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There remains a remarkable controversy on the anti-cancer stand of β-carotene specifically when high concentrations are used. This study aimed to examine the growth inhibitory effect of the lower concentrations of β-carotene in HepG2 cells. Beta-carotene purified from Gracillaria sp. inhibited viability of HepG2 cells in a concentration-dependent manner. This growth inhibitory effect was well correlated with increased apoptotic cell death. Apoptosis induction by β-carotene was associated with increased caspase-3 activity and reduced mitochondrial membrane potential. Western blot analysis showed a significant decrease in the expression of Bcl-2, PARP, and NF-kB protein, and an increase in the protein expression of Bax. Beta-carotene also blocked the activation of intracellular growth signaling proteins, Akt and ERK1/2. Further, it down-regulated the expression of endogenous antioxidant enzymes, SOD-2 and HO-1, and its transactivation factor, Nrf-2. The current study emphasizes the role of β-carotene as an effective inhibitor of the growth of hepatocarcinoma cell.

Cancer is one of the leading causes of death worldwide, accountable for a total of 10 million deaths in the year 2020, according to GLOBOCAN 2020. The advancements in the field of cancer research indicate the need for direction towards the development of new drug candidates that are instrumental in a tumour-specific action. The pool of natural compounds proves to be a promising avenue for the discovery of groundbreaking cancer therapeutics. Elaeocarpus ganitrus (Rudraksha) is known to possess antioxidant properties and after a thorough review of literature, it was speculated to possess significant biomedical potential. Green synthesis of nanoparticles is an environmentally friendly approach intended to eliminate toxic waste and reduce energy consumption. This approach was reported for the synthesis of silver nanoparticles from two different solvent extracts: aqueous and methanolic. These were characterized by biophysical and spectroscopic techniques, namely, UV-Visible Spectroscopy, FTIR, XRD, EDX, DLS, SEM, and GC-MS. The results showed that the nanoconjugates were spherical in geometry. Further, the assessment of antibacterial, antifungal, and antiproliferative activities was conducted which yielded results that were qualitatively positive at the nanoscale. The nanoconjugates were also evaluated for their anticancer properties using a standard MTT Assay. The interactions between the phytochemicals (ligands) and selected cancer receptors were also visualized in silico using the PyRx tool for molecular docking.

The term DSD (Disorders of Sex development) is issued to define those conditions in whom disharmony exists between chromosomal, hormonal and anatomical sex. Parental and patient mental health and quality of life are adversely affected by these conditions. Moreover, individuals with an underlying DSD, especially those with specific Y chromosomal material in their karyotype have an increased risk for developing a germ cell tumor. Here, we present a unique case of 46XY DSD with bilateral dysgerminomas presenting with abdominal mass at the age of 24 years, who was treated with one cycle of chemotherapy comprising of Carboplatin and Etoposide, following which he developed tumor lysis syndrome and later underwent exploratory laparotomy.

Hundreds of mutations in a single gene result in rare diseases, but why mutations induce severe or attenuated states remains poorly understood. Defect in glycine decarboxylase (GLDC) causes Non-ketotic Hyperglycinemia (NKH), a neurological disease associated with elevation of plasma glycine. We unified a human multiparametric NKH mutation scale that separates severe from attenuated neurological disease with new in silico tools for murine and human genome level-analyses, gathered in vivo evidence from mice engineered with top-ranking attenuated and a highly pathogenic mutation, and integrated the data in a model of pre- and post-natal disease outcomes, relevant for over a hundred major and minor neurogenic mutations. Our findings suggest that highly severe neurogenic mutations predict fatal, prenatal disease that can be remedied by metabolic supplementation of dams, without amelioration of persistent plasma glycine. The work also provides a systems approach to identify functional consequences of mutations across hundreds of genetic diseases. Our studies provide a new framework for a large scale understanding of mutation functions and the prediction that severity of a neurogenic mutation is a direct measure of pre-natal disease in neurometabolic NKH mouse models. This framework can be extended to analyses of hundreds of monogenetic rare disorders where the underlying genes are known but understanding of the vast majority of mutations and why and how they cause disease, has yet to be realized.

Introduction: Tocilizumab (TCZ) is an anti-interleukin-6 antibody that has been used for the treatment of severe coronavirus disease 2019 (COVID-19). However, concrete evidence of its benefit in reducing mortality in severe COVID-19 is lacking. Therefore, we performed a systematic review and meta-analysis of relevant studies that compared the efficacy of TCZ in severe COVID-19 vs. standard of care (SOC) alone.Methods: A literature search for studies that compared “tocilizumab” and “standard of care” in the treatment of COVID-19 was done using major online databases from December 2019 to June 14, 2020. Search words “Tocilizumab,” “anti-interleukin-6 antibody,” and “COVID-19” or “coronavirus 2019” in various combinations were used. Articles in the form of abstracts, letters without original data, case reports, and reviews were excluded. Data were gathered on an Excel sheet, and statistical analysis was performed using Review Manager 5.3.Results: Sixteen studies were eligible from 693 initial studies, including 3,641 patients (64% males). There were 13 retrospective studies and three prospective studies. There were 2,488 patients in the SOC group (61.7%) and 1,153 patients (68.7%) in the TCZ group. The death rate in the TCZ group, 22.4% (258/1,153), was lower than in the SOC group, 26.21% (652/2,488) [pooled odds ratio 0.57 (95% CI 0.36–0.92), p = 0.02]. There was a significant heterogeneity (inconsistency index = 80%) among the included studies.Conclusion: The addition of TCZ to the SOC might reduce mortality in severe COVID-19. More extensive randomized clinical trials are needed to validate these findings.

Patients of chronic obstructive pulmonary disease (COPD) during COVID-19 pandemic have higher morbidity. Treatment of these patients require aerosolization procedures like nebulization and noninvasive modalities for ventilation like non-invasive ventilation (NIV) and high flow nasal cannula (HFNC). Role of these procedures in corona virus transmission when treating a case of acute exacerbation of chronic obstructive pulmonary disease should be further studied.

Declarative memory abilities may be important for children who are learning to read in a second language. In the present study, we investigated declarative memory in a recognition memory task in 7-to-13-year-old, Kannada native-speaking, good (n = 22) and poor (n = 22) readers of English, in Karnataka, India. Recognition memory was tested shortly (∼10 min) after encoding (day 1) and again on the next (day 2). Analyses revealed that the two groups did not differ in recognition memory performance on day 1. On day 2, the good readers improved from day 1, whereas poor readers did not. A partial correlation analysis suggests that consolidation – the change in performance in recognition memory between the 2 days – is associated with reading skills in good readers, but not in poor readers. Taken together, these results suggest that children who struggle to read in a second language may have deficits in declarative memory consolidation.

We describe an autopsy case of a 45-year-old male diagnosed with autosomal dominant polycystic kidney disease who presented with complaints of altered sensorium. The autopsy revealed multiple tumor-like masses in the liver, which on histological examination depicted multiple large suppurative granulomas with the presence of variable acid-fast coccobacilli (consistent with Brucella spp.). Interestingly, extensive amyloid deposition in multiple organs was noted. To the best of our knowledge, this is the first case of chronic brucellosis causing tumor-like abscesses in the liver accompanied by secondary systemic amyloidosis in a patient with underlying autosomal dominant polycystic kidney disease.

Background and Aim: Cancer is a devastating disease with a severe impact on the physical and psychological well-being of patients. Hepatocellular carcinoma (HCC) has been reported in various species of animals including dogs, cats, sheep, and pigs. The present study aimed to study the immunohistochemical and histopathological changes and chemoprotective effect of aqueous and alcoholic extracts of Solanum nigrum on N-nitrosodiethylamine (NDEA)-induced HCC rat model. Materials and Methods: Eighty-two male Wistar rats of 15 weeks of age weighing 200-250 g were selected for the experiment. They were randomly divided into ten groups. Group I served as normal control consisted of healthy rats. HCC was induced in Group II, IV, V, VI, VII, and X rats using NDEA as inducing agent followed by phenobarbitone as a promoter for 16 weeks. Group II rats were kept untreated as HCC control. Group III rats were kept as vehicle control (0.05% Sodium bicarbonate). Group IV and V rats were treated with aqueous extract of S. nigrum at 200 mg/kg and 400 mg/kg, respectively, and Group VI and VII rats were treated with an alcoholic extract of S. nigrum at 200 mg/kg and 400 mg/kg, respectively, daily orally for 28 days. Group X rats were treated with sorafenib as reference drug at a dose of 11.4 mg/kg daily orally for 28 days. Group VIII and IX rats were kept as aqueous and alcoholic extract control for studying the effect of the same on normal rats. Liver samples were collected to study the gross and histopathological lesions and the activity of cleaved caspase-3 and chemopreventive effect of aqueous and alcoholic extracts of S. nigrum on HCC. Results: The liver sections of rats from HCC control (Group II) showed loss of lobular architecture, necrosis, fatty change, enlarged and darkened nuclei with variable size, dilatation of hepatic sinusoids with Kupffer cell hyperplasia, dilatation and proliferation of bile duct, and intranuclear vacuoles and also showed the presence of more than one nucleolus. Administration of alcoholic extract of S. nigrum and sorafenib to NDEA/phenobarbital-treated rats reduced the severity of lesions in the liver. Immunohistochemical analysis of liver sections for caspase-3-positive cells of hepatic cancer-induced group showed immunoreactivity to rarely few. The immunoreactivity of the hepatocytes treated with a higher dose of alcoholic extract of S. nigrum was limited and was comparable to a standard drug, sorafenib. Conclusion: Oral administration of aqueous and alcoholic extracts of S. nigrum for 28 days showed significant rejuvenation in the structure of the liver in the histopathological section in a dose-dependent manner in rats.

Acute renal cortical necrosis is a rare cause of intrinsic acute kidney injury (AKI) which is commonly associated with obstetric complications such as placental abruption and some serious systemic disorders such as hemolytic-uremic syndrome, sepsis, severe burns, and snake bite. Acute pancreatitis is an extremely rare cause of renal cortical necrosis, and only less than 10 cases are reported in the literature. Here, we present a 24-year-old male presented with features of acute pancreatitis and oliguric AKI. His pancreatic enzymes were above 1000 IU/mL at admission. He was initiated on hemodialysis. Percutaneous renal biopsy done at 4th week of illness showed features of diffuse renal cortical necrosis. Contrast-enhanced computed tomography demonstrated hypoattenuation of cortex compared to medulla consistent with renal cortical necrosis. He developed complications such as acute necrotic collection, pleural effusion and retinal detachment in addition to renal cortical necrosis which was managed conservatively. Since there was no improvement in the renal function now, he is being evaluated for renal transplantation.

This research work has been focusing on Hemp fibers has an alternative reinforcement for fiber reinforced polymer composites due to its eco-friendly and biodegradable characteristics. This work has been carried out to evaluate the mechanical properties of hemp/E-glass fabrics reinforced polyester hybrid composites. Vacuum bagging method was used for the preparation of six different kinds of hemp/glass fabrics reinforced polyester composite laminates as per layering sequences. The tensile, flexural, impact and water absorption tests of these hybrid composites were carried out experimentally according to ASTM standards. It reveals that an addition of E-glass fabrics with hemp fabrics can increase the mechanical properties of composites and decrease the water absorption of the hybrid composites.

Nephrotic syndrome is well known for its hypercoagulable state and thrombotic complications. This warrants cautious management of the disease with meticulous monitoring for thrombotic complications as it carries high morbidity and mortality than the original disease itself most often. Here, we present a case of adult onset nephrotic syndrome referred to our centre for further evaluation and management. On detailed work up, she turned out to be a case of minimal change disease. During her recovery period in the hospital she developed headache and diplopia. Sudden onset of neurological symptoms in the background of a thrombotic milieu prompted us to do further neurological evaluation which revealed extensive cerebral venous sinus thrombosis. She received anticoagulation without delay. Although the condition is associated with high mortality our patient showed very good response to anticoagulation and she was discharged in a stable condition.

The halophilic bacterial strain WB1 isolated from a hydrothermal vent was taxonomically characterized using multiple proxies, as Halomonas nitroreducens strain WB1. When grown on malt extract/yeast extract (MY) medium, it produced large quantities of exopolysaccharide (EPS). The polymer was synthesized at a higher rate during the log and early stationary phases. The anionic polysaccharide is primarily composed of glucose, mannose, and galactose. The studied EPS was highly viscous and had pseudoplastic nature. The EPS was found to be a mixture of three polysaccharides under FT-IR, which makes it less labile to environmental diagenesis. It also has emulsifying and antioxidant activity along with the binding capacity to heavy metals. The EPS has unique and interesting physical and chemical properties, which are different from earlier reported exo-polysaccharides produced by different bacterial genus. This suggests that the extreme geological niches like hypersaline, hyperthermal, hypothermal, and oligophilic environments, which are not well studied so far, can offer extensive and potential resources for medical, biotechnological and industrial applications. The study clearly showed that the thermal springs from the temperate region can be a potent source of many such industrially important microbial genera and need further detailed studies to be carried out.

Introduction: Malignancies constitute a wide variety of disorders having high mortality and morbidity rates. Current protocols for management include surgical intervention, chemotherapy, and radiation which possess numerous adverse effects. Many phytochemicals are available with anticancer properties similar to anticancer drugs. Major benefit of these compounds is apparent lack of toxicity to normal tissues. Graviola (botanical name: Annona Muricata) contain bioactive compound “annonaceous acetogenins” known for anticancer activity on cancer cell lines. Aims: To determine cytotoxicity of Graviola and percentage cell inhibition at G2M phase of cell cycle. Settings and Design: The cytotoxicity of aqueous extract of Graviola leaves on squamous cell carcinoma (SCC-25) cell lines at various concentrations evaluated using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The percentage of SCC-25 cell inhibition at G2M phase of cell cycle determined using flow cytometry. Methods: Graviola Leaves, American Type Culture Collection SCC-25 cell lines were procured from Skanda Laboratories, Bengaluru. The cytotoxicity of aqueous extract of Graviola on SCC-25 cells at various concentrations evaluated using MTT assay. The percentage of SCC-25 cell inhibition at G2M phase of cell cycle determined using flow cytometry. Statistical Analysis: Statistical analysis was done using one-way ANOVA. Results: MTT assay showed statistically significant (P < 0.001) dose-dependent inhibition of SCC-25 cell lines by Graviola with IC50 value of 12.42 μg/ml. Flow cytometry revealed that Graviola at 25 and 50 g/ml arrested 53.39% and 52.09% cells in G2M phase of cell cycle respectively, which was statistically significant. Conclusion: Graviola showed significant cytotoxic activity and percentage of cell inhibition at G2M phase cell cycle against SCC-25 cell lines.

Introduction: Diabetes is known to be associated with greater production of reactive oxygen species (ROS) like malondialdehyde (MDA) and decreased antioxidants like total thiols and its estimation in plasma is used in monitoring the redox status. The aim of this study was to analyse the association between plasma lipid profile parameters like HDL and LDL with salivary MDA and thiols in diabetic patients. Materials and Methods: Sixty subjects between the age group 35- 70 years who were diagnosed with type II diabetes mellitus based on fasting blood glucose and glycated haemoglobin levels who attended the dental OPD at Kasturba Medical College and Hospital, Manipal consented to participate in this study. Plasma glucose, HDL and LDL were estimated in Cobas autoanalyser by hexokinase method, homogenous enzymatic colorimetric assay and Friedwald’s formula respectively. Assessment of glycated hemoglobin was by ion exchange chromatography, MDA by thiobarbituric acid as a substrate and thiols by Ellmann’s manual method in plasma and saliva. Results: The association of plasma LDL with salivary MDA was found to be positive and significant and that with salivary thiols was negative and significant. Also, the association of plasma HDL with salivary MDA was found to be negative and significant and that with salivary thiols was positive and significant. Conclusion: Results indicate the potential of saliva as a tool to monitor prognosis of diabetes.

Context: Facial palsy is a common problem encountered in clinical practice. These patients suffer serious functional, cosmetic & psychological problems with impaired ability to communicate. Functional problems around the eye are usually a first priority for the patient with facial palsy.The nerve to masseter as a direct nerve transfer to the zygomatic branch of facial nerve to reinnervate viable facial muscles within a year after the onset of paralysis has been scarcely reported. This study was contemplated to evaluate the feasibility of neurotisation of zygomatic branch of facial nerve with masseteric nerve branch of the trigeminal nerve Objectives: Establishing the anatomic relationship of masseteric nerve to masseteric muscle, determining feasibility of neurotisation of zygomatic branch of facial nerve using the nerve to the masseter and establishing fascicular correlation of the donor and the recipient nerves. Materials and Methods: Ninteen hemi-faces in ten fresh cadavers (6 Male and 4 Female)were dissected in a forensic morgue and access was by a standard preauricular incision and anterior skin flap is elevated in a subcutaneous plane. Facial nerve and its two main divisions are dissected in its full A*/extratemporal course. Zygomatic branch dissected upto zygomatic arch and the nerve to the masseter is identified within the masseter muscle, dissected proximodistally to isolate it. Feasibility of transfer of this masseteric nerve to the zygomatic branch without using nerve graft is determined. At the completion of dissection, the ends of both nerves are sent for HPE analysis to determine fascicular anatomy and count. Results: The dissection of masseteric nerve was done taking into consideration of 3 axes, that is anteroposterior (x), vertical (y) axes and mediolateral (z) for locating the nerve and for standardization of the dissection. The nerve was cut and stored in glutaraldehyde solution and subjected to histopathologic examination after fixing and staining with Haematoxilin-eosin stain. Donor masseter neve has 7-10 fascicles. Recipient zygomatic branch has 2-3 fascicles. And the buccal branch has 5-6 fascicles. Conclusion: The use of the nerve to masseter offers a simpler ipsilateral alternative for neurotisation of the facial nerve branches in patients who have an early facial nerve paralysis.

Introduction: Apheresis is practiced widely to collect single donor platelets (SDPs). This procedure utilizes an anticoagulant acid citrate dextrose to prevent clotting of blood in the extracorporeal circuit which chelates divalent ions like calcium. This alters the calcium homeostasis resulting in hypocalcemia causing acute adverse events., Aim: The study aimed to know the calcium homeostasis in apheresis platelet donors., Materials and Methods: This cross-sectional study was conducted from January 2020 to December 2020 in the department of transfusion medicine. The sample size was 50. Donors who walk in for voluntary SDP donation were selected. Total and ionized calcium, pH, and serum albumin for all the donors at baseline and ionic calcium at the end of the procedure and 30 min after the procedure were measured., Results: According to statistical analysis of the ionic calcium level at pre procedure, immediate post procedure and 30 minutes post procedure, there was decrease in the value immediate post procedure and values returned to baseline within 30 minutes. The levels of pH change were analyzed. On comparing the preprocedure and immediate postprocedure values, there was a significant lowering of pH value from the baseline ( P = 0.5), indicating acute lowering of pH immediate postprocedure. Hence, most of the citrate metabolism can be achieved within 30 min after completion of the apheresis procedure., Conclusion: SDP collection is essentially a safe procedure with minimal adverse effects. Toxicity of citrate is not much pronounced. Recovery of calcium levels is within 30 min of completion of plateletpheresis., Competing Interests: There are no conflicts of interest., (Copyright: © 2023 Asian Journal of Transfusion Science.)

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab's Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.

Purpose: Although cyclin-dependent kinase 5 (Cdk5) inhibits the formation of junctions containing N-cadherin, the effect of Cdk5 on junctions containing E-cadherin is less clear. The present study investigates the functional significance of Cdk5 in forming and maintaining cell-cell stability in corneal epithelial cells., Methods: A Cdk5-deficient human corneal limbal epithelial cell line was generated by lentiviral transduction of small hairpin RNA specific for Cdk5 (shCdk5-HCLE cells). A blasticidin-inducible vector for expression of Cdk5-specific short hairpin RNA (ShCdk5) was generated by recombination and packaged into non-replicative lentiviral particles for transduction of human corneal limbal epithelial (HCLE) cells. Blasticidin-resistant cells were isolated for analysis. Cell aggregations were performed using HCLE, Cdk5 inhibitor olomoucine, ShCdk5, and MDA-MB 231 cells in the presence and absence of calcium, and particle size was measured using image analysis software. Relative protein concentrations were measured with immunoblotting and quantitative densitometry. Total internal reflection fluorescence (TIRF) microscopy was performed on cells transfected with green fluorescent protein (GFP)-E-cadherin or GFP-p120, and internalization of boundary-localized proteins was analyzed with particle tracking software. The stability of surface-exposed proteins was determined by measuring the recovery of biotin-labeled proteins with affinity chromatography. Rho and Rac activity was measured with affinity chromatography and immunoblotting., Results: Examining the effect of Cdk5 on E-cadherin containing epithelial cell-cell adhesions using a corneal epithelial cell line (HCLE), we found that Cdk5 and Cdk5 (pY15) coimmunoprecipitate with E-cadherin and Cdk5 (pY15) colocalizes with E-cadherin at cell-cell junctions. Inhibiting Cdk5 activity in HCLE or suppressing Cdk5 expression in a stable HCLE-derived cell line (ShHCLE) decreased calcium-dependent cell adhesion, promoted the cytoplasmic localization of E-cadherin, and accelerated the loss of surface-biotinylated E-cadherin. TIRF microscopy of GFP-E-cadherin in transfected HCLE cells showed an actively internalized sub-population of E-cadherin, which was not bound to p120 as it was trafficked away from the cell-cell boundary. This population increased in the absence of Cdk5 activity, suggesting that Cdk5 inhibition promotes dissociation of p120/E-cadherin junctional complexes. These effects of Cdk5 inhibition or suppression were accompanied by decreased Rac activity, increased Rho activity, and enhanced binding of E-cadherin to the Rac effector Ras GTPase-activating-like protein (IQGAP1). Cdk5 inhibition also reduced adhesion in a cadherin-deficient cell line (MDA-MB-231) expressing exogenous E-cadherin, although Cdk5 inhibition promoted adhesion when these cells were transfected with N-cadherin, as previous studies of Cdk5 and N-cadherin predicted. Moreover, Cdk5 inhibition induced N-cadherin expression and formation of N-cadherin/p120 complexes in HCLE cells., Conclusions: These results indicate that loss of Cdk5 activity destabilizes junctional complexes containing E-cadherin, leading to internalization of E-cadherin and upregulation of N-cadherin. Thus, Cdk5 activity promotes stability of E-cadherin-based cell-cell junctions and inhibits the E-cadherin-to-N-cadherin switch typical of epithelial-mesenchymal transitions.

Purpose: The purpose of this study was to evaluate the subset of limbal epithelial cells with greater nucleus-to-cytoplasm (N/C) ratio expressing high levels of p63 for their slow-cycling property, a characteristic feature of stem cells (SCs)., Methods: Limbal and peripheral corneal explant cultures were pulse labeled with 5-5-bromo-2'-deoxyuridine (BrdU) for 5 days, followed by a period of 3-week chase. Cultured explants were cryosectioned and stained for BrdU. The epithelial cells in the outgrowth and those remaining on the explant were isolated and subjected to cytospin and double immunostaining for BrdU and p63, followed by identification of label-retaining cells (LRCs) and quantification of p63 expression using confocal microscopy., Results: A distinct population of small cells with large N/C ratio expressing high levels of p63 retained the BrdU label after 21-day chase. Further, this population of LRCs, negative for the differentiation marker K3, was observed in the epithelial outgrowth of limbal but not in that of peripheral cornea. LRCs were seen to migrate along the cut edge of limbal explants in culture and were also observed as clusters of small cells in the outgrowth, which contained cells with the ability to form holoclone colonies., Conclusions: These results demonstrate that the small cells with large N/C ratio and high levels of p63 have BrdU label retaining slow-cycling property, thus confirming that these 2 parameters in combination may serve as a precise marker for identification and quantification of ex vivo-expanded limbal SCs. This method would be useful to standardize the optimal culture conditions that can maintain and expand SCs for therapeutic applications.

The objectives were to develop method of isolating viable human limbal basal cells in order to enrich a subset of small cells with a large Nucleus/Cytoplasm (N/C) ratio expressing high levels of p63, nuclear protein. Limbal tissues were treated with trypsin for 50 min at 37 degrees C in an orbital shaker at 100 rpm with epithelial side down followed by additional 5 min with epithelial side up and then with Dispase II to obtain various epithelial fractions. Isolated cell fractions were assessed for colony forming efficiency and DeltaNp63alpha, connexin (Cx43) mRNA levels. Cytospin smears were double-immunostained for p63 and any one of the stem cell (SC) related markers and analyzed using a laser scanning confocal microscope and advanced image analysis software (Leica Confocal software, 2.61 build 1537 version) for quantification of fluorescence intensity. The isolated limbal basal cells were highly positive for DeltaNp63alpha mRNA but expressing low Cx43 mRNA. They gave rise to higher number of large colonies with compact morphology in contrast to the limbal suprabasal/superficial (LS/S) colonies. Furthermore, a subset with a large N/C ratio expressing high levels of p63 was observed, as much as 25% among the limbal basal cell fraction, in contrast to only about 4% in the total limbal epithelial cells. Such cells were positive for K5 and negative for Ki67, Cx43, and 14-3-3s and were absent in the LS/S fraction. These results collectively substantiate our method of isolation of limbal basal layer cells containing an enriched population of cells with SC phenotype., ((c) 2008 Wiley-Liss, Inc.)

Purpose: To characterize human limbal epithelial cells based on the expression levels of nuclear protein p63 and the nucleus-to-cytoplasm (N/C) ratio., Methods: Limbal, peripheral, and central corneal epithelia were separated from the stroma by Dispase II and subsequently were treated with trypsin to obtain single-cell suspensions. Cytospin smears of the cell suspensions were double immunostained for p63 and then stained for any one of the markers (acidic cytokeratins [AE1], K5, K3, or connexin 43 [Cx43]). They were counterstained with propidium iodide. More than 100 cells from each zone were analyzed for p63 expression levels and nuclear/cellular area using quantitative confocal microscopy., Results: A gradient of p63-positive cells was observed in corneal and limbal epithelial cells. The percentage of p63-positive cells and the level of p63 expression were significantly higher in the limbal than in the peripheral or central corneal epithelium. Two-parameter (p63 levels and N/C ratio) analysis revealed the presence of a distinct population of small cells with higher levels of p63 and a large N/C ratio in the limbal epithelium. Such limbal epithelial cells were positive for AE1 and K5 but negative for K3 and Cx43., Conclusions: These results suggest that this distinct group of small cells in the limbal epithelium with greater N/C ratio, expressing high levels of nuclear protein p63, probably represent corneal epithelial stem cells.