IMC29 Plays an Important Role in Toxoplasma Endodyogeny and Reveals New Components of the Daughter-Enriched IMC Proteome.

The Toxoplasma inner membrane complex (IMC) is a unique organelle that plays critical roles in parasite motility, invasion, egress, and replication. The IMC is delineated into the apical, body, and basal regions, defined by proteins that localize to these distinct subcompartments. The IMC can be further segregated by proteins that localize specifically to the maternal IMC, the daughter bud IMC, or both. While the function of the maternal IMC has been better characterized, the precise roles of most daughter IMC components remain poorly understood. Here, we demonstrate that the daughter protein IMC29 plays an important role in parasite replication. We show that Δimc29 parasites exhibit severe replication defects, resulting in substantial growth defects and loss of virulence. Deletion analyses revealed that IMC29 localization is largely dependent on the N-terminal half of the protein containing four predicted coiled-coil domains while IMC29 function requires a short C-terminal helical region. Using proximity labeling, we identify eight novel IMC proteins enriched in daughter buds, significantly expanding the daughter IMC proteome. We additionally report four novel proteins with unique localizations to the interface between two parasites or to the outer face of the IMC, exposing new subregions of the organelle. Together, this work establishes IMC29 as an important early daughter bud component of replication and uncovers an array of new IMC proteins that provides important insights into this organelle. IMPORTANCE The inner membrane complex (IMC) is a conserved structure across the Apicomplexa phylum, which includes obligate intracellular parasites that cause toxoplasmosis, malaria, and cryptosporidiosis. The IMC is critical for the parasite to maintain its intracellular lifestyle, particularly in providing a scaffold for daughter bud formation during parasite replication. While many IMC proteins in the later stages of division have been identified, components of the early stages of division remain unknown. Here, we focus on the early daughter protein IMC29, demonstrating that it is crucial for faithful parasite replication and identifying specific regions of the protein that are important for its localization and function. We additionally use proximity labeling to reveal a suite of daughter-enriched IMC proteins, which represent promising candidates to further explore this IMC subcompartment.

Reversal of High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease by Metformin Combined with PGG, an Inducer of Glycine N-Methyltransferase.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidities and mortality, and no effective drug treatment currently exists. We aimed to develop a novel treatment strategy to induce the expression of glycine N-methyltransferase (GNMT), which is an important enzyme regulating S-adenosylmethionine metabolism whose expression is downregulated in patients with NAFLD. Because 1,2,3,4,6-pentagalloyl glucose (PGG) is a GNMT inducer, and metformin was shown to upregulate liver mitochondrial GNMT protein expression, the effect of PGG and metformin was evaluated. Biochemical analysis, histopathological examination, immunohistochemical staining, reverse transcription-quantitative PCR (RT-qPCR), Western blotting (WB), proteomic analysis and Seahorse XF Cell Mito Stress Test were performed. The high-fat diet (HFD)-induced NAFLD mice were treated with PGG and metformin. Combination of PGG and metformin nearly completely reversed weight gain, elevation of serum aminotransferases, and hepatic steatosis and steatohepatitis. In addition, the downregulated GNMT expression in liver tissues of HFD-induced NAFLD mice was restored. The GNMT expression was further confirmed by RT-qPCR and WB analysis using both in vitro and in vivo systems. In addition, PGG treatment was shown to increase oxygen consumption rate (OCR) maximum capacity in a dose-dependent manner, and was capable of rescuing the suppression of mitochondrial OCR induced by metformin. Proteomic analysis identified increased expression of glutathione S-transferase mu 4 (GSTM4), heat shock protein 72 (HSP72), pyruvate carboxylase (PYC) and 40S ribosomal protein S28 (RS28) in the metformin plus PGG treatment group. Our findings show that GNMT expression plays an important role in the pathogenesis of NAFLD, and combination of an inducer of GNMT and metformin can be of therapeutic potential for patients with NAFLD.

Genetic variants in DDO and PEX5L in peroxisome-related pathways predict non-small cell lung cancer survival.

Peroxisomes play a role in lipid metabolism and regulation of reactive oxygen species, but its role in development and progression of non-small cell lung cancer (NSCLC) is not well understood. Here, we investigated the associations between 9708 single-nucleotide polymorphisms (SNPs) in 113 genes in the peroxisome-related pathways and survival of NSCLC patients from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) and the Harvard Lung Cancer Susceptibility (HLCS) study. In 1185 NSCLC patients from the PLCO trial, we found that 213 SNPs were significantly associated with NSCLC overall survival (OS) (p ≤ 0.05, Bayesian false discovery probability [BFDP] ≤ 0.80), of which eight SNPs were validated in the HLCS data set. In a multivariate Cox proportional hazards regression model, two independent SNPs (rs9384742 DDO and rs9825224 PEX5L) were significantly associated with NSCLC survival (hazards ratios [HR] of 1.17 with 95% CI [confidence interval] of 1.06-1.28 and 0.86 with 95% CI of 0.77-0.96, respectively). Patients with one or two protective genotypes had a significantly higher OS (HR: 0.787 [95% CI: 0.620-0.998] and 0.691 [95% CI: 0.543-0.879], respectively). Further expression quantitative trait loci analysis using whole blood and lung tissue showed that the minor allele of rs9384742 DDO was significantly associated with decreased messenger RNA (mRNA) expression levels and that DDO expression was also decreased in NSCLC tumor tissue. Additionally, high PEX5L expression levels were significantly associated with lower survival of NSCLC. Our data suggest that variants in these peroxisome-related genes may influence gene regulation and are potential predictors of NSCLC OS, once validated by additional studies.

A Novel Toxoplasma Inner Membrane Complex Suture-Associated Protein Regulates Suture Protein Targeting and Colocalizes with Membrane Trafficking Machinery.

The cytoskeleton of Toxoplasma gondii is composed of the inner membrane complex (IMC) and an array of underlying microtubules that provide support at the periphery of the parasite. Specific subregions of the IMC carry out distinct roles in replication, motility, and host cell invasion. Building on our previous in vivo biotinylation (BioID) experiments of the IMC, we identified here a novel protein that localizes to discrete puncta that are embedded in the parasite's cytoskeleton along the IMC sutures. Gene knockout analysis showed that loss of the protein results in defects in cytoskeletal suture protein targeting, cytoskeletal integrity, parasite morphology, and host cell invasion. We then used deletion analyses to identify a domain in the N terminus of the protein that is critical for both localization and function. Finally, we used the protein as bait for in vivo biotinylation, which identified several other proteins that colocalize in similar spot-like patterns. These putative interactors include several proteins that are implicated in membrane trafficking and are also associated with the cytoskeleton. Together, these data reveal an unexpected link between the IMC sutures and membrane trafficking elements of the parasite and suggest that the suture puncta are likely a portal for trafficking cargo across the IMC. IMPORTANCE The inner membrane complex (IMC) is a peripheral membrane and cytoskeletal system that is organized into intriguing rectangular plates at the periphery of the parasite. The IMC plates are delimited by an array of IMC suture proteins that are tethered to both the membrane and the cytoskeleton and are thought to provide structure to the organelle. Here, we identified a protein that forms discrete puncta that are embedded in the IMC sutures, and we show that it is important for the proper sorting of a group of IMC suture proteins as well as maintaining parasite shape and IMC cytoskeletal integrity. Intriguingly, proximity labeling experiments identified several proteins that are involved in membrane trafficking or endocytosis, suggesting that the IMC puncta provide a gateway for transporting molecules across the structure.

A Case of Rapid Development of Methicillin­Resistant Staphylococcus aureus Mechanical Aortic Root Abscess Despite Appropriate Antibiotic Use.

A 74-year-old male with a past medical history of mechanical aortic valve replacement presented with abdominal pain and fever. Blood cultures revealed methicillin-resistant Staphylococcus aureus (MRSA) and the patient was started on target antibiotics. Initial transthoracic echocardiogram and transesophageal echocardiogram (TEE) did not show any vegetations or significant valvular regurgitation. No other sources of infection were identified. Five days after the initial TEE, a repeat TEE revealed new areas of thickening and echolucency seen anterior to the mechanical aortic valve, suggestive of aortic root abscess (AoRA). It also extended down the mitral-aortic intervalvular fibrosa and was associated with mitral valvular vegetation.  Due to worsening clinical status and persistent bacteremia on appropriate antibiotics, a high index of suspicion for infective endocarditis (IE) remained after the initial TEE. As such, the repeat TEE was obtained only five days after and demonstrated clear evidence of rapidly growing endocarditis and abscess formation. This case uniquely demonstrates how rapid MRSA endocarditis may progress and emphasizes its high mortality. This case highlights the importance of a low threshold for repeat imaging when the index of suspicion for endocarditis remains high despite negative imaging.

Mechanisms linking hypoxia to phosphorylation of insulin-like growth factor binding protein-1 in baboon fetuses with intrauterine growth restriction and in cell culture.

Hypoxia increases fetal hepatic insulin-like growth factor binding protein-1 (IGFBP-1) phosphorylation mediated by mechanistic target of rapamycin (mTOR) inhibition. Whether maternal nutrient restriction (MNR) causes fetal hypoxia remains unclear. We used fetal liver from a baboon (Papio sp.) model of intrauterine growth restriction due to MNR (70% global diet of Control) and liver hepatocellular carcinoma (HepG2) cells as a model for human fetal hepatocytes and tested the hypothesis that mTOR-mediated IGFBP-1 hyperphosphorylation in response to hypoxia requires hypoxia-inducible factor-1α (HIF-1α) and regulated in development and DNA-damage responses-1 (REDD-1) signaling. Western blotting (n = 6) and immunohistochemistry (n = 3) using fetal liver indicated greater expression of HIF-1α, REDD-1 as well as erythropoietin and its receptor, and vascular endothelial growth factor at GD120 (GD185 term) in MNR versus Control. Moreover, treatment of HepG2 cells with hypoxia (1% pO2 ) (n = 3) induced REDD-1, inhibited mTOR complex-1 (mTORC1) activity and increased IGFBP-1 secretion/phosphorylation (Ser101/Ser119/Ser169). HIF-1α inhibition by echinomycin or small interfering RNA silencing prevented the hypoxia-mediated inhibition of mTORC1 and induction of IGFBP-1 secretion/phosphorylation. dimethyloxaloylglycine (DMOG) induced HIF-1α and also REDD-1 expression, inhibited mTORC1 and increased IGFBP-1 secretion/phosphorylation. Induction of HIF-1α (DMOG) and REDD-1 by Compound 3 inhibited mTORC1, increased IGFBP-1 secretion/ phosphorylation and protein kinase PKCα expression. Together, our data demonstrate that HIF-1α induction, increased REDD-1 expression and mTORC1 inhibition represent the mechanistic link between hypoxia and increased IGFBP-1 secretion/phosphorylation. We propose that maternal undernutrition limits fetal oxygen delivery, as demonstrated by increased fetal liver expression of hypoxia-responsive proteins in baboon MNR. These findings have important implications for our understanding of the pathophysiology of restricted fetal growth.

IGFBP-1 hyperphosphorylation in response to nutrient deprivation is mediated by activation of protein kinase Cα (PKCα).

Fetal growth restriction (FGR) is associated with decreased nutrient availability and reduced insulin-line growth factor (IGF)-I bioavailability via increased IGF binding protein (IGFBP)-1 phosphorylation. While protein kinase C (PKC) is implicated in IGFBP-1 hyperphosphorylation in nutrient deprivation, the mechanisms remain unclear. We hypothesised that the interaction of PKCα with protein kinase CK2ß and activation of PKCα under leucine deprivation (L0) mediate fetal hepatic IGFBP-1 hyperphosphorylation. Parallel Reaction Monitoring Mass Spectrometry (PRM-MS) followed by PKCα knockdown demonstrated the PKCα isoform interacts with IGFBP-1 and CK2ß under L0. Pharmacological PKCα activation with phorbol 12-myristate 13-acetate (PMA) increased whereas inhibition with bisindolylmaleimide II (Bis II) decreased IGFBP-1 phosphorylation (Ser101/119/169, Ser98 + 101 and Ser169 + 174), respectively. Furthermore, PMA mimicked L0-induced PKCα translocation and IGFBP-1 expression. PKCα expression was increased in baboon fetal liver in FGR, providing biological relevance in vivo. In summary, we report a novel nutrient-sensitive mechanism for PKCα in mediating IGFBP-1 hyperphosphorylation in FGR.

Prognostic Value of 18F-FDG PET/CT Volume-Based Metabolic Parameters in Patients with Node-Negative Stage II Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a major cancer prevalent in Asian males. Pretreatment tumor burden can be prognostic for ESCC. We studied the prognostic value of metabolic parameters of 2-deoxy-2-[18F] fluoro-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) and the serum squamous cell carcinoma antigen (SCC-Ag) level in node-negative stage II ESCC patients. Eighteen males underwent staging evaluation were included. The volume-based metabolic parameters derived from 18F-FDG PET/CT, including metabolic tumor volume (MTV) and total lesion glycolysis (TLG), were obtained using the PET Volume Computer Assisted Reading application. The Spearman correlation coefficients were calculated to assess the relationship between metabolic parameters and pretreatment serum SCC-Ag levels. Based on the 5-year follow-up, patients were sub-divided into the demised and the stable groups. Potential prognostic value was assessed by independent t-test and the Mann-Whitney U test. The association of overall survival was assessed using univariate and multivariate Cox regression analyses. The demised group showed significant higher values in serum SCC-Ag, as well as in MTV and TLG, but not SUVmax and SUVmean. The SUVmax, MTV, TLG, and serum SCC-Ag showed significant association with overall survival. Our findings suggest potential usage of pretreatment volume-based metabolic parameters of 18F-FDG PET/CT and serum SCC-Ag as prognostic factors for node-negative stage II ESCC patients.

Successful frameless radiosurgery for glossopharyngeal neuralgia - Case report.

Idiopathic glossopharyngeal neuralgia (GPN) is a rare disorder of the ninth cranial nerve characterized by severe paroxysmal pain affecting the ear, tongue and throat. Frame-based radiosurgery (SRS) has been shown to be an effective option. We report the first successful pain alleviation by frameless SRS in a GPN patient that failed both medical and surgical interventions.

Ivosidenib induction therapy complicated by myopericarditis and cardiogenic shock: A case report and literature review.

INTRODUCTION: Ivosidenib is a novel oral inhibitor of mutated isocitrate dehydrogenase 1 approved for the treatment of refractory or relapsed acute myeloid leukemia in patients with isocitrate dehydrogenase 1 mutations or as first-line agent in patients unable to tolerate chemotherapy. It is known to commonly cause differentiation syndrome, but an association with cardiovascular complications is not well established. CASE REPORT: We present the case of a 34-year-old female with relapsed acute myeloid leukemia post-allogeneic transplant who developed ivosidenib-induced differentiation syndrome complicated by myopericarditis and cardiogenic shock. MANAGEMENT AND OUTCOME: Ivosidenib was discontinued, and aggressive management was pursued with high-dose steroids, ventilatory and pressure support and diuresis. She had significant improvement and later tolerated reintroduction of ivosidenib without recurrent episodes of differentiation syndrome or cardiac complications. DISCUSSION: To the best of our knowledge, this is the first reported case of myopericarditis and cardiogenic shock related to ivosidenib use. This case highlights the high index of suspicion required to recognize early signs of targeted therapy-related complications and exemplifies the beneficial collaborative role a cardio-oncology team provides in improving patient care.

A lipid-binding protein mediates rhoptry discharge and invasion in Plasmodium falciparum and Toxoplasma gondii parasites.

Members of the Apicomplexa phylum, including Plasmodium and Toxoplasma, have two types of secretory organelles (micronemes and rhoptries) whose sequential release is essential for invasion and the intracellular lifestyle of these eukaryotes. During invasion, rhoptries inject an array of invasion and virulence factors into the cytoplasm of the host cell, but the molecular mechanism mediating rhoptry exocytosis is unknown. Here we identify a set of parasite specific proteins, termed rhoptry apical surface proteins (RASP) that cap the extremity of the rhoptry. Depletion of RASP2 results in loss of rhoptry secretion and completely blocks parasite invasion and therefore parasite proliferation in both Toxoplasma and Plasmodium. Recombinant RASP2 binds charged lipids and likely contributes to assembling the machinery that docks/primes the rhoptry to the plasma membrane prior to fusion. This study provides important mechanistic insight into a parasite specific exocytic pathway, essential for the establishment of infection.

The Role of 3' to 5' Reverse RNA Polymerization in tRNA Fidelity and Repair.

The tRNAHis guanylyltransferase (Thg1) superfamily includes enzymes that are found in all three domains of life that all share the common ability to catalyze the 3' to 5' synthesis of nucleic acids. This catalytic activity, which is the reverse of all other known DNA and RNA polymerases, makes this enzyme family a subject of biological and mechanistic interest. Previous biochemical, structural, and genetic investigations of multiple members of this family have revealed that Thg1 enzymes use the 3' to 5' chemistry for multiple reactions in biology. Here, we describe the current state of knowledge regarding the catalytic features and biological functions that have been so far associated with Thg1 and its homologs. Progress toward the exciting possibility of utilizing this unusual protein activity for applications in biotechnology is also discussed.

Giant Tricuspid Valve Fibroelastoma Incidentally Diagnosed During Routine Stress Testing.

Papillary fibroelastomas (PFEs) are the most common valvular tumor, typically occurring on left-sided valves. We describe the evaluation and treatment of a giant tricuspid PFE in a healthy 43-year-old police officer who was referred for evaluation of frequent premature ventricular contractions during job-related treadmill stress testing. (Level of Difficulty: Beginner.).

Successful stereotactic body radiation therapy for stage I non-small cell lung cancer with malignant hypercalcemia - Case report.

In patients with lung malignancy, the development of malignant hypercalcemia (MH) carries a dismal prognosis and represents a major therapeutic challenge given that conventional medical treatments have limited durability. Robust and lasting hypercalcemic reversal and symptomatic relief have been documented following surgical tumor resection. Stereotactic body radiation therapy (SBRT) has emerged as an effective treatment modality in medically inoperable patients with Stage I non-small cell lung cancer (NSCLC). In the current report, SBRT achieved durable tumor control and calcemic correction in a medically inoperable 79 year-old man with Stage I NSCLC patient with parathyroid hormone-related peptide (PTHrP)-induced MH.

Retraction Note: New molecular tools in Neospora caninum for studying apicomplexan parasite proteins.

This paper has been retracted at the request of the authors.

Minimal requirements for reverse polymerization and tRNA repair by tRNAHis guanylyltransferase.

tRNAHis guanylyltransferase (Thg1) has unique reverse (3'-5') polymerase activity occurring in all three domains of life. Most eukaryotic Thg1 homologs are essential genes involved in tRNAHis maturation. These enzymes normally catalyze a single 5' guanylation of tRNAHis lacking the essential G-1 identity element required for aminoacylation. Recent studies suggest that archaeal type Thg1, which includes most archaeal and bacterial Thg1 enzymes is phylogenetically distant from eukaryotic Thg1. Thg1 is evolutionarily related to canonical 5'-3' forward polymerases but catalyzes reverse 3'-5'polymerization. Similar to its forward polymerase counterparts, Thg1 encodes the conserved catalytic palm domain and fingers domain. Here we investigate the minimal requirements for reverse polymerization. We show that the naturally occurring minimal Thg1 enzyme from Ignicoccus hospitalis (IhThg1), which lacks parts of the conserved fingers domain, is catalytically active. And adds all four natural nucleotides to RNA substrates, we further show that the entire fingers domain of Methanosarcina acetivorans Thg1 and Pyrobaculum aerophilum Thg1 (PaThg1) is dispensable for enzymatic activity. In addition, we identified residues in yeast Thg1 that play a part in preventing extended polymerization. Mutation of these residues with alanine resulted in extended reverse polymerization. PaThg1 was found to catalyze extended, template dependent tRNA repair, adding up to 13 nucleotides to a truncated tRNAHis substrate. Sequencing results suggest that PaThg1 fully restored the near correct sequence of the D- and acceptor stem, but also produced incompletely and incorrectly repaired tRNA products. This research forms the basis for future engineering efforts towards a high fidelity, template dependent reverse polymerase.

New molecular tools in Neospora caninum for studying apicomplexan parasite proteins.

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system as a heterologous platform for the expression of foreign genes. Plasmid constructs containing fluorescent proteins or targeted genes of Toxoplasma gondii, driven by N. caninum promoters, have yielded robust expression and correct trafficking of target gene products as assessed by immunofluorescence assays and Western blot analyses. Using this approach, we here demonstrated that N. caninum expressing T. gondii's GRA15 and ROP16 kinase are biologically active and induced immunological phenotypes consistent with T. gondii strains. N. caninum expressing TgGRA15 differentially disturbed the NF-κB pathway, inducing an increased IL-12 production. On the other hand, N. caninum expressing TgROP16 induced host STAT3 phosphorylation and consequent reduction of IL-12 synthesis. These results indicate that heterologous gene expression in N. caninum is a useful tool for the study of specific gene functions and may allow the identification of antigenic targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. Additionally, these observations may prove to be useful for the development of vaccine protocols to control toxoplasmosis and/or neosporosis.

NEUTRON AND PHOTON DOSE MAPPING OF A DD NEUTRON GENERATOR.

Neutron generators are an excellent tool that can be effectively utilized in educational institutions for applications such as neutron activation analysis, neutron radiography, and profiling and irradiation effects. For safety purposes, it is imperative that appropriate measures are taken in order to minimize the radiation dose from such devices to the operators, students and the public. This work presents the simulation and measurement results for the neutron and photon dose rates in the vicinity of the neutron generator installed at the University of Sharjah. A very good agreement is found between the simulated and measured dose rates. All of the public dose constraints were found to be met. The occupational dose constraint was also met after imposing a 200 cm no entry zone around the generator room.

Novel insights into the composition and function of the Toxoplasma IMC sutures.

The Toxoplasma inner membrane complex (IMC) is a specialized organelle underlying the parasite's plasma membrane that consists of flattened rectangular membrane sacs that are sutured together and positioned atop a supportive cytoskeleton. We have previously identified a novel class of proteins localizing to the transverse and longitudinal sutures of the IMC, which we named IMC sutures components (ISCs). Here, we have used proximity-dependent biotin identification at the sutures to better define the composition of this IMC subcompartment. Using ISC4 as bait, we demonstrate biotin-dependent labeling of the sutures and have uncovered two new ISCs. We also identified five new proteins that exclusively localize to the transverse sutures that we named transverse sutures components (TSCs), demonstrating that components of the IMC sutures consist of two groups: those that localize to the transverse and longitudinal sutures (ISCs) and those residing only in the transverse sutures (TSCs). In addition, we functionally analyze the ISC protein ISC3 and demonstrate that ISC3-null parasites have morphological defects and reduced fitness in vitro. Most importantly, Δisc3 parasites exhibit a complete loss of virulence in vivo. These studies expand the known composition of the IMC sutures and highlight the contribution of ISCs to the ability of the parasite to proliferate and cause disease.