Design and evaluation of a multiepitope vaccine for pancreatic cancer using immune-dominant epitopes derived from the signature proteome in expression datasets.

Pancreatic cancer is a highly aggressive and often lethal malignancy with limited treatment options. Its late-stage diagnosis and resistance to conventional therapies make it a significant challenge in oncology. Immunotherapy, particularly cancer vaccines, has emerged as a promising avenue for treating pancreatic cancer. Multi-epitope vaccines, designed to target multiple epitopes derived from various antigens associated with pancreatic cancer, have gained attention as potential candidates for improving therapeutic outcomes. In this study, we have explored transcriptomics and protein expression databases to identify potential upregulated proteins in pancreatic cancer cells. After examining a total of 21,054 proteins from various databases, it was discovered that 143 proteins expressed differently in malignant and healthy cells. The CTL, HTL and BCE epitopes were predicted for the shortlisted proteins. 51,840 vaccine constructs were created by concatenating CTL, HTL, and B-cell epitopes in the respective sequences. The best 86 structures were selected from a set of 51,840 designs after they were analyzed for vaxijenicity, allergenicity, toxicity, and antigenicity scores. In further simulation of the immune response using constructs, it was found that 41417, 37961, and 40841 constructs could produce a strong immune response when injected. Further, it was found that construct 37961 showed stronger interaction and stability with TLR-9 as determined from the large-scale molecular dynamics simulations. Moreover, the 37961 construct has shown interactions with TLR-9 suggests its potential in inducing immune response. In addition, construct 37961 has shown 100% predicted solubility in the E. coli expression system. Overall, the study indicates the designed construct 37961 has the potential to induce an anti-tumor immune response and long-standing protection pending further studies.

Hemin acts as CD36 ligand to activate down-stream signalling to disturb immune responses and cytokine secretion from macrophages.

Inflammatory responses to hemin are believed to play an important role in tissue damage and cerebral malaria pathology. Macrophage exposed to hemin exhibits modulation of non-opsonic phagocytosis of aged RBCs, ability to kill bacteria and secretion of cytokines. Immuno-fluorescence study indicates translocation and sequestration of CD36 within the intracellular storage in the hemin treated macrophages. It in-turn modulates the global cytokine secretion from macrophages. CD36 has strong affinity for hemin with a dissociation constant of 1.26±0.24 µM. CD36 has hemin bio-phoric environment involving R292, D372 and Q382. The mutation in biophoric residues significantly reduced the affinity towards hemin. Hemin stimulated MG63 cells (transfected with CD36) showed several folds increment in cytokines TNFα, MCP-1, RANTES and CCL1 and CD36-hemin interaction is crucial for aberrant cytokine secretion. CD-36: Hemin interaction is driving down-stream signalling and subsequent recruitment of adaptor proteins to the cytosolic domain of CD36. Immunoprecipitation of membrane bound CD36 gives Lyn kinase as potential adaptor protein down-stream to CD36: hemin signalling. Interestingly, disruption of Lyn kinase abolishes the hemin mediated dysregulation of immune responses. In summary, hemin-CD36-Lyn kinase signalling axis could be a contribution factor to severe malaria pathology and prognosis.

CD36 Ectodomain Detects Apoptosis in Mammalian Cells.

The cells that undergo apoptosis show phosphatidylserine (PS) on the cell membrane. The fluorescently labeled hCD36_ecto is staining and detecting apoptotic cells in a flow-based assay with several advantages over Annexin V. The human CD36 ectodomain (hCD36_ecto) is stable for a range of temperatures and experimental conditions and doesn't require Ca2+ for detecting apoptosis and specific towards PS compared to other lipids. The blocking with unlabeled hCD36_ecto reduces the staining of Annexin V-FITC for apoptotic cells, whereas R63A does not affect the binding of Annexin V- FITC to apoptotic cells. It indicates the role of CD36-PS interaction in detecting apoptotic cells. Dual-staining with hCD36_ecto-FITC/PI is universally detecting apoptosis in different nucleated cells or eryptosis in non-nucleated RBCs. Hence, our study highlights the utility of CD36 as a probe to detect apoptosis in mammalian cells. It might be a robust, economical reagent for the scientific community to facilitate their research.

Therapeutic Potentials of Scavenger Receptor CD36 Mediated Innate Immune Responses Against Infectious and Non-Infectious Diseases.

CD36 is a multifunctional glycoprotein, expressed in different types of cells and known to play a significant role in the pathophysiology of the host. The structural studies revealed that the scavenger receptor consists of short cytosolic domains, two transmembrane domains, and a large ectodomain. The ectodomain serves as a receptor for a diverse number of endogenous and exogenous ligands. The CD36-specific ligands are involved in regulating the immune response during infectious and non-infectious diseases in the host. The role of CD36 in regulating the innate immune response during Pneumonia, Tuberculosis, Malaria, Leishmaniasis, HIV, and Sepsis in a ligand- mediated fashion. Apart from infectious diseases, it is also considered to be involved in metabolic disorders such as Atherosclerosis, Alzheimer's, cancer, and Diabetes. The ligand binding to scavenger receptor modulates the CD36 down-stream innate immune response, and it can be exploited to design suitable immuno-modulators. Hence, the current review focused on the role of the CD36 in innate immune response and therapeutic potentials of novel heterocyclic compounds as CD36 ligands during infectious and non-infectious diseases.

Selective and Sensitive Sensing of Hydrogen Peroxide by a Boronic Acid Functionalized Metal-Organic Framework and Its Application in Live-Cell Imaging.

A new boronic acid functionalized Zr(IV) metal-organic framework having the capability of sensing H2O2 in live cells is reported. The Zr-MOF bears a UiO-66 structure and contains 2-boronobenzene-1,4-dicarboxylic acid (BDC-B(OH)2) as a framework linker. The activated Zr-UiO-66-B(OH)2 compound (called 1') is highly selective for the fluorogenic detection of H2O2 in HEPES buffer at pH 7.4, even in the presence of interfering ROS (ROS = reactive oxygen species) and other biologically relevant analytes. The fluorescent probe was found to display extraordinary sensitivity for H2O2 (detection limit 0.015 µM) in HEPES buffer, which represents a lower value in comparison to those of the MOF probes documented so far for sensing H2O2 using other analytical methods. Taking advantage of its high selectivity and sensitivity for H2O2 in HEPES buffer, the probe was successfully employed for the imaging of intracellular H2O2. Imaging studies with MDAMB-231 cells revealed the emergence of bright blue fluorescence after loading with probe 1' and subsequent treatment with H2O2 solution.

Mapping of phosphatidylserine recognition region on CD36 ectodomain.

CD36-PS interaction is an important affair to identify and remove dead/aged cells to control inflammation. CD36 ectodomain was cloned, over-expressed in bacterial expression system and purified to homogeneity. The dot-blot analysis shows that the CD36_ecto selectively binds PS vesicles blotted on the nitrocellulose membrane. PS binds strongly to CD36_ecto with a dissociation constant KD of 53.7 ±â€¯0.48 µM. The stoichiometry of interaction between CD36 and PS is 1:2. The hCD36_ecto-PS thermogram revealed that the hydrophobic and salt bridge interactions play crucial role in their interactions. PS docked nicely into the predicted pharmacophoric site with a binding energy of 5.1 kcal/mol. Analysis of CD36-PS molecular model showed that the residues R63, R96, N118, D270 and E418 were forming hydrogen bonds with PS. Molecular dynamics simulations indicate that R63 mutation has disrupted the integrity of biophoric constituents, directly affecting the hydrogen bonding from R96, N118 and D270. ITC thermogram analysis of mutant protein with PS vesicles indicate complete loss of binding with R63A and very low affinity of PS vesicles with D270A. Dot blot analysis further confirmed the ITC results. These finding may help to design suitable agents mimicking PS biophore with potentials in diagnostics of apoptotic cells and cardiovascular intervention.

A dinitro-functionalized metal-organic framework featuring visual and fluorogenic sensing of H2S in living cells, human blood plasma and environmental samples.

Here, we describe a new dinitro-functionalized Zr(iv) MOF (MOF = metal-organic framework) having a UiO-66 (UiO = University of Oslo) framework topology called UiO-66-(NO2)2 (1). It shows fluorescence turn-on behavior towards H2S in simulated biological medium (HEPES buffer, pH = 7.4). By employing solvothermal conditions, 1 was successfully synthesized by reacting ZrCl4, H2BDC-(NO2)2 [H2BDC-(NO2)2 = 2,5-dinitro-1,4-benzenedicarboxylic acid] ligand and benzoic acid with a molar ratio of 1 : 1 : 10 in DMF (DMF = N,N-dimethylformamide) at 130 °C for 24 h. The material was characterized by infrared spectroscopy, X-ray powder diffraction (XRPD) and thermogravimetric (TG) analyses. The compound not only displays highly sensitive fluorometric sensing of H2S but also exhibits a visually detectable colorimetric change towards H2S in daylight. Moreover, the high selectivity of 1' towards H2S is retained even when several other biologically intrusive species co-exist in the sensing medium. The limit of detection (LOD) of the compound is 14.14 µM which lies in the range of the H2S concentration found in biological systems. Fluorescence microscopy studies on J774A.1 cells revealed the efficacy of the probe for imaging H2S in living cells. Moreover, this material can detect H2S in human blood plasma (HBP) and monitor the sulfide concentration in real water samples. All these features clearly demonstrate that the material has huge potential for highly selective sensing of both extracellular and intracellular H2S.

Extraordinary sensitivity for H2S and Fe(iii) sensing in aqueous medium by Al-MIL-53-N3 metal-organic framework: in vitro and in vivo applications of H2S sensing.

An Al(iii) metal-organic framework (MOF) called Al-MIL-53-N3 (1) was synthesized under solvothermal reaction conditions using Al(NO3)3·9H2O and H2BDC-N3 (H2BDC-N3 = 2-azido-1,4-benzenedicarboxylic acid) ligand in a DMF/water (DMF = N,N-dimethylformamide) mixture. Phase purity was checked by performing X-ray powder diffraction, infrared spectroscopy and thermogravimetric analysis. Thermogravimetric analysis suggests that 1 is highly stable up to 300 °C under air atmosphere. The activated 1 (called 1') showed a very fast fluorescence response to H2S (turn-on) and Fe(iii) ions (turn-off) in an aqueous medium with excellent sensitivity and selectivity even in the presence of other potentially intrusive analytes. In the presence of H2S, the conversion of the azide moiety to amine is responsible for the fluorescence turn-on properties. On the other hand, the partial replacement of framework Al(iii) ions by Fe(iii) can be assigned for the selective detection behavior to Fe(iii) ions. The detection limits (90.47 nM for H2S and 0.03 µM for Fe(iii) ions in water) of 1' are lower than those of the formerly reported MOF type of fluorescent sensors. The 1'-loaded J774A.1 macrophage cells are healthy and respond to intracellular H2S to exhibit strong blue fluorescence, confirming its suitability to detect H2S inside the cells. In addition, 1' can detect H2S in human blood plasma (HBP) and sulfide ions in real water samples. These features make 1' a very promising candidate for the on-site sensing of Fe(iii) ions and the detection of intracellular and extracellular H2S.

Rapid and highly sensitive detection of extracellular and intracellular H2S by an azide-functionalized Al(iii)-based metal-organic framework.

A new, azide-functionalized Al(iii)-based metal-organic framework (MOF) denoted as CAU-10-N3 (1, CAU = Christian-Albrechts-University) and consisting of the 5-azido-isophthalic acid (H2IPA-N3) ligand was employed as a reaction-based fluorescent turn-on probe for the detection of H2S. The activated compound (1') showed fast, selective and highly sensitive sensing properties for extracellular H2S in HEPES buffer (10 mM, pH = 7.4). The material retained its high selectivity even in the presence of possibly competing biological species. The limit of detection of 1' for H2S is 2.65 µM, which is lower than the earlier reports on MOFs for H2S sensing. The material displayed a short response time (420 s) and a significant increase (20-fold and 26-fold after 1 and 7 min of addition of Na2S, respectively) in the fluorescence intensity towards H2S. Macrophage cells loaded with probe 1' exhibited blue fluorescence with a response time of 15 min after Na2S addition, indicating the suitability of the probe for intracellular H2S detection. Moreover, CAU-10-N3 featured excellent detection performance (quick response and 32-fold increment in fluorescence intensity after 7 min of Na2S addition) in water. Hence, it can be utilized to regulate the H2S level in aqueous samples collected from the environment.

Chemoenzymatic Route for the Synthesis of (S)-Moprolol, a Potential ß-Blocker.

A biocatalytic route for the synthesis of a potential ß-blocker, (S)-moprolol is reported here. Enantiopure synthesis of moprolol is mainly dependent on the chiral intermediate, 3-(2-methoxyphenoxy)-propane-1,2-diol. Various commercial lipases were screened for the enantioselective resolution of (RS)-3-(2-methoxyphenoxy)propane-1,2-diol to produce the desired enantiomer. Among them, Aspergillus niger lipase (ANL) was selected on the basis of both stereo- and regioselectivity. The optimized values of various reaction parameters were determined such as enzyme (15 mg/mL), substrate concentration (10 mM), organic solvent (toluene), reaction temperature (30 °C), and time (18 h).The optimized conditions led to achieving >49% yield with high enantiomeric excess of (S)-3-(2-methoxyphenoxy)propane-1,2-diol. The lipase-mediated catalysis showed regioselective acylation with dual stereoselectivity. Further, the enantiopure intermediate was used for the synthesis of (S)-moprolol, which afforded the desired ß-blocker.