Swarnendu Bag

Epithelio-mesenchymal transitional attributes in oral sub-mucous fibrosis.

Evaluating molecular attributes in association with its epithelial and sub-epithelial changes of oral sub-mucous fibrosis is meaningful in exploring the plausibility of an epithelio-mesenchymal transition (EMT) and malignant potentiality of this pathosis. In this study histopathological and histochemical attributes for basement membrane and connective tissue in biopsies of oral sub-mucous fibrosis (n = 55) and normal oral mucosa (n = 16) were assessed and expressions of p63, E-cadherin, β-catenin, N-cadherin and TWIST were analyzed immunohistochemically. The p63 and its isoforms (TA and ∆N), PARD3, E-cadherin and β-catenin were also assessed transcriptomically by q-PCR and EMT players like TWIST1, ZEB1, MMP9 and micro-RNA 205 were searched in gene expression microarrays. Oral epithelium demonstrating impairment in progressive maturation in oral sub-mucous fibrosis concomitantly experienced an increase in basement membrane thickness and collagen deposition along with alteration in target molecular expressions. In comparison to non-dysplastic conditions dysplastic stages exhibited significant increase in p63 and p63∆N expressions whereas, E-cadherin and β-catenin exhibited loss from the membrane with concurrent increase in cytoplasm. Further the N-cadherin and TWIST were gained remarkably along with the appearance of nuclear accumulation features of β-catenin. The microarray search had noticed the up-regulation of TWIST1, ZEB1 and MMP9 along with down regulation of micro-RNA 205. The simultaneous increase in basement membrane thickness and sub-epithelial collagen deposition were the plausible indicators for increased matrix stiffness with expected impact on oral epithelial functional homoeostasis. This was corroborated with the increase in expressions of epithelial master regulator p63 and its oncogenic isoform (∆N) along with membranous loss of E-cadherin (EMT hallmark) and its associate β-catein and gain of mesenchymal markers like N-cadherin and TWIST. These also became indicative for the induction of epithelial to mesenchymal transitional mechanism in oral sub-mucous fibrosis when connoted here with the relevant modulation in expressions of EMT regulators.

NMR (1H and 13C) based signatures of abnormal choline metabolism in oral squamous cell carcinoma with no prominent Warburg effect

At functional levels, besides genes and proteins, changes in metabolome profiles are instructive for a biological system in health and disease including malignancy. It is understood that metabolomic alterations in association with proteomic and transcriptomic aberrations are very fundamental to unravel malignant micro-ambient criticality and oral cancer is no exception. Hence deciphering intricate dimensions of oral cancer metabolism may be contributory both for integrated appreciation of its pathogenesis and to identify any critical but yet unexplored dimension of this malignancy with high mortality rate. Although several methods do exist, NMR provides higher analytical precision in identification of cancer metabolomic signature. Present study explored abnormal signatures in choline metabolism in oral squamous cell carcinoma (OSCC) using (1)H and (13)C NMR analysis of serum. It has demonstrated down-regulation of choline with concomitant up-regulation of its break-down product in the form of trimethylamine N-oxide in OSCC compared to normal counterpart. Further, no significant change in lactate profile in OSCC possibly indicated that well-known Warburg effect was not a prominent phenomenon in such malignancy. Amongst other important metabolites, malonate has shown up-regulation but d-glucose, saturated fatty acids, acetate and threonine did not show any significant change. Analyzing these metabolomic findings present study proposed trimethyl amine N-oxide and malonate as important metabolic signature for oral cancer with no prominent Warburg effect.

Computer-aided molecular pathology interpretation in exploring prospective markers for oral submucous fibrosis progression

Evaluation of molecular pathology markers using a computer‐aided quantitative assessment framework would help to assess the altered states of cellular proliferation, hypoxia, and neoangiogenesis in oral submucous fibrosis and could improve diagnostic interpretation in gauging its malignant potentiality.

Honey dilution impact on in vitro wound healing: Normoxic and hypoxic condition

Honey is known as a popular healing agent against tropical infections and wounds. However, the effects of honey dilutions on keratinocyte (HaCaT) wound healing under hypoxic condition is still not explored. In this study, we examined whether honey dilution have wound healing potential under hypoxic stress. The antioxidant potential and healing efficacy of honey dilution on in vitro wound of human epidermal keratinocyte (HaCaT cells) under hypoxia (3% O2), and normoxia is explored by nitro blue tetrazolium assay. The cell survival % quantified by MTT assay to select four honey dilutions like 10, 1, 0.1, and 0.01 v/v% and the changes in cellular function was observed microscopically. Further, the cell proliferation, migration, cell–cell adhesion, and relevant gene expression were studied by flow cytometry, migration/scratch assay, immunocytochemistry, and reverse transcription‐polymerase chain reaction, respectively. The expression pattern of cardinal molecular features viz. E‐cadherin, cytoskeletal protein F‐actin, p63, and hypoxia marker Hif 1α were examined. Honey dilution in 0.1% v/v combat wound healing limitations in vitro under normoxia and hypoxia (3%). Its wound healing potential was quantified by immunocytochemistry and real‐time PCR for the associated molecular features that were responsible for cell proliferation and migration. Our data showed that honey dilution can be effective in hypoxic wound healing. Additionally, it reduced superoxide generation and supplied favorable bioambience for cell proliferation, migration, and differentiation during hypoxic wound healing. These findings may reveal the importance of honey as an alternative and cost effective therapeutic natural product for wound healing in hypoxic condition.

Computational analysis of p63+ nuclei distribution pattern by graph theoretic approach in an oral pre-cancer (sub-mucous fibrosis)

Background: Oral submucous fibrosis (OSF) is a pre-cancerous condition with features of chronic, inflammatory and progressive sub-epithelial fibrotic disorder of the buccal mucosa. In this study, malignant potentiality of OSF has been assessed by quantification of immunohistochemical expression of epithelial prime regulator-p63 molecule in correlation to its malignant (oral squamous cell carcinoma [OSCC] and normal counterpart [normal oral mucosa [NOM]). Attributes of spatial extent and distribution of p63 + expression in the epithelium have been investigated. Further, a correlated assessment of histopathological attributes inferred from H&E staining and their mathematical counterparts (molecular pathology of p63) have been proposed. The suggested analytical framework envisaged standardization of the immunohistochemistry evaluation procedure for the molecular marker, using computer-aided image analysis, toward enhancing its prognostic value. Subjects and Methods: In histopathologically confirmed OSF, OSCC and NOM tissue sections, p63 + nuclei were localized and segmented by identifying regional maxima in plateau-like intensity spatial profiles of nuclei. The clustered nuclei were localized and segmented by identifying concave points in the morphometry and by marker-controlled watersheds. Voronoi tessellations were constructed around nuclei centroids and mean values of spatial-relation metrics such as tessellation area, tessellation perimeter, roundness factor and disorder of the area were extracted. Morphology and extent of expression are characterized by area, diameter, perimeter, compactness, eccentricity and density, fraction of p63 + expression and expression distance of p63 + nuclei. Results: Correlative framework between histopathological features characterizing malignant potentiality and their quantitative p63 counterparts was developed. Statistical analyses of mathematical trends were evaluated between different biologically relevant combinations: (i) NOM to oral submucous fibrosis without dysplasia (OSFWT) (ii) NOM to oral submucous fibrosis with dysplasia (OSFWD) (iii) OSFWT-OSFWD (iv) OSFWD-OSCC. Significant histopathogical correlates and their corroborative mathematical features, inferred from p63 staining, were also investigated into. Conclusion: Quantitative assessment and correlative analysis identified mathematical features related to hyperplasia, cellular stratification, differentiation and maturation, shape and size, nuclear crowding and nucleocytoplasmic ratio. It is envisaged that this approach for analyzing the p63 expression and its distribution pattern may help to establish it as a quantitative bio-marker to predict the malignant potentiality and progression. The proposed work would be a value addition to the gold standard by incorporating an observer-independent framework for the associated molecular pathology.

Design and synthesis of dual probes for detection of metal ions by LALDI MS and fluorescence: application in Zn(II) imaging in cells

A Label-Assisted Laser Desorption/Ionization (LALDI) technique has recently been applied to the detection of metal ions through a time of flight (TOF) mass spectrometric measurement. In this paper, we report the synthesis of two terpyridine based ligands L1 and L2 containing a pyrene moiety. The presence of the latter helped the ligand metal complex to desorb from the surface and ionize upon laser irradiation and finally show up in the TOF MS. Both ligands were able to detect Zn2+, Ni2+ and Co2+ ions while Cu2+, Fe2+ and Mg2+ remained LDI silent. Complexation with the ligands also caused a red fluorescence only with Zn2+ ions that allowed Zn2+ imaging in cells. Thus the pyrene moiety acted as a dual probe for metal ion detection exploiting both LALDI MS and fluorescence techniques.

Transfer learning of tissue photon interaction in optical coherence tomography towardsin vivo histology of the oral mucosa.

Oral cancer evolves from different premalignant conditions and the key to save lives is through diagnosis of early symptoms. The conventional practice of post biopsy histopathology reporting is dependent on specificity of sampling site and optical coherence tomography (OCT) imaging is clinically used for guidance. Clinicians infer the tissue constitution by interpreting intensity images and are challenged by inter-and intra-observer variability. In this paper we propose transfer learning of tissue specific photon interaction statistical physics in swept-source OCT for characterizing the oral mucosa with the aim of reducing this reporting variability. The source task models statistical physics of ballistic and near-ballistic photons and its intensity attenuation and target task learns the parameters obtained by solving the source task to identify co-located heterogeneity of tissues. Performance is compared with conventional histopathology of healthy, premalignant and malignant oral lesions supporting its use towards in vivo histology of the oral mucosa for pre-biopsy screening.

NanoLC MALDI MS/MS based quantitative metabolomics reveals the alteration of membrane biogenesis in oral cancer

Cancer cells use aberrant metabolic process for proliferation and metastasis. High-throughput separation and quantification of metabolites from bio-samples is crucial in this aspect. Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), a prime technique used for metabolite identifications, suffers from several limitations in quantification studies. In this study, we have established a novel approach using conventional nanoLC-MALDI MS/MS interface for separation, identification and label free quantifications of the metabolites from biopsies. Quantification of metabolite using MALDI requires a homogeneous distribution of the matrix mixed samples on target plate for proportional time-of-flight (ToF) with the concentration of a particular metabolite present in that sample under same laser intensity. Here, crude metabolites extracted from cancer biopsies are separated and eluted as ‘fraction spots’ on MALDI target plate using nanoLC. Introducing a novel parameter, %aAUC (percentage of average area under the curve), comparative quantification of separated metabolites isolated from normal, pre-cancer and cancer biopsies has been explored. Such comparative quantification is only possible with multiple AUCs of a particular metabolite obtained from this nanoLC-MALDI MS approach. Further, selected metabolite peaks have been analyzed through MS/MS fragmentation. This approach was validated using known concentrations of an internal standard (thiourea) with its corresponding aAUC (average area under the curve). Interestingly, such comparative quantification has revealed a significant change in expression of crucial lipid metabotypes like triglyceride, phospatidyl inositol, phosphatidyl choline, glycerophospholipid, cytidine diphosphate-diacylglycerol and phosphatidylinositol bisphosphate indicating altered lipid metabolism associated membrane biogenesis in oral pre-cancer (oral submucous fibrosis) and cancer. Hence, our study successfully established an altered membrane biogenesis in oral cancer using a novel and distinct approach for separation, identification and label-free quantification of metabolites in fast growing metabotype-biomarker identification era.

Connecting cyto-nano-architectural attributes and epithelial molecular expression in oral submucous fibrosis progression to cancer

Objective Problems in pre-cancer diagnosis complicate cancer theragnosis as well as life expectancy. There is uncertainty regarding malignant transformation of oral submucous fibrosis (OSF), an oral pre-cancer with dysplastic (OSFWD) and non-dysplastic (OSFWT) subtypes. Understanding the structural, molecular and physical aspects of epithelial homeostasis may be useful. Materials and methods Histopathological grading of biopsy sections was performed using H&E staining. Alterations in epithelial surface architecture in different groups was evaluated using scanning electron microscopy (SEM). The expression of crucial epithelial genes (p63, CK-5/6, CK-10, E-cadherin and β-catenin) was studied by immunohistochemistry, Western blot and RT-PCR analysis. Results SEM observations revealed that the surface epithelial ridge pattern became thick and dense, and pit pattern gradually decreased in OSFWD and oral squamous cell carcinoma (OSCC). p63, ΔNp63 and CK-5/6 were up-regulated in OSFWD and OSCC but down-regulated in OSFWT. CK-10 was down-regulated in OSFWD compared to OSFWT. Cytoplasmic expression of E-cadherin and β-catenin was elevated in dysplastic and cancerous conditions. Moreover, statistical correlation between SEM features (ridges and pits) and molecular attributes demonstrated a significant positive relationship between the ridge-to-pit ratio and p63 population density (r=0.85) and the ridge-to-pit ratio and CK-5/6 intensity (r=0.63). Conclusions Molecular changes related to epithelial progressive maturation and cellular proliferation are correlated with concomitant alteration of epithelial surface architecture which helps to predict the malignant potentiality of OSF.

Honey Extracted Polyphenolics Reduce Experimental Hypoxia in Human Keratinocytes Culture

Hypoxic assault affects fundamental cellular processes and generates oxidative stress on healthy cells/molecules. Honey extracted polyphenolics (HEP) as a natural antioxidant reduced hypoxic cytotoxicity in this study. Different honey samples were physicochemically characterized to identify preferred (jamun) honey [pH 3.55 ± 0.04, conductivity (μs/cm) = 6.66 ± 0.14, water content % (w/w) = 14.70 ± 0.35, total solid content % (w/w) = 85.30 ± 0.35, phenol content (mg GAE/100 g) = 403.55 ± 0.35, flavonoid content (mg QE/100 g) = 276.76 ± 4.10, radical scavenging activity (% 500 μL) = 147.75 ± 3.13, catalase activity (absorbance at 620 nm) = 0.226 ± 0.01]. HEP was tested in different doses on hypoxic and normoxic cells (HaCaT) using viability and antioxidant assays. Cardinal molecular expressions such as cadherin-catenin-cytoskeleton complex (namely, E-cadherin, β-catenin, and F-actin), hypoxia marker (Hif 1 α), proliferation marker (Ki67), and epithelial master regulator (p63) were studied by immuno-cytochemisty (ICC) and qRT-PCR. The 0.063 mg/mL HEP demonstrated better vitality and functionality of HaCaT cells as per viability assay (*, P < 0.01) even under hypoxia. ICC and qRT-PCR observations indicated restoration of cellular survival and homeostasis under 0.063 mg/mL HEP after hypoxic assault. Furthermore, major spectral changes for nucleic acid and membrane phospholipid reorganizations by Fourier transform infrared spectroscopy illustrated a positive impact of 0.063 mg/mL HEP on hypoxic cells considering proliferation and cellular integrity. It was concluded that a specific dose of jamun HEP reduces hypoxic cytotoxicity.