Template-assisted covalent modification underlies activity of covalent molecular glues.

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.

Polymerization of ZBTB transcription factors regulates chromatin occupancy.

BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.

UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability.

Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.

The human E3 ligase RNF185 is a regulator of the SARS-CoV-2 envelope protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks multiple human proteins during infection and viral replication. To examine whether any viral proteins employ human E3 ubiquitin ligases, we evaluated the stability of SARS-CoV-2 proteins with inhibition of the ubiquitin proteasome pathway. Using genetic screens to dissect the molecular machinery involved in the degradation of candidate viral proteins, we identified human E3 ligase RNF185 as a regulator of protein stability for the SARS-CoV-2 envelope protein. We found that RNF185 and the SARS-CoV-2 envelope co-localize to the endoplasmic reticulum (ER). Finally, we demonstrate that the depletion of RNF185 significantly increases SARS-CoV-2 viral titer in a cellular model. Modulation of this interaction could provide opportunities for novel antiviral therapies.

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders.

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

Five-Year Follow-up of Third-Generation Percutaneous Chevron and Akin Osteotomies (PECA) for Hallux Valgus.

BACKGROUND: Recent large studies of third-generation minimally invasive hallux valgus surgery (MIS) have demonstrated significant improvement in clinical and radiologic outcomes. It remains unknown whether these clinical and radiologic outcomes are maintained in the medium to long term. The aim of this study was to investigate the minimum 5-year clinical and radiologic outcomes following third-generation MIS hallux valgus surgery in the hands of a high-volume MIS surgeon. METHODS: A retrospective observational single highly experienced MIS surgeon case series of consecutive patients undergoing primary isolated third-generation percutaneous chevron and Akin osteotomies (PECA) for hallux valgus with a minimum 60-month clinical and radiographic follow-up. Primary outcome was radiographic assessment of the hallux valgus angle (HVA) and intermetatarsal angle (IMA) preoperatively, 6 months, and ≥60 months following PECA. Secondary outcomes included the Manchester-Oxford Foot Questionnaire, patient satisfaction, EuroQol-5D visual analog scale and the visual analog scale for pain. RESULTS: Between 2012 and 2014, 126 consecutive feet underwent isolated third-generation PECA, with complete data available for 78 (61.9%) feet. The median follow-up was 65.0 (IQR 64-69; range 60-88) months. There was a significant improvement in radiographic deformity correction; the median IMA improved from 12.0 degrees (interquartile range [IQR]: 10.8-14.2) to 6.0 degrees (IQR: 4.2-7.3) (P < .001), and the median HVA improved from 27.2 degrees (IQR: 20.6-34.4) to 7.2 degrees (IQR: 3.4-11.6). Median MOXFQ Index score at ≥60-month follow-up was 2.3 (IQR: 0.0-7.8). The radiographic recurrence rate (defined as HVA >15 degrees) was 7.7% at final follow-up. The complication rate was 4.8%. CONCLUSION: Radiologic deformity correction for the 78 feet we were able to follow that had third-generation PECA performed by a single highly experienced MIS surgeon was found to be maintained at a mean follow-up of average 66.8 months, with a radiographic recurrence rate of 7.7%. Clinical PROMs and patient satisfaction levels were high and comparable to other third-generation studies with shorter duration of follow-up. LEVEL OF EVIDENCE: Level IV, retrospective cohort study.

Valgus Second Toe Deformity Treated With a Percutaneous Extracapsular Closing-Wedge Osteotomy of the Proximal Phalanx.

BACKGROUND: Coronal and sagittal plane deformities of the lesser toes are common yet challenging to treat. Traditional open releases and translational Weil osteotomies can be unpredictable and lead to postoperative stiffness. We present the results of a percutaneous closing wedge extracapsular osteotomy of the proximal phalanx to treat valgus deformity of the second toe. METHODS: Thirty-one patients underwent 40 percutaneous osteotomies at a median age of 58.6±9.4 years. Using a small dorsomedial incision, a percutaneous proximal metaphyseal medial closing-wedge extracapsular osteotomy of the second toe is performed, leaving the dorsolateral cortex intact. An irrigated low-speed, high-torque 2- × 8-mm burr is used under image guidance. The osteotomy is then closed to correct deformity and taped for 2 weeks. Patient-reported outcomes and weightbearing radiographs were obtained. RESULTS: Questionnaire data was available for 89.7% (n=35) of cases. Most cases (91.4%) were either satisfied or extremely satisfied with the procedure. Radiographs were available for 90.0% of osteotomies, with a median length from surgery to radiographic follow-up of 1.6 years (range 0.5-6.3; SD ±1.5). Median second-toe valgus angle (STVA) decreased from 16.2±10.7 degrees to 5.0±7.0 degrees (P < .001) at final follow-up. All osteotomies united with no delayed union. There were no wound complications or infections. We found 2 cases of radiographic recurrence. CONCLUSION: Percutaneous proximal phalanx base metaphyseal closing wedge extracapsular osteotomies of lesser toes to correct coronal plane deformity is useful adjunct to first-ray corrective surgery and is associated with high levels of patient satisfaction. LEVEL OF EVIDENCE: Level IV, retrospective case series.

BET-bromodomain and EZH2 inhibitor-treated chronic GVHD mice have blunted germinal centers with distinct transcriptomes.

Despite advances in the field, chronic graft-versus-host-disease (cGVHD) remains a leading cause of morbidity and mortality following allogenic hematopoietic stem cell transplant. Because treatment options remain limited, we tested efficacy of anticancer, chromatin-modifying enzyme inhibitors in a clinically relevant murine model of cGVHD with bronchiolitis obliterans (BO). We observed that the novel enhancer of zeste homolog 2 (EZH2) inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 each improved pulmonary function; impaired the germinal center (GC) reaction, a prerequisite in cGVHD/BO pathogenesis; and JQ5 reduced EZH2-mediated H3K27me3 in donor T cells. Using conditional EZH2 knockout donor cells, we demonstrated that EZH2 is obligatory for the initiation of cGVHD/BO. In a sclerodermatous cGVHD model, JQ5 reduced the severity of cutaneous lesions. To determine how the 2 drugs could lead to the same physiological improvements while targeting unique epigenetic processes, we analyzed the transcriptomes of splenic GCB cells (GCBs) from transplanted mice treated with either drug. Multiple inflammatory and signaling pathways enriched in cGVHD/BO GCBs were reduced by each drug. GCBs from JQ5- but not JQ1-treated mice were enriched for proproliferative pathways also seen in GCBs from bone marrow-only transplanted mice, likely reflecting their underlying biology in the unperturbed state. In conjunction with in vivo data, these insights led us to conclude that epigenetic targeting of the GC is a viable clinical approach for the treatment of cGVHD, and that the EZH2 inhibitor JQ5 and the BET-bromodomain inhibitor JQ1 demonstrated clinical potential for EZH2i and BETi in patients with cGVHD/BO.

Unravelling direct and indirect contact patterns between duck farms in France and their association with the 2016-2017 epidemic of Highly Pathogenic Avian Influenza (H5N8).

Live animal movements generate direct contacts (via the exchange of live animals) and indirect contacts (via the transit of transport vehicles) between farms, which can contribute to the spread of pathogens. However, most analyses focus solely on direct contacts and can therefore underestimate the contribution of live animal movements in the spread of infectious diseases. Here, we used French live duck movement data (2016-2018) from one of the largest transport companies to compare direct and indirect contact patterns between duck farms and evaluate how these patterns were associated with the French 2016-2017 epidemic of highly pathogenic avian influenza H5N8. A total number of 614 farms were included in the study, and two directed networks were generated: the animal introduction network (exchange of live ducks) and the transit network (transit of transport vehicles). Following descriptive analyses, these two networks were scrutinized in relation to farm infection status during the epidemic. Results showed that farms were substantially more connected in the transit network than in the animal introduction network and that the transit of transport vehicles generated more opportunities for transmission than the exchange of live animals. We also showed that animal introduction and transit networks' statistics decreased substantially during the epidemic (January-March 2017) compared to non-epidemic periods (January-March 2016 and January-March 2018). We estimated a probability of 33.3 % that a farm exposed to the infection through either of the two live duck movement networks (i.e. that was in direct or indirect contact with a farm that was reported as infected in the following seven days) becomes infected within seven days after the contact. However, we also demonstrated that the level of exposure of farms by these two contact patterns was low, leading only to a handful of transmission events through these routes. As a consequence, we showed that live animal movement patterns are efficient transmission routes for HPAI but have been efficiently reduced to limit the spread during the French 2020-2021 epidemic. These results underpin the relevance of studying indirect contacts resulting from the movement of animals to understand their transmission potential and the importance of accounting for both routes when designing disease control strategies.

Titanium dioxide fiber saturable absorber for Q-switched fiber laser generation in the 1-micrometer region: publisher's note.

This publisher's note serves to correct an error in Appl. Opt. 58, 3495 (2019)APOPAI0003-693510.1364/AO.58.003495.

EP300 Selectively Controls the Enhancer Landscape of MYCN-Amplified Neuroblastoma.

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2ß, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed "JQAD1" that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells. SIGNIFICANCE: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2ß. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587.

Highly efficient mode-locked and Q-switched Er3+-doped fiber lasers using a gold nanorod saturable absorber.

Mode-locked and Q-switched pulsed fiber laser sources with wavelengths of 1.55 µm are widely used in various fields. Gold nanorods (GNRs) have been applied in biomedicine and optics owing to their biocompatibility, easy fabrication, and unique optical properties. This paper presents the analysis of a saturable absorber based on a colloidal gold nanorod (GNR) thin film for dual-function passively mode-locked and Q-switched 1.55-µm fiber lasers. The colloidal GNR thin film possesses superior properties such as a wide operating wavelength range, large nonlinear absorption coefficient, and a picosecond-order recovery time. Its modulation depth and saturation intensity at 1.55 µm are 7.8% and 6.55 MW/cm2, respectively. Passive mode-locked or Q-switched laser operation is achieved by changing the number of GNR thin-film layers. The advantages of these high-quality GNRs in mode-locked and Q-switched fiber lasers with record-high slope efficiency are verified by conducting comprehensive material and laser dynamic analyses. The self-starting mode-locked fiber laser with an efficiency as high as 24.91% and passively Q-switched fiber laser with the maximum energy of 0.403 µJ are successfully demonstrated. This paper presents the novel demonstration of reconfigurable mode-locked and Q-switched all-fiber lasers by incorporating colloidal GNR thin films.

Continuous-wave planar laser induced fluorescence with a fast camera.

We present planar, laser induced fluorescence (PLIF) measurements of the velocity-resolved distribution function of ions in a plasma using a modulated, narrow linewidth, continuous-wave laser. Plasma emission is acquired with a high frame rate camera, and the laser light is spread into a thin sheet so that an entire plane of the plasma is imaged at each interrogation wavelength. Fourier analysis is conducted on each pixel of the images to separate the modulated fluorescent emission from the background light. Argon ion temperatures and bulk flow maps are reported in a helicon plasma source, and standard single-point LIF measurements provide validation of the PLIF measurement.

Lysine Demethylase 5A is Required for MYC Driven Transcription in Multiple Myeloma.

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo. Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)- and P-TEFb (CDK9)-mediated phosphorylation of RNAPII. These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM.

Percutaneous Chevron and Akin (PECA) Osteotomies for Severe Hallux Valgus Deformity With Mean 3-Year Follow-up.

BACKGROUND: Patients with severe hallux valgus deformity present technical and operative challenges with high rates of recurrence and residual deformity. The clinical and radiologic outcomes of percutaneous surgery for severe hallux valgus are not known. METHODS: A retrospective review of consecutive patients with a hallux valgus angle (HVA) >40 degrees or intermetatarsal angle (IMA) >20 degrees who underwent third-generation percutaneous chevron and Akin osteotomy (PECA) for hallux valgus deformity correction. RESULTS: Between December 2012 and August 2019, 59 feet in 50 patients underwent PECA. Preoperative and follow-up radiographic data were available for 53 feet (89.8%). Postoperative clinical patient-reported outcome measures and satisfaction results were available for 51 feet (86.4%). The mean clinical and radiographic follow-up was 3.1 years and the mean postoperative Manchester-Oxford Foot Questionnaire Index score was 15.1. There was a statistically significant improvement (P < .001) in both IMA and HVA following surgery (IMA 17.5-5.1 degrees; HVA 44.1-11.5 degrees). All patients reported they were satisfied with their outcome, with 76.8% reporting they were highly satisfied. The hallux valgus recurrence rate was 7.5%. CONCLUSION: Percutaneous surgery for severe hallux valgus deformity can achieve a large deformity correction, patient satisfaction, and quality of life, with reasonable rates of residual deformity and low rates of recurrence. LEVEL OF EVIDENCE: Level IV, case series.

High-efficiency picosecond mode-locked laser using a thulium-doped nanoengineered yttrium-alumina-silica fiber as the gain medium.

We report the theoretical and experimental investigation of a self-starting mode-locked fiber laser with a nanoengineered Tm3+-doped yttrium-alumina-silica (YAS) fiber as the gain medium. The YAS fiber exhibits a higher capability of Tm3+ cluster elimination than commercial silica fibers. The Tm3+ fluorescence properties and YAS dispersion are well characterized. As a result, an efficient picosecond mode-locked fiber laser is demonstrated with a slope efficiency of 14.14% and maximum pulse energy of 1.27 nJ. To the best of our knowledge, this is the first mode-locked fiber laser based on a Tm3+-doped YAS fiber. The experimental observation is also supported by the numerical analysis.

Randomized Controlled Trial of Topical Skin Adhesive vs Nylon Sutures for Incision Closure in Forefoot Surgery.

BACKGROUND: There are many options for incision closure in forefoot surgery. The aim of this study was to compare topical skin adhesive (2-octyl-cyanoacrylate) to simple interrupted nylon sutures. METHODS: A prospective randomized controlled trial comparing topical skin adhesive (TSA) and nylon sutures (NSs) for elective open forefoot surgery. Primary outcome was Hollander Wound Evaluation Scale (HWES) assessed 2 weeks following surgery. Secondary objectives included time taken for wound closure, wound assessment, patient satisfaction with wound cosmesis, incision pain, and infection rate. RESULTS: Between January and December 2018, 84 feet (70 patients) underwent hallux valgus scarf/Akin osteotomy or first metatarsophalangeal arthrodesis and were randomized to receive either intervention (topical skin adhesive) or control (3/0 nylon sutures). We found worse HWES scores when using TSA compared to NSs (1.07 vs 0.60). Incision closure time was slower for TSA (mean, 272 vs 229 seconds). At 2 weeks postoperatively, wound care was faster for TSA (mean 71 secs) vs NSs (mean 120), and patient-reported pain was less with TSA (visual analog scale: TSA 1.2 vs NSs 2.1). A high degree of overall patient satisfaction was reported in both groups, without significant difference. CONCLUSION: Closure of elective forefoot surgery incisions with topical skin adhesive or interrupted nylon sutures offers high satisfaction rates, low pain scores, and low complications. However, topical skin adhesive was associated with more inflammation and areas of wound separation compared to nylon sutures. We recommend the use of sutures for wound closure in forefoot surgery. LEVEL OF EVIDENCE: Level I, randomized controlled trial.

Compact, portable, laser induced fluorescence diagnostic for laboratory plasma sources.

As diagnostic groups are increasingly called upon to participate in experimental campaigns at remote facilities, there is a need to develop portable versions of plasma diagnostic systems. One such diagnostic is laser induced fluorescence (LIF). Here, we describe a portable LIF apparatus that eliminates the need for an optical table, beam splitters, and an optical chopper. All of the light exiting the laser system is coupled through optical fibers to the experiment and housekeeping diagnostics. The collected light is coupled through an optical fiber as well. A key feature is modulation of the tapered amplifier current instead of physical modulation of the laser output. Using this portable LIF system, measurements of ion temperature, ion flow, and relative metastable ion density are reported for two different remote experiments.

Clinical and Radiological Outcomes of Forefoot Offloading Versus Rigid Flat Shoes in Patients Undergoing Surgery of the First Ray.

BACKGROUND: It is common clinical practice to use either flat or reverse camber shoes to protect the foot for up to 6 weeks after surgery for hallux valgus or hallux rigidus. To date there is a paucity of evidence as to whether there is any difference between these 2 postoperative shoes, in either patient satisfaction or clinical outcomes. METHODS: One hundred consecutive patients undergoing scarf/Akin osteotomies or first metatarsophalangeal joint (MTPJ) arthrodesis were recruited. Patients were randomized 50:50 to either flat or reverse camber postoperative shoes. Patients undergoing ancillary lesser toe procedures were not excluded. Patient satisfaction was assessed by visual analog scale (VAS) pain score and Likert satisfaction survey. Radiographic outcomes were reviewed at 1 year observing differences in fusion rates or deformity recurrence. There were 47 patients in the reverse cam and 43 in the flat shoe group. No difference in primary forefoot operation, additional operation, age at surgery, or preop VAS pain score was seen. RESULTS: At 6 weeks, there was no significant difference in postop VAS pain score. The flat shoe group was significantly more likely to be satisfied with their general mobility (86.0% vs 61.7%; P = .01) and with their stability in the shoe (90.7% vs 69.6%; P = .03). No significant difference was seen between groups for nonunion or hallux valgus recurrence rates. CONCLUSION: Both forms of postoperative footwear were effective in enabling patients to mobilize and in preventing adverse outcomes. Patients were more likely to be satisfied with a flat postoperative shoe due to improved stability and ease of mobilizing. The results of this study aid surgeon decision making for postoperative footwear in forefoot surgery. LEVEL OF EVIDENCE: Level II, prospective randomized controlled trial.