Improved Multiprotein Microcontact Printing on Plasma Immersion Ion Implanted Polystyrene.

Multiprotein micropatterning allows the creation of complex, controlled microenvironments for single cells that can be used for the study of the localized effects of various proteins and signals on cell survival, development, and functions. To enable analysis of cell interactions with microprinted proteins, the multiprotein micropattern must have low cross-contamination and high long-term stability in a cell culture medium. To achieve this, we employed an optimized plasma ion immersion implantation (PIII) treatment to provide polystyrene (PS) with the ability to covalently immobilize proteins on contact while retaining sufficient transparency and suitable surface properties for contact printing and retention of protein activity. The quality and long-term stability of the micropatterns on untreated and PIII treated PS were compared with those on glass using confocal microscopy. The protein micropattern on the PIII treated PS was more uniform and had a significantly higher contrast that was not affected by long-term incubation in cell culture media because the proteins were covalently bonded to PIII treated PS. The immunostaining of mouse pancreatic ß cells interacting with E-cadherin and fibronectin striped surfaces showed phosphorylated paxillin concentrated on cell edges over the fibronectin stripes. This indicates that multiprotein micropatterns printed on PIII treated PS can be used for high-resolution studies of local influence on cell morphology and protein production.

ESHRE guideline: routine psychosocial care in infertility and medically assisted reproduction-a guide for fertility staff.

STUDY QUESTION: Based on the best available evidence in the literature, what is the optimal management of routine psychosocial care at infertility and medically assisted reproduction (MAR) clinics? SUMMARY ANSWER: Using the structured methodology of the Manual for the European Society of Human Reproduction and Embryology (ESHRE) Guideline Development, 120 recommendations were formulated that answered the 12 key questions on optimal management of routine psychosocial care by all fertility staff. WHAT IS ALREADY KNOWN: The 2002 ESHRE Guidelines for counselling in infertility has been a reference point for best psychosocial care in infertility for years, but this guideline needed updating and did not focus on routine psychosocial care that can be delivered by all fertility staff. STUDY, DESIGN, SIZE, DURATION: This guideline was produced by a group of experts in the field according to the 12-step process described in the ESHRE Manual for Guideline Development. After scoping the guideline and listing a set of 12 key questions in PICO (Patient, Intervention, Comparison and Outcome) format, thorough systematic searches of the literature were conducted; evidence from papers published until April 2014 was collected, evaluated for quality and analysed. A summary of evidence was written in a reply to each of the key questions and used as the basis for recommendations, which were defined by consensus within the guideline development group (GDG). Patient and additional clinical input was collected during the scoping and the review phase of the guideline development. PARTICIPANTS/MATERIALS, SETTING, METHODS: The guideline group, comprising psychologists, two medical doctors, a midwife, a patient representative and a methodological expert, met three times to discuss evidence and reach consensus on the recommendations. MAIN RESULTS AND THE ROLE OF CHANCE THE GUIDELINE PROVIDES: 120 recommendations that aim at guiding fertility clinic staff in providing optimal evidence-based routine psychosocial care to patients dealing with infertility and MAR. The guideline is written in two sections. The first section describes patients' preferences regarding the psychosocial care they would like to receive at clinics and how this care is associated with their well-being. The second section of the guideline provides information about the psychosocial needs patients experience across their treatment pathway (before, during and after treatment) and how fertility clinic staff can detect and address these. Needs refer to conditions assumed necessary for patients to have a healthy experience of the fertility treatment. Needs can be behavioural (lifestyle, exercise, nutrition and compliance), relational (relationship with partner if there is one, family friends and larger network, and work), emotional (well-being, e.g. anxiety, depression and quality of life) and cognitive (treatment concerns and knowledge). LIMITATIONS, REASONS FOR CAUTION: We identified many areas in care for which robust evidence was lacking. Gaps in evidence were addressed by formulating good practice points, based on the expert opinion of the GDG, but it is critical for such recommendations to be empirically validated. WIDER IMPLICATIONS OF THE FINDINGS: The evidence presented in this guideline shows that providing routine psychosocial care is associated with or has potential to reduce stress and concerns about medical procedures and improve lifestyle outcomes, fertility-related knowledge, patient well-being and compliance with treatment. As only 45 (36.0%) of the 125 recommendations were based on high-quality evidence, the guideline group formulated recommendations to guide future research with the aim of increasing the body of evidence.

Differential cross sections for intermediate-energy electron scattering from α-tetrahydrofurfuryl alcohol: excitation of electronic-states.

We report on measurements of differential cross sections (DCSs) for electron impact excitation of a series of Rydberg electronic-states in α-tetrahydrofurfuryl alcohol (THFA). The energy range of these experiments was 20-50 eV, while the scattered electron was detected in the 10°-90° angular range. There are currently no other experimental data or theoretical computations against which we can directly compare the present measured results. Nonetheless, we are able to compare our THFA DCSs with earlier cross section measurements for Rydberg-state electronic excitation for tetrahydrofuran, a similar cyclic ether, from Do et al. [J. Chem. Phys. 134, 144302 (2011)]. In addition, "rotationally averaged" elastic DCSs, calculated using our independent atom model with screened additivity rule correction approach are also reported. Those latter results give integral cross sections consistent with the optical theorem, and supercede those from the only previous study of Milosavljevic et al. [Eur. Phys. J. D 40, 107 (2006)].

A dynamical (e,2e) investigation of the structurally related cyclic ethers tetrahydrofuran, tetrahydropyran, and 1,4-dioxane.

Triple differential cross section measurements for the electron-impact ionization of the highest occupied molecular orbitals of tetrahydropyran and 1,4-dioxane are presented. For each molecule, experimental measurements were performed using the (e,2e) technique in asymmetric coplanar kinematics with an incident electron energy of 250 eV and an ejected electron energy of 20 eV. With the scattered electrons being detected at -5°, the angular distributions of the ejected electrons in the binary and recoil regions were observed. These measurements are compared with calculations performed within the molecular 3-body distorted wave model. Here, reasonable agreement was observed between the theoretical model and the experimental measurements. These measurements are compared with results from a recent study on tetrahydrofuran [D. B. Jones, J. D. Builth-Williams, S. M. Bellm, L. Chiari, C. G. Ning, H. Chaluvadi, B. Lohmann, O. Ingolfsson, D. Madison, and M. J. Brunger, Chem. Phys. Lett. 572, 32 (2013)] in order to evaluate the influence of structure on the dynamics of the ionization process across this series of cyclic ethers.

An inquiry-based practical for a large, foundation-level undergraduate laboratory that enhances student understanding of basic cellular concepts and scientific experimental design.

Student-centered education involving research experiences or inquiry have been shown to help undergraduate students understand, and become excited about, the process of scientific investigation. These benefits are particularly important for students in the early stages of their degree (Report and Kenny, http://naplesccsunysbedu/Pres/boyernsf/1998). However, embedding such experiences into the curriculum is particularly difficult when dealing with early stage students, who are in larger cohorts and often lack the background content knowledge necessary to engage with primary research literature and research level methods and equipment. We report here the design, delivery, assessment, and subsequent student learning outcomes of a 4-week practical module for 120 students at the beginning of their second year of university, which successfully engages students in designing cell culture experiments and in understanding the molecular processes and machinery involved in the basic cellular process of macropinocytosis.

Semen donors in Germany: a study exploring motivations and attitudes.

BACKGROUND: Germany is one of the countries where donor insemination (DI) is shrouded in secrecy and where, until recently, donors were assured of anonymity, and clinics were able to destroy documents after 10 years. For many years, preparation seminars for recipients have been conducted. Almost all participants of these seminars intend to disclose the nature of conception to their child, thus representing the beginning of a culture change. This study sought the views of donors regarding their willingness to be identified and therefore meet these expectations. METHODS AND RESULTS: Thirteen of 15 clinics in Germany agreed to participate and of 153 anonymous questionnaires sent, 41% (n = 63, from eight clinics) were returned. Thirty-seven per cent of donors suggested that parents should disclose the nature of the conception to their child, 34% uncertain and 29% opposed. Forty-three percentage were willing to meet offspring, 22% uncertain and 35% opposed. CONCLUSIONS: One-third of the donors supported parental disclosure and just under half of the donors are willing to be identifiable, despite a climate and history of secrecy. This study indicates that there are donors who are agreeable to be part of the move away from secrecy, and this will have implications for professionals involved in providing DI services in Germany.

Confidence in the use of donor insemination: an evaluation of the impact of participating in a group preparation programme.

This paper argues that infertility can result in a range of negative emotional reactions, including a lack of confidence. This is more marked when donor insemination (DI) is used because of the additional shame this is associated with. Parents who do not feel confident may be less likely to share information about their use of DI with others and their child. Legislation in several countries has been introduced to abolish donor anonymity, but this has does not necessarily have an impact on parental confidence. In order to evaluate whether educational preparation programmes can impact on parental confidence and their intention to share information about DI, the confidence levels of 60 participants of three seminars were examined at three different points in time: before, immediately after and several months after attending the programme. Results indicate that most participants' confidence increased markedly as a result of attending an educational seminar. Furthermore, this increased confidence also impacted positively on the intention to share information about DI with future children. Both remained high several months after having attended the programme. These results point out that group preparation programmes can enhance levels of confidence, both as treatment is begun and as family building is undertaken.

Cross sections and oscillator strengths for electron-impact excitation of the A 1B1 electronic state of water.

The authors report absolute differential and integral cross section measurements for electron-impact excitation of the A (1)B(1) electronic state of water. This is an important channel for the production of the OH (X (2)Pi) radical, as well as for understanding the origin of the atmospheric Meinel [Astrophys. J. 111, 555 (1950)] bands. The incident energy range of our measurements is 20-200 eV, while the angular range of the differential cross section data is 3.5 degrees -90 degrees . This is the first time such data are reported in the literature and, where possible, comparison to existing theoretical work, and new scaled Born cross sections calculated as a part of the current study, is made. The scaled Born cross sections are in good agreement with the integral cross sections deduced from the experimental differential cross sections. In addition they report (experimental) generalized oscillator strength data at the incident energies of 100 and 200 eV. These data are used to derive a value for the optical oscillator strength which is found to be in excellent agreement with that from an earlier dipole (e,e) experiment and an earlier photoabsorption experiment.

Sharing information with donor insemination offspring. A child-conception versus a family-building approach.

This paper suggests an innovative approach to the sharing of information between parents and their donor-conceived offspring. The 'family-building' approach is offered in the hope that it will stimulate discussion and development. Traditionally, the emphasis has been on telling the child about his/her donor conception. This has the potential to unintentionally separate the child from the parents. The family-building approach presents donor conception as an issue concerning all members of the family, thus encouraging the child to see him/herself as an integral part of this family's history. Within this approach, the semen provider is acknowledged for his contribution and for having an ongoing significance in the family. It is important, however, to clearly differentiate between him as the genitor and the child's father as the loving and nurturing male in the family. Implications for professionals include the need to acknowledge that donor insemination does not only enable a woman to become pregnant but that it creates a family with a past, present and future. Therefore it will be helpful to provide information about and explore attitudes towards this type of family-building before couples start treatment.

Guidelines for counselling in infertility: outline version.

The Guidelines for Counselling in Infertility describe the purpose, objectives, typical issues and communication skills involved in providing psychosocial care to individuals using fertility services. The Guidelines are presented in six sections. The first section describes how infertility consultations differ from other medical consultations in obstetrics and gynaecology, whereas the second section addresses fundamental issues in counselling, such as what is counselling in infertility, who should counsel and who is likely to need counselling. Section 3 focuses on how to integrate patient-centred care and counselling into routine medical treatment and section 4 highlights some of the special situations which can provoke the need for counselling (e.g. facing the end of treatment, sexual problems). Section 5 deals exclusively with third party reproduction and the psychosocial implications of gamete donation, surrogacy and adoption for heterosexual and gay couples and single women without partners. The final section of the Guidelines is concerned with psychosocial services that can be used to supplement counselling services in fertility clinics: written psychosocial information, telephone counselling, self-help groups and professionally facilitated group work. This paper summarizes the different sections of the Guidelines and describes how to obtain the complete text of the Guidelines for Counselling in Infertility.

Calcium signalling: IP3 rises again...and again.

Recent results indicate that 'regulators of G-protein signalling' may contribute to the generation of receptor-specific patterns of cytosolic Ca2+ oscillations by associating with specific receptors, accelerating G-protein inactivation and responding to changes in cytosolic Ca2+.

The properties of the secretagogue-evoked chloride current in mouse pancreatic acinar cells.

In this paper we describe the properties of the secretagogue-evoked chloride current from mouse pancreatic acinar cells. Single cells were patch-clamped in the whole-cell configuration with solutions that excluded cation currents and then stimulated with 1 mM carbachol (CCh). This resulted in a current that rose to a peak and then decayed to a plateau level. The current/voltage relationship of the peak current was linear whereas that of the plateau phase was rectified and showed time and voltage dependence. To determine if the CCh evoked current was strictly Ca2+ dependent, we compared the properties of the plateau current with those of currents evoked by directly raising cytosolic [Ca2+] The properties of the two currents were the same, with both currents showing outward rectification, development at depolarised potentials, similar time constants of activation, permeability sequences and sensitivity to the Cl- channel inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS). We conclude that the agonist-evoked rise in Ca2+ is alone a sufficient signal to activate the CL- current.

Mechanisms underlying InsP3-evoked global Ca2+ signals in mouse pancreatic acinar cells.

In secretory epithelial cells, complex patterns of Ca2+ signals regulate physiological processes. How these patterns are generated is still not fully understood. In particular, the basis of global Ca2+ waves is not clear. We have studied regional differences in InsP3-evoked Ca2+ release in single mouse pancreatic acinar cells, using high-speed (approximately 90 frames s-1), high-sensitivity Ca2+ imaging combined with rapid (10 ms) spot photolysis (2 micrometer diameter) of caged InsP3. Within a single region we measured Ca2+ response latency and rate of rise to construct an InsP3 dose-response relationship. Spot InsP3 liberation in the secretory pole region consistently elicited a dose-dependent, rapid release of Ca2+. Spot InsP3 liberation in the basal pole region of approximately 50% of cells elicited a similar dose-response relationship but with a lower apparent InsP3 affinity than in the secretory pole. In the other cells, basal pole InsP3 liberation did not elicit active Ca2+ release, even at the highest stimulus intensities we employed, although these same cells did respond when the stimulus spot was moved to different regions. We conclude that in the basal pole active sites of rapid Ca2+ release have a lower functional affinity for InsP3 than those in the secretory pole and are spread out in discrete sites across the basal pole. These properties explain the propagation of Ca2+ waves across the basal pole that are only observed at higher stimulus levels.

Intracellular Ca2+ and Cl- channel activation in secretory cells.

Molecular and functional evidence indicates that a variety of Ca(2+)-dependent chloride (Cl(Ca)) channels are involved in fluid secretion from secretory epithelial cells in different tissues and species. Most Cl(Ca) channels so far characterized have an I- permeability greater than Cl-, and most are sensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Whole-cell Cl(Ca) currents show outward rectification. Single-channel current voltage relationships are linear with conductances ranging from 2 to 30 pS. Some Cl(Ca) channels are blocked by Ca(2+)-calmodulin-dependent protein kinase (CAMKII) inhibitors. Others, such as the Cl(Ca) channels of parotid and submandibular acinar cells, appear to be directly regulated by Ca2+. In native cells, the Cl(Ca) channels are located on the apical plasma membrane and activated by localized mechanisms of Ca2+ release. This positioning allows the Cl(Ca) channel to respond specifically to localized Ca2+ signals that do not invade other regions of the cell. The Cl(Ca) follows the rising phase of the Ca2+ signal, but in the falling phase hysteresis occurs where the Cl(Ca) current decays more rapidly than the underlying Ca2+. The future elucidation of the identity and mechanisms of regulation of Cl(Ca) channels will be critical to our understanding of stimulus-secretion coupling.

Microtubules regulate local Ca2+ spiking in secretory epithelial cells.

The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 micrometer), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me(2)SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca(2+) signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca(2+) response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca(2+) spikes at least in part because of the local positioning of the endoplasmic reticulum.

A bimodal pattern of InsP(3)-evoked elementary Ca(2+) signals in pancreatic acinar cells.

InsP(3)-evoked elementary Ca(2+) release events have been postulated to play a role in providing the building blocks of larger Ca(2+) signals. In pancreatic acinar cells, low concentrations of acetylcholine or the injection of low concentrations of InsP(3) elicit a train of spatially localized Ca(2+) spikes. In this study we have quantified these responses and compared the Ca(2+) signals to the elementary events shown in Xenopus oocytes. The results demonstrate, at the same concentrations of InsP(3), Ca(2+) signals consisting of one population of small transient Ca(2+) release events and a second distinct population of larger Ca(2+) spikes. The signal mass amplitudes of both types of events are within the range of amplitudes for the elementary events in Xenopus oocytes. However, the bimodal Ca(2+) distribution of Ca(2+) responses we observe is not consistent with the continuum of event sizes seen in Xenopus. We conclude that the two types of InsP(3)-dependent events in acinar cells are both elementary Ca(2+) signals, which are independent of one another. Our data indicate a complexity to the organization of the Ca(2+) release apparatus in acinar cells, which might result from the presence of multiple InsP(3) receptor isoforms, and is likely to be important in the physiology of these cells.

Carbachol-stimulated chloride secretion in mouse colon: evidence of a role for autocrine prostaglandin E2 release.

We used the short-circuit current technique to investigate the possible facilitatory role of epithelium-derived prostaglandin E2 (PGE2) release on Cl- secretion in the mouse colon. Carbachol- (CCh)-stimulated Cl- secretion was reduced by pretreatment with either indomethacin (10 microM), or TTX (1 microM), and when added together, these inhibitors revealed net CCh-stimulated K+ secretion. CCh-stimulated Cl- secretion was partially restored to TTX/indomethacin-treated colons by addition of a subsecretory concentration of PGE2 (1 nM). In acutely isolated, unstimulated crypt cells, we measured PGE2 release at a similar level. We conclude that autocrine release of PGs from epithelial cells is sufficient to support the CCh-induced Cl- secretory response and is a likely co-factor in this response.

The role of Ca2+ feedback in shaping InsP3-evoked Ca2+ signals in mouse pancreatic acinar cells.

1. Cytosolic Ca2+ has been proposed to act as both a positive and a negative feedback signal on the inositol trisphosphate (InsP3) receptor. However, it is unclear how this might affect the Ca2+ response in vivo. 2. Mouse pancreatic acinar cells were whole-cell patch clamped to record the Ca2+-dependent chloride (Cl(Ca)) current spikes and imaged to record the cytosolic Ca2+ spikes elicited by the injection of Ins(2,4,5)P3. Increasing concentrations of Ca2+ buffer (up to 200 microM EGTA or BAPTA) were associated with the appearance of steps in the current activation phase and a prevalence of smaller-amplitude Cl(Ca) spikes. Imaging experiments showed that with increased buffer the secretory pole cytosolic Ca2+ signal became fragmented and spatially discrete Ca2+ release events were observed. 3. At higher buffer concentrations (200-500 microM), increasing concentrations of EGTA increased spike frequency and reduced spike amplitude. In contrast, BAPTA decreased spike frequency and maintained large spike amplitudes. 4. We conclude that, during InsP3-evoked spiking, long-range Ca2+ feedback ( approximately 2-4 microm) shapes the rising phase of the Ca2+ signal by acting to co-ordinate discrete Ca2+ release events and short-range ( approximately 40 nm) Ca2+ feedback acts to inhibit further Ca2+ release.

Activation of Ca(2+)- and cAMP-sensitive K(+) channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone.

1-Ethyl-2-benzimidazolone (EBIO) caused a sustained increase in electrogenic Cl(-) secretion in isolated mouse colon mucosae, an effect reduced by blocking basolateral K(+) channels. The Ca(2+)-sensitive K(+) channel blocker charybdotoxin (ChTX) and the cAMP-sensitive K(+) channel blocker 293B were more effective when the other had been added first, suggesting that both types of K(+) channel were activated. EBIO did not cause Cl(-) secretion in cystic fibrosis (CF) colonic epithelia. In apically permeabilized colonic mucosae, EBIO increased the K(+) current when a concentration gradient was imposed, an effect that was completely sensitive to ChTX. No current sensitive to trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2, 2-dimethylchromane (293B) was found in this condition. However, the presence of basolateral cAMP-sensitive K(+) channels was demonstrated by the development of a 293B-sensitive K(+) current after cAMP application in permeabilized mucosae. In isolated colonic crypts EBIO increased cAMP content but had no effect on intracellular Ca(2+). It is concluded that EBIO stimulates Cl(-) secretion by activating Ca(2+)-sensitive and cAMP-sensitive K(+) channels, thereby hyperpolarizing the apical membrane, which increases the electrical gradient for Cl(-) efflux through the CF transmembrane conductance regulator (CFTR). CFTR is also activated by the accumulation of cAMP as well as by direct activation.