Unusual ɑ–amino acids, owing to their ability to accommodate diverse substituents while preserving a chiral center, constitute valuable building blocks in peptidomimetic design. Their incorporation enables fine-tuning of physicochemical and biological properties, while also providing versatile functional handles for further structural modification. Coordination with copper ions represents an additional strategy for modulating peptide activity, often enhancing antimicrobial, antioxidant, and therapeutic potential. In this study, we designed and synthesized two functionalized peptides incorporating selected non-canonical amino acid residues, chosen to improve stability and confer predicted biological activity. We investigated their coordination behavior with Cu(II) ions and evaluated the cytotoxic effects of both peptides and their copper complexes in cancer cell lines and normal fibroblasts. Our results show that Cu(II) coordination markedly increases the in vitro cytotoxic activity of one of the tested compounds. No reduction of viability was detected in normal human dermal fibroblasts within the concentration range tested.
The aim of this study was to investigate whether eprenetapopt (APR-246, PRIMA-1MET) could enhance anti-tumor effects of venetoclax (VEN) on cell proliferation and apoptosis in vitro cell lines, and to further explore the potential mechanisms underlying the enhanced effects. The cellular proliferative capacity was assessed using the CCK-8 assay, whereas apoptosis levels were evaluated through Western blot analysis and flow cytometry. Key pathways and genes were identified using the following bioinformatics tools: the Cancer Dependency Map (DepMap) and the Cancer Genome Atlas Program (TCGA) database, gene set enrichment analysis (GSEA), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Both APR-246 and VEN monotherapy demonstrated significant anti-proliferative and pro-apoptotic effects in TP53 R248Q-mutated AML cell lines. Notably, APR-246 enhanced therapeutic efficacy of VEN in TP53-mutated AML cells. From a mechanistic perspective, APR-246 may exert its anti-leukemic effects through dual pathways: (1) Associated with downregulation of PPP1CA expression, which could potentially involve the Hippo signaling pathway, coinciding with observed proliferation inhibition and apoptosis induction; (2) Attenuation of VEN-induced MCL-1 (myeloid cell leukemia-1) overexpression, potentially contributing to the augmented anti-tumor activity with VEN in TP53-mutated AML models. In TP53 R248Q-mutated AML cell lines, APR-246 augmented anti-tumor effects of VEN via proliferation inhibition and apoptosis induction. These observed enhanced effects may relate to multiple factors including: PPP1CA inhibition, Hippo pathway affecting, and MCL-1 expression suppression, warranting further investigation.
Herpes simplex virus (HSV) remains a pervasive global health concern and has the ability to challenge current therapeutic strategies because of its frequent recurrence and drug resistance. This research aimed to disrupt the critical interaction between the viral glycoprotein gD and the host cell receptor of herpes simplex virus, thereby blocking viral infection as a novel antiviral approach. An HVEM-derived native peptide NP (KEACGELTGTVCEP) targeting HSV was rationally designed, with no cytotoxicity and approximately 50
Traumatic brain injury (TBI) is frequently accompanied by neuroendocrine disturbances involving the hypothalamic–pituitary–thyroid (HPT) axis and vitamin D–calcium–phosphorus homeostasis. Despite increasing interest in nutritional strategies after TBI, the effects of branched-chain amino acid (BCAA) supplementation on post-injury neuroendocrine homeostasis, including thyroid hormone levels, vitamin D status, and calcium–phosphorus balance, remain insufficiently characterized. Twenty-eight male Wistar rats were randomly assigned to Control, Sham, TBI, and TBI + BCAA groups. Moderate TBI was induced using a closed-head weight-drop model. BCAA (0.26 g/kg) was administered orally after injury. Motor function was assessed using balance beam and gait analysis, and serum levels of thyroid hormones, 25-hydroxyvitamin D, calcium, and phosphorus were measured. TBI induced a negative body weight change, impaired balance and gait, reduced serum thyroid hormones, 25-hydroxyvitamin D, calcium, and phosphorus levels, and decreased brain and hippocampal morphometric parameters. Post-injury BCAA supplementation significantly attenuated TBI-associated body weight loss and improved balance beam scores and gait parameters relative to the untreated TBI group. In addition, BCAA treatment partially restored serum thyroid hormones, 25-hydroxyvitamin D, calcium, and phosphorus levels, although values did not fully return to control levels. BCAA supplementation also partially attenuated TBI-induced changes in gross brain and hippocampal morphometric parameters. Post-injury BCAA supplementation exerted modulatory effects, as reflected by partial improvement in neuroendocrine parameters and motor performance. However, incomplete normalization indicates that BCAA supplementation supports metabolic and functional adaptation rather than full endocrine or structural recovery. These findings suggest that BCAAs may serve as a complementary nutritional strategy to mitigate post-TBI neuroendocrine dysfunction.
It is generally accepted that amino acids (AAs) digestibility should be evaluated at the ileum level. A study was conducted, to assess the effects of varying levels of rumen-protected methionine (RPMet) supplementation, with low crude protein (CP) diet, on the total AA flow from rumen to the small intestine (ileum) using the rumen simulation technique (Rusitec) along with in vitro abomasum and ileum digestibility methods. The randomized, independent study was carried out twice over 15-day incubation periods. Four diets were tested: (a) a high-protein diet (163.39 g/kg; HP), (b) a low-protein diet (146.33 g/kg; LP), (c) a low-protein diet supplemented with low RPMet (0.11 g/kg; LPLMet), and (d) a low-protein diet supplemented with high RPMet (0.81 g/kg; LPHMet). Each diet was mixed with 20 ± 0.04 g of a basal diet in each fermenter of Rusitec system. The results showed that methionine (Met) flow from the Rusitec and abomasum residue was not significantly affected (P > 0.05) across all groups. However, total AA flow was significantly higher (P < 0.05) in the HP group than in the LP, LPLMet, and LPHMet groups in both the Rusitec and in vitro abomasum studies. The supplementation of RPMet with low CP diets significantly (P < 0.05) influenced the total and Met AA flow from the Rusitec to the ileum in the fluid portion. However, total AA flow was significantly higher (P < 0.05) in the HP group compared to other groups in the Rusitec fluid. Although at the abomasum and ileum sites, it was comparable (P > 0.05) between HP LPHMet and higher than in the other groups. Furthermore, methionine flow remained unaffected among groups (P > 0.05) in the Rusitec fermenter but was significantly higher (P < 0.05) in the LPHMet at the abomasum and ileum than in the other groups. In conclusion,high level of rumen-protected methionine (RPMet) supplementation with low-protein diet, can increase amino acid supply in intestine at the same rate as higher-protein diet (HP). This helps to ensure that low crude protein diet with RPMet, provide adequate delivery of amino acids, and reduce feed expense with nitrogen losses in environment.
N-Homocysteinylation induces immunogenic, thrombogenic, and amyloidogenic properties of proteins. Although important to gain insight into the mechanisms of homocysteine (Hcy) toxicity, proteome-wide studies of the effects of Hcy-thiolactone (HTL) protein modification remain challenging due to the low abundance of N-Hcy-proteins. High-field asymmetric waveform ion mobility spectrometry (FAIMS), as a robust online fractionation method, improves the identification of other PTMs, we therefore expected it to facilitate the detection of N-homocysteinylated proteins (N-Hcy-proteins) and help gain insight into their role in human disease. After extensive measurement optimization, we compared the yield of N-Hcy-protein/peptide identification across mouse liver and brain samples, either native or modified in vitro with HTL. Additionally, we examined the influence of different thiol reduction and blocking agents, namely dithiothreitol (DTT)/iodoacetamide (IAA) and tris(2-carboxyethyl)phosphine (TCEP)/methyl methanethiosulfonate (MMTS), on the number of identified N-Hcy-sites. FAIMS increased the number of N-Hcy-Lys-peptides and N-Hcy-proteins by 1.3–7-fold and 1.1–7-fold, respectively, and was on average higher with the usage of TCEP/MMTS thiol reduction/blocking method. We have identified 69 and 1198 in vivo and in vitro N-Hcy-proteins, respectively. We conclude that FAIMS is a valuable addition to N-Hcy-proteome analysis workflow and facilitates the mapping of N-Hcy-sites. Data are available via ProteomeXchange with identifier PXD062860.
Biogenic polyamines spermidine, spermine and some of their derivatives are small organic polycations vitally important in all domains of life. Recent studies have demonstrated that polyamine biosynthesis is subject to convergent evolution and that some bacteria employ biosynthetic pathways in which aspartate, rather than decarboxylated S -adenosylmethionine, provides the three-carbon moiety required for polyamine biosynthesis. Carboxypolyamines are among key intermediates in these metabolic pathways. At the final step of this biosynthetic route, the specific PLP-dependent carboxyspermidine decarboxylase (CASDC) converts carboxypolyamines into the corresponding polyamines. The investigation of aspartate beta-semialdehyde-dependent pathways requires commercially unavailable substrates and demand potent enzyme inhibitors. Here we report a convenient synthesis of carboxyspermidine, carboxyspermine, and carboxythermospermine allowing to study these poorly understood pathways in bacteria. The use of isosteric hydroxylamine-containing analogs of carboxypolyamines will lead to selective and effective inhibition of CASDC. The synthesis of these O -substituted hydroxylamines is also described.
Exogenous glucocorticoids are extensively used to combat a plethora of medical conditions. However, a sustained excess of exogenous glucocorticoids causes several adverse metabolic conditions, most notably Cushing’s Syndrome which presents with a well-defined phenotype. Herein we determine the ability of the non-proteogenic amino acid, beta alanine (BA) to mitigate the development of Cushing’s Syndrome. Female C57BL/6J mice were treated with high dose corticosterone or a vehicle control with or without BA for 3 weeks. Indirect calorimetry was conducted during the final week of treatment and glucose tolerance was assessed upon completion. BA partially attenuated the development of the Cushingoid phenotype with a reduction in central adipose accumulation, bodyweight gain and trended reduction in liver weight. Despite this reduction, BA also reversed the apparent corticosterone induced increase in energy expenditure (EE), whilst further restricting mice to carbohydrate utilisation. However, BA completely preserved glucose tolerance. We identify a novel therapeutic role of BA in mitigating the development of some of the symptoms associated with exogenous glucocorticoid treatment, most notably glucose intolerance. Further investigation is now required to determine the extent of BA’s protection as well as the mechanisms involved.
Breast cancer (BRCA) is a major health threat to women and often carries a poor prognosis. Succinylation is closely associated with cancer metabolism; however, research on succinylation-related genes (SRGs) in BRCA remains relatively limited. This study first identified differentially expressed genes between normal and BRCA groups in the TCGA cohort and intersected them with known SRGs to obtain differentially expressed SRGs (DE-SRGs). Univariate Cox and LASSO Cox regression analyses were used to screen the DE-SRGs, followed by multivariate Cox regression to develop a succinylation-related prognostic model. Subsequently, patients were categorized into high and low succinylation-related risk score (SRS) groups according to the median score. Two groups were then compared for differences in immune microenvironment characteristics, somatic mutation burden, and drug sensitivity. Additionally, the expression levels of the signature genes were validated via qRT-PCR. This study developed a prognostic risk model for BRCA patients based on 13 SRGs (ACOT4, ALDH3A1, B3GALT1, IFNG, MMP1, NFKB2, PCSK6, PTK2, SERPINA1, SHMT2, SIRT7, ST3GAL1, and SUCLA2). A markedly elevated infiltration of immune cells was observed in the low-SRS group, including B cells, Mast cells, and Macrophages. The tumor mutational burden was notably lower in the low-SRS group. Drug sensitivity analysis suggested that the high-SRS BRCA patients might be more susceptible to FTI-277 and GNF-2. qRT‑PCR results showed that ACOT4, IFNG, MMP1, NFKB2, PCSK6, PTK2, SHMT2, SIRT7, and ST3GAL1 were significantly upregulated in human BRCA cell lines, while ALDH3A1, B3GALT1, and SUCLA2 were significantly downregulated. Our findings highlight the prognostic value of SRGs in BRCA, laying the groundwork for its potential use in guiding personalized treatment.
This study investigated the effects of proline on intestinal injury in piglets infected with porcine epidemic diarrhea virus (PEDV). Twenty-eight 7-day-old piglets were assigned to four groups: control (CON), proline (Pro), PEDV, and Pro + PEDV. Piglets in the Pro and Pro + PEDV groups received oral proline (500 mg/kg body weight) for 7 days. During the last 3 days, piglets in the PEDV and Pro + PEDV groups were inoculated with PEDV. Under PEDV infection, proline administration increased villus height and the villus height/crypt depth ratio in both the jejunum and ileum, and villus width and villus surface area in the ileum. Proline increased PEDV N protein abundance and interferon β (IFN-β) mRNA level in both intestinal segments, suppressed downstream antiviral genes in infected piglets, but upregulated these genes in the jejunum of uninfected piglets. Proline further increased interleukin 1β (IL-1β) concentrations and the expression of inflammation-related genes in the jejunum of infected piglets. Under PEDV infection, proline increased superoxide dismutase (SOD) activity in serum and jejunum, while reducing jejunal H2O2 concentration. Furthermore, proline downregulated genes related to lipid synthesis and ion transport in the jejunum of infected piglets. Transcriptomic and RT-qPCR analyses supported these findings, indicating that proline enhanced immune defense while suppressing lipid metabolism. In conclusion, proline alleviated PEDV-induced intestinal injury by improving villus morphology and enhancing antiviral and antioxidant defenses. However, it promoted viral replication and inflammation, while suppressing lipid metabolism. The multifaceted effects of proline on PEDV infection indicate its practical use warrants careful consideration.
The mammalian polyamines (putrescine, spermidine and spermine) are ubiquitous polycations, long recognized for their indispensable roles in maintaining cell proliferation, differentiation and survival. Traditionally viewed as metabolic supporters of growth, polyamines have recently emerged as active regulators of cell-cell signaling in diverse physiological and pathological settings. Through intercellular polyamine transfer, modulation of ion channels and interactions with cell-surface receptors, polyamines orchestrate intricate signaling networks, from neurotransmission in the nervous system to cytokine signaling in the immune compartment. Many cancers, though most clearly, central nervous systems (CNS) cancers, exploit neuro- and immunomodulatory circuits, effectively hijacking neural and immune signaling pathways to sustain growth and evade surveillance. Thus, an integrated, multi-disciplinary perspective is required to overcome hurdles in the treatment of these aggressive malignancies. In light of ongoing clinical trials aimed at disrupting polyamine synthesis and transport in brain tumors, better defining the role of polyamines in mediating tumor-host interactions is essential for maximizing anti-tumor efficacy while minimizing normal tissue toxicity. This review integrates advances from cancer biology, immunology and neuroscience to comprehensively discuss the mechanisms through which polyamines regulate cell-cell signaling, the role of these pathways in brain tumor progression and the diagnostic and therapeutic opportunities that arise from this knowledge.
This study aims to computationally identify and prioritize anticancer peptide (ACP) candidate sequences derived from the Leishmania major proteins KMP11 and GP63 using an integrated bioinformatics framework that incorporates peptide design, safety assessment, multi-criteria decision-making, and membrane-oriented evaluation. Amino acid sequences of KMP11 and GP63 were obtained from the NCBI database. Peptide fragments ranging from 5 to 25 residues were computationally generated and initially screened for anticancer potential using AntiCP 2.0 and MLACP prediction tools. Candidate peptides were subsequently subjected to systematic amino acid substitutions followed by iterative re-evaluation to optimize predicted anticancer properties. Toxicity, allergenicity, and antigenicity were assessed using TOXINPRED2, ALGPRED2, and VAXIJEN2, respectively. Peptides meeting safety and functional criteria were prioritized using the Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS). Structural modeling, exploratory molecular docking, and membrane interaction analyses were then performed to provide comparative mechanistic insights into membrane association and potential receptor accessibility, rather than to predict specific target binding. Reference datasets of experimentally validated ACPs and non-ACPs were compiled, and motif analysis using MERCI identified sequence patterns associated with anticancer activity, with enriched motifs most frequently observed in the 12–13 residue range. Following iterative screening and safety evaluation, subsets of peptides derived from KMP11 and GP63 were identified as non-toxic and non-allergenic according to in silico prediction tools. These peptides were subsequently prioritized using the TOPSIS multi-criteria decision-making model. The top-ranked candidates were further subjected to exploratory molecular docking against selected cancer-associated receptors and coarse-grained membrane interaction analysis to provide comparative mechanistic context regarding membrane interaction propensity and potential receptor accessibility. Based on integrated computational scoring, ten peptides were prioritized as candidate sequences for further experimental validation. This study demonstrates the feasibility of computationally deriving and prioritizing anticancer peptide candidates from L. major proteins KMP11 and GP63. The proposed framework provides a structured, hypothesis-generating strategy for ACP candidate prioritization, emphasizing comparative evaluation rather than direct prediction of therapeutic efficacy or specific molecular targets. By leveraging evolutionary and physicochemical features associated with host–pathogen interactions, this approach enables systematic exploration of parasite-derived peptide sequence space. Experimental validation will be essential to determine the biological activity, selectivity, and translational relevance of the identified candidates.
Chronic diseases are often associated with increased inflammation and oxidative stress, which contribute to disease progression. Glutamine, a conditionally essential amino acid, has been studied for its potential anti-inflammatory and antioxidant properties in various chronic conditions. The present systematic review evaluates the effects of glutamine supplementation on inflammatory markers and oxidative stress indices in patients with chronic diseases. Systematic searches were performed in web databases; Web of Science, Scopus, and PubMed/Medline until May 2025, to identify related randomized controlled trials (RCTs) according to the Cochrane Library and PICOS criteria (population: individuals > 18 years, intervention: glutamine, Comparison: placebo or control, Outcomes: inflammatory and oxidative stress markers in chronic diseases). The Cochrane collaboration tool was used to assess the risk of bias in clinical trials. Six RCTs that assessed the effect of glutamine supplementation on inflammation and oxidative stress markers were included in the study. In these studies, glutamine was administered to the participants through oral or parenteral routes. In three studies improve inflammation via significant reductions in CRP were observed. However, in three studies that examined TNF-α as an inflammatory marker, only one study found its levels to be significantly reduced. Also, of the two studies that examined oxidative stress levels, only one study significantly decreased the MDA and increased SOD levels, and in the other study, glutamine supplementation had no significant effect on glutathione levels. Our findings showed that glutamine supplementation might have a positive effect on inflammation and oxidative stress indices such as TNF-α, CRP, MDA, and SOD in some chronic diseases, however, these effects have not been shown in all studies, so more carefully designed clinical trial studies with different doses of glutamine on inflammation and oxidative stress in chronic diseases are needed. PROSPERO Code: This study was registered in the PROSPERO international prospective register of systematic reviews registration number: CRD420251049112.
In seed plants, putrescine, spermidine, and spermine are ubiquitously present, whereas a structural isomer of spermine, thermospermine (TSpm), is synthesized mainly in the vascular tissue. Initially identified in the bacterium Thermus thermophilus, TSpm was later shown to be synthesized in Arabidopsis thaliana by ACAULIS5 (ACL5). ACL5 gene homologs may have been acquired early in plant evolution via endosymbiotic gene transfer from a cyanobacterial ancestor. Loss-of-function acl5 mutants exhibit a dwarf phenotype and excessive vascular xylem formation. Subsequent studies, including analysis of suppressor-of-acl5 (sac) mutants, revealed that TSpm exerts a critical role in the repression of vascular xylem proliferation by acting in upstream open-reading-frame (uORF)-dependent translational regulation of specific mRNAs. A recent study revealed functional TSpm binding to the peptidyl transferase center of 25 S rRNA promoted by methylation of residue U2952. Like other polyamines, TSpm has also been shown to participate in stress responses, enhancing tolerance to salt, drought, heat, and pathogen challenges in multiple species. Collectively, TSpm represents a unique polyamine with dual roles in xylem development and stress adaptation, whose evolutionary origin and molecular mechanisms provide insights into the specialization of polyamine biology. Further studies in nonvascular plants and algae are needed to elucidate the ancestral functions of TSpm.
Zinc finger proteins (ZNFs), characterized by zinc ion-binding domains, participate in cell proliferation, differentiation, and metastasis in lung adenocarcinoma (LUAD). However, associations between ZNFs-related genes and clinical outcomes, immune cell infiltration, and immunotherapy remain unclear. To explore feasibility of using ZNFs-related genes as prognostic tools for LUAD risk stratification. Retrospective analyses were conducted utilizing data from TCGA and GSE26939. After screening differentially expressed ZNFs, regression analyses were performed to construct prognostic signature. Enrichment analysis identified biological processes and pathways involved in signature genes, while immune landscape was examined by multiple algorithms. The drug sensitivity analysis identified potential candidate drugs related to the signature genes. Cell experiments indicated the function of the key risk gene CTCFL in promoting the malignant behavior of LUAD cells. A prognostic signature comprising 12 ZNFs-related genes (CBFA2T3, CTCFL, GFI1B, IGF2BP1, RIMS2, TRIM29, TRIML2, ZIC2, ZNF208, KLF10, ZNF750, and ZNF257) stratified LUAD patients into two risk groups, demonstrating robust performance in predicting clinical outcomes. These genes were significantly enriched in epidermal development, intermediate filament cytoskeleton, endopeptidase inhibitor activity, hormone activity, and neuroactive ligand-receptor interactions. Low-risk patients exhibited higher levels of immune cell infiltration (e.g., DCs, B cells, and neutrophils) and superior responses to immunotherapy (anti-CTLA-4 and PD-1/CTLA-4 dual blockade). Possible therapeutic compounds for LUAD patients included SHP-099, Dimethylfasudil, EMD-534085, and PF-2771. The expression of CTCFL enhanced the malignant cellular behavior in LUAD. ZNFs-related gene signature provides predictive insights into LUAD patient survival, immune cell infiltration, and immune checkpoint blockade therapy, serving as a valuable tool to guide clinical decision-making.
Taurine is a semi-essential amino acid with diverse cytoprotective effects and is associated with anti-aging. This study explores the role and molecular mechanism of taurine in muscle aging. Senescence-accelerated mouse prone 8 (SAMP8) and its resistant (senescence-accelerated mouse resistant 1, SAMR1) mice were given 1
Abstract Polyamines (putrescine, spermidine, and spermine) are essential for animal health and development, and their intracellular levels must be tightly regulated to maintain normal cellular functions. Ornithine decarboxylase (ODC) catalyzes the rate-limiting decarboxylation step in polyamine biosynthesis, and thus accurate assessment of its activity is vital for studies of polyamine metabolism. However, conventional ODC assays rely on radiolabeled substrates and require specialized facilities for radioactive handling. To overcome these limitations, we developed a sensitive and non-radioactive ODC assay using stable isotope-labeled ornithine in combination with liquid chromatography–mass spectrometry (LC–MS). In this protocol, animal tissues (e.g., 0.5 g) or cultured cells (e.g., 2.0 × 10 6 cells) are homogenized, and cytosolic fractions are prepared by centrifugation. These fractions are incubated with d 7 -ornithine under 37 °C conditions, and the produced d 7 -putrescine is derivatized with dansyl chloride and quantified by LC–MS analysis. Reaction samples showed a distinct peak corresponding to d 7 -putrescine, whereas negative control displayed negligible signals. Moreover, d 7 -spermidine and d 7 -spermine were not detectable under these conditions, indicating d 7 -putrescine production directly reflects ODC activity. We optimized reaction time and substrate concentrations to ensure linearity and precision, and confirmed that the assay responds appropriately to pharmacological inhibition of ODC. Collectively, this protocol provides a practical, sensitive, and non-radioactive method for quantifying ODC activity in both animal tissues and cultured cells, and it offers an accessible tool for polyamine metabolism research.
The clinical management of HER2-positive breast cancer is undergoing a paradigm shift, recognizing that hormone receptor (HR) co-expression defines a fundamental biological dichotomy rather than a minor clinical variation. This perspective evaluates the profound heterogeneity within the HER2-positive subclass, where HR status acts as a master regulator of genomic landscapes and patient outcomes. Evidence from large-scale database analyses reveals that HR+/HER2 + malignancies are associated with a significant survival advantage, contrasting with the heightened phenotypic aggression and advanced staging characteristic of HR-negative disease. At the molecular level, this divergence is underpinned by distinct transcriptomic profiles; specifically, the enrichment of drug-metabolizing pathways—including cytochrome P450 and retinol metabolism involving CYP2A6 and UGT2B7—suggests a mechanism for superior endocrine sensitivity and modulated apoptotic signaling in the double-positive cohort. Furthermore, the tumor microenvironment reflects this biological disparity, with HR status potentially governing local immune responses, as evidenced by the differential infiltration of resting mast cells versus plasma cells. Of note, while somatic mutations in TP53 and PIK3CA persist across both cohorts, the unique metabolic and immunological milieus confirm that these subtypes represent biologically discrete entities. Moving forward, the “HER2-positive” designation must be refined through integrated, biomarker-driven frameworks. Incorporating these molecular nuances into prospective clinical trials is essential for optimizing therapeutic de-escalation and overcoming the persistent challenges of resistance in HER2-targeted care.
Bacterial vaginosis (BV), a prevalent form of vaginal dysbiosis, is primarily caused by Gardnerella vaginalis. The increasing prevalence of BV has raised global health concerns, prompting the need for a vaccine that can enhance human immunity and mitigate BV transmission. The study developed a multi-epitope vaccine for BV infection using immunoinformatic methodologies. B-cell and T-cell epitopes were identified using the IEDB recommended server and NetCTL. Specifically, HTL epitopes were predicted against HLA-DR alleles (including DRB101:01, DRB103:05, and DRB104:04), while CTL epitopes were predicted against HLA class I supertypes (including HLA-A02:01 and HLA-A*01:01). The selected epitopes were fused using adjuvants and linkers. The peptide sequence MSPSVRHSPSVRH, derived from the heat shock protein 60 (HSP60) of Mycobacterium tuberculosis, was incorporated as an adjuvant to activate innate immunity via TLR2/4 and enhance dendritic cell maturation. The B-cell and (CTL or HTL) epitopes were connected using GGGS linkers, whereas the CTL + HTL epitopes were connected using HEYGAEALERAG linkers. Additional epitopes were chosen based on antigenicity, allergenicity, and immunological features. TLR2 recognizes bacterial lipoproteins and peptidoglycan. TLR2 and TLR-4 showed robust interactions in molecular docking. The results of the present study demonstrate that the produced vaccine displayed stability, shown by a molecular weight of 49924.79 Da and an antigenicity value of 1.37. The Vaccine Construct demonstrated stability and basicity, shown by an instability score of 32.65 and a projected isoelectric point (pI) of 5.09. The expected secondary structure of the vaccine construct consisted of 94.57
