Plant lectins and their usage in preparing targeted nanovaccines for cancer immunotherapy.

Plant lectins, a natural source of glycans with a therapeutic potential may lead to the discovery of new targeted therapies. Glycans extracted from plant lectins are known to act as ligands for C-type lectin receptors (CLRs) that are primarily present on immune cells. Plant-derived glycosylated lectins offer diversity in their N-linked oligosaccharide structures that can serve as a unique source of homogenous and heterogenous glycans. Among the plant lectins-derived glycan motifs, Man9GlcNAc2Asn exhibits high-affinity interactions with CLRs that may resemble glycan motifs of pathogens. Thus, such glycan domains when presented along with antigens complexed with a nanocarrier of choice may bewilder the immune cells and direct antigen cross-presentation - a cytotoxic T lymphocyte immune response mediated by CD8+ T cells. Glycan structure analysis has attracted considerable interest as glycans are looked upon as better therapeutic alternatives than monoclonal antibodies due to their cost-effectiveness, reduced toxicity and side effects, and high specificity. Furthermore, this approach will be useful to understand whether the multivalent glycan presentation on the surface of nanocarriers can overcome the low-affinity lectin-ligand interaction and thereby modulation of CLR-dependent immune response. Besides this, understanding how the heterogeneity of glycan structure impacts the antigen cross-presentation is pivotal to develop alternative targeted therapies. In the present review, we discuss the findings on structural analysis of glycans from natural lectins performed using GlycanBuilder2 - a software tool based on a thorough literature review of natural lectins. Additionally, we discuss how multiple parameters like the orientation of glycan ligands, ligand density, simultaneous targeting of multiple CLRs and design of antigen delivery nanocarriers may influence the CLR targeting efficacy. Integrating this information will eventually set the ground for new generation immunotherapeutic vaccine design for the treatment of various human malignancies.

Dietary Choices Modulate Colorectal Cancer Stem Cells: A Role of FXR Nuclear Receptor.

Intra- and inter-tumor heterogeneity (TMH) among colorectal cancer patients is considered as major hurdles to develop precise, potent, and personalized cancer therapeutics. The discernible factors that contribute to the existence of TMH and associated problems are suggested as genetic, molecular, epigenetic, and environmental pressures including shifts in trend from high-fiber diet to high-fat/processed sugar diet. In essence, components of high fat/processed sugar diet potentiate metabolic re-programing of inherent cellular heterogeneity of cancer stem cells (CSCs) by genetic and epigenetic pathways intersected by the farnesoid X receptor (FXR) nuclear receptor. Therefore, choices of dietary components shape up protumor or antitumor microenvironment by the modulation of FXR regulated transcriptional and epigenetic events in CSCs. In this article, we highlight the major understanding emanated from preclinical and clinical studies that indicate the potential contribution of high fiber/saturated sugar diet toward carcinogenesis of colorectal cancer.

A Leishmania donovani dominant-negative mutant for eIF2α kinase LdeK1 elicits host-protective immune response.

Dominant-negative mutation of LdeK1 gene, an eIF2α kinase from Leishmania donovani, revealed its role in translation regulation in response to nutrient starvation earlier. However, whether the kinase influences the infectivity of the parasites which naturally encounters nutrient deprivation during its life cycle was interesting to investigate. Both in vitro and in vivo experiments resulted in decrease of the parasite burden in peritoneal macrophages and in splenic/ hepatic load, respectively. An insight into the immune response of mice infected with mutant parasite showed enhanced pro-inflammatory cytokines and nitric oxide levels but reduced TH 2 and Treg population. The significantly reduced loss of infectivity of the parasites lacking a functional LdeK1 by modulating the immune response towards host protection makes it a potential vaccine candidate against Leishmaniasis.

Breast cancer stem cells as last soldiers eluding therapeutic burn: A hard nut to crack.

Cancer stem cells (CSCs) are found in many cancer types, including breast carcinoma. Breast cancer stem cells (BCSCs) are considered as seed of cancer formation and they are associated with metastasis and genotoxic drug resistance. Several studies highlighted the presence of BCSCs in tumor microenvironment and they are accentuated with several carcinoma events including metastasis and resistance to genotoxic drugs and they also rebound after genotoxic burn. Stemness properties of a small population of cells in carcinoma have provided clues regarding the role of tumor microenvironment in tumor pathophysiology. Hence, insights in cancer stem cell biology with respect to molecular signaling, genetics and epigenetic behavior of CSCs have been used to modulate tumor drug resistance due to genotoxic drugs and signaling protein inhibitors. This review summarizes major scientific breakthroughs in understanding the contribution of BCSCs towards tumor's capability to endure destruction inflicted by molecular as well as genotoxic drugs.

Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element.

How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.

Non-Coding RNAs in Oral Cancer: Emerging Roles and Clinical Applications.

Oral cancer (OC) is among the most prevalent cancers in the world. Certain geographical areas are disproportionately affected by OC cases due to the regional differences in dietary habits, tobacco and alcohol consumption. However, conventional therapeutic methods do not yield satisfying treatment outcomes. Thus, there is an urgent need to understand the disease process and to develop diagnostic and therapeutic strategies for OC. In this review, we discuss the role of various types of ncRNAs in OC, and their promising clinical implications as prognostic or diagnostic markers and therapeutic targets. MicroRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), PIWI-interacting RNA (piRNA), and small nucleolar RNA (snoRNA) are the major ncRNA types whose involvement in OC are emerging. Dysregulated expression of ncRNAs, particularly miRNAs, lncRNAs, and circRNAs, are linked with the initiation, progression, as well as therapy resistance of OC via modulation in a series of cellular pathways through epigenetic, transcriptional, post-transcriptional, and translational modifications. Differential expressions of miRNAs and lncRNAs in blood, saliva or extracellular vesicles have indicated potential diagnostic and prognostic importance. In this review, we have summarized all the promising aspects of ncRNAs in the management of OC.

Conformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2α kinase, a key translational regulatory molecule.

In mammalian cells, the heme-regulated inhibitor (HRI) plays a critical role in the regulation of protein synthesis at the initiation step through phosphorylation of α-subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have cloned and performed biophysical characterization of the kinase catalytic domain (KD) of rabbit HRI. The KD described here comprises kinase 1, the kinase insertion domain (KI) and kinase 2. We report here the existence of an active and stable monomer of HRI (KD). The HRI (KD) containing three tryptophan residues was examined for its conformational transitions occurring under various denaturing conditions using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The parameter A and phase diagram analysis revealed multi-state unfolding and existence of three stable intermediates during guanidine hydrochloride (Gdn-HCl) induced unfolding of HRI (KD). The protein treated with 6 M Gdn-HCl showed collisional and static mechanism of acrylamide quenching and the constants (K(sv) = 3.08 M(-1) and K(s)= 5.62 M(-1)) were resolved using time resolved fluorescence titration. Based on pH, guanidine hydrochloride and temperature mediated transitions, HRI (KD) appears to exemplify a rigid molten globule-like intermediate with compact secondary structure, altered tertiary structure and exposed hydrophobic patches at pH 3.0. The results indicate the inherent structural stability of HRI (KD), a member of the class of stress response proteins.

Sensing the interactions between carbohydrate-binding agents and N-linked glycans of SARS-CoV-2 spike glycoprotein using molecular docking and simulation studies.

A recent surge in finding new candidate vaccines and potential antivirals to tackle atypical pneumonia triggered by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) needs new and unexplored approaches in solving this global pandemic. The homotrimeric transmembrane spike (S) glycoprotein of coronaviruses which facilitates virus entry into the host cells is covered with N-linked glycans having oligomannose and complex sugars. These glycans provide a unique opportunity for their targeting via carbohydrate-binding agents (CBAs) which have shown their antiviral potential against coronaviruses and enveloped viruses. However, CBA-ligand interaction is not fully explored in developing novel carbohydrate-binding-based antivirals due to associated unfavorable responses with CBAs. CBAs possess unique carbohydrate-binding specificity, therefore, CBAs like mannose-specific plant lectins/lectin-like mimic Pradimicin-A (PRM-A) can be used for targeting N-linked glycans of S glycoproteins. Here, we report studies on the binding and stability of lectins (NPA, UDA, GRFT, CV-N and wild-type and mutant BanLec) and PRM-A with the S glycoprotein glycans via docking and MD simulation. MM/GBSA calculations were also performed for docked complexes. Interestingly, stable BanLec mutant (H84T) also showed similar docking affinity and interactions as compared to wild-type BanLec, thus, confirming that uncoupling the mitogenic activity did not alter the lectin binding activity of BanLec. The stability of the docked complexes, i.e. PRM-A and lectins with SARS-CoV-2 S glycoprotein showed favorable intermolecular hydrogen-bond formation during the 100 ns MD simulation. Taking these together, our predicted in silico results will be helpful in the design and development of novel CBA-based antivirals for the SARS-CoV-2 neutralization.Communicated by Ramaswamy H. Sarma.

Metabolic Ink Lactate Modulates Epigenomic Landscape: A Concerted Role of Pro-tumor Microenvironment and Macroenvironment During Carcinogenesis.

Tumor heterogeneity is influenced by various factors including genetic, epigenetic and axis of metabolic-epigenomic regulation. In recent years, metabolic-epigenomic reprogramming has been considered as one of the many tumor hallmarks and it appears to be driven by both microenvironment and macroenvironment factors including diet, microbiota and environmental pressures. Epigenetically, histone lysine residues are altered by various post-translational modifications (PTMs) such as acetylation, acylation, methylation and lactylation. Furthermore, lactylation is suggested as a new form of PTM that uses a lactate substrate as a metabolic ink for epigenetic writer enzyme that remodels histone proteins. Therefore, preclinical and clinical attempts are warranted to disrupt the pathway of metabolic-epigenomic reprogramming that will turn pro-tumor microenvironment into an anti-tumor microenvironment. This paper highlights the metabolicepigenomic regulation events including lactylation and its metabolic substrate lactate in the tumor microenvironment.

Distance between RBS and AUG plays an important role in overexpression of recombinant proteins.

The spacing between ribosome binding site (RBS) and AUG is crucial for efficient overexpression of genes when cloned in prokaryotic expression vectors. We undertook a brief study on the overexpression of genes cloned in Escherichia coli expression vectors, wherein the spacing between the RBS and the start codon was varied. SDS-PAGE and Western blot analysis indicated a high level of protein expression only in constructs where the spacing between RBS and AUG was approximately 40 nucleotides or more, despite the synthesis of the transcripts in the representative cases investigated.

Role of 5'- and 3'-untranslated regions of mRNAs in human diseases.

Protein synthesis is often regulated at the level of initiation of translation, making it a critical step. This regulation occurs by both the cis-regulatory elements, which are located in the 5'- and 3'-UTRs (untranslated regions), and trans-acting factors. A breakdown in this regulation machinery can perturb cellular metabolism, leading to various physiological abnormalities. The highly structured UTRs, along with features such as GC-richness, upstream open reading frames and internal ribosome entry sites, significantly influence the rate of translation of mRNAs. In this review, we discuss how changes in the cis-regulatory sequences of the UTRs, for example, point mutations and truncations, influence expression of specific genes at the level of translation. Such modifications may tilt the physiological balance from healthy to diseased states, resulting in conditions such as hereditary thrombocythaemia, breast cancer, fragile X syndrome, bipolar affective disorder and Alzheimer's disease. This information tends to establish the crucial role of UTRs, perhaps as much as that of coding sequences, in health and disease.

Carbohydrate-Binding Agents: Potential of Repurposing for COVID-19 Therapy.

With the emergence of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the whole world is suffering from atypical pneumonia, which resulted in more than 559,047 deaths worldwide. In this time of crisis and urgency, the only hope comes from new candidate vaccines and potential antivirals. However, formulating new vaccines and synthesizing new antivirals are a laborious task. Therefore, considering the high infection rate and mortality due to COVID-19, utilization of previous information, and repurposing of existing drugs against valid viral targets have emerged as a novel drug discovery approach in this challenging time. The transmembrane spike (S) glycoprotein of coronaviruses (CoVs), which facilitates the virus's entry into the host cells, exists in a homotrimeric form and is covered with N-linked glycans. S glycoprotein is known as the main target of antibodies having neutralizing potency and is also considered as an attractive target for therapeutic or vaccine development. Similarly, targeting of N-linked glycans of S glycoprotein envelope of CoV via carbohydrate-binding agents (CBAs) could serve as an attractive therapeutic approach for developing novel antivirals. CBAs from natural sources like lectins from plants, marine algae and prokaryotes and lectin mimics like Pradimicin-A (PRM-A) have shown antiviral activities against CoV and other enveloped viruses. However, the potential use of CBAs specifically lectins was limited due to unfavorable responses like immunogenicity, mitogenicity, hemagglutination, inflammatory activity, cellular toxicity, etc. Here, we reviewed the current scenario of CBAs as antivirals against CoVs, presented strategies to improve the efficacy of CBAs against CoVs; and studied the molecular interactions between CBAs (lectins and PRM-A) with Man9 by molecular docking for potential repurposing against CoVs in general, and SARSCoV- 2, in particular.

Stress-induced overexpression of the heme-regulated eIF-2alpha kinase is regulated by Elk-1 activated through ERK pathway.

The heme-regulated eIF-2alpha kinase, also called the heme-regulated inhibitor (HRI), is activated under various cytoplasmic stresses in reticulocytes leading to inhibition of initiation of protein synthesis. Our previous studies indicated that the promoter activity and expression of the human HRI (hHRI) increase in human K562 cells during heat shock and lead exposure. Contrary to this, hemin chloride which inactivates the kinase, downregulates HRI expression. Here, we attempted to understand the mechanism of regulation of hHRI expression in the lead- and hemin-exposed cells. Our results demonstrate the involvement of two transcription factors, Elk-1 and MZF-1 in regulating HRI expression. Chromatin immunoprecipitation assays established further that Elk-1 is involved in upregulating HRI expression during stress along with a co-activator p300, while MZF-1 along with HDAC-1 is instrumental in its downregulation during hemin treatment. We also demonstrate the involvement of ERK pathway in activating Elk-1 during stress resulting in an over expression of hHRI.

Conformational characterization of human eukaryotic initiation factor 2alpha: a single tryptophan protein.

The alpha-subunit of the human eukaryotic initiation factor 2 (heIF2alpha), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2alpha gets phosphorylated by eIF2alpha-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2alpha, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (tau(1)=1.13ns and tau(2)=4.74ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the alpha-helix and beta-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted alpha-helix and rearranged beta-sheet in presence of 4M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 degrees C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions.

Extrachromosomal circular DNAs: an extra piece of evidence to depict tumor heterogeneity.

The tumor microenvironment (TME) comprises a heterogeneous number and type of cellular and noncellular components that vary in the context of molecular, genomic and epigenomic levels. The genotypic diversity and plasticity within cancer cells are known to be affected by genomic instability and genome alterations. Besides genomic instability within the chromosomal linear DNA, an extra factor appears in the form of extrachromosomal circular DNAs (eccDNAs; 2-20 kbp) and microDNAs (200-400 bp). This extra heterogeneity within cancer cells in the form of an abundance of eccDNAs adds another dimension to the expression of procancer players, such as oncoproteins, acting as a driver for cancer cell survival and proliferation. This article reviews research into eccDNAs centering around cancer plasticity and hallmarks, and discusses these facts in light of therapeutics and biomarker development.

Computational insights into the interaction of small molecule inhibitors with HRI kinase domain.

The Heme-Regulated Inhibitor (HRI) kinase regulates globin synthesis in a heme-dependent manner in reticulocytes and erythroid cells in bone marrow. Inhibitors of HRI have been proposed to lead to an increased amount of haemoglobin, benefitting anaemia patients. A series of indeno[1,2-c]pyrazoles were discovered to be the first known in vitro inhibitors of HRI. However, the structural mechanism of inhibition is yet to be understood. The aim of this study was to unravel the binding mechanism of these inhibitors using molecular dynamic simulations and docking. The docking scores were observed to correlate well with experimentally determined pIC50 values. The inhibitors were observed to bind in the ATP-binding site forming hydrogen bonds with the hinge region and van der Waals interactions with non-polar residues in the binding site. Further, quantitative structure-activity relationship (QSAR) studies were performed to correlate the structural features of the inhibitors with their biological activity. The developed QSAR models were found to be statistically significant in terms of internal and external predictabilities. The presence of chlorine atoms and the hydroxymethyl groups were found to correlate with higher activity. The identified binding modes and the descriptors can support future rational identification of more potent and selective small molecule inhibitors for this kinase which are of therapeutic importance in the context of various human pathological disorders.

Engineering anti-cancer nanovaccine based on antigen cross-presentation.

Dendritic cells (DCs) present exogenous antigens on major histocompatibility complex (MHC) class I molecules, thereby activating CD8+ T cells, contributing to tumor elimination through a mechanism known as antigen cross-presentation. A variety of factors such as maturation state of DCs, co-stimulatory signals, T-cell microenvironment, antigen internalization routes and adjuvants regulate the process of DC-mediated antigen cross-presentation. Recently, the development of successful cancer immunotherapies may be attributed to the ability of DCs to cross-present tumor antigens. In this review article, we focus on the underlying mechanism of antigen cross-presentation and ways to improve antigen cross-presentation in different DC subsets. We have critically summarized the recent developments in the generation of novel nanovaccines for robust CD8+ T-cell response in cancer. In this context, we have reviewed nanocarriers that have been used for cancer immunotherapeutics based on antigen cross-presentation mechanism. Additionally, we have also expressed our views on the future applications of this mechanism in curing cancer.

A Single-Step Simultaneous Protein Staining Procedure for Polyacrylamide Gels and Nitrocellulose Membranes by Alta During Western Blot Analysis.

A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS)-polyacrylamide gels and nitrocellulose membranes by Alta during Western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water, viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for Western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining) and cost-effective.

Modulating secreted components of tumor microenvironment: A masterstroke in tumor therapeutics.

The microenvironment in which cancer resides plays an important role in regulating cancer survival, progression, malignancy and drug resistance. Tumor microenvironment (TME) consists of heterogeneous number and types of cellular and non-cellular components that vary in relation to tumor phenotype and genotype. In recent, non-cellular secreted components of microenvironmental heterogeneity have been suggested to contain various growth factors, cytokines, RNA, DNA, metabolites, structural matrix and matricellular proteins. These non-cellular components have been indicated to orchestrate numerous ways to support cancer survival and progression by providing metabolites, energy, growth signals, evading immune surveillance, drug resistance environment, metastatic and angiogenesis cues. Thus, switching action from pro-cancer to anti-cancer activities of these secreted components of TME has been considered as a new avenue in cancer therapeutics and drug resistance. In this report, we summarize the recent pre-clinical and clinical evidences to emphasize the importance of non-cellular components of TME in achieving precision therapeutics and biomarker study.

Activation of HRI is mediated by Hsp90 during stress through modulation of the HRI-Hsp90 complex.

Heme Regulated Inhibitor (HRI) is known to get activated in various stresses such as heme deficiency, heat shock, heavy metal toxicity etc. Heat shock protein 90 (Hsp90), a ubiquitous cytoplasmic protein interacts with HRI in order to regulate protein synthesis. However, it still remains to establish this interaction of HRI and Hsp90 at cellular levels and how this modulation of HRI activity is mediated by Hsp90 during stress. In the present report, using co-immunoprecipitation analysis we show that HRI interacts with Hsp90 and this association is independent of other co-chaperones in in vitro conditions. Further, analysis using truncated domains of HRI revealed that the K1 subdomain is essential for HRI - Hsp90 complex formation. Our in silico protein - protein interaction studies also indicated interaction of Hsp90 with K1 subdomain of HRI. Mammalian two hybrid assay validated this HRI - Hsp90 interaction at cellular levels. When the in vitro kinase assay was carried out with the co-immunoprecipitated complex of HRI - Hsp90, an increase in the kinase activity was observed resulting elevated levels of eIF2α phosphorylation upon heavy metal stress and heat shock. Thus, our results clearly indicate modulation of HRI kinase activity with simultaneous Hsp90 association under stress conditions.