Relative Amounts Of Proteins Research Articles

Time-course studies of osmoregulatory responses in different salinities for the marbled eel (Anguilla marmorata)

Marbled eel (Anguilla marmorata) is a major aquaculture species in temperate, tropical, and subtropical regions. Recent years, the production of critically endangered wild eels has fallen sharply due to immature aquaculture. A huge problem in cultivating seedlings is that osmotic conditions can be hardly simulated for eels between artificial conditions and natural environments. So comprehending the eels' osmoregulatory mechanisms will help aquaculture practitioners improve the production of eel larvae. In the current research, we examined the time-course studies of osmoregulatory responses when eels were transferred from fresh water (FW, 0‰) to FW, brackish water (BW, 10‰) and saline water (SW, 25‰). In FW, the levels of plasma ions, osmolality and cortisol did not change according to the stated hours. While plasma ions, osmolality and cortisol were significantly increased in the first six hours and decreased to stationary phase in BW and SW. And the levels in SW were always higher than BW in the same hour. Meanwhile, we firstly identified 11β-hydroxysteroid dehydrogenase 2 (hsd11b2) in Anguilla marmorata by the rapid amplification of cDNA ends (RACE) approach. In order to estimate the molecular responses when salinity increases, the time-course expressions of sodium potassium chloride co-transporter 1 (NKCC1) and HSD11B2 were tested in the gill of eels in mRNA and protein levels. In FW, the expressions of NKCC1 and HSD11B2 were stable according to the analyzed hours. The expression of NKCC1 initially increased then decreased in BW compared to FW. NKCC1 and HSD11B2 expressed lower in SW within 96 h compared to FW for the relative protein amounts, indicating the different post-translational modifications among different fish species. This study indicated an efficient acclimation of eels to higher salinities by upregulating the eels' physiological parameters quickly in hours and increasing active ion transport and cortisol synthesis in the gill.

Cross-Species Proteomic Comparison of Outer Membrane Vesicles and Membranes of Francisella tularensis subsp. tularensis versus subsp. holarctica.

Release of outer membrane vesicles (OMV) is an important phenomenon in Gram-negative bacteria playing multiple roles in their lifestyle, including in relation to virulence and host-pathogen interaction. Francisella tularensis, unlike other bacteria, releases unusually shaped, tubular OMV. We present a proteomic comparison of OMV and membrane fractions from two F. tularensis strains: moderately virulent subsp. holarctica strain FSC200 and highly virulent subsp. tularensis strain SchuS4. Proteomic comparison studies routinely evaluate samples from the same proteome, but sometimes we must compare samples from closely related organisms. This raises quantification issues. We propose a novel approach to cross-species proteomic comparison based on an intersection protein database from the individual single-species databases. This is less prone to quantification errors arising from differences in the sequences. Consecutively comparing subproteomes of OMV and membranes of the two strains allows distinguishing differences in relative protein amounts caused by global expression changes from those caused by preferential protein packing to OMV or membranes. Among the proteins most differently packed into OMV between the two strains, we detected proteins involved in biosynthesis and metabolism of bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids, as well as some major structural outer membrane proteins. The data are available via ProteomeXchange with identifier PXD022406.

Altered high-density lipoprotein composition and functions during severe COVID-19

Coronavirus disease 2019 (COVID-19) pandemic is affecting millions of patients worldwide. The consequences of initial exposure to SARS-CoV-2 go beyond pulmonary damage, with a particular impact on lipid metabolism. Decreased levels in HDL-C were reported in COVID-19 patients. Since HDL particles display antioxidant, anti-inflammatory and potential anti-infectious properties, we aimed at characterizing HDL proteome and functionality during COVID-19 relative to healthy subjects. HDLs were isolated from plasma of 8 severe COVID-19 patients sampled at admission to intensive care unit (Day 1, D1) at D3 and D7, and from 16 sex- and age-matched healthy subjects. Proteomic analysis was performed by LC-MS/MS. The relative amounts of proteins identified in HDLs were compared between COVID-19 and controls. apolipoprotein A-I and paraoxonase 1 were confirmed by Western-blot analysis to be less abundant in COVID-19 versus controls, whereas serum amyloid A and alpha-1 antitrypsin were higher. HDLs from patients were less protective in endothelial cells stiumalted by TNFα (permeability, VE-cadherin disorganization and apoptosis). In these conditions, HDL inhibition of apoptosis was blunted in COVID-19 relative to controls. In conclusion, we show major changes in HDL proteome and decreased functionality in severe COVID-19 patients.

Effect of hydrogen peroxide concentration on the maintenance and differentiation of cultured skeletal muscle cells

We have studied the effects of hydrogen peroxide (H2O2) on the differentiation and maintenance of C2C12 myoblasts. The effects of H2O2 were evaluated by cell viability, total protein concentration, the relative amount of muscle-related proteins, sarcomere structure, and active tension generation. Oxidative stress is one of the major causes of myopathy after exercise and thus establishing the method to evaluate the effects on muscle function is essential. The primary function of striated muscle is to generate force, thus, the measurement of active tension is important in assessing the effect of chemicals on muscle. Among the indices we tested, the sarcomere structure was the most sensitive to the H2O2 exposure while the cell viability was less sensitive. The effects of H2O2 on active tension correlated with a decrease in the amount of muscle proteins. In this study, our results showed that the effect of chemicals on muscle should be measured in multiple ways, including active tension generation, for a better understanding of its physiological impact.

Mechanisms of acclimation to hypersalinity in two European sea bass lineages: a focus on the kidney function

European sea bass (Dicentrarchus labrax), a major aquaculture species, is distributed along the coasts of the North-Eastern Atlantic Ocean, Mediterranean and Black Sea. D. labrax enter lagoons and estuaries where salinity fluctuates and sometimes reaches levels over 60‰, notably in Mediterranean lagoons. Keeping in mind that European sea bass are genetically subdivided in an Atlantic and a Mediterranean lineage, we compared fish from Atlantic (A) and West Mediterranean (M) populations regarding their capacity to tolerate hypersalinity with a focus on the kidney, a key organ involved in water reabsorption at high salinity. Fish were analyzed following a two-week transfer from seawater (SW, 36‰) to either seawater (SW, 36‰) or hypersaline water (HW, 55‰). Plasma osmolality was significantly increased in the MHW group compared to the other groups. Plasma sodium levels were significantly increased in hypersaline water compared to seawater in both lineages whereas plasma chloride levels showed an opposite trend. In order to estimate water filtration at the kidney level, the size of renal glomeruli was investigated and showed a decreased glomerulus perimeter and area in hypersaline water compared to seawater. NKA was highly expressed in all kidney tubules notably collecting tubules and ducts. There was an effect of salinity on renal nka α1a mRNA expression with slightly lower transcript levels at 55‰ compared to 36‰. Relative protein amounts and activity of NKA however were significantly higher in fish exposed to hypersalinity regardless of their origin. AQP1a immunolabeling differed between proximal tubules subtypes and only faint AQP1a was detected in subapical parts of cells lining collecting ducts. The transcript levels of renal aqp 1a were lower in the HW group than the SW group whereas the expression of other aqp paralogs (aqp 1b, aqp 8b) did not change according to the analyzed conditions. This study showed an efficient acclimation of sea bass to high salinity by increasing active ion transport at the kidney and by decreasing the size of filtering glomeruli to minimize water loss through urine. Despite Mediterranean D. labrax are supposed to more often encounter high salinities in their habitat, their high blood osmolality in hypersaline water indicates that their overall response to hypersalinity seems not improved compared to the Atlantic lineage. However, at the kidney level, the traits analyzed differ slightly between genetic lineages, potentially as a response to high blood osmolalities in MHW.

Genetic and toxinological divergence among populations of Tityus trivittatus Kraepelin, 1898 (Scorpiones: Buthidae) inhabiting Paraguay and Argentina.

Envenoming by scorpions in genus Tityus is a public health problem in Tropical America. One of the most medically significant species is Tityus trivittatus, which is known to occur from southwest Brazil to central-northern and eastern Argentina. In this work, we studied the lethality, composition, antigenicity, and enzymatic activity of venom from a T. trivittatus population found further north in urban areas of eastern Paraguay, where it has caused serious envenomation of children. Our results indicate that the population is of medical importance as it produces a potently toxic venom with an LD50 around 1.19 mg/kg. Venom neutralization in preliminary mouse bioassays was complete when using Brazilian anti-T. serrulatus antivenom but only partial when using Argentinean anti-T. trivittatus antivenom. Venom competitive solid-phase enzyme immunoassays and immunoblotting from Argentinean and Paraguayan T. trivittatus populations indicated that antigenic differences exist across the species range. SDS-PAGE showed variations in type and relative amounts of venom proteins between T. trivitattus samples from Argentina and Paraguay. MALDI-TOF mass spectrometry indicated that while some sodium channel toxins are shared, including β-toxin Tt1g, others are population-specific. Proteolytic activity by zymography and peptide identification through nESI-MS/MS also point out that population-specific proteases may exist in T. trivitattus, which are postulated to be involved in the envenoming process. A time-calibrated molecular phylogeny of mitochondrial COI sequences revealed a significant (8.14%) genetic differentiation between the Argentinean and Paraguayan populations, which appeared to have diverged between the mid Miocene and early Pliocene. Altogether, toxinological and genetic evidence indicate that T. trivitattus populations from Paraguay and Argentina correspond to distinct, unique cryptic species, and suggest that further venom and taxonomic diversity exists in synanthropic southern South American Tityus than previously thought.

Rapid and Easy High-Molecular-Weight Glutenin Subunit Identification System by Lab-on-a-Chip in Wheat (Triticum aestivum L.).

Lab-on-a-chip technology is an emerging and convenient system to easily and quickly separate proteins of high molecular weight. The current study established a high-molecular-weight glutenin subunit (HMW-GS) identification system using Lab-on-a-chip for three, six, and three of the allelic variations at the Glu-A1, Glu-B1, and Glu-D1 loci, respectively, which are commonly used in wheat breeding programs. The molecular weight of 1Ax1 and 1Ax2* encoded by Glu-A1 locus were of 200 kDa and 192 kDa and positioned below 1Dx subunits. The HMW-GS encoded by Glu-B1 locus were electrophoresed in the following order below 1Ax1 and 1Ax2*: 1Bx13 ≥ 1Bx7 = 1Bx7OE > 1Bx17 > 1By16 > 1By8 = 1By18 > 1By9. 1Dx2 and Dx5 showed around 4-kDa difference in their molecular weights, with 1Dy10 and 1Dy12 having 11-kDa difference, and were clearly differentiated on Lab-on-a-chip. Additionally, some of the HMW-GS, including 1By8, 1By18, and 1Dy10, having different theoretical molecular weights showed similar electrophoretic mobility patterns on Lab-on-a-chip. The relative protein amount of 1Bx7OE was two-fold higher than that of 1Bx7 or 1Dx5 and, therefore, translated a significant increase in the protein amount in 1Bx7OE. Similarly, the relative protein amounts of 8 & 10 and 10 & 18 were higher than each subunit taken alone. Therefore, this study suggests the established HMW-GS identification system using Lab-on-a-chip as a reliable approach for evaluating HMW-GS for wheat breeding programs.

Important role of long non-coding RNA UBE2R2-AS1 in diabetes-induced renal injury

IntroductionThe present study examined the effects and mechanisms underlying long non-coding RNA (lncRNA) UBE2R2-AS1 in diabetes-induced renal injury.Material and methodsHigh glucose was used to imitate diabetes-induced renal injury in a cell model. Cell apoptosis rate was measured using flow cytometry, tumour necrosis factor-a (TNF-a) and interleukin-6 (IL-6) concentrations were evaluated using ELISA, and relative protein expression and amount were measured using western blot (WB) analysis and immunofluorescence, respectively. Correlations between lncRNA UBE2R2-AS1, miRNA-877-3p and TLR4 were analysed using the luciferase reporter assay.ResultsCell apoptosis rate and TNF-a and IL-6 concentrations were significantly higher (p < 0.001) in the high glucose (model) group compared with those of the Control group (CG) group. Following shRNA knockdown of lncRNA UBE2R2-AS1, the cell apoptosis rate and TNF-a and IL-6 concentrations were significantly lower (p < 0.001, respectively) compared with those of the model group. However, under high-glucose conditions, shRNA knockdown of UBE2R2-AS1 and miRNA-877-3p significantly increased (p < 0.001) the cell apoptosis rate as well as TNF-a and IL-6 concentrations compared with the shRNA UBE2R2-AS1 knockdown group. WB and immunofluorescence analysis showed that toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kB (p65) (NF-kB(p65)) pathway proteins were significantly stimulated in the model group compared with those in the CG, whereas shRNA transfection with miRNA-877-3p stimulation suppressed the TLR4/MyD88/NF-kB(p65) pathway.ConclusionsKnockdown of lncRNA UBE2R2-AS1 improves diabetes-induced renal injury via regulation of the miRNA-877-3p/TLR4 axis in vitro.

Physical and microbiological properties of powdered Lactobacillus salivarius NRRL B-30514 as affected by relative amounts of dairy proteins and lactose

The objective of this study was to characterize physical and microbiological properties of powders prepared by mixing Lactobacillus salivarius NRRL B-30514 suspensions with skim milk powder (SMP), spray-dried lactose (SDL), milk protein concentrate (MPC), or MPC/SDL at a mass ratio of 1:2, 1:1 or 2:1 to understand the relative significance of proteins and lactose in bacterial survival. The probiotic viability and storage stability were significantly improved with the increase of dairy protein content. Based on water sorption isotherms and X-ray diffraction spectroscopy, MPC was suggested to preferentially absorb water in cell suspensions, which inhibited the hydration of SDL and therefore lowered the hypertonic pressure to the adhered cells. The LIVE/DEAD® BacLight™ and CTC reduction assays detected higher membrane integrity and respiratory activity of bacteria for treatments with more proteins. Findings from the current study indicated the more significant role of milk proteins than lactose protecting bacteria during dehydration.

Proteomic profile of saliva collected directly from ducts: a systematic review.

To present a systematic review that provides updated information about proteins found in salivary fluid extracted strictly from ducts. The systematic review probing strategy was based on electronic databases word search (PubMed, EMBASE, LILACS, Web of Science, and Scopus). Risk of bias was assessed based on Joanna Briggs Institute Critical Appraisal Checklist for Studies Reporting Prevalence Data. After 2 rounds of scrutiny, 12 articles were included, totaling 231 individuals (125 were healthy, 41 were elder individuals with radicular caries, 56 had primary Sjögren's syndrome, and 9 were patients who had received radiotherapy for head and neck cancer). The selected studies had no similarities among proteins found, demonstrating the need of standard reference in experimental methodology to obtain a thorough coverage of proteins. Further studies are required to better determine the relative amount of proteins described in this study. It is essential to increase the number of samples, to perform similar collection techniques, to include other analyses methods such as mass spectrometry, and to perform the validation of some proteins using immunoassay techniques such as Elisa and Western blot. Proteomic profile of saliva collected from ducts is essential to better understand the disease process, enabling the identification of biomarkers for specific clinical situations.

Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain.

In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.

Fatty acids profile of black soldier fly (Hermetia illucens): Influence of feeding substrate based on coffee-waste silverskin enriched with microalgae

The aim of this work was to find alternative low-cost and environmentally friendly rearing substrates for the growth of Hermetia illucens (HI) (Diptera, Stratiomydae), used as feed. At this purpose, insect feeding substrates based on the re-use of coffee silverskin, the main waste product of the coffee-roasting industry, enriched with various percentages of microalgae (i.e., Schizochytrium sp. or Isochrysis sp.), were tested. The fatty acid profile, as well as the relative amount of lipids, proteins and carbohydrates (these latter calculated as ratio to the total biomass of the sample) of ingredients, insect feeding substrates and HI prepupae, were determined for the first-time coupling Gas Chromatography-Mass Spectrometry and Fourier Transform Infrared Spectroscopy. A multivariate statistical analysis (Principal Component Analysis) was performed to better read into results. In general, the inclusion of microalgae caused in both feeding substrates and in HI prepupae an increase in the relative amount of lipids and proteins, improving their nutritional value. Higher amounts of unsaturated fatty acids, particularly of omega-3, and good nutritional indices were detected in HI prepupae reared on substrates enriched with 10%, 20% or 25% of Schizochytrium sp. with respect to HI prepupae fed with coffee silverskin enriched with Isochrysis sp., suggesting them as new nutraceutical ingredients for future functional feed and food. In addition, the substrate enriched with a 10% inclusion level of Schizochytrium sp. has to be considered the most convenient one since a greater inclusion of microalgae did not promote additional benefits in terms of nutritional value of HI prepupae.

Sample Preservation and Storage Significantly Impact Taxonomic and Functional Profiles in Metaproteomics Studies of the Human Gut Microbiome.

With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at −80 °C should be chosen wherever possible.

P11.48 Dose-dependent effects of radiation exposure on the cell culture of glioblastoma G01, obtained from the patient with a long-term survival

Abstract Background: The predictors of a favorable prognosis regarding radiation exposure in patients with glioblastoma have not been studied enough, therefore, the search for factors determining a tumor response to the combined chemoradiation treatment is an actual task at present. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for homologous recombination and its inhibition has been shown to sensitize glioma stem cells to irradiation. Materials and Methods: G01 primary glioblastoma cell culture was obtained from tumor tissues from patient with long term survival (42 month).An irradiation with the energy of 6 MeV was carried out in 1 fraction 20,40,60,80,100,125,150,200,250 Gy with Linac. mRNA expression of the RAD51 gene, XRCC6, XRCC5 genes, relative amount of RAD51 protein and Ku70, Ku80 proteins, proliferative activity of cell culture was evaluated by RT-PCR, Western blotting, MTT-test, respectively.Results: When G01 cells were irradiated with doses from 5 to 180 Gy there was a uniform linear decrease in the population of proliferating cells compared to the control, but at a dose of 200 Gy a rise to 24% was observed. The mRNA expression of the RAD51 gene increased 2 times after irradiation at a dose of 20 Gy compared with control and then increased, reaching a peak at the level of 100,250 Gy. Minimal expression was observed after irradiation at a dose of 20 and also decreased at 200 Gy. The mRNA expression of the XRCC5 gene increased 2-fold after irradiation at a dose of 20 Gy compared with control and then decreased, remaining at the same level. Minimal expression was observed after irradiation at a dose of 200Gy. The relative amount of Ku80 protein reached a peak at a dose of 100 Gy, followed by a decrease. Maximum expression level of mRNA of the XRCC6 gene was observed after irradiation at a dose of 250Gy. The relative amount of Ku70 protein also decreased at 200 Gy and reached a peak at a dose of 250 Gy. Conclusions: After irradiation transplantable G01 cell culture from patient with long term survival a linear decrease in proliferative activity depending on the dose was observed with an decrease in mRNA expression of Rad51 gene and the relative amount of RAD51,Ku70 in dose level of 200Gy that could potentially indicate sensitivity point to radiation exposure in these cell culture. Further studies using cell cultures from patients may help in understanding the mechanisms of radioresistance and radiosensitivity in glioblastoma.The research was supported financially by RFBR (Project No. 18-29-01061).

Signal and reward in wild fleshy fruits: Does fruit scent predict nutrient content?

Plant species with fleshy fruits offer animals rewards such as sugar, protein, and fat, to feed on their fruits and disperse their seeds. They have also evolved visual and olfactory signals indicating their presence and ripeness.In some systems, fruit color serves as a reliable visual signal of nutrient content. Yet even though many volatile chemicals used as olfactory signals derive from nutrients animals seek, it is still unknown whether fruit scent encodes information regarding nutrient content in wild fruits.We examine the relationship between olfactory signals and nutrient rewards in 28 fruiting plant species in Madagascar. We measured the relative amounts of four chemical classes in fruit scent using gas chromatography and mass spectrometry, as well as the relative amounts of sugar and protein in fruit pulp.We found that protein levels are not associated with elevated amounts of chemically related volatile compounds in fruit scent. In contrast, sugar content is strongly associated with the chemical composition of fruit scent.To our knowledge, this is the first research to explore the connection between fruit chemical signals and nutrient rewards. Our results imply that in the case of sugar, fruit scent is predictive of nutrient content and hence an honest signal.

Extracellular matrix composition of connective tissues: a systematic review and meta-analysis

The function of connective tissues depends on the physical and biochemical properties of their extracellular matrix (ECM), which are in turn dictated by ECM protein composition. With the primary objective of obtaining quantitative estimates for absolute and relative amounts of ECM proteins, we performed a systematic review of papers reporting protein composition of human connective tissues. Articles were included in meta-analysis if they contained absolute or relative quantification of proteins found in the ECM of human bone, adipose tissue, tendon, ligament, cartilage and skeletal muscle. We generated absolute quantitative estimates for collagen in articular cartilage, intervertebral disk (IVD), skeletal muscle, tendon, and adipose tissue. In addition, sulfated glycosaminoglycans were quantified in articular cartilage, tendon and skeletal muscle; total proteoglycans in IVD and articular cartilage, fibronectin in tendon, ligament and articular cartilage, and elastin in tendon and IVD cartilage. We identified significant increases in collagen content in the annulus fibrosus of degenerating IVD and osteoarthritic articular cartilage, and in elastin content in degenerating disc. In contrast, collagen content was decreased in the scoliotic IVD. Finally, we built quantitative whole-tissue component breakdowns. Quantitative estimates improve our understanding of composition of human connective tissues, providing insights into their function in physiology and pathology.

Label-Free Proteomic Analysis Reveals Parasite-Specific Protein Alterations in Macrophages Following Leishmania amazonensis, Leishmania major, or Leishmania infantum Infection.

Leishmania is an obligate intracellular parasite known to modulate the host cell to survive and proliferate. However, the complexity of host-parasite interactions remains unclear. Also, the outcome of the disease has been recognized to be species-specific and dependent on the host's immune responses. Proteomics has emerged as a powerful tool to investigate the host-pathogen interface, allowing us to deepen our knowledge about infectious diseases. Quantification of the relative amount of proteins in a sample can be achieved using label-free proteomics, and for the first time, we have used it to quantify Leishmania-specific protein alterations in macrophages. Protein extracts were obtained and digested, and peptides were identified and quantified using nano-LC coupled with tandem mass spectrometry analyses. Protein expression was validated by Western blot analysis. Integrated Proteomics Pipeline was used for peptide/protein identification and for quantification and data processing. Ingenuity Pathway Analysis was used for network analysis. In this work, we investigated how this intracellular parasite modulates protein expression on a host macrophage by comparing three different Leishmania species- L. amazonensis, one of the causative agents of cutaneous disease in the Amazon region; L. major, another causative agent of cutaneous leishmaniasis in Africa, the Middle East, China, and India; L. infantum, the causative agent of visceral leishmaniasis affecting humans and dogs in Latin America-and lipopolysaccharide stimulated macrophages as an in vitro inflammation model. Our results revealed that Leishmania infection downregulates apoptosis pathways while upregulating the activation of phagocytes/leukocytes and lipid accumulation.

Hepatitis Delta Antigen Regulates mRNA and Antigenome RNA Levels during Hepatitis Delta Virus Replication.

Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of acute and chronic liver disease. HDV produces three processed RNAs that accumulate in infected cells: the circular genome; the circular antigenome, which serves as a replication intermediate; and lesser amounts of the mRNA, which encodes the sole viral protein, hepatitis delta antigen (HDAg). The HDV genome and antigenome RNAs form ribonucleoprotein complexes with HDAg. Although HDAg is required for HDV replication, it is not known how the relative amounts of HDAg and HDV RNA affect replication, or whether HDAg synthesis is regulated by the virus. Using a novel transfection system in which HDV replication is initiated using in vitro-synthesized circular HDV RNAs, HDV replication was found to depend strongly on the relative amounts of HDV RNA and HDAg. HDV controls these relative amounts via differential effects of HDAg on the production of HDV mRNA and antigenome RNA, both of which are synthesized from the genome RNA template. mRNA synthesis is favored at low HDAg levels but becomes saturated at high HDAg concentrations. Antigenome RNA accumulation increases linearly with HDAg and dominates at high HDAg levels. These results provide a conceptual model for how HDV antigenome RNA production and mRNA transcription are controlled from the earliest stage of infection onward and also demonstrate that, in this control, HDV behaves similarly to other negative-strand RNA viruses, even though there is no genetic similarity between them.IMPORTANCE Hepatitis delta virus (HDV) is a satellite of hepatitis B virus that increases the severity of liver disease; approximately 15 million people are chronically infected worldwide. There are no licensed therapies available. HDV is not related to any known virus, and few details regarding its replication cycle are known. One key question is whether and how HDV regulates the relative amounts of viral RNA and protein in infected cells. Such regulation might be important because the HDV RNA and protein form complexes that are essential for HDV replication, and the proper stoichiometry of these complexes could be critical for their function. Our results show that the relative amounts of HDV RNA and protein in cells are indeed important for HDV replication and that the virus does control them. These observations indicate that further study of these regulatory mechanisms is required to better understand replication of this serious human pathogen.

Human chorionic gonadotrophin pharmaceutical formulations of urinary origin display high levels of contaminant proteins-A label-free quantitation proteomics study.

To determine whether there is a measurable protein background in different formulations of urinary and recombinant human chorionic gonadotropin (hCG). Primary outcome measures: identification of contaminant proteins in urinary‐derived formulations of hCG; secondary outcome measures: quantitative values of contaminant proteins in different batches of urinary –derived hCG formulations. It was found that urinary‐derived batches have high presence of contaminant proteins beside the active substance. The relative amount of contaminant proteins and hCG differs strongly between different batches.

MapB, the Brucella suis TamB homologue, is involved in cell envelope biogenesis, cell division and virulence

Brucella species are Gram-negative, facultative intracellular pathogens responsible for a worldwide zoonosis. The envelope of Brucella exhibits unique characteristics that make these bacteria furtive pathogens and resistant to several host defence compounds. We have identified a Brucella suis gene (mapB) that appeared to be crucial for cell envelope integrity. Indeed, the typical resistance of Brucella to both lysozyme and the cationic lipopeptide polymyxin B was markedly reduced in a ∆mapB mutant. MapB turned out to represent a TamB orthologue. This last protein, together with TamA, a protein belonging to the Omp85 family, form a complex that has been proposed to participate in the translocation of autotransporter proteins across the outer membrane (OM). Accordingly, we observed that MapB is required for proper assembly of an autotransporter adhesin in the OM, as most of the autotransporter accumulated in the mutant cell periplasm. Both assessment of the relative amounts of other specific outer membrane proteins (OMPs) and a proteome approach indicated that the absence of MapB did not lead to an extensive alteration in OMP abundance, but to a reduction in the relative amounts of a protein subset, including proteins from the Omp25/31 family. Electron microscopy revealed that ∆mapB cells exhibit multiple anomalies in cell morphology, indicating that the absence of the TamB homologue in B. suis severely affects cell division. Finally, ∆mapB cells were impaired in macrophage infection and showed an attenuated virulence phenotype in the mouse model. Collectively, our results indicate that the role of B. suis TamB homologue is not restricted to participating in the translocation of autotransporters across the OM but that it is essential for OM stability and protein composition and that it is involved in cell envelope biogenesis, a process that is inherently coordinated with cell division.