Microfluidic Purification and Concentration of Malignant Pleural Effusions for Improved Molecular and Cytomorphological Diagnostics

Evaluation of pleural fluids for metastatic cells is a key component of diagnostic cytopathology. However, a large background of smaller leukocytes and/or erythrocytes can make accurate diagnosis difficult and reduce specificity in identification of mutations of interest for targeted anti-cancer therapies. Here, we describe an automated microfluidic system (Centrifuge Chip) which employs microscale vortices for the size-based isolation and concentration of cancer cells and mesothelial cells from a background of blood cells. We are able to process non-diluted pleural fluids at 6 mL/min and enrich target cells significantly over the background; we achieved improved purity in all patient samples analyzed. The resulting isolated and viable cells are readily available for immunostaining, cytological analysis, and detection of gene mutations. To demonstrate the utility towards aiding companion diagnostics, we also show improved detection accuracy of KRAS gene mutations in lung cancer cells processed using the Centrifuge Chip, leading to an increase in the area under the curve (AUC) of the receiver operating characteristic from 0.90 to 0.99. The Centrifuge Chip allows for rapid concentration and processing of large volumes of bodily fluid samples for improved cytological diagnosis and purification of cells of interest for genetic testing, which will be helpful for enhancing diagnostic accuracy.     

Fluxes theory in experiments with random distributed channels on vesicles

When channels are randomly distributed in a population of vesicles, disregarding the number of channels per vesicle, these channels follow a Poisson distribution. This has been verified in many cases, determining the average of channels per vesicle. However, to determine kinetic parameters in population studies, a mathematical expression for the mean flux of solute through channels per vesicle is necessary. Hence, here, this mean flux is calculated, assuming Poisson distributed channels in a population of vesicle. Moreover, this result has been generalized to any number of different kinds of channels (i.e., channels with different permeabilities). These results, useful for in vitro experiments with mixed both channels and vesicles, can be supplemented with those from other techniques, in order to understanding how the nature of the lipid membrane affects kinetic parameters of channel.     

Valepotriates accumulation in callus, suspended cells and untransformed root cultures of Valeriana glechomifolia

Summary Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g⁻¹ DW, valtrate (VAL) 10.2mgg⁻¹ DW, and didrovaltrate (DID) 2.9mg g⁻¹ DW were observed in root cultures after 7–8wk of culture.     

On the Relation between the Open‐Circuit Voltage and Quasi‐Fermi Level Splitting in Efficient Perovskite Solar Cells

Today's perovskite solar cells (PSCs) are limited mainly by their open‐circuit voltage (V_OC) due to nonradiative recombination. Therefore, a comprehensive understanding of the relevant recombination pathways is needed. Here, intensity‐dependent measurements of the quasi‐Fermi level splitting (QFLS) and of the V_OC on the very same devices, including pin‐type PSCs with efficiencies above 20%, are performed. It is found that the QFLS in the perovskite lies significantly below its radiative limit for all intensities but also that the V_OC is generally lower than the QFLS, violating one main assumption of the Shockley‐Queisser theory. This has far‐reaching implications for the applicability of some well‐established techniques, which use the V_OC as a measure of the carrier densities in the absorber. By performing drift‐diffusion simulations, the intensity dependence of the QFLS, the QFLS‐V_OC offset and the ideality factor are consistently explained by trap‐assisted recombination and energetic misalignment at the interfaces. Additionally, it is found that the saturation of the V_OC at high intensities is caused by insufficient contact selectivity while heating effects are of minor importance. It is concluded that the analysis of the V_OC does not provide reliable conclusions of the recombination pathways and that the knowledge of the QFLS‐V_OC relation is of great importance.     

Roles of in vitro plantlet age and growing period in the phenolic constituent yields of acclimatized Hypericum polyanthemum

The influences of culture period on growth, plant survival rate and content of phenolic compounds were investigated in vitro and in acclimatized field-grown plants of Hypericum polyanthemum. The growth kinetics of micropropagated plantlets cultured in MS modified medium and the concomitant transference to ex vitro conditions showed that cultures achieved maximum biomass and yield of bioactive compounds after 12 weeks of in vitro growth, with field-grown plants displaying the same survival pattern. Differences in yield among plants cultured in vitro for 8 and 12 weeks that were acclimatized and followed over two years showed that the physiological age of the in vitro cultures influenced biomass production. However, benzopyrans and total phenolic compounds (TPC) contents did not vary significantly, with the exception of the 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) concentration in the reproductive parts, which was higher in the plants grown in vitro for 12 weeks over the two years of the study. All analyzed plant parts from the spring harvest accumulated lower benzopyran levels than plants harvested after 18 weeks of growth in both treatments, except for the levels of 6-isobutyryl-5,7-dimethoxy-2,2-dimethyl-benzopyran (HP1) and 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) in the new vegetative parts of the plants, which did not vary. The concentration of TPC, which was detected at low levels in the old vegetative parts in both treatments, was not altered in other plant parts. The information provided by this work will help structure plant growth and collection periods designed to optimize the yield of each required bioactive metabolite.     

Long-term exposure to high-altitude chronic hypoxia during gestation induces neonatal pulmonary hypertension at sea level

We determined whether postnatal pulmonary hypertension induced by 70% of pregnancy at high altitude (HA) persists once the offspring return to sea level and investigated pulmonary vascular mechanisms operating under these circumstances. Pregnant ewes were divided into two groups: conception, pregnancy, and delivery at low altitude (580 m, LLL) and conception at low altitude, pregnancy at HA (3,600 m) from 30% of gestation until delivery, and return to lowland (LHL). Pulmonary arterial pressure (PAP) was measured in vivo. Vascular reactivity and morphometry were assessed in small pulmonary arteries (SPA). Protein expression of vascular mediators was determined. LHL lambs had higher basal PAP and a greater increment in PAP after N(G)-nitro-L-arginine methyl ester (20.9 ± 1.1 vs. 13.7 ± 0.5 mmHg; 39.9 ± 5.0 vs. 18.3 ± 1.3 mmHg, respectively). SPA from LHL had a greater maximal contraction to K(+) (1.34 ± 0.05 vs. 1.16 ± 0.05 N/m), higher sensitivity to endothelin-1 and nitroprusside, and persistence of dilatation following blockade of soluble guanylate cyclase. The heart ratio of the right ventricle-to-left ventricle plus septum was higher in the LHL relative to LLL. The muscle area of SPA (29.3 ± 2.9 vs. 21.1 ± 1.7%) and the protein expression of endothelial nitric oxide synthase (1.7 ± 0.1 vs. 1.1 ± 0.2), phosphodiesterase (1.4 ± 0.1 vs. 0.7 ± 0.1), and Ca(2+)-activated K(+) channel (0.76 ± 0.16 vs. 0.30 ± 0.01) were greater in LHL compared with LLL lambs. In contrast, LHL had decreased heme oxygenase-1 expression (0.82 ± 0.26 vs. 2.22 ± 0.44) and carbon monoxide production (all P < 0.05). Postnatal pulmonary hypertension induced by 70% of pregnancy at HA promotes cardiopulmonary remodeling that persists at sea level.