Transmission electron microscopy on a case of Cyclospora cayetanensis infection from an immune-competent case confirms and extends prior detailed descriptions of its notably small endogenous stage.

Although infections with Cyclospora cayetanensis are prevalent worldwide, many aspects of this parasite's life cycle remain unknown. Humans are the only known hosts, existing information on its endogenous development has been derived from histological examination of only a few biopsy specimens. In histological sections, its stages are less than 10 µm, making definitive identification difficult. Here, confirmation of cyclosporiasis in a duodenal biopsy specimen from an 80-year-old man without any recognized immunodeficiency patient is reported. Asexual forms (schizonts) and sexual forms (gamonts) were located within enterocytes, including immature and mature schizonts, an immature male gamont and a female gamont. Merozoites were small (<5 µm × 1 µm) and contained two rhoptries, subterminal nucleus and numerous micronemes and amylopectin granules. These parasite stages were like those recently reported in the gallbladder of an immunocompromised patient, suggesting that the general life-cycle stages are not altered by immunosuppression.

Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition.

Severe cardiovascular complications can occur in coronavirus disease of 2019 (COVID-19) patients. Cardiac damage is attributed mostly to the aberrant host response to acute respiratory infection. However, direct infection of cardiac tissue by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also occurs. We examined here the cardiac tropism of SARS-CoV-2 in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These cardiomyocytes express the angiotensin-converting enzyme 2 (ACE2) receptor but not the transmembrane protease serine 2 (TMPRSS2) that mediates spike protein cleavage in the lungs. Nevertheless, SARS-CoV-2 infection of hiPSC-CMs was prolific; viral transcripts accounted for about 88% of total mRNA. In the cytoplasm of infected hiPSC-CMs, smooth-walled exocytic vesicles contained numerous 65- to 90-nm particles with canonical ribonucleocapsid structures, and virus-like particles with knob-like spikes covered the cell surface. To better understand how SARS-CoV-2 spreads in hiPSC-CMs, we engineered an expression vector coding for the spike protein with a monomeric emerald-green fluorescent protein fused to its cytoplasmic tail (S-mEm). Proteolytic processing of S-mEm and the parental spike were equivalent. Live cell imaging tracked spread of S-mEm cell-to-cell and documented formation of syncytia. A cell-permeable, peptide-based molecule that blocks the catalytic site of furin and furin-like proteases abolished cell fusion. A spike mutant with the single amino acid change R682S that disrupts the multibasic furin cleavage motif was fusion inactive. Thus, SARS-CoV-2 replicates efficiently in hiPSC-CMs and furin, and/or furin-like-protease activation of its spike protein is required for fusion-based cytopathology. This hiPSC-CM platform enables target-based drug discovery in cardiac COVID-19. IMPORTANCE Cardiac complications frequently observed in COVID-19 patients are tentatively attributed to systemic inflammation and thrombosis, but viral replication has occasionally been confirmed in cardiac tissue autopsy materials. We developed an in vitro model of SARS-CoV-2 spread in myocardium using induced pluripotent stem cell-derived cardiomyocytes. In these highly differentiated cells, viral transcription levels exceeded those previously documented in permissive transformed cell lines. To better understand the mechanisms of SARS-CoV-2 spread, we expressed a fluorescent version of its spike protein that allowed us to characterize a fusion-based cytopathic effect. A mutant of the spike protein with a single amino acid mutation in the furin/furin-like protease cleavage site lost cytopathic function. Of note, the fusion activities of the spike protein of other coronaviruses correlated with the level of cardiovascular complications observed in infections with the respective viruses. These data indicate that SARS-CoV-2 may cause cardiac damage by fusing cardiomyocytes.

Diagnostic laboratory standardization and validation of platelet transmission electron microscopy.

Platelet transmission electron microscopy (PTEM) is considered the gold standard test for assessing distinct ultrastructural abnormalities in inherited platelet disorders (IPDs). Nevertheless, PTEM remains mainly a research tool due to the lack of standardized procedures, a validated dense granule (DG) count reference range, and standardized image interpretation criteria. The aim of this study was to standardize and validate PTEM as a clinical laboratory test. Based on previously established methods, we optimized and standardized preanalytical, analytical, and postanalytical procedures for both whole mount (WM) and thin section (TS) PTEM. Mean number of DG/platelet (plt), percentage of plts without DG, platelet count (PC), mean platelet volume (MPV), immature platelet fraction (IPF), and plt light transmission aggregometry analyses were measured on blood samples from 113 healthy donors. Quantile regression was used to estimate the reference range for DG/plt, and linear regression was used to assess the association of DG/plt with other plt measurements. All PTEM procedures were standardized using commercially available materials and reagents. DG interpretation criteria were established based on previous publications and expert consensus, and resulted in improved operator agreement. Mean DG/plt was stable for 2 days after blood sample collection. The median within patient coefficient of variation for mean DG/plt was 22.2%; the mean DG/plt reference range (mid-95th %) was 1.2-4.0. Mean DG/plt was associated with IPF (p = .01, R2 = 0.06) but not age, sex, PC, MPV, or plt maximum aggregation or primary slope of aggregation (p > .17, R2 < 0.02). Baseline ultrastructural features were established for TS-PTEM. PTEM was validated using samples from patients with previously established diagnoses of IPDs. Standardization and validation of PTEM procedures and interpretation, and establishment of the normal mean DG/plt reference range and PTEM baseline ultrastructural features, will facilitate implementation of PTEM as a valid clinical laboratory test for evaluating ultrastructural abnormalities in IPDs.

Comprehensive Platelet Phenotypic Laboratory Testing and Bleeding History Scoring for Diagnosis of Suspected Hereditary Platelet Disorders: A Single-Institution Experience.

OBJECTIVES: Patients with hereditary/congenital platelet disorders (HPDs) have a broad range of clinical manifestations and laboratory phenotypes. We assessed the performance characteristics of the International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) and clinically validated platelet laboratory tests for diagnosis of HPDs. METHODS: The records of 61 patients with suspected HPDs were reviewed and ISTH-BAT scores calculated. RESULTS: Nineteen (31%) patients had thrombocytopenia, and 46 (75%) had positive ISTH-BAT scores. Thirteen and 17 patients had prolonged PFA-100 (Dade Behring, Miami, FL) adenosine diphosphate and epinephrine closure times, respectively. Twenty-two had abnormal platelet light transmission aggregation. Twenty-four had platelet transmission electron microscopy (PTEM) abnormalities (10 dense granule deficiency, 14 other ultrastructural abnormalities). Positive ISTH-BAT scores were associated with thrombocytopenia (P < .0001) and abnormal PTEM (P = .002). Twenty-three patients had normal results. CONCLUSIONS: ISTH-BAT identified patients with suspected HPDs but lacked a robust association with laboratory abnormalities. Despite comprehensive laboratory testing, some patients may have normal results.

Calcifying nanoparticles promote mineralization in vascular smooth muscle cells: implications for atherosclerosis.

BACKGROUND: Nano-sized complexes of calcium phosphate mineral and proteins (calcifying nanoparticles [CNPs]) serve as mineral chaperones. Thus, CNPs may be both a result and cause of soft tissue calcification processes. This study determined if CNPs could augment calcification of arterial vascular smooth muscle cells in vitro. METHODS: CNPs 210 nm in diameter were propagated in vitro from human serum. Porcine aortic smooth muscle cells were cultured for up to 28 days in medium in the absence (control) or presence of 2 mM phosphate ([P] positive calcification control) or after a single 3-day exposure to CNPs. Transmission electron-microscopy was used to characterize CNPs and to examine their cellular uptake. Calcium deposits were visualized by light microscopy and von Kossa staining and were quantified by colorimetry. Cell viability was quantified by confocal microscopy of live-/dead-stained cells and apoptosis was examined concurrently by fluorescent labeling of exposed phosphatidylserine. RESULTS: CNPs, as well as smaller calcium crystals, were observed by transmission electron-microscopy on day 3 in CNP-treated but not P-treated cells. By day 28, calcium deposits were visible in similar amounts within multicellular nodules of both CNP- and P-treated cells. Apoptosis increased with cell density under all treatments. CNP treatment augmented the density of apoptotic bodies and cellular debris in association with mineralized multicellular nodules. CONCLUSION: Exogenous CNPs are taken up by aortic smooth muscle cells in vitro and potentiate accumulation of smooth-muscle-derived apoptotic bodies at sites of mineralization. Thus, CNPs may accelerate vascular calcification.

Incorporation of phosphate group modulates bone cell attachment and differentiation on oligo(polyethylene glycol) fumarate hydrogel.

In this work, we have investigated the development of a synthetic hydrogel that contains a negatively charged phosphate group for use as a substrate for bone cell attachment and differentiation in culture. The photoreactive, phosphate-containing molecule, bis(2-(methacryloyloxy)ethyl)phosphate (BP), was incorporated into oligo(polyethylene glycol) fumarate hydrogel and the mechanical, rheological and thermal properties of the resulting hydrogels were characterized. Our results showed changes in hydrogel compression and storage moduli with incorporation of BP. The modification also resulted in decreased crystallinity as recorded by differential scanning calorimetry. Our data revealed that incorporation of BP improved attachment and differentiation of human fetal osteoblast (hFOB) cells in a dose-dependent manner. A change in surface chemistry and mineralization of the phosphate-containing surfaces verified by scanning electron microscopy and energy dispersive X-ray analysis was found to be important for hFOB cell attachment and differentiation. We also demonstrated that phosphate-containing hydrogels support attachment and differentiation of primary bone marrow stromal cells. These findings suggest that BP-modified hydrogels are capable of sustaining attachment and differentiation of both bone marrow stromal cells and osteoblasts that are critical for bone regeneration.

Proteomic evaluation of biological nanoparticles isolated from human kidney stones and calcified arteries.

Calcifying biological nanoparticles (NPs) develop under cell culture conditions from homogenates of diverse tissue samples displaying extraosseous mineralization, including kidney stones and calcified aneurysms. Probes to definitively identify NPs in biological systems are lacking. Therefore, the aim of this study was to begin to establish a proteomic biosignature of NPs in order to facilitate more definitive investigation of their contribution to disease. Biological NPs derived from human kidney stones and calcified aneurysms were completely decalcified by overnight treatment with ethylenediaminetetraacetic acid or brief incubation in HCl, as evidenced by lack of a calcium shell and of Alizarin Red S staining, by transmission electron microscopy and confocal microscopy, respectively. Decalcified NPs contained numerous proteins, including some from bovine serum and others of prokaryotic origin. Most prominent of the latter group was EF-Tu, which appeared to be identical to EF-Tu from Staphylococcus epidermidis. A monoclonal antibody against human EF-Tu recognized a protein in Western blots of total NP lysate, as well as in intact NPs by immunofluorescence and immunogold EM. Approximately 8% of NPs were quantitatively recognized by the antibody using flow cytometry. Therefore, we have defined methods to reproducibly decalcify biological NPs, and identified key components of their proteome. These elements, including EF-Tu, can be used as biomarkers to further define the processes that mediate propagation of biological NPs and their contribution to disease.

In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy.

Fluorescence detection of single molecules provides a means to investigate protein dynamics minus ambiguities introduced by ensemble averages of unsynchronized protein movement or of protein movement mimicking a local symmetry. For proteins in a biological assembly, taking advantage of the single molecule approach could require single protein isolation from within a high protein concentration milieu. Myosin cross-bridges in a muscle fiber are proteins attaining concentrations of approximately 120 muM, implying single myosin detection volume for this biological assembly is approximately 1 attoL (10(-18) L) provided that just 2% of the cross-bridges are fluorescently labeled. With total internal reflection microscopy (TIRM) an exponentially decaying electromagnetic field established on the surface of a glass-substrate/aqueous-sample interface defines a subdiffraction limit penetration depth into the sample that, when combined with confocal microscopy, permits image formation from approximately 3 attoL volumes. Demonstrated here is a variation of TIRM incorporating a nanometer scale metal film into the substrate/glass interface. Comparison of TIRM images from rhodamine-labeled cross-bridges in muscle fibers contacting simultaneously the bare glass and metal-coated interface show the metal film noticeably reduces both background fluorescence and the depth into the sample from which fluorescence is detected. High contrast metal film-enhanced TIRM images allow secondary label visualization in the muscle fibers, facilitating elucidation of Z-disk structure. Reduction of both background fluorescence and detection depth will enhance TIRM's usefulness for single molecule isolation within biological assemblies.

Sebastian platelet syndrome: a hereditary macrothrombocytopenia.

Sebastian platelet syndrome is a rare autosomal dominant disorder characterized by macrothrombocytopenia with granulocyte inclusions similar to those in patients with Fechtner platelet syndrome but without evidence of hereditary nephritis and sensorineural hearing loss that characterizes the latter. Although by light microscopy the granulocyte inclusions in these disorders appear morphologically similar to those found in May-Hegglin anomaly, another autosomal dominant macrothrombocytopenia, by electron microscopy the inclusions are distinct. Studies of platelet function usually suggest normal or near-normal platelet function, although mild bleeding symptoms can be associated with each of these disorders. We describe a 38-year-old woman and her 11-year-old daughter who presented with lifelong histories of mild thrombocytopenia and easy bruising. Detailed hemostatic studies showed prolonged bleeding times in the child and the mother, with the child having absent secondary wave platelet aggregation responses to epinephrine, also reflected by testing with the platelet function analyzer (PFA-100 device). The mother's hemostatic studies were normal including platelet aggregometry, PFA-100 testing, and platelet flow cytometry. By light microscopy the blood smears of both individuals showed neutrophil inclusions, and their platelets were mildly enlarged but were not giant. Electron microscopy showed the neutrophil inclusions seen in classic Sebastian platelet syndrome or Fechtner platelet syndrome. These 2 cases expand the description of Sebastian platelet syndrome to include individuals with large but not giant platelets and mild or minimal thrombocytopenia. The differential diagnosis of hereditary thrombocytopenias is reviewed briefly.