Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates

The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA‐barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4‐fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate‐derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance.     

The central role of selenium in the biochemistry and ecology of the harmful pelagophyte,Aureococcus anophagefferens

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95% during the peak of blooms to 0.05 nℳ. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.     

The Outcome of Patients with Mild Stroke Improves after Treatment with Systemic Thrombolysis

Introduction

In up to one third of patients with mild stroke suitable to receive systemic thrombolysis the treatment is not administered because the treating physicians estimate a good spontaneous recovery. However, it is not settled whether the fate of these patients is equivalent to those who are thrombolysed.

Methods

We analyzed 203 consecutive patients (134 men and 69 women, mean age 69±14 years) without premorbid disability and a NIHSS score ≤5 at admission [median 3 (IQR 2–4)]. Intravenous thrombolysis was administered within 4.5 hours from stroke onset (n = 119), or it was withheld (n = 84) whenever the treating physician predicted a spontaneous recovery. The baseline risk factors, clinical course, infarction volume, bleeding complications, and functional outcome at 3 months were analyzed and declared to a Web-based registry which was accessible to the local Health Authorities.

Results

Expectedly, not thrombolysed patients had the mildest strokes at admission [median 2 (IQR 1–3.75)]. At day 2 to 5, the infarct volume on DWI-MRI was similar in both groups. There were no symptomatic cerebral bleedings in the study. An ordinal regression model adjusted for baseline stroke severity showed that thrombolysis was associated with a greater proportion of patients who shifted down on the modified Rankin Scale score at 3 months (OR 2.66; 95% CI 1.49–4.74, p = 0.001).

Conclusions

Intravenous thrombolysis seems to be safe in patients with mild stroke and may be associated with improved outcome compared with untreated patients. These results support the evaluation of the efficacy of intravenous thrombolysis in mild stroke patients in randomized clinical trials.     

Construction of Improved Tools for Protein Localization Studies in Streptococcus pneumoniae

We have constructed a set of plasmids that allow efficient expression of both N- and C-terminal fusions of proteins of interest to fluorescent proteins mCherry, Citrine, CFP and GFP in the Gram-positive pathogen Streptococcus pneumoniae. In order to improve expression of the fluorescent fusions to levels that allow their detection by fluorescence microscopy, we have introduced a 10 amino acid tag, named i-tag, at the N-terminal end of the fluorescent proteins. This caused increased expression due to improved translation efficiency and did not interfere with the protein localization in pneumococcal bacteria. Localizing fluorescent derivatives of FtsZ, Wzd and Wze in dividing bacteria validated the developed tools. The availability of the new plasmids described in this work should greatly facilitate studies of protein localization in an important clinical pathogen.     

MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.     

Cliffs Used as Communal Roosts by Andean Condors Protect the Birds from Weather and Predators

The quality and availability of resources influence the geographical distribution of species. Social species need safe places to rest, meet, exchange information and obtain thermoregulatory benefits, but those places may also serve other important functions that have been overlooked in research. We use a large soaring bird that roosts communally in cliffs, the Andean condor (Vultur gryphus), as a model species to elucidate whether roost locations serve as a refuge from adverse weather conditions (climatic refuge hypothesis, CRH), and/or from predators or anthropogenic disturbances (threats refuge hypothesis, TRH). The CRH predicts that communal roosts will face in the opposite direction from where storms originate, and will be located in climatically stable, low precipitation areas. The TRH predicts that communal roosts will be large, poorly accessible cliffs, located far from human-made constructions. We surveyed cliffs used as communal roosts by condors in northwestern Patagonia, and compared them with alternative non-roosting cliffs to test these predictions at local and regional scales. We conclude that communal roosting places provide refuge against climate and disturbances such as, for instance, the threats of predators (including humans). Thus, it is not only the benefits gained from being aggregated per se, but the characteristics of the place selected for roosting that may both be essential for the survival of the species. This should be considered in management and conservation plans given the current scenario of global climate change and the increase in environmental disturbances.     

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

Alzheimer''s disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.