Decrease In Cell Density Research Articles

Vestibular nerve neurectomy: Functional and histopathological outcomes in the cochlea of guinea pigs.

Experimentally test the effect of vestibular nerve neurectomy on the function and histopathology of the cochlea. The study was conducted on 20 normal male adult pigmented guinea pigs (Cavia porcellus). First, each animal was tested with Distortion Product Otoacoustic Emissions (DPOAEs) preoperatively then vestibular neurectomy (V.N) was performed on the right side through suboccipital modified retrosigmoid approach. We tested the DPOAEs postoperatively; After 8 weeks, Guinea pigs were euthanized the cochlea of guinea pig was processed for light microscope and transmission electron microscope examination to observe the outer hair cell count, and spiral ganglion cell density in the operated ear compared to contralateral non operated cochlea of the same guinea pig. There was a significant decrease in spiral ganglion cell density in the operated side compared to the non-operated side. However, there was no significant difference in outer hair cell count or DPOAEs between the two sides, except for a significant increase in DPOAEs at 2 KHz in the operated side. The results suggest that vestibular neurectomy may affect spiral ganglion cell density but not necessarily outer hair cell count or DPOAEs, and the relationship between these variables is complex and could be frequency-dependent.

Cryopreservation of primary neonatal rat oligodendrocytes and recapitulation of in vitro oligodendrocyte characteristics.

In vitro, primary rat oligodendrocytes (OLs) are widely used for research on OL development, physiology, and pathophysiology in demyelinating diseases such as multiple sclerosis. Primary culture methods for OLs from rats have been developed and improved over time, but there are still multiple aspects in which efficiency can be boosted. To make use of excess oligodendrocyte progenitor cells (OPCs) from primary cultures, a cryopreservation process utilizing a commercially available serum-free cryopreservation medium was established to passage and freeze OPCs at -80°C for later use. Cryopreserved OPCs stored for up to 6 months were viable, and retained their OL lineage purity of ~98%. While OPCs cryopreserved for 3-6 months showed a decrease in cell density after two days of proliferation, ~17% of cryopreserved OPCs maintained the potential for proliferation comparable to control OPCs that had not frozen. After induction of differentiation for four days, ~43% of both control and cryopreserved OPCs differentiated into mature OLs, and when differentiation was induced on aligned nanofibers mimicking axonal structure, myelin sheath-like structures indicative of in vitro myelination was observed in all experimental groups. The validation of cryopreserved primary OLs as a functionally robust in vitro model can help improve the efficiency of primary OL culture, expand its applications, and reduce the inevitable sacrifice of animals.

Investigating the effects of stereotactic body radiation therapy on pancreatic tumor hypoxia and microvasculature in an orthotopic mouse model using intravital fluorescence microscopy

Despite decades of improvements in cytotoxic therapy, the current standard of care for locally advanced pancreatic cancer (LAPC) provides, on average, only a few months of survival benefit. Stereotactic Body Radiation Therapy (SBRT), a technique that accurately delivers high doses of radiation to tumors in fewer fractions, has emerged as a promising therapy to improve local control of LAPC; however, its effects on the tumor microenvironment and hypoxia remain poorly understood. To explore how SBRT affects pancreatic tumors, we combined an orthotopic mouse model of pancreatic cancer with an intravital microscopy platform to visualize changes to the in vivo tumor microenvironment in real-time. Mice received SBRT (5 × 8 Gy) or were left untreated, and were imaged before and 1, 4, 7, and 14 days after treatment (n = 7/group). A fluorescent human pancreatic cancer cell line (BxPC3-DsRed) engineered to express GFP under hypoxic conditions (driven by hypoxia-inducible factor, HIF) was used to monitor tumor hypoxia. Immunohistochemical staining was also performed on tissues to validate in vivo data. Our findings demonstrate a persistent decrease in pancreatic tumor hypoxia as early as one day after SBRT. This coincided with a decrease in both tumor cell proliferation and cell density in the SBRT group. Reduced demand for oxygen after SBRT (due to cell death and growth arrest from treatment) significantly contributed to reoxygenation of the pancreatic TME. Understanding how this reoxygenation phenomenon occurs in a dose-dependent manner will help improve dosing and fractionation schemes for clinical SBRT.

Co-pathologies modify hippocampal protein accumulation patterns in neurodegenerative diseases.

Limited research has extensively analyzed neurodegenerative disease-related protein deposition patterns in the hippocampus. This study examined the distribution of proteins in hippocampal subregions across major neurodegenerative diseases and explored their relation to each other. The area density of phosphorylated tau (p-tau), amyloid beta (Aβ), α-synuclein, and phosphorylated TDP-43 protein deposits together with pyramidal cell density in each hippocampal subregion, including CA1-4, prosubiculum (ProS), and subiculum was assessed in 166 cases encompassing various neurodegenerative diseases. Alzheimer's disease-associated p-tau predominated in ProS, Aβ in the CA1, and Lewy body-related α-synuclein in the CA2. The area density of protein deposits increased with the pathological stage until a peak, then decreased in cases with high pathology stages along with pyramidal cell density. Comorbid protein pathology influenced protein deposition patterns. This comprehensive evaluation reveals characteristic neurodegenerative disease-related protein accumulation patterns in hippocampal subregions modified by co-pathologies. Alzheimer's disease-related phosphorylated tau predominates in the prosubiculum. Amyloid beta predominates in the CA1 and Lewy body-related α-synuclein in the CA2. The area density of protein deposition increases with the disease stage up to a peak. In the high pathology stage, protein deposition and pyramidal cell density decreases. Comorbid protein pathology affects the pattern of protein accumulation.

Post‐mortem MR imaging of tau pathology – a pilot study

AbstractBackgroundIn‐vivo detection of neuropathology is critical for early diagnosis of neurodegenerative diseases. Post‐mortem brain magnetic resonance imaging (MRI) of pathological protein inclusions could further our ability to detect them in vivo and correlate MRI parameters to histopathological substrates. In this post‐mortem study, we aimed to identify MRI correlates of neurodegenerative disease pathology in a brain with various forms of proteinopathies.MethodOne large cortical section containing the temporal cortex, hippocampus and thalamus was used from an 82‐year‐old male patient, with chronic traumatic encephalopathy (CTE), Alzheimer’s disease (AD), TDP‐43, aging related tau astrogliosis (ARTAG) and cerebral amyloid angiopathy (CAA) pathologies. The post‐mortem tissue sample was scanned using high‐resolution Rapid Acquisition with Relaxation Enhancement (RARE) and multi‐gradient echo (MGE) sequences on an 11.7 Tesla MRI (Bruker, Germany). RARE scan parameters were as follows: field of view: 97.2 x 86.4 x 28.8 mm, rep time: 2 s, RARE factor: 8, echo time: 48 ms, echo spacing: 12 ms, 225 μm isotropic resolution. Tissue samples were subsequently sectioned (0.004 mm thick slices) and stained using AT8 – for Tau, GFAP – for astrogliosis and H&E/Luxol – for distinguishing gray (GM) and white matter (WM) and PERLS – for extracellular iron.ResultPost‐mortem MRI allowed for clear visualization of WM and GM structures. Decreased cell density in WM as visualised on H&E stained slices was associated with hyperintensities on T2w images and decreased measured R2*. AT8 immunoreactivity in GM and WM was associated with hyperintensities on T2w images and decreased measured R2*.ConclusionHigh‐resolution MRI of post‐mortem tissue allowed for visualization of fine WM‐GM details, as well as hyperintensities in areas that potentially correspond to increased densities of tau pathology and decreased WM density. Further studies are needed to investigate the potential for high‐resolution post‐mortem MRI to visualize the pathological process in neurodegenerative diseases.

Comparison of Virulence and Quorum Sensing Gene Expression between Planktonic and Biofilm Cells of Legionella pneumophila

Abstract Legionella pneumophila is a waterborne bacterium that has a life cycle consisting of a transmissive phase in which free-living cells spread in the external environment, and a replicative phase with cells multiplying within biofilms. The transition from one phase to another is regulated by the bacterium’s quorum sensing system. As this system also regulates virulence, laboratory cultures of L. pneumophila were used to compare the different expressions of virulence and quorum sensing genes in planktonic and biofilm cells using RT-qPCR assays. The results showed a down regulation of the icmK gene in biofilm cells but only insignificant differences in the expression of 10 other genes associated with filamentation, conjugation, adherence and motility. The two quorum sensing genes examined, lpg1057 and lpg2731, differed in their expression profiles in planktonic and biofilm cells. While their expression in planktonic cells increased from day 10 until day 15 of incubation, their expression in biofilm cells steadily declined after 10 days of incubation. This decrease in quorum sensing gene expression was shown to be associated with a decrease in cell density in the biofilm, thus suggesting that the trigger for the transition from the replicative phase to the transmissive phase is likely to be nutrient depletion in the biofilm.

Impact of weathered and virgin polyethylene terephthalate (PET) micro- and nanoplastics on growth dynamics and the production of extracellular polymeric substances (EPS) of microalgae

The ever-increasing plastic waste accumulation in the marine environment necessitates a deeper understanding of microalgae interactions with micro- and nanoplastics (MNP), and the role of extracellular polymeric substances (EPS). EPS, known for its adhesive properties and produced as an algal stress response, may facilitate aggregation of both algae and MNPs, thereby impacting ecological and hydrodynamic processes such as the trophic transfer or vertical transport of MNPs. Moreover, gaining a deeper understanding of the impact of weathering processes on the potential toxicological effects of plastic particles, and the comparative significance of plastic-specific effects relative to those of naturally occurring particles such as kaolin clay, is imperative. Therefore, this study investigated the impact of fragmented, polydisperse virgin polyethylene terephthalate (PET, Daverage = 910 nm) and weathered PET (Daverage = 1700 nm) on the growth and the production of EPS of Rhodomonas salina. Algae were exposed to a range of low MNP concentrations (10, 100 and 1000 and 10,000 MNPs ml-1) for 11 days. A natural particle control (kaolin, Daverage = 1600 nm) was deployed to differentiate particle effects from plastic effects. It was observed that exposure to both weathered PET and virgin PET resulted in initially increased growth rates (7.80 % and 7.28 % respectively), followed by significant decreases in algae cell density (−30.1 % and −11.2 % respectively). Furthermore, exposure to weathered PET caused a simultaneous elevation in cellular EPS production (76.51 %). The effects of plastics were significantly larger than the effect of kaolin. Also, the observed effects were amplified by the weathering of the plastics. These observations underscore the interactions between particle type, age and concentration, and their distinct impacts on algae density and growth inhibition. The observations indicate a role for EPS as an algal protection mechanism, potentially affecting the environmental fate of MNP – microalgae aggregates.

Millennial Generation’s Choice of Contact Lens as the Preference Over Spectacles: A Study of Corneal Endothelial Cell Density

ABSTRACT Introduction: The cornea of the eye is the most vulnerable organ to external insult. Contact lens is no exception. Changes in corneal epithelium, stroma, and loss of hexagonal shape in the endothelium have been taught in contact lens classes. We wanted to understand the cell density of endothelium in prolonged contact lens wear. Methods: It was a prospective, observational, cross-sectional study. The volunteers were divided into two groups: group 1 wore their contact lenses for over 5 years and group 2 never wore contact lenses. Specular microscopy was performed using Topcon SP-3000P. Computerized morphometry was used to evaluate the mean cell density of the corneal endothelial cells. Result: A statistically significant decrease in corneal endothelial cell density was observed in contact lens wearers. Conclusion: The use of contact lenses for a prolonged period significantly decreases corneal endothelial cell density. Suitable intervention may prevent such changes in the cornea.

Confocal Microscopy of the Cornea in Aqueous-Deficient Dry Eye Disease-A Literature Review.

In vivo confocal microscopy (IVCM) is a vital tool in studying dry eye disease (DED), providing insights into morphological changes at ocular surface unit levels. This review presents the main differences in corneal structure between aqueous-deficient dry eye disease (AD-DED) and normal eyes. A comprehensive search of PubMed, Web of Science, Embase, and MEDLINE databases from January 2000 to December 2023 was conducted. The study selection process, as well as data selection and examination, were independently performed by two members of the review team. The review reveals a consistent decrease in corneal surface epithelial cell density in AD-DED cases compared to a control group, but conflicting data on basal epithelial cell density. Notably, the abnormal hyperreflectivity of keratocytes in patients with Sjogren's syndrome was recorded, and there was a significant keratocyte density in AD-DED subjects compared to evaporative DED and control groups. Studies also found a decrease in sub-basal nerve density, increased tortuosity, and the fragmentation of nerve fibers. Dendritic cell density and dendritic cell dendrites increase in AD-DED patients compared to healthy subjects. IVCM is a powerful tool for enhancing our understanding of the pathophysiological mechanisms underlying DED. However, the review underscores the urgent need to standardize the terminology, analysis, and units used for accurate interpretation, a crucial step in advancing our knowledge of DED.

Granulosa Cell-Layer Stiffening Prevents Escape of Mural Granulosa Cells from the Post-Ovulatory Follicle.

Ovulation is vital for successful reproduction. Following ovulation, cumulus cells and oocyte are released, while mural granulosa cells (mGCs) remain sequestered within the post-ovulatory follicle to form the corpus luteum. However, the mechanism underlying the confinement of mGCs has been a longstanding mystery. Here, in vitro and in vivo evidence is provided demonstrating that the stiffening of mGC-layer serves as an evolutionarily conserved mechanism that prevents mGCs from escaping the post-ovulatory follicles. The results from spatial transcriptome analysis and experiments reveal that focal adhesion assembly, triggered by the LH (hCG)-cAMP-PKA-CREB signaling cascade, is necessary for mGC-layer stiffening. Disrupting focal adhesion assembly through RNA interference results in stiffening failure, mGC escape, and the subsequent development of an abnormal corpus luteum characterized by decreased cell density or cavities. These findings introduce a novel concept of "mGC-layer stiffening", shedding light on the mechanism that prevents mGC escape from the post-ovulatory follicle.

The Combination of Auranofin and Celecoxib Suppresses Local Synovial Sarcoma Progression In Vivo.

Synovial sarcoma (SS) is a rare malignant tumor with a poor survival rate. We previously reported that a combination of auranofin (AUR), a thioredoxin reductase inhibitor, and celecoxib (CE), an anti-inflammatory drug, significantly impedes the local progression of osteosarcoma (OS). However, the role of redox regulation in SS remains to be elucidated. This study aimed to investigate the efficacy of combined treatment of AUR and CE on the local progression of SS in vivo. Nu/nu mice were implanted with the human SS cell line, Aska-SS, and treated with vehicle control, AUR, or a combination of AUR and CE (AUR-CE). Primary tumor size and weight were evaluated for the study duration and upon resection, respectively. Hematoxylin and eosin (H&E) and Ki-67 staining were performed to assess the local progression of SS. A statistically significant reduction in tumor size and weight was observed in the AUR- and AUR-CE-treated groups upon excision compared to that in the vehicle-treated group. The AUR-CE-treated group showed synergistic inhibition of local tumor growth. H&E staining of local SS tumors revealed decreased cell density and nuclear deformation in the AUR- and AUR-CE-treated groups compared to those in the vehicle-treated group. Immunohistochemical staining revealed a statistically significant decrease in Ki-67-positive cells in the AUR-CE-treated group compared to the vehicle-treated group. The combination of AUR and CE showed significant potential for delaying the local progression of SS. These findings support the repurposing of AUR and CE as early treatment options for SS.

Reservoir regulation-induced variations in water level impacts cyanobacterial bloom by the changing physiochemical conditions

Gaining insight into the impact of reservoir regulation on algal blooms is essential for comprehending the dynamic changes and response mechanisms in the reservoir ecosystem. In this study, we conducted a comprehensive field investigation linking physiochemical parameters, and phytoplankton community to different water regimes in the Three Gorges Reservoir. Our aim was to explore the effects of reservoir regulation on the extinction of cyanobacterial blooms. The results showed that during the four regulatory events, the water levels decreased by 2.02–4.33 m, and the average water velocity increased 68 % compared to before. The average total phosphorus and total nitrogen concentrations reduced by up to 20 %, and the cyanobacterial biomass correspondingly declined dramatically, between 66.94 % and 75.17 %. As the change of water level decline increasing, there was a significant increase of algal diversity and a notable decrease of algal cell density. Additionally, a shift in the dominant phytoplankton community from Cyanobacteria to Chlorophyceae was observed. Our analysis indicated that water level fluctuations had a pronounced effect on cyanobacterial extinction, with hydrodynamic changes resulting in a reduction of cyanobacterial biomass. This research underlined the potential for employing hydrodynamic management as a viable strategy to mitigate the adverse ecological impacts of cyanobacterial blooms, providing a solution for reservoir's eco-environmental management.

Prenatal ethanol and cannabis exposure have sex- and region-specific effects on somatostatin and neuropeptide Y interneurons in the rat hippocampus.

Cannabis is increasingly being legalized and socially accepted around the world and is often used with alcohol in social settings. We recently showed that in utero exposure to both substances can alter the density of parvalbumin-expressing interneurons in the hippocampus. Here we investigate the effects of in utero alcohol and cannabis exposure, alone or in combination, on somatostatin- and neuropeptide Y-positive (NPY) interneurons. These are separate classes of interneurons important for network synchrony and inhibition in the hippocampus. A 2 (Ethanol, Air) × 2 (tetrahydrocannabinol [THC], Vehicle) design was used to expose pregnant Sprague-Dawley rats to either ethanol or air, in addition to either THC or the inhalant vehicle solution, during gestational days 5-20. Immunohistochemistry for somatostatin- and NPY-positive interneurons was performed in 50 μm tissue sections obtained at postnatal day 70. Exposure to THC in utero had region-specific and sex-specific effects on the density of somatostatin-positive interneurons in the adult rat hippocampus. A female-specific decrease in NPY interneuron cell density was observed in the CA1 region following THC exposure. Combined exposure to alcohol and THC reduced NPY neurons selectively in the ventral dentate gyrus hippocampal subfield. However, overall, co-exposure to alcohol and cannabis had neither additive nor synergistic effects on interneuron populations in other areas of the hippocampus. These results illustrate how alcohol and cannabis exposure in utero may affect hippocampal function by altering inhibitory processes in a sex-specific manner.

Sulfated Hydrogels as Primary Intervertebral Disc Cell Culture Systems.

The negatively charged extracellular matrix plays a vital role in intervertebral disc tissues, providing specific cues for cell maintenance and tissue hydration. Unfortunately, suitable biomimetics for intervertebral disc regeneration are lacking. Here, sulfated alginate was investigated as a 3D culture material due to its similarity to the charged matrix of the intervertebral disc. Precursor solutions of standard alginate, or alginate with 0.1% or 0.2% degrees of sulfation, were mixed with primary human nucleus pulposus cells, cast, and cultured for 14 days. A 0.2% degree of sulfation resulted in significantly decreased cell density and viability after 7 days of culture. Furthermore, a sulfation-dependent decrease in DNA content and metabolic activity was evident after 14 days. Interestingly, no significant differences in cell density and viability were observed between surface and core regions for sulfated alginate, unlike in standard alginate, where the cell number was significantly higher in the core than in the surface region. Due to low cell numbers, phenotypic evaluation was not achieved in sulfated alginate biomaterial. Overall, standard alginate supported human NP cell growth and viability superior to sulfated alginate; however, future research on phenotypic properties is required to decipher the biological properties of sulfated alginate in intervertebral disc cells.

Organoruthenium metallocycle induced mutation in gld-1 tumor suppression gene in JK1466 strain and appreciable lifespan expansion

Four Ru(II) complexes (A2-A5) were synthesized from the reaction of coumarin Schiff base ligands (7da2-tsc, 7da3-mtsc, 7da4-etsc and 7da5-ptsc) with [RuHCl(CO)(PPh3)3]. The compounds were characterized by FT-IR, UV–Vis, 1H, 13C and 31P NMR, mass spectrometry and crystallographic analysis. Calf Thymus DNA (CT-DNA) binding studies revealed the intercalative mode of binding of the complexes with DNA. The results of Bovine serum albumin (BSA) binding studies established the interaction between BSA followed static quenching mechanism. The cytotoxic effects of the complexes and the ligands were evaluated against breast (MCF-7 and MDA-MB-231) and lung carcinoma cell lines (A549 and NCI-H460) using MTT assay. Complex A4 demonstrated potent cytotoxic effects on both breast and lung cancer cells. Furthermore, morphological observations and FACS analysis showed the decrease in cell density by complex A4 by induced morphological changes and apoptotic body formation and cell death in both breast and lung cancer cells. Moreover, the invertebrate model Caenorhabditis elegans was employed to assess the in vivo anticancer activity of compound A4. The findings indicated that the treatment with A4 reduced tumor development and significantly extended organismal lifespan by 64 % in the tumoral strain JK1466 without adversely affecting essential physiological functions of the worm. Additionally, A4 demonstrated an upregulation of two crucial antioxidant defense genes. Overall, these results suggested that the compound A4 can be a potential candidate with novel chemotherapeutic applications.

Histological and Molecular Evaluation of the Antiproliferative Activity of Allium ampeloprasum Water Extract Against Oral Mucosa Cell Line (Gingival Cancer)

Gingival carcinoma is a malignant neoplasm affecting the oral mucosa and is associated with significant morbidity and mortality. Allium ampeloprasum var. porrum water extracts have gotten a lot of attention because of their bioactive components, such as polyphenols, flavonoids, and alkaloids, which have a variety of pharmacological activities, including antiproliferative actions. This study aimed to evaluate the histological and molecular effects of Allium ampeloprasum (leek) water extract on the proliferation of the murine gingival cancer cell line. Histological evaluation was conducted to examine morphological changes induced by extract treatment. Molecular mechanisms underlying the observed histological changes were investigated using real-time polymerase chain reaction (PCR). Expression levels of key genes associated with cell proliferation and apoptosis were assessed. Histological findings revealed a dose-dependent decrease (100, 50, 25, 12.5, and 6.25 µg/ml) in cell density and altered cell shape in the treated cell line. Also, the percentage of inhibition for the oral mucosa cell line was high, with a significant P of 0.006, in the treated group compared to the control group. Additionally, water extract has an IC50 value of 61 g/ml. The P53 fold increment of gene expression is 0.6, which means the expression level in the experimental condition is 60% higher than the control. This study provides evidence for the potential antiproliferative activity of Allium ampeloprasum water extract on the oral mucosa cell line. The observed histological changes, coupled with the modulation of key genes involved in proliferation and apoptosis, suggest that leek water extract may have therapeutic implications in managing oral cancer.

Structural insights on anti-biofilm mechanism of heated slightly acidic electrolyzed water technology against multi-resistant Staphylococcus aureus biofilm on food contact surface

Slightly acidic electrolyzed water (SAEW) has proven to be an efficient and novel sanitizer in food and agriculture field. This study assessed the efficacy of SAEW (30 mg/L) at 40 °C on the inactivation of foodborne pathogens and detachment of multi-resistant Staphylococcus aureus (MRSA) biofilm. Furthermore, the underlying mechanism of MRSA biofilm under heated SAEW at 40 °C treatment on metabolic profiles was investigated. The results showed that the heated SAEW at 40 °C significantly effectively against foodborne pathogens of 1.96–7.56 (lg (CFU/g)) reduction in pork, chicken, spinach, and lettuce. The heated SAEW at 40 °C treatment significantly reduced MRSA biofilm cells by 2.41 (lg (CFU/cm2)). The synergistic effect of SAEW treatment showed intense anti-biofilm activity in decreasing cell density and impairing biofilm cell membranes. Global metabolic response of MRSA biofilms, treated by SAEW at 40 °C, revealed the alterations of intracellular metabolites, including amino acids, organic acid, fatty acid, and lipid. Moreover, signaling pathways involved in amino acid metabolism, energy metabolism, nucleotide synthesis, carbohydrate metabolites, and lipid biosynthesis were functionally disrupted by the SAEW at 40 °C treatment. As per our knowledge, this is the first research to uncover the potential mechanism of heated SAEW treatment against MRSA biofilm on food contact surface.

In vitro cytotoxic activity on KATO-III cancer cell lines of mangiferolic acid purified from Thai Tetragonula laeviceps propolis

Gastric cancer is a global health concern, but current treatment with chemotherapy and surgery is often inadequate, prompting the exploration of alternative treatments. Propolis is a natural substance collected by bees known for its diverse properties linked to floral sources. The Dichloromethane Partitioned Extract (DPE) from Tetragonula laeviceps propolis, in Bankha district, Thailand was previously shown to possess significant cytotoxicity against KATO-III gastric cancer cells, while showing lower cytotoxicity toward WI-38 normal fibroblast cells. Here, the DPE was further fractionated by column chromatography, identified active fractions, and subjected to structural analysis using nuclear magnetic resonance spectroscopy. Cytotoxicity against KATO-III cells was reevaluated, and programmed cell death was analyzed using flow cytometry. Expression levels of cancer-related genes were measured using quantitative real-time reverse transcriptase PCR. Cardol C15:2 (compound 1) and mangiferolic acid (MF; compound 2) were discovered in the most active fractions following structural analysis. MF exhibited strong cytotoxicity against KATO-III cells (IC50 of 4.78–16.02 μg/mL), although this was less effective than doxorubicin (IC50 of 0.56–1.55 μg/mL). Morphological changes, including decreased cell density and increased debris, were observed in KATO-III cells treated with 30 μg/mL of MF. Significant induction of late-stage apoptosis and necrosis, particularly at 48 and 72 h, suggested potential DNA damage and cell cycle arrest, evidenced by an increased proportion of sub-G1 and S-phase cells. Doxorubicin, the positive control, triggered late apoptosis but caused more necrosis after 72 h. Furthermore, MF at 30 μg/mL significantly increased the expression level of COX2 and NFκB genes linked to inflammation and cell death pathways. This upregulation was consistent at later time points (48 and 72 h) and was accompanied by increased expression of CASP3 and CASP7 genes. These findings suggest MF effectively induces cell death in KATO-III cells through late apoptosis and necrosis, potentially mediated by upregulated inflammation-related genes.

Characterization of TGFβ1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-β (TGFβ) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFβ signaling in tendon prompted us to utilize TGFβ1 to induce tendon-like structures in 3D tendon constructs. TGFβ1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFβ1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFβ1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

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

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. 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. 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). 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.