The variable lymphocyte receptor (VLR) B of jawless vertebrates functions as a secreted Ab of jawed vertebrates and has emerged as an alternative Ab with a single polypeptide chain. After observing ...an upregulated VLRB response in hagfish immunized with avian influenza virus (AIV) subtype H9N2, we screened AIV H9N2-specific VLRB using a mammalian expression system. To improve the binding avidity of the Ag-specific VLRB to the Ag, we enabled multimerization of the VLRB by conjugating it with C-terminal domain of human C4b-binding protein. To dramatically enhance the expression and secretion of the Ag-specific VLRB, we introduced a glycine-serine linker and the murine Ig κ leader sequence. The practical use of the Ag-specific VLRB was also demonstrated through various immunoassays, detected by anti-VLRB Ab (11G5). Finally, we found that the Ag-specific VLRB decreased the infectivity of AIV H9N2. Together, our findings suggest that the generated Ag-specific VLRB could be used for various immunoapplications.
Streptococcus iniae
causes severe mortalities among cultured marine species, especially in the olive flounder (
Paralichthys olivaceus
), which is economically important in Korea and Japan. Recently, ...there has been growing concern regarding the emergence of
S. iniae
as a zoonotic pathogen. Here, 89
S. iniae
isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of
S. iniae
data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either “no reliable identification” or were incorrectly identified as
Streptococcus pyogenes
at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as
S. iniae
at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between
Streptococcus
species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying
S. iniae
. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.
The olive flounder (Paralichthys olivaceus) is a cultivated marine species that is economically important in Korea and Japan. Several bacterial pathogens have caused severe mortalities in farmed ...olive flounder, especially Streptococcus parauberis. We collected 145 S. parauberis isolates from diseased olive flounders from 2003 to 2008 in Jeju Island, South Korea and characterized them by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and by serology. The serological analysis divided the isolates into serotype I (62.1%) and serotype II (36.6%) and the proteome analysis divided the isolates into cluster 1 (43.4%) and cluster 2 (56.6%). All cluster 1 isolates had serotype I, but cluster 2 consisted of serotype I (32.9%), serotype II (64.6%), and others (2.5%). Further detailed analysis of the mass spectra led to identification of several specific m/z peaks that enabled discrimination between cluster 1 and 2 and between serotype I and II within cluster 2. Our results suggest that MALDI TOF MS analysis has potential as an alternative method for the rapid and reliable identification of the fish pathogen S. parauberis.
•S. parauberis pathogenic to olive flounder was divided into two serotypes.•MALDI TOF MS was applied to identify S. parauberis from spleens of olive flounder.•Expansion of database with additional strains improved accuracy of identification.•Specific peaks classified the serotype I and serotype II within cluster 2.•Several specific m/z peaks can differentiate S. parauberis from fish and cows.
The causative agent of acute hepatopancreatic necrosis disease (AHPND) is the bacterium, Vibrio parahaemolyticus, which secretes toxins into the gastrointestinal tract of its host. Vibrio ...parahaemolyticus toxins A and B (PirAvp/PirBvp) have been implicated in the pathogenesis of this disease, and are, therefore, the focus of studies developing treatments for AHPND. We previously produced recombinant antibodies based on the hagfish variable lymphocyte receptor B (VLRB) capable of neutralizing some viruses, suggesting that this type of antibody may have a potential application for treatment of AHPND. Here, recombinant PirAvp/PirBvp, produced using a bacterial expression system, were used as antigens to screen a hagfish VLRB cDNA library to obtain PirAvp/PirBvp-specific antibodies. A cell line secreting these antibodies was established by screening and cloning the DNA extracted from hagfish B cells. Supernatants collected from cells secreting the PirAvp/PirBvp antibodies were collected and concentrated, and used to passively immunize shrimp to neutralize the toxins PirAvp or PirBvp associated with AHPND. Briefly, 10 μg of PirAvp and PirBvp antibodies, 7C12 and 9G10, respectively, were mixed with the shrimp feed, and fed to shrimp for three days consecutive days prior to experimentally infecting the shrimp with V. parahaemolyticus (containing toxins A and B), and resulting mortalities recorded for six days. Results showed significantly higher level of survival in shrimp fed with the PirBvp-9G10 antibody (60%) compared to the group fed the PirAvp-7C12 antibody (3%) and the control group (0%). This suggests that VLRB antibodies may be a suitable alternative to immunoglobulin-based antibodies, as passive immunization treatments for effective management of AHPND outbreaks within shrimp farms.
Edwardsiella (E.) ictaluri is a major bacterial pathogen that affects commercially farmed striped catfish (Pangasius hypothalamus) in Vietnam. In a previous study, 19 strains of E. ictaluri collected ...from striped catfish were biochemically identified with an API-20E system. Here, the same 19 strains were used to assess the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; applied using a MALDI Biotyper) to conduct rapid, easy and accurate identification of E. ictaluri. MALDI-TOF MS could directly detect the specific peptide patterns of cultured E. ictaluri colonies with high (＞ 2.0, indicating species-level identification) scores. MALDI Biotyper 3.0 software revealed that all of the strains examined in this study possessed highly similar peptide peak patterns. In addition, electrophoresis (SDS-PAGE) and subsequent immuno-blotting using a specific chicken antibody (IgY) against E. ictaluri revealed that the isolates had highly similar protein profiles and antigenic banding profiles. The results of this study suggest that E. ictaluri isolated from striped catfish in Vietnam have homologous protein compositions. This is important, because it indicates that MALDI-TOF MS analysis could potentially outperform the conventional methods of identifying E. ictaluri.
The cellular DEAD-box helicase DDX41, which functions as an initial sensor for cytoplasmic DNA, is involved in the activation of type I interferon (IFN-1) immune response in olive flounder. A plasmid ...encoding DDX41 (pEF-D) was introduced into flounder cells in vitro and in vivo. Immune responses induced by DDX41 were evaluated by relative quantification value (ΔΔCt) method of RT-qPCR using specific IFN-related gene primers. Results in in vitro, transcript levels of IFN-1, IRF-3, ISG-15 and IL-1β were significantly higher in pEF-D- than pEF-A-transfected cells, with 15-, 4-, 10- and 32-fold changes, respectively. In vivo, elevated expression of these genes was observed in the kidney of pEF-D group on days 1 and 3 post-treatment. The viral challenge test revealed higher survival rate in fish treated with pEF-D (67.5%) than controls, PBS- and pEF-A-treated fish (45%). Conclusively DDX41 elicits a robust IFN-mediated immune response, validating its adjuvant property.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Overexpression and hyperactivity of the estrogen receptor drives 67-80% and 90% of breast cancer in women and men, respectively. Resistance to aromatase inhibitor (AI) therapies necessitates ...the continuous search for novel therapies. Previous studies demonstrate AI-resistance is associated with hormone-independence, enhanced motility, and increased growth factor signaling. Here, long-term letrozole-treated (LTLT-Ca) breast cancer cells were used to evaluate polyisoprenylated cysteinyl amide inhibitors (PCAIs) as potential alternative therapies for the treatment of aromatase inhibitor-resistant breast cancer. We determined the potency of PCAIs as anticancer agents by evaluating their effects on cell viability, phosphorylation of MAPK pathway enzymes, G-proteins levels, cell migration and apoptosis. Among the PCAIs tested, NSL-YHJ-2-27 showed significant potency against cell viability with an EC50 of 3.6 µM. MEK (p-MEK1/2), ERK (p-ERK1/2), and p90RSK (p-p90RSK) phosphorylation were significantly increased by 178, 119 and 125%, respectively, over controls. In addition, of the seven G-proteins evaluated, only KRAS and NRAS showed significant increases of 49% and 97%, respectively. Cell proliferation and colony formation were impeded by 82% and 74%, respectively, after PCAIs treatment. Migration of the LTLT-Ca cells was inhibited by 80% following treatment with 5 µM of NSL-YHJ-2-27. Lastly, the PCAIs also caused the degeneration of the spheroids after 10 µM PCAIs treatment, dead cells were very apparent after AO/EB staining. Our findings suggest that the PCAIs’ suppression of cell viability and activation of the MAPK pathway causes apoptosis possibly through the activation of the proapoptotic p-p90RSK isoforms. The results reveal the ability of the PCAIs to effectively cause a negative impact on several cancer hallmarks suggesting their potency as anticancer agents. These findings also support a potential role for PCAIs’ use in treating breast cancers that have become resistant to aromatase inhibitor therapies.
Citation Format: Jassy Mary S. Lazarte, Syreeta L. Tilghman, Nazarius S. Lamango. Treatment of aromatase inhibitor-resistant cells with polyisoprenylated cysteinyl amide inhibitors stimulates the mitogen-activated protein kinase pathway enzymes, impedes cell proliferation and causes cell death. abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5030.
Abstract
Female breast cancer has now surpassed lung cancer as the most common cancer overall in both sexes with an estimated 2.3 million new cases, representing 11.7% of all cancer cases worldwide ...in 2020. Triple negative breast cancer (TNBC) has been the subject of intensive studies due to its aggressive nature which makes it difficult to treat. Since inclusive treatment for this type had been elusive for decades, it is thus imperative to develop new therapeutic agents to mitigate its deadly effects. PCAIs are novel compounds designed to mimic the essential posttranslational modifications of G-proteins such as KRAS. This study elucidates the effect of the PCAIs downstream of KRAS, specifically on the mitogen activated pathway kinase (MAPK) enzymes. Two breast cancer cell lines, MDA-MB-468 and MDA-MB-231, were used to determine the levels of phosphorylation in several MAPK pathway enzymes. After exposure of the cells to varying concentrations of PCAIs (NSL-YHJ-2-27) for 48 hours, western blot results showed remarkable differences in the phosphorylated levels of the respective enzymes in cells treated with 5 µm of the PCAIs as compared to the control (NSL-YHJ-2-62). Interestingly, the cell lines reacted differently after treatment, in which MDA-MB-468 cells showed increased levels of phosphorylated MEK, ERK, and 90RSK by 122, 273 and 242%, respectively. However, in MDA-MB-231, only the phosphorylated 90RSK showed an increase of 105% and no changes in the other two enzymes. Moreover, a very distinct difference was observed in the levels of phosphorylated BRAF (p-BRAF) and CRAF (p-CRAF). In MDA-MB-231 cells, no change in p-BRAF levels while a 184% increase in p-CRAF were observed. In MDA-MB-468, p-BRAF increased by 41% and p-CRAF decreased by 58%. The differences in the response of the cell lines may reflect their unique genetics. These results show the ability of the PCAIs to interfere with the MAPK pathway. As this signaling cascade plays a very important role in cell response as well as in the progression of cancers, therefore, therapeutics targeting this pathway have a great potential in the development of treatments not only for TNBC but also for other kinds of cancers.
Citation Format: Jassy Mary Lazarte, Nada Tawfeeq, Yonghao Jin, Nazarius Lamango. Activation of MAP kinase pathway in breast cancer cell lines by polyisoprenylated cysteinyl amide inhibitors (PCAIs) causes cell death abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2683.
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