Excess Biotin Research Articles

Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells.

Ceramides generated by the activity of the neutral sphingomyelinase 2 (nSMase2) play a pivotal role in stress responses in mammalian cells. Dysregulation of sphingolipid metabolism has been implicated in numerous inflammation-related pathologies. However, its influence on inflammatory cytokine-induced signaling is yet incompletely understood. Here, we used proximity labeling to explore the plasma membrane proximal protein network of nSMase2 and TNFα-induced changes thereof. We established Jurkat cells stably expressing nSMase2 C-terminally fused to the engineered ascorbate peroxidase 2 (APEX2). Removal of excess biotin phenol substantially improved streptavidin-based affinity purification of biotinylated proteins. Using our optimized protocol, we determined nSMase2-proximal biotinylated proteins and their changes within the first 5min of TNFα stimulation by quantitative mass spectrometry. We observed significant dynamic changes in the nSMase2 microenvironment in response to TNFα stimulation consistent with rapid remodeling of protein networks. Our data confirmed known nSMase2 interactors and revealed that the recruitment of most proteins depended on nSMase2 enzymatic activity. We measured significant enrichment of proteins related to vesicle-mediated transport, including proteins of recycling endosomes, trans-Golgi network, and exocytic vesicles in the proximitome of enzymatically active nSMase2 within the first minutes of TNFα stimulation. Hence, the nSMase2 proximal network and its TNFα-induced changes provide a valuable resource for further investigations into the involvement of nSMase2 in the early signaling pathways triggered by TNFα.

FRI518 Good For Hair, Skin, And Nails But Bad For Thyroid Lab Tests

Abstract Disclosure: S. Khan: None. P. Kachhadia: None. S. Aldasouqi: None. A. Abu Limon: None. Introduction: Biotin also known as Vitamin B7 is very commonly used for hair, skin and nail growth. It can cause falsely high levels of free thyroxine (FT4), and free triiodothyronine (FT3) and falsely low thyroid stimulating hormone (TSH) levels. This can lead to a false diagnosis of hyperthyroidism. The ATA has recommended to stop taking biotin at least 2 days prior to lab testing to avoid spurious results. We present a case of a patient with hypothyroidism on a stable dose of levothyroxine who developed abnormal thyroid function tests (TFTs) due to biotin supplements. Case Presentation: A 57 years old female with long-standing hypothyroidism had been a on stable dose of Levothyroxine, 100 mcg daily. She was compliant with treatment. Her TFTs, done on periodic follow ups every 3-6 months were normal. Recently, on semi-annual lab work, thyroid function tests revealed elevated free T4 and T3, but normal TSH values. The patient was clinically euthyroid. Due to the unexpected thyroid function test result which did not portray clinical hyperthyroidism, repeat detailed history was taken which revealed that the patient was taking Biotin 1000 mg twice daily, as a hair-skin-nail beauty product. TFTs were repeated after she was instructed to stop Biotin for 3 days, which resulted in normal values. Patient was well-educated by our team to stop Biotin before labs, in the future. Once again, close to a year later, TFTs showed elevated FT4 and borderline low TSH. When contacted, she admitted forgetting to stop Biotin before labs. Labs off biotin once more showed normalization of FT4. Discussion: A variety of laboratory immunoassay blood tests utilize Biotin (Vitamin B7) in view of its high affinity to Streptavidin. At physiological concentrations, Biotin does not interfere with thyroid function tests. However, excessive Biotin use which is often taken as a beauty supplement is known to interfere with these tests. Typically, excessive Biotin intake will result in falsely elevated T4 and T3 and falsely low TSH. Furthermore, it will result in falsely elevated immunoglobulins that are markers for Graves’ disease. Our patient was taking a total of 2000mcg of biotin daily over the counter. This interference, which is not uncommon in clinical practice, has led to many misdiagnoses with potential for unnecessary workup, and sometimes initiation of inappropriate or potentially harmful treatment regimens. In this case report, though this patient’s thyroid function test results were suggestive of hyperthyroidism, biotin use is what caused in-vitro interference with the Biotin-Streptavidine Immunoassay. For this reason, clinicians should remain attentive to common biotin use in patients and advise them to hold biotin at least 2 days before labwork to avoid such outcomes. Furthermore, lab centers should also be informed of this interference and should avoid lab draw in those patients did that not hold biotin appropriately. Presentation: Friday, June 16, 2023

Abstract #1183681: Biotin-related lab interference: The Prevalence of excessive Biotin use in rural Michigan

Excessive intake of Biotin (Vitamin B7) can interfere with numerous lab tests that are analyzed via Striptavidin-Biotin immunoassays. Increased Biotin use in recent years, for skin-hair-nail health and as energy boosts and nutritional supplements, has lead to a parallel increase in lab interference in several commonly used lab tests, leading to misdiagnosis and unnecessary work up and treatment. Biotin intake of > 5 mg per day can cause such lab interference. A common misdiagnosis in numerous case reports and case series has been Graves' disease (falsely elevated thyroid hormones, coupled with suppressed TSH and positive TSI and TRAB).

Biotin as a masking agent in chorionic gonadotropin assays utilizing biotinylated antibodies.

Biotin interference in streptavidin/biotin-based immunoassays has been recently recognized as a confounding factor in clinical settings. Depending on the nature of the assay, the presence of excess biotin in patient samples can cause falsely high or low results. One of the platforms known to be affected, Roche Cobas, is widely used in anti-doping laboratories to test for intact chorionic gonadotropin (hCG) in urine. While biotin levels in blood have been well studied, less is known about urinary biotin due to its limited clinical significance. Having analyzed over 4,000 urine samples, we have established a reference range for urinary biotin with a median concentration of approximately 12 ng/ml. However, a significant number of samples contain much higher amounts, with a maximum approaching 10μg/ml, suggesting biotin supplementation. Consequently, the tolerance of hCG STAT assay towards biotin was investigated over a wide concentration range. The apparent hCG concentration was found to decrease almost linearly as biotin increased from 100 to 1,000 ng/ml, with only 10% of the expected value reported by the assay as biotin reached 1,000 ng/ml. Further increase of biotin resulted in a progressive, albeit more moderate, decline in measured hCG concentration. To avoid a false negative result in the context of anti-doping analysis, it is highly recommended to monitor biotin in urine and perform diafiltration before hCG measurement in samples with elevated biotin to remove the interference.

45-Year-Old Woman With Fever and Malaise

45-Year-Old Woman With Fever and Malaise

Spurious Thyroid Function Test Results due to Biotin Interference: a Report of Three Cases and a Literature Review

Biotin (vitamin B7) is a water-soluble vitamin used as a co-enzyme for carboxylases essential for human metabolism. The high affinity to streptavidin makes biotin an important substance in immunoassays. Excessive biotin intake due to over-the counter supplements has become problematic because of the effects on laboratory test results. There have been no reports of biotin-induced thyroid immunoassay interference in Korea. We report three patients with papillary thyroid cancer who showed false thyrotoxicosis on follow-up laboratory examinations with a literature review. The patients’ medical history should be thoroughly questioned and patients should be informed to curtail consuming biotin before laboratory tests to avoid assay interference. Non-biotinylated assays can be considered if it is impossible to withhold the supplements. These methods will prevent physicians from making incorrect decisions that could result in an inappropriate treatment for their patients.

XRE-Type Regulator BioX Acts as a Negative Transcriptional Factor of Biotin Metabolism in Riemerella anatipestifer.

Biotin is essential for the growth and pathogenicity of microorganisms. Damage to biotin biosynthesis results in impaired bacterial growth and decreased virulence in vivo. However, the mechanisms of biotin biosynthesis in Riemerella anatipestifer remain unclear. In this study, two R. anatipestifer genes associated with biotin biosynthesis were identified. AS87_RS05840 encoded a BirA protein lacking the N-terminal winged helix-turn-helix DNA binding domain, identifying it as a group I biotin protein ligase, and AS87_RS09325 encoded a BioX protein, which was in the helix-turn-helix xenobiotic response element family of transcription factors. Electrophoretic mobility shift assays demonstrated that BioX bound to the promoter region of bioF. In addition, the R. anatipestifer genes bioF (encoding 7-keto-8-aminopelargonic acid synthase), bioD (encoding dethiobiotin synthase), and bioA (encoding 7,8-diaminopelargonic acid synthase) were in an operon and were regulated by BioX. Quantitative reverse transcription-PCR showed that transcription of the bioFDA operon increased in the mutant Yb2ΔbioX in the presence of excessive biotin, compared with that in the wild-type strain Yb2, suggesting that BioX acted as a repressor of biotin biosynthesis. Streptavidin blot analysis showed that BirA caused biotinylation of BioX, indicating that biotinylated BioX was involved in metabolic pathways. Moreover, as determined by the median lethal dose, the virulence of Yb2ΔbioX was attenuated 500-fold compared with that of Yb2. To summarize, the genes birA and bioX were identified in R. anatipestifer, and BioX was found to act as a repressor of the bioFDA operon involved in the biotin biosynthesis pathway and identified as a bacterial virulence factor. IMPORTANCE Riemerella anatipestifer is a causative agent of diseases in ducks, geese, turkeys, and various other domestic and wild birds. Our study reveals that biotin synthesis of R. anatipestifer is regulated by the BioX through binding to the promoter region of the bioF gene to inhibit transcription of the bioFDA operon. Moreover, bioX is required for R. anatipestifer pathogenicity, suggesting that BioX is a potential target for treatment of the pathogen. R. anatipestifer BioX has thus been identified as a novel negative regulator involved in biotin metabolism and associated with bacterial virulence in this study.

A novel host-guest complex based on biotin functionalized polyamine-β-cyclodextrin for tumor targeted delivery of luteolin

Luteolin (LU) shows significant bioactivities, but inherent poor aqueous solubility and stability severely hamper its applications in pharmaceutical application. Herein, a series of efficient tumor targeted drug carriers were designed and synthesised by binding biotin to a β-cyclodextrin through a polyamine chain. Their characteristics and inclusion behavior with LU in aqueous and solid states were investigated by UV-Vis, NMR, XRD, FT-IR, TG, and SEM. Moreover, tumor targeted effect of inclusion complexes was confirmed by the Confocal Laser Scanning Microscopy (CLSM) images and the decrease of anti-cancer ability owing to biotin receptor saturated with excess biotin added. Besides, anticancer activity and solubility of inclusion complexes were greatly improved by 27 and 1423 folds, respectively, compared with free LU. It suggests biotin-polyamine-β-CDs (BCDs) might be promising tumor targeted carriers, which are expected to be applied in the pharmaceutical field for further research.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

SummaryNeurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

Design and synthesis of a multivalent catch-and-release assay to measure circulating FXIa

BackgroundDecreased blood coagulation factor (F)XIa levels have been shown to protect from thrombosis without bleeding side effects, but less is known on effects of increased FXIa levels. Studies are hampered by lack of a reliable and robust method for FXIa quantification in blood. We aim to develop a new assay employing a unique multivalent catch-and-release system. The system selectively isolates and protects homodimeric FXIa from plasma and releases free FXIa allowing subsequent quantification. MethodsA dynamic multivalent construct was synthesized by complexing four identical FXIa inhibitors from the snake Bungarus Fasxiatus to avidin through desthiobiotin-PEG-linkers, allowing dissociation of FXIa by excess biotin. PEG-linker lengths were optimised for FXIa inhibitory activity and analysed by Michaelis-Menten kinetics. Finally, the catch-and-release assay was validated in buffer and plasma model systems. ResultsMonovalent and multivalent inhibitor constructs were successfully obtained by total chemical synthesis. Multimerisation of Fasxiator resulted in a 30-fold increase in affinity for FXIa from 1.6 nM to 0.05 nM. With use of this system, FXIa could be quantified down to a concentration of 7 pM in buffer and 20 pM in plasma. ConclusionIn this proof-of-concept study, we have shown that the catch-and-release approach is a promising technique to quantify FXIa in plasma or buffer. By binding FXIa to the multivalent construct directly after blood drawing, FXIa is hypothesized to be inaccessible for serpin inhibition or auto inactivation. This results in a close reflection of actual circulating FXIa levels at the moment of blood drawing.

Dietary Opinion Beyond Biotin Intakes

: Biotin (Vitamin-H) intake is usual in individuals taking balanced diet on regular basis as biotin is present in common dietary substances. Regarding its intakes some recommendations must be keep in mind in order to avoid any inconvenience. Although biotin deficiency is rare but may occur under some special circumstances. Biotin creates hindrance in results of many medical tests reports so while taking biotin as diet or supplements, your consultant should aware of your biotin intakes. Some specific diseased conditions can be avoided only by excessive biotin intakes in dietary forms also.

The development of a biotin-guided and mitochondria-targeting fluorescent probe for detecting SO2 precisely in cancer cells

Mitochondrial sulfur dioxide (SO2) is very closely associated with various activities of cancer cell. However, the specific physiological and pathological roles of mitochondrial SO2 in cancer cells are still not well defined. Lacking a powerful molecular tool for detecting mitochondrial SO2 in cancer cells precisely is an essential factor. So it is urgent to develop a specific method for monitoring mitochondrial SO2 in cancer cells. Herein, we described a distinct cancer cell-specific fluorescent probe NS for detecting mitochondrial SO2 accurately in cancer cells. Biotin, possessing of high affinity for cancer cells, was decorated into probe to provide its cancer cell-targeting property. Moreover, the positive charge hemicyanine group was used to anchor mitochondria selectively. A series of spectral results from concentration titration, dynamics and selectivity experiments showed that NS had high sensitivity, fast response and high selectivity to SO2. These properties render NS ability for detecting SO2 in living cells. In biological imaging, the achievements in detecting exogenous and endogenous SO2 displayed the probe had favorable response to SO2 in living cells with well biocompatibility. Significantly, assisted by competitive experiments with excess biotin, NS demonstrated distinct cancer cell-targeting for detecting mitochondrial SO2. Furthermore, NS could locate mitochondria specially and detect mitochondrial SO2 in cancer cells by co-localization. Moreover, NS can trace SO2 in zebrafish with long wavelength emission. Therefore, NS can achieve in tracing mitochondrial SO2 selectively in cancer cells. It would be a powerful tool for well defining the physiological and pathological roles of mitochondrial SO2 in cancer cells.

SUN-493 A “Curve Ball” in the Management of an Infertile Hyperthyroid Patient

Introduction: We present the case of a young female patient referred for hyperthyroidism with persistently detectable TSH despite elevated free thyroxine (FT4) levels which, interestingly, failed to normalize following anti-thyroidal treatment. Further testing elucidated the underlying causation for detectable TSH in presence of hyperthyroxinemia to be due to interfering substances in the thyroid function assays. Case: A 35-year-old female is evaluated for hyperthyroidism discovered in preparation for embryo transfer. History includes transient hyperthyroidism two years ago while undergoing in vitro fertilization. Prior workup included negative antibodies and nuclear uptake scan revealing a right-sided autonomously functioning thyroid nodule with 24-hour uptake of 40.7%. She was briefly treated with PTU in first trimester and lost to follow-up. While once again undergoing fertility treatment with clomiphene, the patient developed tremor and heat intolerance. She was found to have TSH 0.3 (0.45-4.12 mU/L) and analog FT4 of 6.4 (0.8-1.8 ng/dL). She denied use of OTC supplements. PTU 50 mg twice daily was initiated with minimal improvement with TSH 0.7 mU/L, FT4 3.7 ng/dL. On evaluation, slight tremor and mild thyroid enlargement were noted. Our differential expanded to include TSH-secreting pituitary adenoma, thyroid hormone resistance (no family history), and assay interference. Both MRI pituitary and an alpha-subunit level (0.36) were normal. Symptoms improved gradually with repeat TSH 1.79 mU/L, FT4 2.4 ng/DL by standard analog methods. We suspected ‘assay interference’ for which FT4 via direct equilibrium dialysis was obtained and was indeed normal at 0.76 ng/dL. Further lab testing verified interference in both in the standard TSH and FT4 assays presumed secondary to heterophile antibodies. Discussion: Interpretation of thyroid studies discordant with the clinical picture or incongruent with each other requires understanding of thyroid physiology and the intricacies of commonly utilized assays. The differential of elevated thyroxine levels with detectable/normal TSH rests between thyroid hormone resistance syndromes, TSH secreting pituitary tumors and interfering substances in the assay. Most commercially available TSH assays are based on a ‘sandwich’ assay which is notoriously interfered with by heterophile antibodies and excess biotin. Determination of FT4 is also challenging as the assay must detect very low concentrations of free hormone relative to excess of protein-bound analyte. When in question, it is important consider utilization of laboratory expertise and retesting by alternative assays.

Biotin Measurement by Targeted LC-MS/MS: Method Development and Application to the Evaluation of Immunoassay Interference in Thyroid Function Tests

Abstract Introduction While biotin is widely used as a health and beauty supplement, patients rarely report the use of biotin supplements to clinicians. This can negatively impact patient care since biotin can falsely elevate or lower results in immunoassays that use biotinylated components. On the Beckman DxI platform, total T3 (T3), free T3 (FT3), and free T4 (FT4) assays are falsely elevated by excess biotin, while TSH and total T4 (T4) are unaffected. The goals of this study were to develop a method to measure serum biotin, to determine biotin concentrations in samples submitted for thyroid function testing, and to correlate biotin concentrations with immunoassay result patterns suggestive of biotin interference. Methods We developed and validated a targeted LC-MS/MS method to measure serum biotin (AMR: 5.4-500 mcg/L). Two collections of patient samples were tested using automated Beckman DxI assays for TSH (RR: 0.4-5.0 μIU/mL), T3 (RR: 73-178 ng/dL), FT3 (RR: 2.4-4.1 pg/mL), T4 (RR: 4.8-10.8 mcg/dL), and FT4 (RR: 0.6-1.2 ng/dL). The first collection included consecutive samples submitted for TSH and FT4 testing. To increase the probability of identifying samples with biotin interference, a middleware preselection algorithm was used to identify samples with a results pattern suggestive of biotin interference (normal to high TSH with elevated T3, FT3, and/or FT4). Results Two of the 94 samples consecutively submitted for thyroid function tests demonstrated biotin levels >AMR. TSH and FT4 were within normal ranges for one, while the other had normal TSH and elevated FT4 (biotin: 26 mcg/L). The preselection algorithm targeting normal to high TSH with elevated T3, FT3, and/or FT4 results flagged 83 samples, 21.7% of which had measurable biotin (5.71-194.3 mcg/L). Immunoassay results for TSH, T3, FT3, T4, and FT4 were obtained where there was sufficient volume for testing. Of these 38 samples, all samples with biotin >24 mcg/L had elevated results for 2(+) affected assays. Conclusions In this study, a targeted LC-MS/MS method to measure biotin in serum samples was developed and used to estimate the distribution of biotin concentrations in samples submitted for thyroid function testing. Comparison of immunoassay result patterns with biotin concentrations suggested an interference threshold around 24 mcg/L. The evaluation of consecutively submitted samples showed that biotin in excess of this interference threshold in patients receiving thyroid function tests on the Beckman DxI platform is rare. However, combining the preselection algorithm with quantitative biotin measurement by LC-MS/MS demonstrated that biotin interference does occur in these patient samples and should be evaluated when there are confounding results or result patterns suggestive of interference. In addition, the biotin LC-MS/MS method described here has additional clinical utility in the evaluation of suspected interference in any immunoassay that utilizes biotinylated components.

Engineering Corynebacterium glutamicum triggers glutamic acid accumulation in biotin-rich corn stover hydrolysate

BackgroundLignocellulose biomass contains high amount of biotin and resulted in an excessive biotin condition for cellulosic glutamic acid accumulation by Corynebacterium glutamicum. Penicillin or ethambutol triggers cellulosic glutamic acid accumulation, but they are not suitable for practical use due to the fermentation instability and environmental concerns. Efficient glutamic acid production from lignocellulose feedstocks should be achieved without any chemical inductions.ResultsAn industrial strain C. glutamicum S9114 was metabolically engineered to achieve efficient glutamic acid accumulation in biotin-excessive corn stover hydrolysate. Among the multiple metabolic engineering efforts, two pathway regulations effectively triggered the glutamic acid accumulation in lignocellulose hydrolysate. The C-terminal truncation of glutamate secretion channel MscCG (ΔC110) led to the successful glutamic acid secretion in corn stover hydrolysate without inductions. Then the α-oxoglutarate dehydrogenase complex (ODHC) activity was attenuated by regulating odhA RBS sequence, and glutamic acid accumulation was further elevated for more than fivefolds. The obtained C. glutamicum XW6 strain reached a record-high titer of 65.2 g/L with the overall yield of 0.63 g/g glucose using corn stover as the starting feedstock without any chemical induction.ConclusionsMetabolic engineering method was successfully applied to achieve efficient glutamic acid in biotin-rich lignocellulose hydrolysate for the first time. This study demonstrated the high potential of glutamic acid production from lignocellulose feedstock.

Autophagy inhibition by biotin elicits endoplasmic reticulum stress to differentially regulate adipocyte lipid and protein synthesis.

Biotin is an indispensable adipogenic agent, and its ability to coordinate carbohydrate, lipid, and amino acid metabolism sensitizes insulin signaling in adipocytes. This enables the organism to adapt and survive under nutrient stress by synthesis and storage of lipids. Biotin deficiency mimics insulin resistance with alterations in cellular intermediary metabolism. Though the mechanism of lipogenesis is well established across cell types, considering its predisposition to accumulate only lipids, it is necessary to elucidate the mechanism that minimizes the effects of biotin on adipocyte protein synthesis. In order to determine the differential metabolic phenotype by biotin, the primary cultures of adipocytes were induced to differentiate in the presence and absence of excess biotin. Serum pre-incubated with avidin was used to limit biotin availability in cultured cells. Biotin restricts cellular signaling associated with protein synthesis without altering total protein content. The decline in autophagy elicits endoplasmic reticulum stress to inhibit protein synthesis by eIF2α phosphorylation possibly via accumulation of misfolded/long-lived proteins. Furthermore, the compensatory increase in Unc51 like autophagy activating kinase 1 possibly competes with eukaryotic initiation factor 4E-binding protein 1 and ribosomal p70 S6kinase phosphorylation by mechanistic targets of rapamycin complex 1 to uncouple its effect on protein synthesis. In conclusion, autophagy inhibition by biotin uncouples protein synthesis to promote lipogenesis by eliciting endoplasmic reticulum stress and differential phosphorylation of mechanistic targets of rapamycin complex 1 substrates.

Evaluation of Antibody Biotinylation Approaches for Enhanced Sensitivity of Single Molecule Array (Simoa) Immunoassays.

In this study, we evaluated the performance of Single Molecule Array (Simoa) immunoassays based on various detection antibody biotinylation approaches. Simoa immunoassays, like other sandwich ELISAs, are highly dependent on the interaction of a biotinylated detection antibody with an enzyme conjugated to streptavidin. Thus, we sought to assess whether different biotinylation reagents can improve the performance and sensitivity of Simoa assays. We selected three proteins, GM-CSF, IFNγ, and IL-2, that are present at ultralow levels in many biological samples. We compared the performance of these Simoa assays by using five different biotinylation reagents and varying the amount of molar fold excess biotin during the biotinylation process. We found that the choice of biotinylation reagent and the molar fold excess biotin can highly affect the performance of the Simoa assays, with differences of up to an order of magnitude in sensitivity. We also tested the performance of bulk ELISAs using the different biotinylated detection antibodies and observe differences greater than an order of magnitude in sensitivity. We show that evaluating different strategies for detection antibody biotinylation is a simple approach for optimizing immunoassay performance for enhanced sensitivity.

Rich biotin content in lignocellulose biomass plays the key role in determining cellulosic glutamic acid accumulation by Corynebacterium glutamicum

BackgroundLignocellulose is one of the most promising alternative feedstocks for glutamic acid production as commodity building block chemical, but the efforts by the dominant industrial fermentation strain Corynebacterium glutamicum failed for accumulating glutamic acid using lignocellulose feedstock.ResultsWe identified the existence of surprisingly high biotin concentration in corn stover hydrolysate as the determining factor for the failure of glutamic acid accumulation by Corynebacterium glutamicum. Under excessive biotin content, induction by penicillin resulted in 41.7 ± 0.1 g/L of glutamic acid with the yield of 0.50 g glutamic acid/g glucose. Our further investigation revealed that corn stover contained 353 ± 16 μg of biotin per kg dry solids, approximately one order of magnitude greater than the biotin in corn grain. Most of the biotin remained stable during the biorefining chain and the rich biotin content in corn stover hydrolysate almost completely blocked the glutamic acid accumulation. This rich biotin existence was found to be a common phenomenon in the wide range of lignocellulose biomass and this may be the key reason why the previous studies failed in cellulosic glutamic acid fermentation from lignocellulose biomass. The extended recording of the complete members of all eight vitamin B compounds in lignocellulose biomass further reveals that the major vitamin B members were also under the high concentration levels even after harsh pretreatment.ConclusionsThe high content of biotin in wide range of lignocellulose biomass feedstocks and the corresponding hydrolysates was discovered and it was found to be the key factor in determining the cellulosic glutamic acid accumulation. The highly reserved biotin and the high content of their other vitamin B compounds in biorefining process might act as the potential nutrients to biorefining fermentations. This study creates a new insight that lignocellulose biorefining not only generates inhibitors, but also keeps nutrients for cellulosic fermentations.

Utilization of Polymer Based Protein Engineering and ATRP to Modulate Substrate Size Specificity of Avidin

Avidin and biotin interact very strongly and are commonly used to tag and purify proteins or peptides for proteomic analysis. Using a pH dependent reversible biotin tag that targets primary amines, all the proteins can be tagged at a slightly basic pH. After labeling proteins with the biotin tag, any excess unreacted biotin left over in solution must be dialyzed away to prevent competition for binding spots on the Avidin beads. Tagged proteins can then be bound to a solid surface Avidin bead and contaminants washed away. Proteins can then be released from the beads in their original unmodified state by lowering the pH. While this workflow has been shown to be very useful for purifying proteomes, the requirement for dialysis after the initial tagging step makes this workflow laborious and difficult to automate. This step is time consuming, can result in protein loss or precipitation, and makes automation impossible. In this study we seek to utilize polymer based protein engineering to circumvent this issue.Atom transfer radical polymerization (ATRP) has been utilized in recent years to specifically modify and fine tune the behavior of proteins. By adding polymers of a specific composition and length in targeted areas of a protein, the behavior of a protein can be modified. In this study, we utilized ATRP to grow a dense polymer network off the surface of Avidin to act as a molecular sieve; The large polymers allow small molecules of biotin to bind to the binding site, while larger biotinylated proteins are excluded. The resulting “Caged Avidin” can then be used in the proteome purification workflow in the place of dialysis by binding and sequestering any unreacted free biotin, allowing the entire labeling, binding, washing, and eluting workflow to take place in a single tube with no bias and no sample loss. This can revolutionize sample preparation in biomarker and drug discovery by allowing for automation, and thus improved yield and reliability, of sample preparation.Support or Funding InformationThis work was supported in part by the Center for Polymer Based Protein Engineering at Carnegie Mellon UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Insuffisance rénale chronique et biotine : un duo propice aux bilans thyroïdiens et parathyroïdiens erronés, à propos de 2 cas

In clinical chemistry, many immunoassays apply biotin and streptavidin in the assay principle. Presence of high levels of biotin in patient samples can produce negative or positive interference depending on the assay format. In this study, we describe 2 clinical cases with chronic kidney failure and with unusual thyroid and parathyroid function test results due to biotin interference. We studied the impact of biotin levels on thyroid stimulating hormone (TSH), free thyroxine (T4L) and parathormone (PTH) results with a pool of sera loaded with several concentrations of biotin. In sandwich assays (TSH and PTH), excess biotin displaces biotinylated antibodies resulting in apparently low concentration of the analyte. With competitive immunoassays (T4L), excess biotin competes with biotinylated analog for the binding sites on streptavidin resulting in low signal and falsely high concentration of the analyte. In conclusion, chronic kidney failure combined to therapeutic biotin is in favour of high levels of biotin which causes seriously misleading results in assays using biotin-streptavidin mechanisms.