Mitophagy at the crossroads of cancer development: Exploring the role of mitophagy in tumor progression and therapy resistance.

Preserving a functional mitochondrial network is crucial for cellular well-being, considering the pivotal role of mitochondria in ensuring cellular survival, especially under stressful conditions. Mitophagy, the selective removal of damaged mitochondria through autophagy, plays a pivotal role in preserving cellular homeostasis by preventing the production of harmful reactive oxygen species from dysfunctional mitochondria. While the involvement of mitophagy in neurodegenerative diseases has been thoroughly investigated, it is becoming increasingly evident that mitophagy plays a significant role in cancer biology. Perturbations in mitophagy pathways lead to suboptimal mitochondrial quality control, catalyzing various aspects of carcinogenesis, including establishing metabolic plasticity, stemness, metabolic reconfiguration of cancer-associated fibroblasts, and immunomodulation. While mitophagy performs a delicate balancing act at the intersection of cell survival and cell death, mounting evidence indicates that, particularly in the context of stress responses induced by cancer therapy, it predominantly promotes cell survival. Here, we showcase an overview of the current understanding of the role of mitophagy in cancer biology and its potential as a target for cancer therapy. Gaining a more comprehensive insight into the interaction between cancer therapy and mitophagy has the potential to reveal novel targets and pathways, paving the way for enhanced treatment strategies for therapy-resistant tumors in the near future.

Upregulation of ATP6V0D2 benefits intracellular survival of Leishmania donovani in erythrocytes-engulfing macrophages.

Visceral leishmaniasis (VL) is the most severe type of leishmaniasis which is caused by infection of Leishmania donovani complex. In the BALB/c mouse model of VL, multinucleated giant cells (MGCs) with heavy parasite infection consist of the largest population of hemophagocytes in the spleen of L. donovani-infected mice, indicating that MGCs provide the parasites a circumstance beneficial for their survival. Although ATP6V0D2 is a demonstrated factor inducing the formation of hemophagocytic MGCs during L. donovani infection, functions of this protein in shaping the infection outcome in macrophages remain unclear. Here we evaluated the influence of upregulated ATP6V0D2 on intracellular survival of the parasites. L. donovani infection-induced hemophagocytosis of normal erythrocytes by macrophages was suppressed by RNAi-based knockdown of Atp6v0d2. The knockdown of Atp6v0d2 did not improve the survival of amastigotes within macrophages when the cells were cultured in the absence of erythrocytes. On the other hand, reduced intracellular survival of amastigotes in macrophages by the knockdown was observed when macrophages were supplemented with antibody-opsonized erythrocytes before infection. There, increase in cytosolic labile iron pool was observed in the L. donovani-infected knocked-down macrophages. It suggests that ATP6V0D2 plays roles not only in upregulation of hemophagocytosis but also in iron trafficking within L. donovani-infected macrophages. Superior access to iron in macrophages may be how the upregulated expression of the molecule brings benefit to Leishmania for their intracellular survival in the presence of erythrocytes.

Access and utilization of host-derived iron by Leishmania parasites.

Iron is involved in many biochemical processes including oxygen transport, ATP production, DNA synthesis and antioxidant defense. The importance of iron also applies to Leishmania parasites, an intracellular protozoan pathogen causing leishmaniasis. Leishmania are heme-auxotrophs, devoid of iron storage proteins and the heme synthesis pathway. Acquisition of iron and heme from the surrounding niche is thus critical for the intracellular survival of Leishmania inside the host macrophages. Moreover, Leishmania parasites are also exposed to oxidative stress within phagolysosomes of macrophages in mammalian hosts, and they need iron superoxide dismutase for overcoming this stress. Therefore, untangling the strategy adopted by these parasites for iron acquisition and utilization can be good targets for the development of antileishmanial drugs. Here, in this review, we will address how Leishmania parasites acquire and utilize iron and heme during infection to macrophages.

Selective ADA2 inhibition for enhancing anti-tumor immune response in glioma: Insights from computational screening of flavonoid compounds.

Brain tumors, particularly gliomas, remain difficult to treat due to their complex and dynamic microenvironment and high mortality rate. The presence of tumor-associated macrophages (TAMs) is considered one of the primary factors contributing to a poor prognosis in Glioma. Previous reports have linked elevated levels of Adenosine deaminase 2 (ADA2) with immunosuppression, tumor progression, and angiogenesis via MAPK, PDGFß signaling pathway in the glioma microenvironment. In contrast, Adenosine deaminase 1 (ADA1), another type of adenosine deaminase, plays a pivotal role in purine metabolism, which is essential for lymphocyte survival. Hence, selectively targeting ADA2 while preserving ADA1 activity could offer a viable approach for regulating macrophage polarization and enhancing the anti-tumor immune response. In pursuit of this objective, our study employed a computational approach, unveiling the remarkable attributes of Daidzin, characterized by its exceptional specificity, and binding affinity towards ADA2 while displaying minimal affinity towards ADA1. Furthermore, Define Secondary Structure of Proteins (DSSP) analysis revealed that Daidzin elicits conspicuous conformational alterations within the dimerization domain of the ADA2 receptor, which could have a crucial impact on its activity. However, the ADA1 structure remained unaltered. Our study offers the potential use of Daidzin as a specific therapeutic agent for modulating the tumor microenvironment and revolutionizing glioma management.

Full genomic sequence of the HLA-A*02:01:01:241 allele identified by next-generation sequencing.

HLA-A*02:01:01:241 differs from HLA-A*02:01:01:01 by a single nucleotide substitution at position 178 in intron 1.

An overview of kinin mediated events in cancer progression and therapeutic applications.

Kinins are bioactive peptides generated in the inflammatory milieu of the tissue microenvironment, which is involved in cancer progression and inflammatory response. Kinins signals through activation of two G-protein coupled receptors; inducible Bradykinin Receptor B1 (B1R) and constitutive receptor B2 (B2R). Activation of kinin receptors and its cross-talk with receptor tyrosine kinases activates multiple signaling pathways, including ERK/MAPK, PI3K, PKC, and p38 pathways regulating cancer hallmarks. Perturbations of the kinin-mediated events are implicated in various aspects of cancer invasion, matrix remodeling, and metastasis. In the tumor microenvironment, kinins initiate fibroblast activation, mesenchymal stem cell interactions, and recruitment of immune cells. Albeit the precise nature of kinin function in the metastasis and tumor microenvironment are not completely clear yet, several kinin receptor antagonists show anti-metastatic potential. Here, we showcase an overview of the complex biology of kinins and their role in cancer pathogenesis and therapeutic aspects.

Activation of TLR-pathway to induce host Th1 immune response against visceral leishmaniasis: Involvement of galactosylated-flavonoids.

Immunotherapeutic strategies against visceral leishmaniasis (VL) are pertinent because of the emergence of resistance against existing chemotherapy, coupled with their toxicity and high costs. Various bioactive components with potential immunomodulatory activity, such as alkaloids, terpenes, saponins, flavonoids obtained primarily from medicinal plants, have been screened against different disease models. Reports suggested that glycans containing terminal ß-galactose can skew host immune response towards Th1 by engaging TLRs. In this study, two synthesized terminal galactose-containing flavones, Quercetin 3-d-galactoside (Q-gal) and Kaempferol 3-O-d-galactoside (K-gal), are profiled in terms of inducing host protective Th1 response in both in vitro & in vivo animal models of experimental VL individually against antimony-resistant & antimony-susceptible Leishmania donovani. Further, we explored that both Q-gal and K-gal induce TLR4 mediated Th1 response to encounter VL. Molecular docking analysis also suggested strong interaction with TLR4 for both the galactosides, with a slightly better binding potential towards Q-gal. Treatment with both Q-gal and K-gal showed significant antileishmanial efficacy. Each considerably diminished the liver and splenic parasite burden 60 days after post-infection (>90% in AG83 infected mice and >87% in GE1F8R infected mice) when administered at a 5 mg/kg/day body-weight dose for ten consecutive days. However, the treatments failed to clear the parasites in the TLR4 deficient C3H/HeJ mice. Treatment with these compounds favors the elevation of TLR4 dependent host protective Th1 cytokines and suppression of disease-promoting IL-10. Q-gal and K-gal also triggered sufficient ROS generation in macrophages to kill intracellular parasites directly.

Exploring the crosstalk between long non-coding RNAs and microRNAs to unravel potential prognostic and therapeutic biomarkers in ß-thalassemia.

ß-thalassemia is a prevalent monogenic disorder characterized by reduced or absent synthesis of the ß-globin chain. Although great effort has been made to ameliorate the disease severity of ß-thalassemic patients, progress has been stymied due to limited understanding of the detailed molecular mechanism of disease pathogenesis. Recently, non-coding RNAs have been established as key players in regulating various physiological and pathological processes. Many ncRNAs are involved in hematopoiesis and erythroid development. Furthermore, various studies have also reported the complex interplay between different ncRNAs, such as miRNA, lncRNAs, etc. in regulating disease progression and pathogenesis. Both lncRNAs and miRNAs have been identified as independent regulators of globin gene expression and are intricately involved in disease pathogenesis; yet accumulating evidence suggests that the cross-talk between lncRNAs and miRNAs is intricately involved in the underlying globin gene expression, fine-tuning the effect of their independent regulation. In this review, we summarize the current progress of research on the roles of lncRNAs and miRNAs implicated in ß-thalassemia disease, including their interactions and regulatory networks. This can provide important insights into the detailed epigenetic regulation of globin gene switching and has the potential to develop novel therapeutic approaches against ß-thalassemia.

AUF-1 knockdown in mice undermines gut microbial butyrate-driven hypocholesterolemia through AUF-1-Dicer-1-mir-122 hierarchy.

Introduction and objective: Cholesterol homeostasis is a culmination of cellular synthesis, efflux, and catabolism to important physiological entities where short chain fatty acid, butyrate embodied as a key player. This discourse probes the mechanistic molecular details of butyrate action in maintaining host-cholesterol balance. Methods: Hepatic mir-122 being the most indispensable regulator of cholesterol metabolic enzymes, we studied upstream players of mir-122 biogenesis in the presence and absence of butyrate in Huh7 cells and mice model. We synthesized unique self-transfecting GMO (guanidinium-morpholino-oligo) linked PMO (Phosphorodiamidate-Morpholino Oligo)-based antisense cell-penetrating reagent to selectively knock down the key player in butyrate mediated cholesterol regulation. Results: We showed that butyrate treatment caused upregulation of RNA-binding protein, AUF1 resulting in RNase-III nuclease, Dicer1 instability, and significant diminution of mir-122. We proved the importance of AUF1 and sequential downstream players in AUF1-knock-down mice. Injection of GMO-PMO of AUF1 in mouse caused near absence of AUF1 coupled with increased Dicer1 and mir-122, and reduced serum cholesterol regardless of butyrate treatment indicating that butyrate acts through AUF1. Conclusion: The roster of intracellular players was as follows: AUF1-Dicer1-mir-122 for triggering butyrate driven hypocholesterolemia. To our knowledge this is the first report linking AUF-1 with cholesterol biogenesis.

A Novel Bioimpedance-Based Detection of Miltefosine Susceptibility Among Clinical Leishmania donovani Isolates of the Indian Subcontinent Exhibiting Resistance to Multiple Drugs.

The extent of susceptibility towards miltefosine (Mil), amphotericin B (AmpB), and paromomycin (Paro) was measured among 19 clinical isolates of Leishmania donovani (LD). Thirteen of these clinical isolates were reported to exhibit low susceptibility towards sodium stibogluconate (SSG-R), while six of them were highly susceptible (SSG-S). The degree of clearance of amastigotes (EC50) for these predefined SSG-R- and SSG-S-infected macrophages was determined against Mil, AmpB, and Paro. Two out of the 13 SSG-R isolates (BHU575 and BHU814) showed low susceptibility towards all three drugs studied, while the rest of the 11 SSG-R isolates showed varying degrees of susceptibility either towards none or only towards individual drugs. Interestingly, all the SSG-S isolates showed high susceptibility towards Mil/AmpB/Paro. The total intracellular non-protein thiol content of the LD promastigotes, which have been previously reported to be positively co-related with EC50 towards SSG, was found to be independent from the degree of susceptibility towards Mil/AmpB/Paro. Impedance spectra analysis, which quantifies membrane resistance, revealed lower impedimetric values for all those isolates exhibiting low efficacy to Mil (Mil-R). Our analysis points out that while non-protein thiol content can be an attribute of SSG-R, lower impedimetric values can be linked with lower Mil susceptibility, although neither of these parameters seems to get influenced by the degree of susceptibility towards AmpB/Paro. Finally, a correlation analysis with established biological methods suggests that impedance spectral analysis can be used for the accurate determination of lower Mil susceptibility among LD isolates, which is further validated in the LD-infected in vivo hamster model.

Novel Antiplasmodial Compounds Leveraged with Multistage Potency against the Parasite Plasmodium falciparum: In Vitro and In Vivo Evaluations and Pharmacokinetic Studies.

Hydroxyethylamine (HEA)-based novel compounds were synthesized and their activity against Plasmodium falciparum 3D7 was assessed, identifying a few hits without any apparent toxicity. Hits 5c and 5d also exhibited activity against resistant field strains, PfRKL-9 and PfC580Y. A single dose, 50 mg/Kg, of hits administered to the rodent parasite Plasmodium berghei ANKA exhibited up to 70% reduction in the parasite load. Compound 5d tested in combination with artesunate produced an additional antiparasitic effect with a prolonged survival period. Additionally, compound 5d showed 50% inhibition against hepatic P. berghei infection at 1.56 ± 0.56 µM concentration. This compound also considerably delayed the progression of transmission stages, ookinete and oocyst. Furthermore, the toxicity of 5d assessed in mice supported the normal liver and kidney functions. Altogether, HEA analogues (5a-m), particularly 5d, are nontoxic multistage antiplasmodial agents with therapeutic and transmission-blocking efficacy, along with favorable preliminary pharmacokinetic properties.

Structural insights into an atypical secretory pathway kinase crucial for Toxoplasma gondii invasion.

Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.

Linking membrane fluidity with defective antigen presentation in leishmaniasis.

Hampering-surface presentation of immunogenic peptides by class I/II MHCs is a key strategy opted by several intracellular protozoan pathogens including Leishmania to escape CD8/CD4 mediated host-protective T-cell response. Although Leishmania parasites (LP) primarily hijack/inhibit host lysosomal/proteasomal pathways to hamper antigen-processing/presentation machinery, recent pieces of evidence have linked host-membrane fluidity as a major cause of defective antigen presentation in leishmaniasis. Increased membrane fluidity severely compromised peptide-MHC stability in the lipid raft regions, thereby abrogating T-cell mediated-signalling in the infected host. LP primarily achieves this by quenching host cholesterol, which acts as cementing material in maintaining the membrane fluidity. In this review, we have particularly focused on several strategies opted by LP to hijack-host cholesterol resulting in lipid droplets accumulation around leishmania-containing parasitophorous vacuole favouring intracellular survival of LP. In fact, LP infection can result in altered cholesterol and lipid metabolism in the infected host, thereby favouring the establishment and progression of the infection. From our analysis of two genome-wide transcriptomics data sets of LP infected host, we propose a possible molecular network that connects these interrelated events of altered lipid metabolism with eventual compromised antigen presentation, still existing as a gap in our current understanding of Leishmania infection.

Probing the molecular mechanism of aggressive infection by antimony resistant Leishmania donovani.

The disease visceral leishmaniasis (VL) or kala azar is caused by the protozoan parasite, Leishmania donovani (LD). For many decades the pentavalent antimonial drugs countered the successive epidemics of the disease in the Indian sub-continent and elsewhere. With time, antimony resistant LD (LDR) developed and the drug in turn lost its efficacy. Infection of mammals with LDR gives rise to aggressive infection as compared to its sensitive counterpart (LDS) coupled with higher surge of IL-10 and TGF-ß. The IL-10 causes upregulation of multidrug resistant protein-1 which causes efflux of antimonials from LDR infected cells. This is believed to be a key mechanism of antimony resistance. MicroRNAs (miRNAs) are tiny post-transcriptional regulators of gene expression in mammalian cells and in macrophage play a pivotal role in controlling the expression of cytokines involved in infection process. Therefore, a change in miRNA profiles of macrophages infected with LDS or LDR could explain the differential cytokine response observed. Interestingly, the outcome of LD infection is also governed by the critical balance of pro- and anti-inflammatory cytokines which is inturn regulated by miRNA-Ago2 or miRNP complex and its antagonist RNA binding protein HuR. Here Ago2 plays the fulcrum whose phosphorylation and de-phosphorylation dictates the process; which in turn is controlled by PP2A and HuR. LDS and LDR upregulate PP2A and downregulate HuR at different magnitude leading to various levels of anti-inflammatory to proinflammatory cytokine production and resulting pathology in the host. While ectopic HuR expression alone is sufficient to clear LDS infection, simultaneous upregulation of HuR and inhibition of PP2A is required to inhibit LDR mediated infection. Therefore, tampering with miRNA pathway could be a new strategy to control infection caused by LDR parasite.

Short-Course Treatment With Imipramine Entrapped in Squalene Liposomes Results in Sterile Cure of Experimental Visceral Leishmaniasis Induced by Antimony Resistant Leishmania donovani With Increased Efficacy.

Previously we have shown that long term oral treatment of tricyclic-antidepressant-drug, imipramine, against experimental visceral leishmaniasis, results in clearance of organ parasites, regardless of input infection, either with antimony-sensitive (SbS) or antimony-resistant (SbR) Leishmania donovani (LD) clinical isolates. Although continuous imipramine monotherapy for 28 days (5 mg/kg) results in significant clearance of organ parasites in both SbR and SbSLD infected hamsters, the dose for the sterile parasite clearance from visceral organ is comparatively higher (10 mg/kg) and shows signs of toxicity. Hence, to reduce the toxicity, we encapsulated imipramine in squalene-phosphatidylcholine (SP) liposome (Lip-Imi) and tested its efficacy for a short-course treatment (10 days) in the animal model of visceral leishmaniasis. We observed a significant reduction of hepatic toxicity coupled with sterile parasite clearance in case of this short-course treatment of Lip-Imi, which is absent with free Imi treatment. This also correlates with significant increase in serum availability of imipramine in case of Lip-Imi treatment due to sustained release. Clearance of parasite was coupled with the polarization of antileishmanial immune repertoire from Th2 to Th1 after treatment with Lip-Imi in both SbRLD and SbSLD infected mouse models of LD infection. This study showed that imipramine is effective against both SbSLD and SbRLD at a significantly lower dose with reduced time course of treatment without any toxic side effects, when encapsulated in SP-liposome. Thus, the drug has the potential to be repurposed for the treatment of Kala-azar.

Modeling and resistant alleles explain the selectivity of antimalarial compound 49c towards apicomplexan aspartyl proteases.

Toxoplasma gondii aspartyl protease 3 (TgASP3) phylogenetically clusters with Plasmodium falciparum Plasmepsins IX and X (PfPMIX, PfPMX). These proteases are essential for parasite survival, acting as key maturases for secreted proteins implicated in invasion and egress. A potent antimalarial peptidomimetic inhibitor (49c) originally developed against Plasmepsin II selectively targets TgASP3, PfPMIX, and PfPMX To unravel the molecular basis for the selectivity of 49c, we constructed homology models of PfPMIX, PfPMX, and TgASP3 that were first validated by identifying the determinants of microneme and rhoptry substrate recognition. The flap and flap-like structures of several reported Plasmepsins are highly flexible and critically modulate the access to the binding cavity. Molecular docking of 49c to TgASP3, PfPMIX, and PfPMX models predicted that the conserved phenylalanine residues in the flap, F344, F291, and F305, respectively, account for the sensitivity toward 49c. Concordantly, phenylalanine mutations in the flap of the three proteases increase twofold to 15-fold the IC50 values of 49c. Compellingly the selection of mutagenized T. gondii resistant strains to 49c reproducibly converted F344 to a cysteine residue.

A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress.

Regulated exocytosis by secretory organelles is important for malaria parasite invasion and egress. Many parasite effector proteins, including perforins, adhesins, and proteases, are extensively proteolytically processed both pre- and postexocytosis. Here we report the multistage antiplasmodial activity of the aspartic protease inhibitor hydroxyl-ethyl-amine-based scaffold compound 49c. This scaffold inhibits the preexocytosis processing of several secreted rhoptry and microneme proteins by targeting the corresponding maturases plasmepsins IX (PMIX) and X (PMX), respectively. Conditional excision of PMIX revealed its crucial role in invasion, and recombinantly active PMIX and PMX cleave egress and invasion factors in a 49c-sensitive manner.

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.

Antimony-Resistant Leishmania donovani Exploits miR-466i To Deactivate Host MyD88 for Regulating IL-10/IL-12 Levels during Early Hours of Infection.

Infection with antimony-resistant Leishmania donovani (Sb(R)LD) induces aggressive pathology in the mammalian hosts as compared with ones with antimony-sensitive L. donovani (Sb(S)LD) infection. Sb(R)LD, but not Sb(S)LD, interacts with TLR2/TLR6 to induce IL-10 by exploiting p50/c-Rel subunits of NF-κB in infected macrophages (Mϕs). Most of the TLRs exploit the universal adaptor protein MyD88 to activate NF-κB. We now show that infection of Mϕs from MyD88(-/-) mice with Sb(R)LD gave rise to significantly higher intracellular parasite number coupled with elevated IL-10/IL-12 ratio in the culture supernatant as compared with infection in wild type (WT) Mϕs. Τhese attributes were not seen with Sb(S)LD in similar experiments. Further, Sb(R)LD infection upregulated miR-466i, which binds with 3'-untranslated region, leading to the downregulation of MyD88. Infection of MyD88(-/-) Mϕ or IL-12(-/-) Mϕ with Sb(R)LD induced IL-10 surge at 4 h, whereas the same in WT Mϕ started from 12 h. Thus, absence of IL-12 in MyD88(-/-) mice favored early binding of NF-κB subunits to the IL-10 promoter, resulting in IL-10 surge. Infection of MyD88(-/-) mice with Sb(R)LD showed significantly higher organ parasites coupled with ill-defined and immature hepatic granulomas, whereas in WT mice there were less organ parasites and the granulomas were well defined. From the survival kinetics it was observed that Sb(R)LD-infected MyD88(-/-) mice died by 60 d postinfection, whereas the WT mice continued to survive. Our results demonstrate that Sb(R)LD has evolved a unique strategy to evade host antileishmanial immune repertoire by manipulating host MyD88 to its advantage.