RNA Aptamer Delivery through Intact Human Skin Lenn, Jon D.; Neil, Jessica; Donahue, Christine ...
Journal of investigative dermatology,
February 2018, 2018-02-00, Letnik:
138, Številka:
2
Journal Article
Recenzirano
Odprti dostop
It is generally recognized that only relatively small molecular weight (typically < ∼ 500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a ...62-nucleotide (molecular weight = 20,395 Da) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridization assay quantified aptamer from the epidermis and dermis, giving levels far exceeding the cellular half maximal inhibitory concentration values (>100,000-fold), and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A T helper 17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and IL-22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very-large-molecular-weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin-penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23.
Traumatic spinal cord injury (tSCI) resulting in quadriplegia is a life-altering injury for patients and caregivers. We conducted a retrospective review of patients treated for tSCI and quadriplegia ...at a level 1 trauma center to assess quality of life (QOL), socioeconomic factors, and mortality. Patients and caregivers were surveyed. Of the 65 patients included, 33 contacts were made. Seventeen surveys were completed (12 caregivers and 5 patients). Six unreachable patients were confirmed alive via medical record. Mortality rate among these 39 accessible patients was 23% (n = 9). Medicaid and uninsured patients experienced longer hospital length of stay (
< .0001) and discharged to home or nursing facilities (
< .0001) more often than those with private insurance or Medicare. Patients reported overall "good" QOL (80%) while caregivers reported overall decreased QOL markers. Our results reflect the resilience among this patient population, but also highlight the impact of this life-altering injury on the caregiver.
Background
Roughly 5% of patients with blunt abdominal trauma (BAT) have a blunt bowel and/or mesenteric injury (BBMI). Determining the need for operative management in these patients can be ...challenging when hemodynamically stable. Single center studies have proposed scoring systems based on CT findings to guide management. Our study aimed to determine the predictability of abdominopelvic CT scan (CT A/P) findings in conjunction with clinical exam to determine the necessity of operative intervention for BBMI.
Methods
Patients presenting from 2017 to 2022 to the University of South Alabama Level 1 Trauma Center after motor vehicle collision were retrospectively reviewed. Patients with CT findings suggestive of BBMI were further analyzed, noting CT findings, Glasgow coma scale (GCS), shock index, abdominal exam, operative or nonoperative management, and intraoperative intervention.
Results
1098 patients with BAT underwent CT A/P. 139 patients had ≥1 finding suggestive of BBMI. 38 patients underwent surgical exploration and 30 had surgically confirmed BBMI. 27 patients required intervention for BBMI. Univariate analysis indicated that pneumoperitoneum (p < 0.0001), active extravasation of contrast (p = 0.0001), hemoperitoneum without solid organ injury (SOI) (p < 0.0001), peritonitis (p < 0.0001), and mesenteric stranding(p < 0.05) were significantly associated with intervention.
Conclusion
In total, 30 patients had surgically confirmed BBMI. Active extravasation, pneumoperitoneum, hemoperitoneum without SOI, mesenteric stranding, and peritonitis were significant indicators of BBMI requiring intervention. CT and clinical findings cannot reliably predict the need for surgical intervention without ≥1 of these findings. Initial nonoperative management with serial clinical exams should be strongly considered to reduce incidence of nontherapeutic laparotomies.
Cellular pharmacodynamic assays are crucial aspects of lead optimization programs in drug discovery. These assays are sometimes difficult to develop, oftentimes distal from the target and frequently ...low throughput, which necessitates their incorporation in the drug discovery funnel later than desired. The earlier direct pharmacodynamic modulation of a target can be established, the fewer resources are wasted on compounds that are acting via an off-target mechanism. Mass spectrometry is a versatile tool that is often used for direct, proximal cellular pharmacodynamic assay analysis, but liquid chromatography-mass spectrometry methods are low throughput and are unable to fully support structure–activity relationship efforts in early medicinal chemistry programs. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is an ambient ionization method amenable to high-throughput cellular assays, capable of diverse analyte detection, ambient and rapid laser sampling processes, and low cross-contamination. Here, we demonstrate the capability of IR-MALDESI for the detection of diverse analytes directly from cells and report the development of a high-throughput, label-free, proximal cellular pharmacodynamic assay using IR-MALDESI for the discovery of glutaminase inhibitors and a biochemical assay for hit confirmation. We demonstrate the throughput with a ∼100,000-compound cellular screen. Hits from the screening were confirmed by retesting in dose–response with mass spectrometry-based cellular and biochemical assays. A similar workflow can be applied to other targets with minimal modifications, which will speed up the discovery of cell active lead series and minimize wasted chemistry resources on off-target mechanisms.
Inadvertent medication reconciliation discrepancies are common among trauma patient populations. We conducted a prospective study at a level 1 trauma center to assess incidence of inadvertent ...medication reconciliation discrepancies following decreased reliance on short-term nursing staff. Patients and independent sources were interviewed for home medication lists and compared to admission medication reconciliation (AMR) lists. Of the 108 patients included, 37 patients (34%) never received an AMR. Of the 71 patients that had a completed AMR, 42 patients (59%) had one or more errors, with total 154 errors across all patients, for a rate of 3.7 per patient with any discrepancy. Patients taking ≥ 5 medications were significantly more likely to have an incomplete or inaccurate AMR than those taking <5 medications (89% vs 41%, P < .0001). Decreased reliance on short-term nursing staff did not decrease inadvertent admission medication reconciliation discrepancies. Additional interventions to decrease risk of medication administration errors are needed.
Trauma patients are especially vulnerable to inadvertent medication reconciliation discrepancies. We conducted a prospective study to evaluate the USA Health University Hospital’s incidence and type ...of inadvertent medication reconciliation discrepancies among trauma patients. Patients were interviewed for accuracy of their admission medication reconciliation (AMR). Eighty-nine patients were included in this study. Twenty-six patients (29%) never received an AMR. There were 107 inadvertent medication reconciliation errors identified from 30 separate patients (48%), for a rate of 3.6 errors per patient with any error. There was a significant difference in the frequency of inadvertent medication reconciliation discrepancies for patients with >5 medication compared to those with fewer (P = .00029). In conclusion, trauma centers must be adequately staffed to provide timely, accurate, and available medication lists so that patients can be appropriately cared for.
We performed power‐spectral analyses on 133 globally distributed lake‐level time series after removing annual variability. Lake‐level power spectra are found to be power‐law functions of frequency ...over the range of 20
d−1 to 27
yr−1, suggesting that lake levels are globally a
f−β‐type noise. The spectral exponent (
β), i.e., the best‐fit slope of the logarithm of the power spectrum to the logarithm of frequency, is a nonlinear function of lake surface area, indicating that lake size is an important control on the magnitude of water‐level variability over the range of time scales we considered. A simple cellular model for lake‐level fluctuations that reproduces the observed spectral‐scaling properties is presented. The model (an adaptation of a surface‐growth model with random deposition and relaxation) is based on the equations governing flow in an unconfined aquifer with stochastic inputs and outputs of water (e.g., random storms). The agreement between observation and simulation suggests that lake surface area, spatiotemporal stochastic forcing, and diffusion of the groundwater table are the primary factors controlling lake water‐level variability in natural (unmanaged) lakes. Water‐level variability is generally considered to be a manifestation of climate trends or climate change, yet our work shows that an input with short or no memory (i.e., weather) gives rise to a long‐memory nonstationary output (lake water‐level). This work forms the basis for a null hypothesis of lake water‐level variability that should be disproven before water‐level trends are to be attributed to climate.
Key Points:
Lake‐level spectra are globally power‐law over periods of 20 days to 27 years
Spectral slope of lake‐levels are a nonlinear function of logarithm of surface area
Numerical modeling shows lake‐level variability can be internally generated
Phospholipase C (PLC) enzymes are an important family of regulatory proteins involved in numerous cellular functions, primarily through hydrolysis of the polar head group from inositol-containing ...membrane phospholipids. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione), one of only a few small molecules reported to inhibit the activity of these enzymes, has been broadly applied as a pharmacological tool to implicate PLCs in diverse experimental phenotypes. The purpose of this study was to develop a better understanding of molecular interactions between U73122 and PLCs. Hence, the effects of U73122 on human PLCβ3 (hPLCβ3) were evaluated in a cell-free micellar system. Surprisingly, U73122 increased the activity of hPLCβ3 in a concentration- and time-dependent manner; up to an 8-fold increase in enzyme activity was observed with an EC50 = 13.6 ± 5 μm. Activation of hPLCβ3 by U73122 required covalent modification of cysteines as evidenced by the observation that enzyme activation was attenuated by thiol-containing nucleophiles, l-cysteine and glutathione. Mass spectrometric analysis confirmed covalent reaction with U73122 at eight cysteines, although maximum activation was achieved without complete alkylation; the modified residues were identified by LC/MS/MS peptide sequencing. Interestingly, U73122 (10 μm) also activated hPLCγ1 (>10-fold) and hPLCβ2 (∼2-fold); PLCδ1 was neither activated nor inhibited. Therefore, in contrast to its reported inhibitory potential, U73122 failed to inhibit several purified PLCs. Most of these PLCs were directly activated by U73122, and a simple mechanism for the activation is proposed. These results strongly suggest a need to re-evaluate the use of U73122 as a general inhibitor of PLC isozymes.
Vertebrate members of the nuclear receptor NR5A subfamily, which includes steroidogenic factor 1 (SF-1) and liver receptor homolog 1 (LRH-1), regulate crucial aspects of development, endocrine ...homeostasis, and metabolism. Mouse LRH-1 is believed to be a ligand-independent transcription factor with a large and empty hydrophobic pocket. Here we present structural and biochemical data for three other NR5A members—mouse and human SF-1 and human LRH-1—which reveal that these receptors bind phosphatidyl inositol second messengers and that ligand binding is required for maximal activity. Evolutionary analysis of structure-function relationships across the SF-1/LRH-1 subfamily indicates that ligand binding is the ancestral state of NR5A receptors and was uniquely diminished or altered in the rodent LRH-1 lineage. We propose that phospholipids regulate gene expression by directly binding to NR5A nuclear receptors.
As defined by the United States Department of Health and Human Services, the Social Determinants of Health (SDOH) are conditions in the environment that affect health function and outcomes. The SDOH ...are divided into the following categories: economic stability, education access and quality, health care access and quality, neighborhood and built environment, and social and community content. It is known that SDOH impact long-term health outcomes. The influence that SDOH have on physical recovery after acute injury is less understood, however. In this study, patients who suffered a traumatic blunt injury completed a survey 12-14 months post-injury to assess their SDOH and physical health before and after their injury. The results showed that for the cohort of patients studied SDOH was the greatest predictor of long-term recovery, having a stronger correlation with recovery than injury severity score (ISS) or hospital length of stay (HLOS).
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