PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells

We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.     

Fluorescent Bone Viewed through Toenails of Living Animals: a Method to Observe Bone Regrowth

We have developed a new method to observe bone and to document growth in living animals. The technique involves injecting calcein, a fluorescent calcium deposition marker, waiting approximately 4 hr for it to clear the vascular system, and observing bone directly through the toenails of lightly anesthetized living animals. Bone regrowth can be monitored in situ by amputating the digit through the nail plate, waiting the desired number of days, and injecting a second fluorescent label, alizarin red. Bone that has regrown since the amputation appears as a red area distal to the green calcein label on toes of lightly anesthetized animals when viewed under FITC fluorescence. This method has been used to demonstrate blocked bone synthesis and to quantitate significant differences in bone growth in control and experimental toes of individual animals. Advantages of this method include its simplicity, the use of fewer animals to collect sequential data, and increased reliability of repeated microscopic measurements using the same animal.     

Fluorescent Bone Viewed through Toenails of Living Animals: a Method to Observe Bone Regrowth

We have developed a new method to observe bone and to document growth in living animals. The technique involves injecting calcein, a fluorescent calcium deposition marker, waiting approximately 4 hr for it to clear the vascular system, and observing bone directly through the toenails of lightly anesthetized living animals. Bone regrowth can be monitored in situ by amputating the digit through the nail plate, waiting the desired number of days, and injecting a second fluorescent label, alizarin red. Bone that has regrown since the amputation appears as a red area distal to the green calcein label on toes of lightly anesthetized animals when viewed under FITC fluorescence. This method has been used to demonstrate blocked bone synthesis and to quantitate significant differences in bone growth in control and experimental toes of individual animals. Advantages of this method include its simplicity, the use of fewer animals to collect sequential data, and increased reliability of repeated microscopic measurements using the same animal.     

A Theoretical Analysis of the Effects of Tumor-Treating Electric Fields on Single Cells

Tumor-treating fields (TTFields) are low-intensity and intermediate-frequency alternating electric fields that have been found to inhibit tumor cell growth. While effective, the mechanism by which TTFields affect cell growth is not yet clearly understood. Although numerous mathematical studies on the effects of electromagnetic fields on single cells exist, the effect of TTFields on single cells have been analyzed less frequently. The goal of this study is to explore through a mathematical analysis the effects of TTFields on single cells, with particular emphasis on the thermal effect. We examine herein two single-cell models, a simplified spheroidal model and a simulation of a U-87 MG glioblastoma cell model obtained from microscopic images. A finite element method is used to analyze the electric field distribution, electromagnetic loss, and thermal field distribution. The results further prove that the electric field in the cytoplasm is too weak and its thermal damage can be excluded as a mechanism for cell death in TTFields. Bioelectromagnetics. 2020;41:438–446. © 2020 Bioelectromagnetics Society.     

Multiscale Investigation of Sodium-Ion Battery Anodes: Analytical Techniques and Applications

The anode/electrolyte interface behavior, and by extension, the overall cell performance of sodium-ion batteries is determined by a complex interaction of processes that occur at all components of the electrochemical cell across a wide range of size- and timescales. Single-scale studies may provide incomplete insights, as they cannot capture the full picture of this complex and intertwined behavior. Broad, multiscale studies are essential to elucidate these processes. Within this perspectives article, several analytical and theoretical techniques are introduced, and described how they can be combined to provide a more complete and comprehensive understanding of sodium-ion battery (SIB) performance throughout its lifetime, with a special focus on the interfaces of hard carbon anodes. These methods target various length- and time scales, ranging from micro to nano, from cell level to atomistic structures, and account for a broad spectrum of physical and (electro)chemical characteristics. Specifically, how mass spectrometric, microscopic, spectroscopic, electrochemical, thermodynamic, and physical methods can be employed to obtain the various types of information required to understand battery behavior will be explored. Ways are then discussed how these methods can be coupled together in order to elucidate the multiscale phenomena at the anode interface and develop a holistic understanding of their relationship to overall sodium-ion battery function.     

Living on the edge: Multiscale habitat selection by cheetahs in a human‐wildlife landscape

Animals select habitats that will ultimately optimize their fitness through access to favorable resources, such as food, mates, and breeding sites. However, access to these resources may be limited by bottom‐up effects, such as availability, and top‐down effects, such as risk avoidance and competition, including that with humans. Competition between wildlife and people over resources, specifically over space, has played a significant role in the worldwide decrease in large carnivores. The goal of this study was to determine the habitat selection of cheetahs (Acinonyx jubatus) in a human‐wildlife landscape at multiple spatial scales. Cheetahs are a wide‐ranging, large carnivore, whose significant decline is largely attributed to habitat loss and fragmentation. It is believed that 77% of the global cheetah population ranges outside protected areas, yet little is known about cheetahs’ resource use in areas where they co‐occur with people. The selection, or avoidance, of three anthropogenic variables (human footprint density, distance to main roads and wildlife areas) and five environmental variables (open habitat, semiclosed habitat, edge density, patch density and slope), at multiple spatial scales, was determined by analyzing collar data from six cheetahs. Cheetahs selected variables at different scales; anthropogenic variables were selected at broader scales (720–1440 m) than environmental variables (90–180 m), suggesting that anthropogenic pressures affect habitat selection at a home‐range level, whilst environmental variables influence site‐level habitat selection. Cheetah presence was best explained by human presence, wildlife areas, semiclosed habitat, edge density and slope. Cheetahs showed avoidance for humans and steep slopes and selected for wildlife areas and areas with high proportions of semiclosed habitat and edge density. Understanding a species’ resource requirements, and how these might be affected by humans, is crucial for conservation. Using a multiscale approach, we provide new insights into the habitat selection of a large carnivore living in a human‐wildlife landscape.     

Simultaneous Raman Microspectroscopy and Fluorescence Imaging of Bone Mineralization in Living Zebrafish Larvae

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.Understanding fundamental biological processes relies on probing intra- and extracellular environments, targeted delivery inside living cells and tissues, and real-time detection and imaging of chemical markers and biomolecules (1,2). Typically, information about molecules in cellular environments is obtained by fluorescence microscopy (3). This is a powerful imaging tool for localizing and imaging samples but requires fluorescent labels and markers and lacks capabilities for quantitative mapping of the chemical composition in complex systems. In this regard, confocal Raman spectroscopic imaging is becoming increasingly popular for label-free chemical detection, due to the inherent scattering nature of all biomolecules (4,5). However, confocal Raman imaging alone does not allow live, high-resolution imaging of larger regions of interest in complex biological tissues. Transcutaneous Raman spectroscopy has the potential as a tool for in vivo bone quality assessment (6), whereas the time- and space-resolved Raman spectroscopy allows the visualization in vivo of the distributions of molecular species in human and yeast cells (4,5,7). Here we developed a correlative Raman and fluorescence imaging method that combines the strengths and compensates for the shortcomings of each of these imaging modalities and allows studying in vivo processes in complex animal models such as zebrafish larvae. There are two main advantages of this approach over previous studies (8,9): low light intensity and high acquisition rate, making it well suited for real-time investigation of live samples.Fig. 1, a and b, shows a schematic representation of the experimental setup and of the optical path, respectively. The two techniques are implemented on a commercially available Raman microscope body to perform simultaneously confocal Raman spectroscopy and wide-field fluorescence imaging (see the Supporting Material for details of components). Briefly, the multimodality of the setup is provided by a combination of dichroic mirrors (DM 1–3) and filters that at turns reflect or transmit the excitation and emission signals. This combination of optics allows simultaneous collection of fluorescence images (2560 × 2160 pixels at 30 fps) with excitation at 400 and 490 nm and spatially resolved Raman spectra with excitation at 633 nm.Open in a separate windowFigure 1Fluorescence imaging of zebrafish larvae. (a) Cartoon of the experimental setup showing how the different modules are assembled onto the microscope for the simultaneous use of confocal Raman spectroscopy and fluorescence imaging. (b) Schematic representation of the optical path. (c) Fluorescence image of calcium-containing tissues, and fluids stained with calcein blue and excited at 400 nm (top). Endothelial cells of transgenic tg(fli1:EGFP)y1 zebrafish excited at 490 nm (bottom).As a proof of principle, we have studied the different mineral phases involved in bone formation of the zebrafish larvae. The bone development process involves the transport of ions to specific cells (osteoblasts) that are responsible for the subsequent mineral formation and deposition. The mineral phase in these cells is a poorly characterized disordered calcium phosphate (10–12). The mineral-bearing intracellular vesicles release their content into the extracellular collagen fibrils, where the mineral subsequently crystallizes as carbonated hydroxyapatite (13). Very little is known about the phase transformations the mineral undergoes after the deposition into the collagen matrix in vivo. Raman spectroscopy studies of bone tissue in organ cultures evidenced that the inorganic mineral deposition proceeds through transient intermediates including octacalcium phosphate-like (OCP) minerals (14).To assess the feasibility of imaging a vertebrate organism, fluorescence images of an entire zebrafish larva (Fig. 1 c) were acquired with the correlative fluorescence-Raman setup. The two images in Fig. 1 c were composed by merging several low-magnification (10×) fluorescence images. Larvae of transgenic zebrafish Tg(fli:EGFP); nac mutants (albino fish) expressing EGFP in the cytoplasm of endothelial cells was used. The newly formed bones were stained by soaking the live embryo noninvasively in the calcium markers calcein blue 0.2% wt or calcein green 0.2% wt.The calcein blue marker is excited at 400 nm. It is labeling bones and can be also detected as a fluorescent marker not associated with formed bones (e.g., stomach) (Fig. 1 c, top). At 490 nm, calcein green and endothelial cells within blood vessels expressing EGFP are excited (Fig. 1 c, bottom). Because EGFP and calcein blue have significantly different excitation and emissions spectra, dual staining with calcein blue (as a mineral marker) and EGFP allows fast-switching dual-wavelength fluorescence imaging. Furthermore, because the spectra of the calcium markers and EGFP do not extend beyond the Raman laser, these fluorophores are appropriate candidates for experiments requiring Raman and fluorescence imaging. The dual-excitation offers the capability of mapping several tissues in a single experiment at the video rate. This, in principle, could be used to probe different parameters of the microenvironment (e.g., pH (15), temperature (16), viscosity (17), and calcium concentration (18)) using wavelength-ratiometric fluorescence imaging which, in correlation with confocal Raman spectroscopy, could open new strategies in studies of the microenvironmental properties in living tissues.The fin rays of zebrafish are a simple, growing bone-model system, in which the fins are gradually mineralized within spatially resolved regions (19). Raman spectroscopy revealed details of the calcein green-stained fin where new bone is deposited (Fig. 2). In Fig. 2 a, a fluorescence image of a zebrafish larva analogous to the top image in Fig. 1 c is shown. The right inset in Fig. 3 b shows higher-magnification (60× water-immersion objective) details of the calcein green-stained fin typical of newly deposited bone. Raman spectra of progressively mineralized bone tissue were acquired within representative regions (Fig. 2 b; numbered 1–4). The spectra exhibit characteristic bands that can be assigned to the organic protein extracellular matrix (amide I, amide III, Phe, C-H, etc.) and the inorganic mineral content (v₁, v₂, v₄ of PO₄³⁻).Open in a separate windowFigure 2Correlative fluorescence-Raman imaging of zebrafish fin bone maturation. (a) Low-resolution (10×) fluorescence image of zebrafish stained with calcein green, with high-resolution (60×) details (right inset in panel b) of a representative fin ray region where Raman spectra (b) of progressively mineralized bone tissue were acquired (numbered 1–4). (Left inset in panel b) Integral of the orientation independent mineral band (v₂) where a clear drop of the mineral content can be observed.The analyses of the orientation-independent v₂ phosphate band revealed a clear drop in the mineral content based on the intensity integral (left inset in Fig. 2 b). Assuming that the spectrum collected in region 4 contains only organic matrix (very small phosphate-related peaks) and by subtracting it from the spectrum of mineral-rich bone region (spectrum 1, proximal part of the tail bone), spectral features of only the mineral phase can be plotted (black line). In addition to the phosphate (PO₄³⁻) and carbonate (CO₃²⁻) bands assignable to the carbonated apatite phase characteristic of the more mature bone mineral, several peaks related to the hydrogen phosphate (HPO₄²⁻) species can be clearly distinguished.The HPO₄²⁻ peaks are characteristic of the OCP mineral phase that has been postulated, together with amorphous calcium phosphate, as an intermediate mineral phase in the process of bone maturation (10,13,14,20), but never observed directly in living animals. Our findings show in vivo potential of the correlative setup envisioned by Crane et al. (14) and confirm that the mineral maturation indeed proceeds through an OCP-like mineral phase. Further analysis of the mineral spectrum in Fig. 2 b reveals an extremely broad band in the region 800–1100 cm⁻¹. This envelope can be related to hydrogenated phosphate species typical of amorphous calcium phosphate precipitated in an acidic environment (see Fig. S1 in the Supporting Material), suggesting that this phase is also contributing to the maturation process.In conclusion, the methodology developed here allows for unprecedented chemical characterization of fluorescently-labeled biological tissues in vivo. The approach is suitable for long-term in vivo characterization of zebrafish bone mineralization under (patho-)physiological conditions. Furthermore, the setup can be upgraded to host other advance fluorescence imaging techniques such as super-resolution microscopy (e.g., photoactivated localization microscopy), two-photon excitation, and Forster resonance energy transfer or fluorescence lifetime imaging microscopy, and be applied on both in vivo and in vitro specimens. This opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces.     

Simultaneous Raman Microspectroscopy and Fluorescence Imaging of Bone Mineralization in Living Zebrafish Larvae

Confocal Raman microspectroscopy and fluorescence imaging are two well-established methods providing functional insight into the extracellular matrix and into living cells and tissues, respectively, down to single molecule detection. In living tissues, however, cells and extracellular matrix coexist and interact. To acquire information on this cell-matrix interaction, we developed a technique for colocalized, correlative multispectral tissue analysis by implementing high-sensitivity, wide-field fluorescence imaging on a confocal Raman microscope. As a proof of principle, we study early stages of bone formation in the zebrafish (Danio rerio) larvae because the zebrafish has emerged as a model organism to study vertebrate development. The newly formed bones were stained using a calcium fluorescent marker and the maturation process was imaged and chemically characterized in vivo. Results obtained from early stages of mineral deposition in the zebrafish fin bone unequivocally show the presence of hydrogen phosphate containing mineral phases in addition to the carbonated apatite mineral. The approach developed here opens significant opportunities in molecular imaging of metabolic activities, intracellular sensing, and trafficking as well as in vivo exploration of cell-tissue interfaces under (patho-)physiological conditions.     

退休教师骨密度的超声研究

目的：了解老年教师骨质疏松病的情况,用超声骨密度仪对813例老年教师做跟骨超声振幅衰减值研究。方法：超声测量是一种新技术,利用不同密度的物体衰减值不同及穿过不同密度物体声速不同等特性对跟骨进行成像,并计算最具说明性区域的一系列数据。参数中BUA值为最主要的参数,三根等距离曲线的形态和宽度能直观分析出年龄与骨量变化的关系。结果：本文男性平均BUA为63.87dB/MHz,得出回归公式：体重=189.1417 0.2062BUA-0.0224SOS。女性平均BUA为58.9dB/MHz,各年龄分组的BUA值与参考值很接近,统计学显示无意义。结论：调查发现男性和女性的骨质不同,男性BUA骨峰值比女性高。BUA值与其体重有密切关系。在60岁以后（或绝经1-5年后）年龄增长与BUA值无明显关系,说明随着年龄的增加骨量减少很小或不减少。利用这种关系,假如BUA值突然下降则提示近期发生骨质疏松。     

Spectroscopic Investigation of Local Mechanical Impedance of Living Cells

We studied nanoscale mechanical properties of PC12 living cells with a Force Feedback Microscope using two experimental approaches. The first one consists in measuring the local mechanical impedance of the cell membrane while simultaneously mapping the cell morphology at constant force. As the interaction force is increased, we observe the appearance of the sub-membrane cytoskeleton. We compare our findings with the outcome of other techniques. The second experimental approach consists in a spectroscopic investigation of the cell while varying the tip indentation into the membrane and consequently the applied force. At variance with conventional dynamic Atomic Force Microscopy techniques, here it is not mandatory to work at the first oscillation eigenmode of the cantilever: the excitation frequency of the tip can be chosen arbitrary leading then to new spectroscopic AFM techniques. We found in this way that the mechanical response of the PC12 cell membrane is found to be frequency dependent in the 1 kHz - 10 kHz range. In particular, we observe that the damping coefficient consistently decreases when the excitation frequency is increased.     

Donor Exclusion in the National Blood Service Tissue Services Living Bone Donor Programme

National Blood Service (NBS) Tissue Services (TS) operates living donor and deceased donor tissue banking programmes. The living bone donor programme operates in collaboration with 91 orthopaedic departments across the country and collects bone donations, in the form of surgically removed femoral heads (FHs), from over 5000 patients per annum undergoing total hip replacement. Bone donated via the living programme constitutes approximately 55% of the total bone donated to NBS. Non-NBS tissue banks, primarily in hospital orthopaedic departments, also bank donated bone for the UK. A survey of information received from 16 collaborating orthopaedic centres, between April 2003 and August 2004, identified 709 excluded donors. The total number of donations banked from these sites was 1538. Donations can be excluded before collection if there are contraindications noted in a potential donor’s medical history before their operation. Donors may also be excluded after collection of the FH, for instance because of reactive microbiology tests for blood borne viruses, or if the donation storage conditions or related documentation have not met stringent quality requirements. In this survey, bone or joint conditions were the major reasons for excluding potential donors before donation (154 of 709 exclusions, 22%), followed by a current or a past history of malignancy (139 of 709 exclusions, 20%). Local staffing and operational difficulties sometimes resulted in potential donors being missed, or specific reasons for exclusion not being reported (117 exclusions). These out numbered exclusions due to patient refusal (80 exclusions). A small number (< 5) appear to have been excluded erroneously. There was considerable local variation in the reasons given for exclusion and certainly under-reporting. A survey of donations discarded after collection in the same period highlighted that 43% were donor related; 110 of 370 did not provide a follow-up blood sample. More than 30% were due to delays in forwarding blood samples to the microbiological laboratory for testing, resulting in deterioration of the sample quality. Training to ensure that standards are complied with and a firm evidence base for exclusion criteria, applied uniformly, will help focus donor identification efforts on individuals meeting rational criteria so that fewer potential donations are lost.     

现生太行山猕猴与猕猴化石骨指数的比较

为探讨现生太行山猕猴与猕猴化石骨指数的差异,对太行山猕猴与广西崇左早更新世猕猴化石的肢间指数(Intermembral index,IM)、臂指数(Brachial index,BI)、股指数(Crural index,CI)和股骨粗壮指数(Robusticity index,RI)等指数进行比较。结果表明,猕猴化石IM值(96)高于太行山猕猴,BI值(94.5)和CI值(88.5)均低于太行山猕猴,推测该猕猴化石在早更新世时期可能地栖生活,适合于陆地四足行走,同时也验证了太行山猕猴主要为半树栖生活。结合对猕猴化石伴生哺乳动物习性的分析,推测广西崇左早更新世的气候温暖潮湿并有一定的水域,植被以森林和灌木为主,有局部的草地或草坡,其自然环境非常适宜高等灵长类生息繁衍。     

Multiscale,Converging Defects of Macro-Porosity,Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.     

Bone Mineral Density Measurements,Bone Markers and Serum Vitamin D Concentrations in Men with Chronic Non-Cirrhotic Untreated Hepatitis C

Introduction

The high prevalence of chronic hepatitis C (CHC) and its consequent cirrhosis has been associated with bone fragility. Whether CHC may cause bone and mineral abnormalities in the absence of hepatocellular dysfunction is still unknown. In this study we aimed to determine the prevalence of osteoporotic vertebral fractures and low BMD measurements in men with non-cirrhotic CHC. Risk factors for low BMD and fractures were also investigated.

Methods

Morphometric vertebral fractures and BMD measurements were performed in 60 non-cirrhotic untreated men with CHC and 59 healthy controls, matched for age and gender, weight and current smoking. Serum CTx, calcium, phosphate, intact PTH, alkaline phosphatase and vitamin D (25OHD) concentrations were measured in all participants. Clinical risk factors for low BMD and fractures were evaluated by a structured questionnaire as well as details regarding HCV infection.

Results

Trochanter and total femur BMD were significantly lower in CHC patients as compared to healthy men (p = 0.04). In men 50 years and older, the prevalence of osteoporosis was significantly higher among CHC patients (p = 0.01). Lower levels of physical activities and more often report of prolonged immobilization were observed among CHC patients (p<0.05). Liver inflammation and fibrosis, viral load and genotype did not correlate with BMD measurements. Bone markers and 25OHD concentrations were similar in both groups. Only a few vertebral fractures were observed.

Conclusions

Our results demonstrate that non-cirrhotic untreated CHC patients have lower BMD at the femur as compared to healthy men in spite of the absence of significant bone and mineral abnormalities.     

Antidepressant Treatment Outcome Depends on the Quality of the Living Environment: A Pre-Clinical Investigation in Mice

Antidepressants represent the standard treatment for major depression. However, their efficacy is variable and incomplete. A growing number of studies suggest that the environment plays a major role in determining the efficacy of these drugs, specifically of selective serotonin reuptake inhibitors (SSRI). A recent hypothesis posits that the increase in serotonin levels induced by SSRI may not affect mood per se, but enhances neural plasticity and, consequently, renders the individual more susceptible to the influence of the environment. Thus, SSRI administration in a favorable environment would lead to a reduction of symptoms, while in a stressful environment might lead to a worse prognosis. To test this hypothesis, we treated C57BL/6 adult male mice with chronic fluoxetine while exposing them to either (i) an enriched environment, after exposure to a chronic stress period aimed at inducing a depression-like phenotype, or (ii) a stressful environment. Anhedonia, brain BDNF and circulating corticosterone levels, considered endophenotypes of depression, were investigated. Mice treated with fluoxetine in an enriched condition improved their depression-like phenotype compared to controls, displaying higher saccharin preference, higher brain BDNF levels and reduced corticosterone levels. By contrast, when chronic fluoxetine administration occurred in a stressful condition, mice showed a more distinct worsening of the depression-like profile, displaying a faster decrease of saccharin preference, lower brain BDNF levels and increased corticosterone levels. Our findings suggest that the effect of SSRI on depression-like phenotypes in mice is not determined by the drug per se but is induced by the drug and driven by the environment. These findings may be helpful to explain variable effects of SSRI found in clinical practice and to device strategies aimed at enhancing their efficacy by means of controlling environmental conditions.     

老年男性教师骨密度的调查

目的 为了解老年性骨质疏松情况,用超声骨密度仪对150例老年男性教师做跟骨超振幅衰减值的测试研究。方法：用DMS公怀UBIS5000型骨密度超声仪检查跟骨,研究受检者的年龄、体重、身高和巳患病咎与测得超声振幅衰减值（BUA）,声速（SOS）,骨硬度（STI）和相对骨折危险性（RRF）等参数之间的关系。结果：本仪器输出3种结果供医师参考：1.一幅灰阶图,2.一幅彩色图和曲线图,3.感兴趣区所测得的参数,受检者的平均BUA为63.87dB/MHz,SOS为1485.21m/s,本文数据经Statpal软件处理,得出回归公式：体重=189.1417 0.2062BUA-0.0224SOS,R=0.0466,P值有显著意义。结论：BUA值为主要参数,SOS为补充参数,作为评估老年男性教师的骨密度情况,假如BUA值突然下降则提示近期可能发生骨质疏松。     

Dimensionality of Carbon Nanomaterials Determines the Binding and Dynamics of Amyloidogenic Peptides: Multiscale Theoretical Simulations

Experimental studies have demonstrated that nanoparticles can affect the rate of protein self-assembly, possibly interfering with the development of protein misfolding diseases such as Alzheimer''s, Parkinson''s and prion disease caused by aggregation and fibril formation of amyloid-prone proteins. We employ classical molecular dynamics simulations and large-scale density functional theory calculations to investigate the effects of nanomaterials on the structure, dynamics and binding of an amyloidogenic peptide apoC-II(60-70). We show that the binding affinity of this peptide to carbonaceous nanomaterials such as C60, nanotubes and graphene decreases with increasing nanoparticle curvature. Strong binding is facilitated by the large contact area available for π-stacking between the aromatic residues of the peptide and the extended surfaces of graphene and the nanotube. The highly curved fullerene surface exhibits reduced efficiency for π-stacking but promotes increased peptide dynamics. We postulate that the increase in conformational dynamics of the amyloid peptide can be unfavorable for the formation of fibril competent structures. In contrast, extended fibril forming peptide conformations are promoted by the nanotube and graphene surfaces which can provide a template for fibril-growth.     

A Numerical Investigation of the Electric and Thermal Cell Kill Distributions in Electroporation-Based Therapies in Tissue

Electroporation-based therapies are powerful biotechnological tools for enhancing the delivery of exogeneous agents or killing tissue with pulsed electric fields (PEFs). Electrochemotherapy (ECT) and gene therapy based on gene electrotransfer (EGT) both use reversible electroporation to deliver chemotherapeutics or plasmid DNA into cells, respectively. In both ECT and EGT, the goal is to permeabilize the cell membrane while maintaining high cell viability in order to facilitate drug or gene transport into the cell cytoplasm and induce a therapeutic response. Irreversible electroporation (IRE) results in cell kill due to exposure to PEFs without drugs and is under clinical evaluation for treating otherwise unresectable tumors. These PEF therapies rely mainly on the electric field distributions and do not require changes in tissue temperature for their effectiveness. However, in immediate vicinity of the electrodes the treatment may results in cell kill due to thermal damage because of the inhomogeneous electric field distribution and high current density during the electroporation-based therapies. Therefore, the main objective of this numerical study is to evaluate the influence of pulse number and electrical conductivity in the predicted cell kill zone due to irreversible electroporation and thermal damage. Specifically, we simulated a typical IRE protocol that employs ninety 100-µs PEFs. Our results confirm that it is possible to achieve predominant cell kill due to electroporation if the PEF parameters are chosen carefully. However, if either the pulse number and/or the tissue conductivity are too high, there is also potential to achieve cell kill due to thermal damage in the immediate vicinity of the electrodes. Therefore, it is critical for physicians to be mindful of placement of electrodes with respect to critical tissue structures and treatment parameters in order to maintain the non-thermal benefits of electroporation and prevent unnecessary damage to surrounding healthy tissue, critical vascular structures, and/or adjacent organs.