N-glycomic profiling of capsid proteins from Adeno-Associated Virus serotypes.

Adeno-associated virus (AAV) vector has become the leading platform for gene delivery. Each serotype exhibits a different tissue tropism, immunogenicity, and in vivo transduction performance. Therefore, selecting the most suitable AAV serotype is critical for efficient gene delivery to target cells or tissues. Genome divergence among different serotypes is due mainly to the hypervariable regions of the AAV capsid proteins. However, the heterogeneity of capsid glycosylation is largely unexplored. In the present study, the N-glycosylation profiles of capsid proteins of AAV serotypes 1 to 9 have been systemically characterized and compared using a previously developed high-throughput and high-sensitivity N-glycan profiling platform. The results showed that all 9 investigated AAV serotypes were glycosylated, with comparable profiles. The most conspicuous feature was the high abundance mannosylated N-glycans, including FM3, M5, M6, M7, M8, and M9, that dominated the chromatograms within a range of 74 to 83%. Another feature was the relatively lower abundance of fucosylated and sialylated N-glycan structures, in the range of 23%-40% and 10%-17%, respectively. However, the exact N-glycan composition differed. These differences may be utilized to identify potential structural relationships between the 9 AAV serotypes. The current research lays the foundation for gaining better understanding of the importance of N-glycans on the AAV capsid surface that may play a significant role in tissue tropism, interaction with cell surface receptors, cellular uptake, and intracellular processing.

Multi-attribute analysis of adeno-associated virus by size exclusion chromatography with fluorescence and triple-wavelength UV detection.

Adeno-associated virus (AAV) is the leading platform for in vivo gene therapy to treat numerous genetic diseases. Comprehensive analysis of the AAV particles is essential to ensure desired safety and efficacy. An array of techniques is required to evaluate their critical quality attributes. However, many of these techniques are expensive, time-consuming, labour-intensive, and varying in accuracy. Size exclusion chromatography coupled with fluorescence and triple-wavelength ultraviolet detection (SEC-FLD-TWUV) and incorporating an aromatic amino acid of tryptophan as an internal standard offers a simple, rapid, and reliable approach for simultaneous multi-attribute analysis of AAVs. In the current study, we demonstrate its capability for AAV characterization and quantification, that includes capsid concentration, empty to full capsid ratio, vector genome concentration, and the presence of aggregates or fragments. All were performed in 20-min chromatographic runs with minimal sample handling. Data analysis involves the assessment of intrinsic fluorescence and UV absorbance of samples at three wavelengths that can be utilised to determine the content of the capsid protein and genome copy number. The separation efficiency using SEC columns with different pore sizes, and elution buffers of varying compositions, ionic strength, and pH values was also evaluated. This SEC-FLD-TWUV method may serve as a powerful yet cost-effective tool for responsive quality evaluation of AAVs. This may enhance performance, robustness, and safety of bioprocessing for AAV vectors to be used in gene therapy.

Quantitative profiling of N-glycosylation of SARS-CoV-2 spike protein variants.

With the global spread of the corona virus disease-2019 pandemic, new spike variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuously emerge due to increased possibility of virus adaptive amino acid mutations. However, the N-glycosylation profiles of different spike variants are yet to be explored extensively, although the spike protein is heavily glycosylated and surface glycans are well-established to play key roles in viral infection and immune response. Here, we investigated quantitatively the N-glycosylation profiles of seven major emerging spike variants including Original, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.671.1), Delta (B.1.671.2), and Omicron (B.1.1.529). The aim was to understand the changing pattern of N-glycan profiles in SARS-CoV-2 evolution in addition to the widely studied amino acid mutations. Different spike variants exhibit substantial variations in the relative abundance of different glycan peaks and subclasses, although no specific glycan species are exclusively present in or absent from any specific variant. Cluster analysis shows that the N-glycosylation profiles may hold the potential for SARS-CoV-2 spike variants classification. Alpha and Beta variants exhibit the closest similarity to the Original, and the Delta variant displays substantial similarity to Gamma and Kappa variants, while the Omicron variant is significantly different from its counterparts. We demonstrated that there is a quantifiable difference in N-glycosylation profiles among different spike variants. The current study and observations herein provide a valuable framework for quantitative N-glycosylation profiling of new emerging viral variants and give us a more comprehensive picture of COVID-19 evolution.

Identification of New Fusarium sulawense Strains Causing Soybean Pod Blight in China and Their Control Using Carbendazim, Dipicolinic Acid and Kojic Acid.

Soybean plants are highly susceptible to Fusarium species, which significantly reduce soybean production and quality. Several Fusarium species have been reported to synthesize mycotoxins, such as trichothecene, which have been related to major human diseases. In November 2021, soybean pods in Nantong municipality, China, showed black necrotic lesions during the harvest stage. The disease incidence reached 69%. The pathogen was identified as Fusarium sulawense via morphological analysis and sequencing of ITS, EF1-α and RPB2 genes. A PCR assay with primers targeting the trichothecene biosynthesis genes suggested that the three isolates could synthesize trichothecenes. The effectiveness of fungicide carbendazim and natural metabolites dipicolinic acid and kojic acid was screened for the management of F. sulawense on postharvest soybean pods. The highest efficacy was obtained when combining 3.8 mg/mL carbendazim and 0.84 mg/mL dipicolinic acid (curative efficacy: 49.1% lesion length inhibition; preventive efficacy: 82.7% lesion length inhibition), or 1.9 mg/mL carbendazim and 0.71 mg/mL kojic acid (preventive efficacy: 84.9% lesion length inhibition). Collectively, this report will lead to a better understanding of the safety hazards found in soybean products in China and reveals the application of dipicolinic and kojic acids to reduce the use of carbendazim.

Serum N-glycomic profiling may provide potential signatures for surveillance of COVID-19.

Disease development and progression are often associated with aberrant glycosylation, indicating that changes in biological fluid glycome may potentially serve as disease signatures. The corona virus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a significant threat to global human health. However, the effect of SARS-CoV-2 infection on the overall serum N-glycomic profile has been largely unexplored. Here, we extended our 96-well-plate-based high-throughput, high-sensitivity N-glycan profiling platform further with the aim of elucidating potential COVID-19-associated serum N-glycomic alterations. Use of this platform revealed both similarities and differences between the serum N-glycomic fingerprints of COVID-19 positive and control cohorts. Although there were no specific glycan peaks exclusively present or absent in COVID-19 positive cohort, this cohort showed significantly higher levels of glycans and variability. On the contrary, the overall N-glycomic profiles for healthy controls were well-contained within a narrow range. From the serum glycomic analysis, we were able to deduce changes in different glycan subclasses sharing certain structural features. Of significance was the hyperbranched and hypersialylated glycans and their derived glycan subclass traits. T-distributed stochastic neighbor embedding and hierarchical heatmap clustering analysis were performed to identify 13 serum glycomic variables that potentially distinguished the COVID-19 positive from healthy controls. Such serum N-glycomic changes described herein may indicate or correlate to the changes in serum glycoproteins upon COVID-19 infection. Furthermore, mapping the serum N-glycome following SARS-CoV-2 infection may help us better understand the disease and enable "Long-COVID" surveillance to capture the full spectrum of persistent symptoms.

First Report of Fusarium acuminatum Causing Leaf Blight on Garlic in China.

In June 2021, leaf blight symptoms were detected on garlic plants (Allium sativum) in southeastern Jiangsu (Nantong municipality; 120.61° E, 33.25° N) in China. Two-month-old garlic plants exhibited leaf tip die back and light brown lesions in new and old leaves (Figure 1). The symptoms were observed in 40% of the plants in a 60-square-meters commercial field surrounded by rice fields, and were similar to those reported for Botrytis porri, Septoria allii and Stemphylium eturmiunum causing leaf blight on garlic (Dumin et al. 2021; Park et al. 2013; Zhang et al. 2009). Six samples of symptomatic tissue collected in Nantong municipality, approximately 1 cm2 in size, were sterilized in 2% NaOCl for 15 min and washed twice with sterile ddH2O. The pathogen was isolated from all collected samples on PDA medium, containing 50 µg/mL chloramphenicol, at 26°C. Pink colonies with orange pigmentation were observed after 7 days. Internal transcribed spacer (ITS), elongation factor 1-α (EF1-α), RNA polymerase II largest subunit (RPB1) and RNA polymerase II second largest subunit (RPB2) genes were amplified using ITS1/ITS4, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5 and fRPB2-7CF/fRPB2-11aR primers, respectively. A total of 17 isolates were obtained, with nine of the isolates sharing the same sequences (strain NJC21), six of the isolates sharing the same sequences (strain NJC22), and the other two isolates showing different sequences (strains NJC23 and NJC24). The obtained sequences were submitted in GenBank under accession numbers OL655398-OL655401 (ITS), and OL741712-OL741723 (EF1-α, RPB1, RPB2). The obtained ITS sequences shared >99% homology to the ITS gene from F. acuminatum IBE000006 (EF531232), the EF1-α sequences shared 99% homology to the EF1-α gene from F. acuminatum F1514 (LC469785), the RPB1 sequences shared >99% homology to the RPB1 gene from F. acuminatum JW 289003 (MZ921675), and the RPB2 sequences shared 100% homology to the RPB2 gene from F. acuminatum NL19-077002 (MZ921813) or 100% homology to the RPB2 gene from F. acuminatum MD1 (MW164629). A phylogenetic tree was constructed using MEGA7 with related Fusarium strains (Figure 2). Microscope observations after incubation in potato-sucrose-agar (PSA) medium showed the presence of oval microconidia, fusiform macroconidia, septate mycelium and chlamydospores, and agree with the morphology of F. acuminatum (Marek et al. 2013). The pathogenicity was screened with two-week-old wounded and non-wounded garlic plants using a 1 × 106 spores/mL solution (20 µL). Sterile ddH2O was used in the control experiment. The inoculated plants were incubated at 26°C and 60% relative humidity for 3 days, detecting similar lesions compared to those observed in the field. The pathogen was recovered from 5 different lesions, from different plants, and its identity was confirmed by sequence analysis. Recently, F. acuminatum was reported to cause garlic bulb rot in Serbia (Ignjatov et al. 2017). Although F. acuminatum is well known as a causal agent of root rot (Li et al. 2021; Tang et al. 2021), F. acuminatum has also been found causing leaf blight on onion (Parkunan et al. 2013) and muskmelon (Yu et al. 2021). This is the first report of F. acuminatum causing leaf blight on garlic, demonstrating the host and tissue promiscuity of this pathogen. China is the largest producer of garlic in the world with nearly 20 million tons harvested in 2020. This report will help to better understand the pathogens that are affecting garlic production in China.

Construction of an InstantPC-derivatized glycan glucose unit database: A foundation work for high-throughput and high-sensitivity glycomic analysis.

The glycosylation profile of biotherapeutic glycoproteins is a critical quality attribute that is routinely monitored to ensure desired product quality, safety and efficacy. Additionally, as one of the most prominent and complex post-translational modifications, glycosylation plays a key role in disease manifestation. Changes in glycosylation may serve as a specific and sensitive biomarker for disease diagnostics and prognostics. However, the conventional 2-aminobenzamide-based N-glycosylation analysis procedure is time-consuming and insensitive with poor reproducibility. We have evaluated an innovative streamlined 96-well-plate-based platform utilizing InstantPC label for high-throughput, high-sensitivity glycan profiling, which is user-friendly, robust and ready for automation. However, the limited availability of InstantPC-labeled glycan standards has significantly hampered the applicability and transferability of this platform for expedited glycan structural profiling. To address this challenge, we have constructed a detailed InstantPC-labeled glycan glucose unit (GU) database through analysis of human serum and a variety of other glycoproteins from various sources. Following preliminary hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection separation and analysis, glycoproteins with complex glycan profiles were subjected to further fractionation by weak anion exchange HILIC and exoglycosidase sequential digestion for cross-validation of the glycan assignment. Hydrophilic interaction ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry was subsequently utilized for glycan fragmentation and accurate glycan mass confirmation. The constructed InstantPC glycan GU database is accurate and robust. It is believed that this database will enhance the application of the developed platform for high-throughput, high-sensitivity glycan profiling and that it will eventually advance glycan-based biopharmaceutical production and disease biomarker discovery.

First Report of Epicoccum sorghinum Causing Leaf Sheath and Leaf Spot on Maize in China.

In November 2020, leaf sheath on maize (Zea mays) was detected in southeastern Jiangsu (Nantong municipality; 120.54° E, 31.58° N) in China. Physiologically mature plants, 13 weeks of cultivation (at the harvest stage), exhibited red-brown lesions in stem and leaves, and dried-up stem (Figure 1). The symptoms were observed on approximately 95% of the maize plants in a 0.8 ha maize field surrounded by old sorghum fields and the crop yield was decreased by 70-85% with respect previous years, when no disease symptoms were detected. Small pieces, approximately 0.3 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated (one isolate was obtained) and cultured on PDA medium, containing chloramphenicol (50 µg/mL), under darkness at 26 ºC for 3 days. Amplification of internal transcribed spacer (ITS), large subunit (LSU), actin (ACT) and ß-tubulin (TUB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R and T1/Bt2b primers, respectively (Ma et al. 2021). Sequences were submitted to GenBank under accession numbers MW800180 (ITS), MW800361 (LSU), MW845677 (ACT) and MW892439 (TUB2). Blast search revealed that the ITS sequence had 100% (492/492 bp) homology with E. sorghinum LY-D-1-1, MT604999, LSU had 98% (1075/1091 bp) homology with E. sorghinum GZDS2018BXT010, MK516207, ACT had 96% (214/222 bp) homology with E. sorghinum M3, MK044832, and TUB2 had 99% (498/499 bp) homology with E. sorghinum BJ-F1, MF987525. Molecular phylogenetic tree was constructed using MEGA7 to confirm the identity of the pathogen. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest likelihood (-1774.9882) is shown in Figure 2. Bipolaris, Curvularia and Fusarium spp. found causing leaf spot on maize were included in the phylogenetic tree (Liu et al. 2021; Reyes Gaige et al. 2020; Chang et al. 2016). To confirm pathogenicity, a sterilized spatula was used to make wounds (3 mm diameter, 1 mm depth) on the stem and leaves of 2-week old maize plants. A solution containing 1 × 108 spores/mL (20 µL) was injected in the wound, whereas sterilized ddH2O was used in the control experiment. Inoculated plants were maintained in a growth chamber at 28 °C and 60% relative humidity for 3 days, observing fast-growing necrotic lesions in both stem and leaves. The pathogen was recovered from the infected plants and its identity was confirmed by morphological and sequence analyses. Microscope observations indicated the presence of chlamydospores, oval conidia (3 × 5 µm) and round pycnidia (60-100 µm diameter), and agree with those previously reported for the morphology of E. sorghinum (Bao et al. 2019). During last 2 years, E. sorghinum was reported to cause leaf spot on a number of relevant agricultural crops in China, including taro, Brassica parachinensis, tea, rice and wheat (Du et al. 2020; Li et al. 2020; Liu et al. 2020a, 2020b), confirming the expansion and host promiscuity of this pathogen. The pathogen was also reported to cause leaf spot on maize in Brazil in 2004 (Do Amaral et al. 2004); however, this is the first report of E. sorghinum causing leaf sheath and leaf spot on maize in China. Maize an important agricultural crop in China with more than 168 million tons produced in 2019. The observed yield loss and disease incidence of the isolated strain suggest that E. sorghinum may be a threat to maize production in China.

High-throughput and high-sensitivity N-Glycan profiling: A platform for biopharmaceutical development and disease biomarker discovery.

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of disease biomarkers. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-well plate format platform for streamlined glycan profiling that takes advantage of rapid glycoprotein denaturation, deglycosylation, fluorescent derivatization, and on-matrix glycan clean-up. This approach offers high sensitivity with consistent identification and quantification of diverse N-glycans across multiple samples on a high-throughput scale. We demonstrate its capability for N-glycan profiling of glycoproteins from various sources, including two recombinant monoclonal antibodies produced from Chinese Hamster Ovary cells, EG2-hFc and rituximab, polyclonal antibodies purified from human serum, and total glycoproteins from human serum. Combined with the complementary information obtained by sequential digestion from exoglycosidase arrays, this approach allows the detection and identification of multiple N-glycans in these complex biological samples. The reagents, workflow, and Hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD), are simple enough to be implemented into a straightforward user-friendly setup. This improved technology provides a powerful tool in support of rapid advancement of glycan analysis for biopharmaceutical development and biomarker discovery for clinical disease diagnosis.

The novel therapeutic potential of bovine α-lactalbumin made lethal to tumour cells (BALMET) and oleic acid in oral squamous cell carcinoma (OSCC).

BACKGROUND: Since the serendipitous discovery of bovine α-lactalbumin made lethal to tumour cells (BAMLET)/human α-lactalbumin made lethal to tumour cells there has been an increased interest in the ability of the two components, oleic acid and α-lactalbumin, to form anti-cancer complexes. Here we have investigated the in-vitro efficacy of the BAMLET complex in killing oral cancer (OC) cells, determined the active component of the complex and investigated possible biological mechanisms. MATERIALS AND METHODS: Two OC cell lines (±p53 mutation) and one dysplastic cell line were used as a model of progressive oral carcinogenesis. We performed cell viability assays with increasing BAMLET concentrations to determine the cytotoxic potential of the complex. We further analysed the individual components to determine their respective cytotoxicities. siRNA knockdown of p53 was used to determine its functional role in mediating sensitivity to BAMLET. Cell death mechanisms were investigated by flow cytometry, confocal microscopy and the lactate dehydrogenase assay. RESULTS: Our results show that BAMLET is cytotoxic to the OC and dysplastic cell lines in a time and dose-dependent manner. The cytotoxic component was found to be oleic acid, which, can induce cytotoxicity even when not in complex. Our results indicate that the mechanism of cytotoxicity occurs through multiple simultaneous events including cell cycle arrest, autophagy like processes with a minor involvement of necrosis. CONCLUSION: Deciphering the mechanism of cytotoxicity will aid treatment modalities for OC. This study highlights the potential of BAMLET as a novel therapeutic strategy in oral dysplastic and cancerous cells.

Mammalian cell culture for production of recombinant proteins: A review of the critical steps in their biomanufacturing.

The manufacturing of recombinant protein is traditionally undertaken in mammalian cell culture. Today, speed, cost and safety are the primary considerations for process improvements in both upstream and downstream manufacturing. Leaders in the biopharmaceutical industry are striving for continuous improvements to increase throughput, lower costs and produce safer more efficacious drugs. This can be achieved through advances in cell line engineering, process development of cell culture, development of chemically defined media and increased emphasis on product characterization. In the first part, this review provides a historical perspective on approved biotherapeutics by regulatory bodies which pave the way for next-generation products (including gene therapy). In the second part, it focuses on the application of in vitro and in vivo cell line engineering approaches, modern process development improvements including continuous manufacturing, recent developments in media formulation, and improvements in critical quality attribute determinations for products produced predominantly in mammalian cells.

The "regulation resonance" phenomenon in control systems and optimization schemes.

Steam generator water level control incorporates the system that controls the speed of the main water feed pump and the one that controls the degree to which the main water feed valve is open. Although these two control systems are set independently, they have a reciprocal influence. A key problem is that, in the actuator for the regulation of the two systems, the regulated variables tend to oscillate with a relatively large amplitude even under stable conditions. In this paper, a pervasive regulating resonance phenomenon is inspected and analyzed by means of simulating the real working conditions. And using a mathematical model of the main water feed flow, it reveals that the two factors which directly cause the regulation resonance phenomenon are the frequency characteristics of the control system and the phase difference. On the basis of this, three potential optimization schemes have been proposed: the optimization of the regulation parameters; changing the phase difference; and setting a regulation dead-band which can improve the stability of the control system.

NMR studies on mechanism of isomerisation of fructose 6-phosphate to glucose 6-phosphate catalysed by phosphoglucose isomerase from Thermococcus kodakarensis.

The fate of hydrogen atoms at C-2 of glucose 6-phosphate (G6P) and C-1 of fructose 6-phosphate (F6P) was studied in the reaction catalysed by phosphoglucose isomerase from Thermococcus kodakarensis (TkPGI) through 1D and 2D NMR methods. When the reaction was performed in (2)H2O the hydrogen atoms in the aforementioned positions were exchanged with deuterons indicating that the isomerization occurred by a cis-enediol intermediate involving C-1 pro-R hydrogen of F6P. These features are similar to those described for phosphoglucose isomerases from rabbit muscle and Pyrococcus furiosus.

Amyloid Oligomers and Mature Fibrils Prepared from an Innocuous Protein Cause Diverging Cellular Death Mechanisms.

Despite significant advances, the molecular identity of the cytotoxic species populated during in vivo amyloid formation crucial for the understanding of neurodegenerative disorders is yet to be revealed. In this study lysozyme prefibrillar oligomers and fibrils in both mature and sonicated states have been isolated through an optimized ultrafiltration/ultracentrifugation method and characterized with various optical spectroscopic techniques, atomic force microscopy, and transmission electron microscopy. We examined their level and mode of toxicity on rat pheochromocytoma (PC12) cells in both differentiated and undifferentiated states. We find that oligomers and fibrils display cytotoxic capabilities toward cultured cells in vitro, with oligomers producing elevated levels of cellular injury toward undifferentiated PC12 cells (PC12(undiff)). Furthermore, dual flow cytometry staining experiments demonstrate that the oligomers and mature fibrils induce divergent cellular death pathways (apoptosis and secondary necrosis, respectively) in these PC12 cells. We have also shown that oligomers but not sonicated mature fibrils inhibit hippocampal long term potentiation, a form of synaptic plasticity implicated in learning and memory, in vivo. We conclude that our in vitro and in vivo findings confer a level of resistance toward amyloid fibrils, and that the PC 12-based comparative cytotoxicity assay can provide insights into toxicity differences between differently aggregated protein species.

Electrostatic interactions play an essential role in the binding of oleic acid with α-lactalbumin in the HAMLET-like complex: a study using charge-specific chemical modifications.

Human α-lactalbumin made lethal to tumor cells (HAMLET) and its analogs are partially unfolded protein-oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge-specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively-charged lysine residues to negatively-charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild-type α-lactalbumin-oleic acid complex. With the addition of OA, the wild-type and guanidinated α-lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α-lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively-charged basic groups on α-lactalbumin and the negatively-charged carboxylate groups on OA molecules play an essential role in the binding of OA to α-lactalbumin and that these interactions appear to be as important as hydrophobic interactions.

Alternatively folded proteins with unexpected beneficial functions.

HAMLET (human α-lactalbumin made lethal to tumour cells) and its related partially unfolded protein-fatty acid complexes are novel biomolecular nanoparticles that possess relatively selective cytotoxic activities towards tumour cells. One of the key characteristics is the requirement for the protein to be partially unfolded, hence endowing native proteins with additional functions in the alternatively folded states. Beginning with the history of its discovery and development, the cellular targets that appear to be strongly correlated with tumour cell death are introduced in the present article.