scholarly journals Calf Diarrhea Caused by Prolonged Expansion of Autochthonous Gut Enterobacteriaceae and Their Lytic Bacteriophages

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Tae Woong Whon ◽  
Hyun Sik Kim ◽  
Na-Ri Shin ◽  
Hojun Sung ◽  
Min-Soo Kim ◽  
...  
Keyword(s):  
Disease Control ◽  
Body Weight Gain ◽  
Sulfur Metabolism ◽  
Transcriptional Profiling ◽  
Common Disease ◽  
Rrna Gene ◽  
Gut Inflammation ◽  
Calf Diarrhea ◽  
Multiplex Pcr Assay ◽  
Diarrheic Calves

ABSTRACT Neonatal calf diarrhea is a common disease leading to a major economic loss for cattle producers worldwide. Several infectious and noninfectious factors are implicated in calf diarrhea, but disease control remains problematic because of the multifactorial etiology of the disease. Here, we conducted diagnostic multiplex PCR assay and meta-omics analysis (16S rRNA gene-based metataxonomics and untargeted transcriptional profiling) of rectal content of normal and diarrheic beef calves (n = 111). In the diarrheic calf gut, we detected both microbial compositional dysbiosis (i.e., increased abundances of the family Enterobacteriaceae members and their lytic bacteriophages) and functional dysbiosis (i.e., elevated levels of aerobic respiration and virulence potential). The calf diarrheic transcriptome mirrored the gene expression of the bovine host and was enriched in cellular pathways of sulfur metabolism, innate immunity, and gut motility. We then isolated 12 nontoxigenic Enterobacteriaceae strains from the gut of diarrheic calves. Feeding a strain mixture to preweaning mice resulted in a significantly higher level of fecal moisture content, with decreased body weight gain and shortened colon length. The presented findings suggest that gut inflammation followed by a prolonged expansion of nontoxigenic autochthonous Enterobacteriaceae contributes to the onset of diarrhea in preweaning animals. IMPORTANCE Calf diarrhea is the leading cause of death of neonatal calves worldwide. Several infectious and noninfectious factors are implicated in calf diarrhea, but disease control remains problematic because of the multifactorial etiology of the disease. The major finding of the current study centers around the observation of microbial compositional and functional dysbiosis in rectal samples from diarrheic calves. These results highlight the notion that gut inflammation followed by a prolonged expansion of autochthonous Enterobacteriaceae contributes to the onset of calf diarrhea. Moreover, this condition possibly potentiates the risk of invasion of notorious enteric pathogens, including Salmonella spp., and the emergence of inflammation-resistant (or antibiotic-resistant) microbiota via active horizontal gene transfer mediated by lytic bacteriophages.

scholarly journals Characterization and Transcriptome Analysis of a Long-Chain n-Alkane-Degrading Strain Acinetobacter Pittii SW-1

2021 ◽  
Vol 18 (12) ◽  
pp. 6365
Author(s):  
Weina Kong ◽  
Cheng Zhao ◽  
Xingwang Gao ◽  
Liping Wang ◽  
Qianqian Tian ◽  
...  
Keyword(s):  
Unsaturated Fatty Acids ◽  
Sulfur Metabolism ◽  
Quantitative Polymerase Chain Reaction ◽  
Deep Ocean ◽  
Rrna Gene ◽  
Alkane Hydroxylase ◽  
Degradation Rates ◽  
Acinetobacter Pittii ◽  
Ocean Environment ◽  
Long Chain

Strain sw-1, isolated from 7619-m seawater of the Mariana Trench, was identified as Acinetobacter pittii by 16S rRNA gene and whole-genome sequencing. A. pittii sw-1 was able to efficiently utilize long-chain n-alkanes (C18–C36), but not short- and medium-chain n-alkanes (C8–C16). The degradation rate of C20 was 91.25%, followed by C18, C22, C24, C32, and C36 with the degradation rates of 89.30%, 84.03%, 80.29%, 30.29%, and 13.37%, respectively. To investigate the degradation mechanisms of n-alkanes for this strain, the genome and the transcriptome analyses were performed. Four key alkane hydroxylase genes (alkB, almA, ladA1, and ladA2) were identified in the genome. Transcriptomes of strain sw-1 grown in C20 or CH3COONa (NaAc) as the sole carbon source were compared. The transcriptional levels of alkB and almA, respectively, increased 78.28- and 3.51-fold in C20 compared with NaAc, while ladA1 and ladA2 did not show obvious change. The expression levels of other genes involved in the synthesis of unsaturated fatty acids, permeases, membrane proteins, and sulfur metabolism were also upregulated, and they might be involved in n-alkane uptake. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) confirmed that alkB expression was significantly induced by C20, C24, and C32, and almA induction extent by C24 and C32 was higher than that with C20. Furthermore, ladA2 expression was only induced by C32, and ladA1 expression was not induced by any of n-alkanes. In addition, A. pittii sw-1 could grow with 0%–3% NaCl or 8 out of 10 kinds of the tested heavy metals and degrade n-alkanes at 15 °C. Taken together, these results provide comprehensive insights into the degradation of long-chain n-alkanes by Acinetobacter isolated from the deep ocean environment.

scholarly journals A Metagenomics Investigation of Intergenerational Effects of Non-nutritive Sweeteners on Gut Microbiome

2022 ◽  
Vol 8 ◽  
Author(s):  
Weilan Wang ◽  
Jodi E. Nettleton ◽  
Michael G. Gänzle ◽  
Raylene A. Reimer
Keyword(s):  
Body Weight ◽  
Body Fat ◽  
Body Weight Gain ◽  
Lactate Production ◽  
16S Rrna Gene Sequencing ◽  
Liver Weight ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Microbial Composition ◽  
Maternal Consumption

To identify possible mechanisms by which maternal consumption of non-nutritive sweeteners increases obesity risk in offspring, we reconstructed the major alterations in the cecal microbiome of 3-week-old offspring of obese dams consuming high fat/sucrose (HFS) diet with or without aspartame (5–7 mg/kg/day) or stevia (2–3 mg/kg/day) by shotgun metagenomic sequencing (n = 36). High throughput 16S rRNA gene sequencing (n = 105) was performed for dams, 3- and 18-week-old offspring. Maternal consumption of sweeteners altered cecal microbial composition and metabolism of propionate/lactate in their offspring. Offspring daily body weight gain, liver weight and body fat were positively correlated to the relative abundance of key microbes and enzymes involved in succinate/propionate production while negatively correlated to that of lactose degradation and lactate production. The altered propionate/lactate production in the cecum of weanlings from aspartame and stevia consuming dams implicates an altered ratio of dietary carbohydrate digestion, mainly lactose, in the small intestine vs. microbial fermentation in the large intestine. The reconstructed microbiome alterations could explain increased offspring body weight and body fat. This study demonstrates that intense sweet tastants have a lasting and intergenerational effect on gut microbiota, microbial metabolites and host health.

scholarly journals Comparative Analysis of the Gut Microbiota of Adult Mosquitoes From Eight Locations in Hainan, China

2020 ◽  
Vol 10 ◽  
Author(s):  
Xun Kang ◽  
Yanhong Wang ◽  
Siping Li ◽  
Xiaomei Sun ◽  
Xiangyang Lu ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Disease Control ◽  
Bacterial Communities ◽  
Mosquito Species ◽  
Microbial Community Composition ◽  
Variable Region ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Quality Filtering ◽  
And Function

The midgut microbial community composition, structure, and function of field-collected mosquitoes may provide a way to exploit microbial function for mosquito-borne disease control. However, it is unclear how adult mosquitoes acquire their microbiome, how the microbiome affects life history traits and how the microbiome influences community structure. We analyzed the composition of 501 midgut bacterial communities from field-collected adult female mosquitoes, including Aedes albopictus, Aedes galloisi, Culex pallidothorax, Culex pipiens, Culex gelidus, and Armigeres subalbatus, across eight habitats using the HiSeq 4000 system and the V3−V4 hyper-variable region of 16S rRNA gene. After quality filtering and rarefaction, a total of 1421 operational taxonomic units, belonging to 29 phyla, 44 families, and 43 genera were identified. Proteobacteria (75.67%) were the most common phylum, followed by Firmicutes (10.38%), Bacteroidetes (6.87%), Thermi (4.60%), and Actinobacteria (1.58%). The genera Rickettsiaceae (33.00%), Enterobacteriaceae (20.27%), Enterococcaceae (7.49%), Aeromonadaceae (7.00%), Thermaceae (4.52%), and Moraxellaceae (4.31%) were dominant in the samples analyzed and accounted for 76.59% of the total genera. We characterized the midgut bacterial communities of six mosquito species in Hainan province, China. The gut bacterial communities were different in composition and abundance, among locations, for all mosquito species. There were significant differences in the gut microbial composition between some species and substantial variation in the gut microbiota between individuals of the same mosquito species. There was a marked variation in different mosquito gut microbiota within the same location. These results might be useful in the identification of microbial communities that could be exploited for disease control.

scholarly journals Yoghurt Consumption is Associated With Transient Changes in the Composition of the Human Gut Microbiome

2020 ◽  
Author(s):  
Caroline Ivanne Le Roy ◽  
Alexander Kurilshikov ◽  
Emily Leeming ◽  
Alessia Visconti ◽  
Ruth Bowyer ◽  
...  
Keyword(s):  
16S Rrna ◽  
Gut Microbiota ◽  
Visceral Fat ◽  
Gut Microbiome ◽  
Body Weight Gain ◽  
Healthy Eating Index ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Sequencing Data ◽  
Shotgun Metagenomic Sequencing

Abstract Background: Yoghurt contains live bacteria that could contribute via modulation of the gut microbiota to its reported beneficial effects such as reduced body weight gain and lower incidence of type 2 diabetes. To date, the association between yoghurt consumption and the composition of the gut microbiota is underexplored. Here we used clinical variables, metabolomics, 16S rRNA and shotgun metagenomic sequencing data collected on over 1000 predominantly female UK twins to define the link between the gut microbiota and yoghurt-associated health benefits. Results: According to food frequency questionnaires (FFQ), 73% of subjects consumed yoghurt. Consumers presented a healthier diet pattern (healthy eating index: beta = 2.17±0.34; P = 2.72x10-10) and improved metabolic health characterised by reduced visceral fat (beta = -28.18±11.71 g; P = 0.01). According to 16S rRNA gene analyses and whole shotgun metagenomic sequencing approach consistent taxonomic variations were observed with yoghurt consumption. More specifically, we identified higher abundance of species used as yoghurt starters Streptococcus thermophilus (beta = 0.41±0.051; P = 6.14x10-12) and sometimes added Bifidobacterium animalis subsp. lactis (beta = 0.30±0.052; P = 1.49x10-8) in the gut of yoghurt consumers. Replication in 1103 volunteers from the LifeLines-DEEP cohort confirmed the increase of S. thermophilus among yoghurt consumers. Using food records collected the day prior to faecal sampling we showed that increase in these two yoghurt bacteria could be transient. Metabolomics analysis revealed that B. animalis subsp. lactis was associated with 13 faecal metabolites including a 3-hydroxyoctanoic acid, known to be involved in the regulation of gut inflammation.Conclusions: Yoghurt consumption is associated with reduced visceral fat mass and changes in gut microbiome including transient increase of yoghurt-contained species (i.e. S. thermophilus and B. lactis).

scholarly journals Dietary Proteins Relevant to Human Consumption Impact the Development of Obesity and Type 2 Diabetes in Association with Major Changes in the Gut Microbiota in a Mouse Model (OR27-03-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Noëmie Daniel ◽  
Béatrice Choi ◽  
Vanessa Houde ◽  
Thibault Varin ◽  
Cecile Vors ◽  
...  
Keyword(s):  
Animal Models ◽  
Gut Microbiota ◽  
Insulin Signaling ◽  
Body Weight Gain ◽  
Bacterial Species ◽  
Human Consumption ◽  
Oral Glucose ◽  
Rrna Gene ◽  
Dietary Proteins ◽  
The Impact

Abstract Objectives Animal models fed a high-fat high-sucrose (HFHS) diet are commonly used to study obesity and cardiometabolic diseases. While much attention is paid to the impact of fat and carbohydrates sources, very little consideration is given to the composition of dietary proteins. Indeed, casein is often the only source of protein in rodent's diet. This study aimed to evaluate the impact of a dietary protein mix that is more relevant to typical intakes of proteins in humans and its influences on body weight gain, metabolic health and gut microbiota. Methods Our new diet contained a mix of 10 protein sources based on NHANES data that were incorporated into low-fat low-sucrose (LFLS) and HFHS diets. C57BL/6J mice were fed these diets or control diets containing identical amounts of casein as the only source of protein for 12 weeks. Feces were collected for gut microbiota investigation, an oral glucose tolerance test was performed and tissues were harvested for analysis of insulin signaling and mTOR/S6K1 activation. Results 16S rRNA gene sequencing of fecal samples showed that both LFLS and HFHS mice fed the protein mix had increased gut microbiota diversity, and significant changes in the relative abundance of several bacterial species (higher Adlercreutzia or Tyzzerella, lower Bacteroides or Akkermansia) as compared to mice fed casein only. Importantly, inclusion of the protein mix amplified the effects of the HFHS diet on the development of obesity, glucose intolerance and hyperinsulinemia as compared to casein-fed animals, whereas no difference was observed in the context of LFLS feeding. Evaluation of insulin signaling in the liver also revealed that the protein mix potentiated the effect of HFHS feeding on the mTORC1/S6K1 pathway, increasing inhibitory phosphorylation of IRS-1 on Ser1101 and leading to further impairment of Akt activation by insulin. Conclusions Our results reveal that compared to pure casein, feeding a protein mixture causes major changes in the gut microbiota profile and greater impact on HFHS-induced obesity and associated metabolic impairments. This study illustrates the importance of considering a diverse source of dietary proteins when using laboratory animal models to more reliably reproduce the development of metabolic syndrome in humans, and to enhance the clinical relevance of nutritional and therapeutic interventions. Funding Sources N/A.

scholarly journals Cryptosporidium parvum, Cryptosporidium ryanae, and Cryptosporidium bovis in samples from calves in Austria

2020 ◽  
Vol 119 (12) ◽  
pp. 4291-4295
Author(s):  
Katharina Lichtmannsperger ◽  
Josef Harl ◽  
Katharina Freudenthaler ◽  
Barbara Hinney ◽  
Thomas Wittek ◽  
...  
Keyword(s):  
Phase Contrast ◽  
Gene Sequencing ◽  
Rrna Gene ◽  
Phase Contrast Microscopy ◽  
Widespread Occurrence ◽  
Contrast Microscopy ◽  
Pcr Products ◽  
Rrna Gene Sequencing ◽  
Diarrheic Calves ◽  
Pcr Targeting

AbstractFecal samples of 177 calves of up to 180 days of age with diarrhea from 70 farms in Austria were examined to obtain information on the occurrence of Cryptosporidium species. Initially, all samples were examined by phase-contrast microscopy. Cryptosporidium-positive samples (55.4%; n = 98) were screened by gp60 PCR, resulting in 68.4% (n = 67) C. parvum–positive samples. The remaining 31 gp60-PCR-negative and the phase-contrast microscopy negative samples (n = 79) were screened by PCR targeting a 700 bp fragment of the 18S rRNA gene. Sequencing of the PCR products revealed the presence of C. parvum (n = 69), C. ryanae (n = 11), and C. bovis (n = 7). The latter two species have never been described in Austria. C. parvum–positive samples were genotyped at the gp60 gene locus, featuring four subtypes (IIaA15G2R1, IIaA21G2R1, IIaA19G2R1, IIaA14G1R1). The most frequently detected subtype IIaA15G2R1 (n = 52) was present in calves from 30 different farms. IIaA14G1R1 (n = 5) occurred on a single farm, subtype IIaA21G2R1 (n = 4) on two farms, and subtype IIaA19G2R1 (n = 4) on three farms. The results confirm the widespread occurrence of zoonotic C. parvum in diarrheic calves.

scholarly journals Application of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Confirmation of Listeria monocytogenes and Other Listeria Species in Turkey Sample Surveillance†

2002 ◽  
Vol 65 (5) ◽  
pp. 780-785 ◽  
Author(s):  
IRENE V. WESLEY ◽  
KAREN M. HARMON ◽  
JAMES S. DICKSON ◽  
ANN RAMOS SCHWARTZ
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Listeria Species ◽  
Multiplex Pcr Assay ◽  
Polymerase Chain

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.

scholarly journals Anodes Stimulate Anaerobic Toluene Degradation via Sulfur Cycling in Marine Sediments

2015 ◽  
Vol 82 (1) ◽  
pp. 297-307 ◽  
Author(s):  
Matteo Daghio ◽  
Eleni Vaiopoulou ◽  
Sunil A. Patil ◽  
Ana Suárez-Suárez ◽  
Ian M. Head ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Marine Sediments ◽  
Sulfur Metabolism ◽  
Direct Electron Transfer ◽  
Rrna Gene ◽  
Toluene Degradation ◽  
Α Subunit ◽  
Mediated Electron Transfer ◽  
Benzylsuccinate Synthase ◽  
Pcr Targeting

ABSTRACTHydrocarbons released during oil spills are persistent in marine sediments due to the absence of suitable electron acceptors below the oxic zone. Here, we investigated an alternative bioremediation strategy to remove toluene, a model monoaromatic hydrocarbon, using a bioanode. Bioelectrochemical reactors were inoculated with sediment collected from a hydrocarbon-contaminated marine site, and anodes were polarized at 0 mV and +300 mV (versus an Ag/AgCl [3 M KCl] reference electrode). The degradation of toluene was directly linked to current generation of up to 301 mA m−2and 431 mA m−2for the bioanodes polarized at 0 mV and +300 mV, respectively. Peak currents decreased over time even after periodic spiking with toluene. The monitoring of sulfate concentrations during bioelectrochemical experiments suggested that sulfur metabolism was involved in toluene degradation at bioanodes. 16S rRNA gene-based Illumina sequencing of the bulk anolyte and anode samples revealed enrichment with electrocatalytically active microorganisms, toluene degraders, and sulfate-reducing microorganisms. Quantitative PCR targeting the α-subunit of the dissimilatory sulfite reductase (encoded bydsrA) and the α-subunit of the benzylsuccinate synthase (encoded bybssA) confirmed these findings. In particular, members of the familyDesulfobulbaceaewere enriched concomitantly with current production and toluene degradation. Based on these observations, we propose two mechanisms for bioelectrochemical toluene degradation: (i) direct electron transfer to the anode and/or (ii) sulfide-mediated electron transfer.

scholarly journals A new way for punicalagin to alleviate insulin resistance: regulating gut microbiota and autophagy

2021 ◽  
Author(s):  
Yuan Cao ◽  
Guofeng Ren ◽  
Yahui Zhang ◽  
Hong Qin ◽  
Xin An ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Liver Injury ◽  
Gut Microbiota ◽  
Research Study ◽  
Inflammatory Responses ◽  
Body Weight Gain ◽  
Serum Insulin ◽  
Pomegranate Juice ◽  
Rrna Gene ◽  
Lipid Metabolism Disorder

Background: Insulin resistance, defined as a diminished ability to respond to the stimulation of insulin, is the main line for a variety of metabolic-related diseases. Punicalagin (PU), a hydrolyzable tannin of pomegranate juice, exhibits multiple biological properties, including anti-oxidant, anti-cancer and anti-inflammatory activities. Objective: This research study aimed at determining the protective effect of PU on insulin resistance and to uncover the underlying mechanism based on the gut microbiota, IKKβ/NF-κB pathway, and autophagy. Design: An insulin resistance animal model was established using C57BL/6 mice fed with a high-fat diet (HFD) for 8 weeks. The model included two groups continuing a HFD for 12 weeks with or without administering via gavage with PU 20 mg/kg/day. Changes in fasting plasma glucose levels, fasting serum insulin levels, glucose and insulin tolerance, glycolipid metabolism, gut microbiota composition (16S rRNA gene sequencing), inflammatory responses, and autophagy in the liver were evaluated. Body weight gain, glycolipid metabolic disorder, liver injury, as well as systemic and hepatic insulin sensitivity, were significantly attenuated after supplementing with PU. Results: This research study revealed that PU alleviated HFD-induced glucose and lipid disorders, liver injury and insulin resistance; decreased the Firmicutes/Bacteroides ratio, decreased the abundance of Coprococcus and Anaerotruncus, and increased Rikenellaceae; and decreased serum and liver tumor necrosis factor-alpha and interleukin-1β levels, inhibited liver IKKβ and NF-κB phosphorylation; and increased liver autophagy-related proteins LC3-II, P62, and Beclin1, and increased the number of liver autophagosomes. Conclusion: PU can improve HFD-induced insulin resistance, improved liver glucose and lipid metabolism disorder and liver injury, and the potential mechanism is that PU inhibited the IKKβ/NF-κB inflammatory pathway by regulating gut microbiota homeostasis and up-regulating liver autophagy

scholarly journals The international vaccine scene

2004 ◽  
Vol 28 (2) ◽  
pp. 206 ◽  
Author(s):  
Priscilla Robinson
Keyword(s):  
Public Health ◽  
Disease Control ◽  
Bacterial Infections ◽  
Communicable Disease ◽  
Control Measures ◽  
Common Disease ◽  
Communicable Disease Control ◽  
Enteric Diseases ◽  
The World ◽  
Syncytial Virus

INFECTIOUS AND COMMUNICABLE DISEASES account for a large burden of disease worldwide, causing the death of roughly a million people each month (WHO 2004). This book is about some of the ways in which this enormous problem is being addressed. The book comprises a collection of papers from the global Conference on vaccines, prevention, and public health and is as much about public health thinking in communicable disease control as it is specifically about vaccines. Its contributors are practitioners in the field of immunisation and communicable disease control from all over the world. The book is clearly set out in eight sections, with both an introductory summary, Setting the stage, and an Epilogue summary section. The specific sections cover The present, with summaries of measles, rubella and poliovirus control measures, as well as the case of yellow fever which is used as a warning about what can happen if we take our eyes off the public-health-control ball. This is followed by The cutting edge, covering the newest vaccines for Haemophilus influenzae, varicella (chicken pox), hepatitis A, and the two major bacterial infections ? meningococcal and pneumococcal diseases. The future section covers prospects for vaccines to control a variety of infections, including the enteric diseases rotavirus, Shigella and typhoid; two major causes of cancer, Helicobacter pylori (gastric cancers and ulcers) and human papillomavirus (cervical cancer); hepatitis C; and two common causes of viral respiratory disease, influenza and respiratory syncytial virus (RSV). RSV is a good example of a common disease which carries a large burden of morbidity (Chin 2002) all over the world, especially among children, and yet few people have heard of it.

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