Identification of an elongation factor 1Bγ protein with glutathione transferase activity in both yeast and mycelial morphologies from human pathogenic Blastoschizomyces capitatus

Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.     

Time to reconsider the role of ribavirin in Lassa fever

Ribavirin is the only available Lassa fever treatment. The rationale for using ribavirin is based on one clinical study conducted in the early 1980s. However, reanalysis of previous unpublished data reveals that ribavirin may actually be harmful in some Lassa fever patients. An urgent reevaluation of ribavirin is therefore needed.

Fifty years after its discovery, Lassa fever remains uncontrolled, and mortality remains unacceptably high. Since 2015, Nigeria has been experiencing increasingly large outbreaks of Lassa fever, with new peaks reached in 2016, 2017, and 2018. In 1987, McCormick and colleagues reported a case fatality rate (CFR) of 16.5% among 441 patients hospitalized in Sierra Leone [1]. In Nigeria in 2019, 124 deaths were recorded among 554 laboratory-confirmed cases for a CFR of 22% [2].Ribavirin is the only available Lassa fever–specific treatment and has been used routinely for over 25 years. However, intravenous ribavirin is not licensed for Lassa fever. Its mechanism of action is unclear, it is expensive and hard to source, and it has well-known toxicities [3]. Therefore, the evidence for using ribavirin in Lassa fever deserves careful scrutiny. The emergence of potential new therapeutics for Lassa fever, such as favipiravir and monoclonal antibodies, adds further weight to the case for reconsidering the role of ribavirin since the evaluation of new drugs in clinical trials requires a comparison against existing treatments with a known efficacy and safety profile [4,5].The rationale for using ribavirin in Lassa fever is primarily based on one clinical study conducted in Sierra Leone in the late 1970s and early 1980s. McCormick and colleagues [6] reported that in Lassa fever patients with a serum aspartate aminotransferase (AST) level of ≥150 IU/L, the use of intravenous ribavirin within the first 6 days of illness reduced the fatality rate from 61% (11/18) with no ribavirin to 5% (1/20) (p = 0.002). These authors concluded that ribavirin is effective in the treatment of Lassa fever. However, there are long-standing concerns about the methods used in this study. Although randomization was used to assign patients to treatment groups, the comparisons presented were not according to original randomized groups, and we have reconstructed their derivation (Fig 1). Serious limitations to the comparisons presented include the use of historic controls, inclusion of pregnant women in the control group but their exclusion from the ribavirin group (case fatality is around 2-fold higher in pregnant women than nonpregnant patients), and post hoc merging of treatment groups. Despite this and the fact that the results only supported the use of ribavirin in nonpregnant adult patients with AST ≥150 IU/L, this study is the basis upon which ribavirin is now used in all patients with Lassa fever, including children, pregnant women, and people with normal liver function.Open in a separate windowFig 1Reconstruction of the McCormick et al. data.AST, aspartate aminotransferase; PW, pregnant women. † Discrepancy within McCormick et al, with 39 patients reported treated with oral ribavirin but only 38 (14+24) outcomes reported. ‡ Discrepancy within McCormick et al, with table 1 reporting 12/63 but text reporting 13/62.It has been well known among Lassa specialists that the McCormick study reports a subset of a much larger dataset assembled by the Lassa treatment unit in Sierra Leone and that a report on the full dataset was commissioned by the United States Army Medical Research and Development Command. One of us (PH) therefore submitted a freedom of information (FOI) request to access this report. The full report and an accompanying memo are available, and we encourage readers to access and read the materials [7,8]. The memo states that some of the original trial records were unavailable, and the data should be “interpreted with extreme caution.” Nonetheless, the report presents data from 1977 through to 1991 on 807 Lassa fever patients with a known outcome that were assigned to different ribavirin treatment regimens. These newly available data raise important questions about the safety and efficacy of ribavirin for the treatment of Lassa fever.The original data were lost during the civil war in Sierra Leone, but the report contains tables showing the distribution of characteristics of the whole population according to treatment group, an appendix showing individual data for the 405 patients who died, and results of a logistic regression analysis comparing the effect of ribavirin with no treatment for some of the ribavirin regimens, after adjusting for patient characteristics. Based on these data, we derived aggregated datasets containing the number of deaths according to treatment groups and individual characteristics. We combined groups I (“No treatment given”) and X (“Drugs were not available”) as no treatment and all groups in which ribavirin was administered (II, III, and V to IX) as ribavirin. Exhibit III-8 in the FOI report presented case fatality by treatment group and AST, from which we derived crude odds ratios (ORs) comparing ribavirin with no treatment. The logistic regression reported in Exhibit III-9 was restricted to “those treatment groups that yielded the lowest case fatality rates with respect to untreated patients in the high severity patient illness category” (groups II, III, V, and VII). It was adjusted for age, gender, time to admission, time to treatment, length of stay, and log(AST). We also reconstructed analyses by digitizing the data on individuals who died in Appendix D, calculating the number of deaths according to treatment group and AST, and subtracting these numbers from the totals presented in Exhibit III-2. These allowed us to estimate overall mortality ORs before and after adjusting for ribavirin, although the numbers did not entirely match, and so the number of deaths was reduced in some small groups.Estimates of the effect of oral and intravenous ribavirin from the McCormick study and of all ribavirin from the full report are shown in Fig 2. Based on the crude ORs derived from Exhibit III-8, ribavirin reduced mortality only in patients with serum AST ≥150 IU/L, with less benefit (OR 0.48 [95% CI 0.30 to 0.78]) than reported by McCormick and colleagues. However, ribavirin appeared to increase mortality in patients with serum AST <150 IU/L (2.90 [1.42 to 5.95]). In fact, in our analysis, the only stratum in which ribavirin appeared protective (0.38 [0.21 to 0.70]) was serum AST >300 IU/L (Table H in S1 Text). The logistic regression reported in the FOI report suggested a modest reduction in mortality, but the reasons for the choice of treatment groups compared were unclear. In the reconstructed analyses, ribavirin was associated with overall increased mortality (2.12 [1.67, 2.68]), although this was attenuated after adjustment for AST (1.48 [1.05, 2.08]).Open in a separate windowFig 2Forest plot of the OR of death in treatment and risk subgroups.AST, aspartate aminotransferase; FOI, freedom of information; OR, odds ratio.In our view, there is a compelling case to reevaluate the role of ribavirin in the care of patients with Lassa fever. The data suggest that ribavirin treatment may harm Lassa fever patients with AST <150 IU/L. The limitations revealed by the US Army report, such as large amounts of missing data, unclear treatment allocation practices, imbalances in treatment groups, and errors in coding serology results, cast further doubt on the conclusions of the McCormick study. This aligns with 2 recent systematic reviews by Eberhardt and colleagues and Cheng and colleagues, which concluded that the efficacy of ribavirin in Lassa fever was uncertain because of critical risk of bias in existing studies [9,10].Challenging a quarter of century of clinical practice is difficult. The first step is to acknowledge inadequacies in our knowledge and to ensure that treatment recommendations for Lassa fever better reflect the (weak) strength of evidence for ribavirin in different patient populations. Vigorous efforts should be made to engage clinicians and patients in designing a placebo-controlled trial to assess the safety and efficacy of ribavirin treatment in Lassa fever patients, particularly in those with milder disease (as may be indicated by an admission AST <150 IU/L) in whom the available evidence is compatible with ribavirin causing more harm than good.In conclusion, Lassa fever patients are receiving a drug that may lack efficacy or cause harm. It is incumbent on us to ensure that the next 25 years of Lassa fever treatment are built on more solid foundations.     

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

Recent discoveries have suggested the concept that intracellular signals are the sum of multiple, site‐specified subsignals, rather than single, homogeneous entities. In the context of cancer, searching for compounds that selectively block subsignals essential for tumor progression, but not those regulating “house‐keeping” functions, could help in producing drugs with reduced side effects compared to compounds that block signaling completely. The Ras‐ERK pathway has become a paradigm of how space can differentially shape signaling. Today, we know that Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site‐specific regulatory mechanisms, distinctively engaging effector pathways and switching‐on diverse genetic programs to generate different biological responses. The Ras effector pathway leading to ERKs activation is also under strict, space‐related regulatory processes. These findings may open a gate for aiming at the Ras‐ERK pathway in a spatially restricted fashion, in our quest for new anti‐tumor therapies.     

Interactions between quercetin and Warfarin for albumin binding: A new eye on food/drug interference

The interaction between quercetin, a popular antioxidant flavonoid, and human serum albumin (HSA) is investigated and characterized by means of induced circular dichroism and saturation transfer difference NMR. These techiques demonstrate the reversible binding of quercetin to the carrier protein, which is responsible for its dissolution in aqueous medium. Competition experiments with two classical probes for HSA binding sites, namely Ibuprofen and Warfarin (a common anticoagulant coumarin), demonstrate that quercetin has a primary binding site located in the subdomain IIA, where coumarins are hosted. The affinity for this site is large and we found that quercetin may effectively displace warfarin from HSA. This may have relevant consequences in rationalizing the interferences of common dietary compounds and food supplements to anticoagulant treatments. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Biodiversity of Phototrophic Biofilms Dwelling on Monumental Fountains

Among the stone monumental assets, artistic fountains are particularly affected by microbial colonization due to constant contact with water, giving rise to biodegradation processes related with physical–chemical and aesthetical alterations. In this paper, we make an overview of reported biodiversity of the phototrophic patina developed in various fountains of Italy and Spain. The microbial composition of four fountains (two from Florence, Italy and two from Granada, Spain) was investigated using traditional and/or molecular techniques. The results indicated many common similarities with regard the phototrophic biodiversity for all the investigated fountains. Automated ribosomal RNA intergenic spacer analysis (ARISA), a molecular fingerprint tool, was used to examine the eubacterial and cyanobacterial community for two of the investigated fountains. The principal component analysis of ARISA profiles strengthens the results obtained by traditional methods and revealed separate clusters, as a consequence of the differences of micro-environmental conditions for each fountain.     

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.     

Inoculation of cranberry (Vaccinium macrocarpon) with the ericoid mycorrhizal fungus Rhizoscyphus ericae increases nitrate influx

Despite the ubiquitous presence of ericoid mycorrhizal (ERM) fungi in cranberry (Vaccinium macrocarpon), no prior studies have examined the effect of ERM colonization on NO(3)(-) influx kinetics. Here, (15)NO(3)(-) influx was measured in nonmycorrhizal and mycorrhizal cranberry in hydroponics. Mycorrhizal cranberry were inoculated with the ERM fungus Rhizoscyphus (syn. Hymenoscyphus) ericae. (15)NO(3)(-) influx by R. ericae in solution culture was also measured. Rhizoscyphus ericae NO(3)(-) influx kinetics were linear when mycelium was exposed for 24 h to 3.8 mm NH(4)(+), and saturable when pretreated with 3.8 mm NO(3)(-), 50 microm NO(3)(-), or 50 microm NH(4)(+). Both low-N pretreatments induced greater NO(3)(-) influx than either of the high-N pretreatments. Nonmycorrhizal cranberry exhibited linear NO(3)(-) influx kinetics. By contrast, mycorrhizal cranberry had saturable NO(3)(-) influx kinetics, with c. eightfold greater NO(3)(-) influx than nonmycorrhizal cranberry at NO(3)(-) concentrations from 20 microm to 2 mm. There was no influence of pretreatments on cranberry NO(3)(-) influx kinetics, regardless of mycorrhizal status. Inoculation with R. ericae increased the capacity of cranberry to utilize NO(3)(-)-N. This finding is significant both for understanding the potential nutrient niche breadth of cranberry and for management of cultivated cranberry when irrigation water sources contain nitrate.     

Prostaglandins mediate tonic contraction of the guinea pig and human gallbladder

The gallbladder (GB) maintains tonic contraction modulated by neurohormonal inputs but generated by myogenic mechanisms. The aim of these studies was to examine the role of prostaglandins in the genesis of GB myogenic tension. Muscle strips and cells were treated with prostaglandin agonists, antagonists, cyclooxygenase (COX) inhibitors, and small interference RNA (siRNA). The results show that PGE2, thromboxane A2 (TxA2), and PGF(2alpha) cause a dose-dependent contraction of muscle strips and cells. However, only TxA2 and PGE2 (E prostanoid 1 receptor type) antagonists induced a dose-dependent decrease in tonic tension. A COX-1 inhibitor decreased partially the tonic contraction and TxB2 (TxA2 stable metabolite) levels; a COX-2 inhibitor lowered the tonic contraction partially and reduced PGE2 levels. Both inhibitors and the nonselective COX inhibitor indomethacin abolished the tonic contraction. Transfection of human GB muscle strips with COX-1 siRNA partially lowered the tonic contraction and reduced COX-1 protein expression and TxB2 levels; COX-2 siRNA also partially reduced the tonic contraction, the protein expression of COX-2, and PGE2. Stretching muscle strips by 1, 2, 3, and 4 g increased the active tension, TxB2, and PGE2 levels; a COX-1 inhibitor prevented the increase in tension and TxB2; and a COX-2 inhibitor inhibited the expected rise in tonic contraction and PGE2. Indomethacin blocked the rise in tension and TxB2 and PGE2 levels. We conclude that PGE2 generated by COX-2 and TxA2 generated by COX-1 contributes to the maintenance of GB tonic contraction and that variations in tonic contraction are associated with concomitant changes in PGE2 and TxA2 levels.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.