Transcobalamin receptor antibodies in autoimmune vitamin B12 central deficiency.

Vitamin B12 is critical for hematopoiesis and myelination. Deficiency can cause neurologic deficits including loss of coordination and cognitive decline. However, diagnosis relies on measurement of vitamin B12 in the blood, which may not accurately reflect the concentration in the brain. Using programmable phage display, we identified an autoantibody targeting the transcobalamin receptor (CD320) in a patient with progressive tremor, ataxia, and scanning speech. Anti-CD320 impaired cellular uptake of cobalamin (B12) in vitro by depleting its target from the cell surface. Despite a normal serum concentration, B12 was nearly undetectable in her cerebrospinal fluid (CSF). Immunosuppressive treatment and high-dose systemic B12 supplementation were associated with increased B12 in the CSF and clinical improvement. Optofluidic screening enabled isolation of a patient-derived monoclonal antibody that impaired B12 transport across an in vitro model of the blood-brain barrier (BBB). Autoantibodies targeting the same epitope of CD320 were identified in seven other patients with neurologic deficits of unknown etiology, 6% of healthy controls, and 21.4% of a cohort of patients with neuropsychiatric lupus. In 132 paired serum and CSF samples, detection of anti-CD320 in the blood predicted B12 deficiency in the brain. However, these individuals did not display any hematologic signs of B12 deficiency despite systemic CD320 impairment. Using a genome-wide CRISPR screen, we found that the low-density lipoprotein receptor serves as an alternative B12 uptake pathway in hematopoietic cells. These findings dissect the tissue specificity of B12 transport and elucidate an autoimmune neurologic condition that may be amenable to immunomodulatory treatment and nutritional supplementation.

Towards a machine-learning assisted non-invasive classification of dengue severity using wearable PPG data: a prospective clinical study.

BACKGROUND: Dengue epidemics impose considerable strain on healthcare resources. Real-time continuous and non-invasive monitoring of patients admitted to the hospital could lead to improved care and outcomes. We evaluated the performance of a commercially available wearable (SmartCare) utilising photoplethysmography (PPG) to stratify clinical risk for a cohort of hospitalised patients with dengue in Vietnam. METHODS: We performed a prospective observational study for adult and paediatric patients with a clinical diagnosis of dengue at the Hospital for Tropical Disease, Ho Chi Minh City, Vietnam. Patients underwent PPG monitoring early during admission alongside standard clinical care. PPG waveforms were analysed using machine learning models. Adult patients were classified between 3 severity classes: i) uncomplicated (ward-based), ii) moderate-severe (emergency department-based), and iii) severe (ICU-based). Data from paediatric patients were split into 2 classes: i) severe (during ICU stay) and ii) follow-up (14-21 days after the illness onset). Model performances were evaluated using standard classification metrics and 5-fold stratified cross-validation. FINDINGS: We included PPG and clinical data from 132 adults and 15 paediatric patients with a median age of 28 (IQR, 21-35) and 12 (IQR, 9-13) years respectively. 1781 h of PPG data were available for analysis. The best performing convolutional neural network models (CNN) achieved a precision of 0.785 and recall of 0.771 in classifying adult patients according to severity class and a precision of 0.891 and recall of 0.891 in classifying between disease and post-disease state in paediatric patients. INTERPRETATION: We demonstrate that the use of a low-cost wearable provided clinically actionable data to differentiate between patients with dengue of varying severity. Continuous monitoring and connectivity to early warning systems could significantly benefit clinical care in dengue, particularly within an endemic setting. Work is currently underway to implement these models for dynamic risk predictions and assist in individualised patient care. FUNDING: EPSRC Centre for Doctoral Training in High-Performance Embedded and Distributed Systems (HiPEDS) (Grant: EP/L016796/1) and the Wellcome Trust (Grants: 215010/Z/18/Z and 215688/Z/19/Z).

Generation of Human Isogenic Induced Pluripotent Stem Cell Lines with CRISPR Prime Editing.

We developed an efficient CRISPR prime editing protocol and generated isogenic-induced pluripotent stem cell (iPSC) lines carrying heterozygous or homozygous alleles for putatively causal single nucleotide variants at six type 2 diabetes loci (ABCC8, MTNR1B, TCF7L2, HNF4A, CAMK1D, and GCK). Our two-step sequence-based approach to first identify transfected cell pools with the highest fraction of edited cells significantly reduced the downstream efforts to isolate single clones of edited cells. We found that prime editing can make targeted genetic changes in iPSC and optimization of system components and guide RNA designs that were critical to achieve acceptable efficiency. Systems utilizing PEmax, epegRNA modifications, and MLH1dn provided significant benefit, producing editing efficiencies of 36-73%. Editing success and pegRNA design optimization required for each variant differed depending on the sequence at the target site. With attention to design, prime editing is a promising approach to generate isogenic iPSC lines, enabling the study of specific genetic changes in a common genetic background.

Efficient CRISPR genome editing and integrative genomic analyses reveal the mosaicism of Cas-induced mutations and pleiotropic effects of scarlet gene in an emerging model system.

Despite the revolutionary impacts of CRISPR-Cas gene editing systems, the effective and widespread use of CRISPR technologies in emerging model organisms still faces significant challenges. These include the inefficiency in generating heritable mutations at the organismal level, limited knowledge about the genomic consequences of gene editing, and an inadequate understanding of the inheritance patterns of CRISPR-Cas-induced mutations. This study addresses these issues by 1) developing an efficient microinjection delivery method for CRISPR editing in the microcrustacean Daphnia pulex; 2) assessing the editing efficiency of Cas9 and Cas12a nucleases, examining mutation inheritance patterns, and analyzing the local and global mutation spectrum in the scarlet mutants; and 3) investigating the transcriptomes of scarlet mutants to understand the pleiotropic effects of scarlet underlying their swimming behavior changes. Our reengineered CRISPR microinjection method results in efficient biallelic editing with both nucleases. While indels are dominant in Cas-induced mutations, a few on-site large deletions (>1kb) are observed, most likely caused by microhomology-mediated end joining repair. Knock-in of a stop codon cassette to the scarlet locus was successful, despite complex induced mutations surrounding the target site. Moreover, extensive germline mosaicism exists in some mutants, which unexpectedly produce different phenotypes/genotypes in their asexual progenies. Lastly, our transcriptomic analyses unveil significant gene expression changes associated with scarlet knock-out and altered swimming behavior in mutants, including several genes (e.g., NMDA1, ABAT, CNTNAP2) involved in human neurodegenerative diseases. This study expands our understanding of the dynamics of gene editing in the tractable model organism Daphnia and highlights its promising potential as a neurological disease model.

MXene nanosheet-derived N, S-codoped graphene quantum dots for ultrasensitive and selective detection of 3-nitro-l-tyrosine in human serum.

3-Nitro-l-tyrosine (3NT) is an oxidative stress metabolite associated with neurodegenerative diseases such as Parkinson's disease and rheumatoid arthritis. In this study, the N, S-co-doped graphene quantum dots (NSGQDs) derived from nitrogen-doped Ti3C2Tx MXene nanosheet via the hydrothermal method in the presence of mercaptosuccinic acid was synthesized as an optical sensing probe to detect 3NT in human serum. Tetramethyl ammonium hydroxide, the nitrogen source and delamination agent, was used to prepare nitrogen-doped MXene nanosheets via one step at room temperature. The as-prepared NSGQDs are uniform with an average size of 1.2 ± 0.6 nm, and can be stable in aqueous solution for at least 90 d to serve as the fluorescence probe. The N atoms in N-MXene reduce the restacking and aggregation of MXene nanosheets, while the sulfur dopant in NSGQDs increases the quantum yield from 6.2 to 12.1 % as well as enhances the selectivity of 3NT over the other 12 interferences via coordination interaction with nitro group in 3NT. A linear range of 0.02-150 µM in PBS and 0.05-200 µM in human serum with a recovery of 97-108 % for 3NT detection is observed. Moreover, the limit of detection can be lowered to 4.2 and 7 nM in PBS and 1 × diluted human serum, respectively. Results obtained clearly indicate the potential application of the N-Ti3C2Tx derived NSGQD for effective detection of 3NT, which can open a window for the synthesis of doped GQDs via 2D MXene materials for ultrasensitive and selective detection of other biometabolites and biomarkers of neurodegenerative diseases in biological fluids.