Shear-Thinning and Designable Responsive Supramolecular DNA Hydrogels Based on Chemically Branched DNA.

A novel supramolecular DNA hydrogel system was designed based on a directly synthesized chemically branched DNA. For the hydrogel formation, a self-dimer DNA with two sticky ends was designed as the linker to induce the gelation of B-Y. By programing the linker sequence, thermal and metal-ion responsiveness could be introduced into this hydrogel system. This supramolecular DNA hydrogel shows shear-thinning, designable responsiveness, and good biocompatibility, which will simplify the hydrogel composition and preparation process of the supramolecular DNA hydrogel and accelerate its biomedical applications.

Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells.

RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs.

Low-input ATAC&mRNA-seq protocol for simultaneous profiling of chromatin accessibility and gene expression.

We present a simple, fast, and robust protocol (low-input ATAC&mRNA-seq) to simultaneously generate ATAC-seq and mRNA-seq libraries from the same cells in limited cell numbers by coupling a simplified ATAC procedure using whole cells with a novel mRNA-seq approach that features a seamless on-bead process including direct mRNA isolation from the cell lysate, solid-phase cDNA synthesis, and direct tagmentation of mRNA/cDNA hybrids for library preparation. It enables dual-omics profiling from limited material when joint epigenome and transcriptome analyses are needed. For complete details on the use and execution of this protocol, please refer to Li et al. (2021).

A colorimetric aptamer-based method for detection of cadmium using the enhanced peroxidase-like activity of Au-MoS2 nanocomposites.

In this work, a colorimetric aptamer-based method for detection of cadmium using gold nanoparticles modified MoS2 nanocomposites as enzyme mimic is established. In short, biotinylated Cd2+ aptamers are immobilized by biotin-avidin binding on the bottoms of the microplate, the complementary strands of Cd2+ aptamers are connected to the Au-MoS2 nanocomposites which have the function of enhanced peroxidase-like activity. The csDNA-Au-MoS2 signal probe and target Cd2+ compete for binding Cd2+ aptamer, the color change can be observed by addition of chromogenic substrate, thereby realizing visual detection of Cd2+. The absorbance of the solution at 450 nm has a clear linear relationship with the Cd2+ concentration. The linear range is 1-500 ng/mL, and the limit of detection is 0.7 ng/mL. The assay was used to test white wine samples, the results are consistent with those of atomic absorption spectrometry; which prove that this method can be used for detection of Cd2+ in real samples.

Determination and Quantification of Bacterial Virulent Gene Expression Using Quantitative Real-Time PCR.

Polymerase chain reaction (PCR) plays significant roles in modern molecular biology. However, it is relatively cumbersome and less accurate to use the traditional PCR method in quantifying gene expression because it requires first generating a standard curve with multiple input controls showing linearity with amplified control PCR products on a electrophoresis gel to compare with the abundance of the to-be-determined gene transcript PCR amplicons. Quantitative real-time PCR (qRT-PCR) is a time-efficient and reliable tool for accurate quantification and comparison of gene (RNA transcript) expression from various biological samples. Current technology has simplified and expedited the qPCR process significantly. However, proper techniques and standard protocols are required in eliminating potentially erroneous experimental outcome. Here, we provide an example from a drug-treated bacterial gene expression study with detailed protocols to demonstrate real-time qPCR with SYBR™ Green and TaqMan®, two of the most adapted and well-established qPCR technologies. Relative quantification of gene (RNA transcript) expression using qRT-PCR is demonstrated in detail from sample preparations to data analysis.

Towards Reverse Transcription with an Expanded Genetic Alphabet.

Unnatural base pairs (UBPs) strikingly augment the natural genetic alphabet. The development of particular hydrophobic UBPs even allows insertion and stable propagation in bacteria. Those UBPs expand the chemical scope of DNA and RNA, and thus, could enable the evolution of novel aptamers or ribozymes by in vitro selection (systematic evolution of ligands by exponential enrichment, SELEX). However, the application of such UBPs in reverse transcription (rtc), which is a key step for RNA-based SELEX, has not been reported so far. The implication of Romesberg's NaM:TPT3 base pair in rtc reactions is presented by testing five commercially available reverse transcriptases (RTs). The employed RTs predominantly pause at the site of the unnatural nucleotide rTPT3 not being able to accept the dNaM building block as a substrate. This allows verification of the unnatural base position in RNA and an estimation of their abundance. In contrast, primer extension from an rNaM-containing template results in considerably more full-length cDNA. Furthermore, RTs that could potentially be able to handle an expanded genetic alphabet based on NaM:TPT3 are presented.

Multi-triggered Supramolecular DNA/Bipyridinium Dithienylethene Hydrogels Driven by Light, Redox, and Chemical Stimuli for Shape-Memory and Self-Healing Applications.

Multi-triggered DNA/bipyridinium dithienylethene (DTE) hybrid carboxymethyl cellulose (CMC)-based hydrogels are introduced. DTE exhibits cyclic and reversible photoisomerization properties, switching between the closed state (DTEc), the electron acceptor, and the open isomer (DTEo) that lacks electron acceptor properties. One system introduces a dual stimuli-responsive hydrogel containing CMC chains modified with electron donor dopamine sites and self-complementary nucleic acids. In the presence of DTEc and the CMC scaffold, a stiff hydrogel is formed, cooperatively stabilized by dopamine/DTEc donor-acceptor interactions and by duplex nucleic acids. The cyclic and reversible formation and dissociation of the supramolecular donor-acceptor interactions, through light-induced photoisomerization of DTE, or via oxidation and subsequent reduction of the dopamine sites, leads to hydrogels of switchable stiffness. Another system introduces a stimuli-responsive hydrogel triggered by one of three alternative signals. The stiff, multi-triggered hydrogel consists of CMC chains cross-linked by dopamine/DTEc donor-acceptor interactions, and by supramolecular K+-stabilized G-quadruplexes. The G-quadruplexes are reversibly separated in the presence of 18-crown-6 ether and reformed upon the addition of K+. The stiff hydrogel undergoes reversible transitions between high-stiffness and low-stiffness states triggered by light, redox agents, or K+/crown ether. The hybrid donor-acceptor/G-quadruplex cross-linked hydrogel shows shape-memory and self-healing features. By using three different triggers and two alternative memory-codes, e.g., the dopamine/DTEc or the K+-stabilized G-quadruplexes, the guided shape-memory function of the hydrogel matrices is demonstrated.

Salud mental de migrantes centroamericanos indocumentados en tránsito por la frontera sur de México / Mental health of undocumented migrants in transit at the southern border of Mexico

Objetivo. Conocer las necesidades percibidas de salud mental de migrantes centroamericanos indocumentados en tránsito por la ciudad de Tapachula, Chiapas. Material y métodos. Estudio cualitativo realizado en Casa de Migrantes de Tapachula, Chiapas. Se realizaron 20 entrevistas semiestructuradas a diez mujeres y diez hombres migrantes. Se exploró el estado de salud mental y las expectativas de atención. Se retomaron nociones teórico-metodológicas de la fenomenología sociológica. Resultados. Los migrantes presentaban signos y síntomas de daños en su salud mental relacionados con experiencias vividas en el lugar de origen y en el tránsito por México. La percepción sobre su salud mental es influida por el modelo biomédico hegemónico. Las expectativas de servicios se relacionaron con la satisfacción de necesidades básicas. Conclusiones. Es necesario fortalecer la respuesta del sistema de atención en salud mental a partir de estrategias de cooperación y emprender acciones que promuevan la superación de una construcción biomédica de salud mental que estigmatiza, medicaliza, segrega y dificulta el acceso a servicios.

Objective. To identify the perception and needs in mental health of Central American migrants in transit through Tapachula, Chiapas. Materials and methods. Qualitative study in a migrant shelter in Tapachula, Chiapas. In 20 semi-structured interviews with migrant men and women, we explored their perceptions on mental health and expectations on care. We used basic notions of phenomenology to guide the analysis. Results. Migrants had several mental health problems related to the conditions at their country of origin and due to their initial transit through Mexico.Their perception on mental health problems was heavily influenced by the biomedical health paradigm. The expectations they had on the provision of services were related to the satisfaction of basic needs. Conclusions. It is necessary to strengthen the governmental response to mental health needs through collaborative strategies. Also, actions are needed to further the understanding of mental health in order to transcend the biomedical notions that stigmatize, segregate and create a barrier to accessing services.

Reverse genetics system for studying human rhinovirus infections.

Human rhinovirus (HRV) contains a 7.2 kb messenger-sense RNA genome which is the template for reproducing progeny viruses after it enters the cytoplasm of a host cell. Reverse genetics refers to the regeneration of progeny viruses from an artificial cDNA copy of the RNA genome of an RNA virus. It has been a powerful molecular genetic tool for studying HRV and other RNA viruses because the artificial DNA stage makes it practical to introduce specific mutations into the viral RNA genome. This chapter uses HRV-16 as the model virus to illustrate the strategy and methods for constructing and cloning the artificial cDNA copy of a full-length HRV genome, identifying the infectious cDNA clone isolates, and selecting the most vigorous cDNA clone isolate to serve as the standard parental clone for future molecular genetic study of the virus.

Multiplex RT-PCR method for the simultaneous detection of nine grapevine viruses.

Viral diseases are a serious pathological problem for grapevines, and in recent years the need for increasingly specific and rapid diagnostic methods for the selection of propagation materials has grown. Arabis mosaic virus, Grapevine fanleaf virus, Grapevine virus A, Grapevine virus B, Grapevine rupestris stem pitting-associated virus, Grapevine fleck virus, and Grapevine leafroll-associated viruses 1, 2, and 3 are nine of the most widespread viruses that naturally infect grapevines. A multiplex RT-PCR was developed for simultaneous detection of these nine grapevine viruses, in combination with a plant RNA internal control used as an indicator of the effectiveness of the reaction. One to ten fragments specific for the viruses and an internal control were simultaneously amplified from infected samples and identified by their specific molecular sizes in agarose gel. The protocol reported is an update of previously published protocols for RNA extraction and multiplex diagnosis of viruses. After several years of use and hundreds of samples tested, and following validation in several laboratories, this multiplex RT-PCR provides a reliable and rapid method for detecting grapevine viruses from a large number of samples.

Analysis and application of viroid-specific small RNAs generated by viroid-inducing RNA silencing.

Viroids are noncoding RNA pathogens inducing severe to mild disease symptoms on agriculturally important crop plants. Viroid replication is entirely dependent on host transcription machinery, and their replication/accumulation in the infected cells can activate RNA silencing-a host defense mechanism that targets the viroid itself. RNA silencing produces in the cell large amounts of viroid-specific small RNAs of 21-24-nucleotides by cleaving (or "dicing") entire molecules of viroid RNA. However, viroid replication is resistant to the effects of RNA silencing and disrupts the normal regulation of host gene expression, finally resulting in the development of disease symptoms on infected plant. The molecular mechanisms of biological processes involving RNA silencing and underlying various aspects of viroid-host interaction, such as symptom expression, are of special interests to both basic and applied areas of viroid research. Here we present a method to create infectious viroid cDNA clones and RNA transcripts, the starting material for such analyses, using Hop stunt viroid as an example. Next we describe methods for the preparation and analysis of viroid-specific small RNAs by deep sequencing using tomato plants infected with Potato spindle tuber viroid as an example. Finally we introduce bioinformatics tools and methods necessary to process, analyze, and characterize these viroid-specific small RNAs. These bioinformatic methods provide a powerful new tool for the detection and discovery of both known and new viroid species.

SuperSAGE as an analytical tool for host and viral gene expression.

SuperSAGE is a tag-based transcript profiling method, which allows to analyze the expression of thousands of genes at a time. In SuperSAGE, 26 bp tags are extracted from cDNA using the type III restriction enzyme, EcoP15I. In SuperSAGE, the amount of transcripts was represented by tag counts. Taking advantage of uniqueness of the 26 bp tags, host and virus transcripts can be monitored in virus-infected cells. Combining next generation sequencing technology, we established High-throughput SuperSAGE (Ht-SuperSAGE), which allows the analysis of multiple samples with reduced time and cost. In this chapter, we present the protocol of Ht-SuperSAGE involving a recently available benchtop type next generation sequencer.

Microarray analysis of R-gene-mediated resistance to viruses.

The complex process for host-plant resistance to viruses is precisely regulated by a number of genes and signaling compounds. Thus, global gene expression analysis can provide a powerful tool to grasp the complex molecular network for resistance to viruses. The procedures for comparative global gene expression profiling of virus-resistant and control plants by microarray analysis include RNA extraction, cDNA synthesis, cRNA labeling, hybridization, array scanning, and data mining steps. There are several platforms for the microarray analysis. Commercial services for the steps from cDNA synthesis to array scanning are now widely available; however, the data manipulation step is highly dependent on the experimental design and research focus. The protocols presented here are optimized for analyzing global gene expression during the R gene-conferred defense response using commercial oligonucleotide-based arrays. We also demonstrate a technique to screen for differentially expressed genes using Excel software and a simple Internet tool-based data mining approach for characterizing the identified genes.

Construction of Trypanosoma brucei Illumina RNA-Seq libraries enriched for transcript ends.

High-throughput RNA sequencing (RNA-Seq) has quickly occupied center stage in the repertoire of available tools for transcriptomics. Among many advantages, the single-nucleotide resolution of this powerful approach allows mapping on a genome-wide scale of splice junctions and polyadenylation sites, and thus, the precise definition of mature transcript boundaries. This greatly facilitated the transcriptome annotation of the human pathogen Trypanosoma brucei, a protozoan organism in which all mRNA molecules are matured by spliced leader (SL) trans-splicing from longer polycistronic precursors. The protocols described here for the generation of three types of libraries for Illumina RNA-Seq, 5'-SL enriched, 5'-triphosphate-end enriched, and 3'-poly(A) enriched, enabled the discovery of an unprecedented heterogeneity of pre-mRNA-processing sites, a large number of novel coding and noncoding transcripts from previously unannotated genes, and quantify the cellular abundance of RNA molecules. The method for producing 5'-triphosphate-end-enriched libraries was instrumental for obtaining evidence that transcription initiation by RNA polymerase II in trypanosomes is bidirectional and biosynthesis of mRNA precursors is primed not only at the beginning of unidirectional gene clusters, but also at specific internal sites.

Construction of a synthetic infectious cDNA clone of Grapevine Algerian latent virus (GALV-Nf) and its biological activity in Nicotiana benthamiana and grapevine plants.

BACKGROUND: Grapevine Algerian latent virus (GALV) is a tombusvirus first isolated in 1989 from an Algerian grapevine (Vitis spp.) plant and more recently from water samples and commercial nipplefruit and statice plants. No further reports of natural GALV infections in grapevine have been published in the last two decades, and artificial inoculations of grapevine plants have not been reported. We developed and tested a synthetic GALV construct for the inoculation of Nicotiana benthamiana plants and different grapevine genotypes to investigate the ability of this virus to infect and spread systemically in different hosts. METHODS: We carried out a phylogenetic analysis of all known GALV sequences and an epidemiological survey of grapevine samples to detect the virus. A GALV-Nf clone under the control of the T7 promoter was chemically synthesized based on the full-length sequence of the nipplefruit isolate GALV-Nf, the only available sequence at the time the project was conceived, and the infectious transcripts were tested in N. benthamiana plants. A GALV-Nf-based binary vector was then developed for the agroinoculation of N. benthamiana and grapevine plants. Infections were confirmed by serological and molecular analysis and the resulting ultrastructural changes were investigated in both species. RESULTS: Sequence analysis showed that the GALV coat protein is highly conserved among diverse isolates. The first epidemiological survey of cDNAs collected from 152 grapevine plants with virus-like symptoms did not reveal the presence of GALV in any of the samples. The agroinoculation of N. benthamiana and grapevine plants with the GALV-Nf binary vector promoted efficient infections, as revealed by serological and molecular analysis. The GALV-Nf infection of grapevine plants was characterized in more detail by inoculating different cultivars, revealing distinct patterns of symptom development. Ultrastructural changes induced by GALV-Nf in N. benthamiana were similar to those induced by tombusviruses in other hosts, but the cytopathological alterations in grapevine plants were less severe. CONCLUSIONS: This is the first report describing the development of a synthetic GALV-Nf cDNA clone, its artificial transmission to grapevine plants and the resulting symptoms and cytopathological alterations.

A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas. IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results A full-length JcIPI cDNA consisting of 1355 bp was cloned. It encoded a protein of 305 amino acids. Analysis of deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a significant evolutionary relatedness with Ricinus communis. Southern blot analysis showed the presence of an IPI multigene family in J. curcas. Comparative expression analysis of tissue specific JcIPI demonstrated the highest transcript level in flowers. Abiotic factors could induce the expression of JcIPI. Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion This is the first report of cloning and characterization of IPI from J. curcas. Our study will be of significant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further confirmation.

Molecular cloning and expression analysis of the MaASR1 gene in banana and functional characterization under salt stress

Background Abscisic acid (ABA)-, stress- and ripening-induced protein (ASR) is plant-specific hydrophilic transcriptional regulators involved in sucrose stress and wounding in banana. However, it is not known whether banana ASR genes confer salt stress tolerance. The contexts of the study was to analysis the sequence characterization of banana ASR1, and identify its expression patterns and function under salt stress using quantitative real-time PCR (qPCR) and overexpression in Arabidopsis. The purpose was to evaluate the role of banana ASR1 to salt stress tolerance employed by plants. Results A full-length cDNA isolated from banana fruit was named MaASR1, and it had a 432 bp open reading frame (ORF) encoding 143 amino acids. MaASR1 was preferential expression in roots and leaves compared to low expression in fruits, rhizomes and flowers. Under salt stress, the expression of MaASR1 quickly increased and highest expression level was detected in roots and leaves at 4 h, and then gradually decreased. These results suggested that MaASR1 expression was induced under salt stress. MaASR1 protein was localized in the nucleus and plasma membrane. MaASR1 was transformed to Arabidopsis and verified by southern and northern analysis, transgenic lines L14 and L38 integrated one and two copies of MaASR1, respectively, while overexpression in transgenic lines provided evidence for the role of MaASR1 to salt stress tolerance. Conclusions This study demonstrated that overexpression of MaASR1 in Arabidopsis confers salt stress tolerance by reducing the expression of ABA/stress-responsive genes, but does not affect the expression of the ABA-independent pathway and biosynthesis pathway genes.

Cloning, characterization and expression of Peking duck fatty acid synthase during adipocyte differentiation

Background Fatty acid synthase (FAS) is a key enzyme of de novo lipogenesis (DNL), which has been cloned from several species: Gallus gallus, Mus musculus, Homo sapiens, but not from Anas platyrhynchos. The current study was conducted to obtain the full-length coding sequence of Peking duck FAS and investigate its expression during adipocyte differentiation. Results We have isolated a 7654 bp fragment from Peking duck adipocytes that corresponds to the FAS gene. The cloned fragment contains an open reading frame of 7545 bp, encodes a 2515 amino acid protein, and displays high nucleotide and amino acid homology to avian FAS orthologs. Twelve hour treatment of oleic acid significantly up-regulated the expression of FAS in duck preadipocytes (P < 0.05). However, 1000 µM treatment of oleic acid exhibited lipotoxic effect on cell viability (P < 0.05). In addition, during the first 24 h of duck adipocyte differentiation FAS was induced; however, after 24 h its expression level declined (P < 0.05). Conclusion We have successfully cloned and characterized Peking duck FAS. FAS was induced during adipocyte differentiation and by oleic acid treatment. These findings suggest that Peking duck FAS plays a similar role to mammalian FAS during adipocyte differentiation.

Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

BACKGROUND: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. RESULTS: We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. CONCLUSIONS: A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.