Use of Ultrasound in the Evaluation of Cryptorchidism: A Single-Institution Analysis.

INTRODUCTION: The American Urological Association guidelines recommend against the performance of ultrasound and other imaging modalities in the evaluation of patients with cryptorchidism before expert consultation. We aimed to examine our institutional experience with cryptorchidism and measure adherence to currently available guidelines. METHODS: An institutional review board-approved retrospective review of ultrasound utilization in the evaluation of patients with cryptorchidism was performed from June 1, 2016, to June 30, 2019, at a single tertiary level pediatric hospital. RESULTS: We identified 1796 patients evaluated in surgical clinics for cryptorchidism. Surgical intervention was performed in 75.2% (n = 1351) of the entire cohort. Ultrasound was performed in 42% (n = 754), most of which were ordered by referring physicians (91% n = 686). Of those who received an ultrasound, surgical intervention was performed in 78% (n = 588). Those 166 patients (22%) who did not undergo surgical intervention were referred with ultrasounds suggesting inguinal testes; however, all had normal physical examinations or mildly retractile testes at the time of consultation and were discharged from the outpatient clinic. There were 597 patients referred without an ultrasound, 81% (n = 483) were confirmed to have cryptorchidism at the time of specialist physical examination and underwent definitive surgical intervention, the remainder (19%, n = 114) were discharged from the outpatient clinics. CONCLUSIONS: Ultrasound evaluation of cryptorchidism continues despite high-quality evidence-based guidelines that recommend otherwise, as they should have little to no bearing on the surgeon's decision to operate or the type of operation. Instead, physical examination findings should guide surgical planning.

Structure is beauty, but not always truth.

Structural biology, as powerful as it is, can be misleading. We highlight four fundamental challenges: interpreting raw experimental data; accounting for motion; addressing the misleading nature of in vitro structures; and unraveling interactions between drugs and "anti-targets." Overcoming these challenges will amplify the impact of structural biology on drug discovery.

Recommendations for accelerating open preprint peer review to improve the culture of science.

Peer review is an important part of the scientific process, but traditional peer review at journals is coming under increased scrutiny for its inefficiency and lack of transparency. As preprints become more widely used and accepted, they raise the possibility of rethinking the peer-review process. Preprints are enabling new forms of peer review that have the potential to be more thorough, inclusive, and collegial than traditional journal peer review, and to thus fundamentally shift the culture of peer review toward constructive collaboration. In this Consensus View, we make a call to action to stakeholders in the community to accelerate the growing momentum of preprint sharing and provide recommendations to empower researchers to provide open and constructive peer review for preprints.

Artifact suppression and improved signal-to-noise ratio by phase-locked multiplexed coherent imaging.

Laser additive manufacturing (AM) promises direct metal 3D printing, but is held back by defects and process instabilities, giving rise to a need for in situ process monitoring. Inline coherent imaging (ICI) has proven effective for in situ, direct measurements of vapor depression depth and shape in AM and laser welding but struggles to track turbulent interfaces due to poor coupling back into a single-mode fiber and the presence of artifacts. By z-domain multiplexing, we achieve phase-sensitive image consolidation, automatically attenuating autocorrelation artifacts and improving interface tracking rates by 58% in signal-starved applications.

A national survey of current practice on patients with long term and additional needs in paediatric intensive care units.

BACKGROUND: Patients with long term and additional needs (LEAP) in paediatric intensive care units (PICUs) are a growing and heterogenous cohort that provide unique challenges to clinicians. Currently no standard approach to define and manage this cohort exists. AIM: To analyse bed occupancy, examine current practice, and explore ideas to improve PICU care of patients with long term and additional needs. STUDY DESIGN: Patients with LEAP were defined as meeting two or more of the following criteria: length of stay >14 days; life limiting condition; ≥2 failed extubations; hospital stay >1 month prior to PICU admission; likely to require long-term ventilation. An electronic survey was then sent to all UK PICUs, via the UK Paediatric Critical Care Society, to collect quantitative and qualitative data relating to bed occupancy, length of stay, multidisciplinary and family involvement, and areas of possible improvement. Data collection were occurred between 8 February 2022 and 14 March 2022. Quantitative data were analysed using Microsoft Excel 365 and SPSS Statistics version 28.0. Raw data and descriptive statistics were reported, including percentages and median with interquartile range for non-parametric data. Qualitative raw data were examined using thematic analysis. Analysis was undertaken independently by two authors and results assessed for concordance. RESULTS: 70.1% (17/24) PICUs responded. 25% (67/259) of PICU beds were occupied by patients with long term and additional needs. 29% (5/17) of responding units have tailored management plans to this cohort of patient. A further 11% (2/17) have guidelines for children with generic chronic illness. 12% (2/16) of responding units had a designated area and 81% (13/16) of responding units had designated professionals. The majority (68% and 62%) of responding units engaged families and community professionals in multidisciplinary meetings. When asked how the care of long term and additional needs patients might be improved five themes were identified: consistent, streamlined care pathways; designated transitional care units; designated funding and hospital-to-home commissioning; development of roles to facilitate collaboration between hospital and community teams; proactive discharge planning and parallel planning. CONCLUSIONS: This survey provides a snapshot of UK practice for a cohort of patients that occupies a considerable proportion (29%) of PICU beds. While only a minority of responding PICUs offer specifically tailored management plans, the majority of units have designated professionals. RELEVANCE TO CLINICAL PRACTICE: Opportunities exist to improve PICU care in LEAP patients in areas such as: streamlined care pathways, designated clinical areas, designated funding, and development of defined collaborative roles. Next steps may involve working group convention to develop a consensus definition and share good practice examples.

External second opinions: building trust between health professionals and families.

In medicine, external second opinions are frequently sought to inform decisions around a patient's proposed course of treatment. However, they are also sought in more challenging circumstances such as when disagreement arises between the healthcare team and the family, or during complex end-of-life discussions in critically ill children. When done well, external second opinions can help build trust and reduce conflict. However, when done poorly they may antagonise relationships and thwart attempts to bring about consensus. While principles of good medical practice should always be followed, the actual second opinion process itself remains essentially unregulated in all its forms. In this review, we set out what a standardised and transparent second opinion process should look like and recommend key recommendations for healthcare Trusts, Commissioners and professional bodies to support good practice.

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Uncovering Protein Ensembles: Automated Multiconformer Model Building for X-ray Crystallography and Cryo-EM.

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift towards modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g. Coot) and fit can be further improved by refinement using standard pipelines (e.g. Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

Structure-activity relationships of bensulfuron methyl and its derivatives as novel agents against drug-resistant Candida auris.

With the emergence of the human pathogen Candida auris as a threat to human health, there is a strong demand to identify effective medicines to prevent the harm caused by such drug-tolerant human fungi. Herein, a series of 33 new derivatives of bensulfuron methyl (BSM) were synthesized and characterized by 1 H NMR, 13 C NMR, and HRMS. Among the target compounds, 8a possessed the best Ki value of 1.015 µM against C. auris acetohydroxyacid synthase (CauAHAS) and an MIC value of 6.25 µM against CBS10913, a clinically isolated strain of C. auris. Taken together the structures of BSM and the synthesized compounds, it was found that methoxy groups at both meta-position of pyrimidine ring are likely to provide desirable antifungal activities. Quantum calculations and molecular dockings were performed to understand the structure-activity relationships. The present study has hence provided some interesting clues for the discovery of novel antibiotics with this distinct mode of action.

Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia.

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor-positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor-positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. SIGNIFICANCE: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 227 . This article is featured in Selected Articles from This Issue, p. 201.

Determining the Optimal Technique for Bar Fixation in the Repair of Pectus Excavatum.

Introduction: Pectus bar stabilizers are routinely used for bar fixation in the repair of pectus excavatum. We aimed to determine the optimum technique for bar fixation by reviewing our institutional experience with the use of bilateral, unilateral, and no stabilizer placement. Methods: Retrospective single pediatric center review of patients who underwent minimally invasive bar placement for pectus excavatum and subsequent bar removal between December 2001 and July 2019 was performed. Demographic data, details about the surgery, the number of bars and stabilizers used, and follow-up information were collected. Stabilizer-related complications included pain requiring stabilizer removal, surgical site infections (SSIs), and bar displacement. Data are presented as medians with interquartile ranges (IQRs) and frequencies with percentages. Results: A total of 561 patients were included. The cohort was predominantly male (83.1%, n = 466) with a median age at the time of bar placement of 15 years (IQR 12.4, 16.3) and a median Haller index of 3.8 (IQR 3.4, 4.5). Pain attributed to the stabilizer site that required removal was observed only in the bilateral stabilizer group (2.5%, n = 13). SSI related to the stabilizer site occurred in 1.8% (n = 9) of the bilateral stabilizer cases and 2.1% (n = 1) of the unilateral stabilizer cases. Bar displacement was observed in 0.6% (n = 3) of the bilateral stabilizer cases and 2 of those patients also had an SSI. There were no complications in the no stabilizer group. Conclusion: As the trend moves toward unilateral and no stabilizer use, we observe fewer cases of pain requiring stabilizer removal with no increase in bar displacements.

SAGA Complex Subunit Hfi1 Is Important in the Stress Response and Pathogenesis of Cryptococcus neoformans.

The Spt-Ada-Gcn Acetyltransferase (SAGA) complex is a highly conserved co-activator found across eukaryotes. It is composed of a number of modules which can vary between species, but all contain the core module. Hfi1 (known as TADA1 in Homo sapiens) is one of the proteins that forms the core module, and has been shown to play an important role in maintaining complex structural integrity in both brewer's yeast and humans. In this study we successfully identified the gene encoding this protein in the important fungal pathogen, Cryptococcus neoformans, and named it HFI1. The hfi1Δ mutant is highly pleiotropic in vitro, influencing phenotypes, ranging from temperature sensitivity and melanin production to caffeine resistance and titan cell morphogenesis. In the absence of Hfi1, the transcription of several other SAGA genes is impacted, as is the acetylation and deubiquination of several histone residues. Importantly, loss of the gene significantly impacts virulence in a murine inhalation model of cryptococcosis. In summary, we have established that Hfi1 modulates multiple pathways that directly affect virulence and survival in C. neoformans, and provided deeper insight into the importance of the non-enzymatic components of the SAGA complex.

MET variants with activating N-lobe mutations identified in hereditary papillary renal cell carcinomas still require ligand stimulation.

In hereditary papillary renal cell carcinoma (HPRCC), the MET receptor tyrosine kinase (RTK) mutations recorded to date are located in the kinase domain and lead to constitutive MET activation. This contrasts with MET mutations recently identified in non-small cell lung cancer (NSCLC), which lead to exon 14 skipping and deletion of a regulatory domain: in this latter case, the mutated receptor still requires ligand stimulation. Sequencing of MET in samples from 158 HPRCC and 2808 NSCLC patients revealed ten uncharacterized mutations. Four of these, all found in HPRCC and leading to amino acid substitutions in the N-lobe of the MET kinase, proved able to induce cell transformation, further enhanced by HGF stimulation: His1086Leu, Ile1102Thr, Leu1130Ser, and Cis1125Gly. Similar to the variant resulting in MET exon14 skipping, the two N-lobe MET variants His1086Leu, Ile1102Thr further characterized were found to require stimulation by HGF in order to strongly activate downstream signaling pathways and epithelial cell motility. The Ile1102Thr mutation displayed also transforming potential, promoting tumor growth in a xenograft model. In addition, the N-lobe-mutated MET variants were found to trigger a common HGF-stimulation-dependent transcriptional program, consistent with an observed increase in cell motility and invasion. Altogether, this functional characterization revealed that N-lobe variants still require ligand stimulation, in contrast to other RTK variants. This suggests that HGF expression in the tumor microenvironment is important for tumor growth. The sensitivity of these variants to MET TKIs opens the way for use of targeted therapies for patients harboring the corresponding mutations.

Refinement of multiconformer ensemble models from multi-temperature X-ray diffraction data.

Conformational ensembles underlie all protein functions. Thus, acquiring atomic-level ensemble models that accurately represent conformational heterogeneity is vital to deepen our understanding of how proteins work. Modeling ensemble information from X-ray diffraction data has been challenging, as traditional cryo-crystallography restricts conformational variability while minimizing radiation damage. Recent advances have enabled the collection of high quality diffraction data at ambient temperatures, revealing innate conformational heterogeneity and temperature-driven changes. Here, we used diffraction datasets for Proteinase K collected at temperatures ranging from 313 to 363 K to provide a tutorial for the refinement of multiconformer ensemble models. Integrating automated sampling and refinement tools with manual adjustments, we obtained multiconformer models that describe alternative backbone and sidechain conformations, their relative occupancies, and interconnections between conformers. Our models revealed extensive and diverse conformational changes across temperature, including increased bound peptide ligand occupancies, different Ca2+ binding site configurations and altered rotameric distributions. These insights emphasize the value and need for multiconformer model refinement to extract ensemble information from diffraction data and to understand ensemble-function relationships.

Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography.

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

A type 2 immune circuit in the stomach controls mammalian adaptation to dietary chitin.

Dietary fiber improves metabolic health, but host-encoded mechanisms for digesting fibrous polysaccharides are unclear. In this work, we describe a mammalian adaptation to dietary chitin that is coordinated by gastric innate immune activation and acidic mammalian chitinase (AMCase). Chitin consumption causes gastric distension and cytokine production by stomach tuft cells and group 2 innate lymphoid cells (ILC2s) in mice, which drives the expansion of AMCase-expressing zymogenic chief cells that facilitate chitin digestion. Although chitin influences gut microbial composition, ILC2-mediated tissue adaptation and gastrointestinal responses are preserved in germ-free mice. In the absence of AMCase, sustained chitin intake leads to heightened basal type 2 immunity, reduced adiposity, and resistance to obesity. These data define an endogenous metabolic circuit that enables nutrient extraction from an insoluble dietary constituent by enhancing digestive function.

Conserved regulatory motifs in the juxtamembrane domain and kinase N-lobe revealed through deep mutational scanning of the MET receptor tyrosine kinase domain.

MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of MET intracellular kinase domain in two fusion protein backgrounds: wild type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase αC helix, pointing to differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a ß5 motif that acts as a structural pivot for kinase domain activation in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain.

Chemoenzymatic Syntheses of Fluorine-18-Labeled Disaccharides from [18F] FDG Yield Potent Sensors of Living Bacteria In Vivo.

Chemoenzymatic techniques have been applied extensively to pharmaceutical development, most effectively when routine synthetic methods fail. The regioselective and stereoselective construction of structurally complex glycans is an elegant application of this approach that is seldom applied to positron emission tomography (PET) tracers. We sought a method to dimerize 2-deoxy-[18F]-fluoro-d-glucose ([18F]FDG), the most common tracer used in clinical imaging, to form [18F]-labeled disaccharides for detecting microorganisms in vivo based on their bacteria-specific glycan incorporation. When [18F]FDG was reacted with ß-d-glucose-1-phosphate in the presence of maltose phosphorylase, the α-1,4- and α-1,3-linked products 2-deoxy-[18F]-fluoro-maltose ([18F]FDM) and 2-deoxy-2-[18F]-fluoro-sakebiose ([18F]FSK) were obtained. This method was further extended with the use of trehalose (α,α-1,1), laminaribiose (ß-1,3), and cellobiose (ß-1,4) phosphorylases to synthesize 2-deoxy-2-[18F]fluoro-trehalose ([18F]FDT), 2-deoxy-2-[18F]fluoro-laminaribiose ([18F]FDL), and 2-deoxy-2-[18F]fluoro-cellobiose ([18F]FDC). We subsequently tested [18F]FDM and [18F]FSK in vitro, showing accumulation by several clinically relevant pathogens including Staphylococcus aureus and Acinetobacter baumannii, and demonstrated their specific uptake in vivo. Both [18F]FDM and [18F]FSK were stable in human serum with high accumulation in preclinical infection models. The synthetic ease and high sensitivity of [18F]FDM and [18F]FSK to S. aureus including methicillin-resistant (MRSA) strains strongly justify clinical translation of these tracers to infected patients. Furthermore, this work suggests that chemoenzymatic radiosyntheses of complex [18F]FDG-derived oligomers will afford a wide array of PET radiotracers for infectious and oncologic applications.