High incidence of Pneumocystis jirovecii pneumonia in oncological patients: a 19-year study.

AIM: In the past, Pneumocystis jirovecii belonged to the Protozoa group, but is currently taxonomically included in the kingdom Fungi. P. jirovecii is an opportunistic pathogen, responsible for pneumocystis pneumonia with frequent complications of immunocompromised patients. Delayed initiation of appropriate therapy increases the risk of death in immunocompromised patient. The aim of this work was to determine and evaluate the reliability of methods of laboratory diagnosis of pneumocystosis used in routine laboratories as well as the occurrence of this disease in patients from Slovakia during 19 years. MATERIAL AND METHODS: The diagnosis is based on microscopic examination (Giemsa- and Gram-Weigert-staining) and detection of parasite DNA by classical or real-time PCR in bronchoalveolar lavage and sputum. RESULTS: Pneumocysts were detected in 190 persons (5.7%) from the whole group of patients. Cancer patients represented the riskiest group in terms of pneumocystosis, which was confirmed by the highest percentage (57.9%) of individuals infected with P. jirovecii. Compared with the PCR, 33.7% sensitivity and 100% specificity of microscopy was calculated by using a binary classification test. Molecular methods are more sensitive in the detection of P. jirovecii compared to microscopic evidence and currently represent a reliable detection system in the diagnosis of pneumocystosis. CONCLUSION: In view of the increasing number of immunocompromised persons, diagnostics of P. jirovecii in patients with pulmonary complications is essential. This was also confirmed in our study, where the number of examinations and detection of this opportunistic pathogen increased over the years.

Unusual Toxoplasma infection of the eye and central nervous system in an HIV-positive patient.

OBJECTIVE: To report on a unique combination of cerebral toxoplasmosis and ocular toxoplasmosis in an HIV-positive patient in Slovakia. METHODS: A 35-year-old heterosexual patient who presented with headache and major seizures underwent computed tomography (CT) and magnetic resonance imaging (MRI). Based on clinical findings, serological tests for toxoplasmosis were performed on serum and ocular fluid specimens. PCR was also used to detect Toxoplasma gondii and cytomegalovirus DNA. Goldmann and Witmer coefficient calculation was applied to demonstrate the synthesis of intraocular IgG antibodies. RESULTS: CT and MRI revealed cystic lesions suspected of metastasis in the occipital and temporal regions, and we searched for the primary tumor. After vision loss in the left eye, which rapidly progressed to complete blindness, an eye examination detected macular edema. Anti-edema treatment was initiated. HIV positivity with a very low CD4 T-cell count (20/μL) was found, and the viral load was 100 400 HIV-RNA copies/ml. The serum was positive for anti-Toxoplasma IgG antibodies (> 200 IU/mL), IgM negative, and IgA borderline. As toxoplasmic encephalitis and retinitis were suspected, antitoxoplasmic therapy with pyrimethamine, spiramycin, and folinic acid was started. The ophthalmologist considered cytomegalovirus retinitis, which was not confirmed by serology or PCR. In contrast, the presence of IgG antibodies in ocular fluid and serum with the calculation of the Goldmann-Witmer coefficient (GW = 32) as well as PCR DNA positivity pointed to Toxoplasma gondii as the etiological agent. Follow-up MRI scan confirmed regression of the pathological lesions, neurological deficit also improved, CD4 T-lymphocytes increased above 200/μL, but blindness of the left eye persisted. CONCLUSION: CT and MRI scans offered no clue as to Toxoplasma etiology of the brain and eye involvement in an HIV-positive patient, which was only confirmed by laboratory tests. Due to the delay in the diagnosis of toxoplasmosis, time from the epileptic seizure to treatment initiation was 16 days, which assumedly caused irreversible blindness in the patient.

Local- versus broad-scale environmental drivers of continental ß-diversity patterns in subterranean spider communities across Europe.

Macroecologists seek to identify drivers of community turnover (ß-diversity) through broad spatial scales. However, the influence of local habitat features in driving broad-scale ß-diversity patterns remains largely untested, owing to the objective challenges of associating local-scale variables to continental-framed datasets. We examined the relative contribution of local- versus broad-scale drivers of continental ß-diversity patterns, using a uniquely suited dataset of cave-dwelling spider communities across Europe (35-70° latitude). Generalized dissimilarity modelling showed that geographical distance, mean annual temperature and size of the karst area in which caves occurred drove most of ß-diversity, with differential contributions of each factor according to the level of subterranean specialization. Highly specialized communities were mostly influenced by geographical distance, while less specialized communities were mostly driven by mean annual temperature. Conversely, local-scale habitat features turned out to be meaningless predictors of community change, which emphasizes the idea of caves as the human accessible fraction of the extended network of fissures that more properly represents the elective habitat of the subterranean fauna. To the extent that the effect of local features turned to be inconspicuous, caves emerge as experimental model systems in which to study broad biological patterns without the confounding effect of local habitat features.

Continental data on cave-dwelling spider communities across Europe (Arachnida: Araneae).

BACKGROUND: Spiders (Arachnida: Araneae) are widespread in subterranean ecosystems worldwide and represent an important component of subterranean trophic webs. Yet, global-scale diversity patterns of subterranean spiders are still mostly unknown. In the frame of the CAWEB project, a European joint network of cave arachnologists, we collected data on cave-dwelling spider communities across Europe in order to explore their continental diversity patterns. Two main datasets were compiled: one listing all subterranean spider species recorded in numerous subterranean localities across Europe and another with high resolution data about the subterranean habitat in which they were collected. From these two datasets, we further generated a third dataset with individual geo-referenced occurrence records for all these species. NEW INFORMATION: Data from 475 geo-referenced subterranean localities (caves, mines and other artificial subterranean sites, interstitial habitats) are herein made available. For each subterranean locality, information about the composition of the spider community is provided, along with local geomorphological and habitat features. Altogether, these communities account for > 300 unique taxonomic entities and 2,091 unique geo-referenced occurrence records, that are made available via the Global Biodiversity Information Facility (GBIF) (Mammola and Cardoso 2019). This dataset is unique in that it covers both a large geographic extent (from 35° south to 67° north) and contains high-resolution local data on geomorphological and habitat features. Given that this kind of high-resolution data are rarely associated with broad-scale datasets used in macroecology, this dataset has high potential for helping researchers in tackling a range of biogeographical and macroecological questions, not necessarily uniquely related to arachnology or subterranean biology.

The first karyotype study in palpigrades, a primitive order of arachnids (Arachnida: Palpigradi).

Chromosomes of palpigrades (Arachnida: Palpigradi), a rare arachnid order with numerous primitive characters, were studied for the first time. We analysed two species of the genus Eukoenenia, namely E. spelaea and E. mirabilis. Their karyotypes are uniform, consisting of a low number of tiny chromosomes that decrease gradually in size. Study of the palpigrade karyotype did not reveal morphologically differentiated sex chromosomes. Analysis of E. spelaea showed that constitutive heterochromatin is scarce, GC-rich, and restricted mostly to presumed centromeric regions. Meiosis is remarkable for the presence of a short diffuse stage and prominent nucleolar activity. During prophase I, nuclei contain a large nucleolus. Prominent knob at the end of one bivalent formed by constitutive heterochromatin is associated to the nucleolus by an adjacent NOR. Presence of a nucleolus-like body at male prophase II suggests activity of NOR also during beginning of the second meiotic division. The data suggest acrocentric morphology of palpigrade chromosomes. Palpigrades do not display holocentric chromosomes which appear to be apomorphic features of a number of arachnid groups. These are: acariform mites, buthid scorpions, and spiders of the superfamily Dysderoidea. Therefore, cytogenetic data do not support a close relationship of palpigrades and acariform mites as suggested previously.

Analysis of integrons in human isolates of Salmonella enterica serovar typhimurium isolated in the Slovak Republic.

About 110 sporadic, epidemiologically unrelated Salmonella enterica serovar typhimurium strains isolated in the Slovak Republic were analyzed for the presence of integrons. Of these 110 examined strains, 47 were of definitive phage type DT104 and 63 were strains of various phage type, RDNC and untypeable, designated here as non-DT104 strains. All isolates were also tested for antimicrobial resistance to 10 antibiotics as well as for the presence of virulence plasmid. Of 63 non-DT104 strains, 15 isolates were multiple-resistant, independently from phage type, other strains were resistant to one, two or three drugs. Resistance to ampicillin, streptomycin, tetracycline and sulfisoxazole was most frequently observed. Among the DT104 isolates up 65.9% exhibited characteristic pentaresistance--ACSSuT phenotype. The integron content was studied in PCR experiments using a 5'-CS/3'-CS primer pair. Fourteen non-DT104 strains, independently from phage type, were found to carry integrons with amplicons 650-1900 bp in size. Thirty-six DT104 strains contained integrons of 1000 and 1200 bp and 31 of they exhibited the ACSSuT phenotype. No integron was found in 10 DT104 strains, which included strains mostly resistant only to streptomycin, tetracycline and sulfisoxazole. The majority of non-DT104 strains did not possess any integrons. Our findings show the widespread existence of both resistant and multiple-resistant epidemiologically unrelated Salmonella typhimurium strains and suggest that integrons contribute to this antimicrobial resistance. The presence of 90-kb virulence plasmid in the 54 non-DT104 and in the all DT104 strains was found.

Linear mitochondrial genomes: 30 years down the line.

At variance with the earlier belief that mitochondrial genomes are represented by circular DNA molecules, a large number of organisms have been found to carry linear mitochondrial DNA. Studies of linear mitochondrial genomes might provide a novel view on the evolutionary history of organelle genomes and contribute to delineating mechanisms of maintenance and functioning of telomeres. Because linear mitochondrial DNA is present in a number of human pathogens, its replication mechanisms might become a target for drugs that would not interfere with replication of human circular mitochondrial DNA.

Interspecies transplacement of mitochondria in yeasts.

Protoplasts of a respiration-deficient rho(0)strain of Saccharomyces cerevisiae were incubated with mitochondria isolated from various respiration-competent yeast species under conditions enabling transplacement of mitochondria. Respiration-competent cybrids were selected by plating the protoplasts on agar media containing a non-fermentable energy source. The resulting cybrids contained nuclear DNA of the acceptor S. cerevisiae and mitochondrial DNA of the donor species, as detected by pulsed-field gel electrophoresis of chromosomes and restriction analysis of mitochondrial DNA, respectively. Successful restoration of respiration in the S. cerevisiae mutant was achieved by transplacement of mitochondria isolated from the following Saccharomyces species: S. bayanus, S. capensis, S. delbrueckii, S. exiguus, S. italicus and S. oviformis.

Meat quality assessed Ante- and Post mortem by different ryanodine receptor gene status of pigs.

Different breeds and crossbreeds (total 63) of pigs with defined genotype (DNA based test on RYR 1 gene status) were divided in normal (non mutant), heterozygotes (carriers, monomutant) and homozygote positive (dimutant) animals. Muscle biopsy samples were taken at approximately 80 kg live weight using an efficient non-stress spring-loaded biopsy instrument. After incubation of muscle samples (39 °C, 1 hr) analyses pH, WHC (fluid), R value (ITP:ATP) were performed. After slaughter, meat quality values (pH(1), pH(24), R value, colour, drip loss) were also carried out. Heterozygotes were intermediate and between both homozygote pig groups in most meat quality values analysed. Using WHC (fluid volume) values (0.45 and 0.48 respectively) it was possible to divide heterozygotes into two groups, each closer to homozygotes. The values of pH, WHC (fluid), as measured at definite times on the biopsies, were correlated with the pH(1) post-mortem value (r approx 0.6-0.7). The results support previous findings on the possibility of differentiating pigs within DNA test-defined groups. Introducing the gene into a pig population could be useful, in improving leanness and muscling: but meat quality is certain to deteriorate. Further experiments are needed to support the possibility of identifying PSE-prone pigs from among normal non mutant pigs.

An orphan receptor tyrosine kinase family whose members serve as nonintegrin collagen receptors.

Mammalian cells constantly monitor and respond to a myriad of extracellular signals, often by using cell surface receptors. Two important classes of cell surface receptors include the receptor tyrosine kinases, which recognize peptide growth factors such as insulin, and the integrins, which most often mediate binding to components of the extracellular matrix. We report that the collagens serve as ligands for the previously orphan family of discoidin domain-containing receptor-like tyrosine kinases. The unexpected realization that an extracellular matrix molecule can directly serve as a ligand for receptor tyrosine kinases provides an example of ligands shared by integrins and receptor tyrosine kinases, and this finding seems likely to change prevailing views about the mechanisms by which cells perceive and respond to the extracellular matrix.

Why Saccharomyces cerevisiae can oxidize but not decarboxylate external pyruvate.

Protoplasts of the yeast Saccharomyces cerevisiae oxidized externally added pyruvate by pyruvate oxidase system but were not able to decarboxylate it anaerobically by pyruvate decarboxylase at pH 6.4 in isotonic solutions. The decarboxylation set in hypotonic solutions in which the integrity of the plasma membrane was being impaired. Yeast cells incubated with [1-14C]pyruvate accumulated radioactivity under conditions allowing oxidation of pyruvate, but virtually no pyruvate was taken up when the oxidation had been arrested by inhibition or mutation. In view of a large difference between Km for pyruvate of pyruvate decarboxylase (30 mM) and of pyruvate oxidase (0.16 mM), the results may be accounted for by the assumption that transport of pyruvate across the yeast plasma membrane is trans-inhibited by relatively high concentrations of intracellular pyruvate. This arrangement would allow utilization of external pyruvate by the cell energy-transforming machinery and, at the same time, prevent its wastage by futile decarboxylation.

Probing the mechanism of CRP activation by site-directed mutagenesis: the role of serine 128 in the allosteric pathway of cAMP receptor protein activation.

Upon activation by cAMP, Escherichia coli cAMP receptor protein (CRP) controls the expression of a network of catabolite sensitive genes. The activation of CRP by cAMP involves conformational changes such as realignments between subunits and domains within the protein. To understand the molecular events that lead to the activation of CRP, point mutations at position 128 were introduced via site-directed mutagenesis in an attempt to specifically affect the subunit interfacial interactions, as well as the ligand-binding reaction. The biochemical and biophysical properties of these mutants were rigorously tested with the goal of identifying the partial reactions in the activation pathway that are perturbed by this specific mutation. Results from this study suggest that mutation of Ser 128 to Ala or Pro does not significantly disturb the overall secondary structure as monitored by circular dichroism. The energetics of subunit-subunit interaction and protein stability were monitored by sedimentation and spectroscopic techniques. Although these mutants were designed to interrupt intersubunit interactions, the energetics of subunit association and protein stability remain quantitatively the same as those of the wild-type CRP. Nevertheless, the ability of the subunit to be realigned to the DNA-binding form is significantly affected as reflected by the pronounced decrease in the susceptibility of mutant CRP to proteolytic digestion in the presence of cAMP. In addition, the binding affinity of cAMP to the first ligand site in mutants S128A and S128P is the same as that of the wild type, but the affinity to the second ligand site is reduced. This observation indicates that mutation at position 128 affects ligand binding by amplifying the magnitude of negative cooperativity. Mutation at residue 128 does not impair the ability of interdomain interactions as indicated by the quantitative response of a spectroscopic probe in the DNA-binding domain to the binding of cAMP to the ligand-binding domain. The S128A mutant binds to a specific DNA sequence about 50-fold weaker than the wild-type CRP, while the mutant S128P has no measurable DNA affinity under the same conditions. This observation is consistent with the in vivo result that both mutants display an inactive CRP phenotype (CRP-). In summary, these results suggest that communication between domains induced by cAMP binding can be dissociated from the proper subunit realignment of the CRP dimer that is crucial for the activation of CRP.(ABSTRACT TRUNCATED AT 400 WORDS)

Linear mitochondrial DNAs from yeasts: telomeres with large tandem repetitions.

The terminal structure of the linear mitochondrial DNA (mtDNA) from the yeast Candida parapsilosis was investigated. This mtDNA, 30 kb long, has symmetrical ends forming inverted terminal repeats. These repeats are made up of a variable number of tandemly repeating units of 738 bp each; the terminal nucleotide corresponds to a precise position within the last repeat unit sequence. The ends had an open structure accessible to enzymes, with a 5' single-stranded extension of about 110 nucleotides. No circular forms were detected in the DNA preparations. Two other unrelated species, Pichia philodendra and Candida salmanticensis also appear to have a linear mtDNA of similar organization. These linear DNAs (which we name Type 2 linear mtDNAs) are distinct from the previously described linear mtDNAs of yeasts whose termini are formed by a closed hairpin loop (Type 1 linear mtDNA). The terminal structure of C. parapsilosis mtDNA is reminiscent of the linear mitochondrial genomes of the ciliate Tetrahymena although, in the latter, the telomeric tandem repeat unit is considerably shorter.

Identification of halothane genotypes by calcium accumulation and their meat quality using live pigs.

The three halothane genotypes (NN, Nn, and nm) were identified by measuring the capacity for Ca(2+) accumulation by sarcoplasmic reticulum in whole muscle homogenate preparations of M. longissimus dorsi with a Ca(2+) specific electrode at 35°C. Significant differences (P < 0·001) in deterioration (%) of Ca(2+) accumulation, 12% for NN, 35% for Nn, and 81% for nn pigs, were observed after ageing the whole muscle homogenate preparations for 24 h in ice. Predictions of meat quality in live pigs (n = 34) based on the values for water-holding capacity, assessed as fluid (g/0·5 g wet wt LD), and pH (fluid) by using small biopsy LD samples (Cheah et al. 1993) were performed on all the halothane genotypes. The halothane genotype NN (n = 11) showed a fluid value of 0·37 ± 0·01 and a pH (fluid) value of 6·62 ± 0·03 as compared with 0·61 ± 0·02 and 5·84 ± 0·04, respectively, for the halothane genotype nn (n = 13). The Nn pigs (n = 10) showed fluid (0·49 ± 0·03) and pH (fluid) (6·19 ± 0·11) values between those values observed for the two homozygotes (NN and nn). Predictions of meat quality in live pigs from biopsy LD muscles were confirmed from assessments on post-mortem LD muscles based on pH(1) and fibre optic probe (FOP) measurements. The extent of deterioration (%) in Ca(2+) accumulation showed high correlations with fluid (r = -0·861) and pH (fluid) (r = -0·831) in the biopsy LD samples, and with pH(1) (r = 0·663), FOP (r = -0·812), and drip (%) loss (r = -0·777) in the post-mortem LD samples.

Prediction of meat quality in live pigs using stress-susceptible and stress-resistant animals.

A new in-vivo procedure for predicting the potential meat quality in live pigs was devised using stress-susceptible (halothane positive) and stress-resistant (halothone negative) pigs. The potential meat quality in live pigs was determined using small biopsy samples of M. longissimus dorsi (LD). Meat quality was assessed by the combined measurements of pH and water-holding capacity (WHC) on the 12 000 g supernatant after incubation of 500 mg biopsy LD muscle with an equal volume of 150 mm KCl at 39°C for 45 min. With the LD muscles of halothane positive (n = 37) and halothane negative (n = 55) pigs, high correlations (r = -0·854) were observed between the supernatant (i.e. fluid) pH and WHC of the biopsy samples, between fluid pH of the biopsy samples and 1-h post-mortem (pH(60)) LD muscles (r = 0·951), and between pH(60) and WHC (r = -0·956). The experimental data show that our in-vivo test can differentiate halothane positive from halothane negative pigs and can also predict the potential meat quality in live pigs. The test could be applied to select pigs with differences in WHC to improve meat quality.

Respiratory chain defect of myocardial mitochondria in idiopathic dilated cardiomyopathy of Doberman pinscher dogs.

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause. We tested the hypothesis that IDCM was associated with a myocardial metabolic defect by determining a comprehensive biochemical profile of metabolite concentrations and enzyme activities for the major metabolic pathways of the myocardium. We used the Doberman pinscher breed as a naturally occurring canine model of IDCM and compared its myocardial profile with that of healthy adult mongrels. Compared with controls, myocardium in IDCM had markedly reduced mitochondrial electron transport activity and myoglobin concentration, in association with acidosis and energy depletion following anoxic challenge: 60% decreased NADH dehydrogenase and 50% decreased ATP synthetase activities; 90% decreased myoglobin concentration; and 30% reduced ATP and 50% increased lactate and proton concentrations. Sarcoplasmic reticulum Ca(2+)-transport ATPase was decreased by 42%. There was a 15% compensatory increase in fatty acid oxidation and Krebs cycle activity. Other biochemical changes were mild by comparison with the mitochondrial defects. We conclude that IDCM is associated with a marked impairment of mitochondrial production of ATP, arising from decreased activity of the mitochondrial electron transport system, including myoglobin. These changes may be secondary to an underlying genetic defect or may indicate a deficiency of the mitochondrial respiratory chain that predisposes this breed to heart failure.

[The incorporation of macromolecules into the germ cells of male mice via electroporation and dimethyl sulfoxide]. / Vkliuchenie makromolekul v polovye kletki samtsov myshei pri pomoshchi élektroporatsii i dimetilsul'foksida.

Electroporation (incorporation of macromolecules into the living cells by means of electric pulses) provides inclusion of plasmid 14C-DNA into immature cells of spermatogenic epithelium. The highest level of foreign DNA incorporation into spermatocytes and spermatids has been induced by 8kV electric pulses applied 3 times with 20 sec intervals. Meanwhile, mature sperms are found to be exclusively resistant to exogenous DNA irrespective of the voltage level, the number of pulses and Ca++ uptake (contents). Incubation of mature sperms for two hours in the medium with Ca++ (10 mM) and dimethylsulfoxide--(DMSO, 33%) provides highly reliable incorporation of plasmid 14C-DNA into sperm heads. The sperm cells with foreign DNA incorporated by means of Ca++ and DMSO treatment still remain alive and mobile. The possibilities of mature sperms loaded with foreign DNA for the creation of transgenic mammals are discussed.

[The possibility of the incorporation of macromolecules, including exogenous DNA, into the germ cells of male mice. The liposome method and Ca-P coprecipitation method]. / K vozmozhnosti inkorporirovaniia makromolekul, vkluchaia ékzogennuiu DNK, v polovye kletki samtsov myshei. Liposomnyi metod i metod Ca-P kopretsipitatsii.

Liposomes loaded with FITC-labeled albumin in the presence of PEG-1,500 are actively sorbed on the membranes of mature spermatozoa and remain attached even after thorough washing. Immature sperm cells are able to incorporate alien DNA carried by liposomes. In contrast, the mature spermatozoa could not incorporate plasmid DNA loaded with positively charged liposomes. Chlortetracycline in Ca-P coprecipitate crystals is tightly fixed in the postacrosomal region of mature sperms. Intensity of staining of chlortetracycline is stimulated by DNA load in Ca-P coprecipitate as well as by DMSO or EDTA. The method of Ca-P coprecipitation could not provide for plasmid DNA incorporation into taure sperms. Foreign DNA incorporation in postacrosomal regions of sperm heads seems quite possible in experiments with dimethylsulphoxide (DMSO).