Impact of prescribed fire on soil microbial communities in a Southern Appalachian Forest clear-cut.

Escalating wildfire frequency and severity, exacerbated by shifting climate patterns, pose significant ecological and economic challenges. Prescribed burns, a common forest management tool, aim to mitigate wildfire risks and protect biodiversity. Nevertheless, understanding the impact of prescribed burns on soil and microbial communities in temperate mixed forests, considering temporal dynamics and slash fuel types, remains crucial. Our study, conducted at the University of Tennessee Forest Resources AgResearch and Education Center in Oak Ridge, TN, employed controlled burns across various treatments, and the findings indicate that low-intensity prescribed burns have none or minimal short-term effects on soil parameters but may alter soil nutrient concentrations, as evidenced by significant changes in porewater acetate, formate, and nitrate concentrations. These burns also induce shifts in microbial community structure and diversity, with Proteobacteria and Acidobacteria increasing significantly post-fire, possibly aiding soil recovery. In contrast, Verrucomicrobia showed a notable decrease over time, and other specific microbial taxa correlated with soil pH, porewater nitrate, ammonium, and phosphate concentrations. Our research contributes to understanding the intricate relationships between prescribed fire, soil dynamics, and microbial responses in temperate mixed forests in the Southern Appalachian Region, which is valuable for informed land management practices in the face of evolving environmental challenges.

Intervalos de referencia de minerales en cerdos confinados de diferentes genéticas y categorías

En Argentina, no existen datos regionales de intervalos de referencia de minerales en cerdos según las líneas genéticas actuales y distintas categorías de producción en sistema intensivo. Por ello, el objetivo del presente estudio fue determinar intervalos de minerales: calcio, fósforo, magnesio, sodio, potasio, hierro, cobre y cinc, en suero de cerdos en establecimientos del centro de Santa Fe y de Entre Ríos. Se trabajó con 300 muestras de sangre de cerdos sanos de dos líneas genéticas diferentes en las categorías de lechones recién nacidos, lechones destetados, hembras nulíparas gestantes seleccionadas fenotípicamente para la reposición del plantel, y hembras multíparas gestantes. Se determinaron las concentraciones séricas de los minerales por espectrofotometría de absorción atómica (FAAS) con un equipo Perkin Elmer modelo Analys 200, con métodos oficiales de AOAC. Se analizaron también muestras de agua. Los intervalos de referencia se calcularon utilizando parámetros paramétricos o no paramétricos dependiendo de la distribución de los datos. Los valores medios, medianas, valores mínimo y máximo e intervalos de referencia, para los distintos minerales en cada categoría productiva, se presentan en tablas. Los intervalos de referencia calculados serán útiles para el diagnóstico de deficiencias mineral y la vigilancia nutricional en cerdo de producción de carne.

In Argentina, there is no regional data on mineral reference intervals in swine, according to the genetic lines, categories of production in intensive systems. The objective of this study was to establish ranges of the following serum minerals: calcium, phosphorus, magnesium, sodium, potassium, iron, copper and zinc, in swine farmed in Santa Fe and Entre Ríos. Blood samples were collected from 300 healthy pigs of two different genetic lines belonging to the following categories: newborn piglets, weaned piglets, pregnant nulliparous females selected for re-stocking, and pregnant multiparous females. Serum concentrations of minerals were determined by atomic absorption spectrophotometry (FAAS) with a Perkin Elmer model Analys 200 and using official AOAC methods. Water samples were also analyzed. Reference intervals were calculated using parametric or nonparametric methods on basis of data distribution. Mean, median, minimum, maximum values and reference intervals for different minerals were tabulated. The reference intervals calculated in this study will be useful for the diagnosis of mineral deficiencies and nutritional monitoring in meat production pigs.

Developing more open and equitable relationships with industry to improve advancements in clinical research in dermatology.

Relationships between physicians, scientists, and the pharmaceutical industry can be complicated by conflicts of interest. Honest and equitable relationships, however, are essential to the advancement of dermatologic clinical research. Several factors can increase transparency in clinical trials including preregistration of clinical trials, reporting of all data produced from clinical trials, non-industry ownership of clinical trial data, clarity of statistical methods and publication of both positive and negative results. Through collaborative, scientifically rigorous studies, physicians and industry can achieve significant advances in dermatologic care.

Reversed phase and cation exchange liquid chromatography with spectrophotometric and elemental/molecular mass spectrometric detection for S-adenosyl methionine/S-adenosyl homocysteine ratios as methylation index in cell cultures of ovarian cancer.

S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are essential compounds in the carbon metabolic cycle that have clinical implications in a broad range of disease conditions. The measurement of the ratio SAM/SAH also called methylation index, has become a way of monitoring the DNA methylation of a cell which is an epigenetic event with important clinical implications in diagnosis; therefore the development of suitable methods to accurately quantify these compounds is mandatory. This work illustrates the comparison of three independent methods for the determination of the methylation index, all of them based on the chromatographic separation of the two species (SAM and SAH) using either ion-pairing reversed phase or cation exchange chromatography. The species detection was conducted using either molecular absorption spectrophotometry (HPLC-UV) or mass spectrometry with electrospray (ESI-MS/MS) as ionization source or inductively coupled plasma (DF-ICP-MS) by monitoring the S-atom contained in both analytes. The analytical performance characteristics of the three methods were critically compared obtaining best features for the combination of reversed phase HPLC with ESI-MS in the MRM mode. In this case, detection limits of about 0.5ngmL(-1) for both targeted analytes permitted the application of the designed strategy to evaluate the effect of cisplatin on the changes of the methylation index among epithelial ovarian cancer cell lines sensitive (A2780) and resistant (A2780CIS) to this drug after exposition to cisplatin.

Interaction of Mycobacterium tuberculosis cell wall components with the human natural killer cell receptors NKp44 and Toll-like receptor 2.

We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll-like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl-arabinogalactan-peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN-γ production at levels comparable to M. bovis Bacillus Calmette-Guérin-stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG-exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN-γ production were inhibited by anti-TLR2 MAb, but not by anti-NKp44 MAb. Differently, anti-NKp44 MAb partially inhibited CD69 expression on NK cells pre-activated with IL-2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR-2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN-γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

Saponin promotes rapid identification and antimicrobial susceptibility profiling of Gram-positive and Gram-negative bacteria in blood cultures with the Vitek 2 system.

The rapid identification and antimicrobial susceptibility testing (AST) of bacteria in clinical blood cultures is crucial to optimise antimicrobial therapy. A previous study involving small sample numbers revealed that the addition of saponin to blood cultures, further referred to as the new method, shortened considerably the turn-around time for the identification and AST of Gram-positive cocci as compared to the current method involving an overnight subculture. Here, we extend previous results and compare the identification and AST of blood cultures containing Gram-negative bacilli by the new and current methods. The identification and AST of 121 Gram-positive and 109 Gram-negative bacteria in clinical monomicrobial blood cultures by the new and current methods and, in the case of Gram-negative bacilli, by direct (no additions) inoculation into an automated system (rapid method) was assessed using the Vitek 2 system. Discrepancies between the results obtained with the different methods were solved by manual methods. The new method correctly identified 88 % of Gram-positive and 98 % of Gram-negative bacteria, and the rapid method correctly identified 94 % of Gram-negative bacteria. The AST for all antimicrobials by the new method were concordant with the current method for 55 % and correct for an additional 9 % of Gram-positive bacteria, and concordant with the current method for 62 % and correct for an additional 21 % of Gram-negative bacilli. The AST by the rapid method was concordant with the current method for 62 % and correct for an additional 12 % of Gram-negative bacilli. Together, saponin-treated monomicrobial blood cultures allow rapid and reliable identification and AST of Gram-positive and Gram-negative bacteria.

[Irritable bowel syndrome: frequency and phylogenetic relationship of Blastocystis sp. from Mexican patients]. / Síndrome de intestino irritable: frecuencia y relación filogenética de Blastocystis sp. de pacientes mexicanos.

INTRODUCTION: Recent studies reported increased presence of Blastocystis in patients with Irritable Bowel Syndrome (IBS) and an etiologic role has been proposed. The pathogenic role of Blastocystis is controversial, because it is frequently found not only in individuals with enteric symptoms but also in healthy and asymptomatic subjects. Furthermore, there are few studies of blastocistosis in Mexico. OBJECTIVE: To assess the frequency of Blastocystis sp. in IBS patients using molecular techniques and to describe its phylogenetic relationship with sequences of other countries. METHODS: IBS patients according to Rome III criteria were enrolled. In all patients evaluations included: colonoscopies, coproparasitoscopic studies, coproculture, fecal virus screening. PCR and sequencing for Blastocystis sp. were also performed. RESULTS: We recruited 11 men and 51 women with a mean age of 45.6 (SD ± 15.7) years. Eighty-six percent of the IBS patients presented a normal colonoscopy, 8% showed polyps and 6% diverticular disease. Blastocystis sp. was identified in 25% patients (all of them with normal colonoscopy), while two patients had Endolimax nana and Entamoeba histolytica/E. dispar, respectively. Phylogenetic analysis showed that major sequences of Mexican carriers clustered together with sequences of parasites from Japan and Denmark; furthermore, two sequences from IBS patients were grouped in a single cluster. CONCLUSIONS: Blastocystis sp. was identified in 25% of the IBS patients. Our data support the hypothesis of clonal lineages in distinct geographical areas in the world.

Reduced human beta defensin 3 in individuals with periodontal disease.

The human beta defensin 3 (hBD3) is widely expressed in the oral cavity and exerts strong antibacterial and immunomodulatory activities. Hence, we hypothesized that hBD3 could play a protective role in the maintenance of periodontal homeostasis, and that it could be found in gingival crevicular fluid (GCF) of healthy individuals and those with periodontitis at levels correlating with the degree of periodontal health. By using an ELISA assay to quantify hBD3 in GCF, we demonstrated that the peptide is present at levels easily detectable in the majority of healthy individuals, but it is drastically reduced in GCF from those with periodontitis. Furthermore, hBD3 levels inversely correlate with the severity of the disease and the degree of colonization by combinations of bacterial species with elevated periodontopathogenic potential. Both genetic factors and host/bacterial proteases released in diseased sites may be responsible for the observed low/null hBD3 levels in GCF from individuals with periodontitis.

Induced lead binding phytochelatins in Brassica juncea and Sesuvium portulacastrum investigated by orthogonal chromatography inductively coupled plasma-mass spectrometry and matrix assisted laser desorption ionization-time of flight-mass spectrometry.

The accumulation and transport of lead in Brassica juncea and Sesuvium portulacastrum plants and the possible formation of complexes of this element with bioligands such as phytochelatins was studied in roots and shoots of plants exposed to different amounts of Pb(NO(3))(2). Speciation studies on the plant extracts were conducted using size exclusion liquid chromatography and ion pair liquid chromatography coupled to UV and ICP-MS to monitor lead and sulphur. The identification of the species separated by chromatography was performed by MALDI-TOF-MS. In both types of exposed plants it was possible to identify the presence of the phytochelatin isoform PC(3). The results obtained suggest that both types of plants can be useful in studies of phytoremediation but the ability of S. portulacastrum to accumulate and redistribute Pb from root to shoot is more effective than B. juncea.

Rapid identification and antimicrobial susceptibility testing of Gram-positive cocci in blood cultures by direct inoculation into the BD Phoenix system.

Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections are essential for the selection of appropriate antimicrobial therapy. To speed up the identification and AST of the causative agent, the fluid from blood culture bottles of a Bactec 9240 instrument (Becton Dickinson) containing Gram-positive cocci was mixed with saponin. After a 15-min incubation, the bacteria were harvested and transferred to the appropriate panel of a BD Phoenix automated microbiology system (Becton Dickinson) for identification and AST. With this approach (referred to as the direct method), we concordantly/correctly identified 56 (82%) of 68 monomicrobial cultures using the results obtained with the method currently used in our laboratory (current method) as comparator. Two (3%) isolates could not be identified and ten (15%) were misidentified. Complete agreement, concerning clinical susceptibility categories and MIC values, between the AST results determined with the direct method and the current method was found for 32 (55%) of 58 isolates. The E-test indicated that the direct method yielded a correct susceptibility profile for 13 of the remaining 26 blood culture isolates. Therefore, a concordant/correct susceptibility profile (with all antimicrobial agents tested) was obtained for 45 (77%) of 58 cultures. The overall error rate amounted to 1.9%, with the majority (1.3%) of errors being minor. Importantly, the results obtained with the direct method were available 12-24h earlier than those obtained with the current method.

Efficacy of Chromogenic Candida Agar for isolation and presumptive identification of pathogenic yeast species.

Chromogenic Candida Agar is a novel differential culture medium that is claimed to facilitate isolation and identification of Candida albicans, Candida tropicalis and Candida krusei. The performance of this medium was evaluated for presumptive identification of 521 yeast strains, representing 23 different species, for detection of specimens containing yeast mixtures, and for direct isolation of yeast from blood cultures. All yeasts grew well on the medium following a 48-h incubation period at 37 degrees C, and distinctive colonies were produced by C. albicans, C. tropicalis, C. krusei, Candida guilliermondii, Saccharomyces cerevisiae, Trichosporon mucoides and Geotrichum capitatum. The sensitivity and specificity of the medium exceeded 99.4% for each of these species. The medium provided some indication of the presence of Candida dubliniensis and Candida pulcherrima, and allowed the identification of polyfungal samples in 89.4% of the yeast mixtures. Finally, direct isolation on the medium from blood cultures that were positive for yeast according to Gram's stain (n = 42) showed that the expected colour and morphology of each species were not altered in the presence of blood.

Human beta-defensin-3: a promising antimicrobial peptide.

The field of naturally occurring antimicrobial peptides is a research area rapidly expanding due to the high potential of such molecules as new antimicrobial drugs. In this regard, the human beta-defensin-3 is particularly attractive because of its strong antibacterial activity, relative salt-insensitiveness and low toxicity for host cells.

Serological diversity demonstrable by a set of monoclonal antibodies to eight serotypes of the mutans streptococci.

A set of monoclonal antibodies were prepared by the conventional cell fusion of myeloma cells (SP2/0-Ag14) with spleen cells from BALB/c mice immunised with whole cells of a strain of mutans streptococci. Their specificities were examined against 35 reference strains of mutans streptococci, 34 reference strains of other oral streptococci and 8 reference strains of other microorganisms often inhabiting the oral cavity. Specificity was examined by enzyme immunoassay using whole cells. A total of 52 strains, consisting of 19 strains isolated in Japan, 19 strains isolated in Italy and 14 strains isolated in England, were characterised by conventional physiological and biochemical tests and then serotyped by the use of 8 monoclonal antibodies with different specificities. They were also confirmed by guanine-plus-cytosine contents of their nucleic acid and DNA-DNA hybridisation test. The results indicated that all monoclonal antibodies are useful for identification of 8 serotypes of the mutans streptococci responsible for dental caries. They also suggest the existence of more serological varieties among mutans species.

Metal speciation of metallothionein in white sea catfish, Netuma barba, and pearl cichlid, Geophagus brasiliensis, by orthogonal liquid chromatography coupled to ICP-MS detection.

Metal speciation analysis in MTs was carried out in two tropical fish species of Brazil, the freshwater fish pearl cichlid (Geophagus brasiliensis) and the marine fish white sea catfish (Netuma barba), that are presently used to monitor the effects of heavy metal pollution in aquatic ecosystems in Brazil. In order to obtain the MT fraction, liver cytosols from both fish species where subjected to size exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. That MT fraction was then separated by anion-exchange (AE)-FPLC, whose optimal chromatographic conditions were optimized for the separation of the different hepatic MT isoforms existing in both fish species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with the ICP-MS instrument. The analytical results showed that MTs of these fish species, unknown so far, exhibited unique characteristics in comparison with standard MTs and other fish liver MTs. In fact, MT isoforms of N. barba turned out to be very anionic, as indicated by their high retention in the Mono Q column and the strong ionic strength required to separate them. As for G. brasiliensis, cadmium was exclusively present in only one of the peaks of the MT isoforms showing a unique metal-binding behavior for MT in this fish species. The differences between the MTs among these species and the different association of metals in particular MT isoforms display the importance of the metal speciation analysis in these proteins prior to its use as bioindicators.

Human CD56bright and CD56dim natural killer cell subsets respond differentially to direct stimulation with Mycobacterium bovis bacillus Calmette-Guérin.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is capable of directly stimulating several effector functions of human natural killer (NK) cells in the absence of interleukin-12 and professional antigen presenting cells. To assess the contribution of two main human NK-cell subsets (CD56(dim) and CD56(bright)) to the overall in vitro NK-cell response to BCG, peripheral blood mononuclear cells depleted of nylon wool-adherent cells or purified NK cells were stimulated with live BCG. By combining intranuclear bromodeoxyuridine (BrdU) staining and analysis of CD56 marker intensity, statistically higher percentages of BrdU(+) cells were found among the CD56(bright) subset than the CD56(dim) subset after 6 days of stimulation with BCG. Similarly, evaluation of intracellular interferon-gamma (IFN-gamma) revealed that CD56(bright) cells were those mainly involved in IFN-gamma production in response to BCG. In contrast, the CD56(dim) subset contained higher levels of perforin and granzyme A, two key molecules for exocytosis-mediated cytotoxicity, than the CD56(bright) subset. Although 16-20-h stimulation with BCG did not substantially alter the expression of cytotoxic molecules by the two subsets, a decrease in perforin content was observed in the CD56(dim), but not in the CD56(bright) subset, following 4-h incubation with the NK-sensitive target K562 cell line. This decrease in perforin content correlated with the induction by BCG-stimulated NK cells, of early markers of apoptosis on target cells to a greater extent than unstimulated cells suggesting a major role for the CD56(dim) subset in cytotoxic activity in response to BCG. Taken together, these results demonstrate that CD56(bright) and CD56(dim) human NK-cell subsets exert different functional activities in response to a live bacterial pathogen.

Enhanced resolution of random amplified polymorphic DNA genotyping of Pseudomonas aeruginosa.

AIMS: To develop a rapid, sensitive and reproducible screening test for the detection of nosocomial spreading of Pseudomonas aeruginosa. METHODS AND RESULTS: Ps. aeruginosa genomic DNA extraction, RAPD-PCR, electrophoresis on acrylamide gel and silver staining were performed by using standardized reagents and conditions. The results were compared with the agarose gel electrophoresis followed by ethidium bromide staining. CONCLUSIONS: The coupling of acrylamide gel electrophoresis and silver staining gave about 80% more DNA bands than the traditional method, allowing a finer discrimination among different Ps. aeruginosa strains. SIGNIFICANCE AND IMPACT OF THE STUDY: By enhancing the resolution of the electrophoretic separation and the sensitivity of the staining, random amplification could be easily applied to the surveillance and prevention of nosocomial infections by clinical microbiology laboratories.

An alternative and simple method to consistently prepare viable isolated human islets for clinical transplantation.

We describe a method to consistently prepare human islets for transplantation. By combining a simple collagenase digestion method and a density gradient purification system, we were able to obtain successful isolations (>/=200,000 islet equivalents, >/=50% purity) in 69% of processed glands. No reagent of animal source was used. Isolated islets were morphologically well maintained and functionally competent, with sterility confirmed in 97% of cases. Two patients were transplanted with islets prepared by this method; graft function was demonstrated for a few months. Improved simplicity and consistency, together with adequate quality of the preparations, are the main features of this isolation method.

The risk of contracting an infectious disease from blood transfusion.

In the general population, the likelihood of an individual receiving a transfusion has been calculated to be about 0.89% per year, increasing dramatically with age. Massive intraoperative hemorrhage from trauma, cardiopulmonary bypass, and orthotopic liver transplantation need substantial replacement therapy. In renal transplantation, blood transfusion is a debated induction tool for specific allograft tolerance, since it causes a nonspecific down-regulation of immune function. In transplantations, in humoral immune deficiencies, in hematological disorders, and in HIV infection, the intravenous immunoglobulin prophylaxis may alter the monocyte/macrophage system host immunity and immune surveillance against infection, tissue or cell damage, and malignancy. Some persons, like Jehovah's Witnesses, object to transfusion of blood products, posing ethical and practical issues concerning treatment of blood disorders, transplantation, and trauma. In this review we examined the actual risk of contracting an infectious disease from an allogeneic blood transfusion to contribute to an uneasy decision-making process. We have found that the procedure is presently considerably safe.