Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography.

Adeno-associated viruses (AAVs) have acquired a central role in modern medicine as delivery agents for gene therapies targeting rare diseases. While new AAVs with improved tissue targeting, potency, and safety are being introduced, their biomanufacturing technology is lagging. In particular, the AAV purification pipeline hinges on protein ligands for the affinity-based capture step. While featuring excellent AAV binding capacity and selectivity, these ligands require strong acid (pH <3) elution conditions, which can compromise the product's activity and stability. Additionally, their high cost and limited lifetime has a significant impact on the price tag of AAV-based therapies. Seeking to introduce a more robust and affordable affinity technology, this study introduces a cohort of peptide ligands that (i) mimic the biorecognition activity of the AAV receptor (AAVR) and anti-AAV antibody A20, (ii) enable product elution under near-physiological conditions (pH 6.0), and (iii) grant extended reusability by withstanding multiple regenerations. A20-mimetic CYIHFSGYTNYNPSLKSC and AAVR-mimetic CVIDGSQSTDDDKIC demonstrated excellent capture of serotypes belonging to distinct clones/clades - namely, AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. This corroborates the in silico models documenting their ability to target regions of the viral capsid that are conserved across all serotypes. CVIDGSQSTDDDKIC-Toyopearl resin features binding capacity (≈1014 vp mL-1 ) and product yields (≈60%-80%) on par with commercial adsorbents, and purifies AAV2 from HEK293 and Sf9 cell lysates with high recovery (up to 78%), reduction of host cell proteins (up to 700-fold), and high transduction activity (up to 65%).

Whole genome sequence of mulberry crinivirus, a new member of the genus Crinivirus.

The whole genome sequence of mulberry crinivirus (MuCV), a novel member of the genus Crinivirus (family Closteroviridae) identified in mulberry (Morus alba L), was determined. The virus possesses a bipartite genome. RNA1 contains 8571 nucleotides (nt) with four open reading frames (ORFs). ORF1a encodes a putative polyprotein with papain-like protease, methyltransferase, and RNA helicase domains. ORF1b putatively encodes an RNA-dependent RNA polymerase (RdRp), which is probably expressed via a + 1 ribosomal frameshift. RNA2 consists of 8082 nt, containing eight ORFs that are similar in size and position to orthologous genes of other criniviruses. Phylogenetic analysis based on RdRp amino acid sequences of criniviruses placed MuCV in group 1.

Characterization of a strong constitutive promoter from paper mulberry vein banding virus.

Paper mulberry vein banding virus (PMVBV), a member of the genus Badnavirus in the family Caulimoviridae, infects paper mulberry (Broussonetia papyrifera), a dicotyledonous plant. Putative promoter regions in the PMVBV genome were tested using recombinant plant expression vectors, revealing that the promoter activity of three genome fragments was about 1.5-fold higher than that of the 35S promoter of cauliflower mosaic virus in Nicotiana benthamiana. In transformed transgenic Arabidopsis thaliana plants, these promoter constructs showed constitutive expression. Based on the activity and gene expression patterns of these three promoter constructs, a fragment of 384 bp (named PmVP) was deduced to contain the full-length promoter of the PMVBV genome. The results suggest that the PMVBV-derived promoter can be used for the constitutive expression of transgenes in dicotyledonous plants.

Consequences to patients, clinicians, and manufacturers when very serious adverse drug reactions are identified (1997-2019): A qualitative analysis from the Southern Network on Adverse Reactions (SONAR).

BACKGROUND: Adverse drug/device reactions (ADRs) can result in severe patient harm. We define very serious ADRs as being associated with severe toxicity, as measured on the Common Toxicity Criteria Adverse Events (CTCAE)) scale, following use of drugs or devices with large sales, large financial settlements, and large numbers of injured persons. We report on impacts on patients, clinicians, and manufacturers following very serious ADR reporting. METHODS: We reviewed clinician identified very serious ADRs published between 1997 and 2019. Drugs and devices associated with reports of very serious ADRs were identified. Included drugs or devices had market removal discussed at Food and Drug Advisory (FDA) Advisory Committee meetings, were published by clinicians, had sales > $1 billion, were associated with CTCAE Grade 4 or 5 toxicity effects, and had either >$1 billion in settlements or >1,000 injured patients. Data sources included journals, Congressional transcripts, and news reports. We reviewed data on: 1) timing of ADR reports, Boxed warnings, and product withdrawals, and 2) patient, clinician, and manufacturer impacts. Binomial analysis was used to compare sales pre- and post-FDA Advisory Committee meetings. FINDINGS: Twenty very serious ADRs involved fifteen drugs and one device. Legal settlements totaled $38.4 billion for 753,900 injured persons. Eleven of 18 clinicians (61%) reported harms, including verbal threats from manufacturer (five) and loss of a faculty position (one). Annual sales decreased 94% from $29.1 billion pre-FDA meeting to $4.9 billion afterwards (p<0.0018). Manufacturers of four drugs paid $1.7 billion total in criminal fines for failing to inform the FDA and physicians about very serious ADRs. Following FDA approval, the median time to ADR reporting was 7.5 years (Interquartile range 3,13 years). Twelve drugs received Box warnings and one drug received a warning (median, 7.5 years following ADR reporting (IQR 5,11 years). Six drugs and 1 device were withdrawn from marketing (median, 5 years after ADR reporting (IQR 4,6 years)). INTERPRETATION: Because very serious ADRs impacts are so large, policy makers should consider developing independently funded pharmacovigilance centers of excellence to assist with clinician investigations. FUNDING: This work received support from the National Cancer Institute (1R01 CA102713 (CLB), https://www.nih.gov/about-nih/what-we-do/nih-almanac/national-cancer-institute-nci; and two Pilot Project grants from the American Cancer Society's Institutional Grant Award to the University of South Carolina (IRG-13-043-01) https://www.cancer.org/ (SH; BS).

Determination of the Degradation Chemistry of the Antitumor Agent Pemetrexed Disodium.

Stress-testing (forced degradation) studies have been conducted on pemetrexed disodium heptahydrate (1) (LY231514·2Na·7H2O) drug substance in order to identify its likely degradation products and establish its degradation pathways. Solid samples of the drug substance were stressed under various conditions of heat, humidity, and light, and solutions of the drug substance were stressed under various conditions of heat, light, oxidation, and over a wide pH range (1-13). The stressed samples were analyzed using a gradient elution reversed-phase HPLC method. The 7 major degradation products detected in the stress-testing studies were isolated, and the structures were elucidated via spectroscopic characterization. The structures of the degradation products and their proposed mechanisms of formation indicate that 1 degrades via 2 main pathways: oxidation and hydrolysis. Of the 7 identified degradation products, 6 are proposed to result from oxidation and 1 from hydrolysis.

Leaf gas exchange of understory spruce-fir saplings in relict cloud forests, southern Appalachian Mountains, USA.

The southern Appalachian spruce-fir (Picea rubens Sarg. and Abies fraseri (Pursh) Poir.) forest is found only on high altitude mountain tops that receive copious precipitation ( > 2000 mm year(-1)) and experience frequent cloud immersion. These high-elevation, temperate rain forests are immersed in clouds on approximately 65% of the total growth season days and for 30-40% of a typical summer day, and cloud deposition accounts for up to 50% of their annual water budget. We investigated environmental influences on understory leaf gas exchange and water relations at two sites: Mt. Mitchell, NC (MM; 35 degrees 45'53'' N, 82 degrees 15'53'' W, 2028 m elevation) and Whitetop Mtn., VA (WT; 36 degrees 38'19'' N, 81 degrees 36'19'' W, 1685 m elevation). We hypothesized that the cool, moist and cloudy conditions at these sites exert a strong influence on leaf gas exchange. Maximum photosynthesis (A(max)) varied between 1.6 and 4.0 micromol CO(2) m(-2) s(-1) for both spruce and fir and saturated at irradiances between approximately 200 and 400 micromol m(-2) s(-1) at both sites. Leaf conductance (g) ranged between 0.05 and 0.25 mol m(-2) s(-1) at MM and between 0.15 and 0.40 mol m(-2) s(-1) at WT and was strongly associated with leaf-to-air vapor pressure difference (LAVD). At both sites, g decreased exponentially as LAVD increased, with an 80-90% reduction in g between 0 and 0.5 kPa. Predawn leaf water potentials remained between -0.25 and -0.5 MPa for the entire summer, whereas late afternoon values declined to between -1.25 and -1.75 MPa by late summer. Thus, leaf gas exchange appeared tightly coupled to the response of g to LAVD, which maintained high water status, even at the relatively low LAVD of these cloud forests. Moreover, the cloudy, humid environment of these refugial forests appears to exert a strong influence on tree leaf gas exchange and water relations. Because global climate change is predicted to increase regional cloud ceiling levels, more research on cloud impacts on carbon gain and water relations is needed to predict future impacts on these relict forests.

Radiation frost susceptibility and the association between sky exposure and leaf size.

Plants growing in exposed and sheltered habitats have characteristic leaf structure and physiology that are traditionally associated with the total amount of incident sunlight. However, greater sky exposure also increases the susceptibility of leaves to radiation frost. Plants with large horizontal broadleaves are particularly susceptible to both overheating during the day and freezing at night. Moreover, the combined effects of high daytime sun-exposure and nighttime frost susceptibility could be particularly stressful to plant tissues. The purpose of this study was to evaluate the influence of elevation and microsite exposure (i.e. net loss of longwave radiation) on frost susceptibility, as well as the corresponding intraspecific variation in leaf size in the subalpine daisy (Erigeron peregrinus). Measured decreases in upper hemisphere infrared radiation (sky IR) of 0.014 W m-2 m-1 occurred with increasing elevation, beyond decreases predicted due to changes in air temperature and water content, resulting in an average decrease of 0.029 W m-2 m-1. Previous equations of sky IR based on air temperature and humidity were improved by adding this elevational term (r 2 improved from 0.52 to 0.71). In contrast, a mean decrease of 6.5 W m-2 m-1 occurred with increasing sky exposure across a subalpine meadow. Leaf size in Taraxacum officinale decreased linearly with increasing elevation and a corresponding decline in sky IR. No difference in daily solar irradiance was measured across the same elevational gradient. Also, E. peregrinus had smaller leaves at high elevation microsites with greater sky exposure and decreased sky IR, while there was a much weaker association between leaf size and the amount of total daily solar irradiance. Differences in plant leaf structure and physiology traditionally associated with daytime sun-exposure may also be influenced by nighttime sky exposure and the susceptibility to radiation frosts.