Potent Inhibitory Activity of Natural Product Anaephene B and Analogues against Leishmania tarentolae In Vitro.

In this study, a specific alkylphenol natural product, anaephene B, and its unique synthesized derivatives were tested for their inhibitory effect on the protozoan parasite Leishmania tarentolae. In a series of cell viability tests and enzyme assays, these test compounds have produced interesting results with regard to their antibiotic effect, showing similar potency against L. tarentolae as they do against drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). All compounds tested in this study have shown the ability to completely inhibit our model system, L. tarentolae, in vitro. This study helps increase our understanding of the structure-activity relationship (SAR) between anaephene B and its analogues for a new class of potential pharmaceuticals for the treatment of Leishmania infections.

Cancer cells relax and resist cytotoxic attack.

When it comes to cancer evading the immune response, antigen presentation usually gets all the attention. In this issue of Immunity, Tello-Lafoz et al. reveal that cancer cells have another card up their sleeve: by regulating gene expression to "soften" their actin cytoskeleton, cancer cells limit susceptibility to lymphocyte-mediated cytotoxic attack.

Fatal crashes in the 5 years after recreational marijuana legalization in Colorado and Washington.

Colorado and Washington legalized recreational marijuana in 2012, but the effects of legalization on motor vehicle crashes remains unknown. Using Fatality Analysis Reporting System data, we performed difference-in-differences (DD) analyses comparing changes in fatal crash rates in Washington, Colorado and nine control states with stable anti-marijuana laws or medical marijuana laws over the five years before and after recreational marijuana legalization. In separate analyses, we evaluated fatal crash rates before and after commercial marijuana dispensaries began operating in 2014. In the five years after legalization, fatal crash rates increased more in Colorado and Washington than would be expected had they continued to parallel crash rates in the control states (+1.2 crashes/billion vehicle miles traveled, CI: -0.6 to 2.1, p = 0.087), but not significantly so. The effect was more pronounced and statistically significant after the opening of commercial dispensaries (+1.8 crashes/billion vehicle miles traveled, CI: +0.4 to +3.7, p = 0.020). These data provide evidence of the need for policy strategies to mitigate increasing crash risks as more states legalize recreational marijuana.

Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.

Qualifications and Characteristics North American Dental Deans Seek in Department Chairpersons.

The aim of this study was to determine whether deans of North American dental schools perceived that one category of department chairperson skills (leadership or management) was more important than the other for their chairpersons to be successful. A secondary purpose was to determine the professional qualifications and personal characteristics these deans perceived contributed most to the success of department chairpersons and whether those differed by the research emphasis of the school. An email survey was sent in 2016 to all 75 deans of U.S. and Canadian dental schools with graduating classes. Section one of the survey was an open response section asking deans to list the five most essential characteristics of a successful department chairperson. Section two asked deans to rank the importance of eight listed professional qualifications, and the last section asked deans to rate the importance of four leadership and four management traits that could contribute to the success of their chairpersons. Questions about characteristics of the deans and the schools were also included. A response rate of 46.7% was obtained. The most frequent characteristics listed in the open response section were in the categories of vision, academic expertise, and integrity. The three most highly ranked professional qualifications were previous teaching experience, previous administrative experience, and history of external research funding. Four of the eight professional qualifications were ranked differently by deans of high compared to moderate research-intensive schools (p<0.05). Overall, the respondents rated leadership skills more highly than management skills (p=0.002) as important for departmental chairpersons.

HIV-1 Balances the Fitness Costs and Benefits of Disrupting the Host Cell Actin Cytoskeleton Early after Mucosal Transmission.

HIV-1 primarily infects T lymphocytes and uses these motile cells as migratory vehicles for effective dissemination in the host. Paradoxically, the virus at the same time disrupts multiple cellular processes underlying lymphocyte motility, seemingly counterproductive to rapid systemic infection. Here we show by intravital microscopy in humanized mice that perturbation of the actin cytoskeleton via the lentiviral protein Nef, and not changes to chemokine receptor expression or function, is the dominant cause of dysregulated infected T cell motility in lymphoid tissue by preventing stable cellular polarization required for fast migration. Accordingly, disrupting the Nef hydrophobic patch that facilitates actin cytoskeletal perturbation initially accelerates systemic viral dissemination after female genital transmission. However, the same feature of Nef was subsequently critical for viral persistence in immune-competent hosts. Therefore, a highly conserved activity of lentiviral Nef proteins has dual effects and imposes both fitness costs and benefits on the virus at different stages of infection.

Bone degradation machinery of osteoclasts: An HIV-1 target that contributes to bone loss.

Bone deficits are frequent in HIV-1-infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1-induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.

DOCK8 enforces immunological tolerance by promoting IL-2 signaling and immune synapse formation in Tregs.

Patients deficient in the guanine nucleotide exchange factor DOCK8 have decreased numbers and impaired in vitro function of Tregs and make autoantibodies, but they seldom develop autoimmunity. We show that, similarly, Dock8-/- mice have decreased numbers and impaired in vitro function of Tregs but do not develop autoimmunity. In contrast, mice with selective DOCK8 deficiency in Tregs develop lymphoproliferation, autoantibodies, and gastrointestinal inflammation, despite a normal percentage and in vitro function of Tregs, suggesting that deficient T effector cell function might protect DOCK8-deficient patients from autoimmunity. We demonstrate that DOCK8 associates with STAT5 and is important for IL-2-driven STAT5 phosphorylation in Tregs. DOCK8 localizes within the lamellar actin ring of the Treg immune synapse (IS). Dock8-/- Tregs have abnormal TCR-driven actin dynamics, decreased adhesiveness, an altered gene expression profile, an unstable IS with decreased recruitment of signaling molecules, and impaired transendocytosis of the costimulatory molecule CD86. These data suggest that DOCK8 enforces immunological tolerance by promoting IL-2 signaling, TCR-driven actin dynamics, and the IS in Tregs.

Semen amyloids participate in spermatozoa selection and clearance.

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens.

Leishman and Giemsa stain: a new reliable staining technique for blood/bone marrow smears.

INTRODUCTION: Peripheral blood and bone marrow smear examination is an important basic tool for the diagnosis of different haematological conditions including haematological malignancies. We created a newer modification of the conventional Leishman and Giemsa stains as Leishman and Giemsa (L&G) stain and compared the efficacy and reliability of this stain with conventional stains. The study was performed to evaluate the staining efficacy, feasibility, time and cost of L&G stain over the conventional Leishman and Giemsa stains. METHODS: A pilot study was carried out in the Department of Haematology of our hospital from October 2013 to December 2013. Hundred selected cases, each with peripheral blood and bone marrow smears were taken, and three sets of the smears were prepared from each sample--one for L&G stain and other two--one each for conventional Leishman and Giemsa stains. This staining is further incorporated in our routine standard operating protocols for staining of all the peripheral blood smears in automated stainer, Sysmex SP10. RESULT: The average grading score from each staining methods from all the three experts was compiled. The average grading score of L&G staining method was noted to be significantly higher than the other two methods (analysis of variance test, P value < 0.05). When modified L&G stain (C) was compared with stain conventional stains (A and B), a P value of <0.001 was noted in all parameters except between Leishman stain and L&G stain in mature RBC and WBC nucleus and RBC inclusions (P value between 0.05 and 0.001). CONCLUSION: L&G staining is a newer staining technique of immense help in high-throughput haematology laboratories by offering a time-saving, cost-effective and better staining option to conventional staining methods. It gives a better nuclear and cytoplasmic differential staining and can also be used in automated blood counters/stainer.

Lentiviral Nef proteins manipulate T cells in a subset-specific manner.

UNLABELLED: The role of the accessory viral Nef protein as a multifunctional manipulator of the host cell that is required for effective replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) in vivo is well established. It is unknown, however, whether Nef manipulates all or just specific subsets of CD4(+) T cells, which are the main targets of virus infection and differ substantially in their state of activation and importance for a functional immune system. Here, we analyzed the effect of Nef proteins differing in their T cell receptor (TCR)-CD3 downmodulation function in HIV-infected human lymphoid aggregate cultures and peripheral blood mononuclear cells. We found that Nef efficiently downmodulates TCR-CD3 in naive and memory CD4(+) T cells and protects the latter against apoptosis. In contrast, highly proliferative CD45RA(+) CD45RO(+) CD4(+) T cells were main producers of infectious virus but largely refractory to TCR-CD3 downmodulation. Such T cell subset-specific differences were also observed for Nef-mediated modulation of CD4 but not for enhancement of virion infectivity. Our results indicate that Nef predominantly modulates surface receptors on CD4(+) T cell subsets that are not already fully permissive for viral replication. As a consequence, Nef-mediated downmodulation of TCR-CD3, which distinguishes most primate lentiviruses from HIV type 1 (HIV-1) and its vpu-containing simian precursors, may promote a selective preservation of central memory CD4(+) T cells, which are critical for the maintenance of a functional immune system. IMPORTANCE: The Nef proteins of human and simian immunodeficiency viruses manipulate infected CD4(+) T cells in multiple ways to promote viral replication and immune evasion in vivo. Here, we show that some effects of Nef are subset specific. Downmodulation of CD4 and TCR-CD3 is highly effective in central memory CD4(+) T cells, and the latter Nef function protects this T cell subset against apoptosis. In contrast, highly activated/proliferating CD4(+) T cells are largely refractory to receptor downmodulation but are main producers of infectious HIV-1. Nef-mediated enhancement of virion infectivity, however, was observed in all T cell subsets examined. Our results provide new insights into how primate lentiviruses manipulate their target cells and suggest that the TCR-CD3 downmodulation function of Nef may promote a selective preservation of memory CD4(+) T cells, which are critical for immune function, but has little effect on activated/proliferating CD4(+) T cells, which are the main targets for viral replication.

Nef proteins of epidemic HIV-1 group O strains antagonize human tetherin.

Most simian immunodeficiency viruses use their Nef protein to antagonize the host restriction factor tetherin. A deletion in human tetherin confers Nef resistance, representing a hurdle to successful zoonotic transmission. HIV-1 group M evolved to utilize the viral protein U (Vpu) to counteract tetherin. Although HIV-1 group O has spread epidemically in humans, it has not evolved a Vpu-based tetherin antagonism. Here we show that HIV-1 group O Nef targets a region adjacent to this deletion to inhibit transport of human tetherin to the cell surface, enhances virion release, and increases viral resistance to inhibition by interferon-α. The Nef protein of the inferred common ancestor of group O viruses is also active against human tetherin. Thus, Nef-mediated antagonism of human tetherin evolved prior to the spread of HIV-1 group O and likely facilitated secondary virus transmission. Our results may explain the epidemic spread of HIV-1 group O.

Liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances HIV infection.

UNLABELLED: Semen enhances HIV infection in vitro, but how long it retains this activity has not been carefully examined. Immediately postejaculation, semen exists as a semisolid coagulum, which then converts to a more liquid form in a process termed liquefaction. We demonstrate that early during liquefaction, semen exhibits maximal HIV-enhancing activity that gradually declines upon further incubation. The decline in HIV-enhancing activity parallels the degradation of peptide fragments derived from the semenogelins (SEMs), the major components of the coagulum that are cleaved in a site-specific and progressive manner upon initiation of liquefaction. Because amyloid fibrils generated from SEM fragments were recently demonstrated to enhance HIV infection, we set out to determine whether any of the liquefaction-generated SEM fragments associate with the presence of HIV-enhancing activity. We identify SEM1 from amino acids 86 to 107 [SEM1(86-107)] to be a short, cationic, amyloidogenic SEM peptide that is generated early in the process of liquefaction but that, conversely, is lost during prolonged liquefaction due to the activity of serine proteases. Synthetic SEM1(86-107) amyloids directly bind HIV-1 virions and are sufficient to enhance HIV infection of permissive cells. Furthermore, endogenous seminal levels of SEM1(86-107) correlate with donor-dependent variations in viral enhancement activity, and antibodies generated against SEM1(86-107) recognize endogenous amyloids in human semen. The amyloidogenic potential of SEM1(86-107) and its virus-enhancing properties are conserved among great apes, suggesting an evolutionarily conserved function. These studies identify SEM1(86-107) to be a key, HIV-enhancing amyloid species in human semen and underscore the dynamic nature of semen's HIV-enhancing activity. IMPORTANCE: Semen, the most common vehicle for HIV transmission, enhances HIV infection in vitro, but how long it retains this activity has not been investigated. Semen naturally undergoes physiological changes over time, whereby it converts from a gel-like consistency to a more liquid form. This process, termed liquefaction, is characterized at the molecular level by site-specific and progressive cleavage of SEMs, the major components of the coagulum, by seminal proteases. We demonstrate that the HIV-enhancing activity of semen gradually decreases over the course of extended liquefaction and identify a naturally occurring semenogelin-derived fragment, SEM1(86-107), whose levels correlate with virus-enhancing activity over the course of liquefaction. SEM1(86-107) amyloids are naturally present in semen, and synthetic SEM1(86-107) fibrils bind virions and are sufficient to enhance HIV infection. Therefore, by characterizing dynamic changes in the HIV-enhancing activity of semen during extended liquefaction, we identified SEM1(86-107) to be a key virus-enhancing component of human semen.

Direct visualization of HIV-enhancing endogenous amyloid fibrils in human semen.

Naturally occurring fragments of the abundant semen proteins prostatic acid phosphatase (PAP) and semenogelins form amyloid fibrils in vitro. These fibrils boost HIV infection and may play a key role in the spread of the AIDS pandemic. However, the presence of amyloid fibrils in semen remained to be demonstrated. Here, we use state of the art confocal and electron microscopy techniques for direct imaging of amyloid fibrils in human ejaculates. We detect amyloid aggregates in all semen samples and find that they partially consist of PAP fragments, interact with HIV particles and increase viral infectivity. Our results establish semen as a body fluid that naturally contains amyloid fibrils that are exploited by HIV to promote its sexual transmission.

Ocular disorders among schoolchildren in Khartoum State, Sudan.

From December 2005 to June 2007, a total screening of all 1418 government primary schools in Khartoum State, Sudan, was performed to estimate ocular problems among children aged 6-15 years. We screened 671,119 children (56.7% males) for significant refractive error and other eye ailments. Ocular problems were found in 20,321 (3.03%) children. The 3 localities with highest ocular pathology were Karary (26.2%), Ummbada (21.0%) and Jabal Awlia (15.7%). The overall prevalence of refractive error was 2.19%. Myopia was found in 10,064 (1.50%) children while 4661 (0.70%) were hyperopic. Other ocular problems included vernal keratoconjunctivitis, vitamin A deficiency, microbial conjunctivitis, strabismus and corneal opacity. Only 288 (0.04%) children were diagnosed with active trachoma: 86.5% of these were from Ummbada locality, on the periphery ofthe State, where transportation facilities are poor and poverty is widespread. Overall, 99% of the eye ailments identified are either treatable or preventable. To reduce these and to achieve the goals of Vision 2020, an effective and efficient school health programme is needed.

The efficiency of Vpx-mediated SAMHD1 antagonism does not correlate with the potency of viral control in HIV-2-infected individuals.

BACKGROUND: The presence of a vpx gene distinguishes HIV-2 from HIV-1, the main causative agent of AIDS. Vpx degrades the restriction factor SAMHD1 to boost HIV-2 infection of macrophages and dendritic cells and it has been suggested that the activation of antiviral innate immune responses after Vpx-dependent infection of myeloid cells may explain why most HIV-2-infected individuals efficiently control viral replication and become long-term survivors. However, the role of Vpx-mediated SAMHD1 antagonism in the virological and clinical outcome of HIV-2 infection remained to be investigated. RESULTS: Here, we analyzed the anti-SAMHD1 activity of vpx alleles derived from seven viremic and four long-term aviremic HIV-2-infected individuals. We found that effective Vpx-mediated SAMHD1 degradation and enhancement of myeloid cell infection was preserved in most HIV-2-infected individuals including all seven that failed to control the virus and developed AIDS. The only exception were vpx alleles from an aviremic individual that predicted a M68K change in a highly conserved nuclear localization signal which disrupted the ability of Vpx to counteract SAMHD1. We also found that HIV-2 is less effective than HIV-1 in inducing innate immune activation in dendritic cells. CONCLUSIONS: Effective immune control of viral replication in HIV-2-infected individuals is not associated with increased Vpx-mediated degradation of SAMHD1.

Peptide nanofibrils boost retroviral gene transfer and provide a rapid means for concentrating viruses.

Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.

Ocular disorders among schoolchildren in Khartoum State, Sudan

From December 2005 to June 2007, a total screening of all 1418 government primary schools in Khartoum State, Sudan, was performed to estimate ocular problems among children aged 6-15 years. We screened 671 119 children [56.7% males] for significant refractive error and other eye ailments. Ocular problems were found in 20 321 [3.03%] children. The 3 localities with highest ocular pathology were Karary [26.2%], Ummbada [21.0%] and Jabal Awlia [15.7%]. The overall prevalence of refractive error was 2.19%. Myopia was found in 10 064 [1.50%] children while 4661 [0.70%] were hyperopic. Other ocular problems included vernal keratoconjunctivitis, vitamin A deficiency, microbial conjunctivitis, strabismus and corneal opacity. Only 288 [0.04%] children were diagnosed with active trachoma: 86.5% of these were from Ummbada locality, on the periphery of the State, where transportation facilities are poor and poverty is widespread. Overall, 99% of the eye ailments identified are either treatable or preventable. To reduce these and to achieve the goals of Vision 2020, an effective and efficient school health programme is needed

Reacquisition of Nef-mediated tetherin antagonism in a single in vivo passage of HIV-1 through its original chimpanzee host.

The interferon-induced host restriction factor tetherin poses a barrier for SIV transmission from primates to humans. After cross-species transmission, the chimpanzee precursor of pandemic HIV-1 switched from the accessory protein Nef to Vpu to effectively counteract human tetherin. As we report here, the experimental reintroduction of HIV-1 into its original chimpanzee host resulted in a virus that can use both Vpu and Nef to antagonize chimpanzee tetherin. Functional analyses demonstrated that alterations in and near the highly conserved ExxxLL motif in the C-terminal loop of Nef were critical for the reacquisition of antitetherin activity. Strikingly, just two amino acid changes allowed HIV-1 Nef to counteract chimpanzee tetherin and promote virus release. Our data demonstrate that primate lentiviruses can reacquire lost accessory gene functions during a single in vivo passage and suggest that other functional constraints keep Nef ready to regain antitetherin activity.