Single-institution study evaluating the utility of surveillance bronchoscopy after lung transplantation.
Many lung transplant physicians advocate surveillance bronchoscopy with transbronchial lung biopsy and bronchoalveolar lavage (TBB/BAL) to monitor lung recipients despite limited evidence this strategy improves outcomes. This report compares rates of infection (INF), acute rejection (AR), bronchiolitis obliterans syndrome (BOS) and survival in lung allograft recipients managed with surveillance TBB/BAL (SB) versus those with clinically indicated TBB/BAL (CIB). We reviewed 47 consecutive recipients transplanted between March 2002 and August 2005. Of these recipients, 24 consented to a multi-center trial requiring SB and 23 were managed by our usual practice of CIB. Rates of freedom from INF, AR, BOS and survival were compared. BOS and AR were diagnosed according to published guidelines from the International Society for Heart and Lung Transplantation. A total of 240 TBB/BALs were performed. CIB and SB groups underwent 84 (3.7 +/- 3.4/patient) and 156 (6.5 +/- 2.0/patient) TBB/BALs, respectively. In the SB group, 54 (2.2 +/- 1.6/patient) TBB/BALs were true surveillance procedures, whereas 102 (4.2 +/- 2.3/patient) were clinically indicated. No AR episode requiring treatment was detected by true surveillance. Freedom from respiratory INF, AR, BOS and survival in the SB and CIB groups showed no significant differences. Five patients in the CIB group remained stable without requiring TBB/BAL. In the SB group, 4 previously asymptomatic patients developed pneumonia within 2 weeks of surveillance TBB/BAL. With no obvious advantage identified, surveillance bronchoscopy may pose a risk to stable lung transplant recipients. A multi-center, controlled trial is required to validate the utility and safety of surveillance bronchoscopy in lung transplantation.


Derivation of induced pluripotent stem cells from human peripheral circulating T cells.
This unit describes a protocol for the generation of induced pluripotent stem (iPS) cells from human peripheral circulating T cells. Initially, human dermal fibroblasts and retroviral vectors were used to generate human iPS cells. Invasive approaches, such as skin biopsy, and genomic insertion of transgenes into the host genome are not appropriate for routine clinical application. Peripheral circulating T cells are readily available from blood samples of patients and healthy volunteers. For the efficient generation of human iPS cells, efficient introduction of the transgene into host cells is necessary. Using a combination of activated T cell culture and Sendai virus allows for the easy and efficient introduction of transgenes into activated T cells and the generation of human iPS cells without genomic integration of extrinsic genes. The T cell-derived iPS (TiPS) cells exhibit monoclonal T cell receptor (TCR) rearrangement in their genome, a hallmark of mature terminally differentiated T cells.


Tracking the invasive history of the green alga Codium fragile ssp. tomentosoides.
The spread of nonindigenous species into new habitats is having a drastic effect on natural ecosystems and represents an increasing threat to global biodiversity. In the marine environment, where data on the movement of invasive species is scarce, the spread of alien seaweeds represents a particular problem. We have employed a combination of plastid microsatellite markers and DNA sequence data from three regions of the plastid genome to trace the invasive history of the green alga Codium fragile ssp. tomentosoides. Extremely low levels of genetic variation were detected, with only four haplotypes present in the species' native range in Japan and only two of these found in introduced populations. These invasive populations displayed a high level of geographical structuring of haplotypes, with one haplotype localized in the Mediterranean and the other found in Northwest Atlantic, northern European and South Pacific populations. Consequently, we postulate that there have been at least two separate introductions of C. fragile ssp. tomentosoides from its native range in the North Pacific.


Vinorelbine plus cisplatin vs. observation in resected non-small-cell lung cancer.
We undertook to determine whether adjuvant vinorelbine plus cisplatin prolongs overall survival among patients with completely resected early-stage non-small-cell lung cancer. We randomly assigned patients with completely resected stage IB or stage II non-small-cell lung cancer to vinorelbine plus cisplatin or to observation. The primary end point was overall survival; principal secondary end points were recurrence-free survival and the toxicity and safety of the regimen. A total of 482 patients underwent randomization to vinorelbine plus cisplatin (242 patients) or observation (240); 45 percent of the patients had pathological stage IB disease and 55 percent had stage II, and all had an Eastern Cooperative Oncology Group performance status score of 0 or 1. In both groups, the median age was 61 years, 65 percent were men, and 53 percent had adenocarcinomas. Chemotherapy caused neutropenia in 88 percent of patients (including grade 3 febrile neutropenia in 7 percent) and death from toxic effects in two patients (0.8 percent). Nonhematologic toxic effects of chemotherapy were fatigue (81 percent of patients), nausea (80 percent), anorexia (55 percent), vomiting (48 percent), neuropathy (48 percent), and constipation (47 percent), but severe (grade 3 or greater) toxic effects were uncommon (<10 percent). Overall survival was significantly prolonged in the chemotherapy group as compared with the observation group (94 vs. 73 months; hazard ratio for death, 0.69; P=0.04), as was relapse-free survival (not reached vs. 46.7 months; hazard ratio for recurrence, 0.60; P<0.001). Five-year survival rates were 69 percent and 54 percent, respectively (P=0.03). Adjuvant vinorelbine plus cisplatin has an acceptable level of toxicity and prolongs disease-free and overall survival among patients with completely resected early-stage non-small-cell lung cancer.


Changes in arterial distensibility and flow-mediated dilation after acute resistance vs. aerobic exercise.
Previous research has shown significantly lower arterial distensibility (AD) after resistance exercise (RE) yet higher AD after aerobic exercise (AE). These changes may be related to exercise-induced differences in vasodilatory capacity. The purpose of this study was to investigate the vasodilatory and AD responses to acute AE and RE. Forearm blood flow (FBF) during reactive hyperemia (RH) was assessed before and 60 minutes after exercise, whereas aortic and femoral pulse wave velocity was measured as an index of arterial stiffness pre, 40, and 60 minutes after an acute bout of AE (30-minute leg ergometry at 65% of VO2peak) and RE (3 sets, 10 reps; upper and lower body at 65% 1 repetition maximum) in 10 male subjects (24.9 ± 0.86 years). Area under the curve (AUC) was employed to determine differences in flow. After the intervention, we found that central pulse wave velocity decreased 8% after AE and remained depressed at this level through 60 minutes of observation, whereas RE increased central pulse wave velocity 9.8% from pre to 40 and 60 minutes postexercise. Area under the curve for FBF-RH significantly increased 38% after RE, yet there was no significant change after AE. Forearm vasodilatory capacity increased after acute RE but not after acute AE. This suggests that changes in AD may be disassociated from changes in vasodilatory capacity after acute exercise. Further, in a direct comparison of RE vs. AE, we have shown that RE has greater increases in limb blood flow and augments postexercise hypotension greater at 40 minutes postexercise when compared to AE.


Reverse transcriptases and genomic variability: the accuracy of DNA replication is enzyme specific and sequence dependent.
Kinetics of incorporation of correct and incorrect deoxynucleotides by three reverse transcriptases have been followed, by gel assay, on a series of DNA templates, including part of the HIV-1 gag DNA minus strand. Insertion kinetics for the properly matched nucleotide at a given place on the template vary strongly from one enzyme to the next. No significant correlation is found between the site-specific Michaelis constants, while the maximal velocities are more closely connected. For a given reverse transcriptase these parameters are strongly influenced by the DNA sequence. A systematic evaluation of the frequencies of misincorporation was then performed at 46 positions. Again great variability was found, precluding a very accurate evaluation of an average misincorporation frequency for a given enzyme and a given mismatch. Qualitatively however, HIV-1 reverse transcriptase is certainly not more error-prone in this assay than the other enzymes assayed. The patterns of misincorporations were again very dependent on the enzyme used to replicate a given template. The variability of the gag sequence observed in vivo among various HIV-1 isolates was compared with the patterns of misincorporations obtained in vitro on the same sequence with HIV-1, AMV and MoMLV reverse transcriptases. A fair agreement was found with the pattern observed in the polymerization directed by the HIV-1 reverse transcriptase. The correlation is less important in the two other cases. However some specific changes observed in vivo cannot be accounted for by our misincorporation assay, even when performed with the homologous enzyme, suggesting that an important class of mismatches can only be generated during reverse transcription of the RNA strand. Additional data, using a complementary DNA (positive) strand as a gag template support this hypothesis.


Novel and Unexpected Microbial Diversity in Acid Mine Drainage in Svalbard (78° N), Revealed by Culture-Independent Approaches.
Svalbard, situated in the high Arctic, is an important past and present coal mining area. Dozens of abandoned waste rock piles can be found in the proximity of Longyearbyen. This environment offers a unique opportunity for studying the biological control over the weathering of sulphide rocks at low temperatures. Although the extension and impact of acid mine drainage (AMD) in this area is known, the native microbial communities involved in this process are still scarcely studied and uncharacterized. Several abandoned mining areas were explored in the search for active AMD and a culture-independent approach was applied with samples from two different runoffs for the identification and quantification of the native microbial communities. The results obtained revealed two distinct microbial communities. One of the runoffs was more extreme with regards to pH and higher concentration of soluble iron and heavy metals. These conditions favored the development of algal-dominated microbial mats. Typical AMD microorganisms related to known iron-oxidizing bacteria (Acidithiobacillus ferrivorans, Acidobacteria and Actinobacteria) dominated the bacterial community although some unexpected populations related to Chloroflexi were also significant. No microbial mats were found in the second area. The geochemistry here showed less extreme drainage, most likely in direct contact with the ore under the waste pile. Large deposits of secondary minerals were found and the presence of iron stalks was revealed by microscopy analysis. Although typical AMD microorganisms were also detected here, the microbial community was dominated by other populations, some of them new to this type of system (Saccharibacteria, Gallionellaceae). These were absent or lowered in numbers the farther from the spring source and they could represent native populations involved in the oxidation of sulphide rocks within the waste rock pile. This environment appears thus as a highly interesting field of potential novelty in terms of both phylogenetic/taxonomic and functional diversity.


Sex Differences in HIV Prevalence Persist over Time: Evidence from 18 Countries in Sub-Saharan Africa.
The aim of this study was to examine changes over time in the female: male HIV prevalence ratio in 18 countries in Sub-Saharan Africa, overall and when stratified by area of residence, educational attainment and marital status. We used data from the Demographic and Health Surveys, which are nationally representative household surveys. By using data from 18 countries with at least two survey rounds with HIV testing, and dividing the countries into three regions (Western/Central, Eastern and Southern) we were able to examine cross-country and regional changes in the female: male HIV prevalence ratio over time. Logistic regression was used to estimate female: male HIV prevalence ratios in urban versus rural areas and for different categories of education and marital status. To assess changes over time, we compared the confidence intervals of the prevalence ratios. The female: male HIV prevalence ratio was above one in all countries in at least one survey round for both ages 15-24 years and 25-49 years. In 13 out of 18 countries the prevalence ratio was higher for the younger age group compared to the age group 25-49 years (3 significant) and this difference in prevalence ratios between the age groups did not change over time. Overall, there was a higher frequency of increasing than decreasing prevalence ratios. The gender disparity was greater among those who were married/living together than among the never-married, and over time, the ratio was more stable among the married/living together. The study found no clear differential changes by education. Women continue to carry the greater burden of HIV in Sub-Saharan Africa and there is no clear pattern of change in the gap between men and women as the direction and magnitude of change in the prevalence ratios varied greatly.


Evaluation and management of the patient with acute back pain.
Patients with low back pain are among the largest group of health care consumers today. During an episode of acute back pain the patient may be hospitalized for diagnostic workup and medical management, processes to which occupational therapists can contribute. The occupational therapist's evaluation includes assessment of the patient's activities of daily living and understanding of back protection and pain behaviors. The therapist involves the patient in managing his or her back problem by teaching problem-solving skills and identifying appropriate life-style changes. Instruction is provided in proper body mechanics, anatomy of the spine, work simplification, relaxation, and adaptive methods of performing daily activities. Given the short hospitalization period, outpatient follow-up may be provided to facilitate the application of information learned in the hospital to home and work settings. The goal of occupational therapy is to help the patient with low back pain return to a productive life-style.


Comprehensive analysis of CD8(+)-T-cell responses against hepatitis C virus reveals multiple unpredicted specificities.
The hepatitis C virus (HCV)-specific CD8(+)-T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8(+)-T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8(+)-T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8(+)-T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition.


Impact of COVID-19 restrictions on carers of persons with dementia in the UK: a qualitative study.
informal carers provide the majority of the support for persons with dementia living at home. Restrictions imposed due to COVID-19 have had a profound impact on the daily life of the entire population. This study provides insight into the impact of these restrictions on carers of people with dementia living at home. qualitative semi-structured interviews. purposive sample of carers who provide at least 10 hours of care a week for the person with dementia living at home. UK. twenty-three carers were interviewed, and thematic analysis identified three main themes-Changes to daily life, impact on carer health and wellbeing and reduced support from health and social support networks. The results highlight the impact of restrictions imposed on daily life and routines due to the pandemic, wellbeing of carers, reduced social support, lack of access to health and care professionals and respite for carers. The restrictions have had negative consequences on carers' wellbeing, and they have experienced difficulties in accessing formal care services and respite care. carers attempt to continue to provide physical, emotional and practical support for persons with dementia in the community throughout the COVID-19 restrictions. To prevent a future carer crisis, carers need better support systems including formal carer services, telecare solutions that work for them and additional support for respite, as the restrictions from this pandemic continue.


How much DNA is lost? Measuring DNA loss of short-tandem-repeat length fragments targeted by the PowerPlex 16® system using the Qiagen MinElute Purification Kit.
The success in recovering genetic profiles from aged and degraded biological samples is diminished by fundamental aspects of DNA extraction, as well as its long-term preservation, that are not well understood. While numerous studies have been conducted to determine whether one extraction method was superior to others, nearly all of them were initiated with no knowledge of the actual starting DNA quantity in the samples prior to extraction, so they ultimately compared the outcome of all methods relative to the best. Using quantitative PCR to estimate the copy count of synthetic standards before (i.e., "copies in") and after (i.e., "copies out") purification by the Qiagen MinElute PCR Purification Kit, we documented DNA loss within a pool of 16 different-sized fragments ranging from 106 to 409 bp in length, corresponding to those targeted by the PowerPlex 16 System (Promega, Madison, WI). Across all standards from 10(4) to 10(7) copies/μL, loss averaged between 21.75% and 60.56% (mean, 39.03%), which is not congruent with Qiagen's claim that 80% of 70 bp to 4 kb fragments are retained using this product (i.e., 20% loss). Our study also found no clear relationship either between DNA strand length and retention or between starting copy number and retention. This suggests that there is no molecule bias across the MinElute column membrane and highlights the need for manufacturers to clearly and accurately describe on what their claims are based, and should also encourage researchers to document DNA retention efficiencies of their own methods and protocols. Understanding how and where to reduce loss of molecules during extraction and purification will serve to generate clearer and more accurate data, which will enhance the utility of ancient and low-copy-number DNA as a tool for closing forensic cases or in reconstructing the evolutionary history of humans and other organisms.


Mapping the binding of GluN2B-selective N-methyl-D-aspartate receptor negative allosteric modulators.
We have used recent structural advances in our understanding of the N-methyl-d-aspartate (NMDA) receptor amino terminal domain to explore the binding mode of multiple diaryl GluN2B-selective negative allosteric modulators at the interface between the GluN1 and GluN2B amino-terminal domains. We found that interaction of the A ring within the binding pocket seems largely invariant for a variety of structurally distinct ligands. In addition, a range of structurally diverse linkers between the two aryl rings can be accommodated by the binding site, providing a potential opportunity to tune interactions with the ligand binding pocket via changes in hydrogen bond donors, acceptors, as well as stereochemistry. The most diversity in atomic interactions between protein and ligand occur in the B ring, with functional groups that contain electron donors and acceptors providing additional atomic contacts within the pocket. A cluster of residues distant to the binding site also control ligand potency, the degree of inhibition, and show ligand-induced increases in motion during molecular dynamics simulations. Mutations at some of these residues seem to distinguish between structurally distinct ligands and raise the possibility that GluN2B-selective ligands can be divided into multiple classes. These results should help facilitate the development of well tolerated GluN2B subunit-selective antagonists.


The Collaborative Network Approach: a model for advancing patient-centric research for Castleman disease and other rare diseases.
There are ∼7000 rare diseases affecting 30 000 000 individuals in the U.S.A. 95% of these rare diseases do not have a single Food and Drug Administration-approved therapy. Relatively, limited progress has been made to develop new or repurpose existing therapies for these disorders, in part because traditional funding models are not as effective when applied to rare diseases. Due to the suboptimal research infrastructure and treatment options for Castleman disease, the Castleman Disease Collaborative Network (CDCN), founded in 2012, spearheaded a novel strategy for advancing biomedical research, the 'Collaborative Network Approach'. At its heart, the Collaborative Network Approach leverages and integrates the entire community of stakeholders - patients, physicians and researchers - to identify and prioritize high-impact research questions. It then recruits the most qualified researchers to conduct these studies. In parallel, patients are empowered to fight back by supporting research through fundraising and providing their biospecimens and clinical data. This approach democratizes research, allowing the entire community to identify the most clinically relevant and pressing questions; any idea can be translated into a study rather than limiting research to the ideas proposed by researchers in grant applications. Preliminary results from the CDCN and other organizations that have followed its Collaborative Network Approach suggest that this model is generalizable across rare diseases.


Cerebral coherence between communicators marks the emergence of meaning.
How can we understand each other during communicative interactions? An influential suggestion holds that communicators are primed by each other's behaviors, with associative mechanisms automatically coordinating the production of communicative signals and the comprehension of their meanings. An alternative suggestion posits that mutual understanding requires shared conceptualizations of a signal's use, i.e., "conceptual pacts" that are abstracted away from specific experiences. Both accounts predict coherent neural dynamics across communicators, aligned either to the occurrence of a signal or to the dynamics of conceptual pacts. Using coherence spectral-density analysis of cerebral activity simultaneously measured in pairs of communicators, this study shows that establishing mutual understanding of novel signals synchronizes cerebral dynamics across communicators' right temporal lobes. This interpersonal cerebral coherence occurred only within pairs with a shared communicative history, and at temporal scales independent from signals' occurrences. These findings favor the notion that meaning emerges from shared conceptualizations of a signal's use.


A new approach for modelling gene regulatory networks using fuzzy petri nets.
Gene Regulatory Networks are models of genes and gene interactions at the expression level. The advent of microarray technology has challenged computer scientists to develop better algorithms for modeling the underlying regulatory relationship in between the genes. Fuzzy system has an ability to search microarray datasets for activator/repressor regulatory relationship. In this paper, we present a fuzzy reasoning model based on the Fuzzy Petri Net. The model considers the regulatory triplets by means of predicting changes in expression level of the target based on input expression level. This method eliminates possible false predictions from the classical fuzzy model thereby allowing a wider search space for inferring regulatory relationship. Through formalization of fuzzy reasoning, we propose an approach to construct a rulebased reasoning system. The experimental results show the proposed approach is feasible and acceptable to predict changes in expression level of the target gene.


Effect of the specific Src family kinase inhibitor saracatinib on osteolytic lesions using the PC-3 bone model.
The hematogenous metastatic spread of prostate cancer is preferentially to bone and can result in significant patient morbidity. Although these metastatic lesions are typically osteoblastic, bone resorption is believed to have a prerequisite role in their development. Src kinase has been identified to contribute to prostate cancer tumor growth and metastasis. In addition, Src is also essential in bone metabolism, especially in bone resorption. We hypothesized that inhibiting Src activity with the specific Src family kinase inhibitor saracatinib (AZD0530) would inhibit tumor cell growth and osteoclast differentiation in the tumor-bone interface, thus providing a new approach for advanced prostate cancer. We found that saracatinib inhibited PC-3 cell growth and invasion in a dose-dependent manner. Phosphorylation of Src, focal adhesion kinase, and P38 kinases was inhibited by saracatinib at the submicromolar range. Saracatinib also inhibited the expression and secretion of invasion-related molecules interlukin-8, urokinase-type plasminogen activator, and matrix metalloprotease-9. Receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis and signaling were inhibited by saracatinib in both macrophages and PC-3 cells. In in vivo studies, control mice developed more severe osteolytic lesions compared with the treatment group. Immunohistochemical and biochemical assays of bone metabolites confirmed that saracatinib preserved bone architecture in the presence of prostate cancer tumor cells. In summary, we have shown the inhibition of PC3 cell growth and invasion by saracatinib. Src inhibition also blocked the RANKL stimulatory pathway in osteoclasts and PC3 cells. The inhibition of Src thus targets multiple sites involved in prostate cancer bone metastasis, which may offer a therapeutic advantage in treating advanced prostate cancer.


Non-biological lesbian mothers' experiences of accessing healthcare for their children.
Lesbian, gay, bisexual and trans (LGBT) parents may have negative experiences when accessing healthcare for their children as a result of homophobia, transphobia or other forms of prejudice. Patients and their families have reported receiving inappropriate comments from healthcare professionals about sexual orientation, despite policy and government recommendations advising healthcare professionals to provide care that demonstrates respect, dignity and compassion. The aim of this pilot study was to understand the experience of non-biological lesbian mothers accessing healthcare for their children and the types of attitudes and language they encountered. The pilot was also carried out to determine that the style of questioning allowed differentiation between sexuality and gender. This narrative enquiry pilot study used interviews conducted with three non-biological lesbian mothers aged 36-45 years. Two of the participants were a married couple with adopted children; the other had undergone in vitro fertilisation (IVF) with her partner. Of the three participants, two were the authors of this study. The data were coded using thematic analysis. The participants' interviews revealed mixed experiences with healthcare professionals' attitudes. Four common themes emerged: attitudes and managing healthcare experiences; acknowledgement of sexual orientation; professional standards; and family constellation. The study found that non-biological lesbian mothers experience positive and negative interactions with healthcare professionals but feel disenfranchised because of their non-biological status. A reason for the negative interactions could be a lack of training for healthcare professionals about the challenges that lesbian mothers face when accessing healthcare for their children.


Sexual, marital, and social impact of a man's perceived infertility diagnosis.
Male factor infertility is a relatively common problem. This diagnosis may increase sexual, marital, and relationship strain in male partners of infertile couples. To measure the personal, social, sexual, and marital impacts of a male factor infertility diagnosis among men in couples evaluated for infertility. Cross-sectional analysis of 357 men in infertile couples from eight academic and community-based fertility clinics. Participants completed written surveys and face-to-face and telephone interviews at study enrollment. This interview queried each participant's perception of their infertility etiology to determine the primary study exposure (i.e., male factor only, male and female factors, female factor only, unknown). Personal Impact, Social Impact, Marital Impact, and Sexual Impact scales. Among the 357 men, no male factor was reported in 47%, isolated male factor was present in 12%, combined male and female factors were present in 16%, and unexplained infertility was present in 25% of couples. Male factor infertility was independently associated with worse Sexual (mean 39 vs. 30, standard deviation [SD] 2.7, P = 0.004) and Personal (mean 37 vs. 29, SD 3.8, P = 0.04) Impact scores relative to men in couples without male factor infertility. These differences remained statistically significant after controlling for male age, partner age, race, religion, educational level, employment status, prior pregnancy, duration of infertility, and prior paternity. Male partners in couples who perceive isolated male factor infertility have a lower sexual and personal quality of life compared with male partners of couples without perceived male factor infertility. Social strain is highest among couples without a clear etiology for infertility. These findings highlight the clinically significant negative sexual, personal, and social strains of a perceived infertility diagnosis for men.


p300 Influences Butyrate-Mediated WNT Hyperactivation In Colorectal Cancer Cells.
Deregulated WNT/catenin pathway, usually resulting from mutations in the adenomatous polyposis coli and beta-catenin genes, drives colorectal tumorigenesis. Dietary fiber has been shown to have a protective role against colorectal cancer (CRC). We have previously demonstrated that the histone deacetylase inhibitor (HDACi) butyrate, a fermentation product of dietary fiber, induces WNT/catenin hyperactivation, which promotes CRC cell apoptosis. Therefore, the ability of butyrate to induce WNT hyperactivation and thus promote CRC cell apoptosis may in part explain the preventive function of fiber against CRC. The association between beta-catenin and the transcriptional coactivator p300 may influence WNT/catenin signaling and, therefore, colonic cell physiology. p300 functions as a histone acetylase (HAT); therefore, the modulation of WNT/catenin activity by p300 may influence the ability of the HDACi butyrate to hyperinduce WNT signaling and apoptosis in CRC cells. Our findings indicate that p300 affects the hyperinduction of WNT activity by butyrate. Knockdown of p300 levels represses butyrate-mediated WNT/catenin activity; but still allows for butyrate-mediated apoptosis. Overexpression of p300 stimulates basal and butyrate-induced WNT signaling in some, but not all, CRC cell lines. We also evaluate the role of p300 in therapeutic approaches that target CBP. The small molecule ICG-001, in clinical trial, is a specific inhibitor of CBP-mediated WNT signaling, and previous studies have suggested that p300 is required for the activity of ICG-001. However, we report that ICG-001 maintains full activity against CBP-mediated WNT signaling in p300-deficient cell lines, including the butyrate-resistance line HCT-R. In addition, our findings evaluating combinatorial treatment of ICG-001 and butyrate in HCT-R cells may have important therapeutic implications for the treatment of butyrate-resistant CRCs.


Biomechanics of neck injuries resulting from rear-end vehicle collisions.
It has been claimed that 85% of the neck injuries caused by car accidents are the result of rear-end collisions. This type of injury is called a whiplash injury, and its mechanisms are not completely understood due to the limited ability to diagnose them using X-ray or MRI. Biomechanical studies including research on injury mechanisms, injury criteria, neck kinematics and injury epidemiology were reviewed to investigate the details of whiplash injuries. Many different injury mechanisms has been studied and identified such as hyperextension of the neck, facet joint impingement, spine column pressure, and muscle strains. Possible injury criterions have been reported as The Neck Injury Criterion (NIC), Nij criterion, IV-NIC criterio, Nkm criterion, NDC criterion.


Ion-assisted deposition of moisture-stable hafnium oxide films for ultraviolet applications.
A design-of-experiments statistical approach was taken to determine the optimum ion gun operating parameters for the deposition of moisture-stable, low-absorbing hafnium oxide films by ion-assisted electron-beam evaporation. Factors identified as affecting the quality of hafnia films were chamber pressure, deposition rate, ion gun source gas composition, and ion gun current. Both oxygen and argon were used as source gases. High and low levels of the factors were chosen on the basis of our experience with the operating range of the system, and we made a series of 24 runs with all possible combinations of these factors. From a statistical analysis of the data, we find that the best films are obtained with a 1:1 mixture of argon and oxygen, 3-3.5 x 10(-4) Torr chamber pressure, 0.3-nm/s deposition rate, and 0.5-A ion gun current. X-ray diffraction measurements show that the ion-assisted films exhibit a partial monoclinic crystalline structure, whereas the unassisted films are amorphous.


Growth inhibition and chromosomal instability of cultured marsupial (opossum) cells after treatment with DNA polymerase α inhibitor.
The DNA replication mechanism has been well established for eutherian mammals (placental mammals such as humans, mice, and cattle), but not, to date, for metatherian mammals (marsupials such as kangaroos, koalas, and opossums). In this study, we found that dehydroaltenusin, a selective inhibitor of mammalian (eutherian) DNA polymerase α, clearly suppressed the growth of metatherian (opossum and rat kangaroo) cultured cells. In cultured opossum (OK) cells, dehydroaltenusin also suppressed the progression of DNA replication. These results suggest that dehydroaltenusin inhibits metatherian as well as eutherian DNA replication. Dehydroaltenusin treatment of OK cells engendered fluctuations in the numbers of chromosomes in the OK cells as well as inhibition of cell growth and DNA replication. This suggests that partial inhibition of DNA replication by dehydroaltenusin causes chromosomal instability in cultured cells.


Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.
The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells.


Circulating microRNA-122 as Potential Biomarker for Detection of Testosterone Abuse.
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and thus influence many cellular and physiological processes. miRNAs are also present in cell-free body fluids such as plasma or serum, and these circulating miRNAs are very stable, sensitive, and specific biomarkers of pathophysiological states. In this study, we investigated whether circulating miRNAs could serve as biomarkers of exogenous testosterone administration. Misuse of testosterone as a performance-enhancing drug is thought to be widespread in sports. Detection of testosterone through the urinary steroid profile of the Athlete Biological Passport faces several obstacles, indicating that new biomarkers are required. To this end, we analyzed plasma miRNA levels by high-throughput quantitative real-time PCR. Plasma samples were obtained before and at several time points after transdermal and oral testosterone administration. Screening identified three potential candidate miRNAs that were altered by both routes of testosterone administration. Longitudinal monitoring of these candidates revealed that variation in two of them (miR-150 and miR-342), relative to the corresponding levels in control samples, was testosterone-independent. However, levels of the liver-specific miR-122 increased 3.5-fold 1 day after drug intake. Given that testosterone is metabolized by the liver, this observation suggests that miR-122 in cell-free fluids may be used as a sensitive biomarker of testosterone misuse via multiple dosing routes and could therefore be integrated into a blood-based multiparametric follow-up.


Mesoporous silica nanoparticles for 19F magnetic resonance imaging, fluorescence imaging, and drug delivery.
Multifunctional mesoporous silica nanoparticles (MSNs) are good candidates for multimodal applications in drug delivery, bioimaging, and cell targeting. In particular, controlled release of drugs from MSN pores constitutes one of the superior features of MSNs. In this study, a novel drug delivery carrier based on MSNs, which encapsulated highly sensitive 19F magnetic resonance imaging (MRI) contrast agents inside MSNs, was developed. The nanoparticles were labeled with fluorescent dyes and functionalized with small molecule-based ligands for active targeting. This drug delivery system facilitated the monitoring of the biodistribution of the drug carrier by dual modal imaging (NIR/19F MRI). Furthermore, we demonstrated targeted drug delivery and cellular imaging by the conjugation of nanoparticles with folic acid. An anticancer drug (doxorubicin, DOX) was loaded in the pores of folate-functionalized MSNs for intracellular drug delivery. The release rates of DOX from the nanoparticles increased under acidic conditions, and were favorable for controlled drug release to cancer cells. Our results suggested that MSNs may serve as promising 19F MRI-traceable drug carriers for application in cancer therapy and bio-imaging.


Regulation of the IL-21 gene by the NF-κB transcription factor c-Rel.
IL-21 is a member of the common gamma-chain-dependent cytokine family and is a key modulator of lymphocyte development, proliferation, and differentiation. IL-21 is highly expressed in activated CD4(+) T cells and plays a critical role in the expansion and differentiation of the Th cell subsets, Th17 and follicular helper T (T(FH)) cells. Because of its potent activity in both myeloid and lymphoid cell immune responses, it has been implicated in a number of autoimmune diseases and has also been used as a therapeutic agent in the treatment of some cancers. In this study, we demonstrate that c-Rel, a member of the NF-kappaB family of transcription factors, is required for IL-21 gene expression in T lymphocytes. IL-21 mRNA and protein levels are reduced in the CD4(+) cells of rel(-/-) mice when compared with rel(+/+) mice in both in vitro and in vivo models. A c-Rel binding site identified in the proximal promoter of il21 is confirmed to bind c-Rel in vitro and in vivo and to regulate expression from the il21 promoter in T cells. Downstream of IL-21 expression, Th17, T(FH), and germinal center B cell development are also impaired in rel(-/-) mice. The administration of IL-21 protein rescued the development of T(FH) cells but not germinal center B cells. Taken together, c-Rel plays an important role in the expression of IL-21 in T cells and subsequently in IL-21-dependent T(FH) cell development.


Oligomerization-induced differential dephosphorylation of c-Met receptor tyrosine kinase.
Although protein tyrosine phosphatases (PTPs) are significant negative regulators of receptor tyrosine kinase (RTK)-initiated cell signaling, it is unknown how RTK oligomerization modulates the equilibrium established between kinase and phosphatase activity. To determine the impact of oligomerization on the ability of c-MET RTK to undergo dephosphorylation, we examined the relative dephosphorylation kinetics of similarly phosphorylated dimeric TPR-MET and monomeric cytoMET. Notably, we observed that the dephosphorylation kinetics of phosphorylated MET were significantly modulated by its oligomeric state, with the global dephosphorylation rate of TPR-MET severalfold slower than the dephosphorylation rate of monomeric cytoMET. Furthermore, there were important site-specific differences in the dephosphorylation patterns of cytoMET and TPR-MET. Reduced dephosphorylation activity was predicted to eliminate or reduce the requirement of ligand-dependent oligomerization for MET autophosphorylation. This was demonstrated by the rapid phosphorylation of unstimulated c-MET on its activation loop and carboxy-terminal tyrosines following pervanadate treatment of cells expressing c-MET. We conclude that the MET oligomerization state is a critical regulator of its dephosphorylation rate. Thus, oligomerization plays a role in modifying the receptor's kinase and dephosphorylation rates to change the equilibrium levels of phosphorylated and dephosphorylated receptor in response to ligand stimulation, and that this may be a general mechanism utilized by many oligomeric receptor tyrosine kinases for regulation of their activity.


Increased metabolic vulnerability in early-onset Alzheimer's disease is not related to amyloid burden.
Patients with early age-of-onset Alzheimer's disease show more rapid progression, more generalized cognitive deficits and greater cortical atrophy and hypometabolism compared to late-onset patients at a similar disease stage. The biological mechanisms that underlie these differences are not well understood. The purpose of this study was to examine in vivo whether metabolic differences between early-onset and late-onset Alzheimer's disease are associated with differences in the distribution and burden of fibrillar amyloid-beta. Patients meeting criteria for probable Alzheimer's disease (National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's; Disease and Related Disorders Association criteria) were divided based on estimated age at first symptom (less than or greater than 65 years) into early-onset (n = 21, mean age-at-onset 55.2 +/- 5.9 years) and late-onset (n = 18, 72.0 +/- 4.7 years) groups matched for disease duration and severity. Patients underwent positron emission tomography with the amyloid-beta-ligand [(11)C]-labelled Pittsburgh compound-B and the glucose analogue [(18)F]-labelled fluorodeoxyglucose. A group of cognitively normal controls (n = 30, mean age 73.7 +/- 6.4) was studied for comparison. [(11)C]-labelled Pittsburgh compound-B images were analysed using Logan graphical analysis (cerebellar reference) and [(18)F]-labelled fluorodeoxyglucose images were normalized to mean activity in the pons. Group differences in tracer uptake were assessed on a voxel-wise basis using statistical parametric mapping, and by comparing mean values in regions of interest. To account for brain atrophy, analyses were repeated after applying partial volume correction to positron emission tomography data. Compared to normal controls, both early-onset and late-onset Alzheimer's disease patient groups showed increased [(11)C]-labelled Pittsburgh compound-B uptake throughout frontal, parietal and lateral temporal cortices and striatum on voxel-wise and region of interest comparisons (P < 0.05). However, there were no significant differences in regional or global [(11)C]-labelled Pittsburgh compound-B binding between early-onset and late-onset patients. In contrast, early-onset patients showed significantly lower glucose metabolism than late-onset patients in precuneus/posterior cingulate, lateral temporo-parietal and occipital corticies (voxel-wise and region of interest comparisons, P < 0.05). Similar results were found for [(11)C]-labelled Pittsburgh compound-B and [(18)F]-labelled fluorodeoxyglucose using atrophy-corrected data. Age-at-onset correlated positively with glucose metabolism in precuneus, lateral parietal and occipital regions of interest (controlling for age, education and Mini Mental State Exam, P < 0.05), while no correlations were found between age-at-onset and [(11)C]-labelled Pittsburgh compound-B binding. In summary, a comparable burden of fibrillar amyloid-beta was associated with greater posterior cortical hypometabolism in early-onset Alzheimer's disease. Our data are consistent with a model in which both early amyloid-beta accumulation and increased vulnerability to amyloid-beta pathology play critical roles in the pathogenesis of Alzheimer's disease in young patients.


Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule.
In vitro, when the B7 molecule on the surface of antigen-presenting cells binds to the T cell surface molecules CD28 and CTLA-4, a costimulatory signal for T cell activation is generated. CTLA4Ig is a soluble form of the extracellular domain of CTLA-4 and binds B7 with high avidity. CTLA4Ig treatment in vivo suppressed T cell-dependent antibody responses to sheep erythrocytes or keyhole limpet hemocyanin. Large doses of CTLA4Ig suppressed responses to a second immunization. Thus, costimulation by B7 is important for humoral immune responses in vivo, and interference with costimulation may be useful for treatment of antibody-mediated autoimmune disease.


HGF-independent regulation of MET and GAB1 by nonreceptor tyrosine kinase FER potentiates metastasis in ovarian cancer.
Ovarian cancer cells disseminate readily within the peritoneal cavity, which promotes metastasis, and are often resistant to chemotherapy. Ovarian cancer patients tend to present with advanced disease, which also limits treatment options; consequently, new therapies are required. The oncoprotein tyrosine kinase MET, which is the receptor for hepatocyte growth factor (HGF), has been implicated in ovarian tumorigenesis and has been the subject of extensive drug development efforts. Here, we report a novel ligand- and autophosphorylation-independent activation of MET through the nonreceptor tyrosine kinase feline sarcoma-related (FER). We demonstrated that the levels of FER were elevated in ovarian cancer cell lines relative to those in immortalized normal surface epithelial cells and that suppression of FER attenuated the motility and invasive properties of these cancer cells. Furthermore, loss of FER impaired the metastasis of ovarian cancer cells in vivo. Mechanistically, we demonstrated that FER phosphorylated a signaling site in MET: Tyr1349. This enhanced activation of RAC1/PAK1 and promoted a kinase-independent scaffolding function that led to recruitment and phosphorylation of GAB1 and the specific activation of the SHP2-ERK signaling pathway. Overall, this analysis provides new insights into signaling events that underlie metastasis in ovarian cancer cells, consistent with a prometastatic role of FER and highlighting its potential as a novel therapeutic target for metastatic ovarian cancer.


Widespread Pain Is Associated with Increased Risk of No Clinical Improvement After TKA in Women.
When conservative treatments do not work, TKA may be the best option for patients with knee osteoarthritis, although a relatively large proportion of individuals do not have clinically important improvement after TKA. Evidence also suggests that women are less likely to benefit from TKA than men, but the reasons are unclear. Widespread pain disproportionately affects women and has been associated with worse outcomes after joint arthroplasty, yet it is unknown if the effect of widespread pain on TKA outcomes differs by patient gender. (1) Does the association between widespread pain and no clinically important improvement in osteoarthritis-related pain and disability 2 years after TKA differ between men and women? (2) Does the use of pain medications 2 years after TKA differ between those with widespread pain and those without widespread pain before surgery? Osteoarthritis Initiative (https://nda.nih.gov/oai/) study participants were followed annually from March 2005 until October 2015. Participants who underwent TKA up to the 7-year follow-up visit with pain/disability assessment at the protocol-planned visit before TKA and at the second planned annual visit after surgery were included in the analysis. Among 4796 study participants, 391 had a confirmed TKA, including 315 with pain/disability assessment at the protocol-planned visit before TKA. Overall, 95% of participants (298) had the required follow-up assessment; 5% (17) did not have follow-up data. Widespread pain was defined based on the modified American College of Rheumatology criteria. Symptoms were assessed using the WOMAC pain (range 0 to 20; higher score, more pain) and disability (range 0 to 68; higher score, more disability) scores, and the Knee Injury and Osteoarthritis Outcome Score for pain (range 0 to 100; higher score, less pain). Improvements in pain and disability were classified based on improvement from established clinically important differences (decrease in WOMAC pain ≥ 1.5; decrease in WOMAC disability ≥ 6.0; increase in Knee Injury and Osteoarthritis Outcome Score for pain ≥ 9). At baseline, more women presented with widespread pain than men (45% [84 of 184] versus 32% [36 of 114]). Probability and the relative risk (RR) of no clinically important improvement were estimated using a logistic regression analysis in which participants with widespread pain and those without were compared. The analyses were done for men and women separately, then adjusted for depression and baseline outcome scores. Among women, preoperative widespread pain was associated with an increased risk of no clinically important improvement 2 years after TKA, based on WOMAC pain scores (13.5% versus 4.6%; RR 2.93 [95% CI 1.18 to 7.30]; p = 0.02) and the Knee Injury and Osteoarthritis Outcome Score for pain (16.5% versus 4.9%; RR 3.39 [95% CI 1.34 to 8.59]; p = 0.02). Given the lower and upper limits of the confidence intervals, our data are compatible with a broad range of disparate associations between widespread pain and lack of clinically important improvement in WOMAC pain scores (RR 0.77 [95% CI 0.22 to 2.70]; p = 0.68) and the Knee Injury and Osteoarthritis Outcome Score for pain (RR 1.37 [95% CI 0.47 to 4.00]; p = 0.57) among men, as well as clinically important improvement in WOMAC disability scores among men (RR 0.72 [95% CI 0.20 to 2.55]; p = 0.61) and women (RR 1.98 [95% CI 0.92 to 4.26]; p = 0.08). Participants presenting with widespread pain before TKA were more likely than those without widespread pain to use medication for symptoms of knee osteoarthritis most days for at least 1 month 2 years after TKA (51% [61 of 120] versus 32% [57 of 178]; mean difference, 18.8 [95% CI 7.3 to 30.1]; p < 0.01). Widespread pain before TKA was associated with an increased risk of no clinically important improvement in knee pain 2 years postoperatively among women. Because of the small number of men with widespread pain in the sample, the results for men were inconclusive. In clinical practice, screening TKA candidates for widespread pain may be useful, and expectations of surgical outcomes may need to be tempered if patients have a concurrent diagnosis of widespread pain. Future studies should include more men with widespread pain and investigate if treatment of widespread pain before or concurrent with TKA surgery may improve surgical outcomes. Level III, therapeutic study.


Colorectal cancer and genetic alterations in the Wnt pathway.
In colorectal tumours, Wnt pathway genetics continues to be dominated by mutations in the adenomatous polyposis coli (APC) gene. Germline mutations cause familial adenomatous polyposis and at least two-thirds of sporadic colorectal tumours also acquire APC mutations, quite possibly as the initiating events in tumorigenesis. These mutations almost always cause loss of the C-terminal functions of the APC protein - probably involved in microtubule binding, cell polarity and chromosome segregation - and deletion of the SAMP repeats that are important for binding to axin and formation of the beta-catenin phosphorylation complex. The truncated APC proteins are, in general, stable and almost certainly retain some activity in beta-catenin binding. The 'two hits' at APC are coselected so as to produce an optimal activation of Wnt signalling (just-right hypothesis). In a minority of colorectal tumours, Wnt activation can occur through mutations that affect phosphorylation sites within exon 3 of beta-catenin, causing protein stabilization. In other tumours, epigenetic transcriptional silencing or mutation of the secreted frizzled-related proteins may modulate Wnt levels. Mutations in the Wnt components AXIN1, AXIN2 and TCF4 have been found in microsatellite-unstable colon cancers, but it is not clear in every case whether these changes are functional. Therapeutic modulation of the Wnt pathway remains an attractive therapeutic possibility for colorectal carcinomas.


Immunity to Hantavirus challenge in Meriones unguiculatus induced by vaccinia-vectored viral proteins.
Vaccinia virus recombinants were constructed that incorporated genomic sequences coding for the nucleoprotein (N) and glycoproteins (G1 and G2) of the hantavirus R22 strain isolated from a rat in China, and designated as RNV and RMV9, respectively. The proteins expressed by RNV and RMV9 were identified by radioimmunoprecipitation and indirect immunofluorescence assay using a panel of monoclonal antibodies and polyclonal immune sera, and were found to be antigenically indistinguishable from authentic R22 viral proteins. Both RNV and RMV9 elicited an anti-R22 antibody response in Mongolian gerbils (Meriones unguiculatus) with titers ranging from 6,400 to 12,800 by enzyme-linked immunosorbent assay, but only RMV9 produced neutralizing antibodies to R22 virus (titer 1:200) and Hantaan (HTN) virus (titer 1:20). The ability of these recombinants to protect Mongolian gerbils against challenge with R22 and HTN viruses was examined. The RMV9 recombinant induced a complete protective immune response against challenge with 10(4) plaque-forming units (PFU) of both R22 and HTN viruses, while RNV induced partial protection against a challenge with the homologous R22 virus and the heterologous HTN virus at a dose of 10(3) PFU. Our data show that the common antigenic sites responsible for eliciting a protective response are located mainly on hantavirus glycoproteins, and that the nucleoprotein may also confer partial cross-protection that presumably involves cell-mediated as well as humoral mechanisms.


"We're the heroes!": fathers' perspectives on their role in protecting their preteenage children from sexual risk.
Parents can be an important influence on their children's sexual behavior. However, relatively little research has focused on fathers' roles in protecting children from sexual risk or on their discussions with children about sex. Sixteen focus groups comprising 131 parents of children aged 10-12 were conducted in three U.S. cities in 2007. Separate groups were held for mothers and fathers, and for white, black, English-speaking Hispanic and Spanish-speaking Hispanic participants. Content analysis was used to identify core themes and patterns related to fathers' communications with their children about sex. Fathers were highly invested in ensuring that their children are safe and successful in life. Although some had not talked to their children about sex, others had discussed it frequently and openly, and nearly all agreed that fathers' perspectives are important for children to hear. Fathers reported being better suited to discussing some topics (e.g., male puberty, how young men think) than others (menstruation). Fathers also described other strategies that they use to guide their children's development, such as emphasizing future goals and monitoring children's activities and friends. Many fathers, especially Hispanic participants, reported being more protective of daughters than of sons, and some had particular difficulty talking with their daughters about sex. Research and interventions concerning parent-child communication about sex should not neglect the role of fathers. However, some fathers may need support to overcome barriers to effective communication, especially with their daughters.


4-Nitro-phenol-piperazine (2/1).
In the title adduct, C6H5NO3·0.5C4H10N2, the piperazine ring possesses inversion symmetry and has a chair conformation. Its mean plane makes a dihedral angle of 65.45 (7)° with the 4-nitro-phenol ring. In the crystal, the piperazine ring is linked to two 4-nitro-phenol mol-ecules via O-H⋯N hydrogen bonds. The mol-ecules are also linked via bifurcated N-H⋯(O,O) hydrogen bonds involving the NO2 O atoms, forming a two-dimensional network lying parallel to (102). The networks are linked via C-H⋯O hydrogen bonds, forming a three-dimensional structure.


What is second-order vision for? Discriminating illumination versus material changes.
The human visual system is sensitive to second-order modulations of the local contrast (CM) or amplitude (AM) of a carrier signal. Second-order cues are detected independently of first-order luminance signals; however, it is not clear why vision should benefit from second-order sensitivity. Analysis of the first- and second-order contents of natural images suggests that these cues tend to occur together, but their phase relationship varies. We have shown that in-phase combinations of LM and AM are perceived as a shaded corrugated surface whereas the anti-phase combination can be seen as corrugated when presented alone or as a flat material change when presented in a plaid containing the in-phase cue. We now extend these findings using new stimulus types and a novel haptic matching task. We also introduce a computational model based on initially separate first- and second-order channels that are combined within orientation and subsequently across orientation to produce a shading signal. Contrast gain control allows the LM + AM cue to suppress responses to the LM - AM when presented in a plaid. Thus, the model sees LM - AM as flat in these circumstances. We conclude that second-order vision plays a key role in disambiguating the origin of luminance changes within an image.


New approach to the cultivation of methanogenic bacteria: 2-mercaptoethanesulfonic acid (HS-CoM)-dependent growth of Methanobacterium ruminantium in a pressureized atmosphere.
The sensitivity of the requirement of Methanobacterium ruminantium strain M1 to a new coenzyme, 2-mercaptoethanesulfonic acid (HS-CoM) was examined by use of new techniques that were developed for rapid and efficient handling of large numbers of cultures of methanogenic bacteria. The system uses sealed tubes that contain a gas mixture of 80% hydrogen and 20% carbon dioxide under a pressure of 2 to 3 atm. This modification of the Hungate technique reduces variability among replicate cultures and simplifies the dispensing, sterilization, and storage of liquid media as well as the transfer and maintenance of methanogenic bacteria. Results indicate a limit of sensitivity of the assay at 5 nM HS-CoM, with half-maximal growth at 25 nM HS-CoM. Coenzyme activity could be replaced by 2,2'-dithiodiethanesulfonic acid at a half-molar equivalent of the HS-CoM concentration, or by 2-(methylthio)ethanesulfonic acid on an equimolar basis. These data reveal a very sensitive and precise requirement for HS-CoM in the nutrition of this fastidious anaerobe.


Immunization for Streptococcus pneumoniae infections in high-risk children.
Routine use of the pneumococcal conjugate vaccines (PCV7 and PCV13), beginning in 2000, has resulted in a dramatic reduction in the incidence of invasive pneumococcal disease (IPD) attributable to serotypes of Streptococcus pneumoniae contained in the vaccines. The Advisory Committee on Immunization Practices of the Centers for Disease Control and Prevention and the American Academy of Pediatrics recommend the expanded use of PCV13 in children 6 through 18 years of age with certain conditions that place them at elevated risk of IPD. This statement provides recommendations for the use of PCV13 in children 6 through 18 years. A single dose of PCV13 should be administered to certain children in this age group who are at elevated risk of IPD. Recommendations for the use of PCV13 in healthy children and for pneumococcal polysaccharide vaccine (PPSV23) remain unchanged.


Flow cytometry reveals that H5N1 vaccination elicits cross-reactive stem-directed antibodies from multiple Ig heavy-chain lineages.
An understanding of the antigen-specific B-cell response to the influenza virus hemagglutinin (HA) is critical to the development of universal influenza vaccines, but it has not been possible to examine these cells directly because HA binds to sialic acid (SA) on most cell types. Here, we use structure-based modification of HA to isolate HA-specific B cells by flow cytometry and characterize the features of HA stem antibodies (Abs) required for their development. Incorporation of a previously described mutation (Y98F) to the receptor binding site (RBS) causes HA to bind only those B cells that express HA-specific Abs, but it does not bind nonspecifically to B cells, and this mutation has no effect on the binding of broadly neutralizing Abs to the RBS. To test the specificity of the Y98F mutation, we first demonstrated that previously described HA nanoparticles mediate hemagglutination and then determined that the Y98F mutation eliminates this activity. Cloning of immunoglobulin genes from HA-specific B cells isolated from a single human subject demonstrates that vaccination with H5N1 influenza virus can elicit B cells expressing stem monoclonal Abs (MAbs). Although these MAbs originated mostly from the IGHV1-69 germ line, a reasonable proportion derived from other genes. Analysis of stem Abs provides insight into the maturation pathways of IGVH1-69-derived stem Abs. Furthermore, this analysis shows that multiple non-IGHV1-69 stem Abs with a similar neutralizing breadth develop after vaccination in humans, suggesting that the HA stem response can be elicited in individuals with non-stem-reactive IGHV1-69 alleles. Universal influenza vaccines would improve immune protection against infection and facilitate vaccine manufacturing and distribution. Flu vaccines stimulate B cells in the blood to produce antibodies that neutralize the virus. These antibodies target a protein on the surface of the virus called HA. Flu vaccines must be reformulated annually, because these antibodies are mostly specific to the viral strains used in the vaccine. But humans can produce broadly neutralizing antibodies. We sought to isolate B cells whose genes encode influenza virus antibodies from a patient vaccinated for avian influenza. To do so, we modified HA so it would bind only the desired cells. Sequencing the antibody genes of cells marked by this probe proved that the patient produced broadly neutralizing antibodies in response to the vaccine. Many sequences obtained had not been observed before. There are more ways to generate broadly neutralizing antibodies for influenza virus than previously thought.


Multiple food hypersensitivity as a cause of refractory chronic constipation in adults.
Chronic constipation that is unresponsive to laxative treatment is a severe illness, but children unresponsive to laxatives have been successfully treated with an elimination diet. We report the first cases of refractory chronic constipation caused by food hypersensitivity in adults. Four patients with refractory constipation who were unresponsive to high doses of laxatives were put on an oligo-antigenic diet and underwent successive double-blind, placebo-controlled, food challenges (DBPFC). Routine laboratory tests, immunological assays, colonoscopy, esophago-gastroduodenoscopy and rectal and duodenal histology were performed. While on an elimination diet, bowel habits normalized in all patients and a DBPFC challenge triggered the reappearance of constipation. In comparison with another 13 patients with refractory constipation unresponsive to the elimination diet, observed over the same period, the patients with food-hypersensitivity-related constipation had the following characteristics: longer duration of illness (p < 0.03), lower body mass index (p < 0.03), higher frequency of self-reported food intolerance (p < 0.01), higher frequency of nocturnal abdominal pain and anal itching (p < 0.01). In patients with food hypersensitivity, hemoglobin concentrations and peripheral leukocytes were lower than those in controls (p < 0.03). The duodenal and rectal mucosa histology showed lymphocyte and eosinophil infiltration, and the duodenal villi were flattened in two cases. In adult patients, refractory chronic constipation may be caused by food hypersensitivity and an elimination diet is effective in these subjects.


Lipid interaction of the C terminus and association of the transmembrane segments facilitate atlastin-mediated homotypic endoplasmic reticulum fusion.
The homotypic fusion of endoplasmic reticulum (ER) membranes is mediated by atlastin (ATL), which consists of an N-terminal cytosolic domain containing a GTPase module and a three-helix bundle followed by two transmembrane (TM) segments and a C-terminal tail (CT). Fusion depends on a GTP hydrolysis-induced conformational change in the cytosolic domain. Here, we show that the CT and TM segments also are required for efficient fusion and provide insight into their mechanistic roles. The essential feature of the CT is a conserved amphipathic helix. A synthetic peptide corresponding to the helix, but not to unrelated amphipathic helices, can act in trans to restore the fusion activity of tailless ATL. The CT promotes vesicle fusion by interacting directly with and perturbing the lipid bilayer without causing significant lysis. The TM segments do not serve as mere membrane anchors for the cytosolic domain but rather mediate the formation of ATL oligomers. Point mutations in either the C-terminal helix or the TMs impair ATL's ability to generate and maintain ER morphology in vivo. Our results suggest that protein-lipid and protein-protein interactions within the membrane cooperate with the conformational change of the cytosolic domain to achieve homotypic ER membrane fusion.


Induction by leptin of uncoupling protein-2 and enzymes of fatty acid oxidation.
We have studied mechanisms by which leptin overexpression, which reduces body weight via anorexic and thermogenic actions, induces triglyceride depletion in adipocytes and nonadipocytes. Here we show that leptin alters in pancreatic islets the mRNA of the genes encoding enzymes of free fatty acid metabolism and uncoupling protein-2 (UCP-2). In animals infused with a recombinant adenovirus containing the leptin cDNA, the levels of mRNAs encoding enzymes of mitochondrial and peroxisomal oxidation rose 2- to 3-fold, whereas mRNA encoding an enzyme of esterification declined in islets from hyperleptinemic rats. Islet UCP-2 mRNA rose 6-fold. All in vivo changes occurred in vitro in normal islets cultured with recombinant leptin, indicating direct extraneural effects. Leptin overexpression increased UCP-2 mRNA by more than 10-fold in epididymal, retroperitoneal, and subcutaneous fat tissue of normal, but not of leptin-receptor-defective obese rats. By directly regulating the expression of enzymes of free fatty acid metabolism and of UCP-2, leptin controls intracellular triglyceride content of certain nonadipocytes, as well as adipocytes.


Graphene-on-silver substrates for sensitive surface plasmon resonance imaging biosensors.
Taking advantage of the high impermeability property of graphene and the sharp surface plasmon resonance (SPR) curve of silver, we numerically demonstrate that SPR imaging biosensors with a graphene-on-silver substrate can be used to achieve the dramatically high sensitivity as well as to prevent silver oxidation. Results of our numerical study show that a silver substrate with a few graphene layers can significantly increase the imaging sensitivity, compared to the conventional gold-film-based SPR imaging biosensor. In particular, single layered graphene deposited on the 60-nm thick silver film amplifies the SPR imaging signal more than three times. Therefore, the proposed SPR substrate could potentially open a new possibility of SPR imaging detection for sensitive and high-throughput assessment of multiple biomolecular interactions.


Body mass index and breast cancer survival: a Mendelian randomization analysis.
There is increasing evidence that elevated body mass index (BMI) is associated with reduced survival for women with breast cancer. However, the underlying reasons remain unclear. We conducted a Mendelian randomization analysis to investigate a possible causal role of BMI in survival from breast cancer. We used individual-level data from six large breast cancer case-cohorts including a total of 36 210 individuals (2475 events) of European ancestry. We created a BMI genetic risk score (GRS) based on genotypes at 94 known BMI-associated genetic variants. Association between the BMI genetic score and breast cancer survival was analysed by Cox regression for each study separately. Study-specific hazard ratios were pooled using fixed-effect meta-analysis. BMI genetic score was found to be associated with reduced breast cancer-specific survival for estrogen receptor (ER)-positive cases [hazard ratio (HR) = 1.11, per one-unit increment of GRS, 95% confidence interval (CI) 1.01-1.22, P = 0.03). We observed no association for ER-negative cases (HR = 1.00, per one-unit increment of GRS, 95% CI 0.89-1.13, P = 0.95). Our findings suggest a causal effect of increased BMI on reduced breast cancer survival for ER-positive breast cancer. There is no evidence of a causal effect of higher BMI on survival for ER-negative breast cancer cases.


Bioactive components of tea: cancer, inflammation and behavior.
Tea is one of the most widely consumed beverages worldwide. Several studies have suggested that catechins and theaflavins found in tea may reduce the risk of various types of cancers. Major advances have been made to understand the molecular events leading to cancer prevention; however, the evidence is not conclusive. Evidence from pre-clinical and clinical studies also suggests that persistent inflammation can progress to cancer. Several possible mechanisms of action may explain the cancer preventive aspects of tea components specifically anti-inflammatory effects. In regards to brain health, green tea catechins have been recognized as multifunctional compounds for neuroprotection with beneficial effects on vascular function and mental performance. Theanine, a unique amino acid in tea, enhances cognition in humans and has neuroprotective effects. Human interventional studies with well characterized tea products are needed.


Sensitivity of the breastfeeding motivational measurement scale: a known group analysis of first time mothers.
Breastfeeding has immense public health value for mothers, babies, and society. But there is an undesirably large gap between the number of new mothers who undertake and persist in breastfeeding compared to what would be a preferred level of accomplishment. This gap is a reflection of the many obstacles, both physical and psychological, that confront new mothers. Previous research has illuminated many of these concerns, but research on this problem is limited in part by the unavailability of a research instrument that can measure the key differences between first-time mothers and experienced mothers, with regard to the challenges they face when breastfeeding and the instructional advice they require. An instrument was designed to measure motivational complexity associated with sustained breast feeding behaviour; the Breastfeeding Motivational Measurement Scale. It contains 51 self-report items (7 point Likert scale) that cluster into four categories related to perceived value of breast-feeding, confidence to succeed, factors that influence success or failure, and strength of intentions, or goal. However, this scale has not been validated in terms of its sensitivity to profile the motivation of new mothers and experienced mothers. This issue was investigated by having 202 breastfeeding mothers (100 first time mothers) fill out the scale. The analysis reported in this paper is a three factor solution consisting of value, midwife support, and expectancies for success that explained the characteristics of first time mothers as a known group. These results support the validity of the BMM scale as a diagnostic tool for research on first time mothers who are learning to breastfeed. Further research studies are required to further test the validity of the scale in additional subgroups.


Expert consensus statement 'Neonatologist-performed Echocardiography (NoPE)'-training and accreditation in UK.
Targeted echocardiographic assessments of haemodynamic status are increasingly utilised in many settings. Application in the neonatal intensive care units (NICU) is increasingly demanded but challenging given the risk of underlying structural lesions. This statement follows discussions in UK led by the Neonatologists with an Interest in Cardiology and Haemodynamics (NICHe) group in collaboration with the British Congenital Cardiac Association (BCCA) and the Paediatricians with Expertise in Cardiology Special Interest Group (PECSIG). Clear consensus was agreed on multiple aspects of best practice for neonatologist-performed echocardiogram (NoPE)-rigorous attention to infection control and cardiorespiratory/thermal stability, early referral to paediatric cardiology with suspicion of structural disease, reporting on standardised templates, reliable image storage, regular skills maintenance, collaboration with a designated paediatric cardiologist, and regular scan audit/review. It was agreed that NoPE assessments should confidently exclude structural lesions at first scan. Practitioners would be expected to screen and establish gross normality of structure at first scan and obtain confirmation from paediatric cardiologist if required, and subsequently, functional echocardiography can be performed for haemodynamic assessment to guide management of newborn babies. To achieve training, NICHe group suggested that mandatory placements could be undertaken during core registrar training or neonatal subspecialty grid training with a paediatric cardiology placement for 6 months and a neonatology placement for a minimum of 6 months. In the future, we hope to define a precise curriculum for assessments. Technological advances may provide solutions-improvements in telemedicine may have neonatologists assessing haemodynamic status with paediatric cardiologists excluding structural lesions and neonatal echocardiography simulators could increase exposure to multiple pathologies and allow limitless practice in image acquisition. We propose developing training places in specialist paediatric cardiology centres and neonatal units to facilitate training and suggest all UK practitioners performing neonatologist-performed echocardiogram adopt this current best practice statement. Neonatologist-performed echocardiogram (NoPE) also known as targeted neonatal echocardiography (TNE) or functional ECHO is increasingly recognised and utilised in care of sick newborn and premature babies. There are differences in training for echocardiography across continents and formal accreditation processes are lacking. This is the first document of consensus best practice statement for training of neonatologists in neonatologist-performed echocardiogram (NoPE), jointly drafted by Neonatologists with interest in cardiology & haemodynamics (NICHe), paediatric cardiology and paediatricians with expertise in cardiology interest groups in UK. Key elements of a code of practice for neonatologist-performed echocardiogram are suggested.


Benefits of hyperthermic intraperitoneal chemotherapy for patients with serosal invasion in gastric cancer: a meta-analysis of the randomized controlled trials.
In this meta-analysis we aimed to determine the effectiveness and safety of hyperthermic intraperitoneal chemotherapy (HIPC) for patients with advanced gastric cancer who underwent gastrectomy. In accordance with standard meta-analysis procedures, our study included patients who underwent resection for advanced gastric cancer and were randomly allocated to receive either hyperthermic intraperitoneal chemotherapy or control. We searched PubMed (up to November 2011), EMBASE (up to November 2011), Cochrane Database of Systematic Reviews (CDSR), and Cochrane Central Register of Controlled Trials (CCTR) (up to November 2011). Both published and unpublished trials were included in the analysis, and no search restrictions were imposed. There was no language restriction. The results were analyzed using RevMan 5.1 software, which was provided by Cochrane Collaboration. There were ten randomized controlled trials included in the analysis. A total of 1062 patients with gastric cancer in these studies were divided into the HIPC group (n = 518) and control group (n = 544). A significant improvement in survival was observed in the HIPC groups compared to the control group in the mitomycin C (MMC) subgroup (RR = 0.75, 95%CI 0.65-0.86; P < 0.00001) and the 5-FU group (RR = 0.69, 95%CI 0.52-0.90; P < 0.00001); the total RR was 0.73 (95%CI 0.64-0.83; P < 0.00001). Our findings indicated that HIPC potentially exhibited a lower peritoneal recurrence rate in the HIPC group compared to the control group (RR = 0.45, 95%CI 0.28-0.72; P = 0.001). Our meta-analysis demonstrated that HIPC may improve the overall survival rate for patients who receive resection for advance gastric cancer potentially, and help to prevent peritoneal local recurrence among patients with serosal invasion in gastric cancer.


Continuous culture of two lancelets and production of the second filial generations in the laboratory.
The lancelet is considered to be a promising laboratory model animal. To establish laboratory colonies of lancelet, we collected parental lancelets of Branchiostoma belcheri and B. japonicum (previously named as B. belcheri tsingtauense) with fully developed gonads from Xiamen Rare Marine Creature Conservation Areas (Fujian, China) on dates just before their spawning in the field in 2005. Those parental lancelets spawned spontaneously in the laboratory and produced thousands upon thousands of fertilized eggs. After carefully hatching and maintaining for almost 1 year, we successfully obtained about 500 first generation (F1) adults of B. belcheri and 3,300 of B. japonicum. Part of those F1 lancelets ripened and spontaneously spawned in 2006, and several thousands of second generation (F2) individuals of both species were produced. The young F2 lancelets are growing in good condition and some of B. japonicum initiated gonad development in December, 2006. Our experience emphasizes that cleanness of settlement substratum and sufficient food supply are important factors for long-term culture of lancelets in the laboratory.


Targeting tumor cells by enhancing radiation sensitivity.
The work of Al Knudson created the paradigm in which we see cancer as a result of the accumulation of multiple mutations. Our goal has been to exploit these mutations to develop strategies to enhance therapy for cancer by targeting the malignant cell while sparing the normal tissue. In studying the RAS oncogene, we observed that its expression when activated resulted in enhanced radioresistance. Conversely, inhibition of RAS made cells with activated RAS more radiosensitive. Hence, we postulated that it would be possible to sensitize tumors with RAS mutations to radiation without affecting the sensitivity of the normal tissue in patients with such tumors. This proved to be the case in animal models and has led to current clinical trials. These studies raised the question of identifying the downstream effectors of RAS that are responsible for altering the radiosensitivity of cells. We have found that phosphoinositide-3-kinase (PI3 kinase) is a critical component of this pathway. Blocking PI3 kinase enhanced the radiation response in vitro or in vivo of cells actively signaling through that pathway, but did not affect cells not actively signaling through PI3 kinase at the time of irradiation. Identification of tumors with active signaling in this pathway by immunohistochemical staining for phosphorylated AKT, the downstream target of PI3 kinase correlated with those patients for which radiation failed to achieve local control. Thus, characterization of the active signaling pathways in a given tumor might enable the selection of patients likely to respond to radiation. Pathways upstream from RAS may also be useful targets to consider for enhancing radiation therapy. Epidermal growth factor receptor (EGFR), which is upstream of PI3 kinase, may also mediate resistance through a common pathway. In addition to EGFR and RAS, PTEN can also regulate the PI3 kinase pathway. Identifying a common signal for EGFR, RAS, and PTEN that results in radiation resistance may uncover targets for developing molecular-based radiosensitization protocols for tumors resistant to radiation and thus lead to improvement of local control.


Randomized trial comparing primidone initiation schedules for treating essential tremor.
Early side effects are common when primidone is used to treat essential tremor, with as many as one-third of patients failing to tolerate the tablets. Lower doses can be prescribed initially using a suspension formulation. We suspected suspension initiation would result in fewer early side effects, allow better acclimatization, and improve compliance. Forty patients with essential tremor were randomized to begin primidone treatment using either 2.5 mg doses in the suspension form or 25 mg doses in the tablet form. Doses gradually increased over 3 weeks to 150 mg/day. This was a double-blind, double-dummy trial. Medication cessation due to side effects was designated the primary end-point. Four patients in the suspension group and two in the tablet group dropped out due to early side effects, resulting in a relative risk of 1.9 (95% confidence interval 0.4 to 9.2). Side effects in the first 48 hours of treatment were equally common, affecting seven subjects in each group. Treatment benefits were the same in both groups. We concluded that use of a very low initial dose and a graduated titration schedule in suspension formulation did not appear to improve primidone tolerability. If anything, compliance tended to be worse when compared with the tablet formulation, though the study was under-powered to reject the null hypothesis of equivalence.


A cytochrome P450 2B6 meditated gene therapy strategy to enhance the effects of radiation or cyclophosphamide when combined with the bioreductive drug AQ4N.
AQ4N is metabolised in hypoxic cells by cytochrome P450s (CYPs) to the cytotoxin AQ4. Most solid tumours are known to contain regions of hypoxia whereas levels of CYPs have been found to vary considerably. Enhancement of CYP levels may be obtained using gene-directed enzyme prodrug therapy (GDEPT). We have therefore examined the potential of a CYP2B6-mediated GDEPT strategy to enhance the anti-tumour effect of the combination of AQ4N with radiation or cyclophosphamide (CPA). In vitro and in vivo transient transfection of human CYP2B6 +/- CYP reductase (CYPRED) was investigated in RIF-1 mouse tumours. Efficacy in vitro was assessed using the alkaline comet assay (ACA). In vivo, the time to reach 4x the treatment volume (quadrupling time; VQT) was used as the end point. When CYP2B6 was transfected into RIF-1 cells and treated with AQ4N under hypoxic conditions there was a significant increase in DNA damage (measured by the ACA) compared with non-transfected cells. In vivo, a single intra-tumoural injection of a CYP2B6 vector construct significantly enhanced tumour growth delay in combination with AQ4N (100 mg/kg) and 10 Gy X-rays. AQ4N (100 mg/kg) and CPA (100 mg/kg) with CYP2B6 and CYPRED also enhanced tumour growth delay; this effect became significant when the schedule was repeated 14 days later (p = 0.0197). The results show the efficacy of a CYP2B6-mediated GDEPT strategy for bioreduction of AQ4N; this may offer an additional approach to target radiation- and chemo-resistant hypoxic tumours that should enhance overall tumour control.


CD1a function in human skin disease.
The high expression of CD1a on Langerhans cells in normal human skin suggests a central role for this lipid antigen presenting molecule in skin homeostasis and immunity. Although the lipid antigen presenting function of CD1a has been known for years, the physiological and pathological functions of the CD1a system in human skin remain incompletely understood. This review provides an overview of this active area of investigation, and discusses recent insights into the functions of CD1a, CD1a-restricted T cells, and lipid antigens in inflammatory and allergic skin disease. We include recent publications and work presented at the biennial CD1-MR1 EMBO workshop held in 2019 in Oxford, regarding lipids that increase and those that decrease T cell responses to CD1a.


Improving HPV Vaccination Rates Using Maintenance-of-Certification Requirements.
Many pediatricians are now required to participate in American Board of Pediatrics Maintenance of Certification (MOC) Part IV programs focused on improving health care quality, but the benefits of participation are unproven. Twenty-seven primary care pediatricians from 11 primary care practices participated in a 1-year MOC program for human papillomavirus (HPV) vaccine. Participants received education and electronic health record (EHR)-generated performance feedback reports with their rates of captured HPV immunization opportunities (dose given at eligible visit) and those of peers. In each of 3 cycles, clinicians collectively identified a goal for improvement. Rates of captured opportunities among adolescents 11 to <18 years old were tabulated, and statistical process control charts were created to evaluate changes over time among participants compared with 200 nonparticipants. Provider perceptions of the program and time invested were recorded via survey. Participating clinicians missed fewer opportunities for HPV vaccination than nonparticipants. MOC participants significantly increased their captured opportunities relative to nonparticipating clinicians by 5.7 percentage points for HPV dose 1 at preventive visits and by 0.7 and 5.6 percentage points for doses 1 and 2, respectively, at acute visits. There were no significant differences for other doses. The estimated program cost was $662/participant. Of the participating pediatricians, 96% felt the effort to participate was warranted, and half would not have joined the project without the MOC requirement. Participation in MOC Part IV improved vaccination at modest cost and with high pediatrician satisfaction, demonstrating benefits of the program that may help to inform future initiatives.


Dynamics of photoinduced endosomal release of polyplexes.
Endosomal escape is a well-known bottleneck for successful delivery of macromolecular drugs and genes. Photochemical disruption of endosomal membranes is an approach to overcome this bottleneck. In this study, we used the photosensitizer disulphonated meso-tetraphenylporphine with sulfonate groups on adjacent phenyl rings (TPPS(2a)) to investigate photoinduced endosomal release in living cells with high resolution fluorescence wide-field microscopy in real time. We studied the release dynamics of 10 kDa dextran and polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-(L)-lysine (PLL) or poly-(D)-lysine (PDL). By means of dual-color microscopy and the use of double-labeled polyplexes DNA and polymer were imaged simultaneously. We show that the characteristics of the cationic polymer significantly influence the release behavior of the polyplexes. The release of dextran occurred within 100 ms. For LPEI/DNA particles, LPEI quickly spread throughout the cytosol similar to dextran, whereas DNA was released slowly (within 4 s) and remained immobile thereafter. In case of PLL particles, both DNA and polymer showed quick release. PDL particles remained condensed upon photosensitizer activation. In addition, we demonstrate that TPPS(2a) has biological side effects. Besides stop of microtubule dynamics in the dark, the movement of endosomes ceased after photosensitizer activation.


Biological material collection to advance translational research and treatment of children with CNS tumours: position paper from the SIOPE Brain Tumour Group.
Paediatric CNS tumours are the most common cause of childhood cancer-related morbidity and mortality, and improvements in their diagnosis and treatment are needed. New genetic and epigenetic information about paediatric CNS tumours is transforming the field dramatically. For most paediatric CNS tumour entities, subgroups with distinct biological characteristics have been identified, and these characteristics are increasingly used to facilitate accurate diagnoses and therapeutic recommendations. Future treatments will be further tailored to specific molecular subtypes of disease, specific tumour predisposition syndromes, and other biological criteria. Successful biomaterial collection is a key requirement for the application of contemporary methodologies for the validation of candidate prognostic factors, the discovery of new biomarkers, the establishment of appropriate preclinical research models for targeted agents, a quicker clinical implementation of precision medicine, and for other therapeutic uses (eg, for immunotherapies). However, deficits in organisational structures and interdisciplinary cooperation are impeding the collection of high-quality biomaterial from CNS tumours in most centres. Practical, legal, and ethical guidelines for consent, storage, material transfer, biobanking, data sharing, and funding should be established by research consortia and local institutions to allow optimal collection of primary and subsequent tumour tissue, body fluids, and normal tissue. Procedures for the collection and storage of biomaterials and related data should be implemented according to the individual and organisational structures of the local institutions.


Neisseria gonorrhoeae multi-antigen sequence typing using non-cultured clinical specimens.
The Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) system, based on PCR amplification and sequence analysis of the gonococcal porB and tbpB genes, is widely used for molecular typing of gonococcal isolates but is not validated for non-cultured clinical samples. This study sought to examine the performance of the NG-MAST system on a range of non-cultured samples. Nucleic acid extracts of 73 N gonorrhoeae-positive samples, comprising eight cervical swabs, nine urethral swabs, 35 urine samples, one vaginal swab, 13 rectal swabs and seven throat swabs, were analysed by NG-MAST. For 27 specimens, corresponding gonococcal isolates were also analysed and the results compared. A panel of 44 non-gonococcal Neisseria strains and 100 N gonorrhoeae-negative clinical samples were used to investigate further the specificity of the NG-MAST PCR reactions. PCR amplification and DNA sequencing of gonococcal porB and tbpB genes was successful for all N gonorrhoeae-positive urogenital specimens, 11 of 13 rectal swabs and four of seven throat swabs. For the 27 N gonorrhoeae-positive specimens with corresponding gonococcal isolates, the porB and tbpB sequences obtained from the non-cultured specimen were identical to those obtained from the isolate. Cross-reaction with non-gonococcal Neisseria species was observed for both the porB and tbpB PCR reactions, and proved to be problematical for NG-MAST typing of throat swab specimens. The NG-MAST system can successfully be applied directly to non-cultured urogenital samples, but is less suitable for extragenital specimens, particularly throat swabs, due to cross-reaction with commensal Neisseria species.


Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and the resulting disease, coronavirus disease 2019 (Covid-19), have spread to millions of persons worldwide. Multiple vaccine candidates are under development, but no vaccine is currently available. Interim safety and immunogenicity data about the vaccine candidate BNT162b1 in younger adults have been reported previously from trials in Germany and the United States. In an ongoing, placebo-controlled, observer-blinded, dose-escalation, phase 1 trial conducted in the United States, we randomly assigned healthy adults 18 to 55 years of age and those 65 to 85 years of age to receive either placebo or one of two lipid nanoparticle-formulated, nucleoside-modified RNA vaccine candidates: BNT162b1, which encodes a secreted trimerized SARS-CoV-2 receptor-binding domain; or BNT162b2, which encodes a membrane-anchored SARS-CoV-2 full-length spike, stabilized in the prefusion conformation. The primary outcome was safety (e.g., local and systemic reactions and adverse events); immunogenicity was a secondary outcome. Trial groups were defined according to vaccine candidate, age of the participants, and vaccine dose level (10 μg, 20 μg, 30 μg, and 100 μg). In all groups but one, participants received two doses, with a 21-day interval between doses; in one group (100 μg of BNT162b1), participants received one dose. A total of 195 participants underwent randomization. In each of 13 groups of 15 participants, 12 participants received vaccine and 3 received placebo. BNT162b2 was associated with a lower incidence and severity of systemic reactions than BNT162b1, particularly in older adults. In both younger and older adults, the two vaccine candidates elicited similar dose-dependent SARS-CoV-2-neutralizing geometric mean titers, which were similar to or higher than the geometric mean titer of a panel of SARS-CoV-2 convalescent serum samples. The safety and immunogenicity data from this U.S. phase 1 trial of two vaccine candidates in younger and older adults, added to earlier interim safety and immunogenicity data regarding BNT162b1 in younger adults from trials in Germany and the United States, support the selection of BNT162b2 for advancement to a pivotal phase 2-3 safety and efficacy evaluation. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04368728.).


Long-term insulin treatment of vascular smooth muscle cells from rat aorta attenuates the synergistic effect of insulin on angiotensin II- and epidermal growth factor-induced DNA synthesis.
1. In the present study the effects of acute and chronic insulin treatment on growth factor-induced cell growth were investigated in vascular smooth muscle cells. Cell growth was quantified by the incorporation of [3H]thymidine into cell DNA and by determination of total protein. 2. Insulin in a concentration range of 10(-9)-10(-6) mol/l had no effect on cell DNA synthesis. Insulin-like growth factor-1 in a concentration range of 10(-9)-10(-6) mol/l induced a concentration-dependent increase in total cell protein, whereas no changes in DNA synthesis were observed. 3. Acutely, insulin enhanced the mitogenic effect of epidermal growth factor and angiotensin II. Long-term treatment with 10 and 100 ng/ml insulin over 14 weeks was associated with a time-dependent reduction in this potentiating effect of insulin on growth factor-induced DNA synthesis. To evaluate the mechanism of this effect, receptor binding studies were performed with 125I-labelled insulin-like growth factor-1. No difference in the Kd value (2.1 nmol/l) for insulin-like growth factor-1 was found between untreated and chronically insulin-treated cells, whereas the maximal number of binding sites decreased from 5.9 x 10(4) (untreated cells) to 4.7 x 10(4) (10 ng/ml) and to 4.3 x 10(4) (100 ng/ml) binding sites/cell in chronically insulin-treated cells. 4. We conclude that insulin acutely enhances the mitogenic effect of various growth factors such as epidermal growth factor and angiotensin II on vascular smooth muscle cells. Long-term insulin treatment reduced the number of receptor binding sites for insulin-like growth factor-1.(ABSTRACT TRUNCATED AT 250 WORDS)