Digoxigenin-labeled Probes Research Articles

Northern Blotting: Protocols for Radioactive and Nonradioactive Detection of RNA.

Northern blotting is a common technique in RNA biology, allowing to detect and quantify RNAs of interest following separation by gel electrophoresis, transfer to a membrane, and hybridization of specific anti-complementary labelled probes. In this chapter, we describe our protocol for efficient RNA extraction from yeast, separation on agarose gel, and capillary transfer to a membrane. We provide two different methods for strand-specific detection of several types of RNAs using oligonucleotide probes, the first using radioactive 32P-labelled probes, the second based on nonradioactive digoxigenin-labelled probes.

Current state of in situ gene expression studiesin animal tissues

Morphological studies of farm animals are most often conducted using simple preparation and staining techniques. The study of the processes of embryogenesis, postembryonic features of the development of organs and tissues, as well as the effect of various substances remains to be elucidated not only using histochemical and immunohistochemical staining methods, but also using RNA in situ hybridization and transcriptome in situ sequencing. Aspects of many cellular and tissue processes for cattle, pigs and chickens in the context of comparative physiology have not yet been studied. The high productivity of farm animals is associated with the intensive functioning of all organs and systems of the body. The influence of the agricultural management of farm animals and its consequences on the development of an organism in ontogeny deserve a separate area of research from the point of in situ gene expression. Despite rapid development of transcriptome sequencing technologies, which result in the discovery of new candidate genes for many processes, RNA in situ hybridization remains the gold standard for their validation. This review briefly presents modern techniques and their modifications for studying in situ gene expression. Transcriptome studies that have been implemented in cattle, pigs and chickens as model organisms include: in situ RNA hybridization using ZZ-probes, tyramide signal amplification, hybridization chain reaction, digoxigenin-labeled probes, RT-PCR, single cell transcriptome sequencing, in situ transciptome sequencing. This paper is a review of the results of studies on cattle, pigs and chickens. The results of research in this area are relevant for understanding the features of adaptation mechanisms at the transcriptomic level in highly productive animals under industrial conditions in order to search for new markers of valuable agricultural traits. It should be noted that in literature there are very few studies using RNA in situ hybridization, despite the availability and simplicity of the method.

Addressing Current Challenges in Poultry Meat Safety: Development of a Cultivation and Colony Hybridization Approach to Recover Enterotoxigenic Clostridium perfringens from Broiler Chicken Carcasses.

Enterotoxigenic Clostridium perfringens is one of the main causes of foodborne illness in Canada. The use of a conventional bacterial culture approach to isolate enterotoxigenic C. perfringens from poultry meat is common. This approach is based on the phenotype attributable to a double hemolysis phenomenon, whereas few enterotoxigenic strains of C. perfringens produce it, which further complicates the study of the reservoirs of this important pathogen. The objectives of the current study were to validate the ability of a digoxigenin-labeled probe to detect the C. perfringens cpe gene and to validate the use of either a filtration or a direct plating approach, combined with colony hybridization to detect enterotoxigenic C. perfringens. Pure DNA and pure colonies of enterotoxigenic C. perfringens and broiler chicken carcass rinsate samples were subjected to colony hybridization. The results showed that the synthesized DNA probe can detect the cpe gene from both DNA and pure colonies of enterotoxigenic C. perfringens, and from colonies grown from carcass rinsates artificially contaminated with enterotoxigenic C. perfringens. Our study suggests that this isolation method is a promising tool for a better understanding of the epidemiology of this zoonotic pathogen.

Immunohistochemical Detection of the Expressed BRCA1 and BRCA2 Proteins in Microenvironment of Malignant Breast Cancerous Tissues Infected with Human Mammary Tumor Virus.

The objective of the study was to examine the prevalence of HMTV infection, its associations with breast malignant tissues, and the expression of BRCA1 and BRCA2 proteins. One hundred archival breast tissues, 40 biopsies from female patients with breast cancer (BC), and 20 healthy breast tissues from the control group were used in the study. Immunohistochemical analysis was conducted to detect the expressed BRCA1 and BRCA2 proteins. Digoxigenin-labeled HMTV probes were used in chromogenic in situ hybridization for the identification of HMTV in breast tumor tissues. The complementary sequence sites of the HMTV probe sequences were stained by NBT/BCIP as blue signals. There were 12 out of 40 (30%) benign breast tumorous tissues and 14 out of 40 (35%) BC tissues, while healthy control breast tissues were 10% (2 out of 20 tissues). Positive immunohistochemical (IHC) reactions for BRCA2 protein were observed in 12 out of 40 BC tissues (30.0%), 25% of benign breast tumorous tissues, and 5% of the control group. A significant (p < 0.05) statistical difference in the percentages of HMTV in the studied groups was found. HMTV might contribute to the development of subsets of benign and malignant breast tumors. The observed rates of defective or mutated BRCA1 and BRCA2 genes in healthy tissues indicate a role in the development of breast tumors.

First Report of Beet western yellows virus Infecting Wheat in China.

Wheat (Triticum aestivum L.) is an important cereal crop widely cultivated worldwide. Viral disease is a major threat to wheat yield. In April 2022, fifteen winter wheat plants with yellowing and stunting symptoms were collected from wheat fields in Jingjiang, Jiangsu Province. Total RNA of each sample was extracted, and RT-PCR was performed using two pairs of degenerate luteovirus primers Lu-F (5'-CCAGTGGTTRTGGTC-3') and Lu-R (5'-GTCTACCTATTTGG-3'), Leu-F (5'-GCTCTAGAATTGTTAATGARTACGGTCG-3') and Leu-R (5'-CACGCGTCN ACCTATTTNGGRTTNTG-3'). Amplicons with the expected size were obtained from 10 of the 15 samples (using primers Lu-F/Lu-R) and 3 of the 15 samples (using primers Leu-F/Leu-R), respectively. These amplicons were cloned into the pDM18-T vector (TaKaRa) for sequencing. Blastn alignment showed that 10 amplicons (531 bp) from Lu-F/Lu-R primers were essentially identical to one another, which shared 99.62% nucleotide sequence identity to barley yellow dwarf virus-PAV (BYDV-PAV) isolate GJ1 from Avena sativa in South Korea (LC550014). Three amplicons (635 bp) from Leu-F/Leu-R primers had 99.68% nucleotide identity to the corresponding part of an isolate of beet western yellows virus (BWYV) from saffron (Crocus sativus) in China (MG002646). Among the 13 virus-positive samples, none were co-infected by BYDV-PAV and BWYV. Then, amplification using BWYV-specific primers (BWYV-F: 5'-TGCTCCGGTTTTGACTGGAGTGT-3', BWYV-R: 5'-CGTCTACCT ATTTTGGGTTGTGG-3') generated a 1409 bp product, corresponding to the partial sequence of the viral RNA-dependent RNA polymerase gene and complete sequence of the coat protein (CP) gene. The sequences (GenBank accession no. ON924175) of amplicons from 3 BWYV samples were identical to one another, which shared 98.41% nucleotide identity to BWYV isolate Hs from Japanese hop (Humulus scandens) in China (KC210049). The predicted coat protein of the BWYV wheat isolate had 99.51% nucleotide and 100% amino acid identity to BWYV isolate Hs. BWYV infection on wheat samples was also verified by dot-nucleic acid hybridization using a digoxigenin-labeled cDNA probe against the CP gene, as described previously (Liu et al. 2007). Further, RNA-positive samples were tested by enzyme-linked immunosorbent assay (ELISA) using the ELISA reagent kit for BWYV (Catalog No. KS19341, Shanghai Keshun Biotech, Shanghai, China); test results were also BWYV-positive, confirming that both BWYV nucleic and coat protein are present in these wheat samples. BYDV-PAV is a common wheat virus (Chay et al. 1996), while BWYV has never been reported to infect wheat. BWYV, an aphid-transmitted virus belonging to the Polerovirus genus, has an extensive host range, including over 150 plant species from 23 dicotyledonous families, such as Beta vulgaris, Spinacia oleracea, Lactuca sativa, and Brassica oleracea var. italica (Duffus 1964, 1973; Russell 1965; Beuve et al. 2008). In addition, BWYV was reported to infect a monocotyledonous plant, Crocus sativus (Iridaceae) (Zheng et al. 2018). To our knowledge, this is the first report of BWYV in wheat or any other Gramineae crop. The result also implies that BWYV has a potential risk to cereal crops in the field.

Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues.

Porcine circovirus 3 (PCV3) was recently reported in Malaysian commercial pig population in 2020 by conventional polymerase chain reaction (PCR), revealing a molecular prevalence of 17.02% in the sampled domestic pig population. This study aims to describe a chromogenic in situ hybridization (ISH) technique using digoxigenin (DIG)-labeled cloned PCV3 open reading frame 1 (ORF1) fragment DNA to detect and localize the PCV3 antigen in formalin-fixed, paraffin-embedded lung, and lymphoid tissue specimens. Since PCV3 was mainly detected in lung and lymphoid tissues, we obtained tissue specimens from these organs from the previous Malaysian PCV3 study. Digoxigenin-labeled ISH probes were designed to target a 69 bp region of PCV3 ORF1 spanning from the nucleotide positions (282-350). Light microscopy analysis revealed that chromogenic staining of PCV3 antigens was visualized within the cytoplasm of pneumocytes and lymphocytes, indicating positive ISH results. The results of molecular detection of PCV3 using PCR and ISH showed a high agreement of 90.91%, including for the negative PCV3 status for all samples. This study reports a chromogenic ISH technique using DIG-labeled probes targeting PCV3 ORF1 to detect PCV3 antigens in lung and lymphoid tissues. Despite the limited availability of PCV3 antibodies, ISH remains relevant for investigating PCV3 replication and pathogenesis and can be used complementarily with PCR for evaluating the localization of antigens in infected tissues.

Pursuing new periodontal pathogens with an improved RNA-oligonucleotide quantification technique (ROQT)

ObjectiveThe aim of this study was to optimize the sensitivity, specificity and cost-effectiveness of the RNA-Oligonucleotide Quantification Technique (ROQT) in order to identify periodontal pathogens that remain unrecognized or uncultured in the oral microbiome. DesignTotal nucleic acids (TNA) were extracted from subgingival biofilm samples using an automated process. RNA, DNA and Locked Nucleic Acid (LNA) digoxigenin-labeled oligonucleotide probes targeting 5 cultivated/named species and 16 uncultivated or unnamed bacterial taxa were synthesized. Probe specificity was determined by targeting 96 oral bacterial species; sensitivity was assessed using serial dilutions of reference bacterial strains. Different stringency temperatures were compared and new standards were tested. The tested conditions were evaluated analyzing samples from periodontally healthy individuals, and patients with moderate or severe periodontitis. ResultsThe automated extraction method at 63⁰C along with LNA-oligunucleotides probes, and use of reverse RNA sequences for standards yielded stronger signals without cross-reactions. In the pilot clinical study, the most commonly detected uncultivated/unrecognized species were Selenomonas sp. HMT 134, Prevotella sp. HMT 306, Desulfobulbus sp. HMT 041, Synergistetes sp. HMT 360 and Bacteroidetes HMT 274. In the cultivated segment of the microbiota, the most abundant taxa were T. forsythia HMT 613 and Fretibacterium fastidiosum (formerly Synergistetes) HMT 363. ConclusionsIn general, samples from severe patients had the greatest levels of organisms. Classic (T. forsythia, P. gingivalis) and newly proposed (F. alocis and Desulfobulbus sp. HMT 041) pathogens were present in greater amounts in samples from severe periodontitis sites, followed by moderate periodontitis sites.

Fluorescent In Situ Hybridization in Embryos of the African Turquoise Killifish Nothobranchius furzeri.

Precisely where and when a given gene is expressed is crucial for our understanding of developmental and cell biology but determining this is often constrained by detection limits. Here, we describe a technique for visualization of low-copy mRNA in Nothobranchius furzeri embryos using tyramide signal amplification (TSA). In this protocol, an anti-sense digoxigenin-labeled RNA probe is hybridized to mRNA in situ. Anti-digoxigenin antibodies conjugated to horseradish peroxidase (POD) are then bound to the probe and reacted with fluorescently labeled tyramide. Combining this method with a counterstain, such as DAPI, allows for the detection of mRNA at a single-cell resolution.

Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis.

Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results.

Digoxigenin.

In molecular cloning, digoxigenin is used as a ligand that can be incorporated into DNA and RNA probes and detected after hybridization with an anti-digoxigenin-antibody enzyme conjugate. Methods to label nucleic acids with digoxigenin and to detect digoxigenin-labeled probes are introduced here.

A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs, Small RNAs, and miRNAs Using a Non-Radiolabeled Approach.

Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10–100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.

Study of Circular RNA Expression by Nonradioactive Northern Blot Procedure.

Circular RNAs (circRNAs) are covalently closed transcripts generated by back-splicing reaction. The lack of free ends endows these RNA molecules with high stability thus allowing them to accumulate in tissues and body fluids. They are widely expressed in most organisms, are modulated during development and display tissue-specific expression, resulting particularly enriched in the nervous system. Deregulation of circRNA expression has also been associated with several pathological conditions including neurological diseases and cancer.Here we present a Northern blot procedure that allows the analysis of the expression of bona fide circRNAs through the use of a digoxigenin-labeled RNA probe and the immunodetection of the signals.

Bovine Babesiosis Diagnosed in Formalin-Fixed, Paraffin-Embedded Tissues by Using In Situ Hybridization.

Bovine babesiosis, caused by Babesia divergens, is in general a rare disease in Europe. Nonetheless, local outbreaks can cause severe economic damage, and postmortem identification represents a diagnostic challenge. During a recent outbreak in May 2018 in northern Germany, 21 animals of a herd of 150 cattle died within 40 days having had clinical signs of fever and hemoglobinuria. Gross examination of 4 of the 21 deceased animals revealed a tick infestation, jaundice, and dark brown staining of urine and kidneys. Histologically, there were iron-positive deposits, hyperplasia of the red pulp of the spleen, and centrilobular necrosis of hepatocytes. In several locations, small basophilic granules suggestive of intraerythrocytic parasites were visible in hematoxylin-eosin- and Giemsa-stained sections. Peripheral blood smears from a living cow from the herd and polymerase chain reaction (PCR) of feeding ticks revealed B. divergens infection. In situ hybridization (ISH) was applied on formalin-fixed, paraffin-embedded (FFPE) tissue of the necropsied cattle to confirm babesiosis in these animals postmortem. Digoxigenin-labeled DNA probes were generated based on a specific nucleotide sequence for B. divergens, obtained by PCR and sequencing of DNA isolates from infected Ixodes ricinus ticks from deceased cattle. ISH using these probes allowed postmortem diagnosis of B. divergens infection in routinely fixed FFPE tissues.

The use of digoxigenin-labeled oligonucleotide probes for the specific detection of Salmonella in foods

Three oligonucleotide fragment TS11 ,TS21,and TS31 were labeled with digoxigenin, and the hybridization specificities for three oligonucleotides confirmed with Salmonella and non-Salmonella isolates. Three non-radioactive oligonucleotide probes were used for the specific detection of Salmonella in contaminated food samples, including pork, fish and shrimp samples. It was found that enrichment of Salmonella in food samples using lactose combined tetrathionate broth followed by streaking of the culture on Salmonella-Shigella agar and spotting suspected cells on nitrocellulose paper placed on Luria agar prior to colony hybridization yield unambiguous results. Salmomella at 3 cfu per gram of food could be detected.

First Report of Black Currant Reversion Virus and Gooseberry Vein Banding Associated Virus in Currants in Bosnia and Herzegovina

Currants (Ribes spp.) are infected by at least 30 viruses (Koloniuk et al. 2018; Teifion Jones 2002), yet two are associated with the most important virus diseases of the crop: black currant reversion virus (BRV) for currant reversion and gooseberry vein banding associated virus (GVBaV) for the homonymous disease (Teifion Jones et al. 2001). BRV belongs to subgroup C of the genus Nepovirus of the family Comoviridae, whereas GVBaV is a member of genus Badnavirus, family Caulimoviridae (Petrzik et al. 2012). To this date, there is no information on the presence of either viruses in currant in Bosnia and Herzegovina (BiH). Surveys were carried out to assess the occurrence and diversity of the two viruses using high-throughput sequencing (HTS). Thirteen currant specimens (one red, one white, and 11 black) were collected in spring 2018 from commercial orchards in three localities (Travnik, Teslic, and Bugojno). Only three plants exhibited virus-like symptoms: the red currant (Ribes rubrum) from Travnik displayed leaf and flower malformation; the white currant and one black currant (R. nigrum) from Teslic showed yellow patterns and chlorosis on the upper canopy. RNA was extracted from seven samples (white, red, and black currants; both symptomatic and asymptomatic) using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, Germany) and further depleted of ribosomal RNA using the KAPA RNA Hyper+RiboErase HMR kit. Libraries were generated and sequenced on the Illumina NextSeq 500/550 platform in separate reactions. Sequence analysis using Virfind (Ho and Tzanetakis 2014) revealed the presence of BRV in the red currant plant with typical disease symptoms and an asymptomatic black currant (47 and 28 contigs respectively, from 105 to 1,584 bp) and GVBaV in an asymptomatic plant (17 contigs from 350 to 1,903 bp). Nucleic acids were extracted from all 13 samples using the Poudel et al. (2013) protocol that facilitated detection of both DNA and RNA viruses. Reverse transcription PCR (RT-PCR) and PCR analyses were performed with two pairs of virus-specific primers for BRV (F, 5′-GTAATACGCTGGTGTCTC-3′/R, 5′-GAAAGGACATTTCAGACTC-3′ and F, 5′-ATTTCGAGCTGTATGGTCG-3′/R, 5′-CTCGGAAGCAGTAGACCT-3′ [Lemmetty et al. 2001]) and for GVBaV (F, 5′-ACATCAAAGGGAAGGACAAC-3′/R, 5′-TCTAAAAGCATCCACTACCAC-3′ and F, 5′-TCAGACAGGCTCTCAACAATAC-3′/R, 5′-TTCTAAAAGCATCCACTACCAC-3′) (Jones et al. 2001). These assays identified an additional GVBaV-positive sample. All amplicons were sequenced, and BLAST analysis of the BRV sequences (GenBank accession nos. MK501976 to MK501978) and GVBaV (GenBank accession nos. MK519228 to MK519230) revealed 92 to 98% and 96 to 99% nucleotide identity to GenBank accessions AF321572 and JQ388488, respectively. The positive samples were further assessed for the presence of the viruses using northern-blot hybridization with a digoxigenin-labeled probe that targeted the nontranslated region of BRV RNA2 and conserved motifs in the ribonuclease H domain of GVBaV. The results were in line with HTS and PCR detection and with the symptoms expressed in the three symptomatic plants. In addition, the episomal form of GVBaV was confirmed by positive results in RT-PCR using DNase-treated total nucleic acids and rolling circle amplification. To our knowledge, this is the first time that BRV and GVBaV were detected in currants in BiH. Given the lack of a currant certification program in the country, this information will be used in the development of best management practices for control of the two viruses and their associated diseases in both field and nursery operations.

Regulation of Telomere Length and Atherosclerosis by Protection of Telomeres 1 Protein.

The aim of this study was to investigate the manifestation and significance of changes in both telomere length and the expression of human telomere protective protein (hPOT1) in the peripheral blood leukocytes of patients with atherosclerosis (AS). One hundred subjects-excluding those with acute or chronic inflammation, cancer, and autoimmune diseases-were enrolled in this study and divided into two groups: the atherosclerosis group (AS group) and control group. We extracted peripheral blood leukocyte DNA along with its peripheral proteins. After purity testing, a digoxigeninlabeled telomere probe was used for Southern blotting; the length of the telomeres was obtained by image scanning and software analysis. After extracting the peripheral proteins, hPOT1 expression was detected by western blotting and scanned with an image analysis software system. We found that the telomere lengths in the peripheral blood leukocytes of AS and control groups were 7.45 ± 1.15 kb versus 8.11 ± 0.69 kb, respectively, and that the difference between them was statistically significant (p < 0.005). The expression level of hPOT1 protein in the peripheral blood leukocytes of the AS group was significantly higher than that of the control group (t = 3.77, p < 0.01). As can be determined from these results, telomere length in the peripheral blood leukocytes of AS patients was significantly shorter compared with that of the control group. The regulation of telomere length by hPOT1 by negative regulation may be one of the influencing factors in AS. Therefore, it is suggested that change in telomere length may play a role in the occurrence and development of AS.

Genomic characterisation of a colistin-resistant Klebsiella pneumoniae ST11 strain co-producing KPC-2, FloR, CTX-M-55, SHV-12, FosA and RmtB causing a lethal infection.

The aim of this study was to characterise the genomic and phenotypic characteristics of a colistin-resistant Klebsiella pneumoniae isolate causing a lethal infection that was phenotypically resistant to carbapenems and colistin. Whole-genome sequencing was performed on an Illumina HiSeq 2500 platform. Genome annotation was performed by the Rapid Annotation using Subsystem Technology (RAST) server. Antimicrobial resistance genes and plasmid replicons were identified using ResFinder 2.1 and PlasmidFinder 1.3, respectively. The isolate was further characterised by plasmid analysis using S1-PFGE and Southern blot hybridisation with a digoxigenin-labelled probe specific for blaKPC. RESULTS: The genome of K. pneumoniae LSK16 consists of a 6.02-Mbp chromosome and one plasmid. Multilocus sequence typing (MLST) identified the isolate as ST11, a close variant of the international pandemic clone ST258. The isolate was found to harbour blaKPC-2, blaSHV-12, blaCTX-M-55, floR and rmtB genes. Of note, a novel fosfomycin resistance glutathione transferase variant was confirmed by PCR and sequencing, with 98.6% (136/138) identity to fosA. Moreover, amino acid substitutions in PmrB (R256G) and PhoQ (D150G) were identified in LSK16, confirming the polymyxin/colistin resistance, although the isolate was negative for mcr genes. Southern blotting and plasmid analysis revealed that the blaKPC-2 gene was harboured on a non-conjugative IncR plasmid (˜165kb). Here we identified a colistin-resistant K. pneumoniae ST11 strain co-producing KPC-2, FloR, CTX-M-55, SHV-12 and RmtB causing a lethal infection. This study provides new genomic insights into the diversity of K. pneumoniae ST11 prevalent in Zhejiang Province, China.

A novel MKK gene (EcMKK6) in Epinephelus coioides: Identification, characterization and its response to Vibrio alginolyticus and SGIV infection

Mitogen-activated protein kinase 6 (MKK6) is one of the major important central regulatory proteins response to environmental and physiological stimuli. In this study, a novel MKK6, EcMKK6, was isolated from Epinephelus coioides, an economically important cultured fish in China and Southeast Asian counties. The open reading frame (ORF) of EcMKK6 is 1077 bp encoding 358 amino acids. EcMKK6 contains a serine/threonine protein kinase (S_TKc) domain, a tyrosine kinase catalytic domain, a conserved dual phosphorylation site in the SVAKT motif and a conserved DVD domain. By in situ hybridization (ISH) with Digoxigenin-labeled probe, EcMKK6 mainly located at the cytoplasm of cells, and a little appears in the nucleus. EcMKK6 mRNA can be detected in all eleven tissues examined, but the expression level is different in these tissues. After challenge with Vibrio alginolyticus and Singapore grouper iridovirus (SGIV), the transcription level of EcMKK6 was apparently up-regulated in the tissues examined. The data demonstrated that the sequence and the characters of EcMKK6 were conserved, EcMKK6 showed tissue-specific expression profiles in healthy grouper, and the expression was significantly varied after pathogen infection, indicating that EcMKK6 may play important roles in E. coioides during pathogen-caused inflammation.

First Report of Citrus Chlorotic Spot Virus Infecting the Succulent Plant Agave desmettiana

Endemic in the Americas, citrus leprosis is a viral disease that reduces yields and threatens the sustainability of citrus orchards. The disease is caused by a heterogeneous group of ssRNA viruses assigned to the genera Dichorhavirus (family Rhabdoviridae, order Mononegavirales) or Cilevirus (unassigned genus) (Freitas-Astua et al. 2018). The typical dichorhavirus genome encompasses two (–)ssRNA molecules that encode six open reading frames (ORFs) (Dietzgen et al. 2018). Isolates of the dichorhavirus citrus chlorotic spot virus (CiCSV) were previously identified in Teresina, Piaui, Brazil (Chabi-Jesus et al. 2018). Besides affecting sweet orange (Citrus sinensis) trees producing symptoms resembling early lesions of a typical citrus leprosis, CiCSV also infects beach hibiscus (Talipariti tiliaceum syn. Hibiscus tiliaceus L.) plants. In 2018, a dozen of the ornamental Agave desmettiana Jacob plants (Agavaceae) showing chlorotic lesions were detected in the urban region of Teresina city within an area of approximately 30 km², including the region where the original CiCSV-infected plants were described. Analyses of symptomatic agaves’ tissues by transmission electron microscopy revealed the presence of viroplasms in the nucleus of the infected parenchymal cells, as a typical dichorhavirus infection (Dietzgen et al. 2018). Moreover, eight mites that were collected during the examination of the symptomatic leaves were morphologically identified as Brevipalpus aff. yothersi, the same species found in CiCSV-infected citrus (Chabi-Jesus et al. 2018). Total RNA from leaf lesions of three symptomatic agave plants (isolates Trs4, Trs5, and Trs6) was extracted using TRIzol reagent (Life Technologies, Foster City, CA), and the reverse transcription polymerase chain reaction assays performed using all of the primer pairs available for the detection of known dichorhaviruses (Dietzgen et al. 2018) proved the presence of CiCSV. Additionally, the same RNA extracts reacted with a digoxigenin-labeled probe derived from the N gene of CiCSV_Trs1 in an RNA dot-blot assay (Roche Diagnostics, Mannheim, Germany). Ninety-five percent of the isolate Trs4’s genome was recovered after the Sanger sequencing of the overlapping amplicons generated by using 15 primers pairs (Chabi-Jesus et al. 2018). Globally, RNA1 (6,082 nt, GB: MH595621) and RNA2 (5,688 nt, MH595622) shows 96.4% nucleotide sequence identity with the type virus CiCSV_Trs1 (KY700685 and KY700686) and less than 75% nucleotide sequence identity with the dichorhaviruses orchid fleck virus (OFV; AB244417 and AB244418), coffee ringspot virus (CoRSV; KF812525 and KF812526), citrus leprosis virus N (CiLV-N; KX982176 and KX982179), and clerodendrum chlorotic spot virus (ClCSV; MG938506 and MG938507). Comparisons of the deduced amino acid sequences of the putative proteins N, P, MP, M, G, and L (encoded by ORFs 1 to 6, respectively) of CiCSV_Trs4 show 95 to 99% identity with CiCSV_Trs1 and 28 to 90% identity with OFV, CoRSV, CiLV-N, and ClCSV. Sequences of the generated amplicons corresponding to G and L genes from the isolates Trs5 and Trs6 (MH802037, MH802038, MH802039, and MH802040, respectively) showed >93% nucleotide sequence identity with CiCSV_Trs1 (KY700685 and KY700686) and with isolate Trs4. Comprehensively, results indicate that CiCSV_Trs4, -5, and -6 are isolates of the species Citrus chlorotic spot dichorhavirus of the genus Dichorhavirus, whose transmission has been previously associated with the mite B. aff. yothersi. This is the first report of CiCSV naturally infecting the succulent plant A. desmettiana, and, to our knowledge, it is also the first report of a (–)ssRNA virus infecting plants of this genus.

Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization.

A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus-infected patients treated with certain nucleoside reverse transcriptase inhibitors. Further, cell culture studies have demonstrated that exposure to oxidative stress stimulates mtDNA degradation. Here we outline a Southern blot and nonradioactive digoxigenin-labeled probe hybridization method to estimate mtDNA content in human genomic DNA samples. © 2019 by John Wiley & Sons, Inc.