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We report herein near-infrared (NIR)-emitting cyclometallated iridium(III) complexes bearing a heteroaromatic methylsulfone moiety as sulfhydryl-specific reagents; one of the complexes was conjugated to cysteine and cysteine-containing peptides and proteins for bioimaging and photocytotoxic applications.

Language styles used by academics working in disciplines are different, possibly reflecting how different types of knowledge are conceived, communicated and taught. Most of the studies on the communication style of academics are based on analyses of pre-prepared academic writings and formal speeches. It is not known whether such differences in language styles are also practised in informal, colloquial communications. In this paper, we report a Linguistic Inquiry and Word Count (LIWC) analysis of the telephone interviews given by 82 Nobel Prize laureates in the fields of natural sciences (chemistry, physics, physiology, or medicine) and economic sciences. LIWC analysis identifies differences in usage frequency of words in various language categories. The result shows a higher use of money- and power-related words by economic laureates and a higher use of second-person pronouns among science laureates. When second-person pronouns were used, economic laureates tended to use them as a general reference, while scientists tended to use them as courtesy sentences or discourse markers. These observations indicate that different language styles are used by scholars of different research fields even in their impromptu verbal communications. It is possible that word choices by economists are influenced by the content of economic studies, which are concerned with influencing the external environment and by their preference in describing processes and actions. A more involved and high level of cautions, alertness, and precision in conversion may be indicated in the speaking style of natural scientists.

Antimicrobial peptides (AMPs) have emerged as a promising alternative to small molecule antibiotics. Although AMPs have previously been isolated in many organisms, efforts on the systematic identification of AMPs in fish have been lagging. Here, we collected peptides from the plasma of medaka (Oryzias latipes) fish. By using mass spectrometry, 6399 unique sequences were identified from the isolated peptides, among which 430 peptides were bioinformatically predicted to be potential AMPs. One of them, a thermostable 13-residue peptide named BING, shows a broad-spectrum toxicity against pathogenic bacteria including drug-resistant strains, at concentrations that presented relatively low toxicity to mammalian cell lines and medaka. Proteomic analysis indicated that BING treatment induced a deregulation of periplasmic peptidyl-prolyl isomerases in gram-negative bacteria. We observed that BING reduced the RNA level of cpxR, an upstream regulator of envelope stress responses. cpxR is known to play a crucial role in the development of antimicrobial resistance, including the regulation of genes involved in drug efflux. BING downregulated the expression of efflux pump components mexB, mexY and oprM in P. aeruginosa and significantly synergised the toxicity of antibiotics towards these bacteria. In addition, exposure to sublethal doses of BING delayed the development of antibiotic resistance. To our knowledge, BING is the first AMP shown to suppress cpxR expression in Gram-negative bacteria. This discovery highlights the cpxR pathway as a potential antimicrobial target.

The impact of radiation-induced bystander effect (RIBE) is still not well understood in radiotherapy. RIBEs are biological effects expressed by nonirradiated cells near or far from the irradiated cells. Most radiological studies on cancer cells have been based on biochemical characterization. However, biophysical investigation with label-free techniques to analyze and compare the direct irradiation effect and RIBE has lagged. In this work, we employed an electrical cell-indium tin oxide (ITO) substrate impedance system (ECIIS) as a bioimpedance sensor to evaluate the HeLa cells' response. The bioimpedance of untreated/nonirradiated HeLa (N-HeLa) cells, α-particle (Am-241)-irradiated HeLa (I-HeLa) cells, and bystander HeLa (B-HeLa) cells exposed to media from I-HeLa cells was monitored with a sampling interval of 8 s over a period of 24 h. Also, we imaged the cells at times where impedance changes were observed. Different radiation doses (0.5 cGy, 1.2 cGy, and 1.7 cGy) were used to investigate I-HeLa and B-HeLa cells' radiation-dose-dependence. By analyzing the changes in absolute impedance and cell size/number with time, compared to N-HeLa cells, B-HeLa cells mimicked the I-HeLa cells' damage and modification of proliferation rate. Contrary to the irradiated cells, the bystander cells' damage rate and proliferation rate enhancements have an inverse radiation-dose-response. Also, we report multiple RIBEs in HeLa cells in a single measurement and provide crucial insights into the RIBE mechanism without any labeling procedure. Unambiguously, our results have shown that the time-dependent control of RIBE is important during α-radiation-based radiotherapy of HeLa cells.

Sexual differences have been observed in the onset and prognosis of human cardiovascular diseases, but the underlying mechanisms are not clear. Here, we found that zebrafish heart regeneration is faster in females, can be accelerated by estrogen and is suppressed by the estrogen-antagonist tamoxifen. Injuries to the zebrafish heart, but not other tissues, increased plasma estrogen levels and the expression of estrogen receptors, especially esr2a. The resulting endocrine disruption induces the expression of the female-specific protein vitellogenin in male zebrafish. Transcriptomic analyses suggested heart injuries triggered pronounced immune and inflammatory responses in females. These responses, previously shown to elicit heart regeneration, could be enhanced by estrogen treatment in males and reduced by tamoxifen in females. Furthermore, a prior exposure to estrogen preconditioned the zebrafish heart for an accelerated regeneration. Altogether, this study reveals that heart regeneration is modulated by an estrogen-inducible inflammatory response to cardiac injury. These findings elucidate a previously unknown layer of control in zebrafish heart regeneration and provide a new model system for the study of sexual differences in human cardiac repair.

Red blood cells, or erythrocytes, make up approximately a quarter of all cells in the human body with over 2 billion new erythrocytes made each day in a healthy adult human. This massive cellular production system is coupled with a set of cell biological processes unique to mammals, in particular, the elimination of all organelles, and the expulsion and destruction of the condensed erythroid nucleus. Erythrocytes from birds, reptiles, amphibians and fish possess nuclei, mitochondria and other organelles: erythrocytes from mammals lack all of these intracellular components. This review will focus on the dynamic changes that take place in developing erythroid cells that are interacting with specialized macrophages in multicellular clusters termed erythroblastic islands. Proerythroblasts enter the erythroblastic niche as large cells with active nuclei, mitochondria producing heme and energy, and attach to the central macrophage via a range of adhesion molecules. Proerythroblasts then mature into erythroblasts and, following enucleation, in reticulocytes. When reticulocytes exit the erythroblastic island, they are smaller cells, without nuclei and with few mitochondria, possess some polyribosomes and have a profoundly different surface molecule phenotype. Here, we will review, step-by-step, the biophysical mechanisms that regulate the remarkable process of erythropoiesis with a particular focus on the events taking place in the erythroblastic island niche. This is presented from the biological perspective to offer insight into the elements of red blood cell development in the erythroblastic island niche which could be further explored with biophysical modelling systems.

In vitro cytotoxicity screening is a crucial step of anticancer drug discovery. The application of deep learning methodology is gaining increasing attentions in processing drug screening data and studying anticancer mechanisms of chemical compounds. In this work, we explored the utilization of convolutional neural network in modeling the anticancer efficacy of small molecules. In particular, we presented a VGG19 model trained on 2D structural formulae to predict the growth-inhibitory effects of compounds against leukemia cell line CCRF-CEM, without any use of chemical descriptors. The model achieved a normalized RMSE of 15.76% on predicting growth inhibition and a Pearson Correlation Coefficient of 0.72 between predicted and experimental data, demonstrating a strong predictive power in this task. Furthermore, we implemented the Layer-wise Relevance Propagation technique to interpret the network and visualize the chemical groups predicted by the model that contribute to toxicity with human-readable representations.Clinical relevance—This work predicts the cytotoxicity of chemical compounds against human leukemic lymphoblast CCRF-CEM cell lines on a continuous scale, which only requires 2D images of the structural formulae of the compounds as inputs. Knowledge in the structure-toxicity relationship of small molecules will potentially increase the hit rate of primary drug screening assays.

Tumour metastasis is a complex process that strongly influences the prognosis and treatment of cancer. Apart from intracellular factors, recent studies have indicated that metastasis also depends on microenvironmental factors such as the biochemical, mechanical and topographical properties of the surrounding extracellular matrix (ECM) of tumours. In this study, as a proof of concept, we conducted tumour spheroid dissemination assay on engineered surfaces with micrograting patterns. Nasopharyngeal spheroids were generated by the 3D culture of nasopharyngeal carcinoma (NPC43) cells, a newly established cell line that maintains a high level of Epstein–Barr virus, a hallmark of NPC. Three types of collagen I-coated polydimethylsiloxane (PDMS) substrates were used, with 15 μm deep “trenches” that grated the surfaces: (a) 40/10 μm ridges (R)/trenches (T), (b) 18/18 μm (R/T) and (c) 50/50 μm (R/T). The dimensions of these patterns were designed to test how various topographical cues, different with respect to the size of tumour spheroids and individual NPC43 cells, might affect dissemination behaviours. Spreading efficiencies on all three patterned surfaces, especially 18/18 μm (R/T), were lower than that on flat PDMS surface. The outspreading cell sheets on flat and 40/10 μm (R/T) surfaces were relatively symmetrical but appeared ellipsoid and aligned with the main axes of the 18/18 μm (R/T) and 50/50 μm (R/T) grating platforms. Focal adhesions (FAs) were found to preferentially formed on the ridges of all patterns. The number of FAs per spheroid was strongly influenced by the grating pattern, with the least FAs on the 40/10 μm (R/T) and the most on the 50/50 μm (R/T) substrate. Taken together, these data indicate a previously unknown effect of surface topography on the efficiency and directionality of cancer cell spreading from tumour spheroids, suggesting that topography, like ECM biochemistry and stiffness, can influence the migration dynamics in 3D cell culture models.

Despite the widespread observations on the osteogenic effects of magnesium ion (Mg2+), the diverse roles of Mg2+ during bone healing have not been systematically dissected. Here, we reveal a previously unknown, biphasic mode of action of Mg2+ in bone repair. During the early inflammation phase, Mg2+ contributes to an upregulated expression of transient receptor potential cation channel member 7 (TRPM7), and a TRPM7-dependent influx of Mg2+ in the monocyte-macrophage lineage, resulting in the cleavage and nuclear accumulation of TRPM7-cleaved kinase fragments (M7CKs). This then triggers the phosphorylation of Histone H3 at serine 10, in a TRPM7-dependent manner at the promoters of inflammatory cytokines, leading to the formation of a pro-osteogenic immune microenvironment. In the later remodeling phase, however, the continued exposure of Mg2+ not only lead to the over-activation of NF-κB signaling in macrophages and increased number of osteoclastic-like cells but also decelerates bone maturation through the suppression of hydroxyapatite precipitation. Thus, the negative effects of Mg2+ on osteogenesis can override the initial pro-osteogenic benefits of Mg2+. Taken together, this study establishes a paradigm shift in the understanding of the diverse and multifaceted roles of Mg2+ in bone healing., Supplementation of magnesium (Mg2+) or its inclusion in biomaterials has beneficial effects for bone formation, but it has also been reported that it can have detrimental effects. Here, the authors analyse dose- and time-dependent effects of Mg2+ on bone regeneration and show that it can stimulate monocyte-macrophage lineage cells to support bone formation in the early phases of repair, but inhibit bone repair and mineralization in later stages by promoting a pro-inflammatory environment.

One of the most crucial components of regenerative medicine is the controlled differentiation of embryonic or adult stem cells into the desired cell lineage. Although most of the reported protocols of stem cell differentiation involve the use of soluble growth factors, it is increasingly evident that stem cells also undergo differentiation when cultured in the appropriate microenvironment. When cultured in decellularized tissues, for instance, stem cells can recapitulate the morphogenesis and functional specialization of differentiated cell types with speed and efficiency that often surpass the traditional growth factor-driven protocols. This suggests that the tissue microenvironment (TME) provides stem cells with a holistic "instructive niche" that harbors signals for cellular reprogramming. The translation of this into medical applications requires the decoding of these signals, but this has been hampered by the complexity of TME. This problem is often addressed by a reductionist approach, in which cells are exposed to substrates decorated with simple, empirically designed geometries, textures, and chemical compositions ("bottom-up" approach). Although these studies are invaluable in revealing the basic principles of mechanotransduction mechanisms, their physiological relevance is often uncertain. This review examines the recent progress of an alternative, "top-down" approach, in which the TME is treated as a holistic biological entity. This approach is made possible by recent advances in systems biology and fabrication technologies that enable the isolation, characterization, and reconstitution of TME. It is hoped that these new techniques will elucidate the nature of niche signals so that they can be extracted, replicated, and controlled. This review summarizes these emerging techniques and how the data they generated are changing our view on TME. Impact statement This review summarizes the current state of art of the understanding of instructive niche in the field of tissue microenvironment. Not only did we survey the use of different biochemical preparations as stimuli of stem cell differentiation and summarize the recent effort in dissecting the biochemical composition of these preparations, through the application of extracellular matrix (ECM) arrays and proteomics, but we also introduce the use of open-source, high-content immunohistochemistry projects in contributing to the understanding of tissue-specific composition of ECM. We believe this review would be highly useful for our peer researching in the same field. "Mr. Tulkinghorn is always the same… so oddly out of place and yet so perfectly at home." -Charles Dickens, Bleak House.

The use of decellularized tissues or organs as cell culture scaffolds has proven to be a promising approach for tissue engineering and regenerative medicine, as these decellularized tissues can provide the instructive niche for cell differentiation and functions. Cartilage is a largely avascular tissue with limited regenerative capacity. Lesions caused by arthritis can lead to severe cartilage degeneration. Previous studies have indicated that decellularized cartilage can be used as scaffolds that support the chondrogenic differentiation of adult stem cells. However, these decellularization protocols all require the use of denaturing agents, such as high salt and detergents, that lead to the artifactual disruption of the chemical and physical integrity of the tissue microenvironment. Here, we established a new decellularization method for cartilage, through a combined effect of freezing-thawing, sectioning, and sonication in water. This protocol achieved the complete removal of cells within minutes, instead of hours or days required by existing procedures, and does not use any detergent. The resulting decellularized cartilage preserved the native ultrastructure and biochemical contents, including glycosaminoglycans, which is typically depleted by traditional decellularization methods. Human mesenchymal stem cells could readily adhere onto the decellularized cartilage. Together, this work unveils a simple new method for decellularizing cartilage, which will be useful in studying how tissue microenvironment supports chondrocyte growth and functions. Impact statement In this study, we develop a simple, fast cartilage decellularization method that does not require any detergent, so that the decellularized cartilage chemistry is preserved. Traditional detergent-based decellularization removes the tissue biochemical contents (i.e., glycosaminoglycans). In this new water decellularization protocol, the biochemical contents of cartilage can be preserved. This allows the study of biochemistry and physical content in extracellular matrix as a whole, and this protocol would definitely be useful for studying the effect of tissue microenvironment in supporting chondrocyte growth and functions.

A two-layer scaffold platform was designed for drug screening without using fluorescence staining to examine the effectiveness of the tested compounds. Nasopharyngeal epithelial (NP460) cells were used in this work. Typically, NP460 cells could not squeeze into the $10\ \mu \mathrm{m}$ narrow trenches but preferred to migrate on the top ridges. However, NP460 cells could migrate into $10\ \mu \mathrm{m}$ wide trenches after being stimulated by transforming growth factor beta-1 ( $\text{TGF}-\beta 1$ ) as the cells became more active. This double-layer scaffold platform served as a drug screening device by tracking the number of cells migrated into narrow trenches after drug treatment to test and monitor the effectiveness of a compound in a biomimetic microenvironment.

A series of substituted 9-methylenylanthracene photocages for diphenylphosphinothioesters have been synthesized to explore their photo-uncaging properties by visible light. Substituents such as phenyl, p-trifluoromethylphenyl, p-methoxyphenyl, ethyn-1-ylbenzene, and 3,3-dimethylbut-1-yn-1-yl have been introduced in order to extend the π-conjugation of the photocage and to shift the wavelength response of the uncaging process to the visible spectral range. Among these new photocages, the (10-(3,3-dimethylbut-1-yn-1-yl)anthracen-9-yl)methyl has been shown to have the best performance in terms of fast photo-uncaging and minimal byproduct formation. It is responsive to both UV and visible photoexcitation. Quantum yields of the photoinduced heterolytic anthracenylmethyl-phosphorus bond cleavage at 366 and 416 nm were found to be 0.08 and 0.025, respectively. This photocage enables traceless Staudinger ligation to be triggered by photoirradiation in the visible spectral range for bioconjugation applications. We demonstrate this with a series of visible-light-induced oligopeptide syntheses via the conjugation of amino acid/oligopeptide building blocks by the characteristic peptide linkage attained by traceless Staudinger ligation. Yields of the resultant conjugated oligopeptides ranged from 31 to 43%. This new photocage opens up the possibility of in situ synthesis of functional proteins/peptides mediated by visible-light-induced photoclick processes for the regulation of cellular/metabolic functions of life systems.

The development of polymeric materials as drug delivery systems has advanced from systems that rely on classical passive targeting to carriers that can sustain the precisely controlled release of payloads upon physicochemical triggers in desired microenvironment. Molecularly imprinted polymers (MIP), materials designed to capture specific molecules based on their molecular shape and charge distribution, are attractive candidates for fulfilling these purposes. In particular, drug-imprinted polymers coupled with active targeting mechanisms have been explored as potential drug delivery systems. In this review, we have curated important recent efforts in the development of drug-imprinted polymers in a variety of clinical applications, especially oncology and ophthalmology. MIP possesses properties that may complement the traditional delivery systems of these two disciplines, such as passive enhanced permeability and retention effect (EPR) in cancer tumors, and passive drug diffusion in delivering ophthalmic therapeutics. Furthermore, the prospects of MIP integration with the emerging gene therapies will be discussed.

Fish blood is one of the crucial tissues of innate immune system, but the full repertoire of fish serum components involved in antibacterial defense is not fully identified. In this study, we demonstrated that turbot serum, but not the heat-inactivated control, significantly reduced the number of Edwardsiella tarda (E. tarda). By conjugating serum proteins with fluorescent dyes, we showed that E. tarda were coated with multiple fish proteins. In order to identify these proteins, we used E. tarda to capture turbot serum proteins and subjected the samples to shotgun proteomic analysis. A total of 76 fish proteins were identified in high confidence, including known antimicrobial proteins such as immunoglobins and complement components. 34 proteins with no previously known immunological functions were also identified. The expression of one of these proteins, IQ motif containing H (IQCH), was exclusively in fish brain and gonads and was induced during bacterial infection. This approach also allowed the study of the corresponding proteomic changes in E. tarda exposed to turbot serum, which is a general decrease of bacterial protein expression except for an upregulation of membrane components after serum treatment. Interestingly, while most other known stresses stimulate bacterial antioxidant enzymes, fish serum induced a rapid suppression of antioxidant proteins and led to an accumulation of reactive oxygen species. Heat treatment of fish serum eliminated this effect, suggesting that heat labile factors in the fish serum overrode bacterial antioxidant defenses. Taken together, this work offers a comprehensive view of the interactions between fish serum proteins and bacteria, and reveals previously unknown factors and mechanisms in fish innate immunity.

Thermal scanning probe lithography (t-SPL) is a nanofabrication technique in which an immobilized thermolabile resist, such as polyphthalaldehyde (PPA), is locally vaporized by a heated atomic force microscope tip. Compared with other nanofabrication techniques, such as soft lithography and nanoimprinting lithography, t-SPL is more efficient and convenient as it does not involve time-consuming mask productions or complicated etching procedures, making it a promising candidate technique for the fast prototyping of nanoscale topographies for biological studies. Here, we established the direct use of PPA-coated surfaces as a cell culture substrate. We showed that PPA is biocompatible and that the deposition of allylamine by plasma polymerization on a silicon wafer before PPA coating can stabilize the immobilization of PPA in aqueous solutions. When seeded on PPA-coated surfaces, human mesenchymal stem cells (MSC) adhered, spread, and proliferated in a manner indistinguishable from cells cultured on glass surfaces. This allowed us to subsequently use t-SPL to generate nanotopographies for cell culture experiments. As a proof of concept, we analyzed the surface topography of bovine tendon sections, previously shown to induce morphogenesis and differentiation of MSC, by means of atomic force microscopy, and then "wrote" topographical data on PPA by means of t-SPL. The resulting substrate, matching the native tissue topography on the nanoscale, was directly used for MSC culture. The t-SPL substrate induced similar changes in cell morphology and focal adhesion formation in the MSC compared to native tendon sections, suggesting that t-SPL can rapidly generate cell culture substrates with complex and spatially accurate topographical signals. This technique may greatly accelerate the prototyping of models for the study of cell-matrix interactions.

Sexual differences are frequently observed in the onset and prognosis of human cardiovascular diseases, but the underlying mechanisms are not clearly known. Here, we report that zebrafish heart regeneration is faster in females, can be accelerated by estrogen and suppressed by estrogen-antagonist tamoxifen. Transcriptomic analyses suggested heart injuries triggered more pronounced immune and inflammatory responses, previously shown to enhance heart regeneration, in females, leading to increased STAT1 expression and leukocyte filtration. These responses could be enhanced by estrogen treatment in males. We also showed that injuries to the heart, but not other tissues, increased plasma estrogen level and expression of estrogen receptors, especially esr2a, in zebrafish hearts. Although this effect was stronger in female fish, the resulting endocrine disruption was sufficient to induce the expression of female-specific protein vitellogenin in male zebrafish. Altogether, this study reveals that heart regeneration is modulated by an estrogen-inducible inflammatory response to heart injury, which in turn stimulates estrogen signalling predominately in females. These findings elucidate a previously unknown layer of control in zebrafish heart regeneration and provides a new model system for the study of sexual differences in human cardiac repair.

Radiation-induced bystander effects (RIBE) have raised many concerns about radiation safety and protection. In RIBE, unirradiated cells receive signals from irradiated cells and exhibit irradiation effects. Until now, most RIBE studies have been based on morphological and biochemical characterization. However, research on the impact of RIBE on biophysical properties of cells has been lagging. Non-invasive indium tin oxide (ITO)-based impedance systems have been used as bioimpedance sensors for monitoring cell behaviors. This powerful technique has not been applied to RIBE research. In this work, we employed an electrical cell-ITO substrate impedance system (ECIIS) to study the RIBE on Chinese hamster ovary (CHO) cells. The bioimpedance of bystander CHO cells (BCHO), alpha(α)-particle (Am-241) irradiated CHO (ICHO), and untreated/unirradiated CHO (UCHO) cells were monitored with a sampling interval of 8 s over a period of 24 h. Media from ICHO cells exposed to different radiation doses (0.3 nGy, 0.5 nGy, and 0.7 nGy) were used to investigate the radiation dose dependence of BCHO cells' impedance. In parallel, we imaged the cells at times where impedance changes were observed. By analyzing the changes in absolute impedance and cell size/cell number with time, we observed that BCHO cells mimicked ICHO cells in terms of modification in cell morphology and proliferation rate. Furthermore, these effects appeared to be time-dependent and inversely proportional to the radiation dose. Hence, this approach allows a label-free study of cellular responses to RIBE with high sensitivity and temporal resolution and can provide crucial insights into the RIBE mechanism.

This study aimed to examine the relationship between trait loneliness and diurnal rhythms of salivary cortisol. Fifty-One Chinese undergraduates provided six saliva samples on a weekday at immediately, 0.5, 3, 6, and 12 h after waking, and at bedtime. Saliva collection times were monitored using electronic devices (MEMS TrackCaps). Participants were also administered a questionnaire consisting of scales measuring, trait loneliness, depression, and demographics. Relationships between loneliness and the cortisol awakening response (CAR), diurnal slope (DS), and area under the curve with respect to ground (AUCG) were examined using multiple regression analyses. Results showed that a higher loneliness score was associated with an attenuated CAR, a large AUCG, and a steeper DS, with the effects of compliance, waking time, and depression being controlled. As a blunted CAR and a higher diurnal cortisol level have been shown to be associated with poorer health in prior studies, increased attention to the mechanisms translating loneliness into disease endpoints via elevated cortisol is warranted.

Planarian regeneration involves a complex series of cellular events, precisely choreographed in space and time. Time-lapse imaging can provide powerful insights into tissue dynamics, as variously demonstrated in other model systems. However, time-lapse imaging of planarians has proven to be a challenge. Especially the requisite immobilization of the animals over extended periods of time is difficult, owing to their photophobic behavior and soft body architecture. Here, we describe a new embedding method using 2% (w/v) low melting agarose, and demonstrate that this method can effectively immobilize animals as long as 7 days. In combination with cell-permeable fluorescent dyes, this immobilization method allows for the time-lapse imaging of planaria during regeneration and other physiological processes.

Planarian regeneration involves a complex series of cellular events, precisely choreographed in space and time. Time-lapse imaging can provide powerful insights into tissue dynamics, as variously demonstrated in other model systems. However, time-lapse imaging of planarians has proven to be a challenge. Especially the requisite immobilization of the animals over extended periods of time is difficult, owing to their photophobic behavior and soft body architecture. Here, we describe a new embedding method using 2% (w/v) low melting agarose, and demonstrate that this method can effectively immobilize animals as long as 7 days. In combination with cell-permeable fluorescent dyes, this immobilization method allows for the time-lapse imaging of planaria during regeneration and other physiological processes.

Growing evidence suggests that the immune system of teleost is vulnerable to xenoestrogens, which are ubiquitous in the marine environment. This study detected and identified the major circulatory immune proteins deregulated by 17α-ethinylestradiol (EE2), which may be linked to fish susceptibility to pathogens in the marine medaka, Oryzias melastigma. Fish immune competence was determined using a host resistance assay to pathogenic bacteria Edwardsiella tarda. Females were consistently more susceptible to infection-induced mortality than males. Exposure to EE2 could narrow the sex gap of mortality by increasing infection-induced death in male fish. Proteomic analysis revealed that the major plasma immune proteins of adult fish were highly sexually dimorphic. EE2 induced pronounced sex-specific changes in the plasma proteome, with the male plasma composition clearly becoming "feminised". Male plasma was found to contain a higher level of fibrinogens, WAP63 and ependymin-2-like protein, which are involved in coagulation, inflammation and regeneration. For the first time, we demonstrated that expression of C1q subunit B (C1Q), an initiating factor of the classical complement pathway, was higher in males and was suppressed in both sexes in response to EE2 and bacterial challenge. Moreover, cleavage and post-translational modification of C3, the central component of the complement system, could be altered by EE2 treatment in males (C3dg down; C3g up). Multiple regression analysis indicated that C1Q is possibly an indicator of fish survival, which warrants further confirmation. The findings support the potential application of plasma immune proteins for prognosis/diagnosis of fish immune competence. Moreover, this study provides the first biochemical basis of the sex-differences in fish immunity and how these differences might be modified by xenoestrogens.

By virtue of the specific amalgam of mercury with gold and high specific area of a graphene scaffold, an environment-friendly multifunctional graphene–gold nanocomposite (G-AuNPs) has been identified and prepared by a simple one-pot redox reaction. The resultant G-AuNPs can reversibly enrich about 94% of Hg2+ in water samples, which can be further separated by only a simple filtration. Importantly, the color of the G-AuNPs suspension exclusively changes from purple–red to light brown upon the addition of Hg2+ in the presence of ascorbic acid, which can be applied for colorimetric detection of Hg2+ with a detection limit (3σ, n = 20) of 1.6 × 10–8 mol·L–1. Furthermore, using ascorbic acid as reducing agents, both the preparation process and the resultant nanocomposite are nontoxic. To the best of our knowledge, this is the first report to enrich, separate and detect Hg2+ contaminant simultaneously without causing any secondary pollution.

In this study, we explored the concept of introducing asymmetry to cell shapes by patterned cell culture substrates, and investigated the consequence of this induced asymmetry to cell migration behaviors. Three patterns, named "Squares", "Grating", and "Arcs" were fabricated, representing different levels of rotational asymmetry. Using time-lapse imaging, we systematically compared the motility and directionality of mouse osteoblastic cells MC3T3-E1 cultured on these patterns. Cells were found to move progressively faster on "Arcs" than on "Grating", and cells on "Squares" were the slowest, suggesting that cell motility correlates with the level of rotational asymmetry of the repeating units of the pattern. Among these three patterns, on the "Arcs" pattern, the least symmetrical one, cells not only moved with the highest velocity but also the strongest directional persistence. Although this enhanced motility was not associated with the detected number of focal adhesion sites in the cells, the pattern asymmetry was reflected in the asymmetrical cell spreading. Cells on the "Arcs" pattern consistently displayed larger cytoplasmic protrusion on one side of the cell. This asymmetry in cell shape determined the direction and speed of cell migration. These observations suggest that topographical patterns that enhance the imbalance between the leading and trailing fronts of adherent cells will increase cell speed and control movement directions. Our discovery shows that complex cell behaviors such as the direction of cell movement are influenced by simple geometrical principles, which can be utilized as the design foundation for platforms that guide and sort cultured cells.

This study used an intensive protocol to examine the effects of daily hassles and loneliness on diurnal salivary cortisol levels. Fifty Chinese undergraduates (28 females) provided six saliva samples each day for two consecutive days (at 0, 0.5, 3, 6, and 12 h after waking and at bedtime) and completed a questionnaire that included scales to measure daily hassles experienced over the previous month, trait loneliness, and depression. Cortisol data were aggregated over two days and used in subsequent analyses, focusing on the cortisol awakening response, diurnal slope, and overall cortisol output operationalized as the area under the curve with reference to the ground (AUCG). Multiple regression analysis showed that an increase in loneliness had a significant association with an increase in the AUCG and with a steeper diurnal slope. Loneliness also showed a significant interaction with daily hassles in that the positive association between daily hassles and AUCG was accentuated in the participants who reported a greater degree of loneliness. Our findings demonstrate for the first time the importance of trait loneliness in modulating the association between daily hassles and diurnal cortisol levels, which has significant clinical implications. Interventions to reduce loneliness should help college students to better cope with daily stressors. Increased attention should also be paid to the health implications of an elevated cortisol level in this relatively young and healthy population.

We present a new class of phosphorescent cyclometalated iridium(III) polypyridine poly(ethylene glycol) (PEG) complexes [Ir(N(^)C)2(bpy-CONH-PEG)](PF6) (bpy-CONH-PEG = 4-(N-(2-(ω-methoxypoly-(1-oxapropyl))ethyl)aminocarbonyl)-4'-methyl-2,2'-bipyridine, number average molecular weight (Mn) = 5272.23, weight average molecular weight (Mw) = 5317.38, polydispersity index (PDI) = 1.009; HN(^)C = 2-phenylpyridine, Hppy (1a), 2-((1,1'-biphenyl)-4-yl)pyridine, Hpppy (2a), 2-phenylquinoline, Hpq (3a), 2-phenylbenzothiazole, Hbt (4a), 2-(1-naphthyl)benzothiazole, Hbsn (5a)). The photophysical, photochemical, and biological properties of these complexes have been compared with those of their PEG-free counterparts [Ir(N(^)C)2(bpy-CONH-Et)](PF6) (bpy-CONH-Et = 4-(N-ethylaminocarbonyl)-4'-methyl-2,2'-bipyridine; HN(^)C = Hppy (1b), Hpppy (2b), Hpq (3b), Hbt (4b), Hbsn (5b)). Upon irradiation, all the complexes exhibited intense and long-lived green to orange-red emission under ambient conditions. The emission was phosphorescence in nature and can be quenched by O2 with the generation of singlet oxygen ((1)O2). The quantum yields for (1)O2 production of the complexes in aerated DMSO (0.24-0.83) were found to be dependent on the excited-state lifetimes of the complexes, which can be altered using different cyclometalating ligands (N(^)C). Cell-based assays indicated that the PEG complexes were noncytotoxic in the dark (IC50 > 300 μM); however, most of them became significantly cytotoxic upon irradiation (IC50 = 3.4 - 23.2 μM). Laser-scanning confocal microscopy images revealed localization of complex 3a in the mitochondrial region of HeLa cells and the induction of rapid necrotic cell death upon light activation. Additionally, the lack of dark toxicity and potential application of the PEG complexes as a visualizing reagent have been demonstrated using zebrafish (Danio rerio) as an animal model.

Organelle-specific cell-permeable fluorescent dyes are invaluable tools in cell biology as they reveal intracellular dynamics in living cells. Mitrotracker is a family of dyes that strongly label the mitochondrion, a key organelle associated with many crucial cellular functions. Despite the popularity of these dyes, little is known about the molecular mechanism behind their staining specificity. Here, we aimed to identify the protein targets of one member of this dye family, mitotracker red (MTR), by 2DE and MS. MTR bound to cellular proteins covalently, and its fluorescence persisted even after cell lysis, protein solubilization, denaturation, and electrophoresis. This enabled us to display MTR-labeled proteins by 2DE. The MTR-specific fluorescent signals on the gel revealed the spots that contained MTR-conjugated proteins. These spots were analyzed by MS, resulting into the identification of ten proteins. We discovered that one major target is the mitochondrial protein HSP60 and that MTR staining could induce production of HSP60, predisposing cells to heat shock-like responses. The identification of the molecular targets of biological dyes, or "stainomics," can help correlate their intracellular staining properties with biochemical affinities. We believe this approach can be applied to a wide range of fluorescent probes.

The adult zebrafish has been used to model the electrocardiogram (ECG) for human cardiovascular studies. Nonetheless huge variations are observed among studies probably because of the lack of a reliable and reproducible recording method. In our study, an adult zebrafish surface ECG recording technique was improved using a multi-electrode method and by pre-opening the pericardial sac. A convenient ECG data analysis method without wavelet transform was also established. Intraperitoneal injection of KCl in zebrafish induced an arrhythmia similar to that of humans, and the arrhythmia was partially rescued by calcium gluconate. Amputation and cryoinjury of the zebrafish heart induced ST segment depression and affected QRS duration after injury. Only cryoinjury decelerated the heart rate. Different changes were also observed in the QT interval during heart regeneration in these two injury models. We also characterized the electrocardiophysiology of breakdance zebrafish mutant with a prolonged QT interval, that has not been well described in previous studies. Our study provided a reliable and reproducible means to record zebrafish ECG and analyse data. The detailed characterization of the cardiac electrophysiology of zebrafish and its mutant revealed that the potential of the zebrafish in modeling the human cardiovascular system exceeds expectations.

SummaryBacterial flagellar switching between counterclockwise and clockwise directions is mediated by the coupling of the chemotactic system and the motor switch complex. The conformational changes of FliG are closely associated with this switching mechanism. We present two crystal structures of FliGMC from Helicobacter pylori, each showing distinct domain orientations from previously solved structures. A 180° rotation of the charged ridge-containing C-terminal subdomain FliGCα1–6 that is prompted by the rotational freedom of Met245 psi and Phe246 phi at the MFXF motif was revealed. Studies on the swarming and swimming behavior of Escherichia coli mutants further identified the importance of the 245MFXF248 motif and a highly conserved residue, Asn216, in motor switching. Additionally, multiple conformations of FliGCα1–6 were demonstrated by intramolecular cysteine crosslinking. The conformational flexibility of FliGc leads us to propose a model that accounts for the symmetrical torque generation process and for the dynamics of the motor.

Surface topography and texture of cell culture substrata can affect the differentiation and growth of adherent cells. The biochemical basis of the transduction of the physical and mechanical signals to cellular responses is not well understood. The lack of a systematic characterization of cell-biomaterial interaction is the major bottleneck. This study demonstrated the use of a novel subcellular fractionation method combined with quantitative MS-based proteomics to enable the robust and high-throughput analysis of proteins at the adherence interface of Madin-Darby canine kidney cells. This method revealed the enrichment of extracellular matrix proteins and membrane and stress fibers proteins at the adherence surface, whereas it shows depletion of extracellular matrix belonging to the cytoplasmic, nucleus, and lateral and apical membranes. The asymmetric distribution of proteins between apical and adherence sides was also profiled. Apart from classical proteins with clear involvement in cell-material interactions, proteins previously not known to be involved in cell attachment were also discovered. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_OA_fulltext

Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.

A major challenge in cell biology is to identify the subcellular distribution of proteins within cells and to characterize how protein localization changes under different cell growth conditions and in response to stress and other external signals. Protein localization is usually determined either by microscopy or by using cell fractionation combined with protein blotting techniques. Both these approaches are intrinsically low throughput and limited to the analysis of known components. Here we use mass spectrometry-based proteomics to provide an unbiased, quantitative, and high throughput approach for measuring the subcellular distribution of the proteome, termed "spatial proteomics." The spatial proteomics method analyzes a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass-labeled with heavy isotopes. This was used here to measure the relative distribution between cytoplasm, nucleus, and nucleolus of over 2,000 proteins in HCT116 cells. The data show that, at steady state, the proteome is predominantly partitioned into specific subcellular locations with only a minor subset of proteins equally distributed between two or more compartments. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations, shown here by characterizing dynamic changes in protein localization elicited during the cellular response to DNA damage following treatment of HCT116 cells with etoposide. DNA damage was found to cause dissociation of the proteasome from inhibitory proteins and assembly chaperones in the cytoplasm and relocation to associate with proteasome activators in the nucleus.

ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.