Green Oxidation of Aromatic Hydrazide Derivatives Using an Oxoammonium Salt.

Aromatic diazenes are often prepared by oxidation of the corresponding hydrazides using stoichiometric quantities of nonrecyclable oxidants. We developed a convenient alternative protocol for the oxidation of aromatic hydrazides using Bobbitt's salt (1), a metal-free, recyclable, and commercially available oxoammonium reagent. A variety of aryl hydrazides were oxidized within 75 min at room temperature using the developed protocol. Computational insight suggests that this oxidation occurs by a polar hydride transfer mechanism.

3-Chloropropylbis(catecholato)silicate as a Bifunctional Reagent for the One-Pot Synthesis of Tetrahydroquinolines from o-Bromosulfonamides.

Bis(catecholato)silicate salts are easily accessible reagents that can be used to install alkyl fragments through photoredox-enabled cross-coupling. These reagents can incorporate various functional groups including pendant alkyl halides. A halogenated organosilicate reagent was leveraged to develop a one-pot synthesis of tetrahydroquinolines from o-bromosulfonamides, where the bifunctional reagent participates in a nickel/photoredox cross-coupling followed by intramolecular nucleophilic substitution. The functional group tolerance of this cross-coupling strategy allowed for the preparation of a series of substituted tetrahydroquinolines.

Multisite Verification of a Targeted CFTR Polymerase Chain Reaction/Capillary Electrophoresis Assay That Evaluates Pathogenic Variants Across Diverse Ethnic and Ancestral Groups.

CONTEXT.­: Existing targeted cystic fibrosis screening assays miss important pathogenic CFTR variants in the ethnically diverse US population. OBJECTIVE.­: To evaluate the analytic performance of a multiplex polymerase chain reaction (PCR)/capillary electrophoresis (CE) CFTR assay panel that simultaneously interrogates primary pathogenic variants of different ethnic/ancestral groups. DESIGN.­: Performance characteristic assessment and variant coverage comparison of the panel with a focus on ethnicity-specific CFTR variants were performed. Sample DNA was primarily from whole blood or cell lines. Detection of CFTR carriers was compared across several commercially available CFTR kits and recommended variant sets based on panel content. RESULTS.­: The panel interrogated 65 pathogenic CFTR variants representing 92% coverage from a recent genomic sequencing survey of the US population, including 4 variants with top 5 frequency in African or Asian populations not reflected in other targeted panels. In simulation studies, the panel represented 95% of carriers across the global population, resulting in 6.9% to 19.0% higher carrier detection rate compared with 10 targeted panels or variant sets. Precision and sensitivity/specificity were 100% concordant. Multisite sample-level genotyping accuracy was 99.2%. Across PCR and CE instruments, sample-level genotyping accuracy was 97.1%, with greater than 99% agreement for all variant-level metrics. CONCLUSIONS.­: The CFTR assay achieves 92% or higher coverage of CFTR variants in diverse populations and provides improved pan-ethnic coverage of minority subgroups of the US populace. The assay can be completed within 5 hours from DNA sample to genotype, and performance data exceed acceptance criteria for analytic metrics. This assay panel content may help address gaps in ancestry-specific CFTR genotypes while providing a streamlined procedure with rapidly generated results.

Bicyclobutanes: from curiosities to versatile reagents and covalent warheads.

The unique chemistry of small, strained carbocyclic systems has long captivated organic chemists from a theoretical and fundamental standpoint. A resurgence of interest in strained carbocyclic species has been prompted by their potential as bioisosteres, high fraction of sp3 carbons, and limited appearance in the patent literature. Among strained ring systems, bicyclo[1.1.0]butane (BCB) stands apart as the smallest bicyclic carbocycle and is amongst the most strained carbocycles known. Despite the fact that BCBs have been synthesized and studied for well over 50 years, they have long been regarded as laboratory curiosities. However, new approaches for preparing, functionalizing, and using BCBs in "strain-release" transformations have positioned BCBs to be powerful synthetic workhorses. Further, the olefinic character of the bridgehead bond enables BCBs to be elaborated into various other ring systems and function as covalent warheads for bioconjugation. This review will discuss the recent developments in the synthesis and functionalization of BCBs as well as the applications of these strained rings in synthesis and drug discovery. An overview of the properties and the historical context of this interesting structure will be provided.

Recommendations for Interpreting and Reporting Silent Carrier and Disease-Modifying Variants in SMA Testing Workflows.

Genetic testing for SMA diagnosis, newborn screening, and carrier screening has become a significant public health interest worldwide, driven largely by the development of novel and effective molecular therapies for the treatment of spinal muscular atrophy (SMA) and the corresponding updates to testing guidelines. Concurrently, understanding of the underlying genetics of SMA and their correlation with a broad range of phenotypes and risk factors has also advanced, particularly with respect to variants that modulate disease severity or impact residual carrier risks. While testing guidelines are beginning to emphasize the importance of these variants, there are no clear guidelines on how to utilize them in a real-world setting. Given the need for clarity in practice, this review summarizes several clinically relevant variants in the SMN1 and SMN2 genes, including how they inform outcomes for spinal muscular atrophy carrier risk and disease prognosis.

Postoperative Oral Antibiotics and Sinonasal Outcomes Following Endoscopic Transsphenoidal Surgery for Pituitary Tumors Study: A Multicenter, Prospective, Randomized, Double-Blinded, Placebo-Controlled Study.

BACKGROUND: Postoperative prophylactic antibiotics are commonly used in pituitary surgery, but evidence supporting their use is lacking, which has implications for antibiotic stewardship. OBJECTIVE: To evaluate whether receipt of postoperative oral antibiotics results in superior sinonasal quality of life (QOL) compared with placebo among patients who undergo endoscopic endonasal transsphenoidal pituitary surgery. METHODS: Patients were randomized to receive either oral placebo or cefdinir (trimethoprim-sulfamethoxazole in patients intolerant to cefdinir) for 7 d after surgery. They were monitored for 12 wk. The primary outcome measure was sinonasal QOL at 2 wk on the Anterior Skull Base Nasal Inventory-12. Supplementary end points included sinonasal QOL reported on the Sinonasal Outcome Test-22 and objective endoscopy scores to assess nasal healing according to the Lund-Kennedy method. RESULTS: A total of 461 patients were screened, 131 were randomized, and 113 (placebo arm: 55; antibiotic arm: 58) were analyzed. There was no clinically meaningful or statistically significant difference in sinonasal QOL at any measured time point (P ≥ .24) using either instrument. Nasal cavity endoscopy scores were not significantly different at 1 to 2 wk after surgery (P = .25) or at 3 to 4 wk after surgery (P = .08). CONCLUSION: Postoperative prophylactic oral antibiotics did not result in superior sinonasal QOL compared with placebo among patients who underwent standard endoscopic transsphenoidal surgery.

Assessment of the Validity of the Sinonasal Outcomes Test-22 in Pituitary Surgery: A Multicenter Prospective Trial.

OBJECTIVES/HYPOTHESIS: Sinonasal Outcomes Test-22 (SNOT-22) is used widely as a patient-reported sinonasal quality-of-life (QOL) instrument for endoscopic endonasal pituitary surgery. However, it has never been validated in this population. This study explores the psychometric validity of SNOT-22 to determine if it is a valid scale in patients undergoing endoscopic pituitary surgery. STUDY DESIGN: Multicenter prospective trial. METHODS: Adult patients (n = 113) with pituitary tumors undergoing endoscopic surgery were enrolled in a multicenter study. Patient-reported QOL was assessed using SNOT-22 and the Anterior Skull Base Nasal Inventory-12. Face validity, internal consistency, responsiveness to clinical change, test-retest reliability, and concurrent validity were determined using standard statistical methods. RESULTS: Internal consistency using Cronbach's alpha at baseline and 2 weeks postoperatively were 0.911 and 0.922, indicating SNOT-22 performed well as a single construct. Mean QOL scores were significantly worse at 2 weeks than baseline (16.4 ± 15.1 vs. 23.1 ± 16.4, P < .001), indicating the scale is responsive to clinical change. However, only 11/22 items demonstrated significant changes in mean scores at 2 weeks. Correlation between scores at 2 and 3 weeks was high, suggesting good test-retest reliability, r(107) = 0.75, P < .001. Factor analysis suggests the five-factor solution proposed for the SNOT-22 in rhinosinusitis patients is not valid in pituitary surgery patients. CONCLUSIONS: The SNOT-22 is a valid QOL instrument in patients undergoing endoscopic pituitary surgery. However, because it includes 22 items, can be applied only as a single construct, 50% of the items do not demonstrate changes after surgery, and is not as sensitive to change as other scales, shorter instruments developed specifically for this patient population may be preferable. LEVEL OF EVIDENCE: 2 Laryngoscope, 131:E2757-E2763, 2021.

Multisite Evaluation and Validation of a Sensitive Diagnostic and Screening System for Spinal Muscular Atrophy that Reports SMN1 and SMN2 Copy Number, along with Disease Modifier and Gene Duplication Variants.

Spinal muscular atrophy is a severe autosomal recessive disease caused by disruptions in the SMN1 gene. The nearly identical SMN2 gene copy number is associated with disease severity. SMN1 duplication markers, such as c.∗3+80T>G and c.∗211_∗212del, can assess residual carrier risk. An SMN2 disease modifier (c.859G>C) can help inform prognostic outcomes. The emergence of multiple precision gene therapies for spinal muscular atrophy requires accurate and rapid detection of SMN1 and SMN2 copy numbers to enable early treatment and optimal patient outcomes. We developed and evaluated a single-tube PCR/capillary electrophoresis assay system that quantifies SMN1/2 copy numbers and genotypes three additional clinically relevant variants. Analytical validation was performed with human cell lines and whole blood representing varying SMN1/2 copies on four capillary electrophoresis instrument models. In addition, four independent laboratories used the assay to test 468 residual clinical genomic DNA samples. The results were ≥98.3% concordant with consensus SMN1/2 exon 7 copy numbers, determined using multiplex ligation-dependent probe amplification and droplet digital PCR, and were 100% concordant with Sanger sequencing for the three variants. Furthermore, copy number values were 98.6% (SMN1) and 97.1% (SMN2) concordant to each laboratory's own reference results.

Radical-Polar Crossover Annulation: A Platform for Accessing Polycyclic Cyclopropanes.

Photoredox-mediated radical/polar crossover (RPC) processes provide unique solutions to challenging annulations. Herein, we describe an approach to the cyclopropanation of olefins that are embedded within bicyclic scaffolds. Whereas these systems are notoriously recalcitrant toward classical cyclopropanation approaches, RPC cyclopropanation can be executed with ease, leading to polycarbocyclic and polyheterocyclic cyclopropanes. The cyclopropanation proceeds through a photoredox-enabled Giese-type radical addition followed by an intramolecular anionic substitution reaction on a neopentyl leaving group.

Photoredox Radical/Polar Crossover Enables Construction of Saturated Nitrogen Heterocycles.

Photoredox-mediated radical/polar crossover (RPC) processes offer new avenues for the synthesis of cyclic molecules. This process has been realized for the construction of medium-sized saturated nitrogen heterocycles. Photocatalytically generated alkyl radicals possessing pendant leaving groups engage imines in C-C bond formation, and subsequent reduction of the intermediate nitrogen-centered radical triggers anionic ring closure. With the aid of visible light irradiation, substituted pyrrolidines, piperidines, and azepanes can be prepared under mild, redox-neutral conditions.

Alkyl Carbon-Carbon Bond Formation by Nickel/Photoredox Cross-Coupling.

The union of photoredox and nickel catalysis has resulted in a renaissance in radical chemistry as well as in the use of nickel-catalyzed transformations, specifically for carbon-carbon bond formation. Collectively, these advances address the longstanding challenge of late-stage cross-coupling of functionalized alkyl fragments. Empowered by the notion that photocatalytically generated alkyl radicals readily undergo capture by Ni complexes, wholly new feedstocks for cross-coupling have been realized. Herein, we highlight recent developments in several types of alkyl cross-couplings that are accessible exclusively through this approach.

Radical/Polar Annulation Reactions (RPARs) Enable the Modular Construction of Cyclopropanes.

An annulation process for the construction of 1,1-disubstituted cyclopropanes via a radical/polar crossover process is described. The cyclopropanation proceeds by the addition of a photocatalytically generated radical to a homoallylic tosylate. Reduction of the intermediate radical alkylation adduct (via single electron transfer) furnishes an anion that undergoes an intramolecular substitution. The process displays excellent functional group tolerance, characteristic of proceeding through odd-electron intermediates, and occurs under mild conditions with visible light irradiation.

Fumarate hydratase inactivation in hereditary leiomyomatosis and renal cell cancer is synthetic lethal with ferroptosis induction.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a hereditary cancer syndrome characterized by inactivation of the Krebs cycle enzyme fumarate hydratase (FH). HLRCC patients are at high risk of developing kidney cancer of type 2 papillary morphology that is refractory to current radiotherapy, immunotherapy and chemotherapy. Hence, an effective therapy for this deadly form of cancer is urgently needed. Here, we show that FH inactivation (FH-/- ) proves synthetic lethal with inducers of ferroptosis, an iron-dependent and nonapoptotic form of cell death. Specifically, we identified gene signatures for compound sensitivities based on drug responses for 9 different drug classes against the NCI-60 cell lines. These signatures predicted that ferroptosis inducers would be selectively toxic to FH-/- cell line UOK262. Preferential cell death against UOK262-FH-/- was confirmed with 4 different ferroptosis inducers. Mechanistically, the FH-/- sensitivity to ferroptosis is attributed to dysfunctional GPX4, the primary cellular defender against ferroptosis. We identified that C93 of GPX4 is readily post-translationally modified by fumarates that accumulate in conditions of FH-/- , and that C93 modification represses GPX4 activity. Induction of ferroptosis in FH-inactivated tumors represents an opportunity for synthetic lethality in cancer.

Evolution of a Thermophilic Strand-Displacing Polymerase Using High-Temperature Isothermal Compartmentalized Self-Replication.

Strand-displacing polymerases are a crucial component of isothermal amplification (IA) reactions, where the lack of thermal cycling reduces equipment needs and improves the time to answer, especially for point-of-care applications. In order to improve the function of strand-displacing polymerases, we have developed an emulsion-based directed evolution scheme, high-temperature isothermal compartmentalized self-replication (HTI-CSR) that does not rely on thermal cycling. Starting from an algorithm-optimized shuffled library of exonuclease-deficient Family A polymerases from Geobacillus stearothermophilus (Bst LF) and Thermus aquaticus (Klentaq), we have applied HTI-CSR to generate a more thermostable strand-displacing polymerase variant that performs well in loop-mediated isothermal amplification and rolling circle amplification, even after thermal challenges of up to 95 °C that lead to better primer annealing. The new enzyme (v5.9) is also capable of a variety of new reactions, including isothermal hyperbranched rolling circle amplification. The HTI-CSR method should now prove useful for evolving additional beneficial phenotypes in strand-displacing polymerases.

The Minimal Clinically Important Difference of the Anterior Skull Base Nasal Inventory-12.

BACKGROUND: The minimal clinically important difference (MCID) is defined as the smallest change in health-related quality of life (QOL) that patients consider meaningful. The MCID is essential for determining clinically significant changes, rather than simply statistically significant changes, in QOL scores. The Anterior Skull Base Nasal Inventory-12 (ASK Nasal-12), a site-specific sinonasal QOL instrument, has emerged as a standard instrument for assessing QOL in patients who have undergone endonasal transsphenoidal surgery. OBJECTIVE: To determine the MCID for the ASK Nasal-12. METHODS: Distribution- and anchor-based methods were used to determine the MCID for the ASK Nasal-12 based on raw data from a multicenter prospective QOL study of 218 patients. RESULTS: Two distribution-based statistical methods, the one-half standard deviation method and the effect-size method, both yielded MCIDs of 0.37 (medium effect). The first anchor-based method, using the 2-wk postoperative overall nasal functioning item as the anchor, yielded an MCID of 0.31. The second anchor-based method, using the 2-wk postoperative Short Form Health Survey 8 bodily pain item as the anchor, yielded an MCID of 0.29. CONCLUSION: The largest MCID obtained for the ASK Nasal-12 using 4 statistical methods 2 wk postoperatively was 0.37. This information provides clinicians with an essential context for determining the clinical significance of changes in QOL scores after interventions. Our results will help clinicians better interpret QOL scores and design future studies that are powered to detect meaningful QOL changes.

Shuffle Optimizer: A Program to Optimize DNA Shuffling for Protein Engineering.

DNA shuffling is a powerful tool to develop libraries of variants for protein engineering. Here, we present a protocol to use our freely available and easy-to-use computer program, Shuffle Optimizer. Shuffle Optimizer is written in the Python computer language and increases the nucleotide homology between two pieces of DNA desired to be shuffled together without changing the amino acid sequence. In addition we also include sections on optimal primer design for DNA shuffling and library construction, a small-volume ultrasonicator method to create sheared DNA, and finally a method to reassemble the sheared fragments and recover and clone the library. The Shuffle Optimizer program and these protocols will be useful to anyone desiring to perform any of the nucleotide homology-dependent shuffling methods.

Hydrophosphination of Bicyclo[1.1.0]butane-1-carbonitriles.

Hydrophosphination of bicyclo[1.1.0]butyl nitriles with phosphine boranes and phosphites provided novel cyclobutyl-P derivatives. The reaction generally favors the syn-diastereomer, and the nitrile can be reduced and converted to other functional groups, thus enabling the preparation of bidentate ligands that access new conformational space by virtue of their attachment to the torsionally malleable but sterically restrictive cyclobutane scaffold. The enantioselective hydrogenation of dehydrophenylalanine using a bidentate phosphine-phosphite ligand illustrates the synthetic utility of the newly prepared scaffold.

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.