Biology and Life Sciences

Abstract: Hyaluronan (HA) has traditionally been viewed as a structural component of the extracellular matrix, valued primarily for its viscoelastic, lubricating, and hydrating properties. However, accumulating evidence suggests that ultra-high molecular weight hyaluronan (UHMW-HA) functions far beyond passive tissue support, acting as a dynamic regulator of inflammation, mechanotransduction, immune homeostasis, cellular senescence, and regeneration. In this narrative review, we examine HA and specifically focus upon UHMW-HA operating as a “living hydrogel”, an adaptive regenerative macromolecule that continuously interacts with its microenvironment to shape tissue function and resilience. We examine evidence spanning evolutionary biology, glycobiology, biomaterials science, and regenerative medicine to examine how polymer size encodes biological activity and how UHMW-HA influences stem cell behaviour, extracellular matrix remodelling, and responses to injury and ageing. Attention is given to unique biological models, including the naked mole-rat, whose exceptional longevity and cancer resistance have been linked to abundant UHMW-HA, offering insights into the relationship between extracellular matrix architecture and organismal health. We further discuss how advances in synthetic biology and biomaterials engineering are enabling the development of next-generation HA-based hydrogels that mimic these adaptive properties for therapeutic applications in tissue engineering, in addition to potential roles in dynamically optimising wound repair, and musculoskeletal rejuvenation, through precision medicine approaches. Finally, we argue that reclassifying UHMW-HA as a living regenerative material rather than an inert scaffold provides a new concept for designing bioinspired therapeutics and understanding extracellular matrix function. This review demonstrates HA-adaptive macromolecules as critical entities in future regenerative strategies and highlights the specific and unique potential of UHMW-HA to combine material science developments with enhancement of preferable biological outcome.

Abstract: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and poses significant treatment challenges due to fewer available options. Creating highly specific and precise anticancer therapies continues to be a significant challenge for TNBC. Previous studies showed that combining drugs or adding chemotherapy to other therapies has significantly improved patient prognosis and proved to be an effective strategy for treating TNBC compared to using chemotherapeutic drugs alone targeting TNBC pathways. One such combination is a PARP inhibitor (PARPi) with an immune checkpoint inhibitor (ICI). In this study, we explored the transcriptomic profiles of the combination of Olaparib (a PARP inhibitor) and atezolizumab (a monoclonal antibody targeting PD-L1) in cells derived from African American (AA) and White populations. Our results demonstrated that combining Olaparib (Ola) and Atezolizumab (Atz) significantly reduced cell viability in both AA and White cell lines, compared to either agent alone. Our transcriptional profiling results showed that in both up- and down-regulated genes, there was widespread suppression of proliferation and biosynthetic gene networks, and selective activation of immune and stress-related pathways. Our transcriptional profiling revealed that both up- and down-regulated genes exhibited widespread suppression of proliferation and biosynthesis pathways, along with selective activation of immune and stress-related pathways. Our functional enrichment analysis revealed significant changes in DNA damage response, chromatin organization, RNA regulatory processes, immune signaling, and inflammatory responses. In conclusion, our study’s results provide valuable insights into key features of combination therapy, which may help guide the more targeted development of these regimens.

Abstract: This review synthesizes regulatory and scientific evidence accumulated over the past decade supporting the safety of newly expressed proteins (NEPs) in genetically modified (GM) crops. Using five case studies representing diverse protein classes, including dicamba monooxygenase (DMO), protoporphyrinogen IX oxidase (PPO), Cry1B.2/Cry1B.3, Mpp75Aa1.1, and Vip3Cb1, it illustrates how science-based, case-by-case, weight-of-evidence approaches are applied in food and feed safety assessment and ecological risk assessment (ERA) for regulatory submissions. These examples demonstrate how hazard identification, hazard characterization, and exposure assessment are integrated across proteins with diverse biological functions, structural characteristics, and levels of familiarity. It further examines the safety relevance of protein processing and sequence variation arising from protein engineering or biological processing and discusses the scientific basis supporting bridging and read-across approaches, including phylogenetic relationships, sequence similarity, structural conservation, domain architecture, and functional equivalence. The increasing application of safe-by-design principles and bridging strategies is highlighted as an important advancement enabling efficient, scientifically robust evaluations while reducing unnecessary testing. The review also discusses the convergence of food/feed safety assessment and ERA and future directions. Overall, accumulated evidence demonstrates that NEPs in commercialized GM crops have not introduced safety concerns and supports the continued evolution and modernization of protein safety assessment frameworks.

Abstract: Somatic genome editing is emerging as a potential strategy for achieving durable therapeutic effects in cardiovascular disease. This review examines current genome-editing platforms, therapeutic targets, delivery systems, and translational hurdles, contrasting liver-directed and myocardial applications. CRISPR-Cas nucleases, base editors, and prime editors offer complementary capabilities, enabling durable gene disruption or precise nucleotide correction. However, key translational challenges include unintended alterations, immune responses, irreversibility, dose optimization, long-term surveillance, regulatory requirements, and equitable access. Clinical translation has advanced for liver-directed applications, with single-administration editing of hepatocyte targets including PCSK9, ANGPTL3, and TTR producing decreases in circulating proteins and atherogenic lipids in early-phase studies, although evidence of cardiovascular event reduction and lifelong safety remains unavailable. In contrast, direct myocardial editing remains predominantly preclinical, with studies targeting MYH7 and MYBPC3 pathogenic variants demonstrating proof of concept and disease rescue in selected models, while revealing challenges in cardiomyocyte delivery, spatial coverage, allele selectivity, and tissue-level safety. Future progress will require integrating target biology, editor selection, organ-specific delivery, and rigorous molecular and clinical outcome assessment. Cardiovascular genome editing is therefore entering clinical translation in selected liver-directed applications, whereas direct myocardial editing remains at an earlier stage, with delivery, safety, and myocardial coverage as major barriers.

Abstract: Background: Long-term biospecimen preservation requires sustained institutional investment in storage equipment, energy or cryogen, monitoring, maintenance, and inventory management. Although biobank economics and biospecimen utilization have been examined independently, the recurring direct cost associated with maintaining existing inventories in relation to storage capacity and research utilization remains less well characterized. We introduce Annual Carrying Cost (ACC) as an operational metric for quantifying this continuing institutional investment. Methods: A retrospective observational analysis was conducted within a large academic biobank using long-term storage infrastructure and retrieval data from 2023–2025. ACC incorporated annualized equipment and monitoring-probe costs, energy and cryogen consumption, maintenance and repair, environmental monitoring, and inventory management. Storage capacity was characterized according to inventory-bearing, online contingency, and offline/staged reserve capacity. ACC was normalized to available and occupied vial positions and evaluated in relation to annual researchdirected vial-equivalent retrievals. Results: Sixteen storage units provided 832,120 vial positions, of which 272,476 were occupied (32.7%). Eight inventory-bearing units were 56.1% occupied, while 41.6% of total capacity was intentionally maintained as online contingency or offline/staged reserve infrastructure. Estimated ACC was $131,252 annually, comprising annualized equipment and monitoring probes (40.5%), energy and cryogen (30.8%), environmental monitoring (14.4%), maintenance and repair (8.4%), and inventory management (6.0%). ACC corresponded to $0.16 per available vial position and $0.48 per occupied vial position annually. Research-directed retrieval volume varied more than 13-fold across the study period, from 217,348 vial equivalents in 2023 to 16,414 in 2025, resulting in retrieval-normalized ACC ranging from $0.60 to $8.00 per vial-equivalent retrieved. Conclusions: Long-term biospecimen retention creates a measurable recurring institutional obligation that persists despite variation in annual research utilization. ACC provides a practical framework for characterizing this investment and distinguishing routinely occupied storage from infrastructure maintained for resilience and future capacity. When considered alongside scientific value, utilization, uniqueness, and anticipated future demand, ACC may support more informed capacity planning, lifecycle management, resource allocation, and sustainable long-term biospecimen stewardship.

Abstract: Background: Cannabidiol (CBD), isolated from hemp (Cannabis sativa or Cannabis indica), is receiving increasing attention for its potential against different pathologies, including viral infections. CBD has been reported to alter lateral membrane diffusion and cholesterol accessibility. Therefore, it remains unclear which signaling pathways are affected by CBD treatment. Here, we sought to determine the effect of CBD treatment on human cell lines and primary human cells. Methods: We performed bioinformatics analysis to identify differentially expressed genes (DEGs) in published RNA-seq datasets of CBD-treated cells, including A549, THP-1, normal human epidermal keratinocytes (NHEK), monocyte-derived dendritic cells (DC), and monocyte-derived macrophages (MDM). We then performed RT-qPCR to validate A549, THP-1, and MDM sequencing data. Results: Comparative transcriptional analysis across five CBD-treated cell types identified 21 genes shared across all cell types, 142 among four, and 694 among three, enriched in ER stress, lipid metabolism, and cholesterol metabolism pathways. Transcription factor activity analysis identified ATF4, ATF6, XBP1, SREBF1, and SREBF2, which encode key regulators of the ER stress response and cholesterol metabolism. Conclusions: Results highlight CBD’s pivotal role in regulating transcription factor expression, which is crucial for controlling gene expression involved in ER stress, lipid, and cholesterol metabolism. Consequently, CBD treatment modulates gene expression programs in a cell-dependent manner and promotes lipotoxicity by dysregulation ER stress signaling pathways.

Abstract: Macroalgae host complex microbial communities that influence surface colonization, development, nutrient transformation, carbohydrate turnover, environmental responses, and the production of potentially valuable metabolites. Advances in shotgun metagenomics and genome-resolved analysis have greatly expanded access to this functional diversity, but they have also increased the risk of conflating genomic potential with biological activity or host benefit. This concise review evaluates macroalgal microbiomes through a framework that separates what has been proven from what is merely predicted, distinguishing functional prediction from expression, biochemical activity, metabolite exchange, host response, and experimental causality. Particular attention is given to bacteria-dependent morphogenesis, metabolic complementarity, environmental acclimation, carbohydrate-active enzymes and polysaccharide-utilization loci, biosynthetic gene clusters, and the translational potential of seaweed-associated microorganisms. Comparative perspectives from microalgal and cyanobacterial systems are used to identify principles that generalize across algal microbiomes while emphasizing the distinctive spatial and chemical complexity of macroalgal surfaces. Invasive and bloom-forming macroalgae provide an additional ecological context in which microbial functional redundancy, recruitment flexibility, and dominant populations may shape whole-community profiles. An illustrative taxon-attribution case study comparing Rugulopteryx okamurae and Sargassum sp. holobionts in the Azores demonstrates how the disproportionate contribution of the genus Cobetia can substantially alter interpretation of community-level KEGG pathway differences. Together, these analyses argue that macroalgal microbiome research should move beyond inventories of predicted functions toward explicit taxonomic attribution, experimental validation, and ecological context. Such integration will be essential for establishing which microbial functions are ecologically consequential and which can be translated reproducibly into biotechnology.

Abstract: Microalgae are thought to offer attractive biological platforms for the sustainable synthesis of biodegradable polymers, especially polyhydroxybutyrate (PHB), which belongs to the polyhydroxyalkanoate (PHA) family. Rapid and accurate screening techniques for finding high-PHB-producing microalgal strains are needed due to growing environmental issues related to traditional plastics. For the initial identification of intracellular polymer buildup, staining-based methods provide an economical and effective substitute for traditional analytical procedures. This study as first one achieved  in Yemen aimed to evaluate and compare the performance of three staining approaches—Nile Red, Nile Blue A, and Sudan Black B—for the detection of PHB accumulation in microalgae. Nile Red was used for rapid visualization of intracellular hydrophobic compounds, while Nile Blue A was applied for more selective identification of PHB granules under optimized staining conditions. Sudan Black B provided complementary qualitative assessment of lipid and polymeric inclusions using light microscopy. In accordance to results reads above showed that the stain Nile Blue A dissolved in DMSO showed 100% to be the highest ratio of positive read followed by 98.2% of Nile Red dissolved in DMSO of positive read then 92.79% of Nile Blue A dissolved in Acetone of positive read after that 88.29% of Nile Red dissolved in acetone that showed as orange granules and finally 40.54% of Sudan Black B dissolved in ethanol as blue black granules to be the less ratio amongst all ones stains. However, as seen in the appendix's images, the negative findings revealed pink or red granules and cells. In calculating reads of samples the results showed out of 37 samples 32 samples are positive with 86.49% for biopolymer accumulating inside algal tested cells. These samples were selected to be used for other biotechnological application.

Abstract: We present KOREF1-G-TTAGGA, the first Telomere-to-Telomere Accurate and Gapless Genome Assembly, standing as the Korean standard reference genome. The paternal and maternal haplotypes spanned 2.91 and 3.03 Gb. Genome-wide, at least 99.15% of assembled sequences were reliably haplotype-resolved, and over 95% remained accurate even in the most error-prone loci, including centromeric satellite arrays and segmental duplications. Rare-k-mer copy-number concordance within satellite arrays held up 98.9% and 98.8% per haplotype. Bionano optical maps fully spanned all canonical rDNA arrays on the five acrocentric chromosomes. The assembly quality index (AQI) of 99.77 and 99.69 exceeded the reference-quality threshold of 90, and more than 99.99% of gene and cCRE sequence was free of structural error. Both haplotypes further showed high base-level accuracy, with consensus quality value (QV) of 81.19 and 79.03, corresponding to one error per 131 and 80 Mb. KOREF1-G-TTAGGA is among the highest-quality East Asian telomere-to-telomere assemblies. It moreover anchors a decade-spanning multi-ome reference dataset for defining individual’s molecular states. Together, these resources define the personal referenceome as a foundation for individual biology and precision medicine, with the assemblies, annotations, and all multi-omic data openly available at https://koreanreference.org.

Abstract: The range of local sequence–structure correlation is known for coarse-grained descriptors — secondary structure and side-chain burial — but not for the backbone dihedrals themselves. I measure the information local sequence carries about φ and ψ at 10° resolution against how far a translation-equivariant convolutional model may look. Both peak at a window radius of 8 residues — φ recovering 0.330 bits of its 3.669 and ψ 0.429 of its 4.161 on held-out chains — and decline beyond. Controls test the alternatives: rebinning at 5°, 20° and 30° leaves the radius at 8, profiles do not widen it, and capacity does not rescue a wider one — every radius-32 model tried, to 556,324 weights, scores below every radius-8 model. A parameter-free mutual-information estimate finds the signal at the null level by |d| ≈ 10 downstream, ≈ 8 upstream. On a second dataset of 8,589 chains, no 30% cluster shared across splits, both maxima again fall at 8, though ψ is flat to 16.Decomposed by class, coil carries three times what helix does beyond its own marginal (0.376 against 0.120 bits), though all three retain about 90% of what they fit. An earlier version reported that helix retains none; that is withdrawn. It came of scoring a small split against a class marginal fitted on that same split — an oracle whose advantage equals the train-to-split entropy gap, here 0.369 bits, larger than the helix signal. I report three evaluation errors able to invert conclusions; that is the third.

Abstract: Purpose: To investigate whether quantitative nuclear morphology from routine H&E images can be used as a biomarker for glioblastoma (GBM) biology and prognosis. Methods: Nuclear eccentricity, circularity, density, and heterogeneity were measured from H&E whole-slide images in the TCGA-GBM cohort and independently validated in the CPTAC-GBM cohort. A multivariable Cox model was used to test the survival associations in both cohorts, and transcriptome-wide and gene set enrichment analyses were conducted in TCGA and validated using CPTAC RNA and proteomic data. Results: In TCGA-GBM, greater nuclear eccentricity was associated with better survival (HR 0.85; 95% CI, 0.71–1.02; P=0.089), with the same favorable association independently observed in CPTAC-GBM (HR, 0.74; 95% CI, 0.60–0.90; P=0.003). No individual gene association with eccentricity was statistically significant in the TCGA cohort, but pathway analysis identified its association with epithelial-mesenchymal transition (EMT), inflammatory, hypoxic, and stress-related programs. Across TCGA RNA, CPTAC RNA, and proteomic dataset, greater eccentricity was consistently associated with increased NF-κB signaling, collagen biosynthesis, and ER-to-Golgi transport and decreased L1CAM-related and neuronal-projection programs, with EMT showing the strongest cross-cohort consistency. Conclusion: Nuclear eccentricity from patient H&E slide images is a potential biomarker associated with survival and transcriptional/translational programs in GBM. Integrating quantitative pathology with RNA and proteomic data may provide an accessible approach for GBM prognosis and tumor biology characterization.

Abstract: Osteonecrosis of the femoral head (ONFH) is a progressive ischemic bone disorder that may lead to subchondral fracture, femoral-head collapse, and total hip arthroplasty. Impaired regeneration reflects persistent ischemia, endothelial dysfunction, defective angiogenesis and bone remodeling, inflammation, oxidative stress, and dysfunction of endogenous mesenchymal stromal cells (MSCs). Although MSC therapy, particularly with core decompression in precollapse ONFH, is a promising joint-preserving approach, conventional two-dimensional (2D)-expanded MSCs are limited by poor survival and retention, anoikis, disrupted cell-cell and cell-extracellular matrix interactions, and loss of potency during ex vivo expansion. Three-dimensional MSC spheroids may overcome several of these limitations by preserving intercellular and matrix interactions and enhancing resistance to ischemic stress, paracrine signaling, angiogenic activity, immunomodulation, and osteogenic competence. Hypoxia-responsive signaling, autophagy, trophic-factor secretion, and extracellular vesicle-mediated communication may promote angiogenesis-osteogenesis coupling and restoration of the ischemic bone–vascular niche. This review summarizes the pathophysiological barriers to ONFH regeneration, current evidence and limitations of conventional MSC therapy, biological mechanisms underlying MSC spheroid activity, and emerging strategies for therapeutic optimization. Importantly, direct evidence demonstrating the superiority of MSC spheroids over conventional MSC preparations specifically in ONFH remains limited. Future translation will require disease-specific comparative studies, standardized GMP-compatible manufacturing, validated potency assays and clinically practical delivery systems.

Abstract: Spatial transcriptomics (ST) measures gene expression while retaining the location of each measurement in tissue. This makes it possible to study how molecular patterns relate to tissue structure, but it also creates data that depend on both gene expression and spatial relationships. Graph neural networks (GNNs) are increasingly used to model these relationships across tasks such as spatial domain identification, data integration, imputation, deconvolution, cell--cell communication analysis, and expression prediction. Yet methods with similar GNN architectures may represent different biological units, connect them using different evidence, and produce outputs with different biological meanings. This review provides a practical framework for comparing ST-GNN methods from input data to graph construction, graph-based learning, and downstream output. We focus on what each node represents, how relationships between nodes are defined, and how the graph contributes to the final output. Our analysis shows that node definition sets the resolution of the output, edge construction controls which relationships the model can use, and the role of the graph during learning determines how relational information contributes to the model output. This framework helps readers compare methods more consistently and assess what biological conclusions their outputs can support.

Abstract: Microenergy acoustic pulse (MAP) therapy may promote urethral regeneration after childbirth-related injury, but its multicellular mechanisms remain unclear. We investigated how MAP reshapes the injured and aging urethral microenvironment using single-cell RNA sequencing. Eight female Sprague–Dawley rats were assigned to sham, vaginal balloon dilation plus ovariectomy (VBDO), VBDO plus beta-aminopropionitrile (BAPN), or BAPN plus MAP groups, with one 24-week-old and one 48-week-old rat per group. Urethral tissues were analyzed for cell composition, differential gene expression, Gene Ontology enrichment, stem/progenitor features, senescence-associated secretory phenotype activity, and CellChat-inferred intercellular communication. The integrated dataset comprised 51,690 cells and 20 transcriptionally distinct cell states. Injury and aging disrupted epithelial, vascular, stromal, immune, and muscle compartments, whereas MAP induced cellular redistribution and recovery of urethral muscle content. MAP also enhanced extracellular-matrix, adhesion, vascular, developmental, and neural/neuromuscular signaling, including LAMININ, FN1, COLLAGEN, ANGPT, VEGF, WNT, NOTCH, RELN, SLIT, NCAM, and NRXN pathways. Enrichment analysis indicated increased translation, oxidative metabolism, stress adaptation, and synaptic organization, together with partial restoration of stem/progenitor-like states and selective modulation of senescence-related programs. Reduced Ndrg1 expression in striated muscle after MAP suggested attenuation of persistent cellular stress. Overall, MAP shifted the injured urethral ecosystem toward a more regenerative state, requiring further validation.

Abstract: Secretion of recombinant proteins from microbial cell factories is a promising strategy for recombinant protein biomanufacturing as it simplifies downstream processing. In this study, we sought to intensify the Lactococcus lactis high-cell-density culture, using basic fibroblast growth factor 2 (FGF2) as a model recombinant protein. We implemented a perfusion strategy using tangential flow filtration for cell retention, allowing continuous removal of inhibitory metabolites while replenishing fresh nutrients. When conventional 2×GM17 medium was used, the approach outperformed batch cultivation, achieving a 5.2-fold increase in biomass and a 2.6-fold increase in secreted FGF2, reaching a titer of 9000 µg·L⁻¹. Concurrently, we developed a bioprocess model for L. lactis grown in a fortified spent cell culture medium, enabling systematic exploration of operating conditions. A Pareto front was generated for FGF2 titer against media usage, and perfusion profiles balancing both competing objectives were identified. An experimentally selected operating point validated the model predictions, yielding a final OD₆₀₀ of 49.2 and FGF2 titer of 2166 µg·L⁻¹ FGF2, which were 8-fold and 5-fold higher than batch process respectively. Overall, this work demonstrates a perfusion-based intensification strategy for L. lactis and highlights the utility of model-guided process decision making to enhance productivity while reducing waste.

Abstract: The contemporary management of dentin injuries has progressively shifted from approaches based solely on reparative mineralized barrier formation toward biologically guided strategies that preserve pulp vitality and promote endogenous regeneration. This paradigm change has driven the development of bioactive biomaterials capable of modulating the dentin–pulp microenvironment rather than serving exclusively as passive sealing materials. This review critically discusses recent advances in biomaterial-based approaches for dentin tissue engineering, emphasizing naturally derived polymeric scaffolds, nanofibrous membranes, injectable hydrogels, microsphere-based delivery systems, and three-dimensional (3D)-printed scaffolds. The biological mechanisms by which these platforms regulate inflammation, recruit endogenous progenitor cells, stimulate angiogenesis, direct odontoblast-like differentiation, and promote extracellular matrix deposition are highlighted. Particular attention is given to the sequential development of mineral-functionalized nanofibrous scaffolds, from the selection of calcium hydroxide and nano-hydroxyapatite as bioactive phases to the incorporation of fibronectin and, subsequently, the flavonoids quercetin and hesperetin to combine odontogenic signaling with immunomodulation. Emerging concepts, including immunoinstructive biomaterials, cell-homing strategies, smart hydrogels, and biofabrication technologies, are discussed in the context of their translational potential for vital pulp therapy. Rather than focusing exclusively on recreating the native tubular architecture of dentin, current regenerative strategies increasingly seek to restore pulp homeostasis and establish biological conditions that enable predictable reparative dentinogenesis. Collectively, these advances position biomaterials as active regulators of tissue regeneration and provide a framework for next-generation therapies aimed at preserving pulp vitality and improving the long-term outcomes of dentin regeneration.

Abstract: Pangenome graphs embed assemblies as traversals through sequence-labelled segments, but internal graph validity does not establish that these traversals still reproduce the external assemblies from which a graph was constructed. We present panpath-audit, a command-line program that reconstructs GFA1 paths and coordinate-aware GFA1.1 walks and compares them symbol-for-symbol with named FASTA sources. The program performs an all-or-nothing input preflight, handles complete IUPAC reverse complements, distinguishes unembedded walk ranges from divergence, bounds tracked sequence memory, and emits deterministic human, JSON, or TSV reports. We evaluated the program using public tomato and Human Pangenome Reference Consortium graphs. In the tomato chromosome-2 graph, 23 traversals representing 1,280,460,312 source bases were recovered exactly. Prepared controls localized an altered MM#1#chr02 source at both linear and graph coordinates and identified an omitted SL5#1#ch02 source explicitly. Audits of clipped and full Minigraph–Cactus graphs at human whole-pangenome scale independently reported the same 94 substituted bases, which we trace to IUPAC ambiguity codes in GRCh38 that graph construction had replaced with N. These results support sequence auditing as a distinct complement to topological graph validation and as a practical gate for construction, conversion, distribution, and continuous-integration workflows.

Abstract: Objectives: Rhododendron species are widely used in Himalayan traditional medicine, but the phytochemical composition, antioxidant capacity, and antibacterial potential of their flower, particularly against clinically relevant gram-negative bacteria, remain poorly characterized for Nepalese populations. This study aims to evaluate the phytochemical profile of total phenolic/flavonoid content, antioxidant activity, and antibacterial potential of sequential solvent extract of Rhododendron flowers collected from two sites in Nepal. Materials and Methods: Dried flowers from two sites, R1 and R2, were sequentially extracted with hexane, ethyl acetate, methanol, and water. Extracts were screened quantitatively for major phytochemical classes and total phenolic content (TPC) and total flavonoid content (TFC) were determined colorimetrically. Antioxidant activity was assessed by DP-Ph and ABTS radical scavenging assay. Antibacterial activity of the methanol and ethyl acetate extracts was evaluated against Escherichia coli, Klebsiella sp., and Pseudomonas sp. by agar well diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. Results: Methanol gave the highest extraction yield (17-18%) and the richest phytochemical profile, particularly flavonoids and tannins. TPC and TFC were highest in methanol extracts (up to 78.4 mg GAE/g and 46.2 mg QE/g, respectively) and were significantly negatively correlated with DPPH IC₅₀ (r = −0.75 to −0.79, p < 0.05). Methanol and ethyl acetate extracts showed the strongest antioxidant activity (IC50 as low as 18.2 µg/mL), while hexane extracts were the weakest in both assays. The ethyl acetate extract of R2 and the methanol extract of R1 showed the strongest antibacterial activity, with the lowest MIC (12.5 mg/mL) recorded against E. coli, and predominantly bactericidal action (MBC/MIC ≤ 4) against all three bacterial genera. Conclusion: Rhododendron flower extracts, particularly the methanol and ethyl acetate fractions, are rich in phenolic and flavonoid compounds and exhibit notable antioxidant and antibacterial activities, supporting their potential as natural sources of bioactive compounds against oxidative stress and antimicrobial-resistant pathogens.

Abstract: Keyhole limpet hemocyanin (KLH) is a large copper-containing glycoprotein derived from the marine gastropod Megathura crenulata. Originally functioning as an oxygen transport molecule, KLH has gained considerable attention in biomedical research due to its exceptional immunogenic and immunostimulatory properties. Its complex quaternary structure, extensive glycosylation, and xenogeneic origin contribute to its ability to induce robust humoral and cellular immune responses in mammals without significant toxicity. These characteristics have established KLH as one of the most widely used carrier proteins in vaccine development and as a valuable model antigen for the investigation of adaptive immune responses. This review summarizes current knowledge on the biological origin, molecular structure, biosynthesis, and post-translational processing of KLH, with particular emphasis on its unique glycan architecture and its contribution to immunogenicity. Advances in glycomic and structural analyses have revealed an extraordinary diversity of N-linked glycans that distinguish KLH from mammalian glycoproteins and play a central role in immune recognition. The review further discusses methods for KLH isolation, purification, and characterization, as well as its application in experimental and clinical immunology as a standardized tool for assessing antigen-specific immune responses. In addition, the therapeutic and translational potential of KLH is examined across multiple biomedical fields. Particular attention is given to its use as a carrier protein in conjugate vaccines, its role in cancer immunotherapy, and its emerging applications in the development of vaccines and immunotherapeutic strategies targeting neurodegenerative diseases, atherosclerosis, and substance use disorders. Collectively, the available evidence highlights KLH as a unique marine-derived biomolecule that bridges glycobiology, immunology, and translational medicine, and continues to serve as an important platform for the development of next-generation immunotherapeutics and vaccine technologies.

Abstract: Reliable detection of Salmonella is essential for public health surveillance and food safety, particularly given the diversity of circulating serovars and the potential limitations of individual molecular targets. This study comparatively evaluated six candidate targets (hilA, invA, phoP, fimA, rpoS, and spvA) and developed a hilA-targeting TaqMan real-time PCR assay for Salmonella detection. To the best of our knowledge, this is the first study in Kazakhstan to apply a comparative target-selection approach to the development of a hilA-based real-time PCR assay. Among the targets evaluated, hilA demonstrated the broadest amplification coverage across the tested isolate panel, including less common serovars, whereas invA was not amplified in a subset of iso-lates. The developed assay demonstrated limits of detection of 10² CFU/mL for bacterial suspensions and 10 fg/μL for genomic DNA, with an amplification efficiency of 93.4% and an R² of 0.9988 over the linear range from 1 ng/μL to 10 fg/μL. No cross-reactivity was observed within the tested non-target bacterial panel. All 24 whole-genome sequencing-confirmed Salmonella isolates yielded positive amplification within the predefined Ct threshold (≤37), including S. enterica serovar Dublin strain 25S, which showed the highest mean Ct value (36.66 ± 0.14). The Proteus mirabilis comparator, initially identified as Salmonella by conventional bacteriology, showed no specific amplification. Salmonella DNA was also detected in both artificially contaminated food matrices tested. These findings support hilA as a promising molecular target and demonstrate the potential of the developed assay for laboratory detection, food safety investigations, and epidemiological surveillance. Further validation using broader isolate collections, naturally contaminated samples, and independent laboratories is warranted.