10The extraction and immunoassay of fecal steroids is an increasingly common technique, used in both captive and field studies to 11 provide an approximation of an animal's circulating concentration of hormones through non-invasive methods. Storage of fecal 12 samples is of critical concern because fecal bacteria metabolize fecal steroids within hours after deposit. Ethanol is often used as a 13 preservative for fecal samples stored for several hours at room temperature. We examined the stability of fecal estrogen (fE) and 14 glucocorticoid (fGC) metabolites from baboon (Papio cynocephalus) samples in a 95% ethanol solution at ambient temperature and 15 at )20°C over the course of six months, to determine the effect of storage on steroid concentrations. As measured by radioim-16 munoassay, fE metabolite concentrations increased by 122% at 90 days and fGC metabolite concentrations increased by 92% at 120 17 days. After peaking, both hormones declined to near initial concentrations by 180 days in ambient temperature samples. In samples 18 stored at sub-zero temperatures, fGC metabolite concentrations showed a similar but dampened pattern, while fE metabolite 19 concentrations exhibited small and variable changes with no consistent trend. We discuss explanations for the dynamic pattern of 20 changing fecal metabolite concentrations and offer practical and analytical guidance to field workers for situations in which ideal 21 conditions for stabilizing hormones are not available. Ó 2002 Elsevier Science (USA). All rights reserved. 22

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Currently, there are over 150 ongoing clinical trials utilizing adeno-associated viruses (AAVs) to target various genetic diseases, including hemophilia (AAV2 and AAV8), congenital heart failure (AAV1 and AAV6), cystic fibrosis (AAV2), rheumatoid arthritis (AAV2), and Batten disease (AAVrh.10). Prior to patient administration, AAV vectors must have their serotype, concentration, purity, and stability confirmed. Here, we report the application of differential scanning fluorimetry (DSF) as a good manufacturing practice (GMP) capable of determining the melting temperature (Tm) for AAV serotype identification. This is a simple, rapid, cost effective, and robust method utilizing small amounts of purified AAV capsids (∼25 μL of ∼1011 particles). AAV1-9 and AAVrh.10 exhibit specific Tms in buffer formulations commonly used in clinical trials. Notably, AAV2 and AAV3, which are the least stable, have varied Tms, whereas AAV5, the most stable, has a narrow Tm range in the different buffers, respectively. Vector stability was dictated by VP3 only, specifically, the ratio of basic/acidic amino acids, and was independent of VP1 and VP2 content or the genome packaged. Furthermore, stability of recombinant AAVs differing by a single basic or acidic amino acid residue are distinguishable. Hence, AAV DSF profiles can serve as a robust method for serotype identification of clinical vectors.

Summary Paragraph Advances in genetics and sequencing reveal a plethora of disease-associated and disease-causing genetic alterations. Resolving causality between genetics and disease requires generating accurate models for molecular dissection; however, the rapid expansion of single-cell landscapes presents a major challenge to accurate comparisons between mutants and their wild-type equivalents. Here, we generated mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the Growth factor independent-1 (GFI1) transcription factor. To delineate the impact of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes 1 , aligned mutant cells to their wild-type equivalent and identified differentially expressed genes and epigenetic loci. We find that Gfi1-target genes are altered sequentially, as cells traverse successive states during differentiation. These cell-state-specific insights facilitated genetic rescue of granulocytic specification but not post-commitment defects in innate-immune effector function; underscoring the importance of evaluating the impact of mutations and therapy within each relevant cell state.