This document describes two experiments that examined the effects of dietary zinc source and coccidial vaccine exposure on zinc homeostasis and immune status in broiler chickens. The experiments found that coccidial challenge decreased intracellular zinc levels and phagocytic capacity in the jejunum, while increasing phagocytic capacity in the caecal tonsils. Coccidial challenge also increased the ratio of zinc import to export transporters. Dietary zinc source had little impact except on one zinc transporter. The results suggest that during coccidial challenge, intestinal cells attempt to compensate for the drop in intracellular zinc by upregulating zinc import transporters.
The document reports on further characterization of an Escherichia coli K-12 mutant (E. coli MG1655*) that was previously isolated following passage through the mouse intestine and shown to have enhanced intestinal colonization abilities compared to the wild-type parent strain. Whole genome pyrosequencing revealed no changes to the E. coli MG1655* genome aside from a previously identified deletion in the flhDC locus. Microarray and catabolome analyses showed that both E. coli MG1655* and an isogenic flhD mutant constructed in the laboratory had modest yet significant induction of catabolic gene systems and an enhanced ability to utilize a variety of carbon sources, respectively. The results suggest that loss of FlhD, and the
1. The document discusses metagenomics research on the human gut microbiome using a gene-centric approach.
2. It involves sequencing microbial DNA from gut samples to identify genes and characterize functional profiles of the microbiome.
3. Analysis of Japanese gut microbiome samples identified over 600,000 genes, including many novel to the gut microbiome, providing insight into microbiome composition and functions.
Nutrition Research - Decreased ZnT-2 transporter expression in offspring prod...Aroldo Trejo
Rats born to mothers fed a zinc-deficient diet during pregnancy and lactation displayed stunted growth and decreased expression of the ZnT-2 zinc transporter in the small intestine compared to rats born to mothers fed an adequate zinc diet. Offspring of severely zinc-deficient mothers died or had spinal defects. The downregulation of the ZnT-2 transporter, which exports zinc out of cells, helps maintain zinc homeostasis in conditions of deficiency by reducing zinc efflux. The results emphasize the importance of adequate maternal zinc intake to prevent adverse outcomes in offspring such as stunted growth and mortality.
TNF-alpha and LPA promote synergistic expression of COX-2 in human colonic my...Enrique Moreno Gonzalez
Enhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We
recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α.
Discovering the 100 Trillion Bacteria Living Within Each of UsLarry Smarr
This document provides a summary of a lecture on the human microbiome given by Dr. Larry Smarr. Some key points:
- The human microbiome refers to the trillions of bacteria that live within the human body. Each person contains 100 trillion bacteria, outnumbering human cells.
- Research into the microbiome is a rapidly growing field that provides insights into health and disease. The microbiome plays a role in processes like drug metabolism and immunity.
- The microbiome is established early in life and influenced by factors like birth method and antibiotic use in the first years. This early development can impact future health.
- Microbiome composition and function can change with health status, diet, medications and other
The document reports on further characterization of an Escherichia coli K-12 mutant (E. coli MG1655*) that was previously isolated following passage through the mouse intestine and shown to have enhanced intestinal colonization abilities compared to the wild-type parent strain. Whole genome pyrosequencing revealed no changes to the E. coli MG1655* genome aside from a previously identified deletion in the flhDC locus. Microarray and catabolome analyses showed that both E. coli MG1655* and an isogenic flhD mutant constructed in the laboratory had modest yet significant induction of catabolic gene systems and an enhanced ability to utilize a variety of carbon sources, respectively. The results suggest that loss of FlhD, and the
1. The document discusses metagenomics research on the human gut microbiome using a gene-centric approach.
2. It involves sequencing microbial DNA from gut samples to identify genes and characterize functional profiles of the microbiome.
3. Analysis of Japanese gut microbiome samples identified over 600,000 genes, including many novel to the gut microbiome, providing insight into microbiome composition and functions.
Nutrition Research - Decreased ZnT-2 transporter expression in offspring prod...Aroldo Trejo
Rats born to mothers fed a zinc-deficient diet during pregnancy and lactation displayed stunted growth and decreased expression of the ZnT-2 zinc transporter in the small intestine compared to rats born to mothers fed an adequate zinc diet. Offspring of severely zinc-deficient mothers died or had spinal defects. The downregulation of the ZnT-2 transporter, which exports zinc out of cells, helps maintain zinc homeostasis in conditions of deficiency by reducing zinc efflux. The results emphasize the importance of adequate maternal zinc intake to prevent adverse outcomes in offspring such as stunted growth and mortality.
TNF-alpha and LPA promote synergistic expression of COX-2 in human colonic my...Enrique Moreno Gonzalez
Enhanced EGF receptor (EGFR) signaling is a hallmark of many human cancers, though the role of enhanced EGFR signaling within the surrounding tumor stroma has not been well studied. The myofibroblast is an important stromal cell that demonstrates enhanced EGFR expression in the setting of inflammation, though the functional relevance is not known. We
recently reported that TNF-α and the G protein-coupled receptor (GPCR) agonist lysophosphatidic acid (LPA) lead to synergistic cyclo-oxygenase-2 (COX-2) expression, an enzyme strongly associated with the development of colitis-associated cancer. Here, we investigate whether EGFR signaling plays a role in the synergistic COX-2 expression induced by LPA and TNF-α.
Discovering the 100 Trillion Bacteria Living Within Each of UsLarry Smarr
This document provides a summary of a lecture on the human microbiome given by Dr. Larry Smarr. Some key points:
- The human microbiome refers to the trillions of bacteria that live within the human body. Each person contains 100 trillion bacteria, outnumbering human cells.
- Research into the microbiome is a rapidly growing field that provides insights into health and disease. The microbiome plays a role in processes like drug metabolism and immunity.
- The microbiome is established early in life and influenced by factors like birth method and antibiotic use in the first years. This early development can impact future health.
- Microbiome composition and function can change with health status, diet, medications and other
TiF1-gamma Plays an Essential Role in Murine Hematopoiesis and Regulates Tran...Joe Lee
This document summarizes a study examining the role of the transcriptional co-factor TIF1-gamma in murine hematopoiesis and erythropoiesis. The study finds that deletion of TIF1-gamma in mice leads to:
1) A block in erythroid maturation in the bone marrow, compensated by enhanced spleen erythropoiesis.
2) Defects in other blood lineages, including a loss of B cells and expansion of granulocytes.
3) Decreased hematopoietic stem cell function.
The deletion of TIF1-gamma also results in reduced transcription elongation of erythroid genes in bone marrow cells. This establishes T
This document summarizes a presentation on the gut microbiota and the impact of probiotics. It discusses the gut as a rich ecosystem containing bacteria, archaea, viruses, and eukaryotes. The presentation notes that the human microbiome contains over 100 times as many genes as the human genome. It also reviews clinical studies that have examined the effects of consuming various probiotic strains on the composition of the gut microbiota in healthy adults and IBS patients. The studies found mostly minor or no changes to the dominant microbiota with probiotic intake. However, some genera were found to be modulated in some individuals. The presentation concludes by discussing the need for more targeted screening of probiotics and understanding what level of microbiota modulation leads
This document summarizes research on immortalizing bovine pancreatic duct cells and making them tumorigenic through genetic manipulation. Key points:
1) Bovine pancreatic duct cells were immortalized by transfecting them with SV40 large T antigen cDNA. The immortalized cells retained features of normal duct cells.
2) The immortalized duct cells were then transfected with a mutated K-ras gene. This caused the cells to gain the ability to form tumors when injected into nude mice, suggesting K-ras plays an important role in pancreatic carcinogenesis.
3) The research aims to establish a model for the transition from normal duct cell to neoplastic cell in order to study the initial steps of pancreatic cancer development
Human Microbiome is the current project in Research field. The importance of Microorganisms in the human body, the importance and novel roe of the microorganisms on a human body is very effective and helpful. Fecal Transplantation is a unique and helpful technique to cure a dreadful disease naturally by means of microorganisms or introducing the normal flora in to the body again.
The document discusses gut microbiota and various techniques used to study it. It begins by describing the importance of gut microbiota in digestion, development of the immune system, maintenance of the intestinal barrier, and health and disease. It then covers culture-based and molecular-based techniques used to characterize gut microbiota, including their pros and cons. Specific molecular techniques discussed include FISH, DNA microarrays, DGGE/TGGE, 454 pyrosequencing, and quantitative PCR. The document also addresses pitfalls of molecular techniques and provides examples of studies comparing the gut microbiota of normal vs. diseased horses.
This document describes a study that sequenced the 23S rRNA gene of Campylobacter coli VC167 and analyzed its sequence and structure. It found that the gene contained a 147 bp transcribed spacer that resulted in fragmented 23S rRNA. Analysis of 31 other C. coli and C. jejuni strains found that 69% contained a transcribed spacer of 147 bp or 37 bp, also producing fragmented 23S rRNA. Phylogenetic analysis placed Campylobacter in the epsilon subdivision of Proteobacteria based on signatures in the 23S rRNA at positions 270 and 1850.
An Ecophylogenetic Approach to Determine the Evolutionary History of the Mamm...tsharpton
Identifying those gut microbes that co-diversify with mammals is important to our understanding of the mechanisms and health implications of host-microbiome interactions. For example, microbiota that are conserved across mammalian species may express a trait that has been subject to selection throughout the evolution of these mammals, possibly because it is critical to health. While advances in environmental DNA sequencing have transformed our understanding of how enteric microbes are distributed across mammalian species, these data are frequently analyzed using phylogenetically agnostic approaches. Such approaches can obscure the detection of diverged groups of bacteria that have been conserved across mammalian species. To provide enhanced resolution into evolutionary associations between gut microbiota and mammals, we innovated a high-throughput ecophylogenetic method, known as ClaatTU (Cladal Taxonomic Units). ClaaTU analyzes phylogenies assembled from environmental DNA sequences collected from a set of microbial communities and profiles the presence and abundance of each monophyletic clade in each community. As a result, it enables the identification of specific microbial clades that are distributed across host communities in a manner indicative of being associated with mammalian evolution. To demonstrate this, we applied ClaaTU to a mammalian microbiome dataset and (1) identified clades of gut bacteria that are unique to groups of mammals based on their taxonomy or dietary regime, (2)
found that there exists ecophylogenetic structure in the mammalian gut microbiome, indicating that gut bacterial phylogenetic diversity associates with host phylogeny, and
(3) discovered specic clades that are present in a larger number of mammals than expected by chance, some of which may co-diversify with their hosts. Our findings indicate that some mammalian gut microbiota may have been anciently acquired and subsequently retained in extant lineages, indicating that they may play an important role in mediating host-microbiome interactions and maintaining host health.
1) Researchers developed transgenic medaka (fish) carrying multiple copies of a bacteriophage vector containing the cII gene, which serves as a target for detecting mutations.
2) They adapted an assay used in transgenic rodents to efficiently recover cII mutants from fish genomic DNA and detect them by infecting bacteria.
3) Spontaneous mutant frequencies in liver, whole fish, and testes of medaka were comparable to frequencies observed in transgenic rodents. Treatment with ethylnitrosourea induced cII mutations in the medaka in a tissue-specific and time-dependent manner consistent with known mutagen mechanisms.
This document is the curriculum vitae of Prof Dr Ariyanto Harsono dr SpAK. It details his educational background, including graduating from medical school in 1972 and specializing in pediatrics in 1982. It lists his current position as a professor of pediatric health sciences at the University of Airlangga's medical faculty and as a consultant in allergy and immunology. It also provides information on additional training in food allergy he received in the Netherlands from 1990-1991.
- Microbial metabolites from the gut microbiome, especially butyrate, play an important role in modulating intestinal damage and graft-versus-host disease (GvHD) severity.
- In mice that underwent allogeneic stem cell transplantation, butyrate levels decreased significantly in intestinal epithelial cells, likely due to reduced butyrate transporter expression from inflammation.
- Restoring butyrate levels through intragastric gavage improved epithelial integrity, reduced GvHD severity and improved survival. Specific butyrate-producing bacteria were also associated with lower GvHD risk.
This document describes a microarray analysis comparing gene expression profiles in the large intestine, small intestine, liver, and spleen of mice with different gut microbiota colonization models: specific pathogen-free mice, germ-free mice colonized at birth, and germ-free mice colonized at 5 weeks of age. The analysis found hundreds of differentially expressed genes in each tissue and colonization model. Gene set enrichment analysis identified overrepresented gene ontology categories related to immune system development and antigen presentation in intestines of mice colonized at birth, and metabolic processes in intestines of specific pathogen-free mice. Analysis of signaling pathways found prominent changes in toll-like receptor and type 1 interferon signaling pathways in intestines of mice
Integrin V can form heterodimers with several subunits to mediate cell-cell and cell-extracellular matrix interactions. During zebrafish gastrulation, V is expressed maternally and zygotically. Here, we used a morpholino-mediated V knockdown strategy to study V function. Although V morphants displayed vascular defects, they also exhibited left-right body asymmetry defects affecting multiple visceral organs. This was preceded by mislocalization of dorsal forerunner cells (DFCs) and malformation of the Kupffer’s vesicle (KV) laterality organ
This document compares growth rates and gene expression in two species of oysters, the Pacific oyster and Olympia oyster. It finds that the Pacific oyster grows more rapidly than the Olympia oyster. It identifies four genes associated with growth in both species and analyzes their expression levels in different tissues and time periods. The expression of these genes varies between the species and over time, indicating differences in their metabolic processes during growing versus fasting seasons.
Science 2013-schuenemann-179-83 leprosy önemliHazal Sav
This study obtained near-complete genome sequences of Mycobacterium leprae from skeletal remains dating from the 11th to 14th centuries in Europe, as well as from recent patient biopsies. Genome comparisons revealed remarkable genomic conservation of M. leprae over the past 1000 years. The ancient genomes suggest a European origin for leprosy in the Americas and the presence of a genotype in medieval Europe now commonly associated with the Middle East. Exceptional DNA and mycolic acid preservation in the ancient skeletal remains provides insights into pathogen evolution and the disappearance of leprosy in Europe.
This document reports on a study examining the effects of somatostatin (SST) on human B lymphoblasts. The key findings are:
1) SST stimulates phospholipase C activity and increases cytosolic calcium levels in B lymphoblasts, likely through coupling of SSTR2A to the G protein Gα16.
2) SST activates extracellular signal-regulated kinases and induces increased DNA synthesis, proliferation, and immunoglobulin formation in B lymphoblasts.
3) These stimulatory effects of SST on early signal transduction pathways are accompanied by increased cell proliferation and immunoglobulin production in human B lymphoblasts, indicating SST exerts growth factor-like
The document summarizes an experiment that tested the hypothesis that the peripheral aster spreading during metaphase determines the pattern of the cortical F-actin ring that forms prior to polar body extrusion. The experiment used two methods - treatment with lovastatin followed by washout, and treatment with cytochalasin D followed by washout - to induce multiple cortical spreading asters. Both methods resulted in the formation of multiple, overlapping F-actin rings, supporting the hypothesis that microtubules of the peripheral aster provide a positive stimulus that directs F-actin ring formation.
31 октября самый цитируемый ученый русского происхождения, известный эволюционный биолог Евгений Кунин рассказал в Digital October о постмодернистском взгляде на эволюционные процессы.
http://digitaloctober.ru/ru/events/knowledge_stream_evolyutsiya_po_sluchayu
This document describes the development of a genetically encoded fluorescent biosensor for 2-oxoglutarate (2OG) based on fluorescence resonance energy transfer (FRET). The researchers constructed the biosensor by fusing yellow fluorescent protein (YFP), the 2OG-binding GAF domain of the NifA protein, and cyan fluorescent protein (CFP) between restriction sites in a plasmid vector. They tested the biosensor in vitro and found it responded to 2OG concentrations within the physiological range observed in E. coli cells. They also optimized the peptide linker between domains. In vivo, the biosensor detected changes in 2OG levels in E. coli cells in response to different carbon sources added. The biosensor could image
1) The study analyzed data from over 1,000 Western adults and found evidence that some taxa in the human gut microbiome exist in alternative stable states, shifting between high and low abundance profiles.
2) Specifically, around 10% of prevalent taxa showed bimodal abundance distributions and reduced stability at intermediate abundances, indicating bi-stability.
3) These bi-stable "tipping elements" can co-occur in different combinations and have been linked to metabolic syndrome and inflammatory bowel disease.
The document provides a resume for Alexandria Valdivia. It summarizes her objective as obtaining a rewarding career in medical sonography utilizing her associate's degree in diagnostic medical sonography. It lists her qualifications which include ultrasound certifications and medical training. It also outlines her skills such as patient care, communication, and organization. Her education and externship experiences in ultrasound are described. Previous work experiences in retail are listed, and references are provided.
TiF1-gamma Plays an Essential Role in Murine Hematopoiesis and Regulates Tran...Joe Lee
This document summarizes a study examining the role of the transcriptional co-factor TIF1-gamma in murine hematopoiesis and erythropoiesis. The study finds that deletion of TIF1-gamma in mice leads to:
1) A block in erythroid maturation in the bone marrow, compensated by enhanced spleen erythropoiesis.
2) Defects in other blood lineages, including a loss of B cells and expansion of granulocytes.
3) Decreased hematopoietic stem cell function.
The deletion of TIF1-gamma also results in reduced transcription elongation of erythroid genes in bone marrow cells. This establishes T
This document summarizes a presentation on the gut microbiota and the impact of probiotics. It discusses the gut as a rich ecosystem containing bacteria, archaea, viruses, and eukaryotes. The presentation notes that the human microbiome contains over 100 times as many genes as the human genome. It also reviews clinical studies that have examined the effects of consuming various probiotic strains on the composition of the gut microbiota in healthy adults and IBS patients. The studies found mostly minor or no changes to the dominant microbiota with probiotic intake. However, some genera were found to be modulated in some individuals. The presentation concludes by discussing the need for more targeted screening of probiotics and understanding what level of microbiota modulation leads
This document summarizes research on immortalizing bovine pancreatic duct cells and making them tumorigenic through genetic manipulation. Key points:
1) Bovine pancreatic duct cells were immortalized by transfecting them with SV40 large T antigen cDNA. The immortalized cells retained features of normal duct cells.
2) The immortalized duct cells were then transfected with a mutated K-ras gene. This caused the cells to gain the ability to form tumors when injected into nude mice, suggesting K-ras plays an important role in pancreatic carcinogenesis.
3) The research aims to establish a model for the transition from normal duct cell to neoplastic cell in order to study the initial steps of pancreatic cancer development
Human Microbiome is the current project in Research field. The importance of Microorganisms in the human body, the importance and novel roe of the microorganisms on a human body is very effective and helpful. Fecal Transplantation is a unique and helpful technique to cure a dreadful disease naturally by means of microorganisms or introducing the normal flora in to the body again.
The document discusses gut microbiota and various techniques used to study it. It begins by describing the importance of gut microbiota in digestion, development of the immune system, maintenance of the intestinal barrier, and health and disease. It then covers culture-based and molecular-based techniques used to characterize gut microbiota, including their pros and cons. Specific molecular techniques discussed include FISH, DNA microarrays, DGGE/TGGE, 454 pyrosequencing, and quantitative PCR. The document also addresses pitfalls of molecular techniques and provides examples of studies comparing the gut microbiota of normal vs. diseased horses.
This document describes a study that sequenced the 23S rRNA gene of Campylobacter coli VC167 and analyzed its sequence and structure. It found that the gene contained a 147 bp transcribed spacer that resulted in fragmented 23S rRNA. Analysis of 31 other C. coli and C. jejuni strains found that 69% contained a transcribed spacer of 147 bp or 37 bp, also producing fragmented 23S rRNA. Phylogenetic analysis placed Campylobacter in the epsilon subdivision of Proteobacteria based on signatures in the 23S rRNA at positions 270 and 1850.
An Ecophylogenetic Approach to Determine the Evolutionary History of the Mamm...tsharpton
Identifying those gut microbes that co-diversify with mammals is important to our understanding of the mechanisms and health implications of host-microbiome interactions. For example, microbiota that are conserved across mammalian species may express a trait that has been subject to selection throughout the evolution of these mammals, possibly because it is critical to health. While advances in environmental DNA sequencing have transformed our understanding of how enteric microbes are distributed across mammalian species, these data are frequently analyzed using phylogenetically agnostic approaches. Such approaches can obscure the detection of diverged groups of bacteria that have been conserved across mammalian species. To provide enhanced resolution into evolutionary associations between gut microbiota and mammals, we innovated a high-throughput ecophylogenetic method, known as ClaatTU (Cladal Taxonomic Units). ClaaTU analyzes phylogenies assembled from environmental DNA sequences collected from a set of microbial communities and profiles the presence and abundance of each monophyletic clade in each community. As a result, it enables the identification of specific microbial clades that are distributed across host communities in a manner indicative of being associated with mammalian evolution. To demonstrate this, we applied ClaaTU to a mammalian microbiome dataset and (1) identified clades of gut bacteria that are unique to groups of mammals based on their taxonomy or dietary regime, (2)
found that there exists ecophylogenetic structure in the mammalian gut microbiome, indicating that gut bacterial phylogenetic diversity associates with host phylogeny, and
(3) discovered specic clades that are present in a larger number of mammals than expected by chance, some of which may co-diversify with their hosts. Our findings indicate that some mammalian gut microbiota may have been anciently acquired and subsequently retained in extant lineages, indicating that they may play an important role in mediating host-microbiome interactions and maintaining host health.
1) Researchers developed transgenic medaka (fish) carrying multiple copies of a bacteriophage vector containing the cII gene, which serves as a target for detecting mutations.
2) They adapted an assay used in transgenic rodents to efficiently recover cII mutants from fish genomic DNA and detect them by infecting bacteria.
3) Spontaneous mutant frequencies in liver, whole fish, and testes of medaka were comparable to frequencies observed in transgenic rodents. Treatment with ethylnitrosourea induced cII mutations in the medaka in a tissue-specific and time-dependent manner consistent with known mutagen mechanisms.
This document is the curriculum vitae of Prof Dr Ariyanto Harsono dr SpAK. It details his educational background, including graduating from medical school in 1972 and specializing in pediatrics in 1982. It lists his current position as a professor of pediatric health sciences at the University of Airlangga's medical faculty and as a consultant in allergy and immunology. It also provides information on additional training in food allergy he received in the Netherlands from 1990-1991.
- Microbial metabolites from the gut microbiome, especially butyrate, play an important role in modulating intestinal damage and graft-versus-host disease (GvHD) severity.
- In mice that underwent allogeneic stem cell transplantation, butyrate levels decreased significantly in intestinal epithelial cells, likely due to reduced butyrate transporter expression from inflammation.
- Restoring butyrate levels through intragastric gavage improved epithelial integrity, reduced GvHD severity and improved survival. Specific butyrate-producing bacteria were also associated with lower GvHD risk.
This document describes a microarray analysis comparing gene expression profiles in the large intestine, small intestine, liver, and spleen of mice with different gut microbiota colonization models: specific pathogen-free mice, germ-free mice colonized at birth, and germ-free mice colonized at 5 weeks of age. The analysis found hundreds of differentially expressed genes in each tissue and colonization model. Gene set enrichment analysis identified overrepresented gene ontology categories related to immune system development and antigen presentation in intestines of mice colonized at birth, and metabolic processes in intestines of specific pathogen-free mice. Analysis of signaling pathways found prominent changes in toll-like receptor and type 1 interferon signaling pathways in intestines of mice
Integrin V can form heterodimers with several subunits to mediate cell-cell and cell-extracellular matrix interactions. During zebrafish gastrulation, V is expressed maternally and zygotically. Here, we used a morpholino-mediated V knockdown strategy to study V function. Although V morphants displayed vascular defects, they also exhibited left-right body asymmetry defects affecting multiple visceral organs. This was preceded by mislocalization of dorsal forerunner cells (DFCs) and malformation of the Kupffer’s vesicle (KV) laterality organ
This document compares growth rates and gene expression in two species of oysters, the Pacific oyster and Olympia oyster. It finds that the Pacific oyster grows more rapidly than the Olympia oyster. It identifies four genes associated with growth in both species and analyzes their expression levels in different tissues and time periods. The expression of these genes varies between the species and over time, indicating differences in their metabolic processes during growing versus fasting seasons.
Science 2013-schuenemann-179-83 leprosy önemliHazal Sav
This study obtained near-complete genome sequences of Mycobacterium leprae from skeletal remains dating from the 11th to 14th centuries in Europe, as well as from recent patient biopsies. Genome comparisons revealed remarkable genomic conservation of M. leprae over the past 1000 years. The ancient genomes suggest a European origin for leprosy in the Americas and the presence of a genotype in medieval Europe now commonly associated with the Middle East. Exceptional DNA and mycolic acid preservation in the ancient skeletal remains provides insights into pathogen evolution and the disappearance of leprosy in Europe.
This document reports on a study examining the effects of somatostatin (SST) on human B lymphoblasts. The key findings are:
1) SST stimulates phospholipase C activity and increases cytosolic calcium levels in B lymphoblasts, likely through coupling of SSTR2A to the G protein Gα16.
2) SST activates extracellular signal-regulated kinases and induces increased DNA synthesis, proliferation, and immunoglobulin formation in B lymphoblasts.
3) These stimulatory effects of SST on early signal transduction pathways are accompanied by increased cell proliferation and immunoglobulin production in human B lymphoblasts, indicating SST exerts growth factor-like
The document summarizes an experiment that tested the hypothesis that the peripheral aster spreading during metaphase determines the pattern of the cortical F-actin ring that forms prior to polar body extrusion. The experiment used two methods - treatment with lovastatin followed by washout, and treatment with cytochalasin D followed by washout - to induce multiple cortical spreading asters. Both methods resulted in the formation of multiple, overlapping F-actin rings, supporting the hypothesis that microtubules of the peripheral aster provide a positive stimulus that directs F-actin ring formation.
31 октября самый цитируемый ученый русского происхождения, известный эволюционный биолог Евгений Кунин рассказал в Digital October о постмодернистском взгляде на эволюционные процессы.
http://digitaloctober.ru/ru/events/knowledge_stream_evolyutsiya_po_sluchayu
This document describes the development of a genetically encoded fluorescent biosensor for 2-oxoglutarate (2OG) based on fluorescence resonance energy transfer (FRET). The researchers constructed the biosensor by fusing yellow fluorescent protein (YFP), the 2OG-binding GAF domain of the NifA protein, and cyan fluorescent protein (CFP) between restriction sites in a plasmid vector. They tested the biosensor in vitro and found it responded to 2OG concentrations within the physiological range observed in E. coli cells. They also optimized the peptide linker between domains. In vivo, the biosensor detected changes in 2OG levels in E. coli cells in response to different carbon sources added. The biosensor could image
1) The study analyzed data from over 1,000 Western adults and found evidence that some taxa in the human gut microbiome exist in alternative stable states, shifting between high and low abundance profiles.
2) Specifically, around 10% of prevalent taxa showed bimodal abundance distributions and reduced stability at intermediate abundances, indicating bi-stability.
3) These bi-stable "tipping elements" can co-occur in different combinations and have been linked to metabolic syndrome and inflammatory bowel disease.
The document provides a resume for Alexandria Valdivia. It summarizes her objective as obtaining a rewarding career in medical sonography utilizing her associate's degree in diagnostic medical sonography. It lists her qualifications which include ultrasound certifications and medical training. It also outlines her skills such as patient care, communication, and organization. Her education and externship experiences in ultrasound are described. Previous work experiences in retail are listed, and references are provided.
Timmie Peace is looking for a full time position that offers greater challenge, increased benefits for his family, and opportunities to help the company advance efficiently. He has over 15 years of experience in operations roles including currently working as an Operator 1 at Ozburn Hessey Logistics where he safely and efficiently unloads and moves freight, pulls and prepares products for shipment, and performs picking duties. He is also experienced in quality control, printing, and mail sorting from previous roles at Automatic Data Processing, Cunningham Graphics, and the United States Postal Service.
The experts at FATbit Technologies designed an infographic that explains concepts like enhanced experience, better authority and more to help entrepreneurs increase conversion and visitor engagement.
Peter Culpepper has over two decades’ experience in corporate finance. A Certified Public Accountant, Peter Culpepper is the chief financial officer of Provectus Biopharmaceuticals, an organization that actively supports the Pan-Mass Challenge (PMC).
This document describes a study examining the role of the zinc transporter Zip14 in adipose tissue regulation and metabolism. The key findings are:
1) Mice lacking the Zip14 zinc transporter had increased adiposity, hypertrophied adipocytes, impaired insulin signaling, and chronic inflammation in adipose tissue.
2) Adipose tissue from Zip14 knockout mice showed increased cytokine production, activation of inflammatory pathways, and impaired adipocyte differentiation.
3) The metabolic changes in adipose tissue were linked to increased circulating endotoxin levels caused by the Zip14 deletion.
4) Knocking down Zip14 in cultured adipocytes impaired their ability to mobilize zinc and dysregulated inflammatory pathways
The document discusses the nutrigenomic effects of zinc and selenium. It provides an overview of nutrigenomics and how nutrients can regulate gene expression and body functions. It then focuses on the specific roles of zinc and selenium from a nutrigenomic perspective. Zinc is shown to play roles in gene expression, appetite control, fat absorption and antioxidant defense by regulating genes like metallothionein. Studies demonstrate how zinc deficiency and supplementation impact insulin signaling and inflammation-related gene expression. Similarly, selenium is incorporated into selenoproteins that are involved in antioxidant functions. Studies examine how selenium intake influences transcription factors and selenoprotein expression levels in different tissues.
When yeast cells are exposed to anoxia (no oxygen) on a non-fermentable carbon source, they enter a state of suspended animation where all observable life processes reversibly halt until oxygen is restored. Transcriptional profiling revealed differences in gene expression between yeast exposed to carbon monoxide (CO) gas versus nitrogen (N2) gas. CO can mimic oxygen binding and led to derepression of aerobic metabolism genes compared to N2 exposure. Mutants lacking components of the mitochondrial retrograde signaling pathway recovered normally from CO but not N2 exposure, indicating its importance in the cellular response to different anoxic conditions. The study establishes yeast as a model for investigating suspended animation and oxygen-regulated gene expression.
Transgenic pigs have been developed through genetic modification techniques for various applications. Pigs are useful biomedical models for human diseases due to their similar physiology to humans. Various methods can be used to introduce foreign DNA into pig zygotes, including pronuclear injection and somatic cell nuclear transfer. Transgenic pigs have been created that produce human proteins, are rich in omega-3 fatty acids, can digest phosphorus more efficiently to reduce environmental pollution, and have genes knocked out to avoid immune rejection of pig organs in xenotransplantation. Genetically modified pigs show promise for advancing medical research and treatment.
Effects of dietary vitamin E type and level on lipopolysaccharide-induced cyt...Wei Fang
This document summarizes a study that investigated the effects of dietary vitamin E type (synthetic vs natural) and level (22 IU/kg vs 220 IU/kg) on cytokine mRNA expression in broiler chicks injected with lipopolysaccharide (LPS) or saline. The key findings were:
1) Birds fed natural-type vitamin E had significantly lower LPS-induced expression of the pro-inflammatory cytokine IL-6 compared to those fed synthetic vitamin E, suggesting a protective effect of natural vitamin E.
2) LPS injection significantly increased mRNA expression of the pro-inflammatory cytokines IL-6 and IFN-γ, as well as the innate antimicrobial proteins iNOS and AvBD10, compared to
Transgenic pigs have been developed through inserting foreign DNA into pig genomes using various techniques. Pigs are useful biomedical models because their physiology is similar to humans. Transgenic pigs have been created for various purposes, such as producing human proteins in their milk or blood, modeling human diseases, producing organs for xenotransplantation, and reducing phosphorus pollution through modified digestion of phytates. Genetic engineering of pigs continues to be studied for applications in biomedicine and agriculture.
Survivin Immunoreactivity in the Gastric Mucosa of Rats Feedind with Carpet S...IJRTEMJOURNAL
Survivin has been studied many times because of its overexpression in several types of cancer
including lung, kidney, skin, endometrium, stomach, colon, breast, prostate, over, hematologic, head and neck
cancers, histopathology features and polymorphisms in the promoter region which belongs to the inhibitör of
apoptosis gene family by researchers. There is no study of survivin immunoreactivity in the gastric mucosa of the
rats fed with carpet shell clam grown in the Dardanelles. In this study, it was aimed to investigate the effects of
carpet shell clam fed rats on survivin production in the gastric mucosa. The carpet shell clam given as food to the
rats were removed from the Dardanelles Çardak region. Four groups of rats are included in the study, group 1
(n=6), control group fed with standard rat food, group 2 (n=6), 75% carpet shell clam and 25% standard rat
food daily, group 3 (n=6), 75% carpet shell clam and 25% standard rat food every two days, group 4 (n=6), 75%
carpet shell clam and 25% standard rat food every three days. To detect survivin localization in the tissues, the
LAB-SA Detection System was used. Survivin immunoreactivity was detected of epithelial cells in the gastric
mucosa of rats fed with carpet shell clam. After the immunohistochemical staining processing all gastric tissue
samples are evaluated in terms of survivin immunoreactivity with light microscopy and image analysis software.
Survivin immunoreactivity was detected 0% in the first group, 83.33% in the second group, 61.83% in the third
group and 32.67% in the fourth group. There was statistically significant difference between the survivin
immunoreactivity in the gastric gland cells of the rats in the experimental and control groups (p> 0.05). Survivin
production in the gastric mucosa of rats suggests that consumption of carpet shell clam may cause tissue damage.
Alpha-1 Antitrypsin (α-1 AT) deficiency is a common genetic disorder that affects 1 in 2,000 individuals in the USA. Additionally, over 20 million people have been identified as carriers for this genetic disorder. In severe cases, α-1 AT deficiency can cause substantial lung and liver damage, which if left untreated could result in death and there are no current available treatments. Alpha-1 protein is produced in the liver, travels in the bloodstream and utilized in the lungs to protect healthy lung tissue from harmful destruction by elastase. A common single amino acid substitution, located at E342K (ATZ) was identified in α-1 AT deficient humans. When this specific mutation occurs two phenotypes can result: 1) ATZ can polymerize in the liver causing cellular toxicity 2) inhibits alpha-1 antitrypsin from inhibiting elastase which can result in lung disease. Currently; little is known about the cellular mechanisms that clear the accumulated proteins in the liver. Therefore, an investigative study utilizing C. elegans model of ATZ was performed in order to help determine the cellular mechanisms that dispose of accumulated proteins. Specifically RNA interference was utilized to knockdown expression of specific genes. This investigation examined genes involved in the heat-shock pathway (HSP), unfolded protein response (UPR), and insulin signaling pathway (IS). Phenotypic analysis including: embryonic lethality, protein aggregation expression, and longevity, was completed after knockdown of genes to determine effect on ATZ accumulation. Currently with our preliminary data suggests that the heat-shack pathway may play a role in ATZ accumulation. Determining the mechanism of protein accumulation in the investigation of C. elegans may lead to possible drug targets and therefore the development of a treatment which may alleviate those diagnosed with this disorder.
This document reports on a case study where researchers found high mortality in mice given the pain medication ketorolac after embryo transfer surgery. Between October and December 2009, 14 out of 49 mice (29%) died 1-2 days after surgery when treated with ketorolac, compared to only 1 death out of 75 mice treated with the alternative pain medication ketoprofen in a previous period. Autopsies of the deceased ketorolac-treated mice found acute kidney damage and gastrointestinal bleeding, suggesting the postoperative toxicity of ketorolac caused the deaths. The researchers concluded ketorolac analgesia was associated with increased postoperative mortality in mice after embryo transfer surgery.
This study characterized the function of ALL2, a homolog of ALL1, in the fungal pathogen Cryptococcus neoformans. The key findings were:
1) Unlike deletion of ALL1, deletion of ALL2 attenuated virulence in a pulmonary infection model.
2) The all2Δ mutant shed a less viscous exopolysaccharide and was more sensitive to hydrogen peroxide but more resistant to macrophage killing than the wild type.
3) Transcriptome analysis supported distinct functions for ALL1 and ALL2, with ALL2 uniquely involved in maintaining intracellular pH under low-pH conditions.
This document summarizes a study that evaluated the toxic effects of orally administering titanium dioxide nanoparticles (TiO2 NPs) to male Wistar rats. The rats were divided into control and experimental groups that received 50 or 100 mg/kg body weight of TiO2 NPs daily for 14 days. Blood samples were analyzed for biochemical markers of liver and kidney function. The results showed elevated levels of markers indicating liver damage and renal toxicity in the experimental groups, suggesting oral exposure to TiO2 NPs can negatively impact the liver and kidneys.
Oral toxic exposure of titanium dioxide nanoparticles on serum biochemical ch...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Titanium dioxide (TiO2) nanoparticles (NPs) are widely used in commercial food additives and cosmetics worldwide. Uptake of these nanoparticulate into humans by different routes and may exhibit potential side effects, lags behind the rapid development of nanotechnology. Thus, the present study designed to evaluate the toxic effect of mixed rutile and anatase TiO2 NPs on serum biochemical changes in rats.
Materials and Methods:
In this study, adult male Wistar rats were randomly allotted into the experimental and control groups (n=6), which were orally administered with 50 and 100 mg/kg body weight of TiO2 NPs. Toxic effects were assessed by the changes of serum biochemical parameters such as glucose, total protein, albumin, globulin, cholesterol, triglyceride, high density lipoprotein, alanine transaminase, aspartate transaminase, alkaline phosphatase, total bilirubin, blood urea nitrogen, uric acid and creatinine. All the serum biochemical markers were experimented in rats, after 14-days of post exposure.
Results:
Changes of the serum specific parameters indicated that liver and kidney were significantly affected in both experimental groups. The changes between the levels of total protein, glucose, aspartate transaminase, alanine transaminase and alkaline phosphatase indicate that TiO2 NPs induces liver damage. Significant increase in the blood urea nitrogen and uric acid indicates the renal damage in the TiO2 NPs treated rats.
Conclusion:
The data shows that the oral administration of TiO2 NPs (<100nm) may lead to hepatic and renal toxicity in experimental rats.
Abstract
Objective(s):
Zinc oxide nanoparticles (ZNP) are increasingly used in sunscreens, biosensors, food additives and pigments. In this study the effects of ZNP on liver of rats was investigated.
Materials and Methods:
Experimental groups received 5, 50 and 300 mg/kg ZNP respectively for 14 days. Control group received only distilled water. ALT, AST and ALP were considered as biomarkers to indicate hepatotoxicity. Lipid peroxidation (MDA), SOD and GPx were detected for assessment of oxidative stress in liver tissue. Histological studies and TUNEL assay were also done.
Results:
Plasma concentration of zinc (Zn) was significantly increased in 5 mg/kg ZNP-treated rats. Liver concentration of Zn was significantly increased in the 300 mg/kg ZNP-treated animals. Weight of liver was markedly increased in both 5 and 300 mg/kg doses of ZNP. ZNP at the doses of 5 mg/kg induced a significant increase in oxidative stress through the increase in MDA content and a significant decrease in SOD and GPx enzymes activity in the liver tissue. Administration of ZNP at 5 mg/kg induced a significant elevation in plasma AST, ALT and ALP. Histological studies showed that treatment with 5 mg/kg of ZNP caused hepatocytes swelling, which was accompanied by congestion of RBC and accumulation of inflammatory cells. Apoptotic index was also significantly increased in this group. ZNP at the dose of 300 mg/kg had poor hepatotoxicity effect.
Conclusion:
It is concluded that lower doses of ZNP has more hepatotoxic effects on rats, and recommended to use it with caution if there is a hepatological problem.
ZIP10 is a zinc transporter that may be important for cell cycle progression in breast cancer. This study examined ZIP10 cleavage and expression during different cell cycle stages. MCF-7 breast cancer cells were synchronized to specific stages using serum deprivation or nocodazole treatment. Western blot and immunofluorescence microscopy found N-terminal fragments of ZIP10, indicating cleavage, with highest levels during mitosis and G1 phase. ZIP10 was also expressed on mitotic cell membranes. These results suggest ZIP10 activation via cleavage may be important for zinc influx and cell cycle progression in breast cancer.
ZIP10 is a zinc transporter that may be important for cell cycle progression in breast cancer. This study examined ZIP10 cleavage and expression during different cell cycle stages. MCF-7 breast cancer cells were synchronized to specific stages using serum deprivation or nocodazole treatment. Western blot and immunofluorescence microscopy found N-terminal fragments of ZIP10, indicating cleavage, with highest levels during mitosis and G1 phase. ZIP10 was also expressed on mitotic cell membranes. These results suggest ZIP10 activation via cleavage may be important for zinc influx and cell cycle progression in breast cancer.
Symptoms And Treatment Of Trypanosoma BruceiJennifer Baker
Trypanosoma brucei is a protozoan parasite that causes African trypanosomiasis or sleeping sickness in humans and animals through the transmission via tsetse fly bites. It has 3 subspecies that cause different forms of the disease with varying symptoms and onset. Current treatments for the disease include pentamidine, melarsoprol, eflornithine and suramin which target the bloodstream or procyclic forms of the parasite.
Transgenic mice that overexpress the Cux-1 gene in the kidney develop glomerulosclerosis and interstitial fibrosis. The study found that Cux-1 overexpression leads to increased mesangial cell proliferation and expansion of the mesangial matrix. This results in mesangial cell hyperplasia and mesangial matrix expansion. The changes are associated with disruption of podocyte architecture and loss of kidney filtration function. Overexpression of Cux-1 is sufficient to induce early changes characteristic of mesangioproliferative glomerulonephritis.
The document describes an experiment on transforming E. coli bacteria with the pGLO plasmid containing the GFP gene. When the E. coli was transformed with pGLO, it grew on the LB agar plate but not on plates containing antibiotics, as expected. The transformed E. coli would produce GFP and glow under UV light.
1) The C-terminal carboxylate group of integrin β1 is necessary for its association with the kindlin-2 adapter protein. Affinity measurements indicate this interaction is coordinated by a putative carboxylate-binding motif in the FERM subdomain F3 of kindlin-2.
2) Injection of mRNA encoding mutant integrin β1 cytoplasmic tails that lack the C-terminal carboxylate group perturbed laterality organ development in zebrafish, indicating the carboxylate group is required for physiological functions.
3) The unusual interaction between integrin β1 and kindlin-2 identified here represents a novel protein-protein interaction mode governed mainly by the integrin
Titanium dioxide nanoparticles are commonly used as food additives and are ingested by humans. This study examined the effects of oral titanium dioxide nanoparticle exposure on the gut microbiota and intestinal immune response in mice. Mice were orally administered titanium dioxide nanoparticles or a control and cytokine levels in the ileum, colon, and mesenteric lymph nodes were analyzed after 24 hours and 14 days. The results showed that titanium dioxide nanoparticles increased cytokine levels in the ileum after 24 hours but decreased one cytokine after 14 days. In the colon, one cytokine increased after 14 days. Two cytokines in the mesenteric lymph nodes decreased after 14 days. This suggests that oral titanium dioxide nanoparticle exposure can modulate the intestinal immune response over
1. The influence of dietary zinc source and coccidial vaccine exposure on
intracellular zinc homeostasis and immune status in broiler chickens
Catalina Troche1
†, Susan D. Eicher2
and Todd J. Applegate1
*
1
Department of Animal Sciences, Purdue University, West Lafayette, IN, USA
2
Livestock Behavior Research Unit, United States Department of Agriculture, Agricultural Research Service, West Lafayette,
IN, USA
(Submitted 11 September 2014 – Final revision received 1 April 2015 – Accepted 14 April 2015 – First published online 16 June 2015)
Abstract
Coccidia are protozoal parasites which compromise mucosal integrity of the intestine, potentiating poultry morbidity. The host’s Zn status
influences the course of infection. Therefore, two experiments were designed to determine how supplemental Zn regimens impacted jeju-
nal and caecal immune status and Zn transporter expression. Coccivacw
-B was administered weekly at ten times the recommended dose as
a mild coccidial challenge (10CV). Zn was provided through a basal diet, supplemental zinc sulfate (ZnSO4), or a supplemental 1:1 blend of
ZnSO4 and Availaw
-Zn (Blend). Mucosal jejunum (Expt 1) and caecal tonsils (Expt 2) were evaluated for intracellular Zn concentrations and
phagocytic capacity. Messenger expression of Zn transporters ZnT5, ZnT7, Zip9 and Zip13 were investigated to determine Zn trafficking.
With 10CV, phagocytic capacity was decreased in jejunal cells by 2 %. In the caecal tonsils, however, phagocytic capacity increased with
challenge, with the magnitude of increase being more pronounced with higher dietary Zn (10CV £ Zn interaction; P¼0·04). Intracellular Zn
within caecal tonsils was found significantly reduced with 10CV (27 %, P¼0·0001). 10CV also resulted in an overall increase in the ratio of
Zip:ZnT transporters. With the exception of Zip13 transporter expression, dietary Zn source had little impact on any of the measured cel-
lular parameters. Thus, intestinal mucosal tissues had reductions in intracellular free Zn during coccidial challenge, which was coupled
with an upregulation of measured Zip transporters. This suggests that under coccidial challenge, intestinal cells attempt to compensate
for the drop in intracellular Zn.
Key words: Zinc transporters: Coccidia: Broilers: Zinc sources
Coccidiosis is a protozoal infection responsible for high
morbidity and substantial economic loss to the poultry
industry(1,2)
. Pathogenesis of coccidiosis is attributed to
damage of the mucosal lining, which results in growth
depression and malabsorption of macro- and micronutri-
ents(3 –7)
. In broiler chickens, coccidial infection decreased
levels of serum Fe, while increasing levels of Cu(6,7)
. Plasma
Zn was found decreased in birds inoculated with Eimeria
acervulina or Eimeria tenella compared to their unchallenged
cohorts(8– 12)
. This decrease in plasma Zn corresponded with
increased liver Zn content(7,12)
. Metallothionein (MT) is a
cysteine-rich Zn binding protein, which is upregulated
during the acute phase response through a mechanism
associated with oxidative stress(13 –15)
. Infection with E. tenella
increased Zn-bound MT content in the liver by 91 %(12)
.
Observable increases in MT-bound Zn are not exclusive to
coccidiosis. Immune stressors such as bacterial cell wall
components and inflammatory cytokines increased liver Zn
content(16 –18)
. While MT is the major eurokayotic storage
protein for Zn, transport of Zn to cytosolic proteins is
mitigated by two families of contra-directional Zn transporters.
Both Zn/Fe-regulated transporter (ZRT/IRT-like) proteins
(Zip) and cation diffusion facilitator (CDF) Zn transporters
(ZnT) are responsible for intracellular Zn trafficking(19)
. The
evaluation of Zn transporters within the avian species is
relatively new to the literature; however, Zn transporter
function is highly conserved between species(19,20)
. The cur-
rent NCBI 2011 assembly for Gallus gallus has four complete
coding sequences: ZnT5, ZnT7, Zip9 and Zip13. In the context
of gastrointestinal function, ZIP4 and ZnT1 have been exten-
sively studied in mammals(21 –23)
. However, ZnT5, ZnT7,
ZIP9 and ZIP13 transporters are associated with the trans-
Golgi network and are involved in major signalling pathways,
for example phosphorylation of extracellular signal-regulated
† Present address: Department of Food Science and Human Nutrition, University of Florida, Gainesville, FL, USA.
* Corresponding author: T. J. Applegate, fax þ1 765 494 9346, email applegt@purdue.edu
Abbreviations: 10CV, ten times the recommended dosage of Coccivacw
-B; MFI, median fluorescent intensity; MT, metallothionein; Zip, Zn influx transporter;
ZnT, Zn efflux transporter.
British Journal of Nutrition (2015), 114, 202–212 doi:10.1017/S0007114515001592
q The Authors 2015
BritishJournalofNutrition
2. kinase, protein kinase B, and transforming growth factor-
b(24 –27)
. ZnT7 was found to be particularly critical for Zn
uptake in the murine small intestine(28)
. Therefore, it is
likely that these transporters would be influenced by
inflammation and Zn status in poultry. In confirmation of this
observation, ZnT5 and 7 were found to be critical to the function
of alkaline phosphatases (mucosal protective proteins)(29,30)
.
Influx of Zn into the cytosol is coordinated by ZIP proteins
localised to the plasma membrane, vesicles, and/or the Golgi
complex(19,20)
. Efflux of Zn out of the cytosol and into
vesicles and/or the Golgi complex is mediated by ZnT.
Extracellular stimuli including but not limited to cytokines(31)
,
glucose(32)
and estrogens(33)
have been shown to alter Zn
homeostasis via Zn transport proteins. Directionality of net
Zn transport appears to be dependent on cell type: for
example stimulated monocytes and granulocytes increased
free intracellular Zn, activated dendritic cells decreased
intracellular Zn, and lymphocytes appeared to have no sig-
nificant change(34,35)
. The shift in intracellular Zn content
was attributed to a shift in transporter expression(34,35)
. In
dendritic cells, the control ratio of measured Zip:ZnT mRNA
was 0·67, and when stimulated with lipopolysaccharide, this
ratio decreased to 0·25(35)
. This shift led to an overall
movement of Zn out of the cytosol, ultimately reducing
free intracellular Zn(35)
.
These data show that cellular Zn homeostasis, controlled by
ZIP and ZnT transporters as well as MT, is influenced by extra-
cellular stimuli. These cellular changes can then lead to
changes in tissue homeostasis(36)
. The afore-mentioned
studies focus on changes in hepatic Zn during immune stimu-
lus, and make a strong case for Zn sparing within the
liver(37,38)
. However, the liver is the downstream of the intes-
tinal mucosa, which is coccidia’s main effector tissue. Early
studies were unable to determine any effect of coccidial infec-
tion on intestinal Zn content(7)
. These studies were based on
total Zn content within dry tissue, and were thus unable to
differentiate between membrane- and/or protein-bound Zn
and free intracellular Zn. The experiments presented herein
were designed to test the hypothesis that Zn transport and
immune function were altered with exposure to coccidial
challenge and dietary Zn regimen. Flow cytometry was used
to measure phagocytic capacity and intracellular Zn content
in both jejunum and caecal tonsils. Additional measures of
immune status included intracellular peroxidase and
CD3 þ in jejunum and caecal tonsils, respectively. In order
to characterise Zn flux during coccidial exposure, transporter
expression of ZnT5, ZnT7, ZIP9 and ZIP13 were also
measured in both tissues.
Materials and methods
All procedures of animal care and use for this experiment
were approved by the Purdue University Animal Care and
Use Committee. Newly hatched, male Ross broiler chicks
(708; Aviagen, Inc.) were used for the present study. All
chicks were housed in electrically heated battery cages
(model no. SB 4 T; Alternative Design Manufacturing) in an
environmentally controlled room. Battery cage temperature
was maintained at 37 ^ 18C for the first week, and decreased
by 38C each consecutive week until 248C in the third week.
Chicks were weighed and allocated to groups (six chicks/
cage) in such a way that the initial weight of each group
was the same. The chickens were provided ad libitum
access to drinking water and feed.
Experimental design
Expt 1 and 2. In Expt 1, a 3 £ 2 factorial was utilised with
three dietary treatments and two vaccine exposures: (an
unchallenged control or ten times the recommended
dosage of Coccivacw
-B (10CV); each treatment was replicated
six times. In Expt 2, a 7 £ 2 factorial was utilised with seven
dietary treatments and two vaccine exposures (as in Expt 1);
each treatment was replicated six times. Dietary regimens
were designed to provide 90 mg of Zn/kg of diet from one
of two Zn sources: zinc sulphate (ZnSO4) or a 1:1 blend of
ZnSO4 and Availaw
-Zn 100 (Zinpro Corporation). Availaw
-
Zn 100 (10 % Zn) is a proprietary metal amino acid complex.
The Association of American Feed Control Officials describes
the product as a complex of a soluble metal salt with an
amino acid, where one Zn ion is bound within an amino
acid complex. A basal maize–soyabean meal diet was
formulated to provide Zn from feedstuffs alone and was,
on average, 14 % below the National Research Council(39)
recommendations, which is 40 mg/kg for dietary Zn. Ca and
non-phytate phosphorus were provided at 9·8 and 4·3 mg/kg,
respectively. Zn provided from the maize–soyabean meal
basal diet (30 mg/kg of dietary Zn), was taken into account
when formulating Zn premixes, therefore reported inclusion
levels are based on total dietary Zn. Expt 1 provided broilers
with one of three dietary treatments: basal (30 mg/kg of dietary
Zn provided by feedstuffs alone), 90ZnSO4 (90 mg/kg of total
dietary Zn ¼ 30 mg/kg Zn from basal þ 60 mg/kg supple-
mental Zn from ZnSO4), or 90Blend (90 mg/kg of total dietary
Zn ¼ 30 mg/kg Zn from basal þ 30 mg/kg supplemental
Zn from ZnSO4 þ 30 mg/kg supplemental Zn from
Availaw
-Zn 100). Expt 2 introduced additional supplemental
Zn concentration inclusion levels by mixing portions of basal
and 90 mg/kg of Zn to create intermediate concentrations of
45 and 70 mg/kg of total dietary Zn.
Coccivacw
-B (a live oocyst vaccination containing strains
of E. acervulina, Eimeria mivati, Eimeria maxima and
E. tenella; Intervet, Inc.) administered 10CV was used as an
immune stimulus. The vaccine was introduced to day-old
Ross 708 broilers through oral gavage on day 1, 7, 14, and
22 (Expt 1). At 30 and 31 d of age (8 and 9 d post gavage)
one bird per cage from six replicates of each treatment
was CO2 asphyxiated, and proximal jejunal sections
were removed for fluorophore conjugation and mRNA tran-
script analysis.
In a nearly identical experimental design, chicks in Expt 2
were orally gavaged at 1, 7, 11, and 17 d of age. At 26 and
27 d of age one bird per cage (day 9 and 10 post gavage)
was CO2 asphyxiated, and caecal tonsils were removed
for fluorophore conjugation and mRNA transcript analysis,
as in Expt 1.
Intracellular zinc trafficking 203
BritishJournalofNutrition
3. Tissue processing for flow cytometry and
transcript analysis
Expt 1. Six jejunal sections (3 cm on either side of the
midpoint between the bile ducts and the Meckel’s diverticu-
lum) per treatment were collected from the broilers, and
placed in ice-cold Tris(hydroxymethyl)-aminomethane-glycine
buffer (TrisG; Thermo Scientific) before further processing.
From each jejunal section, a 1 cm portion distal to the Meckel’s
diverticulum was placed into Trizolw
(Life Technologies) for
later transcript analysis. Jejunal sections were cut longitudin-
ally and disrupted with a sterile cell sieve (CD-1e, 60 mesh
screen 230 mm pore size; Sigma-Aldrich). Phosphate-free
buffer, i.e. TrisG, was used to minimise Zn chelation during
tissue processing(40)
. Cell homogenates were centrifuged at
800 g for 20 min, and the supernatant was decanted. The
supernatant was centrifuged at 3560 g for 20 min; the pellet
was retained and re-suspended in 2 ml of TrisG. The crude
cell suspension was then incubated for 30 min at 378C. Five
hundred ml of each cell suspension was divided into four
reagent tubes: an unlabelled tube (to determine background
fluorescence) and three separate reagent tubes containing
fluorescently conjugated cells. Fluorescent indicators
included: 12·5 ml of FluoSpheresw
(1·0 mm diameter; Invitro-
gen) for measurement of phagocytic activity(41)
; 100 ml of
dihydrorhodamine-123 to report the presence of H2O2 and
intracellular peroxidase(42)
; 10 ml of Newport Greene DCF
diacetate (Invitrogen) for determination of intracellular Zn
levels(43)
. A typical avian phagocyte ranges between 8 and
10 mm(44)
. The 1·0 mm diameter of carboxylate-modified
microspheres was thought to be of adequate size to stimulate
phagocytic response, yet small enough to allow for mechan-
ical phagocytosis to occur(45)
. All tubes underwent a final
incubation for 30 min at 378C. After incubation, cells were
washed using a final 10 min 3000 g spin. The supernatant
was discarded and the pellet was re-suspended in 1 ml
TrisG. Cells were preserved with 500 ml of 2 % paraformalde-
hyde in Hanks’ balanced salt solution (no Ca, no Mg; Life
Technologies), and were stored at 38C for next day analysis.
Percent positive cells were calculated by bird, as the difference
in fluorescence between cells only and fluorescently conju-
gated cells. Median fluorescent intensity (MFI) of fluorescently
conjugated cells was also determined.
Expt 2. Tissue processing for Expt 2 was nearly identical to
the protocol described in Expt 1; therefore, only the differ-
ences between the two experiments are described in this
section. Caecal tonsils from six birds per treatment were
collected. One caecal tonsil went into Trizolw
for later tran-
script analysis. The remaining tonsils were processed for
flow cytometry. Caecal tonsil cells are more lymphoid in
nature than jejunal cells, and were therefore centrifuged at
lower speeds: 500 g for 20 min. The supernatant was decanted
and centrifuged at 1150 g for 20 min; the pellet was retained
and re-suspended in 2 ml of TrisG. The crude cell suspension
was then incubated for 30 min at 378C. Five hundred ml of
each cell suspension was divided into four reagent tubes:
one unlabelled (to determine background fluorescence) and
three separate reagent tubes (fluorescently conjugated cells).
Fluorescent indicators included: 12·5 ml of FluoSpheresw
(1·0 mm diameter; Invitrogen) for measurement of phagocytic
activity(41)
; 5 ml of mouse anti-chicken CD3þ –fluorescein
isothiocyanate (FITC) conjugate to label the T cell receptor-
associated CD3 complex (SouthernBiochem);10 ml of Newport
Greene DCF diacetate (Invitrogen) for determination of intra-
cellular Zn levels(43)
. Caecal tonsil cells were preserved in 2 %
paraformaldehyde and refrigerated (38C) for next day analysis.
Flow cytometry parameters and data analysis
Expt 1 and 2 utilised a benchtop flow cytometer with 3-blue
and 1-red lasers configured for excitation at 488 and 640 nm,
respectively (C6 BD Accuri Cytometer, Inc.). The FL1 optical
filter with emission detection of 522/30 nm was used for
Newport Greene, CD3þ –FITC, and dihydrorhodamine-123
analysis. The FL2 optical filter with emission at 585/40 nm
was used to detect phagocytic microbeads. Data were col-
lected on 20 000 cells per sample. Total cell population was
examined on a scatter-height (FSC-H) v. side scatter-height
(SSC-H) plot. Histogram overlays were generated using
System II Software (Beckman Coulter Company). Histograms
of optical filter by cell count were generated for cells-only
and each measured fluorophore. For each bird, a fluorescently
labelled cell population was overlaid against the unlabelled
cells-only histogram. The shift in cell population between
fluorescently conjugated cells and cells-only was calculated
as percentage fluorescence difference. Fluorescence of cells-
only and fluorophore-labelled cells were used to estimate
MFI of FCS-generated histograms.
Gene expression analysis
Trizolw
was used to extract total RNA from jejunal and/or
caecal tonsil mucosa according to manufacturer’s instructions.
RNA samples were dissolved in nuclease-free H2O, and con-
centration was determined with a Nanodrop reader (Thermo
Scientific). DNA was enzymatically degraded from RNA
samples, using the TURBO DNA-freee kit (Applied Biosys-
tems). RNA samples underwent gel electrophoresis on 0·8 %
agarose gel in 1 £ Tris-acetate EDTA running buffer to
check for integrity and genomic DNA contamination.
Expression of ZnT genes was assessed through RT-PCR.
Primers for G. gallus-specific ZnT5, ZnT7, ZIP9, and ZIP13
solute carriers were designed from provisional mRNA
sequences from NCBI (http://www.ncbi.nlm.nih.gov). Each
primer pair was designed to be at least 20 bp in length. The
University of California, Santa Cruz (UCSC) database was
used to blast primer pairs back to the chicken genome
(http://genome.ucsc.edu). Primer pairs crossed an intron/
exon boundary, and matched the target template sequence.
Primer sequences, annealing temperatures and efficiencies
are listed in Table 1. RNA samples were reverse transcribed
using the MultiScribee reverse transcriptase kit (Applied
Biosystems). PCR was performed using the Bio-Rad iCycler
(BioRad). The PCR mix was composed of 0·5 mg of
complementary DNA(cDNA), 0·075 nmol of each forward
and reverse primer, and iQ SYBR green master mix (BioRad).
C. Troche et al.204
BritishJournalofNutrition
4. Nuclease-free H2O was added for total reaction volumes of
25 ml. Reactions were initiated with a 5 min, 958C incubation.
Post incubation, reactions were cycled forty times using the
following procedure: 10 s at 958C, 20 s at primer-specific
annealing temperature, 728C. The Pfaffl method(46)
was used
for the relative quantification of real-time RT-PCR. The initial
housekeeping gene, glyceraldehyde 3-phosphate dehydro-
genase (GAPDH), was found to be significantly affected by
exposure to coccidial vaccine. Due to the inconsistency of
housekeeping gene expression, the BestKeeper-Excel-based
tool was used to evaluate potential genes and create an
expression standard(47)
. Pair-wise correlation analysis of all
pairs of candidate genes revealed that both hypoxanthine
phosphoribosyltransferase 1 (HPRT1) and TATA-binding
protein (TBP) had the lowest between-sample variations,
and high correlations with the BestKeeper index. The
geometric mean of these two genes was used to normalise
target gene expression.
Statistical analyses
All data were analysed using the PROC MIXED procedures of
SASw
(SAS Institute, 2012) as a completely randomised design.
Cage was the experimental unit for all experiments discussed.
For Expt 1, a 3 £ 2 factorial was utilised, with three dietary
regimens (maize–soyabean meal basal, 90 mg/kg dietary Zn
from ZnSO4, or 90 mg/kg dietary Zn from a 1:1 blended
source of Availaw
-Zn and ZnSO4), and two vaccine exposures
(an unchallenged control or a 10CV). Each treatment was
replicated six times. For Expt 2, a 7 £ 2 factorial was utilised
with seven dietary regimens (maize–soyabean meal basal,
45, 70 and 90 mg/kg dietary Zn from ZnSO4, or Blend). Treat-
ment comparisons were obtained through Tukey–Kramer
means separation, and significance was established at P#0·05.
Results
Mucosal response
Jejunum. Flow cytometry was used to characterise mucosal
response to 10CV and dietary Zn source. There was no signifi-
cant interaction between challenge and Zn treatment in any
of the measured jejunal mucosal parameters. The data is
expressed as both percentage of cells positive for fluorophore
and the MFI of the fluorophore positive population.
Unlabelled cells, from individual birds, were used to account
for background fluorescence in each measurement. Exposure
to 10CV decreased phagocytic capacity in jejunal cells by
2 %, with no change in MFI (Fig. 1(a) and (b)). In order
to further characterise the mucosal response to 10CV,
dihydrorhodamine-123 was included to measure peroxynitrite
(a reactive oxygen intermediate) produced by phagocytic cells
during oxidative burst. Repeated exposure to 10CV had no
significant impact on percentage positive or MFI (Fig. 1(c)
and (d)). Intracellular-free Zn was highly variable between
birds, and therefore was not significantly different with 10CV
(Fig. 1(e) and (f)). Dietary regimen did not impact any of
the measured flow parameters for jejunal tissue.
Caecal tonsils. In contrast to jejunal cells, a significant
interaction occurred between 10CV and Zn treatment in
caecal tonsil cells (P,0·0001), with no significant change in
MFI (Fig. 2(a) and (b)). While the main effect mean of Zn
treatment was not significant (P¼0·1), 10CV had a significant
impact on phagocytic capacity (P¼0·001). This interaction
indicates that the phagocytic capacity of caecal cells signifi-
cantly increased with the 10CV, with the magnitude of
increase being more pronounced with higher levels of Zn
(70 and 90 mg/kg, regardless of source). The possibility of a
shift in cell population was investigated with a fluorophore,
designed to conjugate to the T-cell specific (CD3þ) receptors.
There was no significant effect of 10CV on percentage of cells
positive for CD3þ conjugation; however, the MFI of CD3þ
was reduced by 94 % (P¼0·001) with 10CV (Fig. 2(c) and
(d)). The percentage of caecal cells positive for Newport
Greene decreased by an average of 27 % with 10CV
(P,0·0001). This reduction in percentage positive was
coupled with an 86 % decrease (P,0·0001) in MFI (Fig. 2(e)
and (f)). As with jejunal cells, Zn treatment had no effect on
the measured flow parameters.
Zinc trafficking
Jejunum. A significant interaction between dietary regimen
and 10CV occurred in Zip13 expression (Fig. 3). Jejunal
tissue from birds consuming 90ZnSO4 had a 4-fold increase
in Zip13 expression with exposure to coccivac. Zip9
expression was not altered by 10CV. Compared to control
tissues, 10CV reduced ZnT7 expression (P¼0·02) by 50 %.
The ratio of Zip:ZnT mRNA was increased by 75 % with 10CV
(Fig. 5). With the exception of Zip13, dietary Zn treatment did
not impact transporter expression.
Caecal tonsil. A significant interaction between dietary
regimen and vaccine exposure occurred in Zip13 expression
Table 1. Primer pairs selected for zinc trafficking
Forward primer Reverse primer Annealing temperature (8C) Efficiency (%)
ZnT5 50
-GGACATTCCCATGTGTCCTC-30
50
-AGAGCAGAGGGGATCAGCTA-30
62·5 99·5
ZnT7 50
-ATTTTGATGCAGCGAACTCC-30
50
-ATCCACCTTCCATCAGCATC-30
52·9 105
Zip9 50
-CATTGGCAGCACCTGTTATG-30
50
-CGCGATTCCTCCTACTTCTG-30
59·5 100
Zip13 50
-AGCTTTGCAATTGGTGGACT-30
50
-GGGCACTCCTCCTCTTCTTT-30
59·5 110
HPRT1 50
-TGCAGACGATCTGGAGAAAG-30
50
-TGGGGATTGACTTGTCACTGT-30
59·5 107
TBP 50
-AAGCGACACAGGGAACATCT-30
50
-AACCAAGATTCACCGTGGAC-30
62·5 107
ZnT5, Zn efflux transporter 5; ZnT7, Zn efflux transporter 7; Zip9, Zn influx transporter 9; Zip13, Zn influx transporter 13; HPRT1, hypoxanthine phosphoribosyltransferase 1;
TBP, TATA-binding protein.
Intracellular zinc trafficking 205
BritishJournalofNutrition
5. (Fig. 4). In contrast to jejunal tissue where 90ZnSO4
maximised Zip13 expression, birds consuming the 90Blend
treatment had a 27-fold increase in Zip13 caecal tonsil
expression. Zip9 expression within caecal tonsil cells was
increased 2-fold with 10CV. Caecal ZnT expression was not
significantly impacted by Zn treatment or 10CV (Fig. 4). The
ratio of Zip:ZnT mRNA was increased 16-fold with 10CV
(Fig. 5). As in the jejunum, Zip13 expression was the only
measured transporter impacted by dietary Zn source.
Discussion
Mucosal response of jejunal and caecal tonsil tissues
It is known that repeated exposure to oocysts results in
increased cellular infiltration, and faster resolution of infec-
tion(48,49)
. In the present study, repeated coccivac exposure
within the jejunum decreased the phagocytic population.
Respiratory burst, a process through which bactericidal super-
oxide and peroxides are produced, is the primary mechanism
through which phagocytes induce lysis(50)
. In Expt 1, there
was no effect of Zn source or challenge on dihydrorhoda-
mine-123 signal (Fig. 1(c) and (d)). In poultry, secondary
coccidial infections are characterised by high concentrations
of CD8 þ T cells within intraepithelial cell infiltrates(51 –54)
.
During the course of infection, T cells produce interferon-g,
a cytokine which recruits leukocytes and enhances the lysoso-
mal activity of macrophages. Laurent et al.(49)
found that
transcript levels of interferon-g peaked 7 d post infection
and returned to baseline levels 13 d post-infection. In the pre-
sent study, tissues were repeatedly exposed to coccivac and
collected on day 8 and 9 post gavage. Therefore, in Expt 1,
the innate macrophage response may have given way to an
adaptive cellular immune response by the time of collection.
Jejunal intracellular Zn, measured by Newport Greene, was
(a) (b)
(c) (d)
(e) (f)
102
100
98
PhagocyticmicrobeadsDHRNewportgreen
96
94
80
60
40
20
0
40
30
20
10
0
Basal 90ZnSO4
Zn Trt: P=0·5
10CV: P=0·002
Zn Trt×10CV: P=0·9
Zn Trt: P=0·4
10CV: P=0·07
Zn Trt×10CV: P=0·9
Zn Trt: P=0·9
10CV: P=0·09
Zn Trt×10CV: P=0·4
Zn Trt: P=0·7
10CV: P=0·1
Zn Trt×10CV: P=0·4
Zn Trt: P=0·7
10CV: P=0·1
Zn Trt×10CV: P=0·9
Zn Trt: P=0·5
10CV: P=0·3
Zn Trt×10CV: P=0·7
90Blend
0
0
7000
3000
2500
2000
1500
1000
500
0
14000
21000
28000
35000
10000
20000
30000
40000
Basal 90ZnSO4 90Blend
Basal 90ZnSO4 90Blend Basal 90ZnSO4 90Blend
Basal 90ZnSO4 90Blend Basal 90ZnSO4 90Blend
Fig. 1. Jejunal mucosal response. The percentage of jejunal cells positive for the measured fluorophore (a, c and e) and median histogram fluorescent intensity of
the cell population (b, d and f). Expt 1 labels include FluoSpheree microbeads to measure percentage of cells positive (a) and median fluorescent intensity (MFI)
(b) of phagocytic microbeads. Dihydrorhodamine-123 (DHR) was used to report the presence of H2O2 and intracellular peroxidase, expressed as both percent
cells positive (c) and MFI (d). Newport Greene indicator measured intracellular zinc content again expressed as both percent of cells positive for the Newport
Greene indicator (e) and the median shift in fluorescent intensity (f). Values represent mean response of six birds per zinc source for both control (Con) and
10CV (10 £ dose of coccidial vaccine Coccivacw
-B) birds. Significant (P#0·05) main effect mean comparisons of zinc source, 10CV, and their interactions are
indicated within each panel. Dietary regimens consisted of a basal maize–soyabean meal diet, or basal diet supplemented with either zinc from zinc sulphate
(ZnSO4), or a blended source (1:1 blend of ZnSO4 and Availaw
-Zn) to achieve 90 mg/kg of total dietary zinc. Birds were unchallenged (Con, A), or exposed to
10CV ( ) on days 1, 7, 14, and 22, and the final gavage occurred 10 d before tissue collection. Trt, treatment.
C. Troche et al.206
BritishJournalofNutrition
6. widely variable between birds, and therefore the drop in intra-
cellular Zn with 10CV was not significant. However, other
studies have reported decreases in intracellular Zn with
infection(55,56)
; it is said that the drop in intracellular Zn acts
as a protective mechanism through the induction of
apoptosis. Thambiayya et al.(56)
found that Zn binding can
inhibit caspase 3, a pro-apoptotic protein. Limiting labile
Zn within the cell may release inhibition of caspase 3 and
promote apoptosis during oxidative stress.
Caecal tonsil cells (Expt 2) expressed a significant inter-
action between Zn treatment and 10CV. Exposure to coccidia
(10CV) increased phagocytic capacity; however, the magni-
tude of increase from unchallenged and challenged groups
was more pronounced with 70 and 90 mg/kg of supplemental
Zn. Dubben et al.(57)
found that chelating Zn out of solution
was found to enhance monocyte (modelled using the HL-60
cell line) differentiation and phagocytic potential. Therefore,
if Zn has a negative impact on monocyte differentiation,
results of Expt 2 may reflect lower steady state monocyte
activity within caecal tonsils. This steady state population of
monocytes did not negatively impact the phagocytic capabili-
ties of 10CV. Furthermore, caecal tonsils are known to contain
a high concentration of macrophages; macrophage inflamma-
tory protein (a macrophage recruiting chemokine) was
upregulated 80-fold in E. tenella-infected caecal cells(49)
.
Laurent et al.(49)
found that the upregulation of inflammatory
cytokine expression was similar between E. tenella-infected
caecal tonsils and E. maxima-infected jejunum; however, the
caecal response was more pronounced. Several studies have
noted that poultry have an increased cellular immune
100
(a) (b)
(c) (d)
(e) (f)
95
90
85PhagocyticmicrobeadsCD3+Newportgreen
80
75 0
0
00
20
40
60
80
100
100000
200000
300000
0
20
40
60
80
100
200000
400000
600000
40000
80000
120000
160000
Basal
45ZnSO
4
70ZnSO
4
90ZnSO
4
45Blend70Blend90Blend
Basal
45ZnSO
4
70ZnSO
4
90ZnSO
4
45Blend70Blend90Blend
Basal
45ZnSO
4
70ZnSO
4
90ZnSO
4
45Blend70Blend90Blend
Basal
45ZnSO
4
70ZnSO
4
90ZnSO
4
45Blend70Blend90Blend
Basal
45ZnSO
4
70ZnSO
4
90ZnSO
4
45Blend70Blend90Blend
Basal
45ZnSO
4
70ZnSO
4
90ZnSO
4
45Blend70Blend90Blend
Zn Trt: P=0·1
10CV: P=0·001
Zn Trt×10CV: P=0·04
Zn Trt: P=0·6
10CV: P=0·2
Zn Trt×10CV: P=0·4
Zn Trt: P=0·9
10CV: P=0·3
Zn Trt×10CV: P=0·3
Zn Trt: P=0·9
10CV: P=0·001
Zn Trt×10CV: P=0·4
Zn Trt: P=0·4
10CV: P=0·0001
Zn Trt×10CV: P=0·8
Zn Trt: P=0·6
10CV: P=0·001
Zn Trt×10CV: P=0·4
Fig. 2. Caecal tonsil mucosal response. The percentage of caecal tonsil cells positive for the measured fluorophore (a, c and e) and median histogram fluorescent
intensity of the cell population (b, d and f). Expt 2 labels include FluoSpheree microbeads to measure percentage of cells positive (a) and median fluorescent
intensity (MFI) (b) of phagocytic microbeads. Mouse-anti-chicken CD3þ –fluorescein isothiocyanate (FITC) conjugate, used to label the CD3 þ T cell receptor
complex, is expressed as both percent cells positive (c) and MFI (d). Newport Greene indicator measured intracellular zinc content again expressed as both per-
cent of cells positive for the Newport Greene indicator (e) and the median shift in fluorescent intensity (f). Values represent mean response of six birds per zinc
regimen for both control (Con) and 10CV (10£ dose of coccidial vaccine Coccivacw
-B) birds. Significant (P#0·05) main effect mean comparisons of zinc regimen,
10CV, and their interactions are indicated within each panel. Dietary regimens consisted of a basal maize–soyabean meal diet, or basal diet supplemented with
either zinc from zinc sulphate (ZnSO4), or a blended source (1:1 blend of ZnSO4 and Availaw
-Zn) to achieve 45, 70, or 90 mg/kg of total dietary zinc. Birds were
unchallenged (Con, A), or exposed to 10CV ( ) on days 1, 7, 11, and 17, with the final gavage occurring 10 d before tissue collection. Trt, treatment.
Intracellular zinc trafficking 207
BritishJournalofNutrition
7. response (systemically and within the small intestine and
caecal tonsils) to coccidial infection(48,49,51 –53)
. Given the
importance of the cellular immune response during coccidial
infection(51 –60)
, we chose to focus on CD3þ expression in
caecal tonsils. Though we found no change in the population
of cells positive for CD3þ, there was a significant reduction
in the MFI. The CD3þ marker is a critical inducer of the
signalling cascade necessary to activate T cells. In human
8 (b)(a)
(d)(c)
Relativeexpressionoftargetincaecaltonsil
6
4
2
0
8
6
4
2
0 0
10
20
30
40
Basal
45ZnSO
470ZnSO
4
90ZnSO
445Blend70Blend90Blend
Basal45ZnSO
4
70ZnSO
4
90ZnSO
445Blend70Blend90Blend
Basal45ZnSO
4
70ZnSO
4
90ZnSO
445Blend70Blend90Blend
Basal45ZnSO
4
70ZnSO
4
90ZnSO
445Blend70Blend90Blend8
6
4
2
0
Zn Trt: P=0·2
10CV: P=0·4
Zn Trt×10CV: P=0·1
Zn Trt: P=0·6
10CV: P=0·3
Zn Trt×10CV: P=0·9
Zn Trt: P=0·8
10CV: P=0·002
Zn Trt×10CV: P=0·8
Zn Trt: P=0·003
10CV: P<0·001
Zn Trt×10CV: P=0·003
Fig. 4. Caecal tonsil zinc transporter expression. (a) ZnT5, (b) ZnT7, (c) ZIP9 and (d) ZIP13. Zinc transporter expressions in caecal tonsil mucosa (Expt 2) from
26 and 27 d-old broilers consuming different dietary zinc sources and exposed to 10£ dose of coccidial vaccine Coccivacw
-B (10CV). Values represent mean
response of six birds per zinc source for both control (Con) and 10CV birds. Significant (P#0·05) main effect mean comparisons of zinc source, 10CV, and their
interactions are indicated within each panel. Dietary regimens consisted of a basal maize–soyabean meal diet, or basal diet supplemented with either zinc from
zinc sulphate (ZnSO4), or a blended source (1:1 blend of ZnSO4 and Availaw
-Zn) to achieve 45, 70, or 90 mg/kg of total dietary zinc. Birds were unchallenged
(Con, A), or exposed to 10CV ( ) on days 1, 7, 11, and 17, with the final gavage occurring 10 d before tissue collection. Figure depicts the expression of the target
genes against the geometric mean of two housekeeper genes hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP) as selected
by the BestKeeper index(47)
. Trt, treatment.
4(a)
(c)
(b)
(d)
3
2
1
0
0
2
4
6
8
Relativeexpressionoftargetinjejunum
0
2
4
6
8
Basal 90ZnSO4 90Blend
4
3
2
1
0
Basal 90ZnSO4 90Blend
Basal 90ZnSO4 90Blend Basal 90ZnSO4 90Blend
Zn Trt: P=0·8
10CV: P=0·06
Zn Trt×10CV: P=0·2
Zn Trt: P=0·9
10CV: P=0·02
Zn Trt×10CV: P=0·4
Zn Trt: P=0·5
10CV: P=0·2
Zn Trt×10CV: P=0·6
Zn Trt: P=0·02
10CV: P=0·001
Zn Trt×10CV: P=0·01
Fig. 3. Jejunal zinc transporter expression. (a) ZnT5, (b) ZnT7, (c) ZIP9 and (d) ZIP13. Zinc transporter expressions in jejunal mucosal (Expt 1) from 30 and 31 d-old
broilers consuming different dietary zinc sources and exposed to 10£ dose of coccidial vaccine Coccivacw
-B (10CV). Values represent mean response of six birds
per zinc source for both control (Con) and 10CV birds. Significant (P#0·05) main effect mean comparisons of zinc source, 10CV, and their interactions are indicated
within each panel. Dietary regimens consisted of a basal maize–soyabean meal diet, or basal diet supplemented with either zinc from zinc sulphate (ZnSO4), or a
blended source (1:1 blend of ZnSO4 and Availaw
-Zn) to achieve 90 mg/kg of total dietary zinc. Birds were unchallenged (Con, A), or exposed to 10CV ( ) on days 1,
7, 14, and 22, and the final gavage occurred 10 d before tissue collection. Figure depicts the expression of the target genes against the geometric mean of two
housekeeper genes hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP) as selected by the BestKeeper index(47)
. Trt, treatment.
C. Troche et al.208
BritishJournalofNutrition
8. T cell lines, the CD3 þ marker becomes internalised and
eventually degraded, resulting in a loss of CD3þ signal(61,62)
.
This mechanism is similar in chickens, as the T cell receptor/
CD3 complex is known to contain an internalisation motif(63)
.
Furthermore, Go¨bel & Dangy(63)
determined that CD3 was
downregulated in ab T cells during stimulation. Therefore,
while the results of the present study reflect no change in total
T cell population, they do suggest an increase in activated T
cells within 10CV caecal tonsils. Caecal tonsils are the largest
lymphoid organ in the chicken, and they can function in
a manner similar to that of mammalian Peyer’s patches(64)
.
Histologically, caecal tonsils contain germinal centres and
IgA-positive B cells, which survey the intestinal immune
environment, and aid in the development of intestinal
immunity(65)
. The jejunum also contains a large amount of
lymphocytes within the epithelium and lamina propria(64)
.
However, between the two organs, caecal tonsils contain
twice the amount of lymphocytes as the jejunum does(66)
. There-
fore, the difference in phagocytosis between jejunum and
caecal tonsils may lie in the variability of cell type within those
organs. The amount of free Zn (Newport Greene) within
caecal tonsils was significantly decreased with 10CV.
The decrease of free Zn within caecal tonsil cells is presumably
a protective mechanism, promoting apoptosis and limiting
tissue damage.
Zinc trafficking within jejunum and caecal tonsils
ZnT measured in Expt 1 and 2 are localised to the endoplas-
mic reticulum/Golgi complex but expressed different direc-
tionality of transport(19,20)
. In mammals, ZnT5 and ZnT7 are
specifically thought to transport Zn from the cytosol to the
Golgi, while ZIP9 and ZIP13 are responsible for vesicular Zn
influx into the cytoplasm(24 –27)
. In Expt 1, Zip13 mRNA was
significantly upregulated in the 90ZnSO4 treatment in the jeju-
num during challenge, but not by the 90Blend treatment. This
finding suggests differences in availability between Zn
sources(67)
. ZnT7 expression in Expt 1 jejunum, was signifi-
cantly decreased with 10CV. This is in contrast to liver
expression where ZnT5 and ZnT7 have been shown to
increase with inflammation induced by lipopolysaccharide(32)
.
Upregulation of Zip transporters coupled with decreased ZnT
expression suggests that trafficking from cytosolic compart-
ments to the Golgi was decreased with exposure to 10CV.
However, these responses are tissue-specific, and the intestine
is known to have a unique ZnT response during inflammation.
Guthrie et al.(68)
noted degradation of ZIP14 with lipopolysac-
charide (as opposed to the upregulation noted in liver); it was
hypothesised that highly challenging immunoenvironment
was responsible for the differential responses between the
jejunum and liver. In keeping with the general downregula-
tion of ZnT during coccidial challenge, a recent report noted
a significant decrease in the expression of ZnT1 with Eimeria
infection(69)
. A decrease in ZnT1 along with the finding of the
present study of a decrease in jejunal ZnT7 would suggest that
cells do limit Zn efflux into intracellular compartments(69)
.
This conclusion is strengthened by the observed trend of
decreased free intracellular Zn in the present study (as
measured by Newport Greene). This indicates that within
the jejunum, movement of Zn into the cytoplasm was upregu-
lated during repeated exposure to coccidial vaccine (10CV).
Caecal tonsils followed a similar pattern. In comparison to
the jejunum, 10CV appeared to have little impact on measured
ZnT mRNA expression in caecal tonsils. Thus, very little has
been reported on ZnT expression within caecal tonsils.
Though the expression of measured ZnT was not downregu-
lated with 10CV, the expression of both Zip9 and Zip13 were
significantly upregulated. In contrast to jejunal cells, the great-
est expression of Zip13 was observed in 90Blend treatment.
10
(a) (b)
8
6
4
Zip:ZnTexpression
ratios
2
0
10
8
6
4
2
0
CON 10CV CON 10CV
Zn Trt: P=0·9
10CV: P=0·02
Zn Trt×10CV: P=0·6
Zn Trt: P=0·4
10CV: P<0·0001
Zn Trt×10CV: P=0·4
Fig. 5. Ratio of zinc influx transporter (Zip):zinc efflux transporter (ZnT)
expression. Zip:ZnT expression in jejunal (a) and caecal (b) cells. Mean
values with their standard errors represent ratios which were calculated
from expression data, by cage as: (Zip9 þ Zip13)/(ZnT5 þ ZnT7). The main
effects of dietary treatment (Expt 1 (a) basal maize–soyabean meal diet, or
basal diet supplemented with either Zn from zinc sulphate (ZnSO4), or a
blended source, 1:1 blend of ZnSO4 and Availaw
-Zn, to achieve 90 mg/kg of
total dietary Zn. Expt 2 (b) was nearly identical with the exception of two
additional supplemental levels at 45 and 70 mg/kg dietary Zn), and vaccine
exposure (unchallenged control (CON), or 10CV (10 £ dose of coccidial
vaccine Coccivacw
-B)) were analysed. Vaccine exposure (10CV) had a
significant effect on Zip:ZnT expression ratio and P values are indicated
within each panel. Trt, treatment.
Changes in jejunal and caecal
tonsil tissues during
coccidiosis
Zip:ZnT ratio
Intracellular
free Zn
Zn incorporated into
intracellular proteins
Or
Cell may be Zn
starved and
upregulating Zip
ZIP
ZnT
Fig. 6. Model of intracellular zinc trafficking during repeated exposure to
coccidial vaccine. Amended mechanism for zinc transporter expression.
Coccidiosis had a larger impact on transporter expression than dietary zinc
regimen. Zinc influx transporter (Zip):zinc efflux transporter (ZnT) ratio
increased with challenge in jejunal cells and caecal tonsils. However, flow
cytometry reported a decrease in intracellular-free zinc with challenge. The
upregulation of Zip transporters coupled with lower intracellular-free zinc
suggests that the cell (1) may have shuttled zinc into cytosolic proteins
during coccidial challenge, or (2) upregulated Zip transporters in an effort to
replenish lost cytosolic zinc.
Intracellular zinc trafficking 209
BritishJournalofNutrition
9. To our knowledge this is the first report that has linked the
expression of ZnT to a dietary Zn source. Star et al.(67)
found that the bioavailability of Availaw
-Zn, as measured
through broiler chick Zn tibia content, was higher than that
of ZnSO4. Castillo et al.(70)
reported the use of an organic
Zn source tended to reduce enterobacteria levels in weanling
pig jejunums. It is therefore possible that the observed
changes in Zip13 expression in Expt 1 and 2 were due to
altered bioavailability and/or altered microbial load. Overall,
our data shows that the ratio of Zip:ZnT expression was
significantly increased due to 10CV (Fig. 5). This suggests
movement of Zn from intracellular compartments to the cyto-
plasm. As Newport Greene only measures free, not bound,
Zn, this increase in Zip:ZnT ratio suggests that Zn may be
incorporated into cytosolic proteins (e.g. MT or other metal-
regulatory protein). Our hypothesis is that cells upregulate
ZIP transporters in an effort to promote cell/tissue protective
processes during coccivac exposure, i.e. apoptosis. However,
this does not exclude the possibility that cells are simply Zn
starved, and upregulate ZIP transporters in an effort to com-
pensate. A model outlining the findings of the present study
and potential hypotheses is included in Fig. 6. In conclusion,
repeated exposure to coccidial challenge decreased free intra-
cellular Zn, and concurrently increased the ratio of measured
ZIP:ZnT transporters. This response appears to be a compen-
satory effect for reductions in intracellular-free Zn.
Acknowledgements
The authors would like to thank Zhengyu Jiang and Liting Xu
for their assistance with tissue processing and sample prep-
aration during the flow cytometry experiments. We also
thank Kolapo Ajuwon for the generous use of his lab facilities
and expertise with PCR analysis. The authors thank Zinpro
Corporation (Eden Prarie, Minnesota, USA) for partial funding
for the present research.
Partial funding for the present research reported herein was
provided by the Zinpro Corporation, Eden Prairie, MN, USA as
an unconditional research gift. Zinpro Corporation had no role
in the design, analysis or writing of this article. Mention of
trade names or commercial products in this publication is
solely for the purpose of providing specific information
and does not imply recommendation or endorsement by
the United States Department of Agriculture. United States
Department of Agriculture is an equal opportunity employer.
The authors’ contributions are as follows: C. T., S. D. E., and
T. J. A. were responsible for the design of the research; C. T.
carried out the research and prepared the manuscript; C. T.,
S. D. E., and T. J. A. reviewed and edited the manuscript. All
authors read and approved the final version of the manuscript.
The authors have no conflicts of interest to declare.
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