Production and loss of O2(1Δ g ) at atmospheric pressure using microwave-driven microplasmas.

We have used arrays of microwave-generated microplasmas operating at atmospheric pressure to generate high concentrations of singlet molecular oxygen, O2(1Δ g ), which is of interest for biomedical applications. The discharge is sustained by a pair of microstrip-based microwave resonator arrays which force helium/oxygen gas mixtures through a narrow plasma channel. We have demonstrated the efficacy of both NO and less-hazardous N2O additives for suppression of ozone and associated enhancement of the O2(1Δ g ) yield. Quenching of O2(1Δ g ) by ozone is sufficiently suppressed such that quenching by ground state molecular oxygen becomes the dominant loss mechanism in the post-discharge outflow. We verified the absence of other significant gas-phase quenching mechanisms by measuring the O2(1Δ g ) decay along a quartz flow tube. These measurements indicated a first-order rate constant of (1.2 ± 0.3) × 10-24 m3 s-1, slightly slower than but consistent with prior measurements of singlet oxygen quenching on ground state oxygen. The discharge-initiated reaction mechanisms and data analysis are discussed in terms of a chemical kinetics model of the system.

A Compact Model for Lyophilizer Equipment Capability Estimation.

This work presents a compact model for the equipment capability limit of a common configuration of pharmaceutical lyophilizers, a product chamber separated from the condenser by a duct and isolation valve, at a wide range of design parameters. The equipment capability limit is one of the most important characteristics determining the lyophilization design space for a particular product, container, and equipment combination. Experimental measurements of equipment capability are time-consuming and expensive, especially at the production scale. Numerical modeling using computational fluid dynamics may reduce the number of experiments and provide insights into the physics of the process with high resolution. The computational fluid dynamics (CFD) modeling has been used in this work to develop a compact model for lyophilizer equipment capability. This eliminates the need for end users to create a full CFD model of the equipment and process. Full CFD and compact model simulations for laboratory and pilot-scale lyophilizers have been compared with tunable diode laser absorption spectroscopy measurements of the water vapor mass flow during ice slab tests. The compact model results average deviation from the experimental data is within 10%, whereas the full CFD simulations are within 5%. The compact model is based on several key parameters which are the main characteristics of a lyophilizer affecting the equipment capability curve. These parameters are discussed, and their effect on the modeling results is shown.

A Software Tool for Lyophilization Primary Drying Process Development and Scale-up Including Process Heterogeneity, I: Laboratory-Scale Model Testing.

Freeze-drying is a deceptively complex operation requiring sophisticated design of a robust and efficient process that includes understanding and planning for heterogeneity across the batch and shifts in parameters due to vial or lyophilizer changes. A software tool has been designed to assist in process development and scale-up based on a model that includes consideration of the process heterogeneity. Two drug formulations were used to test the ability of the new tool to develop a freeze-drying cycle and correctly predict product temperatures and drying times. Model inputs were determined experimentally, and the primary drying heterogeneous freeze-drying model was used to design drying cycles that provided data to verify the accuracy of model-predicted product temperature and primary drying time. When model inputs were accurate, model-predicted primary drying times were within 0.1 to 15.9% of experimentally measured values, and product temperature accuracy was between 0.2 and 1.2°C for three vial locations, center, inner edge, and outer edge. However, for some drying cycles, differences in vial heat transfer coefficients due to changes in shelf and product temperature as well as altered product resistance due to product temperature-dependent microcollapse increased inaccuracy (up to 28.6% difference in primary drying time and 5.1°C difference in product temperature). This highlights the need for careful determination of experimental conditions used to calculate model inputs. In future efforts, full characterization of location- and shelf temperature-dependentKv as well as location- and product temperature-dependentRp will enhance the accuracy of the predictions by the model within the user-friendly software.

A microfluidic array for large-scale ordering and orientation of embryos.

Quantitative studies of embryogenesis require the ability to monitor pattern formation and morphogenesis in large numbers of embryos, at multiple time points and in diverse genetic backgrounds. We describe a simple approach that greatly facilitates these tasks for Drosophila melanogaster embryos, one of the most advanced models of developmental genetics. Based on passive hydrodynamics, we developed a microfluidic embryo-trap array that can be used to rapidly order and vertically orient hundreds of embryos. We describe the physical principles of the design and used this platform to quantitatively analyze multiple morphogen gradients in the dorsoventral patterning system. Our approach can also be used for live imaging and, with slight modifications, could be adapted for studies of pattern formation and morphogenesis in other model organisms.

Lack of Clinically Significant Relationships of Age or Body Mass Index with Merkel Cell Carcinoma Immunotherapy Outcomes.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with a high risk of metastasis. The development of anti-PD-1/PD-L1 immunotherapy has improved outcomes for advanced MCC, yet about 50% of such patients do not achieve durable responses. This study analyzed the effects of age and body mass index (BMI) on immunotherapy response in 183 advanced MCC patients from a single-center longitudinal database. Using Fine-Gray or Cox regression models, treatment response, progression-free survival (PFS), MCC-specific survival, and overall survival (OS) were evaluated. Age showed a significant non-linear relationship with treatment response (p = 0.04), with patients much older or younger than 70 years less likely to respond. However, age was not significantly associated with PFS (p = 0.21), MCC-specific survival (p = 0.72), or OS (p = 0.36). Similarly, BMI was not significantly correlated with treatment response (p = 0.41), PFS (p = 0.52), MCC-specific survival (p = 0.78), or OS (p = 0.71). Unlike previous studies suggesting that obesity and advanced age improve outcomes in other cancers, these associations were not observed in MCC. These findings suggest that age and BMI should not influence eligibility for immunotherapy in MCC patients, emphasizing the importance of unbiased patient selection for this treatment.

Highly sensitive and label-free digital detection of whole cell E. coli with Interferometric Reflectance Imaging.

Bacterial infectious diseases are a major threat to human health. Timely and sensitive pathogenic bacteria detection is crucial in bacterial contaminations identification and preventing the spread of infectious diseases. Due to limitations of conventional bacteria detection techniques there have been concerted research efforts towards developing new biosensors. Biosensors offering label-free, whole bacteria detection are highly desirable over those relying on label-based or pathogenic molecular components detection. The major advantage is eliminating the additional time and cost required for labeling or extracting the desired bacterial components. Here, we demonstrate rapid, sensitive and label-free Escherichia coli (E. coli) detection utilizing interferometric reflectance imaging enhancement allowing visualizing individual pathogens captured on the surface. Enabled by our ability to count individual bacteria on a large sensor surface, we demonstrate an extrapolated limit of detection of 2.2 CFU/ml from experimental data in buffer solution with no sample preparation. To the best of our knowledge, this level of sensitivity for whole E. coli detection is unprecedented in label-free biosensing. The specificity of our biosensor is validated by comparing the response to target bacteria E. coli and non-target bacteria S. aureus, K. pneumonia and P. aeruginosa. The biosensor's performance in tap water proves that its detection capability is unaffected by the sample complexity. Furthermore, our sensor platform provides high optical magnification imaging and thus validation of recorded detection events as the target bacteria based on morphological characterization. Therefore, our sensitive and label-free detection method offers new perspectives for direct bacterial detection in real matrices and clinical samples.

Estrogen deficient male mice develop compulsive behavior.

BACKGROUND: Aromatase converts androgen to estrogen. Thus, the aromatase knockout (ArKO) mouse is estrogen deficient. We investigated the compulsive behaviors of these animals and the protein levels of catechol-O-methyltransferase (COMT) in frontal cortex, hypothalamus and liver. METHODS: Grooming was analyzed during the 20-min period immediately following a water-mist spray. Running wheel activity over two consecutive nights and barbering were analyzed. COMT protein levels were measured by Western analysis. RESULTS: Six-month old male but not female ArKO mice develop compulsive behaviors such as excessive barbering, grooming and wheel-running. Excessive activities were reversed by 3 weeks of 17beta-estradiol replacement. Interestingly, the presentation of compulsive behaviors is accompanied by concomitant decreases (p < .05) in hypothalamic COMT protein levels in male ArKO mice. These values returned to normal upon 17beta-estradiol treatment. In contrast, hepatic and frontal cortex COMT levels were not affected by the estrogen status, indicating region- and tissue-specific regulation of COMT levels by estrogen. No differences in COMT levels were detectable between female animals of both genotypes. CONCLUSIONS: This study describes the novel observation of a possible link between estrogen, COMT and development of compulsive behaviors in male animals which may have therapeutic implications in obsessive compulsive disorder (OCD) patients.

Engineering cell-cell signaling.

Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling on the basis of quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilize synthetic cells, advanced 'chassis' and predictive modeling to engineer the form and function of living tissues.

Microfluidic chamber arrays for whole-organism behavior-based chemical screening.

The nematode Caenorhabditis elegans is an important model organism in genetic research and drug screening because of its relative simplicity, ease of maintenance, amenability to simple genetic manipulation, and relevance to human biology. However, their small size and mobility make nematodes difficult to physically manipulate, particularly with spatial and temporal precision. We have developed a microfluidic device to overcome these challenges and enable fast behavior-based chemical screening in C. elegans. The key components of this easy-to-use device allow rapid loading and housing of C. elegans in a chamber array for chemical screening. A simple two-step loading process enables simultaneous loading of a large number of animals within a few minutes without using any expensive/active off-chip components. In addition, chemicals can be precisely delivered to the worms and exchanged with high temporal precision. To demonstrate this feature and the ability to measure time dependent responses to chemicals, we characterize the transient response of worms exposed to different concentrations of anesthetics. We then use the device to study the effect of chemical signals from hermaphrodite worms on male behavior. The ability of the device to maintain a large number of free moving animals in one field of view over a long period of time permits us to demonstrate an increase in the incidence of a specific behavior in males subjected to worm-conditioned medium. Because our device allows monitoring of a large number of worms with single-animal resolution, we envision that this platform will greatly expedite chemical screening in C. elegans.