Photoreceptor therapy: generation of neurosphere-like cells from human mesenchymal stem cells expressing erythropoietin

The loss of photoreceptors is a major concern implicated in age-macular degeneration (AMD), a type of neurodegenerative disorder. Failure to prescribe a suitable treatment due to the lack of understanding of the molecular pathogenesis, and limited capacity to compensate irreparably damaged photoreceptors in the retina have greatly contributed to the progression of visual dysfunction. Our previous study has shown that Mesenchymal Stem Cells (MSCs) expressing erythropoietin (EPO) could commit into photoreceptor cell lineage. However, the efficiency of cell differentiation is limited. The present study aims to explore the capacity of these MSCs to form neurospheres. The cells were transduced with lentiviral particles encoding for human EPO and green fluorescent protein (GFP) genes, culture-expanded and sorted before subjected for differentiation induction into neural precursor cells. Our results showed that MSC-EPO developed into larger neurosphere and expressed relatively higher expression of nestin compared with MSCs alone when cultured under neural induction medium. These preliminary findings suggested that MSC-EPO have greater neurogenic potential than MSCs alone. Further study is needed to evaluate the possibilities of neurosphere to delete differentiate into functional photoreceptor cells. We believe that the success of neurosphere expansion may potentially be useful in scaling up the manufacturing of photoreceptors in a shorter time and at an efficient cost for retinal cell replacement therapy

Treatment of colorectal cancer cells with nanocarrier encap-sulated camptothecin reveals histone modifier genes in the Wnt signaling pathway as important molecular cues for cancer targeting

Cancer cells are able to proliferate in an unregulated manner. There are several mechanisms involved that propel such neoplastic transformations. One of these processes involves bypassing cell death through changes in gene expression and, consequently, cell growth. This involves a complex epigenetic interaction within the cell, which drives it towards oncogenic transformations. These epigenetic events augment cellular growth by potentially altering chromatin structures and influencing key gene expressions. Therapeutic mechanisms have been developed to combat this by taking advantage of the underlying oncogenic mechanisms through chemical modulation. Camptothecin (CPT) is an example of this type of drug. It is a selective topoisomerase I inhibitor that is effective against many cancers, such as colorectal cancer. Previously, we successfully formulated a magnetic nanocarrier-conjugated CPT with β-cyclodextrin and iron NPs (Fe3O4) cross-linked using EDTA (CPT-CEF). Compared to CPT alone, it boasts higher efficacy due to its selective targeting and increased solubility. In this study, we treated HT29 colon cancer cells with CPT-CEF and attempted to investigate the cytotoxic effects of the formulation through an epigenetic perspective. By using RNA-Seq, several differentially expressed genes were obtained (p < 0.05). Enrichr was then used for the over-representation analysis, and the genes were compared to the epigenetic roadmap and histone modification database. The results showed that the DEGs had a high correlation with epigenetic modifications involving histone H3 acetylation. Furthermore, a subset of these genes was shown to be associated with the Wnt/β-catenin signaling pathway, which is highly upregulated in a large number of cancer cells. These genes could be investigated as downstream therapeutic targets against the uncontrolled proliferation of cancer cells. Further interaction analysis of the identified genes with the key genes of the Wnt/β-catenin signaling pathway in colorectal cancer identified the direct interactors and a few transcription regulators. Further analysis in cBioPortal confirmed their genetic alterations and their distribution across patient samples. Thus, the findings of this study reveal that colorectal cancer could be reversed by treatment with the CPT-CEF nanoparticle-conjugated nanocarrier through an epigenetic mechanism

In-Vitro Helix Opening of M. tuberculosis oriC by DnaA Occurs at Precise Location and Is Inhibited by IciA Like Protein

BACKGROUND: Mycobacterium tuberculosis (M.tb), the pathogen that causes tuberculosis, is capable of staying asymptomatically in a latent form, persisting for years in very low replicating state, before getting reactivated to cause active infection. It is therefore important to study M.tb chromosome replication, specifically its initiation and regulation. While the region between dnaA and dnaN gene is capable of autonomous replication, little is known about the interaction between DnaA initiator protein, oriC origin of replication sequences and their negative effectors of replication. METHODOLOGY/PRINCIPAL FINDINGS: By KMnO(4) mapping assays the sequences involved in open complex formation within oriC, mediated by M.tb DnaA protein, were mapped to position -500 to -518 with respect to the dnaN gene. Contrary to E. coli, the M.tb DnaA in the presence of non-hydrolysable analogue of ATP (ATPgammaS) was unable to participate in helix opening thereby pointing to the importance of ATP hydrolysis. Interestingly, ATPase activity in the presence of supercoiled template was higher than that observed for DnaA box alone. M.tb rRv1985c, a homologue of E.coli IciA (Inhibitor of chromosomal initiation) protein, could inhibit DnaA-mediated in-vitro helix opening by specifically binding to A+T rich region of oriC, provided the open complex formation had not initiated. rIciA could also inhibit in-vitro replication of plasmid carrying the M.tb origin of replication. CONCLUSIONS/SIGNIFICANCE: These results have a bearing on the functional role of the important regulator of M.tb chromosomal replication belonging to the LysR family of bacterial regulatory proteins in the context of latency

Mechanistic Insights into a Novel Exporter-Importer System of Mycobacterium tuberculosis Unravel Its Role in Trafficking of Iron

Elucidation of the basic mechanistic and biochemical principles underlying siderophore mediated iron uptake in mycobacteria is crucial for targeting this principal survival strategy vis-à-vis virulence determinants of the pathogen. Although, an understanding of siderophore biosynthesis is known, the mechanism of their secretion and uptake still remains elusive.Here, we demonstrate an interplay among three iron regulated Mycobacterium tuberculosis (M.tb) proteins, namely, Rv1348 (IrtA), Rv1349 (IrtB) and Rv2895c in export and import of M.tb siderophores across the membrane and the consequent iron uptake. IrtA, interestingly, has a fused N-terminal substrate binding domain (SBD), representing an atypical subset of ABC transporters, unlike IrtB that harbors only the permease and ATPase domain. SBD selectively binds to non-ferrated siderophores whereas Rv2895c exhibits relatively higher affinity towards ferrated siderophores. An interaction between the permease domain of IrtB and Rv2895c is evident from GST pull-down assay. In vitro liposome reconstitution experiments further demonstrate that IrtA is indeed a siderophore exporter and the two-component IrtB-Rv2895c system is an importer of ferrated siderophores. Knockout of msmeg_6554, the irtA homologue in Mycobacterium smegmatis, resulted in an impaired M.tb siderophore export that is restored upon complementation with M.tb irtA.Our data suggest the interplay of three proteins, namely IrtA, IrtB and Rv2895c in synergizing the balance of siderophores and thus iron inside the mycobacterial cell

Enhancing Skin Cancer Immunotheranostics and Precision Medicine through Functionalized Nanomodulators and Nanosensors: Recent Development and Prospects

Skin cancers, especially melanomas, present a formidable diagnostic and therapeutic challenge to the scientific community. Currently, the incidence of melanomas shows a high increase worldwide. Traditional therapeutics are limited to stalling or reversing malignant proliferation, increased metastasis, or rapid recurrence. Nonetheless, the advent of immunotherapy has led to a paradigm shift in treating skin cancers. Many state-of-art immunotherapeutic techniques, namely, active vaccination, chimeric antigen receptors, adoptive T-cell transfer, and immune checkpoint blockers, have achieved a considerable increase in survival rates. Despite its promising outcomes, current immunotherapy is still limited in its efficacy. Newer modalities are now being explored, and significant progress is made by integrating cancer immunotherapy with modular nanotechnology platforms to enhance its therapeutic efficacy and diagnostics. Research on targeting skin cancers with nanomaterial-based techniques has been much more recent than other cancers. Current investigations using nanomaterial-mediated targeting of nonmelanoma and melanoma cancers are directed at augmenting drug delivery and immunomodulation of skin cancers to induce a robust anticancer response and minimize toxic effects. Many novel nanomaterial formulations are being discovered, and clinical trials are underway to explore their efficacy in targeting skin cancers through functionalization or drug encapsulation. The focus of this review rivets on theranostic nanomaterials that can modulate immune mechanisms toward protective, therapeutic, or diagnostic approaches for skin cancers. The recent breakthroughs in nanomaterial-based immunotherapeutic modulation of skin cancer types and diagnostic potentials in personalized immunotherapies are discussed

Genome scale portrait of cAMP-receptor protein (CRP) regulons in Mycobacteria points to their role in pathogenesis

cAMP Receptor Protein (CRP)/Fumarate Nitrate Reductase Regulator (FNR) family proteins are ubiquitous regulators of cell stress in eubacteria. These proteins are commonly associated with maintenance of intracellular oxygen levels, redox-state, oxidative and nitrosative stresses, and extreme temperature conditions by regulating expression of target genes that contain regulatory cognate DNA elements. We describe the use of informatics enabled comparative genomics to identify novel genes under the control of CRP regulator in Mycobacterium tuberculosis (M.tb). An inventory of CRP regulated genes and their operon context in important mycobacterial species such as M. leprae, M. avium subsp. paratuberculosis and M. smegmatis and several common genes within this genus including the important cellular functions, mainly, cell-wall biogenesis, cAMP signaling and metabolism associated with such regulons were identified. Our results provide a possible theoretical framework for better understanding of the stress response in mycobacteria. The conservation of the CRP regulated genes in pathogenic mycobacteria, as opposed to non-pathogenic ones, highlights the importance of CRP-regulated genes in pathogenesis