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Abstract Background Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated AAVS1 (also known as MBS85). Integration at AAVS1 requires the AAV2 replication (Rep) proteins and a DNA sequence within AAVS1 containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site (also referred to as a terminal resolution site, or trs). The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced trs, but the sequences differ from those in AAVS1. These ITR sequences are required for replication and packaging. Results In this study we demonstrate that the AAVS1 RRS and trs can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of AAVS1 DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at AAVS1, as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to AAVS1 sequences. Conclusions The ability of these AAVS1 sequences to substitute for the AAV2 RRS and trs provides indirect evidence that the stable secondary structure encompassing the trs is part of the AAV2 packaging signal.

Abstract Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation.

Abstract Background To determine whether previously reported loci predisposing to nonsyndromic high myopia show linkage to common myopia in pedigrees from two ethnic groups: Ashkenazi Jewish and Amish. We hypothesized that these high myopia loci might exhibit allelic heterogeneity and be responsible for moderate /mild or common myopia. Methods Cycloplegic and manifest refraction were performed on 38 Jewish and 40 Amish families. Individuals with at least -1.00 D in each meridian of both eyes were classified as myopic. Genomic DNA was genotyped with 12 markers on chromosomes 12q21-23 and 18p11.3. Parametric and nonparametric linkage analyses were conducted to determine whether susceptibility alleles at these loci are important in families with less severe, clinical forms of myopia. Results There was no strong evidence of linkage of common myopia to these candidate regions: all two-point and multipoint heterogeneity LOD scores were < 1.0 and non-parametric linkage p-values were > 0.01. However, one Amish family showed slight evidence of linkage (LOD>1.0) on 12q; another 3 Amish families each gave LOD >1.0 on 18p; and 3 Jewish families each gave LOD >1.0 on 12q. Conclusions Significant evidence of linkage (LOD> 3) of myopia was not found on chromosome 18p or 12q loci in these families. These results suggest that these loci do not play a major role in the causation of common myopia in our families studied.

Introduction/background: Frailty identifies patients that have vulnerability to stress. Acute illness and hospitalization are stressors that may result in delirium and further accelerate the negative consequences of frailty., Purpose: The purpose of this study was to determine whether frailty, identified at hospital admission and as measured by a frailty index, is associated with incident delirium., Methods: A retrospective, observational, cohort study was done at a Veterans hospital between January 2013 and March 2014. English-speaking patients over 55 years were eligible. Exclusion criteria included inability to complete baseline assessments due to pre-existing cognitive impairment, emergent surgery; and/or admission from a nursing home, pre-existing delirium, and those with psychiatric disease or substance use disorder., Main Outcomes and Measures: Frailty index (FI) variables included cognitive screening, physical function and comorbidities. The FI was calculated as a proportion of possible deficits (range 0 to 1; higher scores indicate increased frailty). Incident delirium was measured daily by an expert clinician interview., Results: A total of 247 patients were admitted and 218 met inclusion/exclusion criteria, with a mean age of 71.54 years (SD = 9.53 years) and were predominantly white (92.7%) and male (91.7%). Participants were grouped using FI ranges as non-frail (FI <0.25, n=56 (26%)), pre-frail (FI =0.25-0.35, n=86 (39%)), and frail (FI >0.35, n=76 (35%)). Pre-frailty and frailty were associated with incident delirium (non-frail: 3.6% vs pre-frail: 20.9% vs frail: 29.3%, p =0.001) and total delirium days (mean day =non-frail 0.04 vs pre-frail 0.35 vs frail 0.57, p =0.003). After adjustment for sociodemographic factors, pre-frail (adjusted OR=5.64, 95% CI: 1.23, 25.99) and frail status (adjusted OR=6.80, 95% CI: 1.38, 33.45) were independently associated with delirium., Conclusion: This study demonstrates that a frailty index is independently associated with incident delirium and suggests that admission assessments for frailty may identify patients at high risk of developing delirium., Competing Interests: This paper/was presented at the 2019 Gerontological Society of America Annual Meeting as a poster presentation with interim findings entitled “The Association of a Frailty Index and Incident Delirium in Hospitalized Veterans”. The conference abstract was published in Innovations In Aging with DOI: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6845397/]. Dr Culley reports grants from NIGMS, during the conduct of the study; personal fees from American Society of Anesthesiology, personal fees from American Board of Anesthesiology, outside the submitted work. Dr. Arora reports an unrestricted educational grant from Pfizer Canada Inc. and honoraria from Abbott Nutrition, and honorarium from Edwards Lifesciences, outside the submitted work. Dr Rudolph reports being an unpaid board member of the American Delirium Society - a professional society dedicated to the advancement of delirium science. The authors report no other potential conflicts of interest for this work., (© 2020 Sillner et al.)

Information on the size and shape of the neutron skin on (208)Pb is extracted from coherent pion photoproduction cross sections measured using the Crystal Ball detector together with the Glasgow tagger at the MAMI electron beam facility. On exploitation of an interpolated fit of a theoretical model to the measured cross sections, the half-height radius and diffuseness of the neutron distribution are found to be c(n)=6.70±0.03(stat.)  fm and a(n)=0.55±0.01(stat.)(-0.03)(+0.02)(sys.)  fm, respectively, corresponding to a neutron skin thickness Δr(np)=0.15±0.03(stat.)(-0.03)(+0.01)(sys.)  fm. The results give the first successful extraction of a neutron skin thickness with an electromagnetic probe and indicate that the skin of (208)Pb has a halo character. The measurement provides valuable new constraints on both the structure of nuclei and the equation of state for neutron-rich matter.

We present the first detailed measurement of incoherent photoproduction of neutral pions to a discrete state of a residual nucleus. The 12C(gamma,pi(0))(12)C*(4.4 MeV) reaction has been studied with the Glasgow photon tagger at MAMI employing a new technique which uses the large solid angle Crystal Ball detector both as a pi(0) spectrometer and to detect decay photons from the excited residual nucleus. The technique has potential applications to a broad range of future nuclear measurements with the Crystal Ball and similar detector systems elsewhere. Such data are sensitive to the propagation of the Delta in the nuclear medium and will give the first information on matter transition form factors from measurements with an electromagnetic probe. The incoherent cross sections are compared to two theoretical predictions including a Delta-hole model.

Differential cross sections of the reactions (gamma,pi(0)pi(0)) and (gamma,pi(0)pi(+/-)) have been measured for several nuclei (1H,12C, and (nat)Pb) at an incident-photon energy of E(gamma)=400-460 MeV at the tagged-photon facility at MAMI-B using the TAPS spectrometer. A significant nuclear-mass dependence of the pipi invariant-mass distribution is found in the pi(0)pi(0) channel. This dependence is not observed in the pi(0)pi(+/-) channel and is consistent with an in-medium modification of the pipi interaction in the I=J=0 channel. The data are compared to pi-induced measurements and to calculations within a chiral-unitary approach.

The reaction gammap-->pi(+)pi(0)n has been measured at MAMI for photon energies up to 820 MeV. Invariant mass spectra of the particles in the final state (pi(+)n), (pi(0)n), (pi(+)pi(0)) have been determined for several bins of incident photon energy. Differences in pi(+)pi(0) and simultaneously measured pi(0)pi(0) invariant mass distributions are assigned to a rho branch of the D13(1520) nucleon resonance.

The helicity dependence of the single pion photoproduction on the proton has been measured in the energy range from 200 to 450 MeV for the first time. The experiment, performed at the Mainz microtron MAMI, used a 4pi-detector system, a circularly polarized, tagged photon beam, and a frozen-spin target. The data obtained provide new information for multipole analyses of pion photoproduction and determine the main contributions to the Gerasimov-Drell-Hearn sum rule and the forward spin polarizability gamma(0).