nMAT4, a maturase factor required for nad1 pre‐mRNA processing and maturation,is essential for holocomplex I biogenesis in Arabidopsis mitochondria

Group II introns are large catalytic RNAs that are found in bacteria and organellar genomes of lower eukaryotes, but are particularly prevalent within mitochondria in plants, where they are present in many critical genes. The excision of plant mitochondrial introns is essential for respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self‐splicing ribozyme and its own intron‐encoded maturase protein. A hallmark of maturases is that they are intron‐specific, acting as cofactors that bind their intron‐containing pre‐RNAs to facilitate splicing. However, the degeneracy of the mitochondrial introns in plants and the absence of cognate intron‐encoded maturase open reading frames suggest that their splicing in vivo is assisted by ‘trans’‐acting protein factors. Interestingly, angiosperms harbor several nuclear‐encoded maturase‐related (nMat) genes that contain N‐terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. Here we show that nMAT4 (At1g74350) is required for RNA processing and maturation of nad1 introns 1, 3 and 4 in Arabidopsis mitochondria. Seed germination, seedling establishment and development are strongly affected in homozygous nmat4 mutants, which also show modified respiration phenotypes that are tightly associated with complex I defects.     

Comparable Long-Term Efficacy of Lopinavir/Ritonavir and Similar Drug-Resistance Profiles in Different HIV-1 Subtypes

Background

Analysis of potentially different impact of Lopinavir/Ritonavir (LPV/r) on non-B subtypes is confounded by dissimilarities in the conditions existing in different countries. We retrospectively compared its impact on populations infected with subtypes B and C in Israel, where patients infected with different subtypes receive the same treatment.

Methods

Clinical and demographic data were reported by physicians. Resistance was tested after treatment failure. Statistical analyses were conducted using SPSS.

Results

607 LPV/r treated patients (365 male) were included. 139 had HIV subtype B, 391 C, and 77 other subtypes. At study end 429 (71%) were receiving LPV/r. No significant differences in PI treatment history and in median viral-load (VL) at treatment initiation and termination existed between subtypes. MSM discontinued LPV/r more often than others even when the virologic outcome was good (p = 0.001). VL was below detection level in 81% of patients for whom LPV/r was first PI and in 67% when it was second (P = 0.001). Median VL decrease from baseline was 1.9±0.1 logs and was not significantly associated with subtype. Median CD4 increase was: 162 and 92cells/µl, respectively, for patients receiving LPV/r as first and second PI (P = 0.001), and 175 and 98, respectively, for subtypes B and C (P<0.001). Only 52 (22%) of 237 patients genotyped while under LPV/r were fully resistant to the drug; 12(5%) were partially resistant. In48%, population sequencing did not reveal resistance to any drug notwithstanding the virologic failure. No difference was found in the rates of resistance development between B and C (p = 0.16).

Conclusions

Treatment with LPV/r appeared efficient and tolerable in both subtypes, B and C, but CD4 recovery was significantly better in virologically suppressed subtype-B patients. In both subtypes, LPV/r was more beneficial when given as first PI. Mostly, reasons other than resistance development caused discontinuation of treatment.     

Maximal Sum of Metabolic Exchange Fluxes Outperforms Biomass Yield as a Predictor of Growth Rate of Microorganisms

Growth rate has long been considered one of the most valuable phenotypes that can be measured in cells. Aside from being highly accessible and informative in laboratory cultures, maximal growth rate is often a prime determinant of cellular fitness, and predicting phenotypes that underlie fitness is key to both understanding and manipulating life. Despite this, current methods for predicting microbial fitness typically focus on yields [e.g., predictions of biomass yield using GEnome-scale metabolic Models (GEMs)] or notably require many empirical kinetic constants or substrate uptake rates, which render these methods ineffective in cases where fitness derives most directly from growth rate. Here we present a new method for predicting cellular growth rate, termed SUMEX, which does not require any empirical variables apart from a metabolic network (i.e., a GEM) and the growth medium. SUMEX is calculated by maximizing the SUM of molar EXchange fluxes (hence SUMEX) in a genome-scale metabolic model. SUMEX successfully predicts relative microbial growth rates across species, environments, and genetic conditions, outperforming traditional cellular objectives (most notably, the convention assuming biomass maximization). The success of SUMEX suggests that the ability of a cell to catabolize substrates and produce a strong proton gradient enables fast cell growth. Easily applicable heuristics for predicting growth rate, such as what we demonstrate with SUMEX, may contribute to numerous medical and biotechnological goals, ranging from the engineering of faster-growing industrial strains, modeling of mixed ecological communities, and the inhibition of cancer growth.     

Experimental Myocardial Infarction Induces Altered Regulatory T Cell Hemostasis,and Adoptive Transfer Attenuates Subsequent Remodeling

Background

Ischemic cardiac damage is associated with upregulation of cardiac pro-inflammatory cytokines, as well as invasion of lymphocytes into the heart. Regulatory T cells (Tregs) are known to exert a suppressive effect on several immune cell types. We sought to determine whether the Treg pool is influenced by myocardial damage and whether Tregs transfer and deletion affect cardiac remodeling.

Methods and Results

The number and functional suppressive activity of Tregs were assayed in mice subjected to experimental myocardial infarction. The numbers of splenocyte-derived Tregs in the ischemic mice were significantly higher after the injury than in the controls, and their suppressive properties were significantly compromised. Compared with PBS, adoptive Treg transfer to mice with experimental infarction reduced infarct size and improved LV remodeling and functional performance by echocardiography. Treg deletion with blocking anti-CD25 antibodies did not influence infarct size or echocardiographic features of cardiac remodeling.

Conclusion

Treg numbers are increased whereas their function is compromised in mice with that underwent experimental infarction. Transfer of exogeneous Tregs results in attenuation of myocardial remodeling whereas their ablation has no effect. Thus, Tregs may serve as interesting potential interventional targets for attenuating left ventricular remodeling.     

To dare or not to dare? Risk management by owls in a predator–prey foraging game

In a foraging game, predators must catch elusive prey while avoiding injury. Predators manage their hunting success with behavioral tools such as habitat selection, time allocation, and perhaps daring—the willingness to risk injury to increase hunting success. A predator’s level of daring should be state dependent: the hungrier it is, the more it should be willing to risk injury to better capture prey. We ask, in a foraging game, will a hungry predator be more willing to risk injury while hunting? We performed an experiment in an outdoor vivarium in which barn owls (Tyto alba) were allowed to hunt Allenby’s gerbils (Gerbillus andersoni allenbyi) from a choice of safe and risky patches. Owls were either well fed or hungry, representing the high and low state, respectively. We quantified the owls’ patch use behavior. We predicted that hungry owls would be more daring and allocate more time to the risky patches. Owls preferred to hunt in the safe patches. This indicates that owls manage risk of injury by avoiding the risky patches. Hungry owls doubled their attacks on gerbils, but directed the added effort mostly toward the safe patch and the safer, open areas in the risky patch. Thus, owls dared by performing a risky action—the attack maneuver—more times, but only in the safest places—the open areas. We conclude that daring can be used to manage risk of injury and owls implement it strategically, in ways we did not foresee, to minimize risk of injury while maximizing hunting success.     

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.     

Use of In Vitro and Predictive In Silico Models to Study the Inhibition of Cytochrome P4503A by Stilbenes

CYP3A4 is recognized as the main enzyme involved in the metabolism of drugs and xenobiotics in the human body and its inhibition may lead to undesirable consequences. Stilbenes, including resveratrol, belong to a group of dietary health-promoting compounds that also act as inhibitors of CYP3A4. The aim of this study was to examine the use of computer modeling of enzyme-ligand interactions to analyze and predict the inhibition of structurally related compounds. To this end, an aldehyde group was attached to resveratrol and the interactions of CYP3A4 with resveratrol, its aldehyde analogue (RA) and a known synthetic inhibitor were studied and compared in two biological models. Specifically, the metabolism of testosterone was examined in a human intestine cell line (Caco-2/TC7) and in rat liver microsomes (RLM). The results demonstrated a weak inhibitory effect of RA on CYP3A4, as compared to resveratrol itself, in both biological models. Human CYP3A4 was more susceptible to inhibition than the commonly used model isozyme from rat. Modeling of the binding site of CYP3A4 revealed a combination of three types of interactions: hydrophobic interactions, electrostatic interactions and hydrogen bonds. A docking simulation revealed that the RA lacked an important binding feature, as compared to resveratrol, and that that difference may be responsible for its lower level of affinity for CYP3A4. Software analysis of binding affinity may serve as a predictive tool for designing new therapeutic compounds in terms of inhibition of CYP3A4 and help to reveal the biochemical nature of the interactions of dietary compounds, herbal compounds and drugs whose metabolism is mediated by this enzyme.     

Carbohydrate active enzyme domains from extreme thermophiles: components of a modular toolbox for lignocellulose degradation

Lignocellulosic biomass is a promising feedstock for the manufacture of biodegradable and renewable bioproducts. However, the complex lignocellulosic polymeric structure of woody tissue is difficult to access without extensive industrial pre-treatment. Enzyme processing of partly depolymerised biomass is an established technology, and there is evidence that high temperature (extremely thermophilic) lignocellulose degrading enzymes [carbohydrate active enzymes (CAZymes)] may enhance processing efficiency. However, wild-type thermophilic CAZymes will not necessarily be functionally optimal under industrial pre-treatment conditions. With recent advances in synthetic biology, it is now potentially possible to build CAZyme constructs from individual protein domains, tailored to the conditions of specific industrial processes. In this review, we identify a ‘toolbox’ of thermostable CAZyme domains from extremely thermophilic organisms and highlight recent advances in CAZyme engineering which will allow for the rational design of CAZymes tailored to specific aspects of lignocellulose digestion.

     

The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization

Magnetospirillum gryphiswaldense MSR‐1 synthesizes membrane‐enclosed magnetite (Fe₃O₄) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome‐associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome‐directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi‐disciplinary approach to define the role of MamB during magnetosome formation. Using site‐directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo‐electron tomography, we show that MamB is most likely an active magnetosome‐directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport‐independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C‐terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation.