gp130 functions as a shared signal-transducing subunit not only for interleukin (IL)-6 but also for eight other human cytokine receptor complexes. The IL-6 signaling pathway mediated through gp130 encompasses classical, trans, or cluster signaling, intricately regulated by a diverse array of modulators affecting IL-6, its receptor, and gp130. Currently, only a limited number of small molecule antagonists and agonists for gp130 are known. This review aims to comprehensively examine the current knowledge of these modulators and provide insights into their pharmacological properties, particularly in the context of cancer and other diseases. Notably, the prominent gp130 modulators SC144, bazedoxifene, and raloxifene are discussed in detail, with a specific focus on the discovery of SC144’s iron-chelating properties. This adds a new dimension to the understanding of its pharmacological effects and therapeutic potential in conditions where iron homeostasis is significant. Our bioinformatic analysis of gp130 and genes related to iron homeostasis reveals insightful correlations, implicating the role of iron in the gp130 signaling pathway. Overall, this review contributes to the evolving understanding of gp130 modulation and its potential therapeutic applications in various disease contexts.

Over 4 decades of research support the link between Alzheimer disease (AD) and somatostatin [somatotropin-releasing inhibitory factor (SRIF)]. SRIF and SRIF-expressing neurons play an essential role in brain function, modulating hippocampal activity and memory formation. Loss of SRIF and SRIF-expressing neurons in the brain rests at the center of a series of interdependent pathological events driven by amyloid-β peptide (Aβ), culminating in cognitive decline and dementia. The connection between the SRIF and AD further extends to the neuropsychiatric symptoms, seizure activity, and inflammation, whereas preclinical AD investigations show SRIF or SRIF receptor agonist administration capable of enhancing cognition. SRIF receptor subtype-4 activation in particular presents unique attributes, with the potential to mitigate learning and memory decline, reduce comorbid symptoms, and enhance enzymatic degradation of Aβ in the brain. Here, we review the links between SRIF and AD along with the therapeutic implications.

Bile acids are the end products of cholesterol catabolism. Hepatic bile acid synthesis accounts for a major fraction of daily cholesterol turnover in humans. Biliary secretion of bile acids generates bile flow and facilitates biliary secretion of lipids, endogenous metabolites, and xenobiotics. In intestine, bile acids facilitate the digestion and absorption of dietary lipids and fat-soluble vitamins. Through activation of nuclear receptors and G protein-coupled receptors and interaction with gut microbiome, bile acids critically regulate host metabolism and innate and adaptive immunity and are involved in the pathogenesis of cholestasis, metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, type-2 diabetes, and inflammatory bowel diseases. Bile acids and their derivatives have been developed as potential therapeutic agents for treating chronic metabolic and inflammatory liver diseases and gastrointestinal disorders.

Hearing disorders pose significant challenges to individuals experiencing them and their overall quality of life, emphasizing the critical need for advanced pharmacological approaches to address these conditions. Current treatment options often focus on amplification devices, cochlear implants, or other rehabilitative therapies, leaving a substantial gap regarding effective pharmacological interventions. Advancements in our understanding of the molecular and cellular mechanisms involved in hearing disorders induced by noise, aging, and ototoxicity have opened new avenues for drug development, some of which have led to numerous clinical trials, with promising results. The development of optimal drug delivery solutions in animals and humans can also enhance the targeted delivery of medications to the ear. Moreover, large genome studies contributing to a genetic understanding of hearing loss in humans combined with advanced molecular technologies in animal studies have shown a great potential to increase our understanding of the etiologies of hearing loss. The auditory system exhibits circadian rhythms and temporal variations in its physiology, its vulnerability to auditory insults, and its responsiveness to drug treatments. The cochlear clock rhythms are under the control of the glucocorticoid system, and preclinical evidence suggests that the risk/benefit profile of hearing disorder treatments using chronopharmacological approaches would be beneficial. If translatable to the bedside, such approaches may improve the outcome of clinical trials. Ongoing research into the molecular and genetic basis of auditory disorders, coupled with advancements in drug formulation and delivery as well as optimized timing of drug administration, holds great promise of more effective treatments.

Nitric oxide (NO) from endothelial NO synthase importantly contributes to vascular homeostasis. Reduced NO production or increased scavenging during disease conditions with oxidative stress contribute to endothelial dysfunction and NO deficiency. In addition to the classical enzymatic NO synthases (NOS) system, NO can also be generated via the nitrate-nitrite-NO pathway. Dietary and pharmacological approaches aimed at increasing NO bioactivity, especially in the cardiovascular system, have been the focus of much research since the discovery of this small gaseous signaling molecule. Despite wide appreciation of the biological role of NOS/NO signaling, questions still remain about the chemical nature of NOS-derived bioactivity. Recent studies show that NO-like bioactivity can be efficiently transduced by mobile NO-ferroheme species, which can transfer between proteins, partition into a hydrophobic phase, and directly activate the soluble guanylyl cyclase-cGMP-protein kinase G pathway without intermediacy of free NO. Moreover, interaction between red blood cells and the endothelium in the regulation of vascular NO homeostasis have gained much attention, especially in conditions with cardiometabolic disease. In this review we discuss both classical and nonclassical pathways for NO generation in the cardiovascular system and how these can be modulated for therapeutic purposes.

Both preclinical and clinical studies implicate functional impairments of several neuroactive metabolites of the kynurenine pathway (KP), the major degradative cascade of the essential amino acid tryptophan in mammals, in the pathophysiology of neurologic and psychiatric diseases. A number of KP enzymes, such as tryptophan 2,3-dioxygenase (TDO2), indoleamine 2,3-dioxygenases (IDO1 and IDO2), kynurenine aminotransferases (KATs), kynurenine 3-monooxygenase (KMO), 3-hydroxyanthranilic acid oxygenase (3-HAO), and quinolinic acid phosphoribosyltransferase (QPRT), control brain KP metabolism in health and disease and are therefore increasingly considered to be promising targets for the treatment of disorders of the nervous system. Understanding the distribution, cellular expression, and regulation of KP enzymes and KP metabolites in the brain is therefore critical for the conceptualization and implementation of successful therapeutic strategies.

BACKGROUND AND PURPOSE:

Low-field 64 mT portable brain MRI has recently shown diagnostic promise for MS. This study aimed to evaluate the utility of portable MRI (pMRI) in assessing dissemination in space (DIS) in patients presenting with optic neuritis and determine whether deploying pMRI in the MS clinic can shorten the time from symptom onset to MRI.

BACKGROUND AND PURPOSE:

A cleftlike nonenhancing hypointensity was observed repeatedly in the pituitary gland at the adenohypophysis/neurohypophysis border on contrast-enhanced 3D fat-saturated T1-MPRAGE using clinical 7T MRI. Our primary goal was to assess the prevalence of this finding. The secondary goals were to evaluate the frequency of other incidental pituitary lesions, MRI artifacts, and their effect on pituitary imaging on the contrast-enhanced 3D fat-saturated T1 MPRAGE at 7T.

BACKGROUND AND PURPOSE:

Patients with brain tumors have high intersubject variation in putative language regions, which may limit the utility of straightforward application of healthy subject brain atlases in clinical scenarios. The purpose of this study was to develop a probabilistic functional brain atlas that consolidates language functional activations of sentence completion and Silent Word Generation language paradigms using a large sample of patients with brain tumors.

BACKGROUND AND PURPOSE:

Hemangioblastoma is a rare vascular tumor that occurs within the central nervous system in children. Differentiating hemangioblastoma from other posterior fossa tumors can be challenging on imaging, and preoperative diagnosis can change the neurosurgical approach. We hypothesize that a "lightbulb sign" on the arterial spin-labeling (ASL) sequence (diffuse homogeneous intense hyperperfusion within the solid component of the tumor) will provide additional imaging finding to differentiate hemangioblastoma from other posterior fossa tumors.

BACKGROUND AND PURPOSE:

Gadolinium-based contrast agents are widely used for meningioma imaging; however, concerns exist regarding their side effects, cost, and environmental impact. At the standard gadolinium dose, most meningiomas show avid contrast enhancement, suggesting that administering a smaller dose may be feasible. The purpose of this study was to evaluate the impact of a lower gadolinium dose on the differentiation between meningiomas and adjacent intracranial tissues.

BACKGROUND AND PURPOSE:

Preoperative assessment of meningioma consistency is beneficial for optimizing surgical strategy and prognosis of patients. We aim to develop a noninvasive prediction model for meningioma consistency utilizing MR elastography and DTI.

BACKGROUND AND PURPOSE:

Previous studies have reported metal accumulation and microstructure changes in deep gray nuclei (DGN) in Wilson disease (WD). However, there are limited studies that investigate whether there is metal accumulation and microstructure changes in DGN of patients with WD with normal-appearing routine MRI. This study aimed to evaluate multiparametric changes in DGN of WD and whether the findings correlate with clinical severity in patients with WD.

BACKGROUND AND PURPOSE:

There has been a distinction made in the 2001 Canadian C-Spine Rule regarding patients 65 and older and younger than 65 years of age as far as indications for cervical spine CT scanning. We sought to determine if there are differences in the symptoms, mechanisms of injury, fracture locations, and types that are still relevant in 2024.

BACKGROUND AND PURPOSE:

Periprocedural intracranial hemorrhage is one of common complications after stent placement for symptomatic intracranial atherosclerotic stenosis. This study was conducted to demonstrate predictors and long-term outcomes of periprocedural intracranial hemorrhage after stent placement for symptomatic intracranial atherosclerotic stenosis.

BACKGROUND AND PURPOSE:

Mechanical thrombectomy is a fundamental intervention for acute ischemic stroke treatment. While conventional techniques are effective, cyclic aspiration (CyA) shows potential for better recanalization rates. We aim to investigate factors affecting CyA and compare them with static aspiration (StA).

BACKGROUND AND PURPOSE:

Angioplasty and stent placement have been described as a bailout technique in individuals with failed thrombectomy. We aimed to investigate Stent retriever AssIsted Lysis (SAIL) with tirofiban before angioplasty and stent placement.

BACKGROUND AND PURPOSE:

Prediction of aneurysm instability is crucial to guide treatment decisions and to select appropriate patients with unruptured intracranial aneurysms (IAs) for preventive treatment. High-resolution 4D MR flow imaging and 3D quantification of aneurysm morphology could offer insights and new imaging markers for aneurysm instability. In this cross-sectional study, we aim to identify 4D MR flow imaging markers for aneurysm instability by relating hemodynamics in the aneurysm sac to 3D morphologic proxy parameters for aneurysm instability.

BACKGROUND AND PURPOSE:

Incidental findings on brain MRI and variations of the circle of Willis (CoW) are relatively common among the general population. Ethnic differences have been described before, but few studies have explored the prevalence of incidental intracranial cerebrovascular findings and CoW variants in the setting of a single multiethnic cohort. The purpose of this investigation was to describe both incidental cerebrovascular findings and the morphology of the CoW on high-resolution 3T TOF-MRA in a UK tri-ethnic population-based cohort and to present updated prevalence estimates and morphologic reference values.

BACKGROUND AND PURPOSE:

Intracranial vessel wall imaging is technically challenging to implement, given the simultaneous requirements of high spatial resolution, excellent blood and CSF signal suppression, and clinically acceptable gradient times. Herein, we present our preliminary findings on the evaluation of a deep learning–optimized sequence using T1-weighted imaging.

SUMMARY:

The 2021 World Health Organization Classification of Tumors of the Central Nervous System (CNS5), introduced significant changes, impacting tumors ranging from glial to ependymal neoplasms. Ependymal tumors were previously classified and graded based on histopathology, which had limited clinical and prognostic utility. The updated CNS5 classification now divides ependymomas into 10 subgroups based on anatomic location (supratentorial, posterior fossa, and spinal compartment) and genomic markers. Supratentorial tumors are defined by zinc finger translocation associated (ZFTA) (formerly v-rel avian reticuloendotheliosis viral oncogene [RELA]), or yes-associated protein 1 (YAP1) fusion; posterior fossa tumors are classified into groups A (PFA) and B (PFB), spinal ependymomas are defined by MYCN amplification. Subependymomas are present across all these anatomic compartments. The new classification kept an open category of "not elsewhere classified" or "not otherwise specified" if no pathogenic gene fusion is identified or if the molecular diagnosis is not feasible. Although there is significant overlap in the imaging findings of these tumors, a neuroradiologist needs to be familiar with updated CNS5 classification to understand tumor behavior, for example, the higher tendency for tumor recurrence along the dural flap for ZFTA fusion-positive ependymomas. On imaging, supratentorial ZFTA-fused ependymomas are preferentially located in the cerebral cortex, carrying predominant cystic components. YAP1-MAMLD1-fused ependymomas are intra- or periventricular with prominent multinodular solid components and have significantly better prognosis than ZFTA-fused counterparts. PFA ependymomas are aggressive paramedian masses with frequent calcification, seen in young children, originating from the lateral part of the fourth ventricular roof. PFB ependymomas are usually midline, noncalcified solid-cystic masses seen in adolescents and young adults arising from the fourth ventricular floor. PFA has a poorer prognosis, higher recurrence, and higher metastatic rate than PFB. Myxopapillary spinal ependymomas are now considered grade II due to high recurrence rates. Spinal-MYCN ependymomas are aggressive tumors with frequent leptomeningeal spread, relapse, and poor prognosis. Subependymomas are noninvasive, intraventricular, slow-growing benign tumors with an excellent prognosis. Currently, the molecular classification does not enhance the clinicopathologic understanding of subependymoma and myxopapillary categories. However, given the molecular advancements, this will likely change in the future. This review provides an updated molecular classification of ependymoma, discusses the individual imaging characteristics, and briefly outlines the latest targeted molecular therapies.

Spinal CSF leak care has evolved during the past several years due to pivotal advances in its diagnosis and treatment. To the reader of the American Journal of Neuroradiology (AJNR), it has been impossible to miss the exponential increase in groundbreaking research on spinal CSF leaks and spontaneous intracranial hypotension (SIH). While many clinical specialties have contributed to these successes, the neuroradiologist has been instrumental in driving this transformation due to innovations in noninvasive imaging, novel myelographic techniques, and image-guided therapies. In this editorial, we will delve into the exciting advancements in spinal CSF leak diagnosis and treatment and celebrate the vital role of the neuroradiologist at the forefront of this revolution, with particular attention paid to CSF leak–related work published in the AJNR.

The potential for more than one distinct hematolymphoid neoplasm to arise from a common mutated stem or precursor cell has been proposed based on findings in primary human malignancies. Particularly, angioimmunoblastic T-cell lymphoma (AITL), which shares a somatic mutation profile in common with other hematopoietic malignancies, has been reported to occur alongside myeloid neoplasms or clonal B-cell proliferations, with identical mutations occurring in more than one cell lineage. Here we report such a case of an elderly woman who was diagnosed over a period of 8 years with diffuse large B-cell lymphoma, polycythemia vera, and AITL, each harboring identical somatic mutations in multiple genes. Overall, at least five identical nucleotide mutations were shared across multiple specimens, with two identical mutations co-occurring at variable variant allele frequencies in all three specimen types. These findings lend credence to the theory that a common mutated stem cell could give rise to multiple neoplasms through parallel hematopoietic differentiation pathways.

Although the progressive histologic steps leading to endometrial cancer (EndoCA), the most common female reproductive tract malignancy, from endometrial hyperplasia are well-established, the molecular changes accompanying this malignant transformation in a single patient have never been described. We had the unique opportunity to investigate the paired histologic and molecular features associated with the 12-yr development of EndoCA in a postmenopausal female who could not undergo hysterectomy and instead underwent progesterone treatment. Using a specially designed 58-gene next-generation sequencing panel, we analyzed a total of 10 sequential biopsy samples collected over this time frame. A total of eight pathogenic/likely pathogenic mutations in seven genes, APC, ARID1A, CTNNB1, CDKN2A, KRAS, PTEN, and TP53, were identified. A PTEN nonsense mutation p.W111* was present in all samples analyzed except histologically normal endometrium. Apart from this PTEN mutation, the only other recurrent mutation was KRAS G12D, which was present in six biopsy samplings, including histologically normal tissue obtained at the patient's first visit but not detectable in the cancer. The PTEN p.W111* mutant allele fractions were lowest in benign, inactive endometrial glands (0.7%), highest in adenocarcinoma (36.9%), and, notably, were always markedly reduced following progesterone treatment. To our knowledge, this report provides the first molecular characterization of EndoCA development in a single patient. A single PTEN mutation was present throughout the 12 years of cancer development. Importantly, and with potential significance toward medical and nonsurgical management of EndoCA, progesterone treatments were consistently noted to markedly decrease PTEN mutant allele fractions to precancerous levels.

The diagnosis of maturity-onset diabetes of the young (MODY), a monogenic form of diabetes mellitus caused by a mutation in a single gene, is often uncertain until genetic testing is performed. We report a 13-yr-old Korean boy who was initially diagnosed with type 2 diabetes (T2DM). MODY was suspected because of his nonobese body habitus and family history of multiple affected members. Targeted panel sequencing of all MODY-related genes was performed using the NextSeq 550Dx platform (Illumina). Sanger sequencing was performed using blood samples from the parents, siblings, and other relatives. A frameshift variant in the 3' region of the last exon of PDX1 was detected in the patient and his family members with diabetes. PP1_Moderate criterion was applied and this variant was confirmed to be the genetic cause of diabetes in the family and classified as likely pathogenic. The study highlights the importance of genetic testing for nonobese, early-onset diabetic patients with multiple affected family members. Increased awareness and aggressive genetic testing for MODY are needed.

Anorectal malformations (ARMs) constitute a group of congenital defects of the gastrointestinal and urogenital systems. They affect males and females, with an estimated worldwide prevalence of 1 in 5000 live births. These malformations are clinically heterogeneous and can be part of a syndromic presentation (syndromic ARM) or as a nonsyndromic entity (nonsyndromic ARM). Despite the well-recognized heritability of nonsyndromic ARM, the genetic etiology in most patients is unknown. In this study, we describe three siblings with diverse congenital anomalies of the genitourinary system, anemia, delayed milestones, and skeletal anomalies. Genome sequencing identified a novel, paternally inherited heterozygous Caudal type Homeobox 2 (CDX2) variant (c.722A > G (p.Glu241Gly)), that was present in all three affected siblings. The variant identified in this family is absent from population databases and predicted to be damaging by most in silico pathogenicity tools. So far, only two other reports implicate variants in CDX2 with ARMs. Remarkably, the individuals described in these studies had similar clinical phenotypes and genetic alterations in CDX2. CDX2 encodes a transcription factor and is considered the master regulator of gastrointestinal development. This variant maps to the homeobox domain of the encoded protein, which is critical for interaction with DNA targets. Our finding provides a potential molecular diagnosis for this family's condition and supports the role of CDX2 in anorectal anomalies. It also highlights the clinical heterogeneity and variable penetrance of ARM predisposition variants, another well-documented phenomenon. Finally, it underscores the diagnostic utility of genomic profiling of ARMs to identify the genetic etiology of these defects.

Alveolar capillary dysplasia (ACD) is a fatal disorder that typically presents in the neonatal period with refractory hypoxemia and pulmonary hypertension. Lung biopsy is traditionally required to establish the diagnosis. We report a 22-mo-old male who presented with anemia, severe pulmonary hypertension, and right heart failure. He had a complicated hospital course resulting in cardiac arrest and requirement for extracorporeal membrane oxygenation. Computed tomography of the chest showed a heterogenous pattern of interlobular septal thickening and pulmonary edema. The etiology of his condition was unknown, lung biopsy was contraindicated because of his medical fragility, and discussions were held to move to palliative care. Rapid whole-genome sequencing (rWGS) was performed. In 2 d it resulted, revealing a novel FOXF1 gene pathogenic variant that led to the presumptive diagnosis of atypical ACD. Cases of atypical ACD have been reported with survival in patients using medical therapy or lung transplantation. Based on the rWGS diagnosis and more favorable potential of atypical ACD, aggressive medical treatment was pursued. The patient was discharged home after 67 d in the hospital; he is currently doing well more than 30 mo after his initial presentation with only one subsequent hospitalization and no requirement for lung transplantation. Our case reveals the potential for use of rWGS in a critically ill child in which the diagnosis is unknown. rWGS and other advanced genetic tests can guide clinical management and expand our understanding of atypical ACD and other conditions.

Here, we highlight the case of a 31-yr-old man who had clinical features of primary hypertrophic osteoarthropathy (PHOAR) and harbored a homozygous variant (c.38C > A, p.Ala13Glu) in the HPGD gene, as indicated by whole-exome sequencing (WES). This variant has been previously classified by our laboratory as a variant of uncertain significance (VUS). However, another patient with the same phenotype and the same homozygous variant in HPGD was subsequently reported. In reassessing the variant, the absence of this variant in the gnomAD population database, supporting computational predictions, observation in homozygosity in two probands, and specificity of the phenotype for HPGD, all provide sufficient evidence to reclassify the HPGD c.38C > A, p.Ala13Glu variant as likely pathogenic.

Pilocytic astrocytomas are the most common pediatric brain tumors, typically presenting as low-grade neoplasms. We report two cases of pilocytic astrocytoma with atypical tumor progression. Case 1 involves a 12-yr-old boy with an unresectable suprasellar tumor, negative for BRAF rearrangement but harboring a BRAF p.V600E mutation. He experienced tumor size reduction and stable disease following dabrafenib treatment. Case 2 describes a 6-yr-old boy with a thalamic tumor that underwent multiple resections, with no actionable driver detected using targeted next-generation sequencing. Whole-genome and RNA-seq analysis identified an internal tandem duplication in FGFR1 and RAS pathway activation. Future management options include FGFR1 inhibitors. These cases demonstrate the importance of escalating molecular diagnostics for pediatric brain cancer, advocating for early reflexing to integrative whole-genome sequencing and transcriptomic profiling when targeted panels are uninformative. Identifying molecular drivers can significantly impact treatment decisions and improve patient outcomes.

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for biopanning using multiple display methods. This protocol describes selection from affibody libraries using display on Staphylococcus carnosus. Display of affibodies on staphylococci is very efficient and straightforward because of the single cell membrane and the use of a construct with a constitutive promoter. The workflow involves display of affibody libraries on the surface of S. carnosus cells, followed by screening and selection of binders using fluorescence-activated cell sorting (FACS). The transformation of DNA libraries into S. carnosus is less efficient and more complicated than for Escherichia coli. Because of this, staphylococcal display is suitable for affinity maturation or other protein-engineering efforts that are not dependent on very high diversity, and thus magnetic-activated cell sorting (MACS) is often not required before FACS. However, MACS is an option, and MACS procedures used for E. coli can easily be adapted for use in S. carnosus if needed.

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library, which can then be used for selection via multiple display methods. This protocol describes selection from affibody libraries by Escherichia coli cell surface display. With this method, high-diversity libraries of 10¹¹ can be displayed on the cell surface. The method involves two steps for selection of binders from high-diversity libraries: magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). MACS is used first to enrich the library in target-binding clones and to decrease diversity to a size that can be effectively screened and sorted in the flow cytometer in a reasonable time (typically <10⁷ cells). The protocol is based on methodology using an AIDA-I autotransporter for display on the outer membrane, but the general procedures can also be adjusted and used for other types of autotransporters or alternative E. coli display methods.

Affibody molecules are small (6-kDa) affinity proteins generated by directed evolution for specific binding to various target molecules. The first step in this workflow involves the generation of an affibody library. This is then followed by amplification of the library, which can then be used for biopanning using multiple methods. This protocol describes amplification of affibody libraries, followed by biopanning using phage display and analysis of the selection output. The general procedure is mainly for selection of first-generation affibody molecules from large naive (unbiased) libraries, typically yielding affibody hits with affinities in the low nanomolar range. For selection from affinity maturation libraries with the aim of isolating variants of even higher affinities, the procedure is similar, but parameters such as target concentration and washing are adjusted to achieve the proper stringency.

Affibody molecules are small (6-kDa) affinity proteins folded in a three-helical bundle and generated by directed evolution for specific binding to various target molecules. The most advanced affibody molecules are currently tested in the clinic, and data from more than 300 subjects show excellent activity and safety profiles. The generation of affibody molecules against a particular target starts with the generation of an affibody library, which can then be used for panning using multiple methods and selection systems. This protocol describes the molecular cloning of DNA-encoded affibody libraries to a display vector of choice, for either phage, Escherichia coli, or Staphylococcus carnosus display. The DNA library can come from different sources, such as error-prone polymerase chain reaction (PCR), molecular shuffling of mutations from previous selections, or, more commonly, from DNA synthesis using various methods. Restriction enzyme-based subcloning is the most common strategy for affibody libraries of higher diversity (e.g., >10⁷ variants) and is described here.

Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated and what biological functions it plays. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using beam break counts from Drosophila activity monitors (DAMs). The number of laboratories worldwide studying sleep in Drosophila has grown from only a few 20 years ago to hundreds today. The utility of these studies is limited by the quality of the metrics that can be extracted from the data. Many software options exist to help analyze DAM data; however, these are often expensive or have significant limitations. Therefore, we describe here a method for analyzing DAM-based data using the sleep and circadian analysis MATLAB program (SCAMP). This user-friendly software has an advantage of combining several analyses of both sleep and circadian rhythms in one package and produces graphical outputs as well as spreadsheets of the outputs for further statistical analysis. The version of SCAMP described here is also the first published software package that can analyze data from multibeam DAM5Ms, enabling determination of positional preference over time.

The positional preference of an animal can be very informative regarding the choices it makes about how to interact with its environment. The fruit fly Drosophila melanogaster has been used as a robust system for examining neurobiological mechanisms underlying behavior. Fruit fly positional preference can be gathered from TriKinetics Drosophila activity monitors (DAMs), which contain four infrared beams, allowing for tracking the position of individual flies along the length of a tube. Here, we describe a method for using DAM5Ms to examine food preference. Specifically, we show an example in which circadian changes in food preference are compared between different Drosophila species. More information about the evolution of behavior can be gathered by measuring feeding preference relative to time of day. Noni, fruit from Morinda citrifolia, contains octanoic acid, a chemical toxic to many species of Drosophila. D. melanogaster and D. simulans, both food generalists, show high sensitivity to octanoic acid, whereas D. sechellia, a specialist, can tolerate high concentrations. When two different food substrates are provided at each end of a tube, food preference can be inferred at various times of the day, using the sleep and circadian analysis MATLAB program (SCAMP) to extract and analyze positional data from DAM5Ms. Data gathered from these analyses can be used to compare avoidance or attraction to nutrients, tastants, or odors between species and genotypes or after specific different treatments. Additionally, such data can be examined as a function of time of day.

Sleep is a fundamental feature of life for virtually all multicellular animals, but many questions remain about how sleep is regulated by circadian rhythms, homeostatic sleep drive that builds up with wakefulness, and modifying factors such as hunger or social interactions, as well as about the biological functions of sleep. Substantial headway has been made in the study of both circadian rhythms and sleep in the fruit fly Drosophila melanogaster, much of it through studies of individual fly activity using Drosophila activity monitors (DAMs). Here, we describe approaches for the activation of specific neurons of interest using optogenetics (involving genetic modifications that allow for light-based neuronal activation) and thermogenetics (involving genetic modifications that allow for temperature-based neuronal activation) so that researchers can evaluate the roles of those neurons in controlling rest and activity behavior. In this protocol, we describe how to set up a rig for simultaneous optogenetic or thermogenetic stimulation and activity monitoring for analysis of sleep and circadian rhythms in Drosophila, how to raise appropriate flies, and how to perform the experiment. This protocol will allow researchers to assess the causative role in the regulation of sleep and activity rhythms of any genetically tractable subset of cells.

Sleep is important for survival, and the need for sleep is conserved across species. In the past two decades, the fruit fly Drosophila melanogaster has emerged as a promising system in which to study the genetic, neural, and physiological bases of sleep. Through significant advances in our understanding of the regulation of sleep in flies, the field is poised to address several open questions about sleep, such as how the need for sleep is encoded, how molecular regulators of sleep are situated within brain networks, and what the functions of sleep are. Here, we describe key findings, open questions, and commonly used methods that have been used to inform existing theories and develop new ways of thinking about the function, regulation, and adaptability of sleep behavior.

Affibody molecules are small, robust, and versatile affinity proteins currently being explored for therapeutic, diagnostic, and biotechnological applications. Surface-exposed residues on the affibody scaffold are randomized to create large affibody libraries from which novel binding specificities to virtually any protein target can be generated using combinatorial protein engineering. Affibody molecules have the potential to complement—or even surpass—current antibody-based technologies, exhibiting multiple desirable properties, such as high stability, affinity, and specificity, efficient tissue penetration, and straightforward modular extension of functional domains. It has been shown in both preclinical and clinical studies that affibody molecules are safe, efficacious, and valuable alternatives to antibodies for specific targeting in the context of in vivo diagnostics and therapy. Here, we provide a general background of affibody molecules, give examples of reported applications, and briefly summarize the methodology for affibody generation.

BackgroundDuring the COVID-19 pandemic, global trends of reduced healthcare-seeking behaviour were observed. This raises concerns about the consequences of healthcare avoidance for population health.AimTo determine the association between healthcare avoidance during the early stages of the COVID-19 pandemic and all-cause mortality.Design and settingThis was a 32-month follow-up within the population-based Rotterdam Study, after sending a COVID-19 questionnaire at the onset of the pandemic in April 2020 to all communty dwelling participants (n = 6241/8732, response rate 71.5%).MethodCox proportional hazards models assessed the risk of all-cause mortality among respondents who avoided health care because of the COVID-19 pandemic. Mortality status was collected through municipality registries and medical records.ResultsOf 5656 respondents, one-fifth avoided health care because of the COVID-19 pandemic (n = 1143). Compared with non-avoiders, those who avoided health care more often reported symptoms of depression (n = 357, 31.2% versus n = 554, 12.3%) and anxiety (n = 340, 29.7% versus n = 549, 12.2%), and more often rated their health as poor to fair (n = 336, 29.4% versus n = 457, 10.1%) . Those who avoided health care had an increased adjusted risk of all-cause mortality (hazard ratio [HR] 1.30, 95% confidence interval [CI] = 1.01 to 1.67), which remained nearly identical after adjustment for history of any non-communicable disease (HR 1.20, 95% CI = 0.93 to 1.54). However, this association attenuated after additional adjustment for mental and physical self-perceived health factors (HR 0.93, 95% CI = 0.71 to 1.20).ConclusionThis study found an increased risk of all-cause mortality among individuals who avoided health care during COVID-19. These individuals were characterised by poor mental and physical self-perceived health. Therefore, interventions should be targeted to these vulnerable individuals to safeguard their access to primary and specialist care to limit health disparities, inside and beyond healthcare crises.

BackgroundLong-term health conditions are major challenges for care systems. Social prescribing link workers have been introduced via primary care networks (PCNs) across England since 2019 to address the wider determinants of health by connecting individuals to activities, groups, or services within their local community.AimTo assess whether the rollout of social prescribing link workers was in areas with the highest need.Design and settingA retrospective study of social prescribing link workers in England from 2019 to 2023.MethodWorkforce, population, survey, and area-level data at the PCN-level from April 2020 to October 2023 were combined. Population need before the rollout of link workers was measured using reported lack of support from local services in the 2019 General Practice Patient Survey. To assess if rollout reflected need, linear regression was used to relate provision of link workers (measured by full-time equivalent [FTE] per 10 000 patients) in each quarter to population need for support.ResultsPopulations in urban, more deprived areas and with higher proportions of people from minority ethnic groups had the highest reported lack of support. Geographically these were in the North West and London. Initially, there was no association between need and provision; then from July 2022, this became negative and significant. By October 2023, a 10-percentage point higher need for support was associated with a 0.035 (95% confidence interval = −0.634 to −0.066) lower FTE per 10 000 patients.ConclusionRollout of link workers has not been sufficiently targeted at areas with the highest need. Future deployments should be targeted at those areas.

BackgroundAttention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder, for which there are effective pharmacological treatments that improve symptoms and reduce complications. Guidelines published by the National Institute for Health and Care Excellence recommend that primary care practitioners prescribe medication for adult ADHD under shared-care agreements with Adult Mental Health Services (AMHS). However, provision remains uneven, with some practitioners reporting a lack of support.AimThis study aimed to describe elements of support, and their availability/use, in primary care prescribing for adult ADHD medication in England to improve access for this underserved population and inform service improvement.Design and settingCross-sectional surveys were used to elicit data from commissioners, health professionals (HPs), and people with lived experience of ADHD (LE) across England about elements supporting pharmacological treatment of ADHD in primary care.MethodThree interlinked cross-sectional surveys were used to ask every integrated care board in England (commissioners), along with convenience samples of HPs and LEs, about prescribing rates, AMHS availability, wait times, and shared-care agreement protocols/policies for the pharmacological treatment of ADHD in primary care. Descriptive analyses, percentages, and confidence intervals were used to summarise responses by stakeholder group. Variations in reported provision and practice were explored and displayed visually using mapping software.ResultsData from 782 responders (42 commissioners, 331 HPs, 409 LEs) revealed differences in reported provision by stakeholder group, including for prescribing (95% of HPs versus 64% of LEs). In all, >40% of responders reported extended AMHS wait times of ≥2 years. There was some variability by NHS region – for example, London had the lowest reported extended wait time (25%), while East of England had the highest (55%).ConclusionElements supporting appropriate shared-care prescribing of ADHD medication via primary care are not universally available in England. Coordinated approaches are needed to address these gaps.

BackgroundCOVID-19 pandemic restrictions may have influenced behaviours related to weight.AimTo describe patterns of weight change among adults living in England with type 2 diabetes (T2D) and/or hypertension during the pandemic.Design and settingAn observational cohort study using the routinely collected health data of approximately 40% of adults living in England, accessed through the OpenSAFELY service inside TPP.MethodClinical and sociodemographic characteristics associated with rapid weight gain (>0.5 kg/m2/year) were investigated using multivariable logistic regression.ResultsData were extracted on adults with T2D (n = 1 231 455, 43.9% female, and 76.0% White British) or hypertension (n = 3 558 405, 49.7% female, and 84.3% White British). Adults with T2D lost weight overall (median δ = −0.1 kg/m2/year [interquartile range {IQR} −0.7–0.4]). However, rapid weight gain was common (20.7%) and associated with the following: sex (male versus female: adjusted odds ratio [aOR] 0.78 [95% confidence interval {CI} = 0.77 to 0.79]); age (older age reduced odds, for example, aged 60–69 years versus 18–29 years: aOR 0.66 [95% CI = 0.61 to 0.71]); deprivation (least deprived Index of Multiple Deprivation [IMD] quintile versus most deprived IMD quintile: aOR 0.87 [95% CI = 0.85 to 0.89]); White ethnicity (Black versus White: aOR 0.95 [95% CI = 0.92 to 0.98]); mental health conditions (for example, depression: aOR 1.13 [95% CI = 1.12 to 1.15]); and diabetes treatment (non-insulin treatment versus no pharmacological treatment: aOR 0.68 [95% CI = 0.67 to 0.69]). Adults with hypertension maintained stable weight overall (median δ = 0.0 kg/m2/year [IQR −0.6–0.5]); however, rapid weight gain was common (24.7%) and associated with similar characteristics as in T2D.ConclusionAmong adults living in England with T2D and/or hypertension, rapid pandemic weight gain was more common among females, younger adults, those living in more deprived areas, and those with mental health conditions.

BackgroundIncident benzodiazepine prescriptions in primary care for anxiety decreased between 2003 and 2018. However, from 2008, incident prescribing of benzodiazepines for anxiety increased among those aged 18–34 years. There are increasing concerns around prescribing of benzodiazepines. Further, although guidelines state benzodiazepines should only be prescribed short term, in 2017, 44% of incident prescriptions were prescribed for longer than the recommended duration of 2–4 weeks.AimTo understand when and why GPs prescribe benzodiazepines for anxiety in young adults.Design and settingA qualitative study was undertaken using in-depth interviews with 17 GPs from 10 general practices in South West England.MethodInterviews were conducted by telephone or videocall. A topic guide was used to ensure consistency across interviews. Interviews were audio-recorded, transcribed verbatim, and data analysed using reflexive thematic analysis.ResultsGPs described caution in prescribing benzodiazepines for anxiety in young adults, but thought they had an important role in acute situations. GPs described caution in prescribing duration, but some thought longer-term prescriptions could be appropriate. In light of these views, some GPs questioned whether primary care needs to revisit how clinicians are using benzodiazepines. GPs perceived that some young adults requested benzodiazepines and suggested this might be because they wanted quick symptom relief. GPs noted that refusing to prescribe felt uncomfortable and that the number of young adults presenting to general practice, already dependent on benzodiazepines, had increased.ConclusionPatient-driven factors for prescribing benzodiazepines suggest there are current unmet treatment needs among young adults with anxiety. Given increases in prescribing in this age group, it may be timely to revisit the role of benzodiazepines in the management of people with anxiety in primary care.

BackgroundBetween 2003 and 2018, incident prescriptions of beta-blockers for anxiety increased substantially, particularly for young adults. National Institute for Health and Care Excellence guidance for anxiety does not recommend beta-blockers, probably due to a lack of evidence to support such use. Recent reports have highlighted the potential risks of beta-blockers.AimTo understand when and why GPs prescribe beta-blockers for people with anxiety.Design and settingIn-depth interviews with 17 GPs in Bristol and the surrounding areas.MethodInterviews were held by telephone or video call. A topic guide was used to ensure consistency across interviews. Interviews were audio-recorded, transcribed verbatim, and analysed thematically.ResultsMany GPs viewed beta-blockers as ‘low risk’, particularly for young adults. Some GPs viewed beta-blockers as an alternative to benzodiazepines, acting quickly and not leading to dependence. GPs reflected that some patients appeared to want an ‘immediate fix’ to their symptoms, which GPs thought beta-blockers could potentially offer. This is salient in light of substantial waiting lists for talking therapies and delays in antidepressants taking effect. GPs described how some patients seemed more willing to try beta-blockers than antidepressants, as patients did not perceive them as ‘mental health drugs’ and therefore viewed them as potentially more acceptable and less stigmatising. Further, GPs viewed beta-blockers as ‘patient-led’, with patients managing their own dose and frequency, without GP input.ConclusionMany GPs believe that beta-blockers have a role to play in the management of anxiety. Given recent increases in the prescribing of these drugs in primary care, there is a need to assess their safety and effectiveness as a treatment for people with anxiety disorders.