Search Results (291)

In this study, we examined whether dietary ADY improves growth, digestibility of feed nutrients, meat quality, and rumen microbial ecology in lambs. This experiment enrolled 90 healthy, similarly weighted (29.0 ± 0.5 kg) four-month-old Duhan lambs, which were randomly and evenly distributed into two treatment groups: a control group fed the basal diet and an ADY group fed the basal diet supplemented with 0.3 g/d per lamb of active dry yeast. The supplementation amount was adjusted weekly according to feed intake to maintain a constant daily dose. The results showed that, compared with the control group, ADY significantly increased the lambs’ average daily gain (ADG) and enhanced the apparent digestibility of neutral detergent fiber (NDF), crude protein (CP) (p < 0.05), and significantly reduced the feed conversion ratio (F/G) (p < 0.05). These improvements were accompanied by a shift in rumen fermentation toward propionate production, evidenced by higher NH₃-N, Total volatile fatty acids (TVFAs) and propionate proportion and a lower acetate proportion and acetate-to-propionate ratio (p < 0.05). ADY also altered the rumen microbiota, increasing Proteobacteria and Succinivibrionaceae_UCG-001 while decreasing norank_o_Clostridia_UCG-014 (p < 0.05). In muscle, ADY significantly increased the proportions of C14:0 and C18:3n-3 (p < 0.05). In addition, the proportion of C13:0, C18:0 and C18:2n-6t were significantly reduced (p < 0.05). In conclusion, dietary supplementation with ADY enhanced rumen fermentation, improved rumen microbial composition, and promoted nutrient utilization in lambs, thereby improving growth performance and meat quality. In addition, certain rumen microbial taxa may be associated with the formation of specific muscle fatty acids. Full article

This study aimed to investigate the effects of resveratrol (RES) and carboplatin (CPT), alone and in combination, on cell viability, apoptosis, cell cycle progression, mitochondrial function, and oxidative stress in Y79 retinoblastoma (RB) cells. Particular emphasis was placed on evaluating the synergistic potential of the combination and elucidating the interconnected molecular mechanisms underlying its anticancer effects. Y79 cells were treated with RES, CPT, and their combinations. Cell viability and synergy were assessed using the MTT assay and combination index (CI) analysis. Apoptosis (annexin V/PI), cell cycle distribution (propidium iodide (PI) staining), intracellular ROS production (DCFH-DA), and mitochondrial membrane potential (JC-1) were evaluated by flow cytometry. ROS dependency was further examined using N-acetylcysteine (NAC) pretreatment. Expression levels of apoptosis- and cell cycle-related genes (BAX, BCL-2, CASP3, CASP9, CCNB1, and CDK1) were analyzed by RT-qPCR. Cytoskeletal alterations were assessed by immunocytochemistry. In addition, the antitumor effects of the combination were validated in a three-dimensional (3D) tumor spheroid model. RES and CPT reduced cell viability in a dose- and time-dependent manner and demonstrated synergistic effects (CI < 1) at selected concentrations. Combination treatment significantly increased apoptosis, induced G2/M phase arrest, enhanced ROS accumulation, and promoted mitochondrial depolarization compared with single-agent treatments. NAC pretreatment attenuated ROS generation and partially restored cell viability, supporting a contributory role of oxidative stress in combination-induced cytotoxicity. At the transcriptional level, the RES + CPT combination significantly increased the BAX/BCL-2 ratio and upregulated CASP3 and CASP9 expression, while downregulating CCNB1 and CDK1, consistent with mitochondrial apoptotic activation and G2/M arrest. Immunocytochemical analysis revealed pronounced cytoskeletal disruption and apoptotic morphology in the combination group. Importantly, in the 3D spheroid model, co-treatment markedly reduced spheroid size and viability and enhanced cell death compared with monotherapies. The combination of RES and CPT exerts a synergistic anticancer effect in Y79 RB cells through coordinated mechanisms involving ROS accumulation, mitochondrial dysfunction, caspase activation, and G2/M phase arrest. The attenuation of cytotoxicity by NAC and the validation of efficacy in a 3D tumor spheroid model strengthen the mechanistic relevance of these findings. These results support further preclinical investigation of this combination strategy in in vivo models and normal retinal cell systems. Full article

Background/Objectives: Peroxiredoxins (Prxs) are key antioxidant enzymes involved in cellular redox homeostasis. Prx6 is a multifunctional member of the Prx family that has been reported in other organisms to possess glutathione peroxidase and phospholipase A₂ (PLA₂)-related activities. However, the structural and immunological roles of 1-Cys Prx6 in crustaceans remain poorly understood. This study aimed to identify and characterize a Prx6 gene from Penaeus vannamei (PvPrx6) and to evaluate its potential involvement in antioxidant defense. Methods: PvPrx6 cDNA was identified and analyzed using bioinformatics and AlphaFold2 modeling. Tissue distribution and transcriptional responses to lipopolysaccharide (LPS), poly(I:C), and peptidoglycan (PGN) were examined by RT-qPCR. Recombinant PvPrx6 (rPvPrx6) was expressed in Escherichia coli, and its antioxidant activity was evaluated in vitro using a metal-catalyzed oxidation (MCO) assay. Results: PvPrx6 encodes a 219-amino-acid protein containing conserved AhpC/TSA and 1-Cys Prx domains. Sequence comparison and 3D modeling revealed conserved peroxidase (Thr⁴¹, Cys⁴⁴, Arg¹²⁷) and residues (His²³, Lys²⁹, Asp¹³⁵) corresponding to the reported PLA₂-associated motif. Structural analysis suggested that Lys²⁹ occupies a position corresponding to the Ser³² residue of human Prx6, although this did not imply functional equivalence. PvPrx6 transcripts were highly expressed in the lymphoid organ and hepatopancreas and were significantly induced at 12 h following immune challenge. rPvPrx6 exhibited dose-dependent protection against hydroxyl radical-mediated DNA damage under the experimental conditions. Conclusions: Collectively, these findings suggest that PvPrx6 retains conserved structural characteristics of Prx6 proteins and may contribute to antioxidant defense in P. vannamei. However, further studies are required to validate its enzymatic activity and in vivo functional roles. Full article

Background/Objectives: Retinoblastoma represents the most common intraocular malignancy in childhood; however, the clinical applicability of mitomycin C (MMC) is restricted by dose-dependent ocular toxicity. Consequently, the development of pharmacological strategies that sensitize tumor cells to MMC while allowing dose reduction remains an unmet therapeutic objective. In this context, quercetin, a bioactive flavonoid with pleiotropic anticancer properties, has emerged as a potential chemosensitizing agent. Methods: Human retinoblastoma cell lines Y79 and WERI-Rb1 were exposed to MMC and quercetin, administered either individually or in fixed-ratio combinations. Cytotoxic responses were quantified through dose–response modeling and IC₅₀ determination following 24 and 48 h of treatment. Drug–drug interactions were quantitatively characterized using the Chou–Talalay combination index (CI) approach and isobologram analysis. Cell cycle distribution was assessed by propidium iodide (PI)-based flow cytometric analysis to evaluate treatment-associated alterations in cell cycle progression. Apoptotic cell death was assessed by Annexin V-FITC/PI flow cytometry, while transcriptional modulation of genes associated with apoptosis, cell cycle regulation, and oxidative stress (BAX, BCL-2, TP53, CASP3, CDKN1A, and HMOX1) was evaluated by qRT-PCR. Modulation of tumor-supportive signaling was examined by measuring VEGF and IL-6 secretion. Translational relevance was further investigated using a three-dimensional (3D) tumor spheroid model, and the functional contribution of reactive oxygen species (ROS) was interrogated through N-acetyl-L-cysteine (NAC) rescue experiments. Results: Quercetin significantly enhanced the cytotoxic activity of MMC in both retinoblastoma cell lines, with CI values below 1 across IC₅₀–IC₉₀ effect levels, indicating a synergistic pharmacological interaction. PI–FACS analysis revealed that combined MMC and quercetin treatment induced a pronounced accumulation of cells in the G2/M phase, consistent with cell cycle arrest, with a more marked effect observed in Y79 cells compared with WERI-Rb1 cells. Combination treatment resulted in a pronounced increase in apoptotic cell populations compared with single-agent exposure and triggered a coordinated pro-apoptotic transcriptional response, characterized by increased expression of BAX, TP53, CASP3, CDKN1A, and HMOX1, alongside suppression of BCL-2 and a marked shift in the BAX/BCL-2 ratio. Concurrently, VEGF and IL-6 secretion were significantly reduced, reflecting attenuation of pro-angiogenic and pro-inflammatory signaling. Notably, synergistic cytotoxicity was maintained in 3D tumor spheroids, where combined treatment induced spheroid shrinkage, architectural disruption, and reduced viability. NAC pretreatment diminished ROS accumulation and partially restored cell viability, indicating that oxidative stress contributes to, but does not solely account for, the observed synergistic cytotoxic effect. Conclusions: Collectively, these findings indicate that quercetin appears to function as an effective chemosensitizing adjuvant to MMC in retinoblastoma models, through transcriptional changes consistent with p53-associated apoptotic signaling at the transcriptional level, G2/M cell cycle arrest, and partial involvement of ROS-related cellular stress responses, along with suppression of tumor-supportive signaling pathways. The preservation of synergistic activity in 3D tumor spheroids supports the potential preclinical relevance of this combination. However, these findings are based on transcriptional and phenotypic analyses and should be interpreted as hypothesis-generating, requiring further validation through protein-level and in vivo studies before translational application. Full article

Objective: This prospective study investigated the use of H-scan ultrasound (US) imaging as a novel component of a multiparametric radiomic analysis framework for characterizing human breast cancer response to neoadjuvant chemotherapy (NAC) before and early after treatment initiation. Methods: Thirty breast cancer patients scheduled for NAC were scanned using a clinical US system (Logiq E9, GE HealthCare) equipped with a 9L-D linear array transducer. Radiofrequency (RF) data was obtained at baseline (pre-NAC) and after 10% and 30% of the complete dose of chemotherapy. The RF data was analyzed by a bank of 256 frequency-shifted bandpass filters to form H-scan US frequency images. Grayscale texture features were extracted from both B-scan and H-scan US images. In addition, US attenuation coefficient and speckle statistics based on the Nakagami and Burr distributions were estimated from the RF data. Data classification of tumor and peri-tumoral regions was performed using a novel three-dimensional (3D) score map based on support vector machine (SVM) modeling. Unlike conventional classifiers that report only a single prediction score, a 3D score map provides a visual representation of the classifier decision space, enabling interpretation of class separation and treatment-induced shifts in multiparametric US measurements. Results: The dataset was split into 10 disjoint partitions (90% training, 10% testing) to compute area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and accuracy measures. Actual patient response to NAC was assessed at surgery and categorized as either pathologic complete response (pCR) or non-pCR. Multiparametric US and data classification results at pre-NAC found AUC values of 0.78 after using only tumor information (p < 0.01), which increased to 0.81 with inclusion of peri-tumoral information (p < 0.01). Significant differences in multiparametric US measures from both cancer response types was found after integration of patient data collected at 10% completion of the NAC regimen (i.e., first NAC cycle), yielding an improved AUC of 0.86 (p < 0.001). Conclusions: Multiparametric US imaging with radiomic features from both the tumor and peri-tumoral regions is a promising noninvasive approach for monitoring early breast cancer response to NAC. Full article

Background/Objectives: Chimpanzee adenoviral-vectored vaccines have proven to be both safe and effective, with a manufacturing and distribution pipeline capable of rapid global supply, as demonstrated during the COVID-19 pandemic. Yellow fever is a mosquito-borne viral hemorrhagic disease endemic in parts of Africa and Latin America, and although an effective live attenuated vaccine exists, its use is limited by safety and eligibility restrictions. Moreover, large outbreaks continue to expose critical challenges, such as an insufficient vaccine supply, reliance on fractional dosing, and slow and difficult-to-scale manufacturing processes. Here, we report the design, development and in vivo immunogenicity of multiple yellow fever virus (YFV) antigen constructs based on the pre-membrane (prM) and envelope (E) proteins—with or without the transmembrane domain (TM or ΔTM)—delivered using the ChAdOx1 adenoviral vector. Methods: Four ChAdOx1 YF vaccines were developed, and immunogenicity was evaluated. The efficacy of the full-length YF envelope vaccine was also tested in Balb/c mice. Results/Conclusions: In contrast to previously described orthoflavivirus vaccines on the same platform, the full-length antigen elicited superior immunogenicity and conferred protection against intracranial challenge with the YF17D virus in mice. Notably, this protection was comparable to that induced by the licensed YF17D vaccine, highlighting the promise of this platform as a next-generation yellow fever vaccine candidate. Full article

Background/Objectives: Endometrial cancer frequently develops resistance to therapy, partly due to the ability of tumor cells to adapt to cellular stress through non-apoptotic mechanisms. Mitochondrial dysfunction and cytoskeletal remodeling are increasingly recognized as key components of stress adaptation; however, their structural relationship under pharmacological stress in three-dimensional (3D) tumor models remains poorly characterized. The present study aimed to investigate the ultrastructural and phenotypic effects of lithium chloride (LiCl)-induced stress in 3D endometrial cancer spheroids, with a particular focus on mitochondrial alterations and intermediate filament organization. Methods: Three-dimensional spheroids generated from Ishikawa endometrial cancer cells were exposed to lithium chloride at concentrations of 1, 10, or 50 mM for defined time periods. Cell viability, proliferative activity, and clonogenic capacity were assessed using Trypan Blue exclusion, BrdU incorporation, and soft agar assays. Ultrastructural changes were examined by transmission electron microscopy to evaluate mitochondrial morphology, cytoplasmic organization, and intermediate filament distribution. Results: LiCl exposure resulted in a dose- and time-dependent reduction in cell viability, proliferation, and clonogenic potential in 3D spheroids. Ultrastructural analysis revealed pronounced mitochondrial swelling, cristae disorganization, and membrane-associated mitochondrial alterations. These changes were consistently accompanied by conspicuous accumulation and reorganization of intermediate filaments in close spatial proximity to damaged mitochondria, suggesting a structural association between cytoskeletal remodeling and mitochondrial injury. Across all experimental conditions, classical apoptotic ultrastructural features, including chromatin condensation and apoptotic body formation, were not observed. Conclusions: Together, these observations indicate that lithium chloride elicits a stress phenotype in 3D endometrial cancer spheroids that primarily manifests at the organelle and cytoskeletal levels, rather than through classical apoptotic execution. Although descriptive in nature, the present study highlights intermediate filament accumulation as a prominent structural feature of lithium-induced mitochondrial stress and establishes a structural reference point for future studies aimed at further investigating mitochondrial–cytoskeletal relationships during pharmacological stress in endometrial cancer. Full article

Artificial optical radiation, spanning from 100 nm to 1 mm, encompasses ultraviolet (UV) and infrared (IR) light. UV light is well known for its risks on the skin and eyes. Recently, there has been growing interest in light at 405 nm (violet-blue light, VBL) due to its antimicrobial properties and perceived safety for mammalian cells when administered in controlled amounts. This research delved into the impact of 405 nm VBL on corneal and retinal pigment epithelial cell cultures. ARPE-19 and corneal BCE C/D 1b cells were exposed to VBL for varying doses, according at different exposure times, to evaluate cell viability, oxidative stress levels and apoptotic indicators. A 3D printed prototype with 14 LEDs centred at 405 nm wavelength was used to ensure uniform distribution of light during exposure. Cell viability was assessed using the MTT assay, measurement of oxygen species (ROS) production was carried out, and Western blot analysis was employed to study catalase and SOD-1 expression and apoptotic marker activation. Exposure to 405 nm VBL for both term (3 h) and prolonged durations (9 h) led to a weak decrease in cell viability in ARPE-19 cells, whereas the effect on BCE C/D 1b cells was negligible. There was no increase in ROS production, with catalase and SOD-1 expression remaining stable, suggesting no pro-oxidative stress effects in these models. Moreover, no activation of caspase-3 and accumulation of cytochrome C were found. Based on our results, exposure to 405 nm light at regulated levels does not pose a threat to the viability of the tested cell lines and does not lead to oxidative stress and apoptosis under these conditions. These results suggest a favourable cytocompatibility profile for these specific ocular cell models, laying a foundation for further investigations into its ocular safety. Full article

Background/Objectives: 3D printing, particularly fused deposition modeling (FDM), is an emerging technology in pharmaceutical manufacturing, enabling the customization of dose or release rate to individual patient needs. However, finding the appropriate loading method to ensure the stability of the drug and achieve the targeted dose may be challenging. Furthermore, the drug utilization of most loading methods is poor, which results in considerable waste production and increased environmental burden. This study aimed to compare two post-printing drug-loading techniques: electronic syringe deposition and pan coating on FDM-printed polylactic acid (PLA) tablets. PLA is a biodegradable and biocompatible polymer that is widely used in this field due to its mechanical strength and regulatory approval. Methods: Tablets with honeycomb-shaped infill (30% and 60% infill densities) were fabricated using PLA filaments, followed by loading with a 15% paracetamol solution via either electronic syringe deposition or pan coating. The resulting tablets were assessed for drug content, weight variation, friability%, surface morphology (SEM), drug distribution (Raman mapping), solid-state characteristics (DSC and FTIR), and dissolution performance. Results: The results indicated that pan coating and electronic syringe deposition offered drug utilization up to 88% and 91.7%, respectively, which is superior to conventional soaking methods. Nevertheless, there is a significant difference in drug loading and release rate: pan coating yielded up to 10.14% drug loads and fast release (over 80% in 30 min), while electronic syringe deposition showed lower drug loading up to 4.8% and slower release (less than 80% within 60 min), which could be associated with better mechanical film integrity and higher precision. Both methods met USP standards with a weight loss of less than 1% and maintained the drug’s crystalline state and compatibility with PLA. Conclusions: FDM combined with controlled post-printing drug loading presents a rapid, cost-effective, and flexible novel approach for manufacturing personalized immediate-release tablets, with pan coating potentially being more suitable for commercial scalability and electronic syringe offering precise dosing for personalized therapies. Full article

Background/Objectives: The aim of the study was to develop a physiologically based pharmacokinetic (PBPK) model to predict gentamicin therapeutic protocols for dogs with varying degrees of renal function impairment, considering the minimum inhibitory concentrations (MICs) of the infecting bacteria. Methods: The PBPK model was built using PK-Sim^® software (OPEN SYSTEMS PHARMACOLOGY), based on pharmacokinetic data available in the literature and information on the physicochemical properties of the drug. Model evaluation included the calculation of the geometric mean fold error (GMFE), weighted and percentage residuals were calculated, as well as the following measures: AFE, AWRi, MWRi, MAWRi, APE%, MPE%, MAPE%, MdPE%, and MdAPE%. Therapeutic efficacy was assessed according to the Probability of Target Attainment (PTA), considering an MIC distribution of 0.25 to 8 μg/mL for different doses (2, 4, 6, 8, and 10 mg/kg) using the PK/PD indices C_max/MIC ≥ 10, AUC/MIC ≥ 50, and AUC/MIC ≥ 110. To compare the pharmacokinetics of gentamicin between healthy dogs and those with decreased renal function, different GFR values corresponding to stages of renal impairment were used, as determined by clinical biomarkers (microalbuminuria, UPC ≥ 2, sCr ≥ 1.2 mg/dL, sCr ≥ 2.4 mg/dL, and sCr ≥ 5 mg/dL). The risk of toxicity was assessed according to AUC_24h ≥ 700 mg·h/L and C_min ≥ 0.5. Results: The model demonstrated good predictive performance, with a GMFE value of 1.13 meeting the double error criterion, and weighted residuals randomly distributed around 0 (p = 0.3792). Through the calculation of PTA, it was observed that efficacy varied according to the PK/PD index used, but values greater than 90% were obtained for MICs up to 4 μg/mL. The model allowed the estimation of protocols for each stage of renal impairment, considering the GFR of each group and the risk of nephrotoxicity, in association with the optimal dose to ensure therapeutic efficacy. Conclusions: These findings make it possible to propose a dose for the treatment of an infection, considering the MIC and the patient’s GFR stage, thereby reducing the risk of adverse effects without compromising treatment efficacy. Full article

Despite the clinical success of antibody–drug conjugates (ADCs), their efficacy in solid tumors remains constrained by limited tumor penetration of the IgG format. Smaller antibody fragment–drug conjugates (FDCs) present a compelling alternative, potentially offering superior intratumoral distribution and a wider therapeutic window driven by rapid systemic clearance. This study compares therapeutic activity of ganglioside GD2-specific minibody–drug conjugates against full-length ch14.18 antibody–drug conjugates, and biodistribution of the respective minibody (scFv-C_H3 homodimer) and IgG formats in the GD2-positive B78-D14 melanoma syngeneic mouse model. We conjugated the minibody and antibody with MMAE or MMAF via a cathepsin-cleavable linker, generating FDCs with drug–antibody ratio (DAR) of 2 and ADCs with DAR of 2 or 4. The biodistribution analysis showed no significant difference in tumor uptake for both formats early in the analysis (2–4 h) and a higher tumor uptake for the IgG at 24 h post-injection. However, the minibody achieved a superior tumor-to-blood ratio (TBR) at all timepoints, reaching a TBR > 1 compared to ~0.2 for the antibody by 24 h. In vitro studies demonstrated higher cytotoxicity for the ADCs regardless of drug load (DAR 2 or 4) compared to the FDCs, although the difference between conjugates with equal DAR was modest in B78-D14 cells. Critically, superior in vitro ADC potency did not translate in vivo. Minibody–MMAF and minibody–MMAE achieved 74% and 55% tumor growth inhibition, respectively, by the study endpoint—demonstrating comparable efficacy to ADCs with twice the drug load when administered to mice at equimass dosing. Stron/g in vivo efficacy of anti-GD2 FDCs, combined with the superior TBR for the minibody format, underscores the potential of minibody–drug conjugates for treating GD2-positive tumors, particularly when ADC-associated toxicity precludes high-dose regimens. Full article

Shutdown dose rate (SDDR) assessments have been performed for the DEMO tokamak model, including the latest design and environmental configurations. The main objective of this study was to evaluate the shutdown radiation fields and establish dose rate limits to ensure safe personnel access to the Vacuum Vessel (VV) and nearby components. The simulations were based on the DEMO baseline model, further refined with the minor updates of the lower port, equatorial port limiter, and upper port assemblies. The computational approach employed the Monte Carlo particle transport code MCNP for neutron and photon transport calculations, coupled with the activation and decay code FISPACT-II to determine time-dependent decay gamma source terms. The mesh-coupled Rigorous Two-Step (R2Smesh) methodology developed in KIT was applied to achieve spatially resolved decay of photon source distributions and to compute corresponding SDDR 3D maps within the DEMO reactor configuration. The results provide a detailed characterization of the residual radiation environment inside the VV, offering insight into the accumulated activity, shielding performance of different materials, and potential access scenarios for maintenance operations in next-generation fusion devices. Full article

Obesity is a major global health challenge that substantially affects vitamin D metabolism and status. Numerous studies have consistently demonstrated an inverse relationship between body fat and serum 25-hydroxyvitamin D [25(OH)D] concentrations. Emerging evidence suggests that lower serum 25(OH)D in obesity largely reflects altered distribution and metabolism rather than a uniform state of true functional deficiency. Adipose tissue functions both as a storage compartment and as a metabolically active organ capable of modulating vitamin D handling. Mechanisms include the sequestration of vitamin D in fat, volumetric dilution across a larger body mass, and the local expression of enzymes involved in vitamin D metabolism. As a result, obese individuals typically exhibit a blunted increase in serum 25(OH)D in response to supplementation, consistent with altered pharmacokinetics and increased distribution volume. Weight loss, particularly the reduction in visceral fat, is associated with modest increases in circulating 25(OH)D, further supporting a distribution-based mechanism. Although low 25(OH)D levels in obesity have been linked to insulin resistance, inflammation, and metabolic syndrome, randomized controlled trials have not consistently demonstrated that supplementation improves clinically relevant outcomes in this population. Meta-analyses confirm that the increase in serum 25(OH)D after supplementation is smaller in obese individuals, indicating that higher doses are often required to achieve comparable levels to those in normal-weight subjects. Obesity thus represents a major determinant of vitamin D deficiency, highlighting the need for individualized supplementation strategies alongside weight management. Understanding the mechanistic basis for low 25(OH)D in obesity is essential for distinguishing true deficiency from altered distribution, informing clinical decisions, and optimizing interventions to maintain adequate vitamin D status and support metabolic health. Full article

Background/Objectives: Topical treatment of cutaneous tuberculosis (CTB) requires reliable models to evaluate dermal drug release and diffusion, particularly for fixed-dose combinations (FDCs) with contrasting physicochemical properties. Human skin remains the reference standard but poses ethical, logistical, and reproducibility challenges. This study investigated the suitability of Strat-M^® synthetic membranes as an alternative to human skin for assessing the simultaneous release and diffusion of clofazimine (CFZ) and pyrazinamide (PZA) from a topical FDC, and aimed to develop an optimized dermal emulsion using a Quality-by-Design (QbD)-informed formulation development tool. Methods: Self-emulsifying dermal emulsions containing CFZ and PZA were developed following QbD principles. Preformulation studies included drug solubility screening, oil phase selection, and pseudoternary phase diagram construction to identify stable emulsion regions. Formulations were characterized for droplet size, polydispersity index, zeta potential, viscosity, self-emulsification efficiency, and thermodynamic stability. Eight stable emulsions were identified, of which four were selected for in vitro drug release studies. The peppermint oil-based emulsion (PPO415) was further evaluated in comparative diffusion studies using Strat-M^® membranes and ex vivo human skin (Caucasian and African). Results: PPO415 demonstrated favorable physicochemical properties, including high CFZ solubility, uniform droplet distribution, and suitability for dermal application. Comparative diffusion studies showed that Strat-M^® underestimated the partitioning of lipophilic CFZ while overestimating the diffusion of hydrophilic PZA relative to human skin. These differences were attributed to compositional and structural disparities between synthetic membranes and biological skin. Conclusions: Strat-M^® membranes show potential as a reproducible and ethical in vitro screening tool during early-stage formulation development for topical FDCs. However, ex vivo human skin remains essential for accurately predicting dermal drug distribution and therapeutic performance. Full article

Background: Streptococcus pneumoniae is a leading cause of child mortality in Nepal despite the introduction of the 10-valent pneumococcal conjugate vaccine (PCV10). Vaccine effectiveness is threatened by the emergence of non-vaccine serotypes (NVTs) and the multiple serotypes carriage which often fail to be detected by traditional methods. We aimed to study changes in serotype distribution before and after PCV10 immunization among infants, including serotype dominance in Nepalese infants in the post-vaccine era. Methods: We enrolled infants in a longitudinal cohort study (2020–2022) conducted in Bhaktapur, Nepal. Nasopharyngeal swabs were collected before PCV10 dose 1 (6 weeks) and at 9 and 12 months post-immunization. We used a sensitive nanofluidic qPCR platform to detect multiple serotypes and establish their hierarchy by quantifying the bacterial load of each strain. Inverse Probability Weighting (IPW) adjusted risk factor analysis was used to account for loss to follow-up. Results: PCV10 successfully reduced vaccine-type (VT) carriage, declining sharply from 32.8% at 6 weeks to 4.8% at 12 months. VTs were pushed from being the dominant strain to occupying subdominant roles in co-colonization. Conversely, NVTs rapidly filled the vacated niche, showing a significant increase in their dominant status (p < 0.001). The most common replacing NVTs that rose to dominance were 35B, 19A, 6C/6D, and 15B/15C. Significant risk factors for carriage included older infancy (aOR 3.4, 95%CI: 2.6–4.5 at 9 months), a household kitchen in the living area (aOR 1.4, 95%CI: 1.0–1.9), and winter (aOR 1.7, 95%CI: 1.5–2.7) and pre-monsoon seasons (aOR 2.0, 95%CI: 1.5–2.8). Conclusions: While PCV10 reduced overall VT circulation, the persistence of VTs in subdominant niches creates a continuous reservoir for potential re-emergence and antibiotic resistance. This clear hierarchical shift in dominance towards NVTs underscores the urgent need for a public health strategy that includes the adoption of a higher-valent PCV to provide broader protection, and interventions targeting environmental risk factors are essential to sustain long-term reductions in pneumococcal colonization. Full article