Microcarrier-based clinical-grade manufacturing of therapeutic Wharton's jelly mesenchymal stromal cells.

Due to their immunomodulatory and anti-inflammatory properties, tissue repair capabilities and regenerative potential, Wharton's jelly mesenchymal stem/stromal cells (WJMSCs) have been widely investigated as potential treatment for diverse clinical indications. WJMSCs have been found to be well-tolerated and safe, positioning them as a promising candidate for cellular therapy. To address the commercial need for manufacturing WJMSCs for clinical applications, the production scale should be capable of generating large quantities of cells that retain their expected identity, purity and potency. This study aimed to establish a current Good Manufacturing Practice (cGMP) compliant robust and scalable expansion process representing a critical step towards a cGMP-compliant large-scale production platform for WJMSC-based clinical applications. Using our in-house cGMP-manufactured WJMSCs, which are currently being tested in a Phase Ib clinical trial (NCT03158896) using two-dimensional (2D) planar systems, we optimized various culture parameters including type of microcarrier, seeding density, agitation and culture feed regime in a 3D microcarrier-based culture system in spinner flasks. The results showed that cell adhesion was potentiated under intermittent stirring (3 min of agitation at 25 rpm followed by a period of non-agitation for 30 min), with reduced supplementation (0.05%) during the initial 8 h of cultivation with an initial cell concentration of 0.45 × 105 cells/mL. Microcarrier-based WJMSC expansion in spinner flasks achieved greater cell densities of 1.67 × 106 cells/mL with a maximum of 37-fold expansion, yielding ∼84 × 106 cells after 6 days of culture with a 95% harvest efficiency. Additionally, post 3D expansion, WJMSCs maintained their phenotypic characteristics, differentiation potential, normal karyotype, functional properties and sterility in the culture systems evaluated. This cGMP-compliant expansion process described herein demonstrates a successful transition of an established 2D planar culture process of clinical grade WJMSCs to 3D microcarrier-based suspension process generating higher cell yields, is cost-effective and represents an important step toward fulfilling the commercial demand of clinical grade mesenchymal stromal cells.

Crystal layer growth with embedded carbon-based particles from effervescent tablet-based nanofluids.

Crystallization occurs as dissolved substances gradually solidify into crystal layers within a liquid, which can increase the capability of fluids to transfer heat. In this study, the growth of crystal layer in nanofluids produced from carbon-based effervescent tablets was examined. The tablets were fabricated by combining multi-walled carbon nanotubes (MWCNTs), sodium dodecyl sulfate (SDS), sodium phosphate monobasic (NaH2PO4), and sodium carbonate (Na2CO3). The effervescent tablets were formulated with MWCNTs, NaH2PO4, and Na2CO3 at a weight ratio of 1:5.1:2.26, respectively. These tablets were then immersed in distilled water (DW) and seawater (SW) to produce 0.05 vol.% to 0.15 vol.% MWCNT suspensions. Then, the dispersion stability, thermal conductivity, and crystal layer growth of the nanofluids were characterized. The results showed that the DW-based nanofluids were more stable than their SW-based counterparts. Additionally, the 0.05 vol.% DW-based suspension exhibited greater long-term stability than those of the 0.15 vol.% suspensions, whereas the SW-based nanofluid exhibited the opposite behaviour. The greatest increases in thermal conductivity were 3.29% and 3.13% for 0.15 vol.% MWCNTs in DW and SW, respectively. The crystallization process occurred in nanofluids that contained more than 0.05 vol.% MWCNTs and exhibited a greater growth rate in SW-based suspensions with high effervescent agent concentrations.

UV-B Radiation Exhibited Tissue-Specific Regulation of Isoflavone Biosynthesis in Soybean Cell Suspension Cultures.

Isoflavones, a class of substances with high biological activity, are abundant in soybeans. This study investigated isoflavone biosynthesis in soybean cell suspension cultures under UV-B radiation. UV-B radiation enhanced the transcription level and activity of key enzymes involved in isoflavone synthesis in cell suspension cultures. As a result, the isoflavone contents significantly increased by 19.80% and 91.21% in hypocotyl and cotyledon suspension cultures compared with the control, respectively. Meanwhile, a significant difference was observed in the composition of isoflavones between soybean hypocotyl and cotyledon suspension cultures. Genistin was only detected in hypocotyl suspension cultures, whereas glycitin, daidzein, and genistein accumulated in cotyledon suspension cultures. Therefore, UV-B radiation exhibited tissue-specific regulation of isoflavone biosynthesis in soybean cell suspension cultures. The combination of suspension cultures and abiotic stress provides a novel technological approach to isoflavone accumulation.

Robotic-Enhanced Prosthetic Liners for Vibration Therapy: Reducing Phantom Limb Pain in Transfemoral Amputees.

Phantom limb pain, a common challenge for amputees, lacks effective treatment options. Vibration therapy is a promising non-pharmacologic intervention for reducing pain intensity, but its efficacy in alleviating phantom limb pain requires further investigation. This study focused on developing prosthesis liners with integrated vibration motors to administer vibration therapy for phantom limb pain. The prototypes developed for this study addressed previous issues with wiring the electronic components. Two transfemoral amputees participated in a four-week at-home trial, during which they used the vibration liner and rated their initial and final pain intensity on a numeric rating scale each time they had phantom pain. Semi-structured interviews were conducted to gather feedback following the at-home trial. Both participants described relaxing and soothing sensations in their residual limb and phantom limb while using vibration therapy. One participant reported a relaxation of his phantom limb sensations, while both participants noted a decrease in the intensity of their phantom limb pain. Participants said the vibration liners were comfortable but suggested that the vibration could be stronger and that aligning the contacts could be easier. The results of this study highlight the potential effectiveness of using vibration therapy to reduce the intensity of phantom limb pain and suggest a vibration liner may be a feasible mode of administering the therapy. Future research should address optimizing the performance of the vibration liners to maximize their therapeutic benefits.

The standardized procedure, technical key points and latest progress of laparoscopic lateral suspension surgery.

BACKGROUND: Pelvic organ prolapse (POP) is a common condition that can affect up to 30% of women over the age of 50. For a long time, open abdominal and laparoscopic sacrocolpopexy (LSCP) have been considered the gold standard in the treatment of apical pelvic organ prolapse (POP). Promontory dissection may expose patients to potential life-threatening intraoperative vascular injuries, as well as damage to sacral roots or the hypogastric nerve. Laparoscopic lateral suspension could be considered as an alternative to LSCP in the treatment of POP due to its favorable objective and subjective outcomes. The aim of this article is to demonstrate a step-by-step approach to laparoscopic lateral suspension for POP with the goal of standardizing this procedure. Technical key points and the latest progress are summarized to provide a reference for subsequent gynecological and urological surgeons. METHOD: According to our surgical experience of our hospital, demonstrate a step-by-step approach and highlight technical key points for laparoscopic lateral suspension for POP with the aim of standardizing this procedure. CONCLUSION: LLS with mesh is a safe alternative to laparoscopic sacropexy and is very well suited for uterine-preserving POP surgery. Nevertheless, this novel procedure lacks standardization. Standardization of procedures is necessary to reduce failure rates, generate impactful research data, and enhance patient safety. This article contributes to the standardization of this procedure, and we believe our article will be useful in assisting future gynecological and urological surgeons in performing this procedure.

A Novel, Dual-Initiator, Continuous-Suspension Grafting Strategy for the Preparation of PP-g-AA-MAH Fibers to Remove of Indigo from Wastewater.

The indigo dye found in wastewater from printing and dyeing processes is potentially carcinogenic, teratogenic, and mutagenic, making it a serious threat to the health of animals, plants, and humans. Motivated by the growing need to remove indigo from wastewater, this study prepared novel fiber absorbents using melt-blow polypropylene (PP) melt as a matrix, as well as acrylic acid (AA) and maleic anhydride (MAH) as functional monomers. The modification conditions were studied to optimize the double-initiation, continuous-suspension grafting process, and then functional fibers were prepared by melt-blown spinning the modified PP. The results showed that the optimum modification conditions were as follows: a 3.5 wt% interfacial agent, 8 mg/L of dispersant, 80% monomer content, a 0.8 mass ratio of AA to MAH, a 1000 r/min stir speed, 3.5 wt% initiator DBPH grafting at 130 °C for 3 h, and 1 wt% initiator BPO grafting at 90 °C for 2 h. The highest grafting rate of the PP-g-AA-MAH was 31.2%, and the infrared spectrum and nuclear magnetic resonance spectroscopic analysis showed that AA and MAH were successfully grafted onto PP fiber. This modification strategy also made the fibers more hydrophilic. The adsorption capacity of the PP-g-AA-MAH fibers was highly dependent on pH, and the highest indigo adsorption capacity was 110.43 mg/g at pH 7. The fiber adsorption capacity for indigo increased rapidly before plateauing with increasing time or indigo concentration, and the experimental data were well described in a pseudo-second-order kinetic model and a Langmuir isothermal adsorption model. Most impressively, the modified fiber adsorption capacity for indigo remained as high as 91.22 mg/g after eight regeneration and reuse cycles. In summary, the PP-g-AA-MAH fibers, with excellent adsorption-desorption characteristics, could be readily regenerated and reused, and they are a promising material for the removal of indigo from wastewater.

The effect of low-intensity suspension training with blood flow restriction on GH, IGF-1, and their association with physical fitness in young women.

Blood flow restriction (BFR) has been incorporated in resistance training for over 20 years. We aimed to investigate the impact of low-intensity suspension training with BFR (LIST+BFR) on GH, IGF-1, and their association with physical fitness in young women. Thirty-six active women participated and were randomly assigned to either the high-intensity suspension training (HIST), LIST+BFR, or control (CON) groups. Training groups exercised three sessions weekly for 8 weeks. The CON only engaged in regular physical activity. Fasting serum hormones and physical fitness were assessed 48 h before and after the training intervention. GH and IGF-1 levels significantly higher in the LIST+BFR compared to the HIST and CON. These hormones were significantly higher by HIST, compared to CON. LIST+BFR led to significant enhancements in muscular strength and endurance compared to HIST and CON. Additionally, HIST significantly higher than compared to CON. Sprinting and agility time lower in both suspension training groups rather than the CON. No significant between-groups differences were found in weight. There was a large or moderate correlation between GH and IGF-1 and muscular strength, endurance, sprint, and agility performance. LIST+BFR could more enhanced GH, IGF-1, and muscular strength and endurance in females than HIST.

Recurrence Rate during 5-Year Period after Suspension of Anti-Vascular Endothelial Growth Factor Treatment for Neovascular Age-Related Macular Degeneration.

Purpose: To determine the recurrence rate of neovascular age-related macular degeneration (nAMD) during a 5-year period after the suspension of anti-vascular endothelial growth factor (anti-VEGF) treatments. Methods: Thirty-four eyes of 34 nAMD patients who met the inclusion criteria and were treated by anti-VEGF drugs were studied. All met the treatment suspension criteria and were followed for 5 years after the suspension of the anti-VEGF treatment. Patients with a recurrence within one year were placed in Group A, and patients with a recurrence between 1 and 5 years were placed in Group B. The rate and time of a recurrence were analyzed using the Kaplan-Meier method. We also examined whether there were differences in the baseline factors of age, sex, subtype, treatment period, and treatment interval between Groups A and B. Results: Twenty-five of 34 eyes (73.5%) had a recurrence within 5 years of stopping the anti-VEGF treatments. Thirteen (52.0%) of the 25 eyes had a recurrence within 1 year, 4 (16.0%) eyes between 1 and 2 years, 4 (16.0%) eyes between 2 and 3 years, 2 (8%) between 3 and 4 years, and 2 eyes (8%) between 4 and 5 years. The baseline factors were not significantly different between Groups A and B. Conclusions: The results showed that the recurrence rate was highest within one year after the suspension of the anti-VEGF treatments, with a number of recurrences one year after the suspension. Clinicians should remember that nAMD may recur several years after the suspension of anti-VEGF treatments.

Production and Characterization of Novel Anti-HIV Fc-Fusion Proteins in Plant-Based Systems: Nicotiana benthamiana & tobacco BY-2 cell suspension.

Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies.

Internal mucosal resection and LLC suspension: a new method to establish a stable tip rotation angle and reduce the nasal bridge length.

Background/aim: Herein, we describe a new technique to obtain both the appropriate degree of rotation angle and the ideal nasal bridge length. The aim of this study is to investigate the long-term results of this new technique with regard to these two variables. Materials and methods: A total of 76 (27 males, 49 females) patients were operated in accordance with the presented technique. Internal caudal mucosal excision and lower lateral cartilage (LLC) suspension were applied to all the patients included in this prospective study. Preoperative, immediate postoperative, and postoperative 1st-year photographs were taken. NOSE scores were obtained in the postoperative 1st year. Results: The mean nasolabial angle values of the patients preoperatively, at the end of the surgery (immediate postoperative), and at the end of the first year were 94.13° ± 5.1, 113.1° ± 5.3, and 109.6° ± 5.2, respectively. The patients had an average gain of 19° at the nasolabial angle at the end of the surgery and experienced a 3.5° (3.1%) loss at the end of the first year. For the nasal bridge length (n-prn) values; the preoperative, immediate postoperative, and first year mean values were 5.1 ± 0.55 cm, 3.98 ± 0.41 cm, and 4.29 ± 0.39 cm, respectively. The noses of the patients were shortened by 1.11 cm on average at the end of the surgery. Conclusion: Internal caudal mucosal resection with a suspension of the LLC to the caudal edge of the upper lateral cartilages (ULC) offers a reliable method to control the nasal tip rotation and shorten the long noses. This technique's effect is more obvious in long noses compared to the short ones.

Achilles tendon enthesis behavior under cyclic compressive loading: Consequences of unloading and early remobilization.

The Achilles tendon enthesis (ATE) anchors the Achilles tendon into the calcaneus through fibrocartilaginous tissue. The latter is enriched in type II collagen and proteoglycans (PGs), both of which give the enthesis its capacity to withstand compressive stress. Because unloading and reloading induce remodeling of the ATE fibrocartilage (Camy et al., 2022), chronic changes in the mechanical load could modify the mechanical response under compressive stress. Therefore, we investigated the ATE fatigue behavior in mice, under cyclic compressive loading, after 14 days of hindlimb suspension and 6 days of reloading. In addition, we performed a qualitative histological study of PGs in ATE fibrocartilage. The mechanical behavior of ATE was impaired in unloaded mice. A significant loss of 27 % in Δd (difference between the maximum and minimum displacements) was observed at the end of the test. In addition, the hysteresis area decreased by approximately 27 % and the stiffness increased by over 45 %. The increased stiffness and loss of viscosity were thrice and almost twice those of the control, respectively. In the reloaded entheses, where the loss of Δd was not significant, we found a significant 28 % decrease in the hysteresis area and a 26 % increase in stiffness, both of which were higher regarding the control condition. These load-dependent changes in the mechanical response seem partly related to changes in PGs in the uncalficied part of the ATE. These findings highlight the importance of managing compressive loading on ATE when performing prophylactic and rehabilitation exercises.

Corona mortis injury during anterior vaginal wall suspension: A case report.

The corona mortis is a variant vascular anastomosis that crosses behind the superior pubic ramus connecting the external and internal iliac vessels. Its location with respect to key landmarks in the retropubic space varies. This case demonstrates a life-threatening hemorrhage following an anterior vaginal wall suspension due to needle passer injury of the corona mortis successfully managed with endovascular embolization. Surgeons operating in the retropubic space need to be cognizant of this vascular variant and potential for significant bleeding when injured. Prompt vascular control either endovascularly or with open exploration is critical in preventing patient mortality.

A deep-learning-based threshold-free method for automated analysis of rodent behavior in the forced swim test and tail suspension test.

BACKGROUND: The forced swim test (FST) and tail suspension test (TST) are widely used to assess depressive-like behaviors in animals. Immobility time is used as an important parameter in both FST and TST. Traditional methods for analyzing FST and TST rely on manually setting the threshold for immobility, which is time-consuming and subjective. NEW METHOD: We proposed a threshold-free method for automated analysis of mice in these tests using a Dual-Stream Activity Analysis Network (DSAAN). Specifically, this network extracted spatial information of mice using a limited number of video frames and combined it with temporal information extracted from differential feature maps to determine the mouse's state. To do so, we developed the Mouse FSTST dataset, which consisted of annotated video recordings of FST and TST. RESULTS: By using DSAAN methods, we identify immobility states at accuracies of 92.51 % and 88.70 % for the TST and FST, respectively. The predicted immobility time from DSAAN is nicely correlated with a manual score, which indicates the reliability of the proposed method. Importantly, the DSAAN achieved over 80 % accuracy for both FST and TST by utilizing only 94 annotated images, suggesting that even a very limited training dataset can yield good performance in our model. COMPARISON WITH EXISTING METHOD(S): Compared with DBscorer and EthoVision XT, our method exhibits the highest Pearson correlation coefficient with manual annotation results on the Mouse FSTST dataset. CONCLUSIONS: We established a powerful tool for analyzing depressive-like behavior independent of threshold, which is capable of freeing users from time-consuming manual analysis.

Packing in multimodal crystals and particle assemblies as models for dynamic packing during spontaneous and enforced particle movement: Settling, viscosity and elasticity.

Colloid particles (CP, 10-8-10-6 m = 10-1000 nm) are used as models for atom scale processes, such as crystallization since the process is experimentally observable. Packing of atoms in crystals resemble mono-, bi-, and trimodal packing of noncharged hard spheres (particles). When the size of one particle exceeds the two others an excluded volume consisting of small particles is created around large particles. This is also the case when colloid particles are dispersed in water. The formation of an excluded volume does not require attraction forces, but it is enforced by the presence of dissolved primary (cations) and secondary (protons of surface hydroxyls) potential determining ions. The outcome is an interfacial solid-liquid charge. This excluded volume, denoted Stern layer is characterized by the surface potential and charge density. Charge neutrality is identified by point of zero charge (pHpzc and pcpzc). Outside Stern layer another excluded volume is formed of loosely bound counterions which interact with Stern layer. The extent of this diffuse layer is given by inverse Debye length and effective ζ-potential. The overall balance between attractive and repulsive energies is provided by Derjaguin-Landau-Veerwey-Overbeek (DLVO) model. Charge neutrality is identified at isoelectric point (pHiep and pciep). The dependence of viscosity and yield stress on shear rate may be modeled by von Smoluchowski's volumetric collision frequency multiplied by some total interaction energy given by DLVO model. Equilibrium and dynamic models for settling and enforced particle movement (viscosity) are presented. Both compressive yield stress (sedimentation) and cohesive energy (viscoelasticity) are characterized by power law exponents of volume fraction. The transition of disperse suspensions (sols) to spanning clusters (gels) is identified by oscillatory rheology. The slope of linear plots of logarithmic storage (G´) and loss (G") moduli against logarithm of frequency or logarithm of volume fraction provide power law exponents from the slopes. These exponents relate to percolation and fractal dimensions characterizing the particle network. Moreover, it identifies the structure formation process either as diffusion limited cluster-cluster (DLCCA) or as reaction limited cluster-cluster (RLCCA) aggregation.

The long-term consequences of school suspension and expulsion on depressive symptoms.

Exposure to exclusionary discipline has been tied to several deleterious outcomes in adulthood, including contact with the criminal legal system. While this work provides interesting insight into the long-term consequences tied to this form of school punishment, few have attempted to consider whether and how, exclusionary discipline practices, in particular, school suspension and expulsion shape mental health patterning over the life course. Using panel data from the National Longitudinal Study of Adolescent to Adult Health, we contribute to this body of literature by examining whether exposure to school suspension or expulsion shapes depressive symptom trajectories from adolescence to adulthood. Results from our mixed-effects linear growth curve models demonstrate both forms of exclusionary discipline play a significant role in depressive symptom trajectories. We find suspended and expelled youth exhibit significantly higher depressive symptoms in adolescence when compared to their counterparts with no history of suspension or expulsion. Results also show age variation in depressive symptom trajectories by history of exposure to exclusionary discipline. Specifically, results show the depressive symptoms gap between disciplined and non-disciplined youth slightly dissipates as youth age into early adulthood, but as individuals begin to transition out of this stage of the life course, the gap in depressive symptoms widens substantially. Results carry implications for how punitive disciplinary practices in schools shape mental health from adolescence to adulthood.

Biosensor with Microchannel for Broadband Dielectric Characterization of Nanoliter Cell Suspensions up to 110 GHz.

Cell dielectric property measurement holds significant potential for application in cell detection and diagnosis due to its label-free and noninvasive nature. In this study, we developed a biosensor designed to measure the permittivity of liquid samples, particularly cell suspensions at the nanoliter scale, utilizing microwave and millimeter wave coplanar waveguides in conjunction with a microchannel. This biosensor facilitates the measurement of scattering parameters within a frequency domain ranging from 1 GHz to 110 GHz. The obtained scattering parameters are then converted into dielectric constants using specific algorithms. A cell capture structure within the microchannel ensures that cell suspensions remain stable within the measurement zone. The feasibility of this biosensor was confirmed by comparison with a commercial Keysight probe. We measured the dielectric constants of three different cell suspensions (HepG2, A549, MCF-7) using our biosensor. We also counted the number of cells captured in multiple measurements for each cell type and compared the corresponding changes in permittivity. The results indicated that the real part of the permittivity of HepG2 cells is 0.2-0.8 lower than that of the other two cell types. The difference between A549 and MCF-7 was relatively minor, only 0.2-0.4. The fluctuations in the dielectric spectrum caused by changes in cell numbers during measurements were smaller than the differences observed between different cell types. Thus, the sensor is suitable for measuring cell suspensions and can be utilized for label-free, noninvasive studies in identifying biological cell suspensions.

A Systematic Microfluidic Study of the Use of Diluted Silica Sols to Enhance Oil Displacement.

The paper presents the results of a systematic microfluidic study of the application of nanosuspensions for enhanced oil recovery. For the first time, approximately a dozen nanosuspensions prepared by the dilution of silica sols as displacement fluids were considered. The concentration of nanoparticles in the suspensions varied from 0.125 to 2 wt%, and their size ranged from 10 to 35 nm. Furthermore, the silica sols under consideration differed in their compositions of functional groups and pH. The effects of concentration, nanoparticle size, fluid flow rate, and the viscosity of the displaced oil were investigated using microfluidic technology. The microfluidic experiments demonstrated that the application of nanosuspensions for water flooding has significant potential. The efficiency of oil displacement by nanosuspensions was found to increase significantly (up to 30%) with the increasing concentration and decreasing average size of nanoparticles. The application of nanosuspensions for the enhancement of oil recovery is most appropriate for reservoirs with highly viscous oil.

Clinical Ocular Exposure Extrapolation for a Complex Ophthalmic Suspension Using Physiologically Based Pharmacokinetic Modeling and Simulation.

The development of generic ophthalmic drug products with complex formulations is challenging due to the complexity of the ocular system and a lack of sensitive testing to evaluate the interplay of its physiology with ophthalmic drugs. New methods are needed to facilitate the development of ophthalmic generic drug products. Ocular physiologically based pharmacokinetic (O-PBPK) models can provide insight into drug partitioning in eye tissues that are usually not accessible and/or are challenging to sample in humans. This study aims to demonstrate the utility of an ocular PBPK model to predict human exposure following the administration of ophthalmic suspension. Besifloxacin (Bes) suspension is presented as a case study. The O-PBPK model for Bes ophthalmic suspension (Besivance® 0.6%) accounts for nasolacrimal drainage, suspended particle dissolution in the tears, ocular absorption, and distribution in the rabbit eye. A topical controlled release formulation was used to integrate the effect of Durasite® on Bes ocular retention. The model was subsequently used to predict Bes exposure after its topical administration in humans. Drug-specific parameters were used as validated for rabbits. The physiological parameters were adjusted to match human ocular physiology. Simulated human ocular pharmacokinetic profiles were compared with the observed ocular tissue concentration data to assess the OCAT models' ability to predict human ocular exposure. The O-PBPK model simulations adequately described the observed concentrations in the eye tissues following the topical administration of Bes suspension in rabbits. After adjustment of physiological parameters to represent the human eye, the extrapolation of clinical ocular exposure following a single ocular administration of Bes suspension was successful.

Parallel Multifactorial Process Optimization and Intensification for High-Yield Production of Live YF17D-Vectored Zika Vaccine.

The live-attenuated yellow fever 17D strain is a potent vaccine and viral vector. Its manufacture is based on embryonated chicken eggs or adherent Vero cells. Both processes are unsuitable for rapid and scalable supply. Here, we introduce a high-throughput workflow to identify suspension cells that are fit for the high-yield production of live YF17D-based vaccines in an intensified upstream process. The use of an automated parallel ambr15 microbioreactor system for screening and process optimization has led to the identification of two promising cell lines (AGE1.CR.pIX and HEKDyn) and the establishment of optimized production conditions, which have resulted in a >100-fold increase in virus titers compared to the current state of the art using adherent Vero cells. The process can readily be scaled up from the microbioreactor scale (15 mL) to 1 L stirred tank bioreactors. The viruses produced are genetically stable and maintain their favorable safety and immunogenicity profile, as demonstrated by the absence of neurovirulence in suckling BALB/c mice and consistent seroprotection in AG129 mice. In conclusion, the presented workflow allows for the rapid establishment of a robust, scalable, and high-yield process for the production of live-attenuated orthoflavivirus vaccines, which outperforms current standards. The approach described here can serve as a model for the development of scalable processes and the optimization of yields for other virus-based vaccines that face challenges in meeting growing demands.

Integrated Metabolomics and Transcriptomics Analyses of the Biosynthesis of Arbutin and 6'-O-Caffeoylarbutin in Vaccinium dunalianum Cell Suspension Cultures Fed with Hydroquinone.

Arbutin and 6'-O-caffeoylarbutin (CA) from Vaccinium dunalianum Wight are known for their ability to inhibit melanin synthesis. To boost the production of arbutin and CA, precursor feeding with hydroquinone (HQ) was studied in V. dunalianum suspension cells. The effect of HQ on the biosynthesis of arbutin and CA in the suspension cells was investigated using high-performance liquid chromatography (HPLC), and possible molecular mechanisms were analyzed using metabolomics and transcriptomics analyses. HPLC analysis only showed that the addition of HQ significantly enhanced arbutin synthesis in cells, peaking at 15.52 ± 0.28 mg·g-1 after 0.5 mmol·L-1 HQ treatment for 12 h. Subsequently, metabolomics identified 78 differential expression metabolites (DEMs), of which arbutin and CA were significantly up-regulated metabolites. Moreover, transcriptomics found a total of 10,628 differential expression genes (DEGs). The integrated transcriptomics and metabolomics revealed that HQ significantly enhanced the expression of two arbutin synthase (AS) genes (Unigene0063512 and Unigene0063513), boosting arbutin synthesis. Additionally, it is speculated that CA was generated from arbutin and 3,4,5-tricaffeoylquinic acid catalyzed by caffeoyl transferase, with Unigene0044545, Unigene0043539, and Unigene0017356 as potentially associated genes with CA synthesis. These findings indicate that the precursor feeding strategy offers a promising approach for the mass production of arbutin and CA in V. dunalianum suspension cells and provides new insights for CA biosynthesis in V. dunalianum.