Search

In-depth characterization of adeno-associated virus (AAV)-mediated CRISPR delivery is still lacking. Here, the authors show high levels of integration into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in vivo.

Hereditary hearing loss often results from mutation of genes expressed by cochlear hair cells. Gene addition using AAV vectors has shown some efficacy in mouse models, but clinical application requires two additional advances. First, new AAV capsids must mediate efficient transgene expression in both inner and outer hair cells of the cochlea. Second, to have the best chance of clinical translation, these new vectors must also transduce hair cells in non-human primates. Here, we show that an AAV9 capsid variant, PHP.B, produces efficient transgene expression of a GFP reporter in both inner and outer hair cells of neonatal mice. We show also that AAV9-PHP.B mediates almost complete transduction of inner and outer HCs in a non-human primate. In a mouse model of Usher syndrome type 3A deafness (gene CLRN1), we use AAV9-PHP.B encoding Clrn1 to partially rescue hearing. Thus, we have identified a vector with promise for clinical treatment of hereditary hearing disorders, and we demonstrate, for the first time, viral transduction of the inner ear of a primate with an AAV vector. Keywords: inner ear, AAV, non-human primate, cochlea, hair cells, adeno-associated virus vector, hereditary deafness, gene delivery

Adeno-associated viral vectors (AAVs) have demonstrated potential in applications for neurologic disorders, and the discovery that some AAVs can cross the blood-brain barrier (BBB) after intravenous injection has further expanded these opportunities for non-invasive brain delivery. Anc80L65, a novel AAV capsid designed from in silico reconstruction of the viral evolutionary lineage, has previously demonstrated robust transduction capabilities after local delivery in various tissues such as liver, retina, or cochlea, compared with conventional AAVs. Here, we compared the transduction efficacy of Anc80L65 with conventional AAV9 in the CNS after intravenous, intracerebroventricular (i.c.v.), or intraparenchymal injections. Anc80L65 was more potent at targeting the brain and spinal cord after intravenous injection than AAV9, and mostly transduced astrocytes and a wide range of neuronal subpopulations. Although the efficacy of Anc80L65 and AAV9 is similar after direct intraparenchymal injection in the striatum, Anc80L65’s diffusion throughout the CNS was more extensive than AAV9 after i.c.v. infusion, leading to widespread EGFP expression in the cerebellum. These findings demonstrate that Anc80L65 is a highly efficient gene transfer vector for the murine CNS. Systemic injection of Anc80L65 leads to notable expression in the CNS that does not rely on a self-complementary genome. These data warrant further testing in larger animal models. Keywords: adeno-associated, Anc80L65, AAV, central nervous system

The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo—and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.

Time of Flight (ToF) sensors are the source of various errors, including the multi-camera interference artifact caused by the parallel scanning mode of the sensors. This paper presents the novel Importance Map Based Median filtration algorithm for interference artifacts suppression, as the potential 3D filtration method. The approach is based on the processing of multiple depth frames, using the extraction of the interference region and application of the interpolation. Considering the limitations and good functionalities of proposed algorithm, the combination with some standard methods was suggested. Performance of the algorithm was evaluated on the dataset consisting of the real-world objects with different texture and morphology against popular filtering methods based on neural networks and statistics.

Accurate prediction of human clearance (CL) and volume of distribution at steady state (Vd,ss) for small molecule drug candidates is an essential component of assessing likely efficacious dose and clinical safety margins. In 2021, the IQ Consortium Human PK Prediction Working Group undertook a survey of IQ member companies to understand the current PK prediction methods being used to estimate these parameters across the pharmaceutical industry. The survey revealed a heterogeneity in approaches being used across the industry (e.g., the use of allometric approaches, differing incorporation of binding terms, and inconsistent use of empirical correction factors for in vitro-in vivo extrapolation, IVIVE), which could lead to different PK predictions with the same input data. Member companies expressed an interest in improving human PK predictions by identifying the most appropriate compound-class specific methods, as determined by physiochemical properties and knowledge of CL pathways. Furthermore, there was consensus that increased understanding of the uncertainty inherent to the compound class-dependent prediction would be invaluable in aiding communication of human PK and dose uncertainty at the time of candidate nomination for development. The human PK Prediction Working Group is utilizing these survey findings to help interrogate clinical IV datasets from across the IQ consortium member companies to understand PK prediction accuracy and uncertainty from preclinical datasets. [ABSTRACT FROM AUTHOR]

Brain capillary endothelial cells (BCEC) were cultured as an in vitro model of the blood-brain barrier (BBB) and manipulated to investigate how the BBB responds to changes in zinc status. BCEC were grown in minimum essential medium (MEM) with 2% fetal bovine serum and 13% platelet-poor horse serum. A moderate zinc deficiency was imposed by growing the cells in medium containing serums that had previously been dialyzed against EDTA to remove endogenous labile zinc. The control treatment was MEM with undialyzed serums (3 [micro]mol Zn/L); low-Zn was MEM with dialyzed serums (1.5 [micro]mol Zn/L); Zn-back was MEM with dialyzed serums, plus Zn[Cl.sub.2] added back (3 [micro]mol Zn/L); high-Zn was MEM with undialyzed serums, plus Zn[Cl.sub.2] (50 [micro]mol Zn/L). Low-Zn treatment increased (P < 0.02) the rate of zinc uptake into BCEC, relative to control and Zn-back; low-Zn treatment also increased (P < 0.05) the rate of zinc transport across the BCEC into the abluminal chamber (analogous to the brain), relative to control and Zn-back. High-Zn decreased (P < 0.02) the rate of zinc transport across BCEC into the brain, while increasing (P < 0.001) the rate of zinc uptake into BCEC, relative to controls. We conclude that BCEC responded to changes in zinc status by altering the rate of zinc transport in a manner consistent with the BBB actively working to sustain brain zinc homeostasis. KEY WORDS: * blood-brain barrier * brain capillary endothelial cells * zinc deficiency * zinc homeostasis * zinc transport

In this paper, the results of the fatigue tests of the P92 and 15CH2NMFA steels are presented and discussed. The fatigue S – N curves were obtained by means of both standard and miniature test specimens. The Small Fatigue Test (SFT) specimens were manufactured partially by machining and also by waterjet cutting. The fatigue tests were performed on Amsler 10 HFP 5100 ZWICK//Roell high-frequency pulsator at room temperature and R = 0,1 and R = -1 cycle asymmetries. The results obtained on standard and SFT specimens are compared and in case of P92 steel are also compared the two methods of SFT specimens manufacturing. In case of 15CH2NMFA steel, the results of standard and SFT specimens are in a very good agreement both within the time limit fatigue range and the fatigue limit. After taking the stress concentration of SFT specimens into consideration, the results of traditional fatigue test samples and SFT samples are similar. In case of P92 steel, the leaning branch of S-N curves is much steeper for SFT specimens and the fatigue limits lower than it could be expected after the stress concentration is taken into account. The fatigue limits for equal cycle asymmetry show the same ratio of standard to SFT fatigue limits, for R = 0,1 it is 1,31, for R = 1 it is 1,32. This very good agreement can be used for the evaluation of fatigue behaviour of P92 steel by means of SFT specimens. The comparison of the two ways of SFT samples manufacturing revealed a scatter of fatigue test results, nevertheless the fatigue limits differ by 5 MPa only, so that even the influence of the manufacturing seems to be negligible. [ABSTRACT FROM AUTHOR]

Success in the design of targeted covalent inhibitors depends in part on a knowledge of the factors influencing electrophile reactivity. In an effort to further develop an understanding of structure-reactivity relationships among N-arylacrylamides, we determined glutathione (GSH) reaction rates for a family of N-arylacrylamides independently substituted at ortho-, meta-, and para-positions with 11 different groups common to inhibitor design. We find that substituent effects on reaction rates show a linear Hammett correlation for ortho-, meta-, and para-substitution. In addition, we note a correlation between ¹H and 13C NMR chemical shifts of the acrylamide with GSH reaction rates, suggesting that NMR chemical shifts may be a convenient surrogate measure of relative acrylamide reactivity. Density functional theory calculations reveal a correlation between computed activation parameters and experimentally determined reaction rates, validating the use of such methodology for the screening of synthetic candidates in a prospective fashion. [ABSTRACT FROM AUTHOR]

1. Many UDP-glucuronosyltransferases (UGTs) require phosphorylation by protein kinase C (PKC) for glucuronidation activity. Inhibition of UGT phosphorylation by PKC inhibitor drugs may represent a novel mechanism for drug-drug interactions. 2. The potential for PKC-mediated inhibition of human UGT1A6, an isoform involved in the glucuronidation of drugs such as acetaminophen (paracetamol) and endogenous substrates including serotonin, was evaluated using various cell model systems. 3. Of ten different PKC inhibitors screened for their effects on acetaminophen glucuronidation by human LS180 colon cells, only rottlerin (PKC δ selective inhibitor; IC50 = 9.0 ± 1.2 μM) and the non-selective PKC inhibitors (calphostin-C, curcumin and hypericin) decreased glucuronidation by more than 50%. 4. Using UGT1A6-infected Sf9 insect cells, calphostin-C and hypericin showed three times more potent inhibition of serotonin glucuronidation in treated whole cells versus cell lysates. However, both curcumin and rottlerin showed significant direct inhibition and so (indirect) PKC effects could not be differentiated in this model system. 5. Of nine PKC isoforms co-expressed with UGT1A6 in human embryonic kidney 293T cells only PKC δ increased protein-normalized UGT1A6-mediated serotonin glucuronidation significantly (by 63% ± 4%). 6. These results identify an important role for PKC δ in UGT1A6-mediated glucuronidation and suggest that PKC δ inhibitors could interfere with glucuronidation of UGT1A6 substrates. [ABSTRACT FROM AUTHOR]

Studies the effect of Korean littoral environment on acoustic propagation. Description and measurements of the ocean environment; Details on the geoacoustic model of the bottom environment; Measurement of transmission loss; Sensivity of transmission on the environment.

Time of Flight (ToF) sensors are the source of various errors, including the multi-camera interference artifact caused by the parallel scanning mode of the sensors. This paper presents the novel Importance Map Based Median filtration algorithm for interference artifacts suppression, as the potential 3D filtration method. The approach is based on the processing of multiple depth frames, using the extraction of the interference region and application of the interpolation. Considering the limitations and good functionalities of proposed algorithm, the combination with some standard methods was suggested. Performance of the algorithm was evaluated on the dataset consisting of the real-world objects with different texture and morphology against popular filtering methods based on neural networks and statistics. [ABSTRACT FROM AUTHOR]