Clinical phenotypic spectrum of CTNNB1 neurodevelopmental disorder.

Pathogenic heterozygous loss of function variants in CTNNB1 are associated with CTNNB1 neurodevelopmental disorder. We report the clinical phenotype of individuals with CTNNB1 neurodevelopmental disorder using both caregiver-reported data (medical history, adaptive function, quality of life, and behavior issues) and in-person clinical assessments (neurological, motor, and cognitive function) in 32 individuals with likely pathogenic or pathogenic CTNNB1 variants. Most individuals had truncal hypotonia, muscle weakness, hypertonia, dystonia, microcephaly, and many had a history of tethered cord. Visual problems included strabismus, hyperopia, and familial exudative vitreoretinopathy. Half of individuals walked without an assistive device. The mean Gross Motor Functional Measure-66 score was 56.6 (SD = 14.8). Average time to complete Nine-Hole Peg Test was slower than norms. Mean general conceptual ability composite scores from Differential Ability Scales Second Edition were very low (M = 58.3, SD = 11.3). Fifty-five percent of individuals had low adaptive functioning based on the Vineland Adaptive Behavioral Scales. Based upon the Child Behavior Checklist total problems score, the majority (65%) of individuals had behavioral challenges. The mean overall Quality of Life Inventory-Disability score was 81.7 (SD = 11.9). These data provide a detailed characterization of clinical features in individuals with CTNNB1 neurodevelopmental disorder.

Metachronous colorectal cancer metastasis: Who, what, when and what to do about it.

Metachronous colorectal cancer (CRC) metastasis occurs due to micrometastatic disease, in up to 23% of patients who have undergone curative-intent treatment. Metachronous metastasis tends to occur within 2 years of initial treatment. Diagnosis relies on posttreatment surveillance strategies. Care for patients with metachronous CRC metastasis is complex and requires careful multidisciplinary consideration. Those with isolated and technically resectable diseases are recommended to undergo metastasectomy with adjunct chemotherapy, however, survival, even after curative-intent resection, is poor.

Ultra-clean pure shift NMR with optimal water suppression for analysis of aqueous pharmaceutical samples.

Pure shift NMR experiments greatly enhance spectral resolution by collapsing multiplet structures into singlets and, with water suppression, can be used for aqueous samples. Here, we combine ultra-clean pure-shift NMR (SAPPHIRE) with two different internally encoded water suppression schemes to achieve optimal performance for small molecule and macrocyclic peptide pharmaceuticals in water and acetonitrile-water mixtures.

Optimising multiplex immunofluorescence staining for characterising the tumour immune micro-environment.

Exploring the tumour microenvironment provides insight into the unique interaction between the host and tumour. Ultimately, its study improves understanding of how an individual mounts and achieves an anti-tumour immune response. In the context of colorectal cancer, immune biomarkers within the tumour microenvironment outperform traditional histopathological staging in predicting disease recurrence. Multiplex immunofluorescence enables simultaneous assessment of multiple markers to provide a highly accurate classification of immune cells and their spatial characterisation relative to tumour tissue. Further, automated slide staining provides staining consistency and reduces labour costs. Image acquisition using a non-spectral scanner allows more researchers to utilise multiplexed immunofluorescence for translational research. Herein we describe the optimisation process of conducting automated staining using a five-colour, tyramide signal amplification-based multiplex immunofluorescence panel. Using antibodies against CD3, CD8, CD103 and cytokeratin, the panel characterises T cell populations within human colorectal adenocarcinoma tissue. We provide an overview of primary antibody titration and the development of tyramide signal amplification immunofluorescence monoplex assays. We detail the processes of antibody stripping and the role of exogenous horseradish peroxidase inhibition to facilitate multiplexing. An account of determining the staining sequence and fluorophore assignment is provided. We describe image acquisition using a standard fluorescence microscope slide scanner and the management of spectral crosstalk using this system. Finally, we briefly document the digital image analysis required to characterise cells and determine their spatial distribution within the colorectal tumour microenvironment.

Quantitative nuclear magnetic resonance of chloride by an accurate internal standard approach.

Chloride is the most common counterion used to improve aqueous solubility and enhance stability of small molecule active pharmaceutical ingredients. While several analytical techniques, such as titration, HPLC with charged aerosol detection, and ion chromatography, are currently utilized to assay the level of chloride, they have notable limitations, and these instruments may not be readily available. Here, we present a generally applicable 35 Cl solution NMR method to assay the level of chloride in pharmaceutical compounds. The method uses KClO4 as an internal standard for improved accuracy in comparison with external standard methods, and it was found to be robust, linear over three orders of magnitude, precise (<3% RSD), and accurate (<0.5% absolute error).

DELTA50: A Highly Accurate Database of Experimental 1H and 13C NMR Chemical Shifts Applied to DFT Benchmarking.

Density functional theory (DFT) benchmark studies of 1H and 13C NMR chemical shifts often yield differing conclusions, likely due to non-optimal test molecules and non-standardized data acquisition. To address this issue, we carefully selected and measured 1H and 13C NMR chemical shifts for 50 structurally diverse small organic molecules containing atoms from only the first two rows of the periodic table. Our NMR dataset, DELTA50, was used to calculate linear scaling factors and to evaluate the accuracy of 73 density functionals, 40 basis sets, 3 solvent models, and 3 gauge-referencing schemes. The best performing DFT methodologies for 1H and 13C NMR chemical shift predictions were WP04/6-311++G(2d,p) and ωB97X-D/def2-SVP, respectively, when combined with the polarizable continuum solvent model (PCM) and gauge-independent atomic orbital (GIAO) method. Geometries should be optimized at the B3LYP-D3/6-311G(d,p) level including the PCM solvent model for the best accuracy. Predictions of 20 organic compounds and natural products from a separate probe set had root-mean-square deviations (RMSD) of 0.07 to 0.19 for 1H and 0.5 to 2.9 for 13C. Maximum deviations were less than 0.5 and 6.5 ppm for 1H and 13C, respectively.

Exploring Coronavirus Disease 2019 Vaccine Hesitancy on Twitter Using Sentiment Analysis and Natural Language Processing Algorithms.

BACKGROUND: Vaccination can help control the coronavirus disease 2019 (COVID-19) pandemic but is undermined by vaccine hesitancy. Social media disseminates information and misinformation regarding vaccination. Tracking and analyzing social media vaccine sentiment could better prepare health professionals for vaccination conversations and campaigns. METHODS: A real-time big data analytics framework was developed using natural language processing sentiment analysis, a form of artificial intelligence. The framework ingests, processes, and analyzes tweets for sentiment and content themes, such as natural health or personal freedom, in real time. A later dataset evaluated the relationship between Twitter sentiment scores and vaccination rates in the United States. RESULTS: The real-time analytics framework showed a widening gap in sentiment with more negative sentiment after vaccine rollout. After rollout, using a static dataset, an increase in positive sentiment was followed by an increase in vaccination. Lag cross-correlation analysis across US regions showed evidence that once all adults were eligible for vaccination, the sentiment score consistently correlated with vaccination rate with a lag of around 1 week. The Granger causality test further demonstrated that tweet sentiment scores may help predict vaccination rates. CONCLUSIONS: Social media has influenced the COVID-19 response through valuable information and misinformation and distrust. This tool was used to collect and analyze tweets at scale in real time to study sentiment and key terms of interest. Separate tweet analysis showed that vaccination rates tracked regionally with Twitter vaccine sentiment and might forecast changes in vaccine uptake and/or guide targeted social media and vaccination strategies. Further work is needed to analyze the interplay between specific populations, vaccine sentiment, and vaccination rates.

Deuterated Modifiers in Sub/Supercritical Fluid Chromatography for Streamlined NMR Structure Elucidation.

Isolation and chemical characterization of target components in fast-paced pharmaceutical laboratories can often be challenging, especially when dealing with mixtures of closely related, possibly unstable species. Traditionally, this process involves intense labor and manual intervention including chromatographic method development and optimization, fraction collection, and drying processes prior to NMR analyses for unambiguous structure elucidation. To circumvent these challenges, a foundational framework for the proper utilization of supercritical carbon dioxide (scCO2) and deuterated modifiers (CD3OD) in sub/supercritical fluid chromatography (SFC) is herein introduced. This facilitates a streamlined multicomponent isolation with minimized protic residues, further enabling immediate NMR analysis. In addition to bypassing tedious drying processes and minimizing analyte degradation, this approach (complementary to traditional reversed-phase liquid chromatography, RPLC) delivers highly efficient separations and automated fraction collection using readily available analytical/midscale SFC instrumentation. A series of diverse analytes across a wide spectrum of chemical properties (acid, basic, and neutral), combined with different stationary-phase columns in SFC are investigated using both a protic organic modifier (CH3OH) and its deuterated counterpart (CD3OD). The power of this framework is demonstrated with pharmaceutically relevant applications in the context of target characterization and analysis of complex multicomponent reaction mixtures from modern synthetic chemistry, demonstrating high isolation yields while reducing both the environmental footprint and manual intervention. This workflow enables unambiguous fast-paced structure elucidation on the analytical scale, providing results that are comparable to traditional, but time-consuming, RPLC purification approaches.

Selective Plate-Based Assay for Trace EDTA Analysis via Boron Trifluoride-methanol Derivatization UHPLC-QqQ-MS/MS Enabling Biologic and Vaccine Processes.

The employment of ethylenediaminetetraacetic acid (EDTA) across several fields in chemistry and biology has required the creation of a high number of quantitative assays. Nonetheless, the determination of trace EDTA, especially in biologics and vaccines, remains challenging. Herein, we introduce an automated high-throughput approach based on EDTA esterification in 96-well plates using boron trifluoride-methanol combined with rapid analysis by ultra-high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Derivatization of EDTA to its methyl ester (Me-EDTA) serves to significantly improve chromatographic performance (retention, peak shape, and selectivity), while also delivering a tremendous enhancement of sensitivity in the positive ion mode electrospray ionization (ESI+). This procedure, in contrast to previous EDTA methods based on complexation with metal ions, is not affected by high concentration of other metals, buffers, and related salts abundantly present in biopharmaceutical processes (e.g., iron, copper, citrate, etc.). Validation of this assay for the determination of ng·mL-1 level EDTA in monoclonal antibody and vaccine products demonstrated excellent performance (repeatability, precision, and linear range) with high recovery from small sample volumes while also providing an advantageous automation-friendly workflow for high-throughput analysis.

Synthesis of L-cyclic tetrapeptides by backbone amide activation CyClick strategy.

Cyclic tetrapeptides exhibit high cellular permeability and a wide range of biological properties and thus have gained great interest in the field of medicinal chemistry. We synthesized highly strained 12-membered head to tail cyclic peptides with varying reactive amino acids, without oligomerization using the exclusively intramolecular CyClick chemistry. This occurs by a two-step process involving the low-energy formation of a 15 atom-containing cyclic imine, followed by a chemoselective ring contraction of the peptide backbone generating a highly strained 12 atom-containing cyclic tetrapeptide. This reaction exhibited high substrate scope and generated head to tail cyclic tetrapeptides with varying amino acids at the N-terminus, showing chemoselectivity without the need for side group protection.

Application of 1,1-ADEQUATE and DFT to correct 13 C misassignments of carbonyl chemical shifts for carbapenem antibiotics.

Prior to the development of sensitive proton-detected 2D NMR experiments, assigning 13 C signals could be a significant challenge, and mistakes have occurred even for prominent compound classes. In this study, 1,1-ADEQUATE data were used to unambiguously reassign the 13 C chemical shifts for the ß-lactam carbonyl at the C-7 position and the proximal carboxylate at the C-10 position of the carbapenems, meropenem and imipenem. Density functional theory (DFT) was then investigated to provide sufficiently accurate 13 C chemical shift predictions, allowing for the carbonyl signal reassignment of thienamycin.

Trapping-Enrichment Multi-dimensional Liquid Chromatography with On-Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale.

At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE-Dt-mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension (1 D-UV); 2) automated peak trapping-enrichment and buffer removal achieved through a sequence of H2 O and D2 O washes using an independent pump setup; and 3) a second dimension separation (2 D-UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90 % using as little as a few micrograms of material.

Photoenzymatic Synthesis of α-Tertiary Amines by Engineered Flavin-Dependent "Ene"-Reductases.

α-Tertiary amines are a common motif in pharmaceutically important molecules but are challenging to prepare using asymmetric catalysis. Here, we demonstrate engineered flavin-dependent 'ene'-reductases (EREDs) can catalyze radical additions into oximes to prepare this motif. Two different EREDs were evolved into competent catalysts for this transformation with high levels of stereoselectivity. Mechanistic studies indicate that the oxime contributes to the enzyme templated charge-transfer complex formed between the substrate and cofactor. These products can be further derivatized to prepare a variety of motifs, highlighting the versatility of ERED photoenzymatic catalysis for organic synthesis.

A Multifunctional Reagent Designed for the Site-Selective Amination of Pyridines.

We report the development of a multifunctional reagent for the direct conversion of pyridines to Boc-protected 2-aminopyridines with exquisite site selectivity and chemoselectivity. The novel reagent was prepared on 200-g scale in a single step, reacts in the title reaction under mild conditions without precautions toward air or moisture, and is tolerant of nearly all common functionality. Experimental and in situ spectroscopic monitoring techniques provide detailed insights and unexpected findings for the unique reaction mechanism.

Primary local excision of stage 1 rectal cancer is not associated with worse oncological outcomes when compared with major resection.

PURPOSE: Primary local excision (PLE) for early rectal cancers is associated with decreased surgical morbidity and mortality compared with major resection (MR). However, it is thought to be associated with poorer oncological outcomes. There is a paucity of data regarding PLE within the Australasian population. We present comparative post-operative and survival outcomes for stage 1 rectal cancers treated with PLE or MR from three Western Australian hospitals. METHODS: A retrospective analysis was performed on a prospectively maintained database of patients undergoing PLE or MR for stage 1 rectal cancers between February 1996 and May 2019. RESULTS: Of the 533 patients, 81 underwent PLE. Median post-operative admission was shorter for those undergoing PLE, with no significant difference in post-operative complication rate. Five-year overall survival was greater following MR (89.6% CI 86.1-92.3) compared with PLE (84.6% CI 73.8-91.2; p = 0.0003). There was no significant difference in 5-year cancer-specific survival (MR, 94.4% CI 91.5-96.3; PLE, 95.3% CI 86.0-98.5; p = 0.98) or 5-year disease-free survival (MR, 92.3% CI 89.1-94.7; PLE, 89.1% CI 78.5-94.7; p = 0.36). Local excision provided poorer local tumour control with an inferior 5-year local recurrence rate (MR, 2.16% CI 1.08-4.28; PLE, 10.9% CI 5.30-21.6; p = 0.0002). After controlling for confounders, PLE was significantly associated with worse local recurrence but did not significantly impact overall survival, cancer-specific survival, overall recurrence, or metastatic recurrence. CONCLUSION: Local excision of early rectal cancer remains a viable alternative, in those unwilling or unable to undergo MR. Patients should be informed that while PLE is associated with poorer local pelvic control, this does not translate to worse survival.

Rupture of visceral artery aneurysm following elective spinal surgery: a case report and review.

INTRODUCTION: Visceral artery aneurysms (VAA), an uncommon disorder of splanchnic vasculature, are associated with significant morbidity and mortality. Despite largely being amenable to endovascular therapies, the initial management of symptomatic VAA typically falls under the care of general surgeons. It is thus essential to have knowledge of the diagnosis and treatment of VAA and to be cognisant of deviations from normal gastrointestinal vasculature. CASE PRESENTATION: In this paper, we describe the case of a 72-year-old male presenting with a VAA following elective spinal surgery, followed by a review of the clinical diagnosis and management of VAA. CONCLUSION: Visceral artery aneurysm must be considered as a differential diagnosis for acute abdominal pain and anaemia in the post-operative period following all major operations. Knowledge of the clinical features of VAA and indications for specific intervention are essential for all general surgeons. Furthermore, it is imperative to recognise deviations from normal vasculature of the gastrointestinal tract.

Highly Diastereoselective Synthesis of a HCV NS5B Nucleoside Polymerase Inhibitor.

An asymmetric synthesis of HCV NS5B nucleoside polymerase inhibitor (1) is described. This novel route features several remarkably diastereoselective and high-yielding transformations, including construction of the all-carbon quaternary stereogenic center at C-2 via a thermodynamic aldol reaction. A subsequent glycosylation reaction with activated uracil via C-1 phosphate and installation of the cyclic phosphate group using an achiral phosphorus(III) reagent followed by oxidation provides 1.

CyClick Chemistry for the Synthesis of Cyclic Peptides.

Here, we report a novel "CyClick" strategy for the macrocyclization of peptides that works in an exclusively intramolecular fashion thereby precluding the formation of dimers and oligomers via intermolecular reactions. The CyClick chemistry is highly chemoselective for the N-terminus of the peptide with a C-terminal aldehyde. In this protocol, the peptide conformation internally directs activation of the backbone amide bond and thereby facilitates formation of a stable 4-imidazolidinone-fused cyclic peptide with high diastereoselectivity (>99 %). This method is tolerant to a variety of peptide aldehydes and has been applied for the synthesis of 12- to 23-membered rings with varying amino acid compositions in one pot under mild reaction conditions. The reaction generated peptide macrocycles featuring a 4-imidazolidinone in their scaffolds, which acts as an endocyclic control element that promotes intramolecular hydrogen bonding and leads to macrocycles with conformationally rigid turn structures.

Discovery of a Photoinduced Dark Catalytic Cycle Using in Situ LED-NMR Spectroscopy.

We report the use of LED-NMR spectroscopy to study the reaction mechanism of a newly discovered photoinduced iron-catalyzed cycloisomerization of alkynols to cyclic enol ethers. By understanding on/off ligand binding to the catalyst, we were able to appropriately design reaction conditions to balance catalyst activity and stability. LED-NMR was demonstrated to be a powerful tool in elucidating reaction mechanisms of photochemical reactions. Temporal NMR spectroscopic data under visible light illumination (1) revealed the pre-catalyst activation mechanism, (2) proved that photon flux provides a unique external control of the equilibrium distribution between the pre-catalyst and active catalyst, and ultimately the rate of reaction, (3) provided information about the reaction driving forces and the turnover-limiting step, and (4) enabled both real-time structural and kinetic insights into elusive species (e.g., dissolved gases).