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  This is an Open Access Journal Open Access journal
ISSN (Online) 2571-5135
Published by MDPI Homepage  [258 journals]
  • High-Throughput, Vol. 9, Pages 15: Dark Proteome Database: Studies on

    • Authors: Nelson Perdigão, Pedro M. C. Pina, Cátia Rocha, João Manuel R. S. Tavares, Agostinho Rosa
      First page: 15
      Abstract: There is a misconception that intrinsic disorder in proteins is equivalent to darkness. The present study aims to establish, in the scope of the Swiss-Prot and Dark Proteome databases, the relationship between disorder and darkness. Three distinct predictors were used to calculate the disorder of Swiss-Prot proteins. The analysis of the results obtained with the used predictors and visualization paradigms resulted in the same conclusion that was reached before: disorder is mostly unrelated to darkness.
      Citation: High-Throughput
      PubDate: 2020-06-30
      DOI: 10.3390/ht9030015
      Issue No: Vol. 9, No. 3 (2020)
  • High-Throughput, Vol. 9, Pages 16: Influence of the Ovine Genital Tract
           Microbiota on the Species Artificial Insemination Outcome. A Pilot Study
           in Commercial Sheep Farms

    • Authors: Malena Serrano, Eric Climent, Fernando Freire, Juan F. Martínez-Blanch, Carmen González, Luis Reyes, M. Carmen Solaz-Fuster, Jorge H. Calvo, M. Ángeles Jiménez, Francisco M. Codoñer
      First page: 16
      Abstract: To date, there is a lack of research into the vaginal and sperm microbiome and its bearing on artificial insemination (AI) success in the ovine species. Using hypervariable regions V3–V4 of the 16S rRNA, we describe, for the first time, the combined effect of the ovine microbiome of both females (50 ewes belonging to five herds) and males (five AI rams from an AI center) on AI outcome. Differences in microbiota abundance between pregnant and non-pregnant ewes and between ewes carrying progesterone-releasing intravaginal devices (PRID) with or without antibiotic were tested at different taxonomic levels. The antibiotic treatment applied with the PRID only altered Streptobacillus genus abundance, which was significantly lower in ewes carrying PRID with antibiotic. Mageebacillus, Histophilus, Actinobacilllus and Sneathia genera were significantly less abundant in pregnant ewes. In addition, these genera were more abundant in two farms with higher AI failure. Species of these genera such as Actinobacillus seminis and Histophilus somni have been associated with reproductive disorders in the ovine species. These genera were not present in the sperm samples of AI rams, but were found in the foreskin samples of rams belonging to herd 2 (with high AI failure rate) indicating that their presence in ewes’ vagina could be due to prior transmission by natural mating with rams reared in the herd.
      Citation: High-Throughput
      PubDate: 2020-07-06
      DOI: 10.3390/ht9030016
      Issue No: Vol. 9, No. 3 (2020)
  • High-Throughput, Vol. 9, Pages 17: Health Impact and Therapeutic
           Manipulation of the Gut Microbiome

    • Authors: Eric Banan-Mwine Daliri, Fred Kwame Ofosu, Ramachandran Chelliah, Byong Hoon Lee, Deog-Hwan Oh
      First page: 17
      Abstract: Recent advances in microbiome studies have revealed much information about how the gut virome, mycobiome, and gut bacteria influence health and disease. Over the years, many studies have reported associations between the gut microflora under different pathological conditions. However, information about the role of gut metabolites and the mechanisms by which the gut microbiota affect health and disease does not provide enough evidence. Recent advances in next-generation sequencing and metabolomics coupled with large, randomized clinical trials are helping scientists to understand whether gut dysbiosis precedes pathology or gut dysbiosis is secondary to pathology. In this review, we discuss our current knowledge on the impact of gut bacteria, virome, and mycobiome interactions with the host and how they could be manipulated to promote health.
      Citation: High-Throughput
      PubDate: 2020-07-29
      DOI: 10.3390/ht9030017
      Issue No: Vol. 9, No. 3 (2020)
  • High-Throughput, Vol. 9, Pages 7: Colonization Resistance in the Infant
           Gut: The Role of B. infantis in Reducing pH and Preventing Pathogen Growth

    • Authors: Rebbeca M. Duar, David Kyle, Giorgio Casaburi
      First page: 7
      Abstract: Over the past century, there has been a steady increase in the stool pH of infants from industrialized countries. Analysis of historical data revealed a strong association between abundance of Bifidobacterium in the gut microbiome of breasted infants and stool pH, suggesting that this taxon plays a key role in determining the pH in the gut. Bifidobacterium longum subsp. infantis is uniquely equipped to metabolize human milk oligosaccharides (HMO) from breastmilk into acidic end products, mainly lactate and acetate. The presence of these acidic compounds in the infant gut is linked to a lower stool pH. Conversely, infants lacking B. infantis have a significantly higher stool pH, carry a higher abundance of potential pathogens and mucus-eroding bacteria in their gut microbiomes, and have signs of chronic enteric inflammation. This suggests the presence of B. infantis and low intestinal pH may be critical to maintaining a protective environment in the infant gut. Here, we summarize recent studies demonstrating that feeding B. infantis EVC001 to breastfed infants results in significantly lower fecal pH compared to controls and propose that low pH is one critical factor in preventing the invasion and overgrowth of harmful bacteria in the infant gut, a process known as colonization resistance.
      Citation: High-Throughput
      PubDate: 2020-03-27
      DOI: 10.3390/ht9020007
      Issue No: Vol. 9, No. 2 (2020)
  • High-Throughput, Vol. 9, Pages 8: DMETTM Genotyping: Tools for Biomarkers
           Discovery in the Era of Precision Medicine

    • Authors: Giuseppe Agapito, Marzia Settino, Francesca Scionti, Emanuela Altomare, Pietro Hiram Guzzi, Pierfrancesco Tassone, Pierosandro Tagliaferri, Mario Cannataro, Mariamena Arbitrio, Maria Teresa Di Martino
      First page: 8
      Abstract: The knowledge of genetic variants in genes involved in drug metabolism may be translated into reduction of adverse drug reactions, increase of efficacy, healthcare outcomes improvement and economic benefits. Many high-throughput tools are available for the genotyping of Single Nucleotide Polymorphisms (SNPs) known to be related to drugs and xenobiotics metabolism. DMETTM platform represents an example of SNPs panel to discover biomarkers correlated to efficacy or toxicity in common and rare diseases. The difficulty in analyzing the mole of information generated by DMETTM platform led to the development and implementation of algorithms and tools for statistical and data mining analysis. These softwares allow efficient handling of the omics data to validate the explorative SNPs identified by DMET assay and to correlate them with drug efficacy, toxicity and/or cancer susceptibility. In this review we present a suite of bioinformatic frameworks for the preprocessing and analysis of DMET-SNPs data. In particular, we introduce a workflow that uses the GenoMetric Query Language, a high-level query language specifically designed for genomics, able to query public datasets (such as ENCODE, TCGA, GENCODE annotation dataset, etc.) as well as to combine them with private datasets (e.g., output from Affymetrix® DMETTM Platform).
      Citation: High-Throughput
      PubDate: 2020-03-29
      DOI: 10.3390/ht9020008
      Issue No: Vol. 9, No. 2 (2020)
  • High-Throughput, Vol. 9, Pages 9: A Simple, Label-Free, and
           High-Throughput Method to Evaluate the Epigallocatechin-3-Gallate Impact
           in Plasma Molecular Profile

    • Authors: Rúben Araújo, Luís Ramalhete, Helder Da Paz, Edna Ribeiro, Cecília R.C. Calado
      First page: 9
      Abstract: Epigallocatechin-3-gallate (EGCG), the major catechin present in green tea, presents diverse appealing biological activities, such as antioxidative, anti-inflammatory, antimicrobial, and antiviral activities, among others. The present work evaluated the impact in the molecular profile of human plasma from daily consumption of 225 mg of EGCG for 90 days. Plasma from peripheral blood was collected from 30 healthy human volunteers and analyzed by high-throughput Fourier transform infrared spectroscopy. To capture the biochemical information while minimizing the interference of physical phenomena, several combinations of spectra pre-processing methods were evaluated by principal component analysis. The pre-processing method that led to the best class separation, that is, between the plasma spectral data collected at the beginning and after the 90 days, was a combination of atmospheric correction with a second derivative spectra. A hierarchical cluster analysis of second derivative spectra also highlighted the fact that plasma acquired before EGCG consumption presented a distinct molecular profile after the 90 days of EGCG consumption. It was also possible by partial least squares regression discriminant analysis to correctly predict all unlabeled plasma samples (not used for model construction) at both timeframes. We observed that the similarity in composition among the plasma samples was higher in samples collected after EGCG consumption when compared with the samples taken prior to EGCG consumption. Diverse negative peaks of the normalized second derivative spectra, associated with lipid and protein regions, were significantly affected (p < 0.001) by EGCG consumption, according to the impact of EGCG consumption on the patients’ blood, low density and high density lipoproteins ratio. In conclusion, a single bolus dose of 225 mg of EGCG, ingested throughout a period of 90 days, drastically affected plasma molecular composition in all participants, which raises awareness regarding prolonged human exposure to EGCG. Because the analysis was conducted in a high-throughput, label-free, and economic analysis, it could be applied to high-dimension molecular epidemiological studies to further promote the understanding of the effect of bio-compound consumption mode and frequency.
      Citation: High-Throughput
      PubDate: 2020-04-09
      DOI: 10.3390/ht9020009
      Issue No: Vol. 9, No. 2 (2020)
  • High-Throughput, Vol. 9, Pages 10: Microbiota and Human Reproduction: The
           Case of Male Infertility

    • Authors: Rossella Tomaiuolo, Iolanda Veneruso, Federica Cariati, Valeria D’Argenio
      First page: 10
      Abstract: The increasing interest in metagenomics is enhancing our knowledge regarding the composition and role of the microbiota in human physiology and pathology. Indeed, microbes have been reported to play a role in several diseases, including infertility. In particular, the male seminal microbiota has been suggested as an important factor able to influence couple’s health and pregnancy outcomes, as well as offspring health. Nevertheless, few studies have been carried out to date to deeper investigate semen microbiome origins and functions, and its correlations with the partner’s reproductive tract microbiome. Here, we report the state of the art regarding the male reproductive system microbiome and its alterations in infertility.
      Citation: High-Throughput
      PubDate: 2020-04-13
      DOI: 10.3390/ht9020010
      Issue No: Vol. 9, No. 2 (2020)
  • High-Throughput, Vol. 9, Pages 11: Chimeric Virus Made from crTMV RNA and
           the Coat Protein of Potato Leafroll Virus is Targeted to the Nucleolus and
           Can Infect Nicotiana benthamiana Mechanically

    • Authors: Konstantin O. Butenko, Inna A. Chaban, Eugene V. Skurat, Olga A. Kondakova, Yuri F. Drygin
      First page: 11
      Abstract: A genetically engineered chimeric virus crTMV-CP-PLRV composed of the crucifer-infecting tobacco mosaic virus (crTMV) RNA and the potato leafroll virus (PLRV) coat protein (CP) was obtained by agroinfiltration of Nicotiana benthamiana with the binary vector pCambia-crTMV-CPPLRV. The significant levels of the chimeric virus enabled direct visualization of crTMV-CP-PLRV in the cell and to investigate the mechanism of the pathogenesis. Localization of the crTMV-CP-PLRV in plant cells was examined by immunoblot techniques, as well as light, and transmission electron microscopy. The chimera can transfer between vascular and nonvascular tissues. The chimeric virus inoculum is capable to infect N. benthamiana mechanically. The distinguishing feature of the chimeric virus, the RNA virus with the positive genome, was found to localize in the nucleolus. We also investigated the role of the N-terminal sequence of the PLRV P3 coat protein in the cellular localization of the virus. We believe that the gene of the PLRV CP can be substituted with genes from other challenging-to-study plant pathogens to produce other useful recombinant viruses.
      Citation: High-Throughput
      PubDate: 2020-04-26
      DOI: 10.3390/ht9020011
      Issue No: Vol. 9, No. 2 (2020)
  • High-Throughput, Vol. 9, Pages 12: Microbiota and Human Reproduction: The
           Case of Female Infertility

    • Authors: Rossella Tomaiuolo, Iolanda Veneruso, Federica Cariati, Valeria D’Argenio
      First page: 12
      Abstract: During the last decade, the availability of next-generation sequencing-based approaches has revealed the presence of microbial communities in almost all the human body, including the reproductive tract. As for other body sites, this resident microbiota has been involved in the maintenance of a healthy status. As a consequence, alterations due to internal or external factors may lead to microbial dysbiosis and to the development of pathologies. Female reproductive microbiota has also been suggested to affect infertility, and it may play a key role in the success of assisted reproductive technologies, such as embryo implantation and pregnancy care. While the vaginal microbiota is well described, the uterine microbiota is underexplored. This could be due to technical issues, as the uterus is a low biomass environment. Here, we review the state of the art regarding the role of the female reproductive system microbiota in women’s health and human reproduction, highlighting its contribution to infertility.
      Citation: High-Throughput
      PubDate: 2020-05-03
      DOI: 10.3390/ht9020012
      Issue No: Vol. 9, No. 2 (2020)
  • High-Throughput, Vol. 9, Pages 13: Genetic Counseling and NGS Screening
           for Recessive LGMD2A Families

    • Authors: Claudia Strafella, Valerio Caputo, Giulia Campoli, Rosaria Maria Galota, Julia Mela, Stefania Zampatti, Giulietta Minozzi, Cristina Sancricca, Serenella Servidei, Emiliano Giardina, Raffaella Cascella
      First page: 13
      Abstract: Genetic counseling applied to limb–girdle muscular dystrophies (LGMDs) can be very challenging due to their clinical and genetic heterogeneity and the availability of different molecular assays. Genetic counseling should therefore be addressed to select the most suitable approach to increase the diagnostic rate and provide an accurate estimation of recurrence risk. This is particularly true for families with a positive history for recessive LGMD, in which the presence of a known pathogenetic mutation segregating within the family may not be enough to exclude the risk of having affected children without exploring the genetic background of phenotypically unaffected partners. In this work, we presented a family with a positive history for LGMD2A (OMIM #253600, also known as calpainopathy) characterized by compound heterozygosity for two CAPN3 mutations. The genetic specialist suggested the segregation analysis of both mutations within the family as a first-level analysis. Sequentially, next-generation sequencing (NGS) analysis was performed in the partners of healthy carriers to provide an accurate recurrence/reproductive risk estimation considering the genetic background of the couple. Finally, this work highlighted the importance of providing a genetic counseling/testing service even in unaffected individuals with a carrier partner. This approach can support genetic counselors in estimating the reproductive/recurrence risk and eventually, suggesting prenatal testing, early diagnosis or other medical surveillance strategies.
      Citation: High-Throughput
      PubDate: 2020-05-10
      DOI: 10.3390/ht9020013
      Issue No: Vol. 9, No. 2 (2020)
  • High-Throughput, Vol. 9, Pages 14: Intra-Laboratory Evaluation of
           Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to
           Determine Bactericidal Activity of Human Sera against Shigella

    • Authors: Omar Rossi, Eleonora Molesti, Allan Saul, Carlo Giannelli, Francesca Micoli, Francesca Necchi
      First page: 14
      Abstract: Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing.
      Citation: High-Throughput
      PubDate: 2020-06-08
      DOI: 10.3390/ht9020014
      Issue No: Vol. 9, No. 2 (2020)
  • High-Throughput, Vol. 9, Pages 1: Applications of Next Generation
           Sequencing to the Analysis of Familial Breast/Ovarian Cancer

    • Authors: Veronica Zelli, Chiara Compagnoni, Katia Cannita, Roberta Capelli, Carlo Capalbo, Mauro Di Vito Nolfi, Edoardo Alesse, Francesca Zazzeroni, Alessandra Tessitore
      First page: 1
      Abstract: Next generation sequencing (NGS) provides a powerful tool in the field of medical genetics, allowing one to perform multi-gene analysis and to sequence entire exomes (WES), transcriptomes or genomes (WGS). The generated high-throughput data are particularly suitable for enhancing the understanding of the genetic bases of complex, multi-gene diseases, such as cancer. Among the various types of tumors, those with a familial predisposition are of great interest for the isolation of novel genes or gene variants, detectable at the germline level and involved in cancer pathogenesis. The identification of novel genetic factors would have great translational value, helping clinicians in defining risk and prevention strategies. In this regard, it is known that the majority of breast/ovarian cases with familial predisposition, lacking variants in the highly penetrant BRCA1 and BRCA2 genes (non-BRCA), remains unexplained, although several less penetrant genes (e.g., ATM, PALB2) have been identified. In this scenario, NGS technologies offer a powerful tool for the discovery of novel factors involved in familial breast/ovarian cancer. In this review, we summarize and discuss the state of the art applications of NGS gene panels, WES and WGS in the context of familial breast/ovarian cancer.
      Citation: High-Throughput
      PubDate: 2020-01-10
      DOI: 10.3390/ht9010001
      Issue No: Vol. 9, No. 1 (2020)
  • High-Throughput, Vol. 9, Pages 2: Acknowledgement to Reviewers of
           High-Throughput in 2019

    • Authors: High-Throughput Editorial Office High-Throughput Editorial Office
      First page: 2
      Abstract: The editorial team greatly appreciates the reviewers who have dedicated their considerable time and expertise to the journal’s rigorous editorial process over the past 12 months, regardless of whether the papers are finally published or not [...]
      Citation: High-Throughput
      PubDate: 2020-01-16
      DOI: 10.3390/ht9010002
      Issue No: Vol. 9, No. 1 (2020)
  • High-Throughput, Vol. 9, Pages 3: Precision Medicine in Non-Communicable

    • Authors: Giuseppe Novelli, Michela Biancolella, Andrea Latini, Aldo Spallone, Paola Borgiani, Marisa Papaluca
      First page: 3
      Abstract: The increase in life expectancy during the 20th century ranks as one of society’s greatest achievements, with massive growth in the numbers and proportion of the elderly, virtually occurring in every country of the world. The burden of chronic diseases is one of the main consequences of this phenomenon, severely hampering the quality of life of elderly people and challenging the efficiency and sustainability of healthcare systems. Non-communicable diseases (NCDs) are considered a global emergency responsible for over 70% of deaths worldwide. NCDs are also the basis for complex and multifactorial diseases such as hypertension, diabetes, and obesity. The epidemics of NCDs are a consequence of a complex interaction between health, economic growth, and development. This interaction includes the individual genome, the microbiome, the metabolome, the immune status, and environmental factors such as nutritional and chemical exposure. To counteract NCDs, it is therefore essential to develop an innovative, personalized, preventative, early care model through the integration of different molecular profiles of individuals to identify both the critical biomarkers of NCD susceptibility and to discover novel therapeutic targets.
      Citation: High-Throughput
      PubDate: 2020-02-07
      DOI: 10.3390/ht9010003
      Issue No: Vol. 9, No. 1 (2020)
  • High-Throughput, Vol. 9, Pages 4: Comparative Study of NGS Platform Ion
           Torrent Personal Genome Machine and Therascreen Rotor-Gene Q for the
           Detection of Somatic Variants in Cancer

    • Authors: Angela Lombardi, Margherita Russo, Amalia Luce, Floriana Morgillo, Virginia Tirino, Gabriella Misso, Erika Martinelli, Teresa Troiani, Vincenzo Desiderio, Gianpaolo Papaccio, Francesco Iovino, Giuseppe Argenziano, Elvira Moscarella, Pasquale Sperlongano, Gennaro Galizia, Raffaele Addeo, Alois Necas, Andrea Necasova, Fortunato Ciardiello, Andrea Ronchi, Michele Caraglia, Anna Grimaldi
      First page: 4
      Abstract: Molecular profiling of a tumor allows the opportunity to design specific therapies which are able to interact only with cancer cells characterized by the accumulation of several genomic aberrations. This study investigates the usefulness of next-generation sequencing (NGS) and mutation-specific analysis methods for the detection of target genes for current therapies in non-small-cell lung cancer (NSCLC), metastatic colorectal cancer (mCRC), and melanoma patients. We focused our attention on EGFR, BRAF, KRAS, and BRAF genes for NSCLC, melanoma, and mCRC samples, respectively. Our study demonstrated that in about 2% of analyzed cases, the two techniques did not show the same or overlapping results. Two patients affected by mCRC resulted in wild-type (WT) for BRAF and two cases with NSCLC were WT for EGFR according to PGM analysis. In contrast, these samples were mutated for the evaluated genes using the therascreen test on Rotor-Gene Q. In conclusion, our experience suggests that it would be appropriate to confirm the WT status of the genes of interest with a more sensitive analysis method to avoid the presence of a small neoplastic clone and drive the clinician to correct patient monitoring.
      Citation: High-Throughput
      PubDate: 2020-02-11
      DOI: 10.3390/ht9010004
      Issue No: Vol. 9, No. 1 (2020)
  • High-Throughput, Vol. 9, Pages 5: Rational Engineering of the Substrate
           Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase)

    • Authors: Hovsep Aganyants, Pierre Weigel, Yeranuhi Hovhannisyan, Michèle Lecocq, Haykanush Koloyan, Artur Hambardzumyan, Anichka Hovsepyan, Jean-Noël Hallet, Vehary Sakanyan
      First page: 5
      Abstract: D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.
      Citation: High-Throughput
      PubDate: 2020-02-12
      DOI: 10.3390/ht9010005
      Issue No: Vol. 9, No. 1 (2020)
  • High-Throughput, Vol. 9, Pages 6: Factors Influencing Epigenetic
           Mechanisms: Is There A Role for Bariatric Surgery'

    • Authors: Alessio Metere, Claire E. Graves
      First page: 6
      Abstract: Epigenetics is the interaction between the genome and environmental stimuli capable of influencing gene expression during development and aging. A large number of studies have shown that metabolic diseases are highly associated with epigenetic alterations, suggesting that epigenetic factors may play a central role in obesity. To investigate these relationships, we focus our attention on the most common epigenetic modifications that occur in obesity, including DNA methylation and post-translational modifications of histones. We also consider bariatric surgery as an epigenetic factor, evaluating how the anatomic and physiologic modifications induced by these surgical techniques can change gene expression. Here we discuss the importance of epigenetic mechanisms in chronic disease and cancer, and the role of epigenetic disturbances in obesity, with a focus on the role of bariatric surgery.
      Citation: High-Throughput
      PubDate: 2020-03-20
      DOI: 10.3390/ht9010006
      Issue No: Vol. 9, No. 1 (2020)
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