Liver Tissue Proteins Improve the Accuracy of Plasma Proteins as Biomarkers in Diagnosing Metabolic Dysfunction-Associated Steatohepatitis.

BACKGROUND: Biomarkers for metabolic dysfunction-associated steatohepatitis (MASH) have been considered based on proteomic and lipidomic data from plasma and liver tissue without clinical benefits. This study evaluated proteomics-based plasma and liver tissue biomarkers collected simultaneously from patients with metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: Liver tissue and plasma samples were collected during liver biopsy to diagnose MASLD. Untargeted proteomics was performed on 64 patients. RESULTS: Twenty plasma proteins were up- or downregulated in patients with MASH compared with those without MASH. The potential biomarkers utilizing the best combinations of these plasma proteins had an area under the receiver operating curve (AUROC) of 0.671 for detecting those with MASH compared with those without it. However, none of the 20 plasma proteins were represented among the significantly regulated liver tissue proteins in patients with MASH. Ten of them displayed a trend and relevance in liver tissue with MASLD progression. These 10 plasma proteins had an AUROC of 0.793 for MASH identification and higher positive and negative predictive values. CONCLUSION: The plasma and liver protein expressions of patients with MASH were not directly comparable. Plasma protein biomarkers that are also expressed in liver tissue can help improve MASH detection.

Mitochondrial and Proteasome Dysfunction Occurs in the Hearts of Mice Treated with Triazine Herbicide Prometryn.

Prometryn is a methylthio-s-triazine herbicide used to control the growth of annual broadleaf and grass weeds in many cultivated plants. Significant traces of prometryn are documented in the environment, mainly in waters, soil, and plants used for human and domestic consumption. Previous studies have shown that triazine herbicides have carcinogenic potential in humans. However, there is limited information about the effects of prometryn on the cardiac system in the literature, or the mechanisms and signaling pathways underlying any potential cytotoxic effects are not known. It is important to understand the possible effects of exogenous compounds such as prometryn on the heart. To determine the mechanisms and signaling pathways affected by prometryn (185 mg/kg every 48 h for seven days), we performed proteomic profiling of male mice heart with quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. The data suggest that several major pathways, including energy metabolism, protein degradation, fatty acid metabolism, calcium signaling, and antioxidant defense system were altered in the hearts of prometryn-treated mice. Proteasome and immunoproteasome activity assays and expression levels showed proteasome dysfunction in the hearts of prometryn-treated mice. The results suggest that prometryn induced changes in mitochondrial function and various signaling pathways within the heart, particularly affecting stress-related responses.

Protein profiling of forehead epidermal corneocytes distinguishes frontal fibrosing from androgenetic alopecia.

Protein profiling offers an effective approach to characterizing how far epidermis departs from normal in disease states. The present pilot investigation tested the hypothesis that protein expression in epidermal corneocytes is perturbed in the forehead of subjects exhibiting frontal fibrosing alopecia. To this end, samples were collected by tape stripping from subjects diagnosed with this condition and compared to those from asymptomatic control subjects and from those exhibiting androgenetic alopecia. Unlike the latter, which exhibited only 3 proteins significantly different from controls in expression level, forehead samples from frontal fibrosing alopecia subjects displayed 72 proteins significantly different from controls, nearly two-thirds having lower expression. The results demonstrate frontal fibrosing alopecia exhibits altered corneocyte protein expression in epidermis beyond the scalp, indicative of a systemic condition. They also provide a basis for quantitative measures of departure from normal by assaying forehead epidermis, useful in monitoring response to treatment while avoiding invasive biopsy.

Elucidation of familial relationships using hair shaft proteomics.

This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedness. Proteomic profiling and amino acid sequence analysis provide information for quantitative and statistically-based analysis of individualization and sample similarity. Protein expression levels are a function of cell-specific transcriptional and translational programs. These programs are greatly influenced by an individual's genetic background, and are therefore influenced by familial relatedness as well as ancestry and genetic disease. Proteomic profiles should therefore be more similar among related individuals than unrelated individuals. Likewise, profiles of genetically variant peptides that contain single amino acid polymorphisms, the result of non-synonymous SNP alleles, should behave similarly. The proteomically-inferred SNP alleles should also provide a basis for calculation of combined paternity and sibship indices. We test these hypotheses using matching proteomic and genetic datasets from a family of two adults and four siblings, one of which has a genetic condition that perturbs hair structure and properties. We demonstrate that related individuals, compared to those who are unrelated, have more similar proteomic profiles, profiles of genetically variant peptides and higher combined paternity indices and combined sibship indices. This study builds on previous analyses of hair shaft protein profiling and genetically variant peptide profiles in different real-world scenarios including different human hair shaft body locations and pigmentation status. It also validates the inclusion of proteomic information with other biomolecular substrates in forensic hair shaft analysis, including mitochondrial and nuclear DNA.

Genome-wide CRISPRi screening identifies OCIAD1 as a prohibitin client and regulatory determinant of mitochondrial Complex III assembly in human cells.

Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause of human mitochondrial diseases. To identify determinants of mETC function, we screened a genome-wide human CRISPRi library under oxidative metabolic conditions with selective inhibition of mitochondrial Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as a Complex III assembly factor. We find that OCIAD1 is an inner mitochondrial membrane protein that forms a complex with supramolecular prohibitin assemblies. Our data indicate that OCIAD1 is required for maintenance of normal steady-state levels of Complex III and the proteolytic processing of the catalytic subunit cytochrome c1 (CYC1). In OCIAD1 depleted mitochondria, unprocessed CYC1 is hemylated and incorporated into Complex III. We propose that OCIAD1 acts as an adaptor within prohibitin assemblies to stabilize and/or chaperone CYC1 and to facilitate its proteolytic processing by the IMMP2L protease.

Comparative Proteomic Analysis of Walnut (Juglans regia L.) Pellicle Tissues Reveals the Regulation of Nut Quality Attributes.

Walnuts (Juglans regia L.) are a valuable dietary source of polyphenols and lipids, with increasing worldwide consumption. California is a major producer, with 'Chandler' and 'Tulare' among the cultivars more widely grown. 'Chandler' produces kernels with extra light color at a higher frequency than other cultivars, gaining preference by growers and consumers. Here we performed a deep comparative proteome analysis of kernel pellicle tissue from these two valued genotypes at three harvest maturities, detecting a total of 4937 J. regia proteins. Late and early maturity stages were compared for each cultivar, revealing many developmental responses common or specific for each cultivar. Top protein biomarkers for each developmental stage were also selected based on larger fold-change differences and lower variance among replicates, including proteins for biosynthesis of lipids and phenols, defense-related proteins and desiccation stress-related proteins. Comparison between the genotypes also revealed the common and specific protein repertoires, totaling 321 pellicle proteins with differential abundance at harvest stage. The proteomics data provides clues on antioxidant, secondary, and hormonal metabolism that could be involved in the loss of quality in the pellicles during processing for commercialization.

Optimal processing for proteomic genotyping of single human hairs.

The use of hair evidence for human identification is undergoing considerable improvement through the adoption of proteomic genotyping. Unlike traditional microscopic comparisons, protein sequencing provides quantitative and empirically based estimates for random match probability. Non-synonymous SNPs are translated as single amino acid polymorphisms and result in genetically variant peptides. Using high resolution mass spectrometry, these peptides can be detected in hair shaft proteins and used to infer the genotypes of corresponding SNP alleles. We describe experiments to optimize the proteomic genotyping approach to individual identification from a single human scalp hair 2 cm in length (∼100 µg). This is a necessary step to develop a protocol that will be useful to forensic investigators. To increase peptide yield from hair, and to maximize genetically variant peptide and ancestral information, we examined the conditions for reduction, alkylation, and protein digestion that specifically address the distinctive chemistry of the hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 h of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 h of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times increased the average number of genetically variant peptides from 20 ±â€¯5 to 73 ±â€¯5 (p = 1 × 10-13), excluding intractable hair samples. Random match probabilities reached up to 1 in 620 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original hair protein extraction method. Ancestral information was also present in the data. While the number of genetically variant peptides detected were equivalent for both European and African subjects, the estimated random match probabilities for inferred genotypes of European subjects were considerably smaller in African reference populations and vice versa, resulting in a difference in likelihood ratios of 6.8 orders of magnitude. This research will assure uniformity in results across different biogeographic backgrounds and enhance the use of novel peptide analysis in forensic science by helping to optimize genetically variant peptide yields and discovery. This work also introduces two algorithms, GVP Finder and GVP Scout, which facilitate searches, calculate random match probabilities, and aid in discovery of genetically variant peptides.

Age-Related Changes in Hair Shaft Protein Profiling and Genetically Variant Peptides.

Recent reports highlight possible improvements in individual identification using proteomic information from human hair evidence. These reports have stimulated investigation of parameters that affect the utility of proteomic information. In addition to variables already studied relating to processing technique and anatomic origin of hair shafts, an important variable is hair ageing. Present work focuses on the effect of age on protein profiling and analysis of genetically variant peptides (GVPs). Hair protein profiles may be affected by developmental and physiological changes with age of the donor, exposure to different environmental conditions and intrinsic processes, including during storage. First, to explore whether general trends were evident in the population at different ages, hair samples were analyzed from groups of different subjects in their 20's, 40's and 60's. No significant differences were seen as a function of age, but consistent differences were evident between European American and African American hair profiles. Second, samples collected from single individuals at different ages were analyzed. Mostly, these showed few protein expression level differences over periods of 10 years or less, but samples from subjects at 44 and 65 year intervals were distinctly different in profile. The results indicate that use of protein profiling for personal identification, if practical, would be limited to decadal time intervals. Moreover, batch effects were clearly evident in samples processed by different staff. To investigate the contribution of storage (at room temperature) in affecting the outcomes, the same proteomic digests were analyzed for GVPs. In samples stored over 10 years, GVPs were reduced in number in parallel with the yield of identified proteins and unique peptides. However, a very different picture emerged with respect to personal identification. Numbers of GVPs sufficed to distinguish individuals despite the age differences of the samples. As a practical matter, three hair samples per person provided nearly the maximal number obtained from 5 or 6 samples. The random match probability (where the log increased in proportion to the number of GVPs) reached as high as 1 in 108. The data indicate that GVP results are dependent on the single nucleotide polymorphism profile of the donor genome, where environmental/processing factors affect only the yield, and thus are consistent despite the ages of the donors and samples and batchwise effects in processing. This conclusion is critical for application to casework where the samples may be in storage for long periods and used to match samples recently collected.

Prioritization of metabolic genes as novel therapeutic targets in estrogen-receptor negative breast tumors using multi-omics data and text mining.

Estrogen-receptor negative (ERneg) breast cancer is an aggressive breast cancer subtype in the need for new therapeutic options. We have analyzed metabolomics, proteomics and transcriptomics data for a cohort of 276 breast tumors (MetaCancer study) and nine public transcriptomics datasets using univariate statistics, meta-analysis, Reactome pathway analysis, biochemical network mapping and text mining of metabolic genes. In the MetaCancer cohort, a total of 29% metabolites, 21% proteins and 33% transcripts were significantly different (raw p <0.05) between ERneg and ERpos breast tumors. In the nine public transcriptomics datasets, on average 23% of all genes were significantly different (raw p <0.05). Specifically, up to 60% of the metabolic genes were significantly different (meta-analysis raw p <0.05) across the transcriptomics datasets. Reactome pathway analysis of all omics showed that energy metabolism, and biosynthesis of nucleotides, amino acids, and lipids were associated with ERneg status. Text mining revealed that several significant metabolic genes and enzymes have been rarely reported to date, including PFKP, GART, PLOD1, ASS1, NUDT12, FAR1, PDE7A, FAHD1, ITPK1, SORD, HACD3, CDS2 and PDSS1. Metabolic processes associated with ERneg tumors were identified by multi-omics integration analysis of metabolomics, proteomics and transcriptomics data. Overall results suggested that TCA anaplerosis, proline biosynthesis, synthesis of complex lipids and mechanisms for recycling substrates were activated in ERneg tumors. Under-reported genes were revealed by text mining which may serve as novel candidates for drug targets in cancer therapies. The workflow presented here can also be used for other tumor types.

Profiling of proteins secreted in the bovine oviduct reveals diverse functions of this luminal microenvironment.

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility.

Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence.

Integrated Metabolomics and Proteomics Highlight Altered Nicotinamide- and Polyamine Pathways in Lung Adenocarcinoma.

Lung cancer is the leading cause of cancer mortality in the United States with non-small cell lung cancer (NSCLC) adenocarcinoma being the most common histological type. Early perturbations in cellular metabolism are a hallmark of cancer, but the extent of these changes in early stage lung adenocarcinoma remains largely unknown. In the current study, an integrated metabolomics and proteomics approach was utilized to characterize the biochemical and molecular alterations between malignant and matched control tissue from 27 subjects diagnosed with early stage lung adenocarcinoma. Differential analysis identified 71 metabolites and 1102 proteins that delineated tumor from control tissue. Integrated results indicated four major metabolic changes in early stage adenocarcinoma: (1) increased glycosylation and glutaminolysis; (2) elevated Nrf2 activation; (3) increase in nicotinic and nicotinamide salvaging pathways; and (4) elevated polyamine biosynthesis linked to differential regulation of the SAM/nicotinamide methyl-donor pathway. Genomic data from publicly available databases were included to strengthen proteomic findings. Our findings provide insight into the biochemical and molecular biological reprogramming that may accompanies early stage lung tumorigenesis and highlight potential therapeutic targets.

In vivo digestomics of milk proteins in human milk and infant formula using a suckling rat pup model.

Human milk is the optimal mode of infant feeding for the first several months of life, and infant formulas serve as an alternative when breast-feeding is not possible. Milk proteins have a balanced amino acid composition and some of them provide beneficial bioactivities in their intact forms. They also encrypt a variety of bioactive peptides, possibly contributing to infant health and growth. However, there is limited knowledge of how milk proteins are digested in the gastrointestinal tract and bioactive peptides are released in infants. A peptidomic analysis was conducted to identify peptides released from milk proteins in human milk and infant formula, using a suckling rat pup model. Among the major milk proteins targeted, α-lactalbumin and ß-casein in human milk, and ß-lactoglobulin and ß-casein in infant formula were the main sources of peptides, and these peptides covered large parts of the parental proteins' sequences. Release of peptides was concentrated to specific regions, such as residues 70-92 of ß-casein in human milk, residues 39-55 of ß-lactoglobulin in infant formula, and residues 57-96 and 145-161 of ß-CN in infant formula, where resistance to gastrointestinal digestion was suggested. In the context of bioactive peptides, release of fragments containing known bioactive peptides was confirmed, such as ß-CN-derived opioid and antihypertensive peptides. It is therefore likely that these fragments are of biological significance in neonatal health and development.

Proteomic profiling of lung adenocarcinoma indicates heightened DNA repair, antioxidant mechanisms and identifies LASP1 as a potential negative predictor of survival.

BACKGROUND: Lung cancer is the leading cause of cancer mortality in the United States. Non-small cell lung cancer accounts for 85% of all lung cancers for which adenocarcinoma is the most common histological type. Management of lung cancer is hindered by high false-positive rates due to difficulty resolving between benign and malignant tumors. Better molecular analysis comparing malignant and non-malignant tissues will provide important evidence of the underlying biology contributing to tumorigenesis. METHODS: We utilized a proteomics approach to analyze 38 malignant and non-malignant paired tissue samples obtained from current or former smokers with early stage (Stage IA/IB) lung adenocarcinoma. Statistical mixed effects modeling and orthogonal partial least squares discriminant analysis were used to identify key cancer-associated perturbations in the adenocarcinoma proteome. Identified proteins were subsequently assessed against clinicopathological variables. RESULTS: Top cancer-associated protein alterations were characterized by: (1) elevations in APEX1, HYOU1 and PDIA4, indicative of increased DNA repair machinery and heightened anti-oxidant defense mechanisms; (2) increased LRPPRC, STOML2, COPG1 and EPRS, suggesting altered tumor metabolism and inflammation; (3) reductions in SPTB, SPTA1 and ANK1 implying dysregulation of membrane integrity; and (4) decreased SLCA41 suggesting altered pH regulation. Increased protein levels of HYOU1, EPRS and LASP1 in NSCLC adenocarcinoma was independently validated by tissue microarray immunohistochemistry. Immunohistochemistry for HYOU1 and EPRS indicated AUCs of 0.952 and 0.841, respectively, for classifying tissue as malignant. Increased LASP1 correlated with poor overall survival (HR 3.66 per unit increase; CI 1.37-9.78; p = 0.01). CONCLUSION: These results reveal distinct proteomic changes associated with early stage lung adenocarcinoma that may be useful prognostic indicators and therapeutic targets.

The Secreted Protease PrtA Controls Cell Growth, Biofilm Formation and Pathogenicity in Xylella fastidiosa.

Pierce's disease (PD) is a deadly disease of grapevines caused by the Gram-negative bacterium Xylella fastidiosa. Though disease symptoms were formerly attributed to bacteria blocking the plant xylem, this hypothesis is at best overly simplistic. Recently, we used a proteomic approach to characterize the secretome of X. fastidiosa, both in vitro and in planta, and identified LesA as one of the pathogenicity factors of X. fastidiosa in grapevines that leads to leaf scorching and chlorosis. Herein, we characterize another such factor encoded by PD0956, designated as an antivirulence secreted protease "PrtA" that displays a central role in controlling in vitro cell proliferation, length, motility, biofilm formation, and in planta virulence. The mutant in X. fastidiosa exhibited reduced cell length, hypermotility (and subsequent lack of biofilm formation) and hypervirulence in grapevines. These findings are supported by transcriptomic and proteomic analyses with corresponding plant infection data. Of particular interest, is the hypervirulent response in grapevines observed when X. fastidiosa is disrupted for production of PrtA, and that PD-model tobacco plants transformed to express PrtA exhibited decreased symptoms after infection by X. fastidiosa.

The 2012/2013 ABRF Proteomic Research Group Study: Assessing Longitudinal Intralaboratory Variability in Routine Peptide Liquid Chromatography Tandem Mass Spectrometry Analyses.

Questions concerning longitudinal data quality and reproducibility of proteomic laboratories spurred the Protein Research Group of the Association of Biomolecular Resource Facilities (ABRF-PRG) to design a study to systematically assess the reproducibility of proteomic laboratories over an extended period of time. Developed as an open study, initially 64 participants were recruited from the broader mass spectrometry community to analyze provided aliquots of a six bovine protein tryptic digest mixture every month for a period of nine months. Data were uploaded to a central repository, and the operators answered an accompanying survey. Ultimately, 45 laboratories submitted a minimum of eight LC-MSMS raw data files collected in data-dependent acquisition (DDA) mode. No standard operating procedures were enforced; rather the participants were encouraged to analyze the samples according to usual practices in the laboratory. Unlike previous studies, this investigation was not designed to compare laboratories or instrument configuration, but rather to assess the temporal intralaboratory reproducibility. The outcome of the study was reassuring with 80% of the participating laboratories performing analyses at a medium to high level of reproducibility and quality over the 9-month period. For the groups that had one or more outlying experiments, the major contributing factor that correlated to the survey data was the performance of preventative maintenance prior to the LC-MSMS analyses. Thus, the Protein Research Group of the Association of Biomolecular Resource Facilities recommends that laboratories closely scrutinize the quality control data following such events. Additionally, improved quality control recording is imperative. This longitudinal study provides evidence that mass spectrometry-based proteomics is reproducible. When quality control measures are strictly adhered to, such reproducibility is comparable among many disparate groups. Data from the study are available via ProteomeXchange under the accession code PXD002114.

Mitochondrial proteome remodeling in ischemic heart failure.

AIMS: Mitochondrial dysfunction is an important part of the decline in cardiac function in heart failure. We hypothesized for hypothesized that there would be specific abnormalities in mitochondrial function and proteome with the progression of ischemic heart failure (HF). MAIN METHODS: We used a high left anterior descending artery (LAD) ligation in 3-4month old male rats to generate HF. Rats were studied 9weeks post-ligation. KEY FINDINGS: Electron microscopy of left ventricle samples showed mitochondrial changes including decreased size, increased number, abnormal distribution, and cristae loss. Mitochondria in ischemic HF exhibited decreased total ATP, impaired mitochondrial respiration, as well as reduced complex I activity. Analysis of LV mitochondrial proteins by mass spectrometry was performed, and 31 differentially expressed proteins (p<0.05) of more than 500 total proteins were identified. Of these proteins, 15 were up-regulated and 16 were down-regulated in the failing heart. A set of complex I proteins was significantly decreased, consistent with the impairment of complex I activity. There were distinct changes in mitochondrial function and proteome in ischemic HF. Although there were similarities, the distinction between the reported proteomic changed with TAC pressure overload induced HF and ischemic HF in the current study suggested different pathological mechanisms. SIGNIFICANCE: Specific changes in mitochondrial protein expression, which correlate with changes in mitochondrial function, have been identified in ischemic HF for the first time.

Shotgun proteomic analysis unveils survival and detoxification strategies by Caulobacter crescentus during exposure to uranium, chromium, and cadmium.

The ubiquitous bacterium Caulobacter crescentus holds promise to be used in bioremediation applications due to its ability to mineralize U(VI) under aerobic conditions. Here, cell free extracts of C. crescentus grown in the presence of uranyl nitrate [U(VI)], potassium chromate [Cr(VI)], or cadmium sulfate [Cd(II)] were used for label-free proteomic analysis. Proteins involved in two-component signaling and amino acid metabolism were up-regulated in response to all three metals, and proteins involved in aerobic oxidative phosphorylation and chemotaxis were down-regulated under these conditions. Clustering analysis of proteomic enrichment revealed that the three metals also induce distinct patterns of up- or down-regulated expression among different functional classes of proteins. Under U(VI) exposure, a phytase enzyme and an ABC transporter were up-regulated. Heat shock and outer membrane responses were found associated with Cr(VI), while efflux pumps and oxidative stress proteins were up-regulated with Cd(II). Experimental validations were performed on select proteins. We found that a phytase plays a role in U(VI) and Cr(VI) resistance and detoxification and that a Cd(II)-specific transporter confers Cd(II) resistance. Interestingly, analysis of promoter regions in genes associated with differentially expressed proteins suggests that U(VI) exposure affects cell cycle progression.

Cryptococcus neoformans promotes its transmigration into the central nervous system by inducing molecular and cellular changes in brain endothelial cells.

Cryptococcus spp. cause fungal meningitis, a life-threatening infection that occurs predominately in immunocompromised individuals. In order for Cryptococcus neoformans to invade the central nervous system (CNS), it must first penetrate the brain endothelium, also known as the blood-brain barrier (BBB). Despite the importance of the interrelation between C. neoformans and the brain endothelium in establishing CNS infection, very little is known about this microenvironment. Here we sought to resolve the cellular and molecular basis that defines the fungal-BBB interface during cryptococcal attachment to, and internalization by, the human brain endothelium. In order to accomplish this by a systems-wide approach, the proteomic profile of human brain endothelial cells challenged with C. neoformans was resolved using a label-free differential quantitative mass spectrometry method known as spectral counting (SC). Here, we demonstrate that as brain endothelial cells associate with, and internalize, cryptococci, they upregulate the expression of several proteins involved with cytoskeleton, metabolism, signaling, and inflammation, suggesting that they are actively signaling and undergoing cytoskeleton remodeling via annexin A2, S100A10, transgelin, and myosin. Transmission electronic microscopy (TEM) analysis demonstrates dramatic structural changes in nuclei, mitochondria, the endoplasmic reticulum (ER), and the plasma membrane that are indicative of cell stress and cell damage. The translocation of HMGB1, a marker of cell injury, the downregulation of proteins that function in transcription, energy production, protein processing, and the upregulation of cyclophilin A further support the notion that C. neoformans elicits changes in brain endothelial cells that facilitate the migration of cryptococci across the BBB and ultimately induce endothelial cell necrosis.

Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host.

The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3-5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.