Macromolecular condensation buffers intracellular water potential.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

Vimentin intermediate filaments provide structural stability to the mammalian Golgi complex.

The Golgi complex comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region in most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi complex with intermediate filaments (IFs). In this study, we show that the Golgi is in close physical proximity to vimentin IFs in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with vimentin IFs. Loss of vimentin and/or GORAB had a modest effect upon Golgi structure at the steady state. The Golgi underwent more rapid disassembly upon chemical disruption with brefeldin A or nocodazole, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin caused reduced Golgi ribbon integrity when cells were cultured on high-stiffness hydrogels, which was exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi complex and suggest a role for GORAB in this process.

The circadian clock and extracellular matrix homeostasis in aging and age-related diseases.

The extracellular matrix (ECM) is the noncellular scaffolding component present within all tissues and organs. It provides crucial biochemical and biomechanical cues to instruct cellular behavior and has been shown to be under circadian clock regulation, a highly conserved cell-intrinsic timekeeping mechanism that has evolved with the 24-hour rhythmic environment. Aging is a major risk factor for many diseases, including cancer, fibrosis, and neurodegenerative disorders. Both aging and our modern 24/7 society disrupt circadian rhythms, which could contribute to altered ECM homeostasis. Understanding the daily dynamics of ECM and how this mechanism changes with age will have a profound impact on tissue health, disease prevention, and improving treatments. Maintaining rhythmic oscillations has been proposed as a hallmark of health. On the other hand, many hallmarks of aging turn out to be key regulators of circadian timekeeping mechanisms. In this review, we summarize new work linking the ECM with circadian clocks and tissue aging. We discuss how the changes in the biomechanical and biochemical properties of ECM during aging may contribute to circadian clock dysregulation. We also consider how the dampening of clocks with age could compromise the daily dynamic regulation of ECM homeostasis in matrix-rich tissues. This review aims to encourage new concepts and testable hypotheses about the two-way interactions between circadian clocks and ECM in the context of aging.

Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator.

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.

Post-Processing of Raw Data Recorded Continuously Using a FORS-Fibre-Optic Rotational Seismograph.

Modern optoelectronic devices use the advantage of digital systems for data processing aimed at delivering reliable information. However, since commonly used DACs have limited accuracy, some artefacts can be observed in data streams, especially in systems designed for continuous, long-term process monitoring. In this paper, the authors' experience with data enhancement using a fibre-optic rotational seismograph (FORS) operating in a closed-loop mode is presented and discussed. Generally, two kinds of enhancement are described. The first one uses suitable filtering techniques adequate for FORS noise investigation, as well as a suitable data resampling method for transmitted data file size reduction. The second one relates to the artefacts observed during data recording in real time. The recording starting point is triggered when the detected signal exceeds a middle signal level and, therefore, the existence of artefacts generally disturbs the recording process. Although the artefacts are easily recognised by human eyes even at first sight, their automatic elimination is not so easy. In this paper, the authors propose a new concept of signal filtering to solve the above problem.

Tissue physiology revolving around the clock: circadian rhythms as exemplified by the intervertebral disc.

Circadian clocks in the brain and peripheral tissues temporally coordinate local physiology to align with the 24 hours rhythmic environment through light/darkness, rest/activity and feeding/fasting cycles. Circadian disruptions (during ageing, shift work and jet-lag) have been proposed as a risk factor for degeneration and disease of tissues, including the musculoskeletal system. The intervertebral disc (IVD) in the spine separates the bony vertebrae and permits movement of the spinal column. IVD degeneration is highly prevalent among the ageing population and is a leading cause of lower back pain. The IVD is known to experience diurnal changes in loading patterns driven by the circadian rhythm in rest/activity cycles. In recent years, emerging evidence indicates the existence of molecular circadian clocks within the IVD, disruption to which accelerates tissue ageing and predispose animals to IVD degeneration. The cell-intrinsic circadian clocks in the IVD control key aspects of physiology and pathophysiology by rhythmically regulating the expression of ~3.5% of the IVD transcriptome, allowing cells to cope with the drastic biomechanical and chemical changes that occur throughout the day. Indeed, epidemiological studies on long-term shift workers have shown an increased incidence of lower back pain. In this review, we summarise recent findings of circadian rhythms in health and disease, with the IVD as an exemplar tissue system. We focus on rhythmic IVD functions and discuss implications of utilising biological timing mechanisms to improve tissue health and mitigate degeneration. These findings may have broader implications in chronic rheumatic conditions, given the recent findings of musculoskeletal circadian clocks.

Study of Rotational Motions Caused by Multiple Mining Blasts Recorded by Different Types of Rotational Seismometers.

Digging two vertical shafts with the multiple blasts technique gave the opportunity to measure the induced angular motions in a horizontal plane with well-defined positions of sources. Three kinds of rotation rate sensors, sharing an underground location, were used. Two of them-a Fiber-Optic System for Rotational Events & phenomena Monitoring (FOSREM) and a prototypical seismometer housing the liquid-filled torus-sensed the rotation, while a microarray of two double-pendulum seismometers sensed both the rotation and symmetric strain. The FOSREM was sampled at 656.168 Hz, while all the others were only sampled at 100 Hz. There were considerable differences within the results gathered from the mining blasts, which should be attributed to two causes. The first one is the difference in principles of the operation and sampling rates of the devices used, while the other is the complex and spatially variable character of the studied wave fields. Additionally, we established that the liquid-filled sensor, due to its relatively low sensitivity, proved to be viable only during a registration of strong ground motions. Overall, a comparative study of three different rotational seismometers was performed during mining-induced strong ground motions with well-localized sources.

Rotation, Strain, and Translation Sensors Performance Tests with Active Seismic Sources.

Interest in measuring displacement gradients, such as rotation and strain, is growing in many areas of geophysical research. This results in an urgent demand for reliable and field-deployable instruments measuring these quantities. In order to further establish a high-quality standard for rotation and strain measurements in seismology, we organized a comparative sensor test experiment that took place in November 2019 at the Geophysical Observatory of the Ludwig-Maximilians University Munich in Fürstenfeldbruck, Germany. More than 24 different sensors, including three-component and single-component broadband rotational seismometers, six-component strong-motion sensors and Rotaphone systems, as well as the large ring laser gyroscopes ROMY and a Distributed Acoustic Sensing system, were involved in addition to 14 classical broadband seismometers and a 160 channel, 4.5 Hz geophone chain. The experiment consisted of two parts: during the first part, the sensors were co-located in a huddle test recording self-noise and signals from small, nearby explosions. In a second part, the sensors were distributed into the field in various array configurations recording seismic signals that were generated by small amounts of explosive and a Vibroseis truck. This paper presents details on the experimental setup and a first sensor performance comparison focusing on sensor self-noise, signal-to-noise ratios, and waveform similarities for the rotation rate sensors. Most of the sensors show a high level of coherency and waveform similarity within a narrow frequency range between 10 Hz and 20 Hz for recordings from a nearby explosion signal. Sensor as well as experiment design are critically accessed revealing the great need for reliable reference sensors.

Graphene-based hyperbolic metamaterial as a switchable reflection modulator.

A tunable graphene-based hyperbolic metamaterial is designed and numerically investigated in the mid-infrared frequencies. Theoretical analysis proves that by adjusting the chemical potential of graphene from 0.2 eV to 0.8 eV, the reflectance can be blue-shifted up to 2.3 µm. Furthermore, by modifying the number of graphene monolayers in the hyperbolic metamaterial stack, we are able to shift the plasmonic resonance up to 3.6 µm. Elliptic and type II hyperbolic dispersions are shown for three considered structures. Importantly, a blue/red-shift and switching of the reflectance are reported at different incident angles in TE/TM modes. The obtained results clearly show that graphene-based hyperbolic metamaterials with reversibly controlled tunability may be used in the next generation of nonlinear tunable and reversibly switchable devices operating in the mid-IR range.

Measurements of Rotational Events Generated by Artificial Explosions and External Excitations Using the Optical Fiber Sensors Network.

Measurements of artificial events can substantially confirm the data validity of constructed rotational sensors, as well as provide methods for simplifying the measurement process. The above task, especially with international cooperation, can provide full-field measurement results of the target object, which can deliver more significant data and sensor properties. The paper presents vertical rotational velocity recordings gathered during an international experiment that took place at the Geophysical Observatory of the Ludwig Maximilian University of Munich in Fürstenfeldbruck, Germany. Data were obtained during artificial explosions, as well as external excitations induced by a VibroSeis truck. The authors present data recorded by two prototypes of optical fiber rotational sensors. They have been specially designed for rotational seismology needs and are characterized by a theoretical sensitivity equal to 2 × 10-8 rad/s/√Hz and a wide measuring range both in amplitude even up to 10 rad/s, and a frequency from DC to 1000 Hz. Their self-noise investigation during the aforementioned experiment showed that both sensors have precision no worse than 2 × 10-6 rad/s/sqrt (Hz) in all desired frequency range from 0.01 to 100 Hz. A down-sampling and a spectral analysis of the recorded signals are also presented. The recorded data and their analysis confirmed the performance and reliability of the applied optical fiber rotational sensors. Moreover, the presented international experiment underlines a special necessity for specifying the sensors' performance test methodologies in the rotational seismology.

The Fiber-Optic Rotational Seismograph-Laboratory Tests and Field Application.

The paper presents construction and laboratory tests, as well as the first field application of a new fiber-optic rotational seismograph. The system is based on a fiber-optic gyroscope (FOG), with determined Angle Random Walk of the order of 10-8 rad/Sqrt(s) and a few rad/s maximum detectable amplitude of rotation in the frequency range from direct current (DC) to 328.12 Hz. It has been designed for the rotational seismology area of interest. This work also presents exemplary relevant measurements, which were conducted using a set of two devices installed in the geophysical observatory in Ksiaz, Poland.

The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration.

OBJECTIVES: The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. METHODS: Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. RESULTS: Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1ß but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. CONCLUSIONS: We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

Running on time: the role of circadian clocks in the musculoskeletal system.

The night and day cycle governs the circadian (24 hourly) rhythm of activity and rest in animals and humans. This is reflected in daily changes of the global gene expression pattern and metabolism, but also in the local physiology of various tissues. A central clock in the brain co-ordinates the rhythmic locomotion behaviour, as well as synchronizing various local oscillators, such as those found in the musculoskeletal system. It has become increasingly recognized that the internal molecular clocks in cells allow a tissue to anticipate the rhythmic changes in their local environment and the specific demands of that tissue. Consequently, the majority of the rhythmic clock controlled genes and pathways are tissue specific. The concept of the tissue-specific function of circadian clocks is further supported by the diverse musculoskeletal phenotypes in mice with deletions or mutations of various core clock components, ranging from increased bone mass, dwarfism, arthropathy, reduced muscle strength and tendon calcification. The present review summarizes the current understanding of the circadian clocks in muscle, bone, cartilage and tendon tissues, with particular focus on the evidence of circadian rhythms in tissue physiology, their entrainment mechanisms and disease links, and the tissue-specific clock target genes/pathways. Research in this area holds strong potential to advance our understanding of how circadian rhythms control the health and disease of the musculoskeletal tissues, which has major implications in diseases associated with advancing age. It could also have potential implications in sports performance and sports medicine.

Noise suppressed optical diffraction tomography with autofocus correction.

We propose a novel tomographic measurement approach that enables a noise suppressed characterization of microstructures. The idea of this work is based on a finding that coherent noise in the input phase data generates an artificial circular structure whose magnitude is the highest at the centre of tomographic reconstruction. This method decreases the noise level by applying an unconventional tomographic measurement configuration with an object deliberately shifted with respect to the rotation axis. This enables a spatial separation between the reconstructed sample structure and the area of the largest refractive index perturbations. The input phase data defocusing that is a by-product of the introduced modification is numerically corrected with an automatic focus correction algorithm. The proposed method is validated with simulations and experimental measurements of an optical microtip.

The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc.

The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control ∼6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1-Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12 h ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration.

Mechanical loading and hyperosmolarity as a daily resetting cue for skeletal circadian clocks.

Daily rhythms in mammalian behaviour and physiology are generated by a multi-oscillator circadian system entrained through environmental cues (e.g. light and feeding). The presence of tissue niche-dependent physiological time cues has been proposed, allowing tissues the ability of circadian phase adjustment based on local signals. However, to date, such stimuli have remained elusive. Here we show that daily patterns of mechanical loading and associated osmotic challenge within physiological ranges reset circadian clock phase and amplitude in cartilage and intervertebral disc tissues in vivo and in tissue explant cultures. Hyperosmolarity (but not hypo-osmolarity) resets clocks in young and ageing skeletal tissues and induce genome-wide expression of rhythmic genes in cells. Mechanistically, RNAseq and biochemical analysis revealed the PLD2-mTORC2-AKT-GSK3ß axis as a convergent pathway for both in vivo loading and hyperosmolarity-induced clock changes. These results reveal diurnal patterns of mechanical loading and consequent daily oscillations in osmolarity as a bona fide tissue niche-specific time cue to maintain skeletal circadian rhythms in sync.

Extracorporeal Rewarming Is Associated With Increased Survival Rate in Severely Hypothermic Patients With Preserved Spontaneous Circulation.

Treatment recommendations for rewarming patients in severe accidental hypothermia with preserved spontaneous circulation have a weak evidence due to the absence of randomized clinical trials. We aimed to compare the outcomes of extracorporeal versus less-invasive rewarming of severely hypothermic patients with preserved spontaneous circulation. We conducted a multicenter retrospective study. The patient population was compiled based on data from the HELP Registry, the International Hypothermia Registry, and a literature review. Adult patients with a core temperature <28°C and preserved spontaneous circulation were included. Patients who underwent extracorporeal rewarming were compared with patients rewarmed with less-invasive methods, using a matched-pair analysis. The study population consisted of 50 patients rewarmed extracorporeally and 85 patients rewarmed with other, less-invasive methods. Variables significantly associated with survival included: lower age; outdoor cooling circumstances; higher blood pressure; higher PaCO 2 ; higher BE; higher HCO 3 ; and the absence of comorbidities. The survival rate was higher in patients rewarmed extracorporeally ( p = 0.049). The relative risk of death was twice as high in patients rewarmed less invasively. Based on our data, we conclude that patients in severe accidental hypothermia with circulatory instability can benefit from extracorporeal rewarming without an increased risk of complications.

Detection of Organosulfur and Organophosphorus Compounds Using a Hexafluorobutyl Acrylate-Coated Tapered Optical Fibers.

This paper presents the results of a study on the possibility of detecting organosulfur and organophosphorus compounds by means of polymer-assisted optical fiber technology. The detection of the aforementioned compounds can be realized by fabricating a polymer-coated tapered optical fiber (TOF), where the polymer works as an absorber, which changes the light propagation conditions in the TOF. The TOFs were manufactured based on a standard single-mode fiber for telecommunication purposes and, as an absorbing polymer, hexafluorobutyl acrylate was used, which is sensitive to organosulfur and organophosphorus compounds. The spectral measurements were conducted in a wide optical range-500-1800 nm-covering the visible part of the spectrum as well as near infrared part in order to show the versatility of the proposed solution. Additionally, detailed absorption dynamics measurements were provided for a single wavelength of 1310 nm. The analyses were conducted for two concentrations of evaporating compounds, 10 µL and 100 µL, in a volume of 150 mL. Additionally, a temperature dependency analysis and tests with distilled water were carried out to eliminate the influence of external factors. The results presented in this article confirmed the possibility to provide low-cost sensors for dangerous and harmful chemical compounds using optical fiber technology and polymers as sensitive materials.

Synthetic gene circuits for preventing disruption of the circadian clock due to interleukin-1-induced inflammation.

The circadian clock regulates tissue homeostasis through temporal control of tissue-specific clock-controlled genes. In articular cartilage, disruptions in the circadian clock are linked to a procatabolic state. In the presence of inflammation, the cartilage circadian clock is disrupted, which further contributes to the pathogenesis of diseases such as osteoarthritis. Using synthetic biology and tissue engineering, we developed and tested genetically engineered cartilage from murine induced pluripotent stem cells (miPSCs) capable of preserving the circadian clock in the presence of inflammation. We found that circadian rhythms arise following chondrogenic differentiation of miPSCs. Exposure of tissue-engineered cartilage to the inflammatory cytokine interleukin-1 (IL-1) disrupted circadian rhythms and degraded the cartilage matrix. All three inflammation-resistant approaches showed protection against IL-1-induced degradation and loss of circadian rhythms. These synthetic gene circuits reveal a unique approach to support daily rhythms in cartilage and provide a strategy for creating cell-based therapies to preserve the circadian clock.