An enhanced Eco1 retron editor enables precision genome engineering in human cells from a single-copy integrated lentivirus.

Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast; however, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity as major contributors to poor retron editor efficiency. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to liberate the sgRNA and ncRNA. These modifications yielded a ncRNA with 5'- and 3'-end protection and an sgRNA with minimal 5' extension. This strategy increased steady-state ncRNA levels and rescued Cas9 activity leading to enhanced efficiency of the Eco1 retron editor in human cells. The enhanced Eco1 retron editor enabled the insertion of missense mutations in human cells from a single integrated lentivirus, thereby ensuring genotype-phenotype linkage over multiple cell divisions. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells. Here we present an enhanced Eco1 retron editor that enables efficient introduction of missense mutations in human cells from a single heritable genome copy.

Advancing Diagnostic Accuracy and Quality of Patient Care Through the Implementation of a Flow Cytometry Quality Assurance Program.

CONTEXT.­: Flow cytometry immunophenotypic analysis plays an important role in the diagnosis, classification, and disease monitoring of hematologic neoplasms. The interpretation of flow cytometry testing can be challenging. OBJECTIVE.­: To explore ways to improve diagnostic accuracy and in turn enhance the quality of patient care. DESIGN.­: A flow cytometry quality assurance (QA) program was developed. Cases from various complex flow cytometry panels were randomly selected and cross-reviewed. The outcomes of the QA review were categorized into 3 groups: complete agreement, minor discrepancy, and major discrepancy. Each discrepancy underwent a process of documentation, discussion, and resolution. Here we summarize our 3 years of experience with this program. RESULTS.­: In total, 6166 cases were evaluated; 6028 cases (97.7%) showed complete concordance, 120 cases (2.0%) showed minor discrepancies, and 18 cases (0.3%) showed major discrepancies. Among the top 5 panels evaluated, the panel evaluating mature T-cell abnormalities showed the highest rate of discrepancy, whereas the panel for evaluation of myelodysplastic syndromes showed the lowest discrepancy rate. When analyzing the trends of concordance and discrepancy over time, we observed a statistically significant decrease in discrepancy rate over time, from 4% at the beginning of the 6-month period to 1.5% in the final 6-month period. CONCLUSIONS.­: The overall concordance rate was 97.7%. The remaining 2.3% of cases showed discrepancies that required a correction, underscoring the value and necessity of having a QA program. The overall discrepancy rates exhibited a gradual decline over time, indicative of the positive impact of the QA program on enhancing diagnostic competency and accuracy over time.

Integrative immunophenotypic and genetic characterization of acute myeloid leukemia with CBFB rearrangement.

OBJECTIVES: We sought to characterize the immunophenotype of acute myeloid leukemia (AML) with CBFB rearrangement and correlate the results with cytogenetic and molecular data. METHODS: Sixty-one cases of AML with CBFB rearrangement were evaluated. RESULTS: The sample population consisted of 33 men and 28 women, with a median age of 49 years. Flow cytometry immunophenotypic analysis showed that myeloblasts were positive for CD34 and CD117 in all cases, and myeloperoxidase was positive in 52 of 55 (95%) cases. The most common abnormalities included decreased CD38 in 90%, increased CD13 in 85%, increased CD123 in 84%, and decreased HLA-DR in 84% of cases. Monocytes were increased, with a mature immunophenotype, and accounted for 23.7% of total cells. Among 60 cases with available karyotype, inv(16)(p13.1q22) was most common in 50 (83%) cases, followed by t(16;16) (p13.1;q22) in 6 (10%). Type A CBFB::MYH11 transcript was most common, detected in 84% of cases. Mutational analysis showed mutations of NRAS in 37%, FLT3 in 25%, and KIT in 24% of cases. Comparing cases with type A vs non-type A transcripts, blasts in type A cases more frequently exhibited CD64 positivity and increased CD13 levels while showing a lower frequency of CD7 and CD56 expression. Trisomy 22 and mutations in KIT, NF1, and TET2 were identified only in cases with type A transcript. CONCLUSIONS: Myeloblasts of AML with CBFB rearrangement are positive for CD34, CD117, and myeloperoxidase. These neoplasms most frequently carry inv(16)(p13.1q22) and type A fusion transcript. NRAS mutation was the most common mutation. Some immunophenotypic and genetic correlations occurred with different types of transcripts.

Racial disparities in subjective cognitive decline and its implications among Alzheimer's caretakers.

BACKGROUND/OBJECTIVE: Alzheimer's disease is a prominent neurodegenerative disorder characterized by cognitive decline and memory loss. Variations in subjective cognitive decline among Alzheimer's patients, often reported by caregiver, may stem from cultural, socioeconomic, healthcare access, and genetic factors. This study investigates racial disparities in subjective cognitive decline reported by caregivers and their implications. METHODS: In this study, data from 12,627 Alzheimer's caretakers from the CDC's Alzheimer's Disease and Healthy Aging Data Portal were analyzed using JMP software. Caregivers reported patients' cognitive decline for various racial categories: Asian/Pacific Islander, Black, Hispanic, Native American/Native Alaskan, and White. Fit model tests and distribution analyses were employed to assess disparities in symptom severity. The study focused on four key questions regarding symptom prevalence and healthcare communication to assess the degree of symptoms the patients were experiencing. RESULTS: Significant disparities in symptom severity reported by Alzheimer's caretakers were observed among the racial groups analyzed. The symptom severity ranked from least to most severe is the following: White, Asian/Pacific Islander, Black, Native American/Native Alaskan, and Hispanic patients. There was variance when it came to communication with healthcare providers, as the Asian population had the lowest communication rates. These findings underscore the need for targeted interventions considering cultural differences. It is important that tailoring healthcare approaches for different racial backgrounds is happening as a remedy to this gap in communication. CONCLUSION: Due to cultural, socioeconomic, genetic factors, and others, there were significant observed disparities. Tailoring interventions to these diverse populations is crucial to address these inequities.

Streptacidiphilus durhamensis sp. nov., isolated from a spruce forest soil.

The taxonomic position of three acidophilic actinobacteria, strains FGG38, FGG39 and FSCA67(T), isolated from the fermentation litter layer of a spruce forest soil was established using a polyphasic approach. The strains were shown to have chemotaxonomic and morphological properties consistent with their classification in the genus Streptacidiphilus and formed a distinct phyletic line in the Streptacidiphilus 16S rRNA gene tree being most closely related to Streptacidiphilus albus DSM 41753(T) (99.4 % similarity). DNA:DNA relatedness data showed that isolate FSCA67(T) and the type strain of S. albus belonged to markedly distinct genomic species. The isolates had many phenotypic properties in common and were distinguished readily from their closest phylogenetic neighbours in the Streptacidiphilus gene tree using a broad range of these features. Based on the combined genotypic and phenotypic data the three isolates are considered to represent a new Streptacidiphilus species. The name Streptacidiphilus durhamensis sp. nov. is proposed for this taxon with isolate FSCA67(T) (=DSM 45796(T) = KACC 17154(T) = NCIMB 14828(T)) [corrected] as the type strain.

Nocardia aciditolerans sp. nov., isolated from a spruce forest soil.

Actinomycetes growing on acidified starch-casein agar seeded with suspensions of litter and mineral soil from a spruce forest were provisionally assigned to the genus Nocardia based upon colonial properties. Representative isolates were found to grow optimally at pH 5.5, have chemotaxonomic and morphological features consistent with their assignment to the genus Nocardia and formed two closely related subclades in the Nocardia 16S rRNA gene tree. DNA:DNA relatedness assays showed that representatives of the subclades belong to a single genomic species. The isolates were distantly associated with their nearest phylogenetic neighbour, the type strain of Nocardia kruczakiae, and were distinguished readily from the latter based on phenotypic properties. On the basis of these data it is proposed that the isolates merit recognition as a new species, Nocardia aciditolerans sp. nov. The type strain is isolate CSCA68(T) (=KACC 17155(T) = NCIMB 14829(T) = DSM 45801(T)).