A hybrid approach to persistent sciatic artery fusiform aneurysm repair.

A persistent sciatic artery is a rare vascular anomaly that is prone to early atherosclerotic development and aneurysmal degeneration. Repair of the degenerative aneurysm is critical because it can lead to rupture, thrombosis, distal embolization, and sciatic nerve damage from compression. We report a case of a symptomatic unilateral persistent sciatic artery fusiform aneurysm that was treated using a simultaneous open surgical and endovascular approach. The patient underwent right common femoral to below-knee popliteal artery bypass and percutaneous endovascular embolization of the right sciatic artery aneurysm. Proper surgical intervention determined by the patient's comorbidities and unique anatomy achieved favorable outcomes.

The gut microbiome and inflammation in obsessive-compulsive disorder patients compared to age- and sex-matched controls: a pilot study.

OBJECTIVE: To compare the gut microbiome profile (by way of taxon analysis and indices of ß- and α-diversity) and inflammatory markers (C-reactive protein [CRP], interleukin-6[IL-6] and tumour necrosis factor-α [TNF-α]) of obsessive-compulsive disorder (OCD) outpatients and non-psychiatric community controls. METHODS: We collected morning stool and blood samples from 21 non-depressed, medication-free OCD patients and 22 age- and sex-matched non-psychiatric community controls. Microbiota analysis was performed using Illumina sequencing of the V3 region of 16S rRNA; serum CRP samples were analysed using immunoturbidimetry and plasma IL-6/TNF-α were examined by high-sensitivity ELISA. Multiple comparisons were corrected for using the false discovery rate (α = 0.05). RESULTS: Compared to controls, the OCD group presented lower species richness/evenness (α-diversity, Inverse Simpson) and lower relative abundance of three butyrate producing genera (Oscillospira, Odoribacter and Anaerostipes). Compared to controls, mean CRP, but not IL-6 and TNF-α, was elevated OCD patients. CRP revealed moderate to strong associations with psychiatric symptomatology. CONCLUSION: To our knowledge, this is the first investigation of the gut microbiome in OCD. In addition, our findings lend further support for the potential association of inflammation and OCD. These results suggest the gut microbiome may be a potential pathway of interest for future OCD research.

Specificity of gut microbiota in children with autism spectrum disorder in Slovakia and its correlation with astrocytes activity marker and specific behavioural patterns.

Recent research suggests the involvement of bidirectional gut-brain axis in autism spectrum disorder (ASD). The aim of this study was to establish the role of changed gut microbiota in behavioural and gastrointestinal manifestations, but also in astrocyte activation in children with ASD. Distinct faecal microbiota in children with ASD was found to be more heterogeneous compared to that in neurotypical children. Different bacterial abundance and correlation with behavioural and GI manifestations revealed several bacterial genera possibly important for ASD. Microbial-neuronal cross talk could be accomplished through S100B, released by glial cells, activated by low grade inflammation. More complex studies are required to understand ASD pathogenesis.

Complex phenotypes blur conventional borders between Say-Barber-Biesecker-Young-Simpson syndrome and genitopatellar syndrome.

Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) and genitopatellar syndrome (GTPTS) are clinically similar disorders with some overlapping features. Although they are currently considered to be distinct clinical entities, both were found to be caused by de novo truncating sequence variants in the KAT6B (lysine acetyltransferase 6B) gene, strongly suggesting that they are allelic disorders. Herein, we report the clinical and genetic findings in a girl presenting with a serious multiple congenital anomaly syndrome with phenotypic features overlapping both SBBYSS and GTPTS; pointing out that the clinical distinction between these disorders is not exact and there do exist patients, in whom conventional clinical classification is problematic. Genetic analyses revealed a truncating c.4592delA (p.Asn1531Thrfs*18) variant in the last KAT6B exon. Our findings support that phenotypes associated with typical KAT6B disease-causing variants should be referred to as 'KAT6B spectrum disorders' or 'KAT6B related disorders', rather than their current SBBYSS and GTPTS classification.

Proteomic analysis of the TerC interactome: Novel links to tellurite resistance and pathogenicity.

The tellurite resistance gene operon (ter) is widely spread among bacterial species, particularly pathogenic species. The ter operon has been implicated in tellurite resistance, phage inhibition, colicine resistance, and pathogenicity. The TerC protein represents one of the key proteins in tellurite resistance and shows no significant homology to any protein of known function. So far, there is no experimental evidence for TerC interaction partners. In this study, proteomic-based methods, including blue native electrophoresis and co-immunoprecipitation combined with LC-MS/MS, have been used to identify TerC interaction partners and thus providing indirect evidence for tentative functions of TerC in Escherichia coli. An interactome has been constructed and robust physical interaction of integral membrane protein TerC with TerB, DctA, PspA, HslU, and RplK has been shown. The TerC-TerB complex appears to act as a central unit that may link different functional modules with biochemical activities of C4-dicarboxylate transport, inner membrane stress response (phage shock protein regulatory complex), ATPase/chaperone activity, and proteosynthesis. In previous reports, it was hypothesized that a transmembrane unit formed by TerC protein may interact with the TerD family, but herein neither TerD nor TerE proteins were identified as TerC complex components. We propose that TerD/TerE participates in tellurite resistance through TerC-independent action.

Core promoters of the penicillin biosynthesis genes and quantitative RT-PCR analysis of these genes in high and low production strain of Penicillium chrysogenum.

The transcription start points of the penicillin biosynthesis genes from Penicillium chrysogenum were mapped using the primer extension method. For each of the three genes consensus sequences of the core promoter elements were identified, supporting the notion that the basal transcription of these genes is mediated separately. Interestingly, transcription start of the pcbC gene is located within the potential Inr element with no TATA box-like sequence being found at expected position. This is in contrast to pcbAB and penDE genes with proposed TATA boxes or even to Aspergillus nidulans ipnA (pcbC) gene indicating possible differences in basal transcription regulation. Using the quantitative RT-PCR analysis the expression of all three biosynthesis genes was monitored in both the high and low production strain of P. chrysogenum during a 3-d cultivation under production conditions. The differences were found between the strains in time regulation and transcript levels of the biosynthesis genes. Furthermore, we showed that the effect of higher gene dosage on productivity in the production strain is amplified by more efficient transcription of the biosynthesis genes with the RNA levels approximately 37- and 12-times higher, respectively, than in a low production strain.

Association of biochemical parameters and RAGE gene polymorphisms in healthy infants and their mothers.

BACKGROUND: The receptor for advanced glycation end-products (RAGEs) and its gene polymorphisms are implicated in the pathogenesis of different chronic diseases including diabetes and its complications. Infant formulas contain high amounts of advanced glycation end-products (AGEs) - the ligands of RAGE. METHODS: In this cross-sectional study, we examined the impact of G82S and -374 A/T polymorphisms in the gene encoding RAGE on standard blood chemistry, soluble (s)RAGE and inflammatory markers in 244 healthy infants (3-16months of age) and in 119 healthy mothers. Children were subdivided according to age (younger and older than 8months) and for the -374 A/T polymorphism according to the feeding regimen (breast-fed vs. infant formula-fed). RESULTS: Minor allele of the RAGE gene polymorphism G82S was associated with reduced plasma sRAGE in all age groups and with increased sICAM-1 in older children and mothers. Minor allele carrying mothers had decreased insulin sensitivity and HDL. The A allele of the RAGE gene promoter polymorphism -374 A/T was associated with higher indices of insulin resistance in young infant formula-fed, but not breast-fed children. In older, formerly infant formula-fed children signs of insulin resistance diminished, while formerly breast-fed children with A allele were more insulin sensitive. CONCLUSIONS: The phenotype of minor allele carriers in G82S is associated with reduced levels of protective sRAGE in healthy infants. With increasing age sICAM-1 levels increased and insulin resistance developed. In early childhood the phenotype of the -374 A/T polymorphism was diet-dependently associated with changes in glucose metabolism, which diminished with increasing age.

Multiple regulatory genes in the salinomycin biosynthetic gene cluster of Streptomyces albus CCM 4719.

A DNA fragment containing part of the salinomycin biosynthetic gene cluster from industrial strain Streptomyces albus CCM 4719 was cloned. Sequence analysis of the 25.809-kbp fragment revealed the presence of 8 open reading frames (ORFs), including two large ORFs encoding three modular sets of oligoketide synthase, followed by three genes (salRI, salRII, salRIII) encoding transcriptional regulators. The first two regulators, SalRI and SalRII, belonged to the novel LAL family of large transcriptional regulators. SalRIII was highly similar to the NysRIV, AmphRIV, and FscRI transcriptional regulators from the oligoene macrolides nystatin, amphotericin, and R008/candicidin clusters, respectively.

Antibiotic resistance and virulence factors among clinical and food enterococci isolated in Slovakia.

The resistance to antibiotics and the distribution of virulence factors in enterococci isolated from traditional Slovak sheep cheese bryndza was compared with strains from human infections. The occurrence of 4 enterococcal species was observed in 117 bryndza-cheese isolates. The majority of strains were identified as E. faecium (76 %) and E. faecalis (23 %). Several strains of E. durans and 1 strain of E. hirae were also present. More than 90 % of strains isolated from 109 clinical enterococci were E. faecalis, the rest belonged to E. faecium. The resistance to 6 antimicrobial substances (ampicillin, ciprofloxacin, higher concentration of gentamicin, nitrofurantoin, tetracycline and vancomycin) was tested in clinical and food enterococci. A higher level of resistance was found in clinical than in food strains and E. faecium had a higher resistance than E. faecalis; no resistance to vancomycin was detected. The occurrence of 3 virulence-associated genes, cylA (coding for hemolysin), gelE (coding for gelatinase) and esp (coding for surface protein) was monitored. Differences were found in the distribution of cylA gene between clinical and bryndza-cheese E. faecalis strains; in contrast to clinical strains (45 %), cylA gene was detected in 22 % of food isolates. The distribution of 2 other virulence factors, gelE and esp, was not significantly different in the two groups of E. faecalis strains. cylA and gelE genes were not detected in E. faecium but more than 70 % of clinical E. faecium were positive for esp, even thought none of the 79 E. faecium cheese isolates contained this gene.

Typing of food-borne Listeria monocytogenes by the optimized repetitive extragenic palindrome-based polymerase chain reaction.

The repetitive extragenic palindrome-based polymerase chain reaction was optimized for typing Listeria monocytogenes by 1) using the QlAamp method to increase the reproducibility of DNA isolation, 2) running PCR with three different DNA concentrations in parallel, 3) using antibody-protected therrnostable DNA polymerase to reduce non-specific priming, and 4) using an improved temperature programme to increase the amplification yield. When applied to 42 L. monocytogenes strains isolated from food in the Czech and Slovak republics during 1999-2000, profiles of 7-15 DNA fragments of 330-3,310 bp were amplified. Based on REP-profiles, strains (serotypes 1/2 and 4) could be divided into 12 groups.

Cloning of pac gene and its flanking regions using mini-Mu bacteriophage.

An in vitro method for cloning and mapping Escherichia coli genes by means of mini-Mu phage and its application to the penicillin G acylase (pac) gene of E. coli PAC2 strain with its flanking regions is described. The gene was marked by insertion of a fragment bearing kanamycin resistance (Km(r)). Most of Km(r) clones obtained from mini-Mu transductants contained the whole pac gene with its flanking regions. Localization of pac gene to 98.5 min of E. coli PAC2 chromosome was confirmed by an in vivo P1 phage transduction.

PCR-based methods for identification of Enterococcus species.

Two DNA-based techniques were used for species identification of enterococci. PvuII digestion of the genus-specific PCR product yielded four different restriction profiles among 20 enterococcal species; one of them was species-specific for E. faecium. In the second case, 32 reference strains belonging to 20 enterococcal species were divided to 12 groups by amplification of internal transcribed spacer of rRNA operon. Interspecies and some intraspecies profile variability was determined. Both methods gave similar results.

Phylogenetic analysis of the rplA genes encoding ribosomal protein L1.

Previously we have identified the rplA gene encoding ribosomal protein L1 in Streptomyces aureofaciens. Sequence comparison of ribosomal protein L1 among several bacterial genera revealed a high level of conservation. Based on this conservation, these proteins were used as a phylogenetic tool to compare evolutionary relationships among eubacteria and archaebacteria. This phylogenetic analysis of L1 ribosomal proteins including the S. aureofaciens rplA gene product revealed, except similar bacterial groupings, some new evolutionary relationships.

DNA sequence analysis of the tellurite-resistance determinant from clinical strain of Escherichia coli and identification of essential genes.

The tellurite-resistant Escherichia coli strain KL53 was found during testing of the group of clinical isolates for antibiotics and heavy metal ion resistance (Burian et al. 1990). Determinant of the tellurite resistance of the strain was located on the large conjugative plasmid pTE53 and cloned into pACYC184. Three different Ter clones harboring pLK2, pLK18 and pLK20 were isolated (Burian et al. 1998). The smallest functional Ter clone harboring pLK18 was chosen for further analysis. Plasmid pLK18 have been subcloned to obtain convenient DNA fragments for sequencing of tellurite-resistance determinant. Sequencing of this DNA fragments provided complete DNA sequence of the determinant, 5,250 bp in size. The sequence has been compared with nucleotide and protein databank (BLAST programs) and significant homology with the three known operons coding for tellurite resistance has been found (determinat on plasmid pR478 from Serratia marcescens, on plasmid pMER610 from Alcaligenes sp. and chromosomal tellurite resistance genes from Proteus mirabilis). We identified 5 ORFs coding for 5 genes named terB to terF. The clone harboring pLK18 was subjected to the transposition with Tn1737Km to disrupt determinant of the tellurite resistance. Plasmid DNA of several clones containing pLK18 with Tn1737Km was isolated to locate the target site of Tn1737Km. Analyses showed, the genes terB, terC, terD and terE are essential for conservation of the resistance whereas the gene terF is not important in this respect.

Nucleotide sequence of the gene encoding the major capsid protein of herpesvirus of turkeys.

The gene encoding the major capsid protein (MCP) VP5 of herpesvirus of turkeys (HVT) was identified and sequenced. It has a single open reading frame of 4236 nucleotides encoding 1412 aa protein. The gene is flanked by VP23 and UL20 sequences and is localized in the unique long region (UL) within the BamHI-B fragment. Comparison of amino acid homology has shown its clear position among the alpha-herpesviruses rather than beta- or gamma-herpesviruses. The VP5 is expressed from polycistronic mRNA together with the UL20 and the VP23 genes. The 7,2 kb RNA transcript is lacking any promoter elements or polyA signal in intergenomic regions between VP5 and UL20 or VP5 and VP23 genes, respectively. Multiple alignment of known major capsid protein sequences of all herpesvirus groups revealed presence of seven highly homologous clusters suggesting-that the corresponding protein domains might play an important role in folding of MCP and assembly of herpesvirus capsid.

Escherichia coli strain with a deletion of the chromosomal ampC gene marked with TcR, suitable for production of penicillin G acylase.

Escherichia coli strain which contains a marker of tetracycline resistance gene (TcR) placed by P1 transduction beside the chromosomal deletion of ampC gene (delta ampC) coding for beta-lactamase was constructed. Such introduction of TcR marker permits a fast and simple selection for the transfer of delta ampC by P1 transduction into industrial E. coli strains. This approach was used for constructing an E. coli strain suitable for penicillin acylase production.

In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli.

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.

Overexpression of the FNR protein of Escherichia coli with T7 expression system.

We have used the T7 expression system for expression of E. coli FNR protein. The fnr gene was cloned from its initiation codon ATG into the NdeI site of an expression vector and filamentous phage mGP1-2 was used as a donor of T7 RNA polymerase gene. The level of FNR expression attained by this expression arrangement was about 45% of total cell proteins.

Construction of Lactose-Utilizing Xanthomonas campestris with a Mini-Mu Derivative.

Xanthomonas campestris is not able to grow in lactose media. The lactose operon from Escherichia coli as part of a mini-Mu phage was integrated at random sites in the chromosome of this bacterium. Clones expressing (beta)-galactosidase were selected. The resulting strain X. campestris 204, is suitable for production of xanthan gum directly from lactose.