DNA Barcoding for an Undergraduate Class.

DNA technique is a topic mandatorily covered in a biology and biochemistry undergraduate curriculum. Inquiry-based pedagogy is proven to be the most effective way of learning, and DNA barcoding method allows to merge necessary-to-study experimental techniques such as DNA isolation and purification, PCR, and basic BLAST search into a two- or three-week inquiry-based student project. It also provides a research-based experience to the students, who, when organized in groups, can design their own DNA-barcoding project if they wish. Here, we describe how DNA barcoding can be offered in an undergraduate college or advanced high school settings. This chapter is intended to help college and high school instructors to include DNA barcoding in their classes.

Assessment of Fungal Succession in Decomposing Swine Carcasses (Sus scrofa L.) Using DNA Metabarcoding.

The decomposition of animal bodies is a process defined by specific stages, described by the state of the body and participation of certain guilds of invertebrates and microorganisms. While the participation of invertebrates in decomposing is well-studied and actively used in crime scene investigations, information on bacteria and fungi from the scene is rarely collected or used in the identification of important factors such as estimated time of death. Modern molecular techniques such as DNA metabarcoding allow the identification and quantification of the composition of microbial communities. In this study, we used DNA metabarcoding to monitor fungal succession during the decomposition of juvenile pigs in grasslands of New Jersey, USA. Our findings show that decomposition stages differ in a diversity of fungal communities. In particular, we noted increased fungal species richness in the more advanced stages of decomposition (e.g., bloat and decay stages), with unique fungal taxa becoming active with the progression of decay. Overall, our findings improve knowledge of how fungi contribute to forensically relevant decomposition and could help with the assessment of crime scenes.

DNA isolation and genome sequence of the 134-year-old holotype specimen of Boletus subvelutipes Peck.

Molecular characterization of type specimens is a powerful tool used in clarifying species identity/circumscription, as well as establishing the taxonomic and phylogenetic status of organisms in question. However, DNA sequencing of aged herbarium collections can be a challenge due to the quantity and quality of DNA still present in the specimens. Herein, we report a custom DNA isolation protocol suitable for processing minute quantities of old specimen tissue and its utilization via high-throughput sequencing technologies to obtain, for the first time, the genome assembly of the 134-year-old holotype of Boletus subvelutipes Peck, a North American fleshy pored mushroom of taxonomic and historical significance. A side-by-side evaluation of our DNA isolation method with that of a commercial "kit" by Qiagen is also presented. By relying on the type material, we have established the genetic identity of B. subvelutipes, as well as providing preliminary phylogenetic evidence for its generic affinities in Neoboletus within Boletaceae. The reference genome of the B. subvelutipes holotype provides a resource for future comparative genomic studies, taxonomic revisions in Boletaceae, and other evolutionary studies of fungi.

MycoPins: a metabarcoding-based method to monitor fungal colonization of fine woody debris.

The MycoPins method described here is a rapid and affordable protocol to monitor early colonization events in communities of wood-inhabiting fungi in fine woody debris. It includes easy to implement field sampling techniques and sample processing, followed by data processing, and analysis of the development of early dead wood fungal communities. The method is based on fieldwork from a time series experiment on standard sterilized colonization targets followed by the metabarcoding analysis and automated molecular identification of species. This new monitoring method through its simplicity, moderate costs, and scalability paves a way for a broader and scalable project pipeline. MycoPins establishes a standard routine for research stations or regularly visited field sites for monitoring of fungal colonization of woody substrates. The routine uses widely available consumables and therefore presents a unifying method for monitoring of fungi of this type.

Unsaturated Fatty Acids and Their Immunomodulatory Properties.

Oils are an essential part of the human diet and are primarily derived from plant (or sometimes fish) sources. Several of them exhibit anti-inflammatory properties. Specific diets, such as Mediterranean diet, that are high in ω-3 polyunsaturated fatty acids (PUFAs) and ω-9 monounsaturated fatty acids (MUFAs) have even been shown to exert an overall positive impact on human health. One of the most widely used supplements in the developed world is fish oil, which contains high amounts of PUFAs docosahexaenoic and eicosapentaenoic acid. This review is focused on the natural sources of various polyunsaturated and monounsaturated fatty acids in the human diet, and their role as precursor molecules in immune signaling pathways. Consideration is also given to their role in CNS immunity. Recent findings from clinical trials utilizing various fatty acids or diets high in specific fatty acids are reviewed, along with the mechanisms through which fatty acids exert their anti-inflammatory properties. An overall understanding of diversity of polyunsaturated fatty acids and their role in several molecular signaling pathways is useful in formulating diets that reduce inflammation and increase longevity.

Citizen science helps in the study of fungal diversity in New Jersey.

The history of fungal diversity of the Northeastern United States is currently fragmentary and restricted to particular functional groups or limited geospatial scales. Here, we describe a unique by its size, lifespan and data originators dataset, to improve our understanding of species occurrence and distribution across the state and time. Between the years 2007 to 2019, over 30 parks and nature preserves were sampled during forays conducted by members of the New Jersey Mycological Association (USA), a nonprofit organization of fungi enthusiasts. The dataset contains over 400 000 occurrences of over 1400 species across the state, made up mostly of the phylum Basidiomycota (89%) and Ascomycota (11%), with most observations resolved at the species level (>99%). The database is georeferenced and openly accessible through the Global Biodiversity Information Facility (GBIF) repository. This dataset marks a productive endeavor to contribute to our knowledge of the biodiversity of fungi in the Northeastern United States leveraging citizen science to better resolve biodiversity of this critical and understudied kingdom.

Exploring DNA in biochemistry lab courses: DNA barcoding and phylogenetic analysis.

DNA structure has been leveraged in a variety of facets that allow scientists to perform a range of assays, including ones for identification of species, establishing evolutionary relationships between taxa, or even identifying individuals. Here, we present a DNA barcoding method as practical, hands-on approach that connects several experimental techniques in one sequence to teach the principles behind DNA isolation, purification, PCR, sequencing, and phylogeny analysis. Our set of exercises is designed for a teaching university laboratory setting. The three laboratory class assignments utilize DNA from a mushroom (can be purchased at a supermarket) and provide a pipeline to guide students through the process of identifying an unknown sample, like in many research laboratories. The third assignment can be used as a stand-alone exercise on phylogeny analysis and can be taught remotely. Students explore the theory behind the standard molecular techniques and apply it in a hands-on setting that involves experimental design, sample preparation, and use of hallmark molecular instruments.

Alcohol stress on cyanobacterial membranes: New insights revealed by transcriptomics.

Cyanobacteria are model photosynthetic prokaryotic organisms often used in biotechnology to produce biofuels including alcohols. The effect of alcohols on cyanobacterial cell physiology and specifically on membrane fluidity is poorly understood. Previous research on various primary aliphatic alcohols found that alcohols with a short hydrocarbon chain (C1-C3) do not affect expression of genes related to membrane physical state. In addition, less water-soluble alcohols with a hydrocarbon chain longer than C8 are found to have a reduced ability to reach cellular membranes hence do not drastically change membrane physical state or induce expression of stress-responsive genes. Therefore, hexan-1-ol (C6) is suggested to have the most profound effect on cyanobacterial membrane physical state. Here, we studied the effects of hexan-1-ol on the cyanobacterium Synechocystis sp. PCC 6803 transcriptome. The transcriptome data obtained is compared to the previously reported analysis of gene expression induced by benzyl alcohol and butan-1-ol. The set of genes whose expression is induced after exposure to all three studied alcohols is identified. The expression under alcohol stress for several general stress response operons is analyzed, and examples of antisense interactions of RNA are investigated.

Online laboratory exercise on computational biology: Phylogenetic analyses and protein modeling based on SARS-CoV-2 data during COVID-19 remote instruction.

Suspended on-site instruction during the COVID-19 created an exceptional challenge for teaching hands-on laboratory classes. We designed an online laboratory activity using computational biology techniques to overcome this issue. This set of online exercises introduces bioinformatics skills into existing curricula in the form of guided tutorials based on molecular data on SARS-CoV-2 virus.

Construction of prokaryotic strand-specific primary-transcripts saturated RNASeq library by controlled heat magnesium-dependent mRNA degradation.

The main limiting factors for RNA-Seq analysis are quality and quantity of the isolated mRNA. In prokaryotes, the proportion of messenger RNA to total RNA is rather low. Therefore, the main strategy of library preparation for sequencing is mRNA enrichment. Ribosomal and transfer RNAs, both monophosphorylated at the 5'-ends, are the major fractions of total RNA, while the bulk of primary transcripts is triphosphorylated at the 5'-teminus. Due to its low molecular weight, transfer RNA could be easily removed by a quick precipitation in LiCl solution. Ribosomal RNA may be degraded enzymatically by 5'-end terminal exonuclease XRN-1. These steps allow enriching samples in mRNA during the first stages of RNA-Seq library preparation. The desired level of fragmentation of enriched mRNA necessary for the 2nd generation sequencing can be controlled by the duration of incubation at elevated temperatures in the presence of Mg2+-ions. Here, we describe a simple protocol for construction of the primary prokaryotic mRNA-saturated library without long depletion procedures.

Very-long-chain fatty acids (VLCFAs) in plant response to stress.

Plant growth is affected by various stresses leading to changes in metabolism. Stress conditions include a variety of biotic and abiotic factors such as pathogens, drought, high and low temperatures and heavy metals. Among multiple physiological responses to stress, there is an adaptive modification in membrane lipid constituents. In particular, the composition of membrane very-long-chain fatty acids (VLCFAs) changes both qualitatively and quantitatively. Here, we evaluate the current data on the effects of stress on plant VLCFAs composition. In summary, some stress conditions lead to an increase of the total amount of saturated and, in certain cases, unsaturated VLCFAs. Currently, it is not completely clear how these molecules participate in the biology of plant cell membranes. Their possible functional roles are discussed.

Elucidating Carotenoid Biosynthetic Enzyme Localization and Interactions Using Fluorescent Microscopy.

Carotenoids are essential for survival of all plants, where these colorful pigments and derivatives are biosynthesized, as well as for humans and other species that obtain plant-derived carotenoids in their diets and rely upon them for vitamin biosynthesis or antioxidant actions. The plant carotenoid biosynthetic pathway consists of nuclear encoded enzymes that are imported into chloroplasts and other plastids. The pathway structural genes are known and have been targeted for metabolic engineering to improve carotenoid profiles or content. However, results are not always as expected because there remain fundamental gaps in understanding how the pathway is physically organized. Many of the enzymes have been found in high molecular weight complexes which are poorly described. Elucidation of enzyme localization as well as enzyme interactions in vivo are needed for advancing the carotenoid field and facilitating our understanding of the three-dimensional organization of this important pathway. Fluorescent protein fusions with carotenoid enzymes can provide in vivo information when these fusions are introduced and transiently expressed in plant cells. Current advances in fluorescent microscopy, especially confocal microscopy, provide the resolution needed to localize fluorescently tagged carotenoid enzymes within suborganellar locations of plastids. Interactions between carotenoid biosynthetic enzymes can be determined using bimolecular fluorescence complementation (BiFC), a method whereby genes of interest are fused with sequences encoding nonfluorescent N- and C-terminal halves of YFP (yellow fluorescent protein), and then introduced into plant protoplasts to allow expression and visualization by fluorescence microscopy. The YFP fluorescence is restored only if the N and C-terminal regions are brought together by interacting fusion partners. Here we describe the methodology, with extensive tips and notes, for determining in vivo carotenoid enzyme localization and enzyme interactions by transient expression of enzyme-fluorescent protein fusions.

Universal Molecular Triggers of Stress Responses in Cyanobacterium Synechocystis.

Systemic analysis of stress-induced transcription in the cyanobacterium Synechocystis sp. strain PCC 6803 identifies a number of genes as being induced in response to most abiotic stressors (heat, osmotic, saline, acid stress, strong light, and ultraviolet radiation). Genes for heat-shock proteins (HSPs) are activated by all these stresses and form a group that universally responds to all environmental changes. The functions of universal triggers of stress responses in cyanobacteria can be performed by reactive oxygen species (ROS), in particular H2O2, as well as changes in the redox potential of the components of the photosynthetic electron transport chain. The double mutant of Synechocystis sp. PCC 6803 (katG/tpx, or sll1987/sll0755), which is defective in antioxidant enzymes catalase (KatG) and thioredoxin peroxidase (Tpx), cannot grow in the presence of exogenous hydrogen peroxide (H2O2); and it is extremely sensitive to low concentrations of H2O2, especially under conditions of cold stress. Experiments on this mutant demonstrate that H2O2 is involved in regulation of gene expression that responds to a decrease in ambient temperature, and affects both the perception and the signal transduction of cold stress. In addition, they suggest that formation of ROS largely depends on the physical state of the membranes such as fluidity or viscosity. In cyanobacteria, an increase in membrane turnover leads to a decrease in the formation of ROS and an increase in resistance to cold stress. Therefore: (1) H2O2 is the universal trigger of stress responses in cyanobacterial cells; (2) ROS formation (in particular, H2O2) depends on the physical properties of both cytoplasmic and thylakoid membranes; (3) The destructive effect of H2O2 is reduced by increasing of fluidity of biological membranes.

Draft Genome Sequences of a Putative Prokaryotic Consortium (IPPAS B-1204) Consisting of a Cyanobacterium (Leptolyngbya sp.) and an Alphaproteobacterium (Porphyrobacter sp.).

A new presumably simple consortium of a Leptolyngbya sp. and a Porphyrobacter sp. was isolated from Tolbo Lake in Mongolia. The draft genome sequences of both species are reported. The consortium has been deposited in the Collection of Microalgae and Cyanobacteria of the Institute of Plant Physiology, Moscow, Russia, under the accession number IPPAS B-1204.

Membrane physical state and stress regulation in Synechocystis: fluidizing alcohols repress fatty acid desaturation.

Cyanobacteria are prokaryotic photosynthetic organisms widely used in biotechnology, photosynthesis and abiotic stress research. There are several cyanobacterial strains modified to produce biofuels, but the influence of alcohols on cyanobacterial cell physiology is poorly understood. Here, we conducted a systematic study of the effects of nine primary aliphatic alcohols and an aromatic benzyl alcohol on both membrane physical state and the expression of genes for fatty acid desaturases (FADs) in a model cyanobacterium Synechocystis sp. strain PCC 6803. Hexan-1-ol was found to have the most membrane fluidizing action among all alcohols studied, with its efficiency correlating with both duration of treatment and alcohol concentration. A prolonged exposure to alcohol results in a continuous loss of unsaturated fatty acids (FAs) followed by cell death, an undesired challenge that should be considered in cyanobacterial biotechnology. We suggest that membrane fluidization is the key component in alcohol stress causing inactivation of FADs and resulting in a lethal depletion of unsaturated FAs. Due to the most pronounced effects of alcohol- and heat-induced membrane fluidization on desB encoding a terminal ω3-FAD, we propose to call desB a 'viscosity gene' in analogy to heat-induced 'fluidity gene' hspA.

Hydrogen Peroxide Participates in Perception and Transduction of Cold Stress Signal in Synechocystis.

The double mutant ΔkatG/tpx of cyanobacterium Synechocystis sp. strain PCC 6803, defective in the anti-oxidative enzymes catalase (KatG) and thioredoxin peroxidase (Tpx), is unable to grow in the presence of exogenous H2O2. The ΔkatG/tpx mutant is shown to be extremely sensitive to very low concentrations of H2O2, especially when intensified with cold stress. Analysis of gene expression in both wild-type and ΔkatG/tpx mutant cells treated by combined cold/oxidative stress revealed that H2O2 participates in regulation of expression of cold-responsive genes, affecting either signal perception or transduction. The central role of a transmembrane stress-sensing histidine kinase Hik33 in the cold/oxidative signal transduction pathway is discussed.

Polyphasic characterization of the thermotolerant cyanobacterium Desertifilum sp. strain IPPAS B-1220.

A cyanobacterial strain from Lake Shar-Nuur, a freshwater lake in Mongolia, was isolated and characterized by a polyphasic approach. According to the 16S ribosomal RNA gene sequence, this strain (IPPAS B-1220) belongs to a newly described genus Desertifilum. In general, strains of Desertifilum maintain their genetic stability, as seen from the analysis of the 16S rRNA gene and 16S-23S rRNA internal transcribed spacer sequences from strains collected at distant locations. The newly discovered strain is characterized by an unusual fatty acid composition (16:1Δ7 and 16:2Δ7,10). Analysis of its draft genomic sequence reveals the presence of six genes for the acyl-lipid desaturases: two Δ9-desaturases, desC1 and desC2; two Δ12-desaturases, desA1 and desA2; one desaturase of unknown specificity, desX; and one gene for the bacillary-type desaturase, desG, which supposedly encodes an ω9-desaturase. A scheme for a fatty acid desaturation pathway that describes the biosynthesis of 16:1Δ7 and 16:2Δ7,10 fatty acids in Desertifilum is proposed.

The Phytoene synthase gene family of apple (Malus x domestica) and its role in controlling fruit carotenoid content.

BACKGROUND: Carotenoid compounds play essential roles in plants such as protecting the photosynthetic apparatus and in hormone signalling. Coloured carotenoids provide yellow, orange and red colour to plant tissues, as well as offering nutritional benefit to humans and animals. The enzyme phytoene synthase (PSY) catalyses the first committed step of the carotenoid biosynthetic pathway and has been associated with control of pathway flux. We characterised four PSY genes found in the apple genome to further understand their involvement in fruit carotenoid accumulation. RESULTS: The apple PSY gene family, containing six members, was predicted to have three functional members, PSY1, PSY2, and PSY4, based on translation of the predicted gene sequences and/or corresponding cDNAs. However, only PSY1 and PSY2 showed activity in a complementation assay. Protein localisation experiments revealed differential localization of the PSY proteins in chloroplasts; PSY1 and PSY2 localized to the thylakoid membranes, while PSY4 localized to plastoglobuli. Transcript levels in 'Granny Smith' and 'Royal Gala' apple cultivars showed PSY2 was most highly expressed in fruit and other vegetative tissues. We tested the transient activation of the apple PSY1 and PSY2 promoters and identified potential and differential regulation by AP2/ERF transcription factors, which suggested that the PSY genes are controlled by different transcriptional mechanisms. CONCLUSION: The first committed carotenoid pathway step in apple is controlled by MdPSY1 and MdPSY2, while MdPSY4 play little or no role in this respect. This has implications for apple breeding programmes where carotenoid enhancement is a target and would allow co-segregation with phenotypes to be tested during the development of new cultivars.