Cell Density Research Articles

Intramuscular fatty infiltration and its correlation with muscle composition and function in hip osteoarthritis

BackgroundHip osteoarthritis patients display higher levels of fatty infiltration (FI) in the gluteus minimus (GM) compared to other hip muscles. We investigated specific histological factors such as fiber type composition and collagen deposition, and functional outcomes like muscle strength and activation associated with FI in these patients.MethodsIn twelve men (67 ± 6 y) undergoing total hip replacement (THR), hip and knee muscle strength and activation (electromyography, EMG) were assessed bilaterally. Magnetic resonance imaging (MRI) was used to compare the relative FI area and muscle cross sectional area (CSA) of the GM, rectus femoris (RF), tensor fascia latae (TFL) and vastus lateralis (VL). Adipocyte content, fiber type composition, grouping, fiber size, centrally nucleated fiber frequency, collagen deposition, satellite cell density and capillarization were assessed in intraoperative biopsies of the four muscles. Differences between GM and other muscles were assessed with repeated-measures one-way ANOVA followed by Dunnett’s post-hoc test. Pearson coefficients were calculated for the correlations between FI measurements and the other histological and functional parameters.ResultsStrength was lower in the affected limb. Knee extensor weakness was accompanied by lower VL muscle activation. Muscle CSA and FI did not differ between sides. In the affected limb, GM displayed larger relative FI area (MRI) compared to RF and VL. Biopsy adipocyte content was higher in GM than RF and TFL. Compared to the other hip muscles, GM displayed higher type 1 fiber content while its type 2X fiber content was lower. Fiber grouping levels were higher in GM than the other muscles. Collagen content was higher in GM than TFL and VL. FI in GM was associated with type 1 (r = 0.43) and type 2X (r = -0.34) fiber content, fiber grouping (r = 0.39), and collagen deposition (r = 0.37). FI in VL was negatively associated with maximal knee extension strength (r = -0.65).ConclusionsIn patients undergoing THR, the higher FI levels of GM compared to other hip muscles were associated with fiber type composition and grouping, and with higher collagen deposition. Experimental studies exploring these associations could potentially uncover new targets for the treatment of intramuscular FI and related impairments in muscle function.Trial registrationKEK number: 2016–01852, date of registration: 12-4-2017.

Microglia in the aged brain develop a hypoactive molecular phenotype after surgery

BackgroundMicroglia, the resident immune cells of the brain, play a crucial role in maintaining homeostasis in the central nervous system (CNS). However, they can also contribute to neurodegeneration through their pro-inflammatory properties and phagocytic functions. Acute post-operative cognitive deficits have been associated with inflammation, and microglia have been implicated primarily based on morphological changes. We investigated the impact of surgery on the microglial transcriptome to test the hypothesis that surgery produces an age-dependent pro-inflammatory phenotype in these cells.MethodsThree-to-five and 20-to-22-month-old C57BL/6 mice were anesthetized with isoflurane for an abdominal laparotomy, followed by sacrifice either 6 or 48 h post-surgery. Age-matched controls were exposed to carrier gas. Cytokine concentrations in plasma and brain tissue were evaluated using enzyme-linked immunosorbent assays (ELISA). Iba1+ cell density and morphology were determined by immunohistochemistry. Microglia from both surgically treated mice and age-matched controls were isolated by a well-established fluorescence-activated cell sorting (FACS) protocol. The microglial transcriptome was then analyzed using quantitative polymerase chain reaction (qPCR) and RNA sequencing (RNAseq).ResultsSurgery induced an elevation in plasma cytokines in both age groups. Notably, increased CCL2 was observed in the brain post-surgery, with a greater change in old compared to young mice. Age, rather than the surgical procedure, increased Iba1 immunoreactivity and the number of Iba1+ cells in the hippocampus. Both qPCR and RNAseq analysis demonstrated suppression of neuroinflammation at 6 h after surgery in microglia isolated from aged mice. A comparative analysis of differentially expressed genes (DEGs) with previously published neurodegenerative microglia phenotype (MGnD), also referred to disease-associated microglia (DAM), revealed that surgery upregulates genes typically downregulated in the context of neurodegenerative diseases. These surgery-induced changes resolved by 48 h post-surgery and only a few DEGs were detected at that time point, indicating that the hypoactive phenotype of microglia is transient.ConclusionsWhile anesthesia and surgery induce pro-inflammatory changes in the plasma and brain of mice, microglia adopt a homeostatic molecular phenotype following surgery. This effect seems to be more pronounced in aged mice and is transient. These results challenge the prevailing assumption that surgery activates microglia in the aged brain.

GaAs Solar Cells Grown Directly on V-Groove Si Substrates.

The direct epitaxial growth of high-quality III-V semiconductors on Si is a challenging materials science problem with a number of applications in optoelectronic devices, such as solar cells and on-chip lasers. We report the reduction of dislocation density in GaAs solar cells grown directly on nanopatterned V-groove Si substrates by metal-organic vapor-phase epitaxy. Starting from a template of GaP on V-groove Si, we achieved a low threading dislocation density (TDD) of 3 × 106 cm-2 in the GaAs by performing thermal cycle annealing of the GaAs followed by growth of InGaAs dislocation filter layers. This approach eliminates the need for a metamorphic buffer to directly integrate low-TDD GaAs on Si. We used these low-TDD GaAs/V-groove Si templates to grow GaAs double heterostructures that had a minority carrier lifetime of 5.7 ns, as measured by time-resolved photoluminescence, a value consistent with the material quality associated with a 20%+ efficient GaAs solar cell. However, front-junction GaAs solar cells grown on these low-TDD substrates produced a conversion efficiency of only 6.6% without an antireflection coating. Electron channeling contrast imaging measurements on this cell showed a high density of misfit dislocations at the interface between the AlInP/GaInP window layer and the GaAs absorber and between the GaAs absorber and the GaInP back surface field (BSF), likely causing a high surface recombination velocity and thus poor performance. We showed that we could reduce (and in the case of the BSF, eliminate) these dislocations by employing an AlGaAs-based window layer and BSF. Compared to GaInP, AlGaAs has dislocation glide properties that are more similar to those of GaAs, resulting in more even threading dislocation glide between layers. AlGaAs passivation improved the external quantum efficiency and open-circuit voltage of the devices, but the overall device performance was still low at an efficiency of 7.7% without an antireflection coating, likely due to cracking in the devices. This work demonstrates a route to high material quality in GaAs grown directly on Si that can be used for the production of III-V/Si optoelectronic devices.

Morphological and functional observations of a novel model of retinal ischemia injury induced by bilateral carotid artery stenosis in mice.

To investigate the features of retinal ischemic injuries in a novel mouse model with bilateral carotid artery stenosis (BCAS). BCAS was induced with microcoil implantation in 6-8-week-old C57BL6 mice. Cerebral blood flow was monitored at 2, 7, and 28d postoperatively. Retinal morphological changes were evaluated by fundus photography and hematoxylin-eosin staining. Fluorescein fundus angiography (FFA) was performed to detect retinal vascular changes and circulation. The levels of apoptosis, activation of neurogliosis, and expression of hypoxia-inducible factor (HIF)-1α in the retina were assessed by Western blotting and immunofluorescence staining, followed by retinal ganglion cell (RGC) density detection. Additionally, electrophysiological examinations including photopic negative response (PhNR) was also performed. The mice demonstrated an initial rapid decrease in cerebral blood flow, followed by a 4-week recovery period after BCAS. The ratio of retinal artery and vein was decreased under fundus photography and FFA. Compared with the sham mice, BCAS mice showed thinner retinal thickness on day 28. Additionally, apoptosis was increased and RGC density was decreased mainly in peripheral retinal region. Neurogliosis was mainly located in the inner retinal layers, with a stable increase in HIF-1α expression. The dark-adapted electroretinogram showed a notable reduction in the a-, b-, and oscillatory potential (OP) wave amplitudes between days 2 and 7; this gradually recovered over the following 4wk. However, the b- and OP-wave amplitudes were still significantly decreased on PhNR examination on day 28. BCAS can result in relatively mild retinal ischemia injuries in mice, mainly in the inner layer and peripheral region. Our study provides a novel animal model for investigating retinal ischemic diseases.

Rapid and reproducible generation of glioblastoma spheroids for high-throughput drug screening

Identifying new substances that could potentially be used for tumor therapy and the precise analysis of their spectrum of action requires models that are as similar as possible to the tumor present in the patient. Traditionally, two-dimensional (2D) cell cultures are used. However, these only resemble solid tumors to a limited extent. More realistic in vivo models, such as tissue cultures, which are invaluable for the final analysis of the effect of new substances, are unsuitable for high-throughput screening (HTS), such as substance library screening. Therefore, we addressed which parameters need to be optimized to produce 3D cultures suitable for HTS using established tumor cell lines and ultra-low attachment plates, and we tested which experimental parameters need to be considered. In our studies, we have focused on cell lines from gliomas. Gliomas are incurable tumors of the central nervous system and are the subject of intensive research. Our studies used ten glioma cell lines from which we generated spheroids using ultra-low attachment plates. We then determined the spheroid size as a function of the initial cell number and the culture time. We analyzed cell viability using propidium iodide staining, evaluated the effects of temozolomide and radiation on spheroids, and compared the effect to that on 2D cultures. We found that spheroid size correlated linearly with the initial cell number. Fewer cells (250–500) generally resulted in better growth than a higher number. However, not all cell lines produced growing spheroids at all. The spheroids had an outer layer of living cells and an inner core of dead cells. The size of the inner core of dead cells was different in the various cell lines and developed differently during the incubation period. Radiation affected spheroids more than 2D cultures, especially at higher cell densities. Our results provide insight into using glioma cell lines to form spheroids as model systems. We have identified initial cell numbers as a critical parameter for their effective use in research, offering a hopeful outlook for tumor therapy research and drug development.

A Multicenter Study on Clinical Outcomes of Simultaneous Implantable Collamer Lens Removal and Phacoemulsification with Intraocular Lens Implantation in Eyes Developing Cataract.

This study evaluated the clinical outcomes of simultaneous implantable collamer lens (ICL) removal and phacoemulsification with intraocular lens (IOL) implantation in a multicenter study. We retrospectively investigated 83 eyes of 72 patients requiring ICL extraction and cataract surgery at five institutions. Preoperatively and 3months postoperatively, we determined visual acuity (logMAR), spherical equivalent refraction, and endothelial cell density (ECD), in addition to the preoperative backgrounds and the postoperative complications. The patients' age at the time of cataract surgery was 49.8 ± 6.8years, and the axial length was 28.49 ± 1.97mm. The most prevalent type of cataract was anterior subcapsular cataract, followed by nuclear and cortical cataract. Uncorrected and corrected visual acuities significantly improved to 0.09 ± 0.30 and - 0.12 ± 0.12 logMAR, respectively (p < 0.001). Sixty-three (76%) and 78 (94%) eyes were within ± 0.5D and 1.0D, respectively, of the targeted correction. The mean percentage of ECD loss was 1.3 ± 11.3%. No vision-threatening complications were observed in any case. Our multicenter study showed that simultaneous ICL removal and phacoemulsification with IOL implantation is a safe, effective, and predictable procedure, with no significant complications, making it a feasible option for ICL-implanted eyes developing cataracts.

Cell density quantification of high resolution Nissl images of the juvenile rat brain

Nissl histology underpins our understanding of brain anatomy and architecture. Despite its importance, no high-resolution datasets are currently available in the literature for 14-day-old rats. To remedy this issue and demonstrate the utility of such a dataset, we have acquired over 2000 high-resolution images (0.346 μm per pixel) from eight juvenile rat brains stained with cresyl violet. To analyze this dataset, we developed a semi-automated pipeline using open-source software to perform cell density quantification in the primary somatosensory hindlimb (S1HL) cortical column. In addition, we performed cortical layer annotations both manually and using a machine learning model to expand the number of annotated samples. After training the model, we applied it to 262 images of the S1HL, retroactively assigning segmented cells to specific cortical layers, enabling cell density quantification per layer rather than just for entire brain regions. The pipeline improved the efficiency and reliability of cell density quantification while accurately assigning cortical layer boundaries. Furthermore, the method is adaptable to different brain regions and cell morphologies. The full dataset, annotations, and analysis tools are made publicly available for further research and applications.

Effect of Phosphorus Deprivation on Fatty Acid Synthesis in Scenedesmus subspicatus Microalgae from Rostherne Mere

Numerous studies have examined the feasibility of using microalgae as a long-term source of biofuel. This helped us to determine how different amounts of phosphorus changed the growth of lipids in Scenedesmus subspicatus, a freshwater microalga. This study examined the effects of various phosphorus concentrations on the biochemical makeup of algae, particularly the production of proteins and carbohydrates. When there was insufficient phosphorus, the investigation observed a significant increase in lipids and productivity of S. subspicatus. Additionally, gas chromatography was used to examine the fatty acid profiles of the green algae thoroughly. When Scenedesmus species were tested at different cell densities, the highest amount of chlorophyll was found to be 0.89 mg/L. The amounts of fatty acids in algae grown with 0.4 and 0.04 P of phosphorus were strongly correlated, as shown by the Pearson linear correlation coefficients. Gas chromatography analysis revealed that the major saturated fatty acids were stearic acid (C18:0) and palmitic acid (C16:0). In addition, confirming the presence of several unsaturated fatty acids (C16:3, C18:1, C18:2, and C18:3) has helped us learn more about S. subspicatus’s complex lipid profile of S. subspicatus and how well it can be used to produce biofuels.

Green Nanotechnology of Cell Wall Swelling for Nanostructured Transparent Wood of High Optical Performance.

Transparent wood composites provide new functionalities through active additives distributed at the nanoscale. Scalable nanotechnology includes processing where nanoparticles and molecules are brought into the dense wood cell wall. A novel cell wall swelling step through green chemistry is therefore investigated. Sub-zero centigrade NaOH treatment provides extensive cell wall swelling. Cell wall accessibility is vastly increased so that chemicals can readily impregnate the nanostructured cell wall. Transparent wood with a thickness of up to 15mm can therefore be fabricated. The optical transmittance and the attenuation coefficient are improved since the polymer is distributed inside the cell wall as a matrix for the nanoscale cellulose fibrils. The proposed technology paves the way for scalable wood nanoengineering.

Optimizing in vitro spherulation cues in the fungal pathogen Coccidioides.

Coccidioides spp. are part of a group of thermally dimorphic fungal pathogens, which grow as filamentous cells (hyphae) in the soil and transform to a different morphology upon inhalation into the host. The Coccidioides host form, the spherule, is unique and highly undercharacterized due to both technical and biocontainment challenges. Each spherule arises from an environmental spore (arthroconidium), matures, and develops hundreds of internal endospores, which are released from the spherule upon rupture. Each endospore can then go on to form another spherule in a cycle called spherulation. One of the foremost technical challenges has been reliably growing spherules in culture without the formation of contaminating hyphae and consistently inducing endospore release from spherules. Here, we present optimization of in vitro spherule growth and endospore release, by closely controlling starting cell density in the culture, using freshly harvested arthroconidia, and decreasing the concentration of multiple salts in spherulation media. We developed a minimal medium to test spherule growth on various carbon and nitrogen sources. We defined a critical role for the dispersant Tamol in both early spherule formation and prevention of the accumulation of a visible film around spherules. Finally, we examined how the conditions under which arthroconidia are generated influence their transcriptome and subsequent development into spherules, demonstrating that this is an important variable to control when designing spherulation experiments. Together, our data reveal multiple strategies to optimize in vitro spherulation growth, enabling characterization of this virulence-relevant morphology.IMPORTANCECoccidioides spp. are thermally dimorphic fungal pathogens found in the Southwest United States, Mexico, Central America, and South America. Coccidioides can infect both immunocompetent and immunocompromised people and can cause a devastating disseminated infection, including meningitis, with 30% mortality despite all currently available treatments. In this work, we tackle one of the current largest technical barriers to studying the fungus Coccidioides: reliably growing its host form in vitro. Our work is impactful because we have created a set of foundational tools for the burgeoning field of Coccidioides pathogenesis research. We have carefully optimized conditions that allow the development of Coccidioides in vitro into its pathogenic form. This work will open up many lines of investigation into the molecules that underlie Coccidioides pathogenesis.

Heterogeneity of the Alpha-Smooth Muscle Actin Tumor Score in Breast Cancer Cells Significantly Affects Tumor Invasiveness, Recurrence, and Patient Survival.

Alpha-smooth muscle actin (αSMA) has been widely investigated in malignancies, primarily concerning its expression in cancer-associated fibroblasts (CAFs) inside the tumor stroma. Microscopic examination indicates that αSMA expression is not confined to the tumor stromal compartment but is also present in a subset of tumor cells, and this expression correlates with an enhanced invasive phenotype of malignant cells from lung, liver, or ovarian malignancies. Information on actin expression in breast cancer (BC) cells is scarce, and its influence on clinicopathological characteristics remains ambiguous due to conflicting findings in the literature. To examine the αSMA tumor score in breast cancer cells utilizing digital image analysis (DIA) methodologies and to critically analyze the varying effects of αSMA tumor score values on clinicopathologic parameters, particularly focusing on tumor cell invasiveness, recurrence, and survival. Double immunostaining for CD34 and αSMA was conducted on 53 breast cancer cases that were thoroughly characterized in relation to clinicopathologic data. Double immunostaining for CD34 and αSMA demonstrated different distribution patterns of both markers in normal and breast cancer tissues. DIAdata about αSMA tumor cell density, intensity, tumor score, and histological score were correlated with clinicopathological factors. We delineated three unique breast cancer subgroups based on αSMA tumor scores: a 9.43% low-expressing subgroup (αSMA_TSlow, score 4), a 35.07% medium-expressing subgroup (αSMA_TSmed, scores 5 and 6), and a 55.5% high-expressing subgroup (αSMA_TShigh, scores 7 and 8). Stromal immature vessels and tertiary lymphoid structures (TLS) exhibited a strong correlation with αSMA_TSlow, whereas recurrence, perineural, and lymphovascular invasion strongly influenced the αSMA_TSmed and αSMA_TShigh subgroups. The αSMA_TSmed subgroup demonstrated the most heterogeneity with the influence of αSMA-expressing breast cancer cells on tumor size, nodal status, perineural and lymphovascular invasion, menopausal status, recurrence, and survival. Most of the cases from the αSMA_TSlow subgroup had Luminal B and Luminal B-HER2 phenotypes, while triple-negative breast cancer (TNBC) represented one-third of all cases in the αSMA_TShigh subgroup. αSMA-expressing breast cancer cells variably affect malignant growth, invasion, and recurrence, highly contingent upon their density and expression intensity. The current investigation identified an αSMA_TSmed BC subgroup that appears to promote invasiveness, recurrence, and survival in breast cancer. Our data indicate that αSMA BC-expressing cells play a dual role in BC progression, contingent upon their percentage and expression intensity; however, further research is required to elucidate the factors and mechanisms responsible for their accumulation and/or transdifferentiation in malignant breast tissue.

Engineering Nicotiana benthamiana for chrysoeriol production using synthetic biology approaches

Flavonoids are prevalent plant secondary metabolites with a broad range of biological activities. Their antioxidant, anti-inflammatory, and anti-cancer activities make flavonoids widely useful in a variety of industries, including the pharmaceutical and health food industries. However, many flavonoids occur at only low concentrations in plants, and they are difficult to synthesize chemically due to their structural complexity. To address these difficulties, new technologies have been employed to enhance the production of flavonoids in vivo. In this study, we used synthetic biology techniques to produce the methylated flavone chrysoeriol in Nicotiana benthamiana leaves. The chrysoeriol biosynthetic pathway consists of eight catalytic steps. However, using an Agrobacterium-mediated transient expression assay to examine the in planta activities of genes of interest, we shortened this pathway to four steps catalyzed by five enzymes. Co-expression of these five enzymes in N. benthamiana leaves resulted in de novo chrysoeriol production. Chrysoeriol production was unaffected by the Agrobacterium cell density used for agroinfiltration and increased over time, peaking at 10 days after infiltration. Chrysoeriol accumulation in agroinfiltrated N. benthamiana leaves was associated with increased antioxidant activity, a typical property of flavones. Taken together, our results demonstrate that synthetic biology represents a practical method for engineering plants to produce substantial amounts of flavonoids and flavonoid derivatives without the need for exogenous substrates.

The tumor immune microenvironment and therapeutic efficacy of trastuzumab deruxtecan in gastric cancer.

Trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate with a topoisomerase I inhibitor connected by a cleavable linker, has been approved for patients with HER2-positive gastric or gastroesophageal junction tumors. This biomarker study assessed HER2 expression and immune cell infiltration in relation to the therapeutic response to T-DXd. This retrospective analysis included samples from patients treated with T-DXd in three clinical trials. We performed RNA sequencing and multiplex immunohistochemistry on archival tumor samples obtained at baseline, during treatment, and after treatment. Flow cytometry was performed on tumor infiltrating immune cells freshly isolated from tumor tissues. Samples from 28 patients were included in this study. ERBB2 mRNA levels and CD20+ cell infiltration in tumors were significantly higher at baseline in responders than in nonresponders. Patients were classified into three biological groups based on their baseline tumor/stroma-infiltrating immune cell densities. Two groups reported similar response rates, but a trend was observed toward a shorter progression-free-survival in the group with more immunosuppressive regulatory T cells and programmed death-ligand 1 (PD-L1) expression at baseline. T-DXd treatment tended to increase the levels of tumor-infiltrating CD8+ T cells and PD1+CD8+ T cells, particularly in responders. Gene expression signatures of CTL and helper T cells increased during treatment, whereas signatures related to hypoxia, MYC targets, collagen formation, and interleukin-10 were downregulated. Our data suggest that HER2 expression levels and baseline tumor microenvironment (TME) characteristics correlate with T-DXd efficacy. Furthermore, this treatment may modulate TME immune profiles. Further validation using a larger sample size is warranted.

Effect of endonasal dacryocystorhinostomy on conjunctival morphology and visual quality

ABSTRACT Clinical relevance Endonasal dacryocystorhinostomy (EN-DCR) is one of the preferred interventions for occlusion of the nasolacrimal drainage system. Understanding the bulbar conjunctival changes that occur after EN-DCR surgery will provide better insight into the effect of this procedure on visual quality and patient comfort. Background This study aimed to evaluate visual quality and bulbar conjunctival cytologic changes using in vivo confocal microscopy and corneal topography in patients with epiphora resulting from nasolacrimal duct obstruction after EN-DCR. Methods Nineteen eyes of 19 patients with epiphora were enrolled. All patients underwent EN-DCR with silicone tube intubation. Before and six months after EN-DCR, higher-order aberrations (HOAs) were measured using the Sirius corneal topographer/aberrometer, and bulbar conjunctival changes were assessed using in vivo confocal microscopy. Root mean square values of coma, trefoil, spherical, and total HOAs were analysed. Conjunctival superficial and basal epithelial cell and goblet cell densities and bulbar conjunctival changes were evaluated. The severity of epiphora was assessed by the Munk score before and six months after EN-DCR. Results Nineteen eyes of 19 patients (16 women and 3 men) were examined. The mean age of the patients was 40.8 ± 9.8 years. Total HOA and coma aberrations were significantly decreased at six months after EN-DCR compared to preoperatively. Conjunctival superficial and basal epithelial cell densities and epithelial thickness were significantly increased while goblet cell density was unchanged at six months after EN-DCR. There was also a significant improvement in Munk score six months postoperatively. Conclusion EN-DCR can lead to conjunctival remodelling. Alterations in HOAs and point spread function may occur during the postoperative period. Moreover, point spread function may be positively associated with Munk score.

Examination of the Corneal Endothelium in Patients With Congenital Aniridia With a PAX6 Mutation Using In Vivo Confocal Laser Scanning Microscopy.

In PAX6 syndrome, it is still not clear, whether prenatally, parallel to the iris tissue developmental anomaly, there is neural ectodermal, neural crest, or mesodermal cell deposition at the corneal endothelium, affecting endothelial structure and function. In addition, because of the postnatal corneal inflammation and commonly appearing secondary glaucoma, progressive endothelial changes are expected. Our purpose was to study the corneal endothelium in subjects with PAX6 aniridia, using in vivo confocal laser scanning microscopy. Twenty-seven eyes of 16 subjects with congenital aniridia (age 28.25 ± 16.32 [11-59] years, 8 [50%] female) and 40 eyes of 26 healthy subjects (age 33.8 ± 15.2 [14-67] years, 17 [58.6%] females) were examined. Aniridia-associated keratopathy and iris malformation were graded, and means of endothelial cell density, cell area, cell diameter, spatial pattern of cell centroids (Clark-Evans index), polygonality, neighbor count, percentage of hexagonal cells, and endothelial deposit number were determined by in vivo confocal laser scanning microscopy. Mean cell diameter and mean Clark-Evans index were significantly lower (P = 0.049; P = 0.008) in congenital aniridia eyes than in controls. There were hyperreflective endothelial deposits in congenital aniridia eyes but not in controls (P < 0.001). Only aniridia-associated keratopathy grade correlated positively with mean endothelial deposit number (P = 0.017). In congenital aniridia, the corneal endothelium might possess a slightly better quality and greater reserves than in healthy subjects. Corneal endothelial deposition seems to be independent from developmental abnormalities but may be related to up to date undescribed endothelial inflammatory or metabolic changes.

Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation.

Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.IMPORTANCEHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.

Systemic Hypothermia in the Acute Management of Traumatic Optic Neuropathy in a Murine Animal Model.

To examine the effects of systemic hypothermia on retinal ganglion cell survival and visual outcomes after optic nerve trauma in a sonication-inducted traumatic optic neuropathy murine animal model. Twenty mice underwent sonication-inducted traumatic optic neuropathy. Afterward, 10 mice were placed on a warming pad set to 36°C, and 10 mice were placed on a table. General anesthesia was maintained for 3 hours with subcutaneous injections of ketamine. The rectal temperature was measured every 15 minutes. Pattern electroretinograms were obtained at 2, 4, and 6 weeks. Mice were sacrificed at 6 weeks, and retinal ganglion cell counts were performed. The hypothermia group had an average rectal temperature of 23.1°C; the control group was 33.3°C. At 6 weeks, the hypothermia group had larger a-wave amplitudes (18.19 µV) than the control group (12.75 µV) (p < 0.05). At 6 weeks, retinal ganglion cell density over the entire retina was significantly higher in the hypothermia group versus the control (p < 0.0001). The hypothermia treatment group had significantly higher retinal ganglion cell density and pattern electroretinogram a-wave amplitudes 6 weeks after injury than the control group. Systemic hypothermia may have a neuroprotective effect when initiated immediately after sonication-inducted traumatic optic neuropathy.

Prevalence and Severity of Corneal Guttata After Descemet Membrane Endothelial Keratoplasty.

The postoperative occurrence of corneal guttae (CG) in patients after Descemet membrane endothelial keratoplasty (DMEK) can lead to a significant reduction in visual acuity (VA) with the subsequent need for repeat DMEK. Therefore, the aim of this study was to analyze the prevalence and clinical significance of CG in transplanted corneas after DMEK. The prevalence and progression of CG after DMEK of 1657 patients were examined using endothelial specular microscopy images. The severity grade of CG was classified into 3 grades (G1 <40%, G2 40-80%, G3 >80%). Central corneal thickness, VA, and endothelial cell density (ECD) were examined during a postoperative follow-up time of 19.5 ± 17.6 months (range 6-84 months). The prevalence of CG postoperatively was 1.3% (22/1657 eyes) after 4-6 weeks. We could classify 15/22 (68%) as G1, 5/22 (23%) as G2, and 2/22 (9%) as G3. In 12/22 (55%), either preparation and/or implantation was conspicuously difficult. None of the eyes showed an increase in CG during follow-up. 21/22 were stable in VA, central corneal thickness, and ECD during the entire follow-up. One patient with G3 required a repeat DMEK after 12 months (secondary graft failure). The prevalence of CG in our study population was notably lower than previously described and was mainly characterized by mild, low-grade findings without clinical significance and were stable during the follow-up. Preoperative overlook of CG in the tissue bank cannot be completely excluded, but the remarkable proportion of intraoperative complications in this group may have led to the occurrence of these CG.

Corneal Endothelial Cell Morphology in Eyes With Down Syndrome.

To explore the morphological characteristics of corneal endothelial cells in eyes with Down syndrome. We conducted a comparative analysis of corneal endothelial cell density, coefficient of variation, hexagonal cell appearance rate, and corneal thickness using specular microscopy. Our study included 21 eyes of 11 patients with Down syndrome and 37 eyes of 19 control subjects. In all eyes with Down syndrome, corneal endothelial cell density exceeded 2000 cells/mm2, and no abnormalities in cell morphology or significant differences from the control group were observed across all parameters. The structure and function of corneal endothelial cells in eyes with Down syndrome were within the normal range, even in the presence of chromosomal abnormalities of 21-trisomy in somatic cells.

Tumor-infiltrating lymphocytes vary in different canine soft tissue sarcoma histological types.

Soft tissue sarcomas (STSs) are conventionally viewed as poorly immunogenic tumors; however, some human STSs have recently been reported to elicit an immune response, thus representing potential candidates for immunotherapy. Data regarding immune cell infiltrates in canine STSs are limited and reported without tumor-type stratification. The aim of this study was to retrospectively assess tumor-infiltrating lymphocytes (TILs) in canine STSs of 5 different histotypes. Eighty-seven canine STSs were collected: 22 perivascular wall tumors (PWTs), 19 liposarcomas, 17 fibrosarcomas, 16 myxosarcomas, and 13 leiomyosarcomas. The tumors were graded and immunolabeled for CD3, CD20, and FoxP3, and slides were scanned. T-cell, B-cell, Treg, and total TIL densities were quantified with QuPath software and expressed as cells/mm2. The B/T-cells ratio and Treg/T-cell proportions were calculated. Total TIL densities were higher in PWTs and myxosarcomas (median = 225 and 303, respectively). PWTs had higher T-cell density but lower Treg proportion (median = 152 and 7.6% respectively). Myxosarcomas had higher Treg densities and B/T-cell ratios (median = 24.4 and 1.57, respectively). No association with grade was found among STSs as a group. In myxosarcomas, higher grade was significantly associated with higher total TILs, and CD20+ and FoxP3+ cell densities (p < .05). The results suggest that PWTs and myxosarcomas may represent the most immunogenic STS types. Myxosarcomas elicit a B-cell and Treg-rich immune response; PWTs stimulate a T-cell-rich and Treg-poor reaction. The immune system response may contribute to the more aggressive behavior of myxosarcomas and the more indolent course of PWTs.