An efficient and cost-effective method for disrupting genes in RAW264.7 macrophages using CRISPR-Cas9.

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) are widely used for genome editing in cultured cell lines. However, the implementation of genome editing is still challenging due to the complex and often costly multi-step process associated with this technique. Moreover, the efficiency of genome editing varies across cell types, often limiting utility. Herein, we describe pCRISPR-EASY, a vector for simplified cloning of single guide RNAs (sgRNAs) and its simultaneous introduction with CRISPR-Cas9 into cultured cells using a non-viral delivery system. We outline a comprehensive, step-by-step protocol for genome editing in RAW264.7 macrophages, a mouse macrophage cell line widely used in biomedical research for which genome editing using CRISPR-Cas9 has been restricted to lentiviral or expensive commercial reagents. This provides an economical, highly efficient and reliable method for genome editing that can easily be adapted for use in other systems.

Peptidoglycan deacetylation controls type IV secretion and the intracellular survival of the bacterial pathogen Legionella pneumophila.

Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.

Use of risk scores for predicting new atrial fibrillation after ischemic stroke or transient ischemic attack-A systematic review.

BACKGROUND: Newly detected atrial fibrillation (NDAF) following an ischemic stroke or transient ischemic attack is often paroxysmal in nature. While challenging to detect, extended electrocardiographic (ECG) monitoring is often used to identify NDAF which has resource implications. Prognostic risk scores have been derived which may stratify the risk of NDAF and inform patient selection for ECG monitoring approaches after ischemic stroke/transient ischemic attack. AIM: The overall aim was to identify risk scores that were derived and/or validated to predict NDAF after ischemic stroke/transient ischemic attack and evaluate their performance. SUMMARY OF REVIEW: A systematic literature review was undertaken in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement, with application of the Quality Assessment of Diagnostic Accuracy-2 tool. Published studies, which derived and validated clinical risk scores in patients with ischemic stroke/transient ischemic attack, or externally validated an existing score to predict NDAF after ischemic stroke/transient ischemic attack, were considered and independently screened by two reviewers. Twenty-one studies involving 23 separate cohorts were analyzed from which 17 integer-based risk scores were identified. The overall frequency of NDAF was 9.7% (95% confidence intervals 8%-11.5%; I2 = 98%). The performance of the scores varied widely among derivation and validation cohorts (area under the receiver operating characteristic curve (AUC) 0.54-0.94); scores derived from stroke cohorts (12 scores) appeared to perform better (AUC 0.7-0.94) than those derived from non-stroke cohorts (five scores; AUC 0.53-0.79). The scores also varied considerably in their complexity, ascertainment, component variables, participant characteristics, outcome definition, and ease of application limiting their generalizability and utility. CONCLUSION: Overall, the risk scores identified performed variably in their discriminative ability and the utility of these scores to predict NDAF in clinical practice remains uncertain. Further studies are required using larger prospective cohorts and randomized control trials to evaluate the usefulness of such scores for clinical decision making and preventative intervention.

Medical care-seeking behaviours among drowning casualties: Results from a national survey conducted in Bangladesh.

OBJECTIVE: Despite the high magnitude of drowning, medical care-seeking behaviours among drowning casualties remain unexplored in Bangladesh. This study aimed to explore this behaviour among drowning casualties in Bangladesh. METHODS: A population-based cross-sectional study was conducted using a multi-stage cluster sampling method. Data were collected using a structured questionnaire from 299,216 rural and urban residents. RESULTS: From the survey, we found 191 drowning cases: 40.84% (n = 78) were fatal and 59.16% (n = 113) were non-fatal. Among the drowning cases, 71.2% (n = 136) were referred to healthcare providers, while 62.8% (n = 120) received medical care from different health service providers. Further analysis showed that 66.6% (n = 116) of children and 26.6% (n = 4) of adults sought healthcare. As many as 78.9% (n = 120/152) of rural residents sought healthcare, as compared to 61.5% (n = 24/39) of urban residents. Among all drowning casualties, 31.7% (n = 38) received healthcare from a qualified healthcare provider, whereas 68.3% (n = 82) received it from non-qual]ified healthcare providers. About 59 (49%) casualties received care from a pharmacy and 34 (28%) from a recognised hospital. The hospital admission rate for drowning was 11.7%. About 14 (11.7%) drowning casualties were brought to hospitals in motorised or non-motorised vehicles. As many as 97 (80.8%) patients sought healthcare attention and managed to survive. CONCLUSION: A significant number of drowning casualties sought medical care from qualified and non-qualified healthcare providers. In Bangladesh, it is necessary to develop guidelines for providing medical care for drowning casualties.

Structure of the capsule and lipopolysaccharide O-antigen from the channel catfish pathogen, Aeromonas hydrophila.

A hypervirulent A. hydrophila (vAh) pathotype has been identified as the etiologic agent responsible for disease outbreaks in farmed carp species and channel catfish (Ictalurus punctatus) in China and the Southeastern United States, respectively. The possible route of infection has previously been unknown; however, virulence is believed to be multifactorial, involving the production/secretion of several virulence factors, including a high molecular weight group 4 capsular polysaccharide. Here we present chemical structural evidence of a novel capsule- and LPS-associated O-antigen found present in vAh isolated during these disease outbreaks. In this study, the chemical structure of the vAh O-antigen was determined by chemical analysis, Smith degradation, mass spectrometry, and 2D proton and carbon nuclear magnetic resonance (NMR) spectroscopy and found to be unique among described bacterial O-antigens. The O-antigen consists of hexasaccharide repeating units featuring a 4)-α-l-Fucp-(1-3)-ß-d-GlcpNAc-(1-4)-α-l-Fucp-(1-4)-ß-d-Glcp-(1- backbone, substituted with single residue side chains of α-d-Glcp and α-d-Quip3NAc linked to O-3 of the two fucose residues. The polysaccharide is partially O-acetylated on O-6 of the 4-substituted ß-Glcp residue.

Identification of Subsets of Enteroaggregative Escherichia coli Associated with Diarrheal Disease among Under 5 Years of Age Children from Rural Gambia.

Enteroaggregative Escherichia coli (EAEC) cause acute and persistent diarrhea, mostly in children worldwide. Outbreaks of diarrhea caused by EAEC have been described, including a large outbreak caused by a Shiga toxin expressing strain. This study investigated the association of EAEC virulence factors with diarrhea in children less than 5 years. We characterized 428 EAEC strains isolated from stool samples obtained from moderate-to-severe diarrhea cases (157) and healthy controls (217) children aged 0-59 months recruited over 3 years as part of the Global Enteric Multicenter Study (GEMS) in The Gambia. Four sets of multiplex polymerase chain reaction were applied to detect 21 EAEC-virulence genes from confirmed EAEC strains that target pCVD432 (aatA) and AAIC (aaiC). In addition, Kirby-Bauer disc diffusion antimicrobial susceptibility testing was performed on 88 EAEC strains following Clinical Laboratory Standard Institute guidelines. We observed that the plasmid-encoded enterotoxin [odds ratio (OR): 6.9, 95% confidence interval (CI): 2.06-29.20, P < 0.001], aggregative adherence fimbriae/I fimbriae (aggA) [OR: 2.2, 95% CI: 1.16-4.29, P = 0.008], and hexosyltransferase (capU) [OR: 1.9, 95% CI 1.02-3.51, P = 0.028] were associated with moderate-to-severe diarrhea among children < 12 months old but not in the older age strata (> 12 months). Our data suggest that some EAEC-virulent factors have age-specific associations with moderate-to-severe diarrhea in infants. Furthermore, our study showed that 85% and 72% of EAEC strains tested were resistant to sulphamethoxazole-trimethoprim and ampicillin, respectively. Sulphamethoxazole-trimethoprim and ampicillin are among the first-line antibiotics used for the treatment of diarrhea in The Gambia.

Classification of a Hypervirulent Aeromonas hydrophila Pathotype Responsible for Epidemic Outbreaks in Warm-Water Fishes.

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

A sample cell for in situ electric-field-dependent structural characterization and macroscopic strain measurements.

When studying electro-mechanical materials, observing the structural changes during the actuation process is necessary for gaining a complete picture of the structure-property relationship as certain mechanisms may be meta-stable during actuation. In situ diffraction methods offer a powerful and direct means of quantifying the structural contributions to the macroscopic strain of these materials. Here, a sample cell is demonstrated capable of measuring the structural variations of electro-mechanical materials under applied electric potentials up to 10 kV. The cell is designed for use with X-ray scattering techniques in reflection geometry, while simultaneously collecting macroscopic strain data using a linear displacement sensor. The results show that the macroscopic strain measured using the cell can be directly correlated with the microscopic response of the material obtained from diffraction data. The capabilities of the cell have been successfully demonstrated at the Powder Diffraction beamline of the Australian Synchrotron and the potential implementation of this cell with laboratory X-ray diffraction instrumentation is also discussed.

Comparative behavior of root pathogens in stems and roots of southeastern Pinus species.

Root diseases are expected to become a greater threat to trees in the future due to accidental pathogen introductions and predicted climate changes, thus there is a need for accurate and efficient pathogenicity tests. For many root pathogens, these tests have been conducted in stems instead of roots. It, however, remains unclear whether stem and root inoculations are comparable for most fungal species. In this study we compared the growth and damage caused by five root pathogens (Grosmannia huntii, Grosmannia alacris, Leptographium procerum, Leptographium terebrantis, and Heterobasidion irregulare) in root and stem tissue of two Pinus species by inoculating mature trees and tissue amended agar in the laboratory. Most fungal species tested caused greater damage in roots of both pine hosts following inoculation. The relationship between root and stem damage was, however, similar when most combinations of pathogens were compared. These results suggest that although stem inoculations are not suitable for determining the actual damage potential of a given species, they may be viewed as a useful surrogate for root inoculations when comparing the relative pathogenicity of multiple species. When grown on amended agar, fungal species generally had greater growth in stem tissue, contrasting with the findings from tree inoculations.

Complete genome of Staphylococcus aureus Tager 104 provides evidence of its relation to modern systemic hospital-acquired strains.

BACKGROUND: Staphylococcus aureus (S. aureus) infections range in severity due to expression of certain virulence factors encoded on mobile genetic elements (MGE). As such, characterization of these MGE, as well as single nucleotide polymorphisms, is of high clinical and microbiological importance. To understand the evolution of these dangerous pathogens, it is paramount to define reference strains that may predate MGE acquisition. One such candidate is S. aureus Tager 104, a previously uncharacterized strain isolated from a patient with impetigo in 1947. RESULTS: We show here that S. aureus Tager 104 can survive in the bloodstream and infect naïve organs. We also demonstrate a procedure to construct and validate the assembly of S. aureus genomes, using Tager 104 as a proof-of-concept. In so doing, we bridged confounding gap regions that limited our initial attempts to close this 2.82 Mb genome, through integration of data from Illumina Nextera paired-end, PacBio RS, and Lucigen NxSeq mate-pair libraries. Furthermore, we provide independent confirmation of our segmental arrangement of the Tager 104 genome by the sole use of Lucigen NxSeq libraries filled by paired-end MiSeq reads and alignment with SPAdes software. Genomic analysis of Tager 104 revealed limited MGE, and a νSaß island configuration that is reminiscent of other hospital acquired S. aureus genomes. CONCLUSIONS: Tager 104 represents an early-branching ancestor of certain hospital-acquired strains. Combined with its earlier isolation date and limited content of MGE, Tager 104 can serve as a viable reference for future comparative genome studies.

Deciphering the conserved genetic loci implicated in plant disease control through comparative genomics of Bacillus amyloliquefaciens subsp. plantarum.

To understand the growth-promoting and disease-inhibiting activities of plant growth-promoting rhizobacteria (PGPR) strains, the genomes of 12 Bacillus subtilis group strains with PGPR activity were sequenced and analyzed. These B. subtilis strains exhibited high genomic diversity, whereas the genomes of B. amyloliquefaciens strains (a member of the B. subtilis group) are highly conserved. A pairwise BLASTp matrix revealed that gene family similarity among Bacillus genomes ranges from 32 to 90%, with 2839 genes within the core genome of B. amyloliquefaciens subsp. plantarum. Comparative genomic analyses of B. amyloliquefaciens strains identified genes that are linked with biological control and colonization of roots and/or leaves, including 73 genes uniquely associated with subsp. plantarum strains that have predicted functions related to signaling, transportation, secondary metabolite production, and carbon source utilization. Although B. amyloliquefaciens subsp. plantarum strains contain gene clusters that encode many different secondary metabolites, only polyketide biosynthetic clusters that encode difficidin and macrolactin are conserved within this subspecies. To evaluate their role in plant pathogen biocontrol, genes involved in secondary metabolite biosynthesis were deleted in a B. amyloliquefaciens subsp. plantarum strain, revealing that difficidin expression is critical in reducing the severity of disease, caused by Xanthomonas axonopodis pv. vesicatoria in tomato plants. This study defines genomic features of PGPR strains and links them with biocontrol activity and with host colonization.

Phytochemical and biological investigations of methanol extract of leaves of ziziphus mauritiana lam / Investigaciones fitoquímicas y biológicas de extractos metanólicos de hojas de ziziphus mauritiana lam

Seven compounds were isolated from the methanol extract of leaves of Ziziphus mauritiana. The structures of the isolated compounds were elucidated as y-fagarine (1), beta-sitosterol (2), stigmasterol (3), alfa-amyrin (4), lupeol (5), beta-amyrin (6) and betulin (7) by extensive spectroscopic studies and by co-TLC with authentic samples. This is the first report of isolation of compounds 1-7 from this plant species. The extractives from leaves were investigated for thrombolytic, membrane stabilizing, antimicrobial, antidiarrheal and analgesic potentials along with sleep inducing property on animal model. The thrombolytic and membrane stabilizing activities were assessed by using human erythrocyte comparing with standard streptokinase and acetylsalicylic acid, respectively. The methanolic extract reduced frequency of diarrheal faeces when compared to the standard loperamide. In castor oil induced diarrhea in mice a dose dependent decrease of gastrointestinal transits were observed. In radiant heat tail-flick method the methanol extract showed moderate antinociceptive activity, compared to standard morphine.

Siete compuestos fueron aislados del extracto metanólico de hojas de Ziziphus mauritiana. Las estructuras de los compuestos aislados fueron identificadas como y-fagarina (1), beta-sitosterol (2), estigmasterol (3), alfa-amirina (4), lupeol (5), beta-amirina (6) y betulina (7 ) por medio de extensivos estudios espectroscópicos y por co-TLC con muestras auténticas. Este es el primer informe de aislamiento de los compuestos 1-7 a partir de esta especie vegetal. Los extractos de las hojas fueron investigados por efectos trombolíticos, estabilizadores de membrana, antimicrobianos, antidiarreicos y potenciales analgésicos junto a la propiedad de inducir sueño en el modelo animal. Las actividades trombolíticos y la estabilización de la membrana se evaluaron mediante el uso de eritrocitos humanos comparando con estreptoquinasa estándar y el ácido acetilsalicílico, respectivamente. El extracto metanólico redujo la frecuencia de las heces diarreicas en comparación con el estándar de loperamida. En la inducción por aceite de ricino de diarrea en ratones, se observó una disminución dosis dependiente de los tránsitos gastrointestinales. En el método del coletazo producido por calor radiante, el extracto metabólico mostró una actividad antinociceptiva moderada, en comparación con la morfina estándar.

Draft Genome Sequence of Bacillus amyloliquefaciens AP183 with Antibacterial Activity against Methicillin-Resistant Staphylococcus aureus.

Bacillus amyloliquefaciens AP183 expresses secondary metabolites that inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA). Here, we present a ~3.99-Mbp draft genome sequence of AP183 with the aims of providing insights into the genomic basis of its antibacterial mechanisms and exploring its potential use in preventing MRSA skin colonization.

Strategies to avoid wrongly labelled genomes using as example the detected wrong taxonomic affiliation for aeromonas genomes in the GenBank database.

Around 27,000 prokaryote genomes are presently deposited in the Genome database of GenBank at the National Center for Biotechnology Information (NCBI) and this number is exponentially growing. However, it is not known how many of these genomes correspond correctly to their designated taxon. The taxonomic affiliation of 44 Aeromonas genomes (only five of these are type strains) deposited at the NCBI was determined by a multilocus phylogenetic analysis (MLPA) and by pairwise average nucleotide identity (ANI). Discordant results in relation to taxa assignation were found for 14 (35.9%) of the 39 non-type strain genomes on the basis of both the MLPA and ANI results. Data presented in this study also demonstrated that if the genome of the type strain is not available, a genome of the same species correctly identified can be used as a reference for ANI calculations. Of the three ANI calculating tools compared (ANI calculator, EzGenome and JSpecies), EzGenome and JSpecies provided very similar results. However, the ANI calculator provided higher intra- and inter-species values than the other two tools (differences within the ranges 0.06-0.82% and 0.92-3.38%, respectively). Nevertheless each of these tools produced the same species classification for the studied Aeromonas genomes. To avoid possible misinterpretations with the ANI calculator, particularly when values are at the borderline of the 95% cutoff, one of the other calculation tools (EzGenome or JSpecies) should be used in combination. It is recommended that once a genome sequence is obtained the correct taxonomic affiliation is verified using ANI or a MLPA before it is submitted to the NCBI and that researchers should amend the existing taxonomic errors present in databases.

Complete Genome Sequence of Streptomyces sp. Strain CCM_MD2014, Isolated from Topsoil in Woods Hole, Massachusetts.

Here, we present the complete genome sequence of Streptomyces sp. strain CCM_MD2014 (phylum Actinobacteria), isolated from surface soil in Woods Hole, MA. Its single linear chromosome of 8,274,043 bp in length has a 72.13% G+C content and contains 6,948 coding sequences.

Complete Genome Sequence of Curtobacterium sp. Strain MR_MD2014, Isolated from Topsoil in Woods Hole, Massachusetts.

Here, we present the 3,443,800-bp complete genome sequence of Curtobacterium sp. strain MR_MD2014 (phylum Actinobacteria). This strain was isolated from soil in Woods Hole, MA, as part of the 2014 Microbial Diversity Summer Program at the Marine Biological Laboratory in Woods Hole, MA.

Genome modifications and cloning using a conjugally transferable recombineering system.

The genetic modification of primary bacterial disease isolates is challenging due to the lack of highly efficient genetic tools. Herein we describe the development of a modified PCR-based, λ Red-mediated recombineering system for efficient deletion of genes in Gram-negative bacteria. A series of conjugally transferrable plasmids were constructed by cloning an oriT sequence and different antibiotic resistance genes into recombinogenic plasmid pKD46. Using this system we deleted ten different genes from the genomes of Edwardsiella ictaluri and Aeromonas hydrophila. A temperature sensitive and conjugally transferable flp recombinase plasmid was developed to generate markerless gene deletion mutants. We also developed an efficient cloning system to capture larger bacterial genetic elements and clone them into a conjugally transferrable plasmid for facile transferring to Gram-negative bacteria. This system should be applicable in diverse Gram-negative bacteria to modify and complement genomic elements in bacteria that cannot be manipulated using available genetic tools.

Differential disease symptoms and full-length genome sequence analysis for three strains of Tobacco etch virus.

Tobacco etch virus (TEV) strains HAT, Mex21, and N have been the focus of numerous studies to dissect a host resistance mechanism in Capsicum spp. Little is known, however, about their general pathogenicity and genomic sequence data are not available on the TEV strains Mex21 and N. Four Nicotiana spp. were evaluated after inoculation with each TEV strain. Nicotiana tabacum 'Kentucky 14' and N. clevelandii plants expressed varied systemic symptoms dependent on the TEV strain; however, disease severity increased from HAT (mild mosaic symptoms) to Mex21 (more severe mosaic symptoms with stunting) to N (severe chlorosis and stunting). Nicotiana tabacum 'Samsun' plants developed relatively milder symptoms and N. glutinosa plants remained symptomless, although they were systemically infected. The genome of each TEV strain was sequenced and shown to consist of 9,495 nucleotides and a polyprotein of 3,054 amino acids. Comparison of their nucleotide sequences relative to the original HAT sequence (GenBank Accession No. M11458) revealed 95, 92, and 92 % identity for HAT-AU (from Auburn University), Mex21, and N, respectively. HAT-AU had 91 % sequence identity with Mex21 and N, while Mex21 and N were more closely related with 98 % nucleotide sequence identity. Similarly, the amino acid sequence identities for the full-length polyprotein ranged from 95 % for HAT-AU when compared with N to a high of 98 % identity between Mex21 and N.

Taxonomic affiliation of new genomes should be verified using average nucleotide identity and multilocus phylogenetic analysis.

The average nucleotide identity (ANI) determines if two genomes belong to the same species. Using ANI, we detected mislabeled genomes and recommend verifying with ANI and multilocus phylogenetic analysis the species affiliations of the announced genomes. The slightly different results obtained with different ANI calculation software can potentially mislead taxonomic inferences.

Draft genome sequences of two novel Aeromonas species recovered in association with cyanobacterial blooms.

Aeromonas aquatica and Aeromonas lacus are two new species that have been found in association with cyanobacterial blooms from recreational Finnish lakes where adverse human health effects have been recorded. Here, we present the draft genome sequences of their type strains.