scholarly journals Comparative Genomics of Clostridium perfringens Reveals Patterns of Host-Associated Phylogenetic Clades and Virulence Factors

2021 ◽  
Vol 12 ◽  
Author(s):  
Renae R. Geier ◽  
Thomas G. Rehberger ◽  
Alexandra H. Smith
Keyword(s):  
Virulence Factors ◽  
Clostridium Perfringens ◽  
Broiler Chickens ◽  
Food Poisoning ◽  
Virulence Proteins ◽  
Virulence Protein ◽  
Epsilon Toxin ◽  
Opportunistic Pathogenic ◽  
Beta Toxin ◽  
Genome Assemblies

Clostridium perfringens is an opportunistic pathogenic bacterium that infects both animals and humans. Clostridium perfringens genomes encode a diverse array of toxins and virulence proteins, which continues to expand as more genomes are sequenced. In this study, the genomes of 44 C. perfringens strains isolated from intestinal sections of diseased cattle and from broiler chickens from diseased and healthy flocks were sequenced. These newly assembled genomes were compared to 141 publicly available C. perfringens genome assemblies, by aligning known toxin and virulence protein sequences in the assemblies using BLASTp. The genes for alpha toxin, collagenase, a sialidase (nanH), and alpha-clostripain were present in at least 99% of assemblies analyzed. In contrast, beta toxin, epsilon toxin, iota toxin, and binary enterotoxin of toxinotypes B, C, D, and E were present in less than 5% of assemblies analyzed. Additional sequence variants of beta2 toxin were detected, some of which were missing the leader or signal peptide sequences and therefore likely not secreted. Some pore-forming toxins involved in intestinal diseases were host-associated, the netB gene was only found in avian isolates, while netE, netF, and netG were only present in canine and equine isolates. Alveolysin was positively associated with canine and equine strains and only present in a single monophyletic clade. Strains from ruminant were not associated with known virulence factors and, except for the food poisoning associated clade, were present across the phylogenetic diversity identified to date for C. perfringens. Many C. perfringens strains associated with food poisoning lacked the genes for hyaluronidases and sialidases, important for attaching to and digesting complex carbohydrates found in animal tissues. Overall, the diversity of virulence factors in C. perfringens makes these species capable of causing disease in a wide variety of hosts and niches.

scholarly journals Microbiological aspects of polyphosphate injection in the processing and chill storage of poultry

1979 ◽  
Vol 82 (1) ◽  
pp. 133-142 ◽  
Author(s):  
G. C. Mead ◽  
B. W. Adams
Keyword(s):  
Growth Rate ◽  
Shelf Life ◽  
Clostridium Perfringens ◽  
Broiler Chickens ◽  
Food Poisoning ◽  
Cross Contamination ◽  
Injection Solution ◽  
Commercial Processing ◽  
Deep Muscle ◽  
Microbiological Aspects

SUMMARYDuring commercial processing of broiler chickens, injection of polyphosphate (Puron 604 or 6040) resulted in microorganisms being added to the deep breast muscle. The level of contamination was related to the microbiological condition of the injection solution.Injection of polyphosphate had no effect on the shelf-life of fresh chilled carcasses held at 1° or 10°C but changes were observed in the growth rate of microorganisms in the deep muscle and in the composition of the muscle microflora following storage.Cross-contamination of carcasses and the transfer of organisms from the skin to the deep muscle during injection was demonstrated with a marker strain ofClostridium perfringens. However, both processes were influenced by the number of marker organisms applied initially to the skin.The above findings are discussed in relation to the possible behaviour of any food poisoning bacteria present.

scholarly journals Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid

2008 ◽  
Vol 190 (21) ◽  
pp. 7178-7188 ◽  
Author(s):  
Kazuaki Miyamoto ◽  
Jihong Li ◽  
Sameera Sayeed ◽  
Shigeru Akimoto ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Open Reading Frames ◽  
Toxin Gene ◽  
Type B ◽  
Clostridium Perfringens Enterotoxin ◽  
Type D ◽  
Transfer Genes ◽  
Epsilon Toxin ◽  
Beta2 Toxin ◽  
Beta Toxin

ABSTRACT Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.

scholarly journals CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101

2017 ◽  
Vol 85 (3) ◽  
Author(s):  
Jihong Li ◽  
John C. Freedman ◽  
Daniel R. Evans ◽  
Bruce A. McClane
Keyword(s):  
Gene Expression ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Type A ◽  
Toxin Production ◽  
Null Mutant ◽  
Wild Type ◽  
Content Type ◽  
Type D ◽  
Epsilon Toxin

ABSTRACT Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY-null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY-null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY-null mutant strain but significantly increased in the SM101 codY-null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation.

scholarly journals Characterization of Virulence Plasmid Diversity among Clostridium perfringens Type B Isolates

2009 ◽  
Vol 78 (1) ◽  
pp. 495-504 ◽  
Author(s):  
Sameera Sayeed ◽  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Pcr Analysis ◽  
Virulence Plasmid ◽  
Type B ◽  
Gene Encoding ◽  
Virulence Plasmids ◽  
Genes Encoding ◽  
Epsilon Toxin ◽  
Beta Toxin

ABSTRACT The important veterinary pathogen Clostridium perfringens type B is unique for producing the two most lethal C. perfringens toxins, i.e., epsilon-toxin and beta-toxin. Our recent study (K. Miyamoto, J. Li, S. Sayeed, S. Akimoto, and B. A. McClane, J. Bacteriol. 190:7178-7188, 2008) showed that most, if not all, type B isolates carry a 65-kb epsilon-toxin-encoding plasmid. However, this epsilon-toxin plasmid did not possess the cpb gene encoding beta-toxin, suggesting that type B isolates carry at least one additional virulence plasmid. Therefore, the current study used Southern blotting of pulsed-field gels to localize the cpb gene to ∼90-kb plasmids in most type B isolates, although a few isolates carried a ∼65-kb cpb plasmid distinct from their etx plasmid. Overlapping PCR analysis then showed that the gene encoding the recently discovered TpeL toxin is located ∼3 kb downstream of the plasmid-borne cpb gene. As shown earlier for their epsilon-toxin-encoding plasmids, the beta-toxin-encoding plasmids of type B isolates were found to carry a tcp locus, suggesting that they are conjugative. Additionally, IS1151-like sequences were identified upstream of the cpb gene in type B isolates. These IS1151-like sequences may mobilize the cpb gene based upon detection of possible cpb-containing circular transposition intermediates. Most type B isolates also possessed a third virulence plasmid that carries genes encoding urease and lambda-toxin. Collectively, these findings suggest that type B isolates are among the most plasmid dependent of all C. perfringens isolates for virulence, as they usually carry three potential virulence plasmids.

scholarly journals Update on the toxins of Clostridium perfringens and their actions

2017 ◽  
Vol 61 (3) ◽  
pp. 241
Author(s):  
V. S. TSIOURIS (Β.Σ. ΤΣΙΟΥΡΗΣ) ◽  
I. GEORGOPOULOU (ΓΕΩΡΓΟΠΟΥΛΟΥ) ◽  
E. PETRIDOU (Ε. ΠΕΤΡΙΔΟΥ)
Keyword(s):  
Clostridium Perfringens ◽  
Genomic Analysis ◽  
Cytolytic Activity ◽  
Bacterial Toxin ◽  
Necrotic Enteritis ◽  
Alpha Toxin ◽  
The Third ◽  
Epsilon Toxin ◽  
Beta Toxin

Clostridia appeared as a distinct class, approximately 2.7 billion years ago, before the initial formation of oxygen. Clostridium perfringens is widely distributed throughout the environment due to its ability to form spores. Furthermore, it is a member of intestinal microbiota in animals and human. In 2002, the complete genome of C perfringens strain 13 was published. Genomic analysis has revealed that C. perfringens lacks the genetic machinery to produce 13 essential amino acids and it obtains these in vivo via the action of its toxins. Toxins of C perfringens can be divided into major, minor and enterotoxin. C perfringens strains are classified into five toxinotypes (A, B, C, D and E), based on the production of four major toxins. Alpha toxin is the best and most studied major toxin of C perfringens and it was the first bacterial toxin established to possess enzymatic activity. It has haemolytic, necrotic and cytolytic activity, it can lyse platelets and leukocytes and it can damage fibroblasts and muscle cell membranes. Expression of epa gene, which is responsible for the production of alpha toxin by C perfringens, is down-regulated in the normal healthy gut, but it is upregulated to initiate enteric disease in response to an environmental signal. C perfringens appears to be regulated in a quorum sensing manner, using oligopeptides, AI-2 or both, to regulate expression of the epa gene, and thus the synthesis of alpha toxin. Beta toxin is recognized as an important agent in necrotic enteritis of humans and it is the second most lethal C. perfringens toxin following epsilon toxin. Beta toxin is a membrane spanning protein that oligomerizes to form channels in susceptible cells or it primarily acts as a neurotoxin. Epsilon toxin is the most potent of the C. perfringens toxins and the third most potent neurotoxin from the Clostridium spp., following botulinum and tetanus toxins. Epsilon toxin of C perfringens type D causes enterotoxaemia and pulpy kidneys disease of lambs. Iota toxin causes disruption of the actin cytoskeleton and cell barrier integrity and it is the less toxic of the major toxins of C perfringens. Although C perfringens enterotoxin is not classified as one of the major toxins of C perfringens, it is the third most common cause of food poisoning in industrialized nations. It is not secreted by the cells of growing bacteria, but it is released only with the sporulation of C perfringens. Not all strains of C perfringens carry the epe gene, which is responsible for the production of enterotoxin. Theta toxin is a pore-forming cytolysin that can lyse red blood cells. It is produced by all types of C perfringens. Together with alpha-toxin, theta-toxin modulates the host inflammatory response. ß2 toxin is a pore forming toxin which is involved in necrotic enteritis of swine and horse, in haemorragic enteritis of bovine in diarrhea cases of dogs and along with enterotoxin in diarrhea cases of humans. Recently, -NetB, a novel toxin that is associated with broiler necrotic enteritis, has been described. The mechanism of its action seems to involve the formation of small hydrophilic pores. Other toxins of C. perfringens include λ-toxin, ô-toxin, μ-toxin, v-toxin, κ-toxin, a-clostripain like protease and neuraminidase/sialidase. These toxins can act as enzymes, while many of them can act synergically or supplementally with major pore forming toxins. Potentially, C. perfringens might produce more toxins, which have not been identified. Finally, the actions of C. perfringens toxins, major or minor, in some diseases have not been figured out.

scholarly journals Native or Proteolytically Activated NanI Sialidase Enhances the Binding and Cytotoxic Activity of Clostridium perfringens Enterotoxin and Beta Toxin

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
James R. Theoret ◽  
Jihong Li ◽  
Mauricio A. Navarro ◽  
Jorge P. Garcia ◽  
Francisco A. Uzal ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Clostridium Perfringens ◽  
Full Length ◽  
Host Cells ◽  
Proteolytic Activation ◽  
Clostridium Perfringens Enterotoxin ◽  
Content Type ◽  
Sialidase Activity ◽  
Epsilon Toxin ◽  
Beta Toxin

ABSTRACTManyClostridium perfringensstrains produce NanI as their major sialidase. Previous studies showed that NanI could potentiateC. perfringensepsilon toxin cytotoxicity by enhancing the binding of this toxin to host cells. The present study first determined that NanI exerts similar cytotoxicity-enhancing effects onC. perfringensenterotoxin and beta toxin, which are also important toxins forC. perfringensdiseases (enteritis and enterotoxemia) originating in the gastrointestinal (GI) tract. Building upon previous work demonstrating that purified trypsin can activate NanI activity, this study next determined that purified chymotrypsin or mouse intestinal fluids can also activate NanI activity. Amino acid sequencing then showed that this effect involves the N-terminal processing of the NanI protein. Recombinant NanI (rNanI) species corresponding to major chymotrypsin- or small intestinal fluid-generated NanI fragments possessed more sialidase activity than did full-length rNanI, further supporting the proteolytic activation of NanI activity. rNanI species corresponding to proteolysis products also promoted the cytotoxic activity and binding of enterotoxin and beta toxin more strongly than did full-length rNanI. Since enterotoxin and beta toxin are produced in the intestines during human and animal disease, these findings suggest that intestinal proteases may enhance NanI activity, which in turn could further potentiate the activity of intestinally active toxins during disease. Coupling these new results with previous findings demonstrating that NanI is important for the adherence ofC. perfringensto enterocyte-like cells, NanI sialidase is now emerging as a potential auxiliary virulence factor forC. perfringensenteritis and enterotoxemia.

scholarly journals Both Epsilon-Toxin and Beta-Toxin Are Important for the Lethal Properties of Clostridium perfringens Type B Isolates in the Mouse Intravenous Injection Model

2007 ◽  
Vol 75 (3) ◽  
pp. 1443-1452 ◽  
Author(s):  
Mariano E. Fernandez-Miyakawa ◽  
Derek J. Fisher ◽  
Rachael Poon ◽  
Sameera Sayeed ◽  
Vicki Adams ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Clinical Signs ◽  
Toxin Production ◽  
Intestinal Damage ◽  
Type B ◽  
Trypsin Treatment ◽  
Epsilon Toxin ◽  
Type C ◽  
Beta Toxin

ABSTRACT Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.

scholarly journals Supplementing Garlic Nanohydrogel Optimized Growth, Gastrointestinal Integrity and Economics and Ameliorated Necrotic Enteritis in Broiler Chickens Using a Clostridium perfringens Challenge Model

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 2027
Author(s):  
Doaa Ibrahim ◽  
Tamer Ahmed Ismail ◽  
Eman Khalifa ◽  
Shaimaa A. Abd El-Kader ◽  
Dalia Ibrahim Mohamed ◽  
...  
Keyword(s):  
Growth Performance ◽  
Clostridium Perfringens ◽  
Broiler Chickens ◽  
Body Weight Gain ◽  
Economic Benefits ◽  
Production Costs ◽  
Broiler Chicks ◽  
Necrotic Enteritis ◽  
Feed Conversion ◽  
Intestinal Lesion

Necrotic enteritis (NE) caused by Clostridium perfringens (C. perfringens) results in impaired bird growth performance and increased production costs. Nanotechnology application in the poultry industry to control NE outbreaks is still not completely clarified. Therefore, the efficacy of dietary garlic nano-hydrogel (G-NHG) on broilers growth performance, intestinal integrity, economic returns and its potency to alleviate C. perfringens levels using NE challenge model were addressed. A total of 1200 male broiler chicks (Ross 308) were assigned into six groups; four supplemented with 100, 200, 300 or 400 mg of G-NHG/kg diet and co-challenged with C. perfringens at 21, 22 and 23 d of age and two control groups fed basal diet with or without C. perfringens challenge. Over the total growing period, the 400 mg/kg G-NHG group had the most improved body weight gain and feed conversion efficiency regardless of challenge. Parallel with these results, the mRNA expression of genes encoding digestive enzymes (alpha 2A amylase (AMY2A), pancreatic lipase (PNLIP) and cholecystokinin (CCK)) and intestinal barriers (junctional adhesion molecule-2 (JAM-2), occludin and mucin-2 (Muc-2)) were increased in groups fed G-NHG at higher levels to be nearly similar to those in the unchallenged group. At 14 d post challenge, real-time PCR results revealed that inclusion of G-NHG led to a dose-dependently decrease in the C. perfringens population, thereby decreasing the birds’ intestinal lesion score and mortality rates. Using 400 mg/kg of G-NHG remarkably ameliorated the adverse effects of NE caused by C. perfringens challenge, which contributed to better growth performance of challenged birds with rational economic benefits.

scholarly journals A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 245
Author(s):  
Hiroshi Sekiya ◽  
Maho Okada ◽  
Eiji Tamai ◽  
Toshi Shimamoto ◽  
Tadashi Shimamoto ◽  
...  
Keyword(s):  
Cell Wall ◽  
Clostridium Perfringens ◽  
Antimicrobial Agents ◽  
Catalytic Domain ◽  
Food Poisoning ◽  
Binding Activity ◽  
Lytic Activity ◽  
Intestinal Bacterium ◽  
Terminal Domain ◽  
Cell Wall Binding

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

scholarly journals Innovative and Highly Sensitive Detection of Clostridium perfringens Enterotoxin Based on Receptor Interaction and Monoclonal Antibodies

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 266
Author(s):  
Thea Neumann ◽  
Maren Krüger ◽  
Jasmin Weisemann ◽  
Stefan Mahrhold ◽  
Daniel Stern ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Receptor Interaction ◽  
Clostridium Perfringens Enterotoxin ◽  
Fast Detection ◽  
Highly Sensitive ◽  
Basic And Applied Research ◽  
Highly Sensitive Detection

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research.

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