Feruloyl esterases (FAEs) are a crucial component of the hemicellulose-degrading enzyme family that facilitates the degradation of lignocellulose while releasing hydroxycinnamic acids such as ferulic acid with high added value. Currently, the low enzyme yield of FAEs is one of the primary factors limiting its application. Therefore, in this paper, we optimized the fermentation conditions for the expression of FAE BpFaeT132C-D143C with excellent thermal stability in Escherichia coli by experimental design. Firstly, we explored the effects of 11 factors such as medium type, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration, and inoculum size on BpFaeT132C-D143C activity separately by the single factor design. Then, the significance of the effects of seven factors, such as post-induction temperature, shaker rotational speed, and inoculum size on BpFaeT132C-D143C activity, was analyzed by Plackett-Burman design. We identified the main factors affecting the fermentation conditions of E. coli expressing BpFaeT132C-D143C as post-induction temperature, pre-induction period, and post-induction period. Finally, we used the steepest ascent path design and response surface method to optimize the levels of these three factors further. Under the optimal conditions, the activity of BpFaeT132C-D143C was 3.58 U/ml, which was a significant 6.6-fold increase compared to the pre-optimization (0.47 U/ml), demonstrating the effectiveness of this optimization process. Moreover, BpFaeT132C-D143C activity was 1.52 U/ml in a 3-l fermenter under the abovementioned optimal conditions. It was determined that the expression of BpFaeT132C-D143C in E. coli was predominantly intracellular in the cytoplasm. This study lays the foundation for further research on BpFaeT132C-D143C in degrading agricultural waste transformation applications.

• MeSH
• Escherichia coli * genetics   metabolism   enzymology   MeSH
• Fermentation * MeSH
• Isopropyl Thiogalactoside metabolism   MeSH
• Carboxylic Ester Hydrolases * genetics   metabolism   chemistry   biosynthesis   MeSH
• Culture Media chemistry   MeSH
• Coumaric Acids metabolism   MeSH
• Lignin MeSH
• Recombinant Proteins genetics   metabolism   biosynthesis   chemistry   MeSH
• Enzyme Stability MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH

Glioblastomas are aggressive brain tumors for which effective therapy is still lacking, resulting in dismal survival rates. These tumors display significant phenotypic plasticity, harboring diverse cell populations ranging from tumor core cells to dispersed, highly invasive cells. Neuron navigator 3 (NAV3), a microtubule-associated protein affecting microtubule growth and dynamics, is downregulated in various cancers, including glioblastoma, and has thus been considered a tumor suppressor. In this study, we challenge this designation and unveil distinct expression patterns of NAV3 across different invasion phenotypes. Using glioblastoma cell lines and patient-derived glioma stem-like cell cultures, we disclose an upregulation of NAV3 in invading glioblastoma cells, contrasting with its lower expression in cells residing in tumor spheroid cores. Furthermore, we establish an association between low and high NAV3 expression and the amoeboid and mesenchymal invasive phenotype, respectively, and demonstrate that overexpression of NAV3 directly stimulates glioblastoma invasive behavior in both 2D and 3D environments. Consistently, we observed increased NAV3 expression in cells migrating along blood vessels in mouse xenografts. Overall, our results shed light on the role of NAV3 in glioblastoma invasion, providing insights into this lethal aspect of glioblastoma behavior.

• MeSH
• Phenotype * MeSH
• Glioblastoma * pathology   genetics   metabolism   MeSH
• Neoplasm Invasiveness * genetics   MeSH
• Humans MeSH
• Membrane Proteins MeSH
• Microtubules metabolism   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Brain Neoplasms * pathology   genetics   metabolism   MeSH
• Cell Movement genetics   physiology   MeSH
• Nerve Tissue Proteins metabolism   genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Androgen receptor-targeting agents, particularly enzalutamide, show promise in enhancing prostate cancer diagnostic and therapeutic strategies by modulating prostate-specific membrane antigen (PSMA). Methods: A retrospective clinical cohort study investigated 9 men with metastatic castration-resistant prostate cancer on enzalutamide. PSMA PET/CT scans were obtained before and after enzalutamide initiation to assess PSMA expression changes. Lesions and organs at risk were evaluated visually and semiquantitatively. The flare phenomenon was characterized by a significant increase (≥20%) in the SUVmax of existing lesions or the appearance of new PSMA-positive lesions. Results: Exposure to enzalutamide led to a significant PSMA expression increase in 56% of assessed lesions (n = 42), with new lesions detected in 1 patient (11%). PSMA expression in organs at risk remained largely unaffected, indicating a tumor-specific response. Conclusion: Enzalutamide induces PSMA upregulation in metastatic castration-resistant prostate cancer, potentially enhancing diagnostic and therapeutic strategies. Further exploration of the flare phenomenon's clinical implications is warranted.

• MeSH
• Antigens, Surface * metabolism   MeSH
• Benzamides * MeSH
• Phenylthiohydantoin * therapeutic use   analogs & derivatives   MeSH
• Glutamate Carboxypeptidase II * metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• Prostatic Neoplasms, Castration-Resistant * drug therapy   diagnostic imaging   metabolism   MeSH
• Nitriles * therapeutic use   MeSH
• Positron Emission Tomography Computed Tomography * MeSH
• Retrospective Studies MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Up-Regulation * MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Publication type
• Journal Article MeSH

The immunohistochemical (IHC) or fluorescence/chromogenic in situ hybridization (FISH/CISH) assays for assessing HER2 are now recommended by the American Society of Clinical Oncologists and the College of American Pathologists, but there are an increasing number of published studies describing alternative diagnoses at the molecular level. Inspired by these studies, we established a laboratory-developed test (LDT) to analyze HER2 status not only at the gene expression level but also at the gene copy number. A precise copy number calculation was fulfilled including the Control Genomic DNA of known concentration, which allowed subsequent assay validation at the DNA level. The results were reported according to the concordant results of the DNA and RNA approaches. By comparing with IHC determination, completely identical results were found in ten blank samples, which underlines the legitimacy of molecular biological approaches in this diagnostic field. An equivocal sample that was positive by IHC and qPCR was found to be negative by the FISH and so it may change the choice of personalized medicine. The topic of this short communication will hopefully contribute to allowing IVD-certified diagnostics based on the HER2 gene expression profile or copy number to be tested in the Czech Republic as well.

• MeSH
• DNA genetics   metabolism   MeSH
• Gene Dosage * MeSH
• In Situ Hybridization, Fluorescence * methods   MeSH
• Immunohistochemistry * methods   MeSH
• Humans MeSH
• Biomarkers, Tumor genetics   metabolism   MeSH
• Breast Neoplasms genetics   metabolism   diagnosis   MeSH
• Receptor, ErbB-2 * genetics   metabolism   MeSH
• RNA metabolism   genetics   analysis   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

The RNA chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal architecture of the Hfq hexamer presents three well-characterized surfaces that allow it to bind sRNAs to stabilize them and engage target transcripts. Hfq-interacting sRNAs are categorized into two classes based on the surfaces they use to bind Hfq. By characterizing a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen (N) starvation, we corroborated the important role of the three RNA-binding surfaces for Hfq function. We uncovered two, previously uncharacterized, conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq's RNA-binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilization of both sRNA classes, to the same extent as seen in bacteria devoid of Hfq. However, the alanine substitutions at these residues resulted in only modest alteration in stability and structure of Hfq. We propose that V22 and G34 have impact on Hfq function, especially critical under cellular conditions when there is an increased demand for Hfq, such as N starvation.

• MeSH
• RNA, Bacterial * metabolism   genetics   chemistry   MeSH
• Nitrogen metabolism   MeSH
• Escherichia coli * genetics   metabolism   MeSH
• Hydrophobic and Hydrophilic Interactions * MeSH
• Conserved Sequence MeSH
• RNA, Small Untranslated * metabolism   genetics   chemistry   MeSH
• Mutation MeSH
• Host Factor 1 Protein * metabolism   genetics   chemistry   MeSH
• Escherichia coli Proteins * metabolism   genetics   chemistry   MeSH
• Gene Expression Regulation, Bacterial MeSH
• RNA Stability * genetics   MeSH
• Gene Expression Profiling MeSH
• Transcriptome genetics   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH

WBP1L is a broadly expressed transmembrane adaptor protein involved in regulating hematopoietic stem cell function and T cell development. It interacts with NEDD4-family E3 ubiquitin ligases and regulates important chemokine receptor CXCR4. Using tandem affinity purification coupled with mass spectrometry, we identified novel WBP1L interactions with the IFNγ receptor and the Cullin-RING ubiquitin ligases CRL1β-TrCP1/2. We found that WBP1L interaction with the IFNγ receptor serves to downregulate proximal IFNγ receptor signaling in female macrophages, while the interaction with CRL1β-TrCP1/2 ubiquitin ligases regulates WBP1L protein levels. Disrupting this interaction, as well as inhibiting proteasome activity or neddylation, increased WBP1L protein levels, demonstrating that CRL1β-TrCP1/2 ubiquitin ligases regulate WBP1L protein abundance. These data provide important insights into the mechanisms controlling WBP1L function.

• MeSH
• Adaptor Proteins, Signal Transducing metabolism   MeSH
• HEK293 Cells MeSH
• Hematopoiesis * MeSH
• Humans MeSH
• Macrophages metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• Mice MeSH
• beta-Transducin Repeat-Containing Proteins metabolism   MeSH
• Signal Transduction MeSH
• Ubiquitin-Protein Ligases * metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

PURPOSE: Prostate-specific membrane antigen (PSMA) radioligand therapy is a promising treatment for metastatic castration-resistant prostate cancer (mCRPC). Several beta or alpha particle-emitting radionuclide-conjugated small molecules have shown efficacy in late-stage mCRPC and one, [[177Lu]Lu]Lu-PSMA-617, is FDA approved. In addition to tumor upregulation, PSMA is also expressed in kidneys and salivary glands where specific uptake can cause dose-limiting xerostomia and potential for nephrotoxicity. The PSMA inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) can prevent kidney uptake in mice, but also blocks tumor uptake, precluding its clinical utility. Preferential delivery of 2-PMPA to non-malignant tissues could improve the therapeutic window of PSMA radioligand therapy. METHODS: A tris(isopropoxycarbonyloxymethyl) (TrisPOC) prodrug of 2-PMPA, JHU-2545, was synthesized to enhance 2-PMPA delivery to non-malignant tissues. Mouse pharmacokinetic experiments were conducted to compare JHU-2545-mediated delivery of 2-PMPA to plasma, kidney, salivary glands, and C4-2 prostate tumor xenograft. Imaging studies were conducted in rats and mice to measure uptake of PSMA PET tracers in kidney, salivary glands, and prostate tumor xenografts with and without JHU-2545 pre-treatment. RESULTS: JHU-2545 resulted in approximately 3- and 53-fold greater exposure of 2-PMPA in rodent salivary glands (18.0 ± 0.97 h*nmol/g) and kidneys (359 ± 4.16 h*nmol/g) versus prostate tumor xenograft (6.79 ± 0.19 h*nmol/g). JHU-2545 also blocked rodent kidneys and salivary glands uptake of the PSMA PET tracers [68Ga]Ga-PSMA-11 and [18 F]F-DCFPyL by up to 85% with little effect on tumor. CONCLUSIONS: JHU-2545 pre-treatment may enable greater cumulative administered doses of PSMA radioligand therapy, possibly improving safety and efficacy.

• MeSH
• Antigens, Surface * metabolism   MeSH
• Glutamate Carboxypeptidase II * metabolism   MeSH
• Rats MeSH
• Kidney * diagnostic imaging   metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Organophosphorus Compounds MeSH
• Salivary Glands * diagnostic imaging   metabolism   radiation effects   MeSH
• Tissue Distribution MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The adaptive immune response critically hinges on the functionality of T cell receptors, governed by complex molecular mechanisms, including ubiquitination. In this study, we delved into the role of in T cell immunity, focusing on T cell-B cell conjugate formation and T cell activation. Using a CRISPR-Cas9 screening approach targeting deubiquitinases genes in Jurkat T cells, we identified BAP1 as a key positive regulator of T cell-B cell conjugate formation. Subsequent investigations into BAP1 knockout cells revealed impaired T cell activation, evidenced by decreased MAPK and NF-kB signaling pathways and reduced CD69 expression upon T cell receptor stimulation. Flow cytometry and qPCR analyses demonstrated that BAP1 deficiency leads to decreased surface expression of T cell receptor complex components and reduced mRNA levels of the co-stimulatory molecule CD28. Notably, the observed phenotypes associated with BAP1 knockout are specific to T cells and fully dependent on BAP1 catalytic activity. In-depth RNA-seq and mass spectrometry analyses further revealed that BAP1 deficiency induces broad mRNA and protein expression changes. Overall, our findings elucidate the vital role of BAP1 in T cell biology, especially in T cell-B cell conjugate formation and T cell activation, offering new insights and directions for future research in immune regulation.

• MeSH
• Lymphocyte Activation * immunology   MeSH
• B-Lymphocytes * immunology   metabolism   MeSH
• Jurkat Cells MeSH
• Humans MeSH
• Tumor Suppressor Proteins * metabolism   genetics   MeSH
• Receptors, Antigen, T-Cell * metabolism   MeSH
• Signal Transduction MeSH
• T-Lymphocytes * immunology   metabolism   MeSH
• Ubiquitin Thiolesterase * genetics   metabolism   deficiency   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Immune checkpoint inhibitors (ICIs), including those targeting PD-1, are currently used in a wide range of tumors, but only 20-40% of patients achieve clinical benefit. The objective of our study was to find predictive peripheral blood-based biomarkers for ICI treatment. METHODS: In 41 patients with advanced malignant melanoma (MM) and NSCLC treated with PD-1 inhibitors, we analyzed peripheral blood-based immune subsets by flow cytometry before treatment initialization and the second therapy dose. Specifically, we assessed basic blood differential count, overall T cells and their subgroups, B cells, and myeloid-derived suppressor cells (MDSC). In detail, CD4 + and CD8 + T cells were assessed according to their subtypes, such as central memory T cells (TCM), effector memory T cells (TEM), and naïve T cells (TN). Furthermore, we also evaluated the predictive value of CD28 and ICOS/CD278 co-expression on T cells. RESULTS: Patients who achieved disease control on ICIs had a significantly lower baseline proportion of CD4 + TEM (p = 0.013) and tended to have a higher baseline proportion of CD4 + TCM (p = 0.059). ICI therapy-induced increase in Treg count (p = 0.012) and the proportion of CD4 + TN (p = 0.008) and CD28 + ICOS- T cells (p = 0.012) was associated with disease control. Patients with a high baseline proportion of CD4 + TCM and a low baseline proportion of CD4 + TEM showed significantly longer PFS (p = 0.011, HR 2.6 and p ˂ 0.001, HR 0.23, respectively) and longer OS (p = 0.002, HR 3.75 and p ˂ 0.001, HR 0.15, respectively). Before the second dose, the high proportion of CD28 + ICOS- T cells after ICI therapy initiation was significantly associated with prolonged PFS (p = 0.017, HR 2.51) and OS (p = 0.030, HR 2.69). Also, a high Treg count after 2 weeks of ICI treatment was associated with significantly prolonged PFS (p = 0.016, HR 2.33). CONCLUSION: In summary, our findings suggest that CD4 + TEM and TCM baselines and an early increase in the Treg count induced by PD-1 inhibitors and the proportion of CD28 + ICOS- T cells may be useful in predicting the response in NSCLC and MM patients.

• MeSH
• Inducible T-Cell Co-Stimulator Protein metabolism   MeSH
• Programmed Cell Death 1 Receptor antagonists & inhibitors   MeSH
• CD28 Antigens MeSH
• CD8-Positive T-Lymphocytes immunology   drug effects   metabolism   MeSH
• Adult MeSH
• Immune Checkpoint Inhibitors * therapeutic use   pharmacology   MeSH
• Middle Aged MeSH
• Humans MeSH
• Melanoma * drug therapy   immunology   blood   pathology   MeSH
• Lung Neoplasms * drug therapy   immunology   blood   pathology   MeSH
• Carcinoma, Non-Small-Cell Lung * drug therapy   immunology   blood   pathology   MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

The ApxIVA protein belongs to a distinct class of a "clip and link" activity of Repeat-in-ToXin (RTX) exoproteins. Along with the three other pore-forming RTX toxins (ApxI, ApxII and ApxIII), ApxIVA serves as a major virulence factor of Actinobacillus pleuropneumoniae, the causative agent of porcine pneumonia. The gene encoding ApxIVA is located on a bicistronic operon downstream of the orf1 gene and is expressed exclusively under in vivo conditions. Both ApxIVA and ORF1 are essential for full virulence of A. pleuropneumoniae, but the molecular mechanisms by which they contribute to the pathogenicity are not yet understood. Here, we provide a comprehensive structural and functional analysis of ApxIVA and ORF1 proteins. Our findings reveal that the N-terminal segment of ApxIVA shares structural similarity with colicin M (ColM)-like bacteriocins and exhibits an antimicrobial activity. The ORF1 protein resembles the colicin M immunity protein (Cmi) and, like Cmi, is exported to the periplasm through its N-terminal signal peptide. Additionally, ORF1 can protect bacterial cells from the antimicrobial activity of ApxIVA, suggesting that ORF1 and ApxIVA function as an antibacterial toxin-immunity pair. Moreover, we demonstrate that fetal bovine serum could elicit ApxIVA and ORF1 production under in vitro conditions. These findings highlight the coordinated action of various RTX determinants, where the fine-tuned spatiotemporal production of ApxIVA may enhance the fitness of A. pleuropneumoniae, facilitating its invasion to a resident microbial community on the surface of airway mucosa.

• MeSH
• Actinobacillus pleuropneumoniae * genetics   immunology   MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Proteins * genetics   metabolism   immunology   MeSH
• Bacterial Toxins genetics   metabolism   immunology   MeSH
• Virulence Factors genetics   MeSH
• Actinobacillus Infections microbiology   veterinary   MeSH
• Colicins genetics   metabolism   MeSH
• Operon * MeSH
• Swine MeSH
• Gene Expression Regulation, Bacterial MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH