NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

Acute myeloid leukemias with UBTF tandem duplications are sensitive to menin inhibitors.

ABSTRACT: UBTF tandem duplications (UBTF-TDs) have recently emerged as a recurrent alteration in pediatric and adult acute myeloid leukemia (AML). UBTF-TD leukemias are characterized by a poor response to conventional chemotherapy and a transcriptional signature that mirrors NUP98-rearranged and NPM1-mutant AMLs, including HOX-gene dysregulation. However, the mechanism by which UBTF-TD drives leukemogenesis remains unknown. In this study, we investigated the genomic occupancy of UBTF-TD in transformed cord blood CD34+ cells and patient-derived xenograft models. We found that UBTF-TD protein maintained genomic occupancy at ribosomal DNA loci while also occupying genomic targets commonly dysregulated in UBTF-TD myeloid malignancies, such as the HOXA/HOXB gene clusters and MEIS1. These data suggest that UBTF-TD is a gain-of-function alteration that results in mislocalization to genomic loci dysregulated in UBTF-TD leukemias. UBTF-TD also co-occupies key genomic loci with KMT2A and menin, which are known to be key partners involved in HOX-dysregulated leukemias. Using a protein degradation system, we showed that stemness, proliferation, and transcriptional signatures are dependent on sustained UBTF-TD localization to chromatin. Finally, we demonstrate that primary cells from UBTF-TD leukemias are sensitive to the menin inhibitor SNDX-5613, resulting in markedly reduced in vitro and in vivo tumor growth, myeloid differentiation, and abrogation of the UBTF-TD leukemic expression signature. These findings provide a viable therapeutic strategy for patients with this high-risk AML subtype.

Interplay between Magnetoresistance and Kondo Resonance in Radical Single-Molecule Junctions.

We report transport measurements on tunable single-molecule junctions of the organic perchlorotrityl radical molecule, contacted with gold electrodes at low temperature. The current-voltage characteristics of a subset of junctions shows zero-bias anomalies due to the Kondo effect and in addition elevated magnetoresistance (MR). Junctions without Kondo resonance reveal a much stronger MR. Furthermore, we show that the amplitude of the MR can be tuned by mechanically stretching the junction. On the basis of these findings, we attribute the high MR to an interference effect involving spin-dependent scattering at the metal-molecule interface and assign the Kondo effect to the unpaired spin located in the center of the molecule in asymmetric junctions.

Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning.

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an "inward" position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 "inward", like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work.

Raised initial total white cell count and lower post-operative decline of C reactive protein increases the risk of secondary surgery in septic arthritis of the native knee.

PURPOSE: Septic arthritis is an orthopaedic emergency with high morbidity and mortality. The aim of this study is to determine the risk factors associated with secondary surgery for septic arthritis of the native knee joint. METHODS: This is a retrospective study reviewing all patients who underwent surgery for septic arthritis of the knee from 2012 to 2019 in a single institution. A total of 117 patients were recruited. Patients were divided into 2 groups: Group I (79/117, 67.5%) underwent one surgery and Group II (38/117, 32.5%) underwent more than one surgery. RESULTS: Patients with a raised initial total white (TW) cell count of > 20 × 109/L had a significantly higher risk of secondary surgery. (Adjusted hazard ratio 2.42, p < 0.05) A decline of CRP level of less than 20% within 24 h from initial operation was also a risk for secondary surgery. (Adjusted hazard ratio 0.34, p < 0.01) Patients in group II also had significantly higher post-operative median TW cell count and neutrophil count. There was no significant difference in the offending microbe, surgical approach, and duration of operation from initial presentation between the groups. CONCLUSIONS: Patients with septic arthritis of the native knee joint who present with raised initial total white cell count of > 20 × 109/L and decline of CRP level of less than 20% within 24 h from initial operation are at higher risk of secondary operation. In these patients, more aggressive treatment strategies and appropriate counselling on the risks of repeated surgery are recommended. LEVEL OF EVIDENCE: Level IV.

Life cycle assessment of single-use surgical and embedded filtration layer (EFL) reusable face mask.

BACKGROUND: The outbreak of the COVID-19 pandemic has led to an unprecedented amount of face mask consumption around the world. The increase in face mask consumption has brought focus to their environmental impact. To keep up with the increased demand for face masks, different variations of reusable face masks such as the embedded filtration layer (EFL) reusable face mask have emerged in the market. This study quantifies the environmental impact of the EFL reusable face mask and the single-use surgical face mask. METHODS: The life cycle assessment (LCA) study of the entire value chain from cradle-to-grave is applied to each face mask. Both face masks are evaluated over 1 functional unit (FU) of 31 12-h days for a single person. The ReCiPe method with the Hierachist perspective was applied. A total of nine impact categories as well as the generated waste of each face mask are evaluated. RESULTS: The results show that for 1 functional unit, the use of single-use surgical face mask and EFL reusable face mask will contribute 0.580 kg CO2-eq and 0.338 kg CO2-eq to climate change and generate 0.004 kg and 0.0004 kg of waste respectively. CONCLUSION: Comparing both face masks, the EFL reusable face mask will have a lower emission of at least 30% in terms of the generated waste and the impact categories considered, except for water depletion, freshwater eutrophication, marine eutrophication, and human toxicity.

The Environment-Dependent Behavior of the Blatter Radical at the Metal-Molecule Interface.

Stable organic radicals have potential applications for building organic spintronic devices. To fulfill this potential, the interface between organic radicals and metal electrodes must be well characterized. Here, through a combined effort that includes synthesis, scanning tunneling microscopy, X-ray spectroscopy, and single-molecule conductance measurements, we comprehensively probe the electronic interaction between gold metal electrodes and a benchtop stable radical-the Blatter radical. We find that despite its open-shell character and having a half-filled orbital close to the Fermi level, the radical is stable on a gold substrate under ultrahigh vacuum. We observe a Kondo resonance arising from the radical and spectroscopic signatures of its half-filled orbitals. By contrast, in solution-based single-molecule conductance measurements, the radical character is lost through oxidation with charge transfer occurring from the molecule to metal. Our experiments show that the stability of radical states can be very sensitive to the environment around the molecule.

Breaking Down Resonance: Nonlinear Transport and the Breakdown of Coherent Tunneling Models in Single Molecule Junctions.

The promise of the field of single-molecule electronics is to reveal a new class of quantum devices that leverages the strong electronic interactions inherent to subnanometer scale systems. Here, we form Au-molecule-Au junctions using a custom scanning tunneling microscope and explore charge transport through current-voltage measurements. We focus on the resonant tunneling regime of two molecules, one that is primarily an electron conductor and one that conducts primarily holes. We find that in the high bias regime, junctions that do not rupture demonstrate reproducible and pronounced negative differential resistance (NDR)-like features followed by hysteresis with peak-to-valley ratios exceeding 100 in some cases. Furthermore, we show that both junction rupture and NDR are induced by charging of the molecular orbital dominating transport and find that the charging is reversible at lower bias and with time with kinetic time scales on the order of hundreds of milliseconds. We argue that these results cannot be explained by existing models of charge transport and likely require theoretical advances describing the transition from coherent to sequential tunneling. Our work also suggests new rules for operating single-molecule devices at high bias to obtain highly nonlinear behavior.

Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase.

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.

Resolving the Unpaired-Electron Orbital Distribution in a Stable Organic Radical by Kondo Resonance Mapping.

The adsorption geometry and the electronic structure of a Blatter radical derivative on a gold surface were investigated by a combination of high-resolution noncontact atomic force microscopy and scanning tunneling microscopy. While the hybridization with the substrate hinders direct access to the molecular states, we show that the unpaired-electron orbital can be probed with Ångström resolution by mapping the spatial distribution of the Kondo resonance. The Blatter derivative features a peculiar delocalization of the unpaired-electron orbital over some but not all moieties of the molecule, such that the Kondo signature can be related to the spatial fingerprint of the orbital. We observe a direct correspondence between these two quantities, including a pronounced nodal plane structure. Finally, we demonstrate that the spatial signature of the Kondo resonance also persists upon noncovalent dimerization of molecules.

Prior Band-Resisted Squat Jumps Improves Running and Neuromuscular Performance in Middle-Distance Runners.

Post-activation potentiation (PAP) conditioning has been reported to increase performance. Most research has examined PAP effects on strength/power activities, whereas the effects on endurance sports are understudied. The aim of this study was to characterize PAP conditioning stimulus effects on a subsequent 5x1 km running trial. A randomized, within subjects, repeated measures study utilized 12 male, endurance-trained athletes, who performed a full warm-up, conditioning exercise intervention (4x5 repetition maximum band-resisted squat jumps) or a control condition prior to a 5x1 km time trial run. Tests were conducted immediately prior to the intervention, after each kilometer, immediately following the 5x1 km run, and at seven and ten minutes post 5 km run. Measures included the interpolated twitch technique (ITT), evoked contractile properties, maximum voluntary isometric contractions (MVIC) plantar flexor force, drop jump, rating of perceived exertion, and heart rate. The PAP stimulus reduced the time to complete the run (3.6%; p = 0.07, d = 0.51), and decreased the time to complete kilometer one (8%; d = 1.08, p = 0.014). Jump height (p = 0.02; 9.2%) and reactive strength index (p = 0.035; 16%) increased with PAP. F100 (force produced in the first 100ms of the MVIC) and MVIC force with PAP increased at kilometers 3 (p = 0.04, d=0.84), 4 (p = 0.034, d = 0.29), and 7min post-run (p = 0.03, d = 0.60). Voluntary activation (ITT) increased at 7min post-run (p = 0.04, d = 0.59) with PAP, yet decreased at 7min post-run in the control condition (p = 0.03, d = 0.36). A prior band-resisted squat protocol decreased running time and improved neuromuscular properties in endurance athletes running 5x1 km.

Diastereoselective synthesis of fused cyclopropyl-3-amino-2,4-oxazine ß-amyloid cleaving enzyme (BACE) inhibitors and their biological evaluation.

The diastereoselective synthesis and structure activity relationship (SAR) of a series of fused cyclopropyl-3-amino-2,4-oxazine (2-oxa-4-azabicyclo[4.1.0]hept-3-en-3-amine)-containing BACE inhibitors is described. Through these efforts compound 2 was identified as a potent (cell IC50 = 15 nM) BACE inhibitor with acceptable ADME properties. When tested in vivo, compound 2 demonstrated a significant reduction of brain and cerebral spinal fluid (CSF) Aß40 levels (46% and 66%, respectively) in a rat pharmacodynamic study and thus represents a suitable starting point for the further development of in vivo efficacious compounds for the treatment of Alzheimer's disease.

Exploiting a water network to achieve enthalpy-driven, bromodomain-selective BET inhibitors.

Within the last decade, the Bromodomain and Extra-Terminal domain family (BET) of proteins have emerged as promising drug targets in diverse clinical indications including oncology, auto-immune disease, heart failure, and male contraception. The BET family consists of four isoforms (BRD2, BRD3, BRD4, and BRDT/BRDT6) which are distinguished by the presence of two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine (KAc) residues and appear to have distinct biological roles. BET BD1 and BD2 bromodomains differ at five positions near the substrate binding pocket: the variation in the ZA channel induces different water networks nearby. We designed a set of congeneric 2- and 3-heteroaryl substituted tetrahydroquinolines (THQ) to differentially engage bound waters in the ZA channel with the goal of achieving bromodomain selectivity. SJ830599 (9) showed modest, but consistent, selectivity for BRD2-BD2. Using isothermal titration calorimetry, we showed that the binding of all THQ analogs in our study to either of the two bromodomains was enthalpy driven. Remarkably, the binding of 9 to BRD2-BD2 was marked by negative entropy and was entirely driven by enthalpy, consistent with significant restriction of conformational flexibility and/or engagement with bound waters. Co-crystallography studies confirmed that 9 did indeed stabilize a water-mediated hydrogen bond network. Finally, we report that 9 retained cytotoxicity against several pediatric cancer cell lines with EC50 values comparable to BET inhibitor (BETi) clinical candidates.

Superior Training-Specific Adaptations With an 8-Week Yoak Push-up Training Program.

Lima, C, Li, Y, Low, JL, Herat, N, and Behm, DG. Superior training-specific adaptations with an 8-week yoak push-up training program. J Strength Cond Res 32(9): 2409-2418, 2018-There are few progressive metastability training programs in the literature. The purpose of this study was to investigate changes in strength, endurance, muscle activation, and neuromuscular efficiency after an 8-week progressive, push-up training program under stable and unstable conditions. Nineteen male and female recreationally trained participants performed twice per week, an 8-week push-up training program, using either a relatively unstable suspension system (Yoak) or under stable conditions. Participants were tested in 2 separate sessions before and after training for chest press maximal voluntary isometric contraction (MVIC) forces, and unstable and stable push-up endurance. Participants were tested during all testing measures for anterior deltoid, biceps brachii (BB), triceps brachii (TB), and serratus anterior (SA) electromyography (EMG) activity. The training progression consisted of altering the suspension configurations, push-up height, and increasing the number of sets (1-3 sets). The stable group performed 153.3 and 33.8% less repetitions than the Yoak group when performing push-ups on the Yoak device or stable floor, respectively (p = 0.03). Training-induced MVIC forces were 9.2% (p = 0.03) greater for the Yoak vs. the stable group. Regarding neuromuscular efficiency, the Yoak group decreased (30.4%; p = 0.01) and stable group increased (97.8%; p = 0.02) antagonist BB EMG activity from pre- to post-training. Both groups decreased the TB fatigue index from pre- to post-training. Nevertheless, Yoak group demonstrated 12.5% (p = 0.09) and 8.9% (p = 0.02) lower fatigue indexes with TB and SA, respectively, than the stable group. These findings suggest that Yoak training demonstrates superior improvements over stable training for push-up endurance, neuromuscular efficiency, MVIC, and fatigue index.

Tuning Singlet Fission in π-Bridge-π Chromophores.

We have designed a series of pentacene dimers separated by homoconjugated or nonconjugated bridges that exhibit fast and efficient intramolecular singlet exciton fission (iSF). These materials are distinctive among reported iSF compounds because they exist in the unexplored regime of close spatial proximity but weak electronic coupling between the singlet exciton and triplet pair states. Using transient absorption spectroscopy to investigate photophysics in these molecules, we find that homoconjugated dimers display desirable excited-state dynamics, with significantly reduced recombination rates as compared to conjugated dimers with similar singlet fission rates. In addition, unlike conjugated dimers, the time constants for singlet fission are relatively insensitive to the interplanar angle between chromophores, since rotation about σ bonds negligibly affects the orbital overlap within the π-bonding network. In the nonconjugated dimer, where the iSF occurs with a time constant >10 ns, comparable to the fluorescence lifetime, we used electron spin resonance spectroscopy to unequivocally establish the formation of triplet-triplet multiexcitons and uncoupled triplet excitons through singlet fission. Together, these studies enable us to articulate the role of the conjugation motif in iSF.

Mapping the Transmission Functions of Single-Molecule Junctions.

Charge transport phenomena in single-molecule junctions are often dominated by tunneling, with a transmission function dictating the probability that electrons or holes tunnel through the junction. Here, we present a new and simple technique for measuring the transmission functions of molecular junctions in the coherent tunneling limit, over an energy range of 1.5 eV around the Fermi energy. We create molecular junctions in an ionic environment with electrodes having different exposed areas, which results in the formation of electric double layers of dissimilar density on the two electrodes. This allows us to electrostatically shift the molecular resonance relative to the junction Fermi levels in a manner that depends on the sign of the applied bias, enabling us to map out the junction's transmission function and determine the dominant orbital for charge transport in the molecular junction. We demonstrate this technique using two groups of molecules: one group having molecular resonance energies relatively far from EF and one group having molecular resonance energies within the accessible bias window. Our results compare well with previous electrochemical gating data and with transmission functions computed from first principles. Furthermore, with the second group of molecules, we are able to examine the behavior of a molecular junction as a resonance shifts into the bias window. This work provides a new, experimentally simple route for exploring the fundamentals of charge transport at the nanoscale.

A design strategy for intramolecular singlet fission mediated by charge-transfer states in donor-acceptor organic materials.

The ability to advance our understanding of multiple exciton generation (MEG) in organic materials has been restricted by the limited number of materials capable of singlet fission. A particular challenge is the development of materials that undergo efficient intramolecular fission, such that local order and strong nearest-neighbour coupling is no longer a design constraint. Here we address these challenges by demonstrating that strong intrachain donor-acceptor interactions are a key design feature for organic materials capable of intramolecular singlet fission. By conjugating strong-acceptor and strong-donor building blocks, small molecules and polymers with charge-transfer states that mediate population transfer between singlet excitons and triplet excitons are synthesized. Using transient optical techniques, we show that triplet populations can be generated with yields up to 170%. These guidelines are widely applicable to similar families of polymers and small molecules, and can lead to the development of new fission-capable materials with tunable electronic structure, as well as a deeper fundamental understanding of MEG.

Quantitative Intramolecular Singlet Fission in Bipentacenes.

Singlet fission (SF) has the potential to significantly enhance the photocurrent in single-junction solar cells and thus raise the power conversion efficiency from the Shockley-Queisser limit of 33% to 44%. Until now, quantitative SF yield at room temperature has been observed only in crystalline solids or aggregates of oligoacenes. Here, we employ transient absorption spectroscopy, ultrafast photoluminescence spectroscopy, and triplet photosensitization to demonstrate intramolecular singlet fission (iSF) with triplet yields approaching 200% per absorbed photon in a series of bipentacenes. Crucially, in dilute solution of these systems, SF does not depend on intermolecular interactions. Instead, SF is an intrinsic property of the molecules, with both the fission rate and resulting triplet lifetime determined by the degree of electronic coupling between covalently linked pentacene molecules. We found that the triplet pair lifetime can be as short as 0.5 ns but can be extended up to 270 ns.

Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid.

Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo consolidation therapy-a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RA's overall potency. We then discovered that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RA's ability to clear neuroblastoma cells specifically from the bone marrow, seemingly mimicking interactions between BMP and RA during normal development.

A Qualitative Analysis of Access to Healthcare Among African American Adults in South Carolina.

Access to healthcare is a Social Determinant of Health that is associated with public health outcomes and barriers to access disproportionately affect African American adults. This study used a health and financial literacy approach to qualitatively assess how African American adults access healthcare and potential barriers faced (n = 20). Results indicated a wide range of experiences generally split between positive and negative experiences in access to healthcare. Specific themes that emerged included scheduling issues and appointment availability, expense of care, lack of transparency in insurance coverage, the need for more primary care clinics and enhanced community outreach and education on how to access healthcare. This research identifies a need for increased education surrounding health insurance coverage and an identified need for more local physicians or ease of scheduling. All participants in this study stated they were covered by health insurance of some form. Future research should examine these issues in the context of socioeconomic and insurance status.