Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction

In vivo induction of the Escherichia coli lactose operon as a function of inducer concentration generates a sigmoidal curve, indicating a non-linear response. Suggested explanations for this dependence include a 2:1 inducer–repressor stoichiometry of induction, which is the currently accepted view. It is, however, known for decades that, in vitro, operator binding as a function of inducer concentration is not sigmoidal. This discrepancy between in vivo and in vitro data has so far not been resolved. We demonstrate that the in vivo non-linearity of induction is due to cooperative repression of the wild-type lac operon through DNA loop formation. In the absence of DNA loops, in vivo induction curves are hyperbolic. In the light of this result, we re-address the question of functional molecular inducer–repressor stoichiometry in induction of the lac operon.     

Investigating the Roles of the C-Terminal Domain of Plasmodium falciparum GyrA

Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping.     

Role of hypermutability in the evolution of the genus Oenococcus

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium primarily responsible for malolactic fermentation in wine. A recent comparative genomic analysis of O. oeni PSU-1 with other sequenced lactic acid bacteria indicates that PSU-1 lacks the mismatch repair (MMR) genes mutS and mutL. Consistent with the lack of MMR, mutation rates for O. oeni PSU-1 and a second oenococcal species, O. kitaharae, were higher than those observed for neighboring taxa, Pediococcus pentosaceus and Leuconostoc mesenteroides. Sequence analysis of the rpoB mutations in rifampin-resistant strains from both oenococcal species revealed a high percentage of transition mutations, a result indicative of the lack of MMR. An analysis of common alleles in the two sequenced O. oeni strains, PSU-1 and BAA-1163, also revealed a significantly higher level of transition substitutions than were observed in other Lactobacillales species. These results suggest that the genus Oenococcus is hypermutable due to the loss of mutS and mutL, which occurred with the divergence away from the neighboring Leuconostoc branch. The hypermutable status of the genus Oenococcus explains the observed high level of allelic polymorphism among known O. oeni isolates and likely contributed to the unique adaptation of this genus to acidic and alcoholic environments.     

A study of the mechanism of the antiarrhythmic action of Allapinin

Allapinin (lappaconitine hydrobromide) is a drug used for the treatment of cardiac rhythm disturbances; its properties are characteristic of class IC antiarrhythmics. The mechanism of its electrophysiological action involves the blockade of Na⁺ channels with a subsequent decrease of depolarization rate leading to a slowing of impulse propagation and a decrease of excitability in the conductive system of the heart. Factors underlying the side effects of Allapinin (tachycardia, arterial hypertension, impaired coordination, etc.) are currently unknown, and therefore a study of the molecular mechanisms of its action seems relevant. The target genes of the drug were identified in rats with induced aconitine arrhythmia using the commercially available Rat Neuroscience Ion Channels & Transporters RT² Profiler? PCR Array kit (SA Biosciences). A comparison of expression levels of 84 genes in rats treated with Allapinin, after the induction of arrhythmia by aconitine (experiment) and in physiological saline-treated arrhythmic rats (control), revealed 18 mRNAs which were up- or downregulated twofold or more in the experiment relative to the control. Allapinin was shown to stimulate the expression of genes coding for various types of K⁺ channels (kcna6, kcnj1, kcnj4, kcnq2, and kcnq4), Ca²⁺ channel (cacna1g), and vesicular acetylcholine transporter (slc18a3). A decrease in mRNA levels was detected for genes coding for K⁺ channels (kcne1, kcns1), a Na⁺ channel (scn8a), and membrane transporter genes (atp4a, slc6a9). Our data shows that Allapinin administered to animals with aconitine arrhythmia modulates the expression of genes accounting for ion current conductances involved in the formation of various phases of action potential (I _Na , I _to , I _Ks , I _K1 , I _CaT ). The effect of the drug on the levels of mRNAs coding for acetylcholine and glycine transporters suggests the involvement of these neuromediators in the mechanisms underlying the antiarrhythmic effect of Allapinin.     

Species-Specificity of the BamA Component of the Bacterial Outer Membrane Protein-Assembly Machinery

The BamA protein is the key component of the Bam complex, the assembly machinery for outer membrane proteins (OMP) in gram-negative bacteria. We previously demonstrated that BamA recognizes its OMP substrates in a species-specific manner in vitro. In this work, we further studied species specificity in vivo by testing the functioning of BamA homologs of the proteobacteria Neisseria meningitidis, Neisseria gonorrhoeae, Bordetella pertussis, Burkholderia mallei, and Escherichia coli in E. coli and in N. meningitidis. We found that no BamA functioned in another species than the authentic one, except for N. gonorrhoeae BamA, which fully complemented a N. meningitidis bamA mutant. E. coli BamA was not assembled into the N. meningitidis outer membrane. In contrast, the N. meningitidis BamA protein was assembled into the outer membrane of E. coli to a significant extent and also associated with BamD, an essential accessory lipoprotein of the Bam complex.Various chimeras comprising swapped N-terminal periplasmic and C-terminal membrane-embedded domains of N. meningitidis and E. coli BamA proteins were also not functional in either host, although some of them were inserted in the OM suggesting that the two domains of BamA need to be compatible in order to function. Furthermore, conformational analysis of chimeric proteins provided evidence for a 16-stranded β-barrel conformation of the membrane-embedded domain of BamA.     

Differential expression of Tenomodulin and Chondromodulin-1 at the insertion site of the tendon reflects a phenotypic transition of the resident cells

The insertion site of the tendon to the skeletal element is hypovascular and is one of the most common sites of dysfunction in the musculoskeletal system. However, the resident cells have been poorly defined due to a lack of a specific marker for tenocytes. We previously reported that Tenomodulin (Tnmd) and Chondromodulin-1 (Chm1) are homologous angiogenesis inhibitors and predominantly expressed in the avascular region of tendons and cartilage, respectively. In this study, we analyzed the expression of Tnmd, Chm1, alpha 1 chain of the type I collagen (Col1a1) and alpha 1 chain of the type II collagen (Col2a1) at the insertion site of the Achilles, patellar, or rotator cuff tendons of 1-week-old rabbits by in situ hybridization analysis. Tnmd was co-expressed with Col1a1 in tenocytes of these tendons, while Chm1 and Col2a1 were detected in chondrocytes of the hyaline cartilage. Interestingly, the cell population between Tnmd/Col1a1 positive tenocytes and Chm1/Col2a1 positive chondrocytes expressed Col1a1 but none of the other markers (Tnmd, Chm1, and Col2a1). Red blood cells were exclusively present at the interface between the tendon substance and cartilage in the insertion site of the Achilles tendon. Lack of Tnmd and Chm1 in this newly characterized cell population may allow the transitional zone between the poorly vascularized tendon and cartilage to establish the unique vascular pattern for blood supply.     

Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons, parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, and parCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or both parB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss of parCBA activity. For P. aeruginosa PAO1161Rif^r, the parCBA operon provided little if any plasmid stability, but for P. aeruginosa PAC452Rif^r, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parA and res alone were sufficient for stabilization. The cer resolvase system of plasmid ColE1 and the loxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three E. coli strains. These observations indicate that the parA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of the parC and parB genes but that in at least one E. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or the cer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers.     

Studies of the transmissibility of the agent of bovine spongiform encephalopathy to the domestic chicken

Background

Escherichia coli O157:H7 is the most common serovar of enterohemorrhagic E. coli associated with serious human disease outbreaks. Cattle are the main reservoir with E. coli O157:H7 inducing hemorrhagic enteritis in persistent shedding beef cattle, however little is known about how this pathogen affects cattle health. Jejunal Hemorrhage Syndrome (JHS) has unclear etiology but the pathology is similar to that described for E. coli O157:H7 challenged beef cattle suggestive that E. coli O157:H7 could be involved. There are no effective treatments for JHS however new approaches to managing pathogen issues in livestock using prebiotics and probiotics are gaining support. The first objective of the current study was to characterize pathogen colonization in hemorrhaged jejunum of dairy cattle during natural JHS outbreaks. The second objective was to confirm the association of mycotoxigenic fungi in feeds with the development of JHS and also to identify the presence of potential mycotoxins. The third objective was to determine the impact of a prebiotic, Celmanax?, or probiotic, Dairyman's Choice? paste, on the cytotoxicity associated with feed extracts in vitro. The fourth objective was to determine the impact of a prebiotic or a probiotic on E. coli O157:H7 colonization of mucosal explants and a bovine colonic cell line in vitro. The final objective was to determine if prebiotic and probiotic feed additives could modify the symptoms that preceded JHS losses and the development of new JHS cases.

Findings

Dairy cattle developed JHS after consuming feed containing several types of mycotoxigenic fungi including Fusarium culmorum, F. poae, F. verticillioides, F. sporotrichioides, Aspergillus flavus, Penicillium roqueforti, P. crustosum, P. paneum and P. citrinum. Mixtures of Shiga toxin - producing Escherichia coli (STEC) colonized the mucosa in the hemorrhaged tissues of the cattle and no other pathogen was identified. The STECs expressed Stx1 and Stx2, but more significantly, Stxs were also present in the blood clot blocking the jejunum. Mycotoxin analysis of the corn crop confirmed the presence of fumonisin, NIV, ZEAR, DON, 15-ADON, 3-ADON, NEO, DAS, HT-2 and T-2. Feed extracts were toxic to enterocytes and 0.1% Celmanax? removed the cytotoxicity in vitro. There was no effect of Dairyman's Choice? paste on feed-extract activity in vitro. Fumonisin, T-2, ZEAR and DON were toxic to bovine cells and 0.1% Celmanax? removed the cytotoxicity in vitro. Celmanax? also directly decreased E. coli O157:H7 colonization of mucosal explants and a colonic cell line in a dose-dependent manner. There was no effect of Dairyman's Choice? paste on E. coli O157:H7 colonization in vitro. The inclusion of the prebiotic and probiotic in the feed was associated with a decline in disease.

Conclusion

The current study confirmed an association between mycotoxigenic fungi in the feed and the development of JHS in cattle. This association was further expanded to include mycotoxins in the feed and mixtures of STECs colonizing the severely hemorrhaged tissues. Future studies should examine the extent of involvement of the different STEC in the infection process. The prebiotic, Celmanax?, acted as an anti-adhesive for STEC colonization and a mycotoxin binder in vitro. Future studies should determine the extent of involvement of the prebiotic in altering disease.     

Influence of the acoustic environment on the distribution of the frog Ranidella riparia

At its southern edge the distribution of the frog Ranidella riparia abuts, with only a narrow zone of overlap, that of its allopatric sibling species R. signifera. In previous reports there was no evidence for any reduced survival of R. riparia in the creeks occupied by R. signifera immediately adjacent to the edge of its distribution. Here we examine the hypothesis that the acoustic environment in those creeks might inhibit successful communication by R. riparia. Although the structural characteristics of the R. riparia call were less suitable than those of R. signifera for transmission through the heavily vegetated habitat characteristic of those creeks, this alone did not inhibit successful reproduction by R. riparia. At a distance of 25 cm the average intensity of a call from an R. riparia male was 24 dB lower than one from R. signifera. In addition R. signifera form a continuous chorus in which the minimum sound intensity always exceeds that of single R. riparia calls at 25 cm. We propose that R. riparia cannot colonize creeks adjacent to their present distribution because the loud noise from R. signifera choruses either suppresses their calling activity, or makes them inaudible to potentially receptive females.     

The evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneine

Ergothioneine (EGT) is a histidine betaine derivative that exhibits antioxidant action in humans. EGT is primarily synthesized by fungal species and a number of bacterial species. A five-gene cluster (egtA, egtB, egtC, egtD & egtE) responsible for EGT production in Mycobacteria smegmatis has recently been identified. The first fungal biosynthetic EGT gene (NcEgt-1) has also been identified in Neurospora crassa. NcEgt-1 contains domains similar to those found in M. smegmatis egtB and egtD. EGT is biomembrane impermeable. Here we inferred the evolutionary history of the EGT cluster in prokaryotes as well as examining the phyletic distribution of Egt-1 in the fungal kingdom. A genomic survey of 2509 prokaryotes showed that the five-gene EGT cluster is only found in the Actinobacteria. Our survey identified more than 400 diverse prokaryotes that contain genetically linked orthologs of egtB and egtD. Phylogenetic analyses of Egt proteins show a complex evolutionary history and multiple incidences of horizontal gene transfer. Our analysis also identified two independent incidences of a fusion event of egtB and egtD in bacterial species. A genomic survey of over 100 fungal genomes shows that Egt-1 is found in all fungal phyla, except species that belong to the Saccharomycotina subphylum. This analysis provides a comprehensive analysis of the distribution of the key genes involved in the synthesis of EGT in prokaryotes and fungi. Our phylogenetic inferences illuminate the complex evolutionary history of the genes involved in EGT synthesis in prokaryotes. The potential to synthesize EGT is a fungal trait except for species belonging to the Saccharomycotina subphylum.     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica.     

Effect of atoms evaporated from the anode on the structure of the vacuum arc space charge sheath

The structure of the anode space charge sheath of a vacuum arc is studied with allowance for the dependence of the negative anode fall on the ratio of the directed electron velocity v ₀ to the electron thermal velocity v _T for different values of the flux density of atoms evaporated from the anode. Poisson’s equation for the sheath potential is solved taking into account the electron space charge, fast cathode ions, and slow ions produced due to the ionization of atoms evaporated from the anode. The kinetic equation for atoms and slow anode ions is solved with allowance for ionization in the collision integral. Analytic solutions for the velocity distribution functions of atoms and slow ions and the density of slow ions are obtained. It is shown that the flux of slow ions substantially affects the spatial distribution of the electric field E(z) in the sheath. As the flux density increases, the nonmonotonic dependence E(z) transforms into a monotonic one and the sheath narrows. For a given flux of evaporated atoms Πa, the increase in the ratio of the directed electron velocity to the electron thermal velocity leads again to a nonmonotonic dependence E(z). As z increases, the electric field first increases, passes through the maximum, decreases, passes through the minimum E _min, and then again increases toward the anode. There is a limiting value of the ratio (v ₀/v _T )* at which E _min(z) vanishes. At v ₀/v _T > (v ₀/V _T )*, the condition for the existence of a steady-state sheath is violated and the profiles of the field and potential in the sheath become oscillating. The dependence of (v ₀/v _T )* on the flux density of evaporated atoms Π _a is obtained. It is shown that the domain of existence of steady-state solutions in the sheath broadens with increasing Π _a .     

Cloning and Characterization of the Gene Cluster Involved in the Production of the Circular Bacteriocin Carnocyclin A

Carnocyclin A is a circular bacteriocin of 60 amino acids produced by Carnobacterium maltaromaticum UAL307. A region of 12 kb that contained the structural gene for carnocyclin A, cclA, was sequenced using a fosmid library, and 10 genes were identified that could be responsible for carnocyclin A production and immunity. Five of those genes, cclBITCD, were found upstream of cclA: one encodes a protein containing a conserved ATP-binding domain and four encode proteins with putative membrane-spanning domains. CclC shows homology with a family of membrane proteins that contain the domain of unknown function 95 (DUF95). Downstream of cclA four additional genes, cclEFGH, were identified that show similarity to the last four genes, as-48EFGH, of the enterocin AS-48 bacteriocin gene cluster. CclFGH shows sequence homology with As-48FGH. Transformation of C. maltaromaticum UAL26 with cclBITCDA resulted in production of carnocyclin A, indicating that these genes form the minimal requirement for the secretion of fully matured bacteriocin. cclI encodes for a small hydrophobic protein with a high pI, which are characteristic features of known immunity proteins for other circular bacteriocins. Indeed, cloning of cclI behind a constitutive promoter in UAL26 resulted in immunity although the level of resistance was lower than that of UAL26 containing cclBITCDA, indicating that CclI alone is not enough to confer full immunity to carnocyclin A.     

pha-2 encodes the C. elegans ortholog of the homeodomain protein HEX and is required for the formation of the pharyngeal isthmus

The pha-2 mutant was isolated in 1993 by Leon Avery in a screen for worms with visible defects in pharyngeal feeding behavior. In pha-2 mutant worms, the pharyngeal isthmus is abnormally thick and short and, in contrast to wild-type worms, harbors several cell nuclei. We show here that pha-2 encodes a homeodomain protein and is homologous to the vertebrate homeobox gene, Hex (also known as Prh). Consistent with a function in pharyngeal development, the pha-2 gene is expressed in the pharyngeal primordium of Caenorhabditis elegans embryos, particularly in pm5 cells that form the bulk of the isthmus. We show that in the pha-2 mutant there is a failure of the pm5 cells to elongate anteriorly while keeping their nuclei within the nascent posterior bulb to form the isthmus during the 3-fold embryonic stage. We also present evidence that pha-2 regulates itself positively in pm5 cells, that it is a downstream target of the forkhead gene pha-4, and that it may also act in the isthmus as an inhibitor of the ceh-22 gene, an Nkx2.5 homolog. Finally, we have begun characterizing the regulation of the pha-2 gene and find that intronic sequences are essential for the complete pha-2 expression profile. The present report is the first to examine the expression and function of an invertebrate Hex homolog, that is, the C. elegans pha-2 gene.     

Resolution of the long-standing controversy over the type species of the genus Pseudotypotherium (Notoungulata,Typotheria, Mesotheriidae)

Mesotheres (Notoungulata: Typotheria) are among the most common mammals found in upper Miocene to Pliocene deposits of central Argentina, including the classic type Monte Hermoso locality, which defines the Montehermosan South American Land Mammal “Age”. Nevertheless, the correct name for the mesothere species from this site has been shrouded in uncertainty for well over a century due to questions of taxonomic priority, specimen provenance, and ontogenetic changes in dental formula. Since the mesotheres from Monte Hermoso were named, three distinct species have been formally considered as the type species of the genus: (1) Pseudotypotherium bravardi; (2) “Pseudotypotherium” maendrum; and (3) Pseudotypotherium exiguum. However, none of these species is a nominal species of the Pseudotypotherium genus; all three were originally referred to Typotherium. Article 67.2 of the International Code of Zoological Nomenclature (ICZN, 1999) indicates that only species considered as nominal species are eligible to set the type; in the case of Pseudotypotherium, these include: P. pulchrum, P. carlesi, P. hystatum, and P. carhuense. We conclude that Pseudotypotherium pulchrum F. Ameghino, 1904 (holotype MACN A 10299, Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Ameghino Collection), is the type species of the mesotheriid notoungulate genus from Monte Hermoso. According to Article 68.2, F. Ameghino fixed the type by original designation in 1904 when he described P. pulchrum and included “n. g., n. sp.”. Two of the other species previously considered species P. (= T.) bravardi and P. (= T.) exiguum are invalid as type species according to Article 70.2, since their designations overlooked the previous type fixation. The third species (M. (= T.) maendrum) represents a different mesothere genus (Mesotherium) that only occurs in younger (Pleistocene) deposits. Our analysis puts an end to a historical debate that has been ongoing for more than a century regarding the identity of this well-represented late Miocene–Pliocene mesotheriine genus (Pseudotypotherium). This study provides a solid taxonomic foundation for future studies on intraspecific and ontogenetic variation of Pseudotypotherium pulchrum.     

Engineering of the Yeast Yarrowia lipolytica for the Production of Glycoproteins Lacking the Outer-Chain Mannose Residues of N-Glycans

In an attempt to engineer a Yarrowia lipolytica strain to produce glycoproteins lacking the outer-chain mannose residues of N-linked oligosaccharides, we investigated the functions of the OCH1 gene encoding a putative α-1,6-mannosyltransferase in Y. lipolytica. The complementation of the Saccharomyces cerevisiae och1 mutation by the expression of YlOCH1 and the lack of in vitro α-1,6-mannosyltransferase activity in the Yloch1 null mutant indicated that YlOCH1 is a functional ortholog of S. cerevisiae OCH1. The oligosaccharides assembled on two secretory glycoproteins, the Trichoderma reesei endoglucanase I and the endogenous Y. lipolytica lipase, from the Yloch1 null mutant contained a single predominant species, the core oligosaccharide Man₈GlcNAc₂, whereas those from the wild-type strain consisted of oligosaccharides with heterogeneous sizes, Man₈GlcNAc₂ to Man₁₂GlcNAc₂. Digestion with α-1,2- and α-1,6-mannosidase of the oligosaccharides from the wild-type and Yloch1 mutant strains strongly supported the possibility that the Yloch1 mutant strain has a defect in adding the first α-1,6-linked mannose to the core oligosaccharide. Taken together, these results indicate that YlOCH1 plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in Y. lipolytica. Therefore, the Yloch1 mutant strain can be used as a host to produce glycoproteins lacking the outer-chain mannoses and further developed for the production of therapeutic glycoproteins containing human-compatible oligosaccharides.