Evaluation of tumour-associated antigens to optically label cutaneous basal cell carcinoma for surgical excision

Basal cell carcinoma (BCC) is the most common skin cancer worldwide, with South Africa having the highest incidence rate only after Australia. The most effective treatment modality for BCC is tumor excision via Mohs surgery (pioneered by Dr. Frederic Mohs of the University of Wisconsin in 1930), a microscopically controlled surgery that removes a tumor piecemeal in layers until each layer is free of any neoplastic tissue. The major drawback of Mohs excision is that the surgeon might miss any neoplastic tissue as the tumor margin is not always well defined, and the tumor often could extend beyond the superficial layers of skin. Moreover, it's a time-consuming, expensive procedure that takes generally 3-4 h, at times even more, if several rounds of excisions are warranted. In South Africa, at the time of writing, therapy using the surgery cost around R45,000. The status quo thus necessitates identifying BCC cells both in the superficial layers and beyond the layers of the skin in individual patients. Our aim was to identify BCC-specific cell surface proteins and design, engineer, and test a range of SNAPtag–based antibody fusion proteins that would specifically bind to and detect such BCC cell surface receptors. The SNAP-tag antibody technology is based on the genetic fusion of a disease-specific ligand to a protein tag derived from O6-alkylguanine-DNA alkyltransferase, which would allow for covalent auto-labeling of the corresponding antibody based fusion proteins with benzylguanine-modified (BG) substrates (e.g., fluorophores) under physiological conditions with high efficiency at 1:1 stoichiometry. This would allow to develop a unique immunological screening modality which should allow to visually label BCC lesions for a more precise surgical excision. The best-performing SNAP-tag–based diagnostic antibodies resulting from these studies would be further evaluated in the future in suitable mouse models, thus aiming to reduce the time needed for surgical removal of BCC lesions and complete removal of the tumor from both superficial and deep layers of the skin by a single-excision procedure. We used an integrated computational tool to re-analyze publicly available cDNA microarray data in combination with theoretical search to identify BCC-associated antigens. Accordingly, six different antigens were selected and single-chain variable fragments (scFv) targeting these antigens were cloned in fusion with SNAP-tag encoding gene into a custom expression vector for production in a secretory mammalian system. scFv-SNAP-tag protein was isolated from the cell culture supernatant by immobilized metal affinity chromatography and eluted protein samples were analyzed by gel electrophoresis and immunoblotting. The absolute amount of the full-length protein was quantified by densitometry. Purified scFv-SNAP-tag proteins were validated for specific binding to corresponding antigen-positive cells by flow cytometry and confocal microscopy. Of the six different scFv-SNAP-tag fusion proteins cloned, four were successfully expressed in HEK293T cells. The specific binding to EpCAM, EMA, CSPG4, and CD138 antigenexpressing cell lines was observed on incubation with scFvUBS54-SNAP-tag, scFvID405- SNAP-tag, 9.2.27scFv-SNAP-tag, and scFvh-STL002-SNAP-tag, respectively. In addition, we showed the selective cell killing effect of scFvUBS54-SNAP after conjugating it with the cytotoxic drug BG-modified auristatin-F (BG-AF). In conclusion, we identified various cell surface antigens along with one possibly novel antigen for BCC detection and therapy. Further, we successfully designed and synthesized SNAP tag based antibody fusion proteins and showed their functional activity by selective binding to the corresponding antigens on the surface of tumor cells. Based on these findings, we presume that these antibodies can effectively bind to BCC and can confirm EpCAM as one of the target antigens, which has already been reported to be a standard immunophenotypic marker for differential BCC diagnosis

A Hybrid Optimized Weighted Minimum Spanning Tree for the Shortest Intrapath Selection in Wireless Sensor Network

Wireless sensor network (WSN) consists of sensor nodes that need energy efficient routing techniques as they have limited battery power, computing, and storage resources. WSN routing protocols should enable reliable multihop communication with energy constraints. Clustering is an effective way to reduce overheads and when this is aided by effective resource allocation, it results in reduced energy consumption. In this work, a novel hybrid evolutionary algorithm called Bee Algorithm-Simulated Annealing Weighted Minimal Spanning Tree (BASA-WMST) routing is proposed in which randomly deployed sensor nodes are split into the best possible number of independent clusters with cluster head and optimal route. The former gathers data from sensors belonging to the cluster, forwarding them to the sink. The shortest intrapath selection for the cluster is selected using Weighted Minimum Spanning Tree (WMST). The proposed algorithm computes the distance-based Minimum Spanning Tree (MST) of the weighted graph for the multihop network. The weights are dynamically changed based on the energy level of each sensor during route selection and optimized using the proposed bee algorithm simulated annealing algorithm

Field Investigation on Ground and Structural Vibrations During Prototype Pile Driving

As the land is very precious in urban areas and coastal regions, many structures are built in close proximity. When the soil is weak and heavy loads are to be carried, pile foundation is adopted for such structures. The impact caused by pile driving is a potential hazard to neighbourhood structures. Berthing structures are constructed using piles and the effect of pile driving on already installed adjacent piles is important. To predict the effect of pile driving on neighbourhood piles, measurement of ground and structural vibrations during prototype pile driving was carried out at a site in the city of Chennai, India. The soil at the site up to a depth of 19 m is mainly fine to medium sand. A driven cast in-situ pile of 600 mm diameter (D) was driven up to a depth of 15.8 m. The 25mm thick mild steel (M.S) tube casing is driven by 4.1t hammer with a drop height of 2.5m. During the pile driving, for every blow of hammer, the acceleration of the ground at various distances 5D, 8.33 D and 25 D from the pile are measured. Piezoelectric acceleration transducers, power amplifiers and taper recorder are used for measurement. The time history of vertical and horizontal ground accelerations as well as time history of vertical acceleration of an already installed pile at a distance of 6.25 m was analysed using PC based data acquisition systems. The time-histories and spectrum of ground and structural accelerations are presented

MASKING ANTI-PHAGOCYTIC SIGNAL OF TUMOR BY PRO-PHAGOCYTIC SIGNAL-A KEY TO IMMUREMENT OF CANCER CELL

Immune surveillance is a mechanism where cells and tissues are watched constantly by ever alerted immune system. Most incipient cancer cells are recognized and eliminated by the immune surveillance mechanism, but still tumors have the ability to evade immune surveillance and immunological killing. One greater arm that tumor use to evade immune surveillance, is by expressing anti-phagocytic signal (CD47). Here we present a provocative hypothesis where cancer cells are removed alive by phagocytic cell (DC). That in turn will elicit effective and higher immunogenic condition. All this could be possible by addition pro-phagocytic signal (PtdSer) over cancer cell surface (Breast Cancer), that mask the presence of anti-phagocytic signal (CD47). In other words, adding eat me signal (PtdSer) over the breast cancer cell surface that mask the presence of don't eat me signal or anti-phagocytic signal present in breast cancer cell surface. This could be possible by using bi-specific antibody, conjugated to PEG-modified liposomes, which carry (PtdSer) pro-phagocytic signal (or) eat me signal, which target both CD47 and EGFRVIII on breast carcinoma. The simultaneous masking of anti-phagocytic signal, and adding of pro–phagocytic signal over cancer cell, will enhance the phagocytic clearance of live tumor cell and elicit immunological killing

Prototype Piling in Soft Clay — A Case Study of Ground Vibrations: Field Measurement

Ground vibrations are generated either by natural phenomena or by human activities. Among the natural phenomena, earthquake is the principal source of ground vibrations of most interest. Ground vibrations generated by human activities are called manmade vibrations and these vary greatly in intensity depending on the particular source of vibration. The seismic waves associated with man-made vibrations propagate in the ground and inevitably interact with structures that are above-ground or underground. This interaction induces vibrations in the structure and, in extreme cases, affect its serviceability and integrity seriously. Still, there is no method to quantify the levels of piling vibration. Estimation of amplitudes and frequencies of vibration are based on experience and site testing. Therefore, in order to characterize the ground vibration due to piling, field measurement of ground vibration during prototype pile driving in soft clay was carried out. This paper presents the details of field measurement, instrumentation, collection of vibration data, acquisition and processing of data using PC-based data acquisition system made during the pile driving. The measurement of ground and already installed pile vibrations during prototype pile driving at a construction site in Chennai was carried out. The soil at this site is loose soft clay. The pile was of the type driven casing cast-in-situ pile of 500mm (OD) diameter. The depth of penetration of the pile was 13.25m. A 4 tonne hammer with a drop height of 1m drove the 25mm thick Mild Steel (MS) tube casing. During driving, ground acceleration was measured at a distance of 5D and 25D, where D is the diameter of pile, from the centre of pile. The vertical acceleration of an already installed pile situated at a distance of 37D from the driven pile were recorded. Piezoelectric acceleration transducers, power amplifiers, and tape recorder were used for the measurement. Acceleration signals were recorded using TEAC recorder. The recorded signals were processed using PC-based Data Acquisition System with DASYLAB software. The response time-histories and spectra of ground vibrations are presented and discussed