Partial Digestion Research Articles

Detection of Growth Hormone (GH) Gene Polymorphism in Norduz Sheep

Background: The Norduz sheep is a local breed of sheep bred for its meat, milk, progeny and fleece and is one of the genetic resources in the rural areas of Norduz, a district of the Turkish province of Van. The PCR-based method involves amplification of a conserved DNA region and digestion of the PCR products with DNA restriction endonucleases throughout the genome. Based on the resulting polymorphisms, statistical methods can be used to identify the desired genotypes among the alleles found. The objective of this study was to identify the polymorphisms of the growth hormone gene and determine their association with six growth traits in Norduz sheep. Methods: Out of 50 Norduz sheep, three different loci, GHY1, GHY2 and GHY3 (with the length of 599, 690 and 679 bp respectively) of GH gene on chromosome 11 of sheep genome were selected to identify polymorphisms among individuals using the PCR-RFLP technique (GH/MspI). Result: In this research study, no polymorphisms were detected after digesting three loci of the GH gene with the enzyme MspI. The 50 individuals of Norduz sheep had only AB genotypes for all three loci, indicating that no polymorphisms were present in the regions of the GH genes studied.

Magnetic multi-enzyme cascade combined with liquid chromatography tandem mass spectrometry for fast DNA digestion and quantitative analysis of 5-hydroxymethylcytosine in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone

Long-term exposure to halobenzoquinones (HBQs) can induce genomic damages and abnormal epigenetic modifications. High-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) has shown unique advantages in identification and sensitive analysis of these structurally modified DNA lesions. Prior to MS analysis, genomic DNA needs to be fully digested into mono-nucleosides. Here, we prepared Supernuclease (SN)-, snake venom phosphodiesterase (SVP)- and calf intestinal alkaline phosphatase (CIP)- individually immobilized magnetic nanoparticles (MNPs), and combined them according to certain formula to construct a recyclable SN-SVP-CIP magnetic nanoparticles (SNSC-MNPs) cascade for rapid and efficient DNA digestion. The SNSC-MNPs cascade can fully digest genomic DNA into mono-nucleosides within 30 min. The SNSC-MNPs cascade coupled with HPLC-MS/MS method can accurately and sensitively detect 5-hydroxymethylcytosine (5hmC) changes in genome of human bladder cancer T24 cells induced by tetrachlorobenzoquinone. The immobilization of enzymes on MNPs can enhance the stability and enzymatic activity of the three enzymes, which guarantees the reusability and longtime preservation of the cascades. The relative digestive efficiencies are among 86% -106% up to ten times of reuse. The newly synthesized SNSC-MNPs cascade coupled with HPLC-MS/MS method is promising for fast identification and analysis of structural modifications in genomic DNA.

Comparative DNA Methylome of Phytoplasma Associated Retrograde Metamorphosis in Sesame (Sesamum indicum L.).

Simple SummarySesame (Sesamum indicum L.) is an important oilseed crop that is well known for its highly nutritious oil content. Due to its high oil content and nutritional properties, sesame is also known as the ‘queen of oilseeds’. Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. Sesame phyllody is the leading biotic constraint drastically affecting sesame productivity and resulting in yield losses of up to 80% in major sesame-producing countries in the world. In this study, we explored the role of DNA methylation in phytoplasma infection. Whole Genome Bisulfite Sequencing and a Quantitative analysis of DNA methylation using real-time PCR (qAMP) revealed an alteration of methylation pattern upon phytoplasma infection. Few selected development and defense-related loci were either hypo- or hypermethylated. We hereby report the first methylome study in healthy and phytoplasma-infected sesame. Our study provides fundamental insights into the role of DNA methylation in the symptom development of phytoplasma infection in sesame plants.Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. The mechanism of the dramatic conversion of flowers to leafy structures leading to yield losses and the drastic reduction in leaf size due to Phytoplasma infection remains yet to be identified. Cytosine methylation profiles of healthy and infected sesame plants studied using Whole Genome Bisulfite Sequencing (WGBS) and Quantitative analysis of DNA methylation with the real-time PCR (qAMP) technique revealed altered DNA methylation patterns upon infection. Phyllody was associated with global cytosine hypomethylation, though predominantly in the CHH (where H = A, T or C) context. Interestingly, comparable cytosine methylation levels were observed between healthy and little leaf-affected plant samples in CG, CHG and CHH contexts. Among the different genomic fractions, the highest number of differentially methylated Cytosines was found in the intergenic regions, followed by promoter, exonic and intronic regions in decreasing order. Further, most of the differentially methylated genes were hypomethylated and were mainly associated with development and defense-related processes. Loci for STOREKEEPER protein-like, a DNA-binding protein and PP2-B15, an F-Box protein, responsible for plugging sieve plates to maintain turgor pressure within the sieve tubes were found to be hypomethylated by WGBS, which was confirmed by methylation-dependent restriction digestion and qPCR. Likewise, serine/threonine-protein phosphatase-7 homolog, a positive regulator of cryptochrome signaling involved in hypocotyl and cotyledon growth and probable O-methyltransferase 3 locus were determined to be hypermethylated. Phytoplasma infection-associated global differential methylation as well as the defense and development-related loci reported here for the first time significantly elucidate the mechanism of phytoplasma-associated disease development.

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

Macrophages disseminate pathogen associated molecular patterns through the direct extracellular release of the soluble content of their phagolysosomes

Recognition of pathogen-or-damage-associated molecular patterns is critical to inflammation. However, most pathogen-or-damage-associated molecular patterns exist within intact microbes/cells and are typically part of non-diffusible, stable macromolecules that are not optimally immunostimulatory or available for immune detection. Partial digestion of microbes/cells following phagocytosis potentially generates new diffusible pathogen-or-damage-associated molecular patterns, however, our current understanding of phagosomal biology would have these molecules sequestered and destroyed within phagolysosomes. Here, we show the controlled release of partially-digested, soluble material from phagolysosomes of macrophages through transient, iterative fusion-fission events between mature phagolysosomes and the plasma membrane, a process we term eructophagy. Eructophagy is most active in proinflammatory macrophages and further induced by toll like receptor engagement. Eructophagy is mediated by genes encoding proteins required for autophagy and can activate vicinal cells by release of phagolysosomally-processed, partially-digested pathogen associated molecular patterns. We propose that eructophagy allows macrophages to amplify local inflammation through the processing and dissemination of pathogen-or-damage-associated molecular patterns.

Translocation of mitochondrial DNA into the nuclear genome blurs phylogeographic and conservation genetic studies in seabirds.

Mitochondrial DNA (mtDNA) translocated into the nuclear genome (numt), when co-analysed with genuine mtDNA, could plague phylogeographic studies. To evaluate numt-related biases in population genetics parameters in birds, which are prone to accumulating numts, we targeted the mitochondrial mt-cytb gene. We looked at 13 populations of Audubon's shearwater (Puffinus lherminieri), including five mitochondrial lineages. mt-cytb homologue and paralogue (numt) sequences were determined by Sanger sequencing with and without prior exonuclease digestion of nuclear DNA. Numts formed monophyletic clades corresponding to three of the five mitochondrial lineages tested (the remaining two forming a paraphyletic group). Nineteen percent of numt alleles fell outside of their expected mitochondrial clade, a pattern consistent with multiple translocation events, incomplete lineage sorting (ILS), and/or introgression. When co-analysing mt-cytb paralogues and homologues, excluding individuals with ambiguities underestimates genetic diversity (4%) and differentiation (11%) among least-sampled populations. Removing ambiguous sites drops the proportion of inter-lineage genetic variance by 63%. While co-analysing numts with mitochondrial sequences can lead to severe bias and information loss in bird phylogeographic studies, the separate analysis of genuine mitochondrial loci and their nuclear paralogues can shed light on numt molecular evolution, as well as evolutionary processes such as ILS and introgression.

Total vs. Partial Acid Digestion Methods for Trace Element Analysis in Archaeological Sediments

Trace element analysis of sediments from archaeological sites is a valuable method to investigate the anthropic impact and obtain information on the functions of different areas and changes in human activities. One of the most used and effective techniques to carry out this kind of analysis is inductively coupled plasma–mass spectrometry. This technique needs a previous dissolution of the sample by acid attack, but the development of the best method is still a discussed issue. In the present work, total and partial digestion methods were carried out in sediment samples of Cueva de la Cocina (Dos Aguas, Spain), and trace elements were measured and statistically compared. Major elements, soil organic matter amount, and pH data were used to evaluate the main drivers of trace element contents. The differences between the results from the two methods were highlighted. Total digestion is more effective for aluminosilicates and heavy minerals, although the partial digestion results suggested that, in most cases, the difference between the two methods is irrelevant for archaeological interpretations. Furthermore, in some cases, the total digestion of the mineral phases related to the geological contribution could mask the anthropic elemental signals.

The Short Chain Free Fatty Acids and Their Receptors in the Microbiotic Concept for Asthma Development

Asthma is a chronic, heterogeneous inflammatory disease with a high variability of the flow caused by the trigger influence of genetic and environmental factors. The heterogeneity of asthma indicates the involvement of many components in the mechanism of the inflammation of the respiratory tract. Currently, the microbiotic concept of the pathogenesis of respiratory system diseases is studied, based on the existence of a bilateral relationship between the microflora lungs and the intestines. The key mechanism of these relationships is free fatty acids performing signal and regulatory functions in the body. The medium-chain and long-chain free fatty acids are synthesized by de novo or enter the body as a result of consumption of fats, while the short-chain free fatty acids are formed in the intestine as a result of partial digestion of soluble fibre. The mechanism connecting the free fatty acids and inflammatory reactions includes activation of their receptors (Free Fatty Acid Receptor, FFAR) expressed on the cells of the gastrointestinal and respiratory tract, as well as on immune cells. If the role of the medium-chain and long-chain fatty acids receptors (FFAR1, FFAR4) and their ligands in the pathogenesis of asthma is actively studied, the value of the short-chain receptors (FFAR2, FFAR3) only begins to attract the attention of researchers in connection with the emergence of numerous data on the interconnection of the respiratory microbiome paths and intestines and its possible role in the induction of broncho-pulmonary complications. This review includes modern knowledge of the microbiotic concept of asthma, the basic information about the nomenclature, metabolism and transport of the free fatty acids, the value of the individual classes of the free fatty acids in the regulation of immune processes is normal and at bronchopulmonary pathology. The latest data of the FFAR receptors are systematized, features of their activation and expression, the intended role of FFAR2 and FFAR3 in the development and asthma therapy. It is discussed that gastrointestinal microbiota correction can reduce the activity of chronic inflammation of the respiratory tract at asthma.

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Dual Rolling Circle Amplification-Assisted Single-Particle Fluorescence Profiling of Exosome Heterogeneity for Discriminating Lung Adenocarcinoma from Pulmonary Nodules

Combined use of specific length amplified fragment sequencing (SLAF-seq) and bulked segregant analysis (BSA) for rapid identification of genes influencing fiber content of hemp (Cannabis sativa L.)

Hemp (Cannabis sativa L.), an ancient crop, is a significant source of high-quality fiber that primarily caters to the textile industry worldwide. Fiber content is a crucial quantitative trait for evaluating fiber yield in hemp. Understanding the genetic mechanisms involved in hemp breeding is essential for improving yield. In this study, we developed 660 F1 plants from a cross between Jindao-15 (high fiber content fiber-use variety) and Fire No.1 (low fiber content fiber-use variety), and thirty plants each with high and low fiber content were selected from 305 monoecious plants of this population according to 5%-10% of population size for quantitative traits. The DNA from these plants was extracted to establish two bulk DNA pools and then subjected to the restriction digestion by the enzymes RsaI and HaeIII to obtain 314–364 bp digestion fragments and subjected to sequencing using specific length amplified fragment sequencing (SLAF-seq). Finally, we successfully developed 368,404 SLAF tags, which led to the detection of 25,133 high-quality SNPs. Combing with the resequencing results of parents, the SNPs of mixed pools were then subjected to the SNP-Index correlation algorithm, which revealed four candidate regions related to fiber content traits on Chromosome 1, with a length of 8.68 Mb and containing 389 annotated genes. The annotation information and the comparison results identified 15 genes that were highly likely to modulate the fiber content of hemp. Further, qPCR validation identified six genes (LOC115705530, LOC115705875, LOC115704794, LOC115705371, LOC115705688 and LOC115707511) that were highly positively correlated with influencing the hemp fiber content. These genes were involved in the transcription regulation, auxin and water transportion, one carbon and sugar metabolism. And non-synnoumous mutation SNPs which may play vital role in influencing the fiber content were detected in LOC115705875, LOC115704794, LOC115705688 and LOC115707511. Thus, our study highlights the importance of the combined use of SLAF-Seq and Bulked Segregant analysis (BSA) to locate genes related to hemp fiber content rapidly. Hence, our study provides novel mechanistic inputs for the fast identification of genes related to important agronomic traits of hemp and other crops catering to the textile industry.

Fast Algorithms for the Simplified Partial Digest Problem.

The simplified partial digest problem (SPDP) models an effective and robust method for the building of a physical map using restriction site analysis. The best known algorithm requires O(n2n) time, using O(n2n) working space. The high complexities in time and space impede its application to genomes of a large number of sites. This article gives two new algorithms. The first improves the time by a factor of O(n) and significantly reduces the space to O(n2). The second improves both the time and space to O(n1.52n/2). Extensive experiments are conducted on real genomes. For instances that can be solved by the best known algorithm, the new algorithms achieve a speedup of up to 4000 times; in addition, due to the reduction in space, the new algorithms can solve many more instances. Experiments also reveal the following advantage of the SPDP method: almost every instance has at most four feasible solutions and for an instance that does not contain any pair of symmetric restriction sites, in all observed examples, the solution is unique.

Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry.

Large RNA including mRNA (mRNA) has emerged as an important new class of therapeutics. Recently, this has been demonstrated by two highly efficacious vaccines based on mRNA sequences encoding for a modified version of the SARS-CoV-2 spike protein. There is currently significant demand for the development of new and improved analytical methods for the characterization of large RNA including mRNA therapeutics. In this study, we have developed an automated, high-throughput workflow for the rapid characterization and direct sequence mapping of large RNA and mRNA therapeutics. Partial RNase digestions using RNase T1 immobilized on magnetic particles were performed in conjunction with high-resolution liquid chromatography–mass spectrometry analysis. Sequence mapping was performed using automated oligoribonucleotide annotation and identifications based on MS/MS spectra. Using this approach, a >80% sequence of coverage of a range of large RNAs and mRNA therapeutics including the SARS-CoV-2 spike protein was obtained in a single analysis. The analytical workflow, including automated sample preparation, can be completed within 90 min. The ability to rapidly identify, characterize, and sequence map large mRNA therapeutics with high sequence coverage provides important information for identity testing, sequence validation, and impurity analysis.

Rational fabrication of a DNA walking nanomachine on graphene oxide surface for fluorescent bioassay

DNA nanomachines, a delicate type of molecular machines, have been a research hotspot in biotechnology and materials. Here a two-dimensional (2D) DNA walking nanomachine with high working efficiency and low cost was easily assembled by using graphene oxide (GO) as the working platform for precisely fluorescent bioassay through the binding of target hepatitis B virus DNA (HBV-DNA) and the driving force of Exonuclease III (Exo III). The presence of HBV-DNA made continuous Exo III digestion of the FAM-modified DNA (FAM-DNA) in double-strand DNA (dsDNA) part in a burnt-bridge mechanism to output a “one-to-more” amplified signal. Accordingly, a 2D DNA walking nanomachine with simple operation and high cost-performance ratio was constructed. The walking speed of nanomachine was found to be regulated by loading DNA density on single sheet of GO. Furthermore, this nanomachine had low background since the dual energy transfer including fluorescence resonance energy transfer (FRET) from FAM to BHQ1 and the long-range resonance energy transfer (LrRET) from FAM to GO, making the biosensing applications highly promising.

DNA methylation levels of CEBPβ gene differentially affected by saturated and polyunsaturated fatty acids in adipogenesis of 3T3‐L1 cells

In obesity and type 2 diabetes, adipose and muscle tissues are significantly affected. It has been demonstrated that many of the changes are associated with gene expression. Cultured cell models were used to study the involvement of epigenetic modifications in mediating gene expression affected by hyperlipidemia. Cultured L6 muscle cells and differentiating 3T3‐L1 adipocytes were treated with saturated or polyunsaturated fatty acids to mimic hyperlipidemia conditions. After the treatments, methylation sequencing was conducted to identify the changes of the methylation levels of the genomic DNA samples from the cells. And restriction digestion followed PCR were used to validate the methylation sequencing results. In general, methylation levels of most of the genes were increased while methylation levels of some of the genes were decreased and small portion of the genes changed slightly among the genes with altered methylation levels in differentiating 3T3‐L1 cells treated with a saturated or a polyunsaturated fatty acid. We have demonstrated that both saturated type and polyunsaturated type fatty acids enhanced adipogenesis of differentiating 3T3‐L1 cells. While both saturated and polyunsaturated fatty acids decreased methylation levels of PPARγ gene, only polyunsaturated fatty acids increased methylation levels of C/EBPβ gene. These results are consistent with the knowledge that expression of both PPARγ and C/EBPβ gene are increased in adipogenesis and intake of saturated fat is associated with obesity.

Regional and Single Nucleotide Correction of Sequence Bias in Chromatin Accessibility Data

Chromatin accessibility assays directly measure which regions of DNA are actively regulated in the genome. Genomic chromatin accessibility data provide the gold standard of spatial and temporal resolution of regulatory landscapes in various cell types and conditions. Accessibility assays depend on enzymatic digestion of DNA and these enzymes are biased with respect to the sequences they recognize and cut. Sequence biases can lead to incorrect conclusions about the nature of the chromatin landscape within an experiment. Previous work from our group has shown that we can use observed k‐mer cut frequencies and k‐mer representation in the genome to correct the majority of this bias. However, these k‐mer scaling approaches did not effectively correct ATAC‐seq data, which assesses chromatin accessibility using the Tn5 transposase enzyme. Here, we find that the oligomeric state of Tn5 and wide sequence bias window precludes effective k‐mer scaling to account for Tn5 sequence bias. Tn5 sequence specificity is influenced by sequence content up to ten bases away from the Tn5 insertion site in both directions. In comparison, Benzonase, Cyanase, MNase, and DNase are primarily influenced by the sequence within 3 bases of the cleavage site. We could not effectively apply k‐mer scaling to ATAC‐seq data because the majority of insertion sites happen at unique 20‐mers in the genome and the method requires many genomic instances per k‐mer. To overcome this limitation, we applied an interpretable machine learning approach, prediction rules ensemble, to correct Tn5‐digested naked DNA. Application of a prediction rules ensemble effectively corrects local (single nucleotide resolution) sequence bias as well as sequence biases influenced by regional GC content. Correction of Tn5 sequence biases is important as the field continues to generate ATAC‐seq data at accelerating rates and use these data to accurately infer regulatory element activity.

Case study: Perspectives on the use of LEGO® bricks in the biochemistry classroom.

The use of LEGO® bricks in the higher education classroom has increased in the last two decades. This is no different in the STEM classroom and several disciplines, including physics, chemistry, and biology, have all made use of LEGO® bricks in some way to create models for active learning activities. Currently, the discipline to make the greatest use of LEGO® bricks is chemistry; only limited examples exist in biochemistry and the molecular life sciences. Here, we present the use of a LEGO® brick modelling activity in the introductory biochemistry classroom during the teaching of metabolism. We present student comments on the activity and the models that were generated by the students. Additionally, we focus on other instructor and project student-designed models for the teaching of ATP synthesis, gene regulation and restriction digestion. Interestingly, both the gene regulation and restriction digest activities were generated with the help of undergraduate students or recent graduates, by applying a backward design approach. This case study seeks to encourage more molecular life science educators to adopt the use of LEGO® bricks in their classrooms to engage in more active learning.

Having a Same Type IIS Enzyme's Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis.

Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.

Isolation and Characterization of High-Ethanol-Tolerance Lactic Acid Bacteria from Australian Wine.

Lactic acid bacteria are very important in winemaking. In this study, 108 lactic acid bacteria isolates were obtained from high-ethanol-content (~17% (v/v)) Grenache wines during uninoculated malolactic fermentation (MLF). The 16S rRNA and species-specific PCR showed that 104 of these were Oenococcus oeni, three were Lactobacillus hilgardii, and one was Staphylococcus pasteuri. AFLP of HindIII and MseI digests of the genomic DNA of the O. oeni strains was developed for the first time to discriminate the strains. The results showed that the method was a suitable technique for discriminating the O. oeni strains. Based on the cluster analysis, nine O. oeni strains were chosen for inclusion in an ethanol tolerance assay involving monitoring of optical density (ABS600nm) and viable plating. Several O. oeni strains (G63, G46, G71, G39) survived and grew well in MRS-AJ with 17% (v/v) ethanol, while the commercial O. oeni reference strain did not. Strain G63 could also survive and grow for 168 h after inoculation in MRS-AJ medium with 19% (v/v) ethanol. These results suggest that O. oeni G63, G46, G71, and G39 could potentially be used as MLF starters for high-ethanol-content wines. All three L. hilgardii strains could survive and grow in MRS-AJ with 19% (v/v) ethanol, perhaps also indicating their suitability as next-generation MLF starter cultures.

Enhanced Detection of Viral RNA Species Using FokI-Assisted Digestion of DNA Duplexes and DNA/RNA Hybrids.

The accurate detection of nucleic acids from certain biological pathogens is critical for the diagnosis of human diseases. However, amplified detection of RNA molecules from a complex sample by direct detection of RNA/DNA hybrids remains a challenge. Here, we show that type IIS endonuclease FokI is able to digest DNA duplexes and DNA/RNA hybrids when assisted by a dumbbell-like fluorescent sensing oligonucleotide. As proof of concept, we designed a battery of sensing oligonucleotides against specific regions of the SARS-CoV-2 genome and interrogated the role of FokI relaxation as a potential nicking enzyme for fluorescence signal amplification. FokI-assisted digestion of SARS-CoV-2 probes increases the detection signal of ssDNA and RNA molecules and decreases the limit of detection more than 3.5-fold as compared to conventional molecular beacon approaches. This cleavage reaction is highly specific to its target molecules, and no detection of other highly related B-coronaviruses was observed in the presence of complex RNA mixtures. In addition, the FokI-assisted reaction has a high multiplexing potential, as the combined detection of different viral RNAs, including different SARS-CoV-2 variants, was achieved in the presence of multiple combinations of fluorophores and sensing oligonucleotides. When combined with isothermal rolling circle amplification technologies, FokI-assisted digestion reduced the detection time of SARS-CoV-2 in COVID-19-positive human samples with adequate sensitivity and specificity compared to conventional reverse transcription polymerase chain reaction approaches, highlighting the potential of FokI-assisted signal amplification as a valuable sensing mechanism for the detection of human pathogens.

Partial enzymatic cell wall disruption of Oocystis sp. for simultaneous cultivation and extraction

A green extraction process is proposed to reduce energy and petroleum solvent’s consumption while maintaining environmental safety and quality that allows multiple product recovery. The goal of the study was to generate a highly permeable cell, through the use of crude fungal enzymes, that could permit simultaneous cultivation and extraction. A novel pretreatment method is developed which combines partial enzymatic digestion of cell walls of microalgae, while maintaining their viability, thus enabling further product recovery from the same biomass and has not been reported in literature yet. Partial digestion of the cell wall was accomplished through regulation of the contact time of crude enzymes with the microalgal cells leading to a highly permeable cell that is viable and actively growing. Experimentally this was achieved through controlling the time of incubation of microalgal cells with the crude enzymes. The effects of enzymatic treatment using crude fungal enzymes from Trichoderma reesei (cellulase 10.98 ± 0.17 U/ml and amylase 20.28 ± 0.13 U/ml) were investigated on the structural properties of marine microalga Oocystis sp. Results showed over 2-fold increase in protein efflux; 41% increase in released sugars and 24% decrease in cell count after 1 h of enzyme treatment at 33% (v/v) enzyme concentration. Controlled hydrolysis of algal cells could be achieved through optimizing the contact time (1 h) of enzymes with microalgal cells which retained their viability. This was validated through FTIR, SEM, XRD and Flow Cytometric analysis. The carotenoid content increased by approximately 9% and lipid content rose by nearly 6.2%.