Screening of breast cancer from thermogram images by edge detection aided deep transfer learning model.

Breast cancer, the most common invasive cancer, causes deaths of thousands of women in the world every year. Early detection of the same is a remedy to lessen the death rate. Hence, screening of breast cancer in its early stage is utmost required. However, in the developing nations not many can afford the screening and detection procedures owing to its cost. Hence, an effective and less expensive way of detecting breast cancer is performed using thermography which, unlike other methods, can be used on women of various ages. To this end, we propose a computer aided breast cancer detection system that accepts thermal breast images to detect the same. Here, we use the pre-trained DenseNet121 model as a feature extractor to build a classifier for the said purpose. Before extracting features, we work on the original thermal breast images to get outputs using two edge detectors - Prewitt and Roberts. These two edge-maps along with the original image make the input to the DenseNet121 model as a 3-channel image. The thermal breast image dataset namely, Database for Mastology Research (DMR-IR) is used to evaluate performance of our model. We achieve the highest classification accuracy of 98.80% on the said database, which outperforms many state-of-the-art methods, thereby confirming the superiority of the proposed model. Source code of this work is available here: https://github.com/subro608/thermogram_breast_cancer.

Vaccination with Tumor-Ganglioside Glycomimetics Activates a Selective Immunity that Affords Cancer Therapy.

Immune targeting of (glyco)protein tumor markers has been useful to develop cancer and virus vaccines. However, the ganglioside family of tumor-associated glycolipids remains intractable to vaccine approaches. Here we show that synthetic antigens mimicking the carbohydrate moiety of GD2 or GD3 gangliosides can be used as vaccines to activate a selective humoral and cellular immunity that is therapeutic against several cancers expressing GD2 or GD3. Adoptive transfer of T cells generated after vaccination elicits tumor-infiltrating lymphocytes of the γδ T cell receptor and CD8+ phenotypes; and affords a high therapeutic index. The glycomimetic vaccine principles can be expanded to target the family of tumor gangliosides and other carbohydrates expressed primarily in pathological states.

Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions.

Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated with Leishmania major and trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated with L. major alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected with L. major alone, coinoculated with carrier beads and L. major, or coinoculated with trimannose-coated beads and L. major Trimannose treatment of L. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR-/-) mice lack the ability to detect trimannose. When MR-/- mice were infected with L. major and treated with trimannose beads, they did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.

Functionalization promotes pathogen-mimicking characteristics of polyanhydride nanoparticle adjuvants.

Rational design of adjuvants and delivery systems will promote development of next-generation vaccines to control emerging and re-emerging diseases. To accomplish this, understanding the immune-enhancing properties of new adjuvants relative to those induced by natural infections can help with the development of pathogen-mimicking materials that will effectively initiate innate immune signaling cascades. In this work, the surfaces of polyanhydride nanoparticles composed of sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy) hexane were decorated with an ethylene diamine spacer partially modified with either a glycolic acid linker or an α-1,2-linked di-mannopyranoside (di-mannose) to confer "pathogen-like" properties and enhance adjuvanticity. Co-incubation of linker-modified nanoparticles with dendritic cells (DCs) elicited significant increases in surface expression of MHC I, MHC II, CD86, and CD40, and enhanced secretion of IL-6, IL-12p40, and TNF-α. An 800% increase in uptake of ethylene-diamine-spaced, linker and di-mannose functionalized polyanhydride nanoparticles was also observed. Together, our data showed that linker-functionalized polyanhydride nanoparticles demonstrate similar patterns of uptake, intracellular trafficking, particle persistence, and innate activation as did DCs exposed to Yersinia pestis or Escherichia coli. These results set the stage for rational selection of adjuvant chemistries to induce pathogen-mimicking immune responses. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2762-2771, 2017.

Co-localization of catalysts within a protein cage leads to efficient photochemical NADH and/or hydrogen production.

Using the interior of the P22 virus-like particle (VLP) we have co-localized and constrained multiple copies of a photosensitizer (Eosin-Y) and a NADH/hydrogen catalyst (cobaloxime). These small molecules were conjugated to an amine bearing polymer framework synthesized within the confines of the P22 capsid by atom transfer radical polymerization (ATRP). Using aminoethyl methacrylate (AEMA) and bis-acrylamide as the monomers we introduced a crosslinked polymer framework with addressable amines and conjugated each of the small molecules through an isothiocyanate moiety. With precise control over the average labeling stoichiometry, we conjugated the Eosin-Y and cobaloxime catalysts to the polymer such that they were co-localized on the interior of the P22 VLP. This co-localization facilitated the photochemical production of NADH from NAD+ under aqueous conditions with a maximum turnover of 11.40 × 10-3 s-1. The reaction products could be switched from NADH to H2 production by increasing the relative stoichiometry of the cobaloxime labeling. The co-confinement of this coupled catalytic system within the VLP P22 creates a nano-material whose turnover activity is independent of the bulk concentration. These constructs are an example of a biomimetic materials design and synthesis approach in which efficient photochemical production of both NADH and hydrogen can be controlled by co-localizing catalysts within a virus-like particle.

Acid-Triggered Degradable Reagents for Differentiation of Adaptive and Innate Immune Responses to Leishmania-Associated Sugars.

Lipopolysaccharides (LPS) of Leishmania spp are known to alter innate immune responses. However, the ability of these sugars to specifically alter adaptive T-cell responses is unclear. To study cap sugar-T-cell interactions, pathogen mimics (namely glycodendrimer-coated latex beads with acid-labile linkers) were synthesized. Upon lysosomal acidification, linker breakdown releases glycodendrimers for possible loading on antigen presenting molecules to induce T-cell growth. T-cell proliferation was indeed higher after macrophage exposure to mannobioside or -trioside-containing glycodendrimers than to non-functionalized beads. Yet, blocking phagolysosomal acidification only reduced T-cell proliferation with macrophages exposed to beads with an acid-labile-linker and not to covalently-linked beads. These sugar-modified reagents show that oligosaccharides alone can drive T-cell proliferation by acidification-requiring presentation, most significantly in NKT receptor (CD160)-restricted T cells.

Safety and biocompatibility of carbohydrate-functionalized polyanhydride nanoparticles.

Carbohydrate functionalization of nanoparticles allows for targeting of C-type lectin receptors. This family of pattern recognition receptors expressed on innate immune cells, such as macrophages and dendritic cells, can be used to modulate immune responses. In this work, the in vivo safety profile of carbohydrate-functionalized polyanhydride nanoparticles was analyzed following parenteral and intranasal administration in mice. Polyanhydride nanoparticles based on 1,6-bis-(p-carboxyphenoxy)hexane and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane were used. Nanoparticle functionalization with di-mannose (specifically carboxymethyl-α-D-mannopyranosyl-(1,2)-D-mannopyranoside), galactose (specifically carboxymethyl-ß-galactoside), or glycolic acid induced no adverse effects after administration based on histopathological evaluation of liver, kidneys, and lungs. Regardless of the polymer formulation, there was no evidence of hepatic or renal damage or dysfunction observed in serum or urine samples. The histological profile of cellular infiltration and the cellular distribution and kinetics in the lungs of mice administered with nanoparticle treatments followed similar behavior as that observed in the lungs of animals administered with saline. Cytokine and chemokine profiles in bronchoalveolar lavage fluid indicated surface chemistry dependence on modest secretion of IL-6, IP-10, and MCP-1; however, there was no evidence of any deleterious histopathological changes. Based on these analyses, carbohydrate-functionalized nanoparticles are safe for in vivo applications. These results provide foundational information towards the evaluation of the capabilities of these surface-modified nanoparticles as vaccine delivery formulations.

Synthesis of fluorous photolabile aldehyde and carbamate and alkyl carbamate protecting groups for carbohydrate-associated amines.

Two new fluorous photolabile-protecting groups (FNBC and FNB) and a new base-labile protecting group (FOC) for the masking of amines are reported. The protecting groups survive a wide range of common reaction conditions used in oligosaccharide synthesis and render the attached molecules amenable to fluorous solid-phase extraction (FSPE). A glycosyl acceptor containing the FNB group is shown to be useful in the synthesis of carbohydrates tagged with free deactivated secondary amines.

Functionalization of polyanhydride microparticles with di-mannose influences uptake by and intracellular fate within dendritic cells.

Innovative vaccine delivery platforms can facilitate the development of effective single-dose treatment regimens to control emerging and re-emerging infectious diseases. Polyanhydride microparticles are promising vaccine delivery vehicles due to their ability to stably maintain antigens, provide tailored release kinetics and function as adjuvants. A major obstacle for the use of microparticle-based vaccines, however, is their limited uptake by dendritic cells (DCs). In this study, we functionalized the microparticle surface with di-mannose in order to target C-type lectin receptors (CLRs) on DCs. Polyanhydride particles based on sebacic acid (SA), 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) were evaluated. Co-incubation of di-mannose-functionalized microparticles up-regulated the expression of CLRs on DCs. More importantly, di-mannose functionalization increased the uptake, as measured by the percentage of cells internalizing particles. The uptake of CPH:SA microparticles increased ∼20-fold, from 0.82% (non-functionalized) to 20.2%, and internalization of CPTEG:CPH microparticles increased ∼7-fold from 1.35% (non-functionalized) to 9.3% upon di-mannose functionalization. Both di-mannose-functionalized and non-functionalized particles trafficked to lysosomes. Together, these studies demonstrate that employing rational vaccine design principles, such as the targeting of CLRs on antigen-presenting cells, can enhance delivery of encapsulated antigens and potentially induce a more robust adaptive immune response.

High-throughput synthesis of carbohydrates and functionalization of polyanhydride nanoparticles.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity. Several biodegradable polymers have been studied as vaccine delivery vehicles(1); in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses. The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices. The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses. C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods. Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

Tailoring the immune response by targeting C-type lectin receptors on alveolar macrophages using "pathogen-like" amphiphilic polyanhydride nanoparticles.

C-type lectin receptors (CLRs) offer unique advantages for tailoring immune responses. Engagement of CLRs regulates antigen presenting cell (APC) activation and promotes delivery of antigens to specific intracellular compartments inside APCs for efficient processing and presentation. In these studies, we have designed an approach for targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide pathogen-like properties. Two conserved carbohydrate structures often found on the surface of respiratory pathogens, galactose and di-mannose, were used to functionalize the surface of polyanhydride nanoparticles and target CLRs on alveolar macrophages (AMϕ), a principle respiratory tract APC. Co-culture of functionalized nanoparticles with AMϕ significantly increased cell surface expression of MHC I and II, CD86, CD40 and the CLR CIRE over non-functionalized nanoparticles. Di-mannose and galactose functionalization also enhanced the expression of the macrophage mannose receptor (MMR) and the macrophage galactose lectin, respectively. This enhanced AMϕ activation phenotype was found to be dependent upon nanoparticle internalization. Functionalization also promoted increased AMϕ production of the pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α. Additional studies demonstrated the requirement of the MMR for the enhanced cellular uptake and activation provided by the di-mannose functionalized nanoparticles. Together, these data indicate that targeted engagement of MMR and other CLRs is a viable strategy for enhancing the intrinsic adjuvant properties of nanovaccine adjuvants and promoting robust pulmonary immunity.

New structures, chemical functions, and inhibitors for glycosyltransferases.

Glycosyltransferases are key enzymes in a range of biological processes. The last two years have seen a new appreciation for the range of structural folds that can be adopted by this class of enzymes in addition to continued work in solving the structures of more varieties of these proteins, delineating their chemical function, and designing inhibitors of their catalytic function.