Biomarker discovery in cardiac allograft vasculopathy using targeted aptamer proteomics.

Cardiac allograft vasculopathy (CAV) limits long-term survival after heart transplantation. Non-invasive evaluation is challenging, and currently, there is no validated biomarker for CAV diagnosis or prognostication. To identify potential candidate CAV biomarkers, we utilized the Slow Off-rate Modified Aptamer (SOMAscan) assay, which evaluates over 1000 serum proteins, including many relevant to biological pathways in CAV. We evaluated three heart transplant patient groups according to angiographic ISHLT CAV grade: CAV1-2 (mild-moderate CAV), CAV3 (severe CAV), and CAV0 (normal control). SOMAscan assays were performed and proteins quantitated. Comparisons of proteins between study groups were performed using one-way ANOVA (false discovery rate q-value < 0.10). Thirty-one patients (12 mild-moderate CAV, 9 severe CAV, 10 controls) were included: 81% male, median age 57 years and median 1.1 years post-transplant. Compared to controls, patients with mild-moderate CAV had similar characteristics, while patients with severe CAV had longer time from transplant and increased allosensitization. Statistical/bioinformatics analysis identified 14 novel biomarkers for CAV, including 4 specific for mild-moderate CAV. These proteins demonstrated important actions including apoptosis, inflammation, and platelet/coagulation activation. Upon preliminary receiver operating characteristics curve analysis, our protein biomarkers showed moderate-to-high discriminative ability for CAV (area under curve: 0.72 to 0.94). These candidate biomarkers are being validated in prospective studies.

Phosphatidylinositol-3,4-Bisphosphate and Its Binding Protein Lamellipodin Regulate Chemotaxis of Malignant B Lymphocytes.

Cell migration is controlled by PI3Ks, which generate lipid messengers phosphatidylinositol-3,4,5-trisphosphate and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] and consequently recruit pleckstrin homology (PH) domain-containing signaling proteins. PI3K inhibition impairs migration of normal and transformed B cells, an effect thought to partly underlie the therapeutic efficacy of PI3K inhibitors in treatment of B cell malignancies such as chronic lymphocytic leukemia. Although a number of studies have implicated phosphatidylinositol-3,4,5-trisphosphate in cell migration, it remains unknown whether PI(3,4)P2 plays a distinct role. Using the PI(3,4)P2-specific phosphatase inositol polyphosphate 4-phosphatase, we investigate the impact of depleting PI(3,4)P2 on migration behavior of malignant B cells. We find that cells expressing wild-type, but not phosphatase dead, inositol polyphosphate 4-phosphatase show impaired SDF-induced PI(3,4)P2 responses and reduced migration in Transwell chamber assays. Moreover, PI(3,4)P2 depletion in primary chronic lymphocytic leukemia cells significantly impaired their migration capacity. PI(3,4)P2 depletion reduced both overall motility and migration directionality in the presence of a stable chemokine gradient. Within chemotaxing B cells, the PI(3,4)P2-binding cytoskeletal regulator lamellipodin (Lpd) was found to colocalize with PI(3,4)P2 on the plasma membrane via its PH domain. Overexpression and knockdown studies indicated that Lpd levels significantly impact migration capacity. Moreover, the ability of Lpd to promote directional migration of B cells in an SDF-1 gradient was dependent on its PI(3,4)P2-binding PH domain. These results demonstrate that PI(3,4)P2 plays a significant role in cell migration via binding to specific cytoskeletal regulators such as Lpd, and they suggest that impairment of PI(3,4)P2-dependent processes may contribute to the therapeutic efficacy of PI3K inhibitors in B cell malignancies.

High-throughput Functional Genomics Identifies Regulators of Primary Human Beta Cell Proliferation.

The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing the proliferation of non-transformed human cells. Here, we report the development of a high-throughput RNAi screening strategy specifically for use in primary cells and demonstrate that silencing the cell cycle-dependent kinase inhibitors CDKN2C/p18 or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21 as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens for dissecting therapeutically relevant state changes in primary human cells.

Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

Integrated photothermal decontamination device for N95 respirators.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the COVID-19 global pandemic has infected over 25 million people worldwide and resulted in the death of millions. The COVID-19 pandemic has also resulted in a shortage of personal protective equipment (PPE) in many regions around the world, particularly in middle- and low-income countries. The shortages of PPE, such as N95 respirators, is something that will persist until an effective vaccine is made available. Thus, devices that while being easy to operate can also be rapidly deployed in health centers, and long-term residences without the need for major structural overhaul are instrumental to sustainably use N95 respirators. In this report, we present the design and validation of a decontamination device that combines UV-C & B irradiation with mild-temperature treatment. The device can decontaminate up to 20 masks in a cycle of < 30 min. The decontamination process did not damage or reduce the filtering capacity of the masks. Further, the efficacy of the device to eliminate microbes and viruses from the masks was also evaluated. The photothermal treatment of our device was capable of eradicating > 99.9999% of the bacteria and > 99.99% of the virus tested.

PPP1R9B (Neurabin 2): involvement and dynamics in the NK immunological synapse.

The NK immunological synapse (NKIS) is a dynamic structure dependent on the assembly of membrane, cytoskeletal and signaling components. These serve to focus and generate stimuli for adhesion and orientation of the cytoskeleton for targeted cytolytic granule release. Previous studies have demonstrated the importance of the cytoskeleton in these processes. We previously identified PPP1R9B (neurabin 2, spinophilin) as a cytoskeletal component of the NK-like cell line YTS. We demonstrate that (i) PPP1R9B gradually accumulates at the NKIS in a maturation stage-dependent manner; (ii) it mimics the early kinetics of actin recruitment to the NKIS but it precedes actin departure from the site; (iii) it is recruited by CD18 stimulation but not by CD28 ligation; (iv) it is required for the maintenance of the cortical F-actin organization in the YTS cells and knocking down PPP1R9B reduces the frequency of YTS-target cell conjugation, possibly due to the collapsed F-actin cytoskeleton in these cells. These results indicate that PPP1R9B is required for synapse formation in the NK cells and suggest that it may be involved in the maintenance of cellular architecture by regulation of actin assembly, possibly acting to stabilize the NKIS until granule release is eminent.

Phylogenetic and phenotypic analysis of HIV type 1 env gp120 in cases of subtype C mother-to-child transmission.

Human immunodeficiency virus type 1 (HIV-1) subtype C constitutes a majority of the newly transmitted cases of HIV-1 in many developing countries. There is growing evidence suggesting that subtype C viruses may display characteristics that make them distinct from subtype B and other subtypes, and such differences may affect its transmission and pathogenesis in infected individuals. In this study, HIV-1 sequences from the C2-V4 region of the envelope gene were analyzed from four infected mother-infant pairs (MIPs) after perinatal transmission in Zambia. We found that all viral isolates from four Zambian MIPs were of the subtype C HIV-1 subgroup. All tested isolates were macrophage tropic and did not infect any tested T cell lines or form syncytia in vitro. In addition, the isolates appear to use exclusively the CCR5 coreceptor. Phylogenetic analyses clearly indicated two contrasting phylogenetic transmission patterns for the four MIPs analyzed in the present study. Three cases displayed a pattern of selective transmission of a single or a few variants, and one case was found to transmit multiple maternal HIV-1 variants to her infant. The differences in the mode and timing of transmission may account for the observed transmission pattern among four MIPs.

Genome-wide RNAi screen identifies ATPase inhibitory factor 1 (ATPIF1) as essential for PARK2 recruitment and mitophagy.

Mitochondrial dysfunction is a hallmark of aging and numerous human diseases, including Parkinson disease (PD). Multiple homeostatic mechanisms exist to ensure mitochondrial integrity, including the selective autophagic program mitophagy, that is activated during starvation or in response to mitochondrial dysfunction. Following prolonged loss of potential across the inner mitochondrial membrane (ΔΨ), PTEN-induced putative kinase 1 (PINK1) and the E3-ubiquitin ligase PARK2 work in the same pathway to trigger mitophagy of dysfunctional mitochondria. Mutations in PINK1 and PARK2, as well as PARK7/DJ-1, underlie autosomal recessive Parkinsonism and impair mitochondrial function and morphology. In a genome-wide RNAi screen searching for genes that are required for PARK2 translocation to the mitochondria, we identified ATPase inhibitory factor 1 (ATPIF1/IF1) as essential for PARK2 recruitment and mitophagy in cultured cells. During uncoupling, ATPIF1 promotes collapse of ΔΨ and activation of the PINK-PARK2 mitophagy pathway by blocking the ATPase activity of the F 1-Fo ATP synthase. Restoration of ATPIF1 in Rho0 cells, which lack mtDNA and a functional electron transport chain, lowers ΔΨ and triggers PARK2 recruitment. Our findings identified ATPIF1 and the ATP synthase as novel components of the PINK1-PARK2 mitophagy pathway and provide genetic evidence that loss of ΔΨ is an essential trigger for mitophagy.

The identification and characterization of a novel protein, c19orf10, in the synovium.

Joint inflammation and destruction have been linked to the deregulation of the highly synthetic fibroblast-like synoviocytes (FLSs), and much of our current understanding of the mechanisms that underlie synovitis has been collected from studies of FLSs. During a proteomic analysis of FLS cells, we identified a novel protein, c19orf10 (chromosome 19 open reading frame 10), that was produced in significant amounts by these cells. The present study provides a partial characterization of c19orf10 in FLSs, synovial fluid, and the synovium. Murine monoclonal and chicken polyclonal antibodies were produced against recombinant human c19orf10 protein and used to examine the distribution of c19orf10 in cultured FLSs and in synovial tissue sections from patients with rheumatoid arthritis or osteoarthritis. The intracellular staining pattern of c19orf10 is consistent with localization in the endoplasmic reticulum/Golgi distribution. Sections of rheumatoid arthritis and osteoarthritis synovia expressed similar patterns of c19orf10 distribution with perivascular and synovial lining staining. Double-staining in situ analysis suggests that fibroblast-like synovial cells produced c19orf10, whereas macrophages, B cells, or T cells produced little or none of this protein. There is evidence of secretion into the vascular space and the extracellular matrix surrounding the synovial lining. A competitive enzyme-linked immunosorbent assay confirmed the presence of microgram levels of c19orf10 in the synovial fluids of patients with one of various arthropathies. Collectively, these results suggest that c19orf10 is an FLS-derived protein that is secreted into the synovial fluid. However, the significance of this protein in synovial biology remains to be determined. The absence of known structural motifs or domains and its relatively late evolutionary appearance raise interesting questions about its function.

Follicular dendritic cell secreted protein (FDC-SP) regulates germinal center and antibody responses.

We previously identified follicular dendritic cell secreted protein (FDC-SP), a small secreted protein of unknown function expressed in human tonsillar germinal centers (GC). To assess potential in vivo activities of FDC-SP, transgenic mice were generated to constitutively express FDC-SP in lymphoid tissues. FDC-SP transgenic mice show relatively normal development of immune cell populations, with the exception of a small increase in mature follicular B cells, and normal lymphoid tissue architecture. Upon immunization with a T-dependent Ag, FDC-SP transgenic mice were capable of producing an Ag-specific Ab; however, the titers of Ag-specific IgG2a and IgE were significantly reduced. GC responses after immunization were markedly diminished, with transgenic mice showing decreased numbers and sizes of GCs but normal development of follicular dendritic cell networks and normal positioning of GCs. FDC-SP transgenic mice also showed reduced production of Ag-specific IgG3 Ab after immunization with a type II T-independent Ag, suggesting that the FDC-SP can also regulate the induction of B cell responses outside the GC. Purified FDC-SP transgenic B cells function normally in vitro, with the exception of blunted chemotaxis responses to CXCL12 and CXCL13. FDC-SP can induce the chemotaxis of CD40-stimulated nontransgenic B cells and can significantly enhance B cell migration in combination with chemokines, indicating that FDC-SP may function in part by regulating B cell chemotaxis. These results provide the first evidence for immunomodulatory activities of FDC-SP and implicate this molecule as a regulator of B cell responses.