Offline Handwritten Kannada Numerals Recognition

Handwritten Character Recognition (HCR) is one of the essential aspect in academic and production fields. The recognition system can be either online or offline. There is a large scope for character recognition on hand written papers. India is a multilingual and multi script country, where eighteen official scripts are accepted and have over hundred regional languages. Recognition of unconstrained hand written Indian scripts is difficult because of the presence of numerals, vowels, consonants, vowel modifiers and compound characters. In this paper, recognition of handwritten Kannada numeral characters is implemented and the different Wavelet features are used as feature extraction in this paper. The zonal densities of different region of an image have been extracted in the database. The database consists of 50 samples of each Kannada numeral character. For classification, the K-Nearest Neighbor method is used. Recognition accuracy of 88% has been achieved

Acquisition of Images using Neural Network

The application of computer vision to the image retrieval problem is Content-based image retrieval (CBIR). The interest in digital images is growing day by day. Users in professional fields are make use of the opportunities offered by the ability to access and manipulate remotely-stored images in different ways. The problems in image retrieval are becoming widely accepted, and the finding solution is an active area for research and development. This dissertation work aims at developing a hybrid scheme for intelligent image retrieval system using neural networks. Each image in the database is indexed by a visual feature vector, which is extracted using color moments and discrete cosine transform coefficients. The query is characterized by a set of predefined semantic labels. A novel method of similarity measure using dot product is used for ranking and retrieval for improved performance of the system DOI: 10.17762/ijritcc2321-8169.15050

Characterisation of spontaneous proximal tubule formation in vitro and development of a metastatic clear cell renal cell carcinoma (ccRCC) model

Epithelial cells enable essential physiological functions, including absorption, morphogenesis, secretion, and transport. To execute these functions, epithelial cells often form three-dimensional shapes that include curved sheets of cells surrounding a pressurized fluid-filled lumen. These three-dimensional tissues (called domes) are essential for organ function, but when they are not working properly, developmental defects, inflammation, and cancer can ensue. Recently, it has been shown that the cells that form domes show active superelasticity on micropatterned plates. We show here that the immortalized renal proximal tubule epithelial cell line, LLC-PK1, stereotypically forms tubules in 10 days. Tubule formation takes place in 4 stages. When cells are plated on a culture dish, they form a monolayer on the 1st day; on the 3rd day, three-dimensional structures are formed, called domes; and after the 4.5th day, these domes start fusing to begin the transition stage and transit to the tubule stage. At the end of the 10th day, differentiated, elongated, and matured tubes form (Figure 3.1). Therefore, tubule formation is a self-organized, stereotypic morphogenetic program under long-term, unperturbed tissue culture conditions. We propose that tubulogenesis is a two-step process in proximal tubules by doming and wrapping. The process begins with dome formation, and as the cell layers come together in the transition stage at the edge of the dome, this leads to the formation of the lumen of the eventual tubule. We also found that F-actin provides the mechanical strength during the formation of these three-dimensional structures during tubule formation. To better understand this 4-step process on a molecular level, we performed proteomics of tubule formation to identify the different proteins that play a significant role in proximal tubule development. Importantly, we identified proximal tubule markers like synaptopondin, angiotensin 1-10, collectrin, polycystin 1, and polycystin 2. These proteins play an important role in renal tube formation and differentiation. Cell division is carried out by highly conserved cyclin-CDK complexes, which phosphorylate various cellular components. Cyclin-CDKs act differently depending on the cell cycle phase and work cooperatively to create DNA replication and cytokinesis. Therefore, we identified that cyclin-B1, marker of proliferation Ki-67, the RAD51 recombinase, and proliferating cell nuclear antigen (PNCA) are upregulated in the monolayer stage, and the expression decreases as tubule formation takes place. The proximal tubule reabsorbs 60-65% of the glomerulus filtrate. Therefore, it requires a lot of energy generated by using the fatty acid oxidation (FAO) pathway. In our model, we found FAO expression is higher than that of the other metabolic pathways. We found expression of an intricate protein network in mitochondria, which we interpret as a sign of mitochondrial homeostasis being vital for the FAO pathway to work. Furthermore, we also identified different types of transporters at each stage of proximal tubule formation, and we could recognize different cytoskeletal components playing a significant role in each stage of proximal tubule formation, for instance, at the monolayer stage, vimentin expression is high, and its expression is reduced as tubules form. Hence, this 2D system, at this step of characterization, seems suitable to use to study differential transport protein expression and how this might relate to physiological functions and syndromes. Next, we inhibited different transporters using specific inhibitors and analyzed the effect on dome and tubule formation. We identified that Na+/K+ ATPase and vacuolar H+ ATPase play a significant role in the process of epithelial dynamics. Digoxin (a Na+/K+ ATPase inhibitor) treatment inhibits dome and tubule formation. Bafilomycin (a v-ATPase inhibitor) treatment demonstrated a delay in dome and tube formation. Therefore, this study shows that this 2D proximal tubule novel system can be used for screening of pharmacological leads in the context of specific aspects of kidney physiology. Despite the recent success in growing kidney organoids, they are not well suited to investigate various pathophysiological conditions in vitro for several reasons: They grow in 3D and form a tissue that later needs to be dissected/cleared and stained to investigate pathophysiological changes. Moreover, organoids require complex and expensive protocols for generation and are challenging to use in screening approaches. Therefore, we set out to demonstrate feasibility for our 2D system using normal renal epithelial cells, which are the origin of various pathological conditions, to study pathophysiological conditions.Epithelzellen übernehmen essentielle physiologische Funktionen, die die Absorption, Morphogenese, Sekretion und den Transport einschließen. Um diese Funktionen auszuführen, bilden Epithelzellen oft eine dreidimensionale Form, welche eine gekrümmte Zellschicht umfasst, die ein mit Flüssigkeit gefülltes Lumen umgibt. Diese dreidimensionalen Gewebeteile, Halbzysten (englisch dome) genannt, sind für Organfunktionen unerlässlich. Wenn sie nicht richtig arbeiten, kann dies zu Entwicklungsstörungen, Entzündungen und Krebs führen. Kürzlich wurde gezeigt, dass dome-bildende Zellen auf mikrostrukturierten Platten eine aktive Superelastizität aufweisen. Wir zeigen hier, dass die immortalisierte Epithelzelllinie des proximalen Nierentubulus, LLC-PK1, stereotypisch innerhalb von 10 Tagen Tubuli, (Geweberöhrchen) ausbildet. Die Röhrenbildung oder Tubulogenese erfolgt in vier Stufen: (1) Wenn die Zellen auf eine Kulturschale plattiert werden, bilden sie am ersten Tag eine eine Zelle dicke Schicht (monolayer); (2) am dritten Tag bilden sich dreidimensionale Strukturen, Halbzysten (domes); und (3) nach dem vierten bis fünften Tag beginnen diese Halbzysten zu verschmelzen, was ein Übergangsstadium darstellt, dass in (4) die Tubulogense übergeht. Am Ende des zehnten Tages bilden sich differenzierte, verlängerte und ausgereifte Röhren aus. Daher ist die Tubulogenese ein selbstorganisiertes, stereotypisch morphogenetisches Programm unter langfristigen, ungestörten Gewebekulturbedingungen. Die Tubulogenese könne wir in diesem Zusammenhang als einen zweistufigen Prozess beschrieben, der aus Halbzystenbildung (doming) und Einfaltung (wrapping) besteht. Der Prozess beginnt mit einer Halbzystenbildung und sobald die Zellschichten in der Übergangsphase am Rande der Halbzyste zusammenkommen, führt dies zur Bildung des Lumens des späteren Tubulus. Die Zellen erstrecken sich über die gesamte Länge des Epithels, das eine apikale Verengung erfährt. Dies führt zu einer Einstülpung, welche sich vertieft bevor sich aus den Zellen eine neue Röhre formt. Durch eine Proteomanalyse der vier Stadien der proximalen Tubulusbildung , konnten die verschiedenen Proteine identifiziert werden, die bei der Entwicklung der proximalen Tubuli eine wichtige Rolle spielen. Dabei konnten wir proximale Tubulusmarker wie synaptopondin, angiotensin 1-10, collectrin, polycystin 1 und polycystin 2 in einer Stufenabhängigen Muster nachweisen. Diese Proteine spielen eine wichtige Rolle bei der Bildung und Differenzierung der Nierenkanäle, was die physiologische Relevanz des hier dargestellten zellulären Systems unterstreicht. Bei der hier verwendeten Zelllinie handelt es sich um eine nicht viral oder durch Onkogene immortalisierte Linie. Daher wurde untersucht, wie sich die Regulation des Zellzyklus während der vier Stufen der Morphogenese ändern. Wir konnten zeigen, dass Cyclin-B1, der Proliferationsmarker Ki-67, die Rekombinase RAD51 und das proliferating cell nuclear antigen (PCNA) im Einzellschicht-Stadium hochreguliert sind und deren Expression mit der Tubulusbildung abnimmt, somit verlassen die Zellen während der Morphogenese kontinuierlich den Zellzyklus in eine gap phase. In der Niere reabsorbieren die proximalen Tubuli 60-65 % des Glomerulusfiltrats. Daher benötigen sie viel Energie, die über den Fettsäureoxidationsweg (FAO) gewonnen wird. In unserem Modell ist die FAO-Expression höher als die der anderen Stoffwechselwege. Im Vergleich zu anderen zytosolischen Organellen fanden wir in den Mitochondrien außerdem ein kompliziertes Proteinnetzwerk, da die mitochondriale Homöostase für die Funktion des FAO-Stoffwechselwegs unerlässlich ist. Neben der Expression organspezifischer Marker, dem Verlassen des Zellzyklus ist dies ein dritter Indikator für das Nachvollziehen eines entwicklungsbiologisch regulierten Vorgangs in Form eines selbstorganisierten Prozesses in vitro. Zudem konnten wir als vierten Indikator verschiedene Arten von Transportern in jedem Stadium der proximalen Tubulusbildung identifizieren, die für die Nierenphysiologie von Bedeutung sind. Das Zytoskelett spielt eine wesentliche Rolle bei der Bildung des proximalen Tubulus. Wir haben festgestellt, dass verschiedene Zytoskelette in jeder Phase der Bildung des proximalen Tubulus eine wichtige Rolle spielen. Zum Beispiel ist die Expression von Vimentin im Einzellschicht-Stadium hoch und nimmt mit der Bildung des Tubulus ab. In unseren Augen kann dieses 2D-System daher verwendet werden, um die Rolle der einzelnen Transporter und die mit der Transporteraktivität verbundenen Krankheiten zu untersuchen

Resistance a major hindrance to chemotherapy in hepatocellular carcinoma: an insight

Abstract Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, accounting for almost 90% of total liver cancer burden. Surgical resection followed by adjuvant and systemic chemotherapy are the most meticulously followed treatment procedures but the complex etiology and high metastatic potential of the disease renders surgical treatment futile in majority of the cases. Another hindrance to the scenario is the acquired resistance to drugs resulting in relapse of the disease. Hence, to provide insights into development of novel therapeutic targets and diagnostic biomarkers, this review focuses on the various molecular mechanisms underlying chemoresistance in HCC. We have provided a comprehensive summary of the various strategies adopted by HCC cells, extending from apoptosis evasion, autophagy activation, drug expulsion to epigenetic transformation as modes of therapy resistance. The role of stem cells in imparting chemoresistance is also discussed. Furthermore, the review also focuses on how this knowledge might be exploited for the development of an effective, prospective therapy against HCC

INSILICO INVESTIGATION OF MISSENSE MUTATIONS IN SDH5 GENE USING DIFFERENT GENOMIC ALGORITHMS

Objective: Nonsynonymous single nucleotide polymorphism (nsSNP) has a deleterious effect on a protein, thereby leading to a disease. Succinate dehydrogenase complex 5 (SDH5) gene, which encodes for a mitochondrial protein is responsible for the flavination of succinate dehydrogenase complex and also plays a major role in Kreb's cycle. Mutations in this gene lead to the cancerous diseases such as paraganglioma and pheochromocytoma. The aim of this paper is to excavate the deleterious mutations in SDH5.Method: The deleterious mutations in SDH5is evaluated by assorted genomic algorithmsand to find the drug binding affinity by docking the current drug against the mutated protein using Molecular docking Server. A total of 20 mutation were retrieved from SNP NCBI. The structural and the functional aspectsof these 20 mutations were analysed by using various genomics algorithmssuch as SIFT, PolyPhen2.0, I-Mutant 2.0, SNPs & GO, PANTHER and PhD-SNP, which helped us narrowing down our search to G78R and L80S as the deleterious missense mutations. The drug cyclophosphamide, used for the treatment of these cancerous diseases was considered for our study. Drug-protein interactions were studied using protein docking server. Binding efficiency of the cyclophosphamide drug with the most deleterious mutations was calculated.Result: G78Rwas found to be deleterious and confirmed that the mutation decrease the stability of the protein.Conclusion: Our findings lead to the better understanding of the deleterious mutations in SDH5, providing immense knowledge on the cancerous diseases, such as paraganglioma and pheochromocytoma, and drug docking mechanisms which will be extremely useful in the discovery of new treatmentsagainst such diseases.Keywords: Cyclophosphamide, Molecular docking, Paraganglioma, Pheochromocytoma, SDH5 gen

The dynamic role of autophagy and MAPK signaling in determining cell fate under cisplatin stress in osteosarcoma cells

<div><p>Osteosarcoma (OS) is an aggressive bone malignancy commonly observed in children and adolescents. Sub-optimal therapy for years has irretrievably compromised the chances of OS patient survival; also, lack of extensive research on this rare disease has hindered therapeutic development. Cisplatin, a common anti-tumor drug, is currently an integral part of treatment regime for OS along with methotrexate and doxorubicin. However, toxicity issues associated with combination module impede OS therapy. Also, despite the proven benefits of cisplatin, acquisition of resistance remains a concern with cisplatin-based therapy. This prompted us to investigate the molecular effects of cisplatin exposure and changes associated with acquired resistance in OS cells. Cisplatin shock was found to activate MAPK signaling and autophagy in OS cells. An activation of JNK and autophagy acted as pro-survival strategy, while ERK1/2 triggered apoptotic signals upon cisplatin stress. A crosstalk between JNK and autophagy was observed. Maximal sensitivity to cisplatin was obtained with simultaneous inhibition of both autophagy and JNK pathway. Cisplatin resistant cells were further developed by repetitive drug exposure followed by clonal selection. The resistant cells showed an altered signaling circuitry upon cisplatin exposure. Our results provide valuable cues to possible molecular alterations that can be considered for development of improved therapeutic strategy against osteosarcoma.</p></div

Development and characterization of HOS-resistant model.

<p>(A) Cells were seeded at a density of 4000 per well in triplicates. MTT assay was performed following CDDP exposure for 24h. Percentage viability of OS-R cells compared to parental cells is represented in the figure. (B) Phase contrast images of—i. HOS parental untreated cells; ii. HOS-CDDP-treated cells, images were captured after 30min of 1mg/ml CDDP shock; iii. HOS cells reviving post shock when maintained in drug free media for around 3–4 weeks; iv. Resistant cells (OS-R) maintained in IC₅₀ dose of CDDP. The scale bar represents 30μm. (C) cDNA was synthesized from DNase-treated RNA isolated from HOS and OS-R cells. RT-PCR was performed with ABCB1 specific primers. Fold change in expression level of ABCB1 gene is represented in the figure with untreated control taken as “1”. (D) Single cell colony formation assay was performed in HOS and OS-R cells. Cells were counted and serially diluted until distinct single cells were observed. The cells were then monitored for their colony forming capacity. Fold increase in number of colonies formed after 24 and 48h in OS-R cells compared to HOS is represented. (E) Levels of cleaved PARP-1 was monitored by immunoblot after IC₅₀ dose of CDDP exposure for 24h in HOS cells and in OS-R cells maintained in IC₅₀ dose of CDDP. (F) The HOS cells were maintained in conditioned media (CM) for 48h and survivability upon exposure to various concentrations of CDDP was measured by MTT assay. Here, control (Ctrl) represents regular media in which HOS cells were cultured. The symbol (*) signifies a significant difference in CM with respect to control.</p

Role of p53 status in modulating autophagic response.

<p>(A) CMV-driven wild type p53 expression construct was transfected to HOS cells. Around 24h and 48h post transfection cells were exposed to specific treatments—SP (25μM) for 2h followed by CDDP (IC₅₀) for 24h. Immunoblot analysis was performed to analyze LC3B-II levels. β-Actin served as a loading control and un-transfected cells served as an experimental control. (B) Transfected and un-transfected cells were analyzed for expression of p53 protein. (C) As representative of cells harboring p53 wild type protein, HepG2 cells were treated with SP (25μM) for 2h followed by CDDP (IC₅₀) for 24h and harvested for analysis of LC3B by immunoblot analysis.</p

Effect of autophagy inhibition in HOS and OS-R cells.

<p>(A) Phase contrast images showing morphology of HOS untreated (i), CDDP (IC₅₀) treated (ii), only CQDP (10μM)-treated (iii) and cells treated for 24h with both CQDP and CDDP (iv). The scale bar represents 30μm. (B) Flow cytometric analysis of apoptosis with AnnexinV/PI upon autophagy inhibition. HOS cells were either untreated (i) or treated with CDDP (IC₅₀) (ii), only CQDP (10μM) (iii) or both CQDP and CDDP (iv) for 24h. CQDP was added 24h prior to CDDP treatment. Cells present in the lower right (LR) and upper right (UR) quadrant represent early and late apoptotic cells respectively. A fold increase in total number of apoptotic cells upon different treatment is represented through bar diagram. (C) Immunoblot showing cleaved PARP-1 expression levels in HOS and OS-R cells after 24h of CDDP exposure in presence or absence of CQDP (10μM). Densitometric scanning was performed with ImageJ software. β-Actin served as a loading control. (D) Caspase-3 assay was performed to determine apoptosis induction in cells after various treatments. A prior treatment of CQDP (10μM, 24h) was given to the cells before CDDP (IC₅₀) exposure. To inhibit caspase activity Z-VAD-FMK was used at 50μM concentration and was added 1h prior to administration of various treatments. Activity in control HOS and OS-R cells was taken as "1". (E) Autophagic flux representative of difference in protein level of LC3B-II between CQDP treated HOS cells and CQDP plus CDDP (IC₅₀) treated cells was checked by immunoblot. The same is represented with bar diagram. A prior treatment of CQDP (10μM, 24h) was given to cells before CDDP exposure.</p

Effect of autophagy inhibition on MAPK signaling.

<p>(A) Levels of phosphorylated-JNK were analyzed by immunoblot after CDDP exposure (IC₅₀) for 1h (A), 6h (A) and 24h (B) in HOS cells. A prior treatment of CQDP (10μM, 24h) was given wherever mentioned before CDDP exposure. Densitometric scanning was performed with ImageJ. β-Actin served as a loading control. (C and D) Levels of phosphorylated-ERK1/2 were analyzed by immunoblot after CDDP exposure (IC₅₀) for 1h (C), 6h (C) and 24h (D) in HOS cells. A prior treatment of CQDP (10μM, 24h) was given wherever mentioned before CDDP exposure. Densitometric scanning was performed with ImageJ software. β-Actin served as a loading control. (E) Cell viability was measured 24h after treatment by trypan blue assay. Cells were either untreated or treated with the following:- SP (25μM); CQDP (10μM); SP (25μM) plus CQDP (10μM); CDDP (IC₅₀); CQDP (10μM) plus CDDP (IC₅₀); SP (25μM) plus CDDP (IC₅₀); or SP (25μM) plus CQDP (10μM) plus CDDP (IC₅₀). (F) A fold change in caspase-3 activity was analyzed colorimetrically 24h after treatment in HOS cells. Cells were either untreated or treated with the following:- SP (25μM); CQDP (10μM); SP (25μM) plus CQDP (10μM); CDDP (IC₅₀); SP (25μM) plus CDDP (IC₅₀); or SP (25μM) plus CQDP (10μM) plus CDDP (IC₅₀). Activity in control HOS cells was considered as "1". (G) Representative phase contrast images of HOS cells treated with CDDP (IC₅₀) for 24h or along with combinations of inhibitors, SP600125 (25μM) or CQDP (10μM) or both (i-v). The scale bar represents 30μm.</p