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Diverse assays spanning from immunohistochemistry (IHC), to microarrays (protein, DNA), to high-throughput screens rely on probe-target hybridization to detect analytes. These large-format 'chips' array numerous hybridization sites across centimeter-scale areas. However, the reactions are prone to intra-assay spatial variation in hybridization efficiency. The mechanism of spatial bias in hybridization efficiency is poorly understood, particularly in IHC and in-gel immunoassays, where immobilized targets are heterogeneously distributed throughout a tissue or hydrogel network. In these systems, antibody probe hybridization to a target protein antigen depends on the interplay of dilution, thermodynamic partitioning, diffusion, and reaction. Here, we investigate parameters governing antibody probe transport and reaction (i.e., immunoprobing) in a large-format hydrogel immunoassay. Using transport and bimolecular binding theory, we identify a regime in which immunoprobing efficiency (η) is sensitive to the local concentration of applied antibody probe solution, despite the antibody probe being in excess compared to antigen. Sandwiching antibody probe solution against the hydrogel surface yields spatially nonuniform dilution. Using photopatterned fluorescent protein targets and a single-cell immunoassay, we identify regimes in which nonuniformly distributed antibody probe solution causes intra-assay variation in background and η. Understanding the physicochemical factors affecting probe-target hybridization reduces technical variation in large-format chips, improving measurement precision.

HIV-1 Tat is essential for HIV-1 replication and appears to play an important role in the pathogenesis of HIV-associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor-mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV-1 LTR promoter and facilitate HIV-1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high-affinity rhodamine-dextran or chelating cytosolic calcium with BAPTA-AM attenuated Tat endolysosome escape and LTR transactivation. Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome-resident two-pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium-mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV-1 infection and HIV-associated neurological complications.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. The outcomes for aggressive forms of NB remain poor. The aim of this study was to develop a new molecular-targeted therapy for NB using an antisense oligonucleotide (ASO) and superparamagnetic iron oxide (SPIO) nanoparticles (NPs), as a delivery vehicle, targeting the transcription regulator MAX dimerization protein 3 (MXD3). We previously discovered that MXD3 was highly expressed in high-risk NB, acting as an anti-apoptotic factor; therefore, it can be a good therapeutic target. In this study, we developed two ASO-NP complexes using electrostatic conjugation to polyethylenimine-coated SPIO NPs and chemical conjugation to amphiphilic polymers on amine-functionalized SPIO NPs. Both ASO-NP complexes demonstrated MXD3 knockdown, which resulted in apoptosis in NB cells. ASO chemically-conjugated NP complexes have the potential to be used in the clinic as they showed great efficacy with minimum NP-associated cytotoxicity.

Immunoassays and mass spectrometry are powerful single-cell protein analysis tools; however, interfacing and throughput bottlenecks remain. Here, we introduce three-dimensional single-cell immunoblots to detect both cytosolic and nuclear proteins. The 3D microfluidic device is a photoactive polyacrylamide gel with a microwell array-patterned face (xy) for cell isolation and lysis. Single-cell lysate in each microwell is "electrophoretically projected" into the 3rd dimension (z-axis), separated by size, and photo-captured in the gel for immunoprobing and confocal/light-sheet imaging. Design and analysis are informed by the physics of 3D diffusion. Electrophoresis throughput is > 2.5 cells/s (70× faster than published serial sampling), with 25 immunoblots/mm2 device area (>10× increase over previous immunoblots). The 3D microdevice design synchronizes analyses of hundreds of cells, compared to status quo serial analyses that impart hours-long delay between the first and last cells. Here, we introduce projection electrophoresis to augment the heavily genomic and transcriptomic single-cell atlases with protein-level profiling.

Introduction. Macrophages occupy a special place in the formation and coordination of the immune response, their population is characterized by heterogeneity, however, two main types are distinguished among them: pro-inflammatory (classically activated) macrophages M1, which are activated by interferon-γ (IFN-γ) or lipopolysaccharide (LPS) of bacteria, produce pro-inflammatory cytokines, nitric oxide (NO) and initiate an immune response, and anti-inflammatory (alternatively activated) macrophages M2, which are activated by such cytokines as interleukin 1 (IL-1), IL-10 or IL-13 and are associated with suppression of the immune response and tissue healing. The proinflammatory properties of macrophages are most often associated with the activation of an intracellular signaling cascade with the participation of the IKK-2 kinase complex and nuclear factor-κB (NF-κB), which is the most important signal transduction molecule. Previously, the authors showed that humic acids (HA) of peat cause the appearance of pro-inflammatory properties in macrophages; however, the intracellular mechanisms of such activation have not been studied.Aim. Study of the contribution of the intracellular signaling cascade IKK-2/NF-κB to the activation of pro-inflammatory properties of macrophages by humic acids of peat.Materials and methods. The studies were carried out using cultural methods and western blotting.Results and discussion. It was shown that the addition of the IKK-2 inhibitor to the macrophage culture reduced the ability of HA to stimulate the production of nitric oxide by the cells. Western blotting revealed that after 30 minutes simultaneous incubation of HA with macrophages, the content of the NF-κB p65 molecule in the cells increased, after 60 minutes it decreased to the values of intact control, and after 17 hours it decreased 3 times compared to the control level. However, the IKK-2 inhibitor after a 30-minutes incubation did not have a blocking effect on the IKK kinase, whereas in an hour of the co-incubation of the cells with the inhibitor and substances, a decrease in the level of NF-κB p65 was observed. This trend continued in 17 hours.Conclusion. The stimulating effect of peat humic acids on macrophages is realized, among other things, due to the activation of the IKK-2 kinase complex with further release of the p65 subunit of the NF-κB molecule. The dependence of the degree of NF-κB activation on the period of interaction of the cell with activating agents is traced. HA start the intracellular signaling cascade of the nuclear factor NF-κB already within the first 30 minutes.

Neuroinflammation plays an important role in the onset and progression of neurodegenerative diseases. The multicomponent and multitarget approach may provide a practical strategy to address the complex pathological mechanisms of neuroinflammation. This study aimed to develop synergistic herbal compound formulas to attenuate neuroinflammation using integrated network pharmacology, molecular docking, and experimental bioassays. Eight phytochemicals with anti-neuroinflammatory potential were selected in the present study. A compound-gene target-signaling pathway network was constructed to illustrate the mechanisms of action of each phytochemical and the interactions among them at the molecular level. Molecular docking was performed to verify the binding affinity of each phytochemical and its key gene targets. An experimental study was conducted to identify synergistic interactions among the eight phytochemicals, and the associated molecular mechanisms were examined by immunoblotting based on the findings from the network pharmacology analysis. Two paired combinations, andrographolide and 6-shogaol (AN-SG) (IC50 = 2.85 μg/mL), and baicalein-6-shogaol (BA-SG) (IC50 = 3.28 μg/mL), were found to synergistically (combination index <1) inhibit the lipopolysaccharides (LPS)-induced nitric oxide production in microglia N11 cells. Network pharmacology analysis suggested that MAPK14, MAPK8, and NOS3 were the top three relevant gene targets for the three phytochemicals, and molecular docking demonstrated strong binding affinities of the phytochemicals to their coded proteins. Immunoblotting suggested that the AN-SG and BA-SG both showed prominent effects in inhibiting inducible nitric oxide synthase (iNOS) (p < 0.01 and p < 0.05 , respectively) and MAPKp-p38 (both p < 0.05) compared with those induced by the LPS stimulation only. The AN-SG combination exhibited greater inhibitions of the protein expressions of iNOS (p < 0.05 vs. individual components), which may partly explain the mechanisms of the synergy observed. This study established a practical approach to developing novel herbal-compound formulations using integrated network pharmacology analysis, molecular docking, and experimental bioassays. The study provides a scientific basis and new insight into the two synergistic combinations against neuroinflammation. [ABSTRACT FROM AUTHOR]

Objective: To develop a multianalyte assay for the detection of dermatomyositis (DM)-related autoantibodies using immunoprecipitation (IP) combined with immunoblotting (IB). Methods: Sera from 116 DM patients were subjected to RNA and protein immunoprecipitation assays as well as commercial enzyme-linked immunosorbent assays (ELISAs) for anti-aminoacyl transfer RNA synthetase, anti-melanoma differentiation antigen 5 (MDA5), anti-Mi-2, anti-transcriptional intermediary factor-1? (TIF-1?), and anti-U1 ribonucleoprotein antibodies. The IP/IB assay was developed by immunoprecipitation of autoantigens from HeLa cell extracts using patient sera, followed by immunoblotting with an antibody against Mi-2, TIF-1?, OJ, nuclear matrix protein (NXP)-2, MDA5, PM/Scl, small ubiquitin-like modifier activating enzyme (SAE), or Ku. A multianalyte assay was designed by mixing primary antibodies in the IP/IB assay. Results: IP assays identified any DM-related autoantibodies in 100 patients (86%), of which 82% were covered by commercial ELISAs, with a false-positive result in two sera and a false-negative result in one serum. The results obtained from the multianalyte IP/IB assay and ‘gold-standard’ IP assays were concordant in terms of the presence or absence of anti-MDA5, anti-TIF-1?, anti-OJ, anti-NXP-2, anti-PM/Scl, anti-SAE, anti-Mi-2, and anti-Ku antibodies. Conclusion: This multianalyte IP/IB assay combined with commercial ELISAs is an alternative to ‘gold-standard’ IP assays for the detection of DM-related autoantibodies. [ABSTRACT FROM AUTHOR]

Objective: To develop a multianalyte assay for the detection of dermatomyositis (DM)-related autoantibodies using immunoprecipitation (IP) combined with immunoblotting (IB). Methods: Sera from 116 DM patients were subjected to RNA and protein immunoprecipitation assays as well as commercial enzyme-linked immunosorbent assays (ELISAs) for anti-aminoacyl transfer RNA synthetase, anti-melanoma differentiation antigen 5 (MDA5), anti-Mi-2, antitranscriptional intermediary factor-1γ (TIF-1γ), and anti-U1 ribonucleoprotein antibodies. The IP/IB assay was developed by immunoprecipitation of autoantigens from HeLa cell extracts using patient sera, followed by immunoblotting with an antibody against Mi-2, TIF-1γ, OJ, nuclear matrix protein (NXP)-2, MDA5, PM/Scl, small ubiquitin-like modifier activating enzyme (SAE), or Ku. A multianalyte assay was designed by mixing primary antibodies in the IP/IB assay. Results: IP assays identified any DM-related autoantibodies in 100 patients (86%), of which 82% were covered by commercial ELISAs, with a false-positive result in two sera and a false-negative result in one serum. The results obtained from the multianalyte IP/IB assay and ‘goldstandard’ IP assays were concordant in terms of the presence or absence of anti-MDA5, anti-TIF-1γ, anti-OJ, anti-NXP-2, anti-PM/Scl, anti-SAE, anti-Mi-2, and anti-Ku antibodies. Conclusion: This multianalyte IP/IB assay combined with commercial ELISAs is an alternative to ‘gold-standard’ IP assays for the detection of DM-related autoantibodies. [ABSTRACT FROM AUTHOR]

Objective To explore whether the variants in non MHC proteasome gene are associated with AS and explain the role of the variant in the disease. Material and methods Case-control analysis to identify AS predisposition genes; dual-luciferase reporter assay, immunoblot analysis and osteoclastogenesis assays to detect the function of the positive variant. Affected individuals were diagnosed according to the modified New York Criteria by at least two experienced rheumatologists, and rechecked by another rheumatologist. Results The study included 1037 AS patients and 1014 no rheumatic and arthritis disease controls. The main age of AS onset is between 16 and 35 years old. HLA-B27-positive subjects comprised 90.0% of patients. A nonsynonymous SNP rs12717 in proteasome gene PSMB1 significantly associated with AS. Individuals with CC genotype had a higher onset risk compared with those with GG/GC genotypes (OR = 1.89, P  = 0.0047). We also discovered that PSMB1 regulates the receptor activator of nuclear factor-κB (RANK)/RANK ligand (RANKL) signalling pathway and the disease-associated variant PSMB1-Pro11 significantly inhibits RANKL-induced NF-κB pathway in osteoclast differentiation via the degradation of IKK-β compared with PSMB1-Ala11. RANKL induced osteoclast differentiation was significantly lower in primary monocyte osteoclast precursor from individuals with genotype PSMB1 31C/31C compared with individuals with genotype PSMB1 31G/31G . Conclusions These results reveal a novel understanding of the bone formation and reabsorbing imbalance in AS. The new bone formation phenotype can be attributed to the inhibition of osteoclast differentiation by a more functional PSMB1 gene. [ABSTRACT FROM AUTHOR]

Simple Summary: Treatment of Merkel cell carcinoma—a deadly skin cancer frequently caused by the Merkel cell polyomavirus—still poses challenges. Since the tumor cells depend on expression of viral oncogenes—named T antigens—targeting the expression of these T antigens appears as a potential therapeutic strategy. Therefore, we screened a kinase inhibitor library for compounds repressing growth of Merkel cell carcinoma cells specifically by inhibiting expression of these viral proteins and identified a compound previously described as an inhibitor of Aurora kinase A (AURKA). However, we provide evidence that the T-antigen repressing effect is not related to inhibition of AURKA but probably to a hitherto unknown GSK3-inhibitory activity of the compound, whose potential use a therapeutic is demonstrated in immunocompromised mice transplanted with human MCC. Merkel cell carcinoma (MCC) is frequently caused by the Merkel cell polyomavirus (MCPyV), and MCPyV-positive tumor cells depend on expression of the virus-encoded T antigens (TA). Here, we identify 4-[(5-methyl-1H-pyrazol-3-yl)amino]-2H-phenyl-1-phthalazinone (PHT)—a reported inhibitor of Aurora kinase A—as a compound inhibiting growth of MCC cells by repressing noncoding control region (NCCR)-controlled TA transcription. Surprisingly, we find that TA repression is not caused by inhibition of Aurora kinase A. However, we demonstrate that β-catenin—a transcription factor repressed by active glycogen synthase kinase 3 (GSK3)—is activated by PHT, suggesting that PHT bears a hitherto unreported inhibitory activity against GSK3, a kinase known to function in promoting TA transcription. Indeed, applying an in vitro kinase assay, we demonstrate that PHT directly targets GSK3. Finally, we demonstrate that PHT exhibits in vivo antitumor activity in an MCC xenograft mouse model, suggesting a potential use in future therapeutic settings for MCC. [ABSTRACT FROM AUTHOR]

Simple Summary: PRMT5 is overexpressed and activated in various human cancers, including breast cancer. This study aims to dissect the mechanism underlying how PRMT5 is dysregulated in cancers. Our results demonstrate that TRAF6-mediated ubiquitination plays an important role in the regulation of PRMT5 activity and cell proliferation. Thus, inhibition of TRAF6 is a possible strategy for improving PRMT5 targeted therapy. Protein arginine methyltransferase 5 (PRMT5) is the primary enzyme generating symmetric dimethylarginine (sDMA) on numerous substrates, through which it regulates many cellular processes, such as transcription and DNA repair. Aberrant expression and activation of PRMT5 is frequently observed in various human cancers and associated with poor prognosis and survival. However, the regulatory mechanisms of PRMT5 remain poorly understood. Here, we report that TRAF6 serves as an upstream E3 ubiquitin ligase to promote PRMT5 ubiquitination and activation. We find that TRAF6 catalyzes K63-linked ubiquitination of PRMT5 and interacts with PRMT5 in a TRAF6-binding-motif-dependent manner. Moreover, we identify six lysine residues located at the N-terminus as the primarily ubiquitinated sites. Disruption of TRAF6-mediated ubiquitination decreases PRMT5 methyltransferase activity towards H4R3 in part by impairing PRMT5 interaction with its co-factor MEP50. As a result, mutating the TRAF6-binding motifs or the six lysine residues significantly suppresses cell proliferation and tumor growth. Lastly, we show that TRAF6 inhibitor enhances cellular sensitivity to PRMT5 inhibitor. Therefore, our study reveals a critical regulatory mechanism of PRMT5 in cancers. [ABSTRACT FROM AUTHOR]

Background and Aim: This study investigated the risk of the development of primary biliary cholangitis (PBC) in individuals who were incidentally identified as having positive antimitochondrial antibodies (AMA)-M2. Materials and Methods: We retrospectively reviewed extractable nuclear antibody (ENA) panel test results to identify the incidental AMA-M2- positive patients. Patients who filled the diagnostic criteria for PBC were excluded. AMA-M2-positive patients were further evaluated by physical examination, liver biochemistry, liver ultrasonography, and transient elastography (TE) and were also closely followed. Results: We included 48 (n=45, 93% female) individuals with a median age of 49 (range: 20–69) years. The median follow-up duration was 27 months (range: 9–42) after the detection of AMA-M2. Thirty-three (69%) patients had concomitant autoimmune/inflammatory disorders. Twenty-eight (58%) individuals showed seropositivity for ANA, and 21 had (43%) positive AMA. Fifteen (31%) patients developed typical PBC according to the international PBC diagnostic criteria during the follow-up, and five of them (18%) had significant fibrosis (≥8.2 kPA) by TE at the time of PBC diagnosis. Conclusion: Two-thirds of the incidental AMA-M2-positive patients developed typical features of PBC after a median 27-month follow-up. Our results suggest that AMA-M2 patients should be closely followed up to detect the late development of PBC [ABSTRACT FROM AUTHOR]

RNF43 is an important negative regulator of β-catenin signaling by removing Wnt-receptors from the membrane. It is often mutated in cancers, leading to aberrant Wnt-dependent nuclear translocation of β-catenin. RNF43 has also been suggested to regulate β-catenin signaling directly within the nucleus, among other proposed nuclear functions. Given the importance of RNF43 in regulating Wnt/β-catenin signaling and its potential therapeutic relevance, a proper understanding of RNF43 biology is required. However, the presumed nuclear location is mainly based on available antibodies. These same antibodies have also been used extensively for immunoblotting or immunohistochemical purposes. However, a proper evaluation of their quality to reliably detect endogenous RNF43 has not been performed. Here, using genome editing we have generated a cell line that entirely misses RNF43 exons 8 and 9, encoding the epitopes of commonly used RNF43 antibodies. Using this clone in addition to various other cell line tools, we show that four RNF43 antibodies only yield non-specific signals when applied in immunoblotting, immunofluorescence and immunohistochemical experiments. In other words, they cannot reliably detect endogenous RNF43. Our results suggest that the nuclear staining patterns are an antibody artifact and that RNF43 is unlikely to localize within the nucleus. More generally, reports using RNF43 antibodies should be interpreted with caution, at least for the RNF43 protein aspects described in these papers. [ABSTRACT FROM AUTHOR]

Background: With cancer cases escalation, an urgent request to develop novel combating strategies arise. Pathogen-based cancer-immunotherapy is getting more consideration. Autoclaved parasitic antigens seem promising candidates, taking steadily their first steps. Our aim was to examine the prophylactic antineoplastic activity of autoclaved Toxoplasma vaccine (ATV) and to test for the shared antigen theory between Toxoplasma gondii and cancer cells. Methods: Mice were immunized with ATV followed by Ehrlich solid carcinoma (ESC) inoculation. Tumor weight, volume, histopathology, and immunohistochemistry for CD8+ T cells, Treg cells and VEGF were assessed. In addition, the proposed shared antigen theory between parasites and cancer was also verified using SDS-PAGE and immunoblotting. Results: Results revealed powerful prophylactic activity of ATV with 13.3% inhibition of ESC incidence, significant reduction in tumor weight and volume in ATV vaccinated mice. Immunologically, significantly higher CD8+T cells and lower FOXP3+ Treg cells surrounded and infiltrated ESC in ATV immunized mice with higher CD8+T/Treg cells ratio and significant antiangiogenic effect. Moreover, SDS-PAGE and immunoblotting showed four shared bands between Ehrlich carcinoma and ATV of approximate molecular weights 60, 26, 22 and 12.5 KDa. Conclusion: Exclusively, we demonstrated a prophylactic antineoplastic activity of autoclaved Toxoplasma vaccine against ESC. Moreover, to the best of our knowledge this is the first report highlighting the existence of cross-reactive antigens between Toxoplasma gondi parasite and cancer cells of Ehrlich carcinoma. [ABSTRACT FROM AUTHOR]

Nuclear actin has been implicated in dynamic chromatin rearrangements in diverse eukaryotes. In mammalian cells, it is required to reposition double-strand DNA breaks to enable homologous recombination repair and to enhance transcription by facilitating RNA Pol II recruitment to gene promoters. In the yeast Saccharomyces cerevisiae, nuclear actin modulates interphase chromosome dynamics and is required to reposition the induced INO1 gene to the nuclear periphery. Here, we have investigated the role of actin in driving intergenic interactions between Heat Shock Factor 1 (Hsf1)-regulated Heat Shock Protein (HSP) genes in budding yeast. These genes, dispersed on multiple chromosomes, dramatically reposition following exposure of cells to acute thermal stress, leading to their clustering within dynamic biomolecular condensates. Using an auxin-induced degradation strategy, we found that conditional depletion of nucleators of either linear or branched F-actin (Bni1/Bnr1 and Arp2, respectively) had little or no effect on heat shock-induced HSP gene coalescence or transcription. In addition, we found that pretreatment of cells with latrunculin A, an inhibitor of both filamentous and monomeric actin, failed to affect intergenic interactions between activated HSP genes and their heat shock-induced intragenic looping and folding. Moreover, latrunculin A pretreatment had little effect on HSP gene expression at either RNA or protein levels. In notable contrast, we confirmed that repositioning of activated INO1 to the nuclear periphery and its proper expression do require actin. Collectively, our work suggests that transcriptional activation and 3D genome restructuring of thermally induced, Hsf1-regulated genes can occur in the absence of actin. [ABSTRACT FROM AUTHOR]

Background: Cervical cancer (CC) is the second most common type of cancer among women. A key factor in developing the disease is the human papillomavirus (HPV) infection. Aberrant DNA hypermethylation in gene promoter regions is one of the most well-fined epigenetic alterations in tumors. This study aimed to investigate cell adhesion molecule 1 (CADM1), promoter methylation in HPV serological positive samples in the Iranian population. Method: Genomic DNA was extracted from cervical smears from patients and healthy patients acting as a control group for this analytical observational case-control investigation. Reverse dot blotting was performed to first identify the presence of HPV DNA infection. After that, each sample was transformed by being treated with sodium bisulfite, and MSP was then used to determine the CADM1 gene's level of methylation. Results: Among total number of positive HPV samples (n = 52), 63.5% methylated, 23% hemi-methylated, and 13.5% were unmethylated. On the contrary, in the control group (n=38), 11% methylated, 71% hemi-methylated, and 18% were unmethylated (P < 0.0001). Furthermore, comparing the methylation status among high, low and high/low patients indicated that methylation was (66.66, 24.24, and 9.09%), (25, 66.66, and 8.33%), and (14.28, 57.14%, and 28.57%), respectively (P < 0.0001). Conclusion: The present study confirmed that the methylation status of CADM1 gene was significantly higher in HPV-positive patients than in patients negative for HPV DNA. Moreover, the CADM1 pattern of the gene was associated with the high-risk subtypes of HPV, not with the low-risk ones. Therefore, CADM1 methylation appeared to be a promising biomarker for future studies. [ABSTRACT FROM AUTHOR]

Objective: Cancer‐associated fibroblasts (CAFs) are abundantly infiltrated in oral squamous cell carcinoma (OSCC), but the contact‐dependent mechanisms that regulate CAFs phenotype in precursor cells, such as paracancerous fibroblasts (PFs), remain unclear. Here, a fibroblast‐attached organoid (FAO) model was initiated to determine phenotype transition of fibroblasts triggered by contact with OSCC. Material and methods: Organoids and fibroblasts were generated using OSCC and adjacent tissues. Cell‐clusters containing fibroblasts and tumour cells were aggregated to allow for FAOs expansion. Immunoblotting assay was performed to compare expression of Notch intracellular domain (NICD) in CAFs and PFs. Colony formation assay was employed to evaluate morphological activation of fibroblasts. Results: Compared to traditional 3D co‐culture, FAOs better modulated the spatial distribution of fibroblasts with tumour nests. The presence of CAFs with multiple branches was stably observed in FAOs during serial passage. Incorporation with organoids promoted the ability of PFs to form multiple branches. Immunoblotting assay confirmed higher NICD level in CAFs than PFs. Treatment with Notch inhibitor, N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (i.e. DAPT) blocked morphological activation of fibroblasts incorporated into FAO. Conclusion: We developed a robust strategy to study contact‐dependent mechanisms underlying tumour‐stromal interaction, and suggested that Notch activity contributes to biogenesis of OSCC‐associated fibroblasts. [ABSTRACT FROM AUTHOR]

Immediate allergy caused by natto, a popular Japanese food prepared by fermenting soybeans with Bacillus subtilis var. natto , has been reported. Polygamma glutamic acid (PGA) in the sticky substance around natto beans has been reported to be a causative allergen of natto allergy. However, some of our patients with natto allergy were negative for PGA in the skin prick test (SPT). The sticky substance of natto beans contains a subtilisin family serine protease, nattokinase, along with PGA. In this study, we aimed to examine the antigenicity of nattokinase in natto allergy. Eight patients, who developed symptoms after ingesting natto and positively reacted to natto (seven to the sticky substance in natto and one to the whole natto product) in their SPT, were enrolled in this study. To analyze IgE reactivity, we performed immunoblotting, ELISA, and SPT for natto (bean and sticky substance), and/or PGA, and/or nattokinase and/or cultured B. subtilis var. natto extract. In the SPT, four cases each were PGA-positive and PGA-negative. Immunoblotting of the sera from PGA-negative patients showed a protein band at 30 kDa, which was identified as nattokinase. Three PGA-negative cases, but not three PGA-positive cases, showed a positive reaction to nattokinase in the SPT and had a history of atopic dermatitis. The ELISA for nattokinase revealed a positive reaction of PGA-negative cases and negative reaction of PGA-positive cases in the SPT. We identified a subtilisin family serine protease, nattokinase, as a novel allergen in natto allergy patients unsensitized to PGA. [Display omitted] [ABSTRACT FROM AUTHOR]

Obesity increases the risk of advanced prostate cancer (PCa). The calcium sensing receptor (CaSR) has been shown to be responsive to obesity-mediated cytokines and is upregulated in metastatic PCa. This study used a novel in vitro approach, involving the exposure of PCa cells to sera, from obese or normal weight males, and to CaSR inhibitor NPS-2143. Cell viability was determined using MTT assay. MMP-9 activity and invasion were assessed using zymography and invasion chambers, respectively. Microscopy was used to visualize EMT proteins. qRT-PCR and immunoblot analysis were used to quantify changes in genes and proteins important for tumorigenesis. Exposure to obese sera increased the proliferation, and the invasive capacity of PCa cells and de-localized epithelial-mesenchymal transition markers, which were attenuated with CaSR inhibition. Exposure to obese sera upregulated mRNA expression of PTHrP and protein expression of COX-2, IL-6, and CaSR. Inhibition of CaSR downregulated the mRNA expression of PTHrP and RANK, and protein expression of pERK and TNF-α. Obesity was shown to increase invasion and upregulate the expression of genes and proteins involved in PCa tumorigenesis. CaSR inhibition downregulated the expression of several of these factors. Thus, CaSR is a potentially important protein to target in obesity-mediated PCa progression. [ABSTRACT FROM AUTHOR]

Background Possible associations of localized scleroderma (LS) with Lyme borreliosis (LB) were reported in certain countries, though denied in others. Therefore, it is still under investigation, whether the association is real or fictional. Objective The aims of the current study was to determine percentage of serologically positive patients among suspected LB patients, as well as to investigate etiological differences between confirmed LB patients with and without LS in Ternopil region. Methods An observational study of 196 patients with complaints related to LB treated at Ternopil healthcare institutions (2017-2022) was conducted. LB serological confirmation was done using two-stage procedure (ELISA and immunoblotting). The confirmed LB patients were split into two groups, with or without localized scleroderma (LS), and a wide spectra of specific IgG were analyzed to check for significant differences between the two groups. Results We confirmed the necessity of the two-stage procedure, as some ELISA screening results, 11.4% (95% CI 3.1-29.3) for IgM and 11.5% (95% CI 4.6-23.6) for IgG, appeared to be false positives during stage two immunoblotting tests. VlsE antigens of B. afzelii and p83 were significantly more often detected in the LB patients with LS compared to LB patients without LS, p < 0.05. VlsE antigens of B. burgdorferi s.s. and p39 were diagnosed more often in LB alone patients compared to LB with LS patients, p <0.05. Conclusion We can speculate, that failures to detect borreliosis-associated morphea in some countries may be related to the absence of B. afzelii in those areas. [ABSTRACT FROM AUTHOR]

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Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses. [ABSTRACT FROM AUTHOR]

Background: Many studies have shown effective protection from myocardial ischemia–reperfusion injury (MIRI) in animal models, but few, if any, treatments have yielded a substantial reduction in clinical. Several studies showed significant therapeutic effects for the Chinese patent medicine Suxiao Jiuxin Pill (SJP) in MIRI, although the specific molecular mechanisms remain undefined. Recently, increasing evidence indicates an important role for m6A modification in autophagy regulation in MIRI, and SJP has not been investigated in this regard. Methods: In vivo experiments were performed in a Wistar rat MIRI model. In vitro assays were conducted in hypoxia/reoxygenation (H/R)-treated H9c2 cells. H9c2 cells with ALKBH5 and GSK3β silencing were constructed by lentivirus transfection. TUNEL and Annexin V/PI assays were carried out for apoptosis detection. Then, m6A modification was detected with the EpiQuik m6A RNA methylation quantification kit, and GFP-RFP-LC3B was used to observe dynamic changes in autophagy. The autophagosome structure was assessed by Transmission electron microscopy. qPCR and immunoblot were performed for mRNA and protein analyses, receptively. Results: SJP significantly mitigated MIRI in rats, reducing infarct size and myocardial apoptosis, and improving left ventricular function. In addition, SJP inhibited autophagy through the GSK3β/mTOR pathway in MIRI rats. In cultured H9c2 cells, SJP significantly inhibited H/R- related apoptosis and autophagic activity through the GSK3β/mTOR pathway. Additionally, SJP enhanced ALKBH5 expression in H/R cardiomyocytes, which is important in impaired m6A modification. Interestingly, ALKBH5 knockdown enhanced autophagy and apoptosis in H/R-induced cells, whereas SJP reversed these effects. Further experiments showed that autophagic activity and apoptosis enhanced by ALKBH5 deficiency are GSK3β/mTOR pathway dependent in H/R-treated H9c2 cells. After SJP administration the above effects were alleviated, suggesting SJP inhibited autophagy through the ALKBH5/GSK3β/mTOR pathway in H/R-induced cardiomyocytes. These effects of SJP were common to its two main constituents, including tetra-methylpyrazine (TMP) and borneol (BOR). Conclusion: SJP improves MIRI in rats and alleviates autophagy and apoptosis in H9c2 cells through the ALKBH5/GSK3β/mTOR pathway, thanks to its two major constituents TMP and BOR. [ABSTRACT FROM AUTHOR]

Purpose: Resistance exercise training (RET) attenuates age-related muscle and strength loss ("sarcopenia"). However, compared with machine-based RET, the efficacy of cost-effective, accessible elastic band RET (EB-RET) for muscle adaptive remodeling lacks supporting mechanistic evidence. Methods: Eight young (YM; 24 ± 4 yr) and eight older (OM; 68 ± 6 yr) untrained males consumed an oral stable isotope tracer (D2O) combined with serial vastus lateralis muscle biopsies to measure integrated myofibrillar protein synthesis (iMyoPS) and regulatory signaling over ~48 h before (habitual) and after an acute bout of EB-RET (6 × 12 repetitions at ~70% of one-repetition maximum). iMyoPS was determined via gas chromatography–pyrolysis–isotope ratio mass spectroscopy and regulatory signaling expression by immunoblot. Results: Habitual iMyoPS did not differ between YM and OM (1.62% ± 0.21% vs 1.43% ± 0.47%·d−1, respectively, P = 0.128). There was a significant increase in iMyoPS after EB-RET in YM (2.23% ± 0.69%·d−1, P = 0.02), but not OM (1.75% ± 0.54%·d−1, P = 0.30). EB-RET increased the phosphorylation of key anabolic signaling proteins similarly in YM and OM at 1 h postexercise, including p-IRS-1Ser636/639, p-AktSer473, p-4EBP-1Thr37/46, p-P70S6KThr389, and p-RPS6Ser240/244, whereas p-TSC2Thr1462 and p-mTORSer2448 increased only in YM (all P < 0.05). There were no differences in the expression of amino acid transporters/sensors or proteolytic markers after EB-RET. Conclusions: iMyoPS was elevated after EB-RET in YM but not OM. However, the increase in acute anabolic signaling with EB-RET was largely similar between groups. In conclusion, the capacity for EB-RET to stimulate iMyoPS may be impaired in older age. Further work may be necessary to optimize prescriptive programming in YM and OM. [ABSTRACT FROM AUTHOR]

We evaluated effects of calorie restriction (CR; consuming 65% of ad libitum (AL) intake) for 8 weeks on female wildtype (WT) and Akt substrate of 160 kDa knockout (AS160-KO) rats. Insulin-stimulated glucose uptake (ISGU) was determined in isolated epitrochlearis muscles incubated with 0, 50, 100, or 500 µU/mL insulin. Phosphorylation of key insulin signaling proteins that control ISGU (Akt and AS160) was assessed by immunoblotting (Akt phosphorylation on Threonine-308, pAktThr308 and Serine-473, pAktSer473; AS160 phosphorylation on Serine-588, pAS160Ser588, and Threonine-642, pAS160Thr642). Abundance of proteins that regulate ISGU (GLUT4 glucose transporter protein and hexokinase II) was also determined by immunoblotting. The major results were as follows: (i) WT-CR versus WT-AL rats had greater ISGU with 100 and 500 µU/mL insulin; (ii) CR versus WT-AL rats had greater GLUT4 protein abundance; (iii) WT-CR versus WT-AL rats had greater pAktThr308 with 500 µU/mL insulin; (iv) WT-CR versus WT-AL rats did not differ for pAktSer473, pAS160Ser588, or pAS160Thr642 at any insulin concentration; (v) AS160-KO versus WT rats with each diet had lower ISGU at each insulin concentration, but not lower pAkt on either phosphosite; (vi) AS160-KO versus WT rats had lower muscle GLUT4 abundance regardless of diet; and (vii) AS160-KO-CR versus AS160-KO-AL rats did not differ for ISGU, GLUT4 abundance, pAkt on either phosphosite, or pAS160 on either phosphosite. These novel results demonstrated that AS160 expression, but not greater pAS160 on key phosphosites, was essential for the CR-induced increases in muscle ISGU and GLUT4 abundance of female rats. [ABSTRACT FROM AUTHOR]

Objective: To identify the molecular basis of a severe systemic autoinflammatory disorder (SAID) and define its main phenotypic features, and to functionally assess the sequence variations identified in LYN, a gene encoding a nonreceptor tyrosine kinase. Methods: We used targeted next‐generation sequencing and in vitro functional studies of Lyn phosphorylation state and Lyn‐dependent NF‐κB activity after expression of recombinant Lyn isoforms carrying different sequence variations. Results: We identified a de novo LYN variation (p.Tyr508His) in a patient presenting since birth with recurrent fever, chronic urticaria, atopic dermatitis, arthralgia, increased inflammatory biomarkers, and elevated plasma cytokine levels. We studied the consequences on Lyn phosphorylation state of the p.Tyr508His variation and of the 2 LYN variations reported so far (p.Tyr508Phe and p.Tyr508*), and found that all 3 variations prevent phosphorylation of residue 508 and lead to autophosphorylation of Tyr397. Additionally, these 3 LYN variations activate the NF‐κB pathway. These results show a gain‐of‐function effect of the variations involving Tyr508 on Lyn activity. Conclusion: This study demonstrates the pathogenicity of the first 3 LYN variations identified in SAID patients and delineates the phenotypic spectrum of a disease entity characterized by severe, early‐onset, systemic inflammatory disease affecting neonates with no family history of SAID. All 3 LYN variations affect the same tyrosine residue located in the C‐terminus of Lyn, thereby demonstrating the critical role of this residue in the proper regulation of Lyn activity in humans. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most common cancer worldwide. The main obstacle in treating advanced CRC is tumor recurrence and metastasis due to chemoresistance. S-phase kinase associated protein 2 (Skp2), an E3 ligase, is highly associated with tumor resistance and a poor prognosis. The results of immunoblotting, immunohistochemical staining, ubiquitination analysis, and co-immunoprecipitation (co-IP) assay revealed that the plant curcuma, curcumol, is a novel Skp2 inhibitor for CRC treatment. Curcumol inhibits aerobic glycolysis in CRC by inducing Skp2 degradation. Co-immunoprecipitation results showed that curcumol enhanced the interaction between cadherin-1 (Cdh1) and Skp2 and led to the ubiquitination and degradation of Skp2. Curcumol exhibited significant antitumor effects against CRC, such as increased intrinsic apoptosis and decreased tumorigenic properties, both in vivo and in vitro. Furthermore, curcumol overcame 5-fluorouracil (5-Fu) resistance in CRC and induced apoptosis in 5-Fu-resistant CRC cells. The present data revealed a novel antitumor mechanism of glycolytic regulation by curcumol, suggesting that curcumol may be a potential chemical candidate for treating 5-Fu-resistant CRC. [ABSTRACT FROM AUTHOR]

Background: Amyloid‐β peptide is well‐known as a pathogen of Alzheimer's disease, but its precursor, amyloid‐beta precursor protein (APP), remains unexplained 30 years after its discovery. APP has two homologues called amyloid precursor‐like protein 1 (APLP1) and amyloid precursor‐like protein 2 (APLP2), and shares a similar structural organisation with them and has partially overlapping functions. APP family proteins are essential for survival, shown by the crossbreeding analysis of knockout mice of APP family molecules, including APLP1 and APLP2. APLP2 is known to play the most important role among them, but the molecular metabolism of APLP2 is only partially understood. Here, we analysed ectodomain shedding and γ‐secretase cleavage of APLP2 by molecular biological and biochemical techniques. Method: We analysed the culture supernatant of HEK293 cells overexpressing APLP2 and human cerebrospinal fluid. For the analysis of secreted APLP2 fragments, we raised the OA603 antibody that reacts with the juxtamembrane domain of APLP2. Substrate cleavage sites were identified by matrix assisted laser desorption/ionisation mass spectrometry. Results: By overexpressing in HEK293 cells, APLP2 undergoes ectodomain shedding at three sites in the extracellular region by α‐ and β‐secretase‐like activity and then is intramembranously cleaved at three sites by γ‐secretase. In particular, in shedding, α‐secretase‐like activity was dominant in HEK cells. Surprisingly, in human cerebrospinal fluid, APLP2‐derived metabolic fragments were mainly cleaved by β‐secretase‐like activity, not by α‐secretase‐like activity. Because APP is also mainly cleaved by beta‐site amyloid precursor protein cleaving enzyme 1 in neurons and APLP1 is expressed exclusively in neurons, these findings suggest that APP family proteins may play a common role via β‐secretase‐like cleavage in the central nerve system. Conclusions: Thus, these findings may contribute to a better understanding of the role of APP family proteins in Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Objectives Lung fibrosis is the leading cause of death in SSc, with no cure currently available. Antifibrotic Endostatin (ES) production does not reach therapeutic levels in SSc patients, suggesting a deficit in its release from Collagen XVIII by the main cleavage enzyme, Cathepsin L (CTSL). Thus, elucidating a potential deficit in CTSL expression and activity unravels an underlying molecular cause for SSc-driven lung fibrosis. Methods Fibrosis was induced experimentally using TGF-β in vitro , in primary human lung fibroblasts (pLFs), and ex vivo , in human lung tissues. ES and CTSL expression was quantified using ELISA, RT-qPCR, immunoblotting or immunofluorescence. Recombinant NC1-FLAG peptide was used to assess CTSL cleavage activity. CTSL expression was also compared between SSc vs normal (NL)-derived pLFs and lung tissues. Results ES levels were significantly reduced in media conditioned by TGF-β-induced pLFs. TGF-β-stimulated pLFs significantly reduced expression and secretion of CTSL into the extracellular matrix (ECM). CTSL was also sequestered in its inactive form into extracellular vesicles, further reducing its availability in the ECM. Media conditioned by TGF-β-induced pLFs showed reduced cleavage of NC1-Flag and reduced release of the antifibrotic ES fragment. SSc-derived pLFs and lung tissues expressed significantly lower levels of CTSL compared with NL. Conclusions Our findings identify CTSL as a protein protective against lung fibrosis via its activation of antifibrotic ES, and whose expression in SSc pLFs and lung tissues is suppressed. Identifying strategies to boost CTSL endogenous levels in SSc patients could serve as a viable therapeutic strategy. [ABSTRACT FROM AUTHOR]

Simple Summary: Natural products derived from plants have numerous clinical applications, including anti-cancer activity. In the present study, we identified three different plant extracts as strong inducers of cell death that were not reported previously. We focused on the most potent of these plants, Vernonanthura nudiflora (Vern). We demonstrated that the plant extracts obtained by treatment with a water and ethanol mixture killed tumor cells via multiple routes. These include impairing cell energy and metabolism, generating reactive oxygen species, increasing intracellular Ca2+, and inducing mitochondria-mediated apoptosis. We connected these activities to increased levels of the mitochondrial gatekeeper protein, VDAC1, which is associated with metabolism and apoptosis regulation. In a glioblastoma mouse model, Vern extract strongly inhibited tumor growth and induced massive tumor cell death, including cancer stem cells, by inhibiting blood supply and modulating the tumor microenvironment. The multipronged effects of hydroethanolic Vern extract make it a promising candidate for treating cancer. The mitochondrial voltage-dependent anion channel 1 (VDAC1) protein is involved in several essential cancer hallmarks, including energy and metabolism reprogramming and apoptotic cell death evasion. In this study, we demonstrated the ability of hydroethanolic extracts from three different plants, Vernonanthura nudiflora (Vern), Baccharis trimera (Bac), and Plantago major (Pla), to induce cell death. We focused on the most active Vern extract. We demonstrated that it activates multiple pathways that lead to impaired cell energy and metabolism homeostasis, elevated ROS production, increased intracellular Ca2+, and mitochondria-mediated apoptosis. The massive cell death generated by this plant extract's active compounds involves the induction of VDAC1 overexpression and oligomerization and, thereby, apoptosis. Gas chromatography of the hydroethanolic plant extract identified dozens of compounds, including phytol and ethyl linoleate, with the former producing similar effects as the Vern hydroethanolic extract but at 10-fold higher concentrations than those found in the extract. In a xenograft glioblastoma mouse model, both the Vern extract and phytol strongly inhibited tumor growth and cell proliferation and induced massive tumor cell death, including of cancer stem cells, inhibiting angiogenesis and modulating the tumor microenvironment. Taken together, the multiple effects of Vern extract make it a promising potential cancer therapeutic. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastomas (GBM) are aggressive brain tumors with poor prognosis that need effective treatment. GBMs are characterized by extensive cellular and molecular heterogeneity which are reflected in patient-derived cell cultures, frequently used in testing potential therapeutics. Here, we established GBM-derived cell cultures from fresh tumor specimens and characterized them at the protein and molecular levels. We confirmed the considerable intertumor heterogeneity of GBMs. As the epidermal growth factor receptor (EGFR) is a subject of common oncogenic alterations in GBM, we tested anti-EGFR therapy combined with temozolomide (first-choice medication for GBM) or with doxorubicin (common therapeutic for various solid and blood cancers). We found that GBM-derived cells were more sensitive to a combined therapy than to monotherapy, particularly cells with inactive DNA repair mechanisms. Glioblastomas (GBM) are the most common, primary brain tumors in adults. Despite advances in neurosurgery and radio- and chemotherapy, the median survival of GBM patients is 15 months. Recent large-scale genomic, transcriptomic and epigenetic analyses have shown the cellular and molecular heterogeneity of GBMs, which hampers the outcomes of standard therapies. We have established 13 GBM-derived cell cultures from fresh tumor specimens and characterized them molecularly using RNA-seq, immunoblotting and immunocytochemistry. Evaluation of proneural (OLIG2, IDH1R132H, TP53 and PDGFRα), classical (EGFR) and mesenchymal markers (CHI3L1/YKL40, CD44 and phospho-STAT3), and the expression of pluripotency (SOX2, OLIG2, NESTIN) and differentiation (GFAP, MAP2, β-Tubulin III) markers revealed the striking intertumor heterogeneity of primary GBM cell cultures. Upregulated expression of VIMENTIN, N-CADHERIN and CD44 at the mRNA/protein levels suggested increased epithelial-to-mesenchymal transition (EMT) in most studied cell cultures. The effects of temozolomide (TMZ) or doxorubicin (DOX) were tested in three GBM-derived cell cultures with different methylation status of the MGMT promoter. Amongst TMZ- or DOX-treated cultures, the strongest accumulation of the apoptotic markers caspase 7 and PARP were found in WG4 cells with methylated MGMT, suggesting that its methylation status predicts vulnerability to both drugs. As many GBM-derived cells showed high EGFR levels, we tested the effects of AG1478, an EGFR inhibitor, on downstream signaling pathways. AG1478 caused decreased levels of phospho-STAT3, and thus inhibition of active STAT3 augmented antitumor effects of DOX and TMZ in cells with methylated and intermediate status of MGMT. Altogether, our findings show that GBM-derived cell cultures mimic the considerable tumor heterogeneity, and that identifying patient-specific signaling vulnerabilities can assist in overcoming therapy resistance, by providing personalized combinatorial treatment recommendations. [ABSTRACT FROM AUTHOR]

Simple Summary: Human tumors often establish a local hypercoagulant state that promotes vascular complications, such as venous thromboembolism. The concept of the tumor «coagulome» refers to the repertoire of tumor-expressed genes that locally regulate coagulation and fibrinolysis. Recent systems studies have helped to define the landscape of the coagulome across the spectrum of human tumors, unveiling its link with the tumor microenvironment. Understanding the key elements that regulate the expression of the coagulome is therefore essential. In this study, we explored the dynamic regulation of the tumor coagulome by glucocorticoids. We found that glucocorticoids regulate the coagulome through a combination of direct transcriptional and indirect effects. We show that this transcriptional regulation applies to human tumors, and we suggest that the direct transcriptional regulation of PAI-1 expression by the glucocorticoid receptor may regulate the tumor microenvironment. The transcriptional regulation of the coagulome by glucocorticoids that we report here may have vascular consequences and may account for some of the effects of glucocorticoids on the tumor microenvironment. Background: The coagulome, defined as the repertoire of genes that locally regulate coagulation and fibrinolysis, is a key determinant of vascular thromboembolic complications of cancer. In addition to vascular complications, the coagulome may also regulate the tumor microenvironment (TME). Glucocorticoids are key hormones that mediate cellular responses to various stresses and exert anti-inflammatory effects. We addressed the effects of glucocorticoids on the coagulome of human tumors by investigating interactions with Oral Squamous Cell Carcinoma, Lung Adenocarcinoma, and Pancreatic Adenocarcinoma tumor types. Methods: We analyzed the regulation of three essential coagulome components, i.e., the tissue factor (TF), urokinase-type plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) in cancer cell lines exposed to specific agonists of the glucocorticoid receptor (GR) (dexamethasone and hydrocortisone). We used QPCR, immunoblots, small-interfering RNA, Chromatin immunoprecipitation sequencing (ChIPseq) and genomic data from whole tumor and single-cell analyses. Results: Glucocorticoids modulate the coagulome of cancer cells through a combination of indirect and direct transcriptional effects. Dexamethasone directly increased PAI-1 expression in a GR-dependent manner. We confirmed the relevance of these findings in human tumors, where high GR activity/high SERPINE1 expression corresponded to a TME enriched in active fibroblasts and with a high TGF-β response. Conclusion: The transcriptional regulation of the coagulome by glucocorticoids that we report may have vascular consequences and account for some of the effects of glucocorticoids on the TME. [ABSTRACT FROM AUTHOR]

Background: Cow’s Milk protein allergy (CMA) is a common finding in infants and young children (2-3%). Its diagnosis is a multifaceted aspect including medical history, clinical examination, diagnostic elimination diets, oral challenge tests (OCT), skin prick tests (SPTs) and specific immunoglobulin E (sIgE) measurements. We aimed to assess the value of SPT and sIgE for diagnosis of infants with CMA in routine clinical practice. Methods: This cross-sectional study included 102 infants with suspected CMA. They were subjected to OCT, SPT with pasteurized cow's milk and measurement of serum sIgE for cow’s milk by immunoblot technique. Results: Seventy-two infants (70.59%) showed positive allergic reactions with OCT. Comparing SPT to OCT, sensitivity was 75%, specificity was 68.7%, predictive value for negative (PVN) was 59% and predictive value for positive (PVP) was 93.1%. Comparing sIgE to OCT, sensitivity was 68.1%, specificity was 96.7%, PVN was 69% and PVP was 98%. Comparing both SPT and sIgE together to OCT, sensitivity was 62.5%, specificity was 96.6%, PVN was 51.8% and PVP was 97.8%. Conclusions: For clinical practice, our findings suggest that correlation between SPT and sIgE is significant regarding CMA diagnosis. Therefore, these tests can be used together for diagnosis of CMA. However, still some cases can be only diagnosed with positive OCT with non-detectable sensitization. Therefore, a detailed history is a major factor in assessing CMA. In addition, definition of new optimal cut-offs for sIgE and SPT to cow’s milk can improve the accuracy of these tests, hoping to avoid unnecessary and potentially dangerous OTC. [ABSTRACT FROM AUTHOR]

Goat's milk thought to be a good substitute for cow's milk protein allergic (CMPA) individuals. However, there is growing evidence that their proteins have cross-reactivities with cow's milk allergens. This study aimed to profile and compare milk proteins from different goat breeds that have cross-reactivity to cow's milk allergens. Proteomics was used to compare protein extracts of skim milk from Saanen, Jamnapari, and Toggenburg. Cow's milk was used as a control. IgE-immunoblotting and mass spectrometry were used to compare and identify proteins that cross-reacted with serum IgE from CMPA patients (n = 10). The analysis of IgE-reactive proteins revealed that the protein spots identified with high confidence were proteins homologous to common cow's milk allergens such as α-S1-casein (αS1-CN), β-casein (β-CN), κ-casein (κ-CN), and beta-lactoglobulin (β-LG). Jamnapari's milk proteins were found to cross-react with four major milk allergens: α-S1-CN, β-CN, κ-CN, and β-LG. Saanen goat's milk proteins, on the other hand, cross-reacted with two major milk allergens, α-S1-CN and β-LG, whereas Toggenburg goat's milk proteins only react with one of the major milk allergens, κ-CN. These findings may help in the development of hypoallergenic goat milk through cross-breeding strategies of goat breeds with lower allergenic milk protein contents. • Goat's milk has cross-reactivities with cow's milk allergens. • Jamnapari's breed milk proteins cross-react with four major milk allergens: α-S1-CN, β-CN, κ-CN, and β-LG. • Saanen goat's milk proteins, on the other hand, cross-reacted with two major milk allergens, α-S1-CN and β-LG. • Toggenburg goat's milk proteins only react with one of the major milk allergens, κ-CN. [ABSTRACT FROM AUTHOR]

Objective: There is significant interest in developing early passage cell lines with matched normal reference DNA to facilitate a precision medicine approach in assessing drug response. This study aimed to establish early passage cell lines, and perform whole exome sequencing and short tandem repeat profiling on matched normal reference DNA, primary tumour and corresponding cell lines. Methods: A cell culture based, in vitro study was conducted of patients with primary human papillomavirus positive and human papillomavirus negative tumours. Results: Four early passage cell lines were established. Two cell lines were human papillomavirus positive, confirmed by sequencing and p16 immunoblotting. Short tandem repeat profiling confirmed that all cell lines were established from their index tumours. Whole exome sequencing revealed that the matched normal reference DNA was critical for accurate mutational analysis: a high rate of false positive mutation calls were excluded (87.6 per cent). Conclusion: Early passage cell lines were successfully established. Patient-matched reference DNA is important for accurate cell line mutational calls. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer. Methods: Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay. Results: Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G1/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05). Conclusion: Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway. [ABSTRACT FROM AUTHOR]

Gestational exposure of mice to valproic acid (VPA) is one currently used experimental model for the investigation of typical failure symptoms associated with autism spectrum disorder (ASD). In the present study we hypothesized that the reduction of dopaminergic source neurons of the VTA, followed by perturbed growth of the mesotelencephalic dopamine pathway (MT), should also modify pattern formation in the dopaminoceptive target regions (particularly its mesoaccumbens/mesolimbic portion). Here, we investigated VPA-evoked cellular morphological (apoptosis-frequency detected by Caspase-3, abundance of Ca-binding proteins, CaBP), as well as synaptic proteomic (western blotting) changes, in selected dopaminoceptive subpallial, as compared to pallial, regions of mice, born to mothers treated with 500 mg/kg VPA on day 13.5 of pregnancy. We observed a surge of apoptosis on VPA treatment in nearly all investigated subpallial and pallial regions; with a non-significant trend of similar increase the nucleus accumbens (NAc) at P7, the age at which the MT pathway reduction has been reported (also supplemented by current findings). Of the CaBPs, calretinin (CR) expression was decreased in pallial regions, most prominently in retrosplenial cortex, but not in the subpallium of P7 mice. Calbindin-D 28K (CB) was selectively reduced in the caudate-putamen (CPu) of VPA exposed animals at P7 but no longer at P60, pointing to a potency of repairment. The VPA-associated overall increase in apoptosis at P7 did not correlate with the abundance and distribution of CaBPs, except in CPu, in which the marked drop of CB was negatively correlated with increased apoptosis. Abundance of parvalbumin (PV) at P60 showed no significant response to VPA treatment in any of the observed regions we did not find colocalization of apoptotic (Casp3+) cells with CaBP-immunoreactive neurons. The proteomic findings suggest reduction of tyrosine hydroxylase in the crude synaptosome fraction of NAc, but not in the CPu, without simultaneous decrease of the synaptic protein, synaptophysin, indicating selective impairment of dopaminergic synapses. The morpho-functional changes found in forebrain regions of VPA-exposed mice may signify dendritic and synaptic reorganization in dopaminergic target regions, with potential translational value to similar impairments in the pathogenesis of human ASD.

Background: Atopic dermatitis (AD) is a common allergic disorder. Detection of responsible pathogenic allergens in AD patients by reliable methods has a fundamental role in the prevention, management, and treatment of AD. This study was conducted to determine the most common allergens by the skin prick test (SPT) and immunoblotting among AD patients referring to an allergy clinic in Birjand City, Iran. Methods: The presence of AD was confirmed by an expert allergist. Serum levels of total and specific immunoglobulin E (sIgE) against 30 food and inhalant allergens were evaluated by a commercial immunoblotting kit (AlleisaScreen). Results: The skin prick test was performed by a battery of 17 allergens. In total, 34 AD patients (mean age, 28.76 ± 17.36 years; range, 1-60 years; F/M ratio: 0.88) were enrolled in this study. The sensitization rates to at least 1 fungus, pollen, food, or indoor allergen by the immunoblotting method were 32.35%, 61.76%, 52.94%, and 47.05%, respectively. The most prevalent allergens were ragweed (52.94), Olive tree (41.16), Eucalyptus (35.29), date palm (35.29), and grass mix (32.28). Conclusion: The study found that 85.29% of the studied population were sensitized to at least 1 allergen. Pollens and date palms were the most common allergens among AD patients, but the pattern of sensitization in SPT and immunoblotting was not exactly similar. Detection of allergens to which patients are sensitized and avoidance can help in the management of the disease and its symptoms. [ABSTRACT FROM AUTHOR]

Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models.IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

BackgroundChronic exposure to ethanol (EtOH) and other drugs of abuse can alter the expression and activity of cyclin-dependent kinase 5 (CDK5) and its cofactor p35, but the functional implication of CDK5 signaling in the regulation of EtOH-related behaviors remains unknown. In the present study, we sought to determine whether CDK5 activity plays a role in the escalation of EtOH self-administration triggered by dependence.MethodsWe tested the effect of systemically administered (S)-CR8, a nonselective CDK inhibitor, on operant responding for EtOH or saccharin, a highly palatable reinforcer, in adult male Wistar rats. Half of the rats were made EtOH-dependent via chronic intermittent EtOH inhalation (CIE). We then sought to identify a possible neuroanatomical locus for the behavioral effect of (S)-CR8 by quantifying protein levels of CDK5 and p35 in subregions of the extended amygdala and prefrontal cortex from EtOH-naïve, nondependent, and dependent rats at the expected time of EtOH self-administration. We also analyzed the phosphorylation of 4 CDK5 substrates and of the CDK substrate consensus motif.Results(S)-CR8 dose-dependently reduced EtOH self-administration in dependent rats. It had no effect on water or saccharin self-administration, nor in nondependent rats. The abundance of CDK5 or p35 was not altered in any of the brain regions analyzed. In the bed nucleus of the stria terminalis, CDK5 abundance was negatively correlated with intoxication levels during EtOH vapor exposure but there was no effect of dependence on the phosphorylation ratio of CDK5 substrates. In contrast, EtOH dependence increased the phosphorylation of low-molecular-weight CDK substrates in the basolateral amygdala (BLA).ConclusionsThe selective effect of (S)-CR8 on excessive EtOH intake has potential therapeutic value for the treatment of alcohol use disorders. Our data do not support the hypothesis that this effect would be mediated by the inhibition of up-regulated CDK5 activity in the extended amygdala nor prefrontal cortex. However, increased activity of CDKs other than CDK5 in the BLA may contribute to excessive EtOH consumption in alcohol dependence. Other (S)-CR8 targets may also be implicated.

How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain (StarD)5 leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in WT but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides, which leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of 6-dodecanoyl-2-dimethylaminonaphthalene fluorescence lifetime imaging microscopy data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress-responsive protein that regulates PM cholesterol and intracellular cholesterol homeostasis.

PURPOSE: Recent evidence suggests that retinal photoreceptor cells have an important role in the pathogenesis of retinal microvascular lesions in diabetes. We investigated the role of rod cell phototransduction on the pathogenesis of early diabetic retinopathy (DR) using Gnat1-/- mice (which causes permanent inhibition of phototransduction in rod cells without degeneration). METHODS: Retinal thickness, oxidative stress, expression of inflammatory proteins, electroretinograms (ERG) and optokinetic responses, and capillary permeability and degeneration were evaluated at up to 8 months of diabetes. RESULTS: The diabetes-induced degeneration of retinal capillaries was significantly inhibited in the Gnat1-/- diabetics. The effect of the Gnat1 deletion on the diabetes-induced increase in permeability showed a nonuniform accumulation of albumin in the neural retina; the defect was inhibited in diabetic Gnat1-/- mice in the inner plexiform layer (IPL), but neither in the outer plexiform (OPL) nor inner nuclear (INL) layers. In Gnat1-deficient animals, the diabetes-induced increase in expression of inflammatory associated proteins (iNOS and ICAM-1, and phosphorylation of IĸB) in the retina, and the leukocyte mediated killing of retinal endothelial cells were inhibited, however the diabetes-mediated induction of oxidative stress was not inhibited. CONCLUSIONS: In conclusion, deletion of transducin1 (and the resulting inhibition of phototransduction in rod cells) inhibits the development of retinal vascular pathology in early DR.

Background and Aim: Feline platynosomiasis, also known as lizard poisoning, is a feline hepatic disease caused by the parasitic trematode Platynosomum fastosum. Since this helminth resides in biliary ducts and gallbladder, the heavy infection can lead to failure of the hepatobiliary system and can be associated with cholangiocarcinoma. The primary diagnostic tool currently used is conventional fecal microscopy. However, low sensitivity of detection could occur in the case of light infection or biliary obstruction. This study aimed to determine the antibody-specific pattern of P. fastosum crude antigen and to identify immunoreactive proteins to develop the immunodiagnostic techniques. Materials and Methods: We investigated potential antigens specific to P. fastosum infection using western blotting. Forty-six samples of cat serum, including 16 P. fastosum-infected sera, eight healthy control sera, and 22 sera infected with other endoparasites were used. The sensitivity, specificity, positive predictive value, and negative predictive value of each band were calculated. Immunoreactive bands with high diagnostic values were further analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the protein components. Results: Using immunoblotting, three proteins of 72 kDa, 53 kDa, and 13 kDa were found to be immunogenic. LC-MS/MS identified these proteins as a 70 kDa heat shock protein, a hypothetical protein (CRM22_002083) (adenosine triphosphate synthase subunit beta), and histone H2B, respectively. Conclusion: This study is the first to reveal three proteins that could be candidates for developing diagnostic tools for feline platynosomiasis.

Western blotting (WB), also known as immunoblotting, is a well-known molecular biology method that biologists often use to investigate many features of the protein, ranging from basic protein analysis to disease detection. WB is simple, unique, rapid, widely used routine tool with easy interpretation and definite results. It is being used in various fields of science, research and development, diagnostic labs and hospitals. The principle of WB is to accomplish the separation of proteins based on molecular weight and charge. This review addresses in detail the individual steps involved in the WB technique, its troubleshooting, internal loading controls, total protein staining and its diverse applications in scientific research and clinical settings, along with its future perspectives.

Simple Summary: EGFR upregulation plays an important role in lung cancer as a well-established target for lung cancer therapy. However, the role and mechanism of EGFR upregulation due to chronic arsenic exposure remain to be elucidated. Here, we demonstrated that miR-218-5p was dramatically downregulated in arsenic-induced transformed (As-T) cells. It served as a tumor suppressor to suppress cell proliferation, migration, colony formation, and tube formation, and inhibit tumor growth and angiogenesis by directly targeting EGFR. Our results suggest that the 218-5p/EGFR signaling pathway may be a potential therapeutic target for the treatment of lung cancer induced by chronic arsenic exposure. Background: Arsenic is a well-known carcinogen inducing lung, skin, bladder, and liver cancer. Abnormal epidermal growth factor receptor (EGFR) expression is common in lung cancer; it is involved in cancer initiation, development, metastasis, and treatment resistance. However, the underlying mechanism for arsenic-inducing EGFR upregulation remains unclear. Methods: RT-PCR and immunoblotting assays were used to detect the levels of miR-218-5p and EGFR expression. The Luciferase assay was used to test the transcriptional activity of EGFR mediated by miR-218-5p. Cell proliferation, colony formation, wound healing, migration assays, tube formation assays, and tumor growth assays were used to study the function of miR-218-5p/EGFR signaling. Results: EGFR and miR-218-5p were dramatically upregulated and downregulated in arsenic-induced transformed (As-T) cells, respectively. MiR-218-5p acted as a tumor suppressor to inhibit cell proliferation, migration, colony formation, tube formation, tumor growth, and angiogenesis. Furthermore, miR-218-5p directly targeted EGFR by binding to its 3′-untranslated region (UTR). Finally, miR-218-5p exerted its antitumor effect by inhibiting its direct target, EGFR. Conclusion: Our study highlights the vital role of the miR-218-5p/EGFR signaling pathway in arsenic-induced carcinogenesis and angiogenesis, which may be helpful for the treatment of lung cancer induced by chronic arsenic exposure in the future. [ABSTRACT FROM AUTHOR]