Capture of armA by a novel ISCR element, ISCR28.

ISCR28 is a fully functional and active member of the IS91-like family of insertion sequences. ISCR28 is 1,708-bp long and contains a 1,293-bp long putative open reading frame that codes a transposase. Sixty ISCR28-containing sequences from GenBank generated 27 non-repeat genetic contexts, all of which represented naturally occurring biological events that had occurred in a wide range of gram-negative organisms. Insertion of ISCR28 into target DNA preferred the presence of a 5'-GXXT-3' sequence at its terIS (replication terminator) end. Loss of the first 4 bp of its oriIS (origin of replication) likely caused ISCR28 to be trapped in ISApl1-based transposons or similar structures. Loss of terIS and fusion with a mobile element upstream likely promoted co-transfer of ISCR28 and the downstream resistance genes. ArmA and its downstream intact ISCR28 can be excised from recombinant pKD46 plasmids forming circular intermediates, further elucidating its activity as a transposase.

Phylogeny of Transferable Oxazolidinone Resistance Genes and Homologs.

Oxazolidinone resistance, especially transmissible resistance, is a major public health concern, and the origin of this resistance mechanism is not yet resolved. This study aims to delve into the phylogenetic origin of the transmissible oxazolidinone resistance mechanisms conferring cross-resistance to other drugs of human and veterinary importance. The amino acid sequences of the five cfr ribosomal methylases and optrA and poxtA were used as queries in searches against 219,549 bacterial proteomes in the NCBI RefSeq database. Hits with >40% amino acid identity and >80% query coverage were aligned, and phylogenetic trees were reconstructed. All five cfr genes yielded highly similar trees, with rlmN housekeeping ribosomal methylases located basal to the sister groups of S-adenosyl-methionine-dependent methyltransferases from various Deltaproteobacteria and Actinomycetia, including antibiotic-producing Streptomyces species, and the monophyletic group of cfr genes. The basal branches of the latter contained paenibacilli and other soil bacteria; they then could be split into the clades [cfr(C):cfr(E)] and [[cfr:cfr(B)]:cfr(D)], always with different Bacillaceae in their stems. Lachnospiraceae were encountered in the basal branches of both optrA and poxtA trees. The ultimate origin of the cfr genes is the rlmN housekeeping ribosomal methylases, which evolved into a suicide-avoiding methylase in antibiotic producers; a soil organism (Lachnospiraceae, Paenibacilli) probably acted as a transfer organism into pathogenic bacteria. In the case of optrA, the porcine pathogenic Streptococcus suis was present in all branches, while the proteins closest to poxtA originated from Clostridia.

Molecular Analysis of Carbapenem and Aminoglycoside Resistance Genes in Carbapenem-Resistant Pseudomonas aeruginosa Clinical Strains: A Challenge for Tertiary Care Hospitals.

Carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa) strains have become a global threat due to their remarkable capability to survive and disseminate successfully by the acquisition of resistance genes. As a result, the treatment strategies have been severely compromised. Due to the insufficient available data regarding P. aeruginosa resistance from Pakistan, we aimed to investigate the resistance mechanisms of 249 P. aeruginosa strains by antimicrobial susceptibility testing, polymerase chain reaction for the detection of carbapenemases, aminoglycoside resistance genes, extended-spectrum beta-lactamases (ESBLs), sequence typing and plasmid typing. Furthermore, we tested silver nanoparticles (AgNPs) to evaluate their in vitro sensitivity against antimicrobial-resistant P. aeruginosa strains. We observed higher resistance against antimicrobials in the general surgery ward, general medicine ward and wound samples. Phenotypic carbapenemase-producer strains comprised 80.7% (201/249) with 89.0% (179/201) demonstrating genes encoding carbapenemases: blaNDM-1 (32.96%), blaOXA48 (37.43%), blaIMP (7.26%), blaVIM (5.03%), blaKPC-2 (1.12%), blaNDM-1/blaOXA48 (13.97%), blaOXA-48/blaVIM (1.68%) and blaVIM/blaIMP (0.56%). Aminoglycoside-modifying enzyme genes and 16S rRNA methylase variants were detected in 43.8% (109/249) strains: aac(6')-lb (12.8%), aac(3)-lla (12.0%), rmtB (21.1%), rmtC (11.0%), armA (12.8%), rmtD (4.6%), rmtF (6.4%), rmtB/aac(3)-lla (8.2%), rmtB/aac(6')-lla (7.3%) and rmtB/armA (3.6%). In total, 43.0% (77/179) of the strains coharbored carbapenemases and aminoglycoside resistance genes with 83.1% resistant to at least 1 agent in 3 or more classes and 16.9% resistant to every class of antimicrobials tested. Thirteen sequence types (STs) were identified: ST235, ST277, ST234, ST170, ST381, ST175, ST1455, ST1963, ST313, ST207, ST664, ST357 and ST348. Plasmid replicon types IncFI, IncFII, IncA/C, IncL/M, IncN, IncX, IncR and IncFIIK and MOB types F11, F12, H121, P131 and P3 were detected. Meropenem/AgNPs and Amikacin/AgNPs showed enhanced antibacterial activity. We reported the coexistence of carbapenemases and aminoglycoside resistance genes among carbapenem-resistant P. aeruginosa with diverse clonal lineages from Pakistan. Furthermore, we highlighted AgNP's potential role in handling future antimicrobial resistance concerns.

Molecular epidemiological characteristics of carbapenem-resistant Pseudomonas aeruginosa clinical isolates in southeast Shanxi, China.

OBJECTIVES: Infection by carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a serious clinical problem worldwide. However, the molecular epidemiology of the clinical isolates varies depending on the region. This study was conducted to analyse the resistance phenotype and clarify the genetic and epidemiological properties of CRPA clinical isolates from southeast Shanxi, China. METHODS: Fifty-seven isolates of CRPA were collected from a hospital in this region. These isolates were reidentified by MALDI-TOF and subjected to whole-genome sequencing by next-generation sequencing. Phylogenetic trees were constructed based on single nucleotide polymorphisms (SNPs), after which multilocus sequence typing (MLST) was performed and antimicrobial resistance genes were identified. RESULTS: All the 57 CRPA isolates carried at least one kind of gene encoding carbapenemase, such as blaIMP-1, blaIMP-10, blaOXA-10, blaOXA-395, blaOXA-396, blaOXA-485, blaOXA-486, blaOXA-488, blaOXA-494, and blaOXA-50. The isolates harboured AIM-1, CMY-51, mecD, and NmcR genes and carried one kind of Pseudomonas-derived cephalosporinase (PDC) ß-lactamase-encoding gene, such as blaPCD-1 to blaPCD-3, blaPCD-5, or blaPCD-7 to blaPCD-10. Two isolates were found to harbour the aminoglycoside-modifying enzyme genes aadA1 and aadA7; however, no isolates were found to harbour genes encoding 16S rRNA methylase or quinolone resistance-related genes. These CRPA isolates belonged to various sequence types (STs), two of which, namely, ST235 and ST277, were high-risk types. CONCLUSIONS: Our findings indicate that CRPA isolates carrying resistance genes with unique regional characteristics are spreading in this region, with a high diversity of STs, especially in high-risk clones. These findings highlight the necessity for further measures to prevent CRPA spread in Shanxi.

SETting up the genome: KMT2D and KDM6A genomic function in the Kabuki syndrome craniofacial developmental disorder.

BACKGROUND: Kabuki syndrome is a congenital developmental disorder that is characterized by distinctive facial gestalt and skeletal abnormalities. Although rare, the disorder shares clinical features with several related craniofacial syndromes that manifest from mutations in chromatin-modifying enzymes. Collectively, these clinical studies underscore the crucial, concerted functions of chromatin factors in shaping developmental genome structure and driving cellular transcriptional states. Kabuki syndrome predominantly results from mutations in KMT2D, a histone H3 lysine 4 methylase, or KDM6A, a histone H3 lysine 27 demethylase. AIMS: In this review, we summarize the research efforts to model Kabuki syndrome in vivo to understand the cellular and molecular mechanisms that lead to the craniofacial and skeletal pathogenesis that defines the disorder. DISCUSSION: As several studies have indicated the importance of KMT2D and KDM6A function through catalytic-independent mechanisms, we highlight noncanonical roles for these enzymes as recruitment centers for alternative chromatin and transcriptional machinery.

Crucial roles of trehalose and 5-azacytidine in alleviating salt stress in tomato: Both synergistically and independently.

Trehalose may improve plant stress tolerance by regulating gene expression under different abiotic stresses. DNA methylation is involved in plant growth and development, but also in response to abiotic stresses. 5-azacytidine is a widely used inhibitor of DNA methylation. In this study, tomato (Solanum lycopersicum L. 'Ailsa Craig') was used as experimental material to explore the effects of trehalose and DNA methylation on the growth and development in tomato seedlings under salt stress. 10 mM trehalose, 50 µM 5-azacytidine, and their combined treatments could significantly increase growth parameters in tomato under salt stress, indicating trehalose and 5-azacytidine might play crucial roles in alleviating salt stress both synergistically and independently. Additionally, trehalose significantly down-regulated the expression of DNA methylase genes (SlDRM5, SlDRM1L1, SlCMT3 and SlCMT2) and up-regulated the expression of DNA demethylases genes under salt stress, suggesting that trehalose might regulate DNA methylation under salt stress condition. Under salt stress, trehalose and 5-azacytidine treatments enhanced antioxidant enzyme activity and induced antioxidant enzyme gene expression in tomato seedlings. Meanwhile, trehalose and 5-azacytidine increased ABA content by regulating the expression of ABA metabolism-related genes, thereby enhancing salt tolerance in tomato. Altogether, these results suggest that trehalose conferred salt tolerance in tomato seedlings probably by DNA demethylation and enhancing antioxidant capability and ABA accumulation.

Klebsiella pneumoniae co-harbouring bla NDM-1 , armA and mcr-10 isolated from blood samples in Myanmar.

Background. The spread of Enterobacteriaceae coproducing carbapenemases, 16S rRNA methylase and mobile colistin resistance proteins (MCRs) has become a serious public health problem worldwide. This study describes two clinical isolates of Klebsiella pneumoniae coharbouring bla IMP-1, armA and mcr-10.Methods. Two clinical isolates of K. pneumoniae resistant to carbapenems and aminoglycosides were obtained from two patients at a hospital in Myanmar. Their minimum inhibitory concentrations (MICs) were determined by broth microdilution methods. The whole-genome sequences were determined by MiSeq and MinION methods. Drug-resistant factors and their genomic environments were determined.Results. The two K. pneumoniae isolates showed MICs of ≥4 and ≥1024 µg ml-1 for carbapenems and aminoglycosides, respectively. Two K. pneumonaie harbouring mcr-10 were susceptible to colistin, with MICs of ≤0.015 µg ml-1 using cation-adjusted Mueller-Hinton broth, but those for colistin were significantly higher (0.5 and 4 µg ml-1) using brain heart infusion medium. Whole-genome analysis revealed that these isolates coharboured bla NDM-1, armA and mcr-10. These two isolates showed low MICs of 0.25 µg ml-1 for colistin. Genome analysis revealed that both bla NDM-1 and armA were located on IncFIIs plasmids of similar size (81 kb). The mcr-10 was located on IncM2 plasmids of sizes 220 or 313 kb in each isolate. These two isolates did not possess a qseBC gene encoding a two-component system, which is thought to regulate the expression of mcr genes.Conclusion. This is the first report of isolates of K. pneumoniae coharbouring bla NDM-1, armA and mcr-10 obtained in Myanmar.

Genome-wide identification and comparative analysis of DNA methyltransferase and demethylase gene families in two ploidy Cyclocarya paliurus and their potential function in heterodichogamy.

BACKGROUND: DNA methylation is one of the most abundant epigenetic modifications, which plays important roles in flower development, sex differentiation, and regulation of flowering time. Its pattern is affected by cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase). At present, there are no reports on C5-MTase and dMTase genes in heterodichogamous Cyclocarya paliurus. RESULTS: In this study, 6 CpC5-MTase and 3 CpdMTase genes were identified in diploid (2n = 2 × = 32) C. paliurus, while 20 CpC5-MTase and 13 CpdMTase genes were identified in autotetraploid (2n = 4 × = 64). 80% of identified genes maintained relatively fixed positions on chromosomes during polyploidization. In addition, we found that some DRM subfamily members didn't contain the UBA domain. The transcript abundance of CpC5-MTase and CpdMTase in male and female flowers of two morphs (protandry and protogyny) from diploidy was analyzed. Results showed that all genes were significantly up-regulated at the stage of floral bud break (S2), but significantly down-regulated at the stage of flower maturation (S4). At S2, some CpC5-MTase genes showed higher expression levels in PG-M than in PG-F, whereas some CpdMTase genes showed higher expression levels in PA-M than in PA-F. In addition, these genes were significantly associated with gibberellin synthesis-related genes (e.g. DELLA and GID1), suggesting that DNA methylation may play a role in the asynchronous floral development process through gibberellin signal. CONCLUSIONS: These results broaden our understanding of the CpC5-MTase and CpdMTase genes in diploid and autotetraploid C. paliurus, and provide a novel insight into regulatory mechanisms of DNA methylation in heterodichogamy.

The extremophile Picrophilus torridus carries a DNA adenine methylase M.PtoI that is part of a Type I restriction-modification system.

DNA methylation events mediated by orphan methyltransferases modulate various cellular processes like replication, repair and transcription. Bacteria and archaea also harbor DNA methyltransferases that are part of restriction-modification systems, which serve to protect the host genome from being cleaved by the cognate restriction enzyme. While DNA methylation has been exhaustively investigated in bacteria it remains poorly understood in archaea. Picrophilus torridus is a euryarchaeon that can thrive under conditions of extremely low pH (0.7), and thus far no reports have been published regarding DNA methylation in this extremophile. This study reports the first experimentation examining DNA methylation in P. torridus. We find the genome to carry methylated adenine (m6A) but not methylated cytosine (m5C) residues. The m6A modification is absent at GATC sites, indicating the absence of an active Dam methylase even though the dam gene has been annotated in the genome sequence. Two other methylases have also been annotated in the P. torridus genome sequence. One of these is a part of a Type I restriction-modification system. Considering that all Type I modification methylases characterized to date target adenine residues, the modification methylase of this Type I system has been examined. The genes encoding the S subunit (that is responsible for DNA recognition) and M subunit (that is responsible for DNA methylation) have been cloned and the recombinant protein purified from E.coli, and regions involved in M-S interactions have been identified. The M.PtoI enzyme harbors all the motifs that typify Type I modification methylases, and displays robust adenine methylation in in vitro assays under a variety of conditions. Interestingly, magnesium is essential for enzyme activity. The enzyme displays substrate inhibition at higher concentrations of AdoMet. Mutational analyses reveal that Motif I plays a role in AdoMet binding, and Motif IV is critical for methylation activity. The data presented here lays the foundation for further research in the area of DNA methylation and restriction-modification research in this most unusual microorganism.

The Characterization and Differential Analysis of m6A Methylation in Hycole Rabbit Muscle and Adipose Tissue and Prediction of Regulatory Mechanism about Intramuscular Fat.

N6-methyladenosine (m6A) widely participates in various life processes of animals, including disease, memory, growth and development, etc. However, there is no report on m6A regulating intramuscular fat deposition in rabbits. In this study, m6A modification of Hycole rabbit muscle and adipose tissues were detected by MeRIP-Seq. In this case, 3 methylases and 12 genes modified by m6A were found to be significantly different between muscle and adipose tissues. At the same time, we found 3 methylases can regulate the expression of 12 genes in different ways and the function of 12 genes is related to fat deposition base on existing studies. 12 genes were modified by m6A methylase in rabbit muscle and adipose tissues. These results suggest that 3 methylases may regulate the expression of 12 genes through different pathways. In addition, the analysis of results showed that 6 of the 12 genes regulated eight signaling pathways, which regulated intramuscular fat deposition. RT-qPCR was used to validate the sequencing results and found the expression results of RT-qPCR and sequencing results are consistent. In summary, METTL4, ZC3H13 and IGF2BP2 regulated intramuscular fat by m6A modified gene/signaling pathways. Our work provided a new molecular basis and a new way to produce rabbit meat with good taste.

Emergence of NDM-1-producing Pseudomonas aeruginosa Sequence Type 773 Clone: Shift of Carbapenemase Molecular Epidemiology and Spread of 16S rRNA Methylase Genes in Korea.

Imipenemase (IMP)-6-producing Pseudomonas aeruginosa sequence type (ST) 235 is a dominant clone of carbapenemase-producing P. aeruginosa (CPPAE) in Korea. As part of the Antimicrobial Resistance Surveillance System in Korea, we found an increase in the carbapenem resistance rate of P. aeruginosa isolates from blood cultures and a shift in the molecular epidemiology of CPPAE. A total of 212 non-duplicated P. aeruginosa blood isolates were obtained from nine general hospitals and two nursing homes. Twenty-four isolates were identified as CPPAE. We observed the emergence of the NDM-1 P. aeruginosa ST 773 clone (N=10), mostly from Gyeongsang Province. The IMP-6 ST 235 clone (N=11) was detected in all provinces. CPPAE isolates showed very high resistance rates to amikacin, and all NDM-1 P. aeruginosa strains carried rmtB. This is the first nationwide surveillance of the recently emerged NDM-1-producing P. aeruginosa ST773 clone in Korea. Continuous surveillance is necessary to prevent the infection and transmission of carbapenem- and amikacin-resistant P. aeruginosa in Korea.

Epigenetic regulation of LINC01270 in breast cancer progression by mediating LAMA2 promoter methylation and MAPK signaling pathway.

Application of long non-coding RNAs (lncRNAs) for modulation of breast cancer (BC) has attracted much attention. Here, we probed into the role and underlying mechanism of long intergenic non-coding RNA 01270 (LINC01270) in BC. With the help of bioinformatics tools, we identified laminin subunit alpha 2 (LAMA2) as a BC-related differentially expressed gene to discern the effect of LAMA2 in BC cells. LAMA2 was initially poorly expressed while LINC01270 was highly expressed in BC. BC cells were subsequently treated with sh-LINC01270 or/and sh-LAMA2 for exploration of their regulatory mechanism in BC, which unfolded that LINC01270 inhibition up-regulated LAMA2 and inactivated the MAPK signaling pathway to suppress malignant characteristics of BC cells. Functional assays demonstrated that LINC01270 bound to DNMT1, DNMT3a, and DNMT3b promoted the methylation of CpG islands in LAMA2 promoter and inhibited the LAMA2 expression. Moreover, our data suggested that LAMA2 suppressed MAPK signaling pathway to inhibit BC cell malignant characteristics. The in vitro results were re-produced with the help of the in vivo experimentations. In conclusion, LINC01270 silencing inhibited the methylation of LAMA2 promoter to suppress the activation of MAPK signaling pathway, which subsequently restrained the BC progression. 1, Overexpression of LAMA2 inhibits malignant features of BC cells. 2, LINC01270 promotes LAMA2 promoter methylation by recruiting DNMTs to the LAMA2 promoter region. 3, 5-aza-dc reverses the promotion of LAMA2 promoter methylation by LINC01270. 4, LAMA2 inhibits malignant features of BC cells by suppressing the activation of MAPK signaling pathway.

Brazilian Butt Lift Gone Wrong: A Case Series of Non-Tuberculous Mycobacterial Gluteal Infection After Brazilian Butt Lifts.

Cosmetic surgeries are very popular and glamorized by the mainstream media and celebrities. Many individuals perceive certain bodily features as appealing for physical attraction and will attempt to obtain these features by surgery. However, these surgeries are not without risk, and significant consequences can occur if not performed by qualified medical professionals under sterile procedures. The authors present novel cases of two healthy young female patients who underwent a Brazilian butt lift (BBL) procedure a week apart by the same plastic surgeon in Mexico and developed dark painful lesions secondary to Mycobacterium abscessus (M. abscessus), a multidrug-resistant non-tuberculous mycobacterium (NTM). The literature review shows a paucity of data concerning NTM infections via surgical procedures of this type. The first case was of a 31-year-old woman who underwent a BBL and presented with bilateral dark painful buttock lesions weeks later. The patient returned to the plastic surgeon, who drained some lesions and prescribed oral antibiotics. The patient's clinical status continued to deteriorate and presented to the hospital for further assessment. The patient was initially started on broad-spectrum antibiotic therapy. The patient was found to have an HIV infection with a relatively preserved CD4 lymphocyte count and was started on antiretroviral therapy (ART). Intraoperative excisional tissue sample cultures grew M. abscessus. The patient was started on empiric tigecycline, cefoxitin, and linezolid. Preliminary culture susceptibilities showed resistance to linezolid. Linezolid was discontinued, amikacin was started, and cefoxitin and tigecycline were continued. Tigecycline, cefoxitin, and amikacin were continued and final susceptibilities showed sensitivity to the current treatment. The patient received a total of four months of treatment with tigecycline, cefoxitin, and amikacin. The second case was of a 28-year-old woman who underwent a BBL a week after the first patient by the same surgeon and developed multiple gluteal and body abscesses. The patient underwent bilateral thigh and gluteal, right chest wall, and breast surgical debridements with intraoperative cultures at a different hospital facility, which grew M. abscessus. Susceptibilities were not performed there. The patient was transferred to our facility for further care. Intraoperative cultures remained negative, and the patient was treated with a six-month course of tigecycline, cefoxitin, and amikacin.

Prokaryotic expression,purification and activity of CamA protein with methylase activity from Clostridiodies difficile / 中国生物制品学杂志

@#Objective To express CamA(Clostridiodies difficile adenine methltransferase A,CamA)protein with methylase activity in prokaryotic expression system.Methods The gene sequence of CamA protein of the standard strain of C.difficile1870 was amplified by PCR,cloned into plasmid pGEX-4T-1-MBP,transformed into E.coli HB101 and induced by 1 mmol/L IPTG to express the recombinant target protein. After purification with Amylose Resin,CamA protein was digested by tobacco etch virus(TEV)protease,and verified for its methylase activity using MTase-Glo~(TM)Methyltransferase Assay in vitro.Results PCR sequencing showed that the cloned CamA gene sequence was correct,in which no base mutation occurred. The recombinant expression plasmid pGEX-4T-1-MBP-CamA was digested by EcoRⅠand BamHⅠ,which showed that 1 731 bp of CamA gene was connected to the vector plasmid. The relative molecular mass of the recombinant protein was about 108 800(with 1 MBP tag),and the purity was over 90% after purified with Amylose Resin. Under the condition of20 μmol/L DNA and 20 μmol/L SAM at room temperature for 30 min,0. 5 μg CamA produced SAH at a concentration of about 101. 60 nmol/L and the enzyme activity was(0. 339 ± 0. 027)U/mg.Conclusion The CamA protein of C.difficile has been successfully expressed and purified with methylase activity,which lays a foundation of further study on the function of CamA and its role in the occurrence,development and treatment of C.difficile infection.

Enhanced Fusobacterium nucleatum Genetics Using Host DNA Methyltransferases To Bypass Restriction-Modification Systems.

Bacterial restriction-modification (R-M) systems are a first-line immune defense against foreign DNA from viruses and other bacteria. While R-M systems are critical in maintaining genome integrity, R-M nucleases unfortunately present significant barriers to targeted genetic modification. Bacteria of the genus Fusobacterium are oral, Gram-negative, anaerobic, opportunistic pathogens that are implicated in the progression and severity of multiple cancers and tissue infections, yet our understanding of their direct roles in disease have been severely hindered by their genetic recalcitrance. Here, we demonstrate a path to overcome these barriers in Fusobacterium by using native DNA methylation as a host mimicry strategy to bypass R-M system cleavage of transformed plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum type II and III DNA methyltransferase (MTase) enzymes to produce a multifold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726, as well as the first system for efficient gene knockouts and complementations in F. nucleatum subsp. nucleatum 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an Escherichia coli host that constitutively expresses F. nucleatum subsp. nucleatum MTase enzymes. In summary, this proof-of-concept study characterizes specific MTases that are critical for bypassing R-M systems and has enhanced our understanding of enzyme combinations that could be used to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. IMPORTANCE Fusobacterium nucleatum is an oral opportunistic pathogen associated with diseases that include cancer and preterm birth. Our understanding of how this bacterium modulates human disease has been hindered by a lack of genetic systems. Here, we show that F. nucleatum DNA methyltransferase-modified plasmid DNA overcomes the transformation barrier and has allowed the development of a genetic system in a previously inaccessible strain. We present a strategy that could potentially be expanded to enable the genetic modification of highly recalcitrant strains, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium.

Cytosine Methylation in Genomic DNA and Characterization of DNA Methylases and Demethylases and Their Expression Profiles in Viroid-Infected Hop Plants (Humulus lupulus Var. 'Celeia').

Abiotic and biotic stresses can lead to changes in host DNA methylation, which in plants is also mediated by an RNA-directed DNA methylation mechanism. Infections with viroids have been shown to affect DNA methylation dynamics in different plant hosts. The aim of our research was to determine the content of 5-methylcytosine (5-mC) in genomic DNA at the whole genome level of hop plants (Humulus lupulus Var. 'Celeia') infected with different viroids and their combinations and to analyse the expression of the selected genes to improve our understanding of DNA methylation dynamics in plant-viroid systems. The adapted HPLC-UV method used proved to be suitable for this purpose, and thus we were able to estimate for the first time that the cytosine methylation level in viroid-free hop plants was 26.7%. Interestingly, the observed 5-mC level was the lowest in hop plants infected simultaneously with CBCVd, HLVd and HSVd (23.7%), whereas the highest level was observed in plants infected with HLVd (31.4%). In addition, we identified three DNA methylases and one DNA demethylase gene in the hop's draft genome. The RT-qPCR revealed upregulation of all newly identified genes in hop plants infected with all three viroids, while no altered expression was observed in any of the other hop plants tested, except for CBCVd-infected hop plants, in which one DNA methylase was also upregulated.

Targeting Histone Epigenetic Modifications and DNA Damage Responses in Synthetic Lethality Strategies in Cancer?

Synthetic lethality strategies are likely to be integrated in effective and specific cancer treatments. These strategies combine different specific targets, either in similar or cooperating pathways. Chromatin remodeling underlies, directly or indirectly, all processes of tumor biology. In this context, the combined targeting of proteins associated with different aspects of chromatin remodeling can be exploited to find new alternative targets or to improve treatment for specific individual tumors or patients. There are two major types of proteins, epigenetic modifiers of histones and nuclear or chromatin kinases, all of which are druggable targets. Among epigenetic enzymes, there are four major families: histones acetylases, deacetylases, methylases and demethylases. All these enzymes are druggable. Among chromatin kinases are those associated with DNA damage responses, such as Aurora A/B, Haspin, ATM, ATR, DNA-PK and VRK1-a nucleosomal histone kinase. All these proteins converge on the dynamic regulation chromatin organization, and its functions condition the tumor cell viability. Therefore, the combined targeting of these epigenetic enzymes, in synthetic lethality strategies, can sensitize tumor cells to toxic DNA-damage-based treatments, reducing their toxicity and the selective pressure for tumor resistance and increasing their immunogenicity, which will lead to an improvement in disease-free survival and quality of life.

Fibrillarin RNA methylase is an interacting protein of Cryptosporidium parvum calmodulin-like protein (CpCML).

Cryptosporidium parvum is an obligate protozoan parasite invading epithelial cells of small intestine of human and animals, and causing diarrheal disease. In apicomplexan parasites, calcium signaling can regulate many essential biological processes such as invasion and migration. As the main intracellular receptor for calcium ions, calmodulins control the activities of hundreds of enzymes and proteins. Calmodulin-like protein (CML) is an important member of the calmodulin family and may play a key role in C. parvum, however, the actual situation is still not clear. The present study aimed to identify the parasite interaction partner proteins of C. parvum calmodulin-like protein (CpCML). By constructing the cpcml bait plasmid, 5 potential CpCML - interacting proteins in C. parvum oocyst were screened by yeast-two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) were performed as subsequent validations. Fibrillarin RNA methylase (FBL) was identified via this screening method as CpCML interacting protein in C. parvum. The identification of this interaction made it possible to get a further understanding of the function of CpCML and its contribution to the pathogenicity of C. parvum.

Spread of Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates Producing NDM-Type Metallo-ß-Lactamase in Myanmar.

A total of 38 isolates of carbapenem-resistant Klebsiella pneumoniae harboring blaNDM were obtained during surveillance of 10 hospitals in Myanmar. Of these 38 isolates, 19 (50%) harbored genes encoding 16S rRNA methylases, such as armA or rmtB. The K. pneumoniae strains tested belonged to 17 sequence types (STs), including the high-risk clonal lineages ST101 and ST147. The ST101 and ST147 isolates carried IncFII plasmids harboring blaNDM-5 and IncFIB(pQil) plasmids harboring blaNDM-1, respectively. These results indicate that IncFII plasmids harboring blaNDM-5 and IncFIB(pQil) plasmids harboring blaNDM-1 have been spreading in K. pneumoniae ST101 and ST147 isolates, respectively, in Myanmar. IMPORTANCE The emergence of carbapenem-resistant K. pneumoniae has become a serious problem in medical settings worldwide. The present study demonstrated that carbapenem-resistant K. pneumoniae strains have been spreading in medical settings in Myanmar. In particular, plasmid genes encoding NDMs and 16S rRNA methylases have been spreading in K. pneumoniae high-risk clones.

Reactive Oxygen Species Partly Mediate DNA Methylation in Responses to Different Heavy Metals in Pokeweed.

DNA methylation is a rapid response strategy promoting plant survival under heavy metal (HM) stress. However, the roles of DNA methylation underlying plant adaptation to HM stress remain largely unknown. Here, we used pokeweed, a hyperaccumulator of manganese (Mn) and cadmium (Cd), to explore responses of plant to HM stress at phenotypic, transcriptional and DNA methylation levels. Mn- and Cd-specific response patterns were detected in pokeweed. The growth of pokeweed was both inhibited with exposure to excess Mn/Cd, but pokeweed distinguished Mn and Cd with different subcellular distributions, ROS scavenging systems, transcriptional patterns including genes involved in DNA methylation, and differentially methylated loci (DML). The number of DML between Mn/Cd treated and untreated samples increased with increased Mn/Cd concentrations. Meanwhile, pretreatment with NADPH oxidase inhibitors prior to HM exposure markedly reduced HM-induced reactive oxygen species (ROS), which caused reductions in expressions of DNA methylase and demethylase in pretreated samples. The increased levels of HM-induced demethylation were suppressed with alleviated ROS stress, and a series of HM-related methylated loci were also ROS-related. Taken together, our study demonstrates that different HMs affect different DNA methylation sites in a dose-dependent manner and changes in DNA methylation under Mn/Cd stress are partly mediated by HM-induced ROS.