Ex vivo culture resting time impacts transplantation outcomes of genome-edited human hematopoietic stem and progenitor cells in xenograft mouse models.

Ex vivo resting culture is a standard procedure following genome editing in hematopoietic stem and progenitor cells (HSPCs). However, prolonged culture may critically affect cell viability and stem cell function. We investigated whether varying durations of culture resting times impact the engraftment efficiency of human CD34+ HSPCs edited at the BCL11A enhancer, a key regulator in the expression of fetal hemoglobin. We employed electroporation to introduce CRISPR-Cas9 components for BCL11A enhancer editing and compared outcomes with nonelectroporated (NEP) and electroporated-only (EP) control groups. Post-electroporation, we monitored cell viability, death rates, and the frequency of enriched hematopoietic stem cell (HSC) fractions (CD34+CD90+CD45RA- cells) over a 48-hour period. Our findings reveal that while the NEP group showed an increase in cell numbers 24 hours post-electroporation, both EP and BCL11A-edited groups experienced significant cell loss. Although CD34+ cell frequency remained high in all groups for up to 48 hours post-electroporation, the frequency of the HSC-enriched fraction was significantly lower in the EP and edited groups compared to the NEP group. In NBSGW xenograft mouse models, both conditioned with busulfan and nonconditioned, we found that immediate transplantation post-electroporation led to enhanced engraftment without compromising editing efficiency. Human glycophorin A+ (GPA+) red blood cells (RBCs) sorted from bone marrow of all BCL11A edited mice exhibited similar levels of γ-globin expression, regardless of infusion time. Our findings underscore the critical importance of optimizing the culture duration between genome editing and transplantation. Minimizing this interval may significantly enhance engraftment success and minimize cell loss without compromising editing efficiency. These insights offer a pathway to improve the success rates of genome editing in HSPCs, particularly for conditions like sickle cell disease.

A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies.

BACKGROUND: Studies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads. METHODS: A set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested. RESULTS: The primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize. CONCLUSIONS: This is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites.

A De Novo Chromosome-Level Genome Assembly of the White-Tailed Deer, Odocoileus Virginianus.

Cervids are distinguished by the shedding and regrowth of antlers. Furthermore, they provide insights into prion and other diseases. Genomic resources can facilitate studies of the genetic underpinnings of deer phenotypes, behavior, and disease resistance. Widely distributed in North America, the white-tailed deer (Odocoileus virginianus) has recreational, commercial, and food source value for many households. We present a genome generated using DNA from a single Illinois white-tailed sequenced on the PacBio Sequel II platform and assembled using Wtdbg2. Omni-C chromatin conformation capture sequencing was used to scaffold the genome contigs. The final assembly was 2.42 Gb, consisting of 508 scaffolds with a contig N50 of 21.7 Mb, a scaffold N50 of 52.4 Mb, and a BUSCO complete score of 93.1%. Thirty-six chromosome pseudomolecules comprised 93% of the entire sequenced genome length. A total of 20 651 predicted genes using the BRAKER pipeline were validated using InterProScan. Chromosome length assembly sequences were aligned to the genomes of related species to reveal corresponding chromosomes.

Computerized ICU Orders Versus Handwritten ICU Orders: A Prospective, Pharmacy-Based Analysis.

OBJECTIVE: To compare computerized ICU order writing with handwritten ICU physician orders. DESIGN: Prospective study. SETTING: Medical and surgical Intensive Care Units and pharmacy of a Department of Veterans Affairs Medical Center. PATIENTS: Two hundred sixty-four individual sets of orders. INTERVENTIONS: A time study and problem analysis were performed in the pharmacy as orders were received and processed. MEASUREMENTS AND MAIN RESULTS: Two hundred sixty-four sets of orders were evaluated; MICU (handwritten; n = 133) and SICU (computerized; n = 131). Physician length of training are similar in both units. The patient age and number of major diagnoses per patient in the two ICU groups were similar. Significantly less time (min) (MICU 2.5 ± 0.3 versus SICU 1.84 ± 0.1, p < 0.05) is required to review SICU orders. The SICU had significantly fewer order problems (MICU 45 versus SICU 12, p < 0.05). Computerized SICU orders were corrected more rapidly. The majority of order problems in both groups were resolved by telephone. CONCLUSIONS: ICU orders by computer program are processed more rapidly, have fewer errors, and are corrected more rapidly than standard handwritten orders. We conclude that a dedicated ICU computerized order-writing system permits orders to be written with fewer errors and the pharmacy to process them more efficiently than handwritten orders.