Retinal Explant Culture from Mouse, Human, and Nonhuman Primates and Its Applications in Vision Research.

The retinal explant culture system is a valuable tool for studying the pharmacological, toxicological, and developmental aspects of the retina. It is also used for translational studies such as gene therapy. While no photoreceptor-like cell lines are available for in vitro studies of photoreceptor cell biology, the retinal explant culture maintains the laminated retinal structure ex vivo for as long as a month. Human and nonhuman primate (NHP) postmortem retinal explants cut into small pieces offer the possibility of testing multiple conditions for safety and adeno-associated viral (AAV) vector optimization. In addition, the cone-enriched foveal area can be studied using the retinal explants. Here, we present a detailed working protocol for retinal explant isolation and culture from mouse, human, and NHP for testing drug efficacy and AAV transduction. Future applications of this protocol include combining live imaging and multiwell retinal explant culture for high-throughput drug screening systems in rodent and human retinal explants to identify new drugs against retinal degeneration.

Novel pectin-carboxymethylcellulose-based double-layered mucin/chitosan microcomposites successfully protect the next-generation probiotic Akkermansia muciniphila through simulated gastrointestinal transit and alter microbial communities within colonic ex vivo bioreactors.

The rapid acceleration of microbiome research has identified many potential Next Generation Probiotics (NGPs). Conventional formulation processing methods are non-compatible, leading to reduced viability and unconfirmed incorporation into intestinal microbial communities; consequently, demand for more bespoke formulation strategies of such NGPs is apparent. In this study, Akkermansia muciniphila (A.muciniphila) as a candidate NGP was investigated for its growth and metabolism properties, based on which a novel microcomposite-based oral formulation was formed. Initially, a chitosan-based microcomposite was coated with mucin to establish a surface culture of A.muciniphila. This was followed by 'double encapsulation' with pectin (PEC) using a novel Entrapment Deposition by Prilling method to create core-shell double-encapsulated microcapsules. The formulation of A.muciniphila was verified to require no oxygen-restriction properties, and additionally, biopolymers were selected, including carboxymethylcellulose (CMC), that support and enhance its growth; consequently, a high viability (6 log CFU/g) of A.muciniphila microencapsulated in PEC-CMC double-encapsulates was obtained. Subsequently, the high stability of the PEC-CMC double-encapsulates was verified in simulated gastric fluid, successfully protecting and then releasing the A.muciniphila under intestinal conditions. Finally, employing a model of gastrointestinal transit and faecal-inoculated colonic bioreactors, significant alterations in microbial communities following administration and successful establishment of A.muciniphila were demonstrated.

Enhanced bioavailability and efficacy in antimalarial treatment through QbD approach enteric encased inclusion delivery.

Aim: In this study, we aimed to prepare enteric encapsulated spheroids containing inclusion complex using quality by design approach.Methods: A Box-Behnken design was employed to determine effects of variables on selected responses. Risk assessment was conducted using Ishikawa fishbone diagram. A model with a p-value was less than 0.5 for being a significant error of model was determined based on significance 'lack of fit' value. Spheroids were formulated using the extrusion spheronization technique and were characterized using analytical techniques.Results: In vitro release was performed in both acidic (pH 1.2) and simulated intestinal (pH 6.8) conditions. Permeability studies demonstrated tenfold enhancement compared with arteether. In vivo studies further validated increase of 51.8% oral bioavailability. Ex vivo studies revealed 3.4-fold enhancement in antimalarial activity compared with arteether.Conclusion: These findings highlight effectiveness of inclusion complexation technique as a viable approach to enhance solubility and bioavailability for drugs with low aqueous solubility.

[Box: see text].

Manifesting the Dasatinib-gallic acid co-amorphous system to augment anticancer potential: Physicochemical characterization, in silico molecular simulation, ex vivo permeability, and in vitro efficacy.

Dasatinib (DAB) has been explored for repurposing in the treatment of breast cancer (BC) due to its known effectiveness in treating leukemia, in addition to its role as a tyrosine kinase inhibitor. Gallic acid (GA) was chosen as a co-former due to its anticancer potential in BC, as demonstrated in several previous studies. DAB is a low-solubility drug, which is a significant hurdle for its oral bioavailability. To address this limitation, a DAB and GA co-amorphous (DAB-GA-CA) system was developed using liquid-assisted grinding and ball mill technology to enhance solubility, bioavailability, and anti-tumor efficacy. Physical characterization investigation revealed that the emergence of the halo diffractogram in PXRD, single glass transition temperature (Tg) value at 111.7 °C in DSC thermogram, and irregularly shaped blocks with loose, porous surfaces in SEM analysis indicated the formation of the DAB-GA-CA system at 1:1 M ratio. Furthermore, FTIR, Raman spectroscopy, in-silico molecular docking, and molecular dynamic studies confirmed the intermolecular hydrogen connections between DAB and GA. Moreover, the outcomes of the ligands (DAB and GA) and receptors (BCL-2, mTOR, estrogen receptor, and HER-2) docking studies demonstrated that both DAB and GA could interact with those receptors, leading to preventive action on BC cells. Additionally, the solubility and dissolution rate significantly improved at pH 6.8, and the permeability study indicated that DAB-GA-CA showed 1.9 times higher apparent permeability compared to crystalline DAB. Furthermore, in vitro cytotoxicity assessments of the DAB-GA-CA system revealed 3.42 times lower IC50 than free DAB. The mitochondrial membrane depolarization, apoptotic index, and reactive oxygen species formation in MCF-7 cells were also notably higher in the DAB-GA-CA system than in free DAB. Hence, this research suggests that the DAB-GA-CA system could substantially enhance oral delivery, solubility, and therapeutic efficacy.

Unlocking relief: formulation, characterization, and in vivo assessment of salicylic acid-loaded microemulgel for psoriasis management.

Psoriasis, a chronic skin condition, affects around 2-5% of the population. Topical corticosteroids treat the vast majority of cases (> 80%). Because of the physicochemical characteristics of the damaged stratum corneum, all treatments are ineffective. Nevertheless, systemic immunosuppression, the oral strategy, has substantial adverse effects that may be avoided using the topical procedure. The research sought to determine if a salicylic acid-loaded microemulsion-based gel (emulgel) could successfully infiltrate and maintain salicylic acid in skin tissue for psoriasis treatment. The pseudo-ternary phase was generated in different Smix ratios (1:1, 2:1, and 3:1; Labrasol:Transcutol® P). At a 3:1 ratio, the Smix had a substantial microemulsion area. Microemulsion was characterized for particle size, pH, etc. For topical application, the selected microemulsion was combined with Carbopol 940 gel, and ex vivo permeation and drug retention study were conducted. The effectiveness of the developed gel was checked using the IMQ-induced psoriatic plaque model. Salicylic acid microemulsion has an average globule size of 79.72 nm, pH 5.93, and 100% transmittance. In an ex vivo diffusion study, emulgel revealed greater penetration and more drug retention than ordinary salicylic acid gel. The emulgel was non-irritating on the skin of rats. In vivo studies revealed significant antipsoriatic activity of microemulsion-loaded gel compared to the marketed product. Developed emulgel was considered a potential product for an effective and safe way to administer salicylic acid for the treatment of skin diseases such as psoriasis.

HSPiP and QbD oriented optimized stearylamine-elastic liposomes for topical delivery of ketoconazole to treat deep seated fungal infections: In vitro and ex vivo evaluations.

The study explored stearylamine containing cationic elastic liposomes to improve topical delivery and efficacy of ketoconazole (KETO) to treat deeply seated fungal infections. Stearylamine was used for dual functionalities (electrostatic interaction and flexibility in lipid bilayer). Hansen solubility program (HSPiP) estimated Hansen solubility parameters (HSP) based on the SMILE file and structural properties followed by experimental solubility study to validate the predicted values. Various formulations were developed by varying phosphatidylcholine and surfactants (tween 80 and span 80) concentration. To impart cationic properties, stearylamine (1.0 %) was added into the organic phase. Using quality by design (QbD) method, we optimized the formulations and evaluated for vesicle size, polydispersity index, zeta potential, morphology (scanning electron microscopy), in vitro drug release (%), and ex vivo permeation profiles. Result showed that there is a good correlation (0.65) between HSPiP predicted and actual experimental solubility of KETO in water, chloroform, S80, and tween 80. Spherical OKEL1 showed an established correlation between the predicted and the actual formulation parameters (size, zeta potential, and polydispersity index) (259 nm vs 270 nm, +2.4 vs 0.21 mV, and 0.24 vs 0.27). OKEL1 was associated with the highest value of %EE (83.1 %) as compared to liposomes. Finally, OKEL1 exhibited the highest % cumulative permeation (49.9 %) as compared to DS (13 %) and liposomes (25 %). Moreover, OKEL1 resulted in 4-fold increase in permeation flux as compared to DS which may be attributed to vesicular mediated improved permeation and gel based compensated trans epidermal water loss in the skin. The drug deposition elicited OKEL1 and OKEL1-gel as suitable carriers for maximum therapeutic benefit to treat deeply seated fungal infections.

Ex vivo model of pathological calcification of human aortic valve.

The development of drug therapy for the pathological calcification of the aortic valve is still an open issue due to the lack of effective treatment strategies. Currently, the only option for treating this condition is surgical correction and symptom management. The search for models to study the safety and efficacy of anti-calcifying drugs requires them to not only be as close as possible to in vivo conditions, but also to be flexible with regard to the molecular studies that can be applied to them. The ex vivo model has several advantages, including the ability to study the effect of a drug on human cells while preserving the original structure of the valve. This allows for a better understanding of how different cell types interact within the valve, including non-dividing cells. The aim of this study was to develop a reproducible ex vivo calcification model based on valves from patients with calcific aortic stenosis. We aimed to induce spontaneous calcification in valve tissue fragments under osteogenic conditions, and to demonstrate the possibility of significantly suppressing it using a calcification inhibitor. To validate the model, we tested a Notch inhibitor Crenigacestat (LY3039478), which has been previously shown to have an anti-calcifying effect on interstitial cell of the aortic valve. We demonstrate here an approach to testing calcification inhibitors using an ex vivo model of cultured human aortic valve tissue fragments. Thus, we propose that ex vivo models may warrant further investigation for their utility in studying aortic valve disease and performing pre-clinical assessment of drug efficacy.

A New Frontier in Cystic Fibrosis Pathophysiology: How and When Clock Genes Can Affect the Inflammatory/Immune Response in a Genetic Disease Model.

Cystic fibrosis (CF) is a monogenic syndrome caused by variants in the CF Transmembrane Conductance Regulator (CFTR) gene, affecting various organ and systems, in particular the lung, pancreas, sweat glands, liver, gastrointestinal tract, vas deferens, and vascular system. While for some organs, e.g., the pancreas, a strict genotype-phenotype occurs, others, such as the lung, display a different pathophysiologic outcome in the presence of the same mutational asset, arguing for genetic and environmental modifiers influencing severity and clinical trajectory. CFTR variants trigger a pathophysiological cascade of events responsible for chronic inflammatory responses, many aspects of which, especially related to immunity, are not ascertained yet. Although clock genes expression and function are known modulators of the innate and adaptive immunity, their involvement in CF has been only observed in relation to sleep abnormalities. The aim of this review is to present current evidence on the clock genes role in immune-inflammatory responses at the lung level. While information on this topic is known in other chronic airway diseases (chronic obstructive pulmonary disease and asthma), CF lung disease (CFLD) is lacking in this knowledge. We will present the bidirectional effect between clock genes and inflammatory factors that could possibly be implicated in the CFLD. It must be stressed that besides sleep disturbance and its mechanisms, there are not studies directly addressing the exact nature of clock genes' involvement in inflammation and immunity in CF, pointing out the directions of new and deepened studies in this monogenic affection. Importantly, clock genes have been found to be druggable by means of genetic tools or pharmacological agents, and this could have therapeutic implications in CFLD.

Development of Glycerosomal pH Triggered In Situ Gelling System to Ameliorate the Nasal Delivery of Sulpiride for Pediatric Psychosis.

Sulpiride (Sul) is a medication that blocks dopamine D2 receptors. It is used to treat gastrointestinal disturbances and has antipsychotic effects depending on the dose given. Sulpiride is subject to P-glycoprotein efflux, resulting in limited bioavailability and erratic absorption. Hence, the aim of this study was to generate a glycerosomal in situ gel of sulpiride for intranasal administration, specifically targeting children with schizophrenia who may have difficulty swallowing traditional solid medications, for enhancing its bioavailability. This study aimed to demonstrate the efficacy of intranasal administration of glycerin-encapsulated lipid-nanovesicles (glycerosomes) mixed with in situ gels for prolonged release of anti-psychotic medication. A Box-Behnken design was utilized to create sulpiride-loaded glycerosomes (Sul-GMs), with the lipid amount (A), glycerin concentration (B), and sonication time (C) acting as independent variables. Their impact on the entrapment efficiency, EE% (Y1), and in vitro drug release (Y2) were evaluated. The sulpiride EE% showed an increase when the glycerin concentration was raised to 25% v/v. Nevertheless, when the glycerin concentration was raised to 40% v/v, there was a notable decrease in the EE%. The optimized glycerosome was added to pH triggered carbopol 974P in situ gel formulations including HPMC K15M with different concentrations. The in situ gel formulation (G3) comprising 0.6% carbopol 974P and 0.6% hydroxypropyl methyl cellulose-K15M (HPMC K15M) demonstrated suitable pH, viscosity, desired gel strength, spreadability, and mucoadhesive strength. Consequently, it was selected for in vitro study, ex vivo permeation investigation, and in vivo evaluations. The glycerosomal in situ gel exhibited favorable ex vivo permeability of SU when applied to the nasal mucosa. The pharmacokinetic investigation revealed that the optimized Sul-loaded glycerosomal in situ gel exhibited a significant fourfold and twofold enhancement in systemic bioavailability compared to both the control gel and the commercially available formulation. Finally, the intranasal administration of Sul-loaded glycerosomal in situ gel is a promising alternative to oral treatment for pediatric patients with psychosis.

Staurosporine as a Potential Treatment for Acanthamoeba Keratitis Using Mouse Cornea as an Ex Vivo Model.

Acanthamoeba is a ubiquitous genus of amoebae that can trigger a severe and progressive ocular disease known as Acanthamoeba Keratitis (AK). Furthermore, current treatment protocols are based on the combination of different compounds that are not fully effective. Therefore, an urgent need to find new compounds to treat Acanthamoeba infections is clear. In the present study, we evaluated staurosporine as a potential treatment for Acanthamoeba keratitis using mouse cornea as an ex vivo model, and a comparative proteomic analysis was conducted to elucidate a mechanism of action. The obtained results indicate that staurosporine altered the conformation of actin and tubulin in treated trophozoites of A. castellanii. In addition, proteomic analysis of treated trophozoites revealed that this molecule induced overexpression and a downregulation of proteins related to key functions for Acanthamoeba infection pathways. Additionally, the ex vivo assay used validated this model for the study of the pathogenesis and therapies of AK. Finally, staurosporine eliminated the entire amoebic population and prevented the adhesion and infection of amoebae to the epithelium of treated mouse corneas.

Ex Vivo Simultaneous H215O Positron Emission Tomography and Magnetic Resonance Imaging of Porcine Kidneys-A Feasibility Study.

The aim was to establish combined H215O PET/MRI during ex vivo normothermic machine perfusion (NMP) of isolated porcine kidneys. We examined whether changes in renal arterial blood flow (RABF) are accompanied by changes of a similar magnitude in renal blood perfusion (RBP) as well as the relation between RBP and renal parenchymal oxygenation (RPO). METHODS: Pig kidneys (n = 7) were connected to a NMP circuit. PET/MRI was performed at two different pump flow levels: a blood-oxygenation-level-dependent (BOLD) MRI sequence performed simultaneously with a H215O PET sequence for determination of RBP. RESULTS: RBP was measured using H215O PET in all kidneys (flow 1: 0.42-0.76 mL/min/g, flow 2: 0.7-1.6 mL/min/g). We found a linear correlation between changes in delivered blood flow from the perfusion pump and changes in the measured RBP using PET imaging (r2 = 0.87). CONCLUSION: Our study demonstrated the feasibility of combined H215O PET/MRI during NMP of isolated porcine kidneys with tissue oxygenation being stable over time. The introduction of H215O PET/MRI in nephrological research could be highly relevant for future pre-transplant kidney evaluation and as a tool for studying renal physiology in healthy and diseased kidneys.

The Fibrotic Phenotype of Human Precision-Cut Lung Slices Is Maintained after Cryopreservation.

Human precision-cut lung slices (hPCLS) prepared from fibrotic lungs recapitulate the pathophysiological hallmarks of fibrosis. These hallmark features can also be induced by treating non-fibrotic hPCLS with a fibrotic cocktail (FC). As a result, the fibrotic and fibrosis-induced hPCLS are rapidly emerging as preferred models for disease modeling and drug discovery. However, current hPCLS models are limited by tissue viability in culture, as they are usually only viable for one week after harvesting. Here, we demonstrate that the fibrotic hPCLS can be cryopreserved, stored for months, and then thawed on demand without loss of hPCLS viability or protein content for 14 days post-thawing. Cryopreservation also preserves the pro-fibrotic potential of non-fibrotic hPCLS. Specifically, when we treated the thawed non-fibrotic hPCLS with an FC, we observed significant pro-fibrotic cytokine secretion and elevated tissue stiffness. These pro-fibrotic changes were inhibited by the small-molecule tyrosine kinase inhibitor, Nintedanib. Taken together, our work indicates that a feasible solution to prolong the pre-clinical utility of fibrotic and fibrosis-induced hPCLS is cryopreservation. We anticipate that cryopreserved hPCLS will serve as an advantageous predictive model for the evaluation of pro-fibrotic pathways during acute and chronic toxicity testing.

A novel miniaturized roller pump circuit for simulation of extracorporeal circulation.

OBJECTIVES: Extracorporeal circulation induces pronounced effects on haemostasis and rheology. To study these, an ex vivo simulation model is an attractive alternative but often requires large amounts of blood. We sought to create a miniaturized roller pump circuit requiring minimal amounts of blood and to test if the circuit could be used to compare coagulation, platelet function and blood rheology between a dextran-based and a crystalloid-based priming solution. METHODS: A miniaturized roller pump circuit requiring only 27 ml of blood was created. Blood samples from 8 cardiac surgery patients were mixed with either a dextran-based or a crystalloid-based solution and circulated for 60 min. Coagulation was assessed by rotational thromboelastometry, and platelet function by impedance aggregometry and flow cytometry, before and at 5 and 60 min of circulation. RESULTS: A time-dependent impairment of coagulation was observed in both groups. Maximum clot firmness was lower with dextran-based than with crystalloid-based priming at 5 min (HEPTEM 37 ± 4 vs 43 ± 4 mm, P < 0.001; EXTEM 37 ± 4 vs 43 ± 4 mm, P < 0.001; FIBTEM 3 ± 2 vs 9 ± 2 mm, P < 0.001) and at 60 min (HEPTEM 29 ± 9 vs 38 ± 5 mm, P < 0.001; EXTEM 30 ± 7 vs 39 ± 5 mm, P < 0.001; FIBTEM 3 ± 2 vs 8 ± 3 mm, P = 0.002). The EXTEM clotting time was longer with dextran-based solution at 5 (109 ± 19 vs 63 ± 7 sec, P < 0.001) and at 60 min (176 ± 72 vs 73 ± 7 sec, P = 0.004). CONCLUSIONS: The novel miniaturized roller pump circuit can be used to mimic extracorporeal circulation for selected research questions. Dextran-based priming caused a significant impairment in haemostasis compared with a standard crystalloid solution.

Low-Volume Ex Situ Lung Perfusion System for Single Lung Application in a Small Animal Model Enables Optimal Compliance With "Reduction" in 3R Principles of Animal Research.

Ex situ lung perfusion (ESLP) is used for organ reconditioning, repair, and re-evaluation prior to transplantation. Since valid preclinical animal models are required for translationally relevant studies, we developed a 17 mL low-volume ESLP for double- and single-lung application that enables cost-effective optimal compliance "reduction" of the 3R principles of animal research. In single-lung mode, ten Fischer344 and Lewis rat lungs were subjected to ESLP and static cold storage using STEEN or PerfadexPlus. Key perfusion parameters, thermal lung imaging, blood gas analysis (BGA), colloid oncotic pressure (COP), lung weight gain, histological work-up, and cytokine analysis were performed. Significant differences between perfusion solutions but not between the rat strains were detected. Most relevant perfusion parameters confirmed valid ESLP with homogeneous lung perfusion, evidenced by uniform lung surface temperature. BGA showed temperature-dependent metabolic activities with differences depending on perfusion solution composition. COP is not decisive for pulmonary oedema and associated weight gain, but possibly rather observed chemokine profile and dextran sensitivity of rats. Histological examination confirmed intact lung architecture without infarcts or hemorrhages due to optimal organ procurement and single-lung application protocol using our in-house-designed ESLP system.

Measurement and comparison of dielectric properties of human pancreatic tumours, healthy tissues and porcine tissues ex vivo between 1Hz and 1MHz.

The dielectric properties of pancreatic tissues from human healthy and tumour-bearing tissues have been extracted from impedance measurement on ex vivo, freshly excised samples. They are compared to pig pancreas samples, measured following the same protocol. The purpose is to add data to the scarce literature on the properties of the human pancreas and pancreatic tumours, for treatment planning, tissue identification and numerical simulations. The conductivity measured at 500 kHz for human healthy pancreas is 0.26 S/m, while the conductivity of tumour-bearing tissues is 0.44 S/m. Those values differ significantly from that listed in the IT IS database at 0.57 S/m, suggesting an update might be to consider. However, measures of relative permittivity are in accordance with the database with a value of approximately 2.3x103. Ex vivo porcine model, while being less conductive than human pancreas with 0.16 S/m at the same frequency, is deemed a relevant model when studying pancreatic applications of electromagnetic fields-based treatments, such as radiofrequency ablation.

Randomly methylated ß-cyclodextrin improves water - solubility, cellular protection and mucosa permeability of idebenone.

Neurodegenerative diseases such as Alzheimer's are very common today. Idebenone (IDE) is a potent antioxidant with good potential for restoring cerebral efficiency in cases of these and other medical conditions, but a serious drawback for the clinical use of IDE in neurological disorders lies in its scarce water solubility, which greatly inhibits its bioavailability. In this work, we prepared the inclusion complex of IDE with randomly methylated ß-cyclodextrin (RAMEB), resulting in improved water solubility of the included drug; then its in vitro biological activity and ex vivo permeability was evalutated. The solid complex was characterized through FT-IR spectroscopy, Thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC). A 78-fold improvement of the solubility of IDE in water resulted, together with a strong 1:1 host-guest interaction (association constant of 12630 M-1), and dissolution of the complex within 15 min, all evidenced during the in-solution studies. Biological in vitro studies were then performed on differentiated human neuroblastoma cells (SH-SY5Y) subjected to oxidative stress. Pretreatment with IDE/RAMEB positively affected cell viability, promoted the nuclear translocation of Nrf2, and increased the levels of GSH as well as those of the endogenous antioxidant enzymes Mn-SOD and HO-1. Lastly, the complexation significantly improved the permeation of IDE through isolated rat nasal mucosa.

Extensive immunophenotypic sub-population analysis of StemRegenin1 expanded haematopoietic stem/progenitor cells.

BACKGROUND: Ex vivo haematopoietic stem/progenitor cell (HSPCs) expansion constitutes an important area of research, and has the potential to improve access to umbilical cord blood (UCB) as a source of stem cells for haematopoietic stem cell transplantation (HSCT). The ability to improve stem cell dose and thereby reduce delayed engraftment times, which has plagued the use of UCB as a stem cell source since inception, is a recognised advantage. The extent to which cluster of differentiation (CD)34 sub-populations are affected by expansion with StemRegenin1 (SR1), and whether a particular subtype may account for better engraftment than others, is currently unknown. The purpose of this study was to determine the impact of SR1-induced HSPC expansion on CD34+ immunophenotypic subsets and gene expression profiles. METHODS: UCB-derived CD34+ HSPCs were characterised before (D0) and after expansion (D7) with SR1 using an extensive immunophenotypic panel. In addition, gene expression was assessed and differentially expressed genes were categorised into biological processes. RESULTS: A dose-dependent increase in the number of CD34+ HSPCs was observed with SR1 treatment, and unbiased and extensive HSPC immunophenotyping proved to be a powerful tool in identifying unique sub-populations within the HSPC repertoire. In this regard, we found that SR1 promotes the emergence of HSPC subsets which may aid engraftment post expansion. In addition, we observed that SR1 has a minimal effect on the transcriptome of 7-day expanded CD34+ HSPCs when compared to cells expanded without SR1, with only two genes being downregulated in the former. CONCLUSION: This study revealed that SR1 selects for potentially novel immunophenotypic HSPC subsets post expansion and has a minimal effect on the transcriptome of 7-day expanded HSPCs when compared to vehicle controls. Whether these distinct immunophenotypic sub-populations possess greater engraftment capacity remains to be tested in animal models.

Segmentation of supragranular and infragranular layers in ultra-high-resolution 7T ex vivo MRI of the human cerebral cortex.

Accurate labeling of specific layers in the human cerebral cortex is crucial for advancing our understanding of neurodevelopmental and neurodegenerative disorders. Building on recent advancements in ultra-high-resolution ex vivo MRI, we present a novel semi-supervised segmentation model capable of identifying supragranular and infragranular layers in ex vivo MRI with unprecedented precision. On a dataset consisting of 17 whole-hemisphere ex vivo scans at 120 $\mu $m, we propose a Multi-resolution U-Nets framework that integrates global and local structural information, achieving reliable segmentation maps of the entire hemisphere, with Dice scores over 0.8 for supra- and infragranular layers. This enables surface modeling, atlas construction, anomaly detection in disease states, and cross-modality validation while also paving the way for finer layer segmentation. Our approach offers a powerful tool for comprehensive neuroanatomical investigations and holds promise for advancing our mechanistic understanding of progression of neurodegenerative diseases.

Ex-vivo investigation of probiotic bacterial adhesion to the intestinal mucus.

Recent research has promoted considerable interest in the potential health benefits of the new generation of probiotics. Despite the abundance of probiotic supplements, their adhesion and thereby colonization in the intestinal tract of the host, a determining factor of probiotic efficacy, remains questionable. Indeed, the gastrointestinal tract, a multi-component and complex system, obscures the comprehensive understanding of the probiotic adhesion mechanism. This study aimed to investigate the adhesion capacity of probiotic bacteria using two ex-vivo approaches that were specifically developed to investigate the bacteria-mucus agglomeration and the viable adhesion to intestinal mucus. Five probiotic bacterial strains including Escherichia coli, Lactiplantibacillus plantarum, Faecalibacterium duncaniae, Bifidobacterium longum, and Bifidobacterium longum str. infantis were selected for the investigation. In that context, higher adhesion to mucus was demonstrated by E. coli, L. plantarum, and B. infantis, emphasizing strain-specific differences. While total agglomeration capacity ranged from 8 % to 82 %, actual viable adhesion to mucus remained rather low (0.6 %-2.9 %). SEM images revealed that morphological characteristics, chain and/or cluster forming ability, as well as the presence of surface exopolysaccharides, might have an impact on bacterial adhesion. This study contributes knowledge on probiotic adhesion as well as simple and effective ex-vivo approaches to investigate the bacterial adhesion to the intestinal mucus, which is prerequisite for further colonization in the gut of the host.

Importance of the enhanced cooling system for more spherical ablation zones: Numerical simulation, ex vivo and in vivo validation.

INTRODUCTION: This study aimed to investigate the efficacy of a small-gauge microwave ablation antenna (MWA) with an enhanced cooling system (ECS) for generating more spherical ablation zones. METHODS: A comparison was made between two types of microwave ablation antennas, one with ECS and the other with a conventional cooling system (CCS). The finite element method was used to simulate in vivo ablation. Two types of antennas were used to create MWA zones for 5, 8, 10 min at 50, 60, and 80 W in ex vivo bovine livers (n = 6) and 5 min at 60 W in vivo porcine livers (n = 16). The overtreatment ratio, ablation aspect ratio, carbonization area, and other characteristcs of antennas were measured and compared using numerical simulation and gross pathologic examination. RESULTS: In numerical simulation, the ECS antenna demonstrated a lower overtreatment ratio than the CCS antenna (1.38 vs 1.43 at 50 W 5 min, 1.19 vs 1.35 at 50 W 8 min, 1.13 vs 1.32 at 50 W 10 min, 1.28 vs 1.38 at 60 W 5 min, 1.14 vs 1.32 at 60 W 8 min, 1.10 vs 1.30 at 60 W 10 min). The experiments revealed that the ECS antenna generated ablation zones with a more significant aspect ratio (0.92 ± 0.03 vs 0.72 ± 0.01 at 50 W 5 min, 0.95 ± 0.02 vs 0.70 ± 0.01 at 50 W 8 min, 0.96 ± 0.01 vs 0.71 ± 0.04 at 50 W 10 min, 0.96 ± 0.01 vs 0.73 ± 0.02 at 60 W 5 min, 0.94 ± 0.03 vs 0.71 ± 0.03 at 60 W 8 min, 0.96 ± 0.02 vs 0.69 ± 0.04 at 60 W 10 min) and a smaller carbonization area (0.00 ± 0.00 cm2 vs 0.54 ± 0.06 cm2 at 50 W 5 min, 0.13 ± 0.03 cm2 vs 0.61 ± 0.09 cm2 at 50 W 8 min, 0.23 ± 0.05 cm2 vs 0.73 ± 0.05 m2 at 50 W 10 min, 0.00 ± 0.00 cm2 vs 1.59 ± 0.41 cm2 at 60 W 5 min, 0.23 ± 0.22 cm2 vs 2.11 ± 0.63 cm2 at 60 W 8 min, 0.57 ± 0.09 cm2 vs 2.55 ± 0.51 cm2 at 60 W 10 min). Intraoperative ultrasound images revealed a hypoechoic area instead of a hyperechoic area near the antenna. Hematoxylin-eosin staining of the dissected tissue revealed a correlation between the edge of the ablation zone and that of the hypoechoic area. CONCLUSIONS: The ECS antenna can produce more spherical ablation zones with less charring and a clearer intraoperative ultrasound image of the ablation area than the CCS antenna.