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Undoped ZnO nanoparticles (NPs) with size ~12 nm were produced using forced hydrolysis methods using diethylene glycol (DEG) [called ZnO-I] or denatured ethanol [called ZnO-II] as the reaction solvent; both using Zn acetate dehydrate as precursor. Both samples showed weak ferromagnetic behavior at 300K with saturation magnetization Ms=0.077 ± 0.002 memu/g and 0.088 ± 0.013 memu/g for ZnO-I and ZnO-II samples, respectively. Fourier transform infrared (FTIR) spectra showed that ZnO-I nanocrystals had DEG fragments linked to their surface. Photoluminescence (PL) data showed a broad emission near 500nm for ZnO-II which is absent in the ZnO-I samples, presumably due to the blocking of surface traps by the capping molecules. Intentional oxygen vacancies created in the ZnO-I NPs by annealing at 450 °C in flowing Ar gas gradually increased Ms up to 90 min and x-ray photoelectron spectra (XPS) suggested that oxygen vacancies may have a key role in the observed changes in Ms. Finally, PL spectra of ZnO showed the appearance of a blue/violet emission, attributed to Zn interstitials, whose intensity changes with annealing time, similar to the trend seen for Ms. The observed variation in the magnetization of ZnO NP with increasing Ar annealing time seems to depend on the changes in the number of Zn interstitials and oxygen vacancies. [ABSTRACT FROM AUTHOR]

Abstract Background The development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated. Methods Myometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni’s multiple comparisons test was performed. Results We demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10. Conclusions Isolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain their ability to respond to an inflammatory stimulus. These distinct myometrial cell models will provide a useful tool to investigate mechanisms underlying the process of human labour and the concept of functional regionalization of the pregnant uterus.

Early childhood dental caries (ECC) is the most common chronic disease among children, especially among low socioeconomic populations. Streptococcus mutans is most frequently associated with initiation of ECC. Although many studies report children with multiple S. mutans strains (i.e., genotypes) have greater odds of developing ECC, studies investigating intraspecies interactions in dental caries are lacking. This study investigates the impact of intraspecies interactions on cariogenic and fitness traits of clinical S. mutans isolates using in vitro and in vivo approaches. Association analysis evaluated if presence of multiple S. mutans genotypes within the first year of colonization was associated with caries. Initially, clinical S. mutans isolates from 10 children were evaluated. S. mutans strains (G09 and G18, most prevalent) isolated from one child were used for subsequent analysis. Biofilm analysis was performed for single and mixed cultures to assess cariogenic traits, including biofilm biomass, intra-polysaccharide, pH, and glucan. Confocal laser scanning microscopy (CLSM) and time-lapse imaging were used to evaluate spatial and temporal biofilm dynamics, respectively. A Drosophila model was used to assess colonization in vivo . Results showed the mean biofilm pH was significantly lower in co-cultured biofilms versus monoculture. Doubling of S. mutans biofilms was observed by CLSM and in vivo colonization in Drosophila for co-cultured S. mutans . Individual strains occupied specific domains in co-culture and G09 contributed most to increased co-culture biofilm thickness and colonization in Drosophila . Biofilm formation and acid production displayed distinct signatures in time-lapsed experiments. This study illuminates that intraspecies interactions of S. mutans significantly impacts biofilm acidity, architecture, and colonization.IMPORTANCEThis study sheds light on the complex dynamics of a key contributor to early childhood dental caries (ECC) by exploring intraspecies interactions of different S. mutans strains and their impact on cariogenic traits. Utilizing clinical isolates from children with ECC, the research highlights significant differences in biofilm architecture and acid production in mixed versus single genotype cultures. The findings reveal that co-cultured S. mutans strains exhibit increased cell density and acidity, with individual strains occupying distinct domains. These insights, enhanced by use of time-lapsed confocal laser scanning microscopy and a Drosophila model, offer a deeper understanding of ECC pathogenesis and potential avenues for targeted interventions., Competing Interests: The authors declare no conflict of interest.

As climate change and population growth increasingly stress freshwater resources, government agencies and scientists have recognized the importance of water reuse (the beneficial use of reclaimed or recycled water). While end users (municipalities and farmers) are largely open to exploring water reuse, a barrier to using this water source is the public's negative perception, or the 'yuck factor'. Recognizing that water reuse is becoming a necessity, there is an urgent need to determine how to improve public acceptance of this water source. Increasing knowledge has been identified as a critical method for improving the adoption of sustainable practices, but to date, there has been a lack of outcome studies on the ability of water reuse education materials to change perceptions. Previous studies have relied heavily on self-reported concerns and perceptions which are limited by bias and social expectations, and their results have failed to substantially change water reuse education efforts, and by extension failed to substantially improve water reuse perceptions. We propose the integration of psychology for evaluating the effectiveness of water reuse education and outreach efforts. The purpose of this study was to determine if water reuse educational videos modulate perceptions of water reuse terms. Participants were recruited from a university campus and completed pre-intervention surveys on water reuse knowledge, willingness to use recycled water, and preferred water reuse information sources. We then measured changes in neural activity in response to multiple categories of water reuse terms pre- and post-exposure to water reuse education videos using electroencephalogram (EEG). Through EEG we measured event related potential (ERP), which assesses sustained engagement with the cues (late positive potential (LPP) amplitude), and alpha power. Participants also completed a pre/post exposure battery involving five tasks, in randomized order, presented before and after viewing videos. Sixty-eight percent of survey participants had previously heard of water reuse and 91% reported willingness to use recycled water for at least one activity. Participants reported obtaining water reuse information mostly from the internet (77%) and preferred to receive additional information mainly by watching online videos (73%). We observed significant increases in alpha power, corresponding to feelings of relaxation, in response to seven of eight water reuse term categories post-video exposure. These findings show that water reuse educational videos can change perceptions of water reuse. Our results point to the possibility of validating and increasing the effectiveness of water reuse education materials using sophisticated neurocognitive measures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Phenomics has emerged as the technology of choice for understanding quantitative genetic variation in plant physiology and plant breeding. Phenomics has allowed for unmatched precision in exploring plant life cycles and physiological patterns. As new technologies are developed, it is still vital to follow best practices for designing and planning to be able to fully exploit any experimental results. Here we describe the basic - but sometimes overlooked - considerations of a phenomics experiment to help you maximize the value from the data collected: choosing population and location, accounting for sources of variation, establishing a timeline, and leveraging ground-truth measurements., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

We developed a method for directly measuring displacement of a moving surface for use with dynamic or high explosive driven experiments. The technique, called "Modulation Based Ranging" (MBR), overcomes the errors associated with integrating a velocity history of an object undergoing non-radial flow, while also providing the exact displacement of the object with sub 100 µm resolution. A discussion of sources of phase sensitive errors is presented along with a demonstration of the applied corrections. Excellent agreement between MBR and integrated velocity from the Photonic Doppler Velocimetry (PDV) technique was observed when no non-radial flow was present. We then demonstrated the ability of MBR to accurately measure true displacement of a surface subjected to a strong non-radial component.

Service redevelopment has taken place across the NHS in response to the COVID-19 pandemic. At North Bristol NHS Trust, six vulnerable medical staff in non-patient facing roles set up a virtual advice service called 'Ask the Medical Reg'. This service aimed to provide senior medical support to inpatient and community teams for general medical and COVID-19-related queries. Here we outline the structure of our service and present data from the first 4 weeks of operation. We describe how the service has supported both junior doctors working within the hospital and GPs and paramedics, helping with complex decisions to prevent unnecessary admissions., (© Royal College of Physicians 2020. All rights reserved.)

Purpose: To better understand the longitudinal risks and benefits of telephone disclosure of genetic test results in the era of multigene panel testing., Methods: Adults who were proceeding with germline cancer genetic testing were randomized to telephone disclosure (TD) with a genetic counselor or in-person disclosure (IPD) (i.e., usual care) of test results. All participants who received TD were recommended to return to meet with a physician to discuss medical management recommendations., Results: Four hundred seventy-three participants were randomized to TD and 497 to IPD. There were no differences between arms for any cognitive, affective, or behavioral outcomes at 6 and 12 months. Only 50% of participants in the TD arm returned for the medical follow-up appointment. Returning was associated with site (p < 0.0001), being female (p = 0.047), and not having a true negative result (p < 0.002). Mammography was lower at 12 months among those who had TD and did not return for medical follow-up (70%) compared with those who had TD and returned (86%) and those who had IPD (87%, adjusted p < 0.01)., Conclusion: Telephone disclosure of genetic test results is a reasonable alternative to in-person disclosure, but attention to medical follow-up may remain important for optimizing appropriate use of genetic results.

Streptococcus mutans , the primary etiological agent of tooth decay, has developed multiple adhesion and virulence factors which enable it to colonize and compete with other bacteria. The putative glycosyltransferase SMU&#95;833 is important for the virulence of S. mutans by altering the biofilm matrix composition and cariogenicity. In this study, we further characterized the smu&#95;833 mutant by evaluating its effects on bacterial fitness. Loss of SMU&#95;833 led to extracellular DNA-dependent bacterial aggregation. In addition, the mutant was more susceptible to oxidative stress and less competitive against H 2 O 2 producing oral streptococci. Quantitative proteomics analysis revealed that SMU&#95;833 deficiency resulted in the significant downregulation of 10 proteins encoded by a biosynthetic gene cluster responsible for the production of mutanobactin, a compound produced by S. mutans which helps it survive oxidative stress. Tandem affinity purification demonstrated that SMU&#95;833 interacts with the synthetic enzymes responsible for the production of mutanobactin. Similar to the smu&#95;833 mutant, the deletion of the mutanobactin gene cluster rendered the mutant less competitive against H 2 O 2 -producing streptococci. Our studies revealed a new link between SMU&#95;833 virulence and mutanobactin, suggesting that SMU&#95;833 represents a new virulent target that can be used to develop potential anticaries therapeutics. IMPORTANCE Streptococcus mutans is the major bacterium associated with dental caries. In order to thrive on the highly populated tooth surface and cause disease, S. mutans must be able to protect itself from hydrogen peroxide-producing commensal bacteria and compete effectively against the neighboring microbes. S. mutans produces mutacins, small antimicrobial peptides which help control the population of competing bacterial species. In addition, S. mutans produces a peptide called mutanobactin, which offers S. mutans protection against oxidative stress. Here, we uncover a new link between the putative glycosyltransferase SMU&#95;833 and the mutanobactin-synthesizing protein complex through quantitative proteomic analysis and a tandem-affinity protein purification scheme. Furthermore, we show that SMU&#95;833 mediates bacterial sensitivity to oxidative stress and bacterial ability to compete with commensal streptococci. This study has revealed a previously unknown association between SMU&#95;833 and mutanobactin and demonstrated the importance of SMU&#95;833 in the fitness of S. mutans ., (Copyright © 2019 Rainey et al.)

Background: Identification of genetic factors causing predisposition to renal cell carcinoma has helped improve screening, early detection, and patient survival., Methods: We report the characterization of a proband with renal and thyroid cancers and a family history of renal and other cancers by whole-exome sequencing (WES), coupled with WES analysis of germline DNA from additional affected and unaffected family members., Results: This work identified multiple predicted protein-damaging variants relevant to the pattern of inherited cancer risk. Among these, the proband and an affected brother each had a heterozygous Ala45Thr variant in SDHA, a component of the succinate dehydrogenase (SDH) complex. SDH defects are associated with mitochondrial disorders and risk for various cancers; immunochemical analysis indicated loss of SDHB protein expression in the patient's tumor, compatible with SDH deficiency. Integrated analysis of public databases and structural predictions indicated that the two affected individuals also had additional variants in genes including TGFB2, TRAP1, PARP1, and EGF, each potentially relevant to cancer risk alone or in conjunction with the SDHA variant. In addition, allelic imbalances of PARP1 and TGFB2 were detected in the tumor of the proband., Conclusion: Together, these data suggest the possibility of risk associated with interaction of two or more variants., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Streptococcus mutans is a key cariogenic bacterium responsible for the initiation of tooth decay. Biofilm formation is a crucial virulence property. We discovered a putative glycosyltransferase, SMU&#95;833, in S. mutans capable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion of smu&#95;833 decreases glucan and increases eDNA but maintains the overall biofilm biomass. The decrease in glucan is caused by a reduction in GtfB and GtfC, two key enzymes responsible for the synthesis of glucan. The increase in eDNA was accompanied by an elevated production of membrane vesicles, suggesting that SMU&#95;833 modulates the release of eDNA via the membrane vesicles, thereby altering biofilm matrix constituents. Furthermore, glucan and eDNA were colocalized. The complete deletion of gtfBC from the smu&#95;833 mutant significantly reduced the biofilm biomass despite the elevated eDNA, suggesting the requirement of minimal glucans as a binding substrate for eDNA within the biofilm. Despite no changes in overall biofilm biomass, the mutant biofilm was altered in biofilm architecture and was less acidic in vitro Concurrently, the mutant was less virulent in an in vivo rat model of dental caries, demonstrating that SMU&#95;833 is a new virulence factor. Taken together, we conclude that SMU&#95;833 is required for optimal biofilm development and virulence of S. mutans by modulating extracellular matrix components. Our study of SMU&#95;833-modulated biofilm matrix dynamics uncovered a new target that can be used to develop potential therapeutics that prevent and treat dental caries. IMPORTANCE Tooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacterium Streptococcus mutans The bacteria utilize dietary sugars to build and strengthen biofilms, trapping acids onto the tooth's surface and causing demineralization and decay of teeth. As knowledge of our body's microbiomes increases, the need for developing therapeutics targeted to disease-causing bacteria has arisen. The significance of our research is in studying and identifying a novel therapeutic target, a dynamic biofilm matrix that is mediated by a new virulence factor and membrane vesicles. The study increases our understanding of S. mutans virulence and also offers a new opportunity to develop effective therapeutics targeting S. mutans In addition, the mechanisms of membrane vesicle-mediated biofilm matrix dynamics are also applicable to other biofilm-driven infectious diseases., (Copyright © 2019 American Society for Microbiology.)

Telephone disclosure of cancer genetic test results is noninferior to in-person disclosure. However, how patients who prefer in-person communication of results differ from those who agree to telephone disclosure is unclear but important when considering delivery models for genetic medicine. Patients undergoing cancer genetic testing were recruited to a multicenter, randomized, noninferiority trial (NCT01736345) comparing telephone to in-person disclosure of genetic test results. We evaluated preferences for in-person disclosure, factors associated with this preference and outcomes compared to those who agreed to randomization. Among 1178 enrolled patients, 208 (18%) declined randomization, largely given a preference for in-person disclosure. These patients were more likely to be older (P = 0.007) and to have had multigene panel testing (P < 0.001). General anxiety (P = 0.007), state anxiety (P = 0.008), depression (P = 0.011), cancer-specific distress (P = 0.021) and uncertainty (P = 0.03) were higher after pretest counseling. After disclosure of results, they also had higher general anxiety (P = 0.003), depression (P = 0.002) and cancer-specific distress (P = 0.043). While telephone disclosure is a reasonable alternative to in-person disclosure in most patients, some patients have a strong preference for in-person communication. Patient age, distress and complexity of testing are important factors to consider and requests for in-person disclosure should be honored when possible., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Germline genetic testing is standard practice in oncology. Outcomes of telephone disclosure of a wide range of cancer genetic test results, including multigene panel testing (MGPT) are unknown., Methods: Patients undergoing cancer genetic testing were recruited to a multicenter, randomized, noninferiority trial (NCT01736345) comparing telephone disclosure (TD) of genetic test results with usual care, in-person disclosure (IPD) after tiered-binned in-person pretest counseling. Primary noninferiority outcomes included change in knowledge, state anxiety, and general anxiety. Secondary outcomes included cancer-specific distress, depression, uncertainty, satisfaction, and screening and risk-reducing surgery intentions. To declare noninferiority, we calculated the 98.3% one-sided confidence interval of the standardized effect; t tests were used for secondary subgroup analyses. Only noninferiority tests were one-sided, others were two-sided., Results: A total of 1178 patients enrolled in the study. Two hundred eight (17.7%) participants declined random assignment due to a preference for in-person disclosure; 473 participants were randomly assigned to TD and 497 to IPD; 291 (30.0%) had MGPT. TD was noninferior to IPD for general and state anxiety and all secondary outcomes immediately postdisclosure. TD did not meet the noninferiority threshold for knowledge in the primary analysis, but it did meet the threshold in the multiple imputation analysis. In secondary analyses, there were no statistically significant differences between arms in screening and risk-reducing surgery intentions, and no statistically significant differences in outcomes by arm among those who had MGPT. In subgroup analyses, patients with a positive result had statistically significantly greater decreases in general anxiety with telephone disclosure (TD -0.37 vs IPD +0.87, P = .02)., Conclusions: Even in the era of multigene panel testing, these data suggest that telephone disclosure of cancer genetic test results is as an alternative to in-person disclosure for interested patients after in-person pretest counseling with a genetic counselor.

Purpose: Multigene panels (MGPs) are increasingly being used despite questions regarding their clinical utility and no standard approach to genetic counseling. How frequently genetic providers use MGP testing and how patient-reported outcomes (PROs) differ from targeted testing (eg, BRCA1/2 only) are unknown., Methods: We evaluated use of MGP testing and PROs in participants undergoing cancer genetic testing in the multicenter Communication of Genetic Test Results by Telephone study (ClinicalTrials.gov identifier: ), a randomized study of telephone versus in-person disclosure of genetic test results. PROs included genetic knowledge, general and state anxiety, depression, cancer-specific distress, uncertainty, and satisfaction. Genetic providers offered targeted or MGP testing based on clinical assessment., Results: Since the inclusion of MGP testing in 2014, 395 patients (66%) were offered MGP testing. MGP testing increased over time from 57% in 2014 to 66% in 2015 ( P = .02) and varied by site (46% to 78%; P < .01). Being offered MGP testing was significantly associated with not having Ashkenazi Jewish ancestry, having a history of cancer, not having a mutation in the family, not having made a treatment decision, and study site. After demographic adjustment, patients offered MGP testing had lower general anxiety ( P = .04), state anxiety ( P = .03), depression ( P = .04), and uncertainty ( P = .05) pre-disclosure compared with patients offered targeted testing. State anxiety ( P = .05) and cancer-specific distress ( P = .05) were lower at disclosure in the MGP group. There was a greater increase in change in uncertainty ( P = .04) among patients who underwent MGP testing., Conclusion: MGP testing was more frequently offered to patients with lower anxiety, depression, and uncertainty and was associated with favorable outcomes, with the exception of a greater increase in uncertainty compared with patients who had targeted testing. Addressing uncertainty may be important as MGP testing is increasingly adopted., Competing Interests: POTENTIAL CONFLICTS OF INTEREST For more information about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or ascopubs.org/po/author-center.

Background: Women and students of color are widely underrepresented in most STEM fields. In order to investigate this underrepresentation, we interviewed 201 college seniors, primarily women and people of color, who either majored in STEM or started but dropped a STEM major. Here we discuss one section of the longer interview that focused on students' sense of belonging, which has been found to be related to retention. In our analysis, we examine the intersections of race and gender with students' sense of belonging, a topic largely absent from the current literature., Results: We found that white men were most likely to report a sense of belonging whereas women of color were the least likely. Further, we found that representation within one's STEM sub-discipline, namely biology versus the physical sciences, impacts sense of belonging for women. Four key factors were found to contribute to sense of belonging for all students interviewed: interpersonal relationships, perceived competence, personal interest, and science identity., Conclusions: Our findings indicate that students who remain in STEM majors report a greater sense of belonging than those who leave STEM. Additionally, we found that students from underrepresented groups are less likely to feel they belong. These findings highlight structural and cultural features of universities, as well as STEM curricula and pedagogy, that continue to privilege white males., Competing Interests: All interview participants gave informed consent prior to being interviewed. The University of North Carolina at Charlotte’s Institutional Review Board approved all data collection under protocol #13-0112.The manuscript does not include images or videos relating to individual participants.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

A set of nested association mapping (NAM) families was developed by crossing 40 diverse soybean [ (L.) Merr.] genotypes to the common cultivar. The 41 parents were deeply sequenced for SNP discovery. Based on the polymorphism of the single-nucleotide polymorphisms (SNPs) and other selection criteria, a set of SNPs was selected to be included in the SoyNAM6K BeadChip for genotyping the parents and 5600 RILs from the 40 families. Analysis of the SNP profiles of the RILs showed a low average recombination rate. We constructed genetic linkage maps for each family and a composite linkage map based on recombinant inbred lines (RILs) across the families and identified and annotated 525,772 high confidence SNPs that were used to impute the SNP alleles in the RILs. The segregation distortion in most families significantly favored the alleles from the female parent, and there was no significant difference of residual heterozygosity in the euchromatic vs. heterochromatic regions. The genotypic datasets for the RILs and parents are publicly available and are anticipated to be useful to map quantitative trait loci (QTL) controlling important traits in soybean., (Copyright © 2017 Crop Science Society of America.)

Most nuclear-encoded mitochondrial proteins traffic from the cytosol to mitochondria. Some of these proteins localize at mitochondria-associated membranes (MAM), where mitochondria are closely apposed with the endoplasmic reticulum (ER). We have previously shown that the human cytomegalovirus signal-anchored protein known as viral mitochondria-localized inhibitor of apoptosis (vMIA) traffics from the ER to mitochondria and clusters at the outer mitochondrial membrane (OMM). Here, we have examined the host pathways by which vMIA traffics from the ER to mitochondria and clusters at the OMM. By disruption of phosphofurin acidic cluster sorting protein 2 (PACS-2), mitofusins (Mfn1/2), and dynamin related protein 1 (Drp1), we find these conventional pathways for ER to the mitochondria trafficking are dispensable for vMIA trafficking to OMM. Instead, mutations in vMIA that change its hydrophobicity alter its trafficking to mitochondria. Superresolution imaging showed that PACS-2- and Mfn-mediated membrane apposition or hydrophobic interactions alter vMIA's ability to organize in nanoscale clusters at the OMM. This shows that signal-anchored MAM proteins can make use of hydrophobic interactions independently of conventional ER-mitochondria pathways to traffic from the ER to mitochondria. Further, vMIA hydrophobic interactions and ER-mitochondria contacts facilitate proper organization of vMIA on the OMM.

We describe two-step fluorescence microscopy, a new approach to non-linear imaging based on positive reversible photoswitchable fluorescent probes. The protein Padron approximates ideal two-step fluorescent behaviour: it equilibrates to an inactive state, converts to an active state under blue light, and blue light also excites this active state to fluoresce. Both activation and excitation are linear processes, but the total fluorescent signal is quadratic, proportional to the square of the illumination dose. Here, we use Padron's quadratic non-linearity to demonstrate the principle of two-step microscopy, similar in principle to two-photon microscopy but with orders-of-magnitude better cross-section. As with two-photon, quadratic non-linearity from two-step fluorescence improves resolution and reduces unwanted out-of-focus excitation, and is compatible with structured illumination microscopy. We also show two-step and two-photon imaging can be combined to give quartic non-linearity, further improving imaging in challenging samples. With further improvements, two-step fluorophores could replace conventional fluorophores for many imaging applications.

Purpose: Multiplex genetic testing, including both moderate- and high-penetrance genes for cancer susceptibility, is associated with greater uncertainty than traditional testing, presenting challenges to informed consent and genetic counseling. We sought to develop a new model for informed consent and genetic counseling for four ongoing studies., Methods: Drawing from professional guidelines, literature, conceptual frameworks, and clinical experience, a multidisciplinary group developed a tiered-binned genetic counseling approach proposed to facilitate informed consent and improve outcomes of cancer susceptibility multiplex testing., Results: In this model, tier 1 "indispensable" information is presented to all patients. More specific tier 2 information is provided to support variable informational needs among diverse patient populations. Clinically relevant information is "binned" into groups to minimize information overload, support informed decision making, and facilitate adaptive responses to testing. Seven essential elements of informed consent are provided to address the unique limitations, risks, and uncertainties of multiplex testing., Conclusion: A tiered-binned model for informed consent and genetic counseling has the potential to address the challenges of multiplex testing for cancer susceptibility and to support informed decision making and adaptive responses to testing. Future prospective studies including patient-reported outcomes are needed to inform how to best incorporate multiplex testing for cancer susceptibility into clinical practice.Genet Med 17 6, 485-492.

Anti-N-methyl-D-aspartate (NMDA) receptor encephalitis is an important, treatable cause of encephalitis which remains under-recognised despite a growing body of the literature [1]. It is an immune-mediated syndrome which presents with a variety of neurological symptoms including headache, fever, personality change and seizures. Most case reports to date are of young adults, it is much less frequently reported in older adults. The syndrome has been associated with ovarian teratomas. The prognosis is good with early recognition and treatment, though may relapse. We present a case of NMDA encephalitis in an elderly patient who responded well to immunosuppressive therapy., (© The Author 2014. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, traffics to mitochondria-associated membranes (MAM), where the endoplasmic reticulum (ER) contacts the outer mitochondrial membrane (OMM). vMIA association with the MAM has not been visualized by imaging. Here, we have visualized this by using a combination of confocal and superresolution imaging. Deconvolution of confocal microscopy images shows vMIA localizes away from mitochondrial matrix at the Mitochondria-ER interface. By gated stimulated emission depletion (GSTED) imaging, we show that along this interface vMIA is distributed in clusters. Through multicolor, multifocal structured illumination microscopy (MSIM), we find vMIA clusters localize away from MitoTracker Red, indicating its OMM localization. GSTED and MSIM imaging show vMIA exists in clusters of ~100-150 nm, which is consistent with the cluster size determined by Photoactivated Localization Microscopy (PALM). With these diverse superresolution approaches, we have imaged the clustered distribution of vMIA at the OMM adjacent to the ER. Our findings directly compare the relative advantages of each of these superresolution imaging modalities for imaging components of the MAM and sub-mitochondrial compartments. These studies establish the ability of superresolution imaging to provide valuable insight into viral protein location, particularly in the sub-mitochondrial compartments, and into their clustered organization.

Nine Virginia soybeans grown in a single location were evaluated and compared for their antioxidant properties and isoflavone profiles. The total phenolic content (TPC) in the soybean extracts was significantly different among different genotypes. The V01-4937, V03-1144, and MFS-511 soybeans had the highest TPC values of 3.89, 3.63, and 3.53 mg of gallic acid equiv/g of seeds, respectively. The isoflavone composition was also different among the different soybean varieties. Malonylgenistin was the major isoflavone in all soybean seeds, accounting for 75-83% of the total measured isoflavones. The V01-4937 variety had the highest total isoflavones and malonylgenistin content followed by the V03-5794. The antioxidant activities of the soybean extracts were also significantly different. V01-4937 and Teejay showed the strongest ORAC values, which were 70% higher than that of the V00-3493 soybean, which had the lowest ORAC value (115.7 micromol of Trolox equiv/g of seeds). However, their ORAC values were correlated with neither TPC nor total isoflavone content. The MFS-511, V01-4937, and Teejay soybeans had the highest DPPH radical scavenging activities of 4.94, 4.78, and 4.64 micromol of Trolox equiv/g of seeds. Overall, the V01-4937 soybean stood out among the tested Virginia soybeans with regard to having the highest TPC, ORAC value, and isoflavone content as well as the second highest DPPH scavenging activity.

Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-1, causes cardiac, vascular, and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn1, Irp1, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis.

A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1alpha mutant, or a short hairpin RNA directed against HIF-1alpha. In human RCC biopsies, expression of E-cadherin and HIF-1alpha was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1-dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin.

A fundamental problem in biology is the evolutionary transition from single cells to multicellular life forms. During this transition the unit of selection shifts from individual cells to groups of cooperating cells. Although there is much theory, there are few empirical studies. Here we describe an evolutionary transition that occurs in experimental populations of Pseudomonas fluorescens propagated in a spatially heterogeneous environment. Cooperating groups are formed by over-production of an adhesive polymer, which causes the interests of individuals to align with those of the group. The costs and benefits of cooperation, plus evolutionary susceptibility to defecting genotypes, were analysed to determine conformation to theory. Cooperation was costly to individuals, but beneficial to the group. Defecting genotypes evolved in populations founded by the cooperating type and were fitter in the presence of this type than in its absence. In the short term, defectors sabotaged the viability of the group; but these findings nevertheless show that transitions to higher orders of complexity are readily achievable, provide insights into the selective conditions, and facilitate experimental analysis of the evolution of individuality.