Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

Local vasoregulative interventions impact drug concentrations in the skin after topical laser-assisted delivery.

INTRODUCTION: The ability of ablative fractional lasers (AFL) to enhance topical drug uptake is well established. After AFL delivery, however, drug clearance by local vasculature is poorly understood. Modifications in vascular clearance may enhance AFL-assisted drug concentrations and prolong drug dwell time in the skin. Aiming to assess the role and modifiability of vascular clearance after AFL-assisted delivery, this study examined the impact of vasoregulative interventions on AFL-assisted 5-fluorouracil (5-FU) concentrations in in vivo skin. METHODS: 5-FU uptake was assessed in intact and AFL-exposed skin in a live pig model. After fractional CO2 laser exposure (15 mJ/microbeam, 5% density), vasoregulative intervention using topical brimonidine cream, epinephrine solution, or pulsed dye laser (PDL) was performed in designated treatment areas, followed by a single 5% 5-FU cream application. At 0, 1, 4, 48, and 72 h, 5-FU concentrations were measured in 500 and 1500 µm skin layers by mass spectrometry (n = 6). A supplemental assessment of blood flow following AFL ± vasoregulation was performed using optical coherence tomography (OCT) in a human volunteer. RESULTS: Compared to intact skin, AFL facilitated a prompt peak in 5-FU delivery that remained elevated up to 4 hours (1500 µm: 1.5 vs. 31.8 ng/ml [1 hour, p = 0.002]; 5.3 vs. 14.5 ng/ml [4 hours, p = 0.039]). However, AFL's impact was transient, with 5-FU concentrations comparable to intact skin at later time points. Overall, vasoregulative intervention with brimonidine or PDL led to significantly higher peak 5-FU concentrations, prolonging the drug's dwell time in the skin versus AFL delivery alone. As such, brimonidine and PDL led to twofold higher 5-FU concentrations than AFL alone in both skin layers by 1 hour (e.g., 500 µm: 107 ng/ml [brimonidine]; 96.9 ng/ml [PDL], 46.6 ng/ml [AFL alone], p ≤ 0.024), and remained significantly elevated at 4 hours (p ≤ 0.024). A similar pattern was observed for epinephrine, although trends remained nonsignificant (p ≥ 0.09). Prolonged 5-FU delivery was provided by PDL, resulting in sustained drug deposition compared to AFL alone at both 48 and 72 hours in the superficial skin layer (p ≤ 0.024). Supporting drug delivery findings, OCT revealed that increases in local blood flow after AFL were mitigated in test areas also exposed to PDL, brimonidine, or epinephrine, with PDL providing the greatest, sustained reduction in flow over 48 hours. CONCLUSION: Vasoregulative intervention in conjunction with AFL-assisted delivery enhances and prolongs 5-FU deposition in in vivo skin.

[Uncovering rare diseases in medical data-coding]. / Seltene Erkrankungen in den Daten sichtbar machen ­ Kodierung.

The ICD-10-GM coding system used in the German healthcare system only captures a minority of rare disease diagnoses. Therefore, information on the incidence and prevalence of rare diseases as well as necessary (financial) resources for the expert care required for evidence-based decisions by health insurers, care providers, and politicians are lacking. Furthermore, the missing information complicates and sometimes even precludes the generation of scientific knowledge on rare diseases. Therefore, starting in 2023, all in-patient cases in Germany with a rare disease diagnosis must be coded by an ORPHAcode using the Alpha-ID-SE file.The file Alpha-ID-SE links the ICD-10-GM codes to the internationally established ORPHAcodes for rare diseases. Commercially available software tools progressively support the coding of rare diseases. In several centers for rare diseases linked to university hospitals, IT tools and procedures were established to realize a complete coding of rare diseases. These include financial incentives for the institutions providing rare disease codes, systematic queries asking for rare disease codes during the coding process, and a semi-automated coding process for all patients with a rare disease previously seen at the institution. A combination of the different approaches probably results in the most complete coding.To get the complete picture of rare disease epidemiology and care requirements, a specific and unique coding of out-patient cases is also desirable. Furthermore, a structured reporting of phenotype is required, especially for complex rare diseases and for yet undiagnosed cases.

Lidocaine-induced potentiation of thermal damage in skin and carcinoma cells.

OBJECTIVE: Lidocaine acts as a local anesthetic by blocking transmembrane sodium channel permeability, but also induces the synthesis of heat shock proteins and sensitizes cells to hyperthermia. A previous study reported two cases of deep focal skin ulceration at points corresponding to depot local lidocaine injection sites after treatment with non-ablative fractional resurfacing and it was hypothesized that lidocaine had focally sensitized keratinocytes to the thermal damage of laser treatment. The objective of this study was to investigate whether lidocaine potentiates hyperthermia damage to both normal and cancerous skin cells using an in vitro model. METHODS: Normal skin cell lines (fibroblasts, keratinocytes), skin cancer cell lines (melanoma, basal cell carcinoma), and a mucosal cancer cell line (cervical carcinoma) were exposed to various concentrations of lidocaine (0-0.3%) with or without hyperthermia (37°C, 42°C). RESULTS: Compared to normal skin cells, we demonstrate that cancer cell lines show significantly increased cell toxicity when a moderate temperature (42°C) and low lidocaine concentrations (0.1-0.2%) are combined. The toxicity directly correlates with a higher percentage of cells in S-phase (28-57%) in the cancer cell lines compared to normal skin cell lines (13-19%; R-square 0.6752). CONCLUSION: These results suggest that lidocaine potentiates thermal sensitivity of cell cycle active skin cells. The direct correlation between cell toxicity and S-phase cells could be harnessed to selectively treat skin and mucosal cancer cells while sparing the surrounding normal tissue. Additional research pre-clinically and clinically using several different heat sources (e.g., lasers, ultrasound, etc.) and lidocaine concentrations is needed to confirm and optimize these results. Lidocaine-enhanced hyperthermia may provide a non-invasive, alterative treatment option for highly proliferating, superficial skin, and mucosal lesions such as cancer or warts. Lasers Surg. Med. 51:88-94, 2019. © 2018 Wiley Periodicals, Inc.

Diffuse Reflectance Spectroscopy with Infrared Thermography for Accurate Prediction of Cellulitis.

Cellulitis is frequently misdiagnosed owing to its clinical mimickers, collectively known as pseudocellulitis. This study investigated diffuse reflectance spectroscopy (DRS) alone and in combination with infrared thermography (IRT) for the differentiation of cellulitis from pseudocellulitis. A prospective cohort study at an urban academic hospital was conducted from March 2017 to March 2018. Patients presenting to the emergency department with presumed cellulitis were screened for eligibility, and 30 adult patients were enrolled. Dermatology consultation conferred a final diagnosis of cellulitis or pseudocellulitis. DRS measurements yielded a spectral ratio between 556 nm (deoxyhemoglobin peak) and 542 nm (oxyhemoglobin peak), and IRT measurements yielded temperature differentials between the affected and unaffected skin. Of the 30 enrolled patients, 30% were diagnosed with pseudocellulitis. DRS revealed higher spectral ratios in patients with cellulitis (P = 0.005). A single parameter model using logistic regression on DRS measurements alone demonstrated a classification accuracy of 77.0%. A dual parameter model using linear discriminant analysis on DRS and IRT measurements combined demonstrated a 95.2% sensitivity, 77.8% specificity, and 90.0% accuracy for cellulitis prediction. DRS and IRT combined diagnoses cellulitis with an accuracy of 90%. DRS and IRT are inexpensive and noninvasive, and their use may reduce cellulitis misdiagnosis.

Norrin Protects Retinal Ganglion Cells from Excitotoxic Damage via the Induction of Leukemia Inhibitory Factor.

PURPOSE: To investigate whether and how leukemia inhibitory factor (Lif) is involved in mediating the neuroprotective effects of Norrin on retinal ganglion cells (RGC) following excitotoxic damage. Norrin is a secreted protein that protects RGC from N-methyl-d-aspartate (NMDA)-mediated excitotoxic damage, which is accompanied by increased expression of protective factors such as Lif, Edn2 and Fgf2. METHODS: Lif-deficient mice were injected with NMDA in one eye and NMDA plus Norrin into the other eye. RGC damage was investigated and quantified by TUNEL labeling 24 h after injection. Retinal mRNA expression was analyzed by quantitative real-time polymerase chain reaction following retinal treatment. RESULTS: After intravitreal injection of NMDA and Norrin in wild-type mice approximately 50% less TUNEL positive cells were observed in the RGC layer when compared to NMDA-treated littermates, an effect which was lost in Lif-deficient mice. The mRNA expression for Gfap, a marker for Müller cell gliosis, as well as Edn2 and Fgf2 was induced in wild-type mice following NMDA/Norrin treatment but substantially blocked in Lif-deficient mice. CONCLUSIONS: Norrin mediates its protective properties on RGC via Lif, which is required to enhance Müller cell gliosis and to induce protective factors such as Edn2 or Fgf2.

A microscale, full-thickness, human skin on a chip assay simulating neutrophil responses to skin infection and antibiotic treatments.

Human skin models are essential for understanding dermatological diseases and testing new treatment strategies. The use of skin biopsies ex vivo is the most accurate model. However, their use is expensive and exposes the donor to pain and scarring. While bioengineered skin samples provide a cheaper alternative, they have limitations due to their simple structure and functionality compared to human skin. Here, we present a skin-on-a-chip device designed to study neutrophil responses to Staphylococcus aureus skin infections. We integrate human skin microcolumns, which have a cross-section that is ∼100 times smaller than traditional skin biopsies, are full-thickness, and are collected using minimally invasive skin sampling techniques. We use human neutrophils directly from one drop of blood, without the need for blood separation. Using the skin-on-a-chip device with skin and blood samples from healthy donors, we show that the neutrophil responses correlate with the bacteria-load in the skin. A pre-incubation step increases the number of migrating neutrophils in response to a low concentration of bacteria. Antibiotic treatment of S. aureus-infected skin samples reduces the number of neutrophils migrating towards the skin. Overall, we validate a skin on a chip model that enables the study of neutrophil migration to the skin in the presence of microbes and following the administration of antibiotics, two situations relevant to clinical cases of human skin and soft tissue infections.