Insights into hemoglobin assembly through in vivo mutagenesis of α-hemoglobin stabilizing protein

α-Hemoglobin stabilizing protein (AHSP) is believed to facilitate adult Hemoglobin A assembly and protect against toxic free α-globin subunits. Recombinant AHSP binds multiple forms of free α-globin to stabilize their structures and inhibit precipitation. However, AHSP also stimulates autooxidation of αO(2) subunit and its rapid conversion to a partially unfolded bishistidyl hemichrome structure. To investigate these biochemical properties, we altered the evolutionarily conserved AHSP proline 30 in recombinantly expressed proteins and introduced identical mutations into the endogenous murine Ahsp gene. In vitro, the P30W AHSP variant bound oxygenated α chains with 30-fold increased affinity. Both P30W and P30A mutant proteins also caused decreased rates of αO(2) autooxidation as compared with wild-type AHSP. Despite these abnormalities, mice harboring P30A or P30W Ahsp mutations exhibited no detectable defects in erythropoiesis at steady state or during induced stresses. Further biochemical studies revealed that the AHSP P30A and P30W substitutions had minimal effects on AHSP interactions with ferric α subunits. Together, our findings indicate that the ability of AHSP to stabilize nascent α chain folding intermediates prior to hemin reduction and incorporation into adult Hemoglobin A is physiologically more important than AHSP interactions with ferrous αO(2) subunits.     

Heterogeneity across the dorso-ventral axis in zebrafish EVL is regulated by a novel module consisting of sox, snail1a and max genes

In vertebrates, the dorso-ventral (DV) axis is defined by the combinatorial action of localised Wnt, FGF and Nodal signalling along with the antagonizing activities of Chordin and BMP pathways. Our knowledge of the factors that may act in concert with these core pathways to regulate early embryonic patterning is far from complete. Furthermore, while all three germ layers respond to these patterning cues, it is not clear whether in zebrafish the outermost protective epithelium, the enveloping layer (EVL), is also patterned along the DV axis. Here, we have identified a transgenic line driving GFP under a crestin promoter, which specifically labels the dorsal domain of the EVL suggesting heterogeneity in the EVL across the DV axis. Our attempts to understand how the expression from this promoter fragment is regulated specifically in the dorsal domain, have unravelled potential novel players involved in early EVL and embryonic patterning. We show that along with Nodal signalling components, four proteins Sox11b, Sox19b, Snail1a and Max are involved in regulating the size of this EVL domain. However, Chordin-BMP signalling might be dispensable for the dorso-ventral patterning of the EVL. For the first time, this transgenic line unravels the heterogeneity in the EVL and will serve as an important tool in understanding the molecular basis of the DV patterning of the EVL.     

Late-successional biological soil crusts in a biodiversity hotspot: an example of congruency in species richness

Understanding the biodiversity of functionally important communities in Earth’s ecosystems is vital in the apportionment of limited ecosystem management funds and efforts. In southern California shrublands, which lie in a global biodiversity hotspot, biological soil crusts (BSCs) confer critical ecosystem services; however, their biodiversity remains unknown. In this study, six sites (n = 4 each, 25 m²) were established along a mediterranean shrubland environmental gradient in southern California. Here, the biodiversity of all BSC-forming lichens and bryophytes was evaluated, related to environmental traits along the gradient, and compared to species richness among North American ecosystems supporting BSCs (data from previous studies). In total, 59 BSC-forming lichens and bryophytes were observed, including the very rare Sarcogyne crustacea, a rare moss, and five endemic lichen species. Over half (61%) of the species observed were found at a single site. Along the gradient, species evenness of late-successional BSC was related to dew point and elevation, and both evenness and richness were related to distance to coast. Using an ordination analysis, five distinct late-successional BSC communities were identified: Riversidian, Spike moss, Casperian, Alisian, and Lagunian. Twenty-five lichens and 19 bryophytes are newly reported for North American BSC-forming organisms, now comprising ~1/2 of the North American total. BSCs in North American hot and cold deserts were approximately 4.0 and 2.4 times less species rich than BSCs found in southern California shrublands, respectively. Given the anthropogenic impacts on quality and distribution of California mediterranean shrublands, our results show that these sites represent important refugia of BSC species in this globally important region.     

Antiviral activity and synthesis of quaternized promazine derivatives against HSV-1

N-(4-chlorobenzyl)triflupromazinium chloride, a known antitubercular agent, has been found to also be active against HSV-1. A preliminary structure-activity relation has been explored to determine which groups are crucial to viral inhibition. Antiviral assessments such as GFP reduction, plaque reduction, treatment timing and wash-out studies have also been probed to determine a mode of action for QPD-1. Based on this preliminary data, it appears that QPD-1 is a reversible inhibitor, suspected to inhibit early stages of viral replication of HSV-1 at 50μM, equipotent to acyclovir.     

Sutorius: a new genus for Boletus eximius

Sutorius is described as a new genus of Boletaceae to accommodate Boletus robustus originally named illegitimately by C.C. Frost from eastern North America. The legitimate name, Boletus eximius, provided by C.H. Peck, has been used since for a dark purple to chocolate brown bolete with finely scaly stipe and reddish brown spore deposit. This iconic taxon has been documented on five continents. Despite the straightforward species identification from morphology, the interpretation of stipe macro-morphology and spore color has led to equivocal generic placement. Phylogenetic analyses of genes encoding large subunit rRNA and translation elongation factor 1α confirm Sutorius as a unique generic lineage in the Boletaceae. Two species are recognized based on multiple accessions: S. eximius, represented by collections from North America, Costa Rica, Guyana, Indonesia and Japan (molecular data are lacking for only the Guyanan and Japanese material); and S. australiensis, represented by material from Queensland, Australia. Additional collections from Zambia and Thailand represent independent lineages, but sampling is insufficient to describe new species for these entities.     

Overstory and understory vegetation interact to alter soil community composition and activity

Aim

To test if there is an interactive effect between tree and understory species on the soil microbial community (SMC), community level physiological profiles (CLPP) and soil micro-fauna.

Method

A replicate pot experiment with five sapling tree species (Betula pendula, Betula pubescens, Sorbus aucuparia, Quercus petraea and Pinus sylvestris) and a no-tree treatment with and without Calluna vulgaris was established. After 21?months samples were taken for phospholipid fatty acid (PLFA) analysis, CLPP and soil microfauna assessment.

Results

There was an interactive effect of tree species and Calluna on the SMC, CLPP and nematode densities. Calluna addition changed the SMC composition (increase in fungal PLFAs) and the CLPP (lower utilisation of most carbon sources but greater utilisation of phenolic acids). A multivariate test for homogeneity of dispersion showed that while Calluna addition resulted in the presence of an altered microbial composition, it did not result in there being less variability among the samples with Calluna than among the samples without Calluna. Sapling trees with Calluna present grew less well than trees without Calluna. Structural equation modelling showed that it is possible that Calluna had an indirect effect on the SMC via below-ground tree biomass as well as a direct effect.

Conclusion

Interactions between trees and understory vegetation can impact on the composition of soil biota and their activity.     

Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.     

A double-blind, placebo-controlled, randomized, clinical trial of the TLR-3 agonist rintatolimod in severe cases of chronic fatigue syndrome

Background

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a severely debilitating disease of unknown pathogenesis consisting of a variety of symptoms including severe fatigue. The objective of the study was to examine the efficacy and safety of a TLR-3 agonist, rintatolimod (Poly I: C₁₂U), in patients with debilitating CFS/ME.

Methods and Findings

A Phase III prospective, double-blind, randomized, placebo-controlled trial comparing twice weekly IV rintatolimod versus placebo was conducted in 234 subjects with long-standing, debilitating CFS/ME at 12 sites. The primary endpoint was the intra-patient change from baseline at Week 40 in exercise tolerance (ET). Secondary endpoints included concomitant drug usage, the Karnofsky Performance Score (KPS), Activities of Daily Living (ADL), and Vitality Score (SF 36). Subjects receiving rintatolimod for 40 weeks improved intra-patient placebo-adjusted ET 21.3% (p = 0.047) from baseline in an intention-to-treat analysis. Correction for subjects with reduced dosing compliance increased placebo-adjusted ET improvement to 28% (p = 0.022). The improvement observed represents approximately twice the minimum considered medically significant by regulatory agencies. The rintatolimod cohort vs. placebo also reduced dependence on drugs commonly used by patients in an attempt to alleviate the symptoms of CFS/ME (p = 0.048). Placebo subjects crossed-over to receive rintatolimod demonstrated an intra-patient improvement in ET performance at 24 weeks of 39% (p = 0.04). Rintatolimod at 400 mg twice weekly was generally well-tolerated.

Conclusions/Significance

Rintatolimod produced objective improvement in ET and a reduction in CFS/ME related concomitant medication usage as well as other secondary outcomes.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00215800","term_id":"NCT00215800"}}NCT00215800     

Phase I evaluation of intravenous ascorbic acid in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer

Background

Preclinical data support further investigation of ascorbic acid in pancreatic cancer. There are currently insufficient safety data in human subjects, particularly when ascorbic acid is combined with chemotherapy.

Methods and Findings

14 subjects with metastatic stage IV pancreatic cancer were recruited to receive an eight week cycle of intravenous ascorbic acid (three infusions per week), using a dose escalation design, along with standard treatment of gemcitabine and erlotinib. Of 14 recruited subjects enrolled, nine completed the study (three in each dosage tier). There were fifteen non-serious adverse events and eight serious adverse events, all likely related to progression of disease or treatment with gemcitabine or erlotinib. Applying RECIST 1.0 criteria, seven of the nine subjects had stable disease while the other two had progressive disease.

Conclusions

These initial safety data do not reveal increased toxicity with the addition of ascorbic acid to gemcitabine and erlotinib in pancreatic cancer patients. This, combined with the observed response to treatment, suggests the need for a phase II study of longer duration.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00954525","term_id":"NCT00954525"}}NCT00954525