Global Distribution of Bartonella Infections in Domestic Bovine and Characterization of Bartonella bovis Strains Using Multi-Locus Sequence Typing

Bartonella bovis is commonly detected in cattle. One B. bovis strain was recently isolated from a cow with endocarditis in the USA, suggesting its role as an animal pathogen. In the present study, we investigated bartonella infections in 893 cattle from five countries (Kenya, Thailand, Japan, Georgia, and Guatemala) and 103 water buffaloes from Thailand to compare the prevalence of the infection among different regions and different bovid hosts. We developed a multi-locus sequence typing (MLST) scheme based on nine loci (16S rRNA, gltA, ftsZ, groEL, nuoG, ribC, rpoB, ssrA, and ITS) to compare genetic divergence of B. bovis strains, including 26 representatives from the present study and two previously described reference strains (one from French cows and another from a cow with endocarditis in the USA). Bartonella bacteria were cultured in 6.8% (7/103) of water buffaloes from Thailand; all were B. bovis. The prevalence of bartonella infections in cattle varied tremendously across the investigated regions. In Japan, Kenya, and the Mestia district of Georgia, cattle were free from the infection; in Thailand, Guatemala, and the Dusheti and Marneuli districts of Georgia, cattle were infected with prevalences of 10–90%. The Bartonella isolates from cattle belonged to three species: B. bovis (n=165), B. chomelii (n=9), and B. schoenbuchensis (n=1), with the latter two species found in Georgia only. MLST analysis suggested genetic variations among the 28 analyzed B. bovis strains, which fall into 3 lineages (I, II, and III). Lineages I and II were found in cattle while lineage III was restricted to water buffaloes. The majority of strains (17/28), together with the strain causing endocarditis in a cow in the USA, belonged to lineage I. Further investigations are needed to determine whether B. bovis causes disease in bovids.     

Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria.     

Ankle ligament reconstruction after wide resection of the osteosarcoma of the distal fibula: a case report

Background

Restoration of the lateral ankle after distal fibulectomy is a difficult reconstructive procedure. Many surgical techniques have been proposed. This report shows another fibular reconstructive option with promising outcome.

Case presentation

We report the case of a 30-year-old woman who presented with a solitary mass located in the lateral aspect of the ankle. The mass had grown rapidly for 2 months and caused increasing pain. Physical examination showed a 3.0 cm diameter tender, nonmobile hard mass in the lateral malleolus. Radiographs showed an osteolytic lesion involving the lateral cortex at the distal fibula. After incisional biopsy, pathologic examination found a well-differentiated intramedullary osteosarcoma. Neoadjuvant chemotherapy with doxorubicin was provided for 3 months prior to definitive surgical treatment. Magnetic resonance imaging showed persistent tumor in the biopsy site. After distal fibulectomy and wide resection, split tibialis posterior tendon transfer to the remaining peroneus brevis restored the stability of the ankle. The pain resolved within 3 months. The ankle was stable and no recurrence of the cancer was found at a 7 year follow-up.

Conclusion

Reconstruction following distal fibulectomy and surrounding soft tissue resection responds favorably to split tibialis posterior transfer to the remaining peroneus brevis suggesting that this technique can provide a good and functional outcome.

     

Synthesis of solution-phase combinatorial library of 4,6-diamino-1,2-dihydro-1,3,5-triazine and identification of new leads against A16V+S108T mutant dihydrofolate reductase of Plasmodium falciparum

An efficient method to synthesize solution-phase combinatorial library of 1-aryl-4,6-diamino-1,2-dihydro-1,3,5-triazine was developed. The strategy involved an acid-catalyzed cyclocondensation between arylbiguanide hydrochlorides and carbonyl compounds in the presence of triethyl orthoacetate as water scavenger. A 96-membered combinatorial library was constructed from 6 aryl biguanides and 16 carbonyl compounds. Screening of the library by iterative deconvolution method revealed two candidate leads which are equally active against wild-type Plasmodium falciparum dihydrofolate reductase, but are about 100-fold more effective against the A16V+S108T mutant enzyme as compared to cycloguanil.     

Carbonic anhydrase inhibitors. Inhibition of Plasmodium falciparum carbonic anhydrase with aromatic sulfonamides: towards antimalarials with a novel mechanism of action?

The malarial parasite Plasmodium falciparum encodes for an alpha-carbonic anhydrase (CA) enzyme possessing catalytic properties distinct of that of the human host, which was only recently purified. A series of aromatic sulfonamides, most of which were Schiff's bases derived from sulfanilamide/homosulfanilamide/4-aminoethylbenzenesulfonamide and substituted-aromatic aldehydes, or ureido-substituted such sulfonamides, were investigated for in vitro inhibition of the malarial parasite enzyme (pfCA) and the growth of P. falciparum. Several inhibitors with affinity in the micromolar range (K(I)'s in the range of 0.080-1.230 microM) were detected, whereas the most potent such derivatives were the clinically used sulfonamide CA inhibitor acetazolamide, and 4-(3,4-dichlorophenyl-ureidoethyl)-benzenesulfonamide, which showed an inhibition constant of 80 nM against pfCA, being four times more effective an inhibitor as compared to acetazolamide (K(I) of 315 nM). The lipophilic 4-(3,4-dichlorophenylureido-ethyl)-benzenesulfonamide was also an effective in vitro inhibitor for the growth of P. falciparum (IC50 of 2 microM), whereas acetazolamide achieved the same level of inhibition at 20 microM. This is the first study proving that antimalarials possessing a novel mechanism of action can be obtained, by inhibiting a critical enzyme for the life cycle of the parasite. Indeed, by inhibiting pfCA, the synthesis of pyrimidines mediated by carbamoylphosphate synthase is impaired in P. falciparum but not in the human host. Sulfonamide CA inhibitors have the potential for the development of novel antimalarial drugs.     

Ex vivo and in vivo characterization of cold preserved cartilage for cell transplantation

Due to the inconvenient and invasive nature of chondrocyte transplantation, preserved cartilage has been recognized as an alternative source of chondrocytes for implantation. However, there are major concerns, in particular, the viability and quality of the chondrocytes. This study investigated the biochemistry and molecular characterization of chondrocytes isolated from preserved cartilage for purposes of transplantation. Ex vivo characterization was accomplished by storing human cartilage at either 4 or ?80 °C in a preservation medium. Microscopic evaluation of the preserved cartilage was conducted after 1, 2, 3 and 6 weeks. The chondrocytes were isolated from the preserved cartilage and investigated for proliferation capacity and chondrogenic phenotype. Transplantation of chondrocytes from preserved cartilage into rabbit knees was performed for purposes of in vivo evaluation. The serum cartilage degradation biomarker (WF6 epitopes) was evaluated during the transplantation procedure. Human cartilage preserved for 1 week in a 10 % DMSO chondrogenic medium at 4 °C gave the highest chondrocyte viability. The isolated chondrocytes showed a high proliferative capacity and retained chondrogenic gene expression. Microscopic assessment of the implanted rabbit knees showed tissue regeneration and integration with the host cartilage. A decreased level of the serum biomarker after transplantation was evidence of in vivo repair by the implanted chondrocytes. These results suggest that cartilage preservation for 1 week in a 10 % DMSO chondrogenic medium at 4 °C can maintain proliferation capacity and the chondrogenic phenotype of human chondrocytes. These results can potentially be applied to in vivo allogeneic chondrocyte transplantation. Allogeneic chondrocytes from preserved cartilage would be expected to maintain their chondrogenic phenotype and to result in a high rate of success in transplanted grafts.     

Complete Nucleotide Sequence of pSK41: Evolution of Staphylococcal Conjugative Multiresistance Plasmids

The 46.4-kb nucleotide sequence of pSK41, a prototypical multiresistance plasmid from Staphylococcus aureus, has been determined, representing the first completely sequenced conjugative plasmid from a gram-positive organism. Analysis of the sequence has enabled the identification of the probable replication, maintenance, and transfer functions of the plasmid and has provided insights into the evolution of a clinically significant group of plasmids. The basis of deletions commonly associated with pSK41 family plasmids has been investigated, as has the observed insertion site specificity of Tn552-like β-lactamase transposons within them. Several of the resistance determinants carried by pSK41-like plasmids were found to be located on up to four smaller cointegrated plasmids. pSK41 and related plasmids appear to represent a consolidation of antimicrobial resistance functions, collected by a preexisting conjugative plasmid via transposon insertion and IS257-mediated cointegrative capture of other plasmids.     

Effect of Intracluster Correlation on the R-Square Statistic

R²-statistic is a popular and very widely used statistic in regression analysis to estimate the square multiple correlation (SMC), ρ², between a response variable Y and p predictor variables, _X1, …, _Xp. Numerous articles are available in the statistical literature on the properties of R² as an estimator of ρ² when the observations are uncorrelated. However, relatively little is known about the behavior of R² when the available observations are correlated such as the data that result from complex sampling schemes. In this paper, we study the behavior R² in the presence of two-stage sampling data. An approximate expressions for the variance and the bias of R² in the presence of two-stage cluster sampling data with positive intracluster correlation (ρ*) are obtained. It is evident from these formulas and from a simulation study that R² is a poor estimator of ρ² except when ρ* is small. As such, we consider several alternative estimators of ρ² and evaluate their theoretical properties and finite sample performance using a simulation study.     

Raised serum chondroitin sulfate epitope level in ovarian epithelial cancer

OBJECTIVE: To determine the value of serum chondroitin sulfate epitope WF6 and hyaluronan (HA) levels as a biomarker for early detection of ovarian epithelial cancer and other gynecological disorders. METHOD: Serum WF6 CS epitope and HA were measured in 91 patients with ovarian epithelial cancer, 39 patients with non-cancer gynecological disorders and 30 healthy women. Serum chondroitin sulfate (CS) WF6 epitope was determined by a competitive immunoassay with the monoclonal antibodies WF6, which specifically recognizes an epitope in native CS chains. In addition, serum HA concentration was measured by an ELISA-based assay with a biotinylated affinity HA-binding proteins. RESULTS: The serum concentration of CS (WF6) epitope was highly increased in epithelial types of ovarian cancer and at all stages of development (p < 0.005). Serum HA in ovarian cancer patients was significantly higher than normal controls (p < 0.05). CONCLUSION: These results reflect changes in ECM metabolism in progressive ovarian cancer, which cause an increase in serum CS epitopes and HA. Therefore, serum CS epitopes may provide useful biomarkers for cancers and other disorders of the ovary. Measurement of serum HA provided complementary information, which may be useful as a discriminator between benign ovarian disorders and malignant ovarian diseases.