Targeting autophagy impairment improves the phenotype of a novel CLN8 zebrafish model.

CLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease.

Molecular Basis for Chemoselectivity Control in Oxidations of Internal Aryl-Alkenes Catalyzed by Laboratory Evolved P450s.

P450 enzymes naturally perform selective hydroxylations and epoxidations of unfunctionalized hydrocarbon substrates, among other reactions. The adaptation of P450 enzymes to a particular oxidative reaction involving alkenes is of great interest for the design of new synthetically useful biocatalysts. However, the mechanism that these enzymes utilize to precisely modulate the chemoselectivity and distinguishing between competing alkene double bond epoxidations and allylic C-H hydroxylations is sometimes not clear, which hampers the rational design of specific biocatalysts. In a previous work, a P450 from Labrenzia aggregata (P450LA1) was engineered in the laboratory using directed evolution to catalyze the direct oxidation of trans-ß-methylstyrene to phenylacetone. The final variant, KS, was able to overcome the intrinsic preference for alkene epoxidation to directly generate a ketone product via the formation of a highly reactive carbocation intermediate. Here, additional library screening along this evolutionary lineage permitted to serendipitously detect a mutation that overcomes epoxidation and carbonyl formation by exhibiting a large selectivity of 94 % towards allylic C-H hydroxylation. A multiscalar computational methodology was applied to reveal the molecular basis towards this hydroxylation preference. Enzyme modelling suggests that introduction of a bulky substitution dramatically changes the accessible conformations of the substrate in the active site, thus modifying the enzymatic selectivity towards terminal hydroxylation and avoiding the competing epoxidation pathway, which is sterically hindered.

The potential of 5-methoxy-N,N-dimethyltryptamine in the treatment of alcohol use disorder: A first look at therapeutic mechanisms of action.

Alcohol use disorder (AUD) remains one of the most prevalent psychiatric disorders worldwide with high economic costs. Current treatment options show modest efficacy and relapse rates are high. Furthermore, there are increases in the treatment gap and few new medications have been approved in the past 20 years. Recently, psychedelic-assisted therapy with psilocybin and lysergic acid diethylamide has garnered significant attention in the treatment of AUD. Yet, they require significant amounts of therapist input due to prolonged subjective effects (~4-12 h) leading to high costs and impeding implementation. Accordingly, there is an increasing interest in the rapid and short-acting psychedelic 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT). This paper offers a first look at potential therapeutic mechanisms for AUD by reviewing the current literature on 5-MeO-DMT. Primarily, 5-MeO-DMT is able to induce mystical experiences and ego-dissolution together with increases in psychological flexibility and mindfulness. This could decrease AUD symptoms through the alleviation of psychiatric mood-related comorbidities consistent with the negative reinforcement and self-medication paradigms. In addition, preliminary evidence indicates that 5-MeO-DMT modulates neural oscillations that might subserve ego-dissolution (increases in gamma), psychological flexibility and mindfulness (increases in theta), and the reorganization of executive control networks (increases in coherence across frequencies) that could improve emotion regulation and inhibition. Finally, animal studies show that 5-MeO-DMT is characterized by neuroplasticity, anti-inflammation, 5-HT2A receptor agonism, and downregulation of metabotropic glutamate receptor 5 with clinical implications for AUD and psychiatric mood-related comorbidities. The paper concludes with several recommendations for future research to establish the purported therapeutic mechanisms of action.

Efficient Transferase Engineering for SAM Analog Synthesis from Iodoalkanes.

S-Adenosyl-l-methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand the chemistry accessible with SAM-dependent transferases and go beyond methylation reactions. Here we present an efficient enzyme engineering strategy to synthesize different SAM analogs from "off-the-shelf" iodoalkanes through enzymatic alkylation of S-adenosyl-l-homocysteine (SAH). This was achieved by mutating multiple hydrophobic and structurally dynamic amino acids simultaneously. Combinatorial mutagenesis was guided by the natural amino acid diversity and generated a highly functional mutant library. This approach increased the speed as well as the scale of enzyme engineering by providing a panel of optimized enzymes with orders of magnitude higher activities for multiple substrates in just one round of enzyme engineering. The optimized enzymes exhibit catalytic efficiencies up to 31 M-1 s-1, convert various iodoalkanes, including substrates bearing cyclopropyl or aromatic moieties, and catalyze S-alkylation of SAH with very high stereoselectivities (>99 % de). We further report a high throughput chromatographic screening system for reliable and rapid SAM analog analysis. We believe that the methods and enzymes described herein will further advance the field of selective biocatalytic alkylation chemistry by enabling SAM analog regeneration with "off-the-shelf" reagents.

Best practices for managing and disseminating resources and outreach and evaluating the impact of the IDG Consortium.

The Illuminating the Druggable Genome (IDG) consortium generated reagents, biological model systems, data, informatic databases, and computational tools. The Resource Dissemination and Outreach Center (RDOC) played a central administrative role, organized internal meetings, fostered collaboration, and coordinated consortium-wide efforts. The RDOC developed and deployed a Resource Management System (RMS) to enable efficient workflows for collecting, accessing, validating, registering, and publishing resource metadata. IDG policies for repositories and standardized representations of resources were established, adopting the FAIR (findable, accessible, interoperable, reusable) principles. The RDOC also developed metrics of IDG impact. Outreach initiatives included digital content, the Protein Illumination Timeline (representing milestones in generating data and reagents), the Target Watch publication series, the e-IDG Symposium series, and leveraging social media platforms.

Kinome-Wide Virtual Screening by Multi-Task Deep Learning.

Deep learning is a machine learning technique to model high-level abstractions in data by utilizing a graph composed of multiple processing layers that experience various linear and non-linear transformations. This technique has been shown to perform well for applications in drug discovery, utilizing structural features of small molecules to predict activity. Here, we report a large-scale study to predict the activity of small molecules across the human kinome-a major family of drug targets, particularly in anti-cancer agents. While small-molecule kinase inhibitors exhibit impressive clinical efficacy in several different diseases, resistance often arises through adaptive kinome reprogramming or subpopulation diversity. Polypharmacology and combination therapies offer potential therapeutic strategies for patients with resistant diseases. Their development would benefit from a more comprehensive and dense knowledge of small-molecule inhibition across the human kinome. Leveraging over 650,000 bioactivity annotations for more than 300,000 small molecules, we evaluated multiple machine learning methods to predict the small-molecule inhibition of 342 kinases across the human kinome. Our results demonstrated that multi-task deep neural networks outperformed classical single-task methods, offering the potential for conducting large-scale virtual screening, predicting activity profiles, and bridging the gaps in the available data.

ESPEN practical guideline: Nutritional support for polymorbid medical inpatients.

BACKGROUND: Disease-related malnutrition in polymorbid medical inpatients is a highly prevalent syndrome associated with significantly increased morbidity, disability, short- and long-term mortality, impaired recovery from illness, and healthcare costs. AIM: As there are uncertainties in applying disease-specific guidelines to patients with multiple conditions, our aim was to provide evidence-based recommendations on nutritional support for the polymorbid patient population hospitalized in medical wards. METHODS: The 2023 update adheres to the standard operating procedures for ESPEN guidelines. We undertook a systematic literature search for 15 clinical questions in three different databases (Medline, Embase and the Cochrane Library), as well as in secondary sources (e.g., published guidelines), until July 12th, 2022. Retrieved abstracts were screened to identify relevant studies that were used to develop recommendations (including SIGN grading), which was followed by submission to Delphi voting. Here, the practical version of the guideline is presented which has been shortened and equipped with flow charts for patients care. RESULTS: 32 recommendations (7× A, 11× B, 10× O and 4× GPP), which encompass different aspects of nutritional support were included from the scientific guideline including indication, route of feeding, energy and protein requirements, micronutrient requirements, disease-specific nutrients, timing, monitoring and procedure of intervention. Here, the practical version of the guideline is presented which has been shortened and equipped with flow charts for patients care. CONCLUSIONS: Recent high-quality trials have provided increasing evidence that nutritional support can reduce morbidity and other complications associated with malnutrition in polymorbid patients. The timely screening of patients for risk of malnutrition at hospital admission followed by individualized nutritional support interventions for at-risk patients should be part of routine clinical care and multimodal treatment in hospitals worldwide. Use of this updated practical guideline offers an evidence-based nutritional approach to polymorbid medical inpatients and may improve their outcomes.

Prognostic implications of the arginine metabolism in patients at nutritional risk: A secondary analysis of the randomized EFFORT trial.

BACKGROUND: Arginine, a conditionally essential amino acid, is key component in metabolic pathways including immune regulation and protein synthesis. Depletion of arginine contributes to worse outcomes in severely ill and surgical patient populations. We assessed prognostic implications of arginine levels and its metabolites and ratios in polymorbid medical inpatients at nutritional risk regarding clinical outcomes and treatment response. METHODS: Within this secondary analysis of the randomized controlled Effect of early nutritional support on Frailty, Functional Outcomes, and Recovery of malnourished medical inpatients Trial (EFFORT), we investigated the association of arginine, its metabolites and ratios (i.e., ADMA and SDMA, ratios of arginine/ADMA, arginine/ornithine, and global arginine bioavailability ratio) measured on hospital admission with short-term and long-term mortality by means of regression analysis. RESULTS: Among the 231 patients with available measurements, low arginine levels ≤90.05 µmol/l (n = 86; 37 %) were associated with higher all-cause mortality at 30 days (primary endpoint, adjusted HR 3.27, 95 % CI 1.86 to 5.75, p < 0.001) and at 5 years (adjusted HR 1.50, 95 % CI 1.07 to 2.12, p = 0.020). Arginine metabolites and ratios were also associated with adverse outcome, but had lower prognostic value. There was, however, no evidence that treatment response was influenced by admission arginine levels. CONCLUSION: This secondary analysis focusing on medical inpatients at nutritional risk confirms a strong association of low plasma arginine levels and worse clinical courses. The potential effects of arginine-enriched nutritional supplements should be investigated in this population of patients. CLINICAL TRIAL REGISTRATION: clinicaltrials.gov as NCT02517476 (registered 7 August 2015).

ESPEN practical short micronutrient guideline.

BACKGROUND: Trace elements and vitamins, named together micronutrients (MNs), are essential for human metabolism. The importance of MNs in common pathologies is recognized by recent research, with deficiencies significantly impacting the outcome. OBJECTIVE: This short version of the guideline aims to provide practical recommendations for clinical practice. METHODS: An extensive search of the literature was conducted in the databases Medline, PubMed, Cochrane, Google Scholar, and CINAHL for the initial guideline. The search focused on physiological data, historical evidence (for papers published before PubMed release in 1996), and observational and/or randomized trials. For each MN, the main functions, optimal analytical methods, impact of inflammation, potential toxicity, and provision during enteral or parenteral nutrition were addressed. The SOP wording was applied for strength of recommendations. RESULTS: The limited number of interventional trials prevented meta-analysis and led to a low level of evidence for most recommendations. The recommendations underwent a consensus process, which resulted in a percentage of agreement (%): strong consensus required of >90 % of votes. Altogether the guideline proposes 3 general recommendations and specific recommendations for the 26 MNs. Monitoring and management strategies are proposed. CONCLUSION: This short version of the MN guideline should facilitate handling of the MNs in at-risk diseases, whilst offering practical advice on MN provision and monitoring during nutritional support.

ZSCAN10 deficiency causes a neurodevelopmental disorder with characteristic oto-facial malformations.

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1,500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations, however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESC), including a knock-out and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and Western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR), and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-Seq analyses in Zscan10-/- mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry, and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10.

De Novo Variants Found in Three Distinct Schizophrenia Populations Hit a Common Core Gene Network Related to Microtubule and Actin Cytoskeleton Gene Ontology Classes.

Schizophrenia (SZ) is a heterogeneous and debilitating psychiatric disorder with a strong genetic component. To elucidate functional networks perturbed in schizophrenia, we analysed a large dataset of whole-genome studies that identified SNVs, CNVs, and a multi-stage schizophrenia genome-wide association study. Our analysis identified three subclusters that are interrelated and with small overlaps: GO:0007017~Microtubule-Based Process, GO:00015629~Actin Cytoskeleton, and GO:0007268~SynapticTransmission. We next analysed three distinct trio cohorts of 75 SZ Algerian, 45 SZ French, and 61 SZ Japanese patients. We performed Illumina HiSeq whole-exome sequencing and identified de novo mutations using a Bayesian approach. We validated 88 de novo mutations by Sanger sequencing: 35 in French, 21 in Algerian, and 32 in Japanese SZ patients. These 88 de novo mutations exhibited an enrichment in genes encoding proteins related to GO:0051015~actin filament binding (p = 0.0011) using David, and enrichments in GO: 0003774~transport (p = 0.019) and GO:0003729~mRNA binding (p = 0.010) using Amigo. One of these de novo variant was found in CORO1C coding sequence. We studied Coro1c haploinsufficiency in a Coro1c+/- mouse and found defects in the corpus callosum. These results could motivate future studies of the mechanisms surrounding genes encoding proteins involved in transport and the cytoskeleton, with the goal of developing therapeutic intervention strategies for a subset of SZ cases.

ESPEN practical guideline on clinical nutrition in acute and chronic pancreatitis.

Both acute and chronic pancreatitis are frequent diseases of the pancreas, which, despite being of benign nature, are related to a significant risk of malnutrition and may require nutritional support. Acute necrotizing pancreatitis is encountered in 20 % of patients with acute pancreatitis, is associated with increased morbidity and mortality, and may require artificial nutrition by enteral or parenteral route, as well as additional endoscopic, radiological or surgical interventions. Chronic pancreatitis represents a chronic inflammation of the pancreatic gland with development of fibrosis. Abdominal pain leading to decreased oral intake, as well as exocrine and endocrine failure are frequent complications of the disease. All of the above represent risk factors related to malnutrition. Therefore, patients with chronic pancreatitis should be considered at risk, screened and supplemented accordingly. Moreover, osteoporosis and increased facture risk should be acknowledged in patients with chronic pancreatitis, and preventive measures should be considered.

Wastewater based surveillance can be used to reduce clinical testing intensity on a university campus.

Clinical testing has been a vital part of the response to and suppression of the COVID-19 pandemic; however, testing imposes significant burdens on a population. College students had to contend with clinical testing while simultaneously dealing with health risks and the academic pressures brought on by quarantines, changes to virtual platforms, and other disruptions to daily life. The objective of this study was to analyze whether wastewater surveillance can be used to decrease the intensity of clinical testing while maintaining reliable measurements of diseases incidence on campus. Twelve months of human health and wastewater surveillance data for eight residential buildings on a university campus were analyzed to establish how SARS-CoV-2 levels in the wastewater can be used to minimize clinical testing burden on students. Wastewater SARS-CoV-2 levels were used to create multiple scenarios, each with differing levels of testing intensity, which were compared to the actual testing volumes implemented by the university. We found that scenarios in which testing intensity fluctuations matched rise and falls in SARS-CoV-2 wastewater levels had stronger correlations between SARS-CoV-2 levels and recorded clinical positives. In addition to stronger correlations, most scenarios resulted in overall fewer weekly clinical tests performed. We suggest the use of wastewater surveillance to guide COVID-19 testing as it can significantly increase the efficacy of COVID-19 surveillance while reducing the burden placed on college students during a pandemic. Future efforts should be made to integrate wastewater surveillance into clinical testing strategies implemented on college campuses.

Controlling Monoterpene Isomerization by Guiding Challenging Carbocation Rearrangement Reactions in Engineered Squalene-Hopene Cyclases.

The interconversion of monoterpenes is facilitated by a complex network of carbocation rearrangement pathways. Controlling these isomerization pathways is challenging when using common Brønsted and Lewis acid catalysts, which often produce product mixtures that are difficult to separate. In contrast, natural monoterpene cyclases exhibit high control over the carbocation rearrangement reactions but are reliant on phosphorylated substrates. In this study, we present engineered squalene-hopene cyclases from Alicyclobacillus acidocaldarius (AacSHC) that catalyze the challenging isomerization of monoterpenes with unprecedented precision. Starting from a promiscuous isomerization of (+)-ß-pinene, we first demonstrate noticeable shifts in the product distribution solely by introducing single point mutations. Furthermore, we showcase the tuneable cation steering by enhancing (+)-borneol selectivity from 1 % to >90 % (>99 % de) aided by iterative saturation mutagenesis. Our combined experimental and computational data suggest that the reorganization of key aromatic residues leads to the restructuring of the water network that facilitates the selective termination of the secondary isobornyl cation. This work expands our mechanistic understanding of carbocation rearrangements and sets the stage for target-oriented skeletal reorganization of broadly abundant terpenes.

A vibrating capillary for ultrasound rotation manipulation of zebrafish larvae.

Multifunctional micromanipulation systems have garnered significant attention due to the growing interest in biological and medical research involving model organisms like zebrafish (Danio rerio). Here, we report a novel acoustofluidic rotational micromanipulation system that offers rapid trapping, high-speed rotation, multi-angle imaging, and 3D model reconstruction of zebrafish larvae. An ultrasound-activated oscillatory glass capillary is used to trap and rotate a zebrafish larva. Simulation and experimental results demonstrate that both the vibrating mode and geometric placement of the capillary contribute to the developed polarized vortices along the long axis of the capillary. Given its capacities for easy-to-operate, stable rotation, avoiding overheating, and high-throughput manipulation, our system poses the potential to accelerate zebrafish-directed biomedical research.

Reflective multi-immersion microscope objectives inspired by the Schmidt telescope.

Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.

Importance of parental involvement in paediatric palliative care in Hong Kong: qualitative case study.

OBJECTIVE: To compare and contrast the perceived care needs of children with life-limiting conditions (CLLC) from the perspectives of the children, parents and healthcare providers. DESIGN: A qualitative case study method using semistructured interviews was employed with a within-case and across-case analysis. Themes and subthemes emerging from the cases were compared and contrasted in the across-case analysis to explore the similarities and variations in participant perceptions. SETTING/PARTICIPANTS: The setting was the paediatric departments of five regional hospitals in Hong Kong. Twenty-five sets of informants (CLLC-parent-healthcare provider) were recruited, with 65 individual interviews conducted. RESULTS: A total of 3784 units of analysis were identified, resulting in three themes with subthemes. 'Living with the disease' (55.8%) occupied the largest proportion, followed by 'information and understanding about the disease' (27.4%), and 'care support and palliative care' (16.8%). Healthcare provider support mainly focused on physical concerns. Family and social support were present, but carer stress created tension between couples. Doctors were the primary source of medical information, but the parents had to seek further information via the internet and support from patient groups. There was a perceived need for better coordination and collaboration of care. The palliative care approach coordinated by nurses was seen as helpful in addressing the care needs of the CLLC. CONCLUSIONS: This original study identified the importance of palliative care with active engagement of parents which can address the service gap for CLLC.

Targeted degrader technologies as prospective SARS-CoV-2 therapies.

COVID-19 remains a severe public health threat despite the WHO declaring an end to the public health emergency in May 2023. Continual development of SARS-CoV-2 variants with resistance to vaccine-induced or natural immunity necessitates constant vigilance as well as new vaccines and therapeutics. Targeted protein degradation (TPD) remains relatively untapped in antiviral drug discovery and holds the promise of attenuating viral resistance development. From a unique structural design perspective, this review covers antiviral degrader merits and challenges by highlighting key coronavirus protein targets and their co-crystal structures, specifically illustrating how TPD strategies can refine existing SARS-CoV-2 3CL protease inhibitors to potentially produce superior protease-degrading agents.