Adeno-associated virus gene delivery of broadly neutralizing antibodies as prevention and therapy against HIV-1

Vectored gene delivery of HIV-1 broadly neutralizing antibodies (bNAbs) using recombinant adeno-associated virus (rAAV) is a promising alternative to conventional vaccines for preventing new HIV-1 infections and for therapeutically suppressing established HIV-1 infections. Passive infusion of single bNAbs has already shown promise in initial clinical trials to temporarily decrease HIV-1 load in viremic patients, and to delay viral rebound from latent reservoirs in suppressed patients during analytical treatment interruptions of antiretroviral therapy. Long-term, continuous, systemic expression of such bNAbs could be achieved with a single injection of rAAV encoding antibody genes into muscle tissue, which would bypass the challenges of eliciting such bNAbs through traditional vaccination in naïve patients, and of life-long repeated passive transfers of such biologics for therapy. rAAV delivery of single bNAbs has already demonstrated protection from repeated HIV-1 vaginal challenge in humanized mouse models, and phase I clinical trials of this approach are underway. Selection of which individual, or combination of, bNAbs to deliver to counter pre-existing resistance and the rise of escape mutations in the virus remains a challenge, and such choices may differ depending on use of this technology for prevention versus therapy.     

The mitochondrial phosphatase PPTC7 orchestrates mitochondrial metabolism regulating coenzyme Q10 biosynthesis

Coenzyme Q₁₀ (CoQ₁₀) is a redox molecule critical for the proper function of energy metabolism and antioxidant defenses. Despite its essential role in cellular metabolism, the regulation of CoQ₁₀ biosynthesis in humans remains mostly unknown. Herein, we determined that PPTC7 is a regulatory protein of CoQ₁₀ biosynthesis required for human cell survival. We demonstrated by in vitro approaches that PPTC7 is a bona fide protein phosphatase that dephosphorylates the human COQ7. Expression modulation experiments determined that human PPTC7 dictates cellular CoQ₁₀ content. Using two different approaches (PPTC7 over-expression and caloric restriction), we demonstrated that PPTC7 facilitates and improves the human cell adaptation to respiratory conditions. Moreover, we determined that the physiological role of PPTC7 takes place in the adaptation to starvation and pro-oxidant conditions, facilitating the induction of mitochondrial metabolism while preventing the accumulation of ROS. Here we unveil the first post-translational mechanism regulating CoQ₁₀ biosynthesis in humans and propose targeting the induction of PPTC7 activity/expression for the treatment of CoQ₁₀-related mitochondrial diseases.     

Dynamic modelling of limitations on improving leaf CO2 assimilation under fluctuating irradiance

A dynamic model of leaf CO₂ assimilation was developed as an extension of the canonical steady‐state model, by adding the effects of energy‐dependent non‐photochemical quenching (qE), chloroplast movement, photoinhibition, regulation of enzyme activity in the Calvin cycle, metabolite concentrations, and dynamic CO₂ diffusion. The model was calibrated and tested successfully using published measurements of gas exchange and chlorophyll fluorescence on Arabidopsis thaliana ecotype Col‐0 and several photosynthetic mutants and transformants affecting the regulation of Rubisco activity (rca‐2 and rwt43), non‐photochemical quenching (npq4‐1 and npq1‐2), and sucrose synthesis (spsa1). The potential improvements on CO₂ assimilation under fluctuating irradiance that can be achieved by removing the kinetic limitations on the regulation of enzyme activities, electron transport, and stomatal conductance were calculated in silico for different scenarios. The model predicted that the rates of activation of enzymes in the Calvin cycle and stomatal opening were the most limiting (up to 17% improvement) and that effects varied with the frequency of fluctuations. On the other hand, relaxation of qE and chloroplast movement had a strong effect on average low‐irradiance CO₂ assimilation (up to 10% improvement). Strong synergies among processes were found, such that removing all kinetic limitations simultaneously resulted in improvements of up to 32%.     

Complex selection on a regulator of social cognition: Evidence of balancing selection,regulatory interactions and population differentiation in the prairie vole Avpr1a locus

Adaptive variation in social behaviour depends upon standing genetic variation, but we know little about how evolutionary forces shape genetic diversity relevant to brain and behaviour. In prairie voles (Microtus ochrogaster), variants at the Avpr1a locus predict expression of the vasopressin 1a receptor in the retrosplenial cortex (RSC), a brain region that mediates spatial and contextual memory; cortical V1aR abundance in turn predicts diversity in space use and sexual fidelity in the field. To examine the potential contributions of adaptive and neutral forces to variation at the Avpr1a locus, we explore sequence diversity at the Avpr1a locus and throughout the genome in two populations of wild prairie voles. First, we refine results demonstrating balancing selection at the locus by comparing the frequency spectrum of variants at the locus to a random sample of the genome. Next, we find that the four single nucleotide polymorphisms that predict high V1aR expression in the RSC are in stronger linkage disequilibrium than expected by chance despite high recombination among intervening variants, suggesting that epistatic selection maintains their association. Analysis of population structure and a haplotype network for two populations revealed that this excessive LD was unlikely to be due to admixture alone. Furthermore, the two populations differed considerably in the region shown to be a regulator of V1aR expression despite the extremely low levels of genomewide genetic differentiation. Together, our data suggest that complex selection on Avpr1a locus favours specific combinations of regulatory polymorphisms, maintains the resulting alleles at population‐specific frequencies, and may contribute to unique patterns of spatial cognition and sexual fidelity among populations.     

Compromising the plasma membrane as a secondary target in photodynamic therapy-induced necrosis

Photodynamic therapy (PDT) is a non-invasive treatment widely applied to different cancers. The goal of PDT is the photo-induced destruction of cancer cells by the activation of different cell death mechanisms, including apoptosis and/or necrosis. Recent efforts focusing on understanding the mechanisms of cell death activated by PDT find that it depends on the type of photosensitizer (PS), targeted organelles, and nature of the light used. It is generally accepted that very short incubation times are required to direct the PS to the plasma membrane (PM), while longer periods result in the accumulation of the PS in internal compartments such as the endoplasmic reticulum or mitochondria. Glycosylation of the PS targets cancer via saccharide receptors on the cell surface, and is generally assumed that these compounds rapidly internalize and accumulate, e.g. in the endoplasmic reticulum. Herein we demonstrate that a minor fraction of a glycosylated chlorin compound residing at the PM of cancer cells can activate necrosis upon illumination by compromising the PM independently of the length of the incubation period. The results presented here show that the PM can also be targeted by glycosylated PS designed to accumulate in internal organelles. PS activation to induce necrosis by compromising the plasma membrane has the benefits of fast cell death and shorter irradiation times. The findings described here expand our understanding of the cellular damage induced by phototherapies, presenting the possibility of activating another cell death mechanism based on the incubation time and type of light used.     

The many faces of Peridinium cinctum (Peridiniaceae,Peridiniales): morphological and molecular variability in a common dinophyte

Peridinium cinctum is a common freshwater dinophyte with a long history of research. Erich Lindemann was the first to assess intraspecific variability in this species focusing on plate pattern variation. Since then, this issue has been neglected but with the application of DNA sequence diagnostics, a combination of morphological and molecular characters may enable taxonomic delimitations. Our aim was to identify distinct morphotypes using plate pattern as the main characteristic and then compare them to the geographic occurrence of particular ribotypes (as inferred from sequences of the Internal Transcribed Spacer: ITS) in samples from Central Europe. Approximately 200 observations were carried out under the inverse light microscope for each of a total of 15 strains. We observed two main variations from the abundant plate pattern in P. cinctum, namely an unusual position of the 2a plate and the irregular shape of the 1a plate. In 88 (predominantly clonal) strains, we identified five different ribotypes (submitted as 71 new GenBank entries) which had no clear correlation to the defined morphotypes and/or spatial occurrences. In four cases, we detected two distinct ribotypes at the same locality. However, samples collected south of the Danube River presented a different predominant morphotype from the rest of the samples, thus implying a potential biogeographic signal as inferred from morphology. In general, there is morphological and molecular variability in P. cinctum, which is under-studied and which may uncover geographic or ecological correlations or even the existence of cryptic species.     

4-N-Alkanoyl and 4-N-alkyl gemcitabine analogues with NOTA chelators for 68-gallium labelling

The conjugation of 4-N-(3-aminopropanyl)-2′-deoxy-2′,2′-difluorocytidine with 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) ligand in 0.1?M Na₂CO₃ buffer (pH 11) at ambient temperature provided 4-N-alkylgemcitabine-NOTA chelator. Incubation of latter with excess of gallium(III) chloride (GaCl₃) (0.6?N AcONa/H₂O, pH?=?9.3) over 15?min gave gallium 4-N-alkylgemcitabine-NOTA complex which was characterized by HRMS. Analogous [⁶⁸Ga]-complexation of 4-N-alkylgemcitabine-NOTA conjugate proceeded with high labeling efficiency (94%–96%) with the radioligand almost exclusively found in the aqueous layer (～95%). The high polarity of the gallium 4-N-alkylgemctiabine-NOTA complex resulted in rapid renal clearance of the ⁶⁸Ga-labelled radioligand in BALB/c mice.