Biophysical insight into the binding mechanism of epigallocatechin-3-gallate and cholecalciferol to albumin and its preventive effect against AGEs formation: An in vitro and in silico approach.

Advanced glycation end products (AGEs) are produced non-enzymatically through the process of glycation. Increased AGEs production has been linked to several diseases including polycystic ovary syndrome (PCOS). PCOS contributes to the development of secondary comorbidities, such as diabetes, cardiovascular complications, infertility, etc. Consequently, research is going on AGEs-inhibiting phytochemicals for their potential to remediate and impede the progression of hyperglycaemia associated disorders. In this study human serum albumin is used as a model protein, as albumin is predominantly present in follicular fluid. This article focusses on the interaction and antiglycating potential of (-)-Epigallocatechin-3-gallate (EGCG) and vitamin D in combination using various techniques. The formation of the HSA-EGCG and HSA-vitamin D complex was confirmed by UV and fluorescence spectroscopy. Thermodynamic analysis verified the spontaneity of reaction, and presence of hydrogen bonds and van der Waals interactions. FRET confirms high possibility of energy transfer. Cumulative antiglycation resulted in almost 60 % prevention in AGEs formation, decreased alterations at lysine and arginine, and reduced protein carbonylation. Secondary and tertiary structural changes were analysed by circular dichroism, Raman spectroscopy and ANS binding assay. Type and size of aggregates were confirmed by Rayleigh and dynamic light scattering, ThT fluorescence, SEM and SDS-PAGE. Effect on cellular redox status, DNA integrity and cytotoxicity was analysed in lymphocytes using dichlorofluorescein (DCFH-DA), DAPI and MTT assay which depicted an enhancement in antioxidant level by cumulative treatment. These findings indicate that EGCG and vitamin D binds strongly to HSA and have antiglycation ability which enhances upon synergism.

Challenges faced by teachers of postgraduate health professions blended learning programs: a qualitative analysis.

INTRODUCTION: Blended learning, which integrates classroom face-to-face teaching with both asynchronous and synchronous online learning elements, has swiftly gained acceptance in educational environments. However, the implementation of blended learning presents challenges that impact all stakeholders, necessitating thoughtful consideration. Teachers play a central role in shaping the instructional experience among these stakeholders. To fully realize the potential of comprehensive blended learning, it is imperative to identify the challenges faced by these teachers and develop strategies that sensibly address and overcome them. METHODOLOGY: A qualitative exploratory study was conducted with twelve teachers involved in the postgraduate blended learning health professions program at Khyber Medical University, Peshawar in 2022. One-on-one semi-structured Interviews were conducted via WhatsApp/ZOOM, transcribed by Otter AI, coded on NVivo & analyzed using Braun/Clarke's Thematic Analysis. RESULTS: Three themes of challenges faced by teachers of postgraduate blended learning programs were constructed namely (1) Skills, such as (i) digital, (ii) instructional, and (iii) online class management, and (2) Administrative barriers in terms of (iv) resources (iv) training, and (3) Faculty resistance. CONCLUSION: This study provides profound insights into the daunting challenges that postgraduate blended learning program teachers encounter in terms of skills, administrative barriers, and faculty resistance. These findings offer a valuable opportunity for program directors to identify the critical requirements of these faculties in their pursuit of effective teaching and learning, ultimately transforming the landscape of blended education. This study emphasizes the need for ongoing faculty development and institutional support to address the identified challenges and improve the quality of postgraduate blended-learning programs.

Inhibitory effect of baicalein against glycation in HSA: an in vitro approach.

Hyperglycaemia accelerates the aging process significantly. Diabetes problems can be mitigated by inhibiting glycation. To learn more about glycation and antiglycation mediated by methyl glyoxal and baicalein, we studied human serum albumin as a model protein. A Methylglyoxal (MGO) incubation period of seven days at 37 degrees Celsius induced glycation of Human Serum Albumin.s Hyperchromicity, decreased tryptophan and intrinsic fluorescence, increased AGE-specific fluorescence, and reduced mobility were all seen in glycated human serum albumin (MGO-HSA) in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Fourier transform infrared spectroscopy (FT-IR) and then far ultraviolet dichroism were used to detect secondary and tertiary structural perturbations (CD). The Congo red assay (CR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) all verified the presence of amyloid-like clumps. Structure (carbonyl groups on ketoamine moieties) (CO), physiological problems including diabetes mellitus, and cardiovascular disease, etc. are linked to the structural and functional changes in glycated HSA, as proven by these studies.Communicated by Ramaswamy H. Sarma.

Synergistic effect of chlorogenic acid and vitamin D3 (cholecalciferol) on in-vitro glycation may assist in prevention of Polycystic Ovarian Syndrome (PCOS) progression - A biophysical, biochemical and in-silico study.

Advanced glycation end products (AGEs) are formed through non-enzymatic glycation, that have been linked to various diseases, including polycystic ovarian syndrome (PCOS) playing a critical role leading to secondary comorbidities such as diabetes-related problems, cardiovascular complications, infertility, etc. As a result, there has been a lot of research into AGE-inhibiting phytochemicals for the remediation and obstruct progression of glycation-related illnesses. The current study is based on in-vitro protein model, in which human serum albumin have been used to investigate the cumulative effect of chlorogenic acid (CGA) and cholecalciferol (vitamin D) on glycation and evaluate their inhibitory impact on AGEs production in the presence of methylglyoxal. Through the application of several biochemical and biophysical techniques, we were able to examine the synergistic impact of both the compounds on albumin structure and its biochemical properties during different stages of glycation. According to Nitro-blue tetrazolium assay results indicate that CGA and vitamin D inhibited fructosamine (early glycation product) production. Moreover, free thiol and lysine residues were significantly increased whereas protein carbonyl levels were significantly decreased. Additive effect of CGA and vitamin D were associated with reduced AGEs fluorescence and increased tryptophan and tyrosine fluorescence. Amadori-albumin after treatment showed some evidence of regaining its alpha-helicity as measured by far-UV CD spectrum. Furthermore, secondary structural alterations were confirmed by Fourier transform infrared spectroscopy (FTIR). ANS (1-anilinonaphthalene-8-sulfonic acid) fluorescence spectra also displayed less revelation of hydrophobic patches. Bilirubin binding capacity was also restored which showed functional recovery of HSA. The electrophoretic mobility was also restored which is portrayed by SDS-PAGE. Additionally, to predict the anti-aggregation potential of CGA and vitamin D, congo red assay and ThT fluorescence was performed which reveal low aggregate formation after treatment. These results corroborated with scanning electron microscopy and confocal microscopy. Docking and simulation results also reveal spontaneous binding of CGA and vitamin D on subdomain IIA of HSA favoring their binding thermodynamically. All the findings suggest that chlorogenic acid and cholecalciferol given in combination might help in prevention of PCOS progression and its related complications.

Swallowing Function After Treatment of Laryngeal Cancer.

Dysphagia is a common functional outcome following treatment of laryngeal cancer. Despite curative advances in both nonsurgical and surgical approaches, preserving and optimizing swallowing function is critical. Understanding the nature and severity of dysphagia depending on initial tumor staging and treatment modality and intensity is crucial. This chapter explores current evidence on the acute and chronic impacts of treatments for laryngeal cancer on swallow function, as well as the medical and nonmedical management of dysphagia in this population.

Personalized Palliative and Survivorship Care for Patients With Metastatic Cancer Treated With Radiation Therapy.

While the benefits of early palliative care for patients with metastatic cancer are well established, cancer survivorship remains inadequately integrated into the care of patients with distant metastases. Moreover, the optimal model of care delivery is poorly defined. A prognostic model previously developed and validated at Good Samaritan University Hospital identified four groups of patients with metastatic solid tumor malignancy having very favorable, favorable, standard or unfavorable prognoses with median survival of 31, 14, 4 and 1 month, respectively. This framework holds promise for the personalized delivery of supportive, palliative and survivorship care services in the context of radiation therapy. We review the published literature providing the rationale for a novel multidisciplinary care model where the radiation oncology Clinical Nurse Specialist identifies and coordinates interventions to address unmet physical and emotional issues faced by survivors with metastatic cancer with the goal of improving quality of life and overall survival.

Rutin impedes human low-density lipoprotein from non-enzymatic glycation: A mechanistic insight against diabetes-related disorders.

Glycation of human low-density protein (LDL) has an essential contribution to cardiovascular diseases. Natural compounds like rutin have been extensively studied in preventing glycation-induced oxidative stress. This study examined rutin's anti-glycation potential with glycated LDL utilizing spectroscopic and in silico methods. Glycated LDL treated with rutin, showed around 80 % inhibition in advanced glycation end-product production. Carbonyl content and lipid peroxidation like assays were used to establish the development of oxidative stress. Rutin was seen to lower the generation of oxidative stress in a dose-dependent manner. Using thioflavin-T assay and electron microscopy, rutin was suggested to restore the structural disturbances in glycated LDL. Moreover, CD spectroscopy suggested reinstation of secondary structure of glycated LDL treated with rutin. Mechanistic insights between rutin and LDL were observed through spectroscopic measures. Molecular docking study confirmed the LDL-rutin binding with a binding energy of -10.0 kcal/mol. The rutin-LDL complex was revealed to be highly stable by molecular dynamics simulation, with RMSD, RMSF, Rg, SASA, and the secondary structure of LDL remaining essentially unchanged during the simulation period. Our study suggests that rutin possesses strong anti-glycating properties, which can be useful in therapeutics, as glycated LDL has an important role in atherosclerotic cardiovascular diseases.

Retrograde Cricopharyngeus Dysfunction: The Jefferson Experience.

OBJECTIVES: To describe our center's experience with the identification and treatment of retrograde cricopharyngeus dysfunction (R-CPD), a syndrome involving the inability to belch previously described by only one institution. Additionally, because all patients initially learned of their condition and sought treatment as a result of social media posts, we queried their source and comfort with this form of medical referral. METHODS: Retrospective chart review of patients who underwent botulism toxin injection into the cricopharyngeus muscle for treatment of R-CPD from 2019 to 2022. Demographic data, most common symptoms at presentation, and response to treatment and complications were documented. Post-treatment questionnaires were reviewed. RESULTS: A total of 85 patients were identified. Mean age at surgery was 27 years. There were 54 (63.5%) females and 31 (36.5%) males. The inability to burp (98.8%), bloating (92.9%), gurgling noises (31.8%), and excessive flatulence (21.2%) were the most common symptoms. The minimum units of botox utilized were 25, whereas the maximum was 100. The majority of patients (88.2%) had a successful response at initial follow-up visit. The most common complication was mild dysphagia (30.6%), which was transient for all patients. Most patients learned of our practice through social media, with only one patient being referred by a medical provider. CONCLUSIONS: The majority of patients in our cohort were young and female. The inability to burp and bloating were the most common presenting symptoms. Social media was the primary source of referral. Our institution favors 80-100 units for an effective response. Laryngoscope, 133:1081-1085, 2023.

A brief insight into the etiology, genetics, and immunology of polycystic ovarian syndrome (PCOS).

Polycystic ovarian syndrome (PCOS) is a prevailing endocrine and metabolic disorder occurring in about 6-20% of females in reproductive age. Most symptoms of PCOS arise early during puberty. Since PCOS involves a combination of signs and symptoms, thus it is considered as a heterogeneous disorderliness. The most accepted diagnostic criteria is Rotterdam criteria which involves two of the latter three features: (a) hyperandrogenism, (b) oligo- or an-ovulation, and (c) polycystic ovaries. The persistent hormonal imbalance leads to multiple small antral follicles formation and irregular menstrual cycle, ultimately causing infertility among females. Insulin resistance, cardiovascular diseases, abdominal obesity, psychological disorders, infertility, and cancer are also related to PCOS. These pathophysiologies associated with PCOS are interrelated with each other. Hyperandrogenism causes insulin resistance and hyperglycemia, leading to ROS formation, oxidative stress, and abdominal adiposity. In consequence, inflammation, ROS production, insulin resistance, and hyperandrogenemia also increase. Elevation of AGEs in the body either produced endogenously or consumed from diet exaggerates PCOS symptoms and is also related to ovarian dysfunction. This review summarizes how AGE formation, inflammation, and oxidative stress are significantly essential in PCOS progression. Alterations during prenatal development like exposure to excess AMH, androgens, or toxins (bisphenol-A, endocrine disruptors, etc.) may also be the etiologic mechanism behind PCOS. Although the etiology of this disorder is unclear, environmental and genetic factors are primarily involved. Physical inactivity, as well as unhealthy eating habits, has a vital role in the progression of PCOS. This review outlines a collection of specific genes phenotypically linked with PCOS. Furthermore, beneficial effect of metformin in maintaining endocrine abnormalities and ovarian function is also mentioned. Kisspeptin is a protein which helps in onset of puberty and increases GnRH pulsatile release during ovulation as well as role of KNDy neurons in GnRH pulsatile signal required for reproduction are also elaborated. This review also focuses on the immunology related to PCOS involving chronic low-grade inflammation, and how the alterations within the follicular microenvironment are intricated in the development of infertility in PCOS patients. How PCOS develops following antiepileptic and psychiatric medication is also expanded in this review. Initiation of antiandrogen treatment in early age (≤ 25 years) might be helpful in spontaneous conception in PCOS women. The role of BMP (bone morphogenetic proteins) in folliculogenesis and their expression in oocytes and granulosa cells are also explained. GDF8 and SERPINE1 expression in PCOS is given in detail.

Formulation development of directly compressible mebevarine tablets using superdisintegrant: A way to investigate quality atributes, in vitro release kinetics and stability profile.

In order for preparing a solid oral dosage form, tablet quality is of significant concern. Compressibility behavior of different powders and mixtures of formulations and release pattern of any tablets are characteristic measures to define prerequisite quality attributes of any compressed formulations. There are basically two major methods that can be adopted for the preparation of tablets including granulation and direct compression. Later process offer fewer processing steps and agreeable release profile with acceptable quality parameters and hence preferred over granulation method. In this investigation Mebeverine hydrochloride an anti-muscarinic drug is studied for compression and release behavior using various concentrations of filler binders and disintegrants via rotatable central composite design (CCRD) option of design expert (software). Nine formulations were developed from F1 to F9 with Crospovidone (superdisintegrant) as (X1) (-α=1.17% to ± α=6.83%) and microcrystalline cellulose (Avicel 102, Filler/binder) as X2 (-α = 29.82% to ± α = 65.18%). Disintegration Time (DT) as (R1) and Hardness in (kg) as (R2) were determined as two dependent response variables. The performance of powder blends and formulations was analyzed by micromeritic and physico-chemical and assessments. Dissolution comparisons were statistically analyzed by ANOVA and model dependent and in-dependent methods. Best fit model was found to be Hixon-crowell's model (r2 = 0.995) followed by Weibull's model (r2 = 0.985). The Trial formulations F2, F4, F6 and F8 were also studied on accelerated conditions (40±5ºC 75%±5% RH) for stability tests and validity of the formulations in months were also determined between 35-39 months.

Perceived stress may mediate the relationship between antenatal depressive symptoms and preterm birth: A pilot observational cohort study.

BACKGROUND: Screening for changes in pregnancy-related anxiety and depressive symptoms during pregnancy may further our understanding of the relationship between these two variables and preterm birth. OBJECTIVES: To determine whether changes in pregnancy-related anxiety and depressive symptoms during pregnancy influence the risk of preterm birth among Pakistani women; explore whether perceived stress moderates or mediates this relationship, and examine the relationship between the various components of pregnancy-related anxiety and preterm birth. METHODS: A prospective cohort study design was used to recruit a diverse sample of 300 low-risk pregnant women from four centers of Aga Khan Hospital for Women and Children in Karachi, Pakistan. Changes in pregnancy-related anxiety and depressive symptoms during pregnancy were tested. Multiple logistic regression analysis was used to determine a predictive model for preterm birth. We then determined if the influence of perceived stress could moderate or mediate the effect of depressive symptoms on preterm birth. RESULTS: Changes in pregnancy-related anxiety (OR = 1.1, CI 0.97-1.17, p = 0.167) and depressive symptoms (OR = 0.9, CI 0.85-1.03, p = 0.179) were insignificant as predictors of preterm birth after adjusting for the effects of maternal education and family type. When perceived stress was added into the model, we found that changes in depressive symptoms became marginally significant after adjusting for covariates (OR = 0.9, CI 0.82-1.01, p = 0.082). After adjusting for the mediation effect of change in perceived stress, the effect of change in depressive symptoms on preterm birth were marginally significant after adjusting for covariates. Among six different dimensions of pregnancy-related anxiety, mother's concerns about fetal health showed a trend towards being predictive of preterm birth (OR = 1.3, CI 0.97-1.72, p = 0.078). CONCLUSIONS: There may be a relationship between perceived stress and antenatal depressive symptoms and preterm birth. This is the first study of its kind to be conducted in Pakistan. Further research is required to validate these results.

Epithelial miR-141 regulates IL-13-induced airway mucus production.

IL-13-induced goblet cell metaplasia contributes to airway remodeling and pathological mucus hypersecretion in asthma. miRNAs are potent modulators of cellular responses, but their role in mucus regulation is largely unexplored. We hypothesized that airway epithelial miRNAs play roles in IL-13-induced mucus regulation. miR-141 is highly expressed in human and mouse airway epithelium, is altered in bronchial brushings from asthmatic subjects at baseline, and is induced shortly after airway allergen exposure. We established a CRISPR/Cas9-based protocol to target miR-141 in primary human bronchial epithelial cells that were differentiated at air-liquid-interface, and goblet cell hyperplasia was induced by IL-13 stimulation. miR-141 disruption resulted in decreased goblet cell frequency, intracellular MUC5AC, and total secreted mucus. These effects correlated with a reduction in a goblet cell gene expression signature and enrichment of a basal cell gene expression signature defined by single cell RNA sequencing. Furthermore, intranasal administration of a sequence-specific mmu-miR-141-3p inhibitor in mice decreased Aspergillus-induced secreted mucus and mucus-producing cells in the lung and reduced airway hyperresponsiveness without affecting cellular inflammation. In conclusion, we have identified a miRNA that regulates pathological airway mucus production and is amenable to therapeutic manipulation through an inhaled route.

Management of urologic cancers during the pandemic and potential impact of treatment deferrals on outcomes.

The COVID-19 pandemic-related constraints on healthcare access have raised concerns about adverse outcomes from delayed treatment, including the risk of cancer progression and other complications. Further, concerns were raised about a potentially significant backlog of patients in need of cancer care due to the pandemic-related delays in healthcare, further exacerbating any potential adverse outcomes. Delayed access to surgery is particularly relevant to urologic oncology since one-third of new cancers in men (20% overall) arise from the genitourinary (GU) tract and surgery is often the primary treatment. Herein, we summarize the prepandemic literature on deferred surgery for GU cancers and risk of disease progression. The aforementioned data on delayed surgery were gathered in the context of systemic delays present in certain healthcare systems, or occasionally, due to planned deferral in suboptimal surgical candidates. These data provide indirect, but sufficient insight to develop triage schemas for prioritization of uro-oncological cases. Herein, we outline the extent to which the pandemic-related triage guidelines had influenced urologic practice in various regions. To study the adverse outcomes in the pandemic-era, a survey of urologic oncologists was conducted regarding modifications in their initial management of urologic cancers and any delay-related adverse outcomes. While the adverse effects directly from COVID-19 related delays will become apparent in the coming years, the results showing short-term outcomes are quite instructive. Since cancer care was assigned a higher priority at most centers, this strategy may have avoided significant delays in care and limited the anticipated negative impact of pandemic-related constraints.

Discoid Lupus Erythematosus: A Cross-Sectional Study From the Sindh Institute of Skin Diseases, Karachi, Pakistan.

Introduction Discoid lupus erythematosus (DLE) is the most common form of cutaneous lupus erythematosus. It is a chronic, scar-forming, photosensitive autoimmune dermatosis presenting with erythematous and scaly lesions. Predisposed areas include sun-exposed areas like the nose, forehead, and cheeks, as well as the upper body and extremities. The histological findings are typical, with interface dermatitis. Immunoglobulin M (IgM) and immunoglobulin G (IgG) are the most common deposits in the dermoepidermal junction of the involved skin. The most common treatments used are sunscreens, topical corticosteroids, and antimalarials. Immunosuppressive agents, thalidomide, dapsone, and retinoids can be used in refractory cases. The aim of this study was to study the clinicopathologic patterns of DLE in patients presenting to the Institute of Skin Diseases in Sindh, Karachi. Methods A total of 53 consecutive patients with DLE meeting the inclusion criteria were evaluated between February 18, 2018 to March 2, 2019 at the Institute of Skin Diseases. Patients with clinical suspicion of DLE were evaluated and studied prospectively after written informed consent was obtained. Information was then collected from their medical histories, physical examination records, and laboratory investigation reports. Results A total of 53 consecutive patients with clinical and/or histological diagnosis of DLE was included in this study, out of which 75.5% (40) were females with a male to female ratio of 1:3.1. The mean age of the patients at the time of presentation was 36.02 ± 10.04 years, ranging from 14 to 65 years. More than half of the patients (35, 66.0%) were under 40 years of age and 20.8% (11) had a positive family history of DLE. DLE was localized in 36 patients (67.9%) and exposure to the ultraviolet radiation (UVR) was found to be the most frequent induced factor in 46 patients (86.8%), followed by stress which was observed in 14 patients (26.4%). The distribution of commonly affected sites were the face (81.1%), the limbs (71.7%), and the scalp (48.4%) of the patients. Serology antinuclear antibody (ANA) was positive in 56.6% and serology anti-double-stranded deoxyribonucleic acid antibodies (anti-dsDNA) were positive in 45.3% of patients. Smoking, as an induced factor, was more commonly observed among male patients as compared to the female patients with a proportion of 53.8% vs. 2.5%, p < 0.001, while stress was more common among female patients with a proportion of 35% vs. 0%, p = 0.013, respectively. Histopathology with direct immunofluorescence was done in 33 cases which included cases with negative serology or where the diagnosis was in doubt clinically. The main histopathological features observed were periadnexal and perivascular dermal infiltrates, basal cells vacuolization, epidermal atrophy, hyperkeratosis, and follicular plugging. The commonest morphological form observed was the classic discoid plaque form. Conclusion Clinical patterns of DLE in our population comprises of female dominance. Exposure to UVR was the leading inducing factor. The face and limbs were the most commonly involved sites, and the majority of the patients had localized DLE with positive ANA in more than half of those patients. The importance of limiting ultraviolet radiation exposure and toxins (drugs and smoking) should be emphasized in our population.

Initiating psychiatry teleclinics during the COVID-19 pandemic in a tertiary care hospital in Karachi, Pakistan.

The COVID-19 pandemic has overwhelmed hospitals all over the world. To reduce the risk of transmission of COVID-19 and meet the increasing mental health demands due to this trauma, psychiatry outpatient services have gone online in various countries. We describe steps taken at a tertiary care hospital in Karachi, Pakistan, to start these services during this pandemic. We also discuss the obstacles faced. (PsycInfo Database Record (c) 2020 APA, all rights reserved).

Anemia and blood transfusion requirements in endoscopic sinus surgery: A propensity-matched analysis.

OBJECTIVE: Massive bleeding requiring blood transfusion is a feared complication of endoscopic sinus surgery (ESS). In an effort to improve healthcare quality and outcomes, research is focused on identifying the risk factors for complications following surgical procedures. Blood transfusions have been linked to increased complication rates, but their role has not been extensively studied in ESS. METHODS: The American College of Surgeons National Surgery Quality Improvement Program (ACS NSQIP) participant user files 2005 to 2014 were queried for all cases of ESS. Patients were divided into cohorts based on anemia (hematocrit <39.00 in men and <36.00 in women) and hematocrit levels. Univariate analyses and a propensity score-matching algorithm were employed. RESULTS: Of 630 cases of ESS, 117 (18.6%) had preexisting anemia. Anemic patients were more likely to be black (19.8% vs. 6.7%, P ≤ 0.001), undergo longer operation times (117.50 minutes ±155.73 vs. 129.85 minutes ±36.03, P = 0.005), have diabetes (28.2% vs. 10.9%, P ≤ 0.001), and have hypertension (46.2% vs. 33.1%, P = 0.010). Following propensity score matching, 109 cases were selected for each cohort, with no significant differences in demographics, comorbidities, operation time, or outpatient status. The anemic cohort was independently associated with an increase in overall postoperative complications (17.4% vs. 7.3%, P = 0.038). Transfusion use was significantly associated with preoperative hematocrit levels <30 (30.0% vs. 4.5%, P = 0.001) and between 30 and 34.99 (13.9% vs. 2.5%, P = 0.011). CONCLUSION: Preoperative anemia is a significant predictor of overall complications and bleeding requiring transfusion for patients undergoing ESS. These results highlight the need for careful preoperative assessment and management of anemia in this population. LEVEL OF EVIDENCE: NA Laryngoscope, 130:1377-1382, 2020.

Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.

Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.