Long Term Follow-up of an Open Bicondylar Hoffa Fracture with a Disrupted Extensor Mechanism: A Case Report.

This is the first report of a long-term follow-up of an open bicondylar Hoffa with patella fracture. It is interesting to note the radiological changes of osteoarthritis 15 years after global intra-articular injury of the distal femur. The good clinical outcome is possibly due to the integrity of the knee ligaments and reconstruction of the extensor mechanism in addition to stable anatomical reduction and fixation.

Comparative analysis of intracellular metabolites, proteins and their molecular functions in a flor yeast strain under two enological conditions.

Flor yeasts confer a wide range of organoleptic properties to Sherry-type wines during a process called "biological aging" that takes place after alcoholic fermentation. These kinds of yeasts adapt to a biological aging condition by forming a biofilm known as "flor velum" and by changing from fermentative to oxidative metabolism. It has been reported that some functions such as increase of cell surface hydrophobicity or changes to lipid metabolism are enhanced when yeasts switch to biofilm lifestyle. Here, we attempt to reveal intracellular metabolites and protein molecular functions not documented before that are relevant in biofilm formation and in fermentation by an endometabolome and proteome screening. We report that at early stages of biofilm formation, flor yeasts accumulate mannose, trehalose, glycerol, oleic and stearic acids and synthesize high amounts of GTPases, glycosylases and lipoproteins. On the other hand, in early fermentation, flor yeasts rapidly consume glucose and phosphoric acid; and produce abundant proteins related to chromatin binding, transcription factors and methyl transferases.

Assessment of the Brettanomyces bruxellensis metabolome during sulphur dioxide exposure.

Brettanomyces bruxellensis displays a high degree of genotypic and phenotypic polymorphism and is the main yeast species involved in wine spoilage. The innate resistance of 108 B. bruxellensis strains to the antimicrobial agent SO2 used in winemaking was investigated. Nineteen strains (17.6%) were sensitive to SO2 , failing to grow at the lowest concentration tested (0.1 mg L(-1) molecular SO2). Twenty-nine strains (26.8%) grew at 0.1 mg L(-1), 42 strains (38.9%) grew at 0.2 mg L(-1) , and 16 strains (14.8%) were able to grow as high as 0.4 mg L(-1) mol. SO2. Two strains able to grow in the presence of 0.6 mg L(-1) mol. SO2 were further studied by GCMS-TOF analysis to define the metabolic response to SO2 treatment. Two hundred and fifty-three intracellular metabolites were detected. The main effect observed was a decrease in cytoplasmic levels of polyols and an increase in levels of some amino acids, alanine, glutamic acid, glycine, proline, 5-oxoproline, serine and valine, which were significantly accumulated in the presence of SO2. No alteration in the pentose phosphate pathway was observed, suggesting NADPH usage could be diverted to other pathways. Finally, a change in metabolites involved in the glycerophospholipid pathway (glycerol-3-phosphate and myo-inositol) was also found.

Next-generation sequencing reveals significant bacterial diversity of botrytized wine.

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.

Evaluation of bacteria isolated from rice for plant growth promotion and biological control of seedling disease of rice.

Of 102 rhizoplane and endophytic bacteria isolated from rice roots and stems in California, 37% significantly (P < or = 0.05) inhibited the growth in vitro of two pathogens, Achlya klebsiana and Pythium spinosum, causing seedling disease of rice. Four endophytic strains were highly effective against seedling disease in growth pouch assays, and these were identified as Pseudomonas fluorescens (S3), Pseudomonas tolaasii (S20), Pseudomonas veronii (S21), and Sphingomonas trueperi (S12) by sequencing of amplified 16S rRNA genes. Strains S12, S20, and S21 contained the nitrogen fixation gene, nifD, but only S12 was able to reduce acetylene in pure culture. The four strains significantly enhanced plant growth in the absence of pathogens, as evidenced by increases in plant height and dry weight of inoculated rice seedlings relative to noninoculated rice. Three bacterial strains (S3, S20, and S21) were evaluated in pot bioassays and reduced disease incidence by 50%-73%. Strain S3 was as effective at suppressing disease at the lowest inoculum density (106 CFU/mL) as at higher density (10(8) CFU/mL or undiluted suspension). This study indicates that selected endophytic bacterial strains have potential for control of seedling disease of rice and for plant growth promotion.

High resolution HLA class I and II typing and CTLp frequency in unrelated donor transplantation: a single-institution retrospective study of 69 BMTs.

UNLABELLED: The results of unrelated donor transplantation (URD-BMT) are difficult to analyze since the continuous advances in HLA typing technology allow the detection of new mismatches unknown at the time of transplantation. We sought to confirm that matched recipient-donor pairs are in fact often mismatched when advanced HLA typing techniques are used. We retrospectively studied the impact of the results of high resolution HLA typing for HLA class I (-A, -B, -C) and HLA class II (-DR, -DQ, -DP) loci, and cytotoxic T lymphocyte precursor (CTLp) frequency, on the outcome of 69 URD-BMT procedures. At the time of transplant, six (6/69) and two (2/69) donor-recipient pairs were mismatched for HLA class I (-A and -B by serology) and HLA class II, respectively, while one pair was mismatched for both HLA class I and II. Using high resolution DNA typing, HLA class I mismatches were found in 31 (45%) pairs and HLA class II mismatches in nine (13%) pairs. Twenty-three of the 69 pairs were HLA-C mismatched. Low CTLp frequencies were found among the 19 HLA class I matched pairs tested, and also in 5/14 mismatched pairs (of whom three had severe aGVHD). The overall survival of the cohort was 28 +/- 6%. Among the 33 patients who were fully matched with their donors, the survival rate was 66% in the 18 patients with a standard hematological risk and 9% in the 15 high risk patients. Only two of the 33 patients developed severe aGVHD, and only one had graft rejection. Among the 36 mismatched pairs, the survival rate was 31% in the 13 patients with a standard hematological risk and 8% in the 23 high risk patients. Sixteen of these 36 patients died from severe aGVHD and four had graft failure or rejection. Three of the 10 patients with only an HLA-C mismatch died from severe aGVHD, and two had graft rejection. IN CONCLUSION: (1) donor-recipient matching based on high resolution HLA class I and II DNA typing is associated with significantly better outcome after URD-BMT; (2) the results of URD-BMT with classical GVHD prevention are comparable to those of geno-identical BMT when donor and recipient are fully matched for HLA-A, -B, -C, -DRB1 and -DQB1 on the basis of high resolution typing; (3) CTLp frequencies do not correlate constantly with HLA class I matching, and our results fail to show that CTLp assay can distinguish between permissible and non-permissible class I mismatches; (4) clinical trials involving donor-recipient pairs with known HLA class I mismatches are needed to improve aGVHD prevention without increasing graft failure rate.

Identification of lumichrome as a sinorhizobium enhancer of alfalfa root respiration and shoot growth.

Sinorhizobium meliloti bacteria produce a signal molecule that enhances root respiration in alfalfa (Medicago sativa L.) and also triggers a compensatory increase in whole-plant net carbon assimilation. Nuclear magnetic resonance, mass spectrometry, and ultraviolet-visible absorption identify the enhancer as lumichrome, a common breakdown product of riboflavin. Treating alfalfa roots with 3 nM lumichrome increased root respiration 21% (P < 0.05) within 48 h. A closely linked increase in net carbon assimilation by the shoot compensated for the enhanced root respiration. For example, applying 5 nM lumichrome to young alfalfa roots increased plant growth by 8% (P < 0.05) after 12 days. Soaking alfalfa seeds in 5 nM lumichrome before germination increased growth by 18% (P < 0.01) over the same period. In both cases, significant growth enhancement (P < 0.05) was evident only in the shoot. S. meliloti requires exogenous CO2 for growth and may benefit directly from the enhanced root respiration that is triggered by lumichrome. Thus Sinorhizobium-alfalfa associations, which ultimately form symbiotic N2-reducing root nodules, may be favored at an early developmental stage by lumichrome, a previously unrecognized mutualistic signal. The rapid degradation of riboflavin to lumichrome under many physiological conditions and the prevalence of riboflavin release by rhizosphere bacteria suggest that events demonstrated here in the S. meliloti-alfalfa association may be widely important across many plant-microbe interactions.

Occurrence of flavonoids and nucleosides in agricultural soils.

AN ECOLOGICALLY RELEVANT SOIL EXTRACTION PROCEDURE SEPARATED TWO TYPES OF MOLECULES IMPORTANT FOR BACTERIA: flavonoids and small hydrophilic organic compounds. Two flavonoids, identified previously as inducers of nodulation genes in Rhizobium meliloti, were detected in rhizosphere soil from alfalfa (Medicago sativa L.). In addition, biologically significant quantities (micromoles per kilogram) of ribonucleosides and deoxyribonucleosides were found in all soils tested. Long-term wheat (Triticum aestivum L.) plots that had received manure contained elevated amounts of nucleosides, and in a separate experiment, the presence of legumes in a wheat-cropping sequence increased soil nucleosides. Intact bacterial cells accounted for less than 1% of the free nucleosides detected. These results suggest new testable hypotheses for molecular ecologists and differ from those obtained with older, harsher techniques.

Biotin and other water-soluble vitamins are key growth factors for alfalfa root colonization by Rhizobium meliloti 1021.

Rhizosphere growth limitations imposed on Rhizobium meliloti by availability of biotin, thiamine, and riboflavin were overcome by adding nanomolar amounts of these vitamins. Studies done with R. meliloti 1021 showed that both synthesis and uptake of biotin promote colonization of alfalfa roots. Two lines of evidence indicated that plant-derived biotin normally promotes root colonization: (i) adding avidin significantly (P < or = 0.01) reduced rhizosphere growth of R meliloti 1021, and (ii) growth of Tn5-induced biotin auxotrophs still increased 10-fold in the rhizosphere. Synthesis, however, is the more important source of biotin for R. meliloti 1021 because in root colonization tests biotin auxotrophs competed very poorly with the parent strain. Mutations conferring biotin auxotrophy were closely linked on a single restriction fragment, and one was complemented with the Escherichia coli bio operon. Initial nucleotide sequencing and DNA-DNA hybridization tests showed the biotin synthesis genes in R. meliloti are quite different from those in E. coli.

Mechanism of hyperosmolality stimulation of ANP secretion: its dependency on calcium and sodium.

The calcium dependency of hyperosmolality stimulation of atrial natriuretic peptide (ANP) secretion was determined using isolated superfused nonbeating rat left atrium. Increasing osmolality by 65, 85, and 100 mosmol/kgH2O by superfusion with sucrose produced a peak rise in ANP secretion of 1.8-, 2.0-, and 2.7-fold. To determine whether calcium influx played a role in osmolality (osm)-stimulated ANP secretion, atria were superfused with 2 mM lanthanum, a calcium antagonist. Lanthanum inhibited by 85% the response to a 100 mosmol/kgH2O increase in osm. The voltage-dependent calcium channel blocker isradipine had no effect on osm-stimulated ANP secretion, suggesting that calcium influx via voltage-dependent calcium channels was not playing a significant role. Likewise, depleting sarcoplasmic reticulum calcium with 1 microM ryanodine did not block the response to osm, suggesting that calcium influx was not adequate to induce consequential release of calcium from the sarcoplasmic reticulum. To determine whether calcium influx was via Na(+)-Ca2+ exchange, we determined the sodium dependency of osm-stimulated ANP secretion. Replacement of sodium with lithium or choline blocked the secretory response to 100 mosmol/kgH2O. We conclude that osm-stimulated ANP secretion is calcium and sodium dependent. Calcium influx via Na(+)-Ca2+ exchange is highly implicated as the mechanism of cellular calcium entry.

Alfalfa (Medicago sativa L.) Root Exudates Contain Isoflavonoids in the Presence of Rhizobium meliloti.

Root exudates of alfalfa (Medicago sativa L.) inoculated with symbiotic Rhizobium meliloti bacteria contained three isoflavonoids that were not found in exudates of uninoculated plants. Data from proton nuclear magnetic resonance, mass spectrometry, and ultraviolet-visible absorbance analyses indicated that root exudates of inoculated plants contained aglycone and glycoside forms of the phytoalexin medicarpin and a formononetin-7-O-(6"-O-malonylglycoside), a conjugated form of the medicarpin precursor formononetin. The medicarpin molecules did not induce nod gene transcription in R. meliloti, but the formononetin-7-O-(6"-O-malonylglycoside) induced nod genes regulated by both NodD1 and NodD2 proteins in R. meliloti. Hydrolysis of either the malonyl or the glycosyl linkage from the formononetin conjugate eliminated nod gene-inducing activity. The nod gene-inducing activity of crude root exudates was increased 200 and 65% upon inoculation with R. meliloti or R. leguminosarum bv phaseoli, respectively. When root exudate from uninoculated alfalfa was incubated with R. meliloti, high performance liquid chromatography analyses showed no evidence that bacterial metabolism produced medicarpin. These results indicate that alfalfa responds to symbiotic R. meliloti by exuding a phytoalexin normally elicited by pathogens and that the microsymbiont can use a precursor of the phytoalexin as a signal for inducing symbiotic nod genes.

Isolation of Rhizobium meliloti nod Gene Inducers from Alfalfa Rhizosphere Soil.

Methanolic extracts of alfalfa rhizosphere soil induce more nod gene transcription in Rhizobium meliloti than extracts of nonrhizosphere soil. Six peaks of nod-inducing activity were separated by high-performance liquid chromatography from rhizosphere soil extract, and one compound was identified by H nuclear magnetic resonance, mass spectrometry, and UV-visible spectra as a formononetin-7-O-glycoside that activates both NodD1 and NodD2 proteins. The unanticipated presence of a glycoside in rhizosphere soil suggests either that large amounts of the glycoside were exuded by roots or that some glycosides are unexpectedly stable in soil.

Trigonelline and Stachydrine Released from Alfalfa Seeds Activate NodD2 Protein in Rhizobium meliloti.

Spectroscopic data (nuclear magnetic resonance, mass spectrometry, ultraviolet-visible) in this study identify trigonelline and stachydrine as major components of alfalfa (Medicago sativa L.) seed rinse. Moreover, biological assays show that these natural products induce nodulation (nod) gene transcription in Rhizobium meliloti by activating the regulatory protein NodD2, but not the homologous NodD1 protein. These findings contrast with the fact that the only previously identified NodD2 activator, 4,4' -dihydroxy-2' -methoxychalcone (MCh), also activates NodD1 protein. Trigonelline and stachydrine induce nod genes only at much higher concentrations than MCh, but they are released from seeds in correspondingly greater amounts. The existence of these amphoteric, nonflavonoid nod gene inducers broadens our understanding of the biochemical processes and ecological mechanisms that a legume host uses to regulate its microbial symbiont.

Anthocyanidins and Flavonols, Major nod Gene Inducers from Seeds of a Black-Seeded Common Bean (Phaseolus vulgaris L.).

Eleven compounds released from germinating seeds of a black-seeded bean (Phaseolus vulgaris L., cv PI165426CS) induce transcription of nod genes in Rhizobium leguminosarum biovar phaseoli. Aglycones from 10 of those compounds were identified by spectroscopic methods (ultraviolet/visible, proton nuclear magnetic resonance, and mass spectroscopy), and their biological activities were demonstrated by induction of beta-galactosidase activity in R. leguminosarum strains containing nodA-lacZ or nodC-lacZ fusions controlled by R. leguminosarum biovar phaseoli nodD genes. By making comparisons with authentic standards, the chemical structures for aglycones from the 10 molecules were confirmed as being anthocyanidins (delphinidin, petunidin, and malvidin) and flavonols (myricetin, quercetin, and kaempferol). All anthocyanidins and flavonols had 3-O-glycosylation and free hydroxyl groups at the 4', 5, and 7 positions. Hydrolysis experiments showed that the mean concentration required for half-maximum nod gene induction (I(50)) by the 10 glycosides was about half that of the corresponding aglycones. The mean I(50) value for the three anthocyanidins (360 nanomolar) was less (P

Rhizobium nod Gene Inducers Exuded Naturally from Roots of Common Bean (Phaseolus vulgaris L.).

Four compounds exuded from young roots of a black-seeded bean (Phaseolus vulgaris L., cv PI165426CS) induce transcription of nod genes in Rhizobium leguminosarum biovar phaseoli. The three most active nod gene inducers were identified by spectroscopic methods (ultraviolet/visible absorbance, proton nuclear magnetic resonance, and mass spectrometry) as being eriodictyol (5,7,3',4' -tetrahydroxyflavanone), naringenin (5,7,4' -trihydroxyflavanone), and a 7-O-glycoside of genistein (5,7,4' -trihydroxyisoflavone). Comparisons with authentic standards verified the chemical structures of the aglycones and their capacity to induce beta-galactosidase activity in R. leguminosarum strains containing nodA-lacZ or nodC-lacZ fusions controlled by R. leguminosarum biovar phaseoli nodD genes. Roots of 9-day-old seedlings released 42, 281, and 337 nanomoles per plant per day of genistein, eriodictyol, and naringenin, respectively. Genistein and naringenin induced higher maximum beta-galactosidase activities and required lower concentrations for half-maximum induction than eriodictyol. Comparing the nod gene-inducing activity of seed rinses with root exudate from PI165426CS bean showed that root flavonoids were released at about 6% the rate of those from seeds on a molar basis, but on average the individual compounds from roots were approximately three times more active than nod gene inducers from seeds.

Spirometric indices of early airflow obstruction.

Thirty cigarette smokers and 25 non-smoking controls, all men were evaluated by history, physical examination and simple spirometry. The history and physical examination were not of much use in predicting airflow obstruction. Forced mid-expiratory flow (FEF 25-75%) was abnormally low in 23 of the 30 subjects, while forced expiratory volume in 1 second (FEV1) and FEV1/FVC (forced vital capacity) were less sensitive. Thus simple spirometry is a useful screening tool to detect early airflow obstruction even when it is clinically undetectable.

Flavonoids Released Naturally from Alfalfa Seeds Enhance Growth Rate of Rhizobium meliloti.

Alfalfa (Medicago sativa L.) releases different flavonoids from seeds and roots. Imbibing seeds discharge 3',4',5,7-substituted flavonoids; roots exude 5-deoxy molecules. Many, but not all, of these flavonoids induce nodulation (nod) genes in Rhizobium meliloti. The dominant flavonoid released from alfalfa seeds is identified here as quercetin-3-O-galactoside, a molecule that does not induce nod genes. Low concentrations (1-10 micromolar) of this compound, as well as luteolin-7-O-glucoside, another major flavonoid released from germinating seeds, and the aglycones, quercetin and luteolin, increase growth rate of R. meliloti in a defined minimal medium. Tests show that the 5,7-dihydroxyl substitution pattern on those molecules was primarily responsible for the growth effect, thus explaining how 5-deoxy flavonoids in root exudates fail to enhance growth of R. meliloti. Luteolin increases growth by a mechanism separate from its capacity to induce rhizobial nod genes, because it still enhanced growth rate of R. meliloti lacking functional copies of the three known nodD genes. Quercetin and luteolin also increased growth rate of Pseudomonas putida. They had no effect on growth rate of Bacillus subtilis or Agrobacterium tumefaciens, but they slowed growth of two fungal pathogens of alfalfa. These results suggest that alfalfa can create ecochemical zones for controlling soil microbes by releasing structurally different flavonoids from seeds and roots.

Effects of alfalfa nod gene-inducing flavonoids on nodABC transcription in Rhizobium meliloti strains containing different nodD genes.

Transcription of the nodulation genes nodABC in Rhizobium meliloti requires a plant flavonoid signal and nodD, a family of bacterial regulatory genes (nodD1, nodD2, and nodD3). Results from this study show that all previously identified nod gene inducers released by alfalfa seeds and roots induced nodABC-lacZ transcription in R. meliloti containing extra copies of nodD1, but only 4,4'-dihydroxy-2'-methoxychalcone gave high levels of induction with extra copies of nodD2. While mixtures of the methoxychalcone and luteolin showed a positive synergism with extra NodD1 protein, they apparently competed for binding to the NodD2 protein.

Development and Partial Characterization of Nearly Isogenic Pea Lines (Pisum sativum L.) that Alter Uptake Hydrogenase Activity in Symbiotic Rhizobium.

Some Rhizobium bacteria have H(2)-uptake (Hup) systems that oxidize H(2) evolved from nitrogenase in leguminous root nodules. Pea (Pisum sativum L.) cultivars ;JI1205' and ;Alaska' produce high Hup (Hup(++)) and moderate Hup (Hup(+)) phenotypes, respectively, in Rhizobium leguminosarum 128C53. The physiological significance and biochemical basis of this host plant genetic effect are unknown. The purpose of this investigation was to advance basic Hup studies by developing nearly isogenic lines of peas that alter Hup phenotypes in R. leguminosarum strains containing hup genes. Eight pairs of nearly isogenic pea lines that produce Hup(++) and Hup(+) phenotypes in R. leguminosarum 128C53 were identified in 173 F(2)-derived F(6) families produced from crosses between JI1205 and Alaska. Tests with the pea isolines and three strains of hup-containing R. leguminosarum showed that the isolines altered Hup activity significantly (P

Chrysoeriol and Luteolin Released from Alfalfa Seeds Induce nod Genes in Rhizobium meliloti.

Flavonoid signals from alfalfa (Medicago sativa L.) seed and root exudates induce transcription of nodulation (nod) genes in Rhizobium meliloti. The flavone luteolin previously was isolated from alfalfa seeds by other workers and identified as the first nod gene inducer for R. meliloti. Our recent study of ;Moapa 69' alfalfa root exudates found no luteolin but did identify three other nod gene inducers: 4,4'-dihydroxy-2'-methoxychalcone, 4',7-dihydroxyflavone, and 4',7-dihydroxyflavanone. The goal of the current study was to identify and quantify nod gene-inducing flavonoids that may influence Rhizobium populations around a germinating alfalfa seed. Aqueous rinses of Moapa 69 alfalfa seeds were collected and assayed for induction of a nodABC-lacZ fusion in R. meliloti. During the first 4 hours of imbibition, total nod gene-inducing activity was released from seeds at 100-fold higher rates than from roots of 72-hour-old seedlings. Five flavonoids were purified and identified by spectroscopic analyses (ultraviolet/visible absorbance, proton nuclear magnetic resonance, and mass spectroscopy) and comparison with authentic standards. Two very active nod gene-inducing flavonoids, chrysoeriol (3'-methoxyluteolin) and luteolin, were identified in seed rinses. Luteolin required a higher concentration (18 nanomolar) than chrysoeriol (5 nanomolar) for half-maximum induction of nodABC-lacZ in R. meliloti, and both were less active than 4,4'-dihydroxy-2'-methoxychalcone (2 nanomolar) from root exudates. Seeds exuded three other luteolin derivatives: luteolin-7-O-glucoside, 5-methoxyluteolin, and 3',5-dimethoxyluteolin. Their combined quantities were 24-fold greater than that of luteolin plus chrysoeriol. Most nod gene-inducing activity of these luteolin derivatives apparently is associated with degradation to luteolin and chrysoeriol. However, their presence in large quantities suggests that they may contribute significantly to nod gene-inducing activity in the soil. These results indicate the importance of germinating seeds as a source of nod gene-inducing flavonoids and emphasize the quantitative and qualitative differences in those compounds around the seed and root.