Adeno-associated virus serotype 2 capsid variants for improved liver-directed gene therapy.

BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.

CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia.

The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.

Compromised Grid-Cell-like Representations in Old Age as a Key Mechanism to Explain Age-Related Navigational Deficits.

A progressive loss of navigational abilities in old age has been observed in numerous studies, but we have only limited understanding of the neural mechanisms underlying this decline [1]. A central component of the brain's navigation circuit are grid cells in entorhinal cortex [2], largely thought to support intrinsic self-motion-related computations, such as path integration (i.e., keeping track of one's position by integrating self-motion cues) [3-6]. Given that entorhinal cortex is particularly vulnerable to neurodegenerative processes during aging and Alzheimer's disease [7-14], deficits in grid cell function could be a key mechanism to explain age-related navigational decline. To test this hypothesis, we conducted two experiments in healthy young and older adults. First, in an fMRI experiment, we found significantly reduced grid-cell-like representations in entorhinal cortex of older adults. Second, in a behavioral path integration experiment, older adults showed deficits in computations of self-position during path integration based on body-based or visual self-motion cues. Most strikingly, we found that these path integration deficits in older adults could be explained by their individual magnitudes of grid-cell-like representations, as reduced grid-cell-like representations were associated with larger path integration errors. Together, these results show that grid-cell-like representations in entorhinal cortex are compromised in healthy aging. Furthermore, the association between grid-cell-like representations and path integration performance in old age supports the notion that grid cells underlie path integration processes. We therefore conclude that impaired grid cell function may play a key role in age-related decline of specific higher-order cognitive functions, such as spatial navigation.

Mapping of QTL for Fusarium head blight resistance and morphological and developmental traits in three backcross populations derived from Triticum dicoccum × Triticum durum.

Breeding for resistance to Fusarium head blight (FHB) in durum wheat continues to be hindered by the lack of effective resistance sources. Only limited information is available on resistance QTL for FHB in tetraploid wheat. In this study, resistance to FHB of a Triticum dicoccum line in the background of three Austrian T. durum cultivars was genetically characterized. Three populations of BC(1)F(4)-derived RILs were developed from crosses between the resistant donor line T. dicoccum-161 and the Austrian T. durum recipient varieties DS-131621, Floradur and Helidur. About 130 BC(1)F(4)-derived lines per population were evaluated for FHB response using artificial spray inoculation in four field experiments during two seasons. Lines were genetically fingerprinted using SSR and AFLP markers. Genomic regions on chromosomes 3B, 4B, 6A, 6B and 7B were significantly associated with FHB severity. FHB resistance QTL on 6B and 7B were identified in two populations and a resistance QTL on 4B appeared in three populations. The alleles that enhanced FHB resistance were derived from the T. dicoccum parent, except for the QTL on chromosome 3B. All QTL except the QTL on 6A mapped to genomic regions where QTL for FHB have previously been reported in hexaploid wheat. QTL on 3B and 6B coincided with Fhb1 and Fhb2, respectively. This implies that tetraploid and hexaploid wheat share common genomic regions associated with FHB resistance. QTL for FHB resistance on 4B co-located with a major QTL for plant height and mapped at the position of the Rht-B1 gene, while QTL on 7B overlapped with QTL for flowering time.

Four-year evaluation of a resin composite including nanofillers in posterior cavities.

PURPOSE: The aim of this prospective study was to evaluate the clinical performance of the restorative material Ceram.X in combination with an experimental one-bottle etch-and-rinse adhesive (K-0127). MATERIALS AND METHODS: A single operator placed two Class I or II restorations in molars of 43 patients. One molar was restored with Ceram.X/K-0127 (Dentsply DeTrey), the other one with Tetric Ceram/Syntac Classic (Ivoclar Vivadent). At baseline, after one, two, and four years the restorations were evaluated by a second dentist using modified Ryge's criteria. After four years, 27 patients were examined. RESULTS: In one patient, both restorations (Class II) had to be removed for root canal treatment due to pulpitis. Another Class II Ceram.X restoration (3.8%; 4.3% [1 of 23] of Class II restorations) showed score C with regard to wear/anatomical form. Thus, the cumulative failure rate was 7.4% in the Ceram.X group (8.3% of Class II restorations [2 of 24]) and 3.7% in the Tetric Ceram group (4.2% of Class II restorations [1 of 24]). Furthermore, three restorations (11.5%) in each group showed score B for anatomical form and marginal integrity. Slight marginal discoloration (score B) was found at five Ceram.X restorations (19.2%) and four Tetric Ceram restorations (15.4%). Two restorations (7.7%) in each group showed slight changes in color stability (score B). No sensitivity, recurrent caries, or changes in surface texture were recorded after four years. No statistically significant differences were found between the two restorative materials (p > 0.05). CONCLUSION: After four years of clinical service, 92.6% of Ceram.X/K-0127 and 96.3% of Tetric Ceram/Syntac Classic restorations performed clinically well.

The protective nature of pellicle towards toothpaste abrasion on enamel and dentine.

OBJECTIVES: To determine the protective nature of pellicle towards toothpaste abrasion. METHODS: The enamel region of human enamel-dentine blocks was indented with a Knoop diamond and the profile across the enamel-dentine junction was measured. Blocks were either exposed to deionised water or placed onto intra-oral appliances and worn in the mouth to produce in situ pellicles. This was followed by a 10-day period of tooth brushing experiments. Each day, specimens were brushed with a slurry of either Toothpaste A (RDA=90) or Toothpaste B (RDA=204) for 25 cycles (10s) on a brushing machine. This was repeated three times per day for a total of 750 brushing cycles. Between brushing cycles specimens were returned to water or in situ. The geometry of the Knoop indents and the enamel-dentine profile were re-measured and the enamel and dentine wear calculated. Specimens were also prepared for TEM analyses. RESULTS: The mean enamel wear (microm) for Toothpastes A and B (water) was 0.23 and 0.06, and for Toothpastes A and B (in situ) was 0.03 and 0.08, respectively. The mean dentine wear (microm) for Toothpastes A and B (water) was 5.08 and 6.03, and for Toothpastes A and B (in situ) was 1.94 and 1.70, respectively. For Toothpaste A, the presence of in situ pellicle significantly (p<0.05) reduced enamel and dentine wear compared to water and for Toothpaste B, dentine wear was significantly reduced compared to water. After tooth brushing, residues of the in situ pellicle layer could be detected on enamel and dentine surfaces by TEM analysis. CONCLUSIONS: The study has demonstrated for the first time that the presence of pellicle can significantly reduce toothpaste abrasion.

Detection of salivary alpha-amylase and lysozyme exposed on the pellicle formed in situ on different materials.

Amylase and lysozyme are components of the salivary pellicle, exposing considerable enzymatic activity in the immobilized state. The purpose of the present study was to elucidate the influence of different solid substrata on the amount and distribution of amylase and lysozyme exposed on the surface of the salivary pellicle formed in situ. Slabs of titanium, feldspar ceramic, and bovine enamel were fixed on the buccal sites of individual splints worn by three subjects for 3 or 30 min, respectively, to allow pellicle formation. Subsequently, slabs were removed from the splints and rinsed with running water. Detection of amylase and lysozyme was performed by FEI-SEM after gold-immunolabeling of the enzymes. Both enzymes were found to be distributed randomly at the pellicle surface. Irrespective of formation time and substratum, significantly more labeled lysozyme molecules (5.23 +/- 4.5 microm(-2)) were detected compared with amylase (3.4 +/- 2.9 microm(-2)). Neither the substratum nor the pellicle formation time had significant impact on the amount of the respective enzyme that could be detected. This study for the first time provides evidence, that amylase and lysozyme are exposed at the surface of the salivary pellicle formed in situ on titanium and ceramics. Both enzymes are distributed randomly on the surface of the pellicle, irrespective of the underlying substratum.

Influence of different restorative materials on lysozyme and amylase activity of the salivary pellicle in situ.

Lysozyme and amylase are the most abundant enzymatic components in the salivary pellicle. The purpose of the present study was to determine the influence of different substrata on amylase and lysozyme activity in salivary pellicles formed in situ. Slabs (5 mm diameter) of bovine dentine and enamel, of titanium, gold alloy, resin composite, PMMA, amalgam, and feldspar ceramic were fixed on the buccal sites of individual splints worn by six subjects for 30 min to allow pellicle formation. Thereafter, slabs were removed from the trays and rinsed with running water. Lysozyme activity was determined via lysis of Micrococcus lysodeicticus. Amylase activity was measured with a photometric method using 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosylmaltotriosid (GalG2CNP) as substrate. Both pellicle enzymes were evaluated in the immobilized as well as in the desorbed state. Salivary enzyme activities were also measured. All investigated pellicles exhibited lysozyme and amylase activity. Great intraindividual and interindividual differences were observed. Over all samples, immobilized amylase activity amounted to 0.65 +/- 0.64 mU/cm2. Immobilized lysozyme activity was 5.04 +/- 1.55 U/cm2. There were no major effects of the substratum on pellicle-bound amylase and lysozyme activity. Immobilized and desorbed enzyme activities revealed a strong correlation (lysozyme: r = 0.700; amylase: r = 0.990). Salivary enzyme activities had only little impact on pellicle-bound enzyme activities. Amylase and lysozyme are incorporated in the acquired in situ pellicle on different solid surfaces in an active conformation. Dental material and enzyme activity in the saliva have only little impact on enzymatic activity in the pellicle in situ.

A 2-oxoglutarate-dependent dioxygenase is integrated in DIMBOA-biosynthesis.

Benzoxazinoids are secondary metabolites of grasses that function as natural pesticides. While many steps of DIMBOA biosynthesis have been elucidated, the mechanism of the introduction of OCH(3)-group at the C-7 position was unknown. Inhibitor experiments in Triticum aestivum and Zea mays suggest that a 2-oxoglutarate-dependent dioxygenase catalyses the hydroxylation reaction at C-7. Cloning and reverse genetics analysis have identified the Bx6 gene that encodes this enzyme. Bx6 is located in the Bx-gene cluster of maize.