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We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway. Significance This paper describes the development of a computationally designed enzyme that is the cornerstone of a novel metabolic pathway. This enzyme, formolase, performs a carboligation reaction, directly fixing one-carbon units into three-carbon units that feed into central metabolism. By combining formolase with several naturally occurring enzymes, we created a new carbon fixation pathway, the formolase pathway, which assimilates one-carbon units via formate. Unlike native carbon fixation pathways, this pathway is linear, not oxygen sensitive, and consists of a small number of thermodynamically favorable steps. We demonstrate in vitro pathway function as a proof of principle of how protein design in a pathway context can lead to new efficient metabolic pathways.

There is worldwide interest in newborn screening for lysosomal storage diseases because of the development of treatment options that give better results when carried out early in life. Screens with high differentiation between affected and nonaffected individuals are critical because of the large number of potential false positives. This review summarizes 3 screening methods: (a) direct assay of enzymatic activities using tandem mass spectrometry or fluorometry, (b) immunocapture-based measurement of lysosomal enzyme abundance, and (c) measurement of biomarkers. Assay performance is compared on the basis of small-scale studies as well as on large-scale pilot studies of mass spectrometric and fluorometric screens. Tandem mass spectrometry and fluorometry techniques for direct assay of lysosomal enzymatic activity in dried blood spots have emerged as the most studied approaches. Comparative mass spectrometry vs fluorometry studies show that the former better differentiates between nonaffected vs affected individuals. This in turn leads to a manageable number of screen positives that can be further evaluated with second-tier methods.

The Diels-Alder reaction is a cornerstone in organic synthesis, forming two carbon-carbon bonds and up to four new stereogenic centers in one step. No naturally occurring enzymes have been shown to catalyze bimolecular Diels-Alder reactions. We describe the de novo computational design and experimental characterization of enzymes catalyzing a bimolecular Diels-Alder reaction with high stereoselectivity and substrate specificity. X-ray crystallography confirms that the structure matches the design for the most active of the enzymes, and binding site substitutions reprogram the substrate specificity. Designed stereoselective catalysts for carbon-carbon bond-forming reactions should be broadly useful in synthetic chemistry.

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A₂ IIA (sPLA₂-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA₂-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA₂-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA₂-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

Background Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by mutations in the arylsulfatase A gene ( ARSA ) and categorized into three subtypes according to age of onset. The functional effect of most ARSA mutants remains unknown; better understanding of the genotype–phenotype relationship is required to support newborn screening (NBS) and guide treatment. Results We collected a patient data set from the literature that relates disease severity to ARSA genotype in 489 individuals with MLD. Patient-based data were used to develop a phenotype matrix that predicts MLD phenotype given ARSA alleles in a patient’s genotype with 76% accuracy. We then employed a high-throughput enzyme activity assay using mass spectrometry to explore the function of ARSA variants from the curated patient data set and the Genome Aggregation Database (gnomAD). We observed evidence that 36% of variants of unknown significance (VUS) in ARSA may be pathogenic. By classifying functional effects for 251 VUS from gnomAD, we reduced the incidence of genotypes of unknown significance (GUS) by over 98.5% in the overall population. Conclusions These results provide an additional tool for clinicians to anticipate the disease course in MLD patients, identifying individuals at high risk of severe disease to support treatment access. Our results suggest that more than 1 in 3 VUS in ARSA may be pathogenic. We show that combining genetic and biochemical information increases diagnostic yield. Our strategy may apply to other recessive diseases, providing a tool to address the challenge of interpreting VUS within genotype–phenotype relationships and NBS.

Purpose Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by the deficiency of arylsulfatase A (ARSA), which results in the accumulation of sulfatides. Newborn screening for MLD may be considered in the future as innovative treatments are advancing. We carried out a research study to assess the feasibility of screening MLD using dried blood spots (DBS) from de-identified newborns. Methods To minimize the false-positive rate, a two-tier screening algorithm was designed. The primary test was to quantify C16:0-sulfatide in DBS by ultraperformance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The screening cutoff was established based on the results from 15 MLD newborns to achieve 100% sensitivity. The secondary test was to measure the ARSA activity in DBS from newborns with abnormal C16:0-sulfatide levels. Only newborns that displayed both abnormal C16:0-sulfatide abundance and ARSA activity were considered screen positives. Results A total of 27,335 newborns were screened using this two-tier algorithm, and 2 high-risk cases were identified. ARSA gene sequencing identified these two high-risk subjects to be a MLD-affected patient and a heterozygote. Conclusion Our study demonstrates that newborn screening for MLD is highly feasible in a real-world scenario with near 100% assay specificity.

Background Krabbe disease is a rare neurodegenerative genetic disorder caused by deficiency of galactocerebrosidase. Patients with the infantile form of Krabbe disease can be treated at a presymptomatic stage with human stem cell transplantation which improves survival and clinical outcomes. However, without a family history, most cases of infantile Krabbe disease present after onset of symptoms and are ineligible for transplantation. In 2006, New York began screening newborns for Krabbe disease to identify presymptomatic cases. To ensure that those identified with infantile disease received timely treatment, New York public health and medical systems took steps to accurately diagnose and rapidly refer infants for human stem cell transplantation within the first few weeks of life. After 11 years of active screening in New York and the introduction of Krabbe disease newborn screening in other states, new information has been gained which can inform the design of newborn screening programs to improve infantile Krabbe disease outcomes. Findings Recent information relevant to Krabbe disease screening, diagnosis, and treatment were assessed by a diverse group of public health, medical, and advocacy professionals. Outcomes after newborn screening may improve if treatment for infantile disease is initiated before 30 days of life. Newer laboratory screening and diagnostic tools can improve the speed and specificity of diagnosis and help facilitate this early referral. Given the rarity of Krabbe disease, most recommendations were based on case series or expert opinion. Conclusion This report updates recommendations for Krabbe disease newborn screening to improve the timeliness of diagnosis and treatment of infantile Krabbe disease. In the United States, several states have begun or are considering Krabbe disease newborn screening. These recommendations can guide public health laboratories on methodologies for screening and inform clinicians about the need to promptly diagnose and treat infantile Krabbe disease. The timing of the initial referral after newborn screening, the speed of diagnostic confirmation of infantile disease, and the transplantation center’s experience and ability to rapidly respond to a suspected patient with newly diagnosed infantile Krabbe disease are critical for optimal outcomes.

Cerebrotendinous xanthomatosis (CTX) is a treatable hereditary disorder caused by the deficiency of sterol 27-hydroxylase, which is encoded by the CYP27A1 gene. Different newborn screening biomarkers for CTX have been described, including 7α,12α-dihydroxy-4-cholesten-3-one (7α12αC4), 5β-cholestane-3α,7α,12α,25-tetrol glucuronide (GlcA-tetrol), the ratio of GlcA-tetrol to tauro-chenodeoxycholic acid (t-CDCA) (GlcA-tetrol/t-CDCA), and the ratio of tauro-trihydroxycholestanoic acid (t-THCA) to GlcA-tetrol (t-THCA/GlcA-tetrol). We set out to evaluate these screening methods in a research study using over 32,000 newborn dried blood spots (DBS). Metabolites were extracted from DBS with methanol containing internal standard, which was then quantified by ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The measurement of 7α12αC4 was complicated by isobaric interferences and was discontinued. A total of 32,737 newborns were screened based on the GlcA-tetrol concentration in DBS. GlcA-tetrol/t-CDCA and t-THCA/GlcA-tetrol ratios were also calculated. Newborns displaying both elevated GlcA-tetrol and GlcA-tetrol/t-CDCA ratio were considered to be screen positives. The t-THCA/GlcA-tetrol ratio was used to further distinguish CTX screen positives from Zellweger Spectrum Disorder (ZSD) screen positives. Only one newborn displayed both elevated GlcA-tetrol concentration in DBS and a typical CTX biochemical profile. This newborn was interpreted as a CTX-affected patient as CYP27A1 gene sequencing identified two known pathogenic variants. The results indicate that both GlcA-tetrol and the GlcA-tetrol/t-CDCA ratio are excellent CTX biomarkers suitable for newborn screening. By characterizing the relationship of GlcA-tetrol, t-CDCA, and t-THCA as secondary markers, 100% assay specificity can be achieved.