ABSTRACT
Osteosarcomas arising within the pterygomaxillary/infratemporal fossa region are rare among the pediatric population. Survival rates are most influenced by tumor resection with negative margins, which can be dependent on surgical accessibility of the tumor site. The pterygomaxillary/infratemporal fossa location poses several challenges to safe and adequate tumor resection, including proximity of the facial nerve and great vessels and scarring associated with traditional transfacial approaches. In this article, we present the case of a 6-year-old boy with an osteosarcoma of the left pterygomaxillary/infratemporal fossa region successfully managed with an "oncoplastic" approach, incorporating the use of CAD/CAM and mixed reality technologies.
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OBJECTIVES: Fibrinolytic therapy can be effective for management of complex pleural effusions. Tissue plasminogen activator (tPA, 10 mg) and deoxyribonuclease (DNAse) every 12 h with a dwell time of one hour is a common strategy based on published data. We used a simpler protocol of tPA (4 mg) without DNAse but with a longer dwell time of 12 h, repeated daily. We reviewed our results. METHODS: Charts were reviewed and demographics, clinical data and treatment information were abstracted. Outcomes were assessed based on radiographic findings and need for surgery. RESULTS: Two hundred and fifteen effusions in 207 patients (8 bilateral) were identified. 85% were either infectious or malignant. Two hundred and forty nine chest tubes were used: 84% were 10 Fr or 12 Fr and 7% were PleurX®. Five hundred and thirty one doses of tPA were given. The median number of doses per effusion was 2 (range 1-10), and 84% of effusions were treated with three or fewer doses. There were no significant bleeding complications. Median time to chest tube removal was 6 days (range 1 to 98, IQR 4 to 10). Drainage was considered complete for 78% of effusions, while 6% required decortication. CONCLUSIONS: Low dose tPA daily with a 12 h dwell time may be as effective as the standard regimen of tPA and DNAse twice daily with one hour dwell. For most patients only three doses were required, and small pigtail catheters were sufficient. This regimen uses less medication and is logistically much easier than the current standard.
Subject(s)
Empyema, Pleural , Tissue Plasminogen Activator , Humans , Deoxyribonucleases/administration & dosage , Deoxyribonucleases/therapeutic use , Empyema, Pleural/drug therapy , Fibrinolytic Agents/therapeutic use , Retrospective Studies , Tissue Plasminogen Activator/administration & dosage , Tissue Plasminogen Activator/therapeutic use , Drug Administration ScheduleABSTRACT
Photosynthetic acclimation (photoacclimation) is the process whereby leaves alter their morphology and/or biochemistry to optimize photosynthetic efficiency and productivity according to long-term changes in the light environment. The three-dimensional architecture of plant canopies imposes complex light dynamics, but the drivers for photoacclimation in such fluctuating environments are poorly understood. A technique for high-resolution three-dimensional reconstruction was combined with ray tracing to simulate a daily time course of radiation profiles for architecturally contrasting field-grown wheat (Triticum aestivum) canopies. An empirical model of photoacclimation was adapted to predict the optimal distribution of photosynthesis according to the fluctuating light patterns throughout the canopies. While the photoacclimation model output showed good correlation with field-measured gas-exchange data at the top of the canopy, it predicted a lower optimal light-saturated rate of photosynthesis at the base. Leaf Rubisco and protein contents were consistent with the measured optimal light-saturated rate of photosynthesis. We conclude that, although the photosynthetic capacity of leaves is high enough to exploit brief periods of high light within the canopy (particularly toward the base), the frequency and duration of such sunflecks are too small to make acclimation a viable strategy in terms of carbon gain. This suboptimal acclimation renders a large portion of residual photosynthetic capacity unused and reduces photosynthetic nitrogen use efficiency at the canopy level, with further implications for photosynthetic productivity. It is argued that (1) this represents an untapped source of photosynthetic potential and (2) canopy nitrogen could be lowered with no detriment to carbon gain or grain protein content.
Subject(s)
Acclimatization/physiology , Models, Biological , Photosynthesis/physiology , Triticum/physiology , Light , Plant Leaves/physiology , Plant Proteins/metabolism , Ribulose-Bisphosphate Carboxylase/metabolism , United KingdomABSTRACT
Non-photochemical quenching (NPQ) of chlorophyll fluorescence is the process by which excess light energy is harmlessly dissipated within the photosynthetic membrane. The fastest component of NPQ, known as energy-dependent quenching (qE), occurs within minutes, but the site and mechanism of qE remain of great debate. Here, the chlorophyll fluorescence of Arabidopsis thaliana wild type (WT) plants was compared to mutants lacking all minor antenna complexes (NoM). Upon illumination, NoM exhibits altered chlorophyll fluorescence quenching induction (i.e. from the dark-adapted state) characterised by three different stages: (i) a fast quenching component, (ii) transient fluorescence recovery and (iii) a second quenching component. The initial fast quenching component originates in light harvesting complex II (LHCII) trimers and is dependent upon PsbS and the formation of a proton gradient across the thylakoid membrane (ΔpH). Transient fluorescence recovery is likely to occur in both WT and NoM plants, but it cannot be overcome in NoM due to impaired ΔpH formation and a reduced zeaxanthin synthesis rate. Moreover, an enhanced fluorescence emission peak at ~679â¯nm in NoM plants indicates detachment of LHCII trimers from the bulk antenna system, which could also contribute to the transient fluorescence recovery. Finally, the second quenching component is triggered by both ΔpH and PsbS and enhanced by zeaxanthin synthesis. This study indicates that minor antenna complexes are not essential for qE, but reveals their importance in electron stransport, ΔpH formation and zeaxanthin synthesis.
Subject(s)
Arabidopsis/metabolism , Chlorophyll/metabolism , Fluorescence , Light-Harvesting Protein Complexes/metabolism , Plant Leaves/metabolism , Plants, Genetically Modified/metabolism , Zeaxanthins/metabolism , Arabidopsis/genetics , Arabidopsis/radiation effects , Light-Harvesting Protein Complexes/genetics , Photosynthesis , Plant Leaves/genetics , Plant Leaves/radiation effects , Plants, Genetically Modified/genetics , Plants, Genetically Modified/radiation effects , Thylakoids/metabolismABSTRACT
Photoinhibition is the light-induced reduction in photosynthetic efficiency and is usually associated with damage to the D1 photosystem II (PSII) reaction centre protein. This damage must either be repaired, through the PSII repair cycle, or prevented in the first place by nonphotochemical quenching (NPQ). Both NPQ and D1 repair contribute to light tolerance because they ensure the long-term maintenance of the highest quantum yield of PSII. However, the relative contribution of each of these processes is yet to be elucidated. The application of a pulse amplitude modulation fluorescence methodology, called protective NPQ, enabled us to evaluate of the protective effectiveness of the processes. Within this study, the contribution of NPQ and D1 repair to the photoprotective capacity of Arabidopsis thaliana was elucidated by using inhibitors and mutants known to affect each process. We conclude that NPQ contributes a greater amount to the maintenance of a high PSII yield than D1 repair under short periods of illumination. This research further supports the role of protective components of NPQ during light fluctuations and the value of protective NPQ and qPd as unambiguous fluorescence parameters, as opposed to qI and Fv /Fm , for quantifying photoinactivation of reaction centre II and light tolerance of photosynthetic organisms.
Subject(s)
Arabidopsis/physiology , Photosystem II Protein Complex/metabolism , Arabidopsis/radiation effects , Chlorophyll/metabolism , Etiolation/radiation effects , Fluorescence , Photosynthesis/radiation effects , Photosystem II Protein Complex/radiation effectsABSTRACT
The requirements for ascorbate for growth and photosynthesis were assessed under low (LL; 250 µmol m-2 s-1) or high (HL; 1600 µmol m-2 s-1) irradiance in wild-type Arabidopsis thaliana and two ascorbate synthesis mutants (vtc2-1 and vtc2-4) that have 30% wild-type ascorbate levels. The low ascorbate mutants had the same numbers of leaves but lower rosette area and biomass than the wild type under LL. Wild-type plants experiencing HL had higher leaf ascorbate, anthocyanin, and xanthophyll pigments than under LL. In contrast, leaf ascorbate levels were not increased under HL in the mutant lines. While the degree of oxidation measured using an in vivo redox reporter in the nuclei and cytosol of the leaf epidermal and stomatal cells was similar under both irradiances in all lines, anthocyanin levels were significantly lower in the low ascorbate mutants than in the wild type under HL. Differences in the photosynthetic responses of vtc2-1 and vtc2-4 mutants were observed. Unlike vtc2-1, the vtc2-4 mutants had wild-type zeaxanthin contents. While both low ascorbate mutants had lower levels of non-photochemical quenching of chlorophyll a fluorescence (NPQ) than the wild type under HL, qPd values were greater only in vtc2-1 leaves. Ascorbate is therefore essential for growth but not for photoprotection.
Subject(s)
Arabidopsis/metabolism , Ascorbic Acid/metabolism , Etiolation/physiology , Photosynthesis/physiology , Anthocyanins/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/radiation effects , Etiolation/radiation effects , Photosynthesis/radiation effects , Plant Leaves/metabolism , Xanthophylls/metabolismABSTRACT
Background and Aims: Diurnal changes in solar position and intensity combined with the structural complexity of plant architecture result in highly variable and dynamic light patterns within the plant canopy. This affects productivity through the complex ways that photosynthesis responds to changes in light intensity. Current methods to characterize light dynamics, such as ray-tracing, are able to produce data with excellent spatio-temporal resolution but are computationally intensive and the resulting data are complex and high-dimensional. This necessitates development of more economical models for summarizing the data and for simulating realistic light patterns over the course of a day. Methods: High-resolution reconstructions of field-grown plants are assembled in various configurations to form canopies, and a forward ray-tracing algorithm is applied to the canopies to compute light dynamics at high (1 min) temporal resolution. From the ray-tracer output, the sunlit or shaded state for each patch on the plants is determined, and these data are used to develop a novel stochastic model for the sunlit-shaded patterns. The model is designed to be straightforward to fit to data using maximum likelihood estimation, and fast to simulate from. Key Results: For a wide range of contrasting 3-D canopies, the stochastic model is able to summarize, and replicate in simulations, key features of the light dynamics. When light patterns simulated from the stochastic model are used as input to a model of photoinhibition, the predicted reduction in carbon gain is similar to that from calculations based on the (extremely costly) ray-tracer data. Conclusions: The model provides a way to summarize highly complex data in a small number of parameters, and a cost-effective way to simulate realistic light patterns. Simulations from the model will be particularly useful for feeding into larger-scale photosynthesis models for calculating how light dynamics affects the photosynthetic productivity of canopies.
Subject(s)
Algorithms , Carbon/metabolism , Models, Biological , Photosynthesis/radiation effects , Plants/anatomy & histology , Computer Simulation , Imaging, Three-Dimensional , Plant Leaves/anatomy & histology , Plant Leaves/physiology , Plant Leaves/radiation effects , Plant Physiological Phenomena , Plants/radiation effects , Sunlight , Triticum/anatomy & histology , Triticum/physiology , Triticum/radiation effectsABSTRACT
We present an insert-based approach to fabricate scalable and multiplexable microfluidic devices for 3D cell culture and integration with downstream detection modules. Laser-cut inserts with a layer of electrospun fibers are used as a scaffold for 3D cell culture, with the inserts being easily assembled in a 3D-printed fluidic device for flow-based studies. With this approach, the number and types of cells (on the inserts) in one fluidic device can be customized. Moreover, after an investigation (i.e., stimulation) under flowing conditions, the cell-laden inserts can be removed easily for subsequent studies including imaging and cell lysis. In this paper, we first discuss the fabrication of the device and characterization of the fibrous inserts. Two device designs containing two (channel width = 260 µm) and four (channel width = 180 µm) inserts, respectively, were used for different experiments in this study. Cell adhesion on the inserts with flowing media through the device was tested by culturing endothelial cells. Macrophages were cultured and stimulated under different conditions, the results of which indicate that the fibrous scaffolds under flow conditions result in dramatic effects on the amount and kinetics of TNF-α production (after LPS stimulation). Finally, we show that the cell module can be integrated with a downstream absorbance detection scheme. Overall, this technology represents a new and versatile way to culture cells in a more in vivo fashion for in vitro studies with online detection modules. Graphical abstract This paper describes an insert-based microfluidic device for 3D cell culture that can be easily scaled, multiplexed, and integrated with downstream analytical modules.
Subject(s)
Cell Culture Techniques/instrumentation , Lab-On-A-Chip Devices , Single-Cell Analysis/instrumentation , Animals , Cattle , Cell Adhesion , Cell Line , Endothelial Cells/cytology , Equipment Design , Macrophages/cytology , Mice , Printing, Three-Dimensional , RAW 264.7 CellsABSTRACT
Background: Patients with end-stage renal disease (ESRD) secondary to systemic lupus erythematosus (SLE) have historically been deterred from free flap breast reconstruction due to perceived complication risks. Numerous studies examining patients with ESRD have cited free flap complications, including increased incidences of infection and wound breakdown, with some surgeons suggesting ESRD is an independent risk factor for flap failure.15 Due to perceived risks, autologous breast reconstruction has not been extensively explored as an option in patients with ESRD on hemodialysis with comorbid connective tissue/autoimmune disorders, such as SLE. To the authors' knowledge, there are currently no published reports of successful free flap breast reconstruction in patients with ESRD due to SLE. Methods: This case report describes a patient requiring hemodialysis for ESRD caused by SLE who underwent left mastectomy and immediate autologous breast reconstruction. Deep inferior epigastric perforator flap technique was employed. Conclusions: This successful case report suggests the use of free flaps is a feasible option that should be considered for oncologic breast reconstruction in patients with ESRD secondary to SLE who require hemodialysis. The authors believe that further investigation is warranted to evaluate the safety of autologous breast reconstruction as an option for patients with either comorbidity. While ESRD and SLE are not explicit contraindications to free flap reconstruction, careful patient selection and appropriate indication is paramount for immediate surgical and long-term reconstructive success.
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Background: Implant-based breast augmentation is one of the most popular plastic surgery procedures performed worldwide. As the number of patients who have breast implants continues to rise, so does the number of those who request breast implant removal without replacement. There is little in the current scientific literature describing total intact capsulectomy and simultaneous mastopexy procedures. Objectives: Here, the authors present their current method using the mammary imbrication lift and fixation technique after explant and total capsulectomy. Methods: Between 2016 and 2021, a total of 64 patients (mean age: 42.95 years; range, 27-78 years) underwent the described mammary imbrication lift and fixation technique with bilateral breast implant removal and total capsulectomy. Results: Mean follow-up was 6.5 months (range, 1-36 months). Postoperative complications included minor cellulitis in 1 patient (1.6%), late onset hematoma with infection in 1 patient (1.6%), fat necrosis and pulmonary embolism in 1 patient with prior history of thromboembolic events (1.6%), and breast scar irregularity in 4 patients (6.2%) who required subsequent minor scar revision or steroid injections. Two patients (1.6%) underwent revision surgery with bilateral breast fat grafting to improve shape and add volume. Conclusions: The mammary imbrication lift and fixation technique described here can safely and simultaneously be performed with a total intact capsulectomy and explant procedure. This technique avoids wide undermining, intentionally opening the capsule, performing subtotal capsulectomy, and preserving blood supply to the breast tissue and nipple with low complication rates.
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Background: Significant swelling after rhinoplasty can temporarily obscure results and lead to distress for patients and surgeons. We recently developed three dimensional (3D)-printed nasal splints that aim to protect the nose and limit edema by applying gentle compression. This prospective, randomized study compares postoperative nasal edema in patients being treated with traditional taping versus 3D-printed splints. Methods: Patients undergoing primary rhinoplasty (2019-2020) were randomized into two groups: taping versus 3D-printed splinting. For 12 weeks, patients either applied steri-strips to the dorsum and tip, or used 3D-printed splints, which were based on nasal simulations. The percentage change in volume (cm3) was calculated for the total nose, dorsum, and nasal tip at various time points. Results: Nasal taping (nâ =â 34) demonstrated a volume reduction of 4.8%, 9.9%, 10.0%, 10.3%, and 10.6% (compared with baseline) at 2 weeks, 6 weeks, 3 months, 6 months, and 1 year, respectively. In contrast, the resolution of swelling with 3D splints (nâ =â 36) was 5.0%, 8.6%, 11.0%, 14.9%, and 15.1% at the same time points. Inter-group comparison showed that 3D splints led to significantly less edema of the total nose at 6 months and 1 year (P ≤ 0.05), as well as consistent reductions in the tip and dorsum, specifically (1 year, P ≤ 0.1, 0.01, respectively). Conclusions: 3D-printed splints after rhinoplasty leads to a significant reduction of edema, most noticeable at 6 months and 1 year. This study suggests that customized 3D-printed splints offer an effective clinical alternative to traditional taping to reduce postoperative edema after rhinoplasty.
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Background Esophagectomy is the surgical excision of part or all of the esophagus and is associated with both common and serious complications. Various comorbidities, such as diabetes mellitus, smoking, and congestive heart failure (CHF), have been detected in individuals who have undergone esophagectomy. This study investigates the association of baseline characteristics and comorbidities with postoperative complications. Methods A retrospective cohort study based on data from the National Surgical Quality Improvement Program database was conducted, evaluating 2,544 patients who underwent esophagectomy between January 2016 and December 2018. Data included baseline characteristics, established comorbidities, and postoperative complications within 30 days of the procedure. Risk-adjusted and unadjusted logistic regressions were used to assess the odds of postoperative complications against baseline characteristics. Results The majority of our population were white males (80.8% male; 78.2% white), with a mean age of 63.5 years. More than half of the patients were overweight or obese. A minority of our patients had a smoking history, weight loss, diabetes mellitus, chronic obstructive pulmonary disease (COPD), or CHF. The most frequent postoperative complications were as follows: return to the operating room (15.7%), anastomotic leak (12.9%), pneumonia (12.7%), bleeding/transfusions (11.8%), readmissions (11.4%), and unplanned intubation (10.5%). Adjusted associations for odds of experiencing a postoperative complication were found to be statistically significant for age (odds ratio [OR] 1.02, 95% confidence interval [CI] 1.01-1.03, and P < 0.001), operation time (OR 1.002, 95% CI 1.001-1.003, and P < 0.001), race (not white) (OR 1.76, 95% CI 1.26-2.47, and P = 0.001), BMI (underweight) (OR 2.18, 95% CI 1.36-3.50, and P = 0.001), smoking (OR 1.42, 95% CI 1.14-1.76, and P = 0.001), and chemotherapy and/or radiation (OR 0.82, 95% CI 0.68-0.99, P = 0.038). Conclusions Our study found that age, operation time, nonwhite race, underweight BMI, and smoking were independently associated with an increased risk of developing a postoperative complication following esophagectomy. Additionally, neoadjuvant chemotherapy and/or radiation are associated with a decreased risk. Understanding how baseline characteristics and comorbidities can affect rates of postoperative complications may help to adjust care for patients in both pre- and postoperative settings.
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Background: Aesthetic norms fluctuate over time and often result in generational differences in preferred ideal nasal aesthetics. While some traditional concepts of the ideal nasal aesthetic have been suggested in our literature, there has been no study to date that has identified contemporary preferences across different age groups. Objectives: To understand the general population's current perception of ideal nasal profiles. Methods: Two-dimensional images of female noses (n = 10) of varying ethnicities were simulated to alter either the radix height or nasolabial angle (NLA) independently. Radix height was manipulated by increasing or decreasing the height by 5â mm relative to baseline. For NLA, 3 images were created with the following measurements: (1) 90°, (2) 100°, and (3) 110°. Groups were categorized by generation and age at the time of completing the study: Generation Z (Gen Z; age 18-23), Millennial 20s (age 24-30), Millennial 30s (age 31-39), and Generation X (Gen X; age 40-55). Each figure consisted of either 3 variations in radix height (n = 10) or 3 variations in NLA (n = 10). Within each figure, volunteers were asked to choose their preferred nose. Results: The younger generations, Gen Z and Millennial 20s and 30s, preferred a more augmented radix compared to Gen X which preferred a baseline radix height. Gen Z, Millennial 20s, and Gen X preferred a 90° NLA, while Millennial 30s preferred an NLA of 100°. Conclusions: The authors found that younger populations (Gen Z, Millennial 20s, and Millennial 30s) preferred a more augmented appearance to the nasal radix and, on average, a more acute NLA than published data suggest.
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Recent developments in three-dimensional (3D) imaging technology offer a more comprehensive means of assessing facial features. 3D printing allows for the transition of planning from simply a preoperative tool to an intraoperative device with the use of tools such as 3D-printed cutting guides, marking guides, or positioning guides. With the advent of 3D printing technology, 3D surface images can now be used to generate new medical models, devices, or tools to assist with rhinoplasty during preoperative, intraoperative, and postoperative phases. In the field of rhinoplasty, 3D printing can be applied in three main areas: (1) reference models, (2) surgical guides, and (3) nasal splints. The value of 3D imaging extends far beyond the benefits of "conversion" during a preoperative consultation and has the potential to greatly enhance the overall treatment of rhinoplasty patients with enhanced communication and personalized devices that can be used during surgery and in the postoperative phase.
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The purpose of this study was to critically analyze the risk of unplanned readmission following resection of brain metastasis and to identify key risk factors to allow for early intervention strategies in high-risk patients. We analyzed data from the Nationwide Readmissions Database (NRD) from 2010-2014, and included patients who underwent craniotomy for brain metastasis, identified using ICD-9-CM diagnosis (198.3) and procedure (01.59) codes. The primary outcome of the study was unplanned 30-day all-cause readmission rate. Secondary outcomes included reasons and costs of readmissions. Hierarchical logistic regression model was used to identify the factors associated with 30-day readmission following craniotomy for brain metastasis. During the study period, 44,846 index hospitalizations occurred for patients who underwent resection of brain metastasis. In this cohort, 17.8% (n = 7,965) had unplanned readmissions within the first 30 days after discharge from the index hospitalization. The readmission rate did not change significantly during the five-year study period (p-trend = 0.286). The median per-patient cost for 30-day unplanned readmission was $11,109 and this amounted to a total of $26.4 million per year, which extrapolates to a national expenditure of $269.6 million. Increasing age, male sex, insurance status, Elixhauser comorbidity index, length of stay, teaching status of the hospital, neurological complications and infectious complications were associated with 30-day readmission following discharge after an index admission for craniotomy for brain metastasis. Unplanned readmission rates after resection of brain metastasis remain high and involve substantial healthcare expenditures. Developing tools and interventions to prevent avoidable readmissions could focus on the high-risk patients as a future strategy to decrease substantial healthcare expense.
Subject(s)
Brain Neoplasms/surgery , Craniotomy/adverse effects , Patient Readmission/economics , Patient Readmission/statistics & numerical data , Postoperative Complications , Cohort Studies , Cross-Sectional Studies , Databases, Factual , Female , Humans , Male , Risk Factors , United StatesABSTRACT
We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC-derived ATP. These studies incorporate on-chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC-EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum-black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8 ± 0.2 nM NO per endothelial cell (average ± SEM, n = 8). Pretreatment of RBCs with glybenclamide inhibited treprostinil-induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92 ± 0.1 nM NO per endothelial cell, n = 7). In the future, this device can be used to study interactions between many other cell types (both adherent and non-adherent cell lines) and incorporate other detection schemes.
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Fabrication of microchip-based devices using 3-D printing technology offers a unique platform to create separate modules that can be put together when desired for analysis. A 3-D printed module approach offers various advantages such as file sharing and the ability to easily replace, customize, and modify the individual modules. Here, we describe the use of a modular approach to electrochemically detect the ATP-mediated release of nitric oxide (NO) from endothelial cells. Nitric oxide plays a significant role in the vasodilation process; however, detection of NO is challenging due to its short half-life. To enable this analysis, we use three distinct 3-D printed modules: cell culture, sample injection and detection modules. The detection module follows a pillar-based Wall-Jet Electrode design, where the analyte impinges normal to the electrode surface, offering enhanced sensitivity for the analyte. To further enhance the sensitivity and selectivity for NO detection the working electrode (100 µm gold) is modified by the addition of a 27 µm gold pillar and platinum-black coated with Nafion. The use of the pillar electrode leads to three-dimensional structure protruding into the channel enhancing the sensitivity by 12.4 times in comparison to the flat electrode (resulting LOD for NO = 210 nM). The next module, the 3-D printed sample injection module, follows a simple 4-Port injection rotor design made of two separate components that when assembled can introduce a specific volume of analyte. This module not only serves as a cheaper alternative to the commercially available 4-Port injection valves, but also demonstrates the ability of volume customization and reduced dead-volume issues with the use of capillary-free connections. Comparison between the 3-D printed and a commercial 4-Port injection valve showed similar sensitivities and reproducibility for NO analysis. Lastly, the cell culture module contains electrospun polystyrene fibers with immobilized endothelial cells, resulting in 3-D scaffold for cell culture. With the incorporation of all 3 modules, we can make reproducible ATP injections (via the 3-D printed sample injection module) that can stimulate NO release from endothelial cells cultured on a fibrous insert in the cell culture module which can then be quantitated by the pillar WJE module (0.19 ± 0.03 nM/cell, n = 27, 3 inserts analyzed each day, on 9 different days). The modular approach demonstrates the facile creation of custom and modifiable fluidic components that can be assembled as needed.
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Polymer nano/micro fibers have found many applications including 3D cell culture and the creation of wound dressings. The fibers can be produced by a variety of techniques that include electrospinning, the primary disadvantage of which include the requirement for a high voltage supply (which may cause issues such as polymer denaturation) and lack of portability. More recently, solution blow spinning, where a high velocity sheath gas is used instead of high voltage, has been used to generate polymer fibers. In this work, we used blow spinning to create nano/microfibers for microchip-based 3D cell culture. First, we thoroughly investigated fiber generation from a 3D printed gas sheath device using two polymers that are amenable to cell culture (polycaprolactone, PCL and polystyrene, PS) as well as the parameters that can affect PCL and PS fiber quality. Using the 3D printed sheath device, it was found that the pressure of the sheath N2 and the concentration of polymer solutions determine if fibers can be produced as well as the resulting fiber morphology. In addition, we showed how these fibers can be used for 3D cell culture by directly depositing PCL fibers in petri dishes and well plates. It is shown the fibers have good compatibility with RAW 264.7 macrophages and the PCL fiber scaffold can be as thick as 178 ± 14 µm. PCL fibers created from solution blow spinning (with the 3D printed sheath device) were then integrated with a microfluidic device for the first time to fabricate a 3D cell culture scaffold with a flow component. After culturing and stimulating macrophages on the fluidic device, it was found that the integrated 3D fibrous scaffold is a better mimic of the extracellular matrix (as opposed to a flat, 2D substrate), with enhanced nitrite accumulation (product of nitric oxide release) from macrophages stimulated with lipopolysaccharide. PS fibers were also made and integrated in a microfluidic device for 3D culture of endothelial cells, which stayed viable for at least 72 hours (48 hours under the flowing conditions). This approach will be useful for future studies involving more realistic microchip-based culture models for studying cell-to-cell communication.
ABSTRACT
In plant phenotyping, it has become important to be able to measure many features on large image sets in order to aid genetic discovery. The size of the datasets, now often captured robotically, often precludes manual inspection, hence the motivation for finding a fully automated approach. Deep learning is an emerging field that promises unparalleled results on many data analysis problems. Building on artificial neural networks, deep approaches have many more hidden layers in the network, and hence have greater discriminative and predictive power. We demonstrate the use of such approaches as part of a plant phenotyping pipeline. We show the success offered by such techniques when applied to the challenging problem of image-based plant phenotyping and demonstrate state-of-the-art results (>97% accuracy) for root and shoot feature identification and localization. We use fully automated trait identification using deep learning to identify quantitative trait loci in root architecture datasets. The majority (12 out of 14) of manually identified quantitative trait loci were also discovered using our automated approach based on deep learning detection to locate plant features. We have shown deep learning-based phenotyping to have very good detection and localization accuracy in validation and testing image sets. We have shown that such features can be used to derive meaningful biological traits, which in turn can be used in quantitative trait loci discovery pipelines. This process can be completely automated. We predict a paradigm shift in image-based phenotyping bought about by such deep learning approaches, given sufficient training sets.