Direct binding to sterols accelerates endoplasmic reticulum-associated degradation of HMG CoA reductase.

The maintenance of cholesterol homeostasis is crucial for normal function at both the cellular and organismal levels. Two integral membrane proteins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and Scap, are key targets of a complex feedback regulatory system that operates to ensure cholesterol homeostasis. HMGCR catalyzes the rate-limiting step in the transformation of the 2-carbon precursor acetate to 27-carbon cholesterol. Scap mediates proteolytic activation of sterol regulatory element-binding protein-2 (SREBP-2), a membrane-bound transcription factor that controls expression of genes involved in the synthesis and uptake of cholesterol. Sterol accumulation triggers binding of HMGCR to endoplasmic reticulum (ER)-localized Insig proteins, leading to the enzyme's ubiquitination and proteasome-mediated ER-associated degradation (ERAD). Sterols also induce binding of Insigs to Scap, which leads to sequestration of Scap and its bound SREBP-2 in the ER, thereby preventing proteolytic activation of SREBP-2 in the Golgi. The oxygenated cholesterol derivative 25-hydroxycholesterol (25HC) and the methylated cholesterol synthesis intermediate 24,25-dihydrolanosterol (DHL) differentially modulate HMGCR and Scap. While both sterols promote binding of HMGCR to Insigs for ubiquitination and subsequent ERAD, only 25HC inhibits the Scap-mediated proteolytic activation of SREBP-2. We showed previously that 1,1-bisphosphonate esters mimic DHL, accelerating ERAD of HMGCR while sparing SREBP-2 activation. Building on these results, our current studies reveal specific, Insig-independent photoaffinity labeling of HMGCR by photoactivatable derivatives of the 1,1-bisphosphonate ester SRP-3042 and 25HC. These findings disclose a direct sterol binding mechanism as the trigger that initiates the HMGCR ERAD pathway, providing valuable insights into the intricate mechanisms that govern cholesterol homeostasis.

Association of COVID-19 Vaccination Rates and Facility Characteristics on COVID-19 Cases, Hospitalizations, and Deaths in US Nursing Homes.

OBJECTIVES: Nursing home residents have been disproportionately affected by the COVID-19 pandemic. Despite recognition as a priority group for receipt of the COVID-19 vaccine, vaccine uptake and COVID-19 cases, hospitalizations, and deaths in nursing home facilities were variable across nursing homes. This study has 2 objectives: (1) to describe nursing facility characteristics associated with higher vs lower vaccination rates and (2) to estimate facility characteristics associated with COVID-19 cases, hospitalizations, and deaths, stratified by vaccination rate. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: Facility-level data from 12,811 US nursing home facilities. METHODS: Using the CMS's Nursing Home COVID-19 Public File, we analyzed nursing home COVID-19 vaccination rates and outcomes from June 13, 2021, to September 19, 2021. We performed multivariable logistic regressions and identified facility characteristics associated with increased vaccination uptake and COVID-19 outcomes. RESULTS: Nursing homes with average vaccination rates ≤80% experienced higher total average COVID-19 cases, hospitalizations, and deaths compared to facilities with >80% average vaccination rates during the Delta surge. Moreover, facility factors, such as higher average age of residents, proportion of non-white residents, nurse staffing hours, and occupancy rates, were variably associated with increased risk of COVID-19 outcomes. CONCLUSIONS AND IMPLICATIONS: Facilities with higher resident vaccination rates experienced lower average COVID-19 cases, hospitalizations, and deaths in US nursing homes. Access to vaccines may play a role in mitigating harm associated with infectious diseases. Additionally, facility factors associated with increased adverse outcomes were variably associated with increased odds of COVID-19 outcomes, often, irrespective of vaccination level. As the COVID-19 pandemic continues to evolve and as the possibility of other infectious disease variants emerge, this research provides insight into facility factors, including vaccine uptake, that may mitigate adverse outcomes.

Synthesis, function, and regulation of sterol and nonsterol isoprenoids.

Cholesterol, the bulk end-product of the mevalonate pathway, is a key component of cellular membranes and lipoproteins that transport lipids throughout the body. It is also a precursor of steroid hormones, vitamin D, and bile acids. In addition to cholesterol, the mevalonate pathway yields a variety of nonsterol isoprenoids that are essential to cell survival. Flux through the mevalonate pathway is tightly controlled to ensure cells continuously synthesize nonsterol isoprenoids but avoid overproducing cholesterol and other sterols. Endoplasmic reticulum (ER)-localized 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (HMGCR), the rate limiting enzyme in the mevalonate pathway, is the focus of a complex feedback regulatory system governed by sterol and nonsterol isoprenoids. This review highlights transcriptional and post-translational regulation of HMGCR. Transcriptional regulation of HMGCR is mediated by the Scap-SREBP pathway. Post-translational control is initiated by the intracellular accumulation of sterols, which causes HMGCR to become ubiquitinated and subjected to proteasome-mediated ER-associated degradation (ERAD). Sterols also cause a subfraction of HMGCR molecules to bind the vitamin K2 synthetic enzyme, UbiA prenyltransferase domain-containing protein-1 (UBIAD1). This binding inhibits ERAD of HMGCR, which allows cells to continuously synthesize nonsterol isoprenoids such as geranylgeranyl pyrophosphate (GGPP), even when sterols are abundant. Recent studies reveal that UBIAD1 is a GGPP sensor, dissociating from HMGCR when GGPP thresholds are met to allow maximal ERAD. Animal studies using genetically manipulated mice disclose the physiological significance of the HMGCR regulatory system and we describe how dysregulation of these pathways contributes to disease.

Regulated degradation of HMG CoA reductase requires conformational changes in sterol-sensing domain.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme in cholesterol synthesis and target of cholesterol-lowering statin drugs. Accumulation of sterols in endoplasmic reticulum (ER) membranes accelerates degradation of HMGCR, slowing the synthesis of cholesterol. Degradation of HMGCR is inhibited by its binding to UBIAD1 (UbiA prenyltransferase domain-containing protein-1). This inhibition contributes to statin-induced accumulation of HMGCR, which limits their cholesterol-lowering effects. Here, we report cryo-electron microscopy structures of the HMGCR-UBIAD1 complex, which is maintained by interactions between transmembrane helix (TM) 7 of HMGCR and TMs 2-4 of UBIAD1. Disrupting this interface by mutagenesis prevents complex formation, enhancing HMGCR degradation. TMs 2-6 of HMGCR contain a 170-amino acid sterol sensing domain (SSD), which exists in two conformations-one of which is essential for degradation. Thus, our data supports a model that rearrangement of the TMs in the SSD permits recruitment of proteins that initate HMGCR degradation, a key reaction in the regulatory system that governs cholesterol synthesis.

Thermodynamic Surface Analyses to Inform Biofilm Resistance.

Biofilms are the habitat of 95% of bacteria successfully protecting bacteria from many antibiotics. However, inhibiting biofilm formation is difficult in that it is a complex system involving the physical and chemical interaction of both substrate and bacteria. Focusing on the substrate surface and potential interactions with bacteria, we examined both physical and chemical properties of substrates coated with a series of phenyl acrylate monomer derivatives. Atomic force microscopy (AFM) showed smooth surfaces often approximating surgical grade steel. Induced biofilm growth of five separate bacteria on copolymer samples comprising varying concentrations of phenyl acrylate monomer derivatives evidenced differing degrees of biofilm resistance via optical microscopy. Using goniometric surface analyses, the van Oss-Chaudhury-Good equation was solved linear algebraically to determine the surface energy profile of each polymerized phenyl acrylate monomer derivative, two bacteria, and collagen. Based on the microscopy and surface energy profiles, a thermodynamic explanation for biofilm resistance is posited.

Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling.

Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex 'retriever'; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5ß1 integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major endosomal retrieval and recycling pathway.

Underwater noise levels in UK waters.

Underwater noise from human activities appears to be rising, with ramifications for acoustically sensitive marine organisms and the functioning of marine ecosystems. Policymakers are beginning to address the risk of ecological impact, but are constrained by a lack of data on current and historic noise levels. Here, we present the first nationally coordinated effort to quantify underwater noise levels, in support of UK policy objectives under the EU Marine Strategy Framework Directive (MSFD). Field measurements were made during 2013-2014 at twelve sites around the UK. Median noise levels ranged from 81.5-95.5 dB re 1 µPa for one-third octave bands from 63-500 Hz. Noise exposure varied considerably, with little anthropogenic influence at the Celtic Sea site, to several North Sea sites with persistent vessel noise. Comparison of acoustic metrics found that the RMS level (conventionally used to represent the mean) was highly skewed by outliers, exceeding the 97th percentile at some frequencies. We conclude that environmental indicators of anthropogenic noise should instead use percentiles, to ensure statistical robustness. Power analysis indicated that at least three decades of continuous monitoring would be required to detect trends of similar magnitude to historic rises in noise levels observed in the Northeast Pacific.

A review of crustacean sensitivity to high amplitude underwater noise: Data needs for effective risk assessment in relation to UK commercial species.

High amplitude anthropogenic noise is associated with adverse impacts among a variety of organisms but detailed species-specific knowledge is lacking in relation to effects upon crustaceans. Brown crab (Cancer pagurus), European lobster (Homarus gammarus) and Norway lobster (Nephrops norvegicus) together represent the most valuable commercial fishery in the UK (Defra, 2014). Critical evaluation of literature reveals physiological sensitivity to underwater noise among N. norvegicus and closely related crustacean species, including juvenile stages. Current evidence supports physiological sensitivity to local, particle motion effects of sound production in particular. Derivation of correlative relationships between the introduction of high amplitude impulsive noise and crustacean distribution/abundance is hindered by the coarse resolution of available data at the present time. Future priorities for research are identified and argument for enhanced monitoring under current legislative frameworks outlined.

Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling.

Notch family members are transmembrane receptors that mediate essential developmental programs. Upon ligand binding, a proteolytic event releases the intracellular domain of Notch, which translocates to the nucleus to regulate gene transcription. In addition, Notch trafficking across the endolysosomal system is critical in its regulation. In this study we report that Notch recycling to the cell surface is dependent on the COMMD-CCDC22-CCDC93 (CCC) complex, a recently identified regulator of endosomal trafficking. Disruption in this system leads to intracellular accumulation of Notch2 and concomitant reduction in Notch signaling. Interestingly, among the 10 copper metabolism MURR1 domain containing (COMMD) family members that can associate with the CCC complex, only COMMD9 and its binding partner, COMMD5, have substantial effects on Notch. Furthermore, Commd9 deletion in mice leads to embryonic lethality and complex cardiovascular alterations that bear hallmarks of Notch deficiency. Altogether, these studies highlight that the CCC complex controls Notch activation by modulating its intracellular trafficking and demonstrate cargo-specific effects for members of the COMMD protein family.

Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells.

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein's cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.

The Surface of Nanoparticle Silicon as Studied by Solid-State NMR.

The surface structure and adjacent interior of commercially available silicon nanopowder (np-Si) was studied using multinuclear, solid-state NMR spectroscopy. The results are consistent with an overall picture in which the bulk of the np-Si interior consists of highly ordered ("crystalline") silicon atoms, each bound tetrahedrally to four other silicon atoms. From a combination of ¹H, 29Si and ²H magic-angle-spinning (MAS) NMR results and quantum mechanical 29Si chemical shift calculations, silicon atoms on the surface of "as-received" np-Si were found to exist in a variety of chemical structures, with apparent populations in the order (a) (Si-O-)3Si-H > (b) (Si-O-)3SiOH > (c) (HO-)nSi(Si)m(-OSi)4-m-n ≈ (d) (Si-O-)2Si(H)OH > (e) (Si-O-)2Si(-OH)2 > (f) (Si-O-)4Si, where Si stands for a surface silicon atom and Si represents another silicon atom that is attached to Si by either a Si-Si bond or a Si-O-Si linkage. The relative populations of each of these structures can be modified by chemical treatment, including with O2 gas at elevated temperature. A deliberately oxidized sample displays an increased population of (Si-O-)3Si-H, as well as (Si-O-)3SiOH sites. Considerable heterogeneity of some surface structures was observed. A combination of ¹H and ²H MAS experiments provide evidence for a substantial population of silanol (Si-OH) moieties, some of which are not readily H-exchangeable, along with the dominant Si-H sites, on the surface of "as-received" np-Si; the silanol moieties are enhanced by deliberate oxidation. An extension of the DEPTH background suppression method is also demonstrated that permits measurement of the T2 relaxation parameter simultaneously with background suppression.

Saving transverse magnetization.

A magnetization storage sequence, ALT-1 (alternating longitudinal and transverse components), is reported. The ALT-1 sequence is a hybrid of two types of storage sequences, the Carr-Purcell type and store-and-restore sequences. During incremental storage periods within the ALT-1 sequence, essentially half of the initially transverse magnetization is stored along the z-axis and the other half is prolonged by an echo-generating pulse. The portions of initial magnetization that are stored as longitudinal components or transverse components are alternated by a pi/2 pulse during the cycle. Both transverse components of the initial magnetization are treated the same in the ALT-1 sequence and orientational (phase) information of the initial magnetization is kept during the storage period. The ALT-1 sequence can preserve magnetization more effectively than a published class of modified Carr-Purcell type sequences, because essentially half of the magnetization during incremental storage periods is not subjected to relaxation from T2 effects.

Transvaginal biopsy in the diagnosis of ovarian cancer.

Laparotomy and debulking surgery followed by chemotherapy have been the treatment of choice in late stage ovarian carcinoma. Developments in the chemotherapeutic management of ovarian cancer have resulted in a change in opinion as to the optimal management of this disease. Many patients are now receiving initial chemotherapy and trials are in place to compare up front and adjuvant surgery. Tissue diagnosis is required prior to commencing chemotherapy. This article describes one method for accurately obtaining a tumour biopsy. A retrospective case note review of 14 women with a provisional diagnosis of ovarian carcinoma who underwent transvaginal biopsy of their pelvic disease is described. Only 7/12 cases with a positive biopsy had a definite diagnosis of ovarian cancer. The procedure was found to be safe and well tolerated.

Human papillomavirus type 16 E2- and L1-specific serological and T-cell responses in women with vulval intraepithelial neoplasia.

Human papillomavirus type 16 (HPV-16)-associated vulval intraepithelial neoplasia (VIN) is frequently a chronic, multifocal high-grade condition with an appreciable risk of progression to vulval cancer. The requirement to treat women with VIN has recently stimulated the use of immunotherapy with E6/E7 oncogene vaccines. Animal models have shown that E2 may also be a useful vaccine target for HPV-associated disease; however, little is known about E2 immunity in humans. This study investigated the prevalence of HPV-16 E2-specific serological and T-cell responses in 18 women with HPV-16-associated VIN and 17 healthy volunteers. E2 responses were determined by full-length E2-GST ELISA with ELISPOT and proliferation assays using E2 C-terminal protein. As positive controls, HPV-16 L1 responses were measured using virus-like particles (VLPs) and L1-GST ELISA with ELISPOT and proliferation using VLPs as antigen. The VIN patients all showed a strong serological response to L1 compared with the healthy volunteers by VLP (15/18 vs 1/17, P<0.001) and L1-GST ELISA (18/18 vs 1/17, P<0.001). In contrast, L1-specific cellular immune responses were detected in a significant proportion of controls but were more prevalent in the VIN patients by proliferation assay (9/17 vs 17/18, P<0.02) and interferon-gamma ELISPOT (9/17 vs 13/18, P=not significant). Similar and low numbers of patients and controls were seropositive for E2-specific Ig (2/18 vs 1/17). In spite of previous studies showing the immunogenicity of E2 in eliciting primary T-cell responses in vitro, there was a low prevalence of E2 responses in the VIN patients and controls (2/18 vs 0/17).