RÉSUMÉ
Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.
Sujet(s)
Calcium , Endopeptidase K , Stabilité enzymatique , Simulation de dynamique moléculaire , Stabilité protéique , Endopeptidase K/métabolisme , Endopeptidase K/composition chimique , Calcium/métabolisme , Calcium/composition chimique , Conception assistée par ordinateur , Mutation , Sites de fixation , Ingénierie des protéines/méthodes , Conformation des protéinesRÉSUMÉ
The emergence of drug-resistant bacteria, facilitated by metallo-beta-lactamases (MBLs), presents a significant obstacle to the effective use of antibiotics in the management of clinical drug-resistant bacterial infections. AFM-1 is a MBL derived from Alcaligenes faecalis and shares 86% homology with the NDM-1 family. Both AFM-1 and NDM-1 demonstrate the ability to hydrolyze ampicillin and other ß-lactam antibiotics, however, their substrate affinities vary, and the specific reason for this variation remains unknown. We present the high-resolution structure of AFM-1. The active center of AFM-1 binds two zinc ions, and the conformation of the key amino acid residues in the active center is in accordance with that of NDM-1. However, the substrate-binding pocket of AFM-1 is considerably smaller than that of NDM-1. Additionally, the mutation of amino acid residues in the Loop3 region, as compared to NDM-1, results in the formation of a dense hydrophobic patch comprised of hydrophobic amino acid residues in this area, which facilitates substrate binding. Our findings lay the foundation for understanding the molecular mechanism of AFM-1 with a high affinity for substrates and provide a novel theoretical foundation for addressing the issue of drug resistance caused by B1 MBLs.
Sujet(s)
Modèles moléculaires , bêta-Lactamases , bêta-Lactamases/composition chimique , bêta-Lactamases/métabolisme , bêta-Lactamases/ultrastructure , bêta-Lactamases/génétique , Alcaligenes faecalis/enzymologie , Alcaligenes faecalis/composition chimique , Conformation des protéines , Zinc/composition chimique , Zinc/métabolisme , Cristallographie aux rayons X , Domaine catalytique , Protéines bactériennes/composition chimique , Protéines bactériennes/métabolisme , Protéines bactériennes/génétique , Séquence d'acides aminés , Sites de fixationRÉSUMÉ
BACKGROUND: Hypoxic pulmonary hypertension (HPH) is a common type of pulmonary hypertension and characterized by pulmonary vascular remodeling and constriction. A large number of studies have shown that pulmonary vascular endothelial cells (PVECs) dysfunction plays an important role in the initiation and development stages of HPH, but the mechanism of PVECs dysfunction after hypoxia remains unclear. In this study, we explored the exact mechanism of PVECs dysfunction after hypoxia. METHODS: In vitro, we used primary cultured PVECs hypoxia model to mimic HPH injury. We detected the expressions of mitochondrial biogenesis markers, mitochondrial transcription factor A (TFAM) level inside mitochondria, mitochondrial quantity and function, and the components expressions of translocase of outer mitochondrial membrane (TOM) at 24 h after hypoxia. To explore the effects of Tom70 on mitochondrial biogenesis and functions of PVECs after hypoxia, Tom70 overexpression adenovirus was constructed, and the expressions of mitochondrial biogenesis markers, TFAM level inside mitochondria, mitochondrial quantity and function, and the functions of PVECs were detected. And in vivo, we used cre-dependent overexpression adenovirus of Tom70 in the Cdh5-CreERT2 mouse model of HPH to verify the role of upregulating PVECs Tom70 in improving HPH. RESULTS: Hypoxia obviously increased the expressions of mitochondrial biogenesis markers for PGC-1α, NRF-1 and TFAM, but reduced the content of TFAM in mitochondria and the quantity and functions of mitochondria. In addition, only Tom70 expression among the TOM components was significantly decreased after hypoxia, and up-regulation of Tom70 significantly increased the content of TFAM in mitochondria of PVECs by transporting TFAM into mitochondria after hypoxia, enhanced the quantity and functions of mitochondria, improved the functions of PVECs, and ultimately alleviated HPH. CONCLUSION: The findings of present study demonstrated that hypoxia induced the decreased expression of Tom70 in PVECs, reduced the mitochondrial biogenesis-associated TFAM protein transporting into mitochondria, inhibited mitochondrial biogenesis, caused PVECs injury, and prompted the formation of HPH. However, up-regulation of Tom70 abolished the hypoxia-induced injurious effects on PVECs and alleviated HPH.
Sujet(s)
Hypertension pulmonaire , Animaux , Souris , Cellules endothéliales/métabolisme , Hypertension pulmonaire/génétique , Hypertension pulmonaire/métabolisme , Hypoxie/complications , Poumon/métabolisme , Protéines du complexe d'import des protéines précurseurs mitochondriales , Biogenèse des organellesRÉSUMÉ
BACKGROUND: Microwave imaging has been proposed for medical applications, creating maps related to water content of tissues. Breast imaging has emerged as a key application because the signals can be coupled directly into the breast and experience limited attenuation in fatty tissues. While the literature contains reports of tumor detection with microwave approaches, there is limited exploration of treatment monitoring. PURPOSE: This study aims to detect treatment-related changes in breast tissue with a low-resolution microwave scanner. METHODS: Microwave scans of 15 patients undergoing treatment for early-stage breast cancer are collected at up to 4 time points: after surgery (baseline), 6 weeks after accelerated partial breast radiation, as well as 1 and 2 years post-treatment. Both the treated and untreated breast are scanned at each time point. The microwave scanner consists of planar transmit and receive arrays and uses signals from 0.1 to 10 GHz. The average microwave frequency properties (permittivity) are calculated for each scan to enable quantitative comparison. Baseline and 6-week results are analyzed with a two-way ANOVA with blocking. RESULTS: Consistent properties are observed for the untreated breast over time, similar to a previous study. Comparison of the scans of the treated and untreated breast suggests increased properties related to treatment, particularly at baseline and 6-weeks following radiotherapy. Analysis of the average properties of the scans with ANOVA indicates statistically significant differences ( p < 0.05 $p < 0.05$ ) in the treated and untreated breast at these time points. CONCLUSIONS: Microwave imaging has the potential to track treatment-related changes in breast tissues.
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Tumeurs du sein , Imagerie micro-onde , Humains , Femelle , Tumeurs du sein/imagerie diagnostique , Tumeurs du sein/radiothérapie , Micro-ondes/usage thérapeutique , Projets pilotes , Région mammaire/imagerie diagnostique , Région mammaire/anatomopathologieRÉSUMÉ
Polyethylene terephthalate (PET) is one of the most widely used synthetic polyester. It poses serious threat to terrestrial, aquatic ecosystems and human health since it is difficult to be broken down and deposited in the environment. The biodegradation based on enzymatic catalysis offers a sustainable method for recycling PET. A number of PET hydrolases have been discovered in the last 20 years, and protein engineering has increased their degradation capabilities. However, no PET hydrolases that are practical for widespread industrial use have been identified. Screening of PET hydrolase using conventional detection techniques is laborious and inefficient process. Effective detection techniques are required to promote the commercialization of PET hydrolases. Using efficient detection techniques to screen potent industrial enzymes is essential for supporting the widespread industrial implementation of PET hydrolases. To define PET hydrolase, scientists have created a number of analytical techniques recently. The detection techniques that can be used to screen PET hydrolase, including high performance liquid chromatography, ultraviolet absorption spectrometric, and fluorescence activated droplet sorting method, are summarized in this study along with their potential applications.
Sujet(s)
Écosystème , Téréphtalate polyéthylène , Humains , Dépollution biologique de l'environnement , Catalyse , HydrolasesRÉSUMÉ
The process of recycling poly(ethylene terephthalate) (PET) remains a major challenge due to the enzymatic degradation of high-crystallinity PET (hcPET). Recently, a bacterial PET-degrading enzyme, PETase, was found to have the ability to degrade the hcPET, but with low enzymatic activity. Here we present an engineered whole-cell biocatalyst to simulate both the adsorption and degradation steps in the enzymatic degradation process of PETase to achieve the efficient degradation of hcPET. Our data shows that the adhesive unit hydrophobin and degradation unit PETase are functionally displayed on the surface of yeast cells. The turnover rate of the whole-cell biocatalyst toward hcPET (crystallinity of 45%) dramatically increases approximately 328.8-fold compared with that of purified PETase at 30 °C. In addition, molecular dynamics simulations explain how the enhanced adhesion can promote the enzymatic degradation of PET. This study demonstrates engineering the whole-cell catalyst is an efficient strategy for biodegradation of PET.
Sujet(s)
Acides phtaliques , Téréphtalate polyéthylène , Téréphtalate polyéthylène/métabolisme , Hydrolases/métabolisme , Acides phtaliques/métabolisme , ÉthylènesRÉSUMÉ
OBJECTIVE: This study aims to investigate the use of single residue substitution to promote the formation of pi-stacking interactions between peptides and Human leukocyte antigen (HLA)-A*2402 molecules to improve the affinity of peptides and HLA molecules, as well as the level of cytotoxic T lymphocyte (CTL) cells activated by peptides-HLA (p-HLA) complex. METHODS: Molecular docking and molecular dynamics simulation were used to simulate and analyze the interactions and binding free energies between HLA-A*2402-restricted antigen peptides and HLA molecules, before and after the single residue substitution. HLA-A*2402 restricted antigen peptides before and after the single residue replacement were loaded into dendritic cells (DCs) in vitro, and further Enzyme-Linked ImmunoSpot (ELispot) test was carried out to evaluate the effect of modified antigen peptides on the immune activation of CTL cells. RESULT: After replacing the antigen peptides with a single residue, some of them could promote the formation of pi-stacking interaction. The binding free energy between the modified antigen peptides and HLA-A*2402, as well as the level of immune activation of CTL cells were mostly higher than before, especially after the replacement of the 9th residue of the polypeptide, such as C9F and C9W. There was a significant negative correlation between the level of activated CTL cells by modified antigen peptides and the total interaction amount of hydrogen bonds and salt bridges. CONCLUSION: Promoting the formation of pi-stacking interaction between antigen peptides and HLA-A*2402 molecules could increase the total binding free energy of p-HLA complex and the level of CTL cells activation. In addition, the amount of hydrogen bonds and salt bridges between peptides and HLA could reduce the level of immune activation. All the characteristics above can improve the immunogenicities of the weak antigens.
RÉSUMÉ
The existing zoonotic coronaviruses (CoVs) and viral genetic variants are important microbiological pathogens that cause severe disease in humans and animals. Currently, no effective broad-spectrum antiviral drugs against existing and emerging CoVs are available. The CoV main protease (Mpro) plays an essential role in viral replication, making it an ideal target for drug development. However, the structure of the Deltacoronavirus Mpro is still unavailable. Porcine deltacoronavirus (PDCoV) is a novel CoV that belongs to the genus Deltacoronavirus and causes atrophic enteritis, severe diarrhea, vomiting and dehydration in pigs. Here, we determined the structure of PDCoV Mpro complexed with a Michael acceptor inhibitor. Structural comparison showed that the backbone of PDCoV Mpro is similar to those of alpha-, beta- and gamma-CoV Mpros. The substrate-binding pocket of Mpro is well conserved in the subfamily Coronavirinae. In addition, we also observed that Mpros from the same genus adopted a similar conformation. Furthermore, the structure of PDCoV Mpro in complex with a Michael acceptor inhibitor revealed the mechanism of its inhibition of PDCoV Mpro. Our results provide a basis for the development of broad-spectrum antivirals against PDCoV and other CoVs.
Sujet(s)
Antiviraux , Coronavirus , Animaux , Antiviraux/composition chimique , Antiviraux/pharmacologie , Coronavirus/génétique , Deltacoronavirus (genre) , Peptide hydrolases/composition chimique , SuidaeRÉSUMÉ
Cytoplasmic incompatibility (CI) results when Wolbachia bacteria-infected male insects mate with uninfected females, leading to embryonic lethality. "Rescue" of viability occurs if the female harbors the same Wolbachia strain. CI is caused by linked pairs of Wolbachia genes called CI factors (CifA and CifB). The co-evolution of CifA-CifB pairs may account in part for the incompatibility patterns documented in insects infected with different Wolbachia strains, but the molecular mechanisms remain elusive. Here, we use X-ray crystallography and AlphaFold to analyze the CI factors from Wolbachia strain wMel called CidAwMel and CidBwMel. Substituting CidAwMel interface residues with those from CidAwPip (from strain wPip) enables the mutant protein to bind CidBwPip and rescue CidBwPip-induced yeast growth defects, supporting the importance of CifA-CifB interaction in CI rescue. Sequence divergence in CidAwPip and CidBwPip proteins affects their pairwise interactions, which may help explain the complex incompatibility patterns of mosquitoes infected with different wPip strains.
Sujet(s)
Wolbachia , Animaux , Cytoplasme/génétique , Cytosol , Drosophila melanogaster/génétique , Femelle , Mâle , Saccharomyces cerevisiae , Symbiose/génétique , Wolbachia/génétique , Wolbachia/métabolismeRÉSUMÉ
The flavivirus nonstructural protein 3 helicase (NS3hel) is a multifunctional domain protein that is associated with DNA/RNA helicase, nucleoside triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase) activities. As an NTPase-dependent superfamily 2 (SF2) member, NS3hel employs an NTP-driven motor force to unwind double-stranded RNA while translocating along single-stranded RNA and is extensively involved in the viral replication process. Although the structures of SF2 helicases are widely investigated as promising drug targets, the mechanism of energy transduction between NTP hydrolysis and the RNA binding sites in ZIKV NS3hel remains elusive. Here, we report the crystal structure of ZIKV NS3hel in complex with its natural substrates ATP-Mn2+ and ssRNA. Distinct from other members of the Flavivirus genus, ssRNA binding to ZIKV NS3hel induces relocation of the active water molecules and ATP-associated metal ions in the NTP hydrolysis active site, which promotes the hydrolysis of ATP and the production of AMP. Our findings highlight the importance of the allosteric role of ssRNA on the modulation of ATP hydrolysis and energy utilization.
Sujet(s)
Infection par le virus Zika , Virus Zika , Adénosine triphosphate , Humains , Hydrolyse , ARN viral/génétique , Protéines virales non structurales/génétiqueRÉSUMÉ
Wolbachia bacteria, inherited through the female germ line, infect a large fraction of arthropod species. Many Wolbachia strains manipulate host reproduction, most commonly through cytoplasmic incompatibility (CI). CI, a conditional male sterility, results when Wolbachia-infected male insects mate with uninfected females; viability is restored if the female is similarly infected (called "rescue"). CI is used to help control mosquito-borne viruses such as dengue and Zika, but its mechanisms remain unknown. The coexpressed CI factors CifA and CifB form stable complexes in vitro, but the timing and function of this interaction in the insect are unresolved. CifA expression in the female germ line is sufficient for rescue. We report high-resolution structures of a CI-factor complex, CinA-CinB, which utilizes a unique binding mode between the CinA rescue factor and the CinB nuclease; the structures were validated by biochemical and yeast growth analyses. Importantly, transgenic expression in Drosophila of a nonbinding CinA mutant, designed based on the CinA-CinB structure, suggests CinA expressed in females must bind CinB imported by sperm in order to rescue embryonic viability. Binding between cognate factors is conserved in an enzymatically distinct CI system, CidA-CidB, suggesting universal features in Wolbachia CI induction and rescue.
Sujet(s)
Drosophila melanogaster/microbiologie , Embryon non mammalien/embryologie , Infertilité masculine/physiopathologie , Reproduction/physiologie , Wolbachia/métabolisme , Animaux , Animal génétiquement modifié , Drosophila melanogaster/génétique , Développement embryonnaire , Femelle , Mâle , Lutte contre les moustiques/méthodes , Complexes multiprotéiques/métabolisme , Liaison aux protéines , Symbiose , Maladies vectorielles/prévention et contrôle , Maladies vectorielles/transmission , Maladies vectorielles/virologieRÉSUMÉ
Excitonic insulators (EIs) arise from the formation of bound electron-hole pairs (excitons)1,2 in semiconductors and provide a solid-state platform for quantum many-boson physics3-8. Strong exciton-exciton repulsion is expected to stabilize condensed superfluid and crystalline phases by suppressing both density and phase fluctuations8-11. Although spectroscopic signatures of EIs have been reported6,12-14, conclusive evidence for strongly correlated EI states has remained elusive. Here we demonstrate a strongly correlated two-dimensional (2D) EI ground state formed in transition metal dichalcogenide (TMD) semiconductor double layers. A quasi-equilibrium spatially indirect exciton fluid is created when the bias voltage applied between the two electrically isolated TMD layers is tuned to a range that populates bound electron-hole pairs, but not free electrons or holes15-17. Capacitance measurements show that the fluid is exciton-compressible but charge-incompressible-direct thermodynamic evidence of the EI. The fluid is also strongly correlated with a dimensionless exciton coupling constant exceeding 10. We construct an exciton phase diagram that reveals both the Mott transition and interaction-stabilized quasi-condensation. Our experiment paves the path for realizing exotic quantum phases of excitons8, as well as multi-terminal exciton circuitry for applications18-20.
RÉSUMÉ
The important role of BV in clinical diagnostics of liver-related diseases has been established in veterinary medicine. However, the sensitivity and selectivity of the current BV assays remain relatively low compromising its wider application in clinical diagnosis. Herein, we developed a rapid and sensitive BV-detecting biosensor based on a novel far-red fluorescent protein smURFP, which produced fluorescence only through specific interaction with its cofactor BV. In our study, the binding of BV to smURFP was then systematically optimized based on the structures of the smURFP + BV complex to increase the sensitivity of our biosensor. A wide linear range from 0 µM to 25 µM was obtained in both chicken and human serum. The limit of detection (LOD) and limit of quantification (LOQ) for BV was as low as 0.4 nM and 1.5 nM in human serum, and 0.4 nM and 1.2 nM in chicken serum. To our knowledge, this is the lowest LOD that has ever been reported for a BV biosensor. Our study sheds light on the biological and clinical analysis of BV.
Sujet(s)
Biliverdine , Techniques de biocapteur , Humains , Limite de détectionRÉSUMÉ
The development of rapid and efficient tools to modulate neurons is vital for the treatment of nervous system diseases. Here, a novel non-invasive neurite outgrowth modulation method based on a controllable acoustic streaming effect induced by an electromechanical gigahertz resonator microchip is reported. The results demonstrate that the gigahertz acoustic streaming can induce cell structure changes within a 10 min period of stimulation, which promotes a high proportion of neurite bearing cells and encourages longer neurite outgrowth. Specifically, the resonator stimulation not only promotes outgrowth of neurites, but also can be combined with chemical mediated methods to accelerate the direct entry of nerve growth factor (NGF) into cells, resulting in higher modulation efficacy. Owing to shear stress caused by the acoustic streaming effect, the resonator microchip mediates stress fiber formation and induces the neuron-like phenotype of PC12 cells. We suggest that this method may potentially be applied to precise single-cell modulation, as well as in the development of non-invasive and rapid disease treatment strategies.
RÉSUMÉ
Chiral drugs are drugs with chiral or asymmetric centres in their molecular structure. Different enantiomers of the same chiral drug have noticeably different pharmacological activities and pharmacokinetic properties. However, its distinction has been perplexing scholars for many years in the qualitative and quantitative detection of antagonistic drugs. Conventional detection methods, such as polarimetry, circular dichroism, and high-performance liquid chromatography, are time consuming, cause sample loss and have cumbersome operations, and they can be applied only to the sampling method. In this paper, we propose a fast, accurate, qualitative and quantitative method for the study of chiral drugs based on linearly polarized terahertz (THz) spectroscopy and imaging technology. Taking ibuprofen as an example, based on the THz absorption spectra of the enantiomers RS-ibuprofen, (R)-(-)-ibuprofen, and (S)-(+)-ibuprofen, their characteristic peak frequencies, peak amplitude differences and peak area differences were extracted to qualitatively and quantitatively distinguish and identify the three substances. THz spectral imaging provides more intuitive results than those obtained from previous methods. In quantitative identification, the stability and detection accuracy of THz spectroscopy are much greater than those of Raman spectroscopy (88.8-99.8% vs. 21.42-94.62%, respectively). The qualitative recognition accuracy was 100%, and the quantitative recognition standard deviation was less than 0.01, and it is also a non-destructive testing method. Furthermore, the above method combined with principal component analysis (PCA) and the support vector machine (SVM) neural network classification algorithm was applied to the analysis of other chiral drugs. These results are significant for the rapid, accurate and non-destructive identification of chiral drugs.
Sujet(s)
Préparations pharmaceutiques , Spectroscopie térahertz , , Analyse en composantes principales , Machine à vecteur de supportRÉSUMÉ
Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP51-486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP51-486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.
Sujet(s)
Protéines G/composition chimique , Multimérisation de protéines , Sites de fixation , Protéines G/génétique , Protéines G/métabolisme , Guanosine triphosphate/composition chimique , Guanosine triphosphate/métabolisme , Cellules HEK293 , Humains , Simulation de dynamique moléculaire , Liaison aux protéines , Produits du gène env du virus de l'immunodéficience humaine/composition chimique , Produits du gène env du virus de l'immunodéficience humaine/métabolismeRÉSUMÉ
Panax quinquefolium is a perennial herbaceous plant that contains many beneficial ginsenosides with diverse pharmacological effects. 24(R)-pseudoginsenoside F11 is specific to P. quinquefolium, a useful biomarker for distinguishing this species from other related plants. However, because of its nonconjugated property and the complexity of existing detection methods, this biomarker cannot be used as the identification standard. We herein present a stable 24(R)-pseudoginsenoside F11 fingerprint spectrum in the terahertz band, thereby proving that F11 can be detected and quantitatively analyzed via terahertz spectroscopy. We also analyzed the sample by high-performance liquid chromatography-triple quadrupole mass spectrometry. The difference between the normalized data for the two analytical methods was less than 5%. Furthermore, P. quinquefolium from different areas and other substances can be clearly distinguished based on these terahertz spectra with a standard principal component analysis. Our method is a fast, simple, and cost-effective approach for identifying and quantitatively analyzing P. quinquefolium.
RÉSUMÉ
Enzymatic hydrolysis of polyethylene terephthalate (PET) is considered to be an environmentally friendly method for the recycling of plastic waste. Recently, a bacterial enzyme named IsPETase was found in Ideonella sakaiensis with the ability to degrade amorphous PET at ambient temperature suggesting its possible use in recycling of PET. However, applying the purified IsPETase in large-scale PET recycling has limitations, i.e., a complicated production process, high cost of single-use, and instability of the enzyme. Yeast cell surface display has proven to be an effectual alternative for improving enzyme degradation efficiency and realizing industrial applications. This chapter deals with the construction and application of a whole-cell biocatalyst by displaying IsPETase on the surface of yeast (Pichia pastoris) cells.
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Hydrolases , Téréphtalate polyéthylène , Burkholderiales , Hydrolases/génétique , SaccharomycetalesRÉSUMÉ
Ninhydrin-based fluorometric quantification of phenylalanine is one of the most widely used methods for hyperphenylalaninemia (HPA) screening in neonates due to its high sensitivity, high accuracy, and low cost. Here we report an increase of false positive cases in neonatal HPA screening with this method, caused by contamination of blood specimen collection devices during the printing process. Through multiple steps of verification, the contaminants were identified from ink circles printed on the collection devices to indicate the positions and sizes of blood drops. Blood specimens from HPA-negative persons collected on these contaminated collection devices showed positive results in the fluorometric tests, but negative results in tandem mass spectroscopy (MS/MS) experiments. Contaminants on the collection devices could be extracted by 80% ethanol and showed an absorption peak around 245 nm, suggesting that these contaminants may contain benzene derivatives with similar structure to phenylalanine. High-performance liquid chromatography (HPLC) analysis of the ethanol extracts from contaminated collection devices identified two prominent peaks specifically from the devices. Methyl-2-benzoylbenzoate (MBB, CAS#606-28-0) was found as one of the major chemicals from contaminated collection devices. This report aims to remind colleagues in the field of this potential contamination and call for tighter regulation and quality control of specimen collection devices.