Knockout of all ErbB-family genes delineates their roles in proliferation, survival and migration.

The ErbB-family receptors play pivotal roles in the proliferation, migration and survival of epithelial cells. Because our knowledge on the ErbB-family receptors has been largely obtained by the exogenous application of their ligands, it remains unknown to what extent each of the ErbB members contributes to these outputs. We here knocked out each ErbB gene, various combinations of ErbB genes or all ErbB genes in Madin-Darby canine kidney cells to delineate the contribution of each gene. ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) activation waves during collective cell migration were mediated primarily by ErbB1 and secondarily by the ErbB2 and ErbB3 heterodimer. Either ErbB1 or the ErbB2 and ErbB3 complex was sufficient for the G1/S progression. The saturation cell density was markedly reduced in cells deficient in all ErbB proteins, but not in cells retaining only ErbB2, which cannot bind to ligands. Thus, a ligand-independent ErbB2 activity is sufficient for preventing apoptosis at high cell density. In short, systematic knockout of ErbB-family genes has delineated the roles of each ErbB receptor.

Cell density impacts the susceptibility to ferroptosis by modulating IRP1-mediated iron homeostasis.

Ferroptosis has been implicated in several neurological disorders and may be therapeutically targeted. However, the susceptibility to ferroptosis varies in different cells, and inconsistent results have been reported even using the same cell line. Understanding the effects of key variables of in vitro studies on ferroptosis susceptibility is of critical importance to facilitate drug discoveries targeting ferroptosis. Here, we showed that increased cell seeding density leads to enhanced resistance to ferroptosis by reducing intracellular iron levels. We further identified iron-responsive protein 1 (IRP1) as the key protein affected by cell density, which affects the expression of ferroportin or transferrin receptor and results in altered iron levels. Such observations were consistent across different cell lines, indicating that cell density should be tightly controlled in studies of ferroptosis. Since cell densities vary in different brain regions, these results may also shed light on selective regional vulnerability observed in neurological disorders.

A novel role for PRL in regulating epithelial cell density by inducing apoptosis at confluence.

Maintaining proper epithelial cell density is essential for the survival of multicellular organisms. Although regulation of cell density through apoptosis is well known, its mechanistic details remain elusive. Here, we report the involvement of membrane-anchored phosphatase of regenerating liver (PRL), originally known for its role in cancer malignancy, in this process. In epithelial Madin-Darby canine kidney cells, upon confluence, doxycycline-induced expression of PRL upregulated apoptosis, reducing cell density. This could be circumvented by artificially reducing cell density via stretching the cell-seeded silicon chamber. Moreover, small interfering RNA-mediated knockdown of endogenous PRL blocked apoptosis, leading to greater cell density. Mechanistically, PRL promoted apoptosis by upregulating the translation of E-cadherin and activating the TGF-ß pathway. Morpholino-mediated inhibition of PRL expression in zebrafish embryos caused developmental defects, with reduced apoptosis and increased epithelial cell density during convergent extension. Overall, this study revealed a novel role for PRL in regulating density-dependent apoptosis in vertebrate epithelia. This article has an associated First Person interview with the first author of the paper.

Ascorbic acid predominantly kills cancer stem cell-like cells in the hepatocellular carcinoma cell line Li-7 and is more effective at low cell density and in small spheroids.

The development of therapies that target cancer stem cells (CSCs) is an important challenge in cancer research. The antioxidant system is enhanced in CSCs, which may lead to resistance to existing therapies. Ascorbic acid (AA) has the potential to act as both an antioxidant and a pro-oxidant agent, but its effects on CSCs are a subject of current research. Here, we investigated the effect of AA focusing specifically on CSCs with the hepatocellular carcinoma cell line Li-7. The Li-7 cell line is heterogenous consisting of CD166- and CD166+ cells; CD166- cells include CSC-like cells (CD13+CD166- cells) and CD13-CD166- cells that can revert to CD13+CD166- cells. The addition of AA to the culture medium caused cell death in both cell populations in CD166- cells in a concentration dependent manner. In contrast, AA administration had a limited effect on CD166+ non-CSC cells. The level of reactive oxygen species after AA treatment was elevated only in CD166- cells. The effect of AA only occurred at low cell densities in 2D and 3D cultures. In a mouse tumor model injected with Li-7 cells, intraperitoneal administration of AA failed to prevent tumor formation but appeared to delay tumor growth. Our findings shed light on why AA administration has not become a mainstream treatment for cancer treatment; however, they also show the possibility that AA can be used in therapies to suppress CSCs.

Comparative responses of flocculating and nonflocculating yeasts to cell density and chemical stress in lactic acid fermentation.

While flocculation has demonstrated its efficacy in enhancing yeast robustness and ethanol production, its potential application for lactic acid fermentation remains largely unexplored. Our study examined the differences between flocculating and nonflocculating Saccharomyces cerevisiae strains in terms of their metabolic dynamics when incorporating an exogenous lactic acid pathway, across varying cell densities and in the presence of lignocellulose-derived byproducts. Comparative gene expression profiles revealed that cultivating a nonflocculant strain at higher cell density yielded a substantial upregulation of genes associated with glycolysis, energy metabolism, and other key pathways, resulting in elevated levels of fermentation products. Meanwhile, the flocculating strain displayed an inherent ability to sustain high glycolytic activity regardless of the cell density. Moreover, our investigation revealed a significant reduction in glycolytic activity under chemical stress, potentially attributable to diminished ATP supply during the energy investment phase. Conversely, the formation of flocs in the flocculating strain conferred protection against toxic chemicals present in the medium, fostering more stable lactic acid production levels. Additionally, the distinct flocculation traits observed between the two examined strains may be attributed to variations in the nucleotide sequences of the flocculin genes and their regulators. This study uncovers the potential of flocculation for enhanced lactic acid production in yeast, offering insights into metabolic mechanisms and potential gene targets for strain improvement.

Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis.

Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi-epitope chimeric antigen (ChA) and screening two classes of protein-assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin-binding protein A of Glaesserella parasuis. Our engineered strain, which co-expresses P17 tagged-ChA and endogenous chaperones groEL-ES, yields a 0.346 g/L highly soluble ChA with the property of HPS-positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5-L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

Apparent diffusion coefficient and tissue stiffness are associated with different tumor microenvironment features of hepatocellular carcinoma.

OBJECTIVES: To investigate associations between tissue diffusion, stiffness, and different tumor microenvironment features in resected hepatocellular carcinoma (HCC). METHODS: Seventy-two patients were prospectively included for preoperative magnetic resonance (MR) diffusion-weighted imaging and MR elastography examination. The mean apparent diffusion coefficient (ADC) and stiffness value were measured on the central three slices of the tumor and peri-tumor area. Cell density, tumor-stroma ratio (TSR), lymphocyte-rich HCC (LR-HCC), and CD8 + T cell infiltration were estimated in resected tumors. The interobserver agreement of MRI measurements and subjective pathological evaluation was assessed. Variables influencing ADC and stiffness were screened with univariate analyses, and then identified with multivariable linear regression. The potential relationship between explored imaging biomarkers and histopathological features was assessed with linear regression after adjustment for other influencing factors. RESULTS: Seventy-two patients (male/female: 59/13, mean age: 56 ± 10.2 years) were included for analysis. Inter-reader agreement was good or excellent regarding MRI measurements and histopathological evaluation. No correlation between tumor ADC and tumor stiffness was found. Multivariable linear regression confirmed that cell density was the only factor associated with tumor ADC (Estimate = -0.03, p = 0.006), and tumor-stroma ratio was the only factor associated with tumor stiffness (Estimate = -0.18, p = 0.03). After adjustment for fibrosis stage (Estimate = 0.43, p < 0.001) and age (Estimate = 0.04, p < 0.001) in the multivariate linear regression, intra-tumoral CD8 + T cell infiltration remained a significant factor associated with peri-tumor stiffness (Estimate = 0.63, p = 0.02). CONCLUSIONS: Tumor ADC surpasses tumor stiffness as a biomarker of cellularity. Tumor stiffness is associated with tumor-stroma ratio and peri-tumor stiffness might be an imaging biomarker of intra-tumoral immune microenvironment. CLINICAL RELEVANCE STATEMENT: Tissue stiffness could potentially serve as an imaging biomarker of the intra-tumoral immune microenvironment of hepatocellular carcinoma and aid in patient selection for immunotherapy. KEY POINTS: Apparent diffusion coefficient reflects cellularity of hepatocellular carcinoma. Tumor stiffness reflects tumor-stroma ratio of hepatocellular carcinoma and is associated with tumor-infiltrating lymphocytes. Tumor and peri-tumor stiffness might serve as imaging biomarkers of intra-tumoral immune microenvironment.

High cell density sequential batch fermentation for enhanced propionic acid production from glucose and glycerol/glucose mixture using Acidipropionibacterium acidipropionici.

BACKGROUND: Propionic acid fermentation from renewable feedstock suffers from low volumetric productivity and final product concentration, which limits the industrial feasibility of the microbial route. High cell density fermentation techniques overcome these limitations. Here, propionic acid (PA) production from glucose and a crude glycerol/glucose mixture was evaluated using Acidipropionibacterium acidipropionici, in high cell density (HCD) batch fermentations with cell recycle. The agro-industrial by-product, heat-treated potato juice, was used as N-source. RESULTS: Using 40 g/L glucose for nine consecutive batches yielded an average of 18.76 ± 1.34 g/L of PA per batch (0.59 gPA/gGlu) at a maximum rate of 1.15 gPA/L.h, and a maximum biomass of 39.89 gCDW/L. Succinic acid (SA) and acetic acid (AA) were obtained as major by-products and the mass ratio of PA:SA:AA was 100:23:25. When a crude glycerol/glucose mixture (60 g/L:30 g/L) was used for 6 consecutive batches with cell recycle, an average of 35.36 ± 2.17 g/L of PA was obtained per batch (0.51 gPA/gC-source) at a maximum rate of 0.35 g/L.h, and reaching a maximum biomass concentration of 12.66 gCDW/L. The PA:SA:AA mass ratio was 100:29:3. Further addition of 0.75 mg/L biotin as a supplement to the culture medium enhanced the cell growth reaching 21.89 gCDW/L, and PA productivity to 0.48 g/L.h, but also doubled AA concentration. CONCLUSION: This is the highest reported productivity from glycerol/glucose co-fermentation where majority of the culture medium components comprised industrial by-products (crude glycerol and HTPJ). HCD batch fermentations with cell recycling are promising approaches towards industrialization of the bioprocess.

C-Kit Immunohistochemical Expression as a Complementary Method to Assess Mast Cell Density in Helicobacter pylori-Mediated Gastritis.

INTRODUCTION: Chronic gastritis is a group of conditions commonly characterized by stomach lining inflammation. The study aimed to investigate the clinical and pathological aspects that play a role in its development. Additionally, the study examines the use of CD117 as an immunohistochemistry marker in evaluating mast cell density (MCD). METHODS: This retrospective, cross-sectional study was conducted in Iraqi Kurdistan with a sample size of 380 patients. Patient data included gastritis type, neutrophil infiltration severity, mononuclear cell infiltration within the lamina propria, intestinal metaplasia, and glandular atrophy, which were categorized and given a score. The CD117 level was identified using an anti-human rabbit polyclonal antibody. RESULTS: A statistically significant association was revealed between Helicobacter pylori-mediated gastritis and non-specific gastritis with age, activity, H. pylori and MCD, dysplasia, and malignancy. Meanwhile, no association was found with gender, inflammatory infiltrate, intestinal metaplasia, and glandular atrophy. C-Kit exhibited a marked increase in MCD in patients with H. pylori-mediated gastritis, intestinal metaplasia, atrophy, and gastric carcinoma. However, a significant decrease in MCD was observed on repeating endoscopy evaluations for patients after treatment. CONCLUSION: Regions that exhibit severe inflammation, metaplasia, atrophy, and carcinoma demonstrated an increase in MCD with H. pylori-mediated gastritis. A detailed investigation in clinical practice to screen early diagnosis and treatment needs to be performed in high H. pylori prevalence.

Adipogenic differentiation effect of human periodontal ligament stem cell initial cell density on autologous cells and human bone marrow stromal cells.

Stem cells demonstrate differentiation and regulatory functions. In this discussion, we will explore the impacts of cell culture density on stem cell proliferation, adipogenesis, and regulatory abilities. This study aimed to investigate the impact of the initial culture density of human periodontal ligament stem cells (hPDLSCs) on the adipogenic differentiation of autologous cells. Our findings indicate that the proliferation rate of hPDLSCs increased with increasing initial cell density (0.5-8 × 104 cells/cm2). After adipogenic differentiation induced by different initial cell densities of hPDLSC, we found that the mean adipose concentration and the expression levels of lipoprotein lipase (LPL), CCAAT/enhancer binding protein α (CEBPα), and peroxisome proliferator-activated receptor γ (PPAR-γ) genes all increased with increasing cell density. To investigate the regulatory role of hPDLSCs in the adipogenic differentiation of other cells, we used secreted exocrine vesicles derived from hPDLSCs cultivated at different initial cell densities of 50 µg/mL to induce the adipogenic differentiation of human bone marrow stromal cells. We also found that the mean adipose concentration and expression of LPL, CEBPα, and PPARγ genes increased with increasing cell density, with an optimal culture density of 8 × 104 cells/cm2. This study provides a foundation for the application of adipogenic differentiation in stem cells.

Corneal endothelial cell density and associated factors among adults at a regional referral hospital in Uganda: a cross-sectional study.

BACKGROUND: To assess the prevalence of low corneal endothelial cell density and correlates of corneal endothelial cell density among adults attending Mbarara University and Referral Hospital Eye Centre in Uganda. METHODS: In this hospital-based cross-sectional study, participants 18 years and older, were enrolled. We obtained informed consent, and basic demographic data. We also conducted visual acuity, a detailed slit lamp examination, intra-ocular pressure, corneal diameter, tear-film break-up time, keratometry, A-scan, and pachymetry on all participants. A confocal microscope Heidelberg HRT3 was used to examine the central cornea and to obtain the mean cell density (cells/mm2). To calculate the proportion of low endothelial cell density, descriptive statistics were used, whereas correlates of endothelial cell density were assessed, using linear regression analyses. RESULTS: We evaluated a total of 798 eyes of 404 participants aged between 18 and 90 years (males = 187, females = 217). The average endothelial cell density was 2763.6 cells/mm2, and there was a decrease in endothelial cell density with increasing age, irrespective of gender. There was no significant difference in endothelial cell density between males and females. Increasing age (adjusted coefficient - 10.1, p < 0.001), history of smoking (adjusted coefficient - 439.6, p = 0.004), history of ocular surgery (adjusted coefficient - 168.0, p = 0.023), having dry eye (adjusted coefficient - 136.0, p = 0.051), and having arcus senilis (adjusted coefficient - 132.0, p = 0.08), were correlated with lower endothelial cell density. However, increasing corneal diameter (adjusted coefficient 134.0, p = 0.006), increasing central corneal thickness (adjusted coefficient 1.2, p = 0.058), and increasing axial length (adjusted coefficient 65.8, p = 0.026), were correlated with higher endothelial cell density. We found five eyes (0.63%) from different participants with a low endothelial cell density (< 1000cells/mm2). CONCLUSION: Our study established baseline normal ranges of ECD in a predominantly black African population, and found that low ECD is rare in our population. The elderly, smokers, and those with past ocular surgery are the most vulnerable. The low prevalence could be due to a lack of reference values for the black African population.

Clinical results with a multifocal intraocular lens with a novel optical design.

BACKGROUND: To evaluate the optical performance and safety of a new multifocal lens with a novel optical design featuring two additional foci (or intensifiers) in patients with cataract and presbyopia. METHODS: In this single-center, non-randomized prospective observational study, 31 patients underwent implantation of the new multifocal IOL between March 2020 and November 2021 at a tertiary clinical center in Buenos Aires and Ramos Mejia, Argentina. Postoperative examinations with emphasis on uncorrected and corrected visual acuity at distance and near and at two different intermediate distances (80 cm and 60 cm) were performed during the 3 postoperative months. RESULTS: Of the 31 patients who underwent implantation of the new IOL, 30 underwent bilateral surgery (61 eyes in total). At 3 months, all 61 eyes had an uncorrected distance visual acuity (UCDVA) of at least 0.15 logMAR; 57 eyes (93%) had an uncorrected distance visual acuity (UCDVA) of 0.1 logMAR and 27 eyes (44%) had an UCDVA of 0.0 logMAR. At 80 cm, 60 eyes (98%) had an uncorrected intermediate visual acuity (UCIVA) of at least 0.1 log MAR and 48 eyes (79%) had an UCIVA of 0.0 logMAR. CONCLUSION: The new multifocal IOL with a novel optical concept (5 foci) showed a wide range of visual acuity especially at intermediate and near distances in patients undergoing cataract surgery. Uncorrected visual acuity was excellent at all tested distances, monocularly and binocularly, spectacle independence and patient satisfaction were high.

Comparison of automated corneal endothelial cell analysis in healthy and postoperative eyes with phakic intraocular lens: a cross-sectional study and literature review.

PURPOSE: Measurement of corneal endothelial cells is critical for postoperative evaluation of phakic intraocular lens (pIOL) surgery. However, inter-instrument differences in corneal endothelial cell density (ECD) after pIOL implantation have not yet been reported. This study aimed to compare automated corneal endothelial cell analysis between CellChek-20 (Konan Medical, Hyogo, Japan) and EM-4000 (Tomey, Nagoya, Japan) in healthy and postoperative eyes with pIOL. METHODS: We retrospectively analyzed 154 healthy and 236 postoperative eyes after pIOL surgery. Endothelial cell measurements were performed using CellChek-20 and EM-4000 with autofocusing and automated image analysis. ECD, percentage of hexagonal cells (HEX), coefficient of variation in cell size (CoV), and central corneal thickness (CCT) were compared between the two devices. RESULTS: The ECDs of the two devices were highly correlated in both healthy (Spearman's correlation coefficient [r] = 0.805; p < 0.001) and postoperative (r = 0.901; p < 0.001) groups. ECD from CellChek-20 was higher than EM-4000 in both healthy (mean difference = 228.9 cells/mm2; p < 0.001) and postoperative (mean difference = 115.6 cells/mm2; p < 0.001) groups. The CCT values also showed a strong correlation in healthy eyes (r = 0.974; p < 0.001) and in postoperative eyes (r = 0.936; p < 0.001); however, significant inter-instrument differences were observed. HEX and CV showed significant differences and relatively weak correlations (r < 0.7) between the two devices in both healthy and postoperative groups. CONCLUSION: The ECD values between the two instruments were correlated, but that of the CellChek-20 was significantly higher than that of the EM-4000 in both healthy and postoperative eyes after pIOL surgery. Most previous studies have also shown that the Konan software overestimated the ECD compared to other products in automatic measurement mode. The possibility of measurement bias should be considered when replacing equipment used for corneal endothelial cell measurements.

Clinical observations of EVO-ICL implantation with single incision without viscoelastic agent.

BACKGROUND: To investigate the safety and effectiveness of non-viscoelastic agent technique for EVO-ICL implantation. METHODS: A total of 181 myopia eyes that underwent non-toric ICL without viscoelastic agent through single incision from Beijing Tongren Hosipital were included. An analysis was conducted on the quantity of haptics that were initially implanted intraoperatively into the posterior chamber. Intraocular pressure (IOP) was evaluated at before and 2 h,24 h,1week,6month after surgery. Anterior chamber volume(ACV), anterior chamber depth(ACD), anterior chamber angle(ACA), pupil diameter(PD) and corneal densitometry density (ECD) were evaluated at before and 24 h postoperatively. Refractive outcomes were investigated at before, 24 h ,7 days and 6months. Vault was evaluated at 24 h ,7 days and 6months. RESULTS: The efficacy and safety indices were 1.30 ± 0.32 and 1.31 ± 0.32, respectively. Of 181 eyes, 99 eyes received 4 haptics on the first attempt without any adjustment, and 72 eyes received lens alignment without an viscoelastic agent. The success rate of the viscoelastic agent free procedure was 94.5%. Two hours postoperatively, IOP was 17.41 ± 3.77 mmHg, which was significantly higher than baseline value (t = 8.930, P < 0.000), however there was no significant difference between preoperative IOP and IOP at 1 day ,1 week and 6 months postoperatively. The ECD changed from 2895.52 ± 253.73 cells/mm2 preoperatively to 2873.66 ± 244.17 cells/mm2 at 1 day and 2882.63 ± 239.97 postoperatively, and the difference was not statistically significant (t = 1.811, P = 0.072). The ACA was narrowed by 42% on the first day. CONCLUSION: The pure viscoelastic agent free technique is an efficient and safe way for ICL implantation. It can be a safer method of ICL implantation because of it reduces the risk of complications associated with ocular hypertension at the early postoperative stages. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR2000036335) at August 20, 2020.

Outcomes of retropupillary iris claw lens implantation in patients with intraocular lens dislocation and low (less than 1000 cells/mm2) corneal endothelial cell density.

BACKGROUND: Posterior chamber intraocular lens (IOL) dislocation is a common complication of cataract surgery. Dislocated IOLs often require surgical intervention due to the potentially severe risks of leaving this condition untreated. If a patient with extremely low corneal endothelial cell density (ECD) presents with IOL dislocation, the surgeon faces a crucial dilemma of choosing the most optimal surgical treatment option. We sought to investigate the efficacy and safety of retropupillary iris claw intraocular lens (R-IOL) implantation in patients with IOL dislocation and extremely low (< 1000 cells/mm2) ECD. METHODS: We retrospectively reviewed the medical records of nine patients (all men) whose pre-operative ECD was < 1000 cells/mm2 and who underwent R-IOL implantation due to intraocular subluxation or total dislocation into the vitreous cavity between 2014 and 2020. We evaluated corneal endothelial function and visual outcomes after surgery. RESULTS: Nine patients were included in this study. The mean age at diagnosis was 64.89 ± 7.15 years (range 57-76 years), and the follow-up duration was 37.93 ± 23.72 months (range 18.07-89.07 months). No patients developed bullous keratopathy during follow-up. Compared to the initial ECD, corneal thickness (CT), coefficient variation of cell area (CV) and percentage of hexagonal cells (HEX), there was no statistically significant decrease in the ECD, CV, and HEX at last follow-up (P = 0.944, 0.778, 0.445, 0.443). There was significant improvement in the mean uncorrected distance visual acuity (UDVA) at the last follow-up (average 0.13 logMAR, 20/27 Snellen) compared to the pre-operative mean UDVA (average 1.09 logMAR, 20/250 Snellen) (P < 0.01). CONCLUSIONS: R-IOL implantation did not result in a statistically significant decline in corneal endothelial function in patients with preoperatively low ECD, and it significantly improved the mean UDVA postoperatively. R-IOL implantation appears to be a safe and effective treatment modality for intraocular lens dislocation in patients with low ECD (< 1000 cells/mm²); however, long-term follow-up studies are warranted to corroborate these findings.

Comprehensive assessment of corneal microstructural changes following V4c implantable collamer lens surgery using in vivo confocal microscopy.

BACKGROUND: Implantable Collamer Lense (ICL) presents a viable alternative to conventional refractive surgeries, but their impact on corneal microstructure remains unclear. By employing in vivo confocal microscopy (IVCM), we examined changes in stromal and endothelial cells following the insertion of V4c ICLs, with the goal of enhancing post-surgical care and outcomes. METHODS: In this longitudinal investigation, we conducted detailed preoperative assessments on 103 eyes from 53 participants. Follow-up evaluations were carried out after surgery at set intervals: one day, one week, one month, three months, six months, and twelve months. We used IVCM to analyze changes in stromal and endothelial cells. To assess differences between pre- and post-surgery variables and to investigate correlations with age, axial length (AL), and spherical equivalent refraction (SER), we applied a repeated measures mixed-effects model, with statistical significance set at P < 0.05. RESULTS: No vision-threatening complications were reported post-surgery. Significant reductions in stromal cell density (SCD) were observed postoperatively, with anterior and mid- SCD reaching their lowest values at 3 months and posterior SCD at 1 month, remaining below baseline at 12 months. endothelial cell density (ECD) and percentage of hexagonal cells (PHC) decreased initially, recovering by 12 months. Conversely, endothelial cellular area (ECA) and coefficient of variation of cell size (CoV) increased postoperatively, with the most significant change at 1 week. Endothelial deposits were detected in 49 of 101 eyes on postoperative day 1, half of them were absorbed within 3 months post-surgery. Changes in posterior SCD were negatively related to AL, while AL, SER, lens thickness showed associated with endothelium changes. CONCLUSION: Our findings elucidate the corneal microstructural changes following V4c ICL implantation, particularly the significant early reductions in stromal and endothelial cell densities. We recommend careful management of viscoelastics during surgery to minimize endothelial deposits that may harm the endothelium. Enhanced early postoperative monitoring and these surgical adjustments can lead to improved surgical and post-surgical care, ultimately supporting better patient recovery.

Canine corneal endothelial cell analysis using vital dyes and light microscopy.

PURPOSE: To evaluate the use of vital dyes and light microscopy for assessing canine corneal endothelial morphology ex vivo. METHODS: The corneas of 40 canine eyes (n = 20 dogs) enucleated <24 h following euthanasia or death were isolated and flat-mounted on a slide. Corneal endothelium was stained via 0.25% trypan blue followed by 0.5% alizarin red (pH 4.2), photographed, then the following morphological features were calculated using ImageJ: mean cell density (MCD), mean cell area (MCA), polymegathism (coefficient of variation of cell area), and pleomorphism (% hexagonality). RESULTS: Mean ± standard deviation (range) outcomes were: MCD, 2544 ± 541 cells/mm2 (1750-3922 cells/mm2); MCA, 431 ± 97 µm2 (251-626 µm2); polymegathism, 17 ± 2% (14%-22%); pleomorphism, 84 ± 3% (80%-90%). No significant differences (p ≥ .122) were noted for any outcome between male versus female or brachycephalic versus non-brachycephalic dogs. Young dogs (<10 years) had lower MCA (p = .044), lower pleomorphism (p = .003), and higher MCD (p = .035) when compared to older dogs (≥10 years). Age was significantly (p ≤ .049) correlated with MCA (r = 0.467), MCD (r = -0.476), polymegathism (r = 0.444), and pleomorphism (r = 0.609). CONCLUSIONS: The combination of vital dyes and light microscopy allowed for clear visualization and evaluation of the corneal endothelium in canine eyes ex vivo. Our findings can be used in future studies to deepen our understanding of the corneal endothelium in health and disease.

A study on twitching motility dynamics in Ralstonia solanacearum microcolonies by live imaging.

Ralstonia solanacearum is a rod-shaped phytopathogenic bacterium that causes lethal wilt disease in many plants. On solid agar growth medium, in the early hour of the growth of the bacterial colony, the type IV pili-mediated twitching motility, which is important for its virulence and biofilm formation, is prominently observed under the microscope. In this study, we have done a detailed observation of twitching motility in R. solanacearum colony. In the beginning, twitching motility in the microcolonies was observed as a density-dependent phenomenon that influences the shape of the microcolonies. No such phenomenon was observed in Escherichia coli, where twitching motility is absent. In the early phase of colony growth, twitching motility exhibited by the cells at the peripheral region of the colony was more prominent than the cells toward the center of the colony. Using time-lapse photography and merging the obtained photomicrographs into a video, twitching motility was observed as an intermittent phenomenon that progresses in layers in all directions as finger-like projections at the peripheral region of a bacterial colony. Each layer of bacteria twitches on top of the other and produces a multilayered film-like appearance. We found that the duration between the emergence of each layer diminishes progressively as the colony becomes older. This study on twitching motility demonstrates distinctly heterogeneity among the cells within a colony regarding their dynamics and the influence of microcolonies on each other regarding their morphology.

Bioprinted High-Cell-Density Laminar Scaffolds Stimulate Extracellular Matrix Production in Osteochondral Co-Cultures.

Many tissues have a laminar structure, but there are limited technologies for establishing laminar co-cultures for in vitro testing. Here, we demonstrate that collagen-alginate-fibrin (CAF) hydrogel scaffolds produced using the reactive jet impingement bioprinting technique can produce osteochondral laminar co-cultures with well-defined interfaces between cell types and high cell densities to support cell-cell interaction across the interfaces. The influence of cell density and the presence of the two cell types on the production of extracellular matrix (ECM) and the emergent mechanical properties of gels is investigated using IHC, ELISA, gel mass, and the compression modulus. The results indicate that high-cell-density cultures and co-cultures with these specific cell types produce greater levels of ECM and a more biomimetic in vitro culture than low-cell-density cultures. In laminar scaffolds produced using TC28a2 chondrocytes and SaoS-2 osteoblasts, both cell density and the presence of the two cell types enhance ECM production and the mechanical properties of the cultures, presenting a promising approach for the production of more biomimetic in vitro models.

Continuous fermentation using high cell density cell recycle system for L-lactic acid production.

For complete utilization of high glucose at ∼100 g/L, a high cell density (HCD) continuous fermentation system was established using Lb. delbrueckii NCIM 2025 for the bioproduction of lactic acid (LA). An integrated membrane cell recycling system coupled with the continuous bioreactor, aided to achieve the highest 34.77 g/L h LA productivity and 0.94-0.98 g/g yield. ∼34 times higher productivity was observed (in comparison to batch fermentation conducted in this study), when the continuous operations were carried out at the maximum dilution rate and wet cell weight i.e. 0.36 h-1 and 230 g/L, respectively. These results show the potential of this method for large-scale lactic acid production because it not only produces high titers but also ensures that glucose is used effectively. The method's superior performance in comparison to earlier studies suggests it as an affordable and sustainable alternative for the production of LA.