Dairy veterinarians' skills in motivational interviewing are linked to client verbal behavior.

Veterinarians often give advice in a persuasive form, a style that has been shown to evoke resistance to change in clients experiencing psychological ambivalence (i.e. those who see both advantages and disadvantages to changing). With this style of communication, veterinarians run the risk of counteracting their purpose to encourage clients to follow recommendations. Motivational interviewing (MI) is a client-centered communication methodology that aims to facilitate clients' internal motivation to change. In MI, Change Talk represents clients' own statements expressing consideration of, motivation for or commitment to behavior change and has been shown to be strongly correlated with behavior change. Sustain Talk is corresponding statements related to maintaining the status quo. The aim of this exploratory study was to evaluate the potential of MI to facilitate behavior change in veterinary herd health management (VHHM) by investigating the effect of dairy cattle veterinarians' MI skills on client Change and Sustain Talk. We recorded VHHM consultancies on 170 Swedish cattle farms performed by 36 veterinarians, randomly distributed into 2 groups: MI veterinarians (n = 18) had received 6-month training in MI and control veterinarians (n = 18) had not received any training. Veterinarians' MI skills were assessed using the Motivational Interviewing Treatment Integrity coding system 4.2.1 and categorized as poor_untrained, poor_trained, near moderate and moderate. Client communication was coded using the Client Language Easy Rating coding system. The effect of MI skills on Change Talk, Sustain Talk and Proportion of Change Talk(Change Talk divided by the sum of Sustain Talk plus Change Talk) was investigated using cross-classified regression models with random intercepts for veterinarian and client (farm). The models also included additional explanatory variables (e.g. type of veterinarian and client's satisfaction with the consultation). The veterinarian's MI skills were associated with the client's Change Talk, but results regarding Sustain Talk or Proportion of Change Talk were inconclusive. Clients of veterinarians reaching the highest (i.e. moderate) MI skills expressed 1.5 times more Change Talk than clients of untrained veterinarians. Clients of general large animal practitioners expressed less Sustain Talk than clients of animal health veterinarians and had higher Proportion of Change Talk. Results indicate that learning to practice MI may be one means to improve adherence to veterinary recommendations and to improve efficiency in VHHM services.

Comparison of sporadic and familial behavioral variant frontotemporal dementia (FTD) in a North American cohort.

INTRODUCTION: Behavioral variant frontotemporal dementia (bvFTD) may present sporadically or due to an autosomal dominant mutation. Characterization of both forms will improve understanding of the generalizability of assessments and treatments. METHODS: A total of 135 sporadic (s-bvFTD; mean age 63.3 years; 34% female) and 99 familial (f-bvFTD; mean age 59.9; 48% female) bvFTD participants were identified. f-bvFTD cases included 43 with known or presumed chromosome 9 open reading frame 72 (C9orf72) gene expansions, 28 with known or presumed microtubule-associated protein tau (MAPT) mutations, 14 with known progranulin (GRN) mutations, and 14 with a strong family history of FTD but no identified mutation. RESULTS: Participants with f-bvFTD were younger and had earlier age at onset. s-bvFTD had higher total Neuropsychiatric Inventory Questionnaire (NPI-Q) scores due to more frequent endorsement of depression and irritability. DISCUSSION: f-bvFTD and s-bvFTD cases are clinically similar, suggesting the generalizability of novel biomarkers, therapies, and clinical tools developed in either form to the other.

Active lifestyles moderate clinical outcomes in autosomal dominant frontotemporal degeneration.

INTRODUCTION: Leisure activities impact brain aging and may be prevention targets. We characterized how physical and cognitive activities relate to brain health for the first time in autosomal dominant frontotemporal lobar degeneration (FTLD). METHODS: A total of 105 mutation carriers (C9orf72/MAPT/GRN) and 69 non-carriers reported current physical and cognitive activities at baseline, and completed longitudinal neurobehavioral assessments and brain magnetic resonance imaging (MRI) scans. RESULTS: Greater physical and cognitive activities were each associated with an estimated >55% slower clinical decline per year among dominant gene carriers. There was also an interaction between leisure activities and frontotemporal atrophy on cognition in mutation carriers. High-activity carriers with frontotemporal atrophy (-1 standard deviation/year) demonstrated >two-fold better cognitive performances per year compared to their less active peers with comparable atrophy rates. DISCUSSION: Active lifestyles were associated with less functional decline and moderated brain-to-behavior relationships longitudinally. More active carriers "outperformed" brain volume, commensurate with a cognitive reserve hypothesis. Lifestyle may confer clinical resilience, even in autosomal dominant FTLD.

Communication styles of Swedish veterinarians involved in dairy herd health management: A motivational interviewing perspective.

Veterinary communication has evolved in the context of traditional veterinary tasks, such as diagnosing and treating sick animals, and this may encourage a directive communication style, in which the veterinarian acts as an expert, directing the client in what to do and how to do it. This type of communication style has been shown to evoke resistance to change in clients who are experiencing psychological ambivalence, a well-known aspect of farmers' herd health decisions. Veterinary herd health management (VHHM) constitutes an increasing proportion of the work of cattle veterinarians and often focuses on behavior change. The present study aimed to characterize communication styles of Swedish dairy cattle veterinarians involved in VHHM in relation to their skills in facilitating behavior change. A secondary aim was to investigate whether these skills differed among veterinarians depending on their experience in the veterinary profession or in VHHM. Audio-recorded role-play conversations (n = 123; reflecting VHHM telephone consultations) with 42 veterinarians involved in VHHM in Swedish dairy herds and audio-recorded on-farm consultations (n = 86) with 18 of those veterinarians were coded using a system developed to evaluate motivational interviewing (MI) skills. Motivational interviewing is a communication methodology aimed at facilitating clients' internal motivation to change. The MI Treatment Integrity (MITI) code identifies frequency counts of 10 verbal behaviors, and assesses 4 global variables on a Likert scale, based on 20 min of conversation. It also suggests 6 summary measurements of MI competency based on these 14 original variables. Of the 42 veterinarians, 39 also responded to a web questionnaire about their age, continuing education, and experience in the profession, in dairy herds and in VHHM. We analyzed associations between the 6 summary MITI variables from the role-play conversations and characteristics of the 39 veterinarians using logistic and linear multivariable regression models. Veterinarians in the role-play and on-farm conversations relied predominantly on giving information, questions, and persuasion in their consultation approaches. They generally did not explore the client's expectations or wishes regarding the agenda for the consultation, or acknowledge the client's right to make decisions about actions. Veterinarians gave advice without exploring the client's need for the advice or how the information was perceived. We found a significant reduction in so-called relational scores (Empathy plus Partnership) and an increase in MI-nonadherent behaviors (Persuasion plus Confront) as years of veterinary experience increased. Results showed that there is room for improvement in the communication style of veterinarians involved in VHHM. Wider literature suggests that training veterinarians in a client-centered communication methodology such as MI may increase the demand for and success of VHHM.

Maternal mood disorders and lithium exposure in utero were not associated with poor cognitive development during childhood.

AIM: This study evaluated whether maternal mood disorders (MMD), particularly bipolar disorder, and lithium treatment during pregnancy influenced the neonatal health and cognition of children born from 2006 to 2010. METHODS: Our study at Karolinska University Hospital, Stockholm, Sweden, focused on women with and without mood disorders and their children. Information on pharmacotherapy, mental health, delivery and neonatal complications was retrospectively collected from electronic patient records. Children were tested in a blinded manner at four to five years of age with the Wechsler Preschool and Primary Scale of Intelligence, 3rd edition. Maternal health, child health and social situations were evaluated. RESULTS: Of the 39 children, 20 were exposed to lithium and MMD during pregnancy, eight were exposed to MMD but not lithium and 11 were not exposed to MMD or lithium. The children's full scale intelligence quotient (IQ), performance IQ and verbal IQ results did not differ significantly between the groups. The processing speed quotient was significantly lower in children exposed to mood disorders, but there was a high level of missing data for this subtest. CONCLUSION: This small, clinical cohort showed no significant association between mothers' prenatal exposure to lithium or mood disorders and their offspring's IQ.

Kingella kingae Expresses Four Structurally Distinct Polysaccharide Capsules That Differ in Their Correlation with Invasive Disease.

Kingella kingae is an encapsulated gram-negative organism that is a common cause of osteoarticular infections in young children. In earlier work, we identified a glycosyltransferase gene called csaA that is necessary for synthesis of the [3)-ß-GalpNAc-(1→5)-ß-Kdop-(2→] polysaccharide capsule (type a) in K. kingae strain 269-492. In the current study, we analyzed a large collection of invasive and carrier isolates from Israel and found that csaA was present in only 47% of the isolates. Further examination of this collection using primers based on the sequence that flanks csaA revealed three additional gene clusters (designated the csb, csc, and csd loci), all encoding predicted glycosyltransferases. The csb locus contains the csbA, csbB, and csbC genes and is associated with a capsule that is a polymer of [6)-α-GlcpNAc-(1→5)-ß-(8-OAc)Kdop-(2→] (type b). The csc locus contains the cscA, cscB, and cscC genes and is associated with a capsule that is a polymer of [3)-ß-Ribf-(1→2)-ß-Ribf-(1→2)-ß-Ribf-(1→4)-ß-Kdop-(2→] (type c). The csd locus contains the csdA, csdB, and csdC genes and is associated with a capsule that is a polymer of [P-(O→3)[ß-Galp-(1→4)]-ß-GlcpNAc-(1→3)-α-GlcpNAc-1-] (type d). Introduction of the csa, csb, csc, and csd loci into strain KK01Δcsa, a strain 269-492 derivative that lacks the native csaA gene, was sufficient to produce the type a capsule, type b capsule, type c capsule, and type d capsule, respectively, indicating that these loci are solely responsible for determining capsule type in K. kingae. Further analysis demonstrated that 96% of the invasive isolates express either the type a or type b capsule and that a disproportionate percentage of carrier isolates express the type c or type d capsule. These results establish that there are at least four structurally distinct K. kingae capsule types and suggest that capsule type plays an important role in promoting K. kingae invasive disease.

Comparative analysis of first-year fingolimod and natalizumab drug discontinuation among Swedish patients with multiple sclerosis.

BACKGROUND: Natalizumab (NTZ) and fingolimod (FGL) are mainly used second line in relapsing-remitting multiple sclerosis (MS), although pivotal trials included mainly treatment-naïve patients. OBJECTIVE: This study aims to provide real-world data on safety and discontinuation rates. METHODS: Using IMSE, a drug monitoring registry for all newer MS drugs in Sweden, we analysed differences in baseline characteristics and 1-year drug survival for patients registered 2011-2013, initiating treatment with NTZ (n=640) or FGL (n=876). Among FGL initiators, n=383 (44%) had previously used NTZ (FGL(afterNTZ)). RESULTS: Compared with NTZ, the FGL cohort was older and more often male (36/38 years, 24%/33% males). Baseline Expanded Disability Status Scale was similar across groups, but MS Severity Score was higher in NTZ patients, and Symbol Digit Modalities Test and MS Impact Scale (MSIS-29) was higher in FGL(afterNTZ) versus FGL(NTZ-naïve) patients. Proportion on drug after 1 year was high, NTZ=87%, FGL(NTZ-naïve)=83% and FGL(afterNTZ)=76%. Adverse events was the most frequent reason for discontinuing FGL (FGL(NTZ-naïve)=9%, FGL(afterNTZ)=12%), and was significantly higher than on NTZ (3%). In contrast, the proportion of patients stopping treatment due to lack of effect was more similar: NTZ=4%, FGL(NTZ-naïve)=3%, FGL(afterNTZ)=8%. CONCLUSION: FGL and NTZ were both well tolerated, but FGL less so than NTZ, especially in patients switching to FGL from NTZ. Group differences were not explained by differences in recorded baseline characteristics.

Profound parental bias associated with chromosome 14 acquired uniparental disomy indicates targeting of an imprinted locus.

Acquired uniparental disomy (aUPD) is a common finding in myeloid malignancies and typically acts to convert a somatically acquired heterozygous mutation to homozygosity. We sought to identify the target of chromosome 14 aUPD (aUPD14), a recurrent abnormality in myeloid neoplasms and population cohorts of elderly individuals. We identified 29 cases with aUPD14q that defined a minimal affected region (MAR) of 11.2 Mb running from 14q32.12 to the telomere. Exome sequencing (n=7) did not identify recurrently mutated genes, but methylation-specific PCR at the imprinted MEG3-DLK1 locus located within the MAR demonstrated loss of maternal chromosome 14 and gain of paternal chromosome 14 (P<0.0001), with the degree of methylation imbalance correlating with the level of aUPD (r=0.76; P=0.0001). The absence of driver gene mutations in the exomes of three individuals with aUPD14q but no known haematological disorder suggests that aUPD14q may be sufficient to drive clonal haemopoiesis. Analysis of cases with both aUPD14q and JAK2 V617F (n=11) indicated that aUPD14q may be an early event in some cases but a late event in others. We conclude that aUPD14q is a recurrent abnormality that targets an imprinted locus and may promote clonal haemopoiesis either as an initiating event or as a secondary change.

The secondary cell wall polysaccharide of Bacillus anthracis provides the specific binding ligand for the C-terminal cell wall-binding domain of two phage endolysins, PlyL and PlyG.

Endolysins are bacteriophage enzymes that lyse their bacterial host for phage progeny release. They commonly contain an N-terminal catalytic domain that hydrolyzes bacterial peptidoglycan (PG) and a C-terminal cell wall-binding domain (CBD) that confers enzyme localization to the PG substrate. Two endolysins, phage lysin L (PlyL) and phage lysin G (PlyG), are specific for Bacillus anthracis. To date, the cell wall ligands for their C-terminal CBD have not been identified. We recently described structures for a number of secondary cell wall polysaccharides (SCWPs) from B. anthracis and B. cereus strains. They are covalently bound to the PG and are comprised of a -ManNAc-GlcNAc-HexNAc- backbone with various galactosyl or glucosyl substitutions. Surface plasmon resonance (SPR) showed that the endolysins PlyL and PlyG bind to the SCWP from B. anthracis (SCWPBa) with high affinity (i.e. in the µM range with dissociation constants ranging from 0.81 × 10(-6) to 7.51 × 10(-6) M). In addition, the PlyL and PlyG SCWPBa binding sites reside with their C-terminal domains. The dissociation constants for the interactions of these endolysins and their derived C-terminal domains with the SCWPBa were in the range reported for other protein-carbohydrate interactions. Our findings show that the SCWPBa is the ligand that confers PlyL and PlyG lysin binding and localization to the PG. PlyL and PlyG also bound the SCWP from B. cereus G9241 with comparable affinities to SCWPBa. No detectable binding was found to the SCWPs from B. cereus ATCC (American Type Culture Collection) 10987 and ATCC 14579, thus demonstrating specificity of lysin binding to SCWPs.

Infrequent attention to dietary and physical activity behaviours in conversations in Swedish child health services.

AIM: To investigate conversations between nurses and parents in Swedish child health services and to assess to what extent attention is directed towards dietary and physical activity behaviours in children. METHODS: Twenty-three nurses audio-recorded one session each. Recordings were assessed and topics were classified according to predetermined categories. RESULTS: The three most frequent topics of conversation concerned physical examinations of the child (30% of session time), talking to the child to establish or maintain contact and interest (15%), and development of language skills (12%). Dietary habits came on fourth place (10%), and physical activity ranked 14 (4%). CONCLUSION: Attention to dietary and physical activity behaviours in children is infrequent in Swedish child health services. Concern is raised about the efficacy of prevention efforts against childhood obesity.

Localization and structural analysis of a conserved pyruvylated epitope in Bacillus anthracis secondary cell wall polysaccharides and characterization of the galactose-deficient wall polysaccharide from avirulent B. anthracis CDC 684.

Bacillus anthracis CDC 684 is a naturally occurring, avirulent variant and close relative of the highly pathogenic B. anthracis Vollum. Bacillus anthracis CDC 684 contains both virulence plasmids, pXO1 and pXO2, yet is non-pathogenic in animal models, prompting closer scrutiny of the molecular basis of attenuation. We structurally characterized the secondary cell wall polysaccharide (SCWP) of B. anthracis CDC 684 (Ba684) using chemical and NMR spectroscopy analysis. The SCWP consists of a HexNAc trisaccharide backbone having identical structure as that of B. anthracis Pasteur, Sterne and Ames, →4)-ß-d-ManpNAc-(1 â†’ 4)-ß-d-GlcpNAc-(1 â†’ 6)-α-d-GlcpNAc-(1→. Remarkably, although the backbone is fully polymerized, the SCWP is the devoid of all galactosyl side residues, a feature which normally comprises 50% of the glycosyl residues on the highly galactosylated SCWPs from pathogenic strains. This observation highlights the role of defective wall assembly in virulence and indicates that polymerization occurs independently of galactose side residue attachment. Of particular interest, the polymerized Ba684 backbone retains the substoichiometric pyruvate acetal, O-acetate and amino group modifications found on SCWPs from normal B. anthracis strains, and immunofluorescence analysis confirms that SCWP expression coincides with the ability to bind the surface layer homology (SLH) domain containing S-layer protein extractable antigen-1. Pyruvate was previously demonstrated as part of a conserved epitope, mediating SLH-domain protein attachment to the underlying peptidoglycan layer. We find that a single repeating unit, located at the distal (non-reducing) end of the Ba684 SCWP, is structurally modified and that this modification is present in identical manner in the SCWPs of normal B. anthracis strains. These polysaccharides terminate in the sequence: (S)-4,6-O-(1-carboxyethylidene)-ß-d-ManpNAc-(1 â†’ 4)-[3-O-acetyl]-ß-d-GlcpNAc-(1 â†’ 6)-α-d-GlcpNH(2)-(1→.

Characterization of galacturonosyl transferase genes rgtA, rgtB, rgtC, rgtD, and rgtE responsible for lipopolysaccharide synthesis in nitrogen-fixing endosymbiont Rhizobium leguminosarum: lipopolysaccharide core and lipid galacturonosyl residues confer membrane stability.

Rhizobium lipopolysaccharide (LPS) contains four terminally linked galacturonic acid (GalA) residues; one attached to the lipid A and three attached to the core oligosaccharide moiety. Attachment of the GalA residues requires the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA), which is synthesized by the GalA transferase RgtE reported here. The galacturonosyl transferases RgtA, -B, and -C utilize Dod-P-GalA to attach GalAs on the LPS core region, and RgtD attaches GalA to the lipid A 4' position. As reported here, the functions of the rgtD and rgtE genes were determined via insertion mutagenesis and structural characterization of the mutant lipid A. The rgtE(-) mutant lacked Dod-P-GalA as determined by mass spectrometry of total lipid extracts and the inability of rgtE(-) mutant membranes to provide the substrate for heterologously expressed RgtA activity. In addition, we created single mutations in each of the rgtA, -B, -C, -D, and -E genes to study the biological function of the GalA residues. The structures of the core oligosaccharide region from each of the rgt mutants were elucidated by glycosyl linkage analysis. Each mutant was assayed for its sensitivity to sodium deoxycholate and to the antimicrobial cationic peptide, polymyxin B (PmxB). The rgt mutants were more sensitive than the parent strain to deoxycholate by varying degrees. However, the rgtA, -B, and -C mutants were more resistant to PmxB, whereas the rgtD and E mutants were less resistant in comparison to the parent strain.

Secondary cell wall polysaccharides from Bacillus cereus strains G9241, 03BB87 and 03BB102 causing fatal pneumonia share similar glycosyl structures with the polysaccharides from Bacillus anthracis.

Secondary cell wall polysaccharides (SCWPs) are important structural components of the Bacillus cell wall and contribute to the array of antigens presented by these organisms in both spore and vegetative forms. We previously found that antisera raised to Bacillus anthracis spore preparations cross-reacted with SCWPs isolated from several strains of pathogenic B. cereus, but did not react with other phylogenetically related but nonpathogenic Bacilli, suggesting that the SCWP from B. anthracis and pathogenic B. cereus strains share specific structural features. In this study, SCWPs from three strains of B. cereus causing severe or fatal pneumonia (G9241, 03BB87 and 03BB102) were isolated and subjected to structural analysis and their structures were compared to SCWPs from B. anthracis. Complete structural analysis was performed for the B. cereus G9241 SCWP using NMR spectroscopy, mass spectrometry and derivatization methods. The analyses show that SCWPs from B. cereus G9241 has a glycosyl backbone identical to that of B. anthracis SCWP, consisting of multiple trisaccharide repeats of: →6)-α-d-GlcpNAc-(1 â†’ 4)-ß-d-ManpNAc-(1 â†’ 4)-ß-d-GlcpNAc-(1→. Both the B. anthracis and pathogenic B. cereus SCWPs are highly substituted at all GlcNAc residues with α- and ß-Gal residues, however, only the SCWPs from B. cereus G9241 and 03BB87 carry an additional α-Gal substitution at O-3 of ManNAc residues, a feature lacking in the B. anthracis SCWPs. Both the B. anthracis and B. cereus SCWPs are pyruvylated, with an approximate molecular mass of ≈12,000 Da. The implications of these findings regarding pathogenicity and cell wall structure are discussed.

Lipopolysaccharides in Rhizobium-legume symbioses.

The establishment of nitrogen-fixing symbiosis between a legume plant and its rhizobial symbiont requires that the bacterium adapt to changing conditions that occur with the host plant that both promotes and allows infection of the host root nodule cell, regulates and resists the host defense response, permits the exchange of metabolites, and contributes to the overall health of the host. This adaptive process involves changes to the bacterial cell surface and, therefore, structural modifications to the lipopolysaccharide (LPS). In this chapter, we describe the structures of the LPSs from symbiont members of the Rhizobiales, the genetics and mechanism of their biosynthesis, the modifications that occur during symbiosis, and their possible functions.

Structural characterization of the primary O-antigenic polysaccharide of the Rhizobium leguminosarum 3841 lipopolysaccharide and identification of a new 3-acetimidoylamino-3-deoxyhexuronic acid glycosyl component: a unique O-methylated glycan of uniform size, containing 6-deoxy-3-O-methyl-D-talose, n-acetylquinovosamine, and rhizoaminuronic acid (3-acetimidoylamino-3-deoxy-D-gluco-hexuronic acid).

Rhizobium are Gram-negative bacteria that survive intracellularly, within host membrane-derived plant cell compartments called symbiosomes. Within the symbiosomes the bacteria differentiate to bacteroids, the active form that carries out nitrogen fixation. The progression from free-living bacteria to bacteroid is characterized by physiological and morphological changes at the bacterial surface, a phase shift with an altered array of cell surface glycoconjugates. Lipopolysaccharides undergo structural changes upon differentiation from the free living to the bacteroid (intracellular) form. The array of carbohydrate structures carried on lipopolysaccharides confer resistance to plant defense mechanisms and may serve as signals that trigger the plant to allow the infection to proceed. We have determined the structure of the major O-polysaccharide (OPS) isolated from free living Rhizobium leguminosarum 3841, a symbiont of Pisum sativum, using chemical methods, mass spectrometry, and NMR spectroscopy analysis. The OPS is composed of several unusual glycosyl residues, including 6-deoxy-3-O-methyl-d-talose and 2-acetamido-2deoxy-l-quinovosamine. In addition, a new glycosyl residue, 3-acetimidoylamino-3-deoxy-d-gluco-hexuronic acid was identified and characterized, a novel hexosaminuronic acid that does not have an amino group at the 2-position. The OPS is composed of three to four tetrasaccharide repeating units of -->4)-beta-dGlcp3NAmA-(1-->4)-[2-O-Ac-3-O-Me-alpha-d-6dTalp-(1-->3)]-alpha-l-Fucp-(1-->3)-alpha-l-QuipNAc-(1-->. The unique 3-amino hexuronate residue, rhizoaminuronic acid, is an attractive candidate for selective inhibition of OPS synthesis.

Influence of genetic dissimilarity in the reproductive success and mate choice of brown trout - females fishing for optimal MHC dissimilarity.

We examined the reproductive success of 48 adult brown trout (Salmo trutta L.) which were allowed to reproduce in a stream that was controlled for the absence of other trout. Parentage analyses based on 11 microsatellites permitted us to infer reproductive success and mate choice preferences in situ. We found that pairs with intermediate major histocompatibility complex (MHC) dissimilarity mated more often than expected by chance. It appears that female choice was the driving force behind this observation because, compared with other individuals, males with intermediate MHC dissimilarity produced a larger proportion of offspring, whereas female reproductive output did not show this pattern. Hence, rather than seeking mates with maximal MHC dissimilarity, as found in several species, brown trout seemed to prefer mates of intermediate MHC difference, thus supporting an optimality-based model for MHC-dependent mate choice.

Parafibromin immunoreactivity: its use as an additional diagnostic marker for parathyroid tumor classification.

Parafibromin is a protein product derived from the hyperparathyroidism 2(HRPT2) tumor suppressor geneand its inactivation has been coupled to familial and sporadic forms of parathyroid malignancy. In this study, we have conducted immunohistochemistry on 33 parathyroid carcinomas (22 unequivocal and 11 equivocal) using four parafibromin antibodies directed to different parts of the protein. Furthermore, for a fraction of cases, the immunohistochemical results were compared with known HRPT2 mutational status. Our findings show that 68% (15 out of 22) of the unequivocal carcinomas exhibited reduced expression of parafibromin while the 25 sporadic adenomas used as controls were entirely positive for parafibromin expression. Additionally, three out of the six carcinomas with known HRPT2 mutations showed reduced expression of parafibromin. Using all four antibodies, comparable results were obtained on the cellular level in individual tumors suggesting that there exists no epitope of choice in parafibromin immunohistochemistry. The results agree with the demonstration of a approximately 60 kDa product preferentially in the nuclear fraction by western blot analysis. We conclude that parafibromin immunohistochemistry could be used as an additional marker for parathyroid tumor classification, where positive samples have low risk of malignancy, whereas samples with reduced expression could be either carcinomas or rare cases of adenomas likely carrying an HRPT2 mutation.

Structural characterization of a K-antigen capsular polysaccharide essential for normal symbiotic infection in Rhizobium sp. NGR234: deletion of the rkpMNO locus prevents synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-non-2-ulosonic acid.

Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.

Structural characterization of a flavonoid-inducible Pseudomonas aeruginosa A-band-like O antigen of Rhizobium sp. strain NGR234, required for the formation of nitrogen-fixing nodules.

Rhizobium (Sinorhizobium) sp. strain NGR234 contains three replicons, the smallest of which (pNGR234a) carries most symbiotic genes, including those required for nodulation and lipo-chito-oligosaccharide (Nod factor) biosynthesis. Activation of nod gene expression depends on plant-derived flavonoids, NodD transcriptional activators, and nod box promoter elements. Nod boxes NB6 and NB7 delimit six different types of genes, one of which (fixF) is essential for the formation of effective nodules on Vigna unguiculata. In vegetative culture, wild-type NGR234 produces a distinct, flavonoid-inducible lipopolysaccharide (LPS) that is not produced by the mutant (NGRomegafixF); this LPS is also found in nitrogen-fixing bacteroids isolated from V. unguiculata infected with NGR234. Electron microscopy showed that peribacteroid membrane formation is perturbed in nodule cells infected by the fixF mutant. LPSs were purified from free-living NGR234 cultured in the presence of apigenin. Structural analyses showed that the polysaccharide portions of these LPSs are specialized, rhamnose-containing O antigens attached to a modified core-lipid A carrier. The primary sequence of the O antigen is [-3)-alpha-L-Rhap-(1,3)-alpha-L-Rhap-(1,2)-alpha-L-Rhap-(1-]n, and the LPS core region lacks the acidic sugars commonly associated with the antigenic outer core of LPS from noninduced cells. This rhamnan O antigen, which is absent from noninduced cells, has the same primary sequence as the A-band O antigen of Pseudomonas aeruginosa, except that it is composed of L-rhamnose rather than the D-rhamnose characteristic of the latter. It is noteworthy that A-band LPS is selectively maintained on the P. aeruginosa cell surface during chronic cystic fibrosis lung infection, where it is associated with an increased duration of infection.