Pattern Of Markers Research Articles

PD35-11 EPITHELIAL DIFFERENTIATION IS ALTERED IN URETHRAL LICHEN SCLEROSUS

You have accessJournal of UrologyCME1 Apr 2023PD35-11 EPITHELIAL DIFFERENTIATION IS ALTERED IN URETHRAL LICHEN SCLEROSUS Ethan Richmond, Caleb Sokovich, Wei Huang, Chad Vezina, and Matthew D Grimes Ethan RichmondEthan Richmond More articles by this author , Caleb SokovichCaleb Sokovich More articles by this author , Wei HuangWei Huang More articles by this author , Chad VezinaChad Vezina More articles by this author , and Matthew D GrimesMatthew D Grimes More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003333.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lichen sclerosus (LS) is an idiopathic chronic inflammatory disease of the genital epithelium resulting in fibrosis and severe urethral stricture disease in affected men. Epithelia are subdivided into basal, intermediate, and superficial cell layers which are defined by protein expression patterns. Loss of basal cell differentiation is a feature of other fibrotic disorders but have not been studied in the urethra or LS. The proteins keratin 5 (KRT5) and p63 have been reliably established as basal cell markers in urothelium. Therefore, we hypothesized that urethral epithelium affected by LS would have decreased basal expression of KRT5 and p63. METHODS: Urethral tissue after urethral biopsy or urethroplasty was examined by a genitourinary pathologist to determine LS status. Using multiplexed fluorescent immunohistochemistry, KRT 5, CD45, p63, and DAPI were stained and three epithelial regions imaged per specimen. Using QuPath analysis software, the epithelium was segmented into basal and luminal compartments and relative abundance (RA) of each primary antibody was measured (number of positive cells/number of nucleated cells). RA was compared with respect to LS status and basal/luminal compartment using a Mann-Whitney U test. RESULTS: We analyzed 14 slides (n=7 LS, n=7 non-LS). The RA of KRT5 and p63 positive cells were similar with respect to epithelial versus basal compartment and LS status. However, we noted a change in patterning such that LS tissues displayed a shift of KRT5 and p63 expression from the basal to the luminal epithelium. To quantify this, we compared the ratio of basal to luminal KRT5 and p63 expression and dichotomized this as “basal predominant” (>1) or “luminal predominant” (<1). LS urethra were significantly more likely to have luminal predominant expression of p63 and KRT5 compared to non-LS (p=0.03, p=0.03) (Figure 1). CONCLUSIONS: We identified a change in expression pattern of basal epithelial markers (KRT5 and p63) in LS urethra. Altered epithelial differentiation is seen in other inflammatory diseases and may be implicated in the pathogenesis of LS. Source of Funding: K12DK100022NIH/NIDDK © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e978 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ethan Richmond More articles by this author Caleb Sokovich More articles by this author Wei Huang More articles by this author Chad Vezina More articles by this author Matthew D Grimes More articles by this author Expand All Advertisement PDF downloadLoading ...

Differential staining patterns of immunohistochemical markers for neurogenesis staging in the premature cerebellum of ferrets and mice.

Immunohistochemical staining patterns of markers for neurogenesis staging were compared at the identical stage of cerebellar histogenesis between ferrets (aged 20 days) and mice (aged 10 days). Proliferating cell nuclear antigen (PCNA) immunostaining was observed largely in the granular precursors of the external granular layer (EGL) in both ferrets and mice. PCNA-immunostaining was also found in brain lipid-binding protein-immunopositive cells in the internal granular layer and was more abundant in ferrets than in mice. Paired box 6 immunostaining appeared largely in the EGL granular precursors in mice, whereas it emerged in the migrating/differentiating granular precursors in ferrets. These findings revealed that the types and neurogenesis stages of the EGL granular precursors detected by immunohistochemical markers differed between ferrets and mice.

Specific Subtypes of Carcinoma-Associated Fibroblasts Are Correlated with Worse Survival in Resectable Pancreatic Ductal Adenocarcinoma

The pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancer entities. Effective therapy options are still lacking. The tumor microenvironment possibly bears further treatment possibilities. This study aimed to describe the expression patterns of four established carcinoma-associated fibroblast (CAFs) markers and their correlation in PDAC tissue samples. This project included 321 patients with PDAC who underwent surgery with a curative intent in one of the PANCALYZE study centers. Immunohistochemical stainings for FAP, PDGFR, periostin, and SMA were performed. The expression patterns of each marker were divided into low- and high-expressing CAFs and correlated with patients' survival. Tumors showing SMAhigh-, PeriostinhighSMAhigh-, or PeriostinhighSMAlowPDGFRlowFAPhigh-positive CAFs demonstrated significantly worse survival. Additionally, a high expression of SMA in PDAC tissue samples was shown to be an independent risk factor for worse survival. This project identified three subgroups of PDAC with different expression patterns of CAF markers which showed significantly worse survival. This could be the base for the further characterization of the fibroblast subgroups in PDAC and contribute to the development of new targeted therapy options against CAFs.

Ontogeny and Trophic Factor Sensitivity of Gastrointestinal Projecting Vagal Sensory Cell Types.

Vagal sensory neurons (VSNs) located in the nodose ganglion provide information, such as stomach stretch or the presence of ingested nutrients, to the caudal medulla via specialized cell types expressing unique marker genes. Here, we leverage VSN marker genes identified in adult mice to determine when specialized vagal subtypes arise developmentally and the trophic factors that shape their growth. Experiments to screen for trophic factor sensitivity revealed that brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) robustly stimulate neurite outgrowth from VSNs in vitro Perinatally, BDNF was expressed by neurons of the nodose ganglion itself, while GDNF was expressed by intestinal smooth muscle cells. Thus, BDNF may support VSNs locally, whereas GDNF may act as a target-derived trophic factor supporting the growth of processes at distal innervation sites in the gut. Consistent with this, expression of the GDNF receptor was enriched in VSN cell types that project to the gastrointestinal tract. Last, the mapping of genetic markers in the nodose ganglion demonstrates that defined vagal cell types begin to emerge as early as embryonic day 13, even as VSNs continue to grow to reach gastrointestinal targets. Despite the early onset of expression for some marker genes, the expression patterns of many cell type markers appear immature in prenatal life and mature considerably by the end of the first postnatal week. Together, the data support location-specific roles for BDNF and GDNF in stimulating VSN growth, and a prolonged perinatal timeline for VSN maturation in male and female mice.

PATTERNS OF SEROPREVALENCE FOR TRANSFUSION-TRANSMISSIBLE INFECTIONS AMONG BLOOD DONORS IN A BLOOD CENTRE OF NORTHEAST INDIA

Introduction: Transfusion– Transmissible Infections (TTIs) are the infections resulting from the introduction of a pathogen into a person through blood transfusion. In an attempt to mitigate the inherent risk of TTIs, the demographic information of blood donors including knowledge on local prevalence of infections and its pattern in the donor population is important for formulating recruitment strategies and planning other precautionary measures. Materials &amp; Methods: This cross-sectional study was conducted in the Blood Centre, Jawaharlal Nehru Institute of Medical Sciences, Imphal between January 2019 and December 2022. All donated blood units were tested for the mandatory TTI markers for HIV 1 &amp; 2, Hepatitis B, Hepatitis C, Syphilis and Malarial parasite. The prevalence and patterns of seropositivity for TTIs were studied based on the donor demographic characteristics such as donation type, gender and age group. Results: The overall seroprevalence among blood donors (n=31,563) was 1.79%. The seroprevalence was lower among voluntary donors (1.08%) than the replacement donors (2.10%). Seropositivities among female and male donors were 0.79% and 1.93% respectively. The lowest TTI seropositivity was seen among female voluntary donors (0.59%) and highest among male replacement donors (2.24%). The seroprevalence for specic TTI markers among the blood donors were 0.15% for HIV; 0.57% for HBV; 0.93% for HCV; 0.14% for Syphilis and no donor was found positive for Malaria. The frequency of TTI seropositivity was increased with increase in the age groups of donors in both sexes. There were 9 (0.029%) donors who had co-infection of TTIs. The patterns of TTI markers for co-infections were HCV+HIV=3; HCV+HBV=3; HCV+Syphilis=2 and HIV+ Syphilis=1. Conclusion: The measures to provide safe blood may include collection of blood from the targeted low risk donor population, using more sensitive testing methods, implementing pathogen reduction technologies and other public health measures.

Different Resistance Exercise Loading Paradigms Similarly Affect Skeletal Muscle Gene Expression Patterns of Myostatin-Related Targets and mTORC1 Signaling Markers.

Although transcriptome profiling has been used in several resistance training studies, the associated analytical approaches seldom provide in-depth information on individual genes linked to skeletal muscle hypertrophy. Therefore, a secondary analysis was performed herein on a muscle transcriptomic dataset we previously published involving trained college-aged men (n = 11) performing two resistance exercise bouts in a randomized and crossover fashion. The lower-load bout (30 Fail) consisted of 8 sets of lower body exercises to volitional fatigue using 30% one-repetition maximum (1 RM) loads, whereas the higher-load bout (80 Fail) consisted of the same exercises using 80% 1 RM loads. Vastus lateralis muscle biopsies were collected prior to (PRE), 3 h, and 6 h after each exercise bout, and 58 genes associated with skeletal muscle hypertrophy were manually interrogated from our prior microarray data. Select targets were further interrogated for associated protein expression and phosphorylation induced-signaling events. Although none of the 58 gene targets demonstrated significant bout x time interactions, ~57% (32 genes) showed a significant main effect of time from PRE to 3 h (15↑ and 17↓, p < 0.01), and ~26% (17 genes) showed a significant main effect of time from PRE to 6 h (8↑ and 9↓, p < 0.01). Notably, genes associated with the myostatin (9 genes) and mammalian target of rapamycin complex 1 (mTORC1) (9 genes) signaling pathways were most represented. Compared to mTORC1 signaling mRNAs, more MSTN signaling-related mRNAs (7 of 9) were altered post-exercise, regardless of the bout, and RHEB was the only mTORC1-associated mRNA that was upregulated following exercise. Phosphorylated (phospho-) p70S6K (Thr389) (p = 0.001; PRE to 3 h) and follistatin protein levels (p = 0.021; PRE to 6 h) increased post-exercise, regardless of the bout, whereas phospho-AKT (Thr389), phospho-mTOR (Ser2448), and myostatin protein levels remained unaltered. These data continue to suggest that performing resistance exercise to volitional fatigue, regardless of load selection, elicits similar transient mRNA and signaling responses in skeletal muscle. Moreover, these data provide further evidence that the transcriptional regulation of myostatin signaling is an involved mechanism in response to resistance exercise.

Methodological progress note: Machine learning methods in healthcare research.

Methodological progress note: Machine learning methods in healthcare research.

Patterns of Dietary Blood Markers Are Related to Frailty Status in the FRAILOMIC Validation Phase.

The influence of nutritional factors on frailty syndrome is still poorly understood. Thus, we aimed to confirm cross-sectional associations of diet-related blood biomarker patterns with frailty and pre-frailty statuses in 1271 older adults from four European cohorts. Principal component analysis (PCA) was performed based on plasma levels of α-carotene, β-carotene, lycopene, lutein + zeaxanthin, β-cryptoxanthin, α-tocopherol, γ-tocopherol and retinol. Cross-sectional associations between biomarker patterns and frailty status, according to Fried's frailty criteria, were assessed by using general linear models and multinomial logistic regression models as appropriate with adjustments for the main potential confounders. Robust subjects had higher concentrations of total carotenoids, β-carotene and β-cryptoxanthin than frail and pre-frail subjects and had higher lutein + zeaxanthin concentrations than frail subjects. No associations between 25-Hydroxyvitamin D3 and frailty status were observed. Two distinct biomarker patterns were identified in the PCA results. The principal component 1 (PC1) pattern was characterized by overall higher plasma levels of carotenoids, tocopherols and retinol, and the PC2 pattern was characterized by higher loadings for tocopherols, retinol and lycopene together and lower loadings for other carotenoids. Analyses revealed inverse associations between PC1 and prevalent frailty. Compared to participants in the lowest quartile of PC1, those in the highest quartile were less likely to be frail (odds ratio: 0.45, 95% CI: 0.25-0.80, p = 0.006). In addition, those in the highest quartile of PC2 showed higher odds for prevalent frailty (2.48, 1.28-4.80, p = 0.007) than those in the lowest quartile. Our findings strengthen the results from the first phase of the FRAILOMIC project, indicating carotenoids are suitable components for future biomarker-based frailty indices.

Immunoexpression Pattern of Autophagy Markers in Developing and Postnatal Kidneys of Dab1−/−(yotari) Mice

The purpose of this study was to compare the immunofluorescence patterns of autophagic markers: Light chain 3 beta (LC3B), Glucose regulating protein 78 (GRP78), Heat shock cognate 71 (HSC70) and Lysosomal-associated membrane protein 2A (LAMP2A) in the developing and postnatal kidneys of Dab1-/- (yotari) mice to those of wild-type samples. Embryos were obtained on gestation days 13.5 and 15.5 (E13.5 and E15.5), and adult animals were sacrificed at postnatal days 4, 11 and 14 (P4, P11, and P14). After fixation and dehydration, paraffin-embedded kidney tissues were sectioned and incubated with specific antibodies. Using an immunofluorescence microscope, sections were analyzed. For statistical analysis, a two-way ANOVA test and a Tukey's multiple comparison test were performed with a probability level of p < 0.05. A significant increase in GRP78 and LAMP2A expression was observed in the renal vesicles and convoluted tubules of yotari in embryonic stages. In postnatal kidneys, all observed proteins showed higher signal intensities in proximal and distal convoluted tubules of yotari, while a higher percentage of LC3B-positive cells was also observed in glomeruli. Our findings suggest that all of the examined autophagic markers play an important role in normal kidney development, as well as the potential importance of these proteins in renal pathology, where they primarily serve a protective function and thus may be used as diagnostic and therapeutic targets.

Genetic architectures of postmating isolation and morphology of two highly diverged rockfishes (genus Sebastes).

Postmating isolation is thought to be an important driver of the late stages of speciation. However, relatively little is empirically known about the process compared with other isolating mechanisms that drive the early stages of speciation, especially in non-model organisms. We characterized the genetic architecture of postmating isolation between 2 rockfishes, Sebastes schlegelii and S. trivittatus, whose reproductive isolation is complete. We examined transmission ratio distortion (TRD) patterns of genetic markers in 2 reciprocal backcross populations. Markers showing either of the 2 types of TRD was widespread across the genome, with some of the distorted markers forming extensive clusters around the recombination coldspots. These suggest that the postmating isolation effectively prevents gene flow across the genome and the recombination landscape contributes to the genetic architecture. Comparisons between 2 backcross families and 2 developmental stages showed little similarity in the distorted markers, suggesting asymmetry and stage specificity of the isolation. This may be due to hybrid incompatibility involving maternal factors or extrinsic selection. The lack of sex-ratio distortion in the mapping families suggested that Haldane's rule in terms of hybrid inviability does not hold. Additionally, quantitative trait loci (QTL) mapping detected significant QTLs for sex and the morphological traits relevant to speciation and convergence of rockfishes, including body coloration. Genes in the melanocortin system, including agouti-signaling protein 1 (asip1) and melanocortin 1 receptor (mc1r), might underlie the horizontal and vertical color patterns on the body, respectively. These findings constitute an essential step toward a comprehensive understanding of speciation and morphological diversification of rockfishes.

J-difference GABA-edited MRS reveals altered cerebello-thalamo-cortical metabolism in patients with hepatic encephalopathy.

Hepatic encephalopathy (HE) is a common neurological manifestation of liver cirrhosis and is characterized by an increase of ammonia in the brain accompanied by a disrupted neurotransmitter balance, including the GABAergic and glutamatergic systems. The aim of this study is to investigate metabolic abnormalities in the cerebello-thalamo-cortical system of HE patients using GABA-edited MRS and links between metabolite levels, disease severity, critical flicker frequency (CFF), motor performance scores, and blood ammonia levels. GABA-edited MRS was performed in 35 participants (16 controls, 19 HE patients) on a clinical 3T MRI system. MRS voxels were placed in the right cerebellum, left thalamus, and left motor cortex. Levels of GABA+ and of other metabolites of interest (glutamine, glutamate, myo-inositol, glutathione, total choline, total NAA, and total creatine) were assessed. Group differences in metabolite levels and associations with clinical metrics were tested. GABA+ levels were significantly increased in the cerebellum of patients with HE. GABA+ levels in the motor cortex were significantly decreased in HE patients, and correlated with the CFF (r = 0.73; p < .05) and motor performance scores (r = -0.65; p < .05). Well-established HE-typical metabolite patterns (increased glutamine, decreased myo-inositol and total choline) were confirmed in all three regions and were closely linked to clinical metrics. In summary, our findings provide further evidence for alterations in the GABAergic system in the cerebellum and motor cortex in HE. These changes were accompanied by characteristic patterns of osmolytes and oxidative stress markers in the cerebello-thalamo-cortical system. These metabolic disturbances are a likely contributor to HE motor symptoms in HE. In patients with hepatic encephalopathy, GABA+ levels in the cerebello-thalamo-cortical loop are significantly increased in the cerebellum and significantly decreased in the motor cortex. GABA+ levels in the motor cortex strongly correlate with critical flicker frequency (CFF) and motor performance score (pegboard test tPEG), but not blood ammonia levels (NH3).

Evaluation of LGR5 Cancer Stem Cell Marker Expression in Breast Cancer and Its Relationship with Hormonal Profile and Clinical Pathological Features.

Due to the high prevalence of breast cancer and the importance of evaluating new prognostic criteria for effective treatment of these patients, this study was performed to investigate the role of LGR5 in breast cancer and its relationship with hormonal and clinicalopathological features of the disease. This cross-sectional study was performed on breast cancer tissue samples in the archives of the pathology department of Firoozabadi Hospital in Tehran between 2019 and 2021. Inclusion criteria included invasive ductal carcinoma and exclusion criteria were preoperative chemotherapy. Blocks were examined for LGR5 marker expression by IHC method using LGR5 monoclonal antibody kits (Abcam). The expression pattern of LGR5 marker was cytoplasmic and cells presenting brown staining in the cytoplasm were considered positive for this marker and in terms of distribution and severity of staining were divided into three groups: mild, moderate and severe. This study was performed on 60 patients with breast cancer with a mean age of 55.5±9.7. Most of the patients (55%) were in grade II. The KI67 marker was positive in 45 cases (75%) and the HER2 marker in 14 cases (23.3%) and 8 cases (13.3%) were triple-negative. The expression severity of staining of LGR5 marker in 41 cases (68.3%) was moderate and the distribution of marker expression in 31 cases (51.7%) was moderate. No significant relationship was observed between LGR5 expression severity and tumor characteristics. LGR5 marker is expressed in a remarkable percentage of breast cancer patients and has no significant relationship with tumor characteristics.

Expression of E4 Protein and HPV Major Capsid Protein (L1) as A Novel Combination in Squamous Intraepithelial Lesions.

We aim to describe the relationship between the immunohistochemical expression patterns of HPV E4 markers and the presence of HPV major capsid protein (L1) in cervical tissues obtained by biopsy of patients with abnormal liquid-based cytology (LBC) results, HR HPV infections, or clinically suspicious cervix. A novel HPV-encoded marker, SILgrade-E4 (XR-E4-1), and an HPV (clone K1H8) antibody were used to demonstrate the expression in terminally differentiated epithelial cells with a productive HPV infection in the material. A semiquantitative analysis was performed based on light microscope images. The level of E4 protein decreased with the disease severity. Patients with LSIL-CIN 1 and HSIL-CIN 2 diagnoses had significantly lower levels of HPV major capsid protein (L1) than those without confirmed cervical lesions. Our analysis confirms a higher incidence of L1 in patients with molecularly diagnosed HPV infections and excluded lesions of LSIL-CIN 1 and HSIL-CIN 2. Further studies on the novel biomarkers might help assess the chances of the remission of lesions such as LSIL-CIN 1 and HSIL-CIN 2. Higher levels of E4 protein and L1 may confirm a greater probability of the remission of lesions and incidental infections. In the cytological verification or HPV-dependent screening model, testing for E4 protein and L1 expression may indicate a group with a lower risk of progression of histopathologically diagnosed lesions.

Cell–Cell Interactions Enhance Cartilage Zonal Development in 3D Gradient Hydrogels

Cartilage tissue is characterized by zonal organization with gradual transitions of biochemical and mechanical cues from superficial to deep zones. We previously reported that 3D gradient hydrogels made of polyethylene glycol and chondroitin sulfate can induce zonal-specific responses of chondrocytes, resulting in zonal cartilage formation that mimics native tissues. While the role of cell-matrix interactions has been studied extensively, how cell-cell interactions across different zones influence cartilage zonal development remains unknown. The goal of this study is to harness gradient hydrogels as a tool to elucidate the role of cell-cell interactions in driving cartilage zonal development. When encapsulated in intact gradient hydrogels, chondrocytes exhibited strong zonal-specific responses that mimic native cartilage zonal organization. However, the separate culture of each zone of gradient hydrogels resulted in a significant decrease in cell proliferation and cartilage matrix deposition across all zones, while the trend of zonal dependence remains. Unexpectedly, mixing the coculture of all five zones of hydrogels in the same culture well largely abolished the zonal differences, with all zones behaving similarly to the softest zone. These results suggest that paracrine signal exchange among cells in different zones is essential in driving cartilage zonal development, and a spatial organization of zones is required for proper tissue zonal development. Intact, separate, or coculture groups resulted in distinct gene expression patterns in mechanosensing and cartilage-specific markers, suggesting that cell-cell interactions can also modulate mechanosensing. We further showed that 7 days of priming in intact gradient culture was sufficient to instruct the cells to complete the zonal development, and the separate or mixed coculture after 7 days of intact culture had minimal effects on cartilage formation. This study highlights the important role of cell-cell interactions in driving cartilage zonal development and validates gradient hydrogels as a useful tool to elucidate the role of cell-matrix and cell-cell interactions in driving zonal development during tissue morphogenesis and regeneration.

Homography Estimation Using Marker Projection Control: A Case of Calibration-Free Projection Mapping

Homography transformation, or planar projective transformation, between camera and projector images, which can be estimated by observing four or more pairs of corresponding points between two images, plays a crucial role in dynamic projection mapping with an uncalibrated projector-camera pair. This study discusses a case of dynamic projection mapping onto a moving textured surface where the point correspondence is provided through an actively controlled (not static) marker pattern projection by the projector along with the movement of the surface. Qualitative advantages are discussed in terms of system implementation, the image processing algorithm, and artifact perception by human observers. In addition, a quantitative analysis of alignment errors in the projection mapping is presented to show the effectiveness of the active control of marker patterns.

System analysis of surface CD markers during the process of granulocytic differentiation.

Plasma membrane proteins with extracellular-exposed domains are responsible for transduction of extracellular signals into intracellular responses, and their accessibility to therapeutic molecules makes them attractive targets for drug development. In this work, using omics technologies and immunochemical methods, we have studied changes in the content of markers of clusters of differentiation (CD markers) of neutrophils (CD33, CD97, CD54, CD38, CD18, CD11b, CD44, and CD71) at the level of transcripts and proteins in NB4, HL-60 and K562 cell lines, induced by the treatment with all-trans-retinoic acid (ATRA). Transcriptomic analysis revealed the induction of CD38, CD54, CD11b, and CD18 markers as early as 3 h after the addition of the inducer in the ATRA-responsive cell lines HL-60 and NB4. After 24 h, a line-specific expression pattern of CD markers could be observed in all cell lines. Studies of changes in the content of CD antigens by means of flow cytometry and targeted mass spectrometry (MS) gave similar results. The proteomic profile of the surface markers (CD38, CD54, CD11b, and CD18), characteristic of the NB4 and HL-60 lines, reflects different molecular pathways for the implementation of ATRA-induced differentiation of leukemic cells into mature neutrophils.

Quantitative Profiling of Alpha-Subunit of IL-3 Receptor on Single Acute Myeloid Leukemia Cells by Super-Resolution Imaging.

Quantitative profiling of specific cell surface markers is a new approach in characterization of tumor heterogeneity and single cell biology. The current tools have dearth in detection and quantification of receptor proteins on single cells. we focused on our newly developed protocol to determine the distribution pattern and density of cell surface markers on single acute myeloid leukemia cells. Cell surface proteins were labeled with quantum dots (Qdots) followed by super resolution Structured Illumination Microscopy (SIM) imaging to imprisonment the optical signals emitted by Qdots which were further analyzed by software imaris to do three dimensional (3D) structure reconstruction and digital simulation. Furthermore, MTT assays and flow cytometry was performed to establish association between expression of cell surface markers and drug response. In the present study, we found that the Molm13 and cytarabine-enriched Molm13 cells exhibit different densities of CD123, an alpha-subunit of interleukin-3 receptor, i.e. 0.92 and 1.73 per μm2 of cell surface respectively. Sub-populations of Molm13 cells expressing higher densities of CD123 on cells membranes showed resistance against cytarabine. Further study revealed that romidepsin sensitized and augmented the cytotoxicity of cytarabine in Molm13 and cytarabine-enriched Molm13 cells. Romidepsin increased the percentage of cell death-induced by cytarabine from 21.6 % to 28.6 % and 37.1 % to 57.2 % in Molm13 and cytarabine-enriched Molm13 cells respectively. Altogether, the study suggests that Molm13 cells have sub-populations with differential expression of CD123+ phenotype. Romidepsin sensitizes and augments the effect of cytarabine in Molm13 and cytarabine-enriched Molm13 cells.

Transcriptomic and physiological analyses reveal temporal changes contributing to the delayed healing response to arterial injury in diabetic rats

ObjectiveAtherosclerosis is a leading cause of mortality in the rapidly growing population with diabetes mellitus. Vascular interventions in patients with diabetes can lead to complications attributed to defective vascular remodeling and impaired healing response in the vessel wall. In this study, we aim to elucidate the molecular differences in the vascular healing response over time using a rat model of arterial injury applied to healthy and diabetic conditions. MethodsWistar (healthy) and Goto-Kakizaki (GK, diabetic) rats (n = 40 per strain) were subjected to left common carotid artery (CCA) balloon injury and euthanized at different timepoints: 0 and 20 hours, 5 days, and 2, 4, and 6 weeks. Noninvasive morphological and physiological assessment of the CCA was performed with ultrasound biomicroscopy (Vevo 2100) and corroborated with histology. Total RNA was isolated from the injured CCA at each timepoint, and microarray profiling was performed (n = 3 rats per timepoint; RaGene-1_0-st-v1 platform). Bioinformatic analyses were conducted using R software, DAVID bioinformatic tool, online STRING database, and Cytoscape software. ResultsSignificant increase in the neointimal thickness (P < .01; two-way analysis of variance) as well as exaggerated negative remodeling was observed after 2 weeks of injury in GK rats compared with heathy rats, which was confirmed by histological analyses. Bioinformatic analyses showed defective expression patterns for smooth muscle cells and immune cell markers, along with reduced expression of key extracellular matrix-related genes and increased expression of pro-thrombotic genes, indicating potential faults on cell regulation level. Transcription factor–protein-protein interaction analysis provided mechanistic evidence with an array of transcription factors dysregulated in diabetic rats. ConclusionsIn this study, we have demonstrated that diabetic rats exhibit impaired arterial remodeling characterized by a delayed healing response. We show that increased contractile smooth muscle cell marker expression coincided with decreased matrix metalloproteinase expression, indicating a potential mechanism for a lack of extracellular matrix reorganization in the impaired vascular healing in GK rats. These results further corroborate the higher prevalence of restenosis in patients with diabetes and provide vital molecular insights into the mechanisms contributing to the impaired arterial healing response in diabetes. Moreover, the presented study provides the research community with the valuable longitudinal gene expression data bank for further exploration of diabetic vasculopathy.

P16INK4a and γ-H2AX as Biomarkers of Senescence in Skin Tissue: A Cadaveric Cross-sectional Study at a Tertiary Care Centre in Punjab, India

Introduction: Baseline expression of p16INK4a characterises cellular senescence. Similarly, the measurement of γ-GammaH2AX (γ-H2AX) foci levels in cells provides a reliable method for quantification of Deoxyribonucleic Acid (DNA) damage. Aim: To explore the role of p16INK4a and γ-H2AX as biomarkers of senescence in skin tissue. Materials and Methods: This was a cross-sectional study conducted in the Department of Anatomy, PGIMER, Chandigarh, Punjab, India from June 2022 to January 2023. Skin tissue was obtained from 30 cadavers, aged 20-90 years, from the anterior abdominal wall. The time duration of sample collection after death varied from six hours to 12 hours. Samples were divided into two groups: Group-I &lt;30 years and Group-II &gt;70 years, with n=15 in each group. The relative change in the expression pattern of p16INK4a and γH2AX markers, as well as the microstructure of the skin (thickness of epidermis and dermis, distribution of collagen I/II/III fibres, architecture of sebaceous glands), were statistically analysed using an unpaired t-test. Results: Intense staining was observed with p16INK4a in Group-II, showing positivity in 60.75% of the cells, while Group-I depicted a weak staining pattern (15%). On immunostaining with γ-H2AX, only Group-II cells showed intense positivity (45%). Significant differences were observed in the epidermal and dermal thickness: Group-I (165.5267±37.73 μm; 2394.6±874.13 μm); Group-II (54.6±22.79 μm; 566.67±242.23 μm), and collagen Type-II/III fibres were found predominantly in aging skin tissue. Conclusion: The present study provides comprehensive data regarding age-associated changes between p16INK4a, γ-H2AX, and skin microstructure, which result in decreased repair and regenerative capacity of skin tissue and various age-related skin diseases.

Prognostic Impact of Atrial Fibrillation Pattern and other Clinical Biomarkers in Patients with Stroke Admitted in a Tertiary Hospital in Cebu City from 2015-2022.

The effect of atrial fibrillation (AF) patterns and clinical biomarkers among patients with AF-related stroke is still controversial. The objective of this study is to determine the association of the pattern of AF and markers on routine blood tests with the outcome of patients after an AF-related stroke. This is a retrospective cohort study of patients with stroke and AF admitted in a tertiary hospital in Cebu City from 2015-2022. Patients' baseline characteristics, laboratory tests, ECG, and radiologic data were collected. Descriptive statistics such as mean and frequency were computed. The Kaplan-Meier method and the log-rank test were used to calculate the incidence time. The Cox regression analysis was used to determine factors associated with survival. A stepwise regression technique was used in model building. The mortality rate of patients with AF-related stroke was 0.02. A Kaplan Meier survival estimate shows that patients with paroxysmal AF have better survival. Upon model building of variables, age, red cell distribution width (RDW), neutrophil-to-lymphocyte ratio (NLR), platelet count, low density lipid cholesterol (LDL-C), and pattern of AF were predictive of mortality in patients with AF-related stroke. Among AF-related stroke patients admitted at a tertiary hospital in Cebu City, pattern of AF, age, RDW, NLR, platelet count, and LDL-C were associated with mortality. The parameters associated with increased mortality could be easily assessed using an ECG, CBC, and lipid profile. These are all readily available and cost-efficient.