Search

Background: This study was designed to assess the efficacy of Bifidobacterium animalis ssp. lactis (Bl-04) for prevention of rhinovirus colds and to explore the interactions between the probiotic, the viral infection, the host response and the host microbiome., Methods: The effect of ingestion of the probiotic Bl-04 was evaluated in a randomized, double-blinded rhinovirus (RV) challenge study. Healthy volunteers recruited from a university community in USA were randomized 1:1 using a computer generated code to ingest either Bl-04 (n=165) or placebo (n=169) for 28 days and were then challenged with RV-A39, and followed for 14 days. All study interactions and sample collection occurred in dedicated clinical research space. The primary analysis was the effect of the probiotic on the incidence of RV-associated illness. (Trial registration: NCT02679807, study complete)., Findings: The first cohort of volunteers was randomized on March 14, 2016 and the last (5 th ) cohort was randomized on March 12, 2018. Sixty-three (56%, 95% CI [47%; 66%]) of the 112 subjects in the active group and 60 (50%,95% CI [41%; 59%]) of the 120 subjects in the placebo group had a protocol-defined rhinovirus-associated illness (χ 2 =0·91, p=0·34). The point estimate of the difference in illness (active-placebo) is 6.3% (95% CI -6.7;19.1). There were no adverse events that were judged as definitely or probably related to the study product., Interpretation: In this study there was no effect of orally administered Bl-04 on the occurrence of RV-associated illness., Funding: Danisco Sweeteners Oy (now IFF Health & Biosciences)., Competing Interests: LL, AH, BZ, FB, NY, and MJL report other from IFF Health & Biosciences, outside the submitted work. LL, AH, and MJL report a patent pending based on the results. Dr. Turner reports grants from Danisco Sweeteners OY, during the conduct of the study; other from PrEP BioPharm, Inc, other from Henkel Corporation, other from G.ST Antivirals GmbH, other from American Cleaning Institute, other from Avrio, other from Bayer Consumer Health, outside the submitted work. All the other authors have nothing to disclose., (© 2021 The Author(s).)

Obesity is a multifactorial disease with many complications and related diseases and has become a global epidemic. To thoroughly understand the impact of obesity on whole organism homeostasis, it is helpful to utilize a systems biological approach combining gene expression and metabolomics across tissues and biofluids together with metagenomics of gut microbial diversity. Here, we present a multi-omics study on liver, muscle, adipose tissue, urine, plasma, and feces on mice fed a high-fat diet (HFD). Gene expression analyses showed alterations in genes related to lipid and energy metabolism and inflammation in liver and adipose tissue. The integration of metabolomics data across tissues and biofluids identified major differences in liver TCA cycle, where malate, succinate and oxaloacetate were found to be increased in HFD mice. This finding was supported by gene expression analysis of TCA-related enzymes in liver, where expression of malate dehydrogenase was found to be decreased. Investigations of the microbiome showed enrichment of Lachnospiraceae, Ruminococcaceae, Streptococcaceae and Lactobacillaceae in the HFD group. Our findings help elucidate how the whole organism metabolome and transcriptome are integrated and regulated during obesity.

The SRY-related high-mobility box 9 (SOX9) gene is expressed in many different tissues. To better understand the DNA elements that control tissue-specific expression, we cloned and sequenced a 2.5 kb fragment lying 5' to the zebrafish sox9b gene transcriptional start site. Three regions of this clone contained stable secondary structures that hindered cloning, sequencing, and amplification. This segment and smaller fragmentswere inserted 5' of an EGFP reporter and transgenic fish were raised with the different reporters. Reporter expression was also observed in embryos directly injected with the constructs to transiently express the reporter. Heart expression required only a very short 5' sequence, as a 0.6 kb sox9b fragment produced reporter expression in heart in transgenic zebrafish, and transient experiments showed heart expression from a minimal sox9b promoter region containing a conserved TATA box and an EGR2 element (-74/+29 bp). Reporter expression in transgenic skeletal muscle was consistently lower than in other tissues. Jaw, brain, and notochord expression was strong with the full-length clone, but was dramatically reduced as the size of the fragment driving the reporter decreased from approximately 1.8 to 0.9 kb. The 2.5 kb region 5' of the sox9b contained 7 conserved non-coding elements (CNEs) that included putative hypoxia inducible factor 1α (HIF1α), CAAT box (CCAAT), early growth response protein 2 (EGR2), and core promoter elements. While a synthetic fragment containing all 7 CNEs produced some degree of reporter expression in muscle, jaw, heart and brain, the degree of reporter expression was considerably lower than that produced by the full length clone. These results can account for the tissue-specific expression of sox9b in the developing zebrafish., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The present study was undertaken to provide the foundation for development of genome-scale resources for the fathead minnow (Pimephales promelas), an important model organism widely used in both aquatic toxicology research and regulatory testing. The authors report on the first sequencing and 2 draft assemblies for the reference genome of this species. Approximately 120× sequence coverage was achieved via Illumina sequencing of a combination of paired-end, mate-pair, and fosmid libraries. Evaluation and comparison of these assemblies demonstrate that they are of sufficient quality to be useful for genome-enabled studies, with 418 of 458 (91%) conserved eukaryotic genes mapping to at least 1 of the assemblies. In addition to its immediate utility, the present work provides a strong foundation on which to build further refinements of a reference genome for the fathead minnow., (© 2015 SETAC.)

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) disrupts craniofacial development in zebrafish larvae. However, the cellular changes responsible for the decreased jaw size remain poorly understood. We show that smaller jaw size is due to a decrease in both the size and number of chondrocytes in the developing craniofacial cartilages. TCDD was found to decrease ossification of osteoblasts in the perichondrium of craniofacial cartilages. We also discovered that TCDD caused clefting of the parasphenoid, an effect with similarity to TCDD-induced cleft palate in mice. Thus, dermal and perichondrial bone development of the craniofacial skeleton are clearly disrupted by TCDD exposure in the zebrafish larvae. This dysmorphic response of the zebrafish craniofacial skeleton after exposure to TCDD is consistent with findings demonstrating disruption of axial bone development in medaka and repression of sox9b in zebrafish., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The transcription factor SOX9 is a member of the SRY-related high-mobility-group box (SOX) superfamily of genes. In mammals, Sox9 plays important roles in many developmental processes including craniofacial, skeletal and heart morphogenesis, retinal and brain development, and gonad differentiation. Human mutations in SOX9 or the SOX9 promoter result in campomelic dysplasia, a severe genetic disorder, which disrupts skeletal, craniofacial, cardiac, neural and reproductive development. Due to the duplication of the teleost fish genome, zebrafish (Danio rerio) have two Sox9 genes: sox9a and sox9b. Loss of sox9b in zebrafish results in loss of function phenotypes that are similar to those observed in humans and mice. In order to generate a transgenic sox9b:EGFP reporter line, we cloned a 2450 bp fragment of the sox9b promoter and fused it to an EGFP reporter. Consistent with reported sox9b expression and function, we observed sox9b:EGFP in the developing heart, skeletal and craniofacial structures, brain, retina, and ovaries. Our resulting transgenic line is a useful tool for identifying and studying sox9b function in development and visualizing a number of zebrafish organs and tissues in which sox9b is normally expressed.

In 2010, the BAX System PCR assay for Salmonella was modified to include a hot start functionality designed to keep the reaction enzyme inactive until PCR begins. To validate the assay's Official Methods of Analysis status to include this procedure modification, an evaluation was conducted on four food types that were simultaneously analyzed with the BAX System and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Identical performance between the BAX System method and the reference methods was observed. Additionally, lysates were analyzed using both the BAX System Classic and BAX System Q7 instruments with identical results using both platforms for all samples tested. Of the 100 samples analyzed, 34 samples were positive for both the BAX System and reference methods, and 66 samples were negative by both the BAX System and reference methods, demonstrating 100% correlation. No instrument platform variation was observed. Additional inclusivity and exclusivity testing using the modified test kit demonstrated the test kit to be 100% accurate in evaluation of test panels of 352 Salmonella strains and 46 non-Salmonella strains.

Australia, like other western nations, is facing the prospect that in the decades ahead the overall population will age dramatically. This article considers legal issues associated with the aged and intergenerationally transmitted debt. It will be argued that the present legal regulation of guarantees is a complex amalgam of case law, statute and finance industry self-regulatory codes which does not necessarily or adequately take into account the particular vulnerabilities of the aged and the very old or the intergenerational pressures which they may confront. It may be necessary to take significant steps to protect vulnerable elders while still recognizing their rights to exercise personal autonomy. Some proposals for reform are made.

A gelatinous otolithic membrane (OM) couples a single calcified otolith to the sensory epithelium in the bluegill sunfish (Lepomis macrochirus) saccule, one of the otolithic organs in the inner ear. Though the OM is an integral part of the anatomic network of endorgan structures that result in vestibular function in the inner ear, the identity of the proteins that make up this sensory accessory membrane in teleosts, or in any vertebrate, is not fully known. Previously, we identified a cDNA from the sunfish saccular otolithic organ that encoded a new member of the collagen family of structural proteins. In this study, we examined biochemical features and the localization of the saccular collagen (SC) protein in vivo using polyclonal antisera that recognize the noncollagenous domains of the SC protein. The SC protein, in vivo, was identified as a 95-kDa glycoprotein in sunfish whole-saccule lysate and in homogenates of microdissected saccular OMs. Immunohistochemical analyses demonstrated that the SC protein was localized within one of the two distinct layers of the sunfish saccular OM. The SC protein was also detected within the cytoplasm of supporting cells at the edges of the saccular sensory epithelium, indicating that these cells are a primary site for the synthesis of this structural protein. Further studies of the organization of this matrix molecule in the OM may help clarify the role of this sensory accessory membrane in vestibular sensory function.

The vertebrate inner ear sensory epithelia contain different types of hair cells and supporting cells. The teleost saccule is anatomically similar to the mammalian saccule and is primarily involved in the detection of translational acceleration and orientation with respect to gravity. To facilitate molecular studies of the teleost saccule cDNA libraries were constructed from microdissected Lepomis macrochirus (bluegill sunfish) saccular maculae. To our knowledge, this is the first report of cDNA libraries constructed from the saccule. In one instance, a non-polymerase chain reaction-based method of amplifying a mRNA population from limited amounts of starting tissue was employed that allowed construction of cDNA libraries from nanogram amounts of tissue mRNA. Conventional cDNA libraries were constructed from the sunfish saccular maculae as well. These cDNA libraries enriched in hair cell and supporting cell transcripts should facilitate molecular biological studies of inner ear sensory epithelia. As an example of their utility, efforts to identify tyrosine kinases expressed in the saccular endorgan using low-stringency hybridization screening of these cDNA libraries and the partial sequence of a cDNA found to encode an erbB-2-related tyrosine kinase are also reported.

Molecular biological studies of the mammalian inner ear have been limited by the relatively small size of the sensory endorgans contained within. The saccular otolithic organ in teleostian fish is structurally similar to its mammalian counterpart but can contain an order of magnitude more sensory cells. The prospect of the evolutionary conservation of proteins utilized in the vertebrate inner ear and the relative abundance of teleostian saccular sensory tissue made this an attractive system for molecular biological studies. A complementary DNA obtained by differential screening of a saccular complementary DNA library was identified that encodes an inner ear-specific collagen molecule.

We have mapped and sequenced the GP42/Basigin gene isolated from a Balb/C mouse genomic library. The genomic organization and upstream, putative regulatory, regions of this gene have not been previously reported. Our data show that exon 5 of the GP42/Basigin gene encodes the carboxy proximal half of the second Ig-like domain, the highly conserved transmembrane region and a portion of the cytoplasmic tail. This inclusion of Ig-like and other functional domains in a single exon is unusual. Splice junction analysis indicates that two reported alternate GP42/Basigin cDNA isoforms are likely due to cloning artifacts. In addition, we find that GP42/Basigin is polymorphic in mice. Our data also support the proposal that the transmembrane domain and portions of the cytoplasmic region of GP42/Basigin have been evolutionarily conserved.

We have identified a 95 kd cell surface protein, DM-GRASP, that is expressed on a restricted population of axons. Its expression begins early in chick embryogenesis, and within the spinal cord it is localized to axons in the dorsal funiculus, midline floorplate cells, and motoneurons. Antibodies to DM-GRASP impair neurite extension on axons, and purified DM-GRASP supports neurite extension from chick sensory neurons. We have cloned and sequenced the cDNA corresponding to this protein and find that it is a new member of the immunoglobulin superfamily of adhesion molecules. Consequently we have named this protein DM-GRASP, since it is an immunoglobulin-like restricted axonal surface protein that is expressed in the dorsal funiculus and ventral midline of the chick spinal cord.

Pseudomonas aeruginosa elastase is a zinc metalloproteinase which is released during P. aeruginosa infections. Pseudomonas keratitis, which occurs following contact lens-induced corneal trauma, can lead to rapid, liquefactive necrosis of the cornea. This destruction has been attributed to the release of both host-derived enzymes and the bacterial products P. aeruginosa elastase, alkaline protease, exotoxin A, and lipopolysaccharide endotoxin. A synthetic metalloproteinase inhibitor, HSCH2 (DL)CH[CH2CH(CH3)2]CO-Phe-Ala-NH2, which we previously showed to be a potent inhibitor of corneal collagenase and alkali-induced corneal ulceration, was tested as a potential inhibitor of P. aeruginosa elastase. Inhibition constants (Kis) for the resolved diastereomers were determined with the chromogenic substrate furylacryloyl-glycyl-L-leucyl-L-alanine. One isomer had a Ki of 0.3 microM, while the other had a Ki of 0.4 microM. The more potent diastereomer was evaluated in vivo in experimentally induced Pseudomonas keratitis in rabbits. Following inoculation of one cornea of each rabbit, topical treatment with a 1 mM solution of the inhibitor significantly delayed the onset of corneal melting and perforation, as compared with the results for the control and gentamicin-treated groups. This protective effect suggests that the inhibitor may have a therapeutic application by delaying the progression of corneal destruction in Pseudomonas keratitis.

Corneal ulceration and perforation following a severe alkali burn occur as a consequence of collagen destruction by locally released enzymes. A thiol peptide, which recently was shown to be a potent inhibitor of corneal collagenase in vitro, was tested in alkali-burned rabbit corneas to determine its effectiveness in inhibiting corneal ulceration. Following a standard alkali burn to one eye of each rabbit, ten animals were treated topically six times daily and subconjunctivally one time daily with a 1 mM solution of the peptide for a period of 3 weeks. A control group of ten rabbits was administered vehicle only using the same regimen as the experimental group. Corneal ulceration occurred in ten out of ten of the control eyes and seven out of ten progressed to perforation. The experimental group demonstrated ulcerations in four out of nine animals, only one of which was deep (one of nine), and no perforations. There was no significant difference when comparing the onset of ulceration between the two groups, but the difference was significant when comparing the total number of ulcerations (0.02 less than P less than 0.05), deep ulcerations (0.01 less than P less than 0.02) and perforations (0.001 less than P less than 0.01) between the two groups. Histologic examination of the corneas after 3 weeks of treatment revealed that the experimental, thiol-treated corneas that did not ulcerate contained relatively few PMNs, whereas the control corneas demonstrated a marked inflammatory infiltrate in the form of PMNs, most notably at sites of corneal ulceration. These findings demonstrate that a synthetic thiol peptide inhibits alkali-induced corneal ulceration and perforation in vivo.

The major encephalitogenic epitope for Lewis rats is the 72-89 sequence of guinea pig basic protein (GP-BP) or rat basic protein (Rt-BP). T cells responsive to this epitope are I-A restricted and preferentially express the V alpha 2:V beta 8 gene combination in their TCR. In this work, we describe for the first time the delayed appearance of T cells specific for additional discrete determinant of BP, the nonencephalitogenic 55-68 sequence of GP-BP restricted by I-A, and the encephalitogenic 87-99 sequence of Rt-BP restricted by I-E. The TCR V alpha 2:V beta 8 gene combination was expressed by both encephalitogenic GP-BP S72-89 and Rt-BP S87-99 T cell specificities but not by GP-BP 44-68-specific T cells. This is the first demonstration of I-E-restricted encephalitogenic T cells in Lewis rats and supports the conclusion that the I-E class II locus is involved in autoimmune diseases.

Prospects for specific immune intervention in T cell-mediated autoimmune disease via anti-idiotypic regulation depend on the degree of diversity of the responder cell antigen receptor repertoire. A highly heterogenous response against self epitopes offers little chance for such regulation. We report here that the Lewis rat autoimmune disease experimental allergic encephalomyelitis, generally considered to be a model of human multiple sclerosis, is caused by T cells that use a limited set of TCR V genes. We have cloned the rat TCR alpha and beta chain cDNAs from the Lewis rat x mouse T cell hybridoma 510, which retains the rat specificity for the encephalitogenic determinant of myelin basic protein (MBP). Using Northern blot analysis of T cell RNA with the cloned V region probes, we have found a specific, and near perfect, correlation between expression of TCR message hybridizing to the V alpha 510 and VB510 probes and specificity for the encephalitogenic determinant of MBP in both T cell hybridomas and encephalitogenic T cell clones. This restricted V gene usage provides a basis for observed idiotypic regulation of auto-reactive T cells, and possible therapy for autoimmune disease. A curious and unexplained observation is that the Lewis rat V alpha/V beta combination that dominates the encephalitogenic response to the 68-88 peptide of MBP is precisely the same V alpha/V beta combination used by the B10.PL mouse response to the encephalitogenic response to the 1-9 peptide of MBP.

Chemical burns to the eye are among the most urgent of ocular emergencies. The clinical outcome of the injury is directly related to the expediency with which treatment is begun. Copious irrigation is the most important emergency treatment of the chemically-burned eye. This irrigation should begin immediately at the scene of the accident with any non-toxic liquid. Removal of any particulate matter must be done to prevent further ocular damage. The subsequent therapy is directed at the treatment of secondary sequelae and at preserving the globe to surgically rehabilitate the eye. Many of the treatments which are used in the intermediate and late phases of the injury are used to prevent corneal ulceration and perforation. These are the most difficult sequelae to threat in alkali injuries; thus, preventing the progression to this stage is of the utmost importance. Again the immediate and continuous irrigation of the eye may help accomplish this goal. The availability of emergency eye-wash equipment dispensing a safe, preserved, pH-balanced, physiologically correct solution in the industrial, agricultural and even the home setting is a necessity. The education of employees and family members in the proper technique of irrigating the eye following a chemical burn is also of extreme importance. Immediate irrigation of the eye, continued during rapid transport to a medical care facility, minimizes the damage to the eye and enhances the eventual clinical outcome.

The inhibitory potency of four classes of compounds that inhibit corneal ulceration (thiols, tetracyclines, sodium citrate and sodium ascorbate) was assessed with collagenase purified from culture medium of alkali-burned rabbit corneas. The most potent inhibitor, a beta-mercaptomethyl tripeptide HSCH2(DL)CH[CH2CH(CH3)2]CO-Phe-Ala-NH2, exhibited 50% inhibition (IC50) at approximately 10 nM using the synthetic metalloproteinase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2. The inhibitor was somewhat less potent with type 1 collagen as substrate (IC50 between 1 and 3 microM), possibly because autooxidation of the essential - SH moiety of the inhibitor occurred during the longer time required for assay with the natural substrate. An N-carboxyalkyl tripeptide, CH3(CH2)2(DL)CH-(COOH)-Leu-Phe-Ala-NH2, was less potent (IC50 = 25 microM) than the thiol peptide. N-acetylcysteine, which is used to treat corneal ulceration, gave IC50 values of 2.7 mM and less than 10 mM with the synthetic and natural substrates, respectively. The IC50 values for the tetracyclines using the synthetic substrate were 15, 190 and 350 microM for doxycycline, minocycline and tetracycline, respectively. Inhibition by sodium citrate, but not the tetracyclines, could be reversed by excess Ca2+. Sodium ascorbate did not inhibit collagenase-mediated hydrolysis of either collagen or the synthetic substrate, thus indicating that the mechanism by which this agent inhibits corneal ulceration is not related to inhibition of collagen degradation by collagenase.