The impact of donating milk on the health of milk donors and their infants: A systematic review and meta-analysis protocol.

Objectives: Breast milk is the recommended nutritional source for newborns and has been associated with decreased morbidity in low-birth-weight and preterm infants. In situations where breast milk is not available, donor breast milk is an alternative. Milk banking is becoming increasingly common worldwide to meet this need. Although the benefits of donor breast milk for the recipient infant are well established, the health impact on the breast milk donor and the infant of the breast milk donor is an area of current research. We aim to synthesize and evaluate the available evidence regarding the impact of donating breast milk on the health, lactation, and well-being of the breast milk donor, and the health and growth of the infant of the breast milk donor. Methods: We will search electronic databases, grey literature, and the websites of relevant international organizations. We will include studies that involve lactating women and their infants, healthy or with health conditions, who donate breast milk, without restrictions on study date, language, or study design. If sufficient homogeneity exists between studies, we will complete meta-analyses. We will evaluate the risk of bias using the Risk of Bias tool or the Cochrane Risk of Bias in Non-Randomized Studies tool. We will evaluate the overall certainty of the evidence using the Grading of Recommendations Assessment, Development, and Evaluation approach. Conclusion: In this systematic review and meta-analysis, we will summarize the current literature regarding the effects of human milk donation on human milk donors and their infants.

The impact of storage, handling, and treatment on nutritional quality and safety of animal milk: A protocol for the systematic review and meta-analysis.

Objectives: Human milk has been shown to reduce severe morbidity in preterm/low-birth-weight infants and is therefore the recommended nutritional source. When infants cannot receive maternal milk, donor human milk (DHM) is recommended. The use of human milk banking facilities is increasing to meet the need for DHM. DHM is unique compared to maternal milk as it must be processed and stored. The processing and storage of animal milk has been more rigorously studied than human milk and can serve as proxy to create DHM banking guidelines. Methods and Analysis: We will search electronic databases, grey literature, and the websites of relevant international organizations. We will include studies that evaluated the impact of storage, handling, and treatment on the nutritional quality and safety of animal milk. We will not restrict study date, language, or design. If sufficient homogeneity exists between studies, we will conduct a meta-analysis. We will evaluate the methodological quality of each study using the SYRCLE's (Systematic Review Centre for Laboratory Animal Experimentation) risk of bias tool. (1) We will evaluate the overall certainty of the evidence using the Grading of Recommendations Assessment, Development, and Evaluation approach. Conclusion: In this systematic review and meta-analysis, commissioned by the World Health Organization, we will synthesize the available literature regarding the impact of various storage, handling, and treatment practices on the nutritional quality and safety of animal milk.

Versatile stochastic model for predictive KMC simulation of fcc metal nanostructure evolution with realistic kinetics.

Stochastic lattice-gas models provide the natural framework for analysis of the surface diffusion-mediated evolution of crystalline metal nanostructures on the appropriate time scale (often 101-104 s) and length scale. Model behavior can be precisely assessed by kinetic Monte Carlo simulation, typically incorporating a rejection-free algorithm to efficiently handle the broad range of Arrhenius rates for hopping of surface atoms. The model should realistically prescribe these rates, or the associated barriers, for a diversity of local surface environments. However, commonly used generic choices for barriers fail, even qualitatively, to simultaneously describe diffusion for different low-index facets, for terrace vs step edge diffusion, etc. We introduce an alternative Unconventional Interaction-Conventional Interaction formalism to prescribe these barriers, which, even with few parameters, can realistically capture most aspects of behavior. The model is illustrated for single-component fcc metal systems, mainly for the case of Ag. It is quite versatile and can be applied to describe both the post-deposition evolution of 2D nanostructures in homoepitaxial thin films (e.g., reshaping and coalescence of 2D islands) and the post-synthesis evolution of 3D nanocrystals (e.g., reshaping of nanocrystals synthesized with various faceted non-equilibrium shapes back to 3D equilibrium Wulff shapes).

Impact of Frailty on Surgical Outcomes of Patients With Cushing Disease Using the Multicenter Registry of Adenomas of the Pituitary and Related Disorders Registry.

BACKGROUND AND OBJECTIVES: Despite growing interest in how patient frailty affects outcomes (eg, in neuro-oncology), its role after transsphenoidal surgery for Cushing disease (CD) remains unclear. We evaluated the effect of frailty on CD outcomes using the Registry of Adenomas of the Pituitary and Related Disorders (RAPID) data set from a collaboration of US academic pituitary centers. METHODS: Data on consecutive surgically treated patients with CD (2011-2023) were compiled using the 11-factor modified frailty index. Patients were classified as fit (score, 0-1), managing well (score, 2-3), and mildly frail (score, 4-5). Univariable and multivariable analyses were conducted to examine outcomes. RESULTS: Data were analyzed for 318 patients (193 fit, 113 managing well, 12 mildly frail). Compared with fit and managing well patients, mildly frail patients were older (mean ± SD 39.7 ± 14.2 and 48.9 ± 12.2 vs 49.4 ± 8.9 years, P < .001) but did not different by sex, race, and other factors. They had significantly longer hospitalizations (3.7 ± 2.0 and 4.5 ± 3.5 vs 5.3 ± 3.5 days, P = .02), even after multivariable analysis (ß = 1.01, P = .007) adjusted for known predictors of prolonged hospitalization (age, Knosp grade, surgeon experience, American Society of Anesthesiologists grade, complications, frailty). Patients with mild frailty were more commonly discharged to skilled nursing facilities (0.5% [1/192] and 4.5% [5/112] vs 25% [3/12], P < .001). Most patients underwent gross total resection (84.4% [163/193] and 79.6% [90/113] vs 83% [10/12]). No difference in overall complications was observed; however, venous thromboembolism was more common in mildly frail (8%, 1/12) than in fit (0.5%, 1/193) and managing well (2.7%, 3/113) patients (P = .04). No difference was found in 90-day readmission rates. CONCLUSION: These results demonstrate that mild frailty predicts CD surgical outcomes and may inform preoperative risk stratification. Frailty-influenced outcomes other than age and tumor characteristics may be useful for prognostication. Future studies can help identify strategies to reduce disease burden for frail patients with hypercortisolemia.

Combined inhibition of KRASG12C and mTORC1 kinase is synergistic in non-small cell lung cancer.

Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.

Cryo-EM Structure of the Mnx Protein Complex Reveals a Tunnel Framework for the Mechanism of Manganese Biomineralization.

The global manganese cycle relies on microbes to oxidize soluble Mn(II) to insoluble Mn(IV) oxides. Some microbes require peroxide or superoxide as oxidants, but others can use O2 directly, via multicopper oxidase (MCO) enzymes. One of these, MnxG from Bacillus sp. strain PL-12, was isolated in tight association with small accessory proteins, MnxE and MnxF. The protein complex, called Mnx, has eluded crystallization efforts, but we now report the 3D structure of a point mutant using cryo-EM single particle analysis, cross-linking mass spectrometry, and AlphaFold Multimer prediction. The ß-sheet-rich complex features MnxG enzyme, capped by a heterohexameric ring of alternating MnxE and MnxF subunits, and a tunnel that runs through MnxG and its MnxE3F3 cap. The tunnel dimensions and charges can accommodate the mechanistically inferred binuclear manganese intermediates. Comparison with the Fe(II)-oxidizing MCO, ceruloplasmin, identifies likely coordinating groups for the Mn(II) substrate, at the entrance to the tunnel. Thus, the 3D structure provides a rationale for the established manganese oxidase mechanism, and a platform for further experiments to elucidate mechanistic details of manganese biomineralization.

SMARCB1-Retained and SMARCB1-Deficient SNUC are Genetically Distinct: A Pilot Study Using RNA Sequencing.

Background Understanding the genetic basis for the molecular classification of sinonasal undifferentiated carcinoma (SNUC) based on SMARCB1 may improve our understating regarding the nature of the disease. The objective of the study was to compare the genetic profile of SMARCB1-retained (SR-SNUC) and SMARCB1-deficient SNUC (SD-SNUC). Methods Formalin-fixed, paraffin-embedded tissue from treatment-naive patients with SNUC were selected. Three cases of SR-SNUC, four cases of SD-SNUC, and four samples of nontumor tissue (control samples) were selected. Ribonucleic acid (RNA) sequencing was performed. Results SR-SNUC had a higher number of variants (1 variant for every 15,000 bases) compared with SD-SNUC (1 variant every 29,000 bases). The ratio of missense to silent mutation ratio was higher for SR-SNUC (0.8) as compared with SD-SNUC (0.7). Approximately 1,500 genes were differentially expressed between SR-SNUC and SD-SNUC. The genes that had a higher expression in SR-SNUC included TPD52L1, B3GNT3, GFY, TJP3, ELL3, CYP4F3, ALDH3B2, CKMT1B, VIPR1, SLC7A5, PPP2R2C, UPK3B, MUC1, ELF5, STY7, and H2AC14. The gene that had a higher expression in SD-SNUC was ZFHX4. Most of these genes were related to either protein translation or immune regulation. The most common ( n = 3, 75%) mechanisms of loss of SMARCB1 gene in SD-SNUC was loss of heterozygosity. Conclusion RNA sequencing is a viable and informative approach for genomic profiling of archival SNUC samples. Both SR-SNUC and SD-SNUC were noted to have distinct genetic profiles underlying the molecular classification of these diseases.

Real-Time KMC Simulation of Vacancy-Mediated Intermixing in Au@Ag Octahedral Core-Cubic Shell Nanocrystals with Ab Initio-Guided Kinetics.

Utilization of core-shell rather than monometallic nanocrystals (NCs) facilitates fine-tuning of NC properties for applications. However, compositional evolution via intermixing can degrade these properties prompting recent experimental studies. We develop an atomistic-level stochastic model for vacancy-mediated intermixing exploiting a formalism which allows incorporation at an ab initio density functional theory level of not just the thermodynamics of vacancy formation, but also relevant diffusion barriers for a vast number of possible local environments (in the core and in the shell, at the interface, and in the intermixed phase). This facilitates a predictive treatment and comprehensive understanding of intermixing on the relevant time scale (e.g., 101-103 s). In contrast, previous modeling at the atomistic level utilized only unrealistic generic prescriptions of barriers or employed simplified continuum treatments. For Au@Ag octahedral core-cubic shell NCs, our modeling not only captures the experimentally observed rate or time scale for intermixing of ∼100 s at 450 °C for 60 nm NCs, but also elucidates the underlying rate controlling processes and the effective intermixing barrier.

Multicenter Registry of Adenomas of the Pituitary and Related Disorders: Initial Description of Cushing Disease Cohort, Surgical Outcomes, and Surgeon Characteristics.

BACKGROUND AND OBJECTIVES: To address the lack of a multicenter pituitary surgery research consortium in the United States, we established the Registry of Adenomas of the Pituitary and Related Disorders (RAPID). The goals of RAPID are to examine surgical outcomes, improve patient care, disseminate best practices, and facilitate multicenter surgery research at scale. Our initial focus is Cushing disease (CD). This study aims to describe the current RAPID patient cohort, explore surgical outcomes, and lay the foundation for future studies addressing the limitations of previous studies. METHODS: Prospectively and retrospectively obtained data from participating sites were aggregated using a cloud-based registry and analyzed retrospectively. Standard preoperative variables and outcome measures included length of stay, unplanned readmission, and remission. RESULTS: By July 2023, 528 patients with CD had been treated by 26 neurosurgeons with varying levels of experience at 9 academic pituitary centers. No surgeon treated more than 81 of 528 (15.3%) patients. The mean ± SD patient age was 43.8 ± 13.9 years, and most patients were female (82.2%, 433/527). The mean tumor diameter was 0.8 ± 2.7 cm. Most patients (76.6%, 354/462) had no prior treatment. The most common pathology was corticotroph tumor (76.8%, 381/496). The mean length of stay was 3.8 ± 2.5 days. The most common discharge destination was home (97.2%, 513/528). Two patients (0.4%, 2/528) died perioperatively. A total of 57 patients (11.0%, 57/519) required an unplanned hospital readmission within 90 days of surgery. The median actuarial disease-free survival after index surgery was 8.5 years. CONCLUSION: This study examined an evolving multicenter collaboration on patient outcomes after surgery for CD. Our results provide novel insights on surgical outcomes not possible in prior single-center studies or with national administrative data sets. This collaboration will power future studies to better advance the standard of care for patients with CD.

Health net-outcome objectives and approaches for spatial planning and development: a scoping review protocol.

OBJECTIVE: The objective of this scoping review is to review the body of knowledge on net gain and no net loss (net-outcome) objectives and approaches applicable to health in spatial planning and development policies and practice. INTRODUCTION: There is an established body of academic and gray literature addressing environmental net-outcome objectives, such as biodiversity net gain, in spatial planning policies and practice. A "health net gain" objective has recently been proposed as a driver for health protection and the realization of health. Such an objective and approach are yet to be scoped and defined. INCLUSION CRITERIA: This review will consider sources in the scientific and gray literature that describe health net-outcome objectives that can be implemented in spatial planning and development policies and practice. Source contexts will not be limited to specific countries, geographical areas, or settings. All types of evidence will be considered. METHODS: This review will follow the JBI methodology for scoping reviews. Databases to be searched include PsycINFO (APA), Embase, HMIC Health Management Information Consortium, MEDLINE (Ovid), Scopus, and selected databases from the ProQuest Social Science Premium Collection. Sources of gray literature to be searched include ProQuest Dissertations and Theses, TRIP Pro, and BASE. No language or date restrictions will be applied. Two independent reviewers will retrieve and review full-text studies and extract data. The results will be presented in tabular or diagrammatic format with a narrative summary. REVIEW REGISTRATION: Open Science Framework https://osf.io/4dbcm.

The annotation of GBA1 has been concealed by its protein-coding pseudogene GBAP1.

Mutations in GBA1 cause Gaucher disease and are the most important genetic risk factor for Parkinson's disease. However, analysis of transcription at this locus is complicated by its highly homologous pseudogene, GBAP1. We show that >50% of short RNA-sequencing reads mapping to GBA1 also map to GBAP1. Thus, we used long-read RNA sequencing in the human brain, which allowed us to accurately quantify expression from both GBA1 and GBAP1. We discovered significant differences in expression compared to short-read data and identify currently unannotated transcripts of both GBA1 and GBAP1. These included protein-coding transcripts from both genes that were translated in human brain, but without the known lysosomal function-yet accounting for almost a third of transcription. Analyzing brain-specific cell types using long-read and single-nucleus RNA sequencing revealed region-specific variations in transcript expression. Overall, these findings suggest nonlysosomal roles for GBA1 and GBAP1 with implications for our understanding of the role of GBA1 in health and disease.

Health Disparities in the Neurosurgical Care of Patients with Trigeminal Neuralgia.

BACKGROUND: There has been limited investigation into how social determinants of health impact treatment outcomes in patients with trigeminal neuralgia (TN). We aimed to investigate how social determinants of health may alter the course of clinical care for patients with TN. METHODS: The electronic medical record was queried for patients with a diagnosis of TN comanaged by neurosurgeons and other facial pain specialists at our medical center. Area Deprivation Index served as a proxy for socioeconomic status (SES). Multivariable linear regression models were performed using RStudio to assess the impact of social determinants on the time to neurosurgical referral and surgical intervention. RESULTS: A total of 229 patients (mean age 50 years, 74% female) were included. Of these, 135 (60%) patients underwent a neurosurgical procedure after referral, the most common being microvascular decompression (n = 84, 62%) (Table 1). Most of the patients were white (76.3%) and insured by Medicare (51.8%), followed by private insurance (38.6%). Age and sex were significant predictors of time to neurosurgical referral after symptom onset, as older patients (P < 0.01, Figure 3) and females (P = 0.02) tended to have a greater delay between symptom onset and specialist referral. Race, SES, and insurance status were not significantly associated with time-to-referral or time-to-treatment. CONCLUSION: This study found that older and female patients with TN had a longer time from symptom onset to specialist referral. Based on these data, there is no association between race, SES, and insurance status with time-to-referral or time-to-treatment in patients with TN.

Treating Benzodiazepine Withdrawal in a Bridge Clinic.

BACKGROUND: Benzodiazepine-involved overdose deaths are rising, driven by increasing use of nonprescribed benzodiazepine pills. For patients who wish to stop nonprescribed benzodiazepine use, rapid inpatient tapers are typically the only option to treat benzodiazepine withdrawal. Substance use disorder bridge clinics can provide the high-touch care needed to manage outpatient benzodiazepine tapers in patients at high risk due to other substance use disorders. OBJECTIVE: Describe the implementation and short-term outcomes of an outpatient benzodiazepine taper protocol to treat benzodiazepine withdrawal in a substance use disorder bridge clinic. METHODS: The clinical team developed a 4- to 6-week intensive outpatient taper protocol using diazepam. Patients with benzodiazepine use disorder were eligible if they had benzodiazepine withdrawal, lacked a prescriber, wanted to stop benzodiazepines completely, and agreed to daily visits. For patients who initiated a taper between April 2021 and December 2022, we evaluated the proportion of patients who completed a taper (i.e., tapered to a last prescribed dose of diazepam 10 mg/d or less); likelihood of remaining on the taper over time; and seizure, overdose, or death documented at the study institution during or within 1 month of taper completion or discontinuation. Other secondary outcomes included HIV testing and prevention, hepatitis C testing, and referrals to recovery coaching or psychiatry. RESULTS: Fifty-four patients initiated a total of 60 benzodiazepine tapers. The population was mostly male (61%) and non-Hispanic White (85%). Nearly all patients had opioid use disorder (96%), and most (80%) were taking methadone or buprenorphine for opioid use disorder before starting the taper. Patients reported using multiple substances in addition to benzodiazepines, most commonly fentanyl (75%), followed by cocaine (41%) and methamphetamine (21%). Fourteen patients (23%) completed a taper with a median duration of 34 days (IQR 27.8-43.5). Most tapers were stopped when the patient was lost to follow-up (57%), or the team recommended inpatient care (18%). Two patients had a seizure, and 4 had a presumed opioid-involved overdose during or within 1 month after the last taper visit, all individuals who did not complete a taper. No deaths occurred during or within 1 month of taper completion or discontinuation. Challenges included frequent loss to follow-up in the setting of other unstable substance use. Patients received other high-priority care during the taper including HIV testing (32%), PrEP initiation (6.7%), hepatitis C testing (30%), and referrals to recovery coaches (18%) and psychiatry (6.7%). CONCLUSIONS: Managing benzodiazepine withdrawal with a 4- to 6-week intensive outpatient taper in patients with benzodiazepine and opioid use disorders is challenging. More work is needed to refine patient selection, balance safety risks with feasibility, and study long-term, patient-centered outcomes.

Predictors of Durable Remission After Successful Surgery for Cushing Disease: Results From the Multicenter RAPID Registry.

BACKGROUND AND OBJECTIVE: Cushing disease (CD) affects mortality and quality of life along with limited long-term remission, underscoring the need to better identify recurrence risk. The identification of surgical or imaging predictors for CD remission after transsphenoidal surgery has yielded some inconsistent results and has been limited by single-center, single-surgeon, or meta-analyses studies. We sought to evaluate the multicenter Registry of Adenomas of the Pituitary and Related Disorders (RAPID) database of academic US pituitary centers to assess whether robust nonhormonal recurrence predictors could be elucidated. METHODS: Patients with treated CD from 2011 to 2023 were included. The perioperative and long-term characteristics of CD patients with and without recurrence were assessed using univariable and multivariable analyses. RESULTS: Of 383 patients with CD from 26 surgeons achieving postoperative remission, 288 (75.2%) maintained remission at last follow-up while 95 (24.8%) showed recurrence (median time to recurrence 9.99 ± 1.34 years). Patients with recurrence required longer postoperative hospital stays (5 ± 3 vs 4 ± 2 days, P = .002), had larger average tumor volumes (1.76 ± 2.53 cm3 vs 0.49 ± 1.17 cm3, P = .0001), and more often previously failed prior treatment (31.1% vs 14.9%, P = .001) mostly being prior surgery. Multivariable hazard prediction models for tumor recurrence found younger age (odds ratio [OR] = 0.95, P = .002) and Knosp grade of 0 (OR = 0.09, reference Knosp grade 4, P = .03) to be protective against recurrence. Comparison of Knosp grade 0 to 2 vs 3 to 4 showed that lower grades had reduced risk of recurrence (OR = 0.27, P = .04). Other factors such as length of stay, surgeon experience, prior tumor treatment, and Knosp grades 1, 2, or 3 failed to reach levels of statistical significance in multivariable analysis. CONCLUSION: This multicenter study centers suggests that the strongest predictors of recurrence include tumor size/invasion and age. This insight can help with patient counseling and prognostication. Long-term follow-up is necessary for patients, and early treatment of small tumors may improve outcomes.

Long-Term Outcomes of Surgery and Radiation Treatment for Adult Patients with Craniopharyngioma.

OBJECTIVE: Treatment of craniopharyngioma typically entails gross total resection (GTR) or subtotal resection with adjuvant radiation (STR-RT). We analyzed outcomes in adults with craniopharyngioma undergoing GTR versus STR-RT. METHODS: This retrospective study enrolled 115 patients with craniopharyngioma in 5 institutions. Patients with STR received postoperative RT with stereotactic radiosurgery or fractionated radiation therapy per institutional preference and ability to spare optic structures. RESULTS: Median age was 44 years (range, 19-79 years). GTR was performed in 34 patients and STR-RT was performed in 81 patients with median follow-up of 78.9 months (range, 1-268 months). For GTR, local control was 90.5% at 2 years, 87.2% at 3 years, and 71.9% at 5 years. For STR-RT, local control was 93.6% at 2 years, 90.3% at 3 years, and 88.4% at 5 years. At 5 years following resection, there was no difference in local control (P = 0.08). Differences in rates of visual deterioration or panhypopituitarism were not observed between GTR and STR-RT groups. There was no difference in local control in adamantinomatous and papillary craniopharyngioma regardless of treatment. Additionally, worse local control was found in patients receiving STR-RT who were underdosed with fractionated radiation therapy (P = 0.03) or stereotactic radiosurgery (P = 0.04). CONCLUSIONS: Good long-term control was achieved in adults with craniopharyngioma who underwent STR-RT or GTR with no significant difference in local control. First-line treatment for craniopharyngioma should continue to be maximal safe resection followed by RT as needed to balance optimal local control with long-term morbidity.

Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.

Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus.

BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 611. Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRASG12X). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRASG12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985).

Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression.

Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers' deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus's central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS's function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion.IMPORTANCEBrown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of "omics" platforms has recently uncovered the key "oxidative-hydrolytic" mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes' catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system's use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

Predictors of Subjective Olfactory Dysfunction and Sinonasal Quality-of-Life After Endoscopic Transsphenoidal Pituitary Surgery.

BACKGROUND: This is the largest study in North America investigating olfactory outcomes after pituitary surgery to date. OBJECTIVE: Characterize factors associated with subjective olfactory dysfunction (OD) and worsened sinonasal quality-of-life (QOL) after endoscopic TSA. METHODS: Patients undergoing primary TSA for secreting and non-secreting pituitary adenomas between 2017 and 2021 with pre- and post-operative SNOT-22 scores were included. Subjective OD was determined by the smell/taste dysfunction question on the SNOT-22 (smell-SNOT). RESULTS: 159 patients with pre- and post-operative SNOT-22 scores were included. Average total SNOT-22 scores worsened from pre-operative (16.91 ± 16.91) to POM1 (25.15 ± 20.83, P < .001), with no difference from pre-operative (16.40 ± 15.88) to POM6 (16.27 ± 17.92, P = .936) or pre-operative (13.63 ± 13.54) to POM12 (12.60 ± 16.45, P = .651). Average smell-SNOT scores worsened from pre-operative (0.40 ± 1.27) to POM1 (2.09 ± 2.01, P < .001), and pre-operative (0.46 ± 1.29) to POM6 (1.13 ± 2.45, P = .002), with no difference from pre-operative (0.40 ± 1.07) to POM12 (0.71 ± 1.32, P = .100). Female gender had a 0.9-point (95% CI 0.1 to 1.6) P = .021, increase in smell-SNOT at POM1, resolving by POM6 (0.1 [-0.9 to 1.1], P = .800) and POM12 (0.0 [-1.0 to 0.9], P = .942). Septoplasty with tunnel approach had a 1.1 [0.2 to 2.0] out of 5-point (P = .023) increase in smell-SNOT at POM1, resolving by POM6 (0.2 [-1.1 to 1.6], P = .764) and POM12 (0.4 [-0.9 to 1.6], P = .567). Female gender had a 9.5 (4.0 to 15.1)-point (P = .001) increase in SNOT-22 scores at POM1, resolving by POM6 (3.4 [-3.0 to 9.8], P = .292) and POM12 (6.4 [-5.4 to 18.2], P = .276). Intra-operative CSF leak had an 8.6 [2.1 to 15.1]-point (P = .009) increase in SNOT-22 scores at POM1, resolving by POM6 (5.4 [-1.7 to 12.5], P = .135), and POM12 (1.1 [-12.9 to 15.1], P = .873). CONCLUSION: Changes in subjective olfaction and sinonasal QOL after TSA may be associated with gender, operative approach, and intra-operative CSF leak, resolving 6-12 months post-operatively.