Role of L2 cysteines in papillomavirus infection and neutralization

Vaccination of mice with minor capsid protein L2 or passive transfer with the L2-specific neutralizing monoclonal antibody RG-1 protects against human papillomavirus type 16 (HPV16) challenge. Here we explored the nature of the RG-1 epitope and its contribution to viral infectivity. RG-1 bound equivalently HPV16 L2 residues 17-36 with or without an intact C22-C28 disulphide bridge. HPV16 L2 mutations K20A, C22A, C22S, C28A, C28S, or P29A prevented RG-1 binding, whereas Y19A, K23A or Q24A had no impact. Mutation of either C22 or C28 to alanine or serine compromises HPV16 pseudoviral infectivity both in vitro and in the murine vaginal tract, but does not impact pseudovirion assembly. Despite their lack of infectivity, HPV16 pseudovirions containing C22S or C28S mutant L2 bind to cell surfaces, are taken up, and expose the 17-36 region on the virion surface as for wild type HPV16 pseudovirions suggesting normal furin cleavage of L2. Mutation of the second cysteine residue in Bovine papillomavirus type 1 (BPV1) L2 to serine (C25S) dramatically reduced the infectivity of BPV1 pseudovirions. Surprisingly, in contrast to the double mutation in HPV16 L2, the BPV1 L2 C19S, C25S double mutation reduced BPV1 pseudovirion infectivity of 293TT cells by only half

Family Communication in a Population at Risk for Hypertrophic Cardiomyopathy

Encouraging family communication is an integral component of genetic counseling; therefore, we sought to identify factors impacting communication to family members at risk for Hypertrophic Cardiomyopathy (HCM). Participants (N = 383) completed an online survey assessing: 1) demographics (gender, genetic test results, HCM family history, and disease severity); 2) illness representations; 3) family functioning and cohesiveness; 4) coping styles; 5) comprehension of HCM autosomal dominant inheritance; and 6) communication of HCM risk information to at‐risk relatives. Participants were a national sample of individuals with HCM, recruited through the Hypertrophic Cardiomyopathy Association. Data from 183 participants were analyzed using a logistic regression analysis, with family communication as a dichotomous dependent variable. We found that female gender and higher comprehension of autosomal dominant inheritance were significant predictors of participants’ communication of HCM risk information to all their siblings and children. Our results suggest that utilizing interventions that promote patient comprehension (e.g., a teaching‐focused model of genetic counseling) are important and may positively impact family communication within families with HCM.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147017/1/jgc40336.pd

A novel member of the BTB/POZ family, PATZ, associates with the RNF4 RING finger protein and acts as a transcriptional repressor.

We have identified a novel human gene encoding a 59-kDa POZ-AT hook-zinc finger protein (PATZ) that interacts with RNF4, a mediator of androgen receptor activity, and acts as a transcriptional repressor. PATZ cDNA was isolated through a two-hybrid interaction screening using the RING finger protein RNF4 as a bait. In vitro and in vivo interaction between RNF4 and PATZ was demonstrated by protein-protein affinity chromatography and coimmunoprecipitation experiments. Such interaction occurred through a small region of PATZ containing an AT-hook DNA binding domain. Immunofluorescence staining and confocal microscopy showed that PATZ localizes in distinct punctate nuclear regions and colocalizes with RNF4. Functional analysis was performed by cotransfection assays: PATZ acted as a transcriptional repressor, whereas its partner RNF4 behaved as a transcriptional activator. When both proteins were overexpressed a strong repression of the basal transcription was observed, indicating that the association of PATZ with RNF4 switches activation to repression. In addition, RNF4 was also found to associate with HMGI(Y), a chromatin-modeling factor containing AT-hook domains

Artificial Modifications of the Coast In Response to the \u3ci\u3eDeepwater Horizon\u3c/i\u3e Oil Spill: Quick Solutions or Long-Term Liabilities?

The Deepwater Horizon oil spill threatened many coastal ecosystems in the Gulf of Mexico during the spring and summer of 2010. Mitigation strategies included the construction of barrier sand berms, the restriction or blocking of inlets, and the diversion of freshwater from rivers to the coastal marshes and into the ocean, in order to flush away the oil, on the premise that these measures could reduce the quantity of oil reaching sensitive coastal environments such as wetlands or estuaries. These projects result in changes to the ecosystems that they were intended to protect. Long-term effects include alterations of the hydrological and ecological characteristics of estuaries, changes in sediment transport along the coastal barrier islands, the loss of sand resources, and adverse impacts to benthic and pelagic organisms. Although there are no easy solutions for minimizing the impacts of the Deepwater Horizon disaster on coastal ecosystems, we recommend that federal, state, and local agencies return to the strategic use of long-term restoration plans for this region

The TREAT-NMD advisory committee for therapeutics (TACT): an innovative de-risking model to foster orphan drug development

Despite multiple publications on potential therapies for neuromuscular diseases (NMD) in cell and animal models only a handful reach clinical trials. The ability to prioritise drug development according to objective criteria is particularly critical in rare diseases with large unmet needs and a limited numbers of patients who can be enrolled into clinical trials. TREAT-NMD Advisory Committee for Therapeutics (TACT) was established to provide independent and objective guidance on the preclinical and development pathway of potential therapies (whether novel or repurposed) for NMD. We present our experience in the establishment and operation of the TACT. TACT provides a unique resource of recognized experts from multiple disciplines. The goal of each TACT review is to help the sponsor to position the candidate compound along a realistic and well-informed plan to clinical trials, and eventual registration. The reviews and subsequent recommendations are focused on generating meaningful and rigorous data that can enable clear go/no-go decisions and facilitate longer term funding or partnering opportunities. The review process thereby acts to comment on viability, de-risking the process of proceeding on a development programme. To date TACT has held 10 review meeting and reviewed 29 program applications in several rare neuromuscular diseases: Of the 29 programs reviewed, 19 were from industry and 10 were from academia; 15 were for novel compounds and 14 were for repurposed drugs; 16 were small molecules and 13 were biologics; 14 were preclinical stage applications and 15 were clinical stage applications. 3 had received Orphan drug designation from European Medicines Agency and 3 from Food and Drug Administration. A number of recurrent themes emerged over the course of the reviews and we found that applicants frequently require advice and education on issues concerned with preclinical standard operating procedures, interactions with regulatory agencies, formulation, repurposing, clinical trial design, manufacturing and ethics. Over the 5 years since its establishment TACT has amassed a body of experience that can be extrapolated to other groups of rare diseases to improve the community's chances of successfully bringing new rare disease drugs to registration and ultimately to marke

Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing

HIV-Nef Protein Persists in the Lungs of Aviremic Patients with HIV and Induces Endothelial Cell Death

It remains a mystery why HIV-associated end-organ pathologies persist in the era of combined antiretroviral therapy (ART). One possible mechanism is the continued production of HIV-encoded proteins in latently HIV-infected T cells and macrophages. The proapoptotic protein HIV-Nef persists in the blood of ART-treated patients within extracellular vesicles (EVs) and peripheral blood mononuclear cells. Here we demonstrate that HIV-Nef is present in cells and EVs isolated from BAL of patients on ART. We hypothesize that HIV-Nef persistence in the lung induces endothelial apoptosis leading to endothelial dysfunction and further pulmonary vascular pathologies. The presence of HIV-Nef in patients with HIV correlates with the surface expression of the proapoptotic endothelial-monocyte–activating polypeptide II (EMAPII), which was implicated in progression of pulmonary emphysema via mechanisms involving endothelial cell death. HIV-Nef protein induces EMAPII surface expression in human embryonic kidney 293T cells, T cells, and human and mouse lung endothelial cells. HIV-Nef packages itself into EVs and increases the amount of EVs secreted from Nef-expressing T cells and Nef-transfected human embryonic kidney 293T cells. EVs from BAL of HIV+ patients and Nef-transfected cells induce apoptosis in lung microvascular endothelial cells by upregulating EMAPII surface expression in a PAK2-dependent fashion. Transgenic expression of HIV-Nef in vascular endothelial–cadherin+ endothelial cells leads to lung rarefaction, characterized by reduced alveoli and overall increase in lung inspiratory capacity. These changes occur concomitantly with lung endothelial cell apoptosis. Together, these data suggest that HIV-Nef induces endothelial cell apoptosis via an EMAPII-dependent mechanism that is sufficient to cause pulmonary vascular pathologies even in the absence of inflammation