minor skeletal muscles, surveyed for the first time, contain a mix of at least two tropomyosins - Tpm2 (~ 50% of total) in pectoral fin, jaw and tongue and another isoform, either Tpm1 (pectoral fin) or alpha-1-like Tpm (jaw and tongue). Cheek muscle contains Tpm1 and alpha 1-like Tpm in varying proportion depending upon the section (light or dark). Of the two tropomyosins in tongue, Tpm2 displays comparatively weaker affinity for troponin-Sepharose. A feature of the major sarcomeric tropomyosins in Atlantic salmon is a pair of neighbouring glycines situated between residues 20-90.

Heat stress response in the closest algal relatives of land plants reveals conserved stress signaling circuits.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8 Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat.

Therapy-Induced MHC I Ligands Shape Neo-Antitumor CD8 T Cell Responses during Oncolytic Virus-Based Cancer Immunotherapy.

Oncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies.

Marine Bacteria, A Source for Alginolytic Enzyme to Disrupt Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa biofilms are typically associated with the chronic lung infection of cystic fibrosis (CF) patients and represent a major challenge for treatment. This opportunistic bacterial pathogen secretes alginate, a polysaccharide that is one of the main components of its biofilm. Targeting this major biofilm component has emerged as a tempting therapeutic strategy for tackling biofilm-associated bacterial infections. The enormous potential in genetic diversity of the marine microbial community make it a valuable resource for mining activities responsible for a broad range of metabolic processes, including the alginolytic activity responsible for degrading alginate. A collection of 36 bacterial isolates were purified from marine water based on their alginolytic activity. These isolates were identified based on their 16S rRNA gene sequences. Pseudoalteromonas sp. 1400 showed the highest alginolytic activity and was further confirmed to produce the enzyme alginate lyase. The purified alginate lyase (AlyP1400) produced by Pseudoalteromonas sp. 1400 showed a band of 23 KDa on a protein electrophoresis gel and exhibited a bifunctional lyase activity for both poly-mannuronic acid and poly-glucuronic acid degradation. A tryptic digestion of this gel band analyzed by liquid chromatography-tandem mass spectrometry confirmed high similarity to the alginate lyases in polysaccharide lyase family 18. The purified alginate lyase showed a maximum relative activity at 30 °C at a slightly acidic condition. It decreased the sodium alginate viscosity by over 90% and reduced the P. aeruginosa (strain PA14) biofilms by 69% after 24 h of incubation. The combined activity of AlyP1400 with carbenicillin or ciprofloxacin reduced the P. aeruginosa biofilm thickness, biovolume and surface area in a flow cell system. The present data revealed that AlyP1400 combined with conventional antibiotics helped to disrupt the biofilms produced by P. aeruginosa and can be used as a promising combinational therapeutic strategy.

Transcriptome and proteome analyses to investigate the molecular underpinnings of cold response in the Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an insect that can cope with prolonged periods of low temperatures exposure. The molecular changes required to adapt to such conditions have not been thoroughly investigated in this insect. The current work aims at characterizing deregulated transcripts and proteins in adult L. decemlineata exposed to 15 °C and -5 °C using RNA-sequencing-based transcriptomics and liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics approaches, respectively. RNA-sequencing highlighted the differential expression of several transcripts, including ubiquilin-1 and ubiquitin carboxyl-terminal hydrolase 5, in insects submitted to low temperatures when compared with control insects. In addition, proteomics approach detected 2840 proteins in cold-exposed beetles including elevated levels for 409 proteins and reduced levels for 200 proteins. Cuticular proteins CP1, CP4, CP5 and CP7 as well as eukaryotic translation initiation factor 4B were notable proteins with elevated levels in cold insects. Functional analysis of targets modulated at low temperatures using DAVID indicated processes likely affected under cold conditions including select metabolic cascades and RNA-associated processes. Overall, this work presents molecular candidates impacted by low temperatures exposure in L. decemlineata and builds on the current knowledge associated with response to these conditions in this insect.

Knockdown of the small heat-shock protein p26 by RNA interference modifies the diapause proteome of Artemia franciscana.

Embryos of the crustacean Artemia franciscana may arrest as gastrulae, forming cysts that enter diapause, which is a state of reduced metabolism and enhanced stress tolerance. Diapausing cysts survive physiological stresses for years due, in part, to molecular chaperones. p26, a small heat-shock protein, is an abundant diapause-specific molecular chaperone in cysts, and it affects embryo development and stress tolerance. p26 is therefore thought to influence many proteins in cysts, and this study was undertaken to determine how the loss of p26 by RNA interference (RNAi) affects the diapause proteome of A. franciscana. The proteome was analyzed by shot-gun proteomics coupled to differential isotopic labeling and tandem mass spectrometry. Proteins in the diapause proteome included metabolic enzymes, antioxidants, binding proteins, structural proteins, transporters, translation factors, receptors, and signal transducers. Proteins within the diapause proteome either disappeared or were reduced in amount when p26 was knocked down, or conversely, proteins appeared or increased in amount. Those proteins that disappeared may be p26 substrates, whereas the synthesis of those proteins that appeared or increased may be regulated by p26. This study provides the first global characterization of the diapause proteome of A. franciscana and demonstrates that the sHsp p26 influences proteome composition.