The aim of this research was to determine the impact of heat stress on cell differentiation in an equine mesenchymal stem cell model (EMSC) through the application of heat stress to primary EMSCs as they progressed through the cell specialization process. A proteomic analysis was performed using mass spectrometry to compare relative protein abundances among the proteomes of three cell types: progenitor EMSCs and differentiated osteoblasts and adipocytes, maintained at 37 °C and 42 °C during the process of cell differentiation. A cell-type and temperature-specific response to heat stress was observed, and many of the specific differentially expressed proteins were involved in cell-signaling pathways such as Notch and Wnt signaling, which are known to regulate cellular development. Furthermore, cytoskeletal proteins profilin, DSTN, SPECC1, and DAAM2 showed increased protein levels in osteoblasts differentiated at 42 °C as compared with 37 °C, and these cells, while they appeared to accumulate calcium, did not organize into a whorl agglomerate as is typically seen at physiological temperatures. This altered proteome composition observed suggests that heat stress could have long-term impacts on cellular development. We propose that this in vitro stem cell culture model of cell differentiation is useful for investigating molecular mechanisms that impact cell development in response to stressors. Full article

Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) and feature the threaded, lariat knot-like topology. The basic post-translational modifications (PTMs) of lasso peptide contain two steps, including the leader peptide removal of the ribosome-derived linear precursor peptide by an ATP-dependent cysteine protease, and the macrolactam cyclization by an ATP-dependent macrolactam synthetase. Recently, advanced bioinformatic tools combined with genome mining have paved the way to uncover a rapidly growing number of lasso peptides as well as a series of PTMs other than the general class-defining processes. Despite abundant reviews focusing on lasso peptide discoveries, structures, properties, and physiological functionalities, few summaries concerned their unique PTMs. In this review, we summarized all the unique PTMs of lasso peptides uncovered to date, shedding light on the related investigations in the future. Full article

Deoxynivalenol (DON) is a secondary fungal metabolite that is associated with many adverse toxicological effects in agriculture as well as human/animal nutrition. Bioremediation efforts in recent years have led to the discovery of numerous bacterial isolates that can transform DON to less toxic derivatives. Both 3-keto-DON and 3-epi-DON were recently shown to exhibit reduced toxicity, compared to DON, when tested using different cell lines and mammalian models. In the current study, the toxicological assessment of 3-keto-DON and 3-epi-DON using in planta models surprisingly revealed that 3-keto-DON, but not 3-epi-DON, retained its toxicity to a large extent in both duckweeds (Lemna minor L.) and common wheat (Triticum aestivum L.) model systems. RNA-Seq analysis revealed that the exposure of L. minor to 3-keto-DON and DON resulted in substantial transcriptomic changes and similar gene expression profiles, whereas 3-epi-DON did not. These novel findings are pivotal for understanding the environmental burden of the above metabolites as well as informing the development of future transgenic plant applications. Collectively, they emphasize the fundamental need to assess both plant and animal models when evaluating metabolites/host interactions. Full article

The components of metabolic syndrome (MetS) and hepatogastrointestinal diseases are widespread worldwide, since many factors associated with lifestyle and diet influence their development and correlation. Due to these growing health problems, it is necessary to search for effective alternatives for prevention or adjuvants in treating them. The positive impact of regulated microbiota on health is known; however, states of dysbiosis are closely related to the development of the conditions mentioned above. Therefore, the role of prebiotics, probiotics, or symbiotic complexes has been extensively evaluated; the results are favorable, showing that they play a crucial role in the regulation of the immune system, the metabolism of carbohydrates and lipids, and the biotransformation of bile acids, as well as the modulation of their central receptors FXR and TGR-5, which also have essential immunomodulatory and metabolic activities. It has also been observed that they can benefit the host by displacing pathogenic species, improving the dysbiosis state in MetS. Current studies have reported that paraprobiotics (dead or inactive probiotics) or postbiotics (metabolites generated by active probiotics) also benefit hepatogastrointestinal health. Full article

Renal cell carcinoma is a prevalent disease associated with high morbidity and mortality rates. Partial nephrectomy is a first-line surgical option because it allows the preservation of renal function. Clear differentiation between normal and cancerous tissues is critical for increasing the negative margin rates. This study investigated the capability of the near-infrared (NIR) fluorescent imaging agent ASP5354 for in vivo fluorescence imaging of renal cell carcinoma. ASP5354 at a single dose of 12 nmol (0.037 mg)/kg body weight was intravenously administered to healthy and orthotopic renal cell carcinoma mice under anesthesia. NIR images of the abdominal cavity were obtained using a near-infrared fluorescence (NIRF) camera system. In addition, the cancerous kidneys were harvested, and the NIRF in their sections was measured using an NIRF microscope. Normal renal tissue emitted strong NIRF but the cancer tissue did not. The difference in NIRF intensity between the normal and cancer tissues clearly presented the boundary between the normal and cancer tissues in macro and micro NIRF imaging. ASP5354 can distinguish cancer tissue from normal tissue using NIRF. Thus, ASP5354 is a promising agent for renal cell carcinoma tissue imaging in partial nephrectomy for renal cell carcinoma patients. Full article

Cisplatin is a platinum-based chemotherapeutic that has long since been effective against a variety of solid-cancers, substantially improving the five-year survival rates for cancer patients. Its use has also historically been limited by its adverse drug reactions, or cisplatin-induced toxicities (CITs). Of these reactions, cisplatin-induced nephrotoxicity (CIN), cisplatin-induced peripheral neuropathy (CIPN), and cisplatin-induced ototoxicity (CIO) are the three most common of several CITs recognised thus far. While the anti-cancer activity of cisplatin is well understood, the mechanisms driving its toxicities have only begun to be defined. Most of the literature pertains to damage caused by oxidative stress that occurs downstream of cisplatin treatment, but recent evidence suggests that the instigator of CIT development is inflammation. Cisplatin has been shown to induce pro-inflammatory signalling in CIN, CIPN, and CIO, all of which are associated with persisting markers of inflammation, particularly from the innate immune system. This review covered the hallmarks of inflammation common and distinct between different CITs, the role of innate immune components in development of CITs, as well as current treatments targeting pro-inflammatory signalling pathways to conserve the use of cisplatin in chemotherapy and improve long-term health outcomes of cancer patients. Full article

In inflammatory diseases, polymorphonuclear neutrophils (PMNs) are known to produce elevated levels of pro-inflammatory cytokines and proteases. To limit ensuing exacerbated cell responses and tissue damage, novel therapeutic agents are sought. 4aa and 4ba, two pyridazinone-scaffold-based phosphodiesterase-IV inhibitors are compared in vitro to zardaverine for their ability to: (1) modulate production of pro-inflammatory mediators, reactive oxygen species (ROS), and phagocytosis; (2) modulate degranulation by PMNs after transepithelial lung migration. Compound 4ba and zardaverine were tested in vivo for their ability to limit tissue recruitment of PMNs in a murine air pouch model. In vitro treatment of lipopolysaccharide-stimulated PMNs with compounds 4aa and 4ba inhibited the release of interleukin-8, tumor necrosis factor-α, and matrix metalloproteinase-9. PMNs phagocytic ability, but not ROS production, was reduced following treatment. Using a lung inflammation model, we proved that PMNs transmigration led to reduced expression of the CD16 phagocytic receptor, which was significantly blunted after treatment with compound 4ba or zardaverine. Using the murine air pouch model, LPS-induced PMNs recruitment was significantly decreased upon addition of compound 4ba or zardaverine. Our data suggest that new pyridazinone derivatives have therapeutic potential in inflammatory diseases by limiting tissue recruitment and activation of PMNs. Full article

Bacterial biofilm formation is a major cause of drug resistance and bacterial persistence; thus, controlling pathogenic biofilms is an important component of strategies targeting infectious bacterial diseases. Cinnamaldehyde (CNMA) has broad-spectrum antimicrobial and antibiofilm activities. In this study, we investigated the antibiofilm effects of ten CNMA derivatives and trans-CNMA against Gram-negative uropathogenic Escherichia coli (UPEC) and Gram-positive Staphylococcus aureus. Among the CNMA analogs tested, 4-nitrocinnamaldehyde (4-nitroCNMA) showed antibacterial and antibiofilm activities against UPEC and S. aureus with minimum inhibitory concentrations (MICs) for cell growth of 100 µg/mL, which were much more active than those of trans-CNMA. 4-NitroCNMA inhibited UPEC swimming motility, and both trans-CNMA and 4-nitroCNMA reduced extracellular polymeric substance production by UPEC. Furthermore, 4-nitroCNMA inhibited the formation of mixed UPEC/S. aureus biofilms. Collectively, our observations indicate that trans-CNMA and 4-nitroCNMA potently inhibit biofilm formation by UPEC and S. aureus. We suggest efforts be made to determine the therapeutic scope of CNMA analogs, as our results suggest CNMA derivatives have potential therapeutic use for biofilm-associated diseases. Full article

Circulating DNA has already proven itself as a valuable tool in translational medicine. However, one of the overlooked areas of circulating DNA research is its association with different proteins, despite considerable evidence that this association might impact DNA’s fate in circulation and its biological role. In this review, we attempt to shed light on current ideas about circulating DNA origins and forms of circulation, known biological effects, and the clinical potential of circulating tumor deoxyribonucleoprotein complexes. Full article

Systemic Sclerosis (SSc) is a chronic autoimmune disease with high morbidity and mortality. Autologous Hematopoietic Stem Cell Transplantation (AHSCT) is the best therapeutic option for rapidly progressive SSc, allowing increased survival with regression of skin and lung fibrosis. The immune determinants of the clinical response after AHSCT have yet to be well characterized. In particular, the pivotal role of the Human Leukocyte Antigen (HLA) system is not well understood, including the role of non-classical immuno-modulatory HLA-E and HLA-G molecules in developing tolerance and the role of Natural Killer cells (NK) in the immunomodulation processes. We retrospectively tested whether the genetic and/or circulating expression of the non-classical HLA-E and HLA-G loci, as well as the imputed classical HLA determinants of HLA-E expression, influence the observed clinical response to AHSCT at 12- and 24-month follow-up. In a phenotypically well-defined sample of 46 SSc patients classified as clinical responders or non-responders, we performed HLA genotyping using next-generation sequencing and circulating levels of HLA-G and quantified HLA-E soluble isoforms by ELISA. The -21HLA-B leader peptide dimorphism and the differential expression level of HLA-A and HLA-C alleles were imputed. We observed a strong trend towards better clinical response in HLA-E*01:03 or HLA-G 14bp Del allele carriers, which are known to be associated with high expression of the corresponding molecules. At 12-month post-AHSCT follow-up, higher circulating levels of soluble HLA-E were associated with higher values of modified Rodnan Skin Score (mRSS) (p = 0.0275), a proxy of disease severity. In the non-responder group, the majority of patients carried a double dose of the HLA-B Threonine leader peptide, suggesting a non-efficient inhibitory effect of the HLA-E molecules. We did not find any correlation between the soluble HLA-G levels and the observed clinical response after AHSCT. High imputed expression levels of HLA-C alleles, reflecting more efficient NK cell inhibition, correlated with low values of the mRSS 3 months after AHSCT (p = 0.0087). This first pilot analysis of HLA-E and HLA-G immuno-modulatory molecules suggests that efficient inhibition of NK cells contributes to clinical response after AHSCT for SSc. Further studies are warranted in larger patient cohorts to confirm our results. Full article

The scenario of neoadjuvant and adjuvant settings in non-small cell lung cancer (NSCLC) is rapidly evolving. As already happened for the advanced disease, also early stages have entered the era of precision medicine, with molecular analysis and Programmed death-ligand 1 (PD-L1) evaluation that by now can be considered a routine assessment. New treatment options have been recently approved, with osimertinib now part of clinical practice for Epidermal Growth Factor Receptor mutated (EGFRm) patients, and immune checkpoint inhibitors (ICIs) available after FDA approval both in the adjuvant (atezolizumab) and neoadjuvant (nivolumab) setting. No mature data on overall survival benefits are available yet, though. Several clinical trials with specific-tyrosine kinase inhibitors (TKIs) and ICIs are currently ongoing, both with and without concomitant chemotherapy. As therapeutic strategies are rapidly expanding, quite a few questions remain unsettled, such as the optimal duration of adjuvant targeted therapy or the effective benefit of ICIs in early-stage EGFRm or ALK (Anaplastic Lymphoma Kinase) rearranged patients, or the possibility to individuate high-risk patients after surgical resection assessing minimal residual disease (MRD) by ctDNA evaluation. We hereby report already available literature data and summarize ongoing trials with targeted therapy and immunotherapy in early-stage NSCLC, focusing on practice-changing results and new perspectives for potentially cured patients. Full article

Neurofilament light chain (Nf-L) is a well-known biomarker for axonal damage; however, the corresponding circulating Nf-L analyte in cerebrospinal fluid (CSF) is poorly characterized. We therefore isolated new monoclonal antibodies against synthetic peptides, and these monoclonals were characterized for their specificity on brain-specific intermediate filament proteins. Two highly specific antibodies, ADx206 and ADx209, were analytically validated for CSF applications according to well-established criteria. Interestingly, using three different sources of purified Nf-L proteins, a significant impact on interpolated concentrations was observed. With a lower limit of analytical sensitivity of 100 pg/mL using bovine Nf-L as the calibrator, we were able to quantify the Nf-L analyte in each sample, and these Nf-L concentrations were highly correlated to the Uman diagnostics assay (Spearman rho = 0.97, p < 0.001). In the clinical diagnostic groups, the new Nf-L ELISA could discriminate patients with Alzheimer’s disease (AD, n = 20) from those with frontotemporal lobe dementia (FTD, n = 20) and control samples with subjective cognitive decline (SCD, n = 20). Henceforth, this novel Nf-L ELISA with well-defined specificity and epitopes can be used to enhance our understanding of harmonizing the use of Nf-L as a clinically relevant marker for neurodegeneration in CSF. Full article

Salmonella Typhimurium is an invasive enteric pathogen that causes gastroenteritis in humans and life-threatening systemic infections in mice. During infection of the intestine, S. Typhimurium can exploit nitrate as an electron acceptor to enhance its growth. However, the roles of nitrate on S. Typhimurium systemic infection are unknown. In this study, nitrate levels were found to be significantly increased in the liver and spleen of mice systemically infected by S. Typhimurium. Mutations in genes encoding nitrate transmembrane transporter (narK) or nitrate-producing flavohemoprotein (hmpA) decreased the replication of S. Typhimurium in macrophages and reduced systemic infection in vivo, suggesting that nitrate utilization promotes S. Typhimurium systemic virulence. Moreover, nitrate utilization contributes to the acidification of the S. Typhimurium cytoplasm, which can sustain the virulence of S. Typhimurium by increasing the transcription of virulence genes encoding on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the growth advantage of S. Typhimurium conferred by nitrate utilization occurred only under low-oxygen conditions, and the nitrate utilization was activated by both the global regulator Fnr and the nitrate-sensing two-component system NarX-NarL. Collectively, this study revealed a novel mechanism adopted by Salmonella to interact with its host and increase its virulence. Full article

We studied SARS-CoV-2-specific T cell responses in 22 subacute MIS-C children enrolled in 2021 and 2022 using peptide pools derived from SARS-CoV-2 spike or nonspike proteins. CD4+ and CD8+ SARS-CoV-2-specific T cells were detected in 5 subjects, CD4+ T helper (Th) responses alone were detected in 12 subjects, and CD8+ cytotoxic T cell (CTL) responses alone were documented in 1 subject. Notably, a sizeable subpopulation of CD4− CD8− double-negative (DN) T cells out of total CD3+ T cells was observed in MIS-C (median: 14.5%; IQR 8.65–25.3) and recognized SARS-CoV-2 peptides. T cells bearing the Vβ21.3 T cell receptor (TcRs), previously reported as pathogenic in the context of MIS-C, were detected in high frequencies, namely, in 2.8% and 3.9% of the CD4+ and CD8+ T cells, respectively. However, Vβ21.3 CD8+ T cells that responded to SARS-CoV-2 peptides were detected in only a single subject, suggesting recognition of nonviral antigens in the majority of subjects. Subjects studied 6–14 months after MIS-C showed T cell epitope spreading, meaning the activation of T cells that recognize more SARS-CoV-2 peptides following the initial expansion of T cells that see immunodominant epitopes. For example, subjects that did not recognize nonspike proteins in the subacute phase of MIS-C showed good Th response to nonspike peptides, and/or CD8+ T cell responses not appreciable before arose over time and could be detected in the 6–14 months’ follow-up. The magnitude of the Th and CTL responses also increased over time. In summary, patients with MIS-C associated with acute lymphopenia, a classical feature of MIS-C, showed a physiological response to the virus with a prominent role for virus-specific DN T cells. Full article