Sensitizing drug-resistant cancer cells from blood using microfluidic electroporator.

Direct assessment of patient samples holds unprecedented potential in the treatment of cancer. Circulating tumor cells (CTCs) in liquid biopsies are a rapidly evolving source of primary cells in the clinic and are ideal candidates for functional assays to uncover real-time tumor information in real-time. However, a lack of routines allowing direct and active interrogation of CTCs directly from liquid biopsy samples represents a bottleneck for the translational use of liquid biopsies in clinical settings. To address this, we present a workflow for using a microfluidic vortex-assisted electroporation system designed for the functional assessment of CTCs purified from blood. Validation of this approach was assessed through drug response assays on wild-type (HCC827 wt) and gefitinib-resistant (HCC827 GR6) non-small cell lung cancer (NSCLC) cells. HCC827 cells trapped within microscale vortices were electroporated to sequentially deliver drug agents into the cytosol. Electroporation conditions facilitating multi-agent delivery were characterized for both cell lines using an automatic single-cell image fluorescence intensity algorithm. HCC827 GR6 cells spiked into the blood to emulate drug-resistant CTCs were able to be collected with high purity, demonstrating the ability of the device to minimize background cell impact for downstream sensitive cell assays. Using our proposed workflow, drug agent combinations to restore gefitinib sensitivity reflected the anticipated cytotoxic response. Taken together, these results represent a microfluidics multi-drug screening panel workflow that can enable functional interrogation of patient CTCs in situ, thereby accelerating the clinical standardization of liquid biopsies.

Specific patterns of vital sign fluctuations predict infection and enable sepsis diagnosis in pediatric burn patients.

Early recognition of the clinical signs of bloodstream infection in pediatric burn patients is key to improving survival rates in the burn unit. The objective of this study was to propose a simple scoring criteria that used readily available temperature, heart rate (HR) and mean arterial pressure (MAP) data to accurately predict bloodstream infection in pediatric burn patients. A retrospective chart review included 100 patients admitted to the pediatric burn unit for >20% total body surface area (TBSA) burn injuries. Each patient had multiple blood culture tests, and each test was treated as a separate and independent "infection event" for analysis. The time at each blood culture draw was time 0 for that event, and temperature, HR and MAP data was collected for 24 hours after the blood culture was drawn. "Infection events" included in this study had at least six complete sets of temperature, HR and MAP data entries. Median temperature, HR and MAP, as well as mean fever spikes, HR spikes and MAP dips, were compared between infection group (positive blood cultures) and control group (negative blood cultures). These vital sign fluctuations were evaluated individually and as a combination of all three as timely predictors of bloodstream infection. In addition, we tested the prediction of Gram-negative bacteria versus Gram-positive or fungi present in blood cultures. Patients in the infection group had significantly higher median temperatures (p<0.001), mean fever spikes (p<0.001) and mean HR spikes (p<0.001), compared to the control group. Using the combination scoring criteria to predict bloodstream infection, the strongest predictive values in the 24-hour timeframe had high sensitivity (93%) and specificity (81%). The predictive test metric based on vital sign spikes predicted Gram-negative bacteria, but with limited sensitivity (57%) and specificity (44%). A simple scoring criteria using a combination of fever spikes, HR spikes and MAP dips predicted bloodstream infection in pediatric burn patients, and can be feasibly implemented in routine clinical care. There is also potential to use the predictive metric to detect a few select organisms based on vital signs, however further work is necessary to enhance accuracy to levels that would allow consideration for clinical use.

Exploring donor demographics effects on hepatocyte yield and viability: Results of whole human liver isolation from one center.

Due to the growth of cell-based therapeutic alternatives addressing the shortage of livers for transplant, there is necessity for a reliable source of human hepatocytes. In addition, pharmaceutical research often requires human hepatocytes to assess new drug therapies during development or to achieve FDA approval. Whole human livers producing large quantities of cells from the same donor are ideal, enhancing reproducibility for all purposes, while also allowing for capturing variances in drug-metabolism across different demographics for pharmaceutical testing and development but are limited in availability and quality for research purposes. The present study investigates the effect of donor and liver procurement factors of 16 human livers on cell viability and yield, showing that typical exclusion criteria for transplant still produce viable hepatocytes with significant yields. Although limited in number of data points, which should be taken into consideration, the conclusions of this study could be utilized as indications, allowing for expansion of liver selection criteria for hepatocyte isolation and provide the necessary quality hepatocytes in large quantities for the growing pharmaceutical, biomedical, and therapeutic research fields.

Specific Patterns of Postoperative Temperature Elevations Predict Blood Infection in Pediatric Burn Patients.

Using readily available temperature data, we seek to propose a scoring criteria that can facilitate accurate and immediate prediction of blood infection. The standard in diagnosing blood infection is a positive blood culture result that may take up to 3 days to process, requiring providers to make a prediction about which febrile patient is actually bacteremic. This prediction is difficult in burned children as systemic inflammation can cause fever in the absence of infection. An ability to make this prediction more accurate using readily available information would be useful. A retrospective chart review was performed for 28 pediatric patients, with a burn size 20% or greater, admitted to the burn unit between 2010 and 2014. All children had blood cultures drawn. They were divided into either infection (positive blood cultures) or control (negative blood cultures) groups. Median temperature and mean number of temperature elevations were compared between the two groups. We evaluated the predictive accuracy of using temperature elevation, pattern, and timing to predict blood infection. A significant difference was seen in the mean number of temperature elevations above 39°C. This was significant for each time stage, especially in the 0- to 24-hour post-surgery period. We found the most predictive accuracy in the 0- to 12-, 12- to 38-, and 12- to 48-hour time periods. We found a strong association between mean number of fever spikes above 39°C and blood infection, especially 12 to 24 hours after surgery. This readily available data can be useful to clinicians as they access children with burns.

Biomechanical Measurement Error Can Be Caused by Fujifilm Thickness: A Theoretical, Experimental, and Computational Analysis.

This is the first study to quantify the measurement error due to the physical thickness of Fujifilm for several material combinations relevant to orthopaedics. Theoretical and experimental analyses were conducted for cylinder-on-flat indentation over a series of forces (750 and 3000 N), cylinder diameters (0 to 80 mm), and material combinations (metal-on-metal, MOM; metal-on-polymer, MOP; metal-on-bone, MOB). For the scenario without Fujifilm, classic Hertzian theory predicted the true line-type contact width as WO = {(8FDcyl)/(πLcyl)[(1 - νcyl2)/Ecyl + (1 - νflat2)/Eflat]}1/2, where F is compressive force, Dcyl is cylinder diameter, Lcyl is cylinder length, νcyl and νflat are cylinder and flat Poisson's ratios, and Ecyl and Eflat are cylinder and flat elastic moduli. For the scenario with Fujifilm, experimental measurements resulted in contact widths of WF = 0.1778 × F0.2273 × D0.2936 for MOM tests, WF = 0.0449 × F0.4664 × D0.4201 for MOP tests, and WF = 0.1647 × F0.2397 × D0.3394 for MOB tests, where F is compressive force and D is cylinder diameter. Fujifilm thickness error ratio WF /WO showed a nonlinear decrease versus cylinder diameter, whilst error graphs shifted down as force increased. Computational finite element analysis for several test cases agreed with theoretical and experimental data, respectively, to within 3.3% and 1.4%. Despite its wide use, Fujifilm's measurement errors must be kept in mind when employed in orthopaedic biomechanics research.

The Association between Work-Related Rumination and Heart Rate Variability: A Field Study.

The objective of this study was to examine the association between perseverative cognition in the form of work-related rumination, and heart rate variability (HRV). We tested the hypothesis that high ruminators would show lower vagally mediated HRV relative to low ruminators during their leisure time. Individuals were classified as being low (n = 17) or high ruminators (n = 19), using the affective scale on the work-related rumination measure. HRV was assessed using a wrist sensor band (Microsoft Band 2). HRV was sampled between 8 pm and 10 pm over three workday evenings (Monday to Wednesday) while individuals carried out their normal evening routines. Compared to the low ruminators, high affective ruminators demonstrated lower HRV in the form of root mean square successive differences (RMSSDs), relative to the low ruminators, indicating lower parasympathetic activity. There was no significant difference in heart rate, or activity levels between the two groups during the recording periods. The current findings of this study may have implications for the design and delivery of interventions to help individuals unwind post work and to manage stress more effectively. Limitations and implications for future research are discussed.

Dynamic features of postnatal subventricular zone cell motility: a two-photon time-lapse study.

Neuroblasts migrate long distances in the postnatal subventricular zone (SVZ) and rostral migratory stream (RMS) to the olfactory bulbs. Many fundamental features of SVZ migration are still poorly understood, and we addressed several important questions using two-photon time-lapse microscopy of brain slices from postnatal and adult eGFP(+) transgenic mice. 1) Longitudinal arrays of neuroblasts, so-called chain migration, have never been dynamically visualized in situ. We found that neuroblasts expressing doublecortin-eGFP (Dcx-eGFP) and glutamic acid decarboxylase-eGFP (Gad-eGFP) remained within arrays, which maintained their shape for many hours, despite the fact that there was a wide variety of movement within arrays. 2) In the dorsal SVZ, neuroblasts migrated rostrocaudally as expected, but migration shifted to dorsoventral orientations throughout ventral regions of the lateral ventricle. 3) Whereas polarized bipolar morphology has been a gold standard for inferring migration in histologic sections, our data indicated that migratory morphology was not predictive of motility. 4) Is there local motility in addition to long distance migration? 5) How fast is SVZ migration? Unexpectedly, one-third of motile neuroblasts moved locally in complex exploratory patterns and at average speeds slower than long distance movement. 6) Finally, we tested, and disproved, the hypothesis that all motile cells in the SVZ express doublecortin, indicating that Dcx is not required for migration of all SVZ cell types. These data show that cell motility in the SVZ and RMS is far more complex then previously thought and involves multiple cell types, behaviors, speeds, and directions.