Antibody Western Blot Research Articles

A novel neutralizing monoclonal antibody recognizes a linear antigenic epitope of the spike protein of swine acute diarrhoea syndrome coronavirus

Swine acute diarrhoea syndrome coronavirus (SADS-CoV) causes vomiting, severe diarrhoea and death in newborn piglets. The spike (S) protein plays a crucial role in promoting virus invasion and inducing neutralizing antibody production. In this study, the extracellular region of the S protein was used as an immunogen to immunize BALB/c mice. After immunization, B cells were collected, fused with SP2/0 myeloma cells, cultured and subcloned, and a cell line capable of secreting neutralizing antibodies was obtained and named as 5D6. Additionally, it was determined that the 5D6 mAb could be used as the primary antibody for western blotting and indirect immunofluorescence assay (IFA) to detect SADS-CoV. Further studies indicated that the 5D6 mAb binds to the 136STSHAAD142 motif, which located in the N-terminal domain (NTD) of the spike protein. This result suggested that the NTD of the S protein can induce the production of neutralizing antibodies. Amino acid sequence alignment revealed that the epitope of the 5D6 mAb was conserved among SADS-CoV strains. This study helps elucidate the S protein function of SADS-CoV, and the 5D6 mAb may be used to develop diagnostic and treatment tools for detecting SADS-CoV infection.

Elevated circulating procathepsin L as a potential biomarker of inflamm-aging

Inflamm-aging is a condition of low-grade and chronic systemic inflammation characterized by a systemic increase in multiple inflammatory biomarkers such as tumor necrosis factor (TNF), interleukin 6 (IL-6), C-reactive protein (CRP), and CXCL9 (MIG) in experimental and clinical settings. However, despite the recent identification of extracellular procathepsin L (pCTS-L) as a novel mediator of inflammatory diseases such as sepsis, its possible role in inflamm-aging was previously not investigated. In the present study, we compared blood levels of pCTS-L and other 62 cytokines and chemokines between young and aged Balb/C mice by Western blotting and Cytokine Antibody Arrays. In light of the surprising finding of a marked increase in blood pCTS-L levels in aged mice, we propose that blood pCTS-L levels may serve as another biomarker of inflamm-aging. Given the capacity of pCTS-L in inducing various cytokines (e.g., TNF and IL-6), it will be important to test the hypothetic role of pCTS-L in inflamm-aging under experimental and clinical conditions.

Knockdown of ZNF280A inhibits cell proliferation and promotes cell apoptosis of bladder cancer.

ZNF280A is a member of the zinc finger protein family, whose role in human cancers is little known and rarely reported. This study aimed to investigate the role of ZNF280A in bladder cancer. Immunohistochemical analysis was performed to detect the expression of ZNF280A in clinical samples. ZNF280A knockdown cell models were constructed by transfection of shRNA-expressing lentivirus. MTT assay and flow cytometry were performed for detecting cell proliferation, apoptosis and cycle. Wound healing and Transwell assays were operated to detect cell migration. Western blotting and Human Apoptosis Antibody Microarray were used to measure expression of related proteins. A mouse xenograft model was constructed for in vivo study. Our study demonstrated that ZNF280A was up-regulated in bladder cancer tissues compared with normal tissues, whose high expression was significantly correlated with advanced malignant grade. Knockdown of ZNF280A inhibited cell proliferation and cell migration, promoted cell apoptosis and G1/G2 phase arrest. The tumor growth in vivo was also proved to be inhibited by ZNF280A. Moreover, ZNF280A may promote bladder cancer through regulation of MAPK9, Cyclin D1 and the Akt pathway. In this study, ZNF280A was shown as a potential tumor promoter and prognosis indicator for bladder cancer. Targeting ZNF280A may be a promising strategy for the development of novel bladder cancer treatment.

Protein Arginine Methylation Patterns in Plasma Small Extracellular Vesicles Are Altered in Patients with Early-Stage Pancreatic Ductal Adenocarcinoma.

Small extracellular vesicles (sEVs) contain lipids, proteins and nucleic acids, which often resemble their cells of origin. Therefore, plasma sEVs are considered valuable resources for cancer biomarker development. However, previous efforts have been largely focused on the level of proteins and miRNAs in plasma sEVs, and the post-translational modifications of sEV proteins, such as arginine methylation, have not been explored. Protein arginine methylation, a relatively stable post-translational modification, is a newly described molecular feature of PDAC. The present study examined arginine methylation patterns in plasma sEVs derived from patients with early-stage PDAC (n = 23) and matched controls. By utilizing the arginine methylation-specific antibodies for western blotting, we found that protein arginine methylation patterns in plasma sEVs are altered in patients with early-stage PDAC. Specifically, we observed a reduction in the level of symmetric dimethyl arginine (SDMA) in plasma sEV proteins derived from patients with early- and late-stage PDAC. Importantly, immunoprecipitation followed by proteomics analysis identified a number of arginine-methylated proteins exclusively present in plasma sEVs derived from patients with early-stage PDAC. These results indicate that arginine methylation patterns in plasma sEVs are potential indicators of PDAC, a new concept meriting further investigation.

Neospora caninum surface antigen 1 is a major determinant of the pathogenesis of neosporosis in nonpregnant and pregnant mice.

NcSAG1 is one of most widely investigated antigens of Neospora caninum in various research fields. Such studies demonstrated the proficiency of NcSAG1 in the regulatory process of parasite adhesion and invasion of host cells. Accordingly, the contribution of NcSAG1 to the pathogenesis of neosporosis can undoubtedly be extrapolated, but direct evidence is lacking. Herein, we provide the first successful attempt at the gene disruption of NcSAG1 and novel data on the invasion and virulence potentials of N. caninum in vitro and in vivo. The disruption of the NcSAG1 gene was applied using the CRISPR/Cas9 system and confirmed by PCR, western blot and indirect fluorescent antibody tests as NcSAG1 knockout parasites (NcSAG1KO). Then, we investigated the role of NcSAG1 in the growth kinetics of the parasite in vitro. The deletion of the NcSAG1 gene significantly decreased the infection rate and reduced the egress rate of the parasite. An in vivo study using nonpregnant female and male BALB/c mice revealed a significantly higher survival rate and lower body weight change in the group infected with the NcSAG1KO parasite than in the parental strain (Nc-1)-infected group. Regarding the vertical transmission model of BALB/c mice, the absence of the NcSAG1 gene significantly enhanced the survival of pups and greatly lowered the parasite burden in the brains of pups. In conclusion, our study suggested NcSAG1 as a key molecule in the pathogenesis of N. caninum.

A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures

Purpose This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures. Methods Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts. Results Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination. Conclusions Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.

PD-L1/PD-1 blockage enhanced the cytotoxicity of natural killer cell on the non-small cell lung cancer (NSCLC) by granzyme B secretion

ObjectiveTo explore the role of PD-L1/PD-1 blockage in the cytotoxicity of natural killer cell in NSCLC.MethodsTwo NSCLC cell lines, Calu-1 and H460, were tested for susceptibility to the cytolytic activity of freshly isolated healthy donor NK cells by a non-radioactive cellular cytotoxicity assay kit. Western blot analysis, FACS, ELISA and antibody blockage experiments were conducted to determine the mechanisms. NK cells isolated from NSCLC patients were also collected for functional assays.ResultsCalu-1 and H460 cells were lysed by NK cells in a dose-dependent manner. H460 cells showed less susceptibility to NK cell-mediated lysis than Calu-1 cells at all ratios. The expression of PD-L1 on H460 cells was higher than that on Calu-1 cells, as determined by FACS and western blot analysis. The specific lysis of H460 cells by NK cells was enhanced when the PD-L1/PD-1 interaction was blocked by anti-PD-L1 antibody. This finding was also demonstrated in NK cells isolated from NSCLC patients.ConclusionsThe present study revealed that PD-L1/PD-1 blockage enhanced the cytotoxicity of natural killer cells in NSCLC via granzyme B secretion. This study will greatly facilitate the precise treatment of lung cancer through determination of PD-L1 expression in tumors.

Periostin promotes ovarian cancer metastasis by enhancing M2 macrophages and cancer-associated fibroblasts via integrin-mediated NF-κB and TGF-β2 signaling

BackgroundOvarian cancer has the highest mortality among gynecological cancers due to late diagnosis and lack of effective targeted therapy. Although the study of interplay between cancer cells with their microenvironment is emerging, how ovarian cancer triggers signaling that coordinates with immune cells to promote metastasis is still elusive.MethodsMicroarray and bioinformatics analysis of low and highly invasive ovarian cancer cell lines were used to reveal periostin (POSTN), a matrix protein with multifunctions in cancer, with elevated expression in the highly invasive cells. Anchorage independent assay, Western blot, RNA interference, confocal analysis and neutralizing antibody treatment were performed to analyze the effects of POSTN on tumor promotion and to explore the underlying mechanism. Chemotaxis, flow cytometry and cytokine array analyses were undertaken to analyze the involvement of POSTN in cancer-associated fibroblast (CAF) and macrophage modulation. Correlations between POSTN expression levels and clinical characteristics were analyzed using the Oncomine, commercial ovarian cancer cDNA and China Medical University Hospital patient cohort. In vivo effect of POSTN on metastasis was studied using a mouse xenograft model.ResultsExpression of POSTN was found to be elevated in highly invasive ovarian cancer cells. We observed that POSTN was co-localized with integrin β3 and integrin β5, which was important for POSTN-mediated activation of ERK and NF-κB. Ectopic expression of POSTN enhanced whereas knockdown of POSTN decreased cancer cell migration and invasion in vitro, as well as tumor growth and metastasis in vivo. POSTN enhanced integrin/ERK/NF-κB signaling through an autocrine effect on cancer cells to produce macrophage attracting and mobilizing cytokines including MIP-1β, MCP-1, TNFα and RANTES resulting in increased chemotaxis of THP-1 monocytes and their polarization to M2 macrophages in vitro. In agreement, tumors derived from POSTN-overexpressing SKOV3 harbored more tumor-associated macrophages than the control tumors. POSTN induced TGF-β2 expression from ovarian cancer cells to promote activation of adipose-derived stromal cells to become CAF-like cells expressing alpha smooth muscle actin and fibroblast activation protein alpha. Consistently, increased CAFs were observed in POSTN overexpressing SKOV3 cells-derived metastatic tumors. In clinical relevance, we found that expression of POSTN was positively correlated with advanced-stage diseases and poor overall survival of patients.ConclusionsOur study revealed a POSTN-integrin-NF-κB-mediated signaling and its involvement in enhancing M2 macrophages and CAFs, which could potentially participate in promoting tumor growth. Our results suggest that POSTN could be a useful prognosis marker and potential therapeutic target.

Diverse actions of sirtuin-1 on ovulatory genes and cell death pathways in human granulosa cells

BackgroundHuman granulosa-lutein cells (hGLCs) amply express sirtuin-1 (SIRT1), a NAD + -dependent deacetylase that is associated with various cellular functions. SIRT1 was shown to elevate cAMP on its own and additively with human chorionic gonadotropin (hCG), it is therefore interesting to examine if SIRT1 affects other essential hGLC functions.MethodsPrimary hGLCs, obtained from the follicular aspirates of women undergoing IVF and SV40-transfected, immortalized hGLCs (SVOG cells), were used. Primary cells were treated with SIRT1 specific activator SRT2104, as well as hCG or their combination. Additionally, siRNA-targeting SIRT1 construct was used to silence endogenous SIRT1 in SVOG cells. PTGS2, EREG, VEGFA and FGF2 expression was determined using quantitative polymerase chain reaction (qPCR). Apoptotic and necroptotic proteins were determined by specific antibodies in western blotting. Cell viability/apoptosis was determined by the XTT and flow cytometry analyses. Data were analyzed using student t-test or Mann–Whitney U test or one-way ANOVA followed by Tukey HSD post hoc test.ResultsIn primary and immortalized hGLCs, SRT2104 significantly upregulated key ovulatory and angiogenic genes: PTGS2, EREG, FGF2 and VEGFA, these effects tended to be further augmented in the presence of hCG. Additionally, SRT2104 dose and time-dependently decreased viable cell numbers. Flow cytometry of Annexin V stained cells confirmed that SIRT1 reduced live cell numbers and increased late apoptotic and necrotic cells. Moreover, we found that SIRT1 markedly reduced anti-apoptotic BCL-XL and MCL1 protein levels and increased cleaved forms of pro-apoptotic proteins caspase-3 and PARP. SIRT1 also significantly induced necroptotic proteins RIPK1 and MLKL. RIPK1 inhibitor, necrostatin-1 mitigated SIRT1 actions on RIPK1 and MLKL but also on cleaved caspase-3 and PARP and in accordance on live and apoptotic cells, implying a role for RIPK1 in SIRT1-induced cell death. SIRT1 silencing produced inverse effects on sorted cell populations, anti-apoptotic, pro-apoptotic and necroptotic proteins, corroborating SIRT1 activation.ConclusionsThese findings reveal that in hGLCs, SIRT1 enhances the expression of ovulatory and angiogenic genes while eventually advancing cell death pathways. Interestingly, these seemingly contradictory events may have occurred in a cAMP-dependent manner.

SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry.

SHANK3 is a scaffolding protein implicated in autism spectrum disorders (ASD). Its function at excitatory glutamatergic synapses has been studied for the last two decades, however, tissue-specific expression patterns as well as its subcellular localization need to be studied in further detail. Especially the close sequence homology of SHANK3 to its protein family members SHANK2 and SHANK1 raises the emerging need for specific antibodies that are validated for the desired methodology. With this study, we aim to validate a set of commercial as well as homemade SHANK3 antibodies in Western Blotting, and synaptic immunocyto- and immunohistochemistry. We found that only a small subset of the antibodies included in this study meet the criteria of quality and specificity. Therefore, we aim to share our findings on SHANK3 antibody validation but also raise awareness of the necessity of antibody specificity testing in the field.

Identification and Characterization of P0 Protein as a Vaccine Candidate Against Babesia divergens, Blood Parasite of Veterinary and Zoonotic Importance.

The molecular identification and antigenic characterization of P0 protein in Babesia divergens, a blood parasite of veterinary and zoonotic importance, were carried out in this study for use in developing subunit vaccines against B. divergens infection. Recombinant protein encoding P0 (BdP0) was developed in Escherichia coli, and its antiserum was generated in mice for further molecular characterization. Anti-rBdP0 serum had a specific interaction with the corresponding legitimate B. divergens protein, as confirmed by Western blotting and indirect fluorescent antibody tests. ELISA was used to assess the immunogenicity of BdP0 in a group of 68 bovine field samples, and significant immunological reactivity was found in 19 and 20 positive samples of rBdp0 and B. divergens lysate, respectively. The in vitro growth of B. divergens cultures treated with anti-rBdP0 serum was significantly inhibited (p < 0.05). Furthermore, after 6 h of incubation with 2 mg/ml anti-rBdP0 serum, the ability of pre-incubated free merozoites to invade bovine erythrocytes was reduced by 59.88%. The obtained data suggest the possible use of rBdP0 as diagnostic antigen and may serve as a vaccine candidate against babesiosis caused by B. divergens either in animal or human.

Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants.

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund's complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16–19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.

Disrupting the Reverse Warburg Effect As a Therapeutic Strategy in Multiple Myeloma

Introduction: Altered cellular metabolism is a hallmark of every cancer cell. Aerobic glycolysis (“The Warburg Effect”) is one of the earliest recognized metabolic abnormalities in cancer cells whereby extracellular glucose is preferentially metabolized and eventually processed to generate lactate and energy in the form of ATP before the former is released extracellularly, irrespective of oxygen availability. While extracellular lactate produced and released from cancer cells has traditionally been considered a waste metabolic by-product, recent understanding of cell metabolism suggests that it can also serve as a primary metabolic fuel for cancer cells via uptake by monocarboxylate transporters (MCTs). Our goal was to evaluate this “Reverse Warburg Effect” phenomenon in multiple myeloma (MM) cells and determine if it can be exploited for therapeutic purposes.Methods: All HMCLs, MM1S, RPMI-8226 and U266, were grown in RPMI-1640 cell culture medium containing 11 mM glucose and supplemented with 10% dialyzed fetal bovine serum (FBS) and 2 mM Glutamine. Primary MM cells were extracted using magnetic bead CD138 positive selection from MM patient bone marrow aspirates. For 13C-labeling experiments, HMCLs and primary MM cells were suspended in RPMI-1640 cell culture media containing 13C-labeled isotopes. Isotopomer analysis of glycolytic and tricarboxylic acid (TCA) cycle metabolites from HMCL and primary MM cell pellets was performed using Agilent Technologies 5975C gas chromatography-mass spectrometry. Small molecule inhibitors, AZD3965 and syrosingopine, were purchased from Selleck Chemicals and Sigma respectively. Cellular viability and proliferation were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide (MTT) and CCK-8 assays respectively. MCT-1 and MCT-4 antibodies for western blotting were utilized to evaluate their cell membrane expression on HMCLs.Results: The HMCLs, MM1S and RPMI-8226 as well as primary CD138+ cells from MM patient bone marrow were cultured in cell culture media containing physiological levels (1 mM) of U-13C-Lactate. The incorporation of extracellular 13C into the intracellular glycolytic and TCA cycle metabolite pool was observed (Fig 1) based on the expected isotopomeric patterns, demonstrating the Reverse Warburg Effect in MM cells. The relative contribution of carbon substrate by extracellular lactate compared to extracellular glucose was assessed in the following HMCLs: MM1S, RPMI-8226 and U266 cells by culturing in cell culture media containing 3-13C-Lactate and U-13C-Glucose. Extracellular lactate (yellow bar) contribution to the formation of TCA metabolites equaled that of glucose (red bar) based on the expected isotopomer patterns, suggesting the relative importance of extracellular lactate as an essential nutrient like glucose (Fig 2). Since MCT-1 and MCT-4 are key bidirectional cell membrane transporters of lactate in and out of cells, we explored the clinical significance of their gene expression level on clinical outcomes using the COMMPASS dataset provided by the Multiple Myeloma Research Foundation (MMRF). When MM patients were dichotomized at above or below the median of the expression levels of fragments per kilobase of transcript per million (FPKM), MCT-1 and MCT-4 overexpression conferred a worse progression free survival and overall survival (Fig 3). The MCT-1/MCT-4 protein expression was detectable across the various HMCLs: MM1S, U266 and RPMI-8226 (Fig 4). Inhibition of MCT-1 by specific inhibitor AZD3965 was able to reduce proliferation but not affect viability of HMCLs at 48 hours (Fig 5). However, dual inhibition of MCT-1/MCT-4 using syrosingopine was able to significantly reduce proliferation and decrease viability of HMCLs in a dose dependent fashion (Fig 6). Finally, dual inhibition of MCT-1/MCT-4 using syrosingopine reduced the utilization of extracellular lactate into the TCA cycle pool by HMCLs in media containing 3-13C-Lactate (Fig 7).Conclusion: Utilization of extracellular lactate via Reverse Warburg Effect phenomenon appears highly active in MM cells. Disrupting the utilization of extracellular lactate by dual inhibition of both MCT-1 and MCT-4 appears therapeutic. In the future, dual inhibition of MCT-1/MCT-4 in combination with other anti-MM therapies should be evaluated to determine synergistic therapeutic potential. [Display omitted] DisclosuresKumar: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Carsgen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Antengene: Consultancy, Honoraria; Beigene: Consultancy; Bluebird Bio: Consultancy; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche-Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding.

Proteomics-based identification of cancer-associated proteins in chronic lymphocytic leukaemia

Background: Chronic lymphocytic leukaemia (CLL) is a neoplasm of B-cells characterized by variable prognosis. Exploring the proteome of CLL cells may provide insights into the disease. Therefore, eleven proteomics experiments were conducted on eleven primary CLL samples. Results: We reported a CLL proteome consisting of 919 proteins (false discovery rate (FDR) ≤ 1%) whose identification was based on the sequencing of two or more distinct peptides (FDR of peptide sequencing ≤ 1%). Mass spectrometry-based protein identification was validated for four different proteins using Western blotting and specific antibodies in different CLL samples. Small sizes of nucleolin (~57 kDa and ~68 kDa) showed a potential association with good prognosis CLL cells (n = 8, p Conclusions: Our findings add to the known proteome of CLL and confirm the prognostic importance of two novel cancer-associated proteins in this disease. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

Schistosoma mansoni Heterochromatin Protein 1 (HP1) nuclear interactome in cercariae

Schistosoma mansoni causes schistosomiasis, which affects 240 million people, and 700 million people are living at risk of infection. Epigenetic mechanisms are important for transcriptional control and are well-known conserved transcriptional co-regulators in evolution, already described in mammal, yeast, protozoa and S. mansoni, responsible for heterochromatization and gene silence mechanisms through the formation of complexes of transcriptional repression in chromatin. Previous results from another group have shown that HP1 (SmCBX) proteins form chromatin complexes with SmMDB2/3 proteins and regulate stem cells and oviposition in parasite adult worms. In addition, results from other groups have shown that cercariae are transcriptionally silent and epigenetic mechanisms are involved in the regulation of gene expression in this stage. In this work, our aim was to give insights into SmHP1 and proteins involved in transcriptional regulation in the cercariae stage. Using monoclonal anti-HP1 antibody for Western blotting, immunoprecipitation, and mass spectrometry, we preliminarily determined nuclear proteins that putatively interact with HP1 to form complexes to regulate gene expression, heterochromatin formation, and translational complexes in the cercariae stage. So far, our data is to give some insights into nuclear interactors in S. mansoni cercariae. SignificanceThe significance of this original paper is the evidence for Heterochromatin Protein (HP1), interaction with nuclear proteins in the cercariae stage. Schistosoma mansoni cercariae are the infective stage of the human beings in endemic areas of schistosomiasis, a neglected disease, most prevalent in Brazil and Africa. While cercariae are waiting for a host, it does not feed, gene expression is silent and protein synthesis is stopped. These biochemical mechanisms are recovered when cercariae find a human host, but all proteins and mechanisms are not still elucidated. Until now, literature shows that these phenomena are regulated by epigenetics mechanisms, dependent of histone posttranslational modifications. But we have few pieces of evidence about the other proteins that participates in these processes and which are the co-regulators of expression.

Molecular Characterization and Immunological Evaluation of Truncated Babesia microti Rhoptry Neck Protein 2 as a Vaccine Candidate.

Babesia microti is a protozoan that infects red blood cells. Babesiosis is becoming a new global threat impacting human health. Rhoptry neck proteins (RONs) are proteins located at the neck of the rhoptry and studies indicate that these proteins play an important role in the process of red blood cell invasion. In the present study, we report on the bioinformatic analysis, cloning, and recombinant gene expression of two truncated rhoptry neck proteins 2 (BmRON2), as well as their potential for incorporation in a candidate vaccine for babesiosis. Western blot and immunofluorescence antibody (IFA) assays were performed to detect the presence of specific antibodies against BmRON2 in infected mice and the localization of N-BmRON2 in B. microti parasites. In vitro experiments were carried out to investigate the role of BmRON2 proteins during the B. microti invasion process and in vivo experiments to investigate immunoprotection. Homologous sequence alignment and molecular phylogenetic analysis indicated that BmRON2 showed similarities with RON2 proteins of other Babesia species. We expressed the truncated N-terminal (33–336 aa, designated rN-BmRON2) and C-terminal (915–1171 aa, designated rC-BmRON2) fragments of the BmRON2 protein, with molecular weights of 70 and 29 kDa, respectively. Western blot assays showed that the native BmRON2 protein is approximately 170 kDa, and that rN-BmRON2 was recognized by serum of mice experimentally infected with B. microti. Immunofluorescence analysis indicated that the BmRON2 protein was located at the apical end of merozoites, at the opposite end of the nucleus. In vitro red blood cell invasion inhibition studies with B. microti rBmRON2 proteins showed that relative invasion rate of rN-BmRON2 and rC-BmRON2 group is 45 and 56%, respectively. Analysis of the host immune response after immunization and B. microti infection showed that both rN-BmRON2 and rC-BmRON2 enhanced the immune response, but that rN-BmRON2 conferred better protection than rC–BmRON2. In conclusion, our results indicate that truncated rhoptry neck protein 2, especially its N-terminal fragment (rN-BmRON2), plays an important role in the invasion of host red blood cells, confers immune protection, and shows good potential as a candidate vaccine against babesiosis.

Considerations on Using Antibodies for Studying the Dynorphins/Kappa Opioid Receptor System.

Antibodies are important tools for protein and peptide research, including for the kappa opioid receptor (KOR) and dynorphins (Dyns). Well-characterized antibodies are essential for rigorous and reproducible research. However, lack of validation of antibody specificity has been thought to contribute significantly to the reproducibility crisis in biomedical research. Since 2003, many scientific journals have required documentation of validation of antibody specificity and use of knockout mouse tissues as a negative control is strongly recommended. Lack of specificity of antibodies against many G protein-coupled receptors (GPCRs) after extensive testing has been well-documented, but antibodies generated against partial sequences of the KOR have not been similarly investigated. For the dynorphins, differential processing has been described in distinct brain areas, resulting in controversial findings in immunohistochemistry (IHC) when different antibodies were used. In this chapter, we summarized accepted approaches for validation of antibody specificity. We discussed two KOR antibodies most commonly used in IHC and described generation and characterization of KOR antibodies and phospho-KOR specific antibodies in western blotting or immunoblotting (IB). In addition, applying antibodies targeting prodynorphin or mature dynorphin A illustrates the diversity of results obtained regarding the distribution of dynorphins in distinct brain areas.

Quantitative Proteomics in Drosophila with Holidic Stable-Isotope Labeling of Amino Acids in Fruit Flies (SILAF).

Protein-focused research has been challenging in Drosophila melanogaster due to few specific antibodies for Western blotting and the lack of effective labeling methods for quantitative proteomics. Herein, we describe the preparation of a holidic medium that allows stable-isotope labeling of amino acids in fruit flies (SILAF). Furthermore, in this chapter, we provide a protocol for mitochondrial enrichments from Drosophila larvae and flies together with a procedure to generate high-quality peptides for further analysis by mass spectrometry. Samples obtained following this protocol can be used for various functional studies such as comprehensive proteome profiling or quantitative analysis of posttranslational modifications upon enrichment. SILAF is based on standard fly routines in a basic wet lab environment and provides a flexible and cost-effective tool for quantitative protein expression analysis.

Abstract 6053: Increased nitric oxide augments the action of CSF-1R inhibition against tumor associated macrophages in castration resistant prostate cancer

Abstract Introduction and Objectives: Prostate cancer (PCa) is the most common non-skin cancer among American men, and growing evidence suggests that targeting the tumor microenvironment (TME) could be essential in combating the progression of metastasis and resistance development in cancer. In this context, the colony-stimulating factor 1 (CSF1)/colony-stimulating factor 1 receptor (CSF1R) axis has gained the most attention, and various approaches targeting either the ligands or the receptor are currently in clinical development. However, as a class, CSF1R inhibitors have proved mostly disappointing in early clinical trials when used as monotherapy. Researchers believe their true potential can be tapped by combining them with other anticancer drugs. Sensitization to CSF-1R blockage therapy is tumor microenvironment (TME) driven. In one of our studies, we demonstrated that increased nitric oxide (NO) reduces tumor burden in murine models for CRPC by targeting TME . Therefore, in the present study, we evaluated the hypothesis that Increased Nitric oxide augments the action of CSF-1R inhibition against tumor associated macrophages in castration resistant prostate cancer. Methods: The castrated SCID mice were treated with CSF1R inhibitor (GS2580) at the dosage of 40mg/kg/day IP or/and GSNO at the dosage of 10mg/kg/day IP. After 4 weeks mice were humanely sacrificed. Tumor RNA and proteins to analyze the markers that are important for prostate cancer progression using qPCR, western blot and cytokine antibody array. RNA library was generated, and RNA sequencing was performed using RNA isolated from tumors. Molecular analyses were performed using standard procedures. GraphPad Prism (GraphPad Software) was used for statistical analysis. All data were presented as the means ± SEM. Results: Mice which recieved CSF1R inhibition showed significant reduction in tumor burden (p&amp;lt;0.05), however, in over 50% of the mice, the expression of markers like AR, pERK, p-GSK, and VEGF was found to be increased. Next, to study if increased Nitric oxide levels are able to augment the action of CSF1Ri against CRPC, we studied the effects of GSNO monotherapy, CSF1Ri monotherapy and GSNO+CSF1Ri combination on overall tumor burden. Results revealed that the most significant reduction in tumor burden were in mice that recieved the combination of GSNO-CSF1Ri, compared to GSNO or CSF1Ri monotherpies. Furthermore, RNA sequencing analysis demonstrated that the combination therapy is capable of targeting (suppressing) tumor immunology (signature of interferon-alpha, gama, and Myc signaling were significantly reduced). This was further supported by cytokine antibody array and immunostaning which showed that several cytokines like CXCL5, FGF4, IGFBP-3, MCP-4, IL-6, TNFalpha, expression of CRRPC markers- AR, ARV7, PSA, TMRPSS2, p-GSK, p-ERK, p90RSK, and markers of anti-inflamatorry macrophages (F4/80, CD206 etc) were suppressed in tumor proteins from mice which received combination therapy. Conclusions: Our findings suggest that CSF1R inhibition induced changes against CRPC are augmented in the presence of increased NO levels therefore demonstrating the therapeutic potential of increased NO levels against CRPC. Citation Format: Yash Soni, Manish Kuchakulla, Rehana Qureshi, Van Booven Derek J, Joshua M. Hare, Ranjith Ramasamy, Himanshu Arora. Increased nitric oxide augments the action of CSF-1R inhibition against tumor associated macrophages in castration resistant prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6053.

The CARMA Study: Early Infant Antiretroviral Therapy-Timing Impacts on Total HIV-1 DNA Quantitation 12 Years Later.

BackgroundStrategies aimed at antiretroviral therapy (ART)–free remission will target individuals with a limited viral reservoir. We investigated factors associated with low reservoir measured as total human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood mononuclear cells (PBMCs) in perinatal infection (PaHIV).MethodsChildren from 7 European centers in the Early Treated Perinatally HIV Infected Individuals: Improving Children’s Actual Life (EPIICAL) consortium who commenced ART aged <2 years, and remained suppressed (viral load [VL] <50 copies/mL) for >5 years were included. Total HIV-1 DNA was measured by quantitative polymerase chain reaction per million PBMCs. Factors associated with total HIV-1 DNA were analyzed using generalized additive models. Age, VL at ART initiation, and baseline CD4% effects were tested including smoothing splines to test nonlinear association.ResultsForty PaHIV, 27 (67.5%) female 21 (52.5%) Black/Black African, had total HIV-1 DNA measured; median 12 (IQR, 7.3–15.4) years after ART initiation. Eleven had total HIV-1 DNA <10 copies/106 PBMCs. HIV-1 DNA levels were positively associated with age and VL at ART initiation, baseline CD4%, and Western blot antibody score. Age at ART initiation presented a linear association (coefficient = 0.10 ± 0.001, P ≤ .001), the effect of VL (coefficient = 0.35 ± 0.1, P ≤ .001) noticeable >6 logs. The effect of CD4% (coefficient = 0.03 ± 0.01, P = .049) was not maintained >40%.ConclusionsIn this PaHIV cohort, reduced total HIV-1 DNA levels were associated with younger age and lower VL at ART initiation. The impact of early-infant treatment on reservoir size persists after a decade of suppressive therapy.