Broad Conservation of Milk Utilization Genes in Bifidobacterium longum subsp. infantis as Revealed by Comparative Genomic Hybridization

Human milk oligosaccharides (HMOs) are the third-largest solid component of milk. Their structural complexity renders them nondigestible to the host but liable to hydrolytic enzymes of the infant colonic microbiota. Bifidobacteria and, frequently, Bifidobacterium longum strains predominate the colonic microbiota of exclusively breast-fed infants. Among the three recognized subspecies of B. longum, B. longum subsp. infantis achieves high levels of cell growth on HMOs and is associated with early colonization of the infant gut. The B. longum subsp. infantis ATCC 15697 genome features five distinct gene clusters with the predicted capacity to bind, cleave, and import milk oligosaccharides. Comparative genomic hybridizations (CGHs) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations. Multilocus sequence typing provided taxonomic subspecies designations and grouped the strains between B. longum subsp. infantis and B. longum subsp. longum. CGH analysis determined that HMO utilization gene regions are exclusively conserved across all B. longum subsp. infantis strains capable of growth on HMOs and have diverged in B. longum subsp. longum strains that cannot grow on HMOs. These regions contain fucosidases, sialidases, glycosyl hydrolases, ABC transporters, and family 1 solute binding proteins and are likely needed for efficient metabolism of HMOs. Urea metabolism genes and their activity were exclusively conserved in B. longum subsp. infantis. These results imply that the B. longum has at least two distinct subspecies: B. longum subsp. infantis, specialized to utilize milk carbon, and B. longum subsp. longum, specialized for plant-derived carbon metabolism.The newborn infant not only tolerates but requires colonization by commensal microbes for its own development and health (3). The relevance of the gut microbiome in health and disease is reflected by its influence in a number of important physiological processes, from physical maturation of the developing immune system (28) to the altered energy homeostasis associated with obesity (51, 52).Human milk provides all the nutrients needed to satisfy the neonate energy expenditure and a cadre of molecules with nonnutritional but biologically relevant functions (6). Neonatal health is likely dependent on the timely and complex interactions among bioactive components in human milk, the mucosal immune system, and specialized gut microbial communities (30). Human milk contains complex prebiotic oligosaccharides that stimulated the growth of select bifidobacteria (24, 25) and are believed to modulate mucosal immunity and protect the newborn against pathogens (23, 33, 41). These complex oligosaccharides, which are abundantly present in human milk (their structures are reviewed by Ninonuevo et al. [31] and LoCascio et al. [24]), arrive intact in the infant colon (5) and modulate the composition of neonatal gastrointestinal (GI) microbial communities.Bifidobacteria and, frequently, Bifidobacterium longum strains often predominate the colonic microbiota of exclusively breast-fed infants (10, 11). Among the three subspecies of B. longum, only B. longum subsp. infantis grows robustly on human milk oligosaccharides (HMOs) (24, 25). The availability of the complete genome sequences of B. longum subsp. infantis ATCC 15697 (40) and two other B. longum subsp. longum strains (22, 39) made possible the analysis of whole-genome diversity across the B. longum species. Analysis of the B. longum subsp. infantis ATCC 15697 genome has identified regions predicted to enable the metabolism of HMOs (40); however, their distribution across the B. longum spp. remains unknown. We predict that these regions are exclusively conserved in B. longum strains adapted to colonization of the infant gut microbiome and are therefore capable of robust growth on HMOs. In this work, whole-genome microarray comparisons (comparative genomic hybridizations [CGHs]) were used to associate genotypic biomarkers among 15 B. longum strains exhibiting various HMO utilization phenotypes and host associations.     

Three-dimensional in vitro follicle growth: overview of culture models,biomaterials, design parameters and future directions

In vitro ovarian follicle culture is a new frontier in assisted reproductive technology with tremendous potential, especially for fertility preservation. Folliculogenesis within the ovary is a complex process requiring interaction between somatic cell components and the oocyte. Conventional two-dimensional culture on tissue culture substrata impedes spherical growth and preservation of the spatial arrangements between oocyte and surrounding granulosa cells. Granulosa cell attachment and migration can leave the oocyte naked and unable to complete the maturation process. Recognition of the importance of spatial arrangements between cells has spurred research in to three-dimensional culture system. Such systems may be vital when dealing with human primordial follicles that may require as long as three months in culture. In the present work we review pertinent aspects of in vitro follicle maturation, with an emphasis on tissue-engineering solutions for maintaining the follicular unit during the culture interval. We focus primarily on presenting the various 3-dimensional culture systems that have been applied for in vitro maturation of follicle:oocyte complexes. We also try to present an overview of outcomes with various biomaterials and animal models and also the limitations of the existing systems.     

The Product of the Herpes Simplex Virus Type 1 UL25 Gene Is Required for Encapsidation but Not for Cleavage of Replicated Viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278–283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246–259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.     

A practical approach to crosslinking

The various aspects of chemical crosslinking are addressed. Crosslinker reactivity, specificity, spacer arm length and solubility characteristics are detailed. Considerations for choosing one of these crosslinkers for a particular application are given as well as reaction conditions and practical tips for use of each category of crosslinkers.Abbreviations ABH azidobenzoyl hydrazide - ANB- NOS N-5-azido-2-nitrobenzoyloxysuccinimide - ASIB 1-(p-azidosalicylamido)-4-(iodoacetamido)butane - ASBA 4-(p-azidosalicylamido)butylamine - APDP N-[4-(p-azidosalicylamido) butyl]-3(2-pyridyldithio)propionamide - APG p-azidophenyl glyoxal monohydrate - BASED bis-[-(4-azidosalicylamido)ethyl] disulfide - BMH bismaleimidohexane - BS³ bis(sulfosuccinimidyl) suberate - BSOCOES bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone - DCC N,N-dicyclohexylcarbodiimide - DFDNB 1,5-difluoro-2,4-dinitrobenzene - DMA dimethyl adipimidate·2HCl - DMP dimethyl pimelimidate·2HCl - DMS dimethyl suberimidate·2HCl - DPDPB 1,4-di-(3,2-pyridyldithio)propionamido butane - DMF dimethylformamide - DMSO dimethylsulfoxide - DSG disuccinimidyl glutarate - DSP dithiobis(succinimidylpropionate) - DSS disuccinimidyl suberate - DST disuccinimidyl tartarate - DTSSP 3,3-dithiobis (sulfosuccinimidylpropionate) - DTBP dimethyl 3,3-dithiobispropionimidate·2HCl - EDC or EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride - EDTA ethylenediaminetetraacetic acid disodium salt, dihydrate - EGS ethylene glycolbis(succinimidylsuccinate) - GMBS N--maleimidobutyryloxysuccinimide ester - HSAB N-hydroxysuccinimidyl-4-azidobenzoate - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester - MES 4-morpholineethanesulfonic acid - NHS N-hydroxysuccinimide - NHS-ASA N-hydroxysuccinimidyl-4-azidosalicylic acid - PMFS phenylmethylsulfonyl fluoride - PNP-DTP p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate - SAED sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide) ethyl-1,3-dithiopropionate - SADP N-succinimdyl (4-azidophenyl)1,3-dithiopropionate - SAND sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl-1,3-dithiopropionate - SANPAH N-succinimidyl-6(4-azido-2-nitrophenyl-amino)hexanoate - SASD sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3-dithiopropionate - SATA N-succinimidyl-S-acetylthioacetate - SDBP N-hydroxysuccinimidyl-2,3-dibromopropionate - SIAB N-succinimidyl(4-iodoacetyl)aminobenzoate - SMCC succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SMPB succinimidyl 4-(p-maleimidophenyl) butyrate - SMPT 4-succinimidyloxycarbonyl--methyl--(2-pyridyldithio)-toluene - sulfo-BSOCOES bis[2-sulfosuccinimidooxycarbonyloxy) ethyl]sulfone - sulfo-DST disulfosuccinimidyl tartarate - sulfo-EGS ethylene glycolbis(sulfosuccinimidylsuccinate) - sulfo-GMBS N--maleimidobutyryloxysulfosuccinimide ester - sulfo-MBS m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester - sulfo-SADP sulfosuccinimidyl(4-azidophenyldithio)propionate - sulfo-SAMCA sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate - sulfo-SANPAH sulfosuccinimidyl 6-(4-azido-2-nitrophenylamino)hexanoate - sulfo-SIAB sulfosuccinimidyl(4-iodoacetyl)aminobenzoate - sulfo-SMPB sulfo-succinimidyl 4-(p-maleimidophenyl)butyrate - sulfo-SMCC sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate - SPDP N-succinimidyl 3-(2-pyridyldithio)propionate     

Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco

Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season. We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene. Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development. Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering. Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines. ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue. All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 g ICP per gram fresh weight in leaves from the mid-section of the plant at flowering. The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.     

Possible Involvement of Phage-Like Structures in Antagonism of Cowpea Rhizobia by Rhizobium trifolii

A reduction in the viability of cowpea rhizobia was observed when Rhizobium trifolii IARI and cowpea Rhizobium strain 3824 were inoculated together in soil. The reduction in number of cowpea rhizobia in soil was found to be associated with the reduction in number of nodules per plant and retardation in plant growth. An antimicrobial substance was isolated from R. trifolii which, on electron microscopic investigation, demonstrated the presence of several phage-like structures.     

Generation of a reproducible nutrient-depleted biofilm of Escherichia coli and Burkholderia cepacia

An in vitro method of growing bacteria as a defined nutrient-depleted biofilm is proposed. The medium was defined nutritionally in terms of the quantitative composition and by the total amount of nutrient required to achieve a defined population size. Escherichia coli and Burkholderia cepacia were incubated on a filter support placed on a defined volume of solid medium. The change of biomass of the biofilm population was compared with the change in a planktonic culture. The size of the population in stationary phase was proportional to the concentration of limiting substrate up to 40 μmol cm⁻² glucose for E. coli and up to 2·7 × 10⁻⁹ mol cm⁻² iron for B. cepacia . Escherichia coli growing exponentially had a growth rate of μ = 0·30 h⁻¹ in a biofilm and μ = 0·96 h⁻¹ in planktonic culture. The growth rate, μ, for exponentially growing B. cepacia in a biofilm was 1·12 h⁻¹ and in planktonic culture 0·78 h⁻¹. This method allows the limitation of the size of a biofilm population to a chosen value.