Genetic Assays Research Articles

Subfamily C7 Raf-like kinases MRK1, RAF26, and RAF39 regulate immune homeostasis and stomatal opening in Arabidopsis thaliana.

The calcium-dependent protein kinase CPK28 regulates several stress pathways in multiple plant species. Here, we aimed to discover CPK28-associated proteins in Arabidopsis thaliana. We used affinity-based proteomics and identified several potential CPK28 binding partners, including the C7 Raf-like kinases MRK1, RAF26, and RAF39. We used biochemistry, genetics, and physiological assays to gain insight into their function. We define redundant roles for these kinases in stomatal opening, immune-triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. We report that CPK28 associates with and trans-phosphorylates RAF26 and RAF39, and that MRK1, RAF26, and RAF39 are active kinases that localize to endomembranes. Although Raf-like kinases share some features with mitogen-activated protein kinase kinase kinases (MKKKs), we found that MRK1, RAF26, and RAF39 are unable to trans-phosphorylate any of the 10 Arabidopsis mitogen-activated protein kinase kinases (MKKs). Overall, our study suggests that C7 Raf-like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal opening and immunity, and possess substrate specificities distinct from canonical MKKKs.

GLABRA3-mediated trichome branching requires transcriptional repression of MICROTUBULE-DESTABILIZING PROTEIN25.

Microtubules play pivotal roles in establishing trichome branching patterns, which is a model system for studying cell-shape control in Arabidopsis (Arabidopsis thaliana). However, the signaling pathway that regulates microtubule reorganization during trichome branching remains poorly understood. In this study, we report that MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) is involved in GLABRA3 (GL3)-mediated trichome branching by regulating microtubule stability. Loss of MDP25 function led to excessive trichome branching, and this phenotype in mdp25 could not be rescued by the MDP25 K7A or MDP25 K18A mutated variants. Pharmacological treatment and live-cell imaging revealed increased microtubule stability in the mdp25 mutant. Furthermore, the microtubule collar observed during trichome branching remained more intact in mdp25 compared to the WT under oryzalin treatment. Results of genetic assays further demonstrated that knocking out MDP25 rescued the reduced branching phenotype of gl3 trichomes. In gl3 trichomes, normal microtubule organization was disrupted, and microtubule stability was significantly compromised. Moreover, GL3 physically bound to the MDP25 promoter, thereby inhibiting its expression. Overexpression of GL3 negated the effects of PMDP25-driven MDP25 or its mutant proteins on trichome branching and microtubules in the mdp25 background. Overall, our study uncovers a mechanism by which GL3 inhibits MDP25 transcription, thereby influencing microtubule stability and regulating trichome branching. This mechanism provides a connection between early regulatory components and microtubules during trichome development.

Genetic Characterization of the Glucose-PTS in Streptococcus sanguinis, examining competitive fitness.

Ecological shifts in dental microbiome from homeostasis to dysbiosis have been hypothesized to underlie several oral diseases including dental caries. Streptococcus sanguinis, a commensal bacterium, is pivotal in maintaining oral health through its metabolic activities, including the production of ammonia and hydrogen peroxide (H2O2). This study aims to elucidate the genetic underpinnings of the glucose::phosphotransferase system (PTS) in the physiology and competitive fitness of S. sanguinis within the oral microbiome, by examining single nucleotide polymorphisms (SNPs) in the EIIABMan (manL) and HPr of PTS in S. sanguinis strain SK36. Employing genetic engineering, bioinformatic analysis, growth and metabolic assays, recreating and characterizing several unique ManL SNPs in SK36 that were identified among clinical strains of S. sanguinis. Mutations in ManL and HPr correlated with altered growth dynamics, H2O2 production, acid production, and pH homeostasis in a manner uncoupled from canonical regulators such as CcpA and Rex. These genetic variations likely contributed to S. sanguinis's competitiveness, although their ecological impact remains to be further elucidated. This research is part of the ongoing effort to understand the genetic mechanisms that contribute to microbial ecology in relation to health, knowledge from which may allow for enhanced diagnostics, pro-health formulations, and other microbial interventions.

Potent Anti-Influenza Synergistic Activity of Theobromine and Arainosine.

Influenza represents one of the biggest health threats facing humanity. Seasonal epidemics can transition to global pandemics, with cross-species infection presenting a continuous challenge. Although vaccines and several anti-viral options are available, constant genetic drifts and shifts vitiate any of the aforementioned prevention and treatment options. Therefore, we describe an approach targeted at the virus's channel to derive new anti-viral options. Specifically, Influenza A's M2 protein is a well-characterized channel targeted for a long time by aminoadamantane blockers. However, widespread mutations in the protein render the drugs ineffective. Consequently, we started by screening a repurposed drug library against aminoadamantane-sensitive and resistant M2 channels using bacteria-based genetic assays. Subsequent in cellulo testing and structure-activity relationship studies yielded a combination of Theobromine and Arainosine, which exhibits stark anti-viral activity by inhibiting the virus's channel. The drug duo was potent against H1N1 pandemic swine flu, H5N1 pandemic avian flu, aminoadamantane-resistant and sensitive strains alike, exhibiting activity that surpassed Oseltamivir, the leading anti-flu drug on the market. When this drug duo was tested in an animal model, it once more outperformed Oseltamivir, considerably reducing disease symptoms and viral RNA progeny. In conclusion, the outcome of this study represents a new potential treatment option for influenza alongside an approach that is sufficiently general and readily applicable to other viral targets.

Understanding the genetic landscape of flowering time variation in Brassica juncea (L.) Czern. and its diploid progenitors: unravelling the role of selection and cytoplasmic backgrounds

Context Brassica juncea germplasm exhibits significant variations in flowering timing and vernalisation requirements. However, knowledge gaps exist with respect to variations in expression and the divergent evolution of flowering genes in B. juncea subgenomes. Aims This study aims to examine the role of flowering genes in defining trait variation and to identify indications of directional selection on these genes. Methods Employing a combination of genome-wide association studies, functional genomics and population genetic assays, we explored the genetic architecture underlying flowering time variation within expansive germplasm collections of this allopolyploid and its progenitor species. Key results Genome-wide association studies aided in predicting 17 and 34 candidate genes in B. rapa and B. juncea, respectively. Seven of these (FT, FLC, BAG4, ELF4-L2, EFM, SEP4, and LSH6) were predicted in both B. juncea and B. rapa. Some genes, GA20OX3, NF-YA1, PI, MMP, RPS10B, CRY2, AGL72, LFY, TOC1, ELF5, EFM, FLC and TFL1 exhibited directional selection as inferred from negative Tajima’s D and Fu’s Fs statistics. Conclusions Common predicted genes are known influencers of flowering time and phenological changes between species as well as across zones of adaptation. An analysis of gene expression patterns indicates that the gene expression bias in resynthesised B. juncea could be influenced by the cytoplasmic background. Most expression variants are found in B genome copies. Some genes lacked expression variation in their diploid progenitors, whereas these genes exhibit expression variation in polyploid species. Implications This study highlights that integrating genome-wide association studies with molecular signals of natural selection can effectively contribute to our understanding of the ecological genetics of adaptive evolution.

Methods used in sea turtle genetic and molecular studies

Sea turtles have a tropical and subtropical distribution and can be found in nearly all seas and oceans. They have been the subject of considerable genetic research. However, it does not yet appear that the molecular techniques used for these genetic studies follow a consensus or universal set of tools to be followed for subsequent studies. This is not desirable since it may preclude data exchange and use among studies. Thus, the aim of this review was to survey the main genetic and molecular methods used for sea turtle research worldwide. To achieve this goal, a total of 95 scientific papers were compiled from online databases. We considered articles that used molecular tools for genetic analysis and provided detailed locality data. The following aspects were assessed: species studied, local of sample collection, type of tissue used for molecular studies and type of genetic material used. The seven known sea turtle species have different distribution patterns, with some overlapping of occurrence. Chelonia mydas has been studied genetically along the coasts of all continents. Skin is the most common type of tissue used for molecular analyses. From genetic studies on sea turtles, it is possible to verify the occurrence of hybridism. This phenomenon is relevant to the conservation of the species and was reported in six articles on this topic. Therefore, it is considered that genetic and molecular assays in sea turtles are important tools for biological evaluation and protection. The aim was to survey the main methods used in genetic and molecular research on marine turtles. Keywords: DNA, Chelonian, Testudines, marine turtles

Genomic Instability in the Lymphocytes of Dogs with Squamous Cell Carcinoma.

Genome instability is a characteristic trait of tumours and includes changes in DNA and in chromosomes. The aim of the study was to identify chromosome damage using the sister chromatid exchange assay and DNA fragmentation by the comet assay in dogs with cancer, as well as to determine the suitability of these techniques for the assessment of chromatin stability in healthy and sick dogs. The assays identified genomic instabilities in dogs with cancer (squamous cell carcinoma) and in healthy dogs. The genetic assays are very sensitive and can be used as biomarkers of normal DNA replication and repair potential and the maintenance of control over the entire cell cycle. The use of the cytogenetic tests will enable the more precise assessment of genome stability and integrity in animals and make it possible to determine the number of chromosomal instabilities generated in a given individual, which can be indicative of its health status. The identification of instabilities can be used in routine diagnostic examination in dogs with cancer for more accurate diagnosis and prognosis.

The mutant mouse resource and research center (MMRRC) consortium: the US-based public mouse repository system

AbstractNow in its 25th year, the Mutant Mouse Resource and Research Center (MMRRC) consortium continues to serve the United States and international biomedical scientific community as a public repository and distribution archive of laboratory mouse models of human disease for research. Supported by the National Institutes of Health (NIH), the MMRRC consists of 4 regionally distributed and dedicated vivaria, offices, and specialized laboratory facilities and an Informatics Coordination and Service Center (ICSC). The overarching purpose of the MMRRC is to facilitate groundbreaking biomedical research by offering an extensive repertoire of mutant mice that are essential for advancing the understanding of human physiology and disease. The function of the MMRRC is to identify, acquire, evaluate, characterize, cryopreserve, and distribute mutant mouse strains to qualified biomedical investigators around the nation and the globe. Mouse strains accepted from the research community are held to the highest scientific standards to optimize reproducibility and enhance scientific rigor and transparency. All submitted strains are thoroughly reviewed, documented, and validated using extensive scientific quality control measures. In addition, the MMRRC conducts resource-related research on cryopreservation, mouse genetics, environmental conditions, and other topics that enhance operations of the MMRRC. Today, the MMRRC maintains an archive of mice, cryopreserved embryos and sperm, embryonic stem (ES) cell lines, and murine hybridomas for nearly 65,000 alleles. Since its inception, the MMRRC has fulfilled more than 20,000 orders from 13,651 scientists at 8441 institutions worldwide. The MMRRC also provides numerous services to assist researchers, including scientific consultation, technical assistance, genetic assays, microbiome analysis, analytical phenotyping, pathology, cryorecovery, husbandry, breeding and colony management, infectious disease surveillance, and disease modeling. The ICSC coordinates MMRRC operations, interacts with researchers, and manages the website (mmrrc.org) and online catalogue. Researchers benefit from an expansive list of well-defined mouse models of disease that meet the highest scientific standards while submitting investigators benefit by having their mouse strains cryopreserved, protected, and distributed in compliance with NIH policies.

Assessment of the In Vitro Phosphatidylinositol Glycan Class A (PIG-A) Gene Mutation Assay Using Human TK6 and Mouse Hepa1c1c7 Cell Lines.

Gene mutations linked to diseases like cancer may be caused by exposure to environmental chemicals. The X-linked phosphatidylinositol glycan class A (PIG-A) gene, required for glycosylphosphatidylinositol (GPI) anchor biosynthesis, is a key target locus for in vitro genetic toxicity assays. Various organisms and cell lines may respond differently to genotoxic agents. Here, we compared the mutagenic potential of directly genotoxic ethyl methane sulfonate (EMS) to metabolically activated pro-mutagenic polycyclic aromatic hydrocarbons (PAHs). The two classes of mutagens were compared in an in vitro PIG-A gene mutation test using the metabolically active murine hepatoma Hepa1c1c7 cell line and the human TK6 cell line, which has limited metabolic capability. Determination of cell viability is required for quantifying mutagenicity. Two common cell viability tests, the MTT assay and propidium iodide (PI) staining measured by flow cytometry, were evaluated. The MTT assay overestimated cell viability in adherent cells at high benzo[a]pyrene (B[a]P) exposure concentrations, so PI-based cytotoxicity was used in calculations. The spontaneous mutation rates for TK6 and Hepa1c1c7 cells were 1.87 and 1.57 per million cells per cell cycle, respectively. TK6 cells exposed to 600 µM and 800 µM EMS showed significantly higher mutation frequencies (36 and 47 per million cells per cell cycle, respectively). Exposure to the pro-mutagen benzo[a]pyrene (B[a]P, 10 µM) did not increase mutation frequency in TK6 cells. In Hepa1c1c7 cells, mutation frequencies varied across exposure groups (50, 50, 29, and 81 per million cells per cell cycle when exposed to 10 µM B[a]P, 5-methylcholanthrene (5-MC), chrysene, or 16,000 µM EMS, respectively). We demonstrate that the choice of cytotoxicity assay and cell line can determine the outcome of the Pig-A mutagenesis assay when assessing a specific mutagen.

Split Reporters Facilitate Monitoring of Gene Expression and Peptide Production in Linear Cell-Free Transcription-Translation Systems.

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements. We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction afforded by split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. We anticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

Multifaceted roles of H2B mono-ubiquitylation in D-loop metabolism during homologous recombination repair.

H2Bubi is epistatic to H2A.Z and INO80 in promoting homology search and D-loop formationH2Bubi stabilizes extended D-loopExcessive resection counteracts D-loop formation and extensionH2Bubi promotes crossover events and limits the frequency of break-induced replication outcomes in HR repair.

Revisiting the role of the spindle assembly checkpoint in the formation of gross chromosomal rearrangements in Saccharomyces cerevisiae.

Multiple pathways are known to suppress the formation of gross chromosomal rearrangements (GCRs), which can cause human diseases including cancer. In contrast, much less is known about pathways that promote their formation. The spindle assembly checkpoint (SAC), which ensures the proper separation of chromosomes during mitosis, has been reported to promote GCR, possibly by delaying mitosis to allow GCR-inducing DNA repair to occur. Here we show that this conclusion is the result of an experimental artifact arising from the synthetic lethality caused by disruption of the SAC and loss of the CIN8 gene, which is often lost in the genetic assay used to select for GCRs. After correcting for this artifact, we find no role of the SAC in promoting GCR.

Diagnostic guidelines for familial hemophagocytic lymphohistiocytosis revisited

AbstractCurrent hemophagocytic lymphohistiocytosis 2004 (HLH-2004)–based diagnostic criteria for familial hemophagocytic lymphohistiocytosis (FHL) are based on expert opinion. Here, we performed a case-control study to test and possibly improve these criteria. We also developed 2 complementary expert opinion–based diagnostic strategies for FHL in patients with signs/symptoms suggestive of HLH, based on genetic and cellular cytotoxicity assays. The cases (N = 366) were children aged <16 years with verified familial and/or genetic FHL (n = 341) or Griscelli syndrome type 2 (n = 25); 276 from the HLH-94/HLH-2004 databases and 90 from the Italian HLH Registry. All fulfilled the HLH-94/HLH-2004 patient inclusion criteria. Controls were 374 children with systemic-onset juvenile idiopathic arthritis (sJIA) and 329 + 361 children in 2 cohorts with febrile infections that could be confused with HLH and sepsis, respectively. To provide complete data sets, multiple imputations were performed. The optimal model, based on 17 variables studied, revealed almost similar diagnostic thresholds as the existing criteria, with accuracy 99.1% (sensitivity 97.1%; specificity 99.5%); the original HLH-2004 criteria had accuracy 97.4% (sensitivity 99.0%; specificity 97.1%). Because cellular cytotoxicity assays here constitute a separate diagnostic strategy, HLH-2004 criteria without natural killer (NK)–cell function was also studied, which showed accuracy 99.0% (sensitivity, 96.2%; specificity, 99.5%). Thus, we conclude that the HLH-2004 criteria (without NK-cell function) have significant validity in their current form when tested against severe infections or sJIA. It is important to exclude underlying malignancies and atypical infections. In addition, complementary cellular and genetic diagnostic guidelines can facilitate necessary confirmation of clinical diagnosis.

Generation and characterization of a depigmented variant of Chlorella pyrenoidosa for application in food products

Microalgae are promising natural resources for an array of essential nutrients and health-promoting compounds. The past decade has witnessed their increasing popularity as a value-added ingredient in the food industry. However, some of their intrinsic properties, especially their typical green color and off-flavor, may compromise consumer acceptability and remain a key challenge for food scientists. This study aimed to generate and characterize a chemically induced pigment-deficient mutant (C49) of Chlorella pyrenoidosa. In contrast to the wild type (WT), C49 could not grow photoautotrophically or mixotrophically. However, when grown heterotrophically, it exhibited a growth curve similar to that of WT. Pigment analysis verified the presence of only trace amounts of chlorophylls (0.27 ± 0.02 mg/g) and carotenoids (0.036 ± 0.003 mg/g) in the C49 which were only ∼1.7% and ∼1% of those in the WT, respectively. C49 also had higher carbohydrate (62.75% ± 2.42%) and lower protein (26.54% ± 1.00%) content than the WT (37.41% ± 2.25% and 36.21% ± 1.97%, respectively). Genetic assays revealed inactivation of the phytoene desaturase gene as a key mechanism underlying the reduced xanthophyll-synthesizing capability of C49, thus causing its failure to accumulate chlorophyll and develop chloroplasts. Transcriptomic and RT-PCR analyses indicated that the high carbohydrate content was likely due to reduced starch degradation. Furthermore, C49 demonstrated an emulsifying capacity comparable to that of WT over a wide pH range (3–8) in oil-in-water emulsions consisting of 5% cell-disrupted homogenate in deionized water and rapeseed oil (1:1, v/v). The results of this study support that depigmented Chlorella pyrenoidosa possesses promising potential for application in the food industry.

The Drosophila histone methyltransferase SET1 coordinates multiple signaling pathways in regulating male germline stem cell maintenance and differentiation.

Many tissue-specific adult stem cell lineages maintain a balance between proliferation and differentiation. Here, we study how the H3K4me3 methyltransferase Set1 regulates early-stage male germ cells in Drosophila. Early-stage germline-specific knockdown of Set1 results in temporally progressive defects, arising as germ cell loss and developing into overpopulated early-stage germ cells. These germline defects also impact the niche architecture and cyst stem cell lineage non-cell-autonomously. Additionally, wild-type Set1, but not the catalytically inactive Set1, rescues the Set1 knockdown phenotypes, highlighting the functional importance of the methyltransferase activity of Set1. Further, RNA-sequencing experiments reveal key signaling pathway components, such as the JAK-STAT pathway gene Stat92E and the BMP pathway gene Mad, which are upregulated upon Set1 knockdown. Genetic interaction assays support the functional relationships between Set1 and JAK-STAT or BMP pathways, as both Stat92E and Mad mutations suppress the Set1 knockdown phenotypes. These findings enhance our understanding of the balance between proliferation and differentiation in an adult stem cell lineage. The phenotype of germ cell loss followed by over-proliferation when inhibiting a histone methyltransferase also raises concerns about using their inhibitors in cancer therapy.

A multi-biomarker micronucleus assay using imaging flow cytometry

Genetic toxicity testing assesses the potential of compounds to cause DNA damage. There are many genetic toxicology screening assays designed to assess the DNA damaging potential of chemicals in early drug development aiding the identification of promising drugs that have low-risk potential for causing genetic damage contributing to cancer risk in humans. Despite this, in vitro tests generate a high number of misleading positives, the consequences of which can lead to unnecessary animal testing and/or the abandonment of promising drug candidates. Understanding chemical Mode of Action (MoA) is vital to identifying the true genotoxic potential of substances and, therefore, the risk translation into the clinic. Here we demonstrate a simple, robust protocol for staining fixed, human-lymphoblast p53 proficient TK6 cells with antibodies against ɣH2AX, p53 and pH3S28 along with DRAQ5™ DNA staining that enables analysis of un-lysed cells via microscopy approaches such as imaging flow cytometry. Here, we used the Cytek® Amnis® ImageStream®X Mk II which provides a high-throughput acquisition platform with the sensitivity of flow cytometry and spatial morphological information associated with microscopy. Using the ImageStream manufacturer’s software (IDEAS® 6.2), a masking strategy was developed to automatically detect and quantify micronucleus events (MN) and characterise biomarker populations. The gating strategy developed enables the generation of a template capable of automatically batch processing data files quantifying cell-cycle, MN, ɣH2AX, p53 and pH3 populations simultaneously. In this way, we demonstrate how a multiplex system enables DNA damage assessment alongside MN identification using un-lysed cells on the imaging flow cytometry platform. As a proof-of-concept, we use the tool chemicals carbendazim and methyl methanesulphonate (MMS) to demonstrate the assay’s ability to correctly identify clastogenic or aneugenic MoAs using the biomarker profiles established.

Association of dietary intake and serum concentration of omega-3 fatty acids on celiac disease: evidence from observational study and Mendelian randomization.

The association between omega-3 fatty acids (O3FA) and celiac disease lacks sufficient investigation. Utilizing data gleaned from the 2009 to 2014 National Health and Nutrition Examination Survey (NHANES), this research comprises a sample of 13 403 adults, each aged 20 years and above. We conducted a multivariable logistic regression analysis to assess the association between dietary intake of O3FA and celiac disease. Subsequently, a two-sample Mendelian randomization was performed to estimate the unconfounded causal relationship between serum O3FA and celiac disease. The principal analytical strategy utilized the inverse-variance weighted methodology. In this cross-sectional study, 48 occurrences (0.36%) of celiac disease were encompassed. In the multivariable model, there was no association between dietary intake of O3FA and cases of celiac disease (odds ratio: 1.12, 95% confidence interval: 0.47-2.66, P = 0.792). However, serum levels of O3FA determined by genetic assay were correlated with celiac disease (inverse-variance weighted, β = 0.2439, P = 0.0287), with no evidence of horizontal pleiotropy ( P = 0.3689). The dietary consumption of O3FA did not exhibit an association with the risk of celiac disease in this cross-sectional investigation. However, a correlation between celiac disease and serum levels of O3FA was observed in the Mendelian randomization. Further investigations, including human clinical trials, are warranted.

The complete plastome of Amaranthus roxburghianus (Amaranthaceae)

Amaranthus roxburghianus H.W. Kung 1935, belonging to the Amaranthaceae family, is recognized for its significant medicinal properties. However, molecular research on this species has been limited. This study represents the inaugural documentation of the sequencing and assembly of the complete plastome of A. roxburghianus. The genome spans a total length of 149,969 base pairs (bp), exhibiting a conventional quadripartite structure. This structure comprises a large single-copy (LSC) region of 83,917 bp, a small single-copy (SSC) region of 18,124 bp, and two inverted repeat (IR) regions, each extending to 23,964 bp. In its entirety, the A. roxburghianus plastome encompasses 128 genes, of which 107 are unique, encompassing 77 individual protein-coding genes, 26 unique tRNA genes, and four unique rRNA genes. Phylogenetic analysis has shown a close resemblance between A. roxburghianus and A. polygonoides, both part of the subgenus Albersia. Although the genus Amaranthus is roughly divided into three subgenera, additional plastid genomic data are required for a more accurate assignment of A. albus and A. blitoides. The sequencing of this plastome is a significant step forward, likely to expedite the development of molecular markers and significantly contribute to genetic assays involving this distinctive species.

Resolution of ribosomal stalling by EF-P and ABCF ATPases YfmR and YkpA/YbiT.

Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis.

Who bit the boat? New DNA collection and genomic methods enable species identification in suspected shark-related incidents

Species identification following shark-related incidents is critical for effective incident management and for collecting data to inform shark-bite mitigation strategies. Witness statements are not always reliable, and species identification is often ambiguous or missing. Alternative methods for species identification include morphological assessments of bite marks, analysis of collected teeth at the scene of the incident, and genetic approaches. However, access to appropriate collection media and robust genetic assays have limited the use of genetic technologies. Here, we present a case study that facilitated a unique opportunity to compare the effectiveness of medical gauze readily available in first-aid kits, and forensic-grade swabs in collecting genetic material for shark-species identification. Sterile medical gauze and forensic-grade swabs were used to collect transfer DNA from the bite margins on a bitten surf ski which were compared to a piece of shark tissue embedded along the bite margin. Witness accounts and the characteristics of the bite mark impressions inferred the involvement of a Carcharodon carcharias (white shark). The morphology of a tooth found on the boat that picked up the surf ski, however, suggested it belonged to an Orectolobus spp. (wobbegong). Genetic analysis of DNA transferred from the shark to the surf ski included the application of a broad-target nested PCR assay followed by Sanger sequencing, with white shark contribution to the ‘total sample DNA’ determined with a species-specific qPCR assay. The results of the genetic analyses were congruent between sampling methods with respect to species identification and the level of activity inferred by the donor-specific DNA contribution. These data also supported the inferences drawn from the bite mark morphology. DNA from the recovered tooth was PCR amplified with a wobbegong-specific primer pair designed for this study to corroborate the tooth’s morphological identification. Following the confirmation of gauze used for sampling in the case study event, two additional isolated incidents occurred and were sampled in situ using gauze, as typically found in a first-aid kit, by external personnel. DNA extracted from these gauze samples resulted in the identification of a white shark as the donor of the DNA collected from the bite marks in both instances. This study, involving three incidents separated by time and location, represents the seminal application of gauze as a sampling media after critical human-shark interactions and strongly supports the practical implementation of these methods in the field.