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Neoadjuvant chemotherapy (NAC) can affect pathological complete response (pCR) in breast cancers; the resection that follows identifies patients with residual disease who are then offered second-line therapies. Circulating tumor cells (CTCs) and cancer-associated macrophage-like cells (CAMLs) in the blood can be used as potential biomarkers for predicting pCR before resection. CTCs are of epithelial origin that undergo epithelial-to-mesenchymal transition to become more motile and invasive, thereby leading to invasive mesenchymal cells that seed in distant organs, causing metastasis. Additionally, CAMLs in the blood of cancer patients are reported to either engulf or aid the transport of cancer cells to distant organs. To study these rare cancer-associated cells, we conducted a preliminary study where we collected blood from patients treated with NAC after obtaining their written and informed consent. Blood was collected before, during, and after NAC, and Labyrinth microfluidic technology was used to isolate CTCs and CAMLs. Demographic, tumor marker, and treatment response data were collected. Non-parametric tests were used to compare pCR and non-pCR groups. Univariate and multivariate models were used where CTCs and CAMLs were analyzed for predicting pCR. Sixty-three samples from 21 patients were analyzed. The median(IQR) pre-NAC total and mesenchymal CTC count/5 mL was lower in the pCR vs. non-pCR group [1(3.5) vs. 5(5.75); p = 0.096], [0 vs. 2.5(7.5); p = 0.084], respectively. The median(IQR) post-NAC CAML count/5 mL was higher in the pCR vs. non-pCR group [15(6) vs. 6(4.5); p = 0.004]. The pCR group was more likely to have >10 CAMLs post-NAC vs. non-pCR group [7(100%) vs. 3(21.4%); p = 0.001]. In a multivariate logistic regression model predicting pCR, CAML count was positively associated with the log-odds of pCR [OR = 1.49(1.01, 2.18); p = 0.041], while CTCs showed a negative trend [Odds Ratio (OR) = 0.44(0.18, 1.06); p = 0.068]. In conclusion, increased CAMLs in circulation after treatment combined with lowered CTCs was associated with pCR.

Significance: Circulating tumor cells (CTCs) are important biomarkers for cancer management. Isolated CTCs from blood are stained to detect and enumerate CTCs. However, the staining process is laborious and moreover makes CTCs unsuitable for drug testing and molecular characterization. Aim: The goal is to develop and test deep learning (DL) approaches to detect unstained breast cancer cells in bright-field microscopy images that contain white blood cells (WBCs). Approach: We tested two convolutional neural network (CNN) approaches. The first approach allows investigation of the prominent features extracted by CNN to discriminate in vitro cancer cells from WBCs. The second approach is based on faster region-based convolutional neural network (Faster R-CNN). Results: Both approaches detected cancer cells with higher than 95% sensitivity and 99% specificity with the Faster R-CNN being more efficient and suitable for deployment presenting an improvement of 4% in sensitivity. The distinctive feature that CNN uses for discrimination is cell size, however, in the absence of size difference, the CNN was found to be capable of learning other features. The Faster R-CNN was found to be robust with respect to intensity and contrast image transformations. Conclusions: CNN-based DL approaches could be potentially applied to detect patient-derived CTCs from images of blood samples.

Introduction Cell proliferation represents a major hallmark of cancer biology, and manifests itself in the assessment of tumor growth, drug resistance and metastasis. Tracking cell proliferation or cell fate at the single-cell level can reveal phenotypic heterogeneity. However, characterization of cell proliferation is typically done in bulk assays which does not inform on cells that can proliferate under given environmental perturbations. Thus, there is a need for single-cell approaches that allow longitudinal tracking of the fate of a large number of individual cells to reveal diverse phenotypes. Methods We fabricated a new microfluidic architecture for high efficiency capture of single tumor cells, with the capacity to monitor cell divisions across multiple daughter cells. This single-cell proliferation (SCP) device enabled the quantification of the fate of more than 1000 individual cancer cells longitudinally, allowing comprehensive profiling of the phenotypic heterogeneity that would be otherwise masked in standard cell proliferation assays. We characterized the efficiency of single cell capture and demonstrated the utility of the SCP device by exposing MCF-7 breast tumor cells to different doses of the chemotherapeutic agent doxorubicin. Results The single cell trapping efficiency of the SCP device was found to be ~ 85%. At the low doses of doxorubicin (0.01 µM, 0.001 µM, 0.0001 µM), we observed that 50–80% of the drug-treated cells had undergone proliferation, and less than 10% of the cells do not proliferate. Additionally, we demonstrated the potential of the SCP device in circulating tumor cell applications where minimizing target cell loss is critical. We showed selective capture of breast tumor cells from a binary mixture of cells (tumor cells and white blood cells) that was isolated from blood processing. We successfully characterized the proliferation statistics of these captured cells despite their extremely low counts in the original binary suspension. Conclusions The SCP device has significant potential for cancer research with the ability to quantify proliferation statistics of individual tumor cells, opening new avenues of investigation ranging from evaluating drug resistance of anti-cancer compounds to monitoring the replicative potential of patient-derived cells.

This study examined the role of a P2 receptor and arachidonic acid (AA) in regulatory volume decrease (RVD) by American alligator red blood cells (RBCs). Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca 2+ concentration were visualized using fluorescence microscopy. Gadolinium (50 μM), hexokinase (2.5 U/ml), and suramin (100 μM) increased osmotic fragility, blocked volume recovery after hypotonic shock, and prevented a rise in intracellular Ca 2+ that normally occurs during cell swelling. The P2X antagonists PPADS (50 μM) and TNP-ATP (10 μM) also increased fragility and inhibited volume recovery. In contrast, ATPγS (10 μM), α,β-methylene-ATP (50 μM) and Bz-ATP (50 μM) had the opposite effect, whereas 2-methylthio-ATP (50 μM) and UTP (10 μM) had no effect. In addition, the phospholipase A 2 (PLA 2 ) inhibitors ONO-RS-082 (10 μM), chlorpromazine (10 μM), and isotetrandrine (10 μM) increased osmotic fragility and blocked volume recovery, whereas AA (10 μM) and its nonhydrolyzable analog eicosatetraynoic acid (ETYA, 10 μM) had the reverse effect. Further, AA (10 μM), but not ATPγS (10 μM), prevented the inhibitory effect of a low Ca 2+ -EGTA Ringer on RVD, whereas both AA (10 μM) and ATPγS (10 μM) caused cell shrinkage under isosmotic conditions. In conclusion, our results are consistent with the presence of a P2-like receptor whose activation stimulated RVD. In addition, AA also was important for volume recovery.

Approach: We tested two convolutional neural network (CNN) approaches. The first approach allows investigation of the prominent features extracted by CNN to discriminate in vitro cancer cells from WBCs. The second approach is based on faster region-based convolutional neural network (Faster R-CNN).

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca 2+ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na + was replaced with K + , or when cells were exposed to the K + channel inhibitor quinine, indicating a requirement of K + efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca 2+ , which resulted from Ca 2+ influx because it was not observed when extracellular Ca 2+ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)- N , N , N ′, N ′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane- N , N , N ′, N ′-tetraacetic acid tetrakis (acetoxymethyl) ester) or exposed to a low Ca 2+ -EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca 2+ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca 2+ . Finally, and surprisingly, the Ca 2+ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K + permeable pathway via a Ca 2+ -dependent mechanism, and this process mediated K + loss during RVD.

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca^sup 2+^ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na^sup +^ was replaced with K^sup +^, or when cells were exposed to the K^sup +^ channel inhibitor quinine, indicating a requirement of K^sup +^ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca^sup 2+^, which resulted from Ca^sup 2+^ influx because it was not observed when extracellular Ca^sup 2+^ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca^sup 2+^-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca^sup 2+^ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca^sup 2+^. Finally, and surprisingly, the Ca^sup 2+^ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K^sup +^ permeable pathway via a Ca^sup 2+^-dependent mechanism, and this process mediated K^sup +^ loss during RVD.[PUBLICATION ABSTRACT]

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca super(2+) concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na super(+) was replaced with K super(+), or when cells were exposed to the K super(+) channel inhibitor quinine, indicating a requirement of K super(+) efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5 Ringer) caused an increase in cytosolic Ca super(2+), which resulted from Ca super(2+) influx because it was not observed when extracellular Ca super(2+) was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N',N'-tetraa cetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca super(2+)-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca super(2+) was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca super(2+). Finally, and surprisingly, the Ca super(2+) ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K super(+) permeable pathway via a Ca super(2+)-dependent mechanism, and this process mediated K super(+) loss during RVD.

AstroSat is India's first dedicated multi-wavelength space observatory launched by the Indian Space Research Organisation (ISRO) on 28 September 2015. After launch, the AstroSat Science Support Cell (ASSC) was set up as a joint venture of ISRO and the Inter-University Centre for Astronomy and Astrophysics (IUCAA) with the primary purpose of facilitating the use of AstroSat, both for making observing proposals and for utilising archival data. The ASSC organises meetings, workshops and webinars to train users in these activities, runs a help desk to address user queries, provides utility tools and disseminates analysis software through a consolidated web portal. It also maintains the AstroSat Proposal Processing System (APPS) which is deployed at ISSDC, a software platform central to the workflow management of AstroSat operations. This paper illustrates the various aspects of ASSC functionality.