The Effectiveness of Cobalamin (B12) Treatment for Autism Spectrum Disorder: A Systematic Review and Meta-Analysis

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder affecting 2% of children in the United States. Biochemical abnormalities associated with ASD include impaired methylation and sulphation capacities along with low glutathione (GSH) redox capacity. Potential treatments for these abnormalities include cobalamin (B12). This systematic review collates the studies using B12 as a treatment in ASD. A total of 17 studies were identified; 4 were double-blind, placebo-controlled studies (2 examined B12 injections alone and 2 used B12 in an oral multivitamin); 1 was a prospective controlled study; 6 were prospective, uncontrolled studies, and 6 were retrospective (case series and reports). Most studies (83%) used oral or injected methylcobalamin (mB12), while the remaining studies did not specify the type of B12 used. Studies using subcutaneous mB12 injections (including 2 placebo-controlled studies) used a 64.5–75 µg/kg/dose. One study reported anemia in 2 ASD children with injected cyanocobalamin that resolved with switching to injected mB12. Two studies reported improvements in markers of mitochondrial metabolism. A meta-analysis of methylation metabolites demonstrated decreased S-adenosylhomocysteine (SAH), and increased methionine, S-adenosylmethionine (SAM), SAM/SAH ratio, and homocysteine (with small effect sizes) with mB12. Meta-analysis of the transsulfuration and redox metabolism metabolites demonstrated significant improvements with mB12 in oxidized glutathione (GSSG), cysteine, total glutathione (GSH), and total GSH/GSSG redox ratio with medium to large effect sizes. Improvements in methylation capacity and GSH redox ratio were significantly associated with clinical improvements (with a mean moderate effect size of 0.59) in core and associated ASD symptoms, including expressive communication, personal and domestic daily living skills, and interpersonal, play-leisure, and coping social skills, suggesting these biomarkers may predict response to B12. Other clinical improvements observed with B12 included sleep, gastrointestinal symptoms, hyperactivity, tantrums, nonverbal intellectual quotient, vision, eye contact, echolalia, stereotypy, anemia, and nocturnal enuresis. Adverse events identified by meta-analysis included hyperactivity (11.9%), irritability (3.4%), trouble sleeping (7.6%), aggression (1.8%), and worsening behaviors (7.7%) but were generally few, mild, not serious, and not significantly different compared to placebo. In one study, 78% of parents desired to continue mB12 injections after the study conclusion. Preliminary clinical evidence suggests that B12, particularly subcutaneously injected mB12, improves metabolic abnormalities in ASD along with clinical symptoms. Further large multicenter placebo-controlled studies are needed to confirm these data. B12 is a promising treatment for ASD.

Salicylic Acid Increases Photosynthesis of Drought Grown Mustard Plants Effectively with Sufficient-N via Regulation of Ethylene, ABA and Nitrogen-Use Efficiency

An essential approach to reduce drought in plants is to maximize the use of most limited resources. The increase in water-use efficiency (WUE) is important to maximally utilize the available water to increase photosynthesis and growth of plants under water-deficit stress. Both WUE and photosynthetic nitrogen use efficiency (PNUE), as the indices of resource-use efficiency were studied in mustard (Brassica juncea L.) plants grown under limited water conditions with low-N (100 mg N kg−1 soil) and sufficient-N (200 mg N kg− 1 soil) N and sprayed with 0- and 0.5 mM salicylic acid (SA). Application of SA increased water potential, osmotic potential, WUE and incorporation of soil N into photosynthetic machinery by enhancing PNUE. It also increased photosynthesis of plants maximally by increasing stomatal conductance and intercellular CO2 concentration under water-deficit stress. This increase was greater in the presence of sufficient-N where 0.5 mM SA maximally enhanced the N-metabolism, redox ratio that mitigated the oxidative stress. The application of SA on plants supplemented with N reduced ethylene and abscisic (ABA) synthesis. It could be inferred that SA enhanced N utilization at its optimal level to maintain redox ratio and inhibit ABA-mediated stomatal closure to enhance the resource utilization and photosynthesis. SA also enhanced glucose utilization which prevented photosynthetic repression by enhanced glucose under stress. Thus, SA application may impart a potential management tool for increasing photosynthetic NUE, WUE and photosynthesis under drought.

Optical Redox Imaging of Treatment Responses to Nampt Inhibition and Combination Therapy in Triple-Negative Breast Cancer Cells

We evaluated the utility of optical redox imaging (ORI) to identify the therapeutic response of triple-negative breast cancers (TNBC) under various drug treatments. Cultured HCC1806 and MDA-MB-231 cells treated with FK866 (nicotinamide phosphoribosyltransferase (Nampt) inhibitor), FX11 (lactate dehydrogenase A inhibitor), paclitaxel, and their combinations were subjected to ORI, followed by imaging fluorescently labeled reactive oxygen species (ROS). Cell growth inhibition was measured by a cell viability assay. We found that both cell lines experienced significant NADH decrease and redox ratio (Fp/(NADH+Fp)) increase due to FK866 treatment; however, HCC1806 was much more responsive than MDA-MB-231. We further studied HCC1806 with the main findings: (i) nicotinamide riboside (NR) partially restored NADH in FK866-treated cells; (ii) FX11 induced an over 3-fold NADH increase in FK866 or FK866+NR pretreated cells; (iii) FK866 combined with paclitaxel caused synergistic increases in both Fp and the redox ratio; (iv) FK866 sensitized cells to paclitaxel treatments, which agrees with the redox changes detected by ORI; (v) Fp and the redox ratio positively correlated with cell growth inhibition; and (vi) Fp and NADH positively correlated with ROS level. Our study supports the utility of ORI for detecting the treatment responses of TNBC to Nampt inhibition and the sensitization effects on standard chemotherapeutics.

Intravital Metabolic Autofluorescence Imaging Captures Macrophage Heterogeneity Across Normal and Cancerous Tissue

Macrophages are dynamic immune cells that govern both normal tissue function and disease progression. However, standard methods to measure heterogeneity in macrophage function within tissues require tissue excision and fixation, which limits our understanding of diverse macrophage function in vivo. Two-photon microscopy of the endogenous metabolic co-enzymes NAD(P)H and flavin adenine dinucleotide (FAD) (metabolic autofluorescence imaging) enables dynamic imaging of mouse models in vivo. Here, we demonstrate metabolic autofluorescence imaging to assess cell-level macrophage heterogeneity in response to normal and cancerous tissue microenvironments in vivo. NAD(P)H and FAD fluorescence intensities and lifetimes were measured for both tissue-resident macrophages in mouse ear dermis and tumor-associated macrophages in pancreatic flank tumors. Metabolic and spatial organization of macrophages were determined by performing metabolic autofluorescence imaging and single macrophage segmentation in mice engineered for macrophage-specific fluorescent protein expression. Tumor-associated macrophages exhibited decreased optical redox ratio [NAD(P)H divided by FAD intensity] compared to dermal macrophages, indicating that tumor-associated macrophages are more oxidized than dermal macrophages. The mean fluorescence lifetimes of NAD(P)H and FAD were longer in dermal macrophages than in tumor-associated macrophages, which reflects changes in NAD(P)H and FAD protein-binding activities. Dermal macrophages had greater heterogeneity in optical redox ratio, NAD(P)H mean lifetime, and FAD mean lifetime compared to tumor-associated macrophages. Similarly, standard markers of macrophage phenotype (CD206 and CD86) assessed by immunofluorescence revealed greater heterogeneity in dermal macrophages compared to tumor-associated macrophages. Ultimately, metabolic autofluorescence imaging provides a novel tool to assess tissue-specific macrophage behavior and cell-level heterogeneity in vivo in animal models.

Patient-derived cancer organoid tracking with widefield one-photon redox imaging to assess treatment response

MotivationAccessible tools are needed for rapid, non-destructive imaging of patient-derived cancer organoid (PCO) treatment response to accelerate drug discovery and streamline treatment planning for individual patients.AimSegment and track individual PCOs with widefield one-photon redox imaging to extract morphological and metabolic variables of treatment response.ApproachRedox imaging of the endogenous fluorophores, NAD(P)H and FAD, was used to monitor the metabolic state and morphology of PCOs. Redox imaging was performed on a widefield one-photon epifluorescence microscope to evaluate drug response in two colorectal PCO lines. An automated image analysis framework was developed to track PCOs across multiple time points over 48 hours. Variables quantified for each PCO captured metabolic and morphological response to drug treatment, including the optical redox ratio and organoid area.ResultsThe optical redox ratio (NAD(P)H/(FAD+NAD(P)H)) was independent of PCO morphology pre-tieatment. Drugs that induced cell death decreased the optical redox ratio and growth rate compared to control. Multivariate analysis of redox and morphology variables identified distinct PCO sub-populations. Single-organoid tracking improved sensitivity to drug treatment compared to pooled organoid analysis.ConclusionWidefield one-photon redox imaging can monitor metabolic and morphological changes on a single organoid-level, providing an accessible, non-destructive tool to screen drugs in patient-matched samples.