Transcriptomic and functional analysis of ANGPTL4 overexpression in pancreatic cancer nominates targets that reverse chemoresistance.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers based on five-year survival rates. Genes contributing to chemoresistance represent novel therapeutic targets that can improve treatment response. Increased expression of ANGPTL4 in tumors correlates with poor outcomes in pancreatic cancer. METHODS: We used statistical analysis of publicly available gene expression data (TCGA-PAAD) to test whether expression of ANGPTL4 and its downstream targets, ITGB4 and APOL1, were correlated with patient survival. We measured the impact of ANGPTL4 overexpression in a common pancreatic cancer cell line, MIA PaCa-2 cells, using CRISPRa for overexpression and DsiRNA for knockdown. We characterized global gene expression changes associated with high levels of ANGPTL4 and response to gemcitabine treatment using RNA-sequencing. Gemcitabine dose response curves were calculated on modified cell lines by measuring cell viability with CellTiter-Glo (Promega). Impacts on cell migration were measured using a time course scratch assay. RESULTS: We show that ANGPTL4 overexpression leads to in vitro resistance to gemcitabine and reduced survival times in patients. Overexpression of ANGPTL4 induces transcriptional signatures of tumor invasion and metastasis, proliferation and differentiation, and inhibition of apoptosis. Analyses revealed an overlapping signature of genes associated with both ANGPTL4 activation and gemcitabine response. Increased expression of the genes in this signature in patient PDAC tissues was significantly associated with shorter patient survival. We identified 42 genes that were both co-regulated with ANGPTL4 and were responsive to gemcitabine treatment. ITGB4 and APOL1 were among these genes. Knockdown of either of these genes in cell lines overexpressing ANGPTL4 reversed the observed gemcitabine resistance and inhibited cellular migration associated with epithelial to mesenchymal transition (EMT) and ANGPTL4 overexpression. CONCLUSIONS: These data suggest that ANGPTL4 promotes EMT and regulates the genes APOL1 and ITGB4. Importantly, we show that inhibition of both targets reverses chemoresistance and decreases migratory potential. Our findings have revealed a novel pathway regulating tumor response to treatment and suggest relevant therapeutic targets in pancreatic cancer.

The Integrative Studies on the Functional A-to-I RNA Editing Events in Human Cancers.

Adenosine-to-inosine (A-to-I) RNA editing, constituting nearly 90% of all RNA editing events in humans, has been reported to contribute to the tumorigenesis in diverse cancers. However, the comprehensive map for functional A-to-I RNA editing events in cancers is still insufficient. To fill this gap, we systematically and intensively analyzed multiple tumorigenic mechanisms of A-to-I RNA editing events in samples across 33 cancer types from The Cancer Genome Atlas. For individual candidate among ∼ 1,500,000 quantified RNA editing events, we performed diverse types of downstream functional annotations. Finally, we identified 24,236 potentially functional A-to-I RNA editing events, including the cases in APOL1, IGFBP3, GRIA2, BLCAP, and miR-589-3p. These events might play crucial roles in the scenarios of tumorigenesis, due to their tumor-related editing frequencies or probable effects on altered expression profiles, protein functions, splicing patterns, and microRNA regulations of tumor genes. Our functional A-to-I RNA editing events (https://ccsm.uth.edu/CAeditome/) will help better understand the cancer pathology from the A-to-I RNA editing aspect.

Treatment potential in APOL1-associated nephropathy.

PURPOSE OF REVIEW: More than 5 million African-Americans, and millions more in Africa and worldwide, possess apolipoprotein L1 gene (APOL1) high-risk genotypes with an increased risk for chronic kidney disease. This manuscript reviews treatment approaches for slowing the progression of APOL1-associated nephropathy. RECENT FINDINGS: Since the 2010 discovery of APOL1 as a cause of nondiabetic nephropathy in individuals with sub-Saharan African ancestry, it has become apparent that aggressive hypertension control, renin-angiotensin system blockade, steroids and conventional immunosuppressive agents are suboptimal treatments. In contrast, APOL1-mediated collapsing glomerulopathy due to interferon treatment and HIV infection, respectively, often resolve with cessation of interferon or antiretroviral therapy. Targeted therapies, including APOL1 small molecule inhibitors, APOL1 antisense oligonucleotides (ASO) and inhibitors of APOL1-associated inflammatory pathways, hold promise for these diseases. Evolving therapies and the need for clinical trials support the importance of increased use of APOL1 genotyping and kidney biopsy. SUMMARY: APOL1-associated nephropathy includes a group of related phenotypes that are driven by the same two genetic variants in APOL1. Clinical trials of small molecule inhibitors, ASO, and inflammatory pathway inhibitors may improve outcomes in patients with primary forms of APOL1-associated nephropathy.

A Four Autophagy-Related Gene-Based Prognostic Signature for Pancreatic Cancer.

This study aimed to screen autophagy-related genes (ARGs) that affect the prognosis of pancreatic cancer patients based on The Cancer Genome Atlas (TCGA) gene expression data and genotype-tissue expression (GTEx) databases. The expression data of pancreatic cancer and normal pancreas were downloaded from TCGA and GTEx databases. Human ARGs list was obtained through the Human Autophagy Database (HADB) and GeneCards database. The Wilcox test was performed to screen differentially expressed ARGs. Differentially expressed ARGs were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses. The CIBER-SORT algorithm was utilized to analyze immune cell infiltration in samples. A total of 21 up-regulated ARGs and 11 down-regulated ARGs were screened in the TCGA-GTEx integrated data set. The enrichment analysis of GO and KEGG showed that 32 differentially expressed ARGs were significantly enriched in autophagy-related pathways. Univariate Cox regression analysis showed that 12 candidate ARGs were significantly related to the prognosis of pancreatic cancer patients. Multivariate Cox regression analysis found that ATG16L2, GNAI3, APOL1, and PTK6 genes may be the key ARGs affecting the prognosis of pancreatic cancer patients. Based on these four key ARGs, a prognostic risk assessment model was constructed, and pancreatic cancer patients were classified into the high-risk and low-risk group according to the risk value. Survival analysis and ROC analysis confirmed that the prognostic risk assessment model can accurately predict the prognosis of patients with pancreatic cancer. Immune infiltration analysis found that B cells naive, B cells memory, plasma cells, T cells CD8, T cells CD4 memory resting, monocytes and macrophages M0 were significantly different in tissue samples of pancreatic cancer patients in the high and low risk groups. Pearson's correlation coefficient showed that the four key ARGs may affect the development of pancreatic cancer by affecting immune cell components in the tumor micro-environment. In conclusion, ATG16L2, GNAI3, APOL1, and PTK6 may be related to the prognosis of pancreatic cancer patients. The prognostic risk assessment model constructed based on these four key ARGs could accurately predict the prognosis of pancreatic cancer patients.

Oncogene APOL1 promotes proliferation and inhibits apoptosis via activating NOTCH1 signaling pathway in pancreatic cancer.

APOL1 encodes a secreted high-density lipoprotein, which has been considered as an aberrantly expressed gene in multiple cancers. Nevertheless, the role of APOL1 in the regulatory mechanisms of pancreatic cancer remains unknown and should be explored. We identified APOL1 was abnormally elevated in human pancreatic cancer tissues compared with that in adjacent tissues and was associated with poor prognosis. The effects of APOL1 in PC cell proliferation, cell cycle, and apoptosis was verified via functional in vitro and in vivo experiments. The results showed that knockdown of APOL1 significantly inhibited the proliferation and promoted apoptosis of pancreatic cancer. In addition, we identified APOL1 could be a regulator of NOTCH1 signaling pathway using bioinformatics tools, qRT-PCR, dual-luciferase reporter assay, and western blotting. In summary, APOL1 could function as an oncogene to promote proliferation and inhibit apoptosis through activating NOTCH1 signaling pathway expression in pancreatic cancer; therefore, it may act as a novel therapeutic target for pancreatic cancer.

Coiled-coil binding of the leucine zipper domains of APOL1 is necessary for the open cation channel conformation.

Apolipoprotein L-I (APOL1) is a channel-forming effector of innate immunity. The common human APOL1 variant G0 provides protection against infection with certain Trypanosoma and Leishmania parasite species, but it cannot protect against the trypanosomes responsible for human African trypanosomiasis. Human APOL1 variants G1 and G2 protect against human-infective trypanosomes but also confer a higher risk of developing chronic kidney disease. Trypanosome-killing activity is dependent on the ability of APOL1 to insert into membranes at acidic pH and form pH-gated cation channels. We previously mapped the channel's pore-lining region to the C-terminal domain (residues 332-398) and identified a membrane-insertion domain (MID, residues 177-228) that facilitates acidic pH-dependent membrane insertion. In this article, we further investigate structural determinants of cation channel formation by APOL1. Using a combination of site-directed mutagenesis and targeted chemical modification, our data indicate that the C-terminal heptad-repeat sequence (residues 368-395) is a bona fide leucine zipper domain (ZIP) that is required for cation channel formation as well as lysis of trypanosomes and mammalian cells. Using protein-wide cysteine-scanning mutagenesis, coupled with the substituted cysteine accessibility method, we determined that, in the open channel state, both the N-terminal domain and the C-terminal ZIP domain are exposed on the intralumenal/extracellular side of the membrane and provide evidence that each APOL1 monomer contributes four transmembrane domains to the open cation channel conformation. Based on these data, we propose an oligomeric topology model in which the open APOL1 cation channel is assembled from the coiled-coil association of C-terminal ZIP domains.

The function of apolipoproteins L (APOLs): relevance for kidney disease, neurotransmission disorders, cancer and viral infection.

The discovery that apolipoprotein L1 (APOL1) is the trypanolytic factor of human serum raised interest about the function of APOLs, especially following the unexpected finding that in addition to their protective action against sleeping sickness, APOL1 C-terminal variants also cause kidney disease. Based on the analysis of the structure and trypanolytic activity of APOL1, it was proposed that APOLs could function as ion channels of intracellular membranes and be involved in mechanisms triggering programmed cell death. In this review, the recent finding that APOL1 and APOL3 inversely control the synthesis of phosphatidylinositol-4-phosphate (PI(4)P) by the Golgi PI(4)-kinase IIIB (PI4KB) is commented. APOL3 promotes Ca2+ -dependent activation of PI4KB, but due to their increased interaction with APOL3, APOL1 C-terminal variants can inactivate APOL3, leading to reduction of Golgi PI(4)P synthesis. The impact of APOLs on several pathological processes that depend on Golgi PI(4)P levels is discussed. I propose that through their effect on PI4KB activity, APOLs control not only actomyosin activities related to vesicular trafficking, but also the generation and elongation of autophagosomes induced by inflammation.

Apolipoprotein L1 is transcriptionally regulated by SP1, IRF1 and IRF2 in hepatoma cells.

Apolipoprotein L1 (APOL1) participates in lipid metabolism. Here, we investigate the mechanisms regulating APOL1 gene expression in hepatoma cells. We demonstrate that the -80-nt to +31-nt region of the APOL1 promoter, which contains one SP transcription factor binding GT box and an interferon regulatory factor (IRF) binding ISRE element, maintains the maximum activity. Mutation of the GT box and ISRE element dramatically reduces APOL1 promoter activity. EMSA and chromatin immunoprecipitation assay reveal that the transcription factors Sp1, IRF1 and IRF2 could interact with their cognate binding sites on the APOL1 promoter. Overexpression of Sp1, IRF1 and IRF2 increases promoter activity, leading to increased APOL1 mRNA and protein levels, while knockdown of Sp1, IRF1 and IRF2 has the opposite effects. These results demonstrate that the APOL1 gene could be regulated by Sp1, IRF1 and IRF2 in hepatoma cells.

ApoL1 induces kidney inflammation through RIG-I/NF-κB activation.

The genetic variations of the apolipoprotein L1 (APOL1) gene are associated with non-diabetic kidney diseases. However, very little is known about the role of ApoL1 in glomerular damage. Here, we aimed to identify the function and mechanism of ApoL1 in glomerular damage. The mice were randomly divided into two groups: one group was intraperitoneally injected with phosphate buffer saline (PBS), while the other group was intraperitoneally injected with recombinant ApoL1 every other day for 3 months. Hematoxylin and eosin (HE) and periodic acid Schiff (PAS) staining were used to demonstrate the effects of ApoL1 on kidney inflammation and injury. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses revealed that ApoL1-treated mice exhibited enhanced expression of various inflammation markers in the kidney and serum compared to the PBS-treated mice. Immunofluorescence staining revealed that ApoL1 accumulated in kidney podocytes. Treatment with ApoL1 dose-dependently increased the expression of inflammation markers and apoptotic markers. The abnormal gene expression associated with ApoL1-mediated podocyte inflammation was evaluated using microarray analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that the upregulated genes were enriched in the inflammation-related processes, such as the RIG-I/NF-κB signaling pathway. Consistently, the knockdown of RIG-I significantly mitigated the ApoL1-induced upregulation of inflammatory and apoptotic markers in the human podocytes. Additionally, the ApoL1-induced glomerular damage was attenuated in AAV-shRIG-I mice. Therefore, the effects of ApoL1 on glomerular damage may be, at least partially, through inducing abnormal expression of inflammatory molecules, which may have important implications for treatment of kidney diseases.

APOL1-Associated Collapsing Focal Segmental Glomerulosclerosis in a Patient With Stimulator of Interferon Genes (STING)-Associated Vasculopathy With Onset in Infancy (SAVI).

Apolipoprotein L1 (APOL1) risk variants G1 and G2 are known to result in risk for kidney disease in patients of African ancestry. APOL1-associated nephropathy typically occurs in association with certain environmental factors or systemic diseases. As such, there has been increasing evidence of the role of interferon (IFN) pathways in the pathogenesis of APOL1-associated collapsing glomerulopathy in patients with human immunodeficiency virus (HIV) infection and systemic lupus erythematosus, 2 conditions that are associated with high IFN levels. Collapsing glomerulopathy has also been described in patients receiving exogenous IFN therapy administered for various medical conditions. We describe a patient with a genetic condition that results in an increased IFN state, stimulator of IFN genes (STING)-associated vasculopathy with onset in infancy (SAVI), who developed collapsing glomerulopathy during a flare of his disease. The patient was found to have APOL1 G1 and G2 risk variants. This case supports the role of IFN in inducing APOL1-associated collapsing glomerulopathy.

Disrupted apolipoprotein L1-miR193a axis dedifferentiates podocytes through autophagy blockade in an APOL1 risk milieu.

We hypothesized that a functional apolipoprotein LI (APOL1)-miR193a axis (inverse relationship) preserves, but disruption alters, the podocyte molecular phenotype through the modulation of autophagy flux. Podocyte-expressing APOL1G0 (G0-podocytes) showed downregulation but podocyte-expressing APOL1G1 (G1-podocytes) and APOL1G2 (G2-podocytes) displayed enhanced miR193a expression. G0-, G1-, and G2-podocytes showed enhanced expression of light chain (LC) 3-II and beclin-1, but a disparate expression of p62 (low in wild-type but high in risk alleles). G0-podocytes showed enhanced, whereas G1- and G2-podocytes displayed decreased, phosphorylation of Unc-51-like autophagy-activating kinase (ULK)1 and class III phosphatidylinositol 3-kinase (PI3KC3). Podocytes overexpressing miR193a (miR193a-podocytes), G1, and G2 showed decreased transcription of PIK3R3 (PI3KC3's regulatory unit). Since 3-methyladenine (3-MA) enhanced miR193a expression but inhibited PIK3R3 transcription, it appears that 3-MA inhibits autophagy and induces podocyte dedifferentiation via miR193a generation. miR193a-, G1-, and G2-podocytes also showed decreased phosphorylation of mammalian target of rapamycin (mTOR) that could repress lysosome reformation. G1- and G2-podocytes showed enhanced expression of run domain beclin-1-interacting and cysteine-rich domain-containing protein (Rubicon); however, its silencing prevented their dedifferentiation. Docking, protein-protein interaction, and immunoprecipitation studies with antiautophagy-related gene (ATG)14L, anti-UV radiation resistance-associated gene (UVRAG), or Rubicon antibodies suggested the formation of ATG14L complex I and UVRAG complex II in G0-podocytes and the formation of Rubicon complex III in G1- and G2-podocytes. These findings suggest that the APOL1 risk alleles favor podocyte dedifferentiation through blockade of multiple autophagy pathways.

Human inborn errors of immunity to infection affecting cells other than leukocytes: from the immune system to the whole organism.

Studies of vertebrate immunity have traditionally focused on professional cells, including circulating and tissue-resident leukocytes. Evidence that non-professional cells are also intrinsically essential (i.e. not via their effect on leukocytes) for protective immunity in natural conditions of infection has emerged from three lines of research in human genetics. First, studies of Mendelian resistance to infection have revealed an essential role of DARC-expressing erythrocytes in protection against Plasmodium vivax infection, and an essential role of FUT2-expressing intestinal epithelial cells for protection against norovirus and rotavirus infections. Second, studies of inborn errors of non-hematopoietic cell-extrinsic immunity have shown that APOL1 and complement cascade components secreted by hepatocytes are essential for protective immunity to trypanosome and pyogenic bacteria, respectively. Third, studies of inborn errors of non-hematopoietic cell-intrinsic immunity have suggested that keratinocytes, pulmonary epithelial cells, and cortical neurons are essential for tissue-specific protective immunity to human papillomaviruses, influenza virus, and herpes simplex virus, respectively. Various other types of genetic resistance or predisposition to infection in human populations are not readily explained by inborn variants of genes operating in leukocytes and may, therefore, involve defects in other cells. The probing of this unchartered territory by human genetics is reshaping immunology, by scaling immunity to infection up from the immune system to the whole organism.

APOL1 renal risk variants promote cholesterol accumulation in tissues and cultured macrophages from APOL1 transgenic mice.

Apolipoprotein L1 (APOL1) genetic variants G1 and G2, compared to the common allele G0, are major risk factors for non-diabetic kidney disease in African descent populations. APOL1 is a minor protein component of HDL, as well as being expressed in podocytes and vascular cells. Reverse cholesterol transport involves the transport of cholesterol to HDL by cellular ATP-binding cassette; ABCA1 and ABCG1 with subsequent delivery from peripheral tissues to the liver. With impaired reverse cholesterol transport, lipid accumulation occurs and macrophages morphologically transform into foam cells, releasing inflammatory factors. We asked whether the APOL1 risk variants alter peripheral cholesterol metabolism and specifically affect macrophage cholesterol efflux. Tissues and bone marrow (BM)-derived monocytes were isolated from wild-type mice (WT) and from BAC/APOL1 transgenic (APOL1-G0, APOL1-G1, and APOL1-G2) mice, which carry a bacterial artificial chromosome that contains the human APOL1 genomic region. Monocytes were differentiated into macrophages using M-CSF, and then polarized into M1 and M2 macrophages. Cholesterol content, cholesterol efflux, and ABCA1 and ABCG1 mRNA expression were measured. Kidney, spleen, and bone marrow-derived macrophages from APOL1-G1 and -G2 mice showed increased cholesterol accumulation and decreased ABCA1 and ABCG1 mRNA levels. BM-derived macrophages from APOL1-G1 and -G2 mice showed significantly reduced cholesterol efflux compared to WT or APOL1-G0 macrophages. Taken together, the evidence suggests that APOL1-G1 and -G2 risk variants impaired reverse cholesterol transport through decreased expression of cholesterol efflux transporters suggesting a possible mechanism to promote macrophage foam cell formation, driving inflammation in the glomerulus and renal interstitium.

Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition.

Human parietal epithelial cells (PECs) are progenitor cells that sustain podocyte homeostasis. We hypothesized that the lack of apolipoprotein (APO) L1 ensures the PEC phenotype, but its induction initiates PEC transition (expression of podocyte markers). APOL1 expression and down-regulation of miR193a coincided with the expression of podocyte markers during the transition. The induction of APOL1 also stimulated transition markers in human embryonic kidney cells (cells with undetectable APOL1 protein expression). APOL1 silencing in PECs up-regulated miR193a expression, suggesting the possibility of a reciprocal feedback relationship between APOL1 and miR193a. HIV, interferon-γ, and vitamin D receptor agonist down-regulated miR193a expression and induced APOL1 expression along with transition markers in PECs. Luciferase assay suggested a putative interaction between miR193a and APOL1. Since silencing of APOL1 attenuated HIV-, vitamin D receptor agonist-, miR193a inhibitor-, and interferon-γ-induced expression of transition markers, APOL1 appears to be a critical functional constituent of the miR193a- APOL1 axis in PECs. This notion was confirmed by further enhanced expression of PEC markers in APOL1 mRNA-silenced PECs. In vivo studies, glomeruli in patients with HIV, and HIV/APOL1 transgenic mice had foci of PECs expressing synaptopodin, a transition marker. APOL1 likely regulates PEC molecular phenotype through modulation of miR193a expression, and APOL1 and miR193a share a reciprocal feedback relationship.

ApoL1 renal risk variants induce aberrant THP-1 monocyte differentiation and increase eicosanoid production via enhanced expression of cyclooxygenase-2.

Apolipoprotein L1 ( ApoL1) genetic variants are strongly associated with kidney diseases. We investigated the role of ApoL1 variants in monocyte differentiation and eicosanoid production in macrophages, as activated tissue macrophages in kidney might contribute to kidney injury. In human monocyte THP-1 cells, transient overexpression of ApoL1 (G0, G1, G2) by transfection resulted in a 5- to 11-fold increase in CD14 and CD68 gene expression, similar to that seen with phorbol-12-myristate acetate treatment. All ApoL1 variants caused monocytes to differentiate into atypical M1 macrophages with marked increase in M1 markers CD80, TNF, IL1B, and IL6 and modest increase in the M2 marker CD163 compared with control cells. ApoL1-G1 transfection induced additional CD206 and TGFB1 expression, and ApoL1-G2 transfection induced additional CD204 and TGFB1 expression. Gene expression of prostaglandin E2 (PGE2) synthase and thromboxane synthase and both gene and protein expression of cyclooxygenase-2 (COX-2) were increased by ApoL1-G1 and -G2 variants compared with -G0 transfection. Higher levels of PGE2 and thromboxane B2, a stable metabolite of thromboxane A2, and transforming growth factor (TGF)-ß1 were released into the supernatant of cultured THP-1 cells transfected with ApoL1-G1 and -G2, but not -G0. The increase in PGE2, thromboxane B2, and TGF-ß1 was inhibited by COX-2-specific inhibitor CAY10404 but not by COX-1-specific inhibitor SC-560. These results demonstrate a novel role of ApoL1 variants in the regulation of monocyte differentiation and eicosanoid metabolism, which could modify the immune response and promote inflammatory signaling within the local targeted organs and tissues including the kidney.

Modulation of apolipoprotein L1-microRNA-193a axis prevents podocyte dedifferentiation in high-glucose milieu.

The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms' tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.

Decoding the network of Trypanosoma brucei proteins that determines sensitivity to apolipoprotein-L1.

In contrast to Trypanosoma brucei gambiense and T. b. rhodesiense (the causative agents of human African trypanosomiasis), T. b. brucei is lysed by apolipoprotein-L1 (apoL1)-containing human serum trypanolytic factors (TLF), rendering it non-infectious to humans. While the mechanisms of TLF1 uptake, apoL1 membrane integration, and T. b. gambiense and T. b. rhodesiense apoL1-resistance have been extensively characterised, our understanding of the range of factors that drive apoL1 action in T. b. brucei is limited. Selecting our bloodstream-form T. b. brucei RNAi library with recombinant apoL1 identified an array of factors that supports the trypanocidal action of apoL1, including six putative ubiquitin modifiers and several proteins putatively involved in membrane trafficking; we also identified the known apoL1 sensitivity determinants, TbKIFC1 and the V-ATPase. Most prominent amongst the novel apoL1 sensitivity determinants was a putative ubiquitin ligase. Intriguingly, while loss of this ubiquitin ligase reduces parasite sensitivity to apoL1, its loss enhances parasite sensitivity to TLF1-dominated normal human serum, indicating that free and TLF1-bound apoL1 have contrasting modes-of-action. Indeed, loss of the known human serum sensitivity determinants, p67 (lysosomal associated membrane protein) and the cathepsin-L regulator, 'inhibitor of cysteine peptidase', had no effect on sensitivity to free apoL1. Our findings highlight a complex network of proteins that influences apoL1 action, with implications for our understanding of the anti-trypanosomal action of human serum.

The effect of normal human serum on the mouse trypanosome Trypanosoma musculi in vitro and in vivo.

Trypanosoma musculi, a common blood flagellate found in mice, is similar in morphology and life cycle to the rat trypanosome T. lewisi. Both species belong to the subgenus Herpetosoma, and as T. lewisi has recently been shown to be a zoonotic pathogen, there is concern that T. musculi could also be potentially infective to humans. To test this hypothesis, a well-established method, the normal human serum (NHS) incubation test, was carried out which distinguishes human and non-human infective trypanosomes. We found that T. musculi could grow in 0.31% NHS in vitro, and even kept their infectivity to mice after incubation with 10% NHS for 24 h. In in vivo experiments, T. musculi were only slightly affected by NHS injection, confirming that it was less sensitive to the NHS than T. b. brucei, but more sensitive than T. lewisi. This resistance probably does not rely on a restricted uptake of ApoL-1. Due to this partial resistance, we cannot definitively confirm that T. musculi has the potential for infection to humans. As resistance is less than that of T. lewisi, our data suggest that it is unlikely to be a zoonotic pathogen although we would advise caution in the case of immunocompromised people such as AIDS and cancer patients.

Association Between APOL1 Genotypes and Risk of Cardiovascular Disease in MESA (Multi-Ethnic Study of Atherosclerosis).

BACKGROUND: APOL1 genetic variants confer an increased risk for kidney disease. Their associations with cardiovascular disease (CVD) are less certain. We aimed to compare the prevalence of subclinical CVD and incidence of atherosclerotic CVD and heart failure by APOL1 genotypes among self-identified black participants of MESA (Multi-Ethnic Study of Atherosclerosis). METHODS AND RESULTS: Cross-sectional associations of APOL1 genotypes (high-risk=2 alleles; low-risk=0 or 1 allele) with coronary artery calcification, carotid-intimal media thickness, and left ventricular mass were evaluated using logistic and linear regression. Longitudinal associations of APOL1 genotypes with incident myocardial infarction, stroke, coronary heart disease, and congestive heart failure were examined using Cox regression. We adjusted for African ancestry, age, and sex. We also evaluated whether hypertension or kidney function markers explained the observed associations. Among 1746 participants with APOL1 genotyping (mean age 62 years, 55% women, mean cystatin C-based estimated glomerular filtration rate 89 mL/min per 1.73 m2, 12% with albuminuria), 12% had the high-risk genotypes. We found no difference in prevalence or severity of coronary artery calcification, carotid-intimal media thickness, or left ventricular mass by APOL1 genotypes. The APOL1 high-risk group was 82% more likely to develop incident heart failure compared with the low-risk group (95% confidence interval, 1.01-3.28). Adjusting for hypertension (hazard ratio, 1.80; 95% confidence interval, 1.00-3.24) but not markers of kidney function (hazard ratio, 1.86; 95% confidence interval, 1.03-3.35) slightly attenuated this association. The APOL1 high-risk genotypes were not significantly associated with other clinical CVD outcomes. CONCLUSIONS: Among blacks without baseline CVD, the APOL1 high-risk variants may be associated with increased risk for incident heart failure but not subclinical CVD or incident clinical atherosclerotic CVD.