The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12.

CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology, and activity-based metabolomics, we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human anti-microbial metabolite and immunomodulator. We report that CLYBL loss leads to a cell-autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analog inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation.

Flexible B12 ecophysiology of Phaeocystis antarctica due to a fusion B12-independent methionine synthase with widespread homologues.

Coastal Antarctic marine ecosystems are significant in carbon cycling because of their intense seasonal phytoplankton blooms. Southern Ocean algae are primarily limited by light and iron (Fe) and can be co-limited by cobalamin (vitamin B12). Micronutrient limitation controls productivity and shapes the composition of blooms which are typically dominated by either diatoms or the haptophyte Phaeocystis antarctica. However, the vitamin requirements and ecophysiology of the keystone species P. antarctica remain poorly characterized. Using cultures, physiological analysis, and comparative omics, we examined the response of P. antarctica to a matrix of Fe-B12 conditions. We show that P. antarctica is not auxotrophic for B12, as previously suggested, and identify mechanisms underlying its B12 response in cultures of predominantly solitary and colonial cells. A combination of proteomics and proteogenomics reveals a B12-independent methionine synthase fusion protein (MetE-fusion) that is expressed under vitamin limitation and interreplaced with the B12-dependent isoform under replete conditions. Database searches return homologues of the MetE-fusion protein in multiple Phaeocystis species and in a wide range of marine microbes, including other photosynthetic eukaryotes with polymorphic life cycles as well as bacterioplankton. Furthermore, we find MetE-fusion homologues expressed in metaproteomic and metatranscriptomic field samples in polar and more geographically widespread regions. As climate change impacts micronutrient availability in the coastal Southern Ocean, our finding that P. antarctica has a flexible B12 metabolism has implications for its relative fitness compared to B12-auxotrophic diatoms and for the detection of B12-stress in a more diverse set of marine microbes.

Structural and mechanistic basis of the central energy-converting methyltransferase complex of methanogenesis.

Methanogenic archaea inhabiting anaerobic environments play a crucial role in the global biogeochemical material cycle. The most universal electrogenic reaction of their methane-producing energy metabolism is catalyzed by N    5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH), which couples the vectorial Na+ transport with a methyl transfer between the one-carbon carriers tetrahydromethanopterin and coenzyme M via a vitamin B12 derivative (cobamide) as prosthetic group. We present the 2.08 Šcryo-EM structure of Mtr(ABCDEFG)3 composed of the central Mtr(ABFG)3 stalk symmetrically flanked by three membrane-spanning MtrCDE globes. Tetraether glycolipids visible in the map fill gaps inside the multisubunit complex. Putative coenzyme M and Na+ were identified inside or in a side-pocket of a cytoplasmic cavity formed within MtrCDE. Its bottom marks the gate of the transmembrane pore occluded in the cryo-EM map. By integrating Alphafold2 information, functionally competent MtrA-MtrH and MtrA-MtrCDE subcomplexes could be modeled and thus the methyl-tetrahydromethanopterin demethylation and coenzyme M methylation half-reactions structurally described. Methyl-transfer-driven Na+ transport is proposed to be based on a strong and weak complex between MtrCDE and MtrA carrying vitamin B12, the latter being placed at the entrance of the cytoplasmic MtrCDE cavity. Hypothetically, strongly attached methyl-cob(III)amide (His-on) carrying MtrA induces an inward-facing conformation, Na+ flux into the membrane protein center and finally coenzyme M methylation while the generated loosely attached (or detached) MtrA carrying cob(I)amide (His-off) induces an outward-facing conformation and an extracellular Na+ outflux. Methyl-cob(III)amide (His-on) is regenerated in the distant active site of the methyl-tetrahydromethanopterin binding MtrH implicating a large-scale shuttling movement of the vitamin B12-carrying domain.

The structure of the rat vitamin B12 transporter TC and its complex with glutathionylcobalamin.

Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine, and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor, and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxocobalamin form can coordinate various nucleophiles, for example, GSH, giving rise to glutathionylcobalamin (GSCbl), a naturally occurring form of vitamin B12. Currently, there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12 transporter and GSCbl, which compared to aquo/hydroxocobalamin, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, haptocorrin and intrinsic factor are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl.

Differential utilization of vitamin B12-dependent and independent pathways for propionate metabolism across human cells.

Propionic acid links the oxidation of branched-chain amino acids and odd-chain fatty acids to the TCA cycle. Gut microbes ferment complex fiber remnants, generating high concentrations of short chain fatty acids, acetate, propionate and butyrate, which are shared with the host as fuel sources. Analysis of vitamin B12-dependent propionate utilization in skin biopsy samples has been used to characterize and diagnose underlying inborn errors of cobalamin (or B12) metabolism. In these cells, the B12-dependent enzyme, methylmalonyl-CoA mutase (MMUT), plays a central role in funneling propionate to the TCA cycle intermediate, succinate. Our understanding of the fate of propionate in other cell types, specifically, the involvement of the ß-oxidation-like and methylcitrate pathways, is limited. In this study, we have used [14C]-propionate tracing in combination with genetic ablation or inhibition of MMUT, to reveal the differential utilization of the B12-dependent and independent pathways for propionate metabolism in fibroblast versus colon cell lines. We demonstrate that itaconate can be used as a tool to investigate MMUT-dependent propionate metabolism in cultured cell lines. While MMUT gates the entry of propionate carbons into the TCA cycle in fibroblasts, colon-derived cell lines exhibit a quantitatively significant or exclusive reliance on the ß-oxidation-like pathway. Lipidomics and metabolomics analyses reveal that propionate elicits pleiotropic changes, including an increase in odd-chain glycerophospholipids, and perturbations in the purine nucleotide cycle and arginine/nitric oxide metabolism. The metabolic rationale and the regulatory mechanisms underlying the differential reliance on propionate utilization pathways at a cellular, and possibly tissue level, warrant further elucidation.

Vitamin B12 ameliorates gut epithelial injury via modulating the HIF-1 pathway and gut microbiota.

Inflammatory bowel diseases (IBDs) are immune chronic diseases characterized by recurrent episodes, resulting in continuous intestinal barrier damage and intestinal microbiota dysbiosis. Safe strategies aimed at stabilizing and reducing IBDs recurrence have been vigorously pursued. Here, we constructed a recurrent intestinal injury Drosophila model and found that vitamin B12 (VB12), an essential co-factor for organism physiological functions, could effectively protect the intestine and reduce dextran sulfate sodium-induced intestinal barrier disruption. VB12 also alleviated microbial dysbiosis in the Drosophila model and inhibited the growth of gram-negative bacteria. We demonstrated that VB12 could mitigate intestinal damage by activating the hypoxia-inducible factor-1 signaling pathway in injured conditions, which was achieved by regulating the intestinal oxidation. In addition, we also validated the protective effect of VB12 in a murine acute colitis model. In summary, we offer new insights and implications for the potential supportive role of VB12 in the management of recurrent IBDs flare-ups.

ThnL, a B12-dependent radical S-adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis.

Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.

Causes and consequences of impaired methionine synthase activity in acquired and inherited disorders of vitamin B12 metabolism.

Methyl-Cobalamin (Cbl) derives from dietary vitamin B12 and acts as a cofactor of methionine synthase (MS) in mammals. MS encoded by MTR catalyzes the remethylation of homocysteine to generate methionine and tetrahydrofolate, which fuel methionine and cytoplasmic folate cycles, respectively. Methionine is the precursor of S-adenosyl methionine (SAM), the universal methyl donor of transmethylation reactions. Impaired MS activity results from inadequate dietary intake or malabsorption of B12 and inborn errors of Cbl metabolism (IECM). The mechanisms at the origin of the high variability of clinical presentation of impaired MS activity are classically considered as the consequence of the disruption of the folate cycle and related synthesis of purines and pyrimidines and the decreased synthesis of endogenous methionine and SAM. For one decade, data on cellular and animal models of B12 deficiency and IECM have highlighted other key pathomechanisms, including altered interactome of MS with methionine synthase reductase, MMACHC, and MMADHC, endoplasmic reticulum stress, altered cell signaling, and genomic/epigenomic dysregulations. Decreased MS activity increases catalytic protein phosphatase 2A (PP2A) and produces imbalanced phosphorylation/methylation of nucleocytoplasmic RNA binding proteins, including ELAVL1/HuR protein, with subsequent nuclear sequestration of mRNAs and dramatic alteration of gene expression, including SIRT1. Decreased SAM and SIRT1 activity induce ER stress through impaired SIRT1-deacetylation of HSF1 and hypomethylation/hyperacetylation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), which deactivate nuclear receptors and lead to impaired energy metabolism and neuroplasticity. The reversibility of these pathomechanisms by SIRT1 agonists opens promising perspectives in the treatment of IECM outcomes resistant to conventional supplementation therapies.

Efficient NADPH-dependent dehalogenation afforded by a self-sufficient reductive dehalogenase.

Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.

Progesterone may be a regulator and B12 could be an indicator of the proximal D4Z4 repeat methylation status on 4q35ter.

Facioscapulohumeral dystrophy (FSHD) has a hypomethylation-related epigenetic background and exhibits a different course in male and female patients. The differences between males and females have been linked to the levels of sex hormones. This study is the first to investigate the possible effect of these hormones on methylation status. We hypothesized that the levels of sex-related hormones, estradiol, testosterone, progesterone, and prolactin might be associated with the methylation status of the proximal part of the D4Z4. We also investigated the effect of fT3, folic acid, and vitamin B12 levels. We collected blood from 28 FSHD patients and 28 controls. DNA was extracted from each individual for bisulfite methylation analysis and serum was separated for biochemical analysis of estradiol, testosterone, progesterone, prolactin, fT3, folic acid, and B12 analysis. Methylation analysis was specified to the DR1, 5P regions and the proximal region covering both DR1 and 5P. Methylation levels were compared between FSHD patients and controls. The correlation of methylation levels with estradiol, testosterone, progesterone, prolactin, fT3, folic acid, and B12 was investigated. We found that the 5P region and the proximal region were significantly hypomethylated in FSHD patients compared to the controls, but not the DR1 region. Male patients exhibited a significant reduction in DNA methylation compared to male controls. Older FSHD patients exhibited a notable decrease in fT3 levels and hypomethylation of the 5P region. Analyses of each CpG revealed seven hypomethylated positions that were significantly different from the control group. Two of the positions demonstrated a correlation with progesterone in the control group. With the exception of one position, the methylation levels were inversely correlated with vitamin B12 in FSHD patients. The results of our study indicate that the methylation of the proximal D4Z4 region, particularly at specific positions, may be associated with progesterone. In addition, vitamin B12 may be an indicator of hypomethylation. We suggest that examining position-specific methylations may be a useful approach for the development of epigenetic treatment modalities.

Efficacy of Vitamin B12 and Adenosine Triphosphate in Enhancing Skin Radiance: Unveiled with a Drug-Target Interaction Deep Learning-Based Model.

Skin radiance is crucial for enhancing facial attractiveness and is negatively affected by factors like hyperpigmentation and aging-related changes. Current treatments often lack comprehensive solutions for improving skin radiance. This study aimed to develop a cosmetic formula that enhances skin radiance by reducing hyperpigmentation and improving skin regeneration by targeting specific receptors-the endothelin receptor type B (EDNRB) for hyperpigmentation and the adiponectin receptor 1 (ADIPOR1) for sagging and wrinkles. To achieve this, we used artificial intelligence technologies to screen and select ingredients with an affinity for EDNRB and ADIPOR1. Vitamin B12 (VitB12) was identified as a molecule that targets EDNRB, which is involved in melanogenesis. Adenosine triphosphate (ATP) targets ADIPOR1, which is associated with skin regeneration. VitB12 successfully inhibited intracellular calcium elevation and melanogenesis induced by endothelin-1. In contrast, ATP increased the mRNA expression of collagen and elastin and promoted wound healing. Moreover, the VitB12 and ATP complex significantly increased the expression of hyaluronan synthases, which are crucial for skin hydration. Furthermore, in human participants, the application of the VitB12 and ATP complex to one-half of the face significantly improved skin radiance, elasticity, and texture. Our findings provide valuable insights for the development of skincare formulations.

Low vitamin B12 blood levels in sickle cell disease: Data from a large cohort study in Tanzania.

Sickle cell disease (SCD) is associated with high rates of undernutrition and stunting. Undernutrition in combination with chronic haemolysis may lead to deficiencies in micronutrients necessary for erythropoiesis. Here we examined selected levels of ferritin, vitamins B2 , B6 , B9 and B12 , and vitamin C that were measured in blood samples from 820 SCD patients from Tanzania with no history of hospital admission, infections or painful episodes in the previous 30 days. We studied children (0-8 years), early adolescents (9-14 years), late adolescents (15-17 years) and adults (≥18 years). Severely low levels of vitamin B12 were observed across the four age groups. Despite the lowered vitamin B12 concentrations, total homocysteine concentrations were normal across both genders in all age groups. We found no significant gender-related differences between the other measured micronutrients. In this large SCD population, spanning the whole life cycle, a low level of vitamin B12 was consistently found across both genders and all age groups. Given the pivotal role of vitamin B12 in cellular metabolism, particularly in erythropoiesis, more studies are required to unravel how to better detect clinically relevant vitamin B12 deficiency among SCD patients, and thus to identify more precisely those who need supplementation of vitamin B12 .

Microangiopathic thrombocytopenia caused by vitamin B12 deficiency responding to plasma exchange.

A young adult African American female presented with normocytic microangiopathic haemolytic anaemia, elevated lactate dehydrogenase and thrombocytopenia. The patient responded to therapeutic plasma exchanges (TPE) for presumed thrombotic microangiopathy caused by thrombotic thrombocytopenic purpura (TTP). After relapsing, the patient was found to have pancytopenia, megaloblastic bone marrow and low vitamin B12 consistent with pernicious anaemia, which improved with intramuscular B12 and discontinuation of TPE. B12-deficient macrocytosis was not seen at presentation due to concomitant alpha-thalassaemia. Initial clinical/laboratory improvement is attributed to B12 present in TPE plasma. B12 deficiency can mimic TTP. Vigilance is needed regarding atypical presentations of pernicious anaemia.

Vitamin B12 deficiency misdiagnosed as TTP: What can we learn from it?

The case report by Dwyre et al. shows that vitamin B12 deficiency may be misdiagnosed as acute thrombotic thrombocytopenic purpura. Together with similar observations, this underlines that acquired vitamin B12 deficiency-besides the inherited disorder of intracellular cobalamin metabolism, cbl C disease-should be listed as a separate entity of the thrombotic microangiopathies. Commentary on: Dwyre et al. Microangiopathic thrombocytopenia caused by vitamin B12 deficiency responding to plasma exchange. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19625.

Vitamin B12 inhibits peptidylarginine deiminases and ameliorates rheumatoid arthritis in CAIA mice.

Rheumatoid arthritis is an autoimmune disease whose early onset correlates with dysregulated citrullination, a process catalyzed by peptidylarginine deiminase isoform 4 (PADI-4). Here, we report that PADI-4 is a novel target of vitamin B12, a water-soluble vitamin that serves as a cofactor in DNA synthesis and the metabolism of fatty and amino acids. Vitamin B12 preferentially inhibited PADI-4 over PADI-2 with comparable inhibitory activity to the reference compound Cl-amidine in enzymatic inhibition assays, and reduced total cellular citrullination levels including that of histone H3 citrullination mediated by PADI-4. We also demonstrated that hydroxocobalamin, a manufactured form of vitamin B12, significantly ameliorated the severity of collagen type II antibody induced arthritis (CAIA) in mice and diminished gene expression of the rheumatoid inflammatory factors and cytokines IL17A, TNFα, IL-6, COX-II and ANXA2, as well PADI-4. Therefore, the use of vitamin B12 to treat rheumatoid arthritis merits further study.

Vitamin B12 status and the risk of developing sepsis in patients with bacterial infection: a prospective observational cohort study.

BACKGROUND: Data have shown that vitamin B12 has immunomodulatory effects via different pathways, which could influence the pathophysiology of sepsis. The objective of this study was to investigate whether vitamin B12 levels, assessed by the measurement of holotranscobalamin (HTC), total vitamin B12 (B12), and methylmalonic acid (MMA, which accumulates in case of B12 deficiency), are associated with the development of sepsis in patients with onset of bacterial infection. METHODS: This was a single-center, prospective observational pilot study. Adult patients who presented to the emergency department with bacterial infection confirmed by a positive microbiological culture result were included in the study and followed up for 6 days to assess whether they developed sepsis or not. The primary objective was to compare HTC concentration in patients who developed sepsis to those who did not develop sepsis. Secondary objectives were the evaluation of B12 and MMA concentrations in those two groups. Multiple logistic regression models were used, with presence of sepsis as the outcome variable, and HTC, B12, and MMA concentrations as predictor variables, separately, and adjusted for potential confounders. RESULTS: From 2019 to 2022, 2131 patients were assessed for eligibility, of whom 100 met the inclusion criteria. One patient was excluded from the analysis due to missing data. Of the 99 patients, 29 developed sepsis. There was no evidence for an association between HTC or B12 concentration and the development of sepsis (OR 0.65, 95% CI 0.31-1.29, p = 0.232, OR 0.84, 95% CI 0.44-1.54, p = 0.584, respectively). There was an association between MMA concentration and the development of sepsis, with a positive effect, i.e. with increasing MMA, the odds for sepsis increased (OR 2.36, 95% CI 1.21-4.87, p = 0.014). This association remained significant when adjusted for confounders (OR 2.72, 95% CI 1.23-6.60, p = 0.018). CONCLUSIONS: Our study found an association between elevated MMA concentration and the development of sepsis. We did not find an association between HTC and B12 concentrations and the development of sepsis. Further, larger studies are warranted, as it could lead to interventional trials investigating whether B12 supplementation provides a clinical benefit to patients with infection or sepsis. TRIAL REGISTRATION: The study was registered on ClinicalTrials.gov under the identifier NCT04008446 on June 17, 2019.

Serum interleukin-17 A and homocysteine levels in children with autism.

BACKGROUND: Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that typically emerges early in childhood. This study aimed to explore the potential link between serum levels of vitamin B12 and homocysteine (Hcy) and the severity of ASD symptoms in children. METHODS: In this study, 50 children diagnosed with ASD comprised the observation group, while 50 healthy children constituted the control group. Serum levels of IL-17 A, Hcy, folate, and vitamin B12 were compared between the study group and control group, as well as among children with different degrees of ASD severity. The correlation between the Childhood Autism Rating Scale (CARS) score and serum levels of IL-17 A, Hcy, folate, and vitamin B12 was examined. Additionally, the relationship between serum IL-17 A and Hcy levels and their association with the severity ASD were explored. RESULTS: Compared to the control group, the observation group demonstrated elevated serum Hcy and IL-17 A levels alongside decreased folate and vitamin B12 levels. Individuals with severe ASD exhibited higher Hcy and IL-17 A levels but lower folate and vitamin B12 levels compared to those with mild to moderate ASD. The CARS score showed negative correlations with serum folate and vitamin B12 levels and positive correlations with serum IL-17 A and Hcy levels in ASD patients. Additionally, serum Hcy and IL-17 A levels were correlated with ASD severity. CONCLUSION: Children diagnosed with ASD presented with reduced serum vitamin B12 levels and increased levels of Hcy, potentially contributing to the onset and severity of ASD.

The MMACHC variant c.158T>C: Mild clinical and biochemical phenotypes and marked hydroxocobalamin response in cblC patients.

Mutations in MMACHC cause cobalamin C disease (cblC, OMIM 277400), the commonest inborn error of vitamin B12 metabolism. In cblC, deficient activation of cobalamin results in methylcobalamin and adenosylcobalamin deficiency, elevating methylmalonic acid (MMA) and total plasma homocysteine (tHcy). We retrospectively reviewed the medical files of seven cblC patients: three compound heterozygotes for the MMACHC (NM_015506.3) missense variant c.158T>C p.(Leu53Pro) in trans with the common pathogenic mutation c.271dupA (p.(Arg91Lysfs*14), "compounds"), and four c.271dupA homozygotes ("homozygotes"). Compounds receiving hydroxocobalamin intramuscular injection monotherapy had age-appropriate psychomotor performance and normal ophthalmological examinations. In contrast, c.271dupA homozygotes showed marked psychomotor retardation, retinopathy and feeding problems despite penta-therapy (hydroxocobalamin, betaine, folinic acid, l-carnitine and acetylsalicylic acid). Pretreatment levels of plasma and urine MMA and tHcy were higher in c.271dupA homozygotes than in compounds. Under treatment, levels of the compounds approached or entered the reference range but not those of c.271dupA homozygotes (tHcy: compounds 9.8-32.9 µM, homozygotes 41.6-106.8 (normal (N) < 14); plasma MMA: compounds 0.14-0.81 µM, homozygotes, 10.4-61 (N < 0.4); urine MMA: compounds 1.75-48 mmol/mol creatinine, homozygotes 143-493 (N < 10)). Patient skin fibroblasts all had low cobalamin uptake, but this was milder in compound cells. Also, the distribution pattern of cobalamin species was qualitatively different between cells from compounds and from homozygotes. Compared to the classic cblC phenotype presented by c.271dupA homozygous patients, c.[158T>C];[271dupA] compounds had mild clinical and biochemical phenotypes and responded strikingly to hydroxocobalamin monotherapy.

S100A4-neutralizing monoclonal antibody 6B12 counteracts the established experimental skin fibrosis induced by bleomycin.

OBJECTIVES: Our previous studies have demonstrated that the Damage Associated Molecular Pattern (DAMP) protein, S100A4, is overexpressed in the involved skin and peripheral blood of patients with SSc. It is associated with skin and lung involvement, and disease activity. By contrast, lack of S100A4 prevented the development of experimental dermal fibrosis. Herein we aimed to evaluate the effect of murine anti-S100A4 mAb 6B12 in the treatment of preestablished experimental dermal fibrosis. METHODS: The effects of 6B12 were assessed at therapeutic dosages in a modified bleomycin-induced dermal fibrosis mouse model by evaluating fibrotic (dermal thickness, proliferation of myofibroblasts, hydroxyproline content, phosphorylated Smad3-positive cell count) and inflammatory (leukocytes infiltrating the lesional skin, systemic levels of selected cytokines and chemokines) outcomes, and transcriptional profiling (RNA sequencing). RESULTS: Treatment with 7.5 mg/kg 6B12 attenuated and might even reduce pre-existing dermal fibrosis induced by bleomycin as evidenced by reduction in dermal thickness, myofibroblast count and collagen content. These antifibrotic effects were mediated by the downregulation of TGF-ß/Smad signalling and partially by reducing the number of leukocytes infiltrating the lesional skin and decrease in the systemic levels of IL-1α, eotaxin, CCL2 and CCL5. Moreover, transcriptional profiling demonstrated that 7.5 mg/kg 6B12 also modulated several profibrotic and proinflammatory processes relevant to the pathogenesis of SSc. CONCLUSION: Targeting S100A4 by the 6B12 mAb demonstrated potent antifibrotic and anti-inflammatory effects on bleomycin-induced dermal fibrosis and provided further evidence for the vital role of S100A4 in the pathophysiology of SSc.

Elevated Vitamin B12, Risk of Cancer, and Mortality: A Systematic Review.

Vitamin B12 (B12) is a molecule involved in several biological. Abnormally high levels are frequently found, but their causes can be multiple, and consequences have not been clearly elucidated. The objective of this review was to summarize the current evidence on the associations of elevated B12 and the development of cancer, and all-cause mortality in adults. Six references looking at mortality and seven looking at cancer risk were included. The evidence suggests an association between elevated B12 with a higher risk of cancer, with risk ratios ranging 1,88 to 5,9. There was less consistent evidence linking vitamin B12 and mortality.

Elevated B12 levels exceeding 1000 pg/L, if sustained and unexplainable, warrant a comprehensive individual assessment of each patient. This evaluation should encompass various potential factors contributing to the elevation, aiming to effectively guide the diagnostic process of neoplastic diseases.Clinical longitudinal observational studies have suggested a potential link between heightened B12 levels and the risks of cancer and mortality. Nonetheless, these studies have been retrospective cohort studies, and lack a defined threshold point of B12 levels.Studies have documented a positive correlation between elevated levels of B12 and the incidence of lung, pancreatic, and liver cancers, as well as certain hematological neoplasms, particularly those related to the myeloid lineage. Conversely, a consistent negative association has been observed in the context of breast cancer. Findings concerning neoplasms of the lower gastrointestinal tract and prostate display contradictory outcomes.The diagnostic significance of elevated B12 levels among patients already diagnosed with cancer remains uncertain and could potentially be linked to reverse causality.