Replication and partitioning of the apicoplast genome of Toxoplasma gondii is linked to the cell cycle and requires DNA polymerase and gyrase

Apicomplexans are the causative agents of numerous important infectious diseases including malaria and toxoplasmosis. Most of them harbour a chloroplast-like organelle called the apicoplast that is essential for the parasites’ metabolism and survival. While most apicoplast proteins are nuclear encoded, the organelle also maintains its own genome, a 35 kb circle. In this study we used Toxoplasma gondii to identify and characterise essential proteins involved in apicoplast genome replication and to understand how apicoplast genome segregation unfolds over time. We demonstrated that the DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the apicoplast. We show in knockdown experiments that apicoplast DNA Gyrase A and B, and Prex are required for apicoplast genome replication and growth of the parasite. Analysis of apicoplast genome replication by structured illumination microscopy in T. gondii tachyzoites showed that apicoplast nucleoid division and segregation initiate at the beginning of S phase and conclude during mitosis. Thus, the replication and division of the apicoplast nucleoid is highly coordinated with nuclear genome replication and mitosis. Our observations highlight essential components of apicoplast genome maintenance and shed light on the timing of this process in the context of the overall parasite cell cycle.     

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.     

Potent Anti-Candida Fraction Isolated from Capsicum chinense Fruits Contains an Antimicrobial Peptide That is Similar to Plant Defensin and is Able to Inhibit the Activity of Different α-Amylase Enzymes

Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 μg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.

     

Short Communication

Anaerobic metabolism and oxygen carrying-capacity of white shrimp (Penaeus vannamei) exposed to short term (three days) and long term (two weeks) moderate hypoxia (2-2.6 mg/L) was investigated. Glucose and lactate levels in hemolymph increased under both hypoxic conditions, indicating an activation of anaerobic pathways during the two-weeks exposure period. In muscle, no differences of glucose and lactate levels were observed between the control group and the exposed groups. In animals exposed to hypoxia for two weeks, hemocyanin and copper in hemolymph were higher than in animals under normoxic conditions or exposed for three days. These results indicate that an increase in oxygen carrying-capacity in shrimp is evident only after a sustained condition of hypoxia. Copper levels in the hepatopancreas decreased in both hypoxic conditions, suggesting a mobilization of copper stores for hemocyanin synthesis. These results indicate that penaeid shrimp can tolerate moderate hypoxic conditions by physiological adaptations, such as anaerobic metabolism and increased oxygen carrying-capacity. These adaptations require an adequate dietary supply of proteins and copper for hemocyanin synthesis and of carbohydrates for anaerobic metabolism.     

Surface EMG-force modelling for the biceps brachii and its experimental evaluation during isometric isotonic contractions

The aim of this study was to evaluate a surface electromyography (sEMG) signal and force model for the biceps brachii muscle during isotonic isometric contractions for an experimental set-up as well as for a simulation. The proposed model includes a new rate coding scheme and a new analytical formulation of the muscle force generation. The proposed rate coding scheme supposes varying minimum and peak firing frequencies according to motor unit (MU) type (I or II). Practically, the proposed analytical mechanogram allows us to tune the force contribution of each active MU according to its type and instantaneous firing rate. A subsequent sensitivity analysis using a Monte Carlo simulation allows deducing optimised input parameter ranges that guarantee a realistic behaviour of the proposed model according to two existing criteria and an additional one. In fact, this proposed new criterion evaluates the force generation efficiency according to neural intent. Experiments and simulations, at varying force levels and using the optimised parameter ranges, were performed to evaluate the proposed model. As a result, our study showed that the proposed sEMG–force modelling can emulate the biceps brachii behaviour during isotonic isometric contractions.     

Arabidopsis RECEPTOR-LIKE PROTEIN30 and Receptor-Like Kinase SUPPRESSOR OF BIR1-1/EVERSHED Mediate Innate Immunity to Necrotrophic Fungi

Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called SCLEROTINIA CULTURE FILTRATE ELICITOR1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to RECEPTOR-LIKE PROTEIN30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1) and SUPPRESSOR OF BIR1-1/EVERSHED (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.     

The Deubiquitinating Enzyme AMSH1 and the ESCRT-III Subunit VPS2.1 Are Required for Autophagic Degradation in Arabidopsis

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit VACUOLAR PROTEIN SORTING2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.