Real-world experience quantifying access to JAK inhibitor care for alopecia areata patients: a patient-centered survey study.

Background: Janus kinase inhibitor (JAKi) therapy has revolutionized the treatment landscape for alopecia areata (AA); however, access may be limited by a lack of insurance coverage and high out-of-pocket costs. Objective: We aimed to evaluate real-world patient experiences regarding access to JAKi therapy. Methods: We conducted an online patient-centered survey using the National Alopecia Areata Foundation listserv. Results: In total 784 individuals initiated our survey, and 600 completed it in full (76.5%). While more non-White patients considered obtaining JAKi therapy, more White patients reported the use of this medication class. In total, 74.2% lacked insurance coverage or had partial coverage for JAKi, and 52% expressed dissatisfaction with available coverage. However, 52.9% reported delays in starting medication due to insurance approval processes, contributing to worsened AA and related stress. In total, 35% of patients did not try to obtain JAKi therapy due to concerns about costs, and 18.2% discontinued therapy due to financial barriers. Also, 19.8% of patients reported utilizing financial savings to pay for medication, and 55.2% reported using a copay assistance card. Further, 12.2% reported forgoing other necessities to pay for AA expenses. Limitations: Our results are limited by the subjective nature of survey studies. The recency of FDA approval for JAKi therapy may also influence patients' perceptions of access to care. Conclusion: Patients with AA face significant barriers when trying to obtain JAKi therapy, and existing racial inequities may be exacerbated by these barriers. Further advocacy work is needed to improve access to care.

Single-cell quantification of ribosome occupancy in early mouse development.

Translation regulation is critical for early mammalian embryonic development1. However, previous studies had been restricted to bulk measurements2, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N6-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells.

East Asian Population.

This article explores the ways East Asian American (EAA) children and adolescents have experienced disparities in the United States throughout the COVID-19 pandemic. The history of racism toward Asian American and Pacific Islanders (AAPI) and the complexities of acculturation are reflected through this contemporary lens. Traditional East Asian (EA) values were disrupted during this period. Implications for children and families are discussed. Persistent underlying xenophobia and racism, such as the model minority myth or perpetual foreigner stereotype, rose to new prominence, furthering emotional distress in EA and EAA youths beyond those already experienced universally by AAPI families during the pandemic.

Isolation of enriched small RNA from cell-lysate using on-chip isotachophoresis.

In spite of the growing interest in the roles and applications of small RNAs (sRNAs), sRNA isolation methods are inconsistent, tedious, and dependent on the starting number of cells. In this work, we employ ITP to isolate sRNAs from the cell-lysate of K562 (chronic myelogenous leukemia) cells in a polydimethylsiloxane (PDMS) mesofluidic device. Our method specifically purifies sRNA of <60 nucleotides from lysate of a wide range of cell number spanning from 100 to 1 000 000 cells. We measured the amount of sRNA using the Agilent Bioanalyzer and further verified the extraction efficiency by reverse transcription quantitative PCR. Our method was shown to be more efficient in sRNA extraction than commercial sRNA isolation kits, especially when using smaller numbers of starting cells. Our assay presents a simple and rapid sRNA extraction method with 20 min assay time and no intermediate transfer steps.

Simultaneous RNA purification and size selection using on-chip isotachophoresis with an ionic spacer.

We present an on-chip method for the extraction of RNA within a specific size range from low-abundance samples. We use isotachophoresis (ITP) with an ionic spacer and a sieving matrix to enable size-selection with a high yield of RNA in the target size range. The spacer zone separates two concentrated ITP peaks, the first containing unwanted single nucleotides and the second focusing RNA of the target size range (2-35 nt). Our ITP method excludes >90% of single nucleotides and >65% of longer RNAs (>35 nt). Compared to size selection using gel electrophoresis, ITP-based size-selection yields a 2.2-fold increase in the amount of extracted RNAs within the target size range. We also demonstrate compatibility of the ITP-based size-selection with downstream next generation sequencing. On-chip ITP-prepared samples reveal higher reproducibility of transcript-specific measurements compared to samples size-selected by gel electrophoresis. Our method offers an attractive alternative to conventional sample preparation for sequencing with shorter assay time, higher extraction efficiency and reproducibility. Potential applications of ITP-based size-selection include sequencing-based analyses of small RNAs from low-abundance samples such as rare cell types, samples from fluorescence activated cell sorting (FACS), or limited clinical samples.

Efficient Production of On-Target Reads for Small RNA Sequencing of Single Cells Using Modified Adapters.

Although single-cell mRNA sequencing has been a powerful tool to explore cellular heterogeneity, the sequencing of small RNA at the single-cell level (sc-sRNA-seq) remains a challenge, as these have no consensus sequence, are relatively low abundant, and are difficult to amplify in a bias-free fashion. We present two methods of single-cell-lysis that enable sc-sRNA-seq. The first method is a chemical-based technique with overnight freezing while the second method leverages on-chip electrical lysis of plasma membrane and physical extraction and separation of cytoplasmic RNA via isotachophoresis. We coupled these two methods with off-chip small RNA library preparation using CleanTag modified adapters to prevent the formation of adapter dimers. We then demonstrated sc-sRNA-seq with single K562 human leukemic cells. Our approaches offer a relatively short hands-on time of 6 h and efficient generation of on-target reads. The sc-sRNA-seq with our approaches showed detection of miRNA with various abundances ranging from 16 000 copies/cell to about 10 copies/cell. We anticipate this approach will create a new opportunity to explore cellular heterogeneity through small RNA expression.

Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring.

Here, we report a method for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and mRNA from a variety of cell types. Cytosolic contents were repeatedly sampled from the same cell or population of cells for more than 5 d through a cell-culture substrate, incorporating hollow 150-nm-diameter nanostraws (NS) within a defined sampling region. Once extracted, the cellular contents were analyzed with conventional methods, including fluorescence, enzymatic assays (ELISA), and quantitative real-time PCR. This process was nondestructive with >95% cell viability after sampling, enabling long-term analysis. It is important to note that the measured quantities from the cell extract were found to constitute a statistically significant representation of the actual contents within the cells. Of 48 mRNA sequences analyzed from a population of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs), 41 were accurately quantified. The NS platform samples from a select subpopulation of cells within a larger culture, allowing native cell-to-cell contact and communication even during vigorous activity such as cardiomyocyte beating. This platform was applied both to cell lines and to primary cells, including CHO cells, hiPSC-CMs, and human astrocytes derived in 3D cortical spheroids. By tracking the same cell or group of cells over time, this method offers an avenue to understand dynamic cell behavior, including processes such as induced pluripotency and differentiation.

Increasing hybridization rate and sensitivity of DNA microarrays using isotachophoresis.

We present an on-chip electrokinetic method to increase the reaction kinetics and sensitivity of DNA microarray hybridization. We use isotachophoresis (ITP) to preconcentrate target molecules in solution and transport them over the immobilized probe sites of a microarray, greatly increasing the binding reaction rate. We show theoretically and experimentally that ITP-enhanced microarrays can be hybridized much faster and with higher sensitivity than conventional methods. We demonstrate our assay using a microfluidic system consisting of a PDMS microchannel superstructure bonded onto a glass slide on which 60 spots of 20-27 nt ssDNA oligonucleotide probes are immobilized. Our 30 min assay results in an 8.2 fold higher signal than the conventional overnight hybridization at 100 fM target concentration. We show rapid and quantitative detection over 4 orders of magnitude dynamic range of target concentration with no increase in the nonspecific signal. Our technique can be further multiplexed for higher density microarrays and extended for other reactions of target-surface immobilized ligands.

17ß-estradiol enhances memory duration in the main olfactory bulb in CD-1 mice.

Rodents rely heavily on odor detection, discrimination, and memory to locate food, find mates, care for pups, and avoid predators. Estrogens have been shown to increase memory retention in rodents performing spatial memory and object placement tasks. Here we evaluate the extent to which 17ß-estradiol modulates memory formation and duration in the olfactory system. Adult CD-1 mice were gonadectomized and given either systemic 17ß-estradiol replacement, local 17ß-estradiol in the main olfactory bulb, or no replacement. Before performing the behavioral task the mice were given saline or PHTPP (an estrogen receptor ß [ER-ß] antagonist) via bilateral infusion into the main olfactory bulb. As the beta-type estrogen receptor (ER-ß) is more abundant than the alpha-type estrogen receptor in the murine main olfactory bulb, the current study focuses on 17ß-estradiol and its interactions with ERß. Habituation, a simple, nonassociative learning task in which an animal is exposed to the same odor over successive presentations, was used to evaluate the animals' ability to detect odors and form an olfactory memory. To evaluate memory duration, we added a final trial of intertrial interval time (30 or 60 min) in which we presented the habituated odor. Neither surgical nor drug manipulation affected the ability of mice to detect or habituate to an odor. After habituation, gonadectomized 17ß-estradiol-treated mice retained memory of an odor for 30 min, whereas non-estradiol-treated, 17ß-estradiol+ERß antagonist (PHTPP), and untreated male mice did not remember an odor 30 min after habituation. The results show that both systemic and local bulbar infusions of 17ß-estradiol enhance odor memory duration in mice.

Integration of rapid DNA hybridization and capillary zone electrophoresis using bidirectional isotachophoresis.

We present a method for rapid, sequence-specific detection of multiple DNA fragments by integrating isotachophoresis (ITP) based DNA hybridization and capillary zone electrophoresis (CZE) using bidirectional ITP. Our method leverages the high preconcentration ability of ITP to accelerate slow, second-order DNA hybridization kinetics, and the high resolving power of CZE to separate and identify reaction products. We demonstrate the speed and sensitivity of our assay by detecting 5 pM, 39 nt ssDNA target within 3 min, using a molecular beacon probe. We also demonstrate the feasibility of our assay for multiplexed detection of multiple-length ssDNA targets by simultaneously detecting 39 and 90 nt ssDNA targets.

Rapid hybridization of nucleic acids using isotachophoresis.

We use isotachophoresis (ITP) to control and increase the rate of nucleic acid hybridization reactions in free solution. We present a new physical model, validation experiments, and demonstrations of this assay. We studied the coupled physicochemical processes of preconcentration, mixing, and chemical reaction kinetics under ITP. Our experimentally validated model enables a closed form solution for ITP-aided reaction kinetics, and reveals a new characteristic time scale which correctly predicts order 10,000-fold speed-up of chemical reaction rate for order 100 pM reactants, and greater enhancement at lower concentrations. At 500 pM concentration, we measured a reaction time which is 14,000-fold lower than that predicted for standard second-order hybridization. The model and method are generally applicable to acceleration of reactions involving nucleic acids, and may be applicable to a wide range of reactions involving ionic reactants.

Extraction of DNA from malaria-infected erythrocytes using isotachophoresis.

We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency. We also implement pressure-driven counterflow during ITP extraction to extend focusing time and increase nucleic acid yield. We show that the purified genomic DNA samples are compatible with polymerase chain reaction (PCR) and demonstrate a clinically relevant limit of detection of 0.5 parasites per nanoliter using quantitative PCR.