Biophysical control of plasticity and patterning in regeneration and cancer.

Cells and tissues display a remarkable range of plasticity and tissue-patterning activities that are emergent of complex signaling dynamics within their microenvironments. These properties, which when operating normally guide embryogenesis and regeneration, become highly disordered in diseases such as cancer. While morphogens and other molecular factors help determine the shapes of tissues and their patterned cellular organization, the parallel contributions of biophysical control mechanisms must be considered to accurately predict and model important processes such as growth, maturation, injury, repair, and senescence. We now know that mechanical, optical, electric, and electromagnetic signals are integral to cellular plasticity and tissue patterning. Because biophysical modalities underly interactions between cells and their extracellular matrices, including cell cycle, metabolism, migration, and differentiation, their applications as tuning dials for regenerative and anti-cancer therapies are being rapidly exploited. Despite this, the importance of cellular communication through biophysical signaling remains disproportionately underrepresented in the literature. Here, we provide a review of biophysical signaling modalities and known mechanisms that initiate, modulate, or inhibit plasticity and tissue patterning in models of regeneration and cancer. We also discuss current approaches in biomedical engineering that harness biophysical control mechanisms to model, characterize, diagnose, and treat disease states.

Functional bioengineered models of the central nervous system.

The functional complexity of the central nervous system (CNS) is unparalleled in living organisms. Its nested cells, circuits and networks encode memories, move bodies and generate experiences. Neural tissues can be engineered to assemble model systems that recapitulate essential features of the CNS and to investigate neurodevelopment, delineate pathophysiology, improve regeneration and accelerate drug discovery. In this Review, we discuss essential structure-function relationships of the CNS and examine materials and design considerations, including composition, scale, complexity and maturation, of cell biology-based and engineering-based CNS models. We highlight region-specific CNS models that can emulate functions of the cerebral cortex, hippocampus, spinal cord, neural-X interfaces and other regions, and investigate a range of applications for CNS models, including fundamental and clinical research. We conclude with an outlook to future possibilities of CNS models, highlighting the engineering challenges that remain to be overcome.

Vitamin D Levels in Newly Diagnosed Breast Cancer Patients according to Tumor Sub-Types.

Vitamin D is an important regulator of bone health. In addition, as a ligand for a nuclear receptor expressed in breast cancer cells, vitamin D exerts neoplasia modulating effects in breast cancer. However, despite extensive investigations, associations of vitamin D levels with breast cancer patient characteristics and disease sub-types are conflicting. A retrospective review of medical records of consecutive breast cancer patients treated and followed in a single cancer center was undertaken. All patients with 25-hydroxyvitamin D (25-OHD, the circulating form of vitamin D) measurements available within 3 months of their diagnosis and before the start of any systemic treatment were included. Characteristics of patients and tumors with sufficient levels of 25-OHD were compared with those of patients with 25-OHD insufficiency. Two hundred ninety-two patients were included in the study. Almost two-thirds of the patients were 25-OHD insufficient, defined as having 25-OHD levels below 75 nmol/L. Compared with the group of patients who were 25-OHD sufficient, patients with 25-OHD insufficiency were younger and more often obese. Tumors of patients with 25-OHD insufficiency were more often ductal, of higher grade, and ER negative. 25-OHD insufficiency is prevalent in breast cancer patients and even more prevalent in younger and obese patients. 25-OHD insufficiency is associated with cancers that have aggressive characteristics, including higher grade and ER negativity.

Studying Protista WBR and Repair Using Physarum polycephalum.

Physarum polycephalum is a protist slime mould that exhibits a high degree of responsiveness to its environment through a complex network of tubes and cytoskeletal components that coordinate behavior across its unicellular, multinucleated body. Physarum has been used to study decision making, problem solving, and mechanosensation in aneural biological systems. The robust generative and repair capacities of Physarum also enable the study of whole-body regeneration within a relatively simple model system. Here we describe methods for growing, imaging, quantifying, and sampling Physarum that are adapted for investigating regeneration and repair.

Proteasome regulators in pancreatic cancer.

Pancreatic adenocarcinoma is one of the most lethal cancers with rising incidence. Despite progress in its treatment, with the introduction of more effective chemotherapy regimens in the last decade, prognosis of metastatic disease remains inferior to other cancers with long term survival being the exception. Molecular characterization of pancreatic cancer has elucidated the landscape of the disease and has revealed common lesions that contribute to pancreatic carcinogenesis. Regulation of proteostasis is critical in cancers due to increased protein turnover required to support the intense metabolism of cancer cells. The proteasome is an integral part of this regulation and is regulated, in its turn, by key transcription factors, which induce transcription of proteasome structural units. These include FOXO family transcription factors, NFE2L2, hHSF1 and hHSF2, and NF-Y. Networks that encompass proteasome regulators and transduction pathways dysregulated in pancreatic cancer such as the KRAS/ BRAF/MAPK and the Transforming growth factor beta/SMAD pathway contribute to pancreatic cancer progression. This review discusses the proteasome and its transcription factors within the pancreatic cancer cellular micro-environment. We also consider the role of stemness in carcinogenesis and the use of proteasome inhibitors as therapeutic agents.

Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis.

Limb regeneration is a frontier in biomedical science. Identifying triggers of innate morphogenetic responses in vivo to induce the growth of healthy patterned tissue would address the needs of millions of patients, from diabetics to victims of trauma. Organisms such as Xenopus laevis-whose limited regenerative capacities in adulthood mirror those of humans-are important models with which to test interventions that can restore form and function. Here, we demonstrate long-term (18 months) regrowth, marked tissue repatterning, and functional restoration of an amputated X. laevis hindlimb following a 24-hour exposure to a multidrug, pro-regenerative treatment delivered by a wearable bioreactor. Regenerated tissues composed of skin, bone, vasculature, and nerves significantly exceeded the complexity and sensorimotor capacities of untreated and control animals' hypomorphic spikes. RNA sequencing of early tissue buds revealed activation of developmental pathways such as Wnt/ß-catenin, TGF-ß, hedgehog, and Notch. These data demonstrate the successful "kickstarting" of endogenous regenerative pathways in a vertebrate model.

Mechanosensation Mediates Long-Range Spatial Decision-Making in an Aneural Organism.

The unicellular protist Physarum polycephalum is an important emerging model for understanding how aneural organisms process information toward adaptive behavior. Here, it is revealed that Physarum can use mechanosensation to reliably make decisions about distant objects in its environment, preferentially growing in the direction of heavier, substrate-deforming, but chemically inert masses. This long-range sensing is abolished by gentle rhythmic mechanical disruption, changing substrate stiffness, or the addition of an inhibitor of mechanosensitive transient receptor potential channels. Additionally, it is demonstrated that Physarum does not respond to the absolute magnitude of strain. Computational modeling reveales that Physarum may perform this calculation by sensing the fraction of its perimeter that is distorted above a threshold substrate strain-a fundamentally novel method of mechanosensation. Using its body as both a distributed sensor array and computational substrate, this aneural organism leverages its unique morphology to make long-range decisions. Together, these data identify a surprising behavioral preference relying on biomechanical features and quantitatively characterize how the Physarum exploits physics to adaptively regulate its growth and shape.

Toward Studying Cognition in a Dish.

Bioengineered neural tissues help advance our understanding of neurodevelopment, regeneration, and neural disease; however, it remains unclear whether they can replicate higher-order functions including cognition. Building upon technical achievements in the fields of biomaterials, tissue engineering, and cell biology, investigators have generated an assortment of artificial brain structures and cocultured circuits. Though they have displayed basic electrochemical signaling, their capacities to generate minimal patterns of information processing suggestive of high-order cognitive analogues have not yet been explored. Here, we review the current state of neural tissue engineering and consider the possibility of a study of cognition in vitro. We adopt a practical definition of minimal cognition, anticipate problems of measurement, and discuss solutions toward a study of cognition in a dish.

Matrix Deformation with Ectopic Cells Induced by Rotational Motion in Bioengineered Neural Tissues.

The brain's extracellular matrix (ECM) is a dynamic protein-based scaffold within which neural networks can form, self-maintain, and re-model. When the brain incurs injuries, microscopic tissue tears and active ECM re-modelling give way to abnormal brain structure and function including the presence of ectopic cells. Post-mortem and neuroimaging data suggest that the brains of jet pilots and astronauts, who are exposed to rotational forces, accelerations, and microgravity, display brain anomalies which could be indicative of a mechanodisruptive pathology. Here we present a model of non-impact-based brain injury induced by matrix deformation following mechanical shaking. Using a bioengineered 3D neural tissue platform, we designed a repetitive shaking paradigm to simulate subtle rotational acceleration. Our results indicate shaking induced ectopic cell clustering that could be inhibited by physically restraining tissue movement. Imaging revealed that the collagen substrate surrounding cells was deformed following shaking. Applied to neonatal rat brains, shaking induced deformation of extracellular spaces within the cerebral cortices and reduced the number of cell bodies at higher accelerations. We hypothesize that ECM deformation may represent a more significant role in brain injury progression than previously assumed and that the present model system contributes to its understanding as a phenomenon.

Ultraweak Photon Emissions as a Non-Invasive, Early-Malignancy Detection Tool: An In Vitro and In Vivo Study.

Early detection of cancer improves treatment options and increases survival. Building upon previous demonstrations that ultraweak photon emissions (UPE) could be measured to detect cancers, we designed an early detection protocol to test malignancy in both in vitro and in vivo systems. Photons were measured for 100 s from plates containing ~1 million malignant or non-malignant cells from 13 different types of human and mouse cell lines. Tumor cells displayed increased photon emissions compared to non-malignant cells. Examining the standardized Spectral Power Density (SPD) configurations for flux densities between 0.1 and 25 Hz (Δf = 0.01 Hz) yielded 90% discriminant accuracy. The emission profiles of mice that had been injected with melanoma cells could be differentiated from a non-malignant reference groups as early as 24 h post-injection. The peak SPD associated with photon emissions was ~20 Hz for both malignant cell cultures and mice with growing tumors. These results extend the original suggestion by Takeda and his colleagues (2004) published in this journal concerning the potential diagnostic value of UPEs for assessing proliferations of carcinoma cells. The specificity of the spectral profile in the 20 Hz range may be relevant to the consistent efficacy reported by several authors that weak magnetic field pulsations within this frequency range can diminish the growth of malignant cells in culture and tumor weights in mice.

Biophotonic markers of malignancy: Discriminating cancers using wavelength-specific biophotons.

Early detection is a critically important factor when successfully diagnosing and treating cancer. Whereas contemporary molecular techniques are capable of identifying biomarkers associated with cancer, surgical interventions are required to biopsy tissue. The common imaging alternative, positron-emission tomography (PET), involves the use of nuclear material which poses some risks. Novel, non-invasive techniques to assess the degree to which tissues express malignant properties are now needed. Recent developments in biophoton research have made it possible to discriminate cancerous cells from normal cells both in vitro and in vivo. The current study expands upon a growing body of literature where we classified and characterized malignant and non-malignant cell types according to their biophotonic activity. Using wavelength-exclusion filters, we demonstrate that ratios between infrared and ultraviolet photon emissions differentiate cancer and non-cancer cell types. Further, we identified photon sources associated with three filters (420-nm, 620-nm., and 950-nm) which classified cancer and non-cancer cell types. The temporal increases in biophoton emission within these wavelength bandwidths is shown to be coupled with intrisitic biomolecular events using Cosic's resonant recognition model. Together, the findings suggest that the use of wavelength-exclusion filters in biophotonic measurement can be employed to detect cancer in vitro.

Right cerebral hemispheric sensitivity to pH and physiological ions in fixed post-mortem Wistar rat brains.

Post-mortem human neural tissues fixed in ethanol and aldehyde-based solutions express modulated frequency-dependent microvolt potentials when probed by chemical and electrical stimuli. These observations run contrary to the assumption that basic tissue functions are irreversibly impaired upon fixation, in the absence of nutrients and sufficient concentrations of physiological ions. The aim of the current study was to investigate the relative effects of pH and specific charged particles relevant to normal cell physiology upon electric potentials associated with fixed post-mortem rat brain tissue. We identified a positive relationship between the total time the brains had been immersed in ethanol-formalin-acetic acid and high-frequency microvolt potentials within the dorsal right hemisphere of the rat cerebrum. Measuring the pH of the fixative solution surrounding the brains indicated that as time increased, a logarithmic trend toward alkalinity could be observed. Further experiments revealed that high-frequency microvolt potentials were related to pH changes within the right hemisphere only. The right ventral cerebrum displayed a unique response to potassium chloride in ways uncounted for by pH alone. The results suggest that the fixed post-mortem right cerebrum of the rat is particularly sensitive to pH and physiological ions which explains a subset of previous findings with respect to stimulus-response patterns in human coronal brain sections. A concluding hypothesis is presented which suggests that brain tissue expresses material properties independent of metabolic activity though perhaps relevant to living brain function.

Synergistic interactions between temporal coupling of complex light and magnetic pulses upon melanoma cell proliferation and planarian regeneration.

Synergisms between a physiologically patterned magnetic field that is known to enhance planarian growth and suppress proliferation of malignant cells in culture and three light emitting diode (LED) generated visible wavelengths (blue, green, red) upon planarian regeneration and melanoma cell numbers were discerned. Five days of hourly exposures to either a physiologically patterned (2.5-5.0 µT) magnetic field, one of three wavelengths (3 kLux) or both treatments simultaneously indicated that red light (680 nm), blue light (470 nm) or the magnetic field significantly facilitated regeneration of planarian compared to sham field exposed planarian. Presentation of both light and magnetic field conditions enhanced the effect. Whereas the blue and red light diminished the growth of malignant (melanoma) cells, the effect was not as large as that produced by the magnetic field. Only the paired presentation of the blue light and magnetic field enhanced the suppression. On the other hand, the changes following green light (540 nm) exposure did not differ from the control condition and green light presented with the magnetic field eliminated its effects for both the planarian and melanoma cells. These results indicate specific colors affect positive adaptation that is similar to weak, physiologically patterned frequency modulated (8-24 Hz) magnetic fields and that the two forms of energy can synergistically summate or cancel.

When Is the Brain Dead? Living-Like Electrophysiological Responses and Photon Emissions from Applications of Neurotransmitters in Fixed Post-Mortem Human Brains.

The structure of the post-mortem human brain can be preserved by immersing the organ within a fixative solution. Once the brain is perfused, cellular and histological features are maintained over extended periods of time. However, functions of the human brain are not assumed to be preserved beyond death and subsequent chemical fixation. Here we present a series of experiments which, together, refute this assumption. Instead, we suggest that chemical preservation of brain structure results in some retained functional capacity. Patterns similar to the living condition were elicited by chemical and electrical probes within coronal and sagittal sections of human temporal lobe structures that had been maintained in ethanol-formalin-acetic acid. This was inferred by a reliable modulation of frequency-dependent microvolt fluctuations. These weak microvolt fluctuations were enhanced by receptor-specific agonists and their precursors (i.e., nicotine, 5-HTP, and L-glutamic acid) as well as attenuated by receptor-antagonists (i.e., ketamine). Surface injections of 10 nM nicotine enhanced theta power within the right parahippocampal gyrus without any effect upon the ipsilateral hippocampus. Glutamate-induced high-frequency power densities within the left parahippocampal gyrus were correlated with increased photon counts over the surface of the tissue. Heschl's gyrus, a transverse convexity on which the primary auditory cortex is tonotopically represented, retained frequency-discrimination capacities in response to sweeps of weak (2µV) square-wave electrical pulses between 20 Hz and 20 kHz. Together, these results suggest that portions of the post-mortem human brain may retain latent capacities to respond with potential life-like and virtual properties.

Annual incidence of mortality related to hypertensive disease in Canada and associations with heliophysical parameters.

Increasing research into heliobiology and related fields has revealed a myriad of potential relationships between space weather factors and terrestrial biology. Additionally, many studies have indicated cyclicity in incidence of various diseases along with many aspects of cardiovascular function. The current study examined annual mortality associated with hypertensive diseases in Canada from 1979 to 2009 for periodicities and linear relationships with a range of heliophysical parameters. Analyses indicated a number of significant lagged correlations between space weather and hypertensive mortality, with solar wind plasma beta identified as the likely source of these relationships. Similar periodicities were observed for geomagnetic activity and hypertensive mortality. A significant rhythm was revealed for hypertensive mortality centered on a 9.6-year cycle length, while geomagnetic activity was fit with a 10.1-year cycle. Cross-correlograms of mortality with space weather demonstrated a 10.67-year periodicity coinciding with the average 10.6-year solar cycle length for the time period examined. Further quantification and potential implications are discussed.

LORETA indicates frequency-specific suppressions of current sources within the cerebrums of blindfolded subjects from patterns of blue light flashes applied over the skull.

An array of eight cloistered (completely covered) 470-nm LEDs was attached to the right caudal scalp of subjects while each sat blindfolded within a darkened chamber. The LEDs were activated by a computer-generated complex (frequency-modulated) temporal pattern that, when applied as weak magnetic fields, has elicited sensed presences and changes in LORETA (low-resolution electromagnetic tomography) configurations. Serial 5-min on to 5-min off presentations of the blue light (10,000lx) resulted in suppression of gamma activity within the right cuneus (including the extrastriate area), beta activity within the left angular and right superior temporal regions, and alpha power within the right parahippocampal region. The effect required about 5min to emerge followed by a transient asymptote for about 15 to 20min when diminished current source density was evident even during no light conditions. Subjective experiences, as measured by our standard exit questionnaire, reflected sensations similar to those reported when the pattern was presented as a weak magnetic field. Given previous evidence that photon flux density of this magnitude can penetrate the skull, these results suggest that properly configured LEDs that generate physiologically patterned light sequences might be employed as noninvasive methods to explore the dynamic characteristics of cerebral activity in epileptic and nonepileptic brains.

Inverse relationship between photon flux densities and nanotesla magnetic fields over cell aggregates: Quantitative evidence for energetic conservation.

The quantitative relationship between local changes in magnetic fields and photon emissions within ∼2 mm of aggregates of 10(5)-10(6) cells was explored experimentally. The vertical component of the earth's magnetic field as measured by different magnetometers was ∼15 nT higher when plates of cells removed from incubation were measured compared to plates containing only medium. Additional experiments indicated an inverse relationship over the first ∼45 min between changes in photon counts (∼10(-12) W·m(-2)) following removal from incubation and similar changes in magnetic field intensity. Calculations indicated that the energy within the aqueous volume containing the cells was equivalent for that associated with the flux densities of the magnetic fields and the photon emissions. For every approximately 1 nT increase in magnetic field intensity value there was a decrease of ∼2 photons (equivalent of 10(-18) J). These results complement correlation studies and suggest there may be a conservation of energy between expression as magnetic fields that are subtracted or added to the adjacent geomagnetic field and reciprocal changes in photon emissions when aggregates of cells within a specific volume of medium (water) adapt to new environments.

Novel Cosic resonance (standing wave) solutions for components of the JAK-STAT cellular signaling pathway: A convergence of spectral density profiles.

Cosic discovered that spectral analyses of a protein sequence after each constituent amino acid had been transformed into an appropriate pseudopotential predicted a resonant energy between interacting molecules. Several experimental studies have verified the predicted peak wavelength of photons within the visible or near-visible light band for specific molecules. Here, this concept has been applied to a classic signaling pathway, JAK-STAT, traditionally composed of nine sequential protein interactions. The weighted linear average of the spectral power density (SPD) profiles of each of the eight "precursor" proteins displayed remarkable congruence with the SPD profile of the terminal molecule (CASP-9) in the pathway. These results suggest that classic and complex signaling pathways in cells can also be expressed as combinations of resonance energies.

Bacterial growth rates are influenced by cellular characteristics of individual species when immersed in electromagnetic fields.

Previous studies have shown that exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) have negative effects on the rate of growth of bacteria. In the present study, two Gram-positive and two Gram-negative species were exposed to six magnetic field conditions in broth cultures. Three variations of the 'Thomas' pulsed frequency-modulated pattern; a strong-static "puck" magnet upwards of 5000G in intensity; a pair of these magnets rotating opposite one another at ∼30rpm; and finally a strong dynamic magnetic field generator termed the 'Resonator' with an average intensity of 250µT were used. Growth rate was discerned by optical density (OD) measurements every hour at 600nm. ELF-EMF conditions significantly affected the rates of growth of the bacterial cultures, while the two static magnetic field conditions were not statistically significant. Most interestingly, the 'Resonator' dynamic magnetic field increased the rates of growth of three species (Staphylococcus epidermidis, Staphylococcus aureus, and Escherichia coli), while slowing the growth of one (Serratia marcescens). We suggest that these effects are due to individual biophysical characteristics of the bacterial species.

Group planarian sudden mortality: Is the threshold around global geomagnetic activity ≥K6?

Sudden deaths in groups of animals have been observed by field and laboratory biologists. We have measured mortalities in large group-housed planarian during the infrequent periods of very intense geomagnetic activity. In 13 separate episodes over the last 5 y we have observed the sudden death in our laboratory of hundreds of planarian if their density was about 1 worm per cc and the global geomagnetic activity was K≥6 the day before or the day of the observation of the mortality. Such mortality never occurred in other conditions or days. Both estimates of the "magnetic moment" of a planarian in magnetic fields above this threshold of sustained magnetic flux density as well as the magnetic energy within the planarian volume predict values that could affect phenomenon associated with the total numbers of pH-dependent charges within each worm. These conditions could affect the Levin-Burr bioelectrical signals and networks that affect patterning information and sustainability in whole living systems. The establishment of a central reservoir for the report of these transient events might allow Life Scientists to more fully appreciate the impact of these pervasive global stimuli upon dense groups of animals.