Gentle Rocking Research Articles

Sleep induced by mechanosensory stimulation provides cognitive and health benefits in Drosophila.

Sleep is a complex phenomenon regulated by various factors, including sensory input. Anecdotal observations have suggested that gentle rocking helps babies fall asleep, and experimental studies have verified that rocking promotes sleep in both humans and mice. Recent studies have expanded this understanding, demonstrating that gentle vibration also induces sleep in Drosophila. Natural sleep serves multiple functions, including learning and memory, synaptic downscaling, and clearance of harmful substances associated with neurodegenerative diseases. Here, we investigated whether vibration-induced sleep provides similar cognitive and health benefits in Drosophila. We administered gentle vibration to flies that slept very little due to a forced activation of wake-promoting neurons and investigated how the vibration influenced learning and memory in the courtship conditioning paradigm. Additionally, we examined the effects of VIS on synaptic downscaling by counting synapse numbers of select neurons. Finally, we determined whether vibration could induce sleep in Drosophila models of Alzheimer's disease (AD) and promote the clearance of Amyloid b (Ab) and Tubulin Associated Unit (TAU). Vibration-induced sleep enhanced performance in a courtship conditioning paradigm and reduced the number of synapses in select neurons. Moreover, vibration improved sleep in Drosophila models of AD, promoting the clearance of Ab and TAU. Mechanosensory stimulation offers a promising non-invasive avenue for enhancing sleep, potentially providing associated cognitive and health benefits.

Simple 3D spheroid cell culture model for studies of prion infection.

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

Abstract PO1-26-04: Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients

Abstract Patient-derived organotypic tissue cultures (PD-OTC) are unique models for probing how individual patient’s tumor microenvironment (TME) influences cancer development and treatment responses. They retain the patient tumor architecture and TME, that are difficult to recapitulate in other models, while affording flexible treatment options and comparison of cancer (CA) versus matched non-cancer (NC) tissue responses to treatments. We have developed a culturing method for establishing PD-OTC of breast cancer patients that exhibit CA tissue growth and extensive metabolic reprogramming between CA versus matched NC tissues while enabling patient-derived organoids (PDO) and fibroblasts to be isolated. Pairs of CA and NC tissues freshly resected from six patients bearing early-stage estrogen receptor/progesterone receptor positive ductal carcinoma were sliced at 750 µm, embedded in Matrigel, and cultured in a 6-well plate with a Biopore membrane insert and custom medium at 37°C/5% CO2 with gentle rocking (16-40 mg wet weight; n=3). Excess tissue slices were cryopreserved and kept at -196 °C. Culture media were sampled periodically for metabolite analysis by NMR. Microscopic examination revealed significant tissue, organoid-like, and fibroblast outgrowth after 1-1.5 month of culturing. Two to three days prior to the harvest of two patients’ (CZ16 and 17) OTC, the culture medium was replaced with [U-2H]-glucose + [U-13C,15N]-glutamine-containing medium to allow the two stable isotope tracers to be metabolized, followed by Stable Isotope-Resolved Metabolomic (SIRM) analysis by NMR and ion chromatography coupled with ultra high-resolution Fourier transform mass spectrometry (IC-UHRFTMS). Two separate pieces of the OTC were cut and each was assayed for glucose uptake and mitochondrial membrane potential by live fluorescence spectroscopy via a portable custom-built fluorescence microscope, and by histochemical analysis. Histological analysis showed that both CA and NC OTC of all patients maintained their structural integrity. SIRM analysis of two patients’ OTC revealed active catabolic and anabolic metabolism including glucose uptake/glycolysis, the Krebs cycle/mitochondrial membrane potential, the pentose phosphate pathway (PPP), gluconeogenesis, glycogen synthesis, and purine/pyrimidine/sugar nucleotides synthesis. The CA OTC of both patients showed enhanced glucose uptake, glycolysis, mitochondrial membrane potential/Krebs cycle, PPP, glycogen synthesis, and purine nucleotide synthesis, when compared with the matched NC OTC. Treatment-naïve CA OTC of CZ16 showed much more significant metabolic reprogramming (NAD+ metabolism, in particular) than CA OTC of CZ17 who has undergone neoadjuvant treatment, chemotherapy, and hormone therapy prior to surgery. Distinct metabolic features of breast CA tissues can be exploited as therapeutic targets. Organoids and fibroblasts were isolated from CA and/or NC OTC cultures and their characterization is in progress. When revived, cryopreserved OTC exhibited active metabolism and tissue/organoid/fibroblast outgrowth similarly to fresh OTC. In conclusion, our culturing method enables PD-OTC models to be established efficiently for early-stage breast cancer patients for downstream functional studies including live cell imaging and SIRM analysis. Organoids and fibroblasts can also be derived from < 20 mg of tissues for further studies. Future direction includes therapeutic studies on these patient-derived models to better understand and predict individual patient’s responses to therapy. Citation Format: Teresa Fan, Carlos Goncalves, Jing Yan, Jahid Islam, Penghui Lin, Mohamed Kaddah, Richard Higashi, Andrew Lane, Caigang Zhu. Patient-derived organotypic tissue cultures for organoid isolation and probing unique metabolic features of individual breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-26-04.

Non-Pharmacological Intervention for Personalizing Sleep Quality through Gentle Rocking Motion.

Achieving restorative sleep is crucial for overall well-being, yet sleep difficulties affect a substantial portion of the adult population. Sleep disturbances are associated with diminished quality of life, physical complaints, cognitive impairment, and emotional regulation challenges. This study explores the influence of an innovative experimental bed designed to generate rocking motions on sleep parameters. A prospective observational study enrolled 60 adult participants, assessing their sleep on a regular stationary bed and the Inoveris bed, providing gentle rocking movements. Polysomnography was conducted, recording electroencephalography, electrooculogram, electromyogram, respiratory effort, and other parameters. The rocking bed significantly increased total sleep time (TST) and reduced N1 sleep stage duration (p < 0.001). Participants also experienced a quicker transition to the N2 sleep stage (p = 0.01), indicative of a faster shift from wakefulness to deeper sleep. Additionally, rocking led to a higher percentage of N1 sleep stages (p = 0.01) and a significant increase in N3 sleep stage duration (p = 0.004). While some results lacked statistical significance, notable trends in the rocking bed group have clinical relevance, consistently improving sleep parameters, including increased TST. The rocking bed also showed a trend towards higher sleep efficiency (SE) and sleep duration percentage, hinting at a potential overall enhancement in sleep quality. This study contributes valuable insights into the potential benefits of rocking motions on sleep architecture. Despite variations in outcomes across studies, our results underscore the potential of rocking beds as a non-pharmacological intervention for enhancing sleep quality. Notable improvements in total sleep time (TST), N1 sleep stage reduction, and accelerated transitions to deeper sleep stages highlight the clinical relevance of rocking interventions. Further research, collaboration, and addressing the identified limitations will advance our understanding of the therapeutic applications of rocking motions in sleep science.

Carbon monoxide: a critical physiological regulator sensitive to light

The mechanism by which humans absorb therapeutic light in winter seasonal and nonseasonal depression is unknown. Bright-light-induced release and generation of blood-borne gasotransmitters such as carbon monoxide (CO) may be one mechanism. Here, 24 healthy female volunteers had peripheral blood samples drawn. Samples were collected in a dimly lit room and protected from light exposure. Samples were analyzed for CO concentrations by gas chromatography after 2 h of continuous exposure to darkness vs. bright white light. In a similar confirmatory study, 11 additional volunteers had samples analyzed for CO concentrations after 2 h of continuous exposure to gentle rocking in darkness vs. in bright white light. In the first study, light-unexposed peripheral blood had a mean CO concentration of 1.8 ± 0.4 SD ppm/g. Identically treated samples with 2 h of rocking and exposure to bright white light at illuminance 10,000 lux had a mean CO of 3.6 ± 1.2 ppm/g (p < 0.0001). Post hoc analysis of that study showed that time of day was significantly inversely associated with increase in CO concentration under bright light vs. dark (p < 0.04). In a smaller confirmatory study of 11 healthy female volunteers, after 2 h of rocking, light-unexposed peripheral blood had a mean CO of 1.4 ± 0.5 SD ppm/g. Identically treated blood samples with 2 h of exposure to bright white light at illuminance 10,000 lux had a mean CO of 2.8 ± 1.7 ppm/g (p < 0.02). In conclusion, bright-light exposure robustly increases human blood CO in vitro. This supports the putative role of CO as a physiological regulator of circadian rhythms and light’s antidepressant effects. This human evidence replicates earlier data from a preclinical in vivo model. This effect may be stronger in the morning than in the afternoon.

Gentle rocking movements during sleep in the elderly.

Vestibular stimulation in the form of rocking movements could be a promising non‐pharmacological intervention for populations with reduced sleep quality, such as the elderly. We hypothesized that rocking movements influence sleep by promoting comfort. We assessed whether gentle rocking movements can facilitate the transition from wake to sleep, increase sleep spindle density and promote deep sleep in elderly people. We assessed self‐reported comfort using a pilot protocol including translational movements and movements along a pendulum trajectory with peak linear accelerations between 0.10 and 0.20 m/s2. We provided whole‐night stimulation using the settings rated most comfortable during the pilot study (movements along a pendulum trajectory with peak linear acceleration of 0.15 m/s2). Sleep measures (polysomnography) of two baseline and two movement nights were compared. In our sample (n = 19; eight female; mean age: 66.7 years, standard deviation: 3 years), vestibular stimulation using preferred stimulation settings did not improve sleep. A reduction of delta power was observed, suggesting reduced sleep depth during rocking movements. Sleep fragmentation was similar in both conditions. We did not observe a sleep‐promoting effect using settings optimized to be comfortable. This finding could imply that comfort is not the underlying mechanism. At frequencies below 0.3 Hz, the otoliths cannot distinguish tilt from translation. Translational movement trajectories, such as used in previous studies reporting positive effects of rocking, could have caused sensory confusion due to a mismatch between vestibular and other sensory information. We propose that this sensory confusion might be essential to the sleep‐promoting effect of rocking movements described in other studies.

Effect of Rocking Movements on Afternoon Sleep.

Study ObjectivesGentle rocking movements provided by a moving bed have been proposed as a promising non-pharmacological way to promote sleep. In rodents the sleep promoting effect of rocking movements depended on the peak acceleration (named “stimulation intensity”) perceived by the vestibular system. We set out to verify previous reports on the sleep promoting effect of rocking movements and to investigate the importance of stimulation intensity in this process.MethodsSide-to-side rocking movements along a pendulum trajectory with different peak accelerations (control: 0 m/s2, low intensity: 0.15 m/s2, medium intensity: 0.25 m/s2, high intensity: 0.35 m/s2) were provided for 45 min during an afternoon nap opportunity. Participants were assigned to a low intensity group (n = 10) experiencing control, low and medium intensity stimulation or a high intensity group (n = 12) experiencing control, medium and high intensity stimulation. Sleep and sleep-related memory performance were assessed using polysomnography and a word-pair memory task, respectively.ResultsParticipants transitioned faster into deep sleep under the influence of medium intensity rocking as was evident by a faster buildup of delta power compared to the control condition (n = 22). The faster buildup did not affect sleep architecture, since e.g., the proportion of the nap spent in deep sleep or latencies did not change. Previously reported effects like a shorter latency to stage N2 and a higher density of sleep spindles were not observed. Sleep quality during control naps of the low intensity group was worse than in the high intensity group. In the low intensity group, we also observed a significant increase in delta power throughout the nap, as well as a higher density of slow oscillations both under the influence of low and medium intensity vestibular stimulation. No such effects were observed in the high intensity group.ConclusionRocking movements may promote nap sleep in young adults. Due to a difference in sleep quality during control naps between the low and high intensity group no conclusion regarding the influence of stimulation intensity were possible. Thus, optimal stimulation settings in humans need further investigation.

Morphological changes of sintered polydopamine coatings

Polydopamine (PDA), produced from the self-polymerization of dopamine under alkaline conditions, is well known for its natural adhesive properties on a broad variety of surfaces. However, there is still ongoing controversy as to its growth, coverage and morphology characteristics. Here, we investigate the growth of PDA on a silicon substrate over various time periods (0–24 h) using a gentle rocking method. The samples were then post-heated at 150 °C under vacuum. Both the as-prepared and post-treated samples were examined with a combination of x-ray photoelectron, UV photoelectron, and neutral impact collision ion scattering spectroscopies, as well as atomic force microscopy. Results indicated that the PDA layer growth plateaus after 5 h, proceeding via island-growth, with incomplete coverage after 24 h. Heating PDA substrates resulted in sintering of the PDA islands to form a more homogenous surface coverage, indicating this is critical for the formation of smooth and homogenous coatings.

Sleep: Rock and Swing versus Toss and Turn

It has been known for centuries that gentle rocking promotes sleep. Two new studies now shed light on the underlying mechanism in both humans and mice.

Whole-Night Continuous Rocking Entrains Spontaneous Neural Oscillations with Benefits for Sleep and Memory

Sensory processing continues during sleep and can influence brain oscillations. We previously showed that a gentle rocking stimulation (0.25Hz), during an afternoon nap, facilitates wake-sleep transition and boosts endogenous brain oscillations (i.e., EEG spindles and slow oscillations [SOs]). Here, we tested the hypothesis that the rhythmic rocking stimulation synchronizes sleep oscillations, a neurophysiological mechanism referred to as "neural entrainment." We analyzed EEG brain responses related to the stimulation recorded from 18 participants while they had a full night of sleep on a rocking bed. Moreover, because sleep oscillations are considered of critical relevance for memory processes, we also investigated whether rocking influences overnight declarative memory consolidation. We first show that, compared to a stationary night, continuous rocking shortened the latency to non-REM (NREM) sleep and strengthened sleep maintenance, as indexed by increased NREM stage 3 (N3) duration and fewer arousals. These beneficial effects were paralleled by an increase in SOs and in slow and fast spindles during N3, without affecting the physiological SO-spindle phase coupling. We then confirm that, during the rocking night, overnight memory consolidation was enhanced and also correlated with the increase in fast spindles, whose co-occurrence with the SO up-state is considered to foster cortical synaptic plasticity. Finally, supporting the hypothesis that a rhythmic stimulation entrains sleep oscillations, we report a temporal clustering of spindles and SOs relative to the rocking cycle. Altogether, these findings demonstrate that a continuous rocking stimulation strengthens deep sleep via the neural entrainment of intrinsic sleep oscillations.

The nature of the biological material and the irreproducibility problem in biomedical research.

Biomedical research has a reproducibility problem since various crucial landmark papers could not be independently reproduced. While there are many causes related to statistical analysis, methodology or insufficient reporting of experimental details, this commentary argues that the complexity of biological material itself is, until now, a largely ignored source of irreproducibility. By discussing examples from evolutionary biology, intrinsically disordered proteins and current biomedical research, it contends that some results are irreproducible because we do not have the knowledge, the tools or the analytical ability to understand biological complexity and how it can give rise to different results. Instead of casting irreproducible research out as bad or sloppy science, they should serve as an inspiration for pioneering research not just to develop such tools but also to attempt to explore what lies beneath our current inability to deal with complexity.

Don't just dump your data and run: Authors should submit as much experimental information as possible when uploading sequence data.

If you have, in any way, been involved with genetic research over the past 10 years, then you have likely heard of the Sequence Read Archive (SRA), which is jointly housed at the National Center for Biotechnology (NCBI), the DNA Data Bank of Japan (DDBJ), and the European Bioinformatics Institute (EBI). And if you regularly work with genome or transcriptome sequence information, then you have probably extracted data from and/or deposited data into the SRA. For those who are unfamiliar with it, the SRA is an international public online archive for next‐generation sequencing (NGS) data, which was established about a decade ago under the guidance of the International Nucleotide Sequence Database Collaboration (INSDC) [1], [2]. Despite nearly being shut down in 2011 [3], it has grown at a staggering rate over the past 10 years. As of September 1, 2017, it housed over five quadrillion (1015) open‐access bases of NGS data, coming from thousands of different species and spanning the entire gamut of cellular and viral life. It contains DNA‐ and RNA‐sequencing (DNA‐seq and RNA‐seq) reads of every kind, from bisulfite‐seq to strand‐specific RNA‐seq to single‐cell DNA‐seq, and it accepts reads from every type of NGS platform, be it Illumina, Ion Torrent, or PacBio sequencing. In other words, the SRA is a crucial and central resource in the fast‐paced and increasingly important domain of contemporary genetic research. > … the SRA is a crucial and central resource in the fast‐paced and increasingly important domain of contemporary genetic research. ### The Sequence Read Archive The SRA can be easily accessed and searched via the NCBI (https://www.ncbi.nlm.nih.gov/sra), DDBJ (http://trace.ddbj.nig.ac.jp/dra/index_e.html), and EBI (http://www.ebi.ac.uk/ena/submit/read-submission) websites. Once there, you will find yourself at a sequencing‐read superstore. With a decent Wi‐Fi connection, a couple of keyword searches, and a few clicks of the trackpad, you can quickly …

Abstract 4271: Beta-glucan activates macrophages in human NSCLC demonstrated by Stable Isotope Resolved Metabolomics (SIRM)

Abstract Introduction: We use SIRM to evaluate metabolic reprogramming of lung cancer cells in monoculture, in mouse xenograft/explant models, and in NSCLC patients in situ (1,2). We have now extended the range of models to fresh human tissue slices, which retain the original tissue architecture and heterogeneity with a paired benign versus cancer tissue design under defined cell culture conditions. β-glucan is a polysaccharide that repolarizes tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype in mice (3). Here we report the activation of TAMs in human NSCLC tissue slices. Experimental: Freshly resected paired tissue slices from individual patients (approx. 1 mm or less thick and 5-40 mg wet weight) were incubated ± particulate β-glucan in standard cell culture conditions, with gentle rocking to enable efficient gas, nutrient, and waste product exchange. Tissue slices could be maintained metabolically viable for at least 48 h of incubation. The metabolic activity was determined by measuring the uptake and transformation of 13C and/or 15N-enriched common nutrient tracers such as glucose and glutamine, using high resolution mass spectrometry, GC-MS, and NMR after a period of incubation (2). Findings: Time-course analysis of the slices by NMR, MS, and histology revealed that NSCLC tissue slices, both benign and tumorous, retained their architecture and a broad spectrum of metabolic activities. Glucose and glutamine metabolism was reprogrammed in the tumor relative to the paired benign tissues ex vivo, as the in vivo case. The paired tissues from different patients showed significantly different metabolic responses to β-glucan, as expected Conclusions: This platform offers a human tissue model for preclinical studies on metabolic reprogramming of human cancer and stromal cells in their tissue context, and response to drug treatment (4). As the microenvironment of the target human tissue is maintained, including the resident immune cells, individualized response to immune-active agents can be determined in a clinically relevant setting. Supported by NCI P01CA163223-01 and NIEHS 1R01ES022191-01 1. Lane, A.N., Fan, T.W.-M., Bousamra II, M., et al. (2011) Clinical Applications of Stable Isotope-Resolved Metabolomics (SIRM) in Non-Small Cell Lung Cancer. Omics, 15, 173-182. 2. Sellers, K., Fox, M.P., Bousamra, M., II, et al. (2015) Pyruvate carboxylase is critical for non-small-cell lung cancer proliferation. Journal of Clinical Investigation, 125, 687-698. 3. Liu, M., Luo, F., Ding, C., et al. (2015) Particulate β-Glucan Converts Immunosuppressive Macrophages into M1 Phenotype Through Dectin-1-induced Syk-Card9-Erk Pathway and Raf-1-c-Maf Pathway. J. Immunol., 195, 5055-5065. 4. Xie, H., Hanai, J., Ren, et al. (2014) Targeting lactate dehydrogenase-A (LDH-A) inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor initiating cells. Cell Metabolism 19, 795-809 Citation Format: Teresa W-M Fan, Andrew N. Lane, Jun Yan, Richard M. Higashi, Jeremiah T. Martin, Michael Bousamra. Beta-glucan activates macrophages in human NSCLC demonstrated by Stable Isotope Resolved Metabolomics (SIRM). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4271.

Use of calcium phosphate- silver nanoparticles in chitosan coatings on titanium and for drug delivery

Event Abstract Back to Event Use of calcium phosphate- silver nanoparticles in chitosan coatings on titanium and for drug delivery Gillen Gonzales1, Joel D. Bumgardner1, Jegdish P. Babu2, Jessica A. Jennings1, Warren O. Haggard1, Hitesh Adhikari3, Diego V. Pulgarin1, Timothy B. Martin1, Vishnu P. Murali1 and Sanjay R. Mishra3 1 University of Memphis, Biomedical Engineering, United States 2 UTHCS, School of Dentistry, United States 3 University of Memphis, Department of Physics, United States Introduction: Calcium-phosphate (CaP) nanoparticles decorated with silver (Ag) have shown potential to kill infectious bacteria in vitro.[1] [2]A coating that localizes the antibacterial CaP- Ag nanoparticles using osteoconductive chitosan may provide a means to inhibit bacterial biofilm formation and subsequent infectious complications for these implants. This work evaluated antimicrobial, cytocompatibility, and protein delivery properties of CaP- Ag in chitosan coatings. Materials and Methods: Porous CaP nanospheres with 0, 15, 37, or 50% Ag were fabricated using a hydrothermal synthesis of CaP followed by microwave heating of CaP-AgNO3 for Ag decoration on CaP. [1] Chitosan (82 DDA) coatings with 30wt% CaP-Ag particles were made via silane-solution casting on to cpTi and sterilized using ethylene oxide gas. Coated and uncoated cpTi coupons (n=3/group) were incubated with 3ml of bacteria (5X106 colony forming units) under anaerobic conditions for common dental pathogens (P. gingivalis, ATC BAA-388; P. denticola, ATCC 35308) and aerobic conditions for orthopedic pathogens (P. aeruginosa: ATCC 15442; S. aureus, ATCC 25923, and S. epidermidis, ATCC 700576). Percent viability of bacteria was determined using MTT Bacterial Viability Assay (Roche Lab) using culture plastic controls. Coated and uncoated cpTi coupons were incubated with 2X104 NIH 3T3 cells for 24 hrs and viability determined using CellTitre-Glo Assay (Promega). For protein release, 20mg of CaP-Ag particles (n=4/group) were incubated in 3mL of 1mg/mL α-chymotrypsin (MW = 25kDa, pI ~ 9.1), used as an analogue to BMP-2 (MW = 26kDa, pI ~8.2), for 24 hrs at 37C under gentle rocking. After 24hrs, supernatant was collected for determining protein loading by subtraction. Samples were incubated in 3mL PBS at 37C and eluates were collected with complete PBS every 2 days for 3 weeks. Protein concentration was determined using Quant-iT Protein Kit (Mol. Probe). Size and Zeta potential of particles was determined using Delsa™ Nano Submicron Particle Size and Zeta Potential Particle Analyzer (Beckmann Coulter). Result:Uncoated cpTi, plain chitosan and chitosan coatings with CaP-0%Ag particles had minimal effect on viability of bacteria (Figure 1). There was a significant and dose-related decrease in viability of all bacteria with increasing Ag content on CaP nanoparticles with the CaP-50%Ag particles having the largest antibacterial properties (p<0.05). There were some differences in the sensitivity of bacteria to the coatings with S. epidermidis showing the highest sensitivity. Figure1: Viability of bacteria on chitosan with CaP- Ag coatings The total loading and percent release of protein was not different for the different CaP-Ag particles (Figure 2). This may be due to the similarly large negative Zeta potential of the particles (Table). Protein release pattern was also similar for the different CaP-Ag particles showing an initial burst release followed by a sustained release of approximately10 ng/µl from day 3 to 21. Figure2: Protein release from CaP- Ag particles in PBS Discussion: There was no difference in the viability of the fibroblasts on the uncoated cpTi, plain chitosan and chitosan coatings with CaP-0%Ag particles. However, cell viability was reduced 100-fold on all chitosan coatings incorporating CaP-Ag particles regardless of the %Ag in the particles (data not shown). Part of reduced viability of the fibroblasts may be due to early release of Ag from particles due to the acetic acid used in the coating process. Previous studies showed that CaP-Ag particles alone had little effect on cell viability.[1] Conclusion: Increasing concentration of Ag in CaP particles in chitosan coatings exhibited a dose dependent antibacterial effect on 5 implant pathogens in vitro. However, low cell viability to CaP-Ag particles in the coatings needs to be addressed. The CaP-Ag particles exhibited high protein loading and sustained protein release profile that may be advantageous for the local delivery of growth factors or other agents that require low and extended release profiles. FedEx Institute of Technology, Memphis USA

Abstract 3199: Stable isotope resolved metabolomics (SIRM) on fresh human tissues as a preclinical drug testing platform

Abstract Introduction: All preclinical drug testing models have advantages and drawbacks. We have been using SIRM to evaluate metabolic reprogramming of lung cancer cells in monoculture, in mouse xenograft/explant models, and in NSCLC patients in situ (1), and to determine the influence of the tumor microenvironment using these models. We have now extended the range of models to fresh human tissue slices, similar to those originally described by Warburg (2), which retain the original tissue architecture and heterogeneity with a paired benign versus cancer design under controlled cell culture conditions. Experimental: Freshly resected tissue slices from individuals (ca. 1 mm or less thick and 5-40 mg wet weight) were incubated in standard cell culture conditions with gentle rocking for efficient gas, nutrient and waste product exchange. The metabolic activity was determined by measuring the uptake and transformation of 13C and/or 15N-enriched nutrient tracers such as glucose and glutamine, using high-resolution MS, GC-MS, and NMR after a period of incubation. Acute metabolic and histologic response to inhibitors or drugs was readily detected in treated tissue slices. Findings: Analysis at different time points by NMR, MS and histology shows that the NSCLC tissue slices, both benign and tumorous, retained their architecture and remained metabolically viable for at least 48 h of incubation. Glucose and glutamine metabolism was reprogrammed in the tumor relative to the paired benign tissues. The paired tissue also showed very different responses to Se-containing compounds when incubated at the IC50 established for cell lines. After 24 h of incubation, large scale necrosis was evident in the tumor, but not in the benign slices, which was accompanied by large changes in metabolic activities observed by SIRM analysis. Conclusions: This platform offers a human tissue model for preclinical studies on metabolic reprogramming of human cancer cells in their tissue context, and response to drug treatment (3). As the microenvironment of the target human tissue is retained and individualized response to drugs is obtained, this platform promises to transcend current limitations of drug selection for clinical trials or treatments. Supported by NCI P01CA163223-01A1 and NIEHS 1R01ES022191-01 1. Lane, A.N., Fan, T.W.-M., Bousamra II, M., Higashi, R.M., Yan, J. and Miller, D.M. (2011) Clinical Applications of Stable Isotope-Resolved Metabolomics (SIRM) in Non-Small Cell Lung Cancer. Omics, 15, 173-182. 2. Warburg, O. (1923) Versuche an überlebendem Carcinomgewebe (Methoden). Biochem. Zeitschr., 142, 317-333. 3. Xie, H., Hanai, J., Ren, J.-G., Kats, L., Burgess, K., Bhargava, P., Signoretti, S., Billiard, J., Duffy, K.J., Grant, A. et al. (2014) Targeting lactate dehydrogenase-A (LDH-A) inhibits tumorigenesis and tumor progression in mouse models of lung cancer and impacts tumor initiating cells. Cell Metabolism 19, 795-809. Citation Format: Andrew N. Lane, Teresa W-M Fan, Alexander C. Belshoff, Richard M. Higashi, Jeremiah Martin, Michael Bousamra. Stable isotope resolved metabolomics (SIRM) on fresh human tissues as a preclinical drug testing platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3199. doi:10.1158/1538-7445.AM2015-3199

Rocking media over ex vivo corneas improves this model and allows the study of the effect of proinflammatory cytokines on wound healing.

The aim of this work was to develop an in vitro cornea model to study the effect of proinflammatory cytokines on wound healing. Initial studies investigated how to maintain the ex vivo models for up to 4 weeks without loss of epithelium. To study the effect of cytokines, corneas were cultured with the interleukins IL-17A, IL-22, or a combination of IL-17A and IL-22, or lipopolysaccharide (LPS). The effect of IL-17A on wound healing was then examined. With static culture conditions, organ cultures deteriorated within 2 weeks. With gentle rocking of media over the corneas and carbon dioxide perfusion, the ex vivo models survived for up to 4 weeks without loss of epithelium. The cytokine that caused the most damage to the cornea was IL-17A. Under static conditions, wound healing of the central corneal epithelium occurred within 9 days, but only a single-layered epithelium formed whether the cornea was exposed to IL-17A or not. With rocking of media gently over the corneas, a multilayered epithelium was achieved 9 days after wounding. In the presence of IL-17A, however, there was no wound healing evident. Characterization of the cells showed that wherever epithelium was present, both differentiated cells and highly proliferative cells were present. We propose that introducing rocking to extend the effective working life of this model and the introduction of IL-17A to this model to induce aspects of inflammation extend its usefulness to study the effects of agents that influence corneal regeneration under normal and inflamed conditions.

Editorial: Introduction and Motivations

If you google for “open system” you get plenty of information about security services and consulting firms, as well as IT companies and industries. They explain about firewalls, vpn, intrusion detection, security standards, violations, protection of corporate assets, anti-spam, anti-virus, authentication, service and maintenance, proxy, and audits. Their mission is “to build and foster relationships with clients by providing dedicated, passionate and unrivaled service”, to design “open and timely communications”, and to this end some of them provide a full range of staffing services and solutions. Their main objective is to make one feel secure. Openness to the external world is viewed as a threat. We pretend to be open and to like open systems, but in fact we do not. We prefer closed systems, as they are more controllable and certainly easier to deal with. Closed systems are a cradle. I wonder whether it is not the same with quantum mechanics. Quantum mechanics originally followed the typical “Western” approach to science, based on Greek philosophy: a system is something separated from the rest of the universe and when the rest of the universe interacts with it we invent all sorts of things to describe these “interactions”. We then go back to the system and analyze it. In the worst case, “external”, God-given forces are time-dependent. Divide and conquer. The gentle rocking of the cradle of closed Hamiltonian systems helped us relax peacefully for more than 50 years. Then Gorini, Kossakowski, Lindblad and Sudarshan woke us up [1, 2, 3]. Not only closed quantum systems do not exist, but the dynamical laws that govern (existing) open quantum

Oligomerization of the Nrdp1 E3 Ubiquitin Ligase Is Necessary for Efficient Autoubiquitination but Not ErbB3 Ubiquitination

Overexpression of the ErbB3 receptor tyrosine kinase protein in breast and other cancers contributes to tumor malignancy and therapeutic resistance. The RBCC/TRIM family RING finger E3 ubiquitin ligase Nrdp1 mediates the ubiquitination of ErbB3 in normal mammary epithelial cells to facilitate receptor degradation and suppress steady-state receptor levels. Post-transcriptional loss of Nrdp1 in patient breast tumors allows ErbB3 overexpression and receptor contribution to tumor progression, and elevated lability through autoubiquitination contributes to the observed loss of Nrdp1 in tumors relative to normal tissue. To begin to understand the mechanisms underlying Nrdp1 protein self-regulation through lability, we investigated the structural determinants required for efficient autoubiquitination and ErbB3 ubiquitination. Using mutagenesis, chemical cross-linking, size exclusion chromatography, and native polyacrylamide gel electrophoresis, we demonstrate that Nrdp1 self-associates into a stable oligomeric complex in cells. Deletion of its coiled-coil domain abrogates oligomerization but does not affect Nrdp1-mediated ErbB3 ubiquitination or degradation. On the other hand, the presence of the coiled-coil domain is necessary for efficient Nrdp1 autoubiquitination via a trans mechanism, indicating that Nrdp1 ubiquitination of its various targets is functionally separable. Finally, a GFP fusion of the coiled-coil domain stabilizes Nrdp1 and potentiates ErbB3 ubiquitination and degradation. These observations point to a model whereby the coiled-coil domain plays a key role in regulating Nrdp1 lability by promoting its assembly into an oligomeric complex, and raise the possibility that inhibition of ligase oligomerization via its coiled-coil domain could be of therapeutic benefit to breast cancer patients by restoring Nrdp1 protein.

MicroRNA-31 Activates the RAS Pathway and Functions as an Oncogenic MicroRNA in Human Colorectal Cancer by Repressing RAS p21 GTPase Activating Protein 1 (RASA1)

MicroRNAs (miRNAs) are known to play a vital role in colorectal cancer. We found a widespread disruption in miRNA expression during colorectal tumorigenesis using microarray and quantitative RT-PCR analysis; of the 161 miRNAs altered in colorectal cancer compared with normal adjacent tissue samples, miR-31 was the most significantly dysregulated. We identified candidate targets of miR-31 using bioinformatics approaches and validated RAS p21 GTPase activating protein 1 (RASA1) as a direct target. First, we found an inverse correlation between miR-31 and RASA1 protein levels in vivo. Second, in vitro evidence demonstrated that RASA1 expression was significantly decreased by treatment with pre-miR-31-LV, whereas anti-miR-31-LV treatment increased RASA1 protein levels. Third, a luciferase reporter assay confirmed that miR-31 directly recognizes a specific location within the 3'-untranslated region of RASA1 transcripts. Furthermore, the biological consequences of miR-31 targeting RASA1 were examined by the cell proliferation assay in vitro and by the immunodeficient mouse xenograft tumor model in vivo. Taken together, our results demonstrate for the first time that miR-31 plays a significant role in activating the RAS signaling pathway through the inhibition of RASA1 translation, thereby improving colorectal cancer cell growth and stimulating tumorigenesis.

Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held

Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (N(Glu)) and its diffusion coefficient (D(Glu)) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of N(Glu) and D(Glu) with an optimum of 7000 and 0.3 μm(2) ms(-1) reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals.