Proximal Target Research Articles

Proximity labeling reveals new functional relationships between meiotic recombination proteins in S. cerevisiae.

Several protein ensembles facilitate crossover recombination and the associated assembly of synaptonemal complex (SC) during meiosis. In yeast, meiosis-specific factors including the DNA helicase Mer3, the "ZZS" complex consisting of Zip4, Zip2, and Spo16, the RING-domain protein Zip3, and the MutSγ heterodimer collaborate with crossover-promoting activity of the SC component, Zip1, to generate crossover-designated recombination intermediates. These ensembles also promote SC formation - the organized assembly of Zip1 with other structural proteins between aligned chromosome axes. We used proximity labeling to investigate spatial relationships between meiotic recombination and SC proteins in S. cerevisiae. We find that recombination initiation and SC factors are dispensable for proximity labeling of Zip3 by ZZS components, but proteins associated with early steps in recombination are required for Zip3 proximity labeling by MutSγ, suggesting that MutSγ joins Zip3 only after a recombination intermediate has been generated. We also find that zip1 separation-of-function mutants that are crossover deficient but still assemble SC fail to generate protein ensembles where Zip3 can engage ZZS and/or MutSγ. The SC structural protein Ecm11 is proximity labeled by ZZS proteins in a Zip4-dependent and Zip1-independent manner, but labeling of Ecm11 by Zip3 and MutSγ requires, at least in part, Zip1. Finally, mass spectrometry analysis of biotinylated proteins in eleven proximity labeling strains uncovered shared proximity targets of SC and crossover-associated proteins, some of which have not previously been implicated in meiotic recombination or SC formation, highlighting the potential of proximity labeling as a discovery tool.

Intrinsic Synergy and Selectivity for the Inhibition of Cancer Cell Growth Generated by a Polymer Ligand of Proximal Enzymes.

A fundamental understanding of the design of polymer ligands of proximal enzymes is essential for the precise targeting of cancer cells, but it is still in its infancy. In this study, we systematically investigated the contribution of the chain length, ligand density, and ligand ratio of proximal enzyme-targeted polymers to the efficacy, synergy, and selectivity for the inhibition of cancer cell proliferation. The results revealed that employing a moderate chain length as a scaffold allowed for an intrinsically high efficacy and synergy of proximal enzyme-targeted polymers, in contrast to single enzyme-targeted polymers that prefer longer chain length for efficacy. The synergy obtained in proximal enzyme targeting was not provided by the combination of the corresponding small molecules. Moreover, the maturation of the synergistic efficacy of the proximal enzyme-targeted polymers also improved selectivity. This study proposes a rational design for polymer inhibitors and/or ligands for cancer cells with a high efficacy and selectivity.

The role of training variability for model-based and model-free learning of an arbitrary visuomotor mapping.

A fundamental feature of the human brain is its capacity to learn novel motor skills. This capacity requires the formation of vastly different visuomotor mappings. Using a grid navigation task, we investigated whether training variability would enhance the flexible use of a visuomotor mapping (key-to-direction rule), leading to better generalization performance. Experiments 1 and 2 show that participants trained to move between multiple start-target pairs exhibited greater generalization to both distal and proximal targets compared to participants trained to move between a single pair. This finding suggests that limited variability can impair decisions even in simple tasks without planning. In addition, during the training phase, participants exposed to higher variability were more inclined to choose options that, counterintuitively, moved the cursor away from the target while minimizing its actual distance under the constrained mapping, suggesting a greater engagement in model-based computations. In Experiments 3 and 4, we showed that the limited generalization performance in participants trained with a single pair can be enhanced by a short period of variability introduced early in learning or by incorporating stochasticity into the visuomotor mapping. Our computational modeling analyses revealed that a hybrid model between model-free and model-based computations with different mixing weights for the training and generalization phases, best described participants' data. Importantly, the differences in the model-based weights between our experimental groups, paralleled the behavioral findings during training and generalization. Taken together, our results suggest that training variability enables the flexible use of the visuomotor mapping, potentially by preventing the consolidation of habits due to the continuous demand to change responses.

Managing Major Peripheral Nerves in Forearm-Level Amputations With TMR and RPNI: What's the Best Recipe?

Targeted muscle reinnervation (TMR) and regenerative peripheral nerve interface (RPNI) prevent symptomatic neuroma formation in amputees. Forearm-level amputations present multiple muscular targets, making it challenging to determine the ideal treatment. The purpose of this study was to evaluate the best TMR targets, role of RPNI, and appropriate patient-selection criteria in forearm-level amputations. We hypothesized that deep and distal TMR targets would best prevent symptomatic neuromas, RPNI would prove a success adjunct, and patients with poorly controlled diabetes would not develop symptomatic neuromas regardless of nerve management. We retrospectively identified forearm-level amputations performed between 2017 and 2022. Patients with TMR by outside providers, follow-up <6 months, or insufficient documentation were excluded. Demographics, surgical nerve management, and postoperative complications were collected. The primary outcome was development of a painful neuroma determined by the Eberlin criteria. Patients undergoing TMR were divided a priori into two groups, superficial and proximal versus deep and distal TMR targets, and were compared. Thirty-nine patients met inclusion criteria, and 16 developed a symptomatic neuroma. No patients with a deep or distal TMR target developed a symptomatic neuroma. One nerve out of 12 treated with RPNI developed a symptomatic neuroma. No patient with poorly controlled diabetes developed a symptomatic neuroma, despite no advanced nerve management. In a case series of forearm amputations, deep and distal TMR targets prevented symptomatic neuroma formation more than superficial and proximal targets. Regenerative peripheral nerve interface is a useful adjunct for neuroma control, especially for the radial sensory nerve. Patients with poorly controlled diabetes may not require advanced nerve management. Level IV retrospective case series.

Molecular programs guiding arealization of descending cortical pathways.

Layer 5 extratelencephalic (ET) neurons are present across neocortical areas and send axons to multiple subcortical targets1-6. Two cardinal subtypes exist7,8: (1) Slco2a1-expressing neurons (ETdist), which predominate in the motor cortex and project distally to the pons, medulla and spinal cord; and (2) Nprs1- or Hpgd-expressing neurons (ETprox), which predominate in the visual cortex and project more proximally to the pons and thalamus. An understanding of how area-specific ETdist and ETprox emerge during development is important because they are critical for fine motor skills and are susceptible to spinal cord injury and amyotrophic lateral sclerosis9-12. Here, using cross-areal mapping of axonal projections in the mouse neocortex, we identify the subtype-specific developmental dynamics of ET neurons. Whereas subsets of ETprox emerge by pruning of ETdist axons, others emerge de novo. We outline corresponding subtype-specific developmental transcriptional programs using single-nucleus sequencing. Leveraging these findings, we use postnatal in vivo knockdown of subtype-specific transcription factors to reprogram ET neuron connectivity towards more proximal targets. Together, these results show the functional transcriptional programs driving ET neuron diversity and uncover cell subtype-specific gene regulatory networks that can be manipulated to direct target specificity in motor corticofugal pathways.

EGR2 is an epigenomic regulator of phagocytosis and antifungal immunity in alveolar macrophages.

Alveolar macrophages (AMs) act as gatekeepers of the lung's immune responses, serving essential roles in recognizing and eliminating pathogens. The transcription factor (TF) early growth response 2 (EGR2) has been recently described as required for mature AMs in mice; however, its mechanisms of action have not been explored. Here, we identified EGR2 as an epigenomic regulator and likely direct proximal transcriptional activator in AMs using epigenomic approaches (RNA sequencing, ATAC sequencing, and CUT&RUN). The predicted direct proximal targets of EGR2 included a subset of AM identity genes and ones related to pathogen recognition, phagosome maturation, and adhesion, such as Clec7a, Atp6v0d2, Itgb2, Rhoc, and Tmsb10. We provided evidence that EGR2 deficiency led to impaired zymosan internalization and reduced the capacity to respond to Aspergillus fumigatus. Mechanistically, the lack of EGR2 altered the transcriptional response, secreted cytokines (i.e., CXCL11), and inflammation-resolving lipid mediators (i.e., RvE1) of AMs during in vivo zymosan-induced inflammation, which manifested in impaired resolution. Our findings demonstrated that EGR2 is a key proximal transcriptional activator and epigenomic bookmark in AMs responsible for select, distinct components of cell identity and a protective transcriptional and epigenomic program against fungi.

Modulation of multisensory nociceptive neurons in monkey cortical area 7b and behavioral correlates.

Wide-range thermoreceptive neurons (WRT-EN) in monkey cortical area 7b that encoded innocuous and nocuous cutaneous thermal and threatening visuosensory stimulation with high fidelity were studied to identify their multisensory integrative response properties. Emphasis was given to characterizing the spatial and temporal effects of threatening visuosensory input on the thermal stimulus-response properties of these multisensory nociceptive neurons. Threatening visuosensory stimulation was most efficacious in modulating thermal evoked responses when presented as a downward ("looming"), spatially congruent, approaching and closely proximal target in relation to the somatosensory receptive field. Both temporal alignment and misalignment of spatially aligned threatening visual and thermal stimulation significantly increased mean discharge frequencies above those evoked by thermal stimulation alone, particularly at near noxious (43°C) and mildly noxious (45°C) temperatures. The enhanced multisensory discharge frequencies were equivalent to the discharge frequency evoked by overtly noxious thermal stimulation alone at 47°C (monkey pain tolerance threshold). A significant increase in behavioral mean escape frequency with shorter escape latency was evoked by multisensory stimulation at near noxious temperature (43°C), which was equivalent to that evoked by noxious stimulation alone (47°C). The remarkable concordance of elevating both neural discharge and escape frequency from a nonnociceptive and prepain level by near noxious thermal stimulation to a nociceptive and pain level by multisensory visual and near noxious thermal stimulation and integration is an elegantly designed defensive neural mechanism that in effect lowers both nociceptive response and pain thresholds to preemptively engage nocifensive behavior and, consequently, avert impending and actual injurious noxious thermal stimulation.NEW & NOTEWORTHY Multisensory nociceptive neurons in cortical area 7b are engaged in integration of threatening visuosensory and a wide range of innocuous and nocuous somatosensory (thermoreceptive) inputs. The enhancement of neuronal activity and escape behavior in monkey by multisensory integration is consistent and supportive of human psychophysical studies. The spatial features of visuosensory stimulation in peripersonal space in relation to somatic stimulation in personal space are critical to multisensory integration, nociception, nocifensive behavior, and pain.

Abstract 151: Single Cell Transcriptional And Epigenetic Map Of Human Coronary Artery Disease In A Multi-ethnic Population

Identifying target genes that are causally linked to coronary artery disease (CAD) remains an elusive problem despite the plethora of CAD genome-wide association study (GWAS) risk loci. Here, we performed single nucleus multiomic (RNA + ATAC) sequencing in 44 human coronary arteries and bulk Hi-C in 16 coronaries from multi-ethnic participants. From sequencing 126,804 nuclei, we identified cell type specific transcriptional signatures, chromatin peaks, and gene regulatory networks through peak to gene linkage analysis. Single-cell quantitative trait loci (QTL) analysis identified cell-specific caQTL’s including cell-type specific caQTLs within CAD GWAS loci. The overlap of CAD GWAS loci with ATAC peaks demonstrated that vascular smooth muscle and endothelial cells harbor the greatest genetic risk. To identify proximal and distal putative target genes for CAD loci in a cell type specific manner, we built a enhancer-gene map by leveraging coronary artery ChiP-seq, cell specific RNA/ATAC-seq, and Hi-C based chromatin contact loops. Using this map, we identified distal target genes linked to CAD GWAS loci. Finally, given the effect of ancestry on CAD GWAS risk interpretation, we leveraged Hi-C data to compare the chromatin architecture in coronary tissue from 8 European Americans vs 8 African Americans. A meta-map, sample level, and local ancestry analysis identified ancestry-specific differences in chromatin looping and topologically associated domains. Collectively, our results provide a cell-type specific transcriptomic and epigenetic map which along with bulk chromatin contacts can be used to interpret and prioritize CAD GWAS candidates and nominate cell specific causal target genes.

Diagnostic CT-Enabled Planning (DART): Results of a Randomized Trial in Palliative Radiation Therapy

Using diagnostic computed tomography (dCT) scans instead of CT simulation (CTsim) scans can increase departmental efficiency and reduce patient burden. The goal of the DART trial was to assess the efficacy and acceptability of dCT-based planning workflows with a focus on patient experiences, plan deliverability and adequacy of target coverage, and workflows. Patients undergoing same-day CTsim and treatment for palliative radiation therapy to thoracic, abdominopelvic, or proximal limb targets with a recent dCT (within 28 days) in a reproducible position were eligible. After stratifying by target type (bone or soft tissue vs. visceral), participants were randomized (1:2 ratio) between CTsim-based (CTsim arm) vs. dCT-based planning (dCT arm). The primary endpoint was time in center (TIC), defined as total time spent in the cancer center on first day of treatment, from first radiation department appointment to first fraction completion. Secondary endpoints included plan deliverability, adequacy of target coverage, and stakeholder acceptability. Thirty-three patients (42 treatment sites) were enrolled between June 2022 and April 2023. The median age was 72 (interquartile range [IQR]: 67-78), 73% were male, and the most common primary cancers were lung (33%), prostate (24%), and breast (12%). The most common dose and fractionations were 8 Gy in 1 and 20 Gy in 5 fractions (50% and 43% of plans, respectively). TIC was 4.7 ± 1.1 hours (mean ± SD) in the CTsim arm vs. 0.41 ± 0.14 hours in the dCT arm (P < .001). All dCT plans were deliverable. All plans in both arms were rated as "acceptable" (80% CTsim; 81% dCT) or "acceptable with minor deviation" (20% CTsim; 19% dCT). Patient perception of acceptability was similar in both arms with the exception of time burden, which was rated as "acceptable" by 50% in the CTsim arm vs. 90% in the dCT arm (P = .025). dCT-based radiation planning substantially reduced TIC without detriment in plan deliverability or quality and had a tangible impact on patient experience with reduced patient-reported time burden.

Picturing self-harm: Investigating flash-forward mental imagery as a proximal and modifiable driver of non-suicidal self-injury.

Non-suicidal self-injury (NSSI) is theorized to be reinforced by its emotional consequences. Mental images of NSSI are commonly reported as occurring prior to NSSI. Based on the known functional properties of anticipatory mental imagery as an emotional and motivational amplifier, this study investigated whether NSSI mental imagery constitutes a proximal and dynamic mechanism underpinning NSSI risk. An intensive ecological momentary assessment (EMA) study was conducted to track the occurrence and characteristics of NSSI mental imagery alongside NSSI urge and behavior in naturalistic settings. A sample of N = 43 individuals aged 17 to 24 with a history of repetitive NSSI completed EMA surveys seven times a day for 14 days. Mental preoccupation in the form of NSSI mental imagery-based flash-forwards to the actions, bodily sensations, and emotional benefits of NSSI was found to occur when NSSI urge was high but not when urge was low. Critically, objective cross-panel analyses showed that higher frequencies of NSSI imagery occurrence predicted greater future NSSI urge and increased likelihood of acting on urge, over and above current urge. Mental imagery of NSSI is not simply an epiphenomenal by-product of NSSI urge and may constitute a dynamic and proximal novel intervention target.

Abstract 524: Using acquired resistance to explore the mechanism of action of the integrated stress response/GCN2 activator NXP800 - A new developmental agent for platinum-resistant ARID1A mutant ovarian cancer

Abstract Background: NXP800 is a potent, oral activator of the Integrated Stress response (ISR), inhibitor of heat shock factor 1 (HSF1) activation and tumor cell proliferation, which is in early clinical studies in ARID1A-mutated, platinum resistant, clear cell ovarian cancer (NCT05226507). We discovered NXP800 using multiparameter medicinal chemistry optimization of a hit identified from a cell-based phenotypic screen. Owing to the unbiased nature of phenotypic screening, target identification is crucial to understand the biological and therapeutic activity of hit compounds. Methods and Results:: By RNAseq profiling human cancer cells treated with NXP800 we identified changes in expression of genes regulated by HSF1 or ATF4 - effects accompanied by eIF2alpha (eIF2a) phosphorylation and resulting activation of the ISR. To further understand this response, we explored whether NXP800 resistance models could inform on its mechanism of action (MoA). We used ARID1A mutant SK-OV-3 human ovarian carcinoma cells that are 1) highly sensitive to NXP800, 2) model the target patient population and 3) MSI-high so likely to have an elevated mutation rate contributing to acquisition of resistance. We generated two independent NXP800-resistant SK-OV-3 cell lines in which NXP800-mediated ATF4 induction and concomitant inhibition of global translation were abolished. By whole exome sequencing, we identified a heterozygous L99P mutation in the alpha subunit of eIF2B (eIF2Ba), the nucleotide exchange factor for eIF2a. Expression of eIF2BaL99P, but not wild-type, in parental SK-OV-3 cells reduced sensitivity to NXP800 to the same level as cells with NXP800-induced resistance. Using fluorescence recovery after photobleaching to monitor the dynamic association of the eIF2B:eIF2a complex, we elucidated that the eIF2BaL99P mutation reduces NXP800-mediated inhibition of eIF2a-GFP recycling through eIF2B bodies. Phosphorylation of eIF2a is regulated by four stress-controlled kinases GCN2, HRI, PKR and PERK. Using systematic siRNA knockdown or small-molecule inhibitors, we showed that GCN2 alone is required for ISR activation by NXP800 and that ISR induction inhibited HSF1 activation. Furthermore, inactivation of GCN2 reduced the antiproliferative activity of NXP800 to the same extent observed in NXP800-resistant SK-OV-3 cells. In contrast, exposure of resistant or parental SK-OV-3 cells expressing eIF2BaL99P to GCN2 inhibitors did not cause further reduction in NXP800 sensitivity - confirming the importance of the eIF2BaL99P mutation in the resistance mechanism. Conclusions: We have used acquired resistance to understand the MoA of NXP800 as a potent activator of GCN2 and the ISR pathway. Further studies are underway to determine the exact proximal molecular target of NXP800 and the mechanism of GCN2/ISR activation. Citation Format: Marissa V. Powers, Rachel Hodgson, Swee Y. Sharp, Toby Roe, K. Elizabeth Allen, Susan Campbell, Robert te Poele, Matthew Cheeseman, Keith Jones, Paul A. Clarke, Paul Workman. Using acquired resistance to explore the mechanism of action of the integrated stress response/GCN2 activator NXP800 - A new developmental agent for platinum-resistant ARID1A mutant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 524.

Abstract 520: circBRIP1 RNA as a non-invasive target engagement pharmacodynamic biomarker for DHX9 inhibition

Abstract DHX9 is a multifunctional DEAH-box ATP-independent RNA helicase which has been reported to play important roles in replication, transcription, translation, RNA splicing and RNA processing which contribute to DHX9’s role in maintenance of genomic stability. DHX9 can bind specifically to inverted repeat Alu elements, preventing back-splicing events that lead to circular RNA and exon skipping of the linear transcript. Upon DHX9 inhibition, a robust induction of Alu-mediated circular RNA is observed. For example, circBRIP1 is an Alu-mediated circular RNA that is robustly elevated upon DHX9 loss, with no observed change to levels of linear BRIP1. Here we describe data demonstrating circBRIP1 as a DHX9 specific target engagement pharmacodynamic (PD) biomarker and its potential utility as a non-invasive clinical PD biomarker. We have previously demonstrated that DHX9 inhibition by novel small molecule inhibitors is efficacious in microsatellite instable (MSI) colorectal cancer (CRC) with defective mismatch repair (dMMR), both in vitro and in vivo. DHX9 inhibition in the HCT116 dMMR CRC cell line results in a robust dose dependent induction of the Alu-mediated circular RNA, circBRIP1, as early as 6 hours after compound treatment. The development of lead series DHX9 inhibitors exhibited a positive correlation between circBRIP1 EC50 and DHX9 biochemical activity, as well as proliferation inhibition in a dMMR CRC cell line. However, dose dependent induction of circBRIP1 following DHX9 inhibition occurs with similar potencies across DHX9i sensitive and insensitive CRC cell lines. This suggests that elevation of circBRIP1 is a unique proximal target engagement PD biomarker of DHX9 inhibition, but not a predictor of DHX9 sensitivity. Importantly, induction of circBRIP1 is observed intratumorally in human xenograft in vivo efficacy studies in mice. Induction in each individual tumor correlates with observed tool compound exposure, providing evidence of a predictable PK/PD relationship. We also observe dose dependent circBRIP1 induction in mouse peripheral blood mononuclear cells (PBMC). We have extended these studies to primary human whole blood, where we see robust dose dependent induction of circBRIP1 after ex vivo treatment with DHX9 inhibitor for 24 hours. Altogether, these studies show evidence for the utility of circBRIP1 as a non-invasive target engagement biomarker for DHX9 inhibitors in the clinic. Citation Format: David Brennan, Jennifer Castro, Matthew H. Daniels, Monique Laidlaw, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. circBRIP1 RNA as a non-invasive target engagement pharmacodynamic biomarker for DHX9 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 520.

Risk Factors for Rerupture After Proximal Hamstring Avulsion Injury Including the Optimal Timing for Surgery

Background: Despite the prevalence of proximal hamstring avulsion injuries (PHAIs), the understanding of rerupture risk factors and the influence of injury chronicity on these rates remain limited. Purpose: To investigate the rerupture rate after PHAI repair and identify its associated risk factors and the optimal time to primary surgery. Study Design: Case-control study; Level of evidence, 3. Method: This is a retrospective analysis of prospectively collected data from the French Proximal Hamstring Avulsion Surgery Cohort Study targeting patients surgically treated for PHAI between 2002 and 2022. The primary outcome measure of this study was the rerupture rate of PHAI repair. The secondary outcome measures included the assessment of the potential risk factors for rerupture as well as the investigation of the incidence rate of rerupture for 100 person-years depending on various injury-surgery delay definitions. Results: This study analyzed 740 patients with a mean age of 45.9 years (SD, 13.6 years) and followed up for a mean of 4.9 years (SD, 3.9 years). The rerupture rate was 4.59% (34/740). Most reruptures (75%) occurred within the first 6 months after surgery (median, 88.5 days; interquartile range, 39.5-182 days), and 74% were atraumatic. Univariate analysis identified potential risk factors: longer initial surgery delay (hazard ratio [HR], 1.03; 95% CI, 1.01-1.04; P = .04) and initial complete ruptures (HR, 4.47; 95% CI, 1.07-18.7; P = .04). Receiver operating characteristic curve analysis found the optimal injury-surgery delay cutoff predicting rerupture to be 32 days (area under the curve, 0.62; 95% CI, 0.53-0.71). The relative Youden index was calculated at 0.24, corresponding to a sensitivity of 65% and a specificity of 59%. Surpassing this cutoff showed the highest HR (2.56), narrowest 95% CI (1.27-5.17), and highest incidence of rerupture (1.42 per 100 person-years) (P = .01). In the multivariate analysis, an injury-surgery delay of >32 days (HR, 2.5; 95% CI, 1.24-5.06; P = .01) and initial complete ruptures (HR, 4.33; 95% CI, 1.04-18.08; P = .04) emerged as significant risk factors for rerupture. Conclusion: This study found a 4.59% rerupture risk after PHAI repair. Most reruptures (75%) occurred within the first 6 months after surgery. Risk factors for rerupture included chronicity and initial complete injury. The optimal threshold for chronicity of PHAI lesions, based on rerupture rate, was marked by an injury-surgery delay of >32 days.

Emotional reactivity linking assaultive trauma and risky behavior: Evidence of differences between cisgender women and men.

Accumulating evidence suggests that trauma exposure is positively associated with future engagement in risky behavior, such as substance misuse, aggression, risky sex, and self-harm. However, the psychological factors driving this association and their relevance across gender groups require further clarification. In a community sample of 375 adults with a high rate of trauma exposure (age range: 18-55 years, M = 32.98 years, SD = 10.64; 76.3% assaultive trauma exposure), we examined whether emotional reactivity linked lifetime assaultive trauma exposure with past-month risky behavior. We also explored whether this model differed for cisgender women (n = 178, 47.6%) and men (n = 197, 52.5%). As hypothesized, assaultive trauma was positively related to emotional reactivity, β =.20, SE = 0.03, t(369) = 3.65, p <.001, which, in turn, partially accounted for the association between assaultive trauma and past-month risky behavior, indirect effect: β =.03, SE = 0.01, 95% bootstrapped CI [0.01, 0.06]. Gender moderated this association such that assaultive trauma was indirectly associated with risky behavior via emotional reactivity for women but not for men, index moderation: B = -0.03, SE = 0.02, 95% bootstrapped CI [-0.07, -0.01]. Cross-sectional results suggest that emotional reactivity may be a proximal target for clinical intervention to aid in the reduction of risky behavior among women.

The Quantitative Biotinylproteomics Studies Reveal a WInd-Related Kinase 1 (Raf-Like Kinase 36) Functioning as an Early Signaling Component in Wind-Induced Thigmomorphogenesis and Gravitropism

Wind is one of the most prevalent environmental forces entraining plants to develop various mechano-responses, collectively called thigmomorphogenesis. Largely unknown is how plants transduce these versatile wind force signals downstream to nuclear events and to the development of thigmomorphogenic phenotype or anemotropic response. To identify molecular components at the early steps of the wind force signaling, two mechanical signaling-related phosphoproteins, identified from our previous phosphoproteomic study of Arabidopsis touch response, mitogen-activated protein kinase kinase 1 (MKK1) and 2 (MKK2), were selected for performing in planta TurboID (ID)-based quantitative proximity-labeling (PL) proteomics. This quantitative biotinylproteomics was separately performed on MKK1-ID and MKK2-ID transgenic plants, respectively, using the genetically engineered TurboID biotin ligase expression transgenics as a universal control. This unique PTM proteomics successfully identified 11 and 71 MKK1 and MKK2 putative interactors, respectively. Biotin occupancy ratio (BOR) was found to be an alternative parameter to measure the extent of proximity and specificity between the proximal target proteins and the bait fusion protein. Bioinformatics analysis of these biotinylprotein data also found that TurboID biotin ligase favorably labels the loop region of target proteins. A WInd-Related Kinase 1 (WIRK1), previously known as rapidly accelerated fibrosarcoma (Raf)-like kinase 36 (RAF36), was found to be a putative common interactor for both MKK1 and MKK2 and preferentially interacts with MKK2. Further molecular biology studies of the Arabidopsis RAF36 kinase found that it plays a role in wind regulation of the touch-responsive TCH3 and CML38 gene expression and the phosphorylation of a touch-regulated PATL3 phosphoprotein. Measurement of leaf morphology and shoot gravitropic response of wirk1 (raf36) mutant revealed that the WIRK1 gene is involved in both wind-triggered rosette thigmomorphogenesis and gravitropism of Arabidopsis stems, suggesting that the WIRK1 (RAF36) protein probably functioning upstream of both MKK1 and MKK2 and that it may serve as the crosstalk point among multiple mechano-signal transduction pathways mediating both wind mechano-response and gravitropism.

The relevance of biologically effective dose for pain relief and sensory dysfunction after Gamma Knife radiosurgery for trigeminal neuralgia: an 871-patient multicenter study.

Recent studies have suggested that biologically effective dose (BED) is an important correlate of pain relief and sensory dysfunction after Gamma Knife radiosurgery (GKRS) for trigeminal neuralgia (TN). The goal of this study was to determine if BED is superior to prescription dose in predicting outcomes in TN patients undergoing GKRS as a first procedure. This was a retrospective study of 871 patients with type 1 TN from 13 GKRS centers. Patient demographics, pain characteristics, treatment parameters, and outcomes were reviewed. BED was compared with prescription dose and other dosimetric factors for their predictive value. The median age of the patients was 68 years, and 60% were female. Nearly 70% of patients experienced pain in the V2 and/or V3 dermatomes, predominantly on the right side (60%). Most patients had modified BNI Pain Intensity Scale grade IV or V pain (89.2%) and were taking 1 or 2 pain medications (74.1%). The median prescription dose was 80 Gy (range 62.5-95 Gy). The proximal trigeminal nerve was targeted in 77.9% of cases, and the median follow-up was 21 months (range 6-156 months). Initial pain relief (modified BNI Pain Intensity Scale grades I-IIIa) was noted in 81.8% of evaluable patients at a median of 30 days. Of 709 patients who achieved initial pain relief, 42.3% experienced at least one pain recurrence after GKRS at a median of 44 months, with 49.0% of these patients undergoing a second procedure. New-onset facial numbness occurred in 25.3% of patients after a median of 8 months. Age ≥ 63 years was associated with a higher probability of both initial pain relief and maintaining pain relief. A distal target location was associated with a higher probability of initial and long-term pain relief, but also a higher incidence of sensory dysfunction. BED ≥ 2100 Gy2.47 was predictive of pain relief at 30 days and 1 year for the distal target, whereas physical dose ≥ 85 Gy was significant for the proximal target, but the restricted range of BED values in this subgroup could be a confounding factor. A maximum brainstem point dose ≥ 29.5 Gy was associated with a higher probability of bothersome facial numbness. BED and physical dose were both predictive of pain relief and could be used as treatment planning goals for distal and proximal targets, respectively, while considering maximum brainstem point dose < 29.5 Gy as a potential constraint for bothersome numbness.

Comparison of mid-outcome among bare metal stent, atherectomy with or without drug-coated balloon angioplasty for femoropopliteal arterial occlusion.

This study evaluated the outcomes of a bare metal stent (BMS), DCB alone, atherectomy plus a drug-coated balloon (AT + DCB) and AT alone for the treatment of femoropopliteal artery occlusion. Four groups were included in this retrospective cohort study: 119 patients underwent the BMS procedure, 89 patients underwent DCB alone, 52 patients underwent AT + DCB, and 61 patients underwent AT alone. Patients were followed-up at 1, 6, 12 and 24months after the procedure, the clinical outcomes and complications were assessed, and the primary outcomes were primary patency and restenosis. AT + DCB showed a lower bailout stent, and BMS displayed a higher retrograde puncture, flow-limiting dissection and postdilation (p < 0.05). For all procedures, the walking distance, ABI and pain score post-procedure were significantly improved compared with the pre-procedure values (p < 0.001). The restenosis rate was higher in BMS (21.0%) and AT alone (24.6%) than in DCB (10.1%) alone and AT + DCB (11.5%) (p = 0.04); there was no difference in amputation or clinically driven target lesion revascularization among procedures. The primary patency rates were 77.7%, 89.4%, 88.0% and 73.7% in the BMS, DCB alone, AT + DCB and AT alone groups at 24months, respectively (p = 0.03), while the secondary patency and main adverse events (stroke, MI and death) were similar. Proximal concavity, proximal target vessel diameter ≥ 5mm, runoff number ≥ 2 and DCB use were protective factors for primary patency. Our results suggested that AT + DCB and DCB alone were associated with higher primary patency, and DCB devices (combined with/without AT) should be the preferred choice for FP lesions.

Reconstruction of Upper Extremity Peripheral Nerve Injuries Using an Epineurial-Like Collagen Device—A Prospective Clinical Study

Abstract Background Epineurium acts as a barrier to protect nerves from injury and maintains its structural and functional integrity. A device was developed to mimic the native structure of epineurium. The aim of this study was to evaluate its biological characteristics and clinical performance in the reconstruction of upper extremity peripheral nerves. Methods Scanning electron microscopy, transmission electron microscopy, and enhanced microcomputed tomography were used to examine the ultrastructural characteristics of the device. A prospective case series with 2-year follow-up was undertaken and reported. Patients who required nerve reconstruction in the upper extremities were included and underwent single or multiple nerve reconstructions in one or both upper limbs. Results The device mimics the structural and biological properties of epineurium. During surgical use, it can form compression-free and self-engaged wrapping around the repaired nerves. A total of 36 peripheral nerve reconstructions were performed using either nerve transfer or nerve grafting in 19 patients. Of these, 14 patients had upper limb nerve injuries and 5 had C5 to C8 spinal cord injuries resulting in tetraplegia. Nerve reconstruction using the device restored peripheral nerve function, with functional motor recovery (FMR) observed in 76% of the most proximal target muscle at 12 months and 85% of most proximal muscles at 24 months post-treatment. FMR was observed in 61% of all target muscles at 12 months and 75% at 24 months post-treatment. Conclusion The device restored FMR in the upper extremities in patients with peripheral nerve or spinal cord injuries. Level of Evidence Therapeutic IV

Exposure-Response Relationships between the Complement Factor B Inhibitor Iptacopan and Lactate Dehydrogenase (LDH) and Hemoglobin (Hb) in Patients (Pts) with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Background: PNH is an ultrarare disease characterized by complement-mediated hemolysis and subsequent anemia, bone marrow failure and thrombosis. In pts not treated with complement inhibitors, intravascular hemolysis (IVH) results from unregulated progression of the complement system and membrane attack complex formation. IVH is controlled in pts treated with anti-C5 therapy, but extravascular hemolysis (EVH) may emerge and cause persistent anemia. High LDH levels are a marker of ongoing IVH, while anemia can result from both IVH and EVH. Iptacopan is the first oral proximal complement inhibitor targeting factor B to selectively inhibit the alternative pathway of the complement cascade. As shown in PNH Phase III trials (APPLY-PNH [NCT04558918] and APPOINT-PNH [NCT04820530]), iptacopan can achieve good control of hemolysis. Aim: To support the dose selection for iptacopan, this analysis characterizes the exposure-response relationships between iptacopan and efficacy measures (LDH and Hb) in pts with PNH. The effect of prior anti-C5 treatment status has also been evaluated. Methods: This analysis is based on data from 4 clinical trials enrolling pts with PNH.Final data were used from the completed Phase II trials X2201 (NCT03439839; pts with active hemolysis despite anti-C5 therapy received concomitant iptacopan) and X2204 (NCT03896152; anti-C5-naïve pts received iptacopan monotherapy), in which iptacopan was administered orally in doses ranging from 25 to 200 mg twice daily (bid). Data up to the cut-off dates of 26 September 2022 and 2 November 2022 were used from the Phase III trials, APPLY-PNH (pts previously treated with anti-C5) and APPOINT-PNH (pts not treated with complement inhibitors including anti-C5), respectively, in which oral iptacopan was administered at 200 mg bid. A longitudinal mixed-effects modeling approach was applied to LDH and Hb. To account for the delay between exposure and response, indirect response models were developed for LDH and Hb. The models were used to predict the long-term (6 months) LDH and Hb responses for various iptacopan bid dose levels and to estimate (using simulation) the proportion of pts achieving LDH ≤1.5 × upper limit of normal (ULN) and Hb ≥12 g/dL. Results: The modeling dataset contained 161 pts with PNH; 94 (58%) were female, and median (range) age was 45 (18-84) years. For modeling of LDH and Hb levels, 2812 and 2587 samples were used, respectively, as well as 1577 pharmacokinetic samples. In total, 53 (33%) pts had not received anti-C5 treatment. Of the remaining 108 pts, 16 (15%) received iptacopan as add-on therapy to anti-C5 (pts from X2201) and 92 (85%) had previously received anti-C5 (pts from APPLY-PNH). The indirect response models were considered biologically plausible and adequately described the data using a common EC 50 value to both anti-C5-naïve pts and pts currently receiving or who previously received anti-C5. The estimated concentration of iptacopan required to achieve 90% of the maximum response (EC 90) of LDH was 268 ng/mL (90% confidence interval [CI] 238, 302), irrespective of prior anti-C5 treatment status ( Figure). The estimated typical LDH value after iptacopan treatment at an exposure &amp;gt;EC 90 was 237 U/L (90% CI 224, 250) within the normal LDH range (&amp;lt;250 U/L). Iptacopan 200 mg bid was the dose at which the highest proportion of pts (86%) achieved LDH ≤1.5 × ULN. While baseline LDH levels differed between anti-C5-naïve pts and pts currently receiving or who previously received anti-C5, similar proportions of these pts achieved LDH ≤1.5 × ULN (84% and 88%, respectively). The estimated EC 90 of Hb ≥12 g/dL was 1968 ng/mL (90% CI 1661, 2333) ( Figure); 89% of all pts were expected to reach EC 90 exposure at iptacopan 200 mg bid. The estimated Hb value after treatment with iptacopan at an exposure &amp;gt;EC 90 was 12.7 g/dL (90% CI 12.4, 13.0). 70% of pts were expected to achieve Hb normalization (≥12 g/dL) with iptacopan 200 mg bid. Similar proportions of anti-C5-naïve pts and pts who were currently receiving or who previously received anti-C5 achieved Hb ≥12 g/dL (71% and 68%, respectively). Conclusions: Indirect response models were developed to describe the exposure-response relationships between iptacopan and efficacy measures (LDH and Hb) in pts with PNH. The analysis demonstrated that 200 mg bid is the dose of oral iptacopan that achieves LDH ≤1.5 × ULN and Hb normalization in the highest proportion of patients regardless of prior treatment status.

Clinical Breakthrough Hemolysis (BTH) during Monotherapy with the Oral Factor B Inhibitor Iptacopan Is Generally Not Severe and Managed without Treatment Discontinuation: 48-Week Data from the Phase III Apply-PNH and Appoint-PNH Trials in Paroxysmal Nocturnal Hemoglobinuria (PNH)

Background: In PNH, BTH is characterized by the re-emergence of intravascular hemolysis (IVH) despite ongoing complement inhibition and is recognized by the reappearance of IVH/PNH signs/symptoms, coupled with a marked increase in lactate dehydrogenase (LDH) and sharp decrease in hemoglobin (Hb). Iptacopan is the first oral proximal complement inhibitor targeting factor B in the alternative pathway and has shown efficacy and safety in complement inhibitor-naïve PNH patients (pts; APPOINT-PNH; NCT04820530) and pts with persistent anemia despite anti-C5 treatment (APPLY-PNH; NCT04558918). In the APPLY-PNH 24-week (wk) analysis, iptacopan monotherapy showed superiority in the adjusted annualized rate of clinical BTH vs C5 inhibitors. Aim: We report the proportion of pts with clinical BTH, exposure-adjusted occurrence rates and BTH characteristics during iptacopan monotherapy in the Phase III APPLY-PNH and APPOINT-PNH trials (study completion [48-wk data]: 6 March and 18 April 2023, respectively). Methods: The prespecified protocol criteria for clinical BTH were LDH &amp;gt;1.5 × upper limit of normal (ULN) and increased vs the last 2 assessments, plus a ≥2 g/dL Hb decrease (vs the last assessment or within 15 days) or significant PNH signs/symptoms. All BTH was classed as an adverse event and evaluated for severity (investigator assessment) and seriousness (per protocol). In APPLY-PNH, adults who had received anti-C5 therapy for ≥6 months and had mean Hb &amp;lt;10 g/dL were randomized to receive oral iptacopan 200 mg twice daily or continue their anti-C5 regimen for 24 wks (randomized treatment period [RTP]). In APPOINT-PNH, adult complement inhibitor-naïve pts with mean Hb &amp;lt;10 g/dL and mean LDH &amp;gt;1.5 × ULN received iptacopan 200 mg twice daily for 24 wks. Both trials then had extension periods in which all pts could receive iptacopan monotherapy for another 24 wks. Results: In APPLY-PNH, 61/62 pts in the iptacopan arm continued iptacopan in the extension period and 34/35 in the anti-C5 arm switched to iptacopan. All 40 pts in APPOINT-PNH entered the extension period. When these 48-wk trials completed, 136 pts had received iptacopan with varying exposure (total exposure ~111 pt-years), of whom 9 had clinical BTH (10 events, Table). Two pts were in APPOINT-PNH (2/40, 2 events) and 7 in APPLY-PNH (7/96, 8 events). Of the iptacopan-treated pts in APPLY-PNH, 2 had BTH in the RTP; 1 of these pts plus 5 others had BTH in the extension period (including 1 who switched from anti-C5 to iptacopan at Wk 24). The exposure-adjusted occurrence rate of clinical BTH on iptacopan was 11.0 events per 100 pt-years in APPLY-PNH (95% confidence interval [CI] 5.2‒23.3) and 5.2 events per 100 pt-years in APPOINT-PNH (95% CI 1.3‒20.5). In the 24-wk APPLY-PNH RTP, 6/35 pts in the anti-C5 arm had clinical BTH (11 events); the exposure-adjusted occurrence rate was 66.9 events per 100 pt-years (95% CI 25.0‒178.9), which is &amp;gt;6 times higher than the rates on iptacopan. Of the 10 clinical BTH events reported on iptacopan, all were mild or moderate except 1 severe (and serious) event despite large PNH RBC clone sizes of &amp;gt;90% reported before 5/8 events with available clone size data. The impact of BTH on Hb was generally transient (Figure). Potential complement-amplifying conditions (CACs) were identified for 8 clinical BTH events during iptacopan treatment; the severe event was associated with COVID-19 and the temporal appearance of cold agglutinins. No pts discontinued iptacopan because of clinical BTH. RBC transfusions were the most common intervention; 1 pt received a 900 mg dose of eculizumab. In the anti-C5 arm in the APPLY-PNH RTP, 1/11 BTH events was severe and 2/11 were associated with potential CACs. Conclusions: In Phase III trials, 136 PNH pts received iptacopan monotherapy and few pts had clinical BTH, with severity being comparable to that with anti-C5 despite PNH RBC clone sizes &amp;gt;90%. Most clinical BTH events on iptacopan were associated with potential CACs; BTH was manageable with no intervention or RBC transfusion and without iptacopan discontinuation. The exposure-adjusted occurrence rates support our prior finding of a significantly lower adjusted annualized rate of clinical BTH with iptacopan vs anti-C5. BTH must be monitored with any complement inhibitor, but these data show that BTH was infrequent on iptacopan and support the positive safety profile of proximal complement inhibition with oral iptacopan monotherapy in hemolytic PNH.