Development of a capillary zone electrophoresis method to monitor magnesium ion consumption during in vitro transcription for mRNA production.

Messenger RNA (mRNA) vaccines represent a landmark in vaccinology, especially with their success in COVID-19 vaccines, which have shown great promise for future vaccine development and disease prevention. As a platform technology, synthetic mRNA can be produced with high fidelity using in vitro transcription (IVT). Magnesium plays a vital role in the IVT process, facilitating the phosphodiester bond formation between adjacent nucleotides and ensuring accurate transcription to produce high-quality mRNA. The development of the IVT process has prompted key inquiries about in-process characterization of magnesium ion (Mg++) consumption, relating to the RNA polymerase (RNAP) activation, fed-batch mode production yield, and mRNA quality. Hence, it becomes crucial to monitor the free Mg++ concentration throughout the IVT process. However, no free Mg++ analysis method has been reported for complex IVT reactions. Here we report a robust capillary zone electrophoresis (CZE) method with indirect UV detection. The assay allows accurate quantitation of free Mg++ for the complex IVT reaction where it is essential to preserve IVT samples in their native-like state during analysis to avoid dissociation of bound Mg complexes. By applying this CZE method, the relationships between free Mg++ concentration, the mRNA yield, and dsRNA impurity level were investigated. Such mechanistic understanding facilitates informed decisions regarding the quantity and timing of feeding starting materials to increase the yield. Furthermore, this approach can serve as a platform method for analyzing the free Mg++ in complex sample matrices where preserving the native-like state of Mg++ binding is key for accurate quantitation.

Refining the Classification of Field Cancerization.

BACKGROUND: Field cancerization is a commonly encountered but poorly defined entity in dermatology. Lack of a consensus definition makes characterizing and studying this condition difficult. OBJECTIVE: Propose a framework for defining and managing field cancerization patients. METHODS: Clinical experience and available data in the literature was used to develop a proposed field cancerization disease classification system and chemoprevention algorithm. RESULTS: The author group developed a field cancerization classification and treatment algorithm that stratifies patients into 5 different classes based on the extent of their field cancerization. They have started to recruit and study the highest risk cohort (Category 4 or 5) and have recruited 57 patients with 5 or more lifetime keratinocyte carcinomas for a prospective study evaluating chemoprevention strategies and disease burden. In these cohorts, the average number of skin cancers was greater than 12; however, less than 39% of these patients had used any chemoprevention in the prior 6 months. CONCLUSION: A meaningful and clinically relevant disease stratification framework with chemoprevention guidance has the potential to highly impact the specialty and patients.

Postoperative Complications After Interpolated Flap Repair for Mohs Defects of the Nose: A Multicenter Prospective Cohort Study.

BACKGROUND: Dermatologists perform most interpolated flaps after skin cancer resection. Prospective, multicenter data on complications after interpolated flap repair in this setting are limited. OBJECTIVE: To determine the rate of physician-reported complications after interpolated flap repair of the nose. METHODS: Multicenter, prospective cohort study of 169 patients undergoing 2-stage interpolated flap repair of post-Mohs nasal defects. Frequency of bleeding, infection, dehiscence, necrosis, hospitalization, and death in the 30 days after flap placement and flap takedown are reported. RESULTS: Patients experienced 23 complications after flap placement (13.61%) and 6 complications after flap takedown (3.55%) that were related to the surgical procedure. The most frequent complication after flap placement was bleeding (9, 5.33%, 95% confidence interval [CI]: 2.83%-9.82%). The most frequent complication after flap takedown was infection (5, 2.96%, 95% CI: 1.27%-6.74%). There was one hospitalization related to an adverse reaction to antibiotics. There were no deaths. CONCLUSION: Most complications after interpolated flap repair for post-Mohs defects of the nose are minor and are associated with flap placement. Interpolated flap repair for post-Mohs defects can be performed safely in the outpatient setting under local anesthesia.

Patient-reported outcome measures to evaluate postoperative quality of life in patients undergoing elective abdominal surgery: a systematic review.

PURPOSE: To appraise the measurement properties of generic patient-reported outcome measures (PROMs) measuring postoperative quality of life in adults undergoing elective abdominal surgery. METHODS: We conducted a systematic review of PROMs administered after elective abdominal surgery. We systematically searched Ovid MEDLINE, Embase, the Cumulative Index to Nursing & Allied Health Literature database, and the Cochrane Library from earliest available dates to July 24, 2021, using relevant search terms. Articles were included if they reported assessment of measurement properties of a generic PROM/s measuring postoperative quality of life in adults who had undergone elective abdominal surgery. We used the Consensus-based Standards for the selection of health status Measurement Instruments (COSMIN) Risk of Bias checklist to assess methodological quality. We synthesized the data and used the COSMIN criteria for good measurement properties and the Grading of Recommendations, Assessment, Development and Evaluations criteria to rate the certainty of evidence. RESULTS: Of 12,121 identified articles, nine articles assessing five PROMs (SF-6D, EQ-5D, SF-36, SF-12, PROMIS-10) met inclusion criteria. Measurement properties assessed included internal consistency (n = 2), construct validity (n = 5), and responsiveness (n = 8). Two PROMs had high quality evidence for a single measurement property each. The SF-6D demonstrated high quality evidence for responsiveness and the EQ-5D had high quality evidence for construct validity. CONCLUSION: There is insufficient evidence to support the choice of a specific generic PROM to evaluate quality of life following elective abdominal surgery. Clinicians and researchers should be aware of the current limitations in knowledge of the measurement properties of available PROMs.

Recurrence Status, Perineural Invasion, and Hypothyroidism Are Associated With Lymph Node Metastasis in Cutaneous Squamous Cell Carcinoma: A Case-Control Study.

BACKGROUND: Lymph node metastasis is a critical prognostic indicator for mortality in patients with cutaneous squamous cell carcinoma (SCC). OBJECTIVE: To identify and characterize key risk factors for SCC lymph node metastasis. METHODS: This was a multi-institutional, case-control study of 65 cutaneous SCCs with known lymph node metastasis matched with 195 cutaneous SCCs without lymph node metastasis (3:1 matching). The cases and controls were matched by anatomic location, age, and sex. Odds ratios (ORs) and their 95% confidence intervals (CIs) were generated to determine the association between specific risk factors and lymph node metastasis in a multivariate analysis. RESULTS: Recurrent tumors (p < .001), perineural invasion (p < .001), lymphovascular invasion (p = .002), size of 2 cm or greater (p = .008), and hypothyroidism (p = .03) were significantly more common in the lymph node metastasis cohort. Recurrence (OR 6.3, 95% CI 2.6-15.3), perineural invasion (OR 4.5, 95% CI 1.7-11.8), and hypothyroidism (OR 2.7, 95% CI 1.04-7.0) remained significant on performing a multivariate analysis. CONCLUSION: Lymph node metastasis in SCC is associated with recurrence, perineural invasion, lymphovascular invasion, size of 2 cm or greater, and hypothyroidism. Clinical consideration of these findings within the context of current staging systems may help improve patient outcomes.

Study of a 532/1064 Fractional Picosecond Laser for Facial Rejuvenation.

BACKGROUND AND OBJECTIVES: Picosecond (ps) fractional lasers create small wounds, presumably by laser-induced optical breakdown. We studied a ps fractional laser in the treatment of wrinkles and mottled pigment. MATERIALS AND METHODS: This was a single center, prospective, open-label clinical trial. Patients with at least 2 facial areas, with visible wrinkles and dyschromia, were enrolled in the study and received 3 treatments at monthly intervals and appeared at 3 follow-up visits at 1, 3, and 6 months after treatment. The laser is an 800 ps fractional system with nominal 10 mm macrospot diameter. Both 532 nm and 1,064 nm wavelengths were applied in each subject. Wrinkle and pigmentation clearance were assessed by 2 blinded investigators using a 5-point clearance scale. Skin improvement was assessed by investigators using the 5-point Global Aesthetic Improvement (GAI) Scale based on before/after photographs for the following categories: (1) fine lines/wrinkles and (2) pigmentation. RESULTS: A total of 18 healthy subjects at a single site were enrolled. At least moderate pigmentation and fine line/wrinkles improvement were observed in 93% and 79% of patients at 1 month after the last treatment according to GAI, respectively. Pigment clearance approached a mean of approximately 40%. CONCLUSION: A ps 1,064/532 fractional laser achieves reduction in fine lines and pigment.

Patient Outcomes and Satisfaction After Mohs Micrographic Surgery in Patients With Nonmelanoma Skin Cancer.

BACKGROUND: Quality in medicine is increasingly being measured through patient-reported outcome measures. Given the rising incidence and costs for nonmelanoma skin cancer (NMSC) treatment, it is imperative to define quality measures specific to dermatologic surgery. OBJECTIVE: This study aims to evaluate patient-reported outcomes and satisfaction with Mohs micrographic surgery (MMS) together with patient and tumor factors to better define their use in developing treatment strategies and quality measures. METHODS AND MATERIALS: A prospective study was conducted among 226 patients undergoing MMS for treatment of NMSC. Patient demographics, quality of life, functional status, satisfaction, and prognostic factors were gathered. Postoperative outcomes were measured at 1 month and included patient-reported problems and provider-reported complications. Relationships between patient factors and outcomes were evaluated through statistical analysis. RESULTS: Average patient satisfaction in the domain of general satisfaction of the Patient Satisfaction Questionnaire-18 was 4.34 of 5. General patient satisfaction did not differ across age, final defect size, sex, or prognostic scores. At 1-month postoperatively, 97 percent of patients expressed willingness to undergo future MMS if indicated. CONCLUSION: Patients are generally satisfied with MMS for treatment of NMSC. Specific patient factors that may affect satisfaction include smoking status and anticoagulation use.

Misclassification of Aggressive Basal Cell Carcinoma Subtypes and Implications for Management.

BACKGROUND: Management of basal cell carcinoma (BCC) varies by histopathologic subtype; however, biopsies may inadequately characterize them as nonaggressive, risking potential suboptimal treatment. OBJECTIVE: To characterize the rate of undetected aggressive BCC subtypes by size, location, and histopathology type. MATERIALS AND METHODS: Retrospective cohort study of 928 BCCs treated with Mohs Micrographic Surgery (MMS) at a tertiary academic institution from 2015 to 2017, comparing patient and tumor characteristics and histopathologic subtype on biopsy versus Mohs. RESULTS: Among the 825 BCCs with known subtypes on biopsy, 68% (561/825) were classified as nonaggressive, 28% (159/561) of which were subsequently found to have aggressive subtypes on MMS. Aggressive features were more often underrepresented in biopsy samples taken from Area H compared with Area M/L (odd ratio [OR] 2.65, 95% confidence interval [CI] 1.73-4.08, p < .001) or those with nodular subtypes (OR 2.19, CI 1.08-4.45, p = .03). Of concern, these unsuspected aggressive BCCs required more Mohs stages for clearance (mean 2.37, SD 0.72, p < .001) compared with BCCs that remained nonaggressive on both biopsy and Mohs (mean 1.50, SD 0.75). CONCLUSION: Given the high percentage of BCCs with unsuspected aggressive subtypes, higher clinical suspicion for undiagnosed high-risk BCCs should be given to nodular BCCs and to BCCs on Area H.

Basal Cell Carcinoma Gene Mutations Differ Between Asian, Hispanic, and Caucasian Patients: A Pilot Study.

BACKGROUND: Basal cell carcinoma (BCC) is the most common malignancy worldwide. While most BCCs are treated surgically, advanced BCCs are often treated with gene-targeted therapies. While there has been a lot of research in BCC from Caucasian patients, research is lacking in patients with skin of color. OBJECTIVE: To identify potential variations in BCC gene mutations between Asian, Hispanic, and Caucasian patients. METHODS: A cohort study was performed from 2015 to 2017 with 23 patients treated for BCC at an urban academic hospital. Gene mutations were assessed using a targeted mutation panel for 76 cancer-associated genes from formalin-fixed paraffin-embedded (FFPE) samples. RESULTS: Groups studied comprised Asian (n=5), Hispanic (n=10), and Caucasian (n=8) patients. The Hispanic cohort had the highest number of mutations per patient on average (3.4 versus 2.8 for both Caucasian and Asian cohorts). GATA3 mutations were more prevalent in Hispanic patients (P=0.02, single factor ANOVA). ARID1A and PTEN mutations co-occurred in the Hispanic cohort (P<0.05). The most common mutation in the Asian cohort was TP53 (2/5). The Caucasian cohort had the highest percent of UVB mutations (68.4%). CONCLUSIONS: This study shows potential differences in the prevalence of somatic gene mutations for BCC patients of different races and ethnicities, which could inform the underlying pathogenesis, impact the efficacy of therapies in specific populations, and may also help identify novel therapeutic targets. J Drugs Dermatol. 20(5): doi:10.36849/JDD.5884.

Using natural deep eutectic solvents for the extraction of metabolites in Byrsonima intermedia leaves.

Natural deep eutectic solvents have been used as an alternative to organic solvents for the extraction of plants metabolites, allowing for the extraction of compounds of different polarities, while being inexpensive, non-toxic, and easy to prepare. This work presents the comparison of the chromatographic profiles by high-performance liquid chromatography with diode-array detection obtained from Byrsonima intermedia (Malpighiaceae) using five choline chloride-based natural deep eutectic solvents, in addition to the most used traditional extraction solvents, methanol/water 7:3 and ethanol/water 7:3 v/v. A reference extract was used to tentatively identify compounds by high-performance liquid chromatography with tandem mass spectrometry. The water content appeared to be important for the extraction efficiency and the mixture choline chloride/glycerol was shown to be the best candidate for efficiently extracting this matrix when compared with the traditional extraction media in addition to being far greener as shown by the environmental analysis tool. Seven phenolic compounds (digalloyl quinic acid, proanthocyanidin dimer, galloylproanthocyanidin dimer, quercetin-O-hexoside, galloyl quercetin hexoside, quercetin-O-pentoside, and galloyl quercetin pentoside) were tentatively identified in all extracts. Moreover, the influence of these solvents on the antioxidant activity of the extracts was studied and the results for choline chloride/glycerol extracts were very similar to that of the traditional extraction solvents.

Launching a Mohs Surgery Program.

BACKGROUND: There is a paucity of data or publications in the literature on best practices for opening a new Mohs surgery unit. OBJECTIVE: The goal of this article is to be a "how to" guide for starting a Mohs and dermatologic surgery practice. MATERIALS AND METHODS: Two academic Mohs surgeons share their personal experiences and data from the literature. RESULTS: Topics discussed include picking a location and identifying space, equipment, staffing, regulatory practices, policies and procedures, marketing and outreach, patient experience, building culture, clinic efficiency, and vision. CONCLUSION: Although opening a new Mohs surgery unit is challenging, it can be rewarding to have the opportunity to develop best practices and systems that create a wonderful working environment and allow for exceptional care of patients.

Natural deep eutectic solvents as the major mobile phase components in high-performance liquid chromatography-searching for alternatives to organic solvents.

Over the past six decades, acetonitrile (ACN) has been the most employed organic modifier in reversed-phase high-performance liquid chromatography (RP-HPLC), followed by methanol (MeOH). However, from the growing environmental awareness that leads to the emergence of "green analytical chemistry," new research has emerged that includes finding replacements to problematic ACN because of its low sustainability. Deep eutectic solvents (DES) can be produced from an almost infinite possible combinations of compounds, while being a "greener" alternative to organic solvents in HPLC, especially those prepared from natural compounds called natural DES (NADES). In this work, the use of three NADES as the main organic component in RP-HPLC, rather than simply an additive, was explored and compared to the common organic solvents ACN and MeOH but additionally to the greener ethanol for separating two different mixtures of compounds, one demonstrating the elution of compounds with increasing hydrophobicity and the other comparing molecules of different functionality and molar mass. To utilize NADES as an organic modifier and overcome their high viscosity monolithic columns, temperatures at 50 °C and 5% ethanol in the mobile phase were used. NADES are shown to give chromatographic performances in between those observed for ACN and MeOH when eluotropic strength, resolution, and peak capacity were taken into consideration, while being less environmentally impactful as shown by the HPLC-Environmental Assessment Tool (HPLC-EAT) metric. With the development of proper technologies, DES could open a new class of mobile phases increasing the possibilities of new separation selectivities while reducing the environmental impact of HPLC analyses. Graphical abstract Natural deep eutectic solvents versus traditional solvents in HPLC.

Performance and selectivity of cationic nanoparticle pseudo-stationary phases in electrokinetic chromatography.

Electrokinetic chromatography (EKC) is a powerful analytical technique that uses an ionic pseudo-stationary phase (PSP) to separate neutral compounds. Although anionic surfactants are the most common choice for PSP, cationic latex nanoparticles are an attractive alternative. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize several types of diblock copolymers that self-assemble into latex nanoparticles, which were characterized by a variety of techniques including diffusion NMR. The performance of each nanoparticle as a PSP was studied by using a homologous series of ketones and linear solvation energy relationships (LSER) analysis. A cationic homopolymer coating was found to be necessary to prevent band broadening caused by analyte interactions with nanoparticles adsorbed to the capillary surface. No significant difference in methylene selectivity or LSER parameters was observed between nanoparticles with different cationic shells, but differences were observed between nanoparticles with different hydrophobic cores. Cationic latex nanoparticles behaved more like anionic latex nanoparticles than like cationic surfactants, suggesting that selectivity is primarily driven by the hydrophobic portion of a PSP. Cationic latex nanoparticles in combination with a homopolymer cationic capillary coating are an excellent choice for EKC analyses where an anodic electroosmotic flow is required.