Probe-free optical chromatin deformation and measurement of differential mechanical properties in the nucleus.

The nucleus is highly organized to facilitate coordinated gene transcription. Measuring the rheological properties of the nucleus and its sub-compartments will be crucial to understand the principles underlying nuclear organization. Here, we show that strongly localized temperature gradients (approaching 1°C/µm) can lead to substantial intra-nuclear chromatin displacements (>1 µm), while nuclear area and lamina shape remain unaffected. Using particle image velocimetry (PIV), intra-nuclear displacement fields can be calculated and converted into spatio-temporally resolved maps of various strain components. Using this approach, we show that chromatin displacements are highly reversible, indicating that elastic contributions are dominant in maintaining nuclear organization on the time scale of seconds. In genetically inverted nuclei, centrally compacted heterochromatin displays high resistance to deformation, giving a rigid, solid-like appearance. Correlating spatially resolved strain maps with fluorescent reporters in conventional interphase nuclei reveals that various nuclear compartments possess distinct mechanical identities. Surprisingly, both densely and loosely packed chromatin showed high resistance to deformation, compared to medium dense chromatin. Equally, nucleoli display particularly high resistance and strong local anchoring to heterochromatin. Our results establish how localized temperature gradients can be used to drive nuclear compartments out of mechanical equilibrium to obtain spatial maps of their material responses.

Longitudinal in vivo imaging of perineuronal nets.

Significance: Perineuronal nets (PNNs) are extracellular matrix structures implicated in learning, memory, information processing, synaptic plasticity, and neuroprotection. However, our understanding of mechanisms governing the evidently important contribution of PNNs to central nervous system function is lacking. A primary cause for this gap of knowledge is the absence of direct experimental tools to study their role in vivo. Aim: We introduce a robust approach for quantitative longitudinal imaging of PNNs in brains of awake mice at subcellular resolution. Approach: We label PNNs in vivo with commercially available compounds and monitor their dynamics with two-photon imaging. Results: Using our approach, we show that it is possible to longitudinally follow the same PNNs in vivo while monitoring degradation and reconstitution of PNNs. We demonstrate the compatibility of our method to simultaneously monitor neuronal calcium dynamics in vivo and compare the activity of neurons with and without PNNs. Conclusion: Our approach is tailored for studying the intricate role of PNNs in vivo, while paving the road for elucidating their role in different neuropathological conditions.

Detective quantum efficiency of intensified CMOS cameras for Cherenkov imaging in radiotherapy.

In this study the metric of detective quantum efficiency (DQE) was applied to Cherenkov imaging systems for the first time, and results were compared for different detector hardware, gain levels and with imaging processing for noise suppression. Intensified complementary metal oxide semiconductor cameras using different image intensifier designs (Gen3 and Gen2+) were used to image Cherenkov emission from a tissue phantom in order to measure the modulation transfer function (MTF) and noise power spectrum (NPS) of the systems. These parameters were used to calculate the DQE for varying acquisition settings and image processing steps. MTF curves indicated that the Gen3 system had superior contrast transfer and spatial resolution than the Gen2+ system, with [Formula: see text] values of 0.52 mm-1 and 0.31 mm-1, respectively. With median filtering for noise suppression, these values decreased to 0.50 mm-1 and 0.26 mm-1. The maximum NPS values for the Gen3 and Gen2+ systems at high gain were 1.3 × 106 mm2 and 9.1 × 104 mm2 respectively, representing a 14x decrease in noise power for the Gen2+ system. Both systems exhibited increased NPS intensity with increasing gain, while median filtering lowered the NPS. The DQE of each system increased with increasing gain, and at the maximum gain levels the Gen3 system had a low-frequency DQE of 0.31%, while the Gen2+ system had a value of 1.44%. However, at a higher frequency of 0.4 mm-1, these values became 0.54% and 0.03%. Filtering improved DQE for the Gen3 system and reduced DQE for the Gen2+ system and had a mix of detrimental and beneficial qualitative effects by decreasing the spatial resolution and sharpness but also substantially lowering noise. This methodology for DQE measurement allowed for quantitative comparison between Cherenkov imaging cameras and improvements to their sensitivity, and yielded the first formal assessment of Cherenkov image formation efficiency.

Exosomes as a storehouse of tissue remodeling proteases and mediators of cancer progression.

Rapidly increasing scientific reports of exosomes and their biological effects have improved our understanding of their cellular sources and their cell-to-cell communication. These nano-sized vesicles act as potent carriers of regulatory bio-macromolecules and can induce regulatory functions by delivering them from its source to recipient cells. The details of their communication network are less understood. Recent studies have shown that apart from delivering its cargo to the cells, it can directly act on extracellular matrix (ECM) proteins and growth factors and can induce various remodeling events. More importantly, exosomes carry many surface-bound proteases, which can cleave different ECM proteins and carbohydrates and can shed cell surface receptors. These local extracellular events can modulate signaling cascades, which consequently influences the whole tissue and organ. This review aims to highlight the critical roles of exosomal proteases and their mechanistic insights within the cellular and extracellular environment.

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Multi-well Fibrillation Assay as a Tool for Analyzing Crosslinking and Stabilization of Collagen.

Collagen is the most widely used substratum in cell culture and biomaterials applications. In this chapter, we describe a simple procedure to isolate collagen, which can be employed to a wide range of tissue sources, and subsequently use it to study the collagen crosslinking and stabilization abilities of various compounds. The protocol is designed for a multi-well format assay and thus can be used for simultaneous assessment of multiple number of compounds and can be easily adapted to a high-throughput screening setup.

Technical Note: Time-gating to medical linear accelerator pulses: Stray radiation detector.

PURPOSE: CCD cameras are employed to image scintillation and Cherenkov radiation in external beam radiotherapy. This is achieved by gating the camera to the linear accelerator (Linac) output. A direct output signal line from the linac is not always accessible and even in cases where such a signal is accessible, a physical wire connected to the output port can potentially alter Linac performance through electrical feedback. A scintillating detector for stray radiation inside the Linac room was developed to remotely time-gate to linac pulses for camera-based dosimetry. METHODS: A scintillator coupled silicon photomultiplier detector was optimized and systematically tested for location sensitivity and for use with both x rays and electron beams, at different energies and field sizes. Cherenkov radiation emitted due to static photon beams was captured using the remote trigger and compared to the images captured using a wired trigger. The issue of false-positive event detection, due to additional neutron activated products with high energy beams, was addressed. RESULTS: The designed circuit provided voltage >2.5 V even for distances up to 3 m from the isocenter with a 6 MV, 5 × 5 cm beam, using a Ø3 × 20 mm3 Bi4 Ge3 O12 (BGO) crystal. With a larger scintillator size, the detector could be placed even beyond 3 m distance. False-positive triggering was reduced by a coincidence detection scheme. Negligible fluctuations were observed in time-gated imaging of Cherenkov intensity emitted from a water phantom, when comparing directly connected vs this remote triggering approach. CONCLUSION: The remote detector provides untethered synchronization to linac pulses. It is especially useful for remote Cherenkov imaging or remote scintillator dosimetry imaging during radiotherapeutic procedures when a direct line signal is not accessible.

Algorithm development for intrafraction radiotherapy beam edge verification from Cherenkov imaging.

Imaging of Cherenkov light emission from patient tissue during fractionated radiotherapy has been shown to be a possible way to visualize beam delivery in real time. If this tool is advanced as a delivery verification methodology, then a sequence of image processing steps must be established to maximize accurate recovery of beam edges. This was analyzed and developed here, focusing on the noise characteristics and representative images from both phantoms and patients undergoing whole breast radiotherapy. The processing included temporally integrating video data into a single, composite summary image at each control point. Each image stack was also median filtered for denoising and ultimately thresholded into a binary image, and morphologic small hole removal was used. These processed images were used for day-to-day comparison computation, and either the Dice coefficient or the mean distance to conformity values can be used to analyze them. Systematic position shifts of the phantom up to 5 mm approached the observed variation values of the patient data. This processing algorithm can be used to analyze the variations seen in patients being treated concurrently with daily Cherenkov imaging to quantify the day-to-day disparities in delivery as a quality audit system for position/beam verification.

Matrix metalloproteinases: The sculptors of chronic cutaneous wounds.

Cutaneous wound healing is a complex mechanism with multiple processes orchestrating harmoniously for structural and functional restoration of the damaged tissue. Chronic non-healing wounds plagued with infection create a major healthcare burden and is one of the most frustrating clinical problems. Chronic wounds are manifested by prolonged inflammation, defective re-epithelialization and haphazard remodeling. Matrix metalloproteinases (MMPs) are zinc dependent enzymes that play cardinal functions in wound healing. Understanding the pathological events mediated by MMPs during wound healing may pave way in identifying novel drug targets for chronic wounds. Here, we discuss the functions and skewed regulation of different MMPs during infection and chronic tissue repair. This review also points out the potential of MMPs and their inhibitors as therapeutic agents in treating chronic wounds during distinct phases of the wound healing. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Dermatopontin augments angiogenesis and modulates the expression of transforming growth factor beta 1 and integrin alpha 3 beta 1 in endothelial cells.

Dermatopontin (DPT) is a matricellular protein with cardinal roles in cutaneous wound healing. The protein is also reported to be altered in various anomalies including cancer. The present study is aimed to unravel the role of DPT in angiogenesis which is imperative in many physiological and pathological processes. DPT's capabilities on promoting angiogenesis were assessed using various in vitro and ex vivo systems. The results indicated that DPT enhances cell motility and induces lamellipodia formation in endothelial cells. Additionally, we noticed that DPT stimulates tube formation in endothelial cells when plated on a matrigel substrate. However, it was observed that DPT had no effect on the proliferation of endothelial cells even at higher concentrations and prolonged treatment periods. Additional experiments on CAM and aortic arch assays apparently depicted that DPT promotes neovascularisation and tube sprouting, thus unraveling its prominent role in angiogenesis. Further, PCR analysis revealed that endothelial cells are devoid of DPT expression, but when exogenously supplied, modulates the expression of transforming growth factor ß1 and integrin α3ß1 which are reported to have crucial roles in endothelial cell behaviour during angiogenesis. In conclusion, DPT possess vital pro-angiogenic properties and thus retains promising therapeutic values in managing chronic wounds and cancer.

Directional Kernel Density Estimation for Classification of Breast Tissue Spectra.

In Breast Conserving Therapy, surgeons measure the thickness of healthy tissue surrounding an excised tumor (surgical margin) via post-operative histological or visual assessment tests that, for lack of enough standardization and reliability, have recurrence rates in the order of 33%. Spectroscopic interrogation of these margins is possible during surgery, but algorithms are needed for parametric or dimension reduction processing. One methodology for tumor discrimination based on dimensionality reduction and nonparametric estimation-in particular, Directional Kernel Density Estimation-is proposed and tested on spectral image data from breast samples. Once a hyperspectral image of the tumor has been captured, a surgeon assists by establishing Regions of Interest where tissues are qualitatively differentiable. After proper normalization, Directional KDE is used to estimate the likelihood of every pixel in the image belonging to each specified tissue class. This information is enough to yield, in almost real time and with 98% accuracy, results that coincide with those provided by histological H&E validation performed after the surgery.

Recent advancements in nanotechnological strategies in selection, design and delivery of biomolecules for skin regeneration.

Skin is a very complex organ and hence designing a bioengineered skin model replicating the essential physiological characteristics for replacing the diseased or damaged parts has been a challenging goal for many. Newer technologies for satisfying most of the criteria are being attempted with the copious efforts of biologists, engineers, physiologists, using multitude of features in combination. Amongst them nanotechnology based biomaterials have gained prominence owing to the enhanced pharmacokinetics, bio-distribution profile, extended half-life and reduced side effects. Designing a matrix that can be assimilated into the body during the regeneration and delivering the essential pharmacological agents in a temporal and spatially specific manner is a tremendous goal. This review essentially deals with the various approaches for designing a multidisciplinary translational smart matrix for addressing the various skin related ailments.

Fabrication of electrospun zein nanofibers for the sustained delivery of siRNA.

In this study, zein nanofibers based siRNA delivery system has been attempted for the first time. Here, the amphiphilic property of zein and the size advantage of nanofibers have been brought together in developing an ideal delivery system for siRNA. The morphological analysis of the GAPDH-siRNA loaded zein nanofibers revealed the proper encapsulation of the siRNA in the polymeric matrix. The loading efficiency of this delivery system was found to be 58.57±2.4% (w/w). The agarose gel analysis revealed that the zein nanofibers preserved the integrity of siRNA for a longer period even at the room temperature. The in vitro release studies not only depicted the sustaining potential of the zein nanofibers but also ensured the release of sufficient quantity of siRNA required to induce the gene silencing effect. The amphiphilic property of zein supported the cell attachment and thereby facilitated the transfection of siRNA into the cells. qRT-PCR analysis confirmed the potential of the developed system in inducing the desired gene silencing effect. Thus, electrospun zein nanofibers have been successfully employed for the delivery of siRNA which has a great therapeutic potential.

A novel FRET 'off-on' fluorescent probe for the selective detection of Fe³âº, Al³âº and Cr³âº ions: its ultrafast energy transfer kinetics and application in live cell imaging.

A rhodamine-naphthalimide dyad probe, 1, that selectively responds to the addition of trivalent metal ions (Fe(3+) or Al(3+) or Cr(3+)) via ultrafast Förster resonance energy transfer (FRET) from naphthalimide to rhodamine is designed and synthesized. 1 is highly selective to the trivalent metal ions and the presence of other monovalent or divalent metal ions do not affect its detection ability. The probe is highly sensitive and it can respond to the presence of trivalent metal ions even at sub-micromolar levels. 1 is stable over a broad range of pH, non-toxic under experimental conditions and suitable to the fluorescence bio-imaging of live cells exposed to trivalent metal ions. The trivalent metal ion induced ultrafast energy transfer kinetics of 1 is explored using time resolved fluorescence experiments.

Role of dermatopontin in re-epithelialization: implications on keratinocyte migration and proliferation.

Re-epithelialization is a key event in wound healing and any impairment in that process is associated with various pathological conditions. Epidermal keratinocyte migration and proliferation during re-epithelialization is largely regulated by the cytokines and growth factors from the provisional matrix and dermis. Extracellular matrix consists of numerous growth factors which mediate cell migration via cell membrane receptors. Dermatopontin (DPT), a non-collagenous matrix protein highly expressed in dermis is known for its striking ability to promote cell adhesion. DPT also enhances the biological activity of transforming growth factor beta 1 which plays a central role in the process of wound healing. This study was designed to envisage the role of DPT in keratinocyte migration and proliferation along with its mRNA and protein expression pattern in epidermis. The results showed that DPT promotes keratinocyte migration in a dose dependant fashion but fail to induce proliferation. Further, PCR and immunodetection studies revealed that the mRNA and protein expression of DPT is considerably negligible in the epidermis in contrast to the dermis. To conclude, DPT has a profound role in wound healing specifically during re-epithelialization by promoting keratinocyte migration via paracrine action from the underlying dermis.

A naphthalimide based PET probe with Fe3+ selective detection ability: theoretical and experimental study.

A naphthalimide based fluorescent probe '1' that operates based on photoinduced electron transfer phenomenon is synthesized and its chemosensory application is explored. Among various metal ions, 1 selectively detects Fe(3+) with a detection limit of 3.0 × 10(-8) M. 1 is stable at physiological pH, nontoxic under experimental conditions and suitable for the detection of Fe(3+) ions present in aqueous samples and live cells.

Expression and integrity of dermatopontin in chronic cutaneous wounds: a crucial factor in impaired wound healing.

Chronic cutaneous wound (CCW) is a major health care burden wherein the healing process is slow or rather static resulting in anatomical and functional restriction of the damaged tissue. Dysregulated expression and degradation of matrix proteins, growth factors and cytokines contribute to the disrupted and uncoordinated healing process of CCW. Therefore, therapeutic approaches for effective management of CCW should be focused towards identifying and manipulating the molecular defects, such as reduced bioavailability of the pro-healing molecules and elevated activity of proteases. This study essentially deals with assessing the expression and integrity of an extracellular matrix protein, Dermatopontin (DPT), in CCW using real-time quantitative reverse transcriptase PCR and immunological techniques. The results indicate that, despite DPT's high mRNA expression, the protein levels are markedly reduced in both CCW tissue and its exudate. To elucidate the cause for this contradiction in mRNA and protein levels, the stability of DPT is analyzed in the presence of wound exudates and various proteases that are naturally elevated in CCW. DPT was observed to be degraded at higher rates when incubated with certain recombinant proteases or chronic wound exudate. In conclusion, the susceptibility of DPT protein to specific proteases present at high levels in the wound milieu resulted in the degradation of DPT, thus leading to impaired healing response in CCW.

Fabrication of highly aligned fibrous scaffolds for tissue regeneration by centrifugal spinning technology.

Centrifugal spinning (C-Spin) is an emerging technology which uses centrifugal force to produce ultrafine fibers. Being a voltage free technique it can overcome the limitations of electrospinning. Owing to the unique characteristic features such as high surface area to volume ratio, porosity, mechanical strength and fiber alignment, centrifugal spun (C-spun) fibrous mat has a wide range of scope in various biomedical applications. Higher degree of fiber alignment can be effortlessly achieved by the C-Spin process. In order to prove the versatility of C-Spin system with respect to fiber alignment, Polycaprolactone (PCL) and gelatin were spun taking them as model polymers. The morphological analysis revealed that highly aligned ultrafine fibers with smooth surface are achieved by C-Spinning. Hydrophilicity, porosity and mechanical property results confirm that the C-spun mat is more suitable for tissue engineering applications. In vitro and in vivo experiments proved that the scaffolds are biocompatible and can be efficiently used as a wound dressing material.

Near-infrared spectral tomography integrated with digital breast tomosynthesis: effects of tissue scattering on optical data acquisition design.

PURPOSE: Design optimization and phantom validation of an integrated digital breast tomosynthesis (DBT) and near-infrared spectral tomography (NIRST) system targeting improvement in sensitivity and specificity of breast cancer detection is presented. Factors affecting instrumentation design include minimization of cost, complexity, and examination time while maintaining high fidelity NIRST measurements with sufficient information to recover accurate optical property maps. METHODS: Reconstructed DBT slices from eight patients with abnormal mammograms provided anatomical information for the NIRST simulations. A limited frequency domain (FD) and extensive continuous wave (CW) NIRST system was modeled. The FD components provided tissue scattering estimations used in the reconstruction of the CW data. Scattering estimates were perturbed to study the effects on hemoglobin recovery. Breast mimicking agar phantoms with inclusions were imaged using the combined DBT∕NIRST system for comparison with simulation results. RESULTS: Patient simulations derived from DBT images show successful reconstruction of both normal and malignant lesions in the breast. They also demonstrate the importance of accurately quantifying tissue scattering. Specifically, 20% errors in optical scattering resulted in 22.6% or 35.1% error in quantification of total hemoglobin concentrations, depending on whether scattering was over- or underestimated, respectively. Limited frequency-domain optical signal sampling provided two regions scattering estimates (for fat and fibroglandular tissues) that led to hemoglobin concentrations that reduced the error in the tumor region by 31% relative to when a single estimate of optical scattering was used throughout the breast volume of interest. Acquiring frequency-domain data with six wavelengths instead of three did not significantly improve the hemoglobin concentration estimates. Simulation results were confirmed through experiments in two-region breast mimicking gelatin phantoms. CONCLUSIONS: Accurate characterization of scattering is necessary for quantification of hemoglobin. Based on this study, a system design is described to optimally combine breast tomosynthesis with NIRST.