Amrita Chaudhary

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.

In situ histology of mice skin through transfer learning of tissue energy interaction in optical coherence tomography

Abstract. Tissue characterization method in optical coherence tomography (OCT) for in situ histology of soft tissues is presented and demonstrated for mice skin. OCT allows direct noninvasive visualization of subsurface anatomy. It is currently used for in situ investigation of lesions in skin, vessels, retinal layers, oral, and bronchial cavitities. Although OCT images present high resolution information about tissue morphology, reporting requires a reader experienced in interpretation of the images, viz., identification of anatomical layers and structures constituting the organ based on OCT speckle appearance. Our approach characterizes tissues through transfer learning of tissue energy interaction statistical physics models of ballistic and near-ballistic photons. The clinical information yield with our approach is comparable to conventional invasive histology. On cross evaluation with a mice model experiment, the epidermis, papillary dermis, dermis, and adipose tissue constituting the mice skin are identified with an accuracy of 99%, 95%, 99%, and 98%, respectively. This high accuracy of characterizing heterogeneous tissues using OCT justifies the ability of our computational approach to perform in situ histology and can be extended to regular clinical practice for diagnosis of vascular, retinal, or oral pathologies.

Nano-patterned honey incorporated silk fibroin membranes for improving cellular compatibility

The desirable properties of a matrix for tissue engineering applications appears to shift from a minimal model of supporting cell growth to a more inclusive role as a matrix to trigger the regeneration process. This requirement has propelled efforts towards bio-functionalization of matrices both by chemical and textural modification of most advanced materials, including silk. Amongst different biomaterials, silk fibroin (SF) has been extensively used for various tissue engineering applications, because of its low immunogenicity and good mechanical strength, although low biological activity and a poor biodegradability rate has remained its major limitation for use in regenerative medicine. In the present study, the physical as well as biological properties of SF have been improved by incorporation of honey, a natural healing agent. Honey-blended SF films (HSF) demonstrated increased mechanical strength together with enhanced wettability, swelling ability and degradability without hampering the original mechanical and biological properties of both honey and SF. In addition, parallel-aligned nanostructures were produced over native SF and HSF using soft lithography to form two-dimensional nano-patterned membranes. HSF films demonstrated enhanced surface roughness in flat membranes and even more so in nano-patterned membranes when compared with native SF membranes which are essential for cellular adhesion and proliferation. Furthermore, biocompatibility and cellular behavior such as adhesion, proliferation, spreading and interaction of 3T3 fibroblast cells were improved using HSF when compared with using native SF membranes. Thus, HSF membranes with enhanced bio-physical properties are a potential material for use in regenerative medicine and tissue engineering applications.

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.

Identification of α-enolase as a prognostic and diagnostic precancer biomarker in oral submucous fibrosis

Aims Diagnostic ambiguities regarding the malignant potentiality of oral submucous fibrosis (OSF), an oral precancerous condition having dysplastic and non-dysplastic isoforms are the major failure for early intervention of oral squamous cell carcinoma (OSCC) patients. Our goal is to identify proteomic signatures from biopsies that can be used as precancer diagnostic marker for patient suffering from OSF. Methods The high throughput techniques adopting de novo peptide sequencing (1D SDS-PAGE coupled nanoLC MALDI tandem mass spectrometry (MS/MS)-based peptide mass fingerprint), immunohistochemistry (IHC), Western blot (WB) and real-time PCR (RT-PCR) analysis are considered for such biomarker identification and multilevel validations. Results Alpha-enolase is identified as an overexpressed protein in biopsies of oral submucous fibrosis with dysplasia (OSFWD) compared with oral submucous fibrosis without dysplasia (OSFWT) and normal oral mucosa (NOM). Total proteome analysis of an overexpressed protein band around 47 kDa of OSFWD identifies 334 peptides corresponding to 61 human proteins. Among them α-enolase is identified as a prime protein with highest number of peptides (44 out of 334 peptides) and sequence coverage (66.4%). Furthermore, RT-PCR, WB and IHC analysis also show mRNA and tissue level upregulation of α-enolase in OSFWD validating α-enolase as precancer marker. Conclusions This study for the first time identifies and validates α-enolase as a novel biomarker for early diagnosis of malignant potentiality of OSF. Hence, the identified protein marker, α-enolase can help in early therapeutic intervention of OSF patients leading to the reduction of patient’s pain, treatment cost and enhancement of patient’s quality of life.

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.