scholarly journals Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins

mSphere ◽  
2022 ◽  
Author(s):  
Tiago W. P. Mineo ◽  
Jessica H. Chern ◽  
Amara C. Thind ◽  
Caroline M. Mota ◽  
Santhosh M. Nadipuram ◽  
...  
Keyword(s):  
Functional Assessment ◽  
Scientific Community ◽  
Neospora Caninum ◽  
Gene Knockout ◽  
Severe Disease ◽  
Reproductive Disorders ◽  
Content Type ◽  
Novel Proteins ◽  
Efficient Gene ◽  
Close Phylogenetic Relationship

Neospora caninum is a parasite with veterinary relevance, inducing severe disease in dogs and reproductive disorders in ruminants, especially cattle, leading to major losses. The close phylogenetic relationship to Toxoplasma gondii and the lack of pathogenicity in humans drives an interest of the scientific community toward using N. caninum as a model to study the pathogenicity of T. gondii .

scholarly journals Apicomplexan Protozoa Responsible for Reproductive Disorders: Occurrence of DNA in Blood and Milk of Donkeys (Equus asinus) and Minireview of the Related Literature

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 111
Author(s):  
Stefania Perrucci ◽  
Lisa Guardone ◽  
Iolanda Altomonte ◽  
Federica Salari ◽  
Simona Nardoni ◽  
...  
Keyword(s):  
Animal Species ◽  
Neospora Caninum ◽  
Reproductive Disorders ◽  
Theileria Equi ◽  
Blood Samples ◽  
Related Literature ◽  
Milk Samples ◽  
Equus Asinus ◽  
Time Periods ◽  
Blood Specimens

Donkeys may be susceptible to many pathological agents and may act as carriers of pathogens for other animal species and humans. This study evaluated the occurrence of potentially abortifacient apicomplexan protozoa DNA in blood and milk samples collected at different time periods during lactation (1, 6, and 10 months) from 33 healthy dairy jennies. A total of 73 blood and 73 milk samples were used for DNA extraction and analysis. Blood specimens from 11/33 (33%) jennies scored positive for Theileria equi, while milk samples scored negative. Blood and milk of 3/33 jennies yielded DNA of Toxoplasma gondii at 6 months (n. 1) and 10 months (n. 2) after parturition. Neospora caninum DNA was found in four milk and in five blood samples only at one month after parturition. This study is the first report about the presence of N. caninum DNA in milk of naturally infected jennies. Moreover, the excretion of N. caninum DNA in some of these jennies at 30 days from the parturition may suggest a possible occurrence of an endogenous cycle, while the presence of T. gondii DNA in the milk collected at 6 and 10 months after parturition may be suggestive of a discontinuous excretion.

scholarly journals An Efficient, Rapid, and Recyclable System for CRISPR-Mediated Genome Editing in Candida albicans

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Namkha Nguyen ◽  
Morgan M. F. Quail ◽  
Aaron D. Hernday
Keyword(s):  
Candida Albicans ◽  
Genome Editing ◽  
Fungal Pathogen ◽  
Gene Knockout ◽  
Strain Engineering ◽  
Molecular Genetic Analysis ◽  
Content Type ◽  
Highly Efficient ◽  
Discovery Research ◽  
Gene Knockouts

ABSTRACT Candida albicans is the most common fungal pathogen of humans. Historically, molecular genetic analysis of this important pathogen has been hampered by the lack of stable plasmids or meiotic cell division, limited selectable markers, and inefficient methods for generating gene knockouts. The recent development of clustered regularly interspaced short palindromic repeat(s) (CRISPR)-based tools for use with C. albicans has opened the door to more efficient genome editing; however, previously reported systems have specific limitations. We report the development of an optimized CRISPR-based genome editing system for use with C. albicans. Our system is highly efficient, does not require molecular cloning, does not leave permanent markers in the genome, and supports rapid, precise genome editing in C. albicans. We also demonstrate the utility of our system for generating two independent homozygous gene knockouts in a single transformation and present a method for generating homozygous wild-type gene addbacks at the native locus. Furthermore, each step of our protocol is compatible with high-throughput strain engineering approaches, thus opening the door to the generation of a complete C. albicans gene knockout library. IMPORTANCE Candida albicans is the major fungal pathogen of humans and is the subject of intense biomedical and discovery research. Until recently, the pace of research in this field has been hampered by the lack of efficient methods for genome editing. We report the development of a highly efficient and flexible genome editing system for use with C. albicans. This system improves upon previously published C. albicans CRISPR systems and enables rapid, precise genome editing without the use of permanent markers. This new tool kit promises to expedite the pace of research on this important fungal pathogen.

scholarly journals Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

2015 ◽  
Vol 81 (20) ◽  
pp. 6953-6963 ◽  
Author(s):  
Zhe Zhao ◽  
Lauren J. Eberhart ◽  
Lisa H. Orfe ◽  
Shao-Yeh Lu ◽  
Thomas E. Besser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Disulfide Bonds ◽  
Gene Knockout ◽  
Single Gene ◽  
Site Directed Mutagenesis ◽  
Content Type ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

scholarly journals Changes in Antigen-Specific Cytokine and Chemokine Responses to Plasmodium falciparum Antigens in a Highland Area of Kenya after a Prolonged Absence of Malaria Exposure

2014 ◽  
Vol 82 (9) ◽  
pp. 3775-3782 ◽  
Author(s):  
Lyticia A. Ochola ◽  
Cyrus Ayieko ◽  
Lily Kisia ◽  
Ng'wena G. Magak ◽  
Estela Shabani ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Severe Disease ◽  
Clinical Malaria ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Highland Area ◽  
Cytokine Responses ◽  
Increased Risk ◽  
Tnf Α ◽  
Ifn Γ

ABSTRACTIndividuals naturally exposed toPlasmodium falciparumlose clinical immunity after a prolonged lack of exposure.P. falciparumantigen-specific cytokine responses have been associated with protection from clinical malaria, but the longevity ofP. falciparumantigen-specific cytokine responses in the absence of exposure is not well characterized. A highland area of Kenya with low and unstable malaria transmission provided an opportunity to study this question. The levels of antigen-specific cytokines and chemokines associated in previous studies with protection from clinical malaria (gamma interferon [IFN-γ], interleukin-10 [IL-10], and tumor necrosis factor alpha [TNF-α]), with increased risk of clinical malaria (IL-6), or with pathogenesis of severe disease in malaria (IL-5 and RANTES) were assessed by cytometric bead assay in April 2008, October 2008, and April 2009 in 100 children and adults. During the 1-year study period, none had an episode of clinicalP. falciparummalaria. Two patterns of cytokine responses emerged, with some variation by antigen: a decrease at 6 months (IFN-γ and IL-5) or at both 6 and 12 months (IL-10 and TNF-α) or no change over time (IL-6 and RANTES). These findings document thatP. falciparumantigen-specific cytokine responses associated in prior studies with protection from malaria (IFN-γ, TNF-α, and IL-10) decrease significantly in the absence ofP. falciparumexposure, whereas those associated with increased risk of malaria (IL-6) do not. The study findings provide a strong rationale for future studies of antigen-specific IFN-γ, TNF-α, and IL-10 responses as biomarkers of increased population-level susceptibility to malaria after prolonged lack ofP. falciparumexposure.

scholarly journals Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases

2013 ◽  
Vol 24 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Y. H. Sung ◽  
J. M. Kim ◽  
H.-T. Kim ◽  
J. Lee ◽  
J. Jeon ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Highly Efficient ◽  
Efficient Gene

scholarly journals Kinome Expansion in theFusarium oxysporumSpecies Complex Driven by Accessory Chromosomes

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Gregory A. DeIulio ◽  
Li Guo ◽  
Yong Zhang ◽  
Jonathan M. Goldberg ◽  
H. Corby Kistler ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Species Complex ◽  
Severe Disease ◽  
Cellular Responses ◽  
Human Pathogens ◽  
Content Type ◽  
Evolutionary Forces ◽  
Environmental Signal ◽  
Wide Range ◽  
Opportunistic Fungal Pathogen

ABSTRACTTheFusarium oxysporumspecies complex (FOSC) is a group of soilborne pathogens causing severe disease in more than 100 plant hosts, while individual strains exhibit strong host specificity. Both chromosome transfer and comparative genomics experiments have demonstrated that lineage-specific (LS) chromosomes contribute to the host-specific pathogenicity. However, little is known about the functional importance of genes encoded in these LS chromosomes. Focusing on signaling transduction, this study compared the kinomes of 12F. oxysporumisolates, including both plant and human pathogens and 1 nonpathogenic biocontrol strain, with 7 additional publicly available ascomycete genomes. Overall,F. oxysporumkinomes are the largest, facilitated in part by the acquisitions of the LS chromosomes. The comparative study identified 99 kinases that are present in almost all examined fungal genomes, forming the core signaling network of ascomycete fungi. Compared to the conserved ascomycete kinome, the expansion of theF. oxysporumkinome occurs in several kinase families such as histidine kinases that are involved in environmental signal sensing and target of rapamycin (TOR) kinase that mediates cellular responses. Comparative kinome analysis suggests a convergent evolution that shapes individualF. oxysporumisolates with an enhanced and unique capacity for environmental perception and associated downstream responses.IMPORTANCEIsolates ofFusarium oxysporumare adapted to survive a wide range of host and nonhost conditions. In addition,F. oxysporumwas recently recognized as the top emerging opportunistic fungal pathogen infecting immunocompromised humans. The sensory and response networks of these fungi undoubtedly play a fundamental role in establishing the adaptability of this group. We have examined the kinomes of 12F. oxysporumisolates and highlighted kinase families that distinguishF. oxysporumfrom other fungi, as well as different isolates from one another. The amplification of kinases involved in environmental signal relay and regulating downstream cellular responses clearly setsFusariumapart from otherAscomycetes. Although the functions of many of these kinases are still unclear, their specific proliferation highlights them as a result of the evolutionary forces that have shaped this species complex and clearly marks them as targets for exploitation in order to combat disease.

scholarly journals Histopathology ofBordetella pertussisin the Baboon Model

2018 ◽  
Vol 86 (11) ◽  
Author(s):  
Lindsey I. Zimmerman ◽  
James F. Papin ◽  
Jason Warfel ◽  
Roman F. Wolf ◽  
Stanley D. Kosanke ◽  
...  
Keyword(s):  
Severe Disease ◽  
Clinical Signs ◽  
Human Infant ◽  
Mucus Production ◽  
Content Type ◽  
Age Related ◽  
Baboon Model ◽  
Relevant Animal Model ◽  
Significant Pathology ◽  
Conducting Airways

ABSTRACTPertussis is a severe respiratory disease caused byBordetella pertussis. The classic symptoms of pertussis include paroxysmal coughing with an inspiratory whoop, posttussive vomiting, cyanosis, and persistent coryzal symptoms. Infants under 2 months of age experience more severe disease, with most deaths occurring in this age group. Most of what is known about the pathology of pertussis in humans is from the evaluation of fatal human infant cases. The baboon model of pertussis provides the opportunity to evaluate the histopathology of severe but nonfatal pertussis. The baboon model recapitulates the characteristic clinical signs of pertussis observed in humans, including leukocytosis, paroxysmal coughing, mucus production, heavy colonization of the airway, and transmission of the bacteria between hosts. As in humans, baboons demonstrate age-related differences in clinical presentation, with younger animals experiencing more severe disease. We examined the histopathology of 5- to 6-week-old baboons, with the findings being similar to those reported for fatal human infant cases. In juvenile baboons, we found that the disease is highly inflammatory and concentrated to the lungs with signs of disease that would typically be diagnosed as acute respiratory distress syndrome (ARDS) and bronchopneumonia. In contrast, no significant pathology was observed in the trachea. Histopathological changes in the trachea were limited to cellular infiltrates and mucus production. Immunohistostaining revealed that the bacteria were localized to the surface of the ciliated epithelium in the conducting airways. Our observations provide important insights into the pathology of pertussis in typical, severe but nonfatal pertussis cases in a very relevant animal model.

scholarly journals MBX-500, a Hybrid Antibiotic withIn VitroandIn VivoEfficacy against Toxigenic Clostridium difficile

2012 ◽  
Vol 56 (9) ◽  
pp. 4786-4792 ◽  
Author(s):  
Michelle M. Butler ◽  
Dean L. Shinabarger ◽  
Diane M. Citron ◽  
Ciarán P. Kelly ◽  
Sofya Dvoskin ◽  
...  
Keyword(s):  
Weight Gain ◽  
Clostridium Difficile ◽  
Severe Disease ◽  
Normal Weight ◽  
New Drugs ◽  
Hamster Model ◽  
Resistance Development ◽  
Content Type

ABSTRACTClostridium difficileinfection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, thein vitroandin vivoefficacies of MBX-500 were explored against the Gram-positive anaerobe,C. difficile. MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.

scholarly journals Cationic Liposomes Extend the Immunostimulatory Effect of CpG Oligodeoxynucleotide against Burkholderia pseudomallei Infection in BALB/c Mice

2012 ◽  
Vol 19 (5) ◽  
pp. 675-683 ◽  
Author(s):  
Apichaya Puangpetch ◽  
Robert Anderson ◽  
Yan Y. Huang ◽  
Rasana W. Sermswan ◽  
Wanpen Chaicumpa ◽  
...  
Keyword(s):  
Burkholderia Pseudomallei ◽  
Severe Disease ◽  
Antimicrobial Activities ◽  
Cationic Liposomes ◽  
Phagocytic Index ◽  
Cpg Odn ◽  
Content Type ◽  
Cpg Oligodeoxynucleotide ◽  
Alternative Approach ◽  
Lethal Infection

ABSTRACTMelioidosis is a severe disease caused by the Gram-negative bacteriumBurkholderia pseudomallei. Previously we showed that pretreatment of mice with CpG oligodeoxynucleotide (CpG ODN) 2 to 10 days prior toB. pseudomalleichallenge conferred as high as 90% protection, but this window of protection was rather short. In the present study, we therefore aimed to prolong this protective window and to gain further insight into the mechanisms underlying the protection induced by CpG ODN againstB. pseudomalleiinfection. It was found that the CpG ODN incorporated with cationic liposomes (DOTAP) but not zwitterionic liposomes (DOPC) provided complete protection against bacterial challenge. Although marked elevation of gamma interferon (IFN-γ) was found in the infected animals 2 days postinfection, it was significantly lowered by the DOTAP-plus-CpG ODN pretreatment. When appropriately activated, the phagocytic index and oxidative burst responses of neutrophils appeared not to be elevated. However, macrophages from stimulated mice showed higher levels of nitric oxide production and exhibited higher levels of antimicrobial activities, judging from lower numbers of viable intracellular bacteria. Taken together, our results demonstrate that DOTAP can enhance the protective window period of CpG ODN to at least 30 days and provide 100% protection againstB. pseudomalleiinfection. The protective effect of DOTAP plus CpG ODN could provide an alternative approach to preventing this lethal infection, for which no vaccine is yet available.

The nature of rapid response to COVID-19 in Latin America: an examination of Argentina, Brazil, Chile, Colombia and Mexico

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Janaina Pamplona da Costa ◽  
André Luiz Sica de Campos ◽  
Paulo Roberto Cintra ◽  
Liz Felix Greco ◽  
Johan Hendrik Poker
Keyword(s):  
Latin American ◽  
Scientific Community ◽  
Scientific Output ◽  
Rapid Response ◽  
Future Research ◽  
Scientific Publications ◽  
Scientific Communities ◽  
Content Type ◽  
Scientific Outputs ◽  
Career Status

PurposeThe coronavirus-19 (COVID-19) pandemic mobilized the international scientific community in the search for its cure and containment. The purpose of this paper is to examine the nature of the rapid response to the COVID-19 of the scientific community in selected Latin American countries (Argentina, Brazil, Chile, Colombia and Mexico) in the period running from January to August 2020. Rapid response is reconceptualized from its original meaning in health policy, as the swift mobilization of existing scientific resources to address an emergency (DeVita et al., 2017).Design/methodology/approachThe paper explores the rapid response of the Argentinian, Brazilian, Chilean, Colombian and Mexican scientific communities from the perspective of bibliometric and altmetric data. The authors will examine scientific publications indexed to the Web of Science (WoS) dealing with COVID-19. Besides patterns of scientific output and impact as measured by citations, the authors complement the analysis with altmetric analysis. The aim is to verify whether or not factors that explain the extent of scientific impact can also be identified with respect to the wider impact made evident by altmetric indicators (Haustein, 2016).Findingshe authors identified a somewhat limited response of the Argentinian, Brazilian, Chilean, Colombian and Mexican scientific communities to COVID-19 in terms of quantity of publications. The authorship of publications in the topic of COVID-19 was associated with authorship of publications dealing with locally relevant diseases. Some factors appear to contribute to visibility of scientific outputs. Papers that involved wider international collaborations and authors with previous publications in arboviruses were associated with higher levels of citations. Previous work on arbovirus was also associated with higher altmetric attention. The country of origin of authors exerted a positive effect on altmetric indicators.Research limitations/implicationsA limitation in the analysis is that, due to the nature of the data source (WoS), the authors were unable to verify the career status and the productivity of the authors in the sample. Nonetheless, the results appear to suggest that there is some overlapping in authors conducting research in Arboviruses and COVID-19. Career status and productivity should be the focus of future research.Practical implicationsIn the context of countries with limited scientific resources, like the ones investigated in our Latin American sample, previous efforts in the study of locally relevant diseases may contribute to the creation of an expertise that can be applied when a health emergency brings about a novel disease.Originality/valueThe originality of the paper rests on the fact that the authors identified that previous work on arbovirus contributed to the scientific visibility of publications on COVID-19.

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