Magnet-in-ferroelectric crystals exhibiting photomultiferroicity.

Growing crystallographically incommensurate and dissimilar organic materials is fundamentally intriguing but challenging for the prominent cross-correlation phenomenon enabling unique magnetic, electronic, and optical functionalities. Here, we report the growth of molecular layered magnet-in-ferroelectric crystals, demonstrating photomanipulation of interfacial ferroic coupling. The heterocrystals exhibit striking photomagnetization and magnetoelectricity, resulting in photomultiferroic coupling and complete change of their color while inheriting ferroelectricity and magnetism from the parent phases. Under a light illumination, ferromagnetic resonance shifts of 910 Oe are observed in heterocrystals while showing a magnetization change of 0.015 emu/g. In addition, a noticeable magnetization change (8% of magnetization at a 1,000 Oe external field) in the vicinity of ferro-to-paraelectric transition is observed. The mechanistic electric-field-dependent studies suggest the photoinduced ferroelectric field effect responsible for the tailoring of photo-piezo-magnetism. The crystallographic analyses further evidence the lattice coupling of a magnet-in-ferroelectric heterocrystal system.

Engineering the IL-4/IL-13 axis for targeted immune modulation.

The structurally and functionally related interleukin-4 (IL-4) and IL-13 cytokines play pivotal roles in shaping immune activity. The IL-4/IL-13 axis is best known for its critical role in T helper 2 (Th2) cell-mediated Type 2 inflammation, which protects the host from large multicellular pathogens, such as parasitic helminth worms, and regulates immune responses to allergens. In addition, IL-4 and IL-13 stimulate a wide range of innate and adaptive immune cells, as well as non-hematopoietic cells, to coordinate various functions, including immune regulation, antibody production, and fibrosis. Due to its importance for a broad spectrum of physiological activities, the IL-4/IL-13 network has been targeted through a variety of molecular engineering and synthetic biology approaches to modulate immune behavior and develop novel therapeutics. Here, we review ongoing efforts to manipulate the IL-4/IL-13 axis, including cytokine engineering strategies, formulation of fusion proteins, antagonist development, cell engineering approaches, and biosensor design. We discuss how these strategies have been employed to dissect IL-4 and IL-13 pathways, as well as to discover new immunotherapies targeting allergy, autoimmune diseases, and cancer. Looking ahead, emerging bioengineering tools promise to continue advancing fundamental understanding of IL-4/IL-13 biology and enabling researchers to exploit these insights to develop effective interventions.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

NEAT-seq: simultaneous profiling of intra-nuclear proteins, chromatin accessibility and gene expression in single cells.

In this work, we describe NEAT-seq (sequencing of nuclear protein epitope abundance, chromatin accessibility and the transcriptome in single cells), enabling interrogation of regulatory mechanisms spanning the central dogma. We apply this technique to profile CD4 memory T cells using a panel of master transcription factors (TFs) that drive T cell subsets and identify examples of TFs with regulatory activity gated by transcription, translation and regulation of chromatin binding. We also link a noncoding genome-wide association study single-nucleotide polymorphism (SNP) within a GATA motif to a putative target gene, using NEAT-seq data to internally validate SNP impact on GATA3 regulation.

The SIAH E3 ubiquitin ligases promote Wnt/ß-catenin signaling through mediating Wnt-induced Axin degradation.

The Wnt/ß-catenin signaling pathway plays essential roles in embryonic development and adult tissue homeostasis. Axin is a concentration-limiting factor responsible for the formation of the ß-catenin destruction complex. Wnt signaling itself promotes the degradation of Axin. However, the underlying molecular mechanism and biological relevance of this targeting of Axin have not been elucidated. Here, we identify SIAH1/2 (SIAH) as the E3 ligase mediating Wnt-induced Axin degradation. SIAH proteins promote the ubiquitination and proteasomal degradation of Axin through interacting with a VxP motif in the GSK3-binding domain of Axin, and this function of SIAH is counteracted by GSK3 binding to Axin. Structural analysis reveals that the Axin segment responsible for SIAH binding is also involved in GSK3 binding but adopts distinct conformations in Axin/SIAH and Axin/GSK3 complexes. Knockout of SIAH1 blocks Wnt-induced Axin ubiquitination and attenuates Wnt-induced ß-catenin stabilization. Our data suggest that Wnt-induced dissociation of the Axin/GSK3 complex allows SIAH to interact with Axin not associated with GSK3 and promote its degradation and that SIAH-mediated Axin degradation represents an important feed-forward mechanism to achieve sustained Wnt/ß-catenin signaling.

Allosteric regulation in SARS-CoV-2 spike protein.

Allosteric regulation is common in protein-protein interactions and is thus promising in drug design. Previous experimental and simulation work supported the presence of allosteric regulation in the SARS-CoV-2 spike protein. Here the route of allosteric regulation in SARS-CoV-2 spike protein is examined by all-atom explicit solvent molecular dynamics simulations, contrastive machine learning, and the Ohm approach. It was found that peptide binding to the polybasic cleavage sites, especially the one at the first subunit of the trimeric spike protein, activates the fluctuation of the spike protein's backbone, which eventually propagates to the receptor-binding domain on the third subunit that binds to ACE2. Remarkably, the allosteric regulation routes starting from the polybasic cleavage sites share a high fraction (39-67%) of the critical amino acids with the routes starting from the nitrogen-terminal domains, suggesting the presence of an allosteric regulation network in the spike protein. Our study paves the way for the rational design of allosteric antibody inhibitors.

Patient-Reported Outcomes on Subjective Nipple Sensation After Double-Incision and Periareolar Mastectomy for Chest Masculinization: Subjective Nipple Sensation After Chest Masculinization.

ABSTRACT: We evaluated patient-reported outcomes to assess for patient and procedural factors associated with postchest masculinization subjective nipple sensation. Patients who underwent double-incision or periareolar mastectomies for chest masculinization by a single senior surgeon (2015-2019) were surveyed at 2 time points regarding postoperative nipple sensation and satisfaction, including patient-reported outcomes using BODY-Q modules (Q-Portfolio.org). Demographic, operative, and postoperative variables were obtained from medical records. Patients were stratified according to survey responses. Univariate and multivariate analyses were performed.Response rate was 42% for survey 1 and 22% for survey 2. Of the 151 survey 1 responders, 138 (91.4%) received double-incision mastectomies and 13 (8.6%) received periareolar mastectomies. Among Survey 1 responders, 84.6% periareolar patients and 69.6% double-incision patients reported "completely" or "a little" nipple sensation preservation, and the difference trended toward significance (P = 0.0719). There was a stepwise increase in proportion of patients reporting sensation with greater recovery time until response to survey 1. Obesity (P = 0.0080) and greater tissue removed (P = 0.0247) were significantly associated with decreased nipple sensation. Nipple satisfaction scores were significantly higher for patients reporting improved nipple sensation (P = 0.0235). Responders to survey 2 who reported greater satisfaction with nipple sensation were significantly more likely to report preserved sensitivity to light touch (P = 0.0277), pressure (P = 0.0046), and temperature (P = 0.0031). Preserved erogenous sensation was also significantly associated with greater satisfaction (P = 0.0018).In conclusion, we found that nipple sensation may be associated with postoperative nipple satisfaction. Operative techniques to optimize nipple sensation preservation may improve this population's postoperative satisfaction.

Does Frailty Predict Outcomes in Patients Undergoing Free or Pedicled Flap Procedures for Lower Extremity Limb Salvage? An Analysis of the American College of Surgeons National Surgical Quality Improvement Program Database.

BACKGROUND: Older and frailer patients are increasingly undergoing free or pedicled tissue transfer for lower extremity (LE) limb salvage. This novel study examines the impact of frailty on postoperative outcomes in LE limb salvage patients undergoing free or pedicled tissue transfer. METHODS: The American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database (2010-2020) was queried for free and pedicled tissue transfer to the LE based on Current Procedural Terminology and the International Classification of Diseases9/10 codes. Demographic and clinical variables were extracted. The five-factor modified frailty index (mFI-5) was calculated using functional status, diabetes, chronic obstructive pulmonary disease, congestive heart failure, and hypertension. Patients were stratified by mFI-5 score: no frailty (0), intermediate frailty (1), and high frailty (2 + ). Univariate analysis and multivariate logistic regression were performed. RESULTS: In total, 5,196 patients underwent free or pedicled tissue transfer for LE limb salvage. A majority were intermediate (n = 1,977) or high (n = 1,466) frailty. High frailty patients had greater rates of comorbidities-including those not in the mFI-5 score. Higher frailty was associated with more systemic and all-cause complications. On multivariate analysis, the mFI-5 score remained the best predictor of all-cause complications-with high frailty associated with 1.74 increased adjusted odds when compared with no frailty (95% confidence interval: 1.47-2.05). CONCLUSION: While flap type, age, and diagnosis were independent predictors of outcomes in LE flap reconstruction, frailty (mFI-5) was the strongest predictor on adjusted analysis. This study validates the mFI-5 score for preoperative risk assessment for flap procedures in LE limb salvage. These results highlight the likely importance of prehabilitation and medical optimization prior to limb salvage.

Pedicled Flaps for High-Risk Open Vascular Procedures of the Lower Extremity: An Analysis of The National Surgical Quality Improvement Project Database.

BACKGROUND: Use of pedicled flaps in vascular procedures is associated with decreased infection and wound breakdown. We evaluated the risk profile and postoperative complications associated with lower extremity open vascular procedures with and without pedicled flaps. METHODS: The American College of Surgeons National Surgical Quality Improvement Program database (2010-2020) was queried for Current Procedural Terminology codes representing lower extremity open vascular procedures, including trunk and lower extremity pedicled flaps. Flap patients were compared with a randomized control group without flaps (1:3 cases to controls). Univariate and multivariate analyses were performed. RESULTS: We identified 132,934 adults who underwent lower extremity open vascular procedures. Concurrent pedicled flaps were rare (0.7%), and patients undergoing bypass procedures were more likely to receive a flap than nonbypass patients (69 vs. 64%, p < 0.0001). Flap patients had greater comorbidities. On univariate analysis, flap patients were more likely to experience wound (p = 0.0026), mild systemic (p < 0.0001), severe systemic (p = 0.0452), and all-cause complications (p < 0.0001). After adjusting for factors clinically suspected to be associated with increased risk (gender, body mass index, procedure type, American Society of Anesthesiologists classification, functional status, diabetes, smoking, and albumin < 3.5 mg/dL), wound (p = 0.096) and severe systemic complications (p = 0.0719) were no longer significantly associated with flap patients. CONCLUSION: Lower extremity vascular procedures are associated with a high risk of complications. Use of pedicled flaps remains uncommon and more often performed in patients with greater comorbid disease. However, after risk adjustment, use of a pedicled flap in high-risk patients may be associated with lower than expected wound and severe systemic complications.

Optimizing Surgical Outcomes and the Role of Preventive Surgery: A Scoping Review.

BACKGROUND: Plastic and reconstructive surgeons are often presented with reconstructive challenges as a sequela of complications in high-risk surgical patients, ranging from exposure of hardware, lymphedema, and chronic pain after amputation. These complications can result in significant morbidity, recovery time, resource utilization, and cost. Given the prevalence of surgical complications managed by plastic and reconstructive surgeons, developing novel preventative techniques to mitigate surgical risk is paramount. METHODS: Herein, we aim to understand efforts supporting the nascent field of Preventive Surgery, including (1) enhanced risk stratification, (2) advancements in postoperative care. Through an emphasis on four surgical cohorts who may benefit from preventive surgery, two of which are at high risk of morbidity from wound-related complications (patients undergoing sternotomy and spine procedures) and two at high risk of other morbidities, including lymphedema and neuropathic pain, we aim to provide a comprehensive and improved understanding of preventive surgery. Additionally, the role of risk analysis for these procedures and the relationship between microsurgery and prophylaxis is emphasized. RESULTS: (1) medical optimization and prehabilitation, (2) surgical mitigation techniques. CONCLUSION: Reconstructive surgeons are ideally placed to lead efforts in the creation and validation of accurate risk assessment tools and to support algorithmic approaches to surgical risk mitigation. Through a paradigm shift, including universal promotion of the concept of "Preventive Surgery," major improvements in surgical outcomes may be achieved.

BREAST-Q Analysis of Reduction Mammaplasty: Do Postoperative Complications of Breast Reduction Surgery Negatively Affect Patient Satisfaction?

BACKGROUND: Reduction mammaplasty can provide symptomatic relief to patients suffering from macromastia, however complications such as dehiscence are common. It is unknown if the presence of complications may affect patient reported outcomes. OBJECTIVES: This study aims to (1) determine risk factors for development of complications, and (2) to examine the correlation between postoperative complications and patient reported outcomes in reduction mammaplasty. METHODS: A single-center retrospective chart review was performed on patients who received reduction mammaplasties (CPT19318) between 1/17-2/23 by thirteen surgeons. Breast cancer cases and oncoplastic reconstructions were excluded. Patients with >1 complication were grouped into the complications cohort. BREAST-Q-survey was used to assess satisfaction. RESULTS: A total of 661 patients were included for analysis, and 131 patients developed at least one complication. Patients in the complication group had significantly higher average ages and body mass indexes, and a higher likelihood of hypertension and diabetes (p<0.01). Among 180 BREAST-Q responders, 41 had at least one complication. There were no significant differences between the two groups across survey outcomes. Although obese patients were more likely to develop infection and require revisions (p<0.01), no significant differences in subgroup analysis of patient-reported outcomes focusing on obese patients were observed. CONCLUSIONS: Obesity, hypertension, and diabetes were associated with postoperative complications of reduction mammaplasty. Patients with complications had similar postoperative Breast-Q satisfaction to patients without complications. While risk optimization is critical, patients and surgeons should be reassured that satisfaction may be achieved even in the event of a complication.

Independent Validation of the International Grading System for Medullary Thyroid Carcinoma: A Single Institution Experience.

Medullary thyroid carcinoma (MTC), an uncommon C cell thyroid malignancy, accounts for a disproportionate number of thyroid cancer deaths. To predict MTC clinical behavior, the recent international MTC grading system (IMTCGS) was published combining features from the Memorial Sloan Kettering Cancer Center and Royal North Shore Hospital grading systems that incorporates mitotic count, necrosis, and Ki67 proliferative index (Ki67PI). The IMTCGS appears promising, but independent validation data are limited. Here, we applied the IMTCGS to our institutional MTC cohort and assessed its ability to predict clinical outcomes. Our cohort comprised 87 MTCs (30 germline and 57 sporadic). Slides for each case were reviewed by 2 pathologists and histologic features recorded. Ki67 immunostaining was performed on all cases. Each MTC was graded with the IMTCGS based on tumor necrosis, Ki67PI, and mitotic count. Cox regression analysis was performed to assess the impact of various clinical and pathological data on disease outcomes, including overall survival (OS), disease-free survival, disease-specific survival (DSS), and distant metastasis-free survival. In our MTC cohort, 18.4% (n = 16/87) were IMTCGS high grade. IMTCGS grade was strongly prognostic for OS, disease-free survival, DSS, and distant metastasis-free survival on univariate analysis and multivariable analysis in both the entire MTC cohort and in the sporadic subset. Among the individual IMTCGS parameters, while all 3 were associated with poorer survival outcomes on univariate analysis, necrosis had the strongest association with all survival parameters on multivariable analysis, whereas Ki67PI or mitotic count was associated only with OS and DSS. This retrospective study independently demonstrates that the IMTCGS is valid for grading MTCs. Our findings support incorporating IMTCGS into routine pathology practice. IMTCGS grading may help clinicians to better predict the prognosis of MTC. Future studies may shed light on how MTC grading should impact treatment protocols.

Impact of the 99DOTS digital adherence technology on tuberculosis treatment outcomes in North India: a pre-post study.

BACKGROUND: 99DOTS is a cellphone-based digital adherence technology. The state of Himachal Pradesh, India, made 99DOTS available to all adults being treated for drug-sensitive tuberculosis (TB) in the public sector in May 2018. While 99DOTS has engaged over 500,000 people across India, few studies have evaluated its effectiveness in improving TB treatment outcomes. METHODS: We compared treatment outcomes of adults with drug-sensitive TB before and after Himachal Pradesh's 99DOTS launch using data from India's national TB database. The pre-intervention group initiated treatment between February and October 2017 (N = 7722), and the post-intervention group between July 2018 and March 2019 (N = 8322). We analyzed engagement with 99DOTS and used multivariable logistic regression to estimate impact on favorable treatment outcomes (those marked as cured or treatment complete). RESULTS: In the post-intervention group, 2746 (33.0%) people called 99DOTS at least once. Those who called did so with a wide variation in frequency (< 25% of treatment days: 24.6% of callers; 25-50% of days: 15.1% of callers, 50-75% of days: 15.7% of callers; 75-100% of days: 44.6% of callers). In the pre-intervention group, 7186 (93.1%) had favorable treatment outcomes, compared to 7734 (92.9%) in the post-intervention group. This difference was not statistically significant (OR = 0.981, 95% CI [0.869, 1.108], p = 0.758), including after controlling for individual characteristics (adjusted OR = 0.970, 95% CI [0.854, 1.102]). CONCLUSIONS: We found no statistically significant difference in treatment outcomes before and after a large-scale implementation of 99DOTS. Additional work could help to elucidate factors mediating site-wise variations in uptake of the intervention.

Depression screening in patients with vascular disease.

OBJECTIVES: Major depression is associated with increased morbidity and mortality in vascular surgery patients. The US Preventive Services Task Force and American Heart Association recommend routine depression screening for adults, especially those with cardiovascular disease. Since routine depression screening has not been implemented in most vascular surgery clinics across the nation, we sought to determine the feasibility of depression screening and understand the prevalence and predictors of depression in patients presenting to a single institution's vascular surgery clinic over a 4 month period. METHODS: From June to September 2020, vascular surgery clinic patients were administered a 26-item survey that included validated scales for depression (PHQ-9), pain, frailty, alcohol dependence, and nicotine dependence. Although not validated, the Rosenberg Self-Esteem Scale was also administered. Patient charts were reviewed for demographic information and medical history. 9-digit patient zip codes were used to determine Area Deprivation Index, a measure of socioeconomic status. Univariate and multivariate analyses were performed to understand the factors associated with increased depression prevalence in the study population. RESULTS: A total of 140 (36.4%) of 385 patients met study inclusion criteria. 35.7% of them screened positive for mild to severe depression (PHQ-9 scores ≥5). On univariate analysis, major depression was significantly associated with lower socioeconomic status (p = 0.007), higher frailty (p < 0.001), lower self-esteem (p < 0.001), higher daily pain (p < 0.001), health problems that interfere with social activities (p < 0.001), fatigue (p < 0.001), unmarried status (p = 0.031), and lack of primary care provider (p = 0.048). Multivariate analyses significantly predicted higher frailty (B= 0.487, p = 0.007) and lower self-esteem (B= -0.413, p < 0.001) in patients with depression. Depression was not associated with gender, age, employment status, smoking status, alcohol use, or type of vascular disease. COCLUSIONS: More than one-third of vascular surgery clinic patients have comorbid depression. Higher frailty and lower self-esteem are significant risk factors for depression. Prevention and early identification of frailty may improve outcomes. Depression screening in vascular surgery clinics is feasible and could be useful in determining which patients may benefit from more frequent follow-up and monitoring for associated comorbidities. Vascular surgeons may play an important role in screening for depression and referring patients for psychotherapy and/or pharmacotherapy.

Adaptive evolution of a melanized fungus reveals robust augmentation of radiation resistance by abrogating non-homologous end-joining.

Fungi have been observed to exhibit resistance to high levels of ionizing radiation despite sharing most DNA repair mechanisms with other eukaryotes. Radioresistance, in fact, is such a common feature in fungi that it is difficult to identify species that exhibit widely different radiosensitivities, which in turn has hampered the identification of genetic elements responsible for this resistance phenotype. Due to the inherent mutagenic properties of radiation exposure, however, this can be addressed through adaptive laboratory evolution for increased ionizing radiation resistance. Here, using the black yeast Exophiala dermatitidis, we demonstrate that resistance to γ-radiation can be greatly increased through repeated rounds of irradiation and outgrowth. Moreover, we find that the small genome size of fungi situates them as a relatively simple functional genomics platform for identification of mutations associated with ionizing radiation resistance. This enabled the identification of genetic mutations in genes encoding proteins with a broad range of functions from 10 evolved strains. Specifically, we find that greatly increased resistance to γ-radiation is achieved in E. dermatitidis through disruption of the non-homologous end-joining pathway, with three individual evolutionary paths converging to abolish this DNA repair process. This result suggests that non-homologous end-joining, even in haploid cells where homologous chromosomes are not present during much of the cell cycle, is an impediment to repair of radiation-induced lesions in this organism, and that the relative levels of homologous and non-homologous repair in a given fungal species may play a major role in its radiation resistance.

Discovery of a ZIP7 inhibitor from a Notch pathway screen.

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.

Current Trainee and Workforce Patterns for Thyroid and Parathyroid Surgery in the United States.

OBJECTIVE: Thyroid and parathyroid surgery is performed by both general surgeons and otolaryngologists. We describe the proportion of surgeries performed by specialty, providing data to support decisions about when and to whom to direct research, education, and quality improvement interventions. METHODS: We tabulated case numbers for privately insured patients undergoing thyroid and parathyroid surgery in Marketscan: 2010-2016 and trainee case logs for residents and fellows in general surgery and otolaryngology. Summary statistics and tests for trends and differences were calculated. RESULTS: Marketscan data captured 114 500 thyroid surgeries. The proportion performed by each specialty was not significantly different. Otolaryngologists performed 58 098 and general surgeons performed 56 402. Otolaryngologists more commonly performed hemithyroidectomy (n = 25 148, 43.29% of all thyroid surgeries performed by otolaryngologists) compared to general surgeons (n = 20 353, 36.09% of all thyroid surgeries performed by general surgeons). Marketscan data captured 21 062 parathyroid surgeries: 6582 (31.25%) were performed by otolaryngologists, and 14 480 (68.75%) were performed by general surgeons. The case numbers of otolaryngology and general surgery trainees completing residency and fellowship varied 6- to 9-fold across different sites. The wide variation may reflect both the level of exposure a particular training program offers and trainee level of interest. CONCLUSION: Thyroid surgical care is equally provided by general surgeons and otolaryngologists. Both specialties contribute significantly to parathyroid surgical care. Both specialties should provide input into and be targets of research, quality, and education interventions.

Airway hyperresponsiveness in patients with normal spirometry results and symptoms compatible with asthma: Primary care retrospective chart review.

OBJECTIVE: To evaluate the proportion of patients with symptoms suggestive of asthma and normal lung function who exhibit airway hyperreactivity with methacholine challenge testing (MCT) in primary care. DESIGN: Retrospective chart review. SETTING: Primary care lung clinic in Toronto, Ont. PARTICIPANTS: A total of 69 patients presenting to the lung clinic who had symptoms compatible with asthma, normal spirometry test results, and were referred for MCT. MAIN OUTCOME MEASURES: Descriptive statistics, frequency counts, independent t tests, and 2 tests were used to examine differences in the proportion of clinical and demographic variables identified in patients with or without a positive MCT result. Effect size was determined between MCT-positive and MCT-negative patients for both categorical ( coefficient) and continuous (Hedges g) data. RESULTS: Twenty-one patients (30.4%) had positive MCT results, and 48 patients (69.6%) had negative MCT results. Family history of asthma and reduced baseline and postbronchodilator forced expiratory volume in 1 second were associated with a positive MCT result. CONCLUSION: The findings of this study provide insight into the utility of simple spirometry for asthma diagnosis and the need to further clarify the role of MCT in the primary care setting.

The response of the melanized yeast Exophiala dermatitidis to gamma radiation exposure.

The melanized yeast Exophiala dermatitidis is resistant to many environmental stresses and is used as a model for understanding the diverse roles of melanin in fungi. Here, we describe the extent of resistance of E. dermatitidis to acute γ-radiation exposure and the major mechanisms it uses to recover from this stress. We find that melanin does not protect E. dermatitidis from γ-radiation. Instead, environmental factors such as nutrient availability, culture age and culture density are much greater determinants of cell survival after exposure. We also observe a dramatic transcriptomic response to γ-radiation that mobilizes pathways involved in morphological development, protein degradation and DNA repair, and is unaffected by the presence of melanin. Together, these results suggest that the ability of E. dermatitidis to survive γ-radiation exposure is determined by the prior and the current metabolic state of the cells as well as DNA repair mechanisms, and that small changes in these conditions can lead to large effects in radiation resistance, which should be taken into account when understanding how diverse fungi recover from this unique stress.

Melanin Produced by the Fast-Growing Marine Bacterium Vibrio natriegens through Heterologous Biosynthesis: Characterization and Application.

Melanin is a pigment produced by organisms throughout all domains of life. Due to its unique physicochemical properties, biocompatibility, and biostability, there has been an increasing interest in the use of melanin for broad applications. In the vast majority of studies, melanin has been either chemically synthesized or isolated from animals, which has restricted its use to small-scale applications. Using bacteria as biocatalysts is a promising and economical alternative for the large-scale production of biomaterials. In this study, we engineered the marine bacterium Vibrio natriegens, one of the fastest-growing organisms, to synthesize melanin by expressing a heterologous tyrosinase gene and demonstrated that melanin production was much faster than in previously reported heterologous systems. The melanin of V. natriegens was characterized as a polymer derived from dihydroxyindole-2-carboxylic acid (DHICA) and, similarly to synthetic melanin, exhibited several characteristic and useful features. Electron microscopy analysis demonstrated that melanin produced from V. natriegens formed nanoparticles that were assembled as "melanin ghost" structures, and the photoprotective properties of these particles were validated by their protection of cells from UV irradiation. Using a novel electrochemical reverse engineering method, we observed that melanization conferred redox activity to V. natriegens Moreover, melanized bacteria were able to quickly adsorb the organic compound trinitrotoluene (TNT). Overall, the genetic tractability, rapid division time, and ease of culture provide a set of attractive properties that compare favorably to current E. coli production strains and warrant the further development of this chassis as a microbial factory for natural product biosynthesis.IMPORTANCE Melanins are macromolecules that are ubiquitous in nature and impart a large variety of biological functions, including structure, coloration, radiation resistance, free radical scavenging, and thermoregulation. Currently, in the majority of investigations, melanins are either chemically synthesized or extracted from animals, which presents significant challenges for large-scale production. Bacteria have been used as biocatalysts to synthesize a variety of biomaterials due to their fast growth and amenability to genetic engineering using synthetic biology tools. In this study, we engineered the extremely fast-growing bacterium V. natriegens to synthesize melanin nanoparticles by expressing a heterologous tyrosinase gene with inducible promoters. Characterization of the melanin produced from V. natriegens-produced tyrosinase revealed that it exhibited physical and chemical properties similar to those of natural and chemically synthesized melanins, including nanoparticle structure, protection against UV damage, and adsorption of toxic compounds. We anticipate that producing and controlling melanin structures at the nanoscale in this bacterial system with synthetic biology tools will enable the design and rapid production of novel biomaterials for multiple applications.