Two-dimensional Fluorescence Difference Gel Electrophoresis Research Articles

Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE.

The present study aimed to identify candidate substrates of ubiquitin-specific protease (USP)13 using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). USP13 is a well-characterized member of the USP family, which regulates diverse cellular functions by cleaving ubiquitin from ubiquitinated protein substrates. However, existing studies indicate that USP13 has no detectable hydrolytic activity in vitro. This finding implies that USP13 likely has different substrate specificity. In this study, a USP cleavage assay was performed using two different types of model substrates (glutathione S-transferase-Ub52 and ubiquitin-β-galactosidase) to detect the deubiquitinating enzyme (DUB) activity of USP13. In addition, a proteomic approach was taken by using 2D-DIGE to detect cellular proteins whose expressoin is significantly altered in 293T cell lines following the overexpression of USP13 or its C345S mutant (the catalytically inactive form). The data indicated that USP13 still has no detectable DUB activity in vitro nor does C345S. The results of 2D-DIGE demonstrated that the expression of several proteins increased or decreased significantly in 293T cells following the overexpression of USP13. Mass spec troscopy analysis of gel spots identified 7 proteins, including 4 proteins with an increased expression, namely vinculin, thimet oligopeptidase, cleavage and polyadenylation specific factor 3, and methylosome protein 50, and 3 proteins with a decreased expression, namely adenylosuccinate synthetase, annexin and phosphoglycerate mutase. In addition, in the samples of 293T cell lines after the overexpression of USP13 and USP13 C345S, vinculin exhibited an increased expression, suggesting that it may be a candidate substrate of USP13. However, sufficient follow-up validation studies are required in order to determine whether vinculin protein directly interacts with USP13.

Proteomic approach to detect changes in hippocampal protein levels in an animal model of type 2 diabetes

In our previous study, we demonstrated that type 2 diabetes affects blood-brain barrier integrity and ultrastructural morphology in Zucker diabetic fatty (ZDF) rats at 40 weeks of age. In the present study, we investigated the possible candidates for diabetes-related proteins in the hippocampus of ZDF rats and their control littermates (Zucker lean control, ZLC), by using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Approximately 2756 protein spots were detected by 2D-DIGE, and an increase or decrease of more than 1.4-fold was observed for 13 proteins in the hippocampal homogenates of ZDF rats relative to those of ZLC rats. Among these proteins, we found four proteins whose levels were significantly lower in the hippocampi of ZDF rats than in those of ZLC rats: glial fibrillary acidic protein (GFAP), apolipoprotein A-I preprotein (apoAI-P), myelin basic protein (MBP), and rCG39881, isoform CRA_a. Among these proteins, apoAI-P protein levels were decreased most prominently in ZDF rats than in ZLC rats, based on Western blot analysis. In addition, immunohistochemical and Western blot studies demonstrated that MBP, not GFAP, immunoreactivity and protein levels were significantly decreased in the hippocampus of ZDF rats compared to ZLC rats. In addition, ultrastructural analysis showed that ZDF rats showed myelin degeneration and disarrangement in the hippocampal tissue. These results suggest that chronic type 2 diabetes affects hippocampal function via reduction of MBP and apoAI-P levels as well as disarrangement of myelin.

Down‐regulation of DAB2IP promotes colorectal cancer invasion and metastasis by translocating hnRNPK into nucleus to enhance the transcription of MMP2

DOC-2/DAB2 interacting protein (DAB2IP) is a RasGAP protein that shows a suppressive effect on cancer progression. Our previous study showed the involvement of transcription regulation of DAB2IP in metastasis of colorectal cancer (CRC). However, the molecular mechanisms of DAB2IP in regulating the progression of CRC need to be further explored. Here, we identified heterogeneous nuclear ribonucleoprotein K (hnRNPK) and matrix metalloproteinase 2 (MMP2) as vital downstream targets of DAB2IP in CRC cells by two-dimensional fluorescence difference gel electrophoresis and cDNA microassay, respectively. Mechanistically, down-regulation of DAB2IP increased the level of hnRNPK through MAPK/ERK signaling pathway. Subsequently, translocation of hnRNPK into nucleus enhanced the transcription activity of MMP2, and therefore promoted invasion and metastasis of CRC. Down-regulation of DAB2IP correlated negatively with hnRNPK and MMP2 expressions in CRC tissues. In conclusion, our study elucidates a novel mechanism of the DAB2IP/hnRNPK/MMP2 axis in the regulation of CRC invasion and metastasis, which may be a potential therapeutic target.

Identification of DJ-1 as a contributor to multidrug resistance in human small-cell lung cancer using proteomic analysis.

Proteomic approaches have been proven to provide an important tool in identifying drug resistance-associated proteins. The aim of this study was to investigate the protein profiling of drug resistance-related proteins in small-cell lung cancer (SCLC) by proteomic analysis. The proteomic profiling was performed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF-TOF of SCLC in the multidrug-resistant cell line H69AR and its parental cell line H69. A total of 11 proteins were identified to be >2-fold up-or downregulated between the two cell lines. DJ-1, one of the differently expressed proteins identified by proteomics, was further examined by immunohistochemistry staining in 116 cases of SCLC tissues. Immunohistochemical results demonstrated that DJ-1 was expressed in 51.7% (60/116) of SCLC. DJ-1 expression was correlated significantly with survival time of SCLC patients (P<0.05), but not with other clinical parameters such as gender, age and clinical stage (P>0.05). Downregulation of DJ-1 using DJ-1-siRNA in H69AR cells sensitized cancer cells to chemotherapeutic drugs through increasing drug-induced cell apoptosis accompanied with G0-G1 phase arrest. These findings suggest DJ-1 may serve as a potential biomarker for chemoresistance and prognostic factor for patients with SCLC.

Transglutaminase 2 is upregulated in primary hepatocellular carcinoma with early recurrence as determined by proteomic profiles.

The mechanism of early recurrence of hepato-cellular carcinoma (HCC) is not well understood. To examine whether early intrahepatic metastasis of HCC can be determined by the reliable molecular characteristics of the primary HCC, we focused on early-stage tumors of primary and solitary HCC cases. Proteomic differences were investigated between two groups, 11 early (recurrence within 12months) and 10 late (no recurrence within 48months) HCC cases, using two-dimensional fluorescence difference gel electrophoresis. Overall, 10upregulated and 9downregulated proteins were identified from a total of 1623protein spots detected in early recurrent HCC. Cluster analysis using the 19proteins successfully divided the 21HCC samples exactly into the two above groups. A multifunctional protein, transglutaminase2 (TGM2), was upregulated in the early recurrence group. Immunohistochemistry revealed the frequent observation of TGM2-positive HCC cells in the early group, with a tendency of TGM2-positive staining in HCC cells adjacent to fibrous stroma. To examine whether two major TGM2-associated pathways, epithelial-mesenchymal transition (EMT) and integrin signaling, were activated in the early recurrence group of HCC, downstream molecules of TGM2 were measured. The mRNA level of EMT-related genes was highly positively correlated with TGM2 mRNA. However, E-cadherin (CDH1) mRNA and protein were not downregulated in correlation with TGM2 expression. The phosphorylation of FAK and Akt and the downregulation of PTEN were not associated with the quantity of TGM2. Therefore, TGM2 might contribute to early HCC recurrence through signaling pathways not related to EMT and integrin signaling. The proteomics of strictly classified HCCs would be useful for characterizing pro-metastatic HCC and for developing a new therapeutic target for treatment of metastasis.

The short- and long-term proteomic effects of sleep deprivation on the cortical and thalamic synapses.

Acute total sleep deprivation (SD) impairs memory consolidation, attention, working memory and perception. Structural, electrophysiological and molecular experimental approaches provided evidences for the involvement of sleep in synaptic functions. Despite the wide scientific interest on the effects of sleep on the synapse, there is a lack of systematic investigation of sleep-related changes in the synaptic proteome. We isolated parietal cortical and thalamic synaptosomes of rats after 8h of total SD by gentle handling and 16h after the end of deprivation to investigate the short- and longer-term effects of SD on the synaptic proteome, respectively. The SD efficiency was verified by electrophysiology. Protein abundance alterations of the synaptosomes were analyzed by fluorescent two-dimensional differential gel electrophoresis and by tandem mass spectrometry. As several altered proteins were found to be involved in synaptic strength regulation, our data can support the synaptic homeostasis hypothesis function of sleep and highlight the long-term influence of SD after the recovery sleep period, mostly on cortical synapses. Furthermore, the large-scale and brain area-specific protein network change in the synapses may support both ideas of sleep-related synaptogenesis and molecular maintenance and reorganization in normal rat brain.

Up-Regulation of Antioxidant Proteins in the Plasma Proteome during Saturation Diving: Unique Coincidence under Hypobaric Hypoxia

Saturation diving (SD) is one of the safest techniques for tolerating hyperbaric conditions for long durations. However, the changes in the human plasma protein profile that occur during SD are unknown. To identify differential protein expression during or after SD, 65 blood samples from 15 healthy Japanese men trained in SD were analyzed by two-dimensional fluorescence difference gel electrophoresis. The expression of two proteins, one 32.4 kDa with an isoelectric point (pI) of 5.8 and the other 44.8 kDa with pI 4.0, were elevated during SD to 60, 100, and 200 meters sea water (msw). The expression of these proteins returned to pre-diving level when the SD training was completed. The two proteins were identified using in-gel digestion and mass spectrometric analysis; the 32.4 kDa protein was transthyretin and the 44.8 kDa protein was alpha-1-acid glycoprotein 1. Oxidation was detected at methionine 13 of transthyretin and at methionine 129 of alpha-1-acid glycoprotein 1 by tandem mass spectrometry. Moreover, haptoglobin was up-regulated during the decompression phase of 200 msw. These plasma proteins up-regulated during SD have a common function as anti-oxidants. This suggests that by coordinating their biological effects, these proteins activate a defense mechanism to counteract the effects of hyperbaric-hyperoxic conditions during SD.

Reduced Tau protein expression is associated with frontotemporal degeneration with progranulin mutation.

Reduction of Tau protein expression was described in 2003 by Zhukareva et al. in a variant of frontotemporal lobar degeneration (FTLD) referred to as diagnosis of dementia lacking distinctive histopathology, then re-classified as FTLD with ubiquitin inclusions. However, the analysis of Tau expression in FTLD has not been reconsidered since then. Knowledge of the molecular basis of protein aggregates and genes that are mutated in the FTLD spectrum would enable to determine whether the “Tau-less” is a separate pathological entity or if it belongs to an existing subclass of FTLD. To address this question, we have analyzed Tau expression in the frontal brain areas from control, Alzheimer’s disease and FTLD cases, including FTLD- Tau (MAPT), FTLD-TDP (sporadic, FTLD-TDP-GRN, FTLD-TDP-C9ORF72) and sporadic FTLD-FUS, using western blot and 2D-DIGE (Two-Dimensional fluorescence Difference Gel Electrophoresis) approaches. Surprisingly, we found that most of the FTLD-TDP-GRN brains are characterized by a huge reduction of Tau protein expression without any decrease in Tau mRNA levels. Interestingly, only cases affected by point mutations, rather than cases with total deletion of one GRN allele, seem to be affected by this reduction of Tau protein expression. Moreover, proteomic analysis highlighted correlations between reduced Tau protein level, synaptic impairment and massive reactive astrogliosis in these FTLD-GRN cases. Consistent with a recent study, our data also bring new insights regarding the role of progranulin in neurodegeneration by suggesting its involvement in lysosome and synaptic regulation. Together, our results demonstrate a strong association between progranulin deficiency and reduction of Tau protein expression that could lead to severe neuronal and glial dysfunctions. Our study also indicates that this FTLD-TDP-GRN subgroup could be part as a distinct entity of FTLD classification.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0345-0) contains supplementary material, which is available to authorized users.

Differential expression profile of membrane proteins in L-02 cells exposed to trichloroethylene.

Trichloroethylene (TCE), a halogenated organic solvent widely used in industries, is known to cause severe hepatotoxicity. However, the mechanisms underlying TCE hepatotoxicity are still not well understood. It is predicted that membrane proteins are responsible for key biological functions, and recent studies have revealed that TCE exposure can induce abnormal levels of membrane proteins in body fluids and cultured cells. The aim of this study is to investigate the TCE-induced alterations of membrane proteins profiles in human hepatic L-02 liver cells. A comparative membrane proteomics analysis was performed in combination with two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 15 proteins were identified as differentially expressed (4 upregulated and 11 downregulated) between TCE-treated cells and normal controls. Among this, 14 of them are suggested as membrane-associated proteins by their transmembrane domain and/or subcellular location. Furthermore, the differential expression of β subunit of adenosine triphosphate synthase (ATP5B) and prolyl 4-hydroxylase, β polypeptide (P4HB) were verified by Western blot analysis in TCE-treated L-02 cells. Our work not only reveals the association between TCE exposure and altered expression of membrane proteins but also provides a novel strategy to discover membrane biomarkers and elucidate the potential mechanisms involving with membrane proteins response to chemical-induced toxic effect.

Identification of Novel Key Molecules Involved in Spatial Memory Impairment in Triple Transgenic Mice of Alzheimer's Disease.

The molecular mechanisms underlying cognitive impairment in Alzheimer's disease (AD) remain largely unclear. In the present study, we were aimed to identify the potential key molecules involved in spatial memory impairment in a triple transgenic (3xTg-AD) mouse model of AD. By employing two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, we revealed a total of 24 differentially expressed proteins in hippocampus of 9-month-old 3xTg-AD mice with significant spatial memory impairment in comparison to the age-matched controls. These differentially expressed proteins can be categorized into several functional classifications that are related to synaptic/memory-, energy metabolism-, intracellular transport-, cell cycle-, cellular defense and structure, and stress response. To further verify the target proteins that may underlie the memory deficits, we pre-treated the 3xTg-AD mice for 3months with coenzyme Q10 (CoQ10) (800mg/kg body weight/day), a powerful endogenous antioxidant that has been shown to be able to prevent memory deficits in several AD mouse models. We found that administration of CoQ10 altered the expression levels of nine proteins in hippocampus of 3xTg-AD mice with simultaneous improvement of spatial memory. Interestingly, complexin-1/2, two molecules which were shown to alter LTP, were modulated (i.e., the levels were reduced in 3xTg-AD mice and CoQ10 restored the levels) in response to CoQ10 treatment among these nine proteins. Furthermore, we found that adeno-associated virus serotype 9 (AAV-9)-mediated overexpression of complexin-1/2 prevented memory impairment in the AD mouse model. Taken together, this study has identified a number of differentially expressed proteins in hippocampus of 3xTg-AD mice and the control in presence or absence of CoQ10. The modulation of complexin-1/2 expression by CoQ10 may contribute to the amelioration of memory impairment in the AD transgenic mice.

Spatial memory impairment by TRPC1 depletion is ameliorated by environmental enrichment.

Canonical transient receptor potential (TRPC) channels are widely expressed throughout the nervous system whereas their functions remain largely unclear. Here we investigated the effects of TRPC1 deletion on spatial memory ability of mice and the potential intervention by environmental enrichment (EE). Significant spatial memory impairment assessed by conditional fearing test, Y maze test and step-down test in TRPC1 knockout mice was revealed. The behavioral abnormality were attenuated by the treatment of EE. Proteomic analysis by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with a matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) and tandem mass spectrometry (MS) revealed that TRPC1 deletion caused differential expression of a total of 10 proteins (8 up-regulated and 2 down-regulated) in hippocampus. EE treatment resulted in differential expression of a total of 22 proteins (2 up-regulated and 20 down-regulated) in hippocampus of TRPC1 knockout mice. Among these differentially expressed proteins, the expression of α-internexin and glia maturation factor β (GMF-β), two proteins shown to impair memory, were significantly down-regulated in hippocampus of TRPC1 knockout mice by EE treatment. Taken together, these data suggested that TRPC1 regulated directly or indirectly the expression of multiple proteins, which may be crucial for the maintenance of memory ability, and that EE treatment modulated spatial memory impairment caused by TRPC1 depletion and the mechanisms may involve the modulation of EE on the expression of those dys-regulated proteins such as α-internexin and GMF-β in hippocampus.

Differential analyses of major allergen proteins in wild-type rice and rice producing a fragment of anti-rotavirus antibody

To develop oral antibody therapy against rotavirus infection, we previously produced a recombinant fragment of llama heavy-chain antibody to rotavirus (ARP1) in rice seeds (MucoRice-ARP1). We intend to use a purification-free rice powder for clinical application but needed to check whether MucoRice-ARP1 had increased levels of known allergen proteins. For this purpose, we used two-dimensional fluorescence difference gel electrophoresis to compare the allergen protein levels in MucoRice-ARP1 and wild-type rice. We detected no notable differences, except in the levels of α-amylase/trypsin inhibitor–like family proteins. Because by this approach we could not completely separate ARP1 from the proteins of this family, we confirmed the absence of changes in the levels of these allergens by using shotgun mass spectrometry as well as immunoblot. By using immunoelectron microscopy, we also showed that RAG2, a member of the α-amylase/trypsin inhibitor–like protein family, was relocated from protein bodies II to the plasma membrane or cell wall in MucoRice-ARP1 seed. The relocation did not affect the level of RAG2. We demonstrated that most of the known rice allergens were not considerably upregulated by the genetic modification in MucoRice-ARP1. Our data suggest that MucoRice-ARP1 is a potentially safe oral antibody for clinical application.

Probiotic (Enterococcus faecium) induced responses of the hepatic proteome improves metabolic efficiency of broiler chickens (Gallus gallus).

BackgroundThe liver plays important roles in nutrient metabolism, detoxification and immunity. Enterococcus faecium (E. faecium) is a probiotic that has been shown to have positive effects on broiler production. However, its molecular effects on liver metabolism have not been characterized. This study aims to further identify the biological roles of E. faecium by characterizing the hepatic proteomic changes of broilers (Gallus gallus) fed E. faecium using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) and mass spectrometry (MS).ResultsThirty-three proteins (50 protein spots) involved in nutrient metabolism, immunity and the antioxidant system were shown to be differentially expressed in the liver of broilers fed E. faecium than from birds not fed the probiotic. The biological processes of sulphur amino acids, vitamin and cellular hormone metabolism, sulphur compound biosynthesis and protein tetramerization were enhanced in the liver of broilers fed E. faecium. However, proteins involved in calcium ion flux, cell redox homeostasis and platelet activation related to hepatic immune responses were down-regulated in broilers fed E. faecium. These results indicate that the supplementation of poultry feed with E. faecium may alter the partitioning of nutrients and promote optimal nutrient utilization.ConclusionsThis study assists in unraveling the molecular effects of the dietary probiotic, E. faecium, in the liver of broiler chickens. It shows that the probiotic improves the metabolism of nutrients and decreases inflammatory responses. Our findings extend previous knowledge of the mechanism of dietary probiotic action and provide new findings for research and future probiotic development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2371-5) contains supplementary material, which is available to authorized users.

Proteomic analysis of hippocampus in mice following long-term exposure to low levels of copper.

Recent studies suggest that copper exposure, even at very low levels, can produce significant toxic effects on the brains of mice. This study is aimed to explore the effects of low levels of copper on the hippocampal proteome of mice. Two-dimensional fluorescence difference gel electrophoresis was performed on hippocampal homogenate obtained from mice, which were given either drinking water only (control) or water supplemented with 0.13 ppm copper (copper-treated) for a period of 8 months beginning at an age of 3 months. A total of 9 differentially expressed proteins between copper-treated mice and control mice were identified. Protein functional analysis revealed that the altered proteins mainly involved energy metabolism-related proteins, synaptic proteins, molecular chaperones and cellular structural components. Among these differentially expressed proteins, serine racemase (SRR) and glial fibrillary acidic protein (GFAP) were significantly down-regulated and up-regulated, respectively, in the hippocampus of copper-treated mice compared with the control mice. SRR was shown to be involved in memory formation. The increased expression of GFAP, an astrocyte marker, indicated that long-term low levels of copper exposure caused activation of the inflammatory response, a process linked to spatial memory impairment. In agreement with the data from proteomic analysis, memory impairment was observed in copper-treated mice as measured by the Morris water maze test. In summary, this study has identified a number of abnormally expressed proteins in the hippocampus of copper-treated mice, and the identified protein, such as SRR, together with inflammatory responses, as evidenced by the increased expression of GFAP, could contribute to memory impairment resulting from copper exposure. Our findings provide insights for a better understanding of copper neurotoxicity at the protein level in response to low levels of copper exposure.

HYOU1, Regulated by LPLUNC1, Is Up-Regulated in Nasopharyngeal Carcinoma and Associated with Poor Prognosis.

Objective: This study aims to investigate the roles and mechanisms of long palate, lung and nasal epithelium clone 1 (LPLUNC1) in nasopharyngeal carcinoma (NPC).Methods: The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-TOF-MS/MS) was applied to identify differentially expressed proteins after over-expressing LPLUNC1 in NPC cells. The qRT-PCR and Western Blot were used to further validate differentially expression of Hypoxia up-regulated 1 (HYOU1). We also applied immunohistochemistry (IHC) to validate the expression of HYOU1 protein in NPC tissues.Results: Totally 44 differentially expressed proteins were identified, among which 19 proteins were up-regulated and 25 proteins were down-regulated. Function annotation indicated that these proteins were involved in molecular chaperone, cytoskeleton, metabolism and signal transduction. It was shown that the expression of HYOU1 both at mRNA level and protein level was up-regulated significantly in NPC tissues, and HYOU1 protein expression was positively correlated with clinical staging and metastasis of NPC. Kaplan-Meier survival curves showed that high expression of HYOU1 protein in NPC patients had shorter progression-free survival (PFS) and overall survival (OS). COX multivariate regression analysis further indicated that over-expressed HYOU1 was one of the predictors for poor prognosis in NPC patients.Conclusion: Through regulating proteins in different pathways, LPLUNC1 may inhibit the growth of NPC through participating in cell metabolism, proliferation, transcription and signaling transduction. HYOU1 can be regarded as potential molecular biomarker for progression and prognosis of NPC.

Gender specific differences in the liver proteome of rats exposed to short term and low-concentration hexabromocyclododecane (HBCD).

The influence of short term (7-day) exposure of male rats to the brominated flame retardant hexabromocyclododecane (HBCD) was studied by investigation of the liver proteome, both in euthyroid and hypothyroid rats and by comparing results with general data on animal physiology and thyroid hormone, leptin, insulin and gonadotropin concentrations determined in parallel. Proteome analysis of liver tissue by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) revealed that only small protein pattern changes were induced by exposure in males, on just a few proteins with different functions and not involved in pathways in common. This is in contrast to previous findings in similarly exposed eu- and hypothyroid female rats, where general metabolic pathways had been shown to be affected. The largest gender-dependent effects concerned basal concentrations of liver proteins already in control and hypothyroid animals, involving mainly the pathways which were also differently affected by HBCD exposure. Among them were differences in lipid metabolism, which - upon exposure to HBCD - may also be the reason for the considerably higher ratio of γ-HBCD accumulated in white adipose tissue of exposed female rats compared to males. The results further elucidate the already suggested different sensitivity of genders towards HBCD exposure on the protein level, and confirm the need for undertaking toxicological animal experiments in both genders.

Analysis of fluoxetine-induced plasticity mechanisms as a strategy for understanding plasticity related neural disorders.

Fluoxetine hydrochloride, better known for its commercial name Prozac, is one of the most widely prescribed antidepressant drugs all over the world. This drug was considered a “breakthrough drug” for the treatment of depression because of its very high selectivity as a serotonin reuptake inhibitor and because it presented a lower side-effect profile than previous drugs (Wong et al., 2005). However, the precise mechanisms of fluoxetine action and how it alleviates mood disorders is still largely unknown.

Bordetella pertussis Isolates from Argentinean Whooping Cough Patients Display Enhanced Biofilm Formation Capacity Compared to Tohama I Reference Strain.

Pertussis is a highly contagious disease mainly caused by Bordetella pertussis. Despite the massive use of vaccines, since the 1950s the disease has become re-emergent in 2000 with a shift in incidence from infants to adolescents and adults. Clearly, the efficacy of current cellular or acellular vaccines, formulated from bacteria grown in stirred bioreactors is limited, presenting a challenge for future vaccine development. For gaining insights into the role of B. pertussis biofilm development for host colonization and persistence within the host, we examined the biofilm forming capacity of eight argentinean clinical isolates recovered from 2001 to 2007. All clinical isolates showed an enhanced potential for biofilm formation compared to the reference strain Tohama I. We further selected the clinical isolate B. pertussis 2723, exhibiting the highest biofilm biomass production, for quantitative proteomic profiling by means of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry, which was accompanied by targeted transcriptional analysis. Results revealed an elevated expression of several virulence factors, including adhesins involved in biofilm development. In addition, we observed a higher expression of energy metabolism enzymes in the clinical isolate compared to the Tohama I strain. Furthermore, all clinical isolates carried a polymorphism in the bvgS gene. This mutation was associated to an increased sensitivity to modulation and a faster rate of adhesion to abiotic surfaces. Thus, the phenotypic biofilm characteristics shown by the clinical isolates might represent an important, hitherto underestimated, adaptive strategy for host colonization and long time persistence within the host.

Isolation and Identification of a Red Pigment from the Antarctic Bacterium Shewanella frigidimarina.

The present study dealt with the isolation, identification and characterization of pigments from red snow samples of the Quito coastal front glacier (S 62º 27,217', W 059º 45,960') in Greenwich, Archipelago South Shetland, Antarctica, during summer 2013. As a strain of Shewanella was found to be the most common and abundant species with maximum red color production, the pigment -contained in the protein fraction- was isolated and characterized by high performance liquid chromatography (HPLC), two-dimensional fluorescence Difference Gel Electrophoresis (2-D DIGE) and matrix- assisted laser desorption/ionization-time of flight mass spectrometry (MALDI/TOF/TOF). The identified pigment is a cytochrome c3 with apparent molecular weight of 10 kDa and apparent pI around 4.5. The maximum pigment concentration was produced at warm temperatures, 28ºC, and with increasing exposure time to UV radiation. Here we demonstrate that the synthesis of cytochrome c3 by the Antarctic bacterium is due to thermal adaptation and/or adaptation to radiation. Further, pigments such as cytochrome c3 enable this bacterial species to use an anaerobic and ferric metabolism. In addition, this study draws attention to the relevance of adaptation investigations; to the study of in vivo monitoring of environmental warming and UV radiation due to global warming; and to the study of the potential habitability of other worlds in the Solar System and beyond.

Novel remodeling of the mouse heart mitochondrial proteome in response to acute insulin stimulation

Background and AimMitochondrial dysfunction contributes to the pathophysiology of diabetic cardiomyopathy. The aim of this study was to investigate the acute changes in the mitochondrial proteome in response to insulin stimulation. Methods and ResultsCardiac mitochondria from C57BL/6 mice after insulin stimulation were analyzed using two-dimensional fluorescence difference gel electrophoresis. MALDI-TOF MS/MS was utilized to identify differences. Two enzymes involved in metabolism and four structural proteins were identified. Succinyl-CoA ligase [ADP forming] subunit beta was identified as one of the differentially regulated proteins. Upon insulin stimulation, a relatively more acidic isoform of this protein was increased by 53% and its functional activity was decreased by ∼32%. ConclusionsThis proteomic remodeling in response to insulin stimulation may play an important role in the normal and diabetic heart.