Transport of amino acids and ammonium in mycelium of Agaricus bisporus.

Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis-Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH(+)(4), with a K(i) of 3.7 microM for the high-velocity system. The K(m) values for glutamate, glutamine and arginine transport were 124, 151 and 32 microM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH(+)(4) concentration. Moreover, increase of the intracellular NH(+)(4) level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH(+)(4) concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH(+)(4) did not inhibit glutamine uptake, suggesting that NH(+)(4) and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed.     

Respiratory pathways in Agaricus bisporus and Scytalidium thermophilum

Abstract The respiratory pathways of Agaricus bisporus and Scytalidium thermophilum were studied. A. bisporus appeared to possess both a cyanide-sensitive and a cyanide-insensitive respiration while in S. thermophilum the cyande-insensitive respiration was absent. Growth experiments showed the ecological advantage for A. bisporus under conditions where cytochrome mediated respiration is inhibited.     

Hyphal and mycelial interactions betweenAgaricus bisporus andScytalidium thermophilum on agar media

The interaction betweenAgaricus bisporus andScytalidium thermophilum on agar media was studied by differential interference contrast and phase contrast microscopy.A. bisporus combatively replacesS. thermophilum in culture on agar media. The antagonistic effect ofA. bisporus is transmissible through a cellophane membrane and causes irreversible disintegration ofS. thermophilum protoplasm, resulting in a total loss of viability after prolonged interaction between the two fungi. On compost extract agar, but not on other media, the growth rate ofA. bisporus increased from 2.7 to 5.3 mm·d^–1 following contact withS. thermophilum mycelium.     

Evolution of Volatile Sulfur Compounds during Laboratory-Scale Incubations and Indoor Preparation of Compost Used as a Substrate in Mushroom Cultivation

Volatile sulfur compounds are known to be produced during the preparation of compost used as a substrate in mushroom cultivation. Because they cause odor problems, attempts have been made to reduce the production of these compounds. The influences of temperature and various additions on the production of volatile sulfur compounds from composting material were tested on laboratory-scale preparations. The production of H₂S, COS, CH₃SH, and (CH₃)₂S was proven to be a biological process with an optimal temperature that coincides with the optimal temperature for biological activity. The formation of CS₂ and (CH₃)₂S₂ was shown to be a nonbiological process. The emission of volatile sulfur compounds during the indoor preparation of mushroom compost appeared to be remarkably reduced (about 90%) as compared with the emission during the conventional outdoor process. Introduction of this indoor composting process would result in a significant reduction in environmental pollution.     

Risk Factors of Treatment-Limiting Anemia after Substitution of Zidovudine for Stavudine in HIV-Infected Adult Patients on Antiretroviral Treatment

Background

Anemia is the main concern among patients using a zidovudine (AZT)-based antiretroviral treatment (ART). Some studies suggested weight-adjusted AZT dosing as a way to reduce toxicity. We analyzed the risk factors associated with AZT-induced anemia in a cohort using AZT as substitution for stavudine (D4T).

Methods

We retrospectively studied HIV-infected patients in a referral hospital in Phnom Penh, Cambodia between 2003 and 2011. Factors associated with AZT-related anemia requiring AZT-discontinuation within the first year after AZT initiation were analyzed using Cox regression.

Results

Overall, 1180 patients, 60.5% female, were included. At AZT initiation, the median hemoglobin was 12.7 g/dL (IQR 11.7–13.9), the median weight: 51 kg (IQR 45–58) and the median time on ART prior to AZT substitution: 1.4 years (IQR 1.0–2.0). Within one year follow-up, 139 patients (11.8%) developed anemia requiring AZT discontinuation. Overall, there was no independent association of body weight with AZT discontinuation. AZT discontinuation was associated with lower hemoglobin level when starting AZT; older age and taking D4T-based ART less than one year prior to AZT. In exploratory analysis, a linear increase in risk of grade 2–4 anemia with lower body weight was seen if starting AZT substitution within less than one year of D4T-based ART.

Conclusion

Our findings argue against the need of weight-based dosing of AZT to reduce anemia among patients using AZT as substitution for D4T. Whether this also applies to ART-naïve individuals remains to be assessed. Future studies with AZT dose reduction should assess efficacy and overall tolerance of AZT.     

Development and growth of fruit bodies and crops of the button mushroom, Agaricus bisporus

We studied the appearance of fruit body primordia, the growth of individual fruit bodies and the development of the consecutive flushes of the crop. Relative growth, measured as cap expansion, was not constant. It started extremely rapidly, and slowed down to an exponential rate with diameter doubling of 1.7 d until fruit bodies showed maturation by veil breaking. Initially many outgrowing primordia were arrested, indicating nutritional competition. After reaching 10 mm diameter, no growth arrest occurred; all growing individuals, whether relatively large or small, showed an exponential increase of both cap diameter and biomass, until veil breaking. Biomass doubled in 0.8 d. Exponential growth indicates the absence of competition. Apparently there exist differential nutritional requirements for early growth and for later, continuing growth. Flushing was studied applying different picking sizes. An ordinary flushing pattern occurred at an immature picking size of 8 mm diameter (picking mushrooms once a day with a diameter above 8 mm). The smallest picking size yielded the highest number of mushrooms picked, confirming the competition and arrested growth of outgrowing primordia: competition seems less if outgrowing primordia are removed early. The flush duration (i.e. between the first and last picking moments) was not affected by picking size. At small picking size, the subsequent flushes were not fully separated in time but overlapped. Within 2 d after picking the first individuals of the first flush, primordia for the second flush started outgrowth. Our work supports the view that the acquisition of nutrients by the mycelium is demand rather than supply driven. For formation and early outgrowth of primordia, indications were found for an alternation of local and global control, at least in the casing layer. All these data combined, we postulate that flushing is the consequence of the depletion of some unknown specific nutrition required by outgrowing primordia.     

Procurement and Supply Management System for MDR-TB in Nigeria: Are the Early Warning Targets for Drug Stock Outs and Over Stock of Drugs Being Achieved?

Background

The World Health Organisation (WHO) introduced the twelve early warning indicators for monitoring and evaluating drug Procurement and Supply management (PSM) systems, intended to prevent drug stock-outs and overstocking. Nigeria- one of the high Multi Drug Resistant Tuberculosis (MDR-TB) burden countries, scaled-up treatment in 2012 with the concurrent implementation of a PSM system.

Method

We evaluated how well this system functioned using the WHO indicators, including all seven MDR-TB treatment centres in the country that were functional throughout 2013.

Results

The quantity of MDR-TB drugs ordered for 2013 matched the annual forecast and all central orders placed during the year were delivered in full and on time. Drug consumption was 81%–106% of the quantity allocated for routine consumption. Timely submission of complete inventory reports ranged from 86–100%, late submissions being 5–15 days late. Forty to 71% of treatment centres placed a drug order when stock was below the minimum level of three months. The proportion of drug orders received at the treatment centres in full and on time ranged from 29–80%, late orders being 1–19 days late.

Conclusion

The PSM was found to be performing well in terms of forecasting and procurement of MDR-TB drugs, but there were shortcomings in drug distribution, reporting at treatment centre level and in drug order placements. Despite these gaps, there were no stock outs. These findings indicate that where it matters most, namely ensuring that no drug stock outs affect patient management, the PSM system is effective. Addressing the observed shortcomings will help to strengthen the existing PSM system in anticipation of a growing MDR-TB case burden in the country.     

Thromboelastometry (TEM) findings in disseminated intravascular coagulation in a pig model of endotoxinemia

Standard coagulation tests have a low specificity and sensitivity for diagnosing disseminated intravascular coagulation. The aim of this study was to determine whether whole blood thromboelastometry (TEM) detects lipopolysaccharide (LPS)-induced changes in coagulation. Blood samples from 10 pigs were drawn at baseline, before and at the end of LPS infusion and 2, 3, 4 and 5 h after the start of endotoxinemia. Simultaneous to TEM, standard coagulation tests and extended coagulation analysis including tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) were performed. Endotoxinemia resulted in a significant acceleration of the nonactivated TEM (NATEM) clotting time 2 h after the end of LPS infusion; in contrast, the changes in international normalized ratio and activated partial thromboplastin time suggested delayed initiation of coagulation. NATEM maximum clot firmness (MCF) and fibrin-based thromboelastometry test (FIBTEM)-MCF decreased significantly from baseline until the last time point (from 64.6 ± 7.8 and 35.1 ± 12.8 mm to 52.8 ± 4.6 and 21.4 ± 11.8 mm, respectively; P = 0.01 for both parameters). A sharp, transient increase of t-PA had no effect on maximum lysis in the NATEM test. PAI-1 increased significantly 3 h after the start of LPS infusion, paralleled by a decrease in maximum lysis. In conclusion, TEM was superior to standard coagulation tests in reflecting initial activation of coagulation during endotoxinemia. TEM further suggested consumption of coagulation substrate; at the same time, inhibition of plasminogen activation was accompanied by improved clot stability. Further investigations are necessary to establish the clinical relevance of these findings.     

Effects of hypothermia and re-warming on the inflammatory response in a murine multiple hit model of trauma

INTRODUCTION: Although, hypothermia is a frequent event after trauma, it is unclear whether its beneficial or detrimental effects are more important. This study aims to quantify the effects of hypothermia and re-warming on the inflammatory response after fracture/hemorrhage and subsequent fracture stabilization with resuscitation. MATERIALS AND METHODS: Eighty-one male C57Bl/6 mice (aged 8-10 weeks, weighing 22.0+/-3.0 g) underwent femoral fracture and hemorrhage followed by resuscitation and splint fixation of the fracture. Animals were sacrificed 3h after induction of hemorrhage and fracture. Besides a sham group (n=6), four experimental groups were created: A: normothermia (n=12), B: hypothermia after trauma (n=21), C: re-warming after resuscitation and before stabilization (n=21), and D: hypothermia before trauma (n=21). Groups B-D were further subdivided into three subgroups according to the degree of hypothermia (subgroup 1: 35-33 degrees C, subgroup 2: 32.9-30.0 degrees C, and subgroup 3: 29.9-27.0 degrees C). Plasma cytokine (TNF-alpha, IL-6, and IL-10) and chemokine (MCP-1) concentrations were determined by ELISA, pulmonary permeability changes were quantified, and histological analysis of lung and liver tissues was performed. RESULTS: Normothermia resulted in a significantly increased early mortality rate. A significantly increased pro-inflammatory and decreased anti-inflammatory responses were also observed in normothermia as compared to hypothermia. The extent of these changes was most pronounced in the severe hypothermic group. Re-warming after mild hypothermia resulted in a pro-inflammatory response comparable to normothermia. CONCLUSION: Hypothermia has a beneficial effect on early survival after trauma, which appears to be independent of the level of hypothermia and re-warming. Re-warming, however, enhanced the pro-inflammatory response. Further studies with a longer posttraumatic observation period are required to investigate the long term effects of the hypothermia and re-warming-induced changes on the pro- and anti-inflammatory responses.     

Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate (DCA) on THP-1 cells: a multiparameter analysis

Two human leukemia cells K562 and THP-1, the breast cancer lines MCF-7 and ZR-75-1, and the melanoma line MDA-MB-435S were compared by flowcytometry for their behaviour at increasing levels of 3BP. K562 and THP-1 responded to 3BP by membrane depolarization and increased ROS; MCF-7 and ZR-75-1 showed decreased polarization and low ROS increase; MDA-MB-435S had limited depolarization and no ROS increase. THP-1 cells exposed to a range of 3BP concentrations in combination with DCA showed increase of polarization, slight ROS increase, and weakened nuclear integrity. 3BP and DCA show no synergism, but have complementary destructive effects on THP-1 cells. The data led to the conclusion that the THP-1 cells do not carry a functional membrane monocarboxylate transporter (MCT) or that 3BP circumvents MCT binding and can enter these cells independently.