International Journal of Molecular Sciences

The review presents extensive data (from the works of the author and literature) on the structure of C- and N-chlorophosphorylated enamines and the related heterocycles obtained by multipulse multinuclear ¹H, ¹³C, and ³¹P NMR spectroscopy. The use of phosphorus pentachloride as a phosphorylating agent for functional enamines enables the synthesis of various C- and N-phosphorylated products that are heterocyclized to form various promising nitrogen- and phosphorus-containing heterocyclic systems. ³¹P NMR spectroscopy is the most convenient, reliable and unambiguous method for the study and identification of organophosphorus compounds with different coordination numbers of the phosphorus atom, as well as for the determination of their Z- and E-isomeric forms. An alteration of the coordination number of the phosphorus atom in the phosphorylated compounds from 3 to 6 leads to a drastic screening of the ³¹P nucleus from about +200 to −300 ppm. The unique structural features of nitrogen–phosphorus-containing heterocyclic compounds are discussed. Full article

A [3 + 2] cycloaddition reaction using dialkyne and diazide comonomers, both bearing explosophoric groups, to synthesize energetic polymers containing furazan and 1,2,3-triazole ring as well as nitramine group in the polymer chain have been described. The developed solvent- and catalyst-free approach is methodologically simple and effective, the comonomers used are easily available, and the resulting polymer does not need any purification. All this makes it a promising tool for the synthesis of energetic polymers. The protocol was utilized to generate multigram quantities of the target polymer, which has been comprehensively investigated. The resulting polymer was fully characterized by spectral and physico-chemical methods. Compatibility with energetic plasticizers, thermochemical characteristics, and combustion features indicate the prospects of this polymer as a binder base for energetic materials. The polymer of this study surpasses the benchmark energetic polymer, nitrocellulose (NC), in a number of properties. Full article

Colorectal cancer (CRC) is one of the most lethal malignancies worldwide, so the attempts to find novel therapeutic approaches are necessary. The aim of our study was to analyze how chemical modifications influence physical, chemical, and biological properties of the two peptides, namely, bradykinin (BK) and neurotensin (NT). For this purpose, we used fourteen modified peptides, and their anti-cancers features were analyzed on the HCT116 CRC cell line. Our results confirmed that the spherical mode of a CRC cell line culture better reflects the natural tumour microenvironment. We observed that the size of the colonospheres was markedly reduced following treatment with some BK and NT analogues. The proportion of CD133+ cancer stem cells (CSCs) in colonospheres decreased following incubation with the aforementioned peptides. In our research, we found two groups of these peptides. The first group influenced all the analyzed cellular features, while the second seemed to include the most promising peptides that lowered the count of CD133+ CSCs with parallel substantial reduction in CRC cells viability. These analogues need further analysis to uncover their overall anti-cancer potential. Full article

Monocarboxylate transporter 8 (MCT8) and organic anion-transporting polypeptide 1C1 (OATP1C1) are thyroid hormone (TH) transmembrane transporters relevant for the availability of TH in neural cells, crucial for their proper development and function. Mutations in MCT8 or OATP1C1 result in severe disorders with dramatic movement disability related to alterations in basal ganglia motor circuits. Mapping the expression of MCT8/OATP1C1 in those circuits is necessary to explain their involvement in motor control. We studied the distribution of both transporters in the neuronal subpopulations that configure the direct and indirect basal ganglia motor circuits using immunohistochemistry and double/multiple labeling immunofluorescence for TH transporters and neuronal biomarkers. We found their expression in the medium-sized spiny neurons of the striatum (the receptor neurons of the corticostriatal pathway) and in various types of its local microcircuitry interneurons, including the cholinergic. We also demonstrate the presence of both transporters in projection neurons of intrinsic and output nuclei of the basal ganglia, motor thalamus and nucleus basalis of Meynert, suggesting an important role of MCT8/OATP1C1 for modulating the motor system. Our findings suggest that a lack of function of these transporters in the basal ganglia circuits would significantly impact motor system modulation, leading to clinically severe movement impairment. Full article

In the modern scientific landscape, natriuretic peptides are a complex and interesting network of molecules playing pleiotropic effects on many organs and tissues, ensuring the maintenance of homeostasis mainly in the cardiovascular system and regulating the water–salt balance. The characterization of their receptors, the understanding of the molecular mechanisms through which they exert their action, and the discovery of new peptides in the last period have made it possible to increasingly feature the physiological and pathophysiological role of the members of this family, also allowing to hypothesize the possible settings for using these molecules for therapeutic purposes. This literature review traces the history of the discovery and characterization of the key players among the natriuretic peptides, the scientific trials performed to ascertain their physiological role, and the applications of this knowledge in the clinical field, leaving a glimpse of new and exciting possibilities for their use in the treatment of diseases. Full article

Macrophages constitute important immune cell targets of the antifolate methotrexate (MTX) in autoimmune diseases, including rheumatoid arthritis. Regulation of folate/MTX metabolism remains poorly understood upon pro-inflammatory (M1-type/GM-CSF-polarized) and anti-inflammatory (M2-type/M-CSF-polarized) macrophages. MTX activity strictly relies on the folylpolyglutamate synthetase (FPGS) dependent intracellular conversion and hence retention to MTX-polyglutamate (MTX-PG) forms. Here, we determined FPGS pre-mRNA splicing, FPGS enzyme activity and MTX-polyglutamylation in human monocyte-derived M1- and M2-macrophages exposed to 50 nmol/L MTX ex vivo. Moreover, RNA-sequencing analysis was used to investigate global splicing profiles and differential gene expression in monocytic and MTX-exposed macrophages. Monocytes displayed six–eight-fold higher ratios of alternatively-spliced/wild type FPGS transcripts than M1- and M2-macrophages. These ratios were inversely associated with a six–ten-fold increase in FPGS activity in M1- and M2-macrophages versus monocytes. Total MTX-PG accumulation was four-fold higher in M1- versus M2-macrophages. Differential splicing after MTX-exposure was particularly apparent in M2-macrophages for histone methylation/modification genes. MTX predominantly induced differential gene expression in M1-macrophages, involving folate metabolic pathway genes, signaling pathways, chemokines/cytokines and energy metabolism. Collectively, macrophage polarization-related differences in folate/MTX metabolism and downstream pathways at the level of pre-mRNA splicing and gene expression may account for variable accumulation of MTX-PGs, hence possibly impacting MTX treatment efficacy. Full article

Besides being a marker of kidney disease severity, albuminuria exerts a toxic effect on renal proximal tubular epithelial cells (RPTECs). We evaluated whether an unfolded protein response (UPR) or DNA damage response (DDR) is elicited in RPTECs exposed to high albumin concentration. The deleterious outcomes of the above pathways, apoptosis, senescence, or epithelial-to-mesenchymal transition (EMT) were evaluated. Albumin caused reactive oxygen species (ROS) overproduction and protein modification, and a UPR assessed the level of crucial molecules involved in this pathway. ROS also induced a DDR evaluated by critical molecules involved in this pathway. Apoptosis ensued through the extrinsic pathway. Senescence also occurred, and the RPTECs acquired a senescence-associated secretory phenotype since they overproduced IL-1β and TGF-β1. The latter may contribute to the observed EMT. Agents against endoplasmic reticulum stress (ERS) only partially alleviated the above changes, while the inhibition of ROS upregulation prevented both UPR and DDR and all the subsequent harmful effects. Briefly, albumin overload causes cellular apoptosis, senescence, and EMT in RPTECs by triggering UPR and DDR. Promising anti-ERS factors are beneficial but cannot eliminate the albumin-induced deleterious effects because DDR also occurs. Factors that suppress ROS overproduction may be more effective since they could halt UPR and DDR. Full article

Antimicrobial fatty acids derived from natural sources and renewable feedstocks are promising surface-active substances with a wide range of applications. Their ability to target bacterial membrane in multiple mechanisms offers a promising antimicrobial approach for combating bacterial infections and preventing the development of drug-resistant strains, and it provides a sustainable strategy that aligns with growing environmental awareness compared to their synthetic counterparts. However, the interaction and destabilization of bacterial cell membranes by these amphiphilic compounds are not yet fully understood. Here, we investigated the concentration-dependent and time-dependent membrane interaction between long-chain unsaturated fatty acids—linolenic acid (LNA, C18:3), linoleic (LLA, C18:2), and oleic acid (OA, C18:1)—and the supported lipid bilayers (SLBs) using quartz crystal microbalance-dissipation (QCM-D) and fluorescence microscopy. We first determined the critical micelle concentration (CMC) of each compound using a fluorescence spectrophotometer and monitored the membrane interaction in real time following fatty acid treatment, whereby all micellar fatty acids elicited membrane-active behavior primarily above their respective CMC values. Specifically, LNA and LLA, which have higher degrees of unsaturation and CMC values of 160 µM and 60 µM, respectively, caused significant changes in the membrane with net |Δf| shifts of 23.2 ± 0.8 Hz and 21.4 ± 0.6 Hz and ΔD shifts of 5.2 ± 0.5 × 10⁻⁶ and 7.4 ± 0.5 × 10⁻⁶. On the other hand, OA, with the lowest unsaturation degree and CMC value of 20 µM, produced relatively less membrane change with a net |Δf| shift of 14.6 ± 2.2 Hz and ΔD shift of 8.8 ± 0.2 × 10⁻⁶. Both LNA and LLA required higher concentrations than OA to initiate membrane remodeling as their CMC values increased with the degree of unsaturation. Upon incubating with fluorescence-labeled model membranes, the fatty acids induced tubular morphological changes at concentrations above CMC. Taken together, our findings highlight the critical role of self-aggregation properties and the degree of unsaturated bonds in unsaturated long-chain fatty acids upon modulating membrane destabilization, suggesting potential applications in developing sustainable and effective antimicrobial strategies. Full article

Alfalfa (Medicago sativa) is an important leguminous forage, known as the “The Queen of Forages”. Abiotic stress seriously limits the growth and development of alfalfa, and improving the yield and quality has become an important research area. However, little is known about the Msr (methionine sulfoxide reductase) gene family in alfalfa. In this study, 15 Msr genes were identified through examining the genome of the alfalfa “Xinjiang DaYe”. The MsMsr genes differ in gene structure and conserved protein motifs. Many cis-acting regulatory elements related to the stress response were found in the promoter regions of these genes. In addition, a transcriptional analysis and qRT-PCR (quantitative reverse transcription PCR) showed that MsMsr genes show expression changes in response to abiotic stress in various tissues. Overall, our results suggest that MsMsr genes play an important role in the response to abiotic stress for alfalfa. Full article

Neurodegeneration is a multifactorial process that involves multiple mechanisms. Examples of neurodegenerative diseases are Parkinson’s disease, multiple sclerosis, Alzheimer’s disease, prion diseases such as Creutzfeldt–Jakob’s disease, and amyotrophic lateral sclerosis. These are progressive and irreversible pathologies, characterized by neuron vulnerability, loss of structure or function of neurons, and even neuron demise in the brain, leading to clinical, functional, and cognitive dysfunction and movement disorders. However, iron overload can cause neurodegeneration. Dysregulation of iron metabolism associated with cellular damage and oxidative stress is reported as a common event in several neurodegenerative diseases. Uncontrolled oxidation of membrane fatty acids triggers a programmed cell death involving iron, ROS, and ferroptosis, promoting cell death. In Alzheimer’s disease, the iron content in the brain is significantly increased in vulnerable regions, resulting in a lack of antioxidant defenses and mitochondrial alterations. Iron interacts with glucose metabolism reciprocally. Overall, iron metabolism and accumulation and ferroptosis play a significant role, particularly in the context of diabetes-induced cognitive decline. Iron chelators improve cognitive performance, meaning that brain iron metabolism control reduces neuronal ferroptosis, promising a novel therapeutic approach to cognitive impairment. Full article

The Chinese softshell turtle (CST; Pelodiscus sinensis) is a freshwater aquaculture species of substantial economic importance that is commercially farmed across Asia, particularly in Taiwan. Although diseases caused by the Bacillus cereus group (Bcg) pose a major threat to commercial CST farming systems, information regarding its pathogenicity and genome remains limited. Here, we investigated the pathogenicity of Bcg strains isolated in a previous study and performed whole-genome sequencing. Pathogenicity analysis indicated that QF108-045 isolated from CSTs caused the highest mortality rate, and whole-genome sequencing revealed that it was an independent group distinct from other known Bcg genospecies. The average nucleotide identity compared to other known Bcg genospecies was below 95%, suggesting that QF108-045 belongs to a new genospecies, which we named Bacillus shihchuchen. Furthermore, genes annotation revealed the presence of anthrax toxins, such as edema factor and protective antigen, in QF108-045. Therefore, the biovar anthracis was assigned, and the full name of QF108-045 was Bacillus shihchuchen biovar anthracis. In addition to possessing multiple drug-resistant genes, QF108-045 demonstrated resistance to various types of antibiotics, including penicillins (amoxicillin and ampicillin), cephalosporins (ceftifour, cephalexin, and cephazolin), and polypeptides, such as vancomycin. Full article

Bcl-2-interacting cell death suppressor (BIS), also called BAG3, plays a role in physiological functions such as anti-apoptosis, cell proliferation, autophagy, and senescence. Whole-body Bis-knockout (KO) mice exhibit early lethality accompanied by abnormalities in cardiac and skeletal muscles, suggesting the critical role of BIS in these muscles. In this study, we generated skeletal muscle-specific Bis-knockout (Bis-SMKO) mice for the first time. Bis-SMKO mice exhibit growth retardation, kyphosis, a lack of peripheral fat, and respiratory failure, ultimately leading to early death. Regenerating fibers and increased intensity in cleaved PARP1 immunostaining were observed in the diaphragm of Bis-SMKO mice, indicating considerable muscle degeneration. Through electron microscopy analysis, we observed myofibrillar disruption, degenerated mitochondria, and autophagic vacuoles in the Bis-SMKO diaphragm. Specifically, autophagy was impaired, and heat shock proteins (HSPs), such as HSPB5 and HSP70, and z-disk proteins, including filamin C and desmin, accumulated in Bis-SMKO skeletal muscles. We also found metabolic impairments, including decreased ATP levels and lactate dehydrogenase (LDH) and creatine kinase (CK) activities in the diaphragm of Bis-SMKO mice. Our findings highlight that BIS is critical for protein homeostasis and energy metabolism in skeletal muscles, suggesting that Bis-SMKO mice could be used as a therapeutic strategy for myopathies and to elucidate the molecular function of BIS in skeletal muscle physiology. Full article

Cleft palate is one of the most common birth defects. Previous studies revealed that multiple factors, including impaired intracellular or intercellular signals, and incoordination of oral organs led to cleft palate, but were little concerned about the contribution of the extracellular matrix (ECM) during palatogenesis. Proteoglycans (PGs) are one of the important macromolecules in the ECM. They exert biological functions through one or more glycosaminoglycan (GAG) chains attached to core proteins. The family with sequence similarity 20 member b (Fam20b) are newly identified kinase-phosphorylating xylose residues that promote the correct assembly of the tetrasaccharide linkage region by creating a premise for GAG chain elongation. In this study, we explored the function of GAG chains in palate development through Wnt1-Cre; Fam20b^f/f mice, which exhibited complete cleft palate, malformed tongue, and micrognathia. In contrast, Osr2-Cre; Fam20b^f/f mice, in which Fam20b was deleted only in palatal mesenchyme, showed no abnormality, suggesting that failed palatal elevation in Wnt1-Cre; Fam20b^f/f mice was secondary to micrognathia. In addition, the reduced GAG chains promoted the apoptosis of palatal cells, primarily resulting in reduced cell density and decreased palatal volume. The suppressed BMP signaling and reduced mineralization indicated an impaired osteogenesis of palatine, which could be rescued partially by constitutively active Bmpr1a. Together, our study highlighted the key role of GAG chains in palate morphogenesis. Full article

MicroRNAs (miRNAs) have gained a prominent role as biomarkers in prostate cancer (PCa). Our study aimed to evaluate the potential suppressive effect of miR-137 in a model of advanced PCa with and without diet-induced hypercholesterolemia. In vitro, PC-3 cells were treated with 50 pmol of mimic miR-137 for 24 h, and gene and protein expression levels of SRC-1, SRC-2, SRC-3, and AR were evaluated by qPCR and immunofluorescence. We also assessed migration rate, invasion, colony-forming ability, and flow cytometry assays (apoptosis and cell cycle) after 24 h of miRNA treatment. For in vivo experiments, 16 male NOD/SCID mice were used to evaluate the effect of restoring miR-137 expression together with cholesterol. The animals were fed a standard (SD) or hypercholesterolemic (HCOL) diet for 21 days. After this, we xenografted PC-3 LUC-MC6 cells into their subcutaneous tissue. Tumor volume and bioluminescence intensity were measured weekly. After the tumors reached 50 mm3, we started intratumor treatments with a miR-137 mimic, at a dose of 6 μg weekly for four weeks. Ultimately, the animals were killed, and the xenografts were resected and analyzed for gene and protein expression. The animals’ serum was collected to evaluate the lipid profile. The in vitro results showed that miR-137 could inhibit the transcription and translation of the p160 family, SRC-1, SRC-2, and SRC-3, and indirectly reduce the expression of AR. After these analyses, it was determined that increased miR-137 inhibits cell migration and invasion and impacts reduced proliferation and increased apoptosis rates. The in vivo results demonstrated that tumor growth was arrested after the intratumoral restoration of miR-137, and proliferation levels were reduced in the SD and HCOL groups. Interestingly, the tumor growth retention response was more significant in the HCOL group. We conclude that miR-137 is a potential therapeutic miRNA that, in association with androgen precursors, can restore and reinstate the AR-mediated axis of transcription and transactivation of androgenic pathway homeostasis. Further studies involving the miR-137/coregulator/AR/cholesterol axis should be conducted to evaluate this miR in a clinical context. Full article

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from Thermococcus sibiricus (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC₉₀ values from 2.8- to 7.4-fold lower than that of the wild-type enzyme. Full article

Liver diseases represent a significant global health burden, necessitating the development of reliable biomarkers for early detection, prognosis, and therapeutic monitoring. Extracellular vesicles (EVs) have emerged as promising candidates for liver disease biomarkers due to their unique cargo composition, stability, and accessibility in various biological fluids. In this study, we present an optimized workflow for the identification of EVs-based biomarkers in liver disease, encompassing EVs isolation, characterization, cargo analysis, and biomarker validation. Here we show that the levels of microRNAs miR-10a, miR-21, miR-142-3p, miR-150, and miR-223 were different among EVs isolated from patients with nonalcoholic fatty liver disease and autoimmune hepatitis. In addition, IL2, IL8, and interferon-gamma were found to be increased in EVs isolated from patients with cholangiocarcinoma compared with healthy controls. By implementing this optimized workflow, researchers and clinicians can improve the identification and utilization of EVs-based biomarkers, ultimately enhancing liver disease diagnosis, prognosis, and personalized treatment strategies. Full article

Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by elevated pulmonary vascular resistance and increased pressure in the distal pulmonary arteries. Systematic analysis of the proteins and pathways involved in the progression of PAH is crucial for understanding the underlying molecular mechanism. In this study, we performed tandem mass tags (TMT)-based relative quantitative proteomic profiling of lung tissues from rats treated with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 6759 proteins were quantified, among which 2660 proteins exhibited significant changes (p-value < 0.05, fold change < 0.83 or >1.2). Notably, these changes included several known PAH-related proteins, such as Retnla (resistin-like alpha) and arginase-1. Furthermore, the expression of potential PAH-related proteins, including Aurora kinase B and Cyclin-A2, was verified via Western blot analysis. In addition, we performed quantitative phosphoproteomic analysis on the lungs from MCT-induced PAH rats and identified 1412 upregulated phosphopeptides and 390 downregulated phosphopeptides. Pathway enrichment analysis revealed significant involvement of pathways such as complement and coagulation cascades and the signaling pathway of vascular smooth muscle contraction. Overall, this comprehensive analysis of proteins and phosphoproteins involved in the development and progression of PAH in lung tissues provides valuable insights for the development of potential diagnostic and treatment targets for PAH. Full article

Multiple abiotic stress is known as a type of environmental unfavourable condition maximizing the yield and growth gap of crops compared with the optimal condition in both natural and cultivated environments. Rice is the world’s most important staple food, and its production is limited the most by environmental unfavourable conditions. In this study, we investigated the pre-treatment of abscisic acid (ABA) on the tolerance of the IAC1131 rice genotype to multiple abiotic stress after a 4-day exposure to combined drought, salt and extreme temperature treatments. A total of 3285 proteins were identified and quantified across the four treatment groups, consisting of control and stressed plants with and without pre-treatment with ABA, with 1633 of those proteins found to be differentially abundant between groups. Compared with the control condition, pre-treatment with the ABA hormone significantly mitigated the leaf damage against combined abiotic stress at the proteome level. Furthermore, the application of exogenous ABA did not affect the proteome profile of the control plants remarkably, while the results were different in stress-exposed plants by a greater number of proteins changed in abundance, especially those which were increased. Taken together, these results suggest that exogenous ABA has a potential priming effect for enhancing the rice seedlings’ tolerance against combined abiotic stress, mainly by affecting stress-responsive mechanisms dependent on ABA signalling pathways in plants. Full article