Biophysical model for high-throughput tumor and epithelial cell co-culture in complex biochemical microenvironments

The in vivo tumor microenvironment is a complex niche that includes heterogeneous physical structures,unique biochemical gradients and multiple cell interactions.Its high-fidelity in vitro reconstruction is of fundamental importance to improve current understandings of cell behavior,efficacy predictions and drug safety.In this study,we have developed a high-throughput biochip with hundreds of composite extracellular matrix(ECM)microchambers to co-culture invasive breast cancer cells(MDA-MB-231-RFP)and normal breast epithelial cells(MCF-10 A-GFP).The composite ECM is composed of type I collagen and Matrigel which provides a heterogeneous microenvironment that is similar to that of in vivo cell growth.Additionally,the growth factors and drug gradients that involve human epidermal growth factor(EGF),discoidin domain receptor 1(DDR1)inhibitor 7 rh and matrix metalloproteinase inhibitor batimastat allow for the mimicking of the complex in vivo biochemical microenvironment to investigate their effect on the spatial-temporal dynamics of cell growth.Our results demonstrate that the MDA-MB-231-RFP cells and MCF-10 A-GFP cells exhibit different spatial proliferation behaviors under the combination of growth factors and drugs.Basing on the experimental data,we have also developed a cellular automata(CA)model that incorporated drug diffusion to describe the experimental phenomenon,as well as employed Shannon entropy(SE)to explore the effect of the drug diffusion coefficient on the spatial-temporal dynamics of cell growth.The results indicate that the uniform cell growth is related to the drug diffusion coefficient,which reveals that the pore size of the ECM plays a key role in the formation of complex biochemical gradients.Therefore,our integrated,biomimetic and high-throughput co-culture platforms,as well as the computational model can be used as an effective tool for investigating cancer pathogenesis and drug development.     

Ultrasound-induced cytolysis of cancer cells is enhanced in the presence of micron-sized alumina particles

Micron-sized alumina particles have been shown to enhance sonochemical free radical formation in aqueous solutions and simultaneously increase the solution temperature and acoustic (white) noise, effects attributable to enhanced inertial cavitation [T. Tuziuti, J. Phys. Chem. A 109 (2005) 4869–4872]. In the current study, the same ultrasound exposure system was applied to in vitro cancer cells as a model system to determine the effect of alumina particles on the long-term survival of cells and on the major pathways of cell death, i.e., either apoptosis or necrosis. Following 6 h of incubation after ultrasound treatment, it was found that the cells died mainly through necrosis, irrespective of whether the exposure was conducted in the presence of alumina particles or not. Alumina particles were non-toxic to cells alone, but were found to decrease the long-term survivability of cells that survived the initial exposure. This effect depended on the size and concentration of particles. These results correlated well with the effect of alumina particles on the sonochemical oxidation of KI under the same exposure conditions. Spin-trapping with 5,5-dimethyl-pyroline N-oxide (DMPO) and electron spin resonance spectroscopy indicated that the sonochemical formation of OH radicals increased in the presence of alumina particles. The current study is consistent with the well known observation that micron-sized particles enhance the acoustic cavitation process.     

In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment

In this study was developed a new nano drug delivery system (NDDS) based on association of biodegradable surfactants with biocompatible magnetic fluid of maguemita citrate derivative. This formulation consists in a magnetic emulsion with nanostructured colloidal particles. Preliminary in vitro experiments showed that the formulation presents a great potential for synergic application in the topical release of photosensitizer drug (PS) and excellent target tissue properties in the photodynamic therapy (PDT) combined with hyperthermia (HPT) protocols. The physical chemistry characterization and in vitro assays were carried out by Zn(II) Phtalocyanine (ZnPc) photosensitizer incorporated into NDDS in the absence and the presence of magnetic fluid, showed good results and high biocompatibility. In vitro experiments were accomplished by tape-stripping protocols for quantification of drug association with different skin tissue layers. This technique is a classical method for analyses of drug release in stratum corneum and epidermis+dermis skin layers. The NDDS formulations were applied directly in pig skin (tissue model) fixed in the cell's Franz device with receptor medium container with a PBS/EtOH 20% solution (10 mM, pH 7.4) at 37 °C. After 12 h of topical administration stratum corneum was removed from fifty tapes and the ZnPc retained was evaluated by solvent extraction in dimetil-sulphoxide under ultrasonic bath. These results indicated that magnetic nanoemulsion (MNE) increase the drug release on the deeper skin layers when compared with classical formulation in the absence of magnetic particles. This could be related with the increase of biocompatibility of NDDS due to the great affinity for the polar extracelullar matrix in the skin and also for the increase in the drug partition inside of corneocites wall.     

In vitro three-dimensional cancer metastasis modeling:Past,present, and future

Metastasis is the leading cause of most cancer deaths, as opposed to dysregulated cell growth of the primary tumor.Molecular mechanisms of metastasis have been studied for decades and the findings have evolved our understanding of the progression of malignancy. However, most of the molecular mechanisms fail to address the causes of cancer and its evolutionary origin, demonstrating an inability to find a solution for complete cure of cancer. After being a neglected area of tumor biology for quite some time, recently several studies have focused on the impact of the tumor microenvironment on cancer growth. The importance of the tumor microenvironment is gradually gaining attention, particularly from the perspective of biophysics. In vitro three-dimensional(3-D) metastatic models are an indispensable platform for investigating the tumor microenvironment, as they mimic the in vivo tumor tissue. In 3-D metastatic in vitro models, static factors such as the mechanical properties, biochemical factors, as well as dynamic factors such as cell–cell, cell–ECM interactions, and fluid shear stress can be studied quantitatively. With increasing focus on basic cancer research and drug development, the in vitro 3-D models offer unique advantages in fundamental and clinical biomedical studies.     

三维微纳米制造技术在癌症生物物理研究中的应用

癌症致命的主要原因是癌细胞在临床上的转移性. 癌细胞的侵袭和转移是一个非常复杂的三维过程, 但现有的癌症研究在活体上有诸多观测和操作上的困难. 而体外实验又通常在培养皿中进行, 其二维的生长环境已完全不能满足对癌细胞空间转移性的深入研究, 故在活体外构建出癌细胞侵袭和转移的三维物理模型具有十分重要的意义. 然而如何在体外尽可能真实地模拟体内癌细胞的生长微环境一直是困扰科学家的难题. 本文系统介绍了三维微纳米制造的几种主流技术, 探讨了它们在癌症生物物理研究中的应用和发展. 在此基础上为了在未来实现对体外三维模型的制造、观测和精确操作, 文章还创新性地提出了一种结合紫外线固化生物型水凝胶的三维成型技术、光片三维成像技术以及微纳米探针控制技术的一体化研究平台. 这些先进的技术和理念, 势必会逐步升级现有传统的癌症研究手段, 为未来理解和治疗癌症揭开全新的篇章.     

胶原纤维网络和癌细胞的力学微环境

文章以第一类胶原纤维网络为例, 着重分析了癌细胞三维微环境的多尺度结构及力学特征. 对于细胞与细胞外介质结合的蛋白集团、单个细胞以及细胞群体, 分别由单个纤维(或亚纤维)、纤维集束以及纤维网络整体来决定相应的力学环境. 文章同时也讨论了胶原纤维(及其类似材料) 的局限性.     

Effect of exposure parameters on cavitation induced by low-level dual-frequency ultrasound

In order to quantify the effects of exposure parameters under therapeutic conditions such as sonodynamic therapy, it is necessary initially to evaluate the inertial cavitation activity in vitro. In this study, the dependence of cavitation activity induced by the low-level dual-frequency ultrasound irradiation on exposure parameters has been studied. Experiments were performed in the near 150 kHz and 1 MHz fields in the progressive wave mode. It has been shown that at constant ultrasound energy the fluorescence intensity for continuous sonication is higher than for pulsed mode. With increasing the duty cycle of pulsed field, the inertial cavitation activity is increased. The activity of cavitation produced by simultaneous combined sonication by two ultrasound fields is remarkably higher than the algebraic sum of effects produced by fields separately (p-value < 0.05). This study shows that simultaneous combined dual-frequency ultrasound sonication in continuous mode is more effective in producing inertial cavitation activity at low-level intensity. Therefore, it is concluded that investigations in this combined ultrasound sonication can be useful in sonodynamic therapy for superficial tumors.     

基于19F化学标记磷酸化泛素的温度传感器开发

泛素是一种真核细胞信号分子,主要参与蛋白质降解和DNA修复等生命活动.泛素Ser65位被磷酸化之后,在溶液中呈现两个稳定的溶液构象,这两种构象的比例能够被pH调控,本研究利用NMR进一步发现它还受到温度影响.基于该发现,对磷酸化泛素进行了¹⁹F化学标记,利用¹⁹F NMR方法表征了不同温度下磷酸化泛素两种构象的比例,发现两者比例变化与温度之间的关系可以通过线性方程来描述,利用该方程可以通过构象比例计算样品内部温度,因此可以作为一种基于NMR检测的温度传感器.本文所开发的基于¹⁹F化学标记磷酸化泛素的温度传感器不仅能够作为体外样品温度检测的有力工具,还有望用于检测细胞内部的温度.从而有助于揭示生物学特性和功能.     

鼠S180肉瘤的体内31P MRS研究

利用表面线圈³¹P NMR研究了小鼠S180肉瘤生长过程中能量代谢和磷脂类变化的特点.结果发现:随着肿瘤体积的增大,(1)Pi和PME升高;(2)PCr降低,在肿瘤体积较大时常检测不到;(3)β-NTP(通常用来表示ATP的量)变化较小;(4)PDE波动性较大;(5)PCr/Pi和β-NTP/Pi比值均下降,且PCr/Pi比β-NTP/Pi下降得快;(6)PME/β-NTP比值升高;(7)肿瘤pH下降,且与PCr/Pi、β-NTP/Pi或(PCr+β-NTP)/Pi比值有相关性.讨论了与这些参数变化相关联的生物学意义.     

Intracellular pH mapping with SNARF-1 and confocal microscopy. I: A quantitative technique for living tissue and isolated cells

A fluorescent pH indicator in conjunction with confocal microscopy, was used to map intracellular pH in a variety of cells and tissues with high spatial resolution. The new pH-sensitive fluorescent probe SNARF-1 was excited with the 488 nm band of the argon ion laser of a Bio-Rad MRC-500 confocal microscope. Ratio images were created with pixel-by-pixel division, with the intensity of these images representing a function of pH, that is independent of dye concentration, photobleaching or path length. Cell cultures of rat aortic smooth muscle were loaded with 20 μм SNARF-1/AM for 20 min at 37°C. Intracellular pH levels were calibrated in situ by treatment of each cell with nigericin (20 μм) in solutions of known pH. The cytosolic pH of the majority of cells was uniform, however, pH gradients were evident between the cytosol and nuclear regions, indicating the ability of this technique to map intracellular and intraorganelle pH. Rat C6 glioblastoma spheroids were cultured then loaded with SNARF-1/AM at 10°C for 90 min. The pH values were calibrated in vitro, using SNARF-1 acid in buffered solutions of known pH. Ratio images of the bisected spheroids showed a marked gradient in pH from the outer cells compared with central necrotic cells. The degree of involvement of acidification in muscle fatigue was investigated by simultaneously determining force generation and intracellular pH in individual fibres of an intact rat muscle. The investigation was performed during a stimulation protocol which induced significant fatigue in the force response of the muscle. The fatigue protocol induced little change in cytosolic pH in the fibres. We show that the use of SNARF-1, in conjunction with confocal microscopy is a powerful technique for accurately mapping pH within single cells, multicellular tissues and intact organs, as well as for accurately recording dynamic changes in pH.     

PEG化氘代单磷酸三苯甲基自由基的高效合成及性能研究

电子顺磁共振（EPR）波谱联用外源性自旋探针技术是测量体内外pH的有效手段.有研究表明,单磷酸取代三苯甲基（p₁TAM）自由基是目前用于检测pH的最理想的EPR探针.然而,这类探针的合成产率低、易受蛋白影响,极大限制了其生物应用.针对上述问题,本文提出了高效合成p₁TAM自由基的方法,使其总产率从文献报道的1.6%提高到了25%;同时采用PEG修饰的方法避免了白蛋白（BSA）对其产生的干扰.进一步氘代实验结果证实：非氘代PEG化（p₁TAM-H）衍生物（POP）虽与氘代PEG化（p₁TAM-D）衍生物（dPOP）具有相似的pH敏感性（其pKa分别为6.80和6.79）,但POP的EPR谱图较为复杂,而dPOP无论在低pH还是在高pH条件下均具有简单的EPR双峰信号;而且,dPOP具有比POP和p₁TAM-H更好的检测灵敏性;此外,dPOP还具有较好的生物稳定性和氧气敏感性.因此,dPOP能够用来同时检测pH和氧气,有望在生物医学方面得到更好的应用.     

Confocal microscopy of whole mount embryonic cartilage: Intracellular localization of F-actin, chick prolyl hydroxylase and type II collagen mRNA

We show that whole mount preparations of embryonic chick sterna can be analyzed with confocal laser scanning microscopy (CLSM). This technique replaces the traditional sectioning of cartilage or culturing of chondrocytes. Whole ‘chunks’ of cartilage can be stained with dyes, used for immunohistochemistry or in situ hybridization. Although other stains have been used, the stains presented include phalloidin and propidium iodide which stain filamentous actin (F-actin) and the DNA and RNA of cells, respectively. Collagen secreting endoplasmic reticulum (ER) was localized with a primary antibody to chick prolyl hydroxylase (CPH) that was detected with a secondary antibody conjugated to FITC. The intracellular localization of type II collagen mRNA was analyzed using in situ hybridization. The cDNA probe specific for the C-propeptide region of the 1 type (II) collagen mRNA was nick translated and labeled with biotin-16-dUTP. Biotin labeled probes were visualized with avidin-FITC. Depending on the intensity of the stain, we were able to analyze approximately 3–10 layers of chondrocytes. Stains penetrated into the cartilage better than antibodies and biotin-avidin labeled cDNA probes. The F-actin was located as bands of filaments in the superficial layers of the cartilage and was associated with the membranes that marked cell boundaries as deep as 10 layers of chondrocytes. The ER stained with anti-chick prolyl hydroxylase was prominent in perinuclear regions of the cells, but the antibody was only able to penetrate 4–5 cell layers. Single label in situ hybridization studies show that chondrocytes are positive for type II collagen mRNA. Similar to the immunohistochemistry, in situ hybridization probes were only able to penetrate 4–5 cell layers. The type II collagen mRNA appeared perinuclear in the chondrocytes, similar to the ER staining pattern.     

Temperature dependence of the calibration factor of radon and radium determination in water samples by SSNTD

The sensitivity of a ²²⁶Ra determination method of water samples by SSNTD was measured as a function of storage temperature during exposure. The method is based on an etched track type radon monitor, which is closed into a gas permeable foil and is immersed in the water sample. The sample is sealed in a glass vessel and stored for an exposure time of 10–30 days. The sensitivity increased more than a factor of two when the storage temperature was raised from 2 °C to 30 °C. Temperature dependence of the partition coefficient of radon between water and air provides explanation for this dependence. For practical radio-analytical application the temperature dependence of the calibration factor is given by fitting the sensitivity data obtained by measuring ²²⁶Ra standard solutions (in the activity concentration range of 0.1–48.5 kBq m⁻³) at different storage temperatures.     

High-precision nuclear magnetic resonance probe suitable for in situ studies of high-temperature metallic melts

High-temperature nuclear magnetic resonance (NMR) has proven to be very useful for detecting the temperature-induced structural evolution and dynamics in melts. However, the sensitivity and precision of high-temperature NMR probes are limited. Here we report a sensitive and stable high-temperature NMR probe based on laser-heating, suitable for in situ studies of metallic melts, which can work stably at the temperature of up to 2000 K. In our design, a well-designed optical path and the use of a water-cooled copper radio-frequency (RF) coil significantly optimize the signal-to-noise ratio (S/NR) at high temperatures. Additionally, a precise temperature controlling system with an error of less than ±1 K has been designed. After temperature calibration, the temperature measurement error is controlled within ±2 K. As a performance testing, ²⁷Al NMR spectra are measured in Zr-based metallic glass-forming liquid in situ. Results show that the S/NR reaches 45 within 90 s even when the sample's temperature is up to 1500 K and that the isothermal signal drift is better than 0.001 ppm per hour. This high-temperature NMR probe can be used to clarify some highly debated issues about metallic liquids, such as glass transition and liquid-liquid transition.     

In situ study of calcite-III dimorphism using dynamic diamond anvil cell

The phase transitions among the high-pressure polymorphic forms of CaCO₃ (cc-I, cc-II, cc-III, and cc-IIIb) are investigated by dynamic diamond anvil cell (dDAC) and in situ Raman spectroscopy. Experiments are carried out at room temperature and high pressures up to 12.8 GPa with the pressurizing rate varying from 0.006 GPa/s to 0.056 GPa/s. In situ observation shows that with the increase of pressure, calcite transforms from cc-I to cc-II at ～ 1.5 GPa and from cc-II to cc-III at ～ 2.5 GPa, and transitions are independent of the pressurizing rate. Further, as the pressure continues to increase, the cc-IIIb begins to appear and coexists with cc-III within a pressure range that is inversely proportional to the pressurizing rate. At the pressurizing rates of 0.006, 0.012, 0.021, and 0.056 GPa/s, the coexistence pressure ranges of cc-III and cc-IIIb are 2.8 GPa-9.8 GPa, 3.1 GPa-6.9 GPa, 2.7 GPa-6.0 GPa, and 2.8 GPa-4.5 GPa, respectively. The dependence of the coexistence on the pressurizing rate may result from the influence of pressurizing rate on the activation process of transition by reducing the energy barrier. The higher the pressurizing rate, the lower the energy barrier is, and the easier it is to pull the system out of the coexistence state. The results of this in situ study provide new insights into the understanding of the phase transition of calcite.     

In vitro ultrasound-mediated leakage from phospholipid vesicles

Interest in using ultrasound energy in wound management and intracellular drug delivery has been growing rapidly. Development and treatment optimization of such non-diagnostic applications requires a fundamental understanding of interactions between the acoustic wave and phospholipid membranes, be they cell membranes or liposome bilayers. This work investigates the changes in membrane permeation (leakage mimicking drug release) in vitro during exposure to ultrasound applied in two frequency ranges: “conventional” (1 MHz and 1.6 MHz) therapeutic ultrasound range and low (20 kHz) frequency range. Phospholipids vesicles were used as controllable biological membrane models. The membrane properties were modified by changes in vesicle dimensions and incorporation of poly(ethylene glycol) i.e. PEGylated lipids. Egg phosphatidylcholine vesicles with 5 mol% PEG were prepared with sizes ranging from 100 nm to 1 μm. Leakage was quantified in terms of temporal fluorescence intensity changes observed during carefully controlled ultrasound ON/OFF time intervals. Custom-built transducers operating at frequencies of 1.6 MHz (focused) and 1.0 MHz (unfocused) were used, the I_spta of which were 46.9 W/cm² and 3.0 W/cm², respectively. A commercial 20 kHz, point-source, continuous wave transducer with an I_spta of 0.13 W/cm² was also used for comparative purposes. Whereas complete leakage was obtained for all vesicle sizes at 20 kHz, no leakage was observed for vesicles smaller than 100 nm in diameter at 1.6 or 1.0 MHz. However, introducing leakage at the higher frequencies became feasible when larger (greater than 300 nm) vesicles were used, and the extent of leakage correlated well with vesicle sizes between 100 nm and 1 μm. This observation suggests that physico-chemical membrane properties play a crucial role in ultrasound mediated membrane permeation and that low frequency (tens of kilohertz) ultrasound exposure is more effective in introducing permeability change than the “conventional” (1 MHz) therapeutic one. The experimental data also indicate that the leakage level is controlled by the exposure time. The results of this work might be helpful to optimize acoustic field and membrane parameters for gene or drug delivery. The outcome of this work might also be useful in wound management.     

Quantitative and qualitative assessment of articular cartilage in the goat knee with magnetization transfer imaging

We investigated the role of collagen in the magnetization transfer (MT) effect in contrast to other macromolecules. By means of phantoms made of collagen, chondroitin sulfate (CS) and albumin, MR parameters have been optimized in order to reduce the acquisition time and improve the sensitivity, as well as to minimize the contributions from CS and albumin to the MT induced signal attenuation. The same method was used to study cartilage ex vivo (bovine articular and nasal cartilage plugs) and in vivo (goat knee femoral chondyle). In phantom samples, the MT signal attenuation depended on the collagen concentration while contributions from the other macromolecules were found to be minimal. In average, analysis of MT images revealed a 25%, 35% and 30% signal attenuation in 10% w/v type I collagen gels, cartilage plugs, and cartilage from the weight-bearing areas of the goat knee, respectively. Biochemical data revealed that treatment of cartilage plugs with bacterial collagenase led to collagen depletion and correspondingly to a decrease of the MT response. In contrast, trypsin-induced proteoglycan loss in cartilage plugs did not alter the MT effect. A significant correlation was observed between the collagen content in these plugs and their respective MT ratios and the rate constant k for the exchange process bound versus free water. Finally, data obtained from in vivo MT measurement of the goat knee demonstrated that intra-articular injection of papain might not only cause degradation of proteoglycans but also a change in collagen integrity in a dose-dependent manner. We conclude that in vivo measurement of MT ratios gives quantitative and qualitative information on the collagen status and may be applied for the routine evaluation of normal and abnormal articular cartilage.     

p型层结构与掺杂对GaInN发光二极管正向电压温度特性的影响

在温度变化时, 如果GaInN发光二极管能够保持相对稳定的工作电压对其实际应用具有重要意义. 本文通过金属有机化学气相沉积生长了一系列包含不同有源区结构、不同p型层结构以及不同掺杂浓度纵向分布的样品, 并对其在不同温度区间内正向电压随温度变化的斜率(dV/dT)进行了研究. 结果表明: 1)有源区中包括插入层设计、量子阱结构以及发光波长等因素的变化对正向电压随温度变化特性影响很小; 2)影响常温区间(300 K± 50 K)正向电压随温度变化斜率的最主要因素为p-AlGaN 电子阻挡层起始生长阶段的掺杂形貌, 具有p-AlGaN陡掺界面的样品电压变化斜率为-1.3 mV·K^-1, 与理论极限值 -1.2 mV·K^-1十分接近; 3) p-GaN主段层的掺Mg浓度对低温区间(<200 K)的正向电压随温度变化斜率有直接影响, 掺Mg浓度越低则dV/dT斜率越大. 以上现象归因于在不同温度区间, p-AlGaN 以及p-GaN 发生Mg受主冻结效应的程度主要取决于各自的掺杂浓度. 因此Mg掺杂浓度纵向分布不同的样品在不同的温度区间具有不同的串联电阻, 最终表现为差异很大的正向电压温度特性.     

Pressure–temperature studies on conductivity of TlX(X=Cl,Br,I) compounds:: I: phase diagram. II: ionic mobility

We have measured the conductivity σ of TlX(X=Cl, Br, I) compounds up to 5.3 GPa and between 300–823 K. The σ–T dependence for all compounds can be divided into three distinct regions: (i) low temperature (LT), <400 K, with unusual negative σ–T dependence, (ii) intermediate temperature (IT), 400<650 K, with positive σ–T dependence and (iii) high temperature (HT), T>650 K, with positive σ–T dependence. The σ–T isobars were used to construct the T–P solid phase diagram for each compound. The LT region data indicate a new meta-stable phase in the 1.0–3.5 GPa range. The LT→IT transition is characterized by an inverse σ–T dependence followed by normal Arrhenius behavior up to and including the HT region. The extrapolation to 1 atm of the P-dependent boundary between IT and HT regions above 3 GPa for each compound in the P–T plot yields a value close to its respective normal (1 atm) T_melt suggesting a solid order–disorder transition type paralleling -AgI behavior. The abrupt drop in conductivity in the LT region for P between 2.5–4.1 GPa of all compounds is at variance with the Arrhenius behavior observed for unperturbed ion migration implying the appearance of a second factor overriding the Arrhenius temperature dependence. Normal Arrhenius σ–T dependence prevails in both IT and HT regions with Q_c values of 85–100 kJ mol⁻¹ and 50–75 kJ mol⁻¹, respectively. The higher conductivities at 0.4 GPa for TlBr and TlI relative to their 1 atm data and the increasing σ with P are in strong contrast to the normal σ-P behavior of TlCl. The dependence of activation volume ΔV^‡ on T for TlCl, i.e. ΔV^‡>0, shows abnormally high values with a maximum at 500 K for P<3.0 GPa but reasonable ΔV^‡ values appear above 3.0 GPa. The ΔV^‡–T dependence for both TlBr and TlI with ΔV^‡<0 is incompatible with an ion transport mechanism suggesting an electronic conduction process and implying an ionic–metallic transition at higher pressures. These contrasting conductivity features are discussed and interpreted in terms of electronegativity differences and bonding character rather than structure.     

Projection structure of photosystem II In vivo determined by cryo-electron crystallography

Photosystem II (PSII) is a protein-pigment complex situated in the thylakoid membranes of plants and cyanobacteria where it catalyses the conversion of light into chemical energy. This energy is used to extract electrons from water, during which process oxygen is evolved. Owing to its extreme fragility and the large number of polypeptides (>20) it is composed of, the complex has so far proven recalcitrant to high-resolution structural studies. Cryo-electron crystallography of 2-D crystals (a = 15.4 nm, b = 23.1 nm, γ = 97.2°, p1) comprising in situ PSII revealed the first projection structure of the native complex. The unit cell contain one monomeric complex in which three central domains straddle an elongated intramolecular cavity. In conjunction with earlier data, these central domains were assigned to the reaction centre core subunits of PSII consisting of CP43, CP47, the reaction centre heterodimer D1/D2 and cytochrome b-559. The data are discussed in view of the evolution of reaction centres from anoxygenic to oxygenic photosynthesis.