Syntaxin 5 is required for copper homeostasis in Drosophila and mammals

Copper is essential for aerobic life, but many aspects of its cellular uptake and distribution remain to be fully elucidated. A genome-wide screen for copper homeostasis genes in Drosophila melanogaster identified the SNARE gene Syntaxin 5 (Syx5) as playing an important role in copper regulation; flies heterozygous for a null mutation in Syx5 display increased tolerance to high dietary copper. The phenotype is shown here to be due to a decrease in copper accumulation, a mechanism also observed in both Drosophila and human cell lines. Studies in adult Drosophila tissue suggest that very low levels of Syx5 result in neuronal defects and lethality, and increased levels also generate neuronal defects. In contrast, mild suppression generates a phenotype typical of copper-deficiency in viable, fertile flies and is exacerbated by co-suppression of the copper uptake gene Ctr1A. Reduced copper uptake appears to be due to reduced levels at the plasma membrane of the copper uptake transporter, Ctr1. Thus Syx5 plays an essential role in copper homeostasis and is a candidate gene for copper-related disease in humans.     

A five‐step risk management process for geriatric dental practice during SARS‐CoV‐2 pandemic

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is an RNA virus that causes coronavirus infection (COVID‐19). COVID‐19 is a highly contagious disease transmitted through respiratory droplets, saliva and other contact routes. Within 10 months of its outbreak, SARS‐CoV‐2 has infected more than 23 million people around the world. Evidence suggests that older adults are the most vulnerable to infection and have an increased risk of mortality. Reduced immunity and underlying medical conditions make them risk‐prone and vulnerable to critical care. Older adults affected with the SARS‐CoV‐2 virus present with distinct clinical manifestations necessitating specific treatment needs and management protocols. While it is crucial to prevent the spread of novel coronavirus (2019‐nCoV), the role of oral healthcare workers in addressing the specific needs of ageing adult patients by adopting specific guidelines and appropriate infection control protocols is timely. This paper aims to develop specific guidelines and protocols for the dental management of geriatric patients during the COVID‐19 pandemic.     

Cryoelectron tomographic analysis of an HIV-neutralizing protein and its complex with native viral gp120

Identifying structural determinants of human immunodeficiency virus (HIV) neutralization is an important component of rational drug and vaccine design. We used cryoelectron tomography and atomic force microscopy to characterize the structure of an extremely potent HIV-neutralizing protein, D1D2-Ig alpha tp (abbreviated as D1D2-IgP), a polyvalent antibody construct that presents dodecameric CD4 in place of the Fab regions. We show that D1D2-IgP has a novel structure, displaying greater flexibility of its antibody arms than the closely related IgM. Using simian immunodeficiency virus in complex with D1D2-IgP, we present unequivocal evidence that D1D2-IgP can cross-link surface spikes on the same virus and on neighboring viruses. The observed binding to the viral envelope spikes is the result of specific CD4-gp120 interaction, because binding was not observed with MICA-IgP, a construct that is identical to D1D2-IgP except that major histocompatibility complex Class I-related Chain A (MICA) replaces the CD4 moiety. CD4-mediated binding was also associated with a significantly elevated proportion of ruptured viruses. The ratio of inactivated to CD4-liganded gp120-gp41 spikes can be much greater than 1:1, because all gp120-gp41 spikes on the closely apposed surfaces of cross-linked viruses should be incapable of accessing the target cell surface and mediating entry, as a result of inter-virus spike cross-linking. These results implicate flexibility rather than steric bulk or polyvalence per se as a structural explanation for the extreme potency of D1D2-IgP and thus suggest polyvalence presented on a flexible scaffold as a key design criterion for small molecule HIV entry inhibitors.     

Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab

The major allergens of honeybee venom, hyaluronidase (Hyal) and phospholipase A2, can induce life-threatening IgE-mediated allergic reactions in humans. Although conventional immunotherapy is effective, up to 40% of patients develop allergic side effects including anaphylaxis and thus, there is a need for an improved immunotherapy. A murine monoclonal anti-Hyal IgG1 antibody (mAb 21E11), that competed for Hyal binding with IgEs from sera of bee venom allergic patients, was raised. The fragment of these IgG antibodies which bind to antigen (Fab) was produced and complexed (1:1) with Hyal. The crystal structure determination of Hyal/Fab 21E11 complex (2.6 A) enabled the identification of the Hyal-IgG interface which provides indirect information on the Hyal-IgE interaction (B-cell epitope). The epitope is composed of a linear array of nine residues (Arg138, His141-Arg148) located at the tip of a helix-turn-helix motive which protrudes away from the globular core and fits tightly into the deep surface pocket formed by the residues from the six complementarity determining regions (CDRs) of the Fab. The epitope is continuous and yet its conformation appears to be essential for Ab recognition, since the synthetic 15-mer peptide comprising the entire epitope (Arg138-Glu152) is neither recognized by mAb 21E11 nor by human IgEs. The structure of the complex provides the basis for the rational design of Hyal derivatives with reduced allergenic activity, which could be used in the development of safer allergen-specific immunotherapy.     

Evaluation of neuroprotective and anti-fatigue effects of sildenafil

Sildenafil, a phosphodiesterase-5 inhibitor is widely used for the treatment of erectile dysfunction. Recently, the FDA approved the use of sildenafil in the therapeutic treatment of pulmonary arterial hypertension. Sildenafil crosses the blood-brain barrier and has been shown to enhance memory. Tremor, rigidity and akinesia are the most common symptoms seen in Parkinson's disease. Fatigue and sexual dysfunction are the other prominent features seen in Parkinson's disease. Interestingly, sildenafil is used therapeutically to treat sexual dysfunction in Parkinson's disease patients. Currently research on Parkinson's disease focuses on developing novel drug therapies for retarding the nigral dopaminergic neurodegeneration. Hence, we investigated the anti-fatigue and neuroprotective effects of sildenafil. In this study, the effect of sildenafil on fatigue was evaluated using forced swim test in mice. Sildenafil had no effect on fatigue as seen by the swim time. With regard to neuroprotective effects, we investigated the effects of sildenafil using two animal models of Parkinson's disease. In this study, 6-hydroxydopamine-lesioned (unilateral) rats and MPTP-treated mice were used as the animal models of Parkinson's disease. 6-Hydroxydopamine-lesioned rats were used to determine the effect of sildenafil on rotational behavior. Ipsilateral or contralateral rotational behavior can indicate the amphetamine-like activity or apomorphine-like activity of sildenafil. Sildenafil did not induce contralateral or ipsilateral rotations in 6-hydroxydopamine-lesioned rats. Sildenafil did not protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion in the striatum.     

Electron tomography of viruses

Understanding the molecular architectures of enveloped and complex viruses is a challenging frontier in structural biology. In these viruses, the structural and compositional variation from one viral particle to another generally precludes the use of either crystallization or image averaging procedures that have been successfully implemented in the past for highly symmetric viruses. While advances in cryo electron tomography of unstained specimens provide new opportunities for identification and molecular averaging of individual subcomponents such as the surface glycoprotein spikes on purified viruses, electron tomography of stained and plunge-frozen cells is being used to visualize the cellular context of viral entry and replication. Here, we review recent developments in both areas as they relate to our understanding of the biology of heterogeneous and pleiomorphic viruses.     

Automated 100-position specimen loader and image acquisition system for transmission electron microscopy

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the "Gatling", permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 h. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science, and nanotechnology that require rapid screening and image analysis of multiple specimens.