Recent Submissions

  • iSCAN-V2: A One-Pot RT-RPA–CRISPR/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection

    Aman, Rashid; Marsic, Tin; Sivakrishna Rao, Gundra; Mahas, Ahmed; Ali, Zahir; Alsanea, Madain; Al-Qahtani, Ahmed; Alhamlan, Fatimah; Mahfouz, Magdy (Frontiers in Bioengineering and Biotechnology, Frontiers Media SA, 2022-01-21) [Article]
    Rapid, specific, and sensitive detection platforms are prerequisites for early pathogen detection to efficiently contain and control the spread of contagious diseases. Robust and portable point-of-care (POC) methods are indispensable for mass screening of SARS-CoV-2. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based nucleic acid detection technologies coupled with isothermal amplification methods provide a straightforward and easy-to-handle platform for detecting SARS-CoV-2 at POC, low-resource settings. Recently, we developed iSCAN, a two-pot system based on coupled loop-mediated isothermal amplification (LAMP) and CRISPR/Cas12a reactions. However, in two-pot systems, the tubes must be opened to conduct both reactions; two-pot systems thus have higher inherent risks of cross-contamination and a more cumbersome workflow. In this study, we developed and optimized iSCAN-V2, a one-pot reverse transcription-recombinase polymerase amplification (RT-RPA)-coupled CRISPR/Cas12b-based assay for SARS-CoV-2 detection, at a single temperature in less than an hour. Compared to Cas12a, Cas12b worked more efficiently in the iSCAN-V2 detection platform. We assessed and determined the critical factors, and present detailed guidelines and considerations for developing and establishing a one-pot assay. Clinical validation of our iSCAN-V2 detection module with reverse transcription-quantitative PCR (RT-qPCR) on patient samples showed 93.75% sensitivity and 100% specificity. Furthermore, we coupled our assay with a low-cost, commercially available fluorescence visualizer to enable its in-field deployment and use for SARS-CoV-2 detection. Taken together, our optimized iSCAN-V2 detection platform displays critical features of a POC molecular diagnostic device to enable mass-scale screening of SARS-CoV-2 in low-resource settings.
  • How to Find the Right RNA-Sensing CRISPR-Cas System for an In Vitro Application

    Díaz-Galicia, Escarlet; Grunberg, Raik; Arold, Stefan T. (Biosensors, MDPI AG, 2022-01-19) [Article]
    CRISPR-Cas systems have a great and still largely untapped potential for in vitro applications, in particular, for RNA biosensing. However, there is currently no systematic guide on selecting the most appropriate RNA-targeting CRISPR-Cas system for a given application among thousands of potential candidates. We provide an overview of the currently described Cas effector systems and review existing Cas-based RNA detection methods. We then propose a set of systematic selection criteria for selecting CRISPR-Cas candidates for new applications. Using this approach, we identify four candidates for in vitro RNA.
  • Investigation of important biochemical compounds from selected freshwater macroalgae and their role in agriculture

    Shah, Zahir; Badshah, Syed Lal; Iqbal, Arshad; Shah, Zamarud; Emwas, Abdul-Hamid M.; Jaremko, Mariusz (Chemical and Biological Technologies in Agriculture, Springer Science and Business Media LLC, 2022-01-15) [Article]
    Background Freshwater macroalgae possess a number of important secondary metabolites. They are an unexplored source of medicinal compounds. In this study, three freshwater macroalgae—$\textit{Chara vulgaris}$, $\textit{Cladophora glomerata}$ and $\textit{Spirogyra crassa}$—were collected from the river Swat and the river Kabul in the Charsadda district of Khyber Pakhtunkhwa, Pakistan. To assess the role of freshwater macroalgae in agriculture, various experiments were performed on their extracts. Methanolic extract of the three macroalgae were first analyzed through gas chromatography–mass spectrometry (GC–MS) for the presence of important medicinal secondary metabolites. The methanol based macroalgae extracts were tested for antibacterial, insecticidal, cytotoxic and phytotoxic activities. Results Initially, the algae were dried, crushed and treated with methanol for the extraction of secondary metabolites. The GC–MS results contained several important long chain fatty acids and other related long-chain hydrocarbons, such as alkanes and alkenes. Several benzene derivatives were also detected during the course of the investigation. Several of these compounds have established roles in the treatment of human ailments and can be supplied to farm animals. For example, phenylephrine is a decongestant, dilates pupils, increases blood pressure and helps in relieving hemorrhoids. Hexahydropseudoionone has uses in perfumes and other cosmetics. Several essential oils were also detected in the methanolic extract of the three macroalgae that can be utilized in various industrial products. Bioassays showed that these algal extracts—especially the $\textit{Spirogyra}$ sp. extract—contain moderate to maximum bioactivity. Conclusions Macroalgae possess important secondary metabolites with medicinal properties. These secondary metabolites can be used as biopesticides, plant growth enhancers, and remedies for various diseases in farm animals and for the control of weeds. They can be further explored for isolation and purification of useful biochemical compounds.
  • In Vitro and In Silico Approaches for the Evaluation of Antimicrobial Activity, Time-Kill Kinetics, and Anti-Biofilm Potential of Thymoquinone (2-Methyl-5-propan-2-ylcyclohexa-2,5-diene-1,4-dione) against Selected Human Pathogens

    Qureshi, Kamal A.; Imtiaz, Mahrukh; Parvez, Adil; Rai, Pankaj K.; Jaremko, Mariusz; Emwas, Abdul-Hamid M.; Bholay, Avinash D.; Fatmi, Muhammad Qaiser (Antibiotics, MDPI AG, 2022-01-10) [Article]
    Thymoquinone (2-methyl-5-propan-2-ylcyclohexa-2,5-diene-1,4-dione; TQ), a principal bioactive phytoconstituent of Nigella sativa essential oil, has been reported to have high antimicrobial potential. Thus, the current study evaluated TQ’s antimicrobial potential against a range of selected human pathogens using in vitro assays, including time-kill kinetics and anti-biofilm activity. In silico molecular docking of TQ against several antimicrobial target proteins and a detailed intermolecular interaction analysis was performed, including binding energies and docking feasibility. Of the tested bacteria and fungi, S. epidermidis ATCC 12228 and Candida albicans ATCC 10231 were the most susceptible to TQ, with 50.3 ± 0.3 mm and 21.1 ± 0.1 mm zones of inhibition, respectively. Minimum inhibitory concentration (MIC) values of TQ are in the range of 12.5–50 µg/mL, while minimum biocidal concentration (MBC) values are in the range of 25–100 µg/mL against the tested organisms. Time-kill kinetics of TQ revealed that the killing time for the tested bacteria is in the range of 1–6 h with the MBC of TQ. Anti-biofilm activity results demonstrate that the minimum biofilm inhibitory concentration (MBIC) values of TQ are in the range of 25–50 µg/mL, while the minimum biofilm eradication concentration (MBEC) values are in the range of 25–100 µg/mL, for the tested bacteria. In silico molecular docking studies revealed four preferred antibacterial and antifungal target proteins for TQ: D-alanyl-D-alanine synthetase (Ddl) from Thermus thermophilus, transcriptional regulator qacR from Staphylococcus aureus, N-myristoyltransferase from Candida albicans, and NADPH-dependent D-xylose reductase from Candida tenuis. In contrast, the nitroreductase family protein from Bacillus cereus and spore coat polysaccharide biosynthesis protein from Bacillus subtilis and UDP-N-acetylglucosamine pyrophosphorylase from Aspergillus fumigatus are the least preferred antibacterial and antifungal target proteins for TQ, respectively. Molecular dynamics (MD) simulations revealed that TQ could bind to all four target proteins, with Ddl and NADPH-dependent D-xylose reductase being the most efficient. Our findings corroborate TQ’s high antimicrobial potential, suggesting it may be a promising drug candidate for multi-drug resistant (MDR) pathogens, notably Gram-positive bacteria and Candida albicans.
  • Synthesis and Organization of Gold-Peptide Nanoparticles for Catalytic Activities

    Abbas, Manzar; Susapto, Hepi Hari; Hauser, Charlotte (ACS Omega, American Chemical Society (ACS), 2022-01-06) [Article]
    A significant development in the synthesis strategies of metal-peptide composites and their applications in biomedical and bio-catalysis has been reported. However, the random aggregation of gold nanoparticles provides the opportunity to find alternative fabrication strategies of gold-peptide composite nanomaterials. In this study, we used a facile strategy to synthesize the gold nanoparticles via a green and simple approach where they show self-alignment on the assembled nanofibers of ultrashort oligopeptides as a composite material. A photochemical reduction method is used, which does not require any external chemical reagents for the reduction of gold ions, and resultantly makes the gold nanoparticles of size ca. 5 nm under mild UV light exposure. The specific arrangement of gold nanoparticles on the peptide nanofibers may indicate the electrostatic interactions of two components and the interactions with the amino group of the peptide building block. Furthermore, the gold-peptide nanoparticle composites show the ability as a catalyst to degradation of environmental pollutant p-nitrophenol to p-aminophenol, and the reaction rate constant for catalysis is calculated as 0.057 min–1 at a 50-fold dilute sample of 2 mg/mL and 0.72 mM gold concentration in the composites. This colloidal strategy would help researchers to fabricate the metalized bioorganic composites for various biomedical and bio-catalysis applications.
  • Unconventional metabolites in chromatin regulation

    Gapa, Liubov; Alfardus, Huda; Fischle, Wolfgang (Bioscience Reports, Portland Press Ltd., 2022-01-06) [Article]
    Chromatin, the complex of DNA and histone proteins, serves as a main integrator of cellular signals. Increasing evidence links cellular functional to chromatin state. Indeed, different metabolites are emerging as modulators of chromatin function and structure. Alterations in chromatin state are decisive for regulating all aspects of genome function and ultimately have the potential to produce phenotypic changes. Several metabolites such as acetyl-CoA, S-adenosyl methionine (SAM) or adenosine triphosphate (ATP) have now been well characterized as main substrates or cofactors of chromatin modifying enzymes. However, there are other metabolites that can directly interact with chromatin influencing its state or that modulate the properties of chromatin regulatory factors. Also, there is a growing list of atypical enzymatic and non-enzymatic chromatin modifications that originate from different cellular pathways that have not been in the limelight of chromatin research. Here, we summarize different properties and functions of uncommon regulatory molecules originating from intermediate metabolism of lipids, carbohydrates and amino acids. Based on the various modes of action on chromatin and the plethora of putative, so far not described chromatin regulating metabolites, we propose that there are more links between cellular functional state and chromatin regulation to be discovered. We hypothesize that these connections could provide interesting starting points for interfering with cellular epigenetic states at a molecular level.
  • Destabilization of the Bacterial Interactome Identifies Nutrient Restriction-Induced Dysbiosis in Insect Guts

    Marasco, Ramona; Fusi, Marco; Callegari, Matteo; Jucker, Costanza; Mapelli, Francesca; Borin, Sara; Savoldelli, Sara; Daffonchio, Daniele; Crotti, Elena (Microbiology Spectrum, American Society for Microbiology, 2022-01-05) [Article]
    Changes in diet play a role in reshaping the gut microbiome in animals, inducing dysbiotic configurations of the associated microbiome. Although studies have reported on the effects of specific nutrient contents on the diet, studies regarding the conditions altering the microbiome configurations and networking in response to diet changes are limited.
  • Thymosin β4 Is an Endogenous Iron Chelator and Molecular Switcher of Ferroptosis

    Lachowicz, Joanna; Pichiri, Gius; Piludu, Marco; Fais, Sara; Orrù, Germano; Congiu, Terenzio; Piras, Monica; Faa, Gavino; Fanni, Daniela; Torre, Gabriele; Lopez, Xabier; Chandra, Kousik; Szczepski, Kacper; Jaremko, Lukasz; Ghosh, Mitra; Emwas , Abdul-Hamid; Castagnola, Massimo; Jaremko, Mariusz; Hannappel, Ewald; Coni, Pierpaolo (International Journal of Molecular Sciences, Research Square Platform LLC, 2022-01-04) [Article]
    Thymosin β4 (Tβ4) was extracted forty years agofrom calf thymus. Since then, it has been identified as a G-actin binding protein involved in blood clotting, tissue regeneration, angiogenesis, and anti-inflammatory processes. Tβ4 has also been implicated in tumor metastasis and neurodegeneration. However, the precise roles and mechanism(s) of action of Tβ4 in these processes remain largely unknown, with the binding of the G-actin protein being insufficient to explain these multi-actions. Here we identify for the first time the important role of Tβ4 mechanism in ferroptosis, an iron-dependent form of cell death, which leads to neurodegeneration and somehow protects cancer cells against cell death. Specifically, we demonstrate four iron2+ and iron3+ binding regions along the peptide and show that the presence of Tβ4 in cell growing medium inhibits erastin and glutamate-induced ferroptosis in the macrophage cell line. Moreover, Tβ4 increases the expression of oxidative stress-related genes, namely BAX, hem oxygenase-1, heat shock protein 70 and thioredoxin reductase 1, which are downregulated during ferroptosis. We state the hypothesis that Tβ4 is an endogenous iron chelator and take part in iron homeostasis in the ferroptosis process. We discuss the literature data of parallel involvement of Tβ4 and ferroptosis in different human pathologies, mainly cancer and neurodegeneration. Our findings confronted with literature data show that controlled Tβ4 release could command on/off switching of ferroptosis and may provide novel therapeutic opportunities in cancer and tissue degeneration pathologies
  • Fluxomics - new metabolomics approaches to monitor metabolic pathways

    Emwas, Abdul-Hamid M.; Szczepski, Kacper; Al-Younis (Elyounis), Inas (Enas) M.; Lachowicz, Joanna Izabela; Jaremko, Mariusz (Frontiers in Pharmacology, Frontiers, 2022) [Article]
    Fluxomics is a new omics research field that measures the rates of all intracellular fluxes in the central metabolism of biological systems. Fluxomics is one of the most relevant approaches to investigate metabolic phenotypes. Metabolic flux using 13C-labeled molecules is increasingly used to monitor metabolic pathways. By determining metabolic pathways, researchers can probe the corresponding gene-RNA and protein-metabolite interaction networks in actual time. Thus, fluxomics can be used to understand metabolic pathways and has many applications in biotechnology and pharmacology. Here, we briefly introduce fluxomics with a review of the most recent studies and common analytical tools.
  • Mechanisms of the interaction between beneficial endophytic bacteria and plants conferring enhanced drought and salt stress tolerance

    Alwutayd, Khairiah Mubarak Saleem (2022-01) [Dissertation]
    Advisor: Hirt, Heribert
    Committee members: Rosado, Alexandre S.; Blilou, Ikram; Schikora, Adam
    Drought and salt stress are the main global factors that reduce the average yield of most major crops. In order to meet global demands, we will need to double food production by 2050 (Tilman, Balzer, Hill, & Befort, 2011). Plant growth-promoting bacteria (PGPB) are a group of bacteria that alleviate the harmful effects of abiotic stresses such as salt, heat and drought stress on plants and decrease the global dependence on hazardous agricultural chemicals. We identified that beneficial microbes isolated from desert plants (indigfera argentea) from Jizan region, in 2012 enhance the tolerance of a variety of crop plants to drought and salt stresses under laboratory conditions and in field trials. We analyzed the interaction of these bacteria with the plants by genetic, biochemical and imaging techniques. The goal of this dissertation is to ultimately improve our understanding of the mechanisms of drought and salt stress tolerance conferred by beneficial microbes that can be used as a sustainable solution for plants and crops in degrading lands (deserts) and land affected by abiotic stresses. Outlines how each of chapter of this dissertation will contribute to the discovery of novel drought and salt stress tolerance strategies using a desert-specific bacterial endophyte.
  • Cyclodextrins: Structural, Chemical, and Physical Properties, and Applications

    Poulson, Benjamin Gabriel; Alsulami, Qana; Sharfalddin, Abeer A.; El Agammy, Emam; Mouffouk , Fouzi; Emwas, Abdul-Hamid M.; Jaremko, Lukasz; Jaremko, Mariusz (Polysaccharides, MDPI AG, 2022) [Article]
    Due to their unique structural, physical and chemical properties, cyclodextrins and their derivatives have been of great interest to scientists and researchers in both academia and industry for over a century. Many of the industrial applications of cyclodextrins have arisen from their ability to encapsulate, either partially or fully, other molecules, especially organic compounds. Cyclodextrins are non-toxic oligopolymers of glucose that help to increase the solubility of organic compounds with poor aqueous solubility, can mask odors from foul-smelling compounds, and have been widely studied in the area of drug delivery. In this review, we explore the structural and chemical properties of cyclodextrins that give rise to this encapsulation (i.e., the formation of inclusion complexes) ability. This review is unique from others written on this subject because it provides powerful insights into factors that affect cyclodextrin encapsulation. It also examines these insights in great detail. Later, we provide an overview of some industrial applications of cyclodextrins, while emphasizing the role of encapsulation in these applications. We strongly believe that cyclodextrins will continue to garner interest from scientists for many years to come, and that novel applications of cyclodextrins have yet to be discovered
  • Natural Polysaccharides as Preventive and Therapeutic Horizon for Neurodegenerative Diseases

    Dhahri, Manel; Alghrably, Mawadda; Mohammed, Hamdoon A.; Badshah, Syed Lal; Noreen, Noreen; Mouffouk, Fouzi; Rayyan, Saleh; Qureshi, Kamal A.; Mahmood, Danish; Lachowicz, Joanna Izabela; Jaremko, Mariusz; Emwas, Abdul-Hamid M. (Pharmaceutics, MDPI AG, 2022) [Article]
    Neurodegenerative diseases are a serious and widespread global public health burden amongst aging populations. The total estimated worldwide global cost of dementia was US$\$$818 billion in 2015 and has been projected to rise to 2 trillion US$\$$ by 2030. While advances have been made to understand different neurodegenerative disease mechanisms, effective therapeutic strategies do not generally exist. Several drugs have been proposed in the last two decades for the treatment of different types of neurodegenerative diseases, with little therapeutic benefit, and often with severe adverse and side effects. Thus, the search for novel drugs with higher efficacy and fewer drawbacks is an ongoing challenge in the treatment of neurodegenerative disease. Several natural compounds including polysaccharides have demonstrated neuroprotective and even therapeutic effects. Natural polysaccharides are widely distributed in plants, animals, algae, bacterial and fungal species, and have received considerable attention for their wide-ranging bioactivity, including their antioxidant, anti-neuroinflammatory, anticholinesterase and anti-amyloidogenic effects. In this review, we summarize different mechanisms involved in neurodegenerative diseases and the neuroprotective effects of natural polysaccharides, highlighting their potential role in the prevention and therapy of neurodegenerative disease.
  • Computational Study of SARS-CoV-2 RNA Dependent RNA Polymerase Allosteric Site Inhibition

    Faisal, Shah; Badshah, Syed Lal; Kubra, Bibi; Sharaf, Mohamed; Emwas, Abdul-Hamid M.; Jaremko, Mariusz; Abdalla, Mohnad (Molecules, MDPI AG, 2022) [Article]
    The COVID-19 pandemic has caused millions of fatalities since 2019. Despite the availability of vaccines for this disease, new strains are causing rapid ailment and are a continuous threat to vaccine efficacy. Here, molecular docking and simulations identify strong inhibitors of the allosteric site of the SARS-CoV-2 virus RNA dependent RNA polymerase (RdRp). More than one hundred different flavonoids were docked with the SARS-CoV-2 RdRp allosteric site through computational screening. The three top hits were Naringoside, Myricetin and Aureusidin 4,6-diglucoside. Simulation analyses confirmed that they are in constant contact during the simulation time course and have strong association with the enzyme’s allosteric site. Absorption, distribution, metabolism, excretion and toxicity (ADMET) data provided medicinal information of these top three hits. They had good human intestinal absorption (HIA) concentrations and were non-toxic. Due to high mutation rates in the active sites of the viral enzyme, these new allosteric site inhibitors offer opportunities to drug SARS-CoV-2 RdRp. These results provide new information for the design of novel allosteric inhibitors against SARS-CoV-2 RdRp.
  • A Novel GEMIN4 Variant in a Consanguineous Family Leads to Neurodevelopmental Impairment with Severe Microcephaly, Spastic Quadriplegia, Epilepsy, and Cataracts

    Aldhalaan, Hesham; AlBakheet, Albandary; Alruways, Sarah; Almutairi, Nouf; Alnakiyah, Maha; Alghofaili, Reema; Cardona-Londoño, Kelly J.; Alahmadi, Khalid Omar; Alqudairy, Hanan; Alrasheed, Maha M.; Colak, Dilek; Arold, Stefan T.; Kaya, Namik (Genes, MDPI AG, 2021-12-30) [Article]
    Pathogenic variants in GEMIN4 contribute to a hereditary disorder characterized by neu-rodevelopmental features, microcephaly, cataracts, and renal abnormalities (known as NEDMCR). To date, only two homoallelic variations have been linked to the disease. Moreover, clinical features associated with the variants have not been fully elucidated yet. Here, we identified a novel variant in GEMIN4 (NM_015721:exon2:c.440A>G:p.His147Arg) in two siblings from a consanguineous Saudi family by using whole exome sequencing followed by Sanger sequence verification. We comprehen-sively investigated the patients’ clinical features, including brain imaging and electroencephalogram findings, and compared their phenotypic characteristics with those of previously reported cases. In silico prediction and structural modeling support that the p.His147Arg variant is pathogenic.
  • DeepSVP: Integration of genotype and phenotype for structural variant prioritization using deep learning

    Althagafi, Azza Th.; Alsubaie, Lamia; Kathiresan, Nagarajan; Mineta, Katsuhiko; Aloraini, Taghrid; Almutairi, Fuad; Alfadhel, Majid; Gojobori, Takashi; Alfares, Ahmad; Hoehndorf, Robert (Bioinformatics, Oxford University Press (OUP), 2021-12-24) [Article]
    Abstract Motivation Structural genomic variants account for much of human variability and are involved in several diseases. Structural variants are complex and may affect coding regions of multiple genes, or affect the functions of genomic regions in different ways from single nucleotide variants. Interpreting the phenotypic consequences of structural variants relies on information about gene functions, haploinsufficiency or triplosensitivity, and other genomic features. Phenotype-based methods to identifying variants that are involved in genetic diseases combine molecular features with prior knowledge about the phenotypic consequences of altering gene functions. While phenotype-based methods have been applied successfully to single nucleotide variants as well as short insertions and deletions, the complexity of structural variants makes it more challenging to link them to phenotypes. Furthermore, structural variants can affect a large number of coding regions, and phenotype information may not be available for all of them. Results We developed DeepSVP, a computational method to prioritize structural variants involved in genetic diseases by combining genomic and gene functions information. We incorporate phenotypes linked to genes, functions of gene products, gene expression in individual celltypes, and anatomical sites of expression, and systematically relate them to their phenotypic consequences through ontologies and machine learning. DeepSVP significantly improves the success rate of finding causative variants in several benchmarks and can identify novel pathogenic structural variants in consanguineous families. Availability https://github.com/bio-ontology-research-group/DeepSVP
  • The Lys-motif receptor LYK4 mediates Enterobacter sp. SA187 triggered salt tolerance in Arabidopsis thaliana

    Rolli, Eleonora; Zélicourt, Axel de; Alzubaidy, Hanin S.; Karampelias, Michael; Parween, Sabiha; Rayapuram, Naganand; Han, Baoda; Froehlich, Katja; Abulfaraj, Aala A.; Alhoraibi, Hanna; Mariappan, Kiruthiga; Andres-Barrao, Cristina; Colcombet, Jean; Hirt, Heribert (Environmental Microbiology, Wiley, 2021-12-23) [Article]
    Root endophytes establish beneficial interactions with plants, improving holobiont resilience and fitness, but how plant immunity accommodates beneficial microbes is poorly understood. The multi-stress tolerance-inducing endophyte Enterobacter sp. SA187 triggers a canonical immune response in Arabidopsis only at high bacterial dosage (>108 CFUs ml−1), suggesting that SA187 is able to evade or suppress the plant defence system at lower titres. Although SA187 flagellin epitopes are recognized by the FLS2 receptor, SA187-triggered salt tolerance functions independently of the FLS2 system. In contrast, overexpression of the chitin receptor components LYK4 and LYK5 compromised the beneficial effect of SA187 on Arabidopsis, while it was enhanced in lyk4 mutant plants. Transcriptome analysis revealed that the role of LYK4 is intertwined with a function in remodelling defence responses with growth and root developmental processes. LYK4 interferes with modification of plant ethylene homeostasis by Enterobacter SA187 to boost salt stress resistance. Collectively, these results contribute to unlock the crosstalk between components of the plant immune system and beneficial microbes and point to a new role for the Lys-motif receptor LYK4 in beneficial plant–microbe interaction.
  • Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2

    Ali, Zahir; Sánchez, Edith; Tehseen, Muhammad; Mahas, Ahmed; Marsic, Tin; Aman, Rashid; Sivakrishna Rao, Gundra; Alhamlan, Fatimah Saeed; Alsanea, Madain S.; Al-Qahtani, Ahmed A.; Hamdan, Samir; Mahfouz, Magdy M. (ACS Synthetic Biology, American Chemical Society (ACS), 2021-12-23) [Article]
    Simple, rapid, specific, and sensitive point-of-care detection methods are needed to contain the spread of SARS-CoV-2. CRISPR/Cas9-based lateral flow assays are emerging as a powerful alternative for COVID-19 diagnostics. Here, we developed Bio-SCAN (biotin-coupled specific CRISPR-based assay for nucleic acid detection) as an accurate pathogen detection platform that requires no sophisticated equipment or technical expertise. Bio-SCAN detects the SARS-CoV-2 genome in less than 1 h from sample collection to result. In the first step, the target nucleic acid sequence is isothermally amplified in 15 min via recombinase polymerase amplification before being precisely detected by biotin-labeled nuclease-dead SpCas9 (dCas9) on commercially available lateral flow strips. The resulting readout is visible to the naked eye. Compared to other CRISPR-Cas-based pathogen detection assays, Bio-SCAN requires no additional reporters, probes, enhancers, reagents, or sophisticated devices to interpret the results. Bio-SCAN is highly sensitive and successfully detected a clinically relevant level (4 copies/μL) of synthetic SARS-CoV-2 RNA genome. Similarly, Bio-SCAN showed 100% negative and 96% positive predictive agreement with RT-qPCR results when using clinical samples (86 nasopharyngeal swab samples). Furthermore, incorporating variant-specific sgRNAs in the detection reaction allowed Bio-SCAN to efficiently distinguish between the α, β, and δSARS-CoV-2 variants. Also, our results confirmed that the Bio-SCAN reagents have a long shelf life and can be assembled locally in nonlaboratory and limited-resource settings. Furthermore, the Bio-SCAN platform is compatible with the nucleic acid quick extraction protocol. Our results highlight the potential of Bio-SCAN as a promising point-of-care diagnostic platform that can facilitate low-cost mass screening for SARS-CoV-2.
  • Propylene and butylene glycol: new alternatives to ethylene glycol in conjugated polymers for bioelectronic applications

    Moser, Maximilian; Wang, Yazhou; Hidalgo, Tania C.; Liao, Hailiang; Yu, Yaping; Chen, Junxin; Duan, Jiayao; Moruzzi, Floriana; Griggs, Sophie; Marks, Adam; Gasparini, Nicola; Wadsworth, Andrew; Inal, Sahika; McCulloch, Iain; Yue, Wan (Materials Horizons, Royal Society of Chemistry (RSC), 2021-12-22) [Article]
    To date, many of the high-performance conjugated polymers employed as OECT channel materials make use of ethylene glycol (EG) chains to confer the materials with mixed ionic-electronic conduction properties, with limited emphasis placed on alternative hydrophilic moieties. While a degree of hydrophilicity is required to facilitate some ionic conduction in hydrated channels, an excess results in excessive swelling, with potentially detrimental effects on charge transport. This is therefore a subtle balance that must be optimised to maximise electrical performance. Herein a series of polymers based on a bithiophene–thienothiophene conjugated backbone was synthesised and the conventional EG chains substituted by their propylene and butylene counterparts. Specifically, the use of propylene and butylene chains was found to afford polymers with a more hydrophobic character, thereby reducing excessive water uptake during OECT operation and in turn significantly boosting the polymers’ electronic charge carrier mobility. Despite the polymers’ lower water uptake, the newly developed oligoether chains retained sufficiently high degrees of hydrophilicity to enable bulk volumetric doping, ultimately resulting in the development of polymers with superior OECT performance.
  • Decoding Cancer Variants of Unknown Significance for Helicase-Nuclease-RPA Complexes Orchestrating DNA Repair During Transcription and Replication.

    Tsutakawa, Susan E; Bacolla, Albino; Katsonis, Panagiotis; Bralic, Amer; Hamdan, Samir; Lichtarge, Olivier; Tainer, John A; Tsai, Chi-Lin (Frontiers in molecular biosciences, Frontiers Media SA, 2021-12-14) [Article]
    All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase-nuclease-RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary "driver" and "passenger" mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.
  • MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples

    Li, Mo; Ramos Mandujano, Gerardo (ZappyLab, Inc., 2021-12-08) [Protocol]
    Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.

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