The detection of rare circulating tumor cells (CTCs) in the blood of cancer patients has the potential to be a powerful and noninvasive method for examining metastasis, evaluating prognosis, assessing tumor sensitivity to drugs, and monitoring therapeutic outcomes. In this study, we have developed an efficient strategy to isolate CTCs from the blood of breast cancer patients using a microfluidic immune-affinity approach. Additionally, to gain further access to these rare cells for downstream characterization, our strategy allows for easy detachment of the captured CTCs from the substrate without compromising cell viability or the ability to employ next generation RNA sequencing for the identification of specific breast cancer genes. To achieve this, a chemical ligand-exchange reaction was engineered to release cells attached to a gold nanoparticle coating bound to the surface of a herringbone microfluidic chip (NP-HBCTC-Chip). Compared to the use of the unmodified HBCTC-Chip, our approach provides several advantages, including enhanced capture efficiency and recovery of isolated CTCs.

Myocyte enhancer factor-2 (MEF2) transcription factors regulate the expression of a variety of genes encoding contractile proteins and other proteins associated with muscle performance. We proposed that changes in MEF2 levels and expression of selected downstream targets would aid the skeletal muscle of thirteen-lined ground squirrels (Spermophilus tridecemlineatus) in meeting metabolic challenges associated with winter hibernation; e.g., cycles of torpor-arousal, body temperature that can fall to near 0°C, long periods of inactivity that could lead to atrophy. MEF2A protein levels were significantly elevated when animals were in torpor (maximally 2.8-fold higher than in active squirrels) and the amount of phosphorylated active MEF2A Thr312 increased during entrance into torpor. MEF2C levels also rose significantly during entrance and torpor as did the amount of phosphorylated MEF2C Ser387. Furthermore, both MEF2 members showed elevated amounts in the nuclear fraction during torpor as well as enhanced binding to DNA indicating that MEF2-mediated gene expression was up-regulated in torpid animals. Indeed, the protein products of two MEF2 downstream gene targets increased in muscle during torpor (glucose transporter isoforms 4; GLUT4) or early arousal (myogenic differentiation; MyoD). Significant increases in Glut4 and MyoD mRNA transcript levels correlated with the rise in protein product levels and provided further support for the activation of MEF2-mediated gene expression in the hibernator. Transcript levels of Mef2a and Mef2c also showed time-dependent patterns with levels of both being highest during arousal from torpor. The data suggest a significant role for MEF2-mediated gene transcription in the selective adjustment of muscle protein complement over the course of torpor-arousal cycles.

The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation 1,2. Standard hypothermic preservation at +4°C 1,3 limits liver preservation to less than 12 hours. Our group previously showed that supercooled ice free storage at −6°C can extend viable preservation of rat livers 4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favourable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at −4°C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 hours. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood. The absence of technology to preserve organs for more than a few hours is one of the fundamental causes of the donor organ shortage crisis 1-3. Subzero preservation has the potential to extend the organ storage limits 1-5 , as the metabolic rate halves for every 10°C Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

SUMMARYHibernation is a crucial strategy of winter survival used by many mammals. During hibernation, thirteen-lined ground squirrels, Ictidomys tridecemlineatus, cycle through a series of torpor bouts, each lasting more than a week, during which the animals are largely immobile. Previous hibernation studies have demonstrated that such natural models of skeletal muscle disuse cause limited or no change in either skeletal muscle size or contractile performance. However, work loop analysis of skeletal muscle, which provides a realistic assessment of in vivo power output, has not previously been undertaken in mammals that undergo prolonged torpor during hibernation. In the present study, our aim was to assess the effects of 3months of hibernation on contractile performance (using the work loop technique) and several biochemical properties that may affect performance. There was no significant difference in soleus muscle power output-cycle frequency curves between winter (torpid) and summer (active) animals. Total antioxidant capacity of gastrocnemius muscle was 156% higher in torpid than in summer animals, suggesting one potential mechanism for maintenance of acute muscle performance. Soleus muscle fatigue resistance was significantly lower in torpid than in summer animals. Gastrocnemius muscle glycogen content was unchanged. However, state 3 and state 4 mitochondrial respiration rates were significantly suppressed, by 59% and 44%, respectively, in mixed hindlimb skeletal muscle from torpid animals compared with summer controls. These findings in hindlimb skeletal muscles suggest that, although maximal contractile power output is maintained in torpor, there is both suppression of ATP production capacity and reduced fatigue resistance.

In the context of normal cell turnover, apoptosis is a natural phenomenon involved in making essential life and death decisions. Apoptotic pathways balance signals which promote cell death (pro-apoptotic pathways) or counteract these signals (anti-apoptotic pathways). We proposed that changes in anti-apoptotic proteins would occur during mammalian hibernation to aid cell preservation during prolonged torpor under cellular conditions that are highly injurious to most mammals (e.g. low body temperatures, ischemia). Immunoblotting was used to analyze the expression of proteins associated with pro-survival in six tissues of thirteen-lined ground squirrels, Ictidomys tridecemlineatus. The brain showed a concerted response to torpor with significant increases in the levels of all anti-apoptotic targets analyzed (Bcl-2, Bcl-xL, BI-1, Mcl-1, cIAP1/2, xIAP) as well as enhanced phosphorylation of Bcl-2 at S70 and T56. Heart responded similarly with most anti-apoptotic proteins elevated significantly during torpor except for Bcl-xL and xIAP that decreased and Mcl-1 that was unaltered. In liver, BI-1 increased whereas cIAP1/2 decreased. In kidney, there was an increase in BI-1, cIAP and xIAP but decreases in Bcl-xL and p-Bcl-2(T56) content. In brown adipose tissue, protein levels of BI-1, cIAP1/2, and xIAP decreased significantly during torpor (compared with euthermia) whereas Bcl-2, Bcl-xL, Mcl-1 were unaltered; however, Bcl-2 showed enhanced phosphorylation at Thr56 but not at Ser70. In skeletal muscle, only xIAP levels changed significantly during torpor (an increase). The data show that anti-apoptotic pathways have organ-specific responses in hibernators with a prominent potential role in heart and brain where coordinated enhancement of anti-apoptotic proteins occurred in response to torpor.

To avoid the harsh conditions of winter climates, hibernating mammals undergo a systematic depression of physiological function by reducing their metabolic rate. During this process, hibernators are exposed to significant stresses (e.g., low body temperature, ischemia-reperfusion) that must be dealt with appropriately to avoid irreversible tissue damage. Consequently, we investigated the contribution of stress-responsive antioxidant enzymes, heat shock proteins, signal transduction pathways (e.g., mitogen-activated protein kinases, MAPK), and transcription factors for their role in conferring tolerance to stress in the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus). Using a combination of multiplex protein panels and traditional immunoblotting procedures, we have focused on these stress factors in brown adipose tissue (BAT) and white adipose tissue (WAT) over cycles of torpor-arousal since they provide the means for heat production as a result of non-shivering thermogenesis and the mobilization of critical energy reserves, respectively. We show the differential and tissue-specific regulation of stress factors including a unified upregulation of the antioxidant enzyme Thioredoxin 1 in both tissues, an upregulation of superoxide dismutase (SOD1 and SOD2) in WAT, and an increase in heat shock proteins during the transitory periods of the torpor-arousal cycle (HSP90α in BAT and HSP60 in WAT). Additionally, an upregulation of the active form of ERK1/2 and p38 in BAT and select transcription factors (e.g., CREB-1 and ELK-1) in both tissues were identified. These data provide us with greater insight into the molecular mechanisms responsible for this animal's natural stress tolerance and outline molecular signatures which define stress resistance.

Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the ice-nucleation characteristics of picoliter-sized drops consisting of different D2O and H2O mixtures with and without the ice-nucleating bacteria Pseudomonas syringae. We also studied the effects of commonly used cryoprotectants such as ethylene glycol, propylene glycol, and trehalose on the nucleation characteristics of D2O and H2O mixtures. The results show that the median freezing temperature of the suspension containing 1 mg/mL of a lyophilized preparation of P. syringae is as high as −4.6 °C for 100% D2O, compared to −8.9 °C for 100% H2O. As the D2O concentration increases every 25% (v/v), the profile of the ice-nucleation kinetics of D2O + H2O mixtures containing 1 mg/mL Snomax shifts by about 1 °C, suggesting an ideal mixing behavior of D2O and H2O. Furthermore, all of the cryoprotectants investigated in this study are found to depress the freezing phenomenon. Both the homogeneous and heterogeneous freezing temperatures of these aqueous solutions depend on the water activity and are independent of the nature of the solute. These findings enrich our fundamental knowledge of D2O-related ice nucleation and suggest that the combination of D2O and ice-nucleating agents could be a potential self-ice-nucleating formulation. The implications of self-nucleation include a higher, precisely controlled ice seeding temperature for slow freezing that would significantly improve the viability of many ice-assisted cryopreservation protocols.

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.