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.

Determining the Oxidation Mechanism through Radical Intermediates in Polysorbates 80 and 20 by Electron Paramagnetic Resonance Spectroscopy.

Polysorbates 20 and 80 (PS20 and PS80) are added to many commercial biologic and vaccine pharmaceuticals. It is commonly known that these polysorbates undergo a radical oxidation mechanism; however, the identity of these radical intermediates has not been clearly determined. Furthermore, PS20 and PS80 differ by the presence of a lauric acid instead of an oleic acid, respectively. The oxidation of PS80 is thought to be centered around the double bond of the oleic acid even though PS20 also undergoes oxidation, making the mechanism of oxidation unclear for PS20. Using commercial stocks of PS20 and PS80 alkyl (R•), alkoxyl (C-O•) and peroxyl (C-OO•) radicals were detected by electron paramagnetic resonance spectroscopy likely originating from radical-initiating species already present in the material. When dissolved in water, the peroxyl radicals (C-OO•) originally in the stocks were not detected but poly(ethylene oxide) radicals were. An oxidative pathway for polysorbates was suggested based on the radical species identified in the polysorbate stock material and solutions.

A case of trichodysplasia spinulosa related to ruxolitinib treated successfully with oral acitretin.

Trichodysplasia spinulosa (TS) is a rare disease that affects immunocompromised patients, characterized by hair-like growths caused by TS-associated polyomavirus infection. Little is known about specific immunosuppressive drugs that can precipitate the condition. We report a case of TS presenting after initiating the oral Janus-associated kinase inhibitor (JAKi) ruxolitinib. A 67-year-old female with a history of allogeneic bone marrow transplant requiring immunosuppression with tacrolimus, prednisone and, more recently, ruxolitinib 5 mg twice daily due to Graft versus Host Disease presented to the clinic with a facial rash. The clinical and histopathological findings in the setting of immunosuppression were consistent with TS. Initial treatments were ineffective, but oral acitretin showed significant improvement after 3 months. Due to the close temporal relationship between the initiation of ruxolitinib and the development of TS, this case suggests that JAKis may contribute to TS development by suppressing the JAK-signal transducer and activator of the transcription pathway's antiviral functions.

Online reading habits can reveal personality traits: towards detecting psychological microtargeting.

Building on big data from Reddit, we generated two computational text models: (i) Predicting the personality of users from the text they have written and (ii) predicting the personality of users based on the text they have consumed. The second model is novel and without precedent in the literature. We recruited active Reddit users (N=1,105) of fiction-writing communities. The participants completed a Big Five personality questionnaire and consented for their Reddit activity to be scraped and used to create a machine learning model. We trained an natural language processing model [Bidirectional Encoder Representations from Transformers (BERT)], predicting personality from produced text (average performance: r=0.33). We then applied this model to a new set of Reddit users (N=10,050), predicted their personality based on their produced text, and trained a second BERT model to predict their predicted-personality scores based on consumed text (average performance: r=0.13). By doing so, we provide the first glimpse into the linguistic markers of personality-congruent consumed content.

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 Nasal Function and Appearance After Interpolation Flap Repair Following Skin Cancer Resection: A Multicenter Prospective Cohort Study.

Objective: Among patients undergoing two-stage interpolated flap repair of nasal defects, nasal function, and appearance before surgery and at 16 weeks after flap takedown were compared using the Nasal Appearance and Function Evaluation Questionnaire (NAFEQ). Design: Multicenter prospective cohort study. Methods: Adult patients with a nasal skin cancer anticipated to require two-stage interpolation flap repair completed the NAFEQ before surgery, at 1 week after flap placement, 4 weeks after flap takedown, and 16 weeks after flap takedown. Results: One hundred sixty-nine patients were enrolled, with 138 patients completing both presurgical and 16-week post-takedown NAFEQs. Overall NAFEQ score increased by 1.09 points (1.91% improvement, confidence interval [95% CI -0.34 to 2.53]). NAFEQ functional subscale increased by 0.72 points (2.58% increase; 95% CI [0.10-1.35]) and appearance subscale increased by 0.37 points (1.28% improvement, 95% CI [-0.65 to 1.39]). Conclusion: At 16 weeks after flap takedown, patients' perceptions of their nasal function and appearance are similar to or slightly improved when compared with their presurgical assessments.

Extensive Characterization of Polysorbate 80 Oxidative Degradation Under Stainless Steel Conditions.

Polysorbate-80 (PS-80) is a common surfactant used in biologics formulations. However, the tendency of oxidation to PS-80 when exposed to stainless steel surfaces brings various challenges during manufacturing processes, such as inconsistent shelf-life of PS-80 solutions, which can further impact the biologics and vaccines production. In this work, the root causes of PS-80 oxidation when in contact with stainless steel conditions were thoroughly investigated through the use of various complementary analytical techniques including U/HPLC-CAD, LC-MS, ICP-MS, peroxide assay, and EPR spectroscopy. The analytical tool kit used in this work successfully revealed a PS-80 degradation mechanism from the perspective of PS-80 content, PS-80 profile, iron content, peroxide production, and radical species. The combined datasets reveal that PS-80 oxidative degradation occurs in the presence of histidine and iron in addition to being combined with the hydroperoxides in PS-80 material. The oxidative pathway and potential degradants were identified by LC-MS. The PS-80 profile based on the U/HPLC-CAD assay provided an effective way to identify early-signs of PS-80 degradation. The results from a peroxide assay observed increased hydroperoxide along with PS-80 degradation. EPR spectra confirmed the presence of histidine-related radicals during PS-80 oxidation identifying how histidine is involved in the oxidation. All assays and findings introduced in this work will provide insight into how PS-80 oxidative degradation can be avoided, controlled, or detected. It will also provide valuable evaluations on techniques that can be used to identify PS-80 degradation related events that occur during the manufacturing process.

A Unique Case of Squamous Cell Carcinoma Presenting Within a Lesion of Granuloma Annulare.

Granuloma annulare (GA) is a common benign granulomatous inflammatory disorder of the dermis or subcutis with classic morphologic and histologic presentation. Common clinical subtypes of granuloma annulare include localized, generalized, subcutaneous, perforating, and patch types. A biopsy is critical in cases of granuloma annulare with atypical features. We present a case of a 58-year-old male who presented with an annular scaly erythematous plaque on the right dorsal hand. Shave biopsy demonstrated irregular nests of mildly atypical squamous epithelium present within the superficial dermis, with abundant histiocytes and multinucleated giant cells arranged in palisades peripherally. These findings were consistent with invasive well-differentiated squamous cell carcinoma (SCC) with surrounding granuloma annulare. This case highlights a unique presentation in which clinicopathologic correlation is critical prior to arriving at a correct diagnosis.

Sequencing of Cutaneous Squamous Cell Carcinoma Primary Tumors and Patient-Matched Metastases Reveals ALK as a Potential Driver in Metastases and Low Mutational Concordance in Immunocompromised Patients.

Cutaneous squamous cell carcinoma is a common skin cancer that is responsible for 1,000,000 cases and up to 9,000 deaths annually in the United States. Metastases occur in 2-5% of patients and are responsible for significant morbidity and mortality. The objective of this study is to perform targeted next-generation sequencing on a cohort of squamous cell carcinoma primary tumors and patient-matched lymph node metastases. An oncology 76-gene panel was run from formalin-fixed paraffin-embedded samples of patient-matched primary squamous cell carcinomas (10) and resultant metastases (10). ALK was discovered to be a driver mutation in metastases using two different algorithms, oncoCLUSTand dNdScv. Mutational concordance between primary tumors and metastases was notably lower in immunosuppressed patients, especially among pathogenic mutations (41.7% vs. 83.3%, P = 0.01). Sequencing of matched squamous cell carcinoma primary tumors and lymph node metastases identified genes and pathways that may have clinical importance, most notably ALK as a potential driver mutation of metastasis. Sequencing of both primary tumors and metastases may improve the efficacy of targeted therapies.