Heme binding to the SARS-CoV-2 spike glycoprotein.

The target for humoral immunity, SARS-CoV-2 spike glycoprotein, has become the focus of vaccine research and development. Previous work demonstrated that the N-terminal domain (NTD) of SARS-CoV-2 spike binds biliverdin-a product of heme catabolism-causing a strong allosteric effect on the activity of a subset of neutralizing antibodies. Herein, we show that the spike glycoprotein is also able to bind heme (KD = 0.5 ± 0.2 µM). Molecular modeling indicated that the heme group fits well within the same pocket on the SARS-CoV-2 spike NTD. Lined by aromatic and hydrophobic residues (W104, V126, I129, F192, F194, I203, and L226), the pocket provides a suitable environment to stabilize the hydrophobic heme. Mutagenesis of N121 has a substantive effect on heme binding (KD = 3000 ± 220 µM), confirming the pocket as a major heme binding location of the viral glycoprotein. Coupled oxidation experiments in the presence of ascorbate indicated that the SARS-CoV-2 glycoprotein can catalyze the slow conversion of heme to biliverdin. The heme trapping and oxidation activities of the spike may allow the virus to reduce levels of free heme during infection to facilitate evasion of the adaptive and innate immunity.

Infrared Spectroscopy as a Potential Diagnostic Tool for Medulloblastoma.

INTRODUCTION: Medulloblastoma (MB) is the most common malignant tumor of the central nervous system in childhood. FTIR spectroscopy provides a holistic view of the chemical composition of biological samples, including the detection of molecules such as nucleic acids, proteins, and lipids. This study evaluated the applicability of FTIR spectroscopy as a potential diagnostic tool for MB. MATERIALS AND METHODS: FTIR spectra of MB samples from 40 children (boys/girls: 31/9; age: median 7.8 years, range 1.5-21.5 years) treated in the Oncology Department of the Children's Memorial Health Institute in Warsaw between 2010 and 2019 were analyzed. The control group consisted of normal brain tissue taken from four children diagnosed with causes other than cancer. Formalin-fixed and paraffin-embedded tissues were sectioned and used for FTIR spectroscopic analysis. The sections were examined in the mid-infrared range (800-3500 cm-1) by ATR-FTIR. Spectra were analysed using a combination of principal component analysis, hierarchical cluster analysis, and absorbance dynamics. RESULTS: FTIR spectra in MB were significantly different from those of normal brain tissue. The most significant differences related to the range of nucleic acids and proteins in the region 800-1800 cm-1. Some major differences were also revealed in the quantification of protein conformations (α-helices, ß-sheets, and others) in the amide I band, as well as in the absorbance dynamics in the 1714-1716 cm-1 range (nucleic acids). It was not, however, possible to clearly distinguish between the various histological subtypes of MB using FTIR spectroscopy. CONCLUSIONS: MB and normal brain tissue can be distinguished from one another to some extent using FTIR spectroscopy. As a result, it may be used as a further tool to hasten and enhance histological diagnosis.

Cell Type Variability in the Incorporation of Lipids in the Dengue Virus Virion.

A lipid bilayer produced from the host membrane makes up around 20% of the weight of the dengue virus (DENV) virion and is crucial for virus entry. Despite its significance, the virion's lipid composition is still poorly understood. In tandem with lipid profiles of the cells utilised to generate the virions, this work determined a partial lipid profile of DENV virions derived from two cell lines (C6/36 and LLC-MK2). The results showed distinctive profiles between the two cell types. In the mammalian LLC-MK2 cells, 30.8% (73/237 identified lipid species; 31 upregulated, 42 downregulated) of lipid species were altered in response to infection, whilst in insect C6/36 cells only 12.0% (25/208; 19 upregulated, 6 downregulated) of lipid species showed alterations in response to infection. For virions from LLC-MK2 cells, 14 lipids were detected specifically in virions with a further seven lipids being enriched (over mock controls). For virions from C6/36 cells, 43 lipids were detected that were not seen in mock preparations, with a further 16 being specifically enriched (over mock control). These results provide the first lipid description of DENV virions produced in mammalian and mosquito cells, as well as the lipid changes in the corresponding infected cells.

MicroRNA gene methylation landscape in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Aberrant DNA methylation is an important mechanism by which the normal patterns of microRNA expression are disrupted in human cancers including B-cell precursor acute lymphoblastic leukemia (BCP ALL), the most common pediatric malignancy. OBJECTIVES: To characterize the methylation profile landscape of microRNA genes in BCP ALL patients. MATERIAL AND METHODS: We employed Infinium® MethylationEPIC BeadChip Arrays to measure the methylation of microRNA genes from bone marrow samples of children with BCP ALL (n = 38) and controls without neoplasms (n = 4). RESULTS: This analysis revealed differential methylation of the microRNA genes in the pediatric BCP ALL when compared to the control. A subcluster amongst BCP ALL patients with TCF3-PBX1 genetic subtype was also observed. No other differences were observed in association with age, gender or risk group. Several interesting leukemia-related phenotypes are enriched by the genes with hyperand hypomethylated sites located in promoters as well as gene bodies. The top 3 miRNA genes, promoters of which were the most statistically significantly hypermethylated in BCP ALL were MIR1273G, MIR1304 and MIR663, and the top 3 hypomethylated were MIR4442, MIR155 and MIR3909. CONCLUSIONS: In this study, a different microRNA genes methylation landscape was shown in pediatric BCP ALL compared to children without neoplasms. A visible subcluster among BCP ALL samples consisted of individuals with TCF3-PBX1 genetic subtype. No other differences were observed in association with age, gender or risk group. Several interesting leukemia-connected phenotypes were found, associated with genes with hyperand hypomethylated sites located on promoters as well as gene bodies.

Chemometric approaches to resolving base oil mixtures.

RATIONALE: In the lubrication industry, commercial base oils are commonly made up of blends of base oil stocks from different sources in different ratios to reduce production costs and modulate rheological properties. This practice introduces complexity in lubricant design because as the chemistry of the base oil becomes more complicated, it can become harder to formulate the base oil - particularly when the ratio of the original base oil stocks is unknown. METHODS: In this study, field ionisation mass spectrometry is used to collect chemical information on a range of base oil mixtures. The resultant data are processed within the Python workspace where molecular formulae are assigned to the components and statistical analyses are performed. A variety of regression techniques including regularised linear models and automated machine learning are evaluated on the data. RESULTS: The use of an automated machine learning pipeline yields insight into effective modelling strategies that could be applied to the data obtained. The best results were obtained using polynomial feature generation combined with ridge cross-validation regression. Overall, with this methodology it is possible to resolve the ratio of group 2 and group 3 base oil within a blended mixture to an accuracy of ±5%. CONCLUSIONS: The strategies outlined in this study show how modern data science and chemometrics can be applied successfully to resolve the ratio of a complex mixture.

Small-residue packing motifs modulate the structure and function of a minimal de novo membrane protein.

Alpha-helical integral membrane proteins contain conserved sequence motifs that are known to be important in helix packing. These motifs are a promising starting point for the construction of artificial proteins, but their potential has not yet been fully explored. Here, we study the impact of introducing a common natural helix packing motif to the transmembrane domain of a genetically-encoded and structurally dynamic de novo membrane protein. The resulting construct is an artificial four-helix bundle with lipophilic regions that are defined only by the amino acids L, G, S, A and W. This minimal proto-protein could be recombinantly expressed by diverse prokaryotic and eukaryotic hosts and was found to co-sediment with cellular membranes. The protein could be extracted and purified in surfactant micelles and was monodisperse and stable in vitro, with sufficient structural definition to support the rapid binding of a heme cofactor. The reduction in conformational diversity imposed by this design also enhances the nascent peroxidase activity of the protein-heme complex. Unexpectedly, strains of Escherichia coli expressing this artificial protein specifically accumulated zinc protoporphyrin IX, a rare cofactor that is not used by natural metalloenzymes. Our results demonstrate that simple sequence motifs can rigidify elementary membrane proteins, and that orthogonal artificial membrane proteins can influence the cofactor repertoire of a living cell. These findings have implications for rational protein design and synthetic biology.

A computational and experimental study of the fragmentation of l-leucine, l-isoleucine and l-allo-isoleucine under collision-induced dissociation tandem mass spectrometry.

The isomeric amino acids l-leucine, l-isoleucine and l-allo-isoleucine, are essential to many vital biological processes and are therefore of interest to the fields of metabolomics and proteomics. Their discrimination can be problematic however due to their isomeric natue. This study demonstrates a systematic investigation of the fragmentations of l-leucine, l-isoleucine and l-allo-isoleucine in combination with a thorough theoretical rationalisation. Collision induced dissociation (CID) tandem mass spectra (MS/MS) of all three amino acids were collected under a range of different collision energies to identify spontaneous and sequential fragmentation processes. We demonstrate that the three structural isomers can be distinguished by their CID MS/MS spectra, and additional computational modelling is used to rationalise these differences.

Identification and quantification of myo-inositol hexakisphosphate in complex environmental matrices using ion chromatography and high-resolution mass spectrometry in comparison to 31P NMR spectroscopy.

Myo-inositol hexakisphosphate, or phytic acid, (myo-IP6) is a key organic phosphorus (P) compound in soils and manures. Determinations of myo-IP6 in soils and manure extracts are frequently performed by 31P NMR spectroscopy. This approach is time-consuming in terms of both sample preparation and instrument time, with uncertainties existing in relation to accuracy of identification and quantification due to potentially interfering resonances from co-extracted P species. In contrast, ion chromatography (IC) in combination with high-resolution mass spectrometry (HRMS) negative ion, electrospray ionisation (ESI) has been shown to enable highly specific identifications of myo-IP6 isolated from complex mixtures. In this paper, IC and ESI-HRMS were applied to the identification and the quantification of myo-IP6 isolated from soils and manures using NaOH-EDTA extraction, and quantifications based on IC. ESI-HRMS analysis of eluate trapped from IC unequivocally confirmed identification of myo-IP6 from a soil extract. The ion suppression cell of the IC instrument provides isolates of the analyte free of ionic components that would interfere with ESI. The myo-IP6 was identified in the NMR by comparing spectra of extracts of soils with and without authentic myo-IP6 "spiked" prior to extraction. Comparison of quantification via standard addition in IC and NMR analysis gave good correlation (r = 0.955). IC with ESI-HRMS was found to be more sensitive, rapid and reliable for the identification and quantification of myo-IP6 with a limit of detection (LOD) of 0.7 mg kg-1 and limit of quantification (LOQ) of 2.1 mg kg-1 using IC versus > 10 mg kg-1 LOD using 31P NMR.

Untargeted characterisation of dissolved organic matter contributions to rivers from anthropogenic point sources using direct-infusion and high-performance liquid chromatography/Orbitrap mass spectrometry.

RATIONALE: Anthropogenic organic inputs to freshwaters can exert detrimental effects on aquatic ecosystems, raising growing concern for both environmental conservation and water security. Current regulation by the EU water framework directive (European Union, 2000/60/EC) relates to organic pollution by monitoring selected micropollutants; however, aquatic ecosystem responses require a comprehensive understanding of dissolved organic matter (DOM) composition. The introduction of high-resolution mass spectrometry (HRMS) is set to greatly increase our understanding of the composition of DOM of both natural and anthropogenic origin derived from diffuse and point sources. METHODS: DOM was extracted from riverine and treated sewage effluent using solid-phase extraction (SPE) and analysed using dissolved organic carbon analysis, direct-infusion high-resolution mass spectrometry (DI-HRMS) and high-performance liquid chromatography (HPLC)/HRMS. The data obtained were analysed using univariate and multivariate statistics to demonstrate differences in background DOM, anthropogenic inputs and in-river mixing. Compound identifications were achieved based on MS2 spectra searched against on-line databases. RESULTS: DI-HRMS spectra showed the highly complex nature of all DOM SPE extracts. Classification and visualisation of extracts containing many thousands of individual compounds were achieved using principal component analysis (PCA) and hierarchical cluster analysis. Kruskal-Wallis analyses highlighted significant discriminating ions originating from the sewage treatment works for more in-depth investigation by HPLC/HRMS. The generation of MS2 spectra in HPLC/HRMS provided the basis for identification of anthropogenic compounds including; pharmaceuticals, illicit drugs, metabolites and oligomers, although many thousands of compounds remain unidentified. CONCLUSIONS: This new approach enables comprehensive analysis of DOM in extracts without any preconceived ideas of the compounds which may be present. This approach has the potential to be used as a high throughput, qualitative, screening method to determine if the composition of point sources differs from that of the receiving water bodies, providing a new approach to the identification of hitherto unrecognised organic contribution to water bodies.

Predicting Ewing Sarcoma Treatment Outcome Using Infrared Spectroscopy and Machine Learning.

BACKGROUND: Improved outcome prediction is vital for the delivery of risk-adjusted, appropriate and effective care to paediatric patients with Ewing sarcoma-the second most common paediatric malignant bone tumour. Fourier transform infrared (FTIR) spectroscopy of tissues allows the bulk biochemical content of a biological sample to be probed and makes possible the study and diagnosis of disease. METHODS: In this retrospective study, FTIR spectra of sections of biopsy-obtained bone tissue were recorded. Twenty-seven patients (between 5 and 20 years of age) with newly diagnosed Ewing sarcoma of bone were included in this study. The prognostic value of FTIR spectra obtained from Ewing sarcoma (ES) tumours before and after neoadjuvant chemotherapy were analysed in combination with various data-reduction and machine learning approaches. RESULTS: Random forest and linear discriminant analysis supervised learning models were able to correctly predict patient mortality in 92% of cases using leave-one-out cross-validation. The best performing model for predicting patient relapse was a linear Support Vector Machine trained on the observed spectral changes as a result of chemotherapy treatment, which achieved 92% accuracy. CONCLUSION: FTIR spectra of tumour biopsy samples may predict treatment outcome in paediatric Ewing sarcoma patients with greater than 92% accuracy.

The distinguishable DNA whole genome methylation profile of 2 cases of pediatric precursor B acute lymphoblastic leukaemia (BCP ALL) with prodromal, preleukemic phase: A case report.

RATIONALE: A prolonged, prodromal phase before definitive paediatric precursor B acute lymphoblastic leukaemia (BCP ALL) diagnosis is rarely observed. PATIENTS CONCERNS: In the first, the patient presented with an aplastic preleukemic phase, whilst the second presented with a rheumatic-like preliminary phase. DIAGNOSES: The case reports of two patients with BCP ALL with a prodromal phase lasting a few weeks are presented. INTERVENTIONS AND OUTCOMES: DNA whole genome profile methylation analysis of bone marrow cells obtained at diagnosis revealed a pattern of methylation that was readily distinguishable from both healthy and standard course BCP ALL bone marrow samples. LESSONS: The biological implication of this observation remains unclear, with many differentially methylated loci involved in many processes like neurogenesis, cell projection organization and adhesion along with leucocyte activation and apoptosis. The prevalence and clinical significance of these methylation changes is unknown but this data indicates that the epigenetic basis of BCP ALL with a prolonged, prodromal phase requires a more detailed assessment.

Distinguishing Ewing sarcoma and osteomyelitis using FTIR spectroscopy.

The differential diagnosis of Ewing sarcoma and osteomyelitis can be challenging and can lead to delays in treatment with possibly devastating results. In this retrospective, small-cohort study we demonstrate, that the Fourier Transformed Infrared (FTIR) spectra of osteomyelitis bone tissue can be differentiated from Ewing sarcoma and normal bone tissue sampled outside tumour area. Significant differences in osteomyelitis samples can be seen in lipid and protein composition. Supervised learning using a quadratic discriminant analysis classifier was able to differentiate the osteomyelitis samples with high accuracy. FTIR spectroscopy, alongside routine radiological and histopathological methods, may offer an additional tool for the differential diagnosis of osteomyelitis and ES.

The de novo design of a biocompatible and functional integral membrane protein using minimal sequence complexity.

The de novo design of integral membrane proteins remains a major challenge in protein chemistry. Here, we describe the bottom-up design of a genetically-encoded synthetic membrane protein comprising only four amino acids (L, S, G and W) in the transmembrane domains. This artificial sequence, which we call REAMP for recombinantly expressed artificial membrane protein, is a single chain of 133 residues arranged into four antiparallel membrane-spanning α-helices. REAMP was overexpressed in Escherichia coli and localized to the cytoplasmic membrane with the intended transmembrane topology. Recombinant REAMP could be extracted from the cell membrane in detergent micelles and was robust and stable in vitro, containing helical secondary structure consistent with the original design. Engineered mono- and bis-histidine residues in the membrane domain of REAMP were able to coordinate heme in vitro, in a manner reminiscent of natural b-type cytochromes. This binding shifted the electrochemical potential of the cofactor, producing a synthetic hemoprotein capable of nascent redox catalysis. These results show that a highly reduced set of amino acids is sufficient to mimic some key properties of natural proteins, and that cellular biosynthesis is a viable route for the production of minimal de novo membrane sequences.

Prediction of Ewing Sarcoma treatment outcome using attenuated tissue reflection FTIR tissue spectroscopy.

Ewing sarcoma is the second most common type of primary bone cancer and predominantly affects children and young people. Improved outcome prediction is key to delivering risk-adjusted, appropriate and effective care to cancer patients. Advances in the Fourier Transform Infrared (FTIR) spectroscopy of tissues enable it to be a non-invasive method to obtain information about the biochemical content of any biological sample. In this retrospective study, attenuated tissue reflection FTIR spectroscopy of biopsy samples from paediatric patients reveals spectral features that are diagnostic for Ewing Sarcoma. Furthermore, our results suggest that spectral features such as these may be of value for the prediction of treatment outcome independent to well-known, routinely used risk factors.

Oxidative Addition, Transmetalation, and Reductive Elimination at a 2,2'-Bipyridyl-Ligated Gold Center.

Three-coordinate bipyridyl complexes of gold, [(κ2-bipy)Au(η2-C2H4)][NTf2], are readily accessed by direct reaction of 2,2'-bipyridine (bipy), or its derivatives, with the homoleptic gold ethylene complex [Au(C2H4)3][NTf2]. The cheap and readily available bipyridyl ligands facilitate oxidative addition of aryl iodides to the Au(I) center to give [(κ2-bipy)Au(Ar)I][NTf2], which undergo first aryl-zinc transmetalation and second C-C reductive elimination to produce biaryl products. The products of each distinct step have been characterized. Computational techniques are used to probe the mechanism of the oxidative addition step, offering insight into both the origin of the reversibility of this process and the observation that electron-rich aryl iodides add faster than electron-poor substrates. Thus, for the first time, all steps that are characteristic of a conventional intermolecular Pd(0)-catalyzed biaryl synthesis are demonstrated from a common monometallic Au complex and in the absence of directing groups.

Whole-genome DNA methylation characteristics in pediatric precursor B cell acute lymphoblastic leukemia (BCP ALL).

In addition to genetic alterations, epigenetic abnormalities have been shown to underlie the pathogenesis of acute lymphoblastic leukemia (ALL)-the most common pediatric cancer. The purpose of this study was to characterize the whole genome DNA methylation profile in children with precursor B-cell ALL (BCP ALL) and to compare this profile with methylation observed in normal bone marrow samples. Additional efforts were made to correlate the observed methylation patterns with selected clinical features. We assessed DNA methylation from bone marrow samples obtained from 38 children with BCP ALL at the time of diagnosis along with 4 samples of normal bone marrow cells as controls using Infinium MethylationEPIC BeadChip Array. Patients were diagnosed and stratified into prognosis groups according to the BFM ALL IC 2009 protocol. The analysis of differentially methylated sites across the genome as well as promoter methylation profiles allowed clear separation of the leukemic and control samples into two clusters. 86.6% of the promoter-associated differentially methylated sites were hypermethylated in BCP ALL. Seven sites were found to correlate with the BFM ALL IC 2009 high risk group. Amongst these, one was located within the gene body of the MBP gene and another was within the promoter region- PSMF1 gene. Differentially methylated sites that were significantly related with subsets of patients with ETV6-RUNX1 fusion and hyperdiploidy. The analyzed translocations and change of genes' sequence context does not affect methylation and methylation seems not to be a mechanism for the regulation of expression of the resulting fusion genes.

High-resolution mass spectrometric analysis of myo-inositol hexakisphosphate using electrospray ionisation Orbitrap.

RATIONALE: The phosphorus storage compound in grains, phytic acid, or myo-inositol hexakisphosphate (IP6), is important for nutrition and human health, and is reportedly the most abundant organic phosphorus compound in soils. Methods for its determination have traditionally relied on complexation with iron and precipitation, acid digestion and measurement of phosphate concentration, or 31 P NMR spectroscopy. Direct determination of phytic acid (and its homologues) using mass spectrometry has, as yet, found limited application to environmental or other complex matrices. The behaviour of phytic acid in electrospray ionisation high-resolution mass spectrometry (ESI-HRMS) and its fragmentation, both in-source and via collision-induced dissociation, have not been studied so far. METHODS: The negative ion mass spectrometry and tandem mass spectrometry (MS/MS) of IP6, and the lower inositol pentakisphosphate (IP5), using an ESI-Orbitrap mass spectrometer is described. The purity of the compounds was investigated using anion-exchange chromatography. RESULTS: IP6 is highly anionic, forming multiply charged ions and sodium adduct ions, which readily undergo dissociation in the ESI source. MS/MS analysis of the phytic acid [M-2H]2- ion and fragment ions and comparison with the full MS of the IP5 reference standard, and the MS/MS spectrum of the pentakisphosphate [M-2H]2- ion, confirm the fragmentation pattern of inositol phosphates in ESI. Further evidence for dissociation in the ion source is shown by the effect of increasing the source voltage on the mass spectrum of phytic acid. CONCLUSIONS: The ESI-HRMS of inositol phosphates is unusual and highly characteristic. The study of the full mass spectrum of IP6 in ESI-HRMS mode indicates the detection of the compound in environmental matrices using this technique is preferable to the use of multiple reaction monitoring (MRM).

Investigation of colloidal graphite as a matrix for matrix-assisted laser desorption/ionisation mass spectrometry of low molecular weight analytes.

The analysis of low molecular weight compounds by matrix-assisted laser desorption/ionisation mass spectrometry is problematic due to the interference and suppression of analyte ionisation by the matrices typically employed - which are themselves low molecular weight compounds. The application of colloidal graphite is demonstrated here as an easy to use matrix that can promote the ionisation of a wide range of analytes including low molecular weight organic compounds, complex natural products and inorganic complexes. Analyte ionisation with colloidal graphite is compared with traditional organic matrices along with various other sources of graphite (e.g. graphite rods and charcoal pencils). Factors such as ease of application, spectra reproducibility, spot longevity, spot-to-spot reproducibility and spot homogeneity (through single spot imaging) are explored. For some analytes, considerable matrix suppression effects are observed resulting in spectra completely devoid of matrix ions. We also report the observation of radical molecular ions [M(-●) ] in the negative ion mode, particularly with some aromatic analytes. Copyright © 2016 John Wiley & Sons, Ltd.

Biochemical characterization of Sfh-I, a subclass B2 metallo-beta-lactamase from Serratia fonticola UTAD54.

The subclass B2 metallo-ß-lactamase (MBL) Sfh-I from Serratia fonticola UTAD54 was cloned and overexpressed in Escherichia coli. The recombinant protein binds one equivalent of zinc, as shown by mass spectrometry, and preferentially hydrolyzes carbapenem substrates. However, compared to other B2 MBLs, Sfh-I also shows limited hydrolytic activity against some additional substrates and is not inhibited by a second equivalent of zinc. These data confirm Sfh-I to be a subclass B2 metallo-ß-lactamase with some distinctive properties.

Molecular characterization of the uncultivatable hemotropic bacterium Mycoplasma haemofelis.

Mycoplasma haemofelis is a pathogenic feline hemoplasma. Despite its importance, little is known about its metabolic pathways or mechanism of pathogenicity due to it being uncultivatable. The recently sequenced M. haemofelis str. Langford 1 genome was analysed and compared to those of other available hemoplasma genomes.Analysis showed that in hemoplasmas genes involved in carbohydrate metabolism are limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source. The majority of the pentose phosphate pathway enzymes that catalyze the de novo synthesis of ribonucleotides were absent, as were cell division protein FtsZ and chaperonins GroEL/ES. Uncharacterized protein paralogs containing putative surface expression motifs, comprised 62% of M. haemofelis and 19% of Mycoplasma suis genome coverage respectively, the majority of which were present in a small number of unstructured islands. Limited mass spectrometry and immunoblot data matched a number of characterized proteins and uncharacterized paralogs, confirming their expression and immunogenicity in vivo.These data have allowed further characterization of these important pathogens, including their limited metabolic capabilities, which may contribute to their uncultivatable status. A number of immunogenic proteins, and a potential mechanism for host immune system evasion, have been identified.