Search Results (1,915)

Herein, we report an unexpected pseudo-multicomponent transformation discovered during attempts to selectively nitrate the pyridine core of 3-acetyl-2,4,6-trimethylpyridine (3). Despite employing standard nitration conditions, including KNO₃–H₂SO₄ and HNO₃–H₂SO₄ mixtures, electrophilic substitution of the aromatic ring did not occur. Instead, the reaction sequence promoted an in situ nitrozation, dehydration to nitrile oxide intermediates, and subsequent [3+2]-cycloaddition involving two substrate molecules. This process yielded a novel, highly functionalized furoxan derivative, precisely identified as 3,4-bis(2,4,6-trimethylnicotinoyl)-1,2,5-oxadiazole 2-oxide (5). The molecular architecture of compound 5 was established by ¹H and ¹³C NMR spectroscopy, mass spectrometry, elemental analysis, and single-crystal X-ray diffraction (XRD) analysis. To elucidate the stereochemical and electronic features governing compound 5, DFT calculations were performed at the ωB97X-D/6-311++G(d,p) level of theory. The experimental crystallographic disorder of the N-oxide oxygen atom was computationally rationalized by the thermodynamic near-degeneracy (ΔG < 0.63 kcal/mol) of two orientational isomers (5a and 5b). Furthermore, frontier molecular orbital analysis within the framework of perturbation theory accounted for the head-to-tail regioselectivity during cyclization, while wide energy gaps (ΔE = 8.13–8.27 eV) and high chemical hardness (η = 4.07–4.14 eV) underscored the kinetic stability of the heterocycle. Phenotypic and target-specific in silico profiling using PASS Online identified Matrix Metalloproteinase-9 (MMP-9) as a relevant target for potential hemorheological and cardioprotective applications. Validated molecular docking simulations across three human MMP-9 crystallographic domains (PDB: 8K5Y, 6ESM, 4XCT) demonstrated competitive binding affinities and balanced Ligand Efficiency metrics (LE = 0.26–0.29 kcal/mol/heavy atom), anchoring compound 5 within the catalytic pocket via conventional hydrogen bonds and π-mediated interactions. Finally, in vitro evaluations using a blood hyperviscosity model confirmed significant hemorheological efficacy, as compound 5 effectively prevented the rise in blood viscosity, outperforming the reference drug pentoxifylline. The convergence of computational insights and experimental functional activity establishes this novel bis(nicotinoyl)furoxan framework as a promising candidate for further hemorheological and cardioprotective applications. Full article

As the prevalence of type 2 diabetes increases rapidly, the demand for natural origin dipeptidyl peptidase-IV (DPP-IV) inhibitors with fewer side effects is increasing. In this study, protein-rich Limnospira platensis was investigated as a source of bioactive peptides to enhance its value as a functional food ingredient. Hydrolysates were prepared using three food-processing proteases, individually and in two-step combinations, followed by in silico analysis and peptide identification via liquid chromatography–tandem mass spectrometry. Subsequently, the thermal stability, gastrointestinal resistance, and intestinal permeability (using Caco-2 cells) of the identified peptides were evaluated. It was revealed that the Orientase 22BF digest exhibited high DPP-IV inhibitory activity. From the digest, three novel peptides—SPSPN (IC₅₀ = 144.1 ± 2.2 μM), VPSV (IC₅₀ = 93.6 ± 5.8 μM), and IPIGG (IC₅₀ = 13.4 ± 2.3 μM)—were identified, exhibiting DPP-IV inhibitory potencies comparable to or higher than previously reported Limnospira-derived peptides. Although VPSV exhibited low epithelial permeability (P_app = 4.02 ± 0.69 × 10⁻⁸ cm/s), it remained stable under simulated gastrointestinal digestion conditions, suggesting potential local luminal inhibitory activity within the small intestine. Overall, these findings highlight Limnospira-derived VPSV as a promising functional ingredient candidate with high bioactivity and digestive stability. Full article

RNA aptamers are often used as ligand-recognition modules in engineered RNA systems, but integration into larger RNA constructs can influence stability and ligand binding. As a result, aptamer sequences may need to be adapted to new environments while preserving essential properties. Here, we examine this sequence editability problem for the theophylline RNA aptamer. Starting from the experimentally determined structure, we introduced targeted mutations in peripheral structural elements while leaving the recognition site unchanged. The native aptamer, mutated variants, a Mg²⁺-depleted system, and a caffeine-bound control were analyzed using three independent 1 $\mathsf{\mu }$s molecular dynamics simulations. Binding energetics were estimated with multiple end-point as well as alchemical free energy approaches. Results were interpreted together with base pair stability, the conformational landscape of the binding pocket, and per-nucleotide energy contributions. This allows us to predict whether an edit is tolerated or disruptive. Some mutations retained structural and energetic profiles comparable to the native aptamer, whereas others reduced ligand affinity by propagating structural distortions into the binding pocket. These results show that sequence changes outside the binding site can modulate ligand binding indirectly, and that the ligand interaction network is useful for evaluating edited aptamers. The introduced workflow provides a novel combination of established computational strategies for efficient in silico screening of aptamer variants before experimental testing and can be integrated into the design of larger RNA structures. This works particularly well when an experimental structure is available and the tested mutations are small enough not to disrupt the folding pathway. Full article

Crinum amoenum Roxb. ex Ker Gawl. (C. amoenum) is traditionally used in Nepal, but its antidiabetic potential remains insufficiently validated. This study characterized the 80% (v/v) ethanol bulb extract of C. amoenum and evaluated its antioxidant, α-amylase inhibitory, hypoglycemic, molecular docking, molecular dynamics (MD) simulation, and ADMET profiles. The ethanolic bulb extract was evaluated through phytochemical screening, thin-layer chromatography, total phenolic content (TPC), total flavonoid content (TFC) estimation, LC-MS analysis, antioxidant assays, α-amylase inhibition assays, acute toxicity testing, and hypoglycemic/oral glucose tolerance test (OGTT) studies in Wistar rats. Lycorine, pratorinine, and palmatine were docked against α-amylase (PDB ID: 5U3A) and GLP-1R (PDB ID: 6GB1), followed by 200 ns MD simulations and ADMET prediction. Qualitative analysis of the extract was positive for flavonoids, phenolic compounds, tannins, saponins, alkaloids, and carbohydrates, and a positive Salkowski reaction indicated the presence of constituents occurring in glycosidic form. The TPC was 173.38 ± 1.22 mg GAE/g, and the TFC was 314.58 ± 0.09 mg QE/g. LC-MS tentatively annotated lycorine, pratorinine, and palmatine based on retention time, m/z values in positive-mode electrospray ionization, and comparison with previously reported spectral information. The extract showed DPPH scavenging activity (IC₅₀: 90.98 μg/mL) and strong α-amylase inhibition (IC₅₀: 49.31 μg/mL) compared with acarbose (IC₅₀: 51.51 μg/mL). No deaths were observed following oral administration at 5000 mg/kg to rats. The 500 mg/kg dose produced the greatest hypoglycemic response, reducing blood glucose by 35.78% at 120 min in non-diabetic Wistar rats and by 19.21% at 60 min in glucose-loaded rats. Among all the compounds tested, pratorinine demonstrated the highest docking affinity with α-amylase (−8.2 kcal/mol; ASP300) and GLP-1R (−7.0 kcal/mol; GLY132). MD simulation supported greater stability of the α-amylase complex, and ADMET prediction identified pratorinine as a comparatively favorable predicted lead candidate (LD₅₀: 1000 mg/kg; Class 4 toxicity category). Pratorinine was identified as the most promising in silico antidiabetic candidate from the C. amoenum extract, primarily due to stable α-amylase interactions and favorable ADMET properties. Full article

Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects mainly peripheral joints because of inflammation of the synovial membrane. Current treatments, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, although effective, are associated with high costs and several adverse effects. In this context, natural products have emerged as promising alternatives because of their potential therapeutic effects and lower toxicity. The objective of this review was to identify and evaluate natural substances with potential applications in RA treatment on the basis of studies published between 2015 and 2020. A literature search was conducted in SciELO, PubMed, and Google Scholar using the keywords “rheumatoid arthritis”, “treatment”, “toxicity”, and “natural products”. Additionally, we applied in silico methods to predict pharmacokinetic and toxicological parameters using ADMET Predictor^® (Simulation Plus) and compared the results with those of commercial drugs such as diclofenac, ibuprofen, and naproxen. Target fishing (reverse docking) was also performed to identify possible molecular targets related to RA. Seven natural compounds were identified, mostly evaluated through in vivo studies. Among them, paeoniflorin, quercetin, resveratrol, and celastrol are in clinical phases and present potential as RA treatments. In silico analysis highlighted curcumin, tetramethylpyrazine, and resveratrol as the most promising candidates, with ADMET profiles comparable or superior to those of current NSAIDs. In conclusion, natural products represent viable alternatives for RA therapy. However, further studies are essential to better understand their safety, pharmacokinetics, and drug interactions to ensure their clinical applicability. Full article

Osteoarthritis (OA) causes irreversible cartilage damage, highlighting the need for early and sensitive assessment. Current imaging modalities are limited in detecting early-stage changes. Ultrasound (US) provides a non-invasive and accessible alternative, but its clinical adoption is limited by the lack of standardized protocols and reliable cartilage assessment. Simulations can be used to address these challenges by enabling system design, acquisition optimization and validation by providing ground truth when in vivo ground truth is unavailable. The aim of this study is to develop an in silico framework for realistic US imaging of healthy and OA cartilage by combining accurate acoustic wave modeling with a 2D microstructural cartilage phantom. The model was calibrated to healthy cartilage using first-order speckle statistics and extended to simulate degeneration through changes in structural and acoustic properties. As a proof-of-concept study, simulations were evaluated against limited ex vivo US data from healthy and OA cartilage and compared with literature data. The simulations reproduced key OA-related features and trends, including changes in reflection coefficient (R), integrated reflection coefficient (IRC), apparent integrated backscatter (AIB), and gray level distributions. These findings demonstrate the feasibility of using microstructure-based tissue phantoms to model healthy and OA cartilage. The framework provides a platform for systematic investigation of cartilage microstructure and US-derived features and may support future generation of synthetic datasets for data-driven and AI-based OA assessment. Full article

Background Aflatoxin B1 (AFB1) is a potent hepatotoxic mycotoxin that induces severe oxidative liver damage. Kaempferol (Kae), a natural flavonoid with known antioxidant properties, has unclear protective effects against AFB1-induced hepatotoxicity. This study aimed to evaluate the hepatoprotective role of Kae and elucidate its underlying mechanism using integrated in vivo, in silico, and in vitro approaches. Methods: In vivo (AFB1-challenged mice) and in vitro (hepatocyte) models were employed, combined with network pharmacology, molecular docking, and molecular dynamics simulations. Liver injury indices, oxidative stress markers, antioxidant enzyme activities, and Keap1/Nrf2 pathway expression were assessed. Results: Kae co-treatment reversed AFB1-induced increases in liver index, serum ALT/AST, histological lesions, and reduced antioxidant capacity in mice. Network pharmacology revealed 59 common targets, with NFE2L2 (Nrf2) as a key node. In vitro, Kae pretreatment significantly lowered AFB1-elevated ROS, MDA, ALT, and AST, while restoring GSH and total antioxidant capacity. Kae reversed AFB1-induced Keap1 upregulation and Nrf2 downregulation, and increased mRNA levels of HO-1, NQO1, SOD, GPX1, and CAT. Molecular docking and simulation showed stable Kae–Keap1 binding (−9.6 kcal/mol) with critical hydrogen bonds (VAL-606) and van der Waals contacts. Conclusions: Kae directly binds Keap1, activates Nrf2 signaling, upregulates antioxidant gene expression, and mitigates AFB1-induced oxidative liver injury. These findings support Kae as a promising candidate for preventing AFB1 hepatotoxicity. Full article

Background/Objectives: Benzimidazole and hydrazone scaffolds are widely investigated in anticancer drug discovery because of their structural versatility and capacity to interact with multiple cancer-related targets. This study aimed to synthesize a series of trifluoromethyl-substituted benzimidazole-hydrazone derivatives, evaluate their antiproliferative activity and cancer-cell selectivity, and explore their potential interactions with breast cancer-associated molecular targets. Methods: A series of benzimidazole-hydrazone derivatives were prepared through a four-step synthetic route using both conventional and microwave-assisted procedures. The structures of the synthesized compounds were characterized using spectroscopic and analytical methods. Antiproliferative activity was evaluated in estrogen receptor-positive MCF-7 human breast cancer cells using the MTT assay after 48 h of exposure. Selected active compounds were additionally tested against non-tumorigenic hTERT cells to determine their selectivity indices. In silico analyses were performed against estrogen receptor alpha in antagonist and selective estrogen receptor degrader conformations, bromodomain-containing protein 4, and the mTOR kinase domain. Results: Microwave irradiation substantially reduced reaction times from 12–24 h to 4–6 min and increased isolated yields by 7–24 percentage points compared with conventional conditions. Compounds 4, 7, 9, and 11 reduced MCF-7 cell viability below 50% at 20 μM. Compound 7 showed the highest antiproliferative activity, with an IC₅₀ value of 7.5 ± 0.8 μM, and the greatest selectivity toward MCF-7 cells over hTERT cells, with a selectivity index of 12.66. Molecular docking, MD simulation, and MM/GBSA calculations predicted that compound 7 would effectively bind to the investigated targets, particularly estrogen receptor alpha, as well as the BRD4 and mTOR kinase domains. Conclusions: Compound 7 represents a selective, mid-micromolar benzimidazole-hydrazone lead against MCF-7 breast cancer cells. Its predicted molecular interactions warrant further target-based, mechanistic, pharmacokinetic, and in vivo evaluation. Full article

Background/Objectives: The clinical management of visceral pain remains a significant challenge. Our previous study confirmed the analgesic efficacy of dexmedetomidine (Dex) in a mouse model of inflammatory visceral pain (IVP). However, the underlying molecular mechanism, particularly regarding immune regulation, has not been fully elucidated. Methods: In the present study, transcriptomic profiling of both physiological and disease states was performed to characterize associated molecular and immune signatures. Key driver genes were identified by integrating differentially expressed genes (DEGs) from the IVP model with potential Dex targets derived from network pharmacology. Molecular docking simulations evaluated binding interactions. The correlations between Dex’s targets and immune cell infiltration patterns, with a focus on macrophages, were analyzed. Results: Multi-tissue analysis revealed a strong association between IVP pathogenesis and pro-inflammatory immune responses, specifically a shift in macrophage polarization. Within this dysregulated network, seven proteins were identified as direct potential targets of Dex. Among these, six core targets, such as ADGRF1, IDO1, JAK3 and NR1H4, demonstrated the most significant correlations with M1-like macrophages and exhibited strong in silico binding potential with Dex. Conclusions: This integrated analysis suggests that Dex may alleviate visceral pain by modulating a specific gene network linked to pro-inflammatory macrophage activation, thereby promoting the restoration of immune balance. Our findings provide a novel mechanism-informed perspective for the application of Dex in visceral pain therapy. Full article

Endophytic bacteria from medicinal plants are increasingly recognised as sources of antimicrobial metabolites. However, the endophytic bacterial community of Mitrephora heyneana remains poorly explored. In the present study, endophytic bacteria were isolated from the leaves of M. heyneana, collected from the Western Ghats of Tamil Nadu, India. Among seven isolates, the plant endophyte strain from leaf 6th strain (PEL6) was identified as Paenibacillus lactis through 16S rRNA gene sequencing. The ethyl acetate extract of PEL6 (EAE-PEL6) was subjected to gas chromatography–mass spectrometry (GC-MS) analysis, which putatively identified 32 metabolites based on GC-MS library matching, including pyrrolo [1,2-a] pyrazine-1,4-dione derivatives and triazole compounds as major constituents. The EAE-PEL6 demonstrated significant in vitro antibacterial activity against Staphylococcus aureus. In silico ADMET (absorption, distribution, metabolism, excretion, and toxicity), profiling predicted drug-likeness and pharmacokinetic properties of selected candidate compounds. Molecular docking suggested favourable binding of selected metabolites to S. aureus target proteins; however, these interactions require experimental validation. Density Functional Theory calculations indicated that CID 70504 had the lowest Highest Occupied Molecular Orbital (HOMO)–Lowest Unoccupied Molecular Orbital (LUMO) energy gap, reflecting higher electronic reactivity. Molecular electrostatic potential mapping further supported its enhanced binding propensity. Molecular dynamics simulations suggested structural stability, with Root Mean Square Deviation, Solvent Accessible Surface Area, radius of gyration, and hydrogen-bond analyses indicating stable interactions throughout the 100 ns trajectory. Overall, this study identifies P. lactis PEL6 as a promising endophytic source of anti-S. aureus metabolites and provides candidates for future purification, structural confirmation, and biological validation. Full article

Four sesquiterpenes, namely tomentosin, 11α,13-dihydrotomentosin, inuviscolide, and α-costic acid, were isolated from the aerial parts of the medicinal plant Dittrichia viscosa (L.) Greuter and evaluated for antiproliferative activity against HCT116 colorectal and A549 lung cancer cells. All compounds reduced cell viability in a concentration-dependent manner, with tomentosin and inuviscolide exhibiting the greatest activity among the tested compounds and IC₅₀ values in the moderate micromolar range. Evaluation in non-malignant human neonatal dermal fibroblasts (HDFn) revealed compound- and cancer-type-dependent selectivity, with inuviscolide showing greater selectivity toward HCT116 cells and tomentosin showing moderate selectivity toward both cancer cell lines. In silico ADMET and DFT analyses characterized the predicted drug-likeness, pharmacokinetic, toxicity-related, and electronic properties of the compounds, particularly tomentosin and inuviscolide. Network pharmacology prioritized STAT3, ESR1, and PTGS2 as candidate hub targets associated with both cancer types, while enrichment analyses identified pathways related to tumor proliferation, inflammation, immune regulation, and cellular stress. Noncovalent molecular docking, 200 ns molecular dynamics simulations, and MM/GBSA calculations supported possible modeled interactions of tomentosin and inuviscolide with these proteins, with tomentosin displaying the most favorable predicted binding profile. Covalent docking further identified structurally feasible STAT3 CYS687-linked poses with the two sesquiterpenes, although covalent adduct formation and direct target engagement remain to be experimentally validated. Overall, the integrated findings prioritize tomentosin and inuviscolide as D. viscosa-derived natural-product scaffolds with moderate antiproliferative activity for further structural optimization, selectivity assessment, and experimental mechanistic validation. Full article

Phosphatidylinositol 5-phosphate 4-kinase type 2 alpha (PIP4K2A) is a key oncogenic driver that regulates the PI5P/PIP2 axis to promote metastatic migration in breast cancer. This study aimed to investigate the therapeutic potential of a crude extract from Tamarix aphylla against breast cancer progression and identify its primary active constituents. The crude extract was initially evaluated against MDA-MB-231 and MCF7 breast cancer cell lines using wound healing assays. Bioassay-guided isolation and screening were deployed to isolate individual components, and the most potent lead compound was structurally characterized using preparative HPLC and FTIR. To analyze its interaction with PIP4K2A, in silico molecular docking, MM/GBSA calculations, and 200 ns molecular dynamics simulations were conducted. In vitro validation was subsequently performed via dose-dependent cytotoxicity assays, scratch assays, single-cell tracking, and RT-qPCR expression analysis. Quercetin was identified as the most potent lead inhibitor against PIP4K2A. Computational modeling revealed that quercetin binds tightly within the PIP4K2A ATP-binding pocket, yielding a superior binding affinity of −10.77 kcal/mol and enhanced thermodynamic stability (ΔGMM/GBSA = −42.6 ± 2.1 kcal/mol) compared to the native ligand (ΔG MM/GBSA = −23.3 ± 1.8 kcal/mol). Molecular dynamics simulations confirmed an induced-fit structural transition that locked the complex into an ultra-stable conformation within a deep global energy minimum basin (−10.8 kcal/mol). In vitro assays demonstrated dose-dependent cytotoxicity, with aggressive triple-negative MDA-MB-231 cells exhibiting higher sensitivity (IC₅₀ = 82.23 µg/mL) than luminal MCF7 cells (IC₅₀ = 97.14 µg/mL). Furthermore, scratch and single-cell tracking assays showed a profound suppression of migration speed and wound closure (reduced to ~40%), while RT-qPCR revealed a near-complete transcriptional knockdown of PIP4K2A mRNA expression (down to 0.025-fold). Collectively, these findings elucidate a unique dual-action mechanism for Tamarix aphylla-derived quercetin—characterized by both direct competitive enzymatic inhibition and downstream transcriptional silencing—positioning it as a promising therapeutic scaffold for targeted anti-metastatic breast cancer interventions. Full article

The discovery of angiotensin-converting enzyme (ACE)-inhibitory peptides from food protein hydrolysates is commonly guided by empirical fractionation or sequence-based prediction, but few screening rules have been evaluated at the hydrolysate level and independently benchmarked across peptide lengths. Here, we developed a length-aware C-terminal screening rule (Rule 5: P1’ ∈ {W, Y, F, P} and P2’ ∈ {L, I, V, K, R, H}) through an experimentally anchored framework. The rule was derived using 24 stratified protein–protease hydrolysates and showed the strongest associations with ACE inhibition (r = 0.711, p < 0.001) and log₁₀(1/IC₅₀) (r = 0.741, p < 0.001) among five evaluated rules. Independent evaluation in 16 composition-weighted commercial hydrolysates retained predictive utility (r = 0.608 for ACE inhibition and r = 0.581 for log₁₀(1/IC₅₀)). Five peptides—VF, GIF, LP, IP, and VP—were selected because they represented the intersection of in silico cleavage prediction, Rule 5 compliance, and corresponding candidate-associated low-mass MALDI features in the experimentally prepared hydrolysates. All five inhibited ACE (IC₅₀ = 19.50–93.19 µM); VF and GIF showed mixed-type inhibition, whereas LP, IP, and VP showed competitive inhibition. External benchmarking against 1429 quantitative ACE-inhibitory peptides established a defined applicability domain: Rule 5 significantly enriched potent peptides among di- and tripeptides (2–3 residues; median IC₅₀, 28.0 vs. 79.0 µM; p_adj < 0.001, Benjamini–Hochberg-corrected; enrichment factor = 2.5 at IC₅₀ ≤ 1 µM), but enrichment attenuated rapidly as longer sequences were included. Molecular dynamics simulations (200 ns) showed persistent peptide–ACE contact for all five candidates under the simulated conditions. Rule 5 is therefore proposed as a transparent first-pass filter for prioritizing short ACE-inhibitory candidates and protein–protease combinations, rather than as a universal predictor across the full peptide-length spectrum. Full article

A previous investigation on limonene (LM), perillyl alcohol (POH), and carveol (CV), focused on the structure–activity relationship and hydroxyl group, documented that the presence of the hydroxyl group influences the pharmacodynamic potency of these agents, inhibiting smooth muscle contraction with the order of potency: POH > CV > LM. That investigation also suggested a mechanism of action, which importantly included activity on the voltage-dependent calcium channel. Here, we investigated whether this structure–activity relationship also applies to nerve excitability (an activity greatly dependent on sodium channels) using compound action potential (CAP) recordings from mouse sciatic nerves and in silico simulations. POH, CV, and LM inhibited both the positive amplitudes and conduction velocities of the two CAP components in a concentration-dependent manner, with IC₅₀ values of 0.8, 1.0, and 4.3 mM (1st component) and 0.6, 0.6, and 3.0 mM (2nd component) for amplitude, and 2.4, 2.4, and 7.1 mM (1st component) and 1.0, 2.6, and 4.3 mM (2nd component) for conduction velocity. The order of pharmacodynamic potency, thus, was POH = CV > LM. In silico simulation demonstrated that POH and CV penetrate the Nav 1.6 and accommodate in the channel at the interface between the selectivity filter and the central cavity, a position very favorable to block the channel pore. In contrast, LM exhibited a markedly different docking profile, suggesting that LM binding is less likely to directly obstruct sodium permeation. In conclusion, the three substances investigated inhibited nerve excitability, but those with a hydroxyl group demonstrated greater pharmacodynamic potency. Full article

Hemp hurds (HHs), an underutilised lignocellulosic biomass (LB) from agricultural waste, offer potential for bioconversion into high-value bioproducts within a circular bioeconomy. Building on prior work involving magnetic nanoparticle-immobilised cellulase hydrolysis of pretreated HH, this study computationally designed candidate phaCAB expression constructs for cellulolytic hosts toward future polyhdroxybutyrate (PHB) production. The objective was to evaluate, in silico, the feasibility of introducing the phaC1, phaA and phaB1 genes from Cupriavidus necator H16 (C. necator) (assembly GCA_000009285.2; loci H16_A1437–H16_A1439) into the cellulolytic hosts Clostridium thermocellum DSM 1313 (C. thermocellum) and Trichoderma reesei RUT C-30 (T. reesei). Coding sequences were retrieved and translated individually and host-specific expression compatibility was assessed via codon adaptation index, effective number of codons, GC content and rare-codon frequency/clustering. Host-specific architectures were designed: three independent tef1-promoter cassettes with fungal Kozak contexts and cbh1 terminators for T. reesei (TrePHB3 integration construct), and a single groEL-promoter operon with graded ribosome-binding sites for the pIKM1-based C. thermocellum construct (pCtPHB1); Escherichia coli BL21 (DE3) (E. coli)/pET-24a(+) served only as an intermediate assembly platform. Clustal Omega alignment, virtual plasmid assembly and simulated restriction digestion (SnapGene) confirmed preservation of the open reading frames and expected fragment sizes. ProtParam analysis indicated instability indices below 40 and negative GRAVY values for PhaC, PhaA and PhaB, indicating overall hydrophilic character. This computational framework links HH valorisation, cellulolytic Consolidated Bioprocessing (CBP) hosts and PHB pathway design within a conceptual biorefinery, supporting future integrated biomass-valorisation platforms; it is computational only and does not demonstrate transformation, expression, PHB accumulation or biomass conversion, which require experimental validation. Full article