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5PRIME Phase Lock Gel

Simplifies organic extraction of nucleic acid template and improves safety
Features & Benefits
  • Eliminates interphase contamination of nucleic acid solution
  • 30% greater yield of nucleic acids over conventional method
  • Gel barrier allows easy sample decanting
  • Reduced contact with hazardous organic solvents


5PRIME Phase Lock Gel is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.

Kit Size
Order Info
Kit Size
Order Info
Phase Lock Gel – Heavy
Kit Size:
Order Info:
200 tubes
Kit Size
Order Info
Phase Lock Gel – Light
Kit Size:
Order Info:
200 tubes


5PRIME Phase Lock Gel (PLG) is a unique product that eliminates interphase-protein contamination during phenol extraction and ensures faster results with improved recoveries. PLG migrates to form a tight seal between the phases of an aqueous/organic extraction during centrifugation. The organic phase and the interphase materials are effectively trapped in or below the barrier, thus enabling complete and easy decanting or pipetting of the entire aqueous phase. The benefits are increased yields of up to 30%, increased protection from exposure to hazardous compounds, and no risk of interphase sample contamination.

PLG can be adapted to virtually any protocol requiring extraction of an aqueous sample with phenol and/or chloroform. For convenience, PLG is provided predispensed into standard 2mL centrifuge tubes and comes in two varieties:


PLG Heavy

Aqueous phase: High density sample (for example, high salt content).

Organic phase: Standard mixtures of phenol, chloroform, isoamyl alcohol with a chloroform fraction of at least 60% (Phenol as the sole organic solvent is not compatible with PLG Heavy).

PLG Light

Aqueous phase: Low-density sample

Organic phase: Standard mixtures of phenol, chloroform, isoamyl alcohol. 


PLG is inert and stable to mild heating, and it does not interfere with any of the standard nucleic acid restriction and modification enzymes. The reactions can be carried out in the presence of PLG at the appropriate temperature and then terminated and extracted by the addition of phenol or phenol/chloroform to the reaction tube. PLG can be present during the heating stage of the deactivation of enzymes (65°C for 10 min.) prior to the organic extraction.


  • Genomic DNA from Mouse Tails: Use PLG Heavy to enhance isolation of genomic DNA from mouse tails using a standard proteinase K/SDS organic extraction protocol.
  • Plasmid DNA Isolation: Use PLG Heavy to enhance isolation of plasmid DNA using standard alkaline lysis/phenol extraction protocols.
  • Total RNA Isolation: Use PLG Heavy to enhance isolation of total RNA from cells and tissues using a modified guanidinium isothiocyanate/acid phenol method.
  • Genomic DNA Isolation: Use PLG Light to enhance isolation of genomic DNA using standard proteinase K/SDS procedures.
  • DNA Isolation from Agarose Gels: Use PLG Light to enhance isolation of DNA fragments from agarose gels using phenol extraction procedures.
  • Lamba Phage or M13 DNA Isolation: Use PLG Light to enhance isolation of bacteriophage DNA using standard purification protocols.



200 x  2 mL tubes with Phase Lock Gel



200 x  2 mL tubes with Phase Lock Gel

Performance Data



Magnetic bead-based separation of pneumococcal serotypes
Anna York - 2023
The separation of pneumococcal serotypes from a complex polymicrobial mixture may be required for different applications. For instance, a minority strain could be present at a low frequency in a clinical sample, making it difficult to identify and isolate by traditional culture-based methods. We therefore developed an assay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. Using qPCR and colony counting methods, we first show that serotypes (12F, 23F, 3, 14, 19A, and 15A) present at 0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. We demonstrate two applications for this method: extraction of known pneumococcal serotypes from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. This method may have further laboratory or clinical applications when the selection of specific serotypes is required.
Postnatal oogenesis leads to an exceptionally large ovarian reserve in naked mole-rats
Miguel Angel Brieño-Enríquez - 2023
In the long-lived naked mole-rat (NMR), the entire process of oogenesis occurs postnatally. Germ cell numbers increase significantly in NMRs between postnatal days 5 (P5) and P8, and germs cells positive for proliferation markers (Ki-67, pHH3) are present at least until P90. Using pluripotency markers (SOX2 and OCT4) and the primordial germ cell (PGC) marker BLIMP1, we show that PGCs persist up to P90 alongside germ cells in all stages of female differentiation and undergo mitosis both in vivo and in vitro. We identified VASA+ SOX2+ cells at 6 months and at 3-years in subordinate and reproductively activated females. Reproductive activation was associated with proliferation of VASA+ SOX2+ cells. Collectively, our results suggest that highly desynchronized germ cell development and the maintenance of a small population of PGCs that can expand upon reproductive activation are unique strategies that could help to maintain the NMR’s ovarian reserve for its 30-year reproductive lifespan.
Antigenic diversity in malaria parasites is maintained on extrachromosomal DNA
Emily R. Ebel - 2023
Sequence variation among antigenic var genes enables Plasmodium falciparum malaria parasites to evade host immunity. Using long sequence reads from haploid clones from a mutation accumulation experiment, we detect var diversity inconsistent with simple chromosomal inheritance. We discover putatively circular DNA that is strongly enriched for var genes, which exist in multiple alleles per locus separated by recombination and indel events. Extrachromosomal DNA likely contributes to rapid antigenic diversification in P. falciparum. Malaria caused by the parasite Plasmodium falciparum is a leading cause of death and disease in tropical regions of the world1 . Adaptive immunity to malaria is limited, even after repeated infection, by extensive variation in P. falciparum antigenic gene families2–4 . In particular, var genes encode PfEMP1 proteins that are exported to the surface of infected red blood cells, where they mediate pathogenic cytoadherence to host endothelial receptors and elicit variant-specific immunity2 . Each parasite genome contains ~60 var genes distributed among 26 subtelomeric and 9 internal loci. Var genes are named after variation in their antigenic properties5 , driven by extreme amino acid divergence6 36 relative to the rest of the genome. For example, pairs of parasites sampled from the same population share almost no var genes with ≥96% sequence identity7 . However, var genes from unrelated parasites share small blocks of homology6,8 consistent with a history of recombination or gene conversion among alleles. Recent studies have reported frequent var recombination during asexual, mitotic reproduction, which may create millions of new alleles during blood-stage infection9 41 . Our current understanding of var diversification relies primarily on short-read sequencing that may yield only a partial picture of var genetic diversity. The primary hurdle is structural variation among var genes6,10 44 , such as copy number variation, which makes it difficult to accurately align short reads to reference genomes. To achieve a more complete understanding of var diversity and mutational mechanisms, we generated long sequence reads from a mutation accumulation (MA) experiment in P. falciparum11 48 (Fig 1A). Specifically, 31 MA lines (MAL) were independently cloned from an isogenic population of the 3D7 reference strain (the ‘Ancestor’) and propagated for 6- 12 months (~90-180 cell divisions). Each MAL was re-cloned to a single cell every 21±4 days (~10 cell divisions) to minimize selection and allow fixation of de novo mutations. Previous Illumina analysis of the core genome of 31 MAL identified an average of 0.55 SNP and 3.42 small indel mutations per MAL over the course of the MA experiment11 53 . To detect large structural mutations at var loci, we initially used long PacBio reads (>16kb, Fig 1B) to build de novo genome assemblies for the Ancestor (ANC) and three MAL (MAL39, MAL47, MAL53). Each of these high-quality assemblies contained few gaps (0-4) and covered ≥99.5% of reference bases with ≥94.4% identity (ANC, Fig 1C; others, EDF 1A; Supplementary Table 1). Nonetheless, we observed that 27 genomic regions were represented by multiple contigs in at least one assembly (Fig 1C; EDF 1AB), which could represent structural variation. These incompletely-resolved regions were highly enriched for var genes, which comprise 9% of the genome but 69% of unresolved regions (p<0.0001, χ2=228.5). To examine the structural layout of var genes on each assembled contig, we developed a Shiny app that draws BLAST homology between contigs and reference genes. As an example, consider an internal var locus on chr12, which was identified on multiple contigs in 3 of 4 assemblies (Fig 1C, EDF 1A). When we applied our app to this locus in the reference genome, it drew a diagonal line of five sequential genes separated by color and vertical position (Fig 1D).
mRNA ageing shapes the Cap2 methylome in mammalian mRNA
Vladimir Despic - 2023
The mRNA cap structure is a major site of dynamic mRNA methylation. mRNA caps exist in either the Cap1 or Cap2 form, depending on the presence of 2′-O-methylation on the first transcribed nucleotide or both the first and second transcribed nucleotides, respectively1,2. However, the identity of Cap2-containing mRNAs and the function of Cap2 are unclear. Here we describe CLAM-Cap-seq, a method for transcriptome-wide mapping and quantification of Cap2. We find that unlike other epitranscriptomic modifications, Cap2 can occur on all mRNAs. Cap2 is formed through a slow continuous conversion of mRNAs from Cap1 to Cap2 as mRNAs age in the cytosol. As a result, Cap2 is enriched on long-lived mRNAs. Large increases in the abundance of Cap1 leads to activation of RIG-I, especially in conditions in which expression of RIG-I is increased. The methylation of Cap1 to Cap2 markedly reduces the ability of RNAs to bind to and activate RIG-I. The slow methylation rate of Cap2 allows Cap2 to accumulate on host mRNAs, yet ensures that low levels of Cap2 occur on newly expressed viral RNAs. Overall, these results reveal an immunostimulatory role for Cap1, and that Cap2 functions to reduce activation of the innate immune response.
Genomic Characterization of Salmonella enterica serovar Weltevreden Associated with Human Diarrhea
Jianmin Zhang - 2023
Salmonella Weltevreden is an emerging pathogen associated with human diarrhea, and knowledge of the genomics and epidemiology of this serovar is still limited. In this study, we performed whole-genome sequencing of 96 S. Weltevreden isolates recovered from diarrheal patients and 62 isolates from food animals in China between 2006 and 2017. Together, with an additional 199 genome sequences of S. Weltevreden published in NCBI, we performed an analysis on all 357 S. Weltevreden genome sequences. Our results demonstrated that the majority of S. Weltevreden from diarrheal patients from China (97.92%, 94/96) and the other regions in the world (94.97%, 189/199) identified in this study were sequence type (ST) 365. The remaining types were ST3771 (n = 3), ST22 (n = 1), ST155 (n = 1), and ST684 (n = 1). In addition, ST365 was also widely recovered from animals, food, and environmental samples in different regions of the world. Phylogenetic analysis and pulsed-field gel electrophoresis (PFGE) revealed that S. Weltevreden from diarrheal patients was closely related to those recovered from food and environmental specimens. We also showed that S. Weltevreden did not exhibit severe antimicrobial resistance profiles, suggesting administering antibiotics is still effective for controlling the agent. Interestingly, we found that S. Weltevreden strains carried a number of virulence factor genes, and a 100.03-kb IncFII(S) type plasmid was widely distributed in S. Weltevreden strains. Elimination of this plasmid decreased the bacterial capacity to infect both Caco-2 cells and C57BL/6 mice, suggesting the importance of this plasmid for bacterial virulence. Our results contribute to the understanding of the epidemiology and virulence of S. Weltevreden. IMPORTANCE Salmonella Weltevreden is a pathogen associated with human diarrheal diseases found across the globe. However, knowledge of the genomics and epidemiology of this pathogen is still limited. In this study, we found S. Weltevreden sequence type (ST) 365 is commonly recovered from diarrheal patients in China and many other regions of the world, and there is no major difference between the Chinese isolates and the global isolates at the phylogenetic level. We also demonstrated that ST365 was widely recovered from animal, food, and environmental samples collected in different, global regions. Importantly, we discovered an IncFII(S) type plasmid commonly carried by S. Weltevreden strains of human, animal, and food origins, and this plasmid is likely to contribute to the bacterial pathogenesis. These findings enhance our understanding of the emergence of S. Weltevreden involved in diarrheal outbreaks and the global spread of S. Weltevreden strains.
Genomic relationships among diploid and polyploid species of the genus Ludwigia L. section Jussiaea using a combination of cytogenetic, morphological, and crossing investigations
D. Barloy - 2023
The genus Ludwigia L. section Jussiaea is composed of a polyploid species complex with 2x, 4x, 6x and 10x ploidy levels, suggesting possible hybrid origins. The aim of the present study is to understand the genomic relationships among diploid and polyploid species in the section Jussiaea. Morphological and cytogenetic observations, controlled crosses, genomic in situ hybridization (GISH), and flow cytometry were used to characterize species, ploidy levels, ploidy patterns, and genomic composition across taxa. Genome sizes obtained were in agreement with the diploid, tetraploid, hexaploid, and decaploid ploidy levels. Results of GISH showed that progenitors of Ludwigia stolonifera (4x) were Ludwigia peploides subsp. montevidensis (2x) and Ludwigia helminthorrhiza (2x), which also participated for one part (2x) to the Ludwigia ascendens genome (4x). Ludwigia grandiflora subsp. hexapetala (10x) resulted from the hybridization between L. stolonifera (4x) and Ludwigia grandiflora subsp. grandiflora (6x). One progenitor of L. grandiflora subsp. grandiflora was identified as L. peploides (2x). Our results suggest the existence of several processes of hybridization, leading to polyploidy, and possibly allopolyploidy, in the section Jussiaea due to the diversity of ploidy levels. The success of GISH opens up the potential for future studies to identify other missing progenitors in Ludwigia L. as well as other taxa.
Diurnal transcriptome landscape of a multi-tissue response to time-restricted feeding in mammals
Shaunak Deota - 2023
Time-restricted feeding (TRF) is an emerging behavioral nutrition intervention that involves a daily cycle of feeding and fasting. In both animals and humans, TRF has pleiotropic health benefits that arise from multiple organ systems, yet the molecular basis of TRF-mediated benefits is not well understood. Here, we subjected mice to isocaloric ad libitum feeding (ALF) or TRF of a western diet and examined gene expression changes in samples taken from 22 organs and brain regions collected every 2 h over a 24-h period. We discovered that TRF profoundly impacts gene expression. Nearly 80% of all genes show differential expression or rhythmicity under TRF in at least one tissue. Functional annotation of these changes revealed tissue- and pathway-specific impacts of TRF. These findings and resources provide a critical foundation for future mechanistic studies and will help to guide human time-restricted eating (TRE) interventions to treat various disease conditions with or without pharmacotherapies.
A reference genome assembly of the declining tricolored blackbird, Agelaius tricolor
Kimberly M. Ballare - 2022
The tricolored blackbird, Agelaius tricolor, is a gregarious species that forms enormous breeding and foraging colonies in wetland and agricultural habitats, primarily in California, USA. Once extremely abundant, species numbers have declined dramatically in the past century, largely due to losses of breeding and foraging habitats. Tricolored blackbirds are currently listed as Endangered by the IUCN, and Threatened under the California Endangered Species Act. Increased genetic information is needed to detail the evolutionary consequences of a species-wide bottleneck and inform conservation management. Here, we present a contiguous tricolored blackbird reference genome, assembled with PacBio HiFi long reads and Dovetail Omni-C data to generate a scaffold-level assembly containing multiple chromosome-length scaffolds. This genome adds a valuable resource for important evolutionary and conservation research on tricolored blackbirds and related species.
Optimized protocol to generate genome-wide inactivated Cas9-expressing murine T cells
Marguerite Laprie-Sentenac - 2023
In vivo genome-wide CRISPR screens in primary T cells allow the systematic and unbiased identification of non-redundant regulatory mechanisms shaping immune responses. Here, we present an optimized protocol for efficient generation of a pool of genome-wide inactivated Cas9-expressing T cells using a retroviral library of sgRNA. We detail the process of large-scale viral production and library integration in activated murine T cells as well as the two-step PCR approach for sgRNA recovery and abundance evaluation.
Chromosome arm length, and a species-specific determinant, define chromosome arm width
Yasutaka Kakui - 2022
Mitotic chromosomes in different organisms adopt various dimensions. What defines these dimensions is scarcely understood. Here, we compare mitotic chromosomes in budding and fission yeasts harboring similarly sized genomes distributed among 16 or 3 chromosomes, respectively. Hi-C analyses and superresolution microscopy reveal that budding yeast chromosomes are characterized by shorter-ranging mitotic chromatin contacts and are thinner compared with the thicker fission yeast chromosomes that contain longer-ranging mitotic contacts. These distinctions persist even after budding yeast chromosomes are fused to form three fission-yeast-length entities, revealing a species-specific organizing principle. Species-specific widths correlate with the known binding site intervals of the chromosomal condensin complex. Unexpectedly, within each species, we find that longer chromosome arms are always thicker and harbor longer-ranging contacts, a trend that we also observe with human chromosomes. Arm length as a chromosome width determinant informs mitotic chromosome formation models.
Branchpoint translocation by fork remodelers as a general mechanism of R-loop removal
Charlotte Hodson - 2022
Co-transcriptional R loops arise from stalling of RNA polymerase, leading to the formation of stable DNA:RNA hybrids. Unresolved R loops promote genome instability but are counteracted by helicases and nucleases. Here, we show that branchpoint translocases are a third class of R-loop-displacing enzyme in vitro. In cells, deficiency in the Fanconi-anemia-associated branchpoint translocase FANCM causes R-loop accumulation, particularly after treatment with DNA:RNA-hybrid-stabilizing agents. This correlates with FANCM localization at R-loop-prone regions of the genome. Moreover, other branchpoint translocases associated with human disease, such as SMARCAL1 and ZRANB3, and those from lower organisms can also remove R loops in vitro. Branchpoint translocases are more potent than helicases in resolving R loops, indicating their evolutionary important role in R-loop suppression. In human cells, FANCM, SMARCAL1, and ZRANB3 depletion causes additive effects on R-loop accumulation and DNA damage. Our work reveals a mechanistic basis for R-loop displacement that is linked to genome stability.
A draft reference genome of the Vernal Pool Fairy Shrimp, Branchinecta lynchi
Shannon Rose Kieran Blair - 2022
We present the reference genome of the Vernal Pool Fairy Shrimp Branchinecta lynchi. This branchiopod crustacean is endemic to California’s freshwater ephemeral ponds. It faces enormous habitat loss and fragmentation as urbanization and agriculture have fundamentally changed the vernal pool landscape over the past three centuries. The assembled genome consists of 22 chromosome-length scaffolds that account for 96.85% of the total sequence. 195 unscaffolded contigs comprise the rest of the genome’s 575.6 Mb length. The genome is substantially complete with a BUSCO score of 90.0%. There is no immediately-identifiable sex chromosome, typical for this class of organism. This new resource will permit researchers to better understand the adaptive capacity of this imperiled species, as well as answer lingering questions about anostracan physiology, sex determination and development.
MiR-302d inhibits TGFB-induced EMT and promotes MET in primary human RPE cells
Xiaonan Hu - 2022
Purpose Transforming growth factor-beta (TGFB)-mediated epithelial-mesenchymal transition (EMT) plays a crucial role in the pathogenesis of retinal fibrosis, which is one of the leading causes of impaired vision. Current approaches to treating retinal fibrosis focus, among other things, on inhibiting the TGFB signaling pathway. Transient expression of microRNAs (miRNAs) is one way to inhibit the TGFB pathway post-transcriptionally. Our previous study identified the miRNA miR-302d as a regulator of multiple TGFB-related genes in ARPE-19 cells. To further explore its effect on primary cells, the effect of miR-302d on TGFB-induced EMT in primary human retinal pigment epithelium (hRPE) was investigated in vitro. Methods hRPE cells were extracted from patients receiving enucleation. Transfection of hRPE cells with miR-302d was performed before or after TGFB1 stimulation. Live-cell imaging, immunocytochemistry staining, Western blot, and ELISA assays were utilized to identify the alterations of cellular morphology and EMT-related factors expressions in hRPE cells. Results hRPE cells underwent EMT by TGFB1 exposure. The transfection of miR-302d inhibited the transition with decreased production of mesenchymal markers and increased epithelial factors. Meanwhile, the phosphorylation of SMAD2 activated by TGFB1 was suppressed. Moreover, miR-302d expression promoted TGFB1-induced fibroblast-like hRPE cells to revert towards an epithelial stage. As confirmed by ELISA, miR-302d reduced TGFB receptor 2 (TGFBR2) and vascular endothelial growth factor A (VEGFA) levels 48 hours after transfection. Conclusions The protective effect of miR-302d might be a promising approach for ameliorating retinal fibrosis and neovascularization. MiR-302d suppresses TGFB-induced EMT in hRPE cells via downregulation of TGFBR2, even reversing the process. Furthermore, miR-302d reduces the constitutive secretion of VEGFA from hRPE cells.
Stress deficits in reward behaviour are associated with and replicated by dysregulated amygdala-nucleus accumbens pathway function
Lorraine Madur - 2022
Reduced reward interest/learning and reward-to-effort valuation are distinct, common symptoms in neuropsychiatric disorders for which chronic stress is a major aetiological factor. Glutamate neurons in basal amygdala (BA) project to various regions including nucleus accumbens (NAc). The BA-NAc neural pathway is activated by reward and aversion, with many neurons being monovalent. In adult male mice, chronic social stress (CSS) led to both reduced discriminative reward learning (DRL) associated with decreased BA-NAc activity, and reduced reward-to-effort valuation (REV) associated, in contrast, with increased BA-NAc activity. Chronic tetanus toxin BA-NAc inhibition replicated the CSS-DRL effect and caused a mild REV reduction, whilst chronic DREADDs BA-NAc activation replicated the CSS effect on REV without affecting DRL. This study provides novel evidence that stress disruption of reward processing involves the BA-NAc neural pathway; the bi-directional effects implicate opposite activity changes in reward (learning) neurons and aversion (effort) neurons in the BA-NAc pathway following chronic stress.
Roberto Arsie - 2022
The intracellular distribution of a specific protein is essential for its function. Proper protein localization is therefore an essential biological process, regulated at many levels and central for eukaryotic compartments organization. Consequently, several methods have been established to globally profile proteomes at different subcellular regions. Many of these approaches rely on the different biochemical features of eukaryotic compartments to separate them and analyse their relative protein composition in parallel. In this study we assessed the protein distribution of HEK293 cells using BirA* proximity labelling activity and restricting its localization at cellular regions difficult to purified with traditional methods: the cytosol-facing sides of the endoplasmic reticulum, mitochondria, and plasma membranes. We obtained high-quality datasets for each of the compartment examined, both recapitulating the protein distribution known from literature and discovering new candidates at the investigated subcellular regions. Interestingly, we also identified proteins localizing at more than one subcellular compartment, providing useful insights on the protein composition shared by different organelles. Proteins with different biological activity were captured by our proximity labelling approach, such as metabolic enzymes and integral receptors. RNA-binding proteins (RBPs) were also found in our datasets, but the direct interactions between them and RNA molecules could not be validated using only BirA* biotinylation. To obtain an activity-based RBP profiling and assess the composition of protein binding to RNA in different cellular compartments, we developed the f-XRNAX protocol. It consists of UV-crosslinking, cellular fractionation and organic-phase enrichment of RBPs coupled with mass spectrometry. We recovered background-corrected proteomes for nuclei, cytoplasm and membranes of HEK293 cells. Protein domain analysis of peptides crosslinked to ribonucleotides showed an enrichment in known RNA-binding domains. We also determined the protein domains where the RNA-protein contacts occurred in vivo using enriched peptides profiles and reported some cases of differential domain usage of the same protein in two different compartments. Surprisingly, many non-canonical RBPs were identified in the membrane fraction, and their peptide profiles were enriched in regions with high density of intrinsically disordered regions, indicating a possibly weak interaction with RNA mediated by these non-structural motives. Lastly, we provided evidence of the differential binding to RNA of the same protein in different HEK293 compartments. In the second part of this thesis, we focused on the determination and quantification of newly transcribed RNAs at the single-cell level. The kinetics of RNA transcription, processing and degradation were until recently not measurable at the single-cell level. Thus, we have developed a novel approach (called SLAM-Drop-seq ) by adapting the published SLAM-seq method to single cells. We used SLAMDrop-seq to estimate time-dependent RNA kinetics rates of transcription and turnover for hundreds ZUSAMMENFASSUNG 3 of oscillating transcripts during the cell cycle of HEK293 cells. We found that genes regulate their expression with different strategies and have specific modes to fine-tune their kinetic rates along the cell cycle.
The reference genome of the Vernal Pool Tadpole Shrimp, Lepidurus packardi
Shannon Rose Kieran Blair - 2022
In this paper we report on the scaffold-level assembled genome for the federally endangered, California endemic crustacean Lepidurus packardi (the Vernal Pool Tadpole Shrimp). Lepidurus packardi is a key food source for other conserved California species including the California Tiger Salamander Ambystoma californiense. It faces significant habitat loss and fragmentation as vernal pools are threatened by urbanization, agricultural conversion and climate change. This resource represents the first scaffold-level genome of any Lepidurus species. The assembled genome spans 108.6 Mbps, with six chromosome-length scaffolds comprising 71% of total genomic length and 444 total contigs. The BUSCO score for this genome is 97.3%, suggesting a high level of completeness. We produced a predicted gene set for this species trained on the Daphnia magna set of genes and predicted 17,650 genes. These tools can aid researchers in understanding the evolution and adaptive potential of alternative reproductive modes within this species.
Plasma contains ultrashort single-stranded DNA in addition to nucleosomal cell-free DNA
Jordan Cheng - 2022
Plasma cell-free DNA is being widely explored as a biomarker for clinical screening. Currently, methods are optimized for the extraction and detection of double-stranded mononucleosomal cell-free DNA of ∼160bp in length. We introduce uscfDNA-seq, a single-stranded cell-free DNA next-generation sequencing pipeline, which bypasses previous limitations to reveal a population of ultrashort single-stranded cell-free DNA in human plasma. This species has a modal size of 50nt and is distinctly separate from mononucleosomal cell-free DNA. Treatment with single-stranded and double-stranded specific nucleases suggests that ultrashort cell-free DNA is primarily single-stranded. It is distributed evenly across chromosomes and has a similar distribution profile over functional elements as the genome, albeit with an enrichment over promoters, exons, and introns, which may be suggestive of a terminal state of genome degradation. The examination of this cfDNA species could reveal new features of cell death pathways or it can be used for cell-free DNA biomarker discovery.
Neuron-specific translational control shift ensures proteostatic resilience during ER stress
Kimberly Wolzak - 2022
Proteostasis is essential for cellular survival and particularly important for highly specialised post-mitotic cells such as neurons. Transient reduction in protein synthesis by protein kinase R-like endoplasmic reticulum (ER) kinase (PERK)-mediated phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α) is a major proteostatic survival response during ER stress. Paradoxically, neurons are remarkably tolerant to PERK dysfunction, which suggests the existence of cell type-specific mechanisms that secure proteostatic stress resilience. Here, we demonstrate that PERK-deficient neurons, unlike other cell types, fully retain the capacity to control translation during ER stress. We observe rescaling of the ATF4 response, while the reduction in protein synthesis is fully retained. We identify two molecular pathways that jointly drive translational control in PERK-deficient neurons. Haem-regulated inhibitor (HRI) mediates p-eIF2α and the ATF4 response and is complemented by the tRNA cleaving RNase angiogenin (ANG) to reduce protein synthesis. Overall, our study elucidates an intricate back-up mechanism to ascertain translational control during ER stress in neurons that provides a mechanistic explanation for the thus far unresolved observation of neuronal resilience to proteostatic stress.
Characterization of the detrimental effects of type IV glandular trichomes on the aphid Macrosiphum euphorbiae in tomato
Lidia Blanco-Sánchez, - 2021
BACKGROUND: Glandular trichomes are essential in plants' defence against pests however, the mechanisms of action are not completely understood. While there is considerable evidence of feeding and movement impairment by trichomes, the effect on other traits is less clear. We combined laboratory and greenhouse experiments with molecular analysis to understand how glandular trichomes affect the behavior, population growth, and the expression of biomarkers involved in detoxification, primary metabolism, and developmental pathways of the aphid Macrosiphum euphorbiae. We used two isogenic tomato lines that differ in the presence of type IV glandular trichomes and production of acylsucroses; i.e.,Solanum lycopersicum cv. ‘Moneymaker’ and an introgressed line from Solanum pimpinellifolium (with trichomes type IV). RESULTS: Type IV glandular trichomes affected host selection and aphid proliferation with aphids avoiding, and showing impaired multiplication on the genotype with trichomes. The exposure to type IV glandular trichomes resulted in the overexpression of detoxication markers (i.e., Hsp70, Hsp17, Hsp10); the repression of the energetic metabolism (GAPDH), and the activation of the ecdysone pathway; all these, underlying the key adaptations and metabolic trade-offs in aphids exposed to glandular trichomes. CONCLUSION: Our results demonstrate the detrimental effect of glandular trichomes (type IV) on the aphid and put forward their mode of action. Given the prevalence of glandular trichomes in wild and cultivated Solanaceae; and of the investigated molecular biomarkers in insects in general, our results provide relevant mechanisms to understand the effect of trichomes not only on herbivorous insects but also on other trophic levels.
Chromatin immunoprecipitation oftranscription factors and histone modificationsin Comma-Dbmammary epithelial cellsChromatin immunoprecipitation (ChIP) is used to study interactions between proteins and DNA.Nuclear lysates are prepared, and chromatin is fr
Holly Holliday - 2021
Chromatin immunoprecipitation (ChIP)is used to study interactions between proteins and DNA. Nuclear lysates are prepared, and chromatin is fragmented by sonication. Antibodies are used to purify a protein of interest (e.g., a transcription factor or histone mark) along with any bound DNA. The genomic binding sites can then be mapped by sequencing the bound DNA (ChIP-seq) or by qPCR if binding sites are already known. ChIP requires optimization for each cell type, and success is highly antibody dependent. This protocol can be adapted to other cell lines with careful optimization. For complete details on the use and execution of this protocol, please refer to Holliday et al. (2021).
Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection
Sara F. Martins Gomes - 2019
Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.
An unconventional RNA-based thermosensor within the 5’ UTR of Staphylococcus aureus cidA
Hebaallaha Hussein - 2019
Staphylococcus aureus is a Gram-positive bacterial pathogen of global concern and a leading cause of bacterial infections worldwide. Asymptomatic carriage of S. aureus on the skin and in the anterior nares is common and recognized as a predisposing factor to invasive infection. Transition of S. aureus from the carriage state to that of invasive infection is often accompanied by a temperature upshift from approximately 33°C to 37°C. Such a temperature shift is known in other pathogens to influence gene expression, often resulting in increased production of factors that promote survival or virulence within the host. One mechanism by which bacteria modulate gene expression in response to temperature is by the regulatory activity of RNA-based thermosensors, cis-acting riboregulators that control translation efficiency. This study was designed to identify and characterize RNA-based thermosensors in S. aureus. Initially predicted by in silico analyses of the S. aureus USA300 genome, reporter-based gene expression analyses and site-specific mutagenesis were performed to demonstrate the presence of a functional thermosensor within the 5’ UTR of cidA, a gene implicated in biofilm formation and survival of the pathogen. The nucleic sequence composing the identified thermosensor are sufficient to confer temperature-dependent post-transcriptional regulation, and activity is predictably altered by the introduction of site-specific mutations designed to stabilize or destabilize the structure within the identified thermosensor. The identified regulator is functional in both the native bacterial host S. aureus and in the distally related species Escherichia coli, suggesting that its regulatory activity is independent of host-specific factors. Interestingly, unlike the majority of bacterial RNA-based thermosensors characterized to date, the cidA thermosensor facilitates increased target gene expression at lower temperatures. In addition to the characterization of the first RNA-based thermosensor in the significant pathogen S. aureus, it highlights the diversity of function within this important class of ribo-regulators.
Symbiotic bacteria associated with ascidian vanadium accumulation identified by 16S rRNA amplicon sequencing
Tatsuya Ueki - 2019
Ascidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (≤ 350 mM). Vanadium levels are strictly regulated and vary among ascidian species; thus, they represent well-suited models for studies on vanadium accumulation. No comprehensive study on metal accumulation and reduction in marine organisms in relation to their symbiotic bacterial communities has been published. Therefore, we performed comparative 16S rRNA amplicon sequence analyses on samples from three tissues (branchial sac, intestine, and intestinal lumen) involved in vanadium absorption, isolated from two vanadium-rich (Ascidia ahodori and Ascidia sydneiensis samea) and one vanadium-poor species (Styela plicata). For each sample, the abundance of every bacteria and an abundance value normalized to their abundance in seawater were calculated and compared. Two bacterial genera, Pseudomonas and Ralstonia, were extremely abundant in the branchial sacs of vanadium-rich ascidians. Two bacterial genera, Treponema and Borrelia, were abundant and enriched in the intestinal content of vanadium-rich ascidians. The results suggest that specific selective forces maintain the bacterial population in the three ascidian tissues examined, which contribute to successful vanadium accumulation. This study furthers the understanding of the relationship between bacterial communities and metal accumulation in marine life.
HCN4 knockdown in dorsal hippocampus promotes anxiety‐like behavior in mice
Anne Gunther - 2019
Hyperpolarization‐activated and cyclic nucleotide‐gated (HCN) channels mediate the Ih current in the murine hippocampus. Disruption of the Ih current by knockout of HCN1, HCN2 or tetratricopeptide repeat‐containing Rab8b‐interacting protein has been shown to affect physiological processes such as synaptic integration and maintenance of resting membrane potentials as well as several behaviors in mice, including depressive‐like and anxiety‐like behaviors. However, the potential involvement of the HCN4 isoform in these processes is unknown. Here, we assessed the contribution of the HCN4 isoform to neuronal processing and hippocampus‐based behaviors in mice. We show that HCN4 is expressed in various regions of the hippocampus, with distinct expression patterns that partially overlapped with other HCN isoforms. For behavioral analysis, we specifically modulated HCN4 expression by injecting recombinant adeno‐associated viral (rAAV) vectors mediating expression of short hairpin RNA against hcn4 (shHcn4) into the dorsal hippocampus of mice. HCN4 knockdown produced no effect on contextual fear conditioning or spatial memory. However, a pronounced anxiogenic effect was evident in mice treated with shHcn4 compared to control littermates. Our findings suggest that HCN4 specifically contributes to anxiety‐like behaviors in mice.
Expression of ELF1, a lymphoid ETS domain‐containing transcription factor, is recurrently lost in classical Hodgkin lymphoma
Julia Paczkowska - 2019
Loss of B cell-specific transcription factors (TFs) and the resulting loss ofB-cell phenotype of Hodgkin and Reed-Sternberg (HRS) cells is a hallmarkof classical Hodgkin lymphoma (cHL). Here we have analysed two mem-bers of ETS domain containing TFs, ELF1 and ELF2,regarding (epi)ge-nomic changes as well as gene and protein expression. We observedabsence or lower levels of ELF1 protein in HRS cells of 31/35 (89%) casescompared to the bystander cells and significant (P<001) downregulationof the gene on mRNA as well as protein level in cHL compared to non-cHL cell lines. However, no recurrent loss of ELF2 protein was observed.Moreover,ELF1was targeted by heterozygous deletions combined withhypermethylation of the remaining allele(s) in 4/7 (57%) cell lines. Indeed,DNA hypermethylation (range 95–99%, mean 98%) detected in the vicinityof theELF1transcription start site was found in all 7/7 (100%) cHL celllines. Similarly, 5/18 (28%) analysed primary biopsies carried heterozygousdeletions of the gene. We demonstrate that expression of ELF1 is impairedin cHL through genetic and epigenetic alterations, and thus, it may repre-sent an additional member of a TF network whose downregulation con-tributes to the loss of B-cell phenotype of HRS cells.
Feiran Zhang - 2018
N6-methyladenosine (m6A) is the most prevalent internal modification of mammalian messenger RNAs (mRNAs) and long non-coding RNAs. The biological functions of this reversible RNA modification can be interpreted by cytoplasmic and nuclear “m6A reader” proteins to fine-tune gene expression, such as mRNA degradation and translation initiation. Here we profiled transcriptome-wide m6A sites in adult mouse cerebral cortex, underscoring that m6A is a widespread epitranscriptomic modification in brain. Interestingly, the mRNA targets of fragile X mental retardation protein (FMRP), a selective RNA-binding protein, are enriched for m6A marks. Loss of functional FMRP leads to Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Transcriptome-wide gene expression profiling identified 2,035 genes differentially expressed in the absence of FMRP in cortex, and 92.5% of 174 downregulated FMRP targets are marked by m6A. Biochemical analyses indicate that FMRP binds to the m6A sites of its mRNA targets and interacts with m6A reader YTHDF2 in an RNA-independent manner. FMRP maintains the stability of its mRNA targets while YTHDF2 promotes the degradation of these mRNAs. These data together suggest that FMRP regulates the stability of its m6A-marked mRNA targets through YTHDF2, which could potentially contribute to the molecular pathogenesis of FXS.
Transcriptome of Xenopus andrei, an octoploid frog, during embryonic development
Mark E. Pownall - 2018
Although polyploidy occurs throughout the fish and amphibian lineages, the Xenopus genus exhibits a high incidence of polyploidy, with 25 out of the 26 known species being polyploid. However, transcriptomic information is currently available for only one of these species, the tetraploid Xenopus laevis. Xenopus andrei, an octoploid species within the Xenopus genus, offers an opportunity for assessing a novel polyploid transcriptome during vertebrate development. RNA-Seq data was generated at nine different developmental stages ranging from unfertilized eggs through swimming tadpole stages and raw FASTQ files were deposited in the NCBI SRA database (accession number SRP134281). Additionally, RNA-seq data from all nine stages were pooled to create a de novo assembly of the transcriptome using Trinity and has been deposited in the NCBI GEO database (accession number GSE111639). To our knowledge, this represents the first published assembly of an octoploid vertebrate transcriptome. In total, 849Mb were assembled, which led to the identification of 1,650,048 transcripts in the assembly with a contig N50 of 630 bases. This RNA-Seq and transcriptome data will be valuable for comparing polyploid transcriptomes across Xenopus species, as well as understanding evolutionary implications of whole-genome duplication and polyploidy in vertebrates.
Maternal obesity during lactation may protect offspring from high fat diet-induced metabolic dysfunction
Jenifer Monks - 2018
Background/Objectives The current obesity epidemic has spurred exploration of the developmental origin of adult heath and disease. A mother’s dietary choices and health can affect both the early wellbeing and lifelong disease-risk of the offspring. Subjects/Methods To determine if changes in the mother’s diet and adiposity have long-term effects on the baby’s metabolism, independently from a prenatal insult, we utilized a mouse model of diet-induced-obesity and cross-fostering. All pups were born to lean dams fed a low fat diet but were fostered onto lean or obese dams fed a high fat diet. This study design allowed us to discern the effects of a poor diet from those of mother’s adiposity and metabolism. The weaned offspring were placed on a high fat diet to test their metabolic function. Results In this feeding challenge, all male (but not female) offspring developed metabolic dysfunction. We saw increased weight gain in the pups nursed on an obesity-resistant dam fed a high fat diet, and increased pathogenesis including liver steatosis and adipose tissue inflammation, when compared to pups nursed on either obesity-prone dams on a high fat diet or lean dams on a low fat diet. Conclusion Exposure to maternal over-nutrition, through the milk, is sufficient to shape offspring health outcomes in a sex- and organ-specific manner, and milk from a mother who is obesity-prone may partially protect the offspring from the insult of a poor diet.
The BPA-substitute bisphenol S alters the transcription of genes related to endocrine, stress response and biotransformation pathways in the aquatic midge Chironomus riparius (Diptera, Chironomidae)
Óscar Herrero - 2018
Bisphenol S (BPS) is an industrial alternative to the endocrine disruptor bisphenol A (BPA), and can be found in many products labeled “BPA-free”. Its use has grown in recent years, and presently it is considered a ubiquitous emerging pollutant. To date there is a lack of information on the effects of BPS on invertebrates, although they represent more than 95% of known species in the animal kingdom and are crucial for the structure and proper function of ecosystems. In this study, real-time RT-PCR was used to determine the early detrimental effects of BPS on the transcriptional rate of genes in the model species Chironomus riparius, specifically those related to the ecdysone pathway (EcR, ERR, E74, Vtg, cyp18a1) crucial for insect development and metamorphosis, stress and biotransformation mechanisms (hsp70, hsp40, cyp4g, GPx, GSTd3) that regulate adaptive responses and determine survival, and ribosome biogenesis (its2, rpL4, rpL13) which is essential for protein synthesis and homeostasis. While 24-hour exposure to 0.5, 5, 50, and 500 μg/L BPS had no effect on larval survival, almost all the studied genes were upregulated following a non-monotonic dose-response curve. Genes with the greatest increases in transcriptional activity (fold change relative to control) were EcR (3.8), ERR (2), E74 (2.4), cyp18a1 (2.5), hsp70 (1.7), hsp40 (2.5), cyp4g (6.4), GPx (1.8), and GST (2.1), while others including Vtg, GAPDH, and selected ribosomal genes remained stable. We also measured the transcriptional activity of these genes 24 hours after BPS withdrawal and a general downregulation compared to controls was observed, though not significant in most cases. Our findings showed that BPS exposure altered the transcriptional profile of these genes, which may have consequences for the hormone system and several metabolic pathways. Although further research is needed to elucidate its mode of action, these results raise new concerns about the safety of BPA alternatives.
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