UNASSIGNED: Asthma, a chronic respiratory disease closely associated with inflammation, presents ongoing treatment challenges. IALLIPF (le-Ala-Leu-Leu-Ile-Pro-Phe) is one of millet prolamins peptides (MPP) which shows anti-oxidant bioactivity by reducing the production of reactive oxygen species (ROS). Tryptophan (Trp, W) is an amino acid that has been demonstrated to possess anti-inflammatory effects. We introduce a novel cathepsin B-activatable bioactive peptides nanocarrier, PEG-IALLIPF-GFLG-W (MPP-Trp), designed for immunotherapy of asthma.
UNASSIGNED: MPP-Trp is synthesized, purified, and its characteristics are investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The yield of nitric oxide (NO) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) are examined to evaluate anti-inflammatory effects of IALLIPF, Trp and MPP-Trp. The immunomodulatory effects of IALLIPF, Trp and MPP-Trp on Th1/Th2 cell populations and cytokines are investigated by flow cytometry, qRT-PCR and ELISA assays. We explore the therapeutic effect of MPP-Trp in the mouse model of asthma by the analysis of lung histology and ELISA. It is necessary to study the biocompatibility of MPP-Trp by CCK8 assay and histopathologic analysis using hematoxylin and eosin (HE) staining.
UNASSIGNED: In asthmatic peripheral blood mononuclear cells (PBMCs), IALLIPF, Trp and MPP-Trp are able to significantly alleviate inflammation by inhibiting the yield of nitric oxide (NO) and pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β), especially MPP-Trp. MPP-Trp significantly upregulates Th1 cell levels while notably reducing Th2 cell levels. Furthermore, MPP-Trp effectively elevates the expression and production of interferon-gamma (IFN-γ), an essential cytokine from Th1 cells. Additionally, MPP-Trp markedly diminishes the mRNA expression and levels of key asthma pathogenesis cytokines, such as interleukin-4 (IL-4), interleukin-13 (IL-13), and interleukin-5 (IL-5), in asthma PBMCs. MPP-Trp ameliorates pulmonary pathological alterations and significantly inhibits OVA-induced inflammation in mice with asthma. It has little influence on the cell viability in Asthma-PBMCs treated with various concentrations or durations of MPP-Trp. No pathological changes, including in the heart, liver, spleen, lung, and kidney tissues, are observed in non-sensitized and non-challenged mice treated with MPP-Trp (20 mg/kg).
UNASSIGNED: Our research demonstrates that MPP-Trp has immunomodulatory effects on Th1/Th2 cell populations, essential in managing asthma. It considerably alleviates OVA-induced asthma by shifting the immune response towards a Th1-dominant profile, thereby reducing Th2-driven inflammation. Therefore, this novel bioactive peptide nanocarrier, MPP-Trp, holds promise as a candidate for asthma immunotherapy.

BACKGROUND: Cellular angiofibroma, a rare benign mesenchymal neoplasm, is classified within the 13q/RB1 family of tumors due to morphological, immunohistochemical, and genetic similarities with spindle cell lipoma. Here, genetic data reveal pathogenetic heterogeneity in cellular angiofibroma.
METHODS: Three cellular angiofibromas were studied using G-banding/Karyotyping, array comparative genomic hybridization, RNA sequencing, and direct cycling sequencing.
RESULTS: The first tumor carried a del(13)(q12) together with heterozygous loss and minimal expression of the RB1 gene. Tumors two and three displayed chromosome 8 abnormalities associated with chimeras of the pleomorphic adenoma gene 1 (PLAG1). In tumor 2, the cathepsin B (CTSB) fused to PLAG1 (CTSB::PLAG1) while in tumor 3, the mir-99a-let-7c cluster host gene (MIR99AHG) fused to PLAG1 (MIR99AHG::PLAG1), both leading to elevated expression of PLAG1 and insulin growth factor 2.
CONCLUSIONS: This study uncovers two genetic pathways contributing to the pathogenetic heterogeneity within cellular angiofibromas. The first aligns with the 13q/RB1 family of tumors and the second involves PLAG1-chimeras. These findings highlight the diverse genetic landscape of cellular angiofibromas, providing insights into potential diagnostic strategies.

Glioblastoma (GBM) is the most prevalent and aggressive malignant primary brain tumor. GBM proximal to the lateral ventricles (LVs) is more aggressive, potentially because of subventricular zone contact. Despite this, cross-talk between GBM and neural stem/progenitor cells (NSC/NPCs) is not well understood. Using cell-specific proteomics, we show that LV-proximal GBM prevents neuronal maturation of NSCs through induction of senescence. In addition, GBM brain tumor-initiating cells (BTICs) increase expression of cathepsin B (CTSB) upon interaction with NPCs. Lentiviral knockdown and recombinant protein experiments reveal that both cell-intrinsic and soluble CTSB promote malignancy-associated phenotypes in BTICs. Soluble CTSB stalls neuronal maturation in NPCs while promoting senescence, providing a link between LV-tumor proximity and neurogenesis disruption. Last, we show LV-proximal CTSB up-regulation in patients, showing the relevance of this cross-talk in human GBM biology. These results demonstrate the value of proteomic analysis in tumor microenvironment research and provide direction for new therapeutic strategies in GBM.

Serine protease inhibitors (serpins) constitute the largest family of protease inhibitors expressed in humans, but their role in infection remains largely unexplored. In infected macrophages, the mycobacterial ESX-1 type VII secretion system permeabilizes internal host membranes and causes leakage into the cytosol of host DNA, which induces type I interferon (IFN) production via the cyclic GMP-AMP synthase (cGAS) and stimulator of IFN genes (STING) surveillance pathway, and promotes infection in vivo. Using the Mycobacterium marinum infection model, we show that ESX-1-mediated type I IFN signaling in macrophages selectively induces the expression of serpina3f and serpina3g, two cytosolic serpins of the clade A3. The membranolytic activity of ESX-1 also caused leakage of cathepsin B into the cytosol where it promoted cell death, suggesting that the induction of type I IFN comes at the cost of lysosomal rupture and toxicity. However, the production of cytosolic serpins suppressed the protease activity of cathepsin B in this compartment and thus limited cell death, a function that was associated with increased bacterial growth in infected mice. These results suggest that cytosolic serpins act in a type I IFN-dependent cytoprotective feedback loop to counteract the inevitable toxic effect of ESX-1-mediated host membrane rupture.
OBJECTIVE: The ESX-1 type VII secretion system is a key virulence determinant of pathogenic mycobacteria. The ability to permeabilize host cell membranes is critical for several ESX-1-dependent virulence traits, including phagosomal escape and induction of the type I interferon (IFN) response. We find that it comes at the cost of lysosomal leakage and subsequent host cell death. However, our results suggest that ESX-1-mediated type I IFN signaling selectively upregulates serpina3f and serpina3g and that these cytosolic serpins limit cell death caused by cathepsin B that has leaked into the cytosol, a function that is associated with increased bacterial growth in vivo. The ability to rupture host membranes is widespread among bacterial pathogens, and it will be of interest to evaluate the role of cytosolic serpins and this type I IFN-dependent cytoprotective feedback loop in the context of human infection.

Milk-derived peptides and milk fat globule membrane (MFGM) have gained interest as health-promoting food ingredients. However, the mechanisms by which these nutraceuticals modulate the function of biological systems often remain unclear. We utilized Caenorhabditis elegans to elucidate how MFGM-containing protein powder (MProPow), previously used in a clinical trial, affect the physiology of this model organism. Our results demonstrate that MProPow does not affect lifespan but promotes the fitness of the animals. Surprisingly, gene expression analysis revealed that MProPow decreases the expression of genes functioning on innate immunity, which also translates into reduced survival on pathogenic bacteria. One of the innate immunity-associated genes showing reduced expression upon MProPow supplementation is cpr-3, the homolog of human cathepsin B. Interestingly, knockdown of cpr-3 enhances fitness, but not in MProPow-treated animals, suggesting that MProPow contributes to fitness by downregulating the expression of this gene. In summary, this research highlights the value of C. elegans in testing the biological activity of food supplements and nutraceuticals. Furthermore, this study should encourage investigations into whether milk-derived peptides and MFGM mediate their beneficial effects through the modulation of cathepsin B expression in humans.

Traumatic brain injury (TBI) affects millions of people each year. Previous studies using the central fluid percussion injury (CFPI) model in adult male rats indicated that elevated intracranial pressure (ICP) was associated with long-term effects, including neuronal cell loss and increased sensory sensitivity post-injury and secondary ICP elevation, which were not seen following injury alone. Investigations also indicated that cathepsin B (Cath B), a lysosomal cysteine protease, may play a role in the pathological progression of neuronal membrane disruption; however, the specific impact of Cath B inhibition following CFPI remained unknown. Thus, the focus of this study was to evaluate the effects of Cath B inhibition via the intracerebroventricular infusion of the Cath B inhibitor to the CA-074 methyl ester (CA-074Me) 2w following injury with or without secondary ICP elevation. This was accomplished using adult male rats continuously infused with CA-074Me or 10% DMSO as a vehicle control for 2w following either sham injury, CFPI only, or CFPI with subsequent ICP elevation to 20 mmHg. We assessed Cath B activity and evaluated the protein levels of Cath B and Cath B-binding partners AIF, Bcl-XL, and Bak. We also conducted histological analyses of the total cell counts to assess for cell loss, membrane disruption, and Cath B localization. Finally, we investigated somatosensory changes with the whisker nuisance task. Overall, this study demonstrated that Cath B is not a direct driver of membrane disruption; however, the administration of CA-074Me alters Cath B localization and reduces hypersensitivity, emphasizing Cath B as an important component in late secondary pathologies.

Targeting the programmed cell death-1/ligand 1 (PD-1/PD-L1) pathway is one of the most promising cancer treatment strategies. Studies have shown that HDAC inhibitors can enhance the antitumor immune response by modulating the expression of PD-L1. Herein, we designed and synthesized a series of novel hydrazide-based small molecule HDAC inhibitors; among them, compound HQ-30 showed selective HDAC3 inhibition (IC50 = 89 nM) and remarkable PD-L1-degrading activity (DC50 = 5.7 μM, Dmax = 80% at 10 μM). Further studies revealed that HQ-30 induced the degradation of PD-L1 by regulating cathepsin B (CTSB) in the lysosomes. Further, HQ-30 could enhance the infiltration of CD3+ CD4+ helper T and CD3+ CD8+ cytotoxic T cells in tumors, thus activating the tumor immune microenvironment. Moreover, HQ-30 possessed a benign toxicity profile (LD50 > 1000 mg/kg) and favorable pharmacokinetic properties (F = 57%). Taken together, HQ-30 is worthy of further investigation as a small molecule-based epigenetic modulator of tumor immunotherapy.

Colorectal cancer (CRC) represents a substantial burden on global healthcare, contributing to significant morbidity and mortality worldwide. Despite advances in screening methodologies, its incidence remains high, necessitating continued efforts in early detection and treatment. Neoplastic invasion and metastasis are primary determinants of CRC lethality, emphasizing the urgency of understanding underlying mechanisms to develop effective therapeutic strategies. This study aimed to explore the potential of serum biomarkers in predicting survival outcomes in CRC patients, with a focus on cathepsin B (CB), leukocytic elastase (LE), total sialic acid (TSA), lipid-associated sialic acid (LASA), antitrypsin activity (ATA), C-reactive protein (CRP), and cystatin C (CC). We recruited 185 CRC patients and 35 healthy controls, assessing demographic variables, tumor characteristics, and 7 serum biomarker levels, including (1) CB, (2) LE, (3) TSA, (4) LASA, (5) ATA, (6) CRP, and (7) CC. Statistical analyses included ANOVA with Tukey\'s post hoc tests and MANOVA for continuous variables. Student\'s t-test was used for dependent samples, while non-parametric tests like Mann-Whitney U and Wilcoxon signed-rank tests were applied for variables deviating from the normal distribution. Categorical variables were assessed using chi-square and Kruskal-Wallis tests. Spearman\'s rank correlation coefficient was utilized to examine variable correlations. Survival analysis employed the Kaplan-Meier method with a log-rank test for comparing survival times between groups. Significant associations were observed between CB (p = 0.04), LE (p = 0.01), and TSA (p = 0.008) levels and survival outcomes in CRC patients. Dukes\' classification stages also showed a significant correlation with survival (p = 0.001). However, no significant associations were found for LASA, ATA, CRP, and CC. Multivariate analysis of LE, TSA, and ATA demonstrated a notable correlation with survival (p = 0.041), notwithstanding ATA\'s lack of significance in univariate analysis (p = 0.13). CB, LE, and TSA emerged as promising diagnostic markers with prognostic value in CRC, potentially aiding in early diagnosis and treatment planning. Further research is needed to validate these findings and explore additional prognostic indicators.

Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus\'s internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro. When HE25 was administered in vivo, either by intravenous injection or inhalation, it accumulated in lungs, veins and arteries, endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro. The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases.

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73-79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA\'s and reduced MdCSTA\'s inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat\'s stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.