Routine Media Research Articles

REVIEW ARTICLES Genitourinary tuberculosis: historical and basic science review: past and present

Genitourinary tuberculosis (GUTB) usually results from the reactivation of old, dormant tuberculous diseases by pathogens of the mycobacterium tuberculosis complex. GUTB is the second most common form of extrapulmonary tuberculosis, with more than 90% of cases occurring in developing countries. In GUTB, the kidneys are the most common sites of infection and are infected through hematogenous spread of the bacilli, which then spread through the renal and urinary tract. Patients with genital and urethral TB present with a superficial tuberculous ulcer on the penis or in the female genital tract develop mainly due to primary contact with mycobacterium exposure during intercourse or inoculation via goods or chattels contaminated with mycobacterium.The diagnosis of TB of the urinary tract is based on the case history, the finding of pyuria in the absence of infection as judged by culture on routine media, and by radiological imaging. However, a positive yellow egg culture and/or histological analysis of biopsy specimens, possibly combined with the polymerase chain reaction (PCR), is still required in most patients to establish a definitive diagnosis of GUTB.The standard antituberculous drug treatment should be administered initially for two months during the intensive phase with three or four drugs daily followed by dual continuation therapy for four months. Surgery as a treatment option in GUTB might be indicated in complicated urinary tuberculosis. After antituberculous treatment of GUTB, surveillance with regular follow-up visits over the next five years is recommended.In cases of drug resistance, additional drugs and prolonged treatment are required. Furthermore, increasing rates of drug-resistant cases and co-infection with HIV pose challenges in the treatment GUTB and other forms of TB.

The use of the water extract of Rosa spp petals as a bacterial growth medium

In a pioneer study, simple water extract for the red petals of Rosa spp. was prepared under sterile conditions, then used for the first time as experimental bacterial culture medium for the growth of the bacteria: Pseudomonas aeruginosa, Proteus vulgaris, Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae and Klebsiella pneumoniae; the medium was used as alternative culture medium for the routine culture media (Nutrient agar, MacConkey agar and blood agar) that used for the growth of these genera in the laboratories.All the genera showed active growth after 24 hours when it used directly as a liquid culture medium. The extract was used also to enrich the agar-agar and cultivated with the same bacteria; it showed a noticeable growth. The results suggest that this extract is a suitable culture medium; it could be used instead ofthe routine culture media that used in the cultivation of these bacteria in the laboratories. It also represents important, rich nutritional medium as those that isused in the routine laboratory work.

Effect of Selenium Supplementation on Glutathione Peroxidase and Catalase Activities in Senescent Cultured Human Fibroblasts

Aims: To investigate the effect of senescence and selenium supplementation on glutathione peroxidase (cGPx) and catalase (CAT) activities, and concurrent hydrogen peroxide (H₂O₂) generation in subcultured human fibroblasts. Methods: cGPx and CAT activities and H₂O₂ levels were assayed in presenescent passage 5 and 10 cells, and in senescent passage 20, 25, 30 and 35 cells cultured in routine medium (MEM1) and supplemented media MEM2 and MEM3 containing normal and triple human plasma levels of Se, respectively. Senescent cells were identified by studying their growth and replication states, and by monitoring their activity of key glucose and glycogen degradative enzymes. Results: cGPx activity showed moderate increases in senescent cells at passages 20–35 subcultured in MEM1 or MEM2. This activity underwent highly significant progressive increases in the same senescent cells subcultured in MEM3. In contrast, CAT activity showed progressive, highly significant increases in senescent cells at passages 20–35 regardless of the culture medium type. Concurrent H₂O₂ generation was significantly increased in passage 15–25 cells and peaked to higher levels in passage 30 and 35 cells cultured in MEM1 or MEM2. These rates, however, were significantly reduced in senescent passage 20–35 cells cultured in MEM3. Conclusions: The highest cGPx activity and coupled lower H₂O₂ generation were achieved in senescent cells cultured in MEM3.

Diagnostic use of Burkholderia pseudomallei selective media in a low prevalence setting

Routine use of selective media improves diagnosis of Burkholderia pseudomallei, but resources may be limited in endemic developing countries. To maximise yield in the relatively low-prevalence setting of Kuala Lumpur, Malaysia, B. pseudomallei selective agar and broth were compared with routine media for 154 respiratory specimens from patients with community-acquired disease. Selective media detected three additional culture-positive specimens and one additional melioidosis patient, at a consumables cost of US$75. Burkholderia pseudomallei was not isolated from 74 diabetic foot ulcer samples. Following careful local evaluation, focused use of selective media may be cost-effective.

Post‐traumatic Pseudallescheria apiosperma osteomyelitis: positive outcome of a young immunocompetent male patient due to surgical intervention and voriconazole therapy

The present case describes thesuccessful treatment of an immunocompetent youngmale patient suffering from a severe, post-traumaticPseudallescheria apiosperma osteomyelitis of the tibia.Cure of the patient was achieved by long-termvoriconazole administration and surgical debridementof infected soft tissue and bone.

Excitotoxicity Triggered by Neurobasal Culture Medium

Neurobasal defined culture medium has been optimized for survival of rat embryonic hippocampal neurons and is now widely used for many types of primary neuronal cell culture. Therefore, we were surprised that routine medium exchange with serum- and supplement-free Neurobasal killed as many as 50% of postnatal hippocampal neurons after a 4 h exposure at day in vitro 12–15. Minimal Essential Medium (MEM), in contrast, produced no significant toxicity. Detectable Neurobasal-induced neuronal death occurred with as little as 5 min exposure, measured 24 h later. D-2-Amino-5-phosphonovalerate (D-APV) completely prevented Neurobasal toxicity, implicating direct or indirect N-methyl-D-aspartate (NMDA) receptor-mediated neuronal excitotoxicity. Whole-cell recordings revealed that Neurobasal but not MEM directly activated D-APV-sensitive currents similar in amplitude to those gated by 1 µM glutamate. We hypothesized that L-cysteine likely mediates the excitotoxic effects of Neurobasal incubation. Although the original published formulation of Neurobasal contained only 10 µM L-cysteine, commercial recipes contain 260 µM, a concentration in the range reported to activate NMDA receptors. Consistent with our hypothesis, 260 µM L-cysteine in bicarbonate-buffered saline gated NMDA receptor currents and produced toxicity equivalent to Neurobasal. Although NMDA receptor-mediated depolarization and Ca2+ influx may support survival of young neurons, NMDA receptor agonist effects on development and survival should be considered when employing Neurobasal culture medium.

Place media events and place image communication: The role of festivals and special events from the perspective of media relations

This article attempts to demonstrate the significant role played by festivals and special events in place image communication from the perspective of media relations through a review of the literature referring to place image and media relations. By developing the term ‘place media event’, the authors arise a number of issues in place image communication. The authors argue that to win satisfactory uncontrolled media coverage, the importance of inner place image, media relations practitioners, media logic and routine media relations practices should been highlighted. These factors determine the successful transformation of standard events into media events.

‘The effect of micronutrients on superoxide dismutase in senescent fibroblasts’

The specific activities of zinc/copper (Zn/Cu)-superoxide dismutase (SOD-1) and manganese (Mn)-superoxide dismutase (SOD-2) were assayed in young passage 5 fibroblasts and in serially subcultured cells that were characterized as senescent at passages 15-35. SOD-1 and SOD-2 activities did not significantly change in senescent and young cells cultured in either routine medium [minimum essential medium 1 (MEM1)], or in Zn, Cu and Mn supplemented medium (MEM2) containing normal human plasma levels of the cations. SOD-1 and SOD-2 activities, however, underwent parallel progressive significant activity increases in senescent passage 20 and 25 cells, which peaked in value in passage 30 and 35 cells subcultured in supplemented medium (MEM3) containing triple human plasma levels of the cations. Concurrently, superoxide radical generation rates underwent progressive significant increases in senescent passage 15-25 cells, which peaked in value in passage 30 and 35 cells subcultured in MEM1 or MEM2. These rates, however, were significantly lowered in senescent cells subcultured in MEM3. We infer that it was only possible to significantly stimulate SOD-1 and SOD-2 activities in senescent MEM3 cultured cells enabling them to combat oxidative stress.

Re-assessment of phenotypic identifications of Bacteroides putredinis to Alistipes species using molecular methods

Alistipes (previously Bacteroides) are strictly anaerobic gram-negative rods that resemble the Bacteroides fragilis group in that most species are bile-resistant and indole-positive; however, they are only weakly saccharolytic and most species produce light brown pigment only on laked rabbit blood agar. In this retrospective study, we re-identified 18 organisms previously identified phenotypically as “ Bacteroides putredinis-like”, but that did not produce pigment on routine media. The strains were identified with 16S rDNA sequencing and pigment production was evaluated on several different culture media. Only 12/18 strains had molecular identifications of Alistipes species, while the remaining strains phylogenetically resembled Butyricimonas and Odoribacter spp. Pigment production was not a reliable test for those Alistipes strains that are described as pigment producers.

Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: Implication on use of higher passage cells

Human umbilical vein endothelial cells (HUVEC) were cultured in two different media, viz. the commonly used M199 containing 20% fetal bovine serum (FBS) and endothelial cell growth factor and a defined media EGM-2 containing 2% FBS along with growth supplements in known concentrations. The purpose of this study was to determine the effect of different media on the growth potential and cell morphology in subsequent passages. We have established that a dual coating of gelatin and human fibronectin extracellular matrix provides optimal cell attachment. Growth rate for primary culture was almost double in defined media. For secondary culture a two fold higher proliferation rate was observed in defined EGM-2 media. Histological studies were done using phase contrast, confocal and scanning electron microscopy which showed that cells cultured in M199 started losing their morphological characteristic from 3rd passage and after 6th passage appeared to come in senescent stage, while in case of defined media there was no change observed in the cells up to 10th passage. A significant difference was found in the expression of soluble intracellular adhesion molecule-1 (sICAM-1) which is an endothelial cell marker on cells cultured in different media. Additionally it was observed that exposure duration to trypsin–EDTA during cell detachment also plays an important role in maintaining cell morphological characteristics. These results show that significant morphological changes appear in higher order passages if cells are grown in routine medium for a long time and therefore may not be suitable for cell signaling experiments.

Klebsiella pneumoniae Outer Membrane Porins OmpK35 and OmpK36 Play Roles in both Antimicrobial Resistance and Virulence

OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. In this study, a virulent clinical isolate was selected to study the role of these two porins in antimicrobial resistance and virulence. The single deletion of ompK36 (ΔompK36) resulted in MIC shifts of cefazolin, cephalothin, and cefoxitin from susceptible to resistant, while the single deletion of ompK35 (ΔompK35) had no significant effect. A double deletion of ompK35 and ompK36 (ΔompK35/36) further increased these MICs to high-level resistance and led to 8- and 16-fold increases in the MICs of meropenem and cefepime, respectively. In contrast to the routine testing medium, which is of high osmolarity, susceptibility tests using low-osmolarity medium showed that the ΔompK35 mutation resulted in a significant (≥ 4-fold) increase in the MICs of cefazolin and ceftazidime, whereas a ΔompK36 deletion conferred a significantly (4-fold) lower increase in the MIC of cefazolin. In the virulence assays, a significant (P < 0.05) defect in the growth rate was found only in the ΔompK35/36 mutant, indicating the effect on metabolic fitness. A significant (P < 0.05) increase in susceptibility to neutrophil phagocytosis was observed in both ΔompK36 and ΔompK35/36 mutants. In a mouse peritonitis model, the ΔompK35 mutant showed no change in virulence, and the ΔompK36 mutant exhibited significantly (P < 0.01) lower virulence, whereas the ΔompK35/36 mutant presented the highest 50% lethal dose of these strains. In conclusion, porin deficiency in K. pneumoniae could increase antimicrobial resistance but decrease virulence at the same time.

Both Juxtacrine and Paracrine Signaling Indispensable in Spermatogonial Stem Cell Cultures

Objective To prove that juxtacrine and paracrine signaling are essential in the culture of spermatogonial stem cells (SSCs) with Sertoli cell feeder layer in vitro. Methods Mice aged 7 d were chosen to harvest testes. A two-step enzyme digestion method was applied in testis suspension. The SSCs and Sertoli cells were separated by adherence distinguishing methods and biologically identified by immunofluorescence and Oil Red O staining methods. Flow cytometry was used to analyze purity of SSCs. Three groups were constructed according to different culture conditions: SSC and Sertoli cell co-culture group, SSC conditional culture group and SSC routine culture group. The conditional medium was collected from supernate of culture Sertoli cell in vitro and double-concentrated with DMEM/F12 and fetal bovine serum in a proportion of 4.5 : 4.5 : 1. The routine medium was DMEM/F12 containing 10% fetal bovine serum. Adherence rates were measured by Trypan blue staining. Absorbance of SSCs of each group was measured by MTT assay and proliferation curves shown to demonstrate proliferative features of SSCs. Proliferative features and colony formation were observed by inverted microscope. With 24 h difference in adherence rates, proliferations were compared and analyzed. Results The adherence rate of co-culture group was greater than that in the others (P 0.05). SSCs of co-culture group showed stable proliferation immediately following inoculation. A stable colony formed within 7–10 d and maintained for 30 d. SSCs in conditional culture group and routine group decreased rapidly following transient proliferation. Conclusion The actions of SSCs in Sertoli cell cultures in vitro depended on both juxtacrine and paracrine signaling, Sertoli cell paracrine signaling was unable to promote SSC adherence and proliferation alone.

Cutaneous fungal granulomas due to Alternaria spp. infection in a horse in New Zealand

Equine cutaneous fungal granulomas have been previously referred to in New Zealand (Fairley 1998), and are described in the veterinary literature from around the world, including North America and Australia (Pascoe and Summers 1981; Genovese et al. 2001; Valentine et al. 2006), but no peer-reviewed reports appear published in the literature in New Zealand. Described here is a case of multiple cutaneous fungal granulomas caused by Alternaria spp. in a horse in New Zealand.

Osteogenic differentiation behaviors of mesenehymal stem cells on chitosan based network composites films

Objective To study the osteogenic differentiation capacity of mesenchymal stem cells（MSCs）on chitosan based biomimetic network composites films.Methods The chitosan-based biomimetic network composites films were prepared using chitosan,gelatin and pectin in certain ratio by biomimetic approach.The experiment was designed as follow：experimental group 1 （composites films＋routine medium）,control group 1 （routine medium）,experimental group 2（composites films＋osteogenic medium）,control group 2（osteogenic medium）.The growth and proliferation of MSCs were observed by inverted phase contrast microscope and scanning electronmicroscopy（SEM） and detected through methyl thiazolyl tetrazolium（MTT） assay.Alkaline phosphatase（ALP） activity was used to investigate the osteogenic differentiation capacity.The formation of mineral was determined by special staining and the energy dispersive X-ray apparatus （EDX）.Results MSCs could well adhere,grow and proliferate on the network composites films.The MTT assay demonstrated that there was no significant difference in cell viability between the experimental and control groups （P〉0.05）.SEM observation showed that MSCs grew assembly on the surface of the films secreted a large number of extracelluar matrix,and formed scattered nodules.The cells on the experimental group 1 showed statistically higher ALP activity level than those cells on the other groups （P〈0.01）.Moreover,the calcified nodules can be seen in the experimental groups by Alizarin red and Von Kossa staining.ALP staining showed blue-staining granules within the cytoplasm,and the deposition of Ca and P was detected on the films by SEM-EDX.Conclusion The novel chitosan-based biomimetic network composites films not only have good biocompatibility,but can also improve MSCs differentiation toward the osteogenic lineage. Key words: Chitosan; Network composites film; Mesenchymal stem cells; Viocompatibility; Osteogenic differentiation

E0173 Isolation culture and identification of rat MSC

AimsTo investigate different ways of isolating and culturing rat MSC and different serum concentrations of medium for the best selection.Materials and methodsDirect adherence and density gradient centrifugation methods are used...

The biological performance of cell-containing phospholipid polymer hydrogels in bulk and microscale form

The biological performances of a cell-containing phospholipid polymer hydrogel in bulk and miniaturized formats without an additional culture medium support were investigated and compared. The cell-containing hydrogel was formed spontaneously when solutions of commercial polyvinyl alcohol (PVA) and the phospholipid polymer poly[2-methacryloyloxyethyl phosphorylcholine (MPC)- co- n-butyl methacrylate (BMA)- co- p-vinylphenylboronic acid (VPBA)] (PMBV) suspended with cells in a cell culture medium are mixed together. Bulk and miniaturized hydrogels, with approximate thicknesses of 3.1 mm and 400 μm, respectively, were prepared in a 96-well microplate and a glass microchip, respectively. In both cases, the hydrogels were homogeneous, and cells were spatially encapsulated. The long-term observation (4 and 8 days) of cell morphology suggested that cells were passively attached to the interface of the hydrogel but were unable to spread and flatten, which inhibited cell growth in both hydrogels. Viability evaluations revealed that cells in both hydrogel formats maintained the same high viability levels after long-term encapsulation. Cytotoxicity assays indicated that the cells in the miniaturized hydrogel maintained a high degree of correlation in cytotoxic sensitivity with the cells in the bulk hydrogel and a routine medium culture. The PMBV/PVA hydrogel not only provides a beneficial cytocompatible microenvironment for long-term cell survival without an additional culture medium support but also creates a static condition for cell sustainment in a microchip similar to that in bulk. The uniform long-term performances of PMBV/PVA hydrogels in bulk and miniaturized formats make them ideal for the development of long-term, flexible, three-dimensional, living cell-based tools for routine cell-based assays and applications on bulk to microscale levels.

Characteristics of pathogenic fungi and antifungal therapy in cystic fibrosis

A defective mucociliary clearance facilitates colonization with bacteria and fungal spores in cystic fibrosis patients. Yeasts and molds are cultured from the cystic fibrosis respiratory tract and often their clinical relevance is unknown. Candida spp. are the most commonly isolated yeasts, whereas Aspergillus spp., Scedosporium apiospermum, as well as Exophiala dermatitidis in some countries, are the most frequent molds recovered from respiratory specimens. Molecular biotyping studies have revealed that some fungal genotypes are capable of chronically colonizing the airways. Persistent Aspergillus fumigatus infection is associated with an increased risk of pulmonary exacerbations requiring hospitalization. The prevalence of non-Aspergillus molds may be underestimated due to overgrowth of Pseudomonas and Aspergillus spp. on routine media. Allergic bronchopulmonary aspergillosis is usually treated by oral steroids and an antifungal azole drug. Interactions with the co-medication have to be considered. A small number of antifungal pharmacokinetic studies indicate a high inter-subject variability for itraconazole, voriconazole and posaconazole, and therefore therapeutic drug monitoring is recommended.

A survey on epidemiology of urinary tract infections and resistance pattern of uropathogens in an Iranian 1000-bed tertiary care hospital.

Urinary tract infections (UTIs) are one of the most common infectious diseases diagnosed in communities and hospitalized patients. The aim of this study was to determine frequency of occurrence and antimicrobial susceptibility patterns of uropathogens in Milad hospital of Tehran, Iran. In a prospective study from March to June 2009, a total of 11308 urine sample from patients admitted in Milad hospital of Tehran were analyzed. All specimens were inoculated on routine culture media. Bacterial isolates were identified by conventional bacteriological methods. Susceptibility testing was performed by standard methods as recommended by clinical laboratory standard institute. 11308 urine samples were cultured and 1020 pathogen were isolated. Escherchia coli with 620 (60.78%) isolates was the most common causative agent of UTI followed by Klebsiella pneumoniae with 115 (11.27%) isolates. Among gram positive CocciEnterococcus spp with 110 (10.78%) isolates and Staphylococcus aureus with 81 (7.94%) isolates were predominant organisms. Of 1020 patients, 227 (22.25%) were male and 793 (77.74%) were female. Of 1020 patients, 224 (21.96%) of patients were hospitalized and 796 (78.03%) were outpatients. Of 224 hospitalized patients, 85% of isolates of E. coli were resistant to ampicillin, while this figure was 90% for K. pneumoniae. Resistant to other antibiotics were also prevalent. Nitrofurantoin was the most effective antibiotics against E. coli andEnterococcus spp. In conclusion, our study revealed that bacterial resistance in uropathogens in our hospital continues to be a great problem and needs drug resistance surveillance periodically. Key words: Urinary tract infections, drug resistance.

β-Sheet-dependent Dimerization Is Essential for the Stability of NhaA Na+/H+ Antiporter

A structural model of the NhaA dimer showed that a beta-hairpin of each monomer combines to form a beta-sheet at the periplasmic side of the membrane. By Cys scanning the entire beta-hairpin and testing each Cys replacement for functionality and intermolecular cross-linking, we found that Gln47 and Arg49 are critical for the NhaA dimer and that K57C causes an acidic shift of 1 pH unit to the pH dependence of NhaA. Comparing the growth of the NhaA variants with the previously isolated beta-hairpin deleted mutant (Delta(P45-N58)) and the wild type validated that NhaA dimers have an advantage over monomers in growth under extreme stress conditions and unraveled that during this growth the apparent Km for Na+ of Delta(P45-N58) was increased 50-fold as compared with the wild type. Remarkably, the effect of the extreme stress on the NhaA variants is reversible. Testing the temperature stability (4-55 degrees C) of the NhaA variants in dodecyl maltoside micells showed that the mutants impaired in dimerization were much less temperature-stable than the wild type. We suggest that NhaA dimers are crucial for the stability of the antiporter under extreme stress conditions.

Unusual infections due to Listeria monocytogenes in the Southern California Desert

During the past 22 years, 14 patients have been hospitalized with infection due to Listeria monocytogenes at the Eisenhower Medical Center, a regional 300-bed hospital in the desert southwest of Southern California. A large number of patients are retired, elderly, and have underlying and often systemic disease. Blood agar and routine media were inoculated with liquid from a sterile site such as blood, cerebrospinal fluid, or joint fluid and observed daily for growth. Appropriate biochemical studies were used to speciate the organism. While bacteremia and meningitis constitute 75% of infections in most studies, they made up only 36% of patients in the current study. Listeriosis occurred mostly in patients with infected aortic aneurysms and brain abscesses, and in prosthetic joint infections. While mortality is generally stated to be around 45% in patients with listeriosis, it was 35% in this study. However, there were no deaths in five patients with bacteremia or meningitis inferring that organ involvement poses a greater hazard for survival. Listeriosis usually presents as a bacteremia or meningitis due to a food-borne invasive infection. In the desert of Southern California most cases are seen in older patients with underlying disease and present with infected aortic aneurysms, prosthetic joints, and brain abscesses. They represent a greater threat to survival due to organ involvement.