Kb In Size Research Articles

Coproduction of Tet(X7) Conferring High-Level Tigecycline Resistance, Fosfomycin FosA4, and Colistin Mcr-1.1 in Escherichia coli Strains from Chickens in Egypt.

The plasmid-mediated tet(X7) conferring high-level tigecycline resistance was identified in five mcr-1.1-positive Escherichia coli strains (ST10 [n = 3] and ST155 [n = 2]) isolated from chickens in Egypt. Two fosfomycin-resistant fosA4-carrying IncFII plasmids (∼79 kb in size) were detected. Transposase ISCR3 (IS91 family) is syntenic with tet(X7) in all isolates, suggesting its role in the mobilization of tet(X7). To our knowledge, this is the first global report of ST4-IncHI2 plasmids cocarrying tet(X7) and mcr-1.1 from chickens.

A rhomboid-like protease gene from an interspecies translocation confers resistance to cyst nematodes.

Plant-parasitic nematodes are severe pests in crop production worldwide. Chemical control of nematodes has been continuously reduced in recent decades owing to environmental and health concerns. Therefore, breeding nematode-resistant crops is an important aim if we are to secure harvests. The beet cyst nematode impairs root development and causes severe losses in sugar beet production. The only sources for resistance are distantly related wild species of the genus Patellifolia. Nematode resistance had been introduced into the beet genome via translocations from P.procumbens. We sequenced three translocations and identified the translocation breakpoints. By comparative sequence analysis of three translocations, we localized the resistance gene Hs4 within a region c.230kb in size. A candidate gene was characterized by CRISPR-Cas-mediated knockout and overexpression in susceptible roots. The gene encodes a rhomboid-like protease, which is predicted to be bound to the endoplasmic reticulum. Gene knockout resulted in complete loss of resistance, while overexpression caused resistance. The data confirm that the Hs4 gene alone protects against the pest. Thus, it constitutes a previously unknown mechanism of plants to combat parasitic nematodes. Its function in a nonrelated species suggests that the gene can confer resistance in crop species from different plant families.

Does an Antibiotic Stewardship Applied in a Pig Farm Lead to Low ESBL Prevalence?

Background. The aim of the present study was to prospectively evaluate the prevalence of intestinal carriage of colistin-resistant and extended-spectrum β-lactamase (ESBL)-producing Enterobacterales among pigs from a Swiss farm attending an animal health and antibiotic stewardship program and to determine the associated mechanisms of resistance. Materials/Methods. Eighty-one fecal samples were recovered and screened for either β-lactam-resistant, colistin-resistant, or aminoglycoside-resistant Enterobacterales, using respective screening media. All recovered isolates were tested for antimicrobial susceptibility and their clonal relationship (PFGE and MLST). Plasmid typing was performed by plasmid-based replicon typing (PBRT). Resistance genes were searched by PCR and sequencing. Results. A total of 38 ESBL-producing Escherichia coli and a single ESBL-producing Enterobacter cloacae were recovered from 81 pigs, corresponding to a prevalence of 50%, no other β-lactamase producer being identified. Among the 38 ESBL-producing E. coli, all belonged to sequence type (ST) ST10, except two ST34 and ST744 isolates. Among the ST10-blaCTX-M-1 isolates, three subclones (n = 22, n = 13, and n = 1, respectively) were identified according to the PFGE analysis. The most commonly identified IncI1 plasmid harboring the blaCTX-M-1 gene was 143 kb in size and coharbored other resistance genes. Only three colistin-resistant Enterobacterales isolates were recovered, namely two Klebsiella pneumoniae isolates and a single E. cloacae isolate. Screening for the plasmid-borne mcr-1 to mcr-9 genes in these three isolates gave negative results. The two K. pneumoniae isolates were clonally related, belonged to ST76, and harbored a truncated mgrB chromosomal gene being the source of colistin resistance. Conclusion. A high prevalence of fecal carriage of ESBL-producing E. coli was found, being mainly caused by the spread of a clonal lineage within the farm. By contrast, a low prevalence of colistin-resistant Enterobacterales was found.

Corona virus and traditional approach for developing immunity through unani herbal medicine

Till date four coronaviruses are found to exist i.e. human coronavirus 229E (HCoV-229E), HCoV-0C43, severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV), fourth human coronavirus, HCoV-NL63 has been reported from a seven-month-old child with bronchiolitis and conjunctival inflammation. Coronaviruses, a genus of the coronaviridae family, are enveloped viruses with large plua- strand RNA genome. The genomic RNA is 27-32 kb in size, capped and polyadenylated. After detailed and extensive research coronaviruses have been found in rats, mice, chickens, swine cattle, horses, dog, cats, rabbits, humans and can cause a wide variety of diseases including gastroenteritis and respiratory tract diseases. The most recent found SARS-CoV can lead to life threating pneumonia and is the most pathogenic human coronavirus identified so far. This fatal and highly contagious virus is likely to decide in an animal reservoir and resulted in recent epidemic in humans through zoonotic transmission.

Biochemical and phylogenetic characterization of the wastewater tolerant Chlamydomonas biconvexa Embrapa|LBA40 strain cultivated in palm oil mill effluent.

The increasing demand for water, food and energy poses challenges for the world´s sustainability. Tropical palm oil is currently the major source of vegetable oil worldwide with a production that exceeds 55 million tons per year, while generating over 200 million tons of palm oil mill effluent (POME). It could potentially be used as a substrate for production of microalgal biomass though. In this study, the microalgal strain Chlamydomonas biconvexa Embrapa|LBA40, originally isolated from a sugarcane vinasse stabilization pond, was selected among 17 strains tested for growth in POME retrieved from anaerobic ponds of a palm oil industrial plant located within the Amazon rainforest region. During cultivation in POME, C. biconvexa Embrapa|LBA40 biomass productivity reached 190.60 mgDW • L-1 • d-1 using 15L airlift flat plate photobioreactors. Carbohydrates comprised the major fraction of algal biomass (31.96%), while the lipidic fraction reached up to 11.3% of dry mass. Reductions of 99% in ammonium and nitrite, as well as 98% reduction in phosphate present in POME were detected after 5 days of algal cultivation. This suggests that the aerobic pond stage, usually used in palm oil industrial plants to reduce POME inorganic load, could be substituted by high rate photobioreactors, significantly reducing the time and area requirements for wastewater treatment. In addition, the complete mitochondrial genome of C. biconvexa Embrapa|LBA40 strain was sequenced, revealing a compact mitogenome, with 15.98 kb in size, a total of 14 genes, of which 9 are protein coding genes. Phylogenetic analysis confirmed the strain taxonomic status within the Chlamydomonas genus, opening up opportunities for future genetic modification and molecular breeding programs in these species.

Cross-Border Emergence of Escherichia coli Producing the Carbapenemase NDM-5 in Switzerland and Germany.

A series of clinical NDM-5-producing Escherichia coli isolates obtained from two surveillance networks for carbapenem-producing Enterobacterales from 2018 to 2019, namely, Switzerland (NARA) and Germany (SurvCARE), were analyzed. The 33 NDM-5-producing E. coli isolates were highly resistant to β-lactams, including novel β-lactam/β-lactamase inhibitor combinations (ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam), and remained susceptible to fosfomycin, colistin, and tigecycline. These isolates were assigned to different sequence types (STs) and indicated a predominance of isolates exhibiting ST167 in Switzerland and Germany (n = 10) (phylogenetic group C), followed by ST405 (n = 4) (phylogenetic group E), ST1284 (n = 4) (phylogenetic group C), and ST361 (n = 4) (phylogenetic group C). The blaNDM-5 gene was predominantly present on an IncF-type plasmid (n = 29) and, to a lesser extent, on the narrow-host-range IncX3 plasmid (n = 4). Sequence analyses of eight NDM-5 plasmids indicated that NDM-5-encoding F-type plasmids varied in size between 86 and 132 kb. The two IncX3 plasmids pCH8NDM5 and pD12NDM5 were 46 and 45 kb in size, respectively. The highly conserved blaNDM-5 genetic surrounding structures (ΔISAba125-blaNDM-5-bleMBL-trpT-dsbD-IS26) of both the F-type and IncX3 plasmids suggested a common genetic origin. The emergence of the NDM-5 carbapenemase was evidenced in particular for the E. coli ST167 clone, which is a successful epidemic clone known to be associated with both multiresistance and virulence traits and is therefore of high public health concern. The occurrence of clonally related NDM-5-producing E. coli isolates in Switzerland and Germany further indicates the international spread of this multidrug-resistant superbug at least throughout Europe.

Benzalkonium chloride and heavy metal resistance profiles of Listeria monocytogenes strains isolated from fish, fish products and food-producing factories in Poland

Phenotypic and genotypic resistance to benzalkonium chloride (BC), cadmium and arsenic was tested (by susceptibility assays and molecular methods) in 287 Listeria monocytogenes strains isolated from fish and fish products, and food-producing factories in Poland. Overall, 40% of the isolates were resistant to BC, 56% to cadmium and 41% to arsenic (57% displayed resistance to more than one of the tested compounds). Among BC-resistant isolates, the most commonly detected resistance determinant was the qacH gene (83%). Three distinct types of cadA gene determining resistance to cadmium were detected, with the cadA1 variant predominant (88%), while most arsenic-resistant isolates (86%) harbored the arsA gene associated with a Tn554-like transposon (one strain harbored two copies of arsA in different arsenic resistance cassettes). 53% of all tested isolates contained plasmids (from 4 kb to > 90 kb in size), which were classified into 11 groups (p1–p11) based on their restriction patterns. Interestingly, 12 isolates harbored the small mobilizable pLMST6-like plasmid pLIS3 encoding multidrug efflux pump EmrC. Clustering analysis of PFGE patterns revealed that these isolates represent several diverse bacterial populations, which strongly suggests mobility of the pLMST6-like plasmids among L. monocytogenes strains and their role in dissemination of BC resistance.

Full-Length Genome and Partial Viral Genes Phylogenetic and Geographical Analysis of Dengue Serotype 3 Isolates.

Dengue fever is among the most common vector-borne diseases. Dengue virus (DENV), responsible for dengue fever as well as dengue hemorrhagic fever, belongs to the genus flavivirus and family Flaviviridae. Flaviviruses infect various vertebrate species and arthropods and are also responsible for diseases in birds, wild animals, and primates. DENV consists of a single-stranded, positive-sense RNA genome ~11 kb in size. Complete genome and partial gene sequences of geographically distinct DENV-3 strains were retrieved from the GenBank database. The evolutionary divergence of the 33 whole-genome and individual gene sequences of the nucleotides and amino acids of DENV-3 strains were generated with the maximum likelihood (ML) and Bayesian phylogenetic study (BEAST) methods using the MEGA 7 software. The genome size varied from 10,484 to 10,724 nucleotides among the strains with distinct geographical backgrounds belonging to Central America, South-Central Asia, and Eastern Asia. A phylogenetic analysis of the nucleotide and amino acid sequences of these DENV-3 isolates revealed extensive differences in the topologies due to PrM/M, NS1, NS2B, and NS3 genes. These results suggest substantial variation in the evolutionary pathways of the studied genes and genomes.

Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases.

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

Characterization of a vancomycin-resistant Enterococcus faecium isolate and a vancomycin-susceptible E. faecium isolate from the same blood culture.

To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture. The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization. Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical. Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates.

Conjugative IncHI2 plasmid harboring novel class 1 integron mediated dissemination of multidrug resistance genes in Salmonella Typhimurium

Salmonella enterica serovar Typhimurium is one of the most important serovars among nontyphoidal Salmonella. The inappropriate and over usage of antimicrobials in clinical and animal husbandry has increased the prevalence of antimicrobial-resistant Salmonella Typhimurium (S. Typhimurium). Diverse mobile genetic elements (MGEs) such as plasmids, insertion sequences and integrons also have a crucial role in the rapid dissemination of multidrug resistance (MDR) bacteria. This study aimed to screen and analyze the genetic environment of MGEs carried by S. Typhimurium, and to clarify the multidrug resistance dissemination mechanism in S. Typhimurium. The results showed that, out of 255 S. Typhimurium isolates, 62 isolates, were screened out for the presence of IncHI2 plasmid by PCR amplification, of which 61 isolates were carrying both IS26 and class 1 integron. All the 61 isolates were resistant to at least one antimicrobial and 58 were MDR. The correlation between plasmids and antimicrobial resistance components was determined by conjugation, antimicrobial susceptibility test, PCR-based replicon typing (PBRT) and S1-PFGE experiment. The results of PBRT revealed that 15 transconjugants harboring two kinds of plasmid incompatibility groups including HI2 and I1. Further, S1-PFGE validated the presence of HI2 plasmid in 13 and I1 plasmid in 2 transconjugants, which was about 200–300 kb and 80–100 kb in size, respectively. Whole genome sequencing revealed that no significant difference (P > 0.05) was found at the number of antimicrobial resistance gene categories among 15 donor isolates, and ST34 was the predominant sequence type. Phylogenetic analysis based on the complete plasmid sequences revealed 15 IncHI2 plasmids in this study clustered in two different clades and clustered together with other IncHI2 plasmids isolated from China, suggesting that the prevalence and dissemination of IncHI2 plasmids from diverse sources and regions. Four kinds of IS elements-class 1 integron arrangement patterns were identified in the 15 conjugative IncHI2-plasmids, of which three patterns were characterized to be complex IS26-class 1 integron arrangement and the other one was IS256-class 1 integron arrangement. Overall, these results suggested that IncHI2 was one of the major plasmid lineages presented in the MDR S. Typhimurium strains. Furthermore, the presence of complex IS elements-class 1 integrons and resistance genes co-located on IncHI2 plasmids contributed to the prevalence and rapid dissemination of multidrug resistance genes in Salmonella.

GENOMICS OF SARS-COV-2: A STUDY

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a type of novel coronavirus responsible for the COVID-19 outbreak, which attained pandemic proportions in 2020. SARS-CoV-2 belongs to that group of coronaviruses which infect the lower respiratory tract. The complete genome was found to be 29.9kb in size. The viral genome encodes 16 non-structural proteins (Nsps), essential for pathogenesis and replication. Structural proteins- envelope (E), membrane (M), nucleocapsid (N), and spike (S) glycoprotein are also encoded, important for virus subtyping and response to vaccines. L and S type of SARS-CoV-2 were identified in Wuhan initially. Later on, as the pandemic spread across the globe, A and C subtypes were identified to me the common type of strains across the Americas and Europe respectively. In Asia, B subtype was found to be common. ACE2 (angiotensin-converting enzyme 2), a receptor is utilised by SARS-CoV-2 for entry into host cell. People of European descent have a higher frequency of alternate allele of rs763395248 SNP in T92I risk variant compared to African and Asian population. In Asian populations, SNPs like rs758278442 and rs759134032 in the region of protective variants (K31R and Y83H) of ACE2 gene show higher frequency of mutant alleles than American and European populations. According to a structural modelling study, certain ACE2 variants may provide potential resistance to SARS-CoV-2 infection.

An ABCA7 partial deletion and a GRN variant in a semantic variant of primary progressive aphasia patient

AbstractBackground ABCA7 gene (ATP‐binding cassette transporter A7) loss‐of‐function mutations are related to increased risk of suffering Alzheimer’s disease (AD). On the other hand, mutations in GRN (Progranulin) gene are causative of frontotemporal dementia (FTD).MethodsThe proband was a patient diagnosed from semantic variant of primary progressive aphasia. Age at onset was at 50 years‐old, presenting progressive cognitive decline with an important language loss. The MRI showed a left temporal atrophy. AD CSF biomarkers were normal and no familial history of dementia was reported. Next generation sequencing was performed with Illumina NextSeq500. Single nucleotide variants were detected using GATK and copy number variants using ExomeDepth algorithm. Sanger sequencing was performed for GRN variant confirmation and MLPA technique for ABCA7 deletion validation. C9orf72 repeat expansion was studied with a repeat primed PCR and fragment analysis. Biological samples from his mother and a brother were obtained. Commercial ELISA kit was used to measure serum PGRN levels (Adipogen).ResultsPatient showed an ABCA7 partial deletion (exons 17‐47) plus 4 contiguous genes, of a total of 105 kb in size (hg19 chr19:g.1048865_1154298). Deletion was confirmed in the proband and discarded in the proband’s mother and brother by MLPA. Patient and his mother (asymptomatic at 81 yo) were also carriers of a reported GRN variant, p.(Asp33Glu; rs63750742). Progranulin serum levels were normal in the patient and his family members. C9orf72 screening was negative.ConclusionsThe patient harbored two genetic alterations in genes related to dementia risk, although it is unlikely that any of them alone could be responsible of the FTD phenotype. ABCA7 deletion should be de novo or father inherited. ABCA7 protein truncating variant at exon 14 (p.Arg578fs), which has a similar protein consequence, is relatively frequent in control population, although has showed a 1.8‐fold enrichment in AD patients. Moreover, GRN variant does not seem to be pathogenic or low penetrance because proband’s mother is unaffected and serum progranulin levels are normal. In conclusion, these variants per se are likely not sufficient to cause the disease, but rather risk variants of intermediate to high penetrance along with other factors.

Present Status and Future Management Strategies for Sugarcane Yellow Leaf Virus: A Major Constraint to the Global Sugarcane Production.

Sugarcane yellow leaf virus (SCYLV) is a distinct member of the Polerovirus genus of the Luteoviridae family. SCYLV is the major limitation to sugarcane production worldwide and presently occurring in most of the sugarcane growing countries. SCYLV having high genetic diversity within the species and presently ten genotypes are known to occur based on the complete genome sequence information. SCYLV is present in almost all the states of India where sugarcane is grown. Virion comprises of 180 coat protein units and are 24-29 nm in diameter. The genome of SCYLV is a monopartite and comprised of single-stranded (ss) positive-sense (+) linear RNA of about 6 kb in size. Virus genome consists of six open reading frames (ORFs) that are expressed by sub-genomic RNAs. The SCYLV is phloem-limited and transmitted by sugarcane aphid Melanaphis sacchari in a circulative and non-propagative manner. The other aphid species namely, Ceratovacuna lanigera, Rhopalosiphum rufiabdominalis, and R. maidis also been reported to transmit the virus. The virus is not transmitted mechanically, therefore, its transmission by M. sacchari has been studied in different countries. SCYLV has a limited natural host range and mainly infect sugarcane (Sachharum hybrid), grain sorghum (Sorghum bicolor), and Columbus grass (Sorghum almum). Recent insights in the protein-protein interactions of Polerovirus through protein interaction reporter (PIR) technology enable us to understand viral encoded proteins during virus replication, assembly, plant defence mechanism, short and long-distance travel of the virus. This review presents the recent understandings on virus biology, diagnosis, genetic diversity, virus-vector and host-virus interactions and conventional and next generation management approaches.

English

Carbapenem-resistant Escherichia coli (CRE) with a multidrug resistant phenotype was isolated from four clinically ill dogs treated with meropenem in different local animal hospitals between 2017 and 2019. IncX3-type plasmids of ca. 46 kb in size carrying blaNDM-5 were present in all CRE strains and their transconjugants. High genetic similarity (>90%) by PFGE analysis was observed among the CRE strains, which were identified as ST410.To the best of our knowledge, blaNDM-5-producing E. coli ST410 clones are emerging sporadically in companion dogs treated with meropenem. The spread of Enterobacteriaceae harboring the NDM-5 gene in companion animals can pose a threat to public health; therefore, extensive monitoring in veterinary hospitals using carbapenem and careful antibiotic use are crucial for managing and monitoring these resistant strains

Integration of Hi-C and Nanopore Sequencing for Structural Variant Analysis in AML with a Complex Karyotype: (Chromothripsis)²

Background. Acute myeloid leukemia (AML) with a complex karyotype (CK-AML) is an AML subtype with a still dismal outcome despite recent therapeutic advances. The prognosis is even worse when the underlying structural variants (SVs) lead to an extremely complex pattern of rearrangements, called chromothripsis, with a median overall survival of only 120 days. Except for the presence of inactivating TP53 aberrations in about 70% of all AML-CK cases, the pathogenesis is poorly understood. To gain novel insights into the molecular mechanisms underlying CK-AML reliable high precision SV delineation is needed, which so far has been a major limitation in cancer research. Aim. We developed a SV detection pipeline by integrating Oxford Nanopore Technology (ONT) based whole genome sequencing (WGS) and Hi-C sequencing. This pipeline generated precise characterization of SVs for which the impact on gene expression and the emergence of novel fusion genes was studied by RNA-seq and ONT transcriptome sequencing. Patients and Methods. We applied our WGS and Hi-C SV detection pipeline to a cohort of 11 AML-CK cases. Nanopore DNA Sequencing was performed until a genomic coverage >10x per patient was reached. The samples of 9 patients were also subjected to Nanopore cDNA sequencing for fusion gene analysis and Illumina based RNA-seq for transcript quantification. As controls for Hi-C and Illumina RNA sequencing, CD34+ hematopoietic stem cell enriched samples from five healthy donors were used. Results. Our SV detection pipeline enabled us to fully reconstruct the derivate chromosome structure even of very complex, chromothriptic rearrangements in CK-AML. This enabled us to identify features of chromothripsis, that could previously not be detected using conventual technologies. We found local clustering of breakpoints in three of the patients with up to 31 Inversions and Translocations located in a genomic region of just 2.7 kb. These breakpoints were present in the Hi-C as well as in our Nanopore SV dataset. Our SV pipeline also showed that in these highly clustered regions, the very small rejoined fragments (in many cases less than 1 kb in size) often showed an elevated copy number (CN) state, i.e. small amplifications. We termed this newly discovered phenomenon chromothripsis-in-chromothripsis or (chromothripsis)². The precise knowledge about these breakpoints, which were validated by two different technologies, enabled us to study the pathogenesis of CK-AML at a so far unprecedented resolution. Fusion transcripts could be very precisely mapped and the impact of the breakpoints and CN changes on gene expression levels could be validated, thereby indicating functional relevance of the respective aberrations. Conclusions. The combination of Hi-C and long-read sequencing for SV detection proved to be a powerful tool for precise SV detection. Our SV pipeline allowed us to discover a new level of complexity in chromothripsis. Application of this pipeline to leukemias as well as other types of cancer can improve the precision of SV detection, thereby raising new opportunities for functional interpretation of complex genomic aberrations of pathogenic relevance. Disclosures Döhner: Sunesis Pharmaceuticals: Research Funding; Astex Pharmaceuticals: Consultancy; Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Arog: Research Funding; Roche: Consultancy; Novartis: Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Abbvie: Consultancy; Agios: Consultancy; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Astellas Pharma: Consultancy; Celgene: Consultancy, Honoraria. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Bullinger:Amgen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees.

Mitochondrial Genome Evolution of Placozoans: Gene Rearrangements and Repeat Expansions.

Placozoans, nonbilaterian animals with the simplest known metazoan bauplan, are currently classified into 20 haplotypes belonging to three genera, Polyplacotoma, Trichoplax, and Hoilungia. The latter two comprise two and five clades, respectively. In Trichoplax and Hoilungia, previous studies on six haplotypes belonging to four different clades have shown that their mtDNAs are circular chromosomes of 32–43 kb in size, which encode 12 protein-coding genes, 24 tRNAs, and two rRNAs. These mitochondrial genomes (mitogenomes) also show unique features rarely seen in other metazoans, including open reading frames (ORFs) of unknown function, and group I and II introns. Here, we report seven new mitogenomes, covering the five previously described haplotypes H2, H17, H19, H9, and H11, as well as two new haplotypes, H23 (clade III) and H24 (clade VII). The overall gene content is shared between all placozoan mitochondrial genomes, but genome sizes, gene orders, and several exon–intron boundaries vary among clades. Phylogenomic analyses strongly support a tree topology different from previous 16S rRNA analyses, with clade VI as the sister group to all other Hoilungia clades. We found small inverted repeats in all 13 mitochondrial genomes of the Trichoplax and Hoilungia genera and evaluated their distribution patterns among haplotypes. Because Polyplacotoma mediterranea (H0), the sister to the remaining haplotypes, has a small mitochondrial genome with few small inverted repeats and ORFs, we hypothesized that the proliferation of inverted repeats and ORFs substantially contributed to the observed increase in the size and GC content of the Trichoplax and Hoilungia mitochondrial genomes.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

Biology and Physics of Heterochromatin-Like Domains/Complexes.

The hallmarks of constitutive heterochromatin, HP1 and H3K9me2/3, assemble heterochromatin-like domains/complexes outside canonical constitutively heterochromatic territories where they regulate chromatin template-dependent processes. Domains are more than 100 kb in size; complexes less than 100 kb. They are present in the genomes of organisms ranging from fission yeast to human, with an expansion in size and number in mammals. Some of the likely functions of domains/complexes include silencing of the donor mating type region in fission yeast, preservation of DNA methylation at imprinted germline differentially methylated regions (gDMRs) and regulation of the phylotypic progression during vertebrate development. Far cis- and trans-contacts between micro-phase separated domains/complexes in mammalian nuclei contribute to the emergence of epigenetic compartmental domains (ECDs) detected in Hi-C maps. A thermodynamic description of micro-phase separation of heterochromatin-like domains/complexes may require a gestalt shift away from the monomer as the “unit of incompatibility” that determines the sign and magnitude of the Flory–Huggins parameter, χ. Instead, a more dynamic structure, the oligo-nucleosomal “clutch”, consisting of between 2 and 10 nucleosomes is both the long sought-after secondary structure of chromatin and its unit of incompatibility. Based on this assumption we present a simple theoretical framework that enables an estimation of χ for domains/complexes flanked by euchromatin and thereby an indication of their tendency to phase separate. The degree of phase separation is specified by χN, where N is the number of “clutches” in a domain/complex. Our approach could provide an additional tool for understanding the biophysics of the 3D genome.

Draft genome sequence of a blaNDM-1- and blaOXA-244-carrying multidrug-resistant Escherichia coli D-ST69 clinical isolate from Egypt

ObjectivesThis study describes the first draft genome sequence of a multidrug-resistant (MDR) Escherichia coli D-ST69 clinical isolate from Egypt carrying blaNDM-1 and blaOXA-244. MethodsThe strain was isolated in December 2014 from a wound pus swab of a male patient in the city of Kafr El-Sheikh using MacConkey agar containing 2 μg/mL meropenem. The strain was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole-genome sequencing using an Illumina MiSeq platform. ResultsThe draft genome of the strain (HR14_AS) was 5.08 Mbp in size containing a total of 90 contigs encoding 4677 predicted genes with an average G+C content of 50.7%. Strain HR14_AS belongs to sequence type 69 (ST69), phylogroup D and exhibits an MDR phenotype, with minimum inhibitory concentrations (MICs) of 64 μg/mL and 32 μg/mL for meropenem and doripenem, respectively. Multiple acquired antimicrobial resistance genes conferring resistance to macrolides [mdf(A)], fluoroquinolones [aac(6')-Ib-cr], quinolones (qnrS1), trimethoprim (dfrA14), β-lactams (blaNDM-1, blaOXA-244, blaCTX-M-15, blaOXA-9 and blaTEM-1B) and aminoglycosides [aac(3)-IId, aac(6')-Ib, aadA1 and aph(3')-VI] were detected. The blaOXA-244 and blaNDM-1 genes were located on the chromosome (Tn6237) and on an IncI1-type self-conjugative plasmid of >93 kb in size, respectively. ConclusionsHere we report the first draft genome sequence of a MDR E. coli D-ST69 isolate carrying blaNDM-1 and blaOXA-244. Besides clonal expansion of the E. coli ST38 pandemic clone, this study further identified that the spread of OXA-244-producing E. coli could be related to mobilisation of the IS1R-made composite transposon (Tn6237) carrying blaOXA-244.