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Objectives: The increase in carbapenemase-producing organism (CPO) transmission among hospitalized patients is a growing concern. Studies investigating the transmission of CPO to epidemiologically linked contacts are scarce. We conducted an interim subgroup analysis of the ongoing multicenter household transmission of CPO in Singapore (CaPES-C) study to identify the acquisition rate of CPO among epidemiologically linked contacts of hospitalized CPO patients. Methods: This multicenter prospective cohort study was conducted between January and December 2021. We recruited CPO-positive patients and their epidemiologically linked contacts. Stool samples were collected from the patients at baseline, day 3, day 7, and at weeks 2, 3, 4, 5, 6, 12, 24, 36, and 48. Additionally, a sample was collected at the time of discharge from the hospital. Xpert Carba-R test was used to detect CPO genotypes in the stool samples. In this interim analysis, we calculated the acquisition rate of CPO among the epidemiologically linked hospital contacts of CPO positive patients using Stata version 15 software. Results: We recruited 22 (56.4%) CPO-positive index patients [blaNDM, n = 7 (31.8%); blaIMP, n = 3 (13.6%); blaOXA-48, n = 10 (45.5%), others, n = 2 (9.1%)] and 14 (35.9%) epidemiologically linked hospital contacts. The median age of CPO-positive patients was 72.5 years (IQR, 62–82) and 15 (68.2%) were female. The median age for the epidemiologically linked contacts was 82.5 years (IQR, 70–85) and 4 (28.6%) were female. After 1,082 patient days, 2 (14.3%) epidemiologically linked contacts tested positive for CPO giving an acquisition rate of 1.85 per 1,000 patient days (95% CI, 0.46 – 7.39). One of these participants acquired a concordant genotype (blaOXA-48) at day 7 and the other acquired a discordant genotype (CPO positive index, blaIMP; epidemiologically linked contact, blaNDM) at week 12 of follow-up. Conclusions: This small interim analysis revealed a high conversion rate among epidemiologically linked hospital contacts. A larger study is needed to understand the influence of genotypes, hospital environment, and human behavior on the transmission of CPO in hospitals.
OBJECTIVES/GOALS: In a familial case where 10 of 17 members inherited EA/LVNC in an autosomal dominant pattern, we discovered a novel, damaging missense variant in the gene KLHL26 that segregates with disease and comprises an altered electrostatic surface profile, likely decoupling the CUL3-interactome. We hypothesize that this KLHL26 variant is etiologic of EA/LVNC. METHODS/STUDY POPULATION: We differentiated a family trio (a heart-healthy daughter and EA/LVNC-affected mother and daughter) of induced pluripotent stem cells into cardiomyocytes (iPSC-CMs) in a blinded manner on three iPSC clones per subject. Using flow cytometry, immunofluorescence, and biomechanical, electrophysiological, and automated contraction methods, we investigated iPSC-CM differentiation efficiency between D10-20, contractility analysis and cell cycle regulation at D20, and sarcomere organization at D60. We further conducted differential analyses following label-free protein and RNA-Seq quantification at D20. Via CRISPR-Cas9 gene editing, we plan to characterize KLHL26 variant-specific iPSC-CM alterations and connect findings to discoveries from patient-specific studies. RESULTS/ANTICIPATED RESULTS: All iPSC lines differentiated into CMs with an increased percentage of cTnT+ cells in the affected daughter line. In comparison to the unaffected, affected iPSC-CMs had fewer contractions per minute and altered calcium transients, mainly a higher amount of total calcium release, faster rate of rise and faster rate of fall. The affected daughter line further had shorter shortening and relaxation times, higher proliferation, lower apoptosis, and a smaller cell surface area per cardiac nucleus. The affected mother line trended in a similar direction to the affected daughter line. There were no gross differences in sarcomere organization between the lines. We also discovered differential expression of candidate proteins such as kinase VRK1 and collagen COL5A1 from proteomic profiling. DISCUSSION/SIGNIFICANCE: These discoveries suggest that EA/LVNC characteristics or pathogenesis may result from decreased contractile ability, altered calcium transients, and cell cycle dysregulation. Through the KLHL26 variant correction and introduction in the daughter lines, we will build upon this understanding to inform exploration of critical clinical targets.
The macaque visual cortex is exquisitely organized into columns,
modules, and streams, much of which can be correlated with its metabolic
organization revealed by cytochrome oxidase (CO). Plasticity in the adult
primate visual system has also been documented by changes in CO activity.
Yet, the molecular mechanism of regulating this enzyme remains not well
understood. Being one of only four bigenomic enzymes in mammalian cells,
the transcriptional regulation of this enzyme necessitates a potential
bigenomic coordinator. Nuclear respiratory factor 2 (NRF-2) or GA-binding
protein is a transcription factor that may serve such a critical role. The
goal of the present study was to determine if the two major subunits of
NRF-2, 2α and 2β, had distinct subcellular distribution in neurons
of the rat and monkey visual cortex, if major metabolic neuronal types in
the macaque exhibited different levels of the two subunits, and if they
would respond differently to monocular impulse blockade. Quantitative
immuno-electron microscopy was used. In both rats and monkeys, nuclear
labeling of α and β subunits was mainly over euchromatin rather
than heterochromatin, consistent with their active participation in
transcriptional activity. Cytoplasmic labeling was over free ribosomes,
the Golgi apparatus, and occasionally the nuclear envelope, signifying
sites of synthesis and possible posttranslational modifications. The
density of both subunits was much higher in the nucleus than in the
cytoplasm for all neurons examined, again indicating that their major
sites of cellular action is in the nucleus. In both layer IVC and
supragranular puffs of the macaque visual cortex, the expression of both
NRF-2α and β was higher in medium-sized, non-pyramidal (type C and
C-like) cells previously shown to have higher CO activity than small, type
A and A-like cells with low CO activity. Pyramidal, type B cells in puffs
had intermediate levels of CO as well as NRF-2α and β labeling.
Monocular impulse blockade induced a greater reduction of NRF-2 labeling
in type C/C-like than type A/A-like cells. These results
substantiate and extend our previous findings that NRF-2 is constitutively
active in adult primate and rat visual cortical neurons, that it is
expressed more strongly in metabolically more active neurons, and that its
level is directly regulated by neuronal activity, the blockade of which
imposes a greater down-regulation of this transcription factor in
metabolically more active than less active neurons.
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