Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

The RAS/MAPK Axis Gets Stressed Out.

In this issue of Molecular Cell, Ritt et al. (2016) describe a stress-induced checkpoint that effectively suppresses RAS-MAPK signaling. This pathway, activated by agents such as Rigosertib that induce mitotic and oxidative stress, results in JNK-mediated inhibition of RAS-MAPK pathway components SOS and RAF.

Waveguide-integrated chip-scale optomechanical magnetometer.

Optomechanical magnetometers enable highly sensitive magnetic field sensing. However, all such magnetometers to date have been optically excited and read-out either via free space or a tapered optical fiber. This limits their scalability and integrability, and ultimately their range of applications. Here, we present an optomechanical magnetometer that is excited and read-out via a suspended optical waveguide fabricated on the same silicon chip as the magnetometer. Moreover, we demonstrate that thermomechanical noise limited sensitivity is possible using portable electronics and laser. The magnetometer employs a silica microdisk resonator selectively sputtered with a magnetostrictive film of galfenol (FeGa) which induces a resonant frequency shift in response to an external magnetic field. Experimental results reveal the retention of high quality-factor optical whispering gallery mode resonances whilst also demonstrating high sensitivity and dynamic range in ambient conditions. The use of off-the-shelf portable electronics without compromising sensor performance demonstrates promise for applications.

Preferential coupling of diamond NV centres in step-index fibres.

Diamonds containing the negatively charged nitrogen-vacancy centre are a promising system for room-temperature magnetometry. The combination of nano- and micro-diamond particles with optical fibres provides an option for deploying nitrogen-vacancy magnetometers in harsh and challenging environments. Here we numerically explore the coupling efficiency from nitrogen-vacancy centres within a diamond doped at the core/clad interface across a range of commercially available fibre types so as to inform the design process for a diamond in fibre magnetometers. We determine coupling efficiencies from nitrogen-vacancy centres to the guided modes of a step-index fibre and predict the optically detected magnetic resonance (ODMR) generated by a ensemble of four nitrogen-vacancy centres in this hybrid fibre system. Our results show that the coupling efficiency is enhanced with a high refractive index difference between the fibre core and cladding and depends on the radial position of the nitrogen-vacancy centres in the fibre core. Our ODMR simulations show that due to the preferential coupling of the nitrogen-vacancy emission to the fibre guided modes, certain magnetometry features such as ODMR contrast can be enhanced and lead to improved sensitivity in such diamond-fibre systems, relative to conventional diamond only ensemble geometries.

Effects of ignoring survey design information for data reuse.

Data are currently being used, and reused, in ecological research at an unprecedented rate. To ensure appropriate reuse however, we need to ask the question: "Are aggregated databases currently providing the right information to enable effective and unbiased reuse?" We investigate this question, with a focus on designs that purposefully favor the selection of sampling locations (upweighting the probability of selection of some locations). These designs are common and examples are those designs that have uneven inclusion probabilities or are stratified. We perform a simulation experiment by creating data sets with progressively more uneven inclusion probabilities and examine the resulting estimates of the average number of individuals per unit area (density). The effect of ignoring the survey design can be profound, with biases of up to 250% in density estimates when naive analytical methods are used. This density estimation bias is not reduced by adding more data. Fortunately, the estimation bias can be mitigated by using an appropriate estimator or an appropriate model that incorporates the design information. These are only available however, when essential information about the survey design is available: the sample location selection process (e.g., inclusion probabilities), and/or covariates used in their specification. The results suggest that such information must be stored and served with the data to support meaningful inference and data reuse.

A modified flavonoid accelerates oligodendrocyte maturation and functional remyelination.

Myelination delay and remyelination failure following insults to the central nervous system (CNS) impede axonal conduction and lead to motor, sensory and cognitive impairments. Both myelination and remyelination are often inhibited or delayed due to the failure of oligodendrocyte progenitor cells (OPCs) to mature into myelinating oligodendrocytes (OLs). Digestion products of the glycosaminoglycan hyaluronan (HA) have been implicated in blocking OPC maturation, but how these digestion products are generated is unclear. We tested the possibility that hyaluronidase activity is directly linked to the inhibition of OPC maturation by developing a novel modified flavonoid that functions as a hyaluronidase inhibitor. This compound, called S3, blocks some but not all hyaluronidases and only inhibits matrix metalloproteinase activity at high concentrations. We find that S3 reverses HA-mediated inhibition of OPC maturation in vitro, an effect that can be overcome by excess recombinant hyaluronidase. Furthermore, we find that hyaluronidase inhibition by S3 accelerates OPC maturation in an in vitro model of perinatal white matter injury. Finally, blocking hyaluronidase activity with S3 promotes functional remyelination in mice with lysolecithin-induced demyelinating corpus callosum lesions. All together, these findings support the notion that hyaluronidase activity originating from OPCs in CNS lesions is sufficient to prevent OPC maturation, which delays myelination or blocks remyelination. These data also indicate that modified flavonoids can act as selective inhibitors of hyaluronidase activity and can promote OPC maturation, making them excellent candidates to accelerate myelination or promote remyelination following perinatal and adult CNS insults.

The APC/C subunit Mnd2/Apc15 promotes Cdc20 autoubiquitination and spindle assembly checkpoint inactivation.

The fidelity of chromosome segregation depends on the spindle assembly checkpoint (SAC). In the presence of unattached kinetochores, anaphase is delayed when three SAC components (Mad2, Mad3/BubR1, and Bub3) inhibit Cdc20, the activating subunit of the anaphase-promoting complex (APC/C). We analyzed the role of Cdc20 autoubiquitination in the SAC of budding yeast. Reconstitution with purified components revealed that a Mad3-Bub3 complex synergizes with Mad2 to lock Cdc20 on the APC/C and stimulate Cdc20 autoubiquitination, while inhibiting ubiquitination of substrates. SAC-dependent Cdc20 autoubiquitination required the Mnd2/Apc15 subunit of the APC/C. General inhibition of Cdc20 ubiquitination in vivo resulted in high Cdc20 levels and a failure to establish a SAC arrest, suggesting that SAC establishment depends on low Cdc20 levels. Specific inhibition of SAC-dependent ubiquitination, by deletion of Mnd2, allowed establishment of a SAC arrest but delayed release from the arrest, suggesting that Cdc20 ubiquitination is also required for SAC inactivation.

A Randomized, Double-Blind, Placebo-Controlled Phase 3 Trial of Oral Brincidofovir for Cytomegalovirus Prophylaxis in Allogeneic Hematopoietic Cell Transplantation.

Cytomegalovirus (CMV) infection is a common complication of allogeneic hematopoietic cell transplantation (HCT). In this trial, we randomized adult CMV-seropositive HCT recipients without CMV viremia at screening 2:1 to receive brincidofovir or placebo until week 14 post-HCT. Randomization was stratified by center and risk of CMV infection. Patients were assessed weekly through week 15 and every third week thereafter through week 24 post-HCT. Patients who developed clinically significant CMV infection (CS-CMVi; CMV viremia requiring preemptive therapy or CMV disease) discontinued the study drug and began anti-CMV treatment. The primary endpoint was the proportion of patients with CS-CMVi through week 24 post-HCT; patients who discontinued the trial or with missing data were imputed as primary endpoint events. Between August 2013 and June 2015, 452 patients were randomized at a median of 15 days after HCT and received study drug. The proportion of patients who developed CS-CMVi or were imputed as having a primary endpoint event through week 24 was similar between brincidofovir-treated patients and placebo recipients (155 of 303 [51.2%] versus 78 of 149 [52.3%]; odds ratio, .95 [95% confidence interval, .64 to 1.41]; P = .805); fewer brincidofovir recipients developed CMV viremia through week 14 compared with placebo recipients (41.6%; P < .001). Serious adverse events were more frequent among brincidofovir recipients (57.1% versus 37.6%), driven by acute graft-versus-host disease (32.3% versus 6.0%) and diarrhea (6.9% versus 2.7%). Week 24 all-cause mortality was 15.5% among brincidofovir recipients and 10.1% among placebo recipients. Brincidofovir did not reduce CS-CMVi by week 24 post-HCT and was associated with gastrointestinal toxicity.

Diastereoselective Synthesis of Arabino- and Ribo-like Nucleoside Analogues Bearing a Stereogenic C3' All-Carbon Quaternary Center.

The synthesis of novel nucleoside analogues bearing a C3' all-carbon quaternary center and a C2'-hydroxy substituent is described. The all-carbon stereogenic center was generated through an intramolecular 7-endo attack of a silyl-tethered allyl moiety on a tertiary radical using photoredox catalysis. Subsequent allylic oxidation and diastereoselective hydride reductions provided the hydroxy substituent at C2', which then controls the stereoselective introduction of pyrimidine nucleobases on the corresponding furanose scaffold. Density functional theory (DFT) calculations provided insights into the origin of the high syn diastereoselectivity resulting from the radical cyclization. This original methodology grants access to a wide range of 1',2'-cis and 1',2'-trans arabino- and ribo-like analogues bearing an all-carbon quaternary center at C3'. These molecules are currently being tested for their antiviral and anticancer properties.

CD44 Transmembrane Receptor and Hyaluronan Regulate Adult Hippocampal Neural Stem Cell Quiescence and Differentiation.

Adult neurogenesis in the hippocampal subgranular zone (SGZ) is involved in learning and memory throughout life but declines with aging. Mice lacking the CD44 transmembrane receptor for the glycosaminoglycan hyaluronan (HA) demonstrate a number of neurological disturbances including hippocampal memory deficits, implicating CD44 in the processes underlying hippocampal memory encoding, storage, or retrieval. Here, we found that HA and CD44 play important roles in regulating adult neurogenesis, and we provide evidence that HA contributes to age-related reductions in neural stem cell (NSC) expansion and differentiation in the hippocampus. CD44-expressing NSCs isolated from the mouse SGZ are self-renewing and capable of differentiating into neurons, astrocytes, and oligodendrocytes. Mice lacking CD44 demonstrate increases in NSC proliferation in the SGZ. This increased proliferation is also observed in NSCs grown in vitro, suggesting that CD44 functions to regulate NSC proliferation in a cell-autonomous manner. HA is synthesized by NSCs and increases in the SGZ with aging. Treating wild type but not CD44-null NSCs with HA inhibits NSC proliferation. HA digestion in wild type NSC cultures or in the SGZ induces increased NSC proliferation, and CD44-null as well as HA-disrupted wild type NSCs demonstrate delayed neuronal differentiation. HA therefore signals through CD44 to regulate NSC quiescence and differentiation, and HA accumulation in the SGZ may contribute to reductions in neurogenesis that are linked to age-related decline in spatial memory.

Catalysis of lysine 48-specific ubiquitin chain assembly by residues in E2 and ubiquitin.

Protein ubiquitination is catalyzed by ubiquitin-conjugating enzymes (E2s) in collaboration with ubiquitin-protein ligases (E3s). This process depends on nucleophilic attack by a substrate lysine on a thioester bond linking the C terminus of ubiquitin to a cysteine in the E2 active site. Different E2 family members display specificity for lysines in distinct contexts. We addressed the mechanistic basis for this lysine selectivity in Ubc1, an E2 that catalyzes the ubiquitination of lysine 48 (K48) in ubiquitin, leading to the formation of K48-linked polyubiquitin chains. We identified a cluster of polar residues near the Ubc1 active site, as well as a residue in ubiquitin itself, that are required for catalysis of K48-specific ubiquitin ligation, but not for general activity toward other lysines. Our results suggest that the active site of Ubc1, as well as the surface of ubiquitin, contains specificity determinants that channel specific lysines to the central residues involved directly in catalysis.

Brg1 directly regulates Olig2 transcription and is required for oligodendrocyte progenitor cell specification.

The Olig2 basic-helix-loop-helix transcription factor promotes oligodendrocyte specification in early neural progenitor cells (NPCs), including radial glial cells, in part by recruiting SWI/SNF chromatin remodeling complexes to the enhancers of genes involved in oligodendrocyte differentiation. How Olig2 expression is regulated during oligodendrogliogenesis is not clear. Here, we find that the Brg1 subunit of SWI/SNF complexes interacts with a proximal Olig2 promoter and represses Olig2 transcription in the mouse cortex at E14, when oligodendrocyte progenitors (OPCs) are not yet found in this location. Brg1 does not interact with the Olig2 promoter in the E14 ganglionic eminence, where NPCs differentiate into Olig2-positive OPCs. Consistent with these findings, Brg1-null NPCs demonstrate precocious expression of Olig2 in the cortex. However, these cells fail to differentiate into OPCs. We further find that Brg1 is necessary for neuroepithelial-to-radial glial cell transition, but not neuronal differentiation despite a reduction in expression of the pro-neural transcription factor Pax6. Collectively, these and earlier findings support a model whereby Brg1 promotes neurogenic radial glial progenitor cell specification but is dispensable for neuronal differentiation. Concurrently, Brg1 represses Olig2 expression and the specification of OPCs, but is required for OPC differentiation and oligodendrocyte maturation.

Analysis of Mutations in the Gene Encoding Cytomegalovirus DNA Polymerase in a Phase 2 Clinical Trial of Brincidofovir Prophylaxis.

Brincidofovir is an oral antiviral in development for prevention of cytomegalovirus disease. Cytomegalovirus genotyping results from a phase 2 trial comparing brincidofovir to placebo for prophylaxis against cytomegalovirus infection in hematopoietic cell transplant recipients provided initial data on the clinical resistance profile for brincidofovir. In this study, no known resistance-associated mutations were detected in brincidofovir-treated subjects; identified genotypic substitutions did not confer resistance to cytomegalovirus antivirals in vitro, suggesting that these changes represent polymorphisms unrelated to brincidofovir resistance. Lack of evidence for genotypic resistance during prophylaxis suggests that first-line use of brincidofovir for prevention of cytomegalovirus infection may preserve downstream options for patients.

Effects of Obesity on Health Related Quality of Life Following Total Hip Arthroplasty.

Obesity is known to negatively impact health related quality of life (HRQoL). Although non-disease specific tools have been used to study HRQoL after THA in obese patients, these do not directly measure health utility improvements. All 435 THA patients in the current study, regardless of BMI, reported improvement in HRQoL as measured by EQ-5D, a universal, standardized, non-disease specific preference-based instrument. These data suggest obese patients value their quality of life improvement following THA as much as non-obese patients. Furthermore, the increased activity level observed following THA in obese patients suggests obese patients may also obtain non-disease specific benefits of a more active lifestyle. This information is important for future assessments of value and cost-effectiveness of THA in the obese population.

In vitro efficacy of brincidofovir against variola virus.

Brincidofovir (CMX001), a lipid conjugate of the acyclic nucleotide phosphonate cidofovir, is under development for smallpox treatment using "the Animal Rule," established by the FDA in 2002. Brincidofovir reduces mortality caused by orthopoxvirus infection in animal models. Compared to cidofovir, brincidofovir has increased potency, is administered orally, and shows no evidence of nephrotoxicity. Here we report that the brincidofovir half-maximal effective concentration (EC50) against five variola virus strains in vitro averaged 0.11 µM and that brincidofovir was therefore nearly 100-fold more potent than cidofovir.

Do communities exist? Complex patterns of overlapping marine species distributions.

Understanding the way in which species are associated in communities is a fundamental question in ecology. Yet there remains a tension between communities as highly structured units or as coincidental collections of individualistic species. We explored these ideas using a new statistical approach that clusters species based on their environmental response: a species archetype, rather than clustering sites based on their species composition. We found groups of species that are consistently highly correlated, but that these groups are not unique to any set of locations and overlap spatially. The species present at a single site are a realization of species from the (multiple) archetype groups that are likely to be present at that location based on their response to the environment.

Digestion products of the PH20 hyaluronidase inhibit remyelination.

OBJECTIVE: Oligodendrocyte progenitor cells (OPCs) recruited to demyelinating lesions often fail to mature into oligodendrocytes (OLs) that remyelinate spared axons. The glycosaminoglycan hyaluronan (HA) accumulates in demyelinating lesions and has been implicated in the failure of OPC maturation and remyelination. We tested the hypothesis that OPCs in demyelinating lesions express a specific hyaluronidase, and that digestion products of this enzyme inhibit OPC maturation. METHODS: Mouse OPCs grown in vitro were analyzed for hyaluronidase expression and activity. Gain of function studies were used to define the hyaluronidases that blocked OPC maturation. Mouse and human demyelinating lesions were assessed for hyaluronidase expression. Digestion products from different hyaluronidases and a hyaluronidase inhibitor were tested for their effects on OPC maturation and functional remyelination in vivo. RESULTS: OPCs demonstrated hyaluronidase activity in vitro and expressed multiple hyaluronidases, including HYAL1, HYAL2, and PH20. HA digestion by PH20 but not other hyaluronidases inhibited OPC maturation into OLs. In contrast, inhibiting HA synthesis did not influence OPC maturation. PH20 expression was elevated in OPCs and reactive astrocytes in both rodent and human demyelinating lesions. HA digestion products generated by the PH20 hyaluronidase but not another hyaluronidase inhibited remyelination following lysolecithin-induced demyelination. Inhibition of hyaluronidase activity lead to increased OPC maturation and promoted increased conduction velocities through lesions. INTERPRETATION: We determined that PH20 is elevated in demyelinating lesions and that increased PH20 expression is sufficient to inhibit OPC maturation and remyelination. Pharmacological inhibition of PH20 may therefore be an effective way to promote remyelination in multiple sclerosis and related conditions.

Naphthyridinone (NTD) integrase inhibitors 4. Investigating N1 acetamide substituent effects with C3 amide groups.

A series of N1 acetamide substituted naphthyridinone HIV-1 integrase inhibitors have been explored to understand structure-activity relationships (SAR) with various C3 amide groups. Investigations were evaluated using integrase enzyme inhibition, antiviral activity and protein binding effects to optimize the sub-structures. Lipophilicity was also incorporated to understand ligand lipophilic efficiency as a function of the structural modifications. Three representative analogs were further examined in a peripheral blood mononuclear cell (PBMC) antiviral assay as well as in vitro and in vivo drug metabolism and pharmacokinetic studies.

The spatialization of decent work and the role of employability empowerment for minority ethnic young people in emerging economies.

Global rises in precarious labour conditions have prompted further empirical work in Decent Work, a special category of employment characterised by equitable pay, treatment, and healthy working conditions. Despite this, research has tended to be conducted in developed countries with privileged groups such as those with typical working arrangements and rely on psychologically framed individual characteristics to explain marginalising factors. We propose a more sociologically framed, spatialised perspective on Decent Work which posits that marginalising factors are spatially variable and determined but moderated by employability empowerment. We measure our propositions across three spatially different sites of Vietnam through (1) a survey of minority ethnic students and graduates (N = 1071) and (2) a survey of stakeholders involved in the recruitment and employment of this group (N = 204). We find support for most of our propositions and call for more spatialised empirical work in the field of Decent Work.

Allosteric Inhibitors of the SARS-COV-2 Papain-like Protease Domain Induce Proteasomal Degradation of Its Parent Protein NSP3.

The papain-like protease of SARS-COV-2 is essential for viral replication and pathogenesis. Its location within a much larger multifunctional protein, NSP3, makes it an ideal candidate for a targeted degradation approach capable of eliminating multiple functions with a single-molecule treatment. In this work, we have developed a HiBiT-based cellular model to study NSP3 degradation and used this platform for the discovery of monovalent NSP3 degraders. We present previously unreported degradation activity of published papain-like protease inhibitors. Follow-up exploration of structure-activity relationships and mechanism-of-action studies points to the recruitment of the ubiquitin-proteasome machinery that is solely driven by site occupancy, regardless of molecular features of the ligand. Supported by HDX data, we hypothesize that binding-induced structural changes in NSP3 trigger the recruitment of an E3 ligase and lead to proteasomal degradation.