Genetic Manipulation in Mucorales and New Developments to Study Mucormycosis.

The study of the Mucoralean fungi physiology is a neglected field that the lack of effective genetic tools has hampered in the past. However, the emerging fungal infection caused by these fungi, known as mucormycosis, has prompted many researchers to study the pathogenic potential of Mucorales. The main reasons for this current attraction to study mucormycosis are its high lethality, the lack of effective antifungal drugs, and its recent increased incidence. The most contemporary example of the emergence character of mucormycosis is the epidemics declared in several Asian countries as a direct consequence of the COVID-19 pandemic. Fortunately, this pressure to understand mucormycosis and develop new treatment strategies has encouraged the blossoming of new genetic techniques and methodologies. This review describes the history of genetic manipulation in Mucorales, highlighting the development of methods and how they allowed the main genetic studies in these fungi. Moreover, we have emphasized the recent development of new genetic models to study mucormycosis, a landmark in the field that will configure future research related to this disease.

Detection of N6-methyladenosine in SARS-CoV-2 RNA by methylated RNA immunoprecipitation sequencing.

N 6 -methylation of adenosine (m6A) is the most abundant internal mRNA modification and is an important post-transcriptional regulator of gene expression. Here, we describe a protocol for methylated RNA immunoprecipitation sequencing (MeRIP-Seq) to detect and quantify m6A modifications in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. The protocol is optimized for low viral RNA levels and is readily adaptable for other applications. For complete details on the use and execution of this protocol, please refer to Li et al. (2021).

Global metabolome profiling of COVID-19 respiratory specimen using high-resolution mass spectrometry (HRMS).

Here we describe a protocol for identifying metabolites in respiratory specimens of patients that are SARS-CoV-2 positive, SARS-CoV-2 negative, or H1N1 positive. This protocol provides step-by-step instructions on sample collection from patients, followed by metabolite extraction. We use ultra-high-pressure liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) for data acquisition and describe the steps for data analysis. The protocol was standardized with specific customization for SARS-CoV-2-containing respiratory specimens. For complete details on the use and execution of this protocol, please refer to Maras et al. (2021).

Protocol for global proteome, virome, and metaproteome profiling of respiratory specimen (VTM) in COVID-19 patient by LC-MS/MS-based analysis.

In this protocol, we describe global proteome profiling for the respiratory specimen of COVID-19 patients, patients suspected with COVID-19, and H1N1 patients. In this protocol, details for identifying host, viral, or bacterial proteome (Meta-proteome) are provided. Major steps of the protocol include virus inactivation, protein quantification and digestion, desalting of peptides, high-resolution mass spectrometry (HRMS)-based analysis, and downstream bioinformatics analysis. For complete details on the use and execution of this profile, please refer to Maras et al. (2021).

CRISPR Screening: Molecular Tools for Studying Virus-Host Interactions.

CRISPR/Cas is a powerful tool for studying the role of genes in viral infections. The invention of CRISPR screening technologies has made it possible to untangle complex interactions between the host and viral agents. Moreover, whole-genome and pathway-specific CRISPR screens have facilitated identification of novel drug candidates for treating viral infections. In this review, we highlight recent developments in the fields of CRISPR/Cas with a focus on the use of CRISPR screens for studying viral infections and identifying new candidate genes to aid development of antivirals.

CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation.

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.

Drug repurposing for coronavirus (SARS-CoV-2) based on gene co-expression network analysis.

Severe acute respiratory syndrome (SARS) is a highly contagious viral respiratory illness. This illness is spurred on by a coronavirus known as SARS-associated coronavirus (SARS-CoV). SARS was first detected in Asia in late February 2003. The genome of this virus is very similar to the SARS-CoV-2. Therefore, the study of SARS-CoV disease and the identification of effective drugs to treat this disease can be new clues for the treatment of SARS-Cov-2. This study aimed to discover novel potential drugs for SARS-CoV disease in order to treating SARS-Cov-2 disease based on a novel systems biology approach. To this end, gene co-expression network analysis was applied. First, the gene co-expression network was reconstructed for 1441 genes, and then two gene modules were discovered as significant modules. Next, a list of miRNAs and transcription factors that target gene co-expression modules' genes were gathered from the valid databases, and two sub-networks formed of transcription factors and miRNAs were established. Afterward, the list of the drugs targeting obtained sub-networks' genes was retrieved from the DGIDb database, and two drug-gene and drug-TF interaction networks were reconstructed. Finally, after conducting different network analyses, we proposed five drugs, including FLUOROURACIL, CISPLATIN, SIROLIMUS, CYCLOPHOSPHAMIDE, and METHYLDOPA, as candidate drugs for SARS-CoV-2 coronavirus treatment. Moreover, ten miRNAs including miR-193b, miR-192, miR-215, miR-34a, miR-16, miR-16, miR-92a, miR-30a, miR-7, and miR-26b were found to be significant miRNAs in treating SARS-CoV-2 coronavirus.

SBSA: an online service for somatic binding sequence annotation.

Efficient annotation of alterations in binding sequences of molecular regulators can help identify novel candidates for mechanisms study and offer original therapeutic hypotheses. In this work, we developed Somatic Binding Sequence Annotator (SBSA) as a full-capacity online tool to annotate altered binding motifs/sequences, addressing diverse types of genomic variants and molecular regulators. The genomic variants can be somatic mutation, single nucleotide polymorphism, RNA editing, etc. The binding motifs/sequences involve transcription factors (TFs), RNA-binding proteins, miRNA seeds, miRNA-mRNA 3'-UTR binding target, or can be any custom motifs/sequences. Compared to similar tools, SBSA is the first to support miRNA seeds and miRNA-mRNA 3'-UTR binding target, and it unprecedentedly implements a personalized genome approach that accommodates joint adjacent variants. SBSA is empowered to support an indefinite species, including preloaded reference genomes for SARS-Cov-2 and 25 other common organisms. We demonstrated SBSA by annotating multi-omics data from over 30,890 human subjects. Of the millions of somatic binding sequences identified, many are with known severe biological repercussions, such as the somatic mutation in TERT promoter region which causes a gained binding sequence for E26 transformation-specific factor (ETS1). We further validated the function of this TERT mutation using experimental data in cancer cells. Availability:http://innovebioinfo.com/Annotation/SBSA/SBSA.php.

Fast, Scalable, and Practical: An Alkaline DNA Extraction Pipeline for Emergency Microbiomics Biosurveillance.

Pandemics and environmental crises evident from the first two decades of the 21st century call for methods innovation in biosurveillance and early detection of risk signals in planetary ecosystems. In crises conditions, conventional methods in public health, biosecurity, and environmental surveillance do not work well. In addition, the standard laboratory amenities and procedures may become unavailable, irrelevant, or simply not feasible, for example, owing to disruptions in logistics and process supply chains. The COVID-19 pandemic has been a wakeup call in this sense to reintroduce point-of-need diagnostics with an eye to limited resource settings and biosurveillance solutions. We report here a methodology innovation, a fast, scalable, and alkaline DNA extraction pipeline for emergency microbiomics biosurveillance. We believe that the presented methodology is well poised for effective, resilient, and anticipatory responses to future pandemics and ecological crises while contributing to microbiome science and point-of-need diagnostics in nonelective emergency contexts. The alkaline DNA extraction pipeline can usefully expand the throughput in emergencies by deployment or to allow backup in case of instrumentation failure in vital facilities. The need for distributed public health genomics surveillance is increasingly evident in the 21st century. This study makes a contribution to these ends broadly, and for future pandemic preparedness in particular. We call for innovation in biosurveillance methods that remain important existentially on a planet under pressure from unchecked human growth and breach of the boundaries between human and nonhuman animal habitats.

Patents and technology transfer in CRISPR technology.

CRISPR technology has revolutionized biological research in the last decade and many academic institutions and companies have patented CRISPR systems and applications. Several patents have been filed for various applications of CRISPR in different industries such as agriculture, synthetic biology, bio-nanotechnology and precision medicine. Despite tremendous pressure on the technology transfer teams, several startups and spin-out companies are already using CRISPR technologies for commercial applications. In this chapter, we discuss the different CRISPR nucleases and their applications. Secondly, we detail our current opinion and perspective on the CRISPR patent and technology landscape for non-mammalian systems. We present two case-studies on CRISPR diagnostics companies, SHERLOCK and Mammoth Biosciences, who are currently at the forefront of establishing diagnostics platforms for coronavirus (SARS-CoV-2) detection. Finally, our chapter identifies future advancements and possible challenges that CRISPR technology might face in non-mammalian systems.

Discover the Microbes Within! The Wolbachia Project: Citizen Science and Student-Based Discoveries for 15 Years and Counting.

The Elizabeth W. Jones Award for Excellence in Education recognizes an individual who has had a significant impact on genetics education at any education level. Seth R. Bordenstein, Ph.D., Centennial Professor of Biological Sciences at Vanderbilt University and Founding Director of the Vanderbilt Microbiome Initiative, is the 2020 recipient in recognition of his cofounding, developing, and expanding Discover the Microbes Within! The Wolbachia Project.

Pathogenic viruses: Molecular detection and characterization.

Pathogenic viruses are viruses that can infect and replicate within human cells and cause diseases. The continuous emergence and re-emergence of pathogenic viruses has become a major threat to public health. Whenever pathogenic viruses emerge, their rapid detection is critical to enable implementation of specific control measures and the limitation of virus spread. Further molecular characterization to better understand these viruses is required for the development of diagnostic tests and countermeasures. Advances in molecular biology techniques have revolutionized the procedures for detection and characterization of pathogenic viruses. The development of PCR-based techniques together with DNA sequencing technology, have provided highly sensitive and specific methods to determine virus circulation. Pathogenic viruses potentially having global catastrophic consequences may emerge in regions where capacity for their detection and characterization is limited. Development of a local capacity to rapidly identify new viruses is therefore critical. This article reviews the molecular biology of pathogenic viruses and the basic principles of molecular techniques commonly used for their detection and characterization. The principles of good laboratory practices for handling pathogenic viruses are also discussed. This review aims at providing researchers and laboratory personnel with an overview of the molecular biology of pathogenic viruses and the principles of molecular techniques and good laboratory practices commonly implemented for their detection and characterization.

Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus-based assay.

Pseudotyped viruses are useful virological tools because of their safety and versatility. On the basis of a vesicular stomatitis virus (VSV) pseudotyped virus production system, we developed a pseudotyped virus-based neutralization assay against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in biosafety level 2 facilities. Compared with the binding antibody test, the neutralization assay could discriminate the protective agents from the antibody family. This protocol includes production and titration of the SARS-CoV-2 S pseudotyped virus and the neutralization assay based on it. Various types of samples targeting virus attachment and entry could be evaluated for their potency, including serum samples derived from animals and humans, monoclonal antibodies and fusion inhibitors (peptides or small molecules). If the pseudotyped virus stock has been prepared in advance, it will take 2 days to get the potency data for the candidate samples. Experience in handling cells is needed before implementing this protocol.