Structure-function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum.

Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the ß-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.

Secretome profiling reveals acute changes in oxidative stress, brain homeostasis, and coagulation following short-duration spaceflight.

As spaceflight becomes more common with commercial crews, blood-based measures of crew health can guide both astronaut biomedicine and countermeasures. By profiling plasma proteins, metabolites, and extracellular vesicles/particles (EVPs) from the SpaceX Inspiration4 crew, we generated "spaceflight secretome profiles," which showed significant differences in coagulation, oxidative stress, and brain-enriched proteins. While >93% of differentially abundant proteins (DAPs) in vesicles and metabolites recovered within six months, the majority (73%) of plasma DAPs were still perturbed post-flight. Moreover, these proteomic alterations correlated better with peripheral blood mononuclear cells than whole blood, suggesting that immune cells contribute more DAPs than erythrocytes. Finally, to discern possible mechanisms leading to brain-enriched protein detection and blood-brain barrier (BBB) disruption, we examined protein changes in dissected brains of spaceflight mice, which showed increases in PECAM-1, a marker of BBB integrity. These data highlight how even short-duration spaceflight can disrupt human and murine physiology and identify spaceflight biomarkers that can guide countermeasure development.

Plasmodium falciparum Alba6 exhibits DNase activity and participates in stress response.

Alba domain proteins, owing to their functional plasticity, play a significant role in organisms. Here, we report an intrinsic DNase activity of PfAlba6 from Plasmodium falciparum, an etiological agent responsible for human malignant malaria. We identified that tyrosine28 plays a critical role in the Mg2+ driven 5'-3' DNase activity of PfAlba6. PfAlba6 cleaves both dsDNA as well as ssDNA. We also characterized PfAlba6-DNA interaction and observed concentration-dependent oligomerization in the presence of DNA, which is evident from size exclusion chromatography and single molecule AFM-imaging. PfAlba6 mRNA expression level is up-regulated several folds following heat stress and treatment with artemisinin, indicating a possible role in stress response. PfAlba6 has no human orthologs and is expressed in all intra-erythrocytic stages; thus, this protein can potentially be a new anti-malarial drug target.

Nanoparticle enrichment mass-spectrometry proteomics identifies protein-altering variants for precise pQTL mapping.

Proteogenomics studies generate hypotheses on protein function and provide genetic evidence for drug target prioritization. Most previous work has been conducted using affinity-based proteomics approaches. These technologies face challenges, such as uncertainty regarding target identity, non-specific binding, and handling of variants that affect epitope affinity binding. Mass spectrometry-based proteomics can overcome some of these challenges. Here we report a pQTL study using the Proteograph™ Product Suite workflow (Seer, Inc.) where we quantify over 18,000 unique peptides from nearly 3000 proteins in more than 320 blood samples from a multi-ethnic cohort in a bottom-up, peptide-centric, mass spectrometry-based proteomics approach. We identify 184 protein-altering variants in 137 genes that are significantly associated with their corresponding variant peptides, confirming target specificity of co-associated affinity binders, identifying putatively causal cis-encoded proteins and providing experimental evidence for their presence in blood, including proteins that may be inaccessible to affinity-based proteomics.

Expanding the DNA editing toolbox: Novel lambda integrase variants targeting microalgal and human genome sequences.

Recombinase enzymes are extremely efficient at integrating very large DNA fragments into target genomes. However, intrinsic sequence specificities curtail their use to DNA sequences with sufficient homology to endogenous target motifs. Extensive engineering is therefore required to broaden applicability and robustness. Here, we describe the directed evolution of novel lambda integrase variants capable of editing exogenous target sequences identified in the diatom Phaeodactylum tricornutum and the algae Nannochloropsis oceanica. These microorganisms hold great promise as conduits for green biomanufacturing and carbon sequestration. The evolved enzyme variants show >1000-fold switch in specificity towards the non-natural target sites when assayed in vitro. A single-copy target motif in the human genome with homology to the Nannochloropsis oceanica site can also be efficiently targeted using an engineered integrase, both in vitro and in human cells. The developed integrase variants represent useful additions to the DNA editing toolbox, with particular application for targeted genomic insertion of large DNA cargos.

Identification of Novel Biomarkers for Alzheimer's Disease and Related Dementias Using Unbiased Plasma Proteomics.

Alzheimer's disease (AD) and related dementias (ADRD) is a complex disease with multiple pathophysiological drivers that determine clinical symptomology and disease progression. These diseases develop insidiously over time, through many pathways and disease mechanisms and continue to have a huge societal impact for affected individuals and their families. While emerging blood-based biomarkers, such as plasma p-tau181 and p-tau217, accurately detect Alzheimer neuropthology and are associated with faster cognitive decline, the full extension of plasma proteomic changes in ADRD remains unknown. Earlier detection and better classification of the different subtypes may provide opportunities for earlier, more targeted interventions, and perhaps a higher likelihood of successful therapeutic development. In this study, we aim to leverage unbiased mass spectrometry proteomics to identify novel, blood-based biomarkers associated with cognitive decline. 1,786 plasma samples from 1,005 patients were collected over 12 years from partcipants in the Massachusetts Alzheimer's Disease Research Center Longitudinal Cohort Study. Patient metadata includes demographics, final diagnoses, and clinical dementia rating (CDR) scores taken concurrently. The Proteograph™ Product Suite (Seer, Inc.) and liquid-chromatography mass-spectrometry (LC-MS) analysis were used to process the plasma samples in this cohort and generate unbiased proteomics data. Data-independent acquisition (DIA) mass spectrometry results yielded 36,259 peptides and 4,007 protein groups. Linear mixed effects models revealed 138 differentially abundant proteins between AD and healthy controls. Machine learning classification models for AD diagnosis identified potential candidate biomarkers including MBP, BGLAP, and APoD. Cox regression models were created to determine the association of proteins with disease progression and suggest CLNS1A, CRISPLD2, and GOLPH3 as targets of further investigation as potential biomarkers. The Proteograph workflow provided deep, unbiased coverage of the plasma proteome at a speed that enabled a cohort study of almost 1,800 samples, which is the largest, deep, unbiased proteomics study of ADRD conducted to date.

Protein Coronas on Functionalized Nanoparticles Enable Quantitative and Precise Large-Scale Deep Plasma Proteomics.

Background: The wide dynamic range of circulating proteins coupled with the diversity of proteoforms present in plasma has historically impeded comprehensive and quantitative characterization of the plasma proteome at scale. Automated nanoparticle (NP) protein corona-based proteomics workflows can efficiently compress the dynamic range of protein abundances into a mass spectrometry (MS)-accessible detection range. This enhances the depth and scalability of quantitative MS-based methods, which can elucidate the molecular mechanisms of biological processes, discover new protein biomarkers, and improve comprehensiveness of MS-based diagnostics. Methods: Investigating multi-species spike-in experiments and a cohort, we investigated fold-change accuracy, linearity, precision, and statistical power for the using the Proteograph™ Product Suite, a deep plasma proteomics workflow, in conjunction with multiple MS instruments. Results: We show that NP-based workflows enable accurate identification (false discovery rate of 1%) of more than 6,000 proteins from plasma (Orbitrap Astral) and, compared to a gold standard neat plasma workflow that is limited to the detection of hundreds of plasma proteins, facilitate quantification of more proteins with accurate fold-changes, high linearity, and precision. Furthermore, we demonstrate high statistical power for the discovery of biomarkers in small- and large-scale cohorts. Conclusions: The automated NP workflow enables high-throughput, deep, and quantitative plasma proteomics investigation with sufficient power to discover new biomarker signatures with a peptide level resolution.

A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells.

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based applications for more effective and affordable therapies.

Nuclease activity of Plasmodium falciparum Alba family protein PfAlba3.

Plasmodium falciparum Alba domain-containing protein Alba3 (PfAlba3) is ubiquitously expressed in intra-erythrocytic stages of Plasmodium falciparum, but the function of this protein is not yet established. Here, we report an apurinic/apyrimidinic site-driven intrinsic nuclease activity of PfAlba3 assisted by divalent metal ions. Surface plasmon resonance and atomic force microscopy confirm sequence non-specific DNA binding by PfAlba3. Upon binding, PfAlba3 cleaves double-stranded DNA (dsDNA) hydrolytically. Mutational studies coupled with mass spectrometric analysis indicate that K23 is the essential residue in modulating the binding to DNA through acetylation-deacetylation. We further demonstrate that PfSir2a interacts and deacetylates K23-acetylated PfAlba3 in favoring DNA binding. Hence, K23 serves as a putative molecular switch regulating the nuclease activity of PfAlba3. Thus, the nuclease activity of PfAlba3, along with its apurinic/apyrimidinic (AP) endonuclease feature identified in this study, indicates a role of PfAlba3 in DNA-damage response that may have a far-reaching consequence in Plasmodium pathogenicity.

Functionally distinct BMP1 isoforms show an opposite pattern of abundance in plasma from non-small cell lung cancer subjects and controls.

Advancements in deep plasma proteomics are enabling high-resolution measurement of plasma proteoforms, which may reveal a rich source of novel biomarkers previously concealed by aggregated protein methods. Here, we analyze 188 plasma proteomes from non-small cell lung cancer subjects (NSCLC) and controls to identify NSCLC-associated protein isoforms by examining differentially abundant peptides as a proxy for isoform-specific exon usage. We find four proteins comprised of peptides with opposite patterns of abundance between cancer and control subjects. One of these proteins, BMP1, has known isoforms that can explain this differential pattern, for which the abundance of the NSCLC-associated isoform increases with stage of NSCLC progression. The presence of cancer and control-associated isoforms suggests differential regulation of BMP1 isoforms. The identified BMP1 isoforms have known functional differences, which may reveal insights into mechanisms impacting NSCLC disease progression.

Honokiol, an inducer of sirtuin-3, protects against non-steroidal anti-inflammatory drug-induced gastric mucosal mitochondrial pathology, apoptosis and inflammatory tissue injury.

BACKGROUND AND PURPOSE: Mitochondrial oxidative stress, inflammation and apoptosis primarily underlie gastric mucosal injury caused by the widely used non-steroidal anti-inflammatory drugs (NSAIDs). Alternative gastroprotective strategies are therefore needed. Sirtuin-3 pivotally maintains mitochondrial structural integrity and metabolism while preventing oxidative stress; however, its relevance to gastric injury was never explored. Here, we have investigated whether and how sirtuin-3 stimulation by the phytochemical, honokiol, could rescue NSAID-induced gastric injury. EXPERIMENTAL APPROACH: Gastric injury in rats induced by indomethacin was used to assess the effects of honokiol. Next-generation sequencing-based transcriptomics followed by functional validation identified the gastroprotective function of sirtuin-3. Flow cytometry, immunoblotting, qRT-PCR and immunohistochemistry were used measure effects on oxidative stress, mitochondrial dynamics, electron transport chain function, and markers of inflammation and apoptosis. Sirtuin-3 deacetylase activity was also estimated and gastric luminal pH was measured. KEY RESULTS: Indomethacin down-regulated sirtuin-3 to induce oxidative stress, mitochondrial hyperacetylation, 8-oxoguanine DNA glycosylase 1 depletion, mitochondrial DNA damage, respiratory chain defect and mitochondrial fragmentation leading to severe mucosal injury. Indomethacin dose-dependently inhibited sirtuin-3 deacetylase activity. Honokiol prevented mitochondrial oxidative damage and inflammatory tissue injury by attenuating indomethacin-induced depletion of both sirtuin-3 and its transcriptional regulators PGC1α and ERRα. Honokiol also accelerated gastric wound healing but did not alter gastric acid secretion, unlike lansoprazole. CONCLUSIONS AND IMPLICATIONS: Sirtuin-3 stimulation by honokiol prevented and reversed NSAID-induced gastric injury through maintaining mitochondrial integrity. Honokiol did not affect gastric acid secretion. Sirtuin-3 stimulation by honokiol may be utilized as a mitochondria-based, acid-independent novel gastroprotective strategy against NSAIDs.

Enhanced Competition at the Nano-Bio Interface Enables Comprehensive Characterization of Protein Corona Dynamics and Deep Coverage of Proteomes.

Introducing engineered nanoparticles (NPs) into a biofluid such as blood plasma leads to the formation of a selective and reproducible protein corona at the particle-protein interface, driven by the relationship between protein-NP affinity and protein abundance. This enables scalable systems that leverage protein-nano interactions to overcome current limitations of deep plasma proteomics in large cohorts. Here the importance of the protein to NP-surface ratio (P/NP) is demonstrated and protein corona formation dynamics are modeled, which determine the competition between proteins for binding. Tuning the P/NP ratio significantly modulates the protein corona composition, enhancing depth and precision of a fully automated NP-based deep proteomic workflow (Proteograph). By increasing the binding competition on engineered NPs, 1.2-1.7× more proteins with 1% false discovery rate are identified on the surface of each NP, and up to 3× more proteins compared to a standard plasma proteomics workflow. Moreover, the data suggest P/NP plays a significant role in determining the in vivo fate of nanomaterials in biomedical applications. Together, the study showcases the importance of P/NP as a key design element for biomaterials and nanomedicine in vivo and as a powerful tuning strategy for accurate, large-scale NP-based deep proteomic studies.

Observational Study on Outcomes after Radioiodine Ablation in Hyperthyroid Patients.

Introduction: Radio-active Iodine (RAI) is a safe, definitive, and cost-effective modality of treatment that is used as the first line of treatment for Graves' hyperthyroidism by most endocrinologists. Very few reports are available from India, observational follow-up data is needed to determine the meaningful prognostic outcomes of RAI ablation in the Indian population. Aims: To study the outcomes in hyperthyroid patients undergoing RAI ablation. Materials and Methods: This observational cohort study was conducted at Department of Endocrinology at Indraprastha Apollo Hospital, New Delhi. A total of 82 hyperthyroid patients who underwent RAI ablation between June 2014 to June 2018 were enrolled. RAI dose was calculated arbitrarily in most cases; often by an empirical fixed dose based on the goiter size and RAIU. The patients were reviewed at 1, 3 and 6 months post-RAI ablation. During follow-up, along with a detailed clinical examination, free T4, free T3 and TSH were checked. Results: The dose of I-131 varied from 6 mCi to 14 mCi. Most of the patients were given RAI in the dose of 7.1-10 mci. About 63.4% of patients achieved hypothyroidism in 6 months, 6.1% in 1 month, 37.8% in 3 months, and 19.5% in 6 months. Gender, age, etiology of hyperthyroidism, baseline thyroid function, goiter, and ophthalmopathy did not affect outcomes after RAI ablation. Those who were not treated with antithyroid drugs prior to RAI therapy were found to have higher rates of conversion to a hypothyroid state. Conclusion: RAI can be given safely as the first line of treatment in Graves' disease and antithyroid drug naïve patients respond better to therapy.

Engineered nanoparticles enable deep proteomics studies at scale by leveraging tunable nano-bio interactions.

SignificanceDeep profiling of the plasma proteome at scale has been a challenge for traditional approaches. We achieve superior performance across the dimensions of precision, depth, and throughput using a panel of surface-functionalized superparamagnetic nanoparticles in comparison to conventional workflows for deep proteomics interrogation. Our automated workflow leverages competitive nanoparticle-protein binding equilibria that quantitatively compress the large dynamic range of proteomes to an accessible scale. Using machine learning, we dissect the contribution of individual physicochemical properties of nanoparticles to the composition of protein coronas. Our results suggest that nanoparticle functionalization can be tailored to protein sets. This work demonstrates the feasibility of deep, precise, unbiased plasma proteomics at a scale compatible with large-scale genomics enabling multiomic studies.

Single-cell RNA-seq reveals ectopic and aberrant lung-resident cell populations in idiopathic pulmonary fibrosis.

We provide a single-cell atlas of idiopathic pulmonary fibrosis (IPF), a fatal interstitial lung disease, by profiling 312,928 cells from 32 IPF, 28 smoker and nonsmoker controls, and 18 chronic obstructive pulmonary disease (COPD) lungs. Among epithelial cells enriched in IPF, we identify a previously unidentified population of aberrant basaloid cells that coexpress basal epithelial, mesenchymal, senescence, and developmental markers and are located at the edge of myofibroblast foci in the IPF lung. Among vascular endothelial cells, we identify an ectopically expanded cell population transcriptomically identical to bronchial restricted vascular endothelial cells in IPF. We confirm the presence of both populations by immunohistochemistry and independent datasets. Among stromal cells, we identify IPF myofibroblasts and invasive fibroblasts with partially overlapping cells in control and COPD lungs. Last, we confirm previous findings of profibrotic macrophage populations in the IPF lung. Our comprehensive catalog reveals the complexity and diversity of aberrant cellular populations in IPF.

Rapid, deep and precise profiling of the plasma proteome with multi-nanoparticle protein corona.

Large-scale, unbiased proteomics studies are constrained by the complexity of the plasma proteome. Here we report a highly parallel protein quantitation platform integrating nanoparticle (NP) protein coronas with liquid chromatography-mass spectrometry for efficient proteomic profiling. A protein corona is a protein layer adsorbed onto NPs upon contact with biofluids. Varying the physicochemical properties of engineered NPs translates to distinct protein corona patterns enabling differential and reproducible interrogation of biological samples, including deep sampling of the plasma proteome. Spike experiments confirm a linear signal response. The median coefficient of variation was 22%. We screened 43 NPs and selected a panel of 5, which detect more than 2,000 proteins from 141 plasma samples using a 96-well automated workflow in a pilot non-small cell lung cancer classification study. Our streamlined workflow combines depth of coverage and throughput with precise quantification based on unique interactions between proteins and NPs engineered for deep and scalable quantitative proteomic studies.

Rab7 of Plasmodium falciparum is involved in its retromer complex assembly near the digestive vacuole.

BACKGROUND: Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate. METHODS: The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined. RESULTS: PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg2+ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly. CONCLUSION: Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum. GENERAL SIGNIFICANCE: This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.

Indomethacin impairs mitochondrial dynamics by activating the PKCζ-p38-DRP1 pathway and inducing apoptosis in gastric cancer and normal mucosal cells.

The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs. Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase Cζ (PKCζ)-p38 MAPK (p38)-dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis. Notably, DRP1 knockdown or SB203580-induced p38 inhibition reduced indomethacin-induced damage to AGSs. Indomethacin impaired mitochondrial dynamics by promoting fissogenic activation and mitochondrial recruitment of DRP1 and down-regulating fusogenic optic atrophy 1 (OPA1) and mitofusins in rat gastric mucosa. Consistent with OPA1 maintaining cristae architecture, its down-regulation resulted in EM-detectable cristae deformity. Deregulated mitochondrial dynamics resulting in defective mitochondria were evident from enhanced Parkin expression and mitochondrial proteome ubiquitination. Indomethacin ultimately induced mitochondrial metabolic and bioenergetic crises in the rat stomach, indicated by compromised fatty acid oxidation, reduced complex I- associated electron transport chain activity, and ATP depletion. Interestingly, Mdivi-1, a fission-preventing mito-protective drug, reversed indomethacin-induced DRP1 phosphorylation on Ser-616, mitochondrial proteome ubiquitination, and mitochondrial metabolic crisis. Mdivi-1 also prevented indomethacin-induced mitochondrial macromolecular damage, caspase activation, mucosal inflammation, and gastric mucosal injury. Our results identify mitochondrial hyper-fission as a critical and common subcellular event triggered by indomethacin that promotes apoptosis in both gastric cancer and normal mucosal cells, thereby contributing to mucosal injury.

Hydrazonophenol, a Food Vacuole-Targeted and Ferriprotoporphyrin IX-Interacting Chemotype Prevents Drug-Resistant Malaria.

The rapid emergence of resistance against frontline antimalarial drugs essentially warrants the identification of new-generation antimalarials. Here, we describe the synthesis of ( E)-2-isopropyl-5-methyl-4-((2-(pyridin-4-yl)hydrazono)methyl)phenol (18), which binds ferriprotoporphyrin-IX (FeIII-PPIX) ( Kd = 33 nM) and offers antimalarial activity against chloroquine-resistant and sensitive strains of Plasmodium falciparum in vitro. Structure-function analysis reveals that compound 18 binds FeIII-PPIX through the -C═N-NH- moiety and 2-pyridyl substitution at the hydrazine counterpart plays a critical role in antimalarial efficacy. Live cell confocal imaging using a fluorophore-tagged compound confirms its accumulation inside the acidic food vacuole (FV) of P. falciparum. Furthermore, this compound concentration-dependently elevates the pH in FV, implicating a plausible interference with FeIII-PPIX crystallization (hemozoin formation) by a dual function: increasing the pH and binding free FeIII-PPIX. Different off-target bioassays reduce the possibility of the promiscuous nature of compound 18. Compound 18 also exhibits potent in vivo antimalarial activity against chloroquine-resistant P. yoelii and P. berghei ANKA (causing cerebral malaria) in mice with negligible toxicity.

Macrophage migration inhibitory factor regulates mitochondrial dynamics and cell growth of human cancer cell lines through CD74-NF-κB signaling.

The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, although which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure. Using different cancer cell lines (AGS, HepG2, HCT116, and HeLa), here we report that the silencing of MIF severely deregulated mitochondrial structural dynamics by shifting the balance toward excess fission, besides inducing apoptosis with increasing sub-G0 cells. Furthermore, enhanced mitochondrial Bax translocation along with cytochrome c release, down-regulation of Bcl-xL, and Bcl-2 as well as up-regulation of Bad, Bax, and p53 indicated the activation of a mitochondrial pathway of apoptosis upon MIF silencing. The data also indicate a concerted down-regulation of Opa1 and Mfn1 along with a significant elevation of Drp1, cumulatively causing mitochondrial fragmentation upon MIF silencing. Up-regulation of Drp1 was found to be further coupled with fissogenic serine 616 phosphorylation and serine 637 dephosphorylation, thus ensuring enhanced mitochondrial translocation. Interestingly, MIF silencing was found to be associated with decreased NF-κB activation. In fact, NF-κB knockdown in turn increased mitochondrial fission and cell death. In addition, the silencing of CD74, the cognate receptor of MIF, remarkably increased mitochondrial fragmentation in addition to preventing cell proliferation, inducing mitochondrial depolarization, and increasing apoptotic cell death. This indicates the active operation of a MIF-regulated CD74-NF-κB signaling axis for maintaining mitochondrial stability and cell growth. Thus, we propose that MIF, through CD74, constitutively activates NF-κB to control mitochondrial dynamics and stability for promoting carcinogenesis via averting apoptosis.