Gram-negative bacteria have an outer membrane inhibiting the entry of antibiotics. Porins, found within the outer membrane, are involved in regulating the permeability of β-lactam antibiotics. ...β-lactamases are enzymes that are able to inactivate the antibacterial properties of β-lactam antibiotics. Interestingly, porins and β-lactamase are found in outer membrane vesicles (OMVs) of β-lactam-resistant
and may be involved in the survival of susceptible strains of
in the presence of antibiotics, through the hydrolysis of the β-lactam antibiotic. In this study, OMVs isolated from β-lactam-resistant
and from mutants, lacking porin or β-lactamase, were evaluated to establish if the porins or β-lactamase in OMVs were involved in the degradation of β-lactam antibiotics. OMVs isolated from
deficient in β-lactamase did not show any degradation ability against β-lactam antibiotics, while OMVs lacking OmpC or OmpF showed significantly lower levels of hydrolyzing activity than OMVs from parent
. These data reveal an important role of OMVs in bacterial defense mechanisms demonstrating that the OmpC and OmpF proteins allow permeation of β-lactam antibiotics into the lumen of OMVs, and antibiotics that enter the OMVs can be degraded by β-lactamase.
The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable ...tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (
) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4
cells, with the majority of the CD4 T cell subpopulations being CD4-1
/CD4-2
cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.
Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and ...access to true normal samples hindered attaining this goal.
We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.
Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.
Aim
Bacteria naturally produce membrane vesicles (MVs), which have been shown to contribute to the spread of multi‐drug resistant bacteria (MDR) by delivering antibiotic‐resistant substances to ...antibiotic‐susceptible bacteria. Here, we aim to show that MVs from Gram‐positive bacteria are capable of transferring β‐lactam antibiotic‐resistant substances to antibiotic‐sensitive Gram‐negative bacteria.
Materials and Methods
MVs were collected from a methicillin‐resistant strain of Staphylococcus aureus (MRSA) and vesicle‐mediated fusion with antimicrobial‐sensitive Escherichia coli (RC85). It was performed by exposing the bacteria to the MVs to develop antimicrobial‐resistant E. coli (RC85‐T).
Results
The RC85‐T exhibited a higher resistance to β‐lactam antibiotics compared to the parent strain. Although the secretion rates of the MVs from RC85‐T and the parent strain were nearly equal, the β‐lactamase activity of the MVs from RC85‐T was 12‐times higher than that of MVs from the parent strain, based on equivalent protein concentrations. Moreover, MVs secreted by RC85‐T were able to protect β‐lactam‐susceptible E. coli from β‐lactam antibiotic‐induced growth inhibition in a dose‐dependent manner.
Conclusion
MVs play a role in transferring substances from Gram‐positive to Gram‐negative bacteria, shown by the release of MVs from RC85‐T that were able to protect β‐lactam‐susceptible bacteria from β‐lactam antibiotics.
Significance and impact of study
MVs are involved in the emergence of antibiotic‐resistant strains in a mixed bacterial culture, helping us to understand how the spread of multidrug‐resistant bacteria could be reduced.
Outer membrane vesicles (OMVs) containing various bacterial compounds are released from mainly gram-negative bacteria. Secreted OMVs play important roles in the ability of a bacterium to defend ...itself, and thus contribute to the survival of bacteria in a community. In this study, we collected OMVs from β-lactam antibiotic-resistant Escherichia coli established by conjugation assay and the parental β-lactam antibiotic-susceptible strain, and performed comparative proteomic analysis to examine whether these OMVs carried β-lactam-resistant compounds. We also investigated whether both types of OMVs could protect susceptible cells from β-lactam-induced death and/or directly degrade β-lactam antibiotics. Several proteins that can be involved in degrading β-lactam antibiotics were more abundant in OMVs from β-lactam-resistant E. coli, and thus OMVs from β-lactam resistant E. coli could directly and dose-dependently degrade β-lactam antibiotics and fully rescue β-lactam-susceptible E. coli and other bacterial species from β-lactam antibiotic-induced growth inhibition. Taken together, present study demonstrate that OMVs from β-lactam-resistant E. coli play important roles in survival of antibiotic susceptible bacteria against β-lactam antibiotics. This finding may pave the way for new efforts to combat the current global spread of antibiotic resistances, which is considered to be a significant public health threat.
There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human ...samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
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•Proteomic profiles of extracellular vesicles and particles (EVPs) from 426 human samples•Identification of pan-EVP markers•Characterization of tumor-derived EVP markers in human tissues and plasma•EVP proteins can be useful for cancer detection and determining cancer type
A comprehensive proteomic analysis of extracellular vesicles and particles (EVPs) from 426 human samples identifies pan-EVP markers, biomarkers for EVP isolation, for cancer detection and determining cancer type.
In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts ...involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder (
) CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder's CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.
Crohn's disease (CD) is a chronic inflammatory intestinal disorder associated with intestinal dysbiosis. Diet modulates the intestinal microbiome and therefore has a therapeutic potential. The aim of ...this study is to determine the potential efficacy of three versions of the specific carbohydrate diet (SCD) in active Crohn's Disease.
18 patients with mild/moderate CD (PCDAI 15-45) aged 7 to 18 years were enrolled. Patients were randomized to either SCD, modified SCD(MSCD) or whole foods (WF) diet. Patients were evaluated at baseline, 2, 4, 8 and 12 weeks. PCDAI, inflammatory labs and multi-omics evaluations were assessed.
Mean age was 14.3 ± 2.9 years. At week 12, all participants (n = 10) who completed the study achieved clinical remission. The C-reactive protein decreased from 1.3 ± 0.7 at enrollment to 0.9 ± 0.5 at 12 weeks in the SCD group. In the MSCD group, the CRP decreased from 1.6 ± 1.1 at enrollment to 0.7 ± 0.1 at 12 weeks. In the WF group, the CRP decreased from 3.9 ± 4.3 at enrollment to 1.6 ± 1.3 at 12 weeks. In addition, the microbiome composition shifted in all patients across the study period. While the nature of the changes was largely patient specific, the predicted metabolic mode of the organisms increasing and decreasing in activity was consistent across patients.
This study emphasizes the impact of diet in CD. Each diet had a positive effect on symptoms and inflammatory burden; the more exclusionary diets were associated with a better resolution of inflammation.
Extracellular vesicles (EVs) containing specific cargo molecules from the cell of origin are naturally secreted from bacteria. EVs play significant roles in protecting the bacterium, which can ...contribute to their survival in the presence of antibiotics. Herein, we isolated EVs from methicillin-resistant Staphylococcus aureus (MRSA) in an environment with or without stressor by adding ampicillin at a lower concentration than the minimum inhibitory concentration (MIC). We investigated whether EVs from MRSA under stress condition or normal condition could defend susceptible bacteria in the presence of several β-lactam antibiotics, and directly degrade the antibiotics. A comparative proteomic approach was carried out in both types of EVs to investigate β-lactam resistant determinants. The secretion of EVs from MRSA under antibiotic stressed conditions was increased by 22.4-fold compared with that of EVs without stress. Proteins related to the degradation of β-lactam antibiotics were abundant in EVs released from the stressed condition. Taken together, the present data reveal that EVs from MRSA play a crucial role in the survival of β-lactam susceptible bacteria by acting as the first line of defense against β-lactam antibiotics, and antibiotic stress leads to release EVs with high defense activity.
The occurrence of CD4 helper T cells has already been established for a number of teleost species, though, it has not been possible to analyze these responses at a cellular level due to a large lack ...of appropriate monoclonal antibodies (mAbs). In the present study, we produced a mAb against olive flounder (Paralichthys olivaceus) CD4-1 lymphocyte to investigate the functional activity of the cells to improve our understanding of the T cell response in this species. This mAb is specifically able to detect CD4-1 lymphocytes in olive flounder proved by immunofluorescence staining and RT-PCR analysis. In flow cytometry analysis, the number of CD4-1-positive lymphocytes was observed to gradually increase from 3 days post infection (dpi) and then reach peak at 7 dpi against two viruses challenge. As a conclusion, both the basic properties of CD4-1 T cells and its response to viral infections in olive flounder are very similar to the helper T cells in terrestrial animals.
•Monoclonal antibody against flounder CD4-1 was produced using bacteria expression system.•This mAb specifically detects the CD4-1 lymphocyte in olive flounder.•CD4-1 T lymphocytes are involved in virus specific cellular immune responses.•The progress of immune response related to CD4-1 depends on the infecting viruses.
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