CHF5074 restores visual memory ability and pre‐synaptic cortical acetylcholine release in pre‐plaque Tg2576 mice

CHF5074, a new microglial modulator, attenuates memory deficit in Alzheimer's disease transgenic mice. In this study, the effect of an acute or subacute CHF5074 treatment on in vivo novel object recognition test and on [³H]Acetylcholine (ACh) and GABA release in pre‐plaque (7‐month‐old) Tg2576 mice have been compared with those induced by the γ‐secretase inhibitor LY450139 (semagacestat). Vehicle‐treated Tg2576 mice displayed an impairment of recognition memory compared with wild‐type animals. This impairment was recovered in transgenic animals acutely treated with CHF5074 (30 mg/kg), while LY450139 (1, 3, 10 mg/kg) was ineffective. In frontal cortex synaptosomes from vehicle‐treated Tg2576 mice, K⁺‐evoked [³H]ACh release was lower than that measured in wild‐type mice. This reduction was absent in transgenic animals subacutely treated with CHF5074 (30 mg/kg daily for 8 days), while it was slightly, not significantly, amplified by LY450139 (3 mg/kg daily for 8 days). There were no differences between the groups on spontaneous [³H]ACh release as well as spontaneous and K⁺‐evoked GABA release. These results suggest that CHF5074 has beneficial effects on visual memory and cortical cholinergic dysfunctions in pre‐plaque Tg2576 mice. Together with previous findings, these data suggest that CHF5074 could be a possible candidate for early Alzheimer's disease therapeutic regimens.     

Insight into Pleiotropic Drug Resistance ATP-binding Cassette Pump Drug Transport through Mutagenesis of Cdr1p Transmembrane Domains

The fungal ATP-binding cassette (ABC) transporter Cdr1 protein (Cdr1p), responsible for clinically significant drug resistance, is composed of two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs). We have probed the nature of the drug binding pocket by performing systematic mutagenesis of the primary sequences of the 12 transmembrane segments (TMSs) found in the TMDs. All mutated proteins were expressed equally well and localized properly at the plasma membrane in the heterologous host Saccharomyces cerevisiae, but some variants differed significantly in efflux activity, substrate specificity, and coupled ATPase activity. Replacement of the majority of the amino acid residues with alanine or glycine yielded neutral mutations, but about 42% of the variants lost resistance to drug efflux substrates completely or selectively. A predicted three-dimensional homology model shows that all the TMSs, apart from TMS4 and TMS10, interact directly with the drug-binding cavity in both the open and closed Cdr1p conformations. However, TMS4 and TMS10 mutations can also induce total or selective drug susceptibility. Functional data and homology modeling assisted identification of critical amino acids within a drug-binding cavity that, upon mutation, abolished resistance to all drugs tested singly or in combinations. The open and closed Cdr1p models enabled the identification of amino acid residues that bordered a drug-binding cavity dominated by hydrophobic residues. The disposition of TMD residues with differential effects on drug binding and transport are consistent with a large polyspecific drug binding pocket in this yeast multidrug transporter.     

Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological–chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.     

Ancient Substructure in Early mtDNA Lineages of Southern Africa

Among the deepest-rooting clades in the human mitochondrial DNA (mtDNA) phylogeny are the haplogroups defined as L0d and L0k, which are found primarily in southern Africa. These lineages are typically present at high frequency in the so-called Khoisan populations of hunter-gatherers and herders who speak non-Bantu languages, and the early divergence of these lineages led to the hypothesis of ancient genetic substructure in Africa. Here we update the phylogeny of the basal haplogroups L0d and L0k with 500 full mtDNA genome sequences from 45 southern African Khoisan and Bantu-speaking populations. We find previously unreported subhaplogroups and greatly extend the amount of variation and time-depth of most of the known subhaplogroups. Our major finding is the definition of two ancient sublineages of L0k (L0k1b and L0k2) that are present almost exclusively in Bantu-speaking populations from Zambia; the presence of such relic haplogroups in Bantu speakers is most probably due to contact with ancestral pre-Bantu populations that harbored different lineages than those found in extant Khoisan. We suggest that although these populations went extinct after the immigration of the Bantu-speaking populations, some traces of their haplogroup composition survived through incorporation into the gene pool of the immigrants. Our findings thus provide evidence for deep genetic substructure in southern Africa prior to the Bantu expansion that is not represented in extant Khoisan populations.     

A plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus

Structure, size, physicochemical properties and production strategies make many plant viruses ideal protein based nanoscaffolds, nanocontainers and nano-building blocks expected to deliver a multitude of applications in different fields such as biomedicine, pharmaceutical chemistry, separation science, catalytic chemistry, crop pest control and biomaterials science. Functionalization of viral nanoparticles through modification by design of their external and internal surfaces is essential to fully exploit the potentiality of these objects. In the present paper we describe the development of a plant derived multifunctional tool for nanobiotechnology based on Tomato bushy stunt virus. We demonstrate the ability of this system to remarkably sustain genetic modifications and in vitro chemical derivatizations of its outer surface, which resulted in the successful display of large chimeric peptides fusions and small chemical molecules, respectively. Moreover, we have defined physicochemical conditions for viral swelling and reversible viral pore gating that we have successfully employed for foreign molecules loading and retention in the inner cavity of this plant virus nanoparticles system. Finally, a production and purification strategy from Nicotiana benthamiana plants has been addressed and optimized.