Computational biomarker pipeline from discovery to clinical implementation: plasma proteomic biomarkers for cardiac transplantation.
Recent technical advances in the field of quantitative proteomics have stimulated a large number of biomarker discovery studies of various diseases, providing avenues for new treatments and diagnostics. However, inherent challenges have limited the successful translation of candidate biomarkers into clinical use, thus highlighting the need for a robust analytical methodology to transition from biomarker discovery to clinical implementation. We have developed an end-to-end computational proteomic pipeline for biomarkers studies. At the discovery stage, the pipeline emphasizes different aspects of experimental design, appropriate statistical methodologies, and quality assessment of results. At the validation stage, the pipeline focuses on the migration of the results to a platform appropriate for external validation, and the development of a classifier score based on corroborated protein biomarkers. At the last stage towards clinical implementation, the main aims are to develop and validate an assay suitable for clinical deployment, and to calibrate the biomarker classifier using the developed assay. The proposed pipeline was applied to a biomarker study in cardiac transplantation aimed at developing a minimally invasive clinical test to monitor acute rejection. Starting with an untargeted screening of the human plasma proteome, five candidate biomarker proteins were identified. Rejection-regulated proteins reflect cellular and humoral immune responses, acute phase inflammatory pathways, and lipid metabolism biological processes. A multiplex multiple reaction monitoring mass-spectrometry (MRM-MS) assay was developed for the five candidate biomarkers and validated by enzyme-linked immune-sorbent (ELISA) and immunonephelometric assays (INA). A classifier score based on corroborated proteins demonstrated that the developed MRM-MS assay provides an appropriate methodology for an external validation, which is still in progress. Plasma proteomic biomarkers of acute cardiac rejection may offer a relevant post-transplant monitoring tool to effectively guide clinical care. The proposed computational pipeline is highly applicable to a wide range of biomarker proteomic studies.


Relationship of the polymerase chain reaction for cytomegalovirus to the development of hepatitis in liver transplant recipients.
In a pilot study, the polymerase chain reaction was found to be more sensitive than standard viral culture methods for the detection of cytomegalovirus, particularly from blood and tissues. We therefore applied this technique to 71 serially collected liver biopsies from 16 orthotopic liver transplant patients. All patients were CMV-seropositive (n = 15) or seroconverted (n = 1). Seven patients (9 biopsies) had histologically proved CMV hepatitis, and all these biopsies were CMV PCR-positive. Six of these 7 patients had a prior liver biopsy that was CMV PCR-positive, but culture and histology-negative, an average of 13.2 +/- 6.9 days before the histologically positive biopsy. The 7th patient was not biopsied prior to the diagnostic biopsy. Three patients had 7 liver biopsies that were CMV PCR-positive, but histologically negative for CMV hepatitis. Two of these three had CMV infection confirmed by viral culture of blood or liver biopsy. The remaining 6 patients had a total of 26 liver biopsies that were negative for CMV by PCR, culture, and histology. Among liver transplant patients, CMV PCR performed on liver biopsy specimens correctly identified all histologically proven cases of CMV hepatitis. CMV PCR positivity in liver tissue did not correlate with latent infection and preceded the development of CMV hepatitis or other meaningful CMV infection in 8 of 10 patients.


RPD3 encodes a second factor required to achieve maximum positive and negative transcriptional states in Saccharomyces cerevisiae.
In Saccharomyces cerevisiae, TRK1 and TRK2 encode the high- and low-affinity K+ transporters, respectively. In cells containing a deletion of TRK1, transcription levels of TRK2 are extremely low and are limiting for growth in media containing low levels of K+ (Trk- phenotype). Recessive mutations in RPD1 and RPD3 suppress the TRK2, conferring an approximately fourfold increase in transcription. rpd3 mutations confer pleiotropic phenotypes, including (i) mating defects, (ii) hypersensitivity to cycloheximide, (iii) inability to sporulate as homozygous diploids, and (iv) constitutive derepression of acid phosphatase. RPD3 was cloned and is predicted to encode a 48-kDa protein with no extensive similarity to proteins contained in current data bases. Deletion of RPD3 is not lethal but confers phenotypes identical to those caused by spontaneous mutations. RPD3 is required for both full repression and full activation of transcription of target genes including PHO5, STE6, and TY2. RPD3 is the second gene required for this function, since RPD1 is also required. The effects of mutations in RPD1 and RPD3 are not additive, suggesting that these genes are involved in the same transcriptional regulatory function or pathway.


Leilani Muir versus the philosopher king: eugenics on trial in Alberta.
The Province of Alberta in Canada was the only jurisdiction in the British Empire where a eugenic sterilization law was passed (in 1928) and vigorously implemented. The pace of sterilization orders accelerated during the Nazi era and remained high after World War II, terminating only in 1972 when the Sexual Sterilization Act was repealed. The Alberta Eugenics Board operated away from public and legislative scrutiny, and many things done in the name of eugenics were clearly illegal. Eugenics was put on trial in Alberta in 1995 and a judge of the Court of Queen's Bench ruled in 1996 that the government had wrongly sterilized Leilani Muir. After hearing evidence about the history of the eugenics movement, the origins of Alberta's Sexual Sterilization Act, the operation of the Eugenics Board, and details of Muir's life, Madam Justice Joanne B. Veit found that 'the damage inflicted by the operation was catastrophic', the 'wrongful stigmatization of Ms. Muir as a moron ... has humiliated Ms. Muir every day of her life', and 'the circumstances of Ms. Muir's sterilization were so high-handed and so contemptuous of the statutory authority to effect sterilization, and were undertaken in an atmosphere that so little respected Ms. Muir's human dignity that the community's, and the court's, sense of decency is offended'. Veit awarded Muir damages of $740,780 CAD and legal costs of $230,000 CAD. The order for Muir's sterilization was signed by John M. MacEachran, founder of the Department of Philosophy and Psychology at the University of Alberta and chairman of the Eugenics Board from 1929 to 1965. An exponent of Platonic idealism, MacEachran believed sterilization of children with a low IQ test score was a means of 'raising and safeguarding the purity of the race'. However, the Alberta Sterilization Act was passed and implemented with cavalier disregard for the principles of genetics as well as the rights of children.


RIPK1 is a critical modulator of both tonic and TLR-responsive inflammatory and cell death pathways in human macrophage differentiation.
In this study, we took advantage of human-induced pluripotent stem cells (hiPSC) and CRISPR/Cas9 technology to investigate the potential roles of RIPK1 in regulating hematopoiesis and macrophage differentiation, proinflammatory activation, and cell death pathways. Knock-out of RIPK1 in hiPSCs demonstrated that this protein is not required for erythro-myeloid differentiation. Using a well-established macrophage differentiation protocol, knock-out of RIPK1 did not block the differentiation of iPSC-derived macrophages, which displayed a similar phenotype to WT hiPSC-derived macrophages. However, knock-out of RIPK1 leads to a TNFα-dependent apoptotic death of differentiated hiPSC-derived macrophages (iPS-MΦ) and progressive loss of iPS-MΦ production irrespective of external pro-inflammatory stimuli. Live video analysis demonstrated that TLR3/4 activation of RIPK1 KO hiPSC-derived macrophages triggered TRIF and RIPK3-dependent necroptosis irrespective of caspase-8 activation. In contrast, TLR3/4 activation of WT macrophages-induced necroptosis only when caspases were inhibited, confirming the modulating effect of RIPK1 on RIPK3-mediated necroptosis through the FADD, Caspase-8 pathway. Activation of these inflammatory pathways required RIPK3 kinase activity while RIPK1 was dispensable. However, loss of RIPK1 sensitizes macrophages to activate RIPK3 in response to inflammatory stimuli, thereby exacerbating a potentially pathological inflammatory response. Taken together, these results reveal that RIPK1 has an important role in regulating the potent inflammatory pathways in authentic human macrophages that are poised to respond to external stimuli. Consequently, RIPK1 activity might be a valid target in the development of novel therapies for chronic inflammatory diseases.


Preclinical Development of a Bispecific Antibody that Safely and Effectively Targets CD19 and CD47 for the Treatment of B-Cell Lymphoma and Leukemia.
CD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal "don't eat me" signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701-induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20-targeted therapy. Mol Cancer Ther; 17(8); 1739-51. ©2018 AACR.


Novel drugs targeting EGFR and HER2 exon 20 mutations in metastatic NSCLC.
Approximately 4% of epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) present EGFR exon 20 in-frame insertions, accounting for 0.3 %-3.7 % of NSCLC. In addition, 2 %-4 % of patients with NSCLC harbor human epidermal growth factor receptor 2 gene (HER2) mutations, being the 90 % of them exon 20 insertions. These mutations confer intrinsic resistance to available EGFR tyrosine kinase inhibitors (TKIs) and anti-HER2 treatments, as they result in steric hindrance of the drug-binding pocket. Therefore, no targeted therapies have been approved for NSCLC patients with EGFR or HER2 exon 20- activating mutations to date and remain an unmet clinical need. Promising efforts to novel treatment development have been made. Early data provide encouraging activity of novel drugs targeting EGFR and HER2 mutations in metastatic NSCLC. In this review we will summarize all the data reported to date about these driver molecular alterations and potential targeted therapies.


Efficacy and effectiveness of community-based treatment of severe malnutrition.
There is a long tradition of community-based rehabilitation for treatment of severe malnutrition: the question is whether it is effective and whether it should be advised for routine health systems. To examine the effectiveness of rehabilitating severely malnourished children in the community in nonemergency situations. A literature search was conducted of community-based rehabilitation programs delivered by day-care nutrition centers, residential nutrition centers, primary health clinics, and domiciliary care with or without provision of food, for the period 1980-2005. Effectiveness was defined as mortality of less than 5% and an average weight gain of at least 5 g/kg/day. Thirty-three studies of community-based rehabilitation were examined and summarized. Eleven (33%) programs were considered effective. Of the sub-sample of programs reported since 1995, 8 of 13 (62%) were effective. None of the programs operating within routine health systems without external assistance was effective. With careful planning and resources, all four delivery systems can be effective. It is unlikely that a single delivery system would suit all situations worldwide. The choice of a system depends on local factors. High energy intakes (> 150 kcal/kg/day), high protein intakes (4-6 g/kg/day), and provision of micronutrients are essential for success. When done well, rehabilitation at home with family foods is more cost-effective than inpatient care, but the cost effectiveness of ready-to-use therapeutic foods (RUTF) versus family foods has not been studied. Where children have access to a functioning primary health-care system and can be monitored, the rehabilitation phase of treatment of severe malnutrition should take place in the community rather than in the hospital but only if caregivers can make energy- and protein-dense food mixtures or are given RUTF. For routine health services, the cost of RUTF, logistics of procurement and distribution, and sustainability need to be carefully considered.


Mortality and morbidity from malaria among children in a rural area of The Gambia, West Africa.
Mortality and morbidity from malaria were measured among 3000 children under the age of 7 years in a rural area of The Gambia, West Africa. Using a post-mortem questionnaire technique, malaria was identified as the probable cause of 4% of infant deaths and of 25% of deaths in children aged 1 to 4 years. The malaria mortality rate was 6.3 per 1000 per year in infants and 10.7 per 1000 per year in children aged 1 to 4 years. Morbidity surveys suggested that children under the age of 7 years experienced about one clinical episode of malaria per year. Calculation of attributable fractions showed that malaria may be responsible for about 40% of episodes of fever in children. Although the overall level of parasitaemia showed little seasonal variation, the clinical impact of malaria was highly seasonal; all malaria deaths and a high proportion of febrile episodes were recorded during a limited period at the end of the rainy season.


Improved intensity-based label-free quantification via proximity-based intensity normalization (PIN).
Researchers are increasingly turning to label-free MS1 intensity-based quantification strategies within HPLC-ESI-MS/MS workflows to reveal biological variation at the molecule level. Unfortunately, HPLC-ESI-MS/MS workflows using these strategies produce results with poor repeatability and reproducibility, primarily due to systematic bias and complex variability. While current global normalization strategies can mitigate systematic bias, they fail when faced with complex variability stemming from transient stochastic events during HPLC-ESI-MS/MS analysis. To address these problems, we developed a novel local normalization method, proximity-based intensity normalization (PIN), based on the analysis of compositional data. We evaluated PIN against common normalization strategies. PIN outperforms them in dramatically reducing variance and in identifying 20% more proteins with statistically significant abundance differences that other strategies missed. Our results show the PIN enables the discovery of statistically significant biological variation that otherwise is falsely reported or missed.


Analysis of Hi-C data using SIP effectively identifies loops in organisms from C. elegans to mammals.
Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome-wide condensation in C. elegans hermaphrodites.


Ability to identify scene-relative object movement is not limited by, or yoked to, ability to perceive heading.
During locomotion humans can judge where they are heading relative to the scene and the movement of objects within the scene. Both judgments rely on identifying global components of optic flow. What is the relationship between the perception of heading, and the identification of object movement during self-movement? Do they rely on a shared mechanism? One way to address these questions is to compare performance on the two tasks. We designed stimuli that allowed direct comparison of the precision of heading and object movement judgments. Across a series of experiments, we found the precision was typically higher when judging scene-relative object movement than when judging heading. We also found that manipulations of the content of the visual scene can change the relative precision of the two judgments. These results demonstrate that the ability to judge scene-relative object movement during self-movement is not limited by, or yoked to, the ability to judge the direction of self-movement.


Central losartan attenuates increases in arterial pressure and expression of FosB/ΔFosB along the autonomic axis associated with chronic intermittent hypoxia.
Chronic intermittent hypoxia (CIH) increases mean arterial pressure (MAP) and FosB/ΔFosB staining in central autonomic nuclei. To test the role of the brain renin-angiotensin system (RAS) in CIH hypertension, rats were implanted with intracerebroventricular (icv) cannulae delivering losartan (1 μg/h) or vehicle (VEH) via miniosmotic pumps and telemetry devices for arterial pressure recording. A third group was given the same dose of losartan subcutaneously (sc). Two groups of losartan-treated rats served as normoxic controls. Rats were exposed to CIH or normoxia for 7 days and then euthanized for immunohistochemistry. Intracerebroventricular losartan attenuated CIH-induced increases in arterial pressure during CIH exposure (0800-1600 during the light phase) on days 1, 6, and 7 and each day during the normoxic dark phase. FosB/ΔFosB staining in the organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus (MnPO), paraventricular nucleus of the hypothalamus (PVN), the rostral ventrolateral medulla (RVLM), and the nucleus of the solitary tract (NTS) was decreased in icv losartan-treated rats. Subcutaneous losartan also reduced CIH hypertension during the last 2 days of CIH and produced bradycardia prior to the effect on blood pressure. Following sc losartan, FosB/ΔFosB staining was reduced only in the OVLT, MnPO, PVN, and NTS. These data indicate that the central and peripheral RAS contribute to CIH-induced hypertension and transcriptional activation of autonomic nuclei and that the contribution of the central RAS is greater during the normoxic dark phase of CIH hypertension.


Serum levels of advanced glycation endproducts and other markers of protein damage in early diabetic nephropathy in type 1 diabetes.
To determine the role of markers of plasma protein damage by glycation, oxidation and nitration in microalbuminuria onset or subsequent decline of glomerular filtration rate (termed "early GFR decline") in patients with type 1 diabetes. From the 1(st) Joslin Kidney Study, we selected 30 patients with longstanding normoalbuminuria and 55 patients with new onset microalbuminuria. Patients with microalbuminuria had 8-12 years follow-up during which 33 had stable GFR and 22 early GFR decline. Mean baseline GFR(CYSTATIN C) was similar between the three groups. Glycation, oxidation and nitration markers were measured in protein and ultrafiltrate at baseline by liquid chromatography-tandem mass spectrometry using the most reliable methods currently available. Though none were significantly different between patients with microalbuminuria with stable or early GFR decline, levels of 6 protein damage adduct residues of plasma protein and 4 related free adducts of plasma ultrafiltrate were significantly different in patients with microalbuminuria compared to normoalbuminuria controls. Three protein damage adduct residues were decreased and 3 increased in microalbuminuria while 3 free adducts were decreased and one increased in microalbuminuria. The most profound differences were of N-formylkynurenine (NFK) protein adduct residue and N(ω)-carboxymethylarginine (CMA) free adduct in which levels were markedly lower in microalbuminuria (P<0.001 for both). Complex processes influence levels of plasma protein damage and related proteolysis product free adducts in type 1 diabetes and microalbuminuria. The effects observed point to the possibility that patients who have efficient mechanisms of disposal of damaged proteins might be at an increased risk of developing microalbuminuria but not early renal function decline. The findings support the concept that the mechanisms responsible for microalbuminuria may differ from the mechanisms involved in the initiation of early renal function decline.


Gonadotropin-releasing hormone agonist induces downregulation of tensin 1 in women with endometriosis.
Many cell migration-related molecules are associated with endometriosis. Tensin 1 (TNS1), which has been implicated in cell migration, may play a role in endometriosis. The study goal was to evaluate the TNS1 expression in endometrial tissue and serum from women with endometriosis treated with gonadotropin-releasing hormone agonist (GnRHa). Tissue and serum samples were collected from women with endometriosis who were treated (n = 29) with GnRHa or untreated (n = 30). TNS1 mRNA was examined using quantitative PCR. TNS1 protein levels in tissue and serum samples were investigated using Western blot, immunohistochemistry and ELISA. Eleven women with endometriosis participated in a follow-up investigation of serum TNS1 before and after GnRHa treatment. TNS1 mRNA (P = 0.006) and protein (P = 0.001) were significantly downregulated in endometriotic tissue from women with endometriosis who received GnRHa. Immunolocalization of TNS1 showed strong expression in the epithelial and stromal cells of endometriotic tissue from women untreated with GnRHa, whereas endometriotic tissue from GnRHa-treated women showed low TNS1 expression. Follow-up monitoring of serum TNS1 concentration in 11 women showed an average decrease in concentration of 53%, from 294.9 ± 66.69 to 140.3 ± 55.21 pg/mL, following GnRHa treatment (P = 0.003). GnRHa induces downregulation of TNS1 in tissue and serum in women with endometriosis. These results emphasize the importance TNS1 as a potential therapeutic molecular target for the treatment of endometriosis with GnRHa.


Molecular analysis of the von Hippel-Lindau disease tumor suppressor gene in human lung cancer cell lines.
The deletion of the short arm of chromosome 3 is frequently observed in lung cancer. To determine whether the von Hippel-Lindau (VHL) disease tumor suppressor gene located at 3p25 is responsible for oncogenesis in lung cancer, we searched the known open reading frame using the single-strand conformation polymorphism (SSCP) technique for mutations in the VHL gene in 72 cancer cell lines including small cell (SCLC) and non-small cell (NSCLC) lung cancers, carcinoids, and mesotheliomas. SSCP analysis showed that four cell lines have altered SSCP patterns within the coding region and one in an intron of the VHL gene. SCLC line NCI-H1672 had a somatic mutation, G to A at nucleotide (nt) 530, leading to amino acid substitution (glycine to aspartic acid) compared to normal DNA from the same patient. Mesothelioma line NCI-H28 had T to A mutation at nt 479 leading to leucine to histidine amino acid change. We found one frequent polymorphism A (0.72) or G (0.28) at nt 19 resulting in either serine or glycine at this position, changes also found in normal peripheral blood cell DNA, often in a heterozygous state. In addition, we found single rare polymorphisms which did not alter the coding region including: C to G at nt 396, G to T at nt 843, and C to T change in an intron. These results suggest that the VHL gene is only rarely mutated in thoracic malignancies.


Neutrophil to lymphocyte ratio at the early phase of acute pancreatitis correlates with serum urokinase-type plasminogen activator receptor and interleukin 6 and predicts organ failure.
In early phase of acute pancreatitis (AP), systemic inflammatory response syndrome may lead to organ failure. The severe form of AP is associated with high mortality that may be prevented by timely diagnosis and treatment of the predicted severe cases. Serum interleukin 6 (IL-6) and urokinase-type plasminogen activator receptor (uPAR) have been proposed as accurate early markers of severe AP. The aim of the study was to assess whether widely available blood count indexes: neutrophil to lymphocyte (NLR), lymphocyte to monocyte (LMR) and platelet to lymphocyte ratios correlate with IL-6 and uPAR and may be utilized to predict organ complications at the early phase of AP. The study included 95 adult patients with AP treated at the Surgical Ward Complex of Health Care Centers in Wadowice, Poland. Organ failure was diagnosed according to modi ed Marshall scoring system, as recommended by 2012 Atlanta classification. Blood samples for laboratory tests were collected on days 1, 2 and 3 following the onset of AP symptoms. Patients with organ failure presented significantly lower LMR on day 1 and significantly higher NLR on days 2 and 3. Strong positive correlations between NLR and IL-6 and moderate correlations between NLR and uPAR were observed throughout the study. Day 2 and 3 NLR values significantly predicted organ failure at the early phase of AP. Taking into account the wide availability of NLR, it may be considered as a surrogate of more expensive tests to help the early assessment of organ failure complicating AP.


Exploring sequence characteristics related to high-level production of secreted proteins in Aspergillus niger.
Protein sequence features are explored in relation to the production of over-expressed extracellular proteins by fungi. Knowledge on features influencing protein production and secretion could be employed to improve enzyme production levels in industrial bioprocesses via protein engineering. A large set, over 600 homologous and nearly 2,000 heterologous fungal genes, were overexpressed in Aspergillus niger using a standardized expression cassette and scored for high versus no production. Subsequently, sequence-based machine learning techniques were applied for identifying relevant DNA and protein sequence features. The amino-acid composition of the protein sequence was found to be most predictive and interpretation revealed that, for both homologous and heterologous gene expression, the same features are important: tyrosine and asparagine composition was found to have a positive correlation with high-level production, whereas for unsuccessful production, contributions were found for methionine and lysine composition. The predictor is available online at http://bioinformatics.tudelft.nl/hipsec. Subsequent work aims at validating these findings by protein engineering as a method for increasing expression levels per gene copy.


Obstetric ultrasonography: wider role for radiographers?
Most obstetric ultrasound scanning is carried out by radiographers, who have professional constraints that forbid them to disclose the result of any investigation to any patient or to hold themselves as professionally qualified to diagnose. A nationwide survey was carried out to explore the paramedical aspects of obstetric ultrasound services and determine how radiographers reconcile their professional constraints with the needs and expectations of patients. It was concluded that over 70% of obstetric scanning in the United Kingdom is carried out by radiographers who tell patients some results and report both numerical and interpretational data to referring clinicians. This survey suggests that constraints on radiographers who perform obstetric ultrasonography are excessively restrictive and conflict with everyday clinical demands. The medical supervision of some obstetric ultrasound units should be improved, and partners of patients should not be encouraged to expect access to obstetric scans unless radiographers have close medical support.


Measuring Acuity and Pediatric Critical Care Nursing Workload by Using ICU CAMEO III.
The Intensive Care Unit Complexity Assessment and Monitoring to Ensure Optimal Outcomes (ICU CAMEO III) acuity tool measures patient acuity in terms of the complexity of nursing cognitive workload. To validate the ICU CAMEO III acuity tool in US children's hospitals. Using a convenience sample, 9 sites enrolled children admitted to pediatric intensive care units (ICUs). Descriptive statistics were used to summarize patient, nursing, and unit characteristics. Concurrent validity was evaluated by correlating the ICU CAMEO III with the Therapeutic Intervention Scoring System-Children (TISS-C) and the Pediatric Risk of Mortality III (PRISM III). Patients (N = 840) were enrolled from 15 units (7 cardiac and 8 mixed pediatric ICUs). The mean number of ICU beds was 23 (range, 12-34). Among the patients, 512 (61%) were diagnosed with cardiac and 328 (39%) with noncardiac conditions; 463 patients (55.1%) were admitted for medical reasons, and 377 patients (44.9%) were surgical. The ICU CAMEO III median score was 99 (range, 59-163). The ICU CAMEO complexity classification was determined for all 840 patients: 60 (7.1%) with level I complexity; 183 (21.8%) with level II; 201 (23.9%), level III; 267 (31.8%), level IV; and 129 (15.4%), level V. Strong correlation was found between ICU CAMEO III and both TISS-C (ρ = .822, P < .001) and PRISM III (ρ = .607, P < .001) scores, and between the CAMEO complexity classifications and the PRISM III categories (ρ = .575, P = .001). The ICU CAMEO III acuity tool and CAMEO complexity classifications are valid measures of patient acuity and nursing cognitive workload compared with PRISM III and TISS-C in academic children's hospitals.


Help-seeking and access to mental health care in a university student population.
University students represent an important population in which to study access to mental health care. Understanding their unmet needs will enhance efforts to prevent and treat mental disorders during a pivotal period in life. To quantify mental health service use and estimate how various factors are associated with help-seeking and access in a university student population. A Web-based survey was administered to a random sample of 2785 students attending a large, public university with a demographic profile similar to the national student population. Nonresponse bias was accounted for using administrative data and a nonrespondent survey. Mental health was measured using the Patient Health Questionnaire screens for depressive and anxiety disorders. Mental health service utilization was measured as having received psychotropic medication or psychotherapy in the past year. Of students with positive screens for depression or anxiety, the proportion who did not receive any services ranged from 37% to 84%, depending on the disorder. Predictors of not receiving services included a lack of perceived need, being unaware of services or insurance coverage, skepticism about treatment effectiveness, low socioeconomic background, and being Asian or Pacific Islander. Even in an environment with universal access to free short-term psychotherapy and basic health services, most students with apparent mental disorders did not receive treatment. Initiatives to improve access to mental health care for students have the potential to produce substantial benefits in terms of mental health and related outcomes.


Electroacupuncture Pretreatment Attenuates Inflammatory Lung Injury After Cardiopulmonary Bypass by Suppressing NLRP3 Inflammasome Activation in Rats.
Cardiopulmonary bypass (CPB) can induce inflammatory lung injury, which is a common complication during cardiac surgery. Nod-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome-induced inflammation plays a crucial role in lung injury after CPB. Previous studies have shown that electroacupuncture (EA) has potential anti-inflammatory activity. However, the role of EA in CPB is poorly understood. The aim of this study was to determine whether EA was associated with CPB-induced inflammatory lung injury. In the present study, rats were treated with EA for 5 days before CPB. Two hours after CPB, the lung tissue, serum, and bronchoalveolar lavage fluid (BALF) were prepared for assessment. Our results showed that the expression of NLRP3 in the lung tissue increased significantly after CPB. The EA pretreatment suppressed NLRP3 inflammasome activation, reduced lung edema, and inhibited IL-1β release into the serum and BALF after CPB. Our findings suggest that EA pretreatment attenuates inflammatory lung injury after CPB by suppressing NLRP3 inflammasome activation.


Cx32 reverses epithelial-mesenchymal transition in doxorubicin-resistant hepatocellular carcinoma.
Recently, epithelial-mesenchymal transition (EMT) has been reported to be an important mechanism of drug resistance in numerous types of cancer cells, including hepatocellular carcinoma (HCC). However, the underlying mechanisms remain to be fully elucidated. Connexin (Cx)32 plays a crucial role in hepatocarcinogenesis. The present study investigated the role of Cx32 in the regulation of chemotherapy-induced EMT in HCC. We found that the expression levels of Cx32 and E-cadherin were clearly decreased in HCC tissues compared with the corresponding paracancerous tissues, while the expression level of vimentin was significantly enhanced in HCC tissues. The expression of Cx32 had a strong correlation with the expression of E-cadherin and vimentin. In an in vitro study, a doxorubicin (DOX)-resistant liver cell line HepG2/DOX was established from parental HepG2 cells. The results showed that HepG2/DOX cells acquired EMT characteristics, with a decreased expression level of E-cadherin and an enhanced expression level of vimentin, and possessed high migratory abilities and invasiveness. Meanwhile, Cx32 was significantly decreased in the HepG2/DOX cells. Knockdown of Cx32 by shRNA in HepG2 cells induced EMT, while overexpression of Cx32 converted EMT to mesenchymal-epithelial transition (MET) in the HepG2/DOX cells. These results suggest that Cx32 is an important regulator of DOX-induced EMT in HCC. Cx32 could be considered as a novel target to reverse DOX resistance in HCC.


Parasitic castration promotes coevolutionary cycling but also imposes a cost on sex.
Antagonistic coevolution between hosts and parasites is thought to drive a range of biological phenomena including the maintenance of sexual reproduction. Of particular interest are conditions that produce persistent fluctuations in the frequencies of genes governing host-parasite specificity (coevolutionary cycling), as sex may be more beneficial than asexual reproduction in a constantly changing environment. Although many studies have shown that coevolutionary cycling can lead to the maintenance of sex, the effects of ecological feedbacks on the persistence of these fluctuations in gene frequencies are not well understood. Here, we use a simple deterministic model that incorporates ecological feedbacks to explore how parasitic reductions in host fecundity affect the maintenance of coevolutionary cycling. We demonstrate that parasitic castration is inherently destabilizing and may be necessary for coevolutionary cycling to persist indefinitely, but also reduces the likelihood that sexually reproducing individuals will find a fertile partner, which may select against sex. These findings suggest that castrators can play an important role in shaping host evolution and are likely to be good targets for observing fluctuations in gene frequencies that govern specificity in host-parasite interactions.


Proteomic profiling reveals the prognostic value of adenomatous polyposis coli-end-binding protein 1 in hepatocellular carcinoma.
Histological differentiation is a major pathological parameter associated with poor prognosis in patients with hepatocellular carcinoma (HCC) and the molecular signature underlying HCC differentiation may involve key proteins potentially affecting the malignant characters of HCC. To develop prognostic biomarkers for HCC, we examined the global protein expression profiles of 45 surgically resected tissues, including 27 HCCs with different degree of histological differentiation, 11 adjacent nontumor tissues, and seven normal liver tissues. Unsupervised classification grouped the 45 samples according to their histological classification based on the protein expression profiles created by laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE). Statistical analysis and mass spectrometry identified 26 proteins with differential expression, of which 14 were functionally linked to c-Myc, AP-1, HIF1A, hepatocyte nuclear factor 4 alpha, or the Ras superfamily (RhoA, CDC42, and Rac1). Among the proteins identified, we focused on APC-binding protein EB1 (EB1) because it was dominantly expressed in poorly differentiated HCCs, which generally correlate with the poor prognosis in patients with HCC. In addition, EB1 is controlled by c-Myc, RhoA, and CDC42, which have all been linked to HCC malignancy. Immunohistochemistry in a further 145 HCC cases revealed that EB1 significantly correlated with the degree of histological differentiation (P < 0.001), and univariate and multivariate analyses indicated that EB1 is an independent prognostic factor for recurrence (hazard ratio, 2.740; 95% confidence interval, 1.771-4.239; P < 0.001) and survival (hazard ratio, 2.256; 95% confidence interval, 1.337-3.807; P = 0.002) of patients with HCC after curative surgery. Proteomic profiling revealed the molecular signature behind the progression of HCC, and the prognostic value of EB1 in HCC.


Community composition has greater impact on the functioning of marine phytoplankton communities than ocean acidification.
Ecosystem functioning is simultaneously affected by changes in community composition and environmental change such as increasing atmospheric carbon dioxide (CO2 ) and subsequent ocean acidification. However, it largely remains uncertain how the effects of these factors compare to each other. Addressing this question, we experimentally tested the hypothesis that initial community composition and elevated CO2 are equally important to the regulation of phytoplankton biomass. We full-factorially exposed three compositionally different marine phytoplankton communities to two different CO2 levels and examined the effects and relative importance (ω(2) ) of the two factors and their interaction on phytoplankton biomass at bloom peak. The results showed that initial community composition had a significantly greater impact than elevated CO2 on phytoplankton biomass, which varied largely among communities. We suggest that the different initial ratios between cyanobacteria, diatoms, and dinoflagellates might be the key for the varying competitive and thus functional outcome among communities. Furthermore, the results showed that depending on initial community composition elevated CO2 selected for larger sized diatoms, which led to increased total phytoplankton biomass. This study highlights the relevance of initial community composition, which strongly drives the functional outcome, when assessing impacts of climate change on ecosystem functioning. In particular, the increase in phytoplankton biomass driven by the gain of larger sized diatoms in response to elevated CO2 potentially has strong implications for nutrient cycling and carbon export in future oceans.


High numerical aperture reflection mode coherent diffraction microscopy using off-axis apertured illumination.
We extend coherent diffraction imaging (CDI) to a high numerical aperture reflection mode geometry for the first time. We derive a coordinate transform that allows us to rewrite the recorded far-field scatter pattern from a tilted object as a uniformly spaced Fourier transform. Using this approach, FFTs in standard iterative phase retrieval algorithms can be used to significantly speed up the image reconstruction times. Moreover, we avoid the isolated sample requirement by imaging a pinhole onto the specimen, in a technique termed apertured illumination CDI. By combining the new coordinate transformation with apertured illumination CDI, we demonstrate rapid high numerical aperture imaging of samples illuminated by visible laser light. Finally, we demonstrate future promise for this technique by using high harmonic beams for high numerical aperture reflection mode imaging.


Combined effects of type and depth of counting chamber, and rate of image frame capture, on bull sperm motility and kinematics.
Semen quality assessment requires accurate, reliable and objective methods for examination of sperm variables including sperm motility. For preparation of semen samples for artificial insemination, as a genetic resource, samples that are used for insemination need to have the capacity to result in a highly acceptable fertility rate. Several methods have been developed for evaluation of bull sperm in laboratory conditions and for preparation of doses for artificial insemination. Computer-assisted semen analyses can provide objective information on various sperm variables. Nevertheless, this equipment requires fine-calibrations considering differences among species, breeds and conditions for sample evaluation and data analyses. In the present study, there was examination of the interaction between factors such as image frame rate and type and depth of counting chamber in which sperm were evaluated, together with differences between bulls of four breeds. The use of the Spermtrack® reusable 10 μm-depth chamber provided more reliable results than results obtained using disposable chambers (10 and 20 μm depth). A capture rate of at least 90 fps is required for assessment of sperm motility percentage, whereas a rate of 250 fps is needed for obtaining consistent kinematic data. Differences among breeds in the present study indicate conditions for sperm analyses should include specific equipment calibrations for each breed. These results contribute to development of more precise conditions for assessments of bull sperm quality taking into account breed differences and the requirement each breed has for the adequate evaluation and preparation of samples for artificial insemination.


An evaluation of a hospital stay regulatory mechanism.
The results of an evaluation of a predischarge utilization review program [PDUR] for Medicaid Patients are presented. A group of hospitals in Allegheny County, Pennsylvania, participated in this program on a voluntary basis prior to the program's being mandated statewide. All other hospitals in the county experienced retrospective review of Medicaid cases. Our analysis incorporates both types of hospitals in a quasi-experimental design. We found that during the period studied the length of stay of Medicaid patients fell proportionately more than that of the Blue Cross patients in both groups of hospitals; the relative decrease in the length of stay began to occur prior to the introduction of the PDUR program, but no differential effect of the PDUR review process could be demonstrated. The decline in the length of stay was, however, more continuous and smooth in those hospitals participating in the program.


Intravenous use of valproic acid in status epilepticus is associated with high risk of hyperammonemia.
The aim of the study was to examine the frequency of hyperammonemia secondary to valproic acid treatment in status epilepticus and to describe the characteristics of the patients. All patients with established status epilepticus during 2014 to 2016 at Ryhov County Hospital were identified in a retrospective case series. Clinical and laboratory findings were collected from electronic medical files and the Metavision database at the intensive care unit (ICU). Hyperammonemia was defined as a concentration of at least 50 μmol/L. 11 of 40 patients developed hyperammonemia. These patients had a significantly longer stay at the ICU (12.6 vs 2.5 days) and at the hospital (22 vs 11 days). All patients with hyperammonemia were treated at the ICU and all received antibiotics. 12 patients were treated with intravenous valproic acid outside the ICU. Hyperammonemia was not related to Body Mass Index, time to initiation of therapy or laboratory abnormalities except anemia (Hemoglobin 104 vs 122 g/l). There was no difference in mortality between groups. The risk of hyperammonemia is almost 40% in patients receiving intravenous valproic acid in the ICU setting. The underlying mechanisms are probably either individual susceptibility or high metabolic demands. A high vigilance should be recommended. These data require further research via prospective designs in which multiple variables are controlled to explore the effects of individual factors on treatment outcome.


Changes in the enteric vasculature of mice infected with Schistosoma mansoni.
Mesenteric and intestinal vasculature of mice infected with S. mansoni was injected transaortically with a silicone elastomer. The mesenteries and intestines were then cleared and examined with a stereomicroscope. Marked tortuosity and dilatation of mesenteric and intestinal veins and arteries were found to be confined to areas of oviposition and were associated with small arteriovenous shunts which were not present in control animals or areas of intestine devoid of eggs. These findings suggest that splanchnic arterial-portal venous shunts add a hyperkinetic component to intrahepatic portal venous obstruction and synergistically result in portal hypertension.


The clinical, cerebrospinal fluid, and histopathologic effects of epidural ketorolac in dogs.
To evaluate the clinical, cerebrospinal fluid (CSF), and histopathologic effects of epidural ketorolac. Blinded, randomized, placebo controlled study. Twenty-two adult mixed breed dogs with 16 treatment and 6 control dogs, weighing 14.4 to 29.8 kg. Dogs were anesthetized and epidural catheters were placed at the lumbosacral space. Catheter placement was evaluated fluoroscopically. Ketorolac (0.4 mg/kg) or placebo (5% ethanol) was administered epidurally over a 52-hour period, with 5 injections given at 12-hour intervals. At 1, 2, 4, or 8 hours after the first and last injection of ketorolac, dogs were anesthetized and CSF was obtained. Control dogs had CSF sampled 1 hour after the first and last ethanol injection. Neurologic function and pain responses were evaluated before and during the study. Selected dogs were then killed and necropsies performed. None of the dogs exhibited any clinical or neurologic abnormalities during the study. No statistical difference was noted in pain response or CSF analysis between treatment and control dogs. Gross necropsy revealed gastrointestinal ulceration of varying degrees in all treatment dogs. Histopathologic analysis of the spinal cord and meninges revealed minimal focal leptomeningeal phlebitis in 2 of 8 treatment dogs and minor subdural inflammation in 1 control dog. No changes to the neural structures were noted in any dogs. Epidural administration of ketorolac did not cause clinical signs, alteration in CSF values, or pathologic changes to the spinal cord when used for short duration. Gastrointestinal ulceration was common when ketorolac was administered epidurally at 0.4 mg/kg every 12 hours for 5 treatments. This study documented the neurologic safety of epidural ketorolac in dogs before an efficacy trial can be performed. Gastrointestinal ulceration may limit use to short duration or a single injection.


The human tRNA(m(2)(2)G(26))dimethyltransferase: functional expression and characterization of a cloned hTRM1 gene.
This paper presents the first example of a complete gene sequence coding for and expressing a biologically functional human tRNA methyltransferase: the hTRM1 gene product tRNA(m(2)(2)G)dimethyltransferase. We isolated a human cDNA (1980 bp) made from placental mRNA coding for the full-length (659 amino acids) human TRM1 polypeptide. The sequence was fairly similar to Saccharomyces cerevisiae Trm1p, to Caenorhabditis elegans TRM1p and to open reading frames (ORFs) found in mouse and a plant (Arabidopsis thaliana) DNA. The human TRM1 gene was expressed at low temperature in Escherichia coli as a functional recombinant protein, able to catalyze the formation of dimethylguanosine in E.coli tRNA in vivo. It targeted solely position G(26) in T7 transcribed spliced and unspliced human tRNA(Tyr) in vitro and in yeast trm1 mutant tRNA. Thus, the human TRM1 protein is a tRNA(m(2)(2)G(26))dimethyltransferase. Compared with yeast Trm1p, hTRM1p has a C-terminal protrusion of approximately 90 amino acids which shows similarities to a mouse protein related to RNA splicing. A deletion of these 90 C-terminal amino acids left the modification activity in vitro intact. Among point mutations in the hTRM1 gene, only those located in conserved regions of hTRM1p completely eliminated modification activity.


Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12.
Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a 'PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.


Molecular determinants of ciliary membrane localization of Trypanosoma cruzi flagellar calcium-binding protein.
The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.


Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling.
Heparin binds to phospholipase A2 myotoxins from Bothrops asper snake venom, inhibiting their toxic activities. This interaction was investigated using purified myotoxin II, a Lys-49 phospholipase A2 of this venom, and a series of heparin variants, fragments, and other glycosaminoglycans. The binding was correlated to toxin neutralization, using endothelial cells as a target. Myotoxin II binds radiolabeled heparin in solution unselectively, and forms macromolecular complexes with an optimum at a heparin:toxin molar ratio of 1:5. Both O-sulfates and N-sulfates play a role in heparin binding, in the order of importance 2-O-sulfates > 6-Osulfates > N-sulfates. The shortest heparin oligosaccharides interacting with myotoxin II are hexasaccharides. The binding of a neutralizing monoclonal antibody (MAb-3) to myotoxin II was not inhibited by heparin, indicating that the two molecules interact with different sites on the toxin. A synthetic peptide (residues 115-129 in the numbering system of Renetseder et al. (Renetseder, R., Brunie, S., Dijkstra, B. W., Drenth, J., and Sigler, P. B. (1985) J. Biol. Chem. 260, 11627-11634) of myotoxin II displays both heparin-binding and cytolytic activities. It is concluded that heparin neutralizes myotoxin II by binding to a strongly cationic site in the region of residues 115-129, a possible contribution of lysines 36 and 38 suggested by molecular modeling studies. As this cationic region appears to be responsible for the cytolytic activity of the toxin, the present report constitutes the first identification of a cytotoxic region on a phospholipase A2 myotoxin.


Next-Generation Sequencing and In Silico Analysis Facilitate Prolonged Response to Pazopanib in a Patient With Metastatic Urothelial Carcinoma of the Renal Pelvis.
With the advent of widespread tumor genetic profiling, an increased number of mutations with unknown significance are being identified. Often, a glut of uninterpretable findings may confuse the clinician and provide little or inappropriate guidance in therapeutic decision-making. This report describes a method of protein modeling by in silico analysis (ie, using computer simulation) that is easily accessible to the practicing clinician without need for further laboratory analysis, which can potentially serve as a guide in therapeutic decisions based on poorly characterized tumor mutations. An example of this model is given wherein poorly characterized KIT, PDGFRB, and ERBB2 mutations were discovered in a patient with treatment-refractory metastatic transitional cell carcinoma of the renal pelvis. The KIT and PDGFRB mutations were predicted to be pathogenic using in silico analysis, whereas the ERBB2 mutation was predicted to be benign. Based on these findings, the patient was treated with pazopanib and achieved a partial response that lasted for 7.5 months. We propose that in silico analysis be explored as a potential means to further characterize genetic abnormalities found by tumor profiling assays, such as next-generation sequencing.


Exercise self-identity: interactions with social comparison and exercise behaviour.
Possible interactions among exercise self-identity, social comparison and exercise behaviour were explored in a sample of 417 undergraduate students (mean age = 21.5, SD = 3.0; 73% female). Two models were examined using self-report data; (1) a mediation model which proposed an association between social comparison and exercise behaviour mediated by exercise self-identity and (2) a moderation model proposing an association between exercise behaviour and self-identity moderated by social comparison. Results of the mediation analyses revealed partial mediation of the social comparison--exercise behaviour relationship by self-identity in females. Results of the moderation analyses revealed in males a significant interaction of social comparison with exercise behaviour in the prediction of self-identity - the positive association between exercise behaviour and exercise self-identity showed only significant among male students who believed to exercise equally much or less than peers. Possible explanations and implications for exercise promotion are discussed.


Immunopolymer Lipid Nanoparticles for Delivery of Macromolecules to Antigen-Expressing Cells.
Macromolecules such as antibodies and antibody fragments have been reported to interfere with intracellular targets, but their use is limited to delivery systems where expression is achieved from vectors such as plasmids or viruses. We have developed PEGylated nanoparticles of poly-lactic acid (PLA), including the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), which are functionalized with monoclonal anti-CD7, anti-CD53, or anti-GPR56 antibodies for receptor-mediated endocytic delivery into T-cell leukemia cell lines. Incorporation of DOTAP as the lipid component of the PLA nanoparticles enhanced the release of the immuno-nanoparticles from the endosomes into the cytosol compared to nanoparticles made from PLA alone. Systemic delivery of these anti-CD7 immuno-nanoparticles into humanized CD7 transgenic mice resulted in localization in the spleen, enhanced uptake into CD7-expressing splenocytes, and release of low amounts of reporter mRNA for translation. These functionalized polymer lipid nanoparticles are the basis for elaboration and optimization for realizing their potential in therapeutic applications to carry specific macromolecules such as mRNAs for translation into therapeutic proteins that can target intracellular proteins which mediate disease.


Expression of brain-derived neurotrophic factor and p145TrkB affects survival, differentiation, and invasiveness of human neuroblastoma cells.
A large number of poor prognosis neuroblastoma (NB) tumors constitutively express brain-derived neurotrophic factor (BDNF) and variably express the gene for its tyrosine kinase (Trk) receptor TrkB. Good prognosis NB tumors typically express high levels of TrkA mRNA, which encodes the signal transducing receptor for nerve growth factor, p140TrkA. These neurotrophins are necessary for neural cell survival and differentiation. This study evaluates the effects of activation of the BDNF-TrkB signal transduction pathway on the growth, survival, morphology, and invasive capacity of NB cells. We find that the addition of BDNF to SY5Y cells induced to express p145TrkB by retinoic acid treatment does not significantly affect cell proliferation yet will support cell survival. Activation of the BDNF-TrkB signal transduction pathway stimulates disaggregation of cells and extension of neuritic processes which can be blocked by a BDNF-neutralizing antibody. Treatment of cells with K252a, an inhibitor of Trk, reverses the cellular disaggregation. An evaluation of the effects of BDNF and nerve growth factor on the ability of NB cells to penetrate basement membrane proteins indicated that BDNF stimulated a 2-fold increase while nerve growth factor inhibited RA-SY5Y cell invasion. Thus, activation of the p145TrkB signal transduction pathway stimulates NB cell survival, disaggregation, and invasion; all characteristics of metastatic cells. Furthermore, these studies indicate that activation of different Trk signal transduction pathways in NB cells results in distinct differences in tumor cell biology and these may be relevant to the clinical course of the patients.


Prior storm experience moderates water surge perception and risk.
How accurately do people perceive extreme water speeds and how does their perception affect perceived risk? Prior research has focused on the characteristics of moving water that can reduce human stability or balance. The current research presents the first experiment on people's perceptions of risk and moving water at different speeds and depths. Using a randomized within-person 2 (water depth: 0.45, 0.90 m) ×3 (water speed: 0.4, 0.8, 1.2 m/s) experiment, we immersed 76 people in moving water and asked them to estimate water speed and the risk they felt. Multilevel modeling showed that people increasingly overestimated water speeds as actual water speeds increased or as water depth increased. Water speed perceptions mediated the direct positive relationship between actual water speeds and perceptions of risk; the faster the moving water, the greater the perceived risk. Participants' prior experience with rip currents and tropical cyclones moderated the strength of the actual-perceived water speed relationship; consequently, mediation was stronger for people who had experienced no rip currents or fewer storms. These findings provide a clearer understanding of water speed and risk perception, which may help communicate the risks associated with anticipated floods and tropical cyclones.


A cytosolic pathway for the conversion of hydroxypyruvate to glycerate during photorespiration in Arabidopsis.
Deletion of any of the core enzymes of the photorespiratory cycle, one of the major pathways of plant primary metabolism, results in severe air-sensitivity of the respective mutants. The peroxisomal enzyme hydroxypyruvate reductase (HPR1) represents the only exception to this rule. This indicates the presence of extraperoxisomal reactions of photorespiratory hydroxypyruvate metabolism. We have identified a second hydroxypyruvate reductase, HPR2, and present genetic and biochemical evidence that the enzyme provides a cytosolic bypass to the photorespiratory core cycle in Arabidopsis thaliana. Deletion of HPR2 results in elevated levels of hydroxypyruvate and other metabolites in leaves. Photosynthetic gas exchange is slightly altered, especially under long-day conditions. Otherwise, the mutant closely resembles wild-type plants. The combined deletion of both HPR1 and HPR2, however, results in distinct air-sensitivity and a dramatic reduction in photosynthetic performance. These results suggest that photorespiratory metabolism is not confined to chloroplasts, peroxisomes, and mitochondria but also extends to the cytosol. The extent to which cytosolic reactions contribute to the operation of the photorespiratory cycle in varying natural environments is not yet known, but it might be dynamically regulated by the availability of NADH in the context of peroxisomal redox homeostasis.


Age differences in the motor control of speech: An fMRI study of healthy aging.
Healthy aging is associated with a decline in cognitive, executive, and motor processes that are concomitant with changes in brain activation patterns, particularly at high complexity levels. While speech production relies on all these processes, and is known to decline with age, the mechanisms that underlie these changes remain poorly understood, despite the importance of communication on everyday life. In this cross-sectional group study, we investigated age differences in the neuromotor control of speech production by combining behavioral and functional magnetic resonance imaging (fMRI) data. Twenty-seven healthy adults underwent fMRI while performing a speech production task consisting in the articulation of nonwords of different sequential and motor complexity. Results demonstrate strong age differences in movement time (MT), with longer and more variable MT in older adults. The fMRI results revealed extensive age differences in the relationship between BOLD signal and MT, within and outside the sensorimotor system. Moreover, age differences were also found in relation to sequential complexity within the motor and attentional systems, reflecting both compensatory and de-differentiation mechanisms. At very high complexity level (high motor complexity and high sequence complexity), age differences were found in both MT data and BOLD response, which increased in several sensorimotor and executive control areas. Together, these results suggest that aging of motor and executive control mechanisms may contribute to age differences in speech production. These findings highlight the importance of studying functionally relevant behavior such as speech to understand the mechanisms of human brain aging. Hum Brain Mapp 38:2751-2771, 2017. © 2017 Wiley Periodicals, Inc.


HIV-1 Envelope Glycoproteins from Diverse Clades Differentiate Antibody Responses and Durability among Vaccinees.
Induction of broadly cross-reactive antiviral humoral responses with the capacity to target globally diverse circulating strains is a key goal for HIV-1 immunogen design. A major gap in the field is the identification of diverse HIV-1 envelope antigens to evaluate vaccine regimens for binding antibody breadth. In this study, we define unique antigen panels to map HIV-1 vaccine-elicited antibody breadth and durability. Diverse HIV-1 envelope glycoproteins were selected based on genetic and geographic diversity to cover the global epidemic, with a focus on sexually acquired transmitted/founder viruses with a tier 2 neutralization phenotype. Unique antigenicity was determined by nonredundancy (Spearman correlation), and antigens were clustered using partitioning around medoids (PAM) to identify antigen diversity. Cross-validation demonstrated that the PAM method was better than selection by reactivity and random selection. Analysis of vaccine-elicited V1V2 binding antibody in longitudinal samples from the RV144 clinical trial revealed the striking heterogeneity among individual vaccinees in maintaining durable responses. These data support the idea that a major goal for vaccine development is to improve antibody levels, breadth, and durability at the population level. Elucidating the level and durability of vaccine-elicited binding antibody breadth needed for protection is critical for the development of a globally efficacious HIV vaccine.IMPORTANCE The path toward an efficacious HIV-1 vaccine will require characterization of vaccine-induced immunity that can recognize and target the highly genetically diverse virus envelope glycoproteins. Antibodies that target the envelope glycoproteins, including diverse sequences within the first and second hypervariable regions (V1V2) of gp120, were identified as correlates of risk for the one partially efficacious HIV-1 vaccine. To build upon this discovery, we experimentally and computationally evaluated humoral responses to define envelope glycoproteins representative of the antigenic diversity of HIV globally. These diverse envelope antigens distinguished binding antibody breadth and durability among vaccine candidates, thus providing insights for advancing the most promising HIV-1 vaccine candidates.


Invited review: decoding the microRNA response to hypoxia.
microRNAs (miRNAs) were discovered nearly two decades ago by researchers who sought to understand how basic developmental mechanisms work in the nematode Caenorhabditis elegans. Since the identification of conserved miRNA families in higher eukaryotes, there has been an explosion of interest into how these tiny RNA molecules function. miRNAs are 20-24 nucleotide non-coding RNA molecules that predominantly regulate transcripts of target genes through translational inhibition. Much recent interest has focused on the influence of miRNAs on homeostatic regulation, and in particular, hypoxic responses. The ability to sense and respond to hypoxia is of fundamental importance to aerobic organisms and dysregulated oxygen homeostasis is a hallmark in the pathophysiology of cancer, neurological dysfunction, myocardial infarction, and lung disease. miRNAs are ideal mediators of hypoxic stress responses as they are able to modify gene expression both rapidly and reversibly. This enables miRNA-mediated gene regulatory circuits to modify metabolic networks with immaculate precision and control. Therefore, one may consider miRNAs as molecular rheostats which effect tuning and switching of regulatory circuits to facilitate survival and adaptation to hypoxic conditions. Such miRNA-mediated regulatory circuits would provide flexible and conditional alternatives to "conventional" transcriptional regulation. Here, I review recent discoveries that have boosted our understanding of miRNA regulation of hypoxia and discuss where future breakthroughs in this area may be made.


Stability of adrenocortical steroidogenesis over time in healthy women and women with polycystic ovary syndrome.
Adrenocortical secretion is up-regulated in women with polycystic ovary syndrome (PCOS), and absolute adrenal androgen (AA) excess is evident in approximately 25% of these patients. We hypothesized that AA biosynthesis is an inherited trait and that, as for other inherited traits, AA biosynthesis remains stable over time. To test this hypothesis, we prospectively studied 23 off-treatment PCOS patients and seven age- and body mass index-matched control women on two separate occasions 3-5 yr apart (45.0 +/- 19.0 months and 47.4 +/- 21.3 months, respectively; P > 0.05). All subjects underwent an acute adrenal stimulation using 0.25 mg ACTH-(1-24), and dehydroepiandrosterone (DHEA), androstenedione, and cortisol (F) were measured 0 and 60 min post ACTH; basal levels of total and free testosterone (T), SHBG, and DHEA sulfate (DHEA-S) were also assessed. Among PCOS patients, the mean DHEA-S levels during the repeat study were significantly lower when compared with the initial assessment (170 +/- 107 microg/dl vs. 134 +/- 79 microg/dl, respectively; P = 0.02). However, only patients with initial DHEA-S levels above the median (high DHEA-S) experienced a net decrease in the levels of this metabolite (252.5 +/- 99.2 microg/dl vs. 174.3 +/- 82.5 microg/dl; P = 0.001) over the time of the study; patients with initial DHEA-S levels in the lower half (low DHEA-S) did not experience a change in DHEA-S (94.6 +/- 28.9 microg/dl vs. 97.7 +/- 56.5 microg/dl; P = 0.85). In patients, the total T levels tended to be higher at the second study, although SHBG levels were also higher, resulting in unchanged free T levels over time. Among controls, no significant changes in basal androgens were observed over the time of the study. There were no significant differences in either the basal or ACTH-stimulated levels of DHEA, androstenedione, or F over the time of the study in either PCOS or control women. We conclude that the adrenocortical secretion of AAs or F in PCOS and control women remains stable over time, supporting the hypothesis that the adrenal response to ACTH may be an inherited trait. Alternatively, a decrease in DHEA-S levels over time was observed but only among PCOS patients whose initial levels of this metabolite were above the group median, suggesting that the activity of sulfotransferase in these patients may be up-regulated by factors other than those affecting adrenocortical biosynthesis and that such regulatory influences attenuate over time.


Influence of dissolved sodium and cesium on uranyl oxide hydrate solubility.
The solubility of uranium-containing minerals can control the mobility of uranium in contaminated soil and groundwater. The identity and solubility of these minerals are strongly influenced by solution composition. The influence of dissolved sodium and cesium on the solubility of uranyl oxide hydrates has been investigated in a series of batch experiments conducted with synthetic metaschoepite ((UO2)8O2(OH)12 x 10H2O). During reaction of metaschoepite in NaNO3, CsNO3, and NaF solutions, an initial increase in the dissolved uranium concentration was followed by a decrease as uranium was incorporated into a secondary solid phase. Given sufficient reaction time, metaschoepite was completely transformed to a clarkeite-like sodium uranyl oxide hydrate or a cesium uranyl oxide hydrate that has not previously been described. These secondary solid phases exhibited X-ray diffraction patterns and Raman spectra that were distinct from those of the original metaschoepite. Dissolved uranium concentrations in equilibrium with the sodium and cesium uranyl oxide hydrates can be more than 2 orders of magnitude lower than those in equilibrium with metaschoepite. Initial changes in metaschoepite solubility may also result from particle growth induced by sodium and cesium incorporation into the solid phase.


Depth-enhanced three-dimensional-two-dimensional convertible display based on modified integral imaging.
A depth-enhanced three-dimensional-two-dimensional convertible display that uses a polymer-dispersed liquid crystal based on the principle of integral imaging is proposed. In the proposed method, a lens array is located behind a transmission-type display panel to form an array of point-light sources, and a polymer-dispersed liquid crystal is electrically controlled to pass or to scatter light coming from these point-light sources. Therefore, three-dimensional-two-dimensional conversion is accomplished electrically without any mechanical movement. Moreover, the nonimaging structure of the proposed method increases the expressible depth range considerably. We explain the method of operation and present experimental results.


Mab-3 is a direct tra-1 target gene regulating diverse aspects of C. elegans male sexual development and behavior.
Sex determination is controlled by global regulatory genes, such as tra-1 in Caenorhabditis elegans, Sex lethal in Drosophila, or Sry in mammals. How these genes coordinate sexual differentiation throughout the body is a key unanswered question. tra-1 encodes a zinc finger transcription factor, TRA-1A, that regulates, directly or indirectly, all genes required for sexual development. mab-3 (male abnormal 3), acts downstream of tra-1 and is known to be required for sexual differentiation of at least two tissues. mab-3 directly regulates yolk protein transcription in the intestine and specifies male sense organ differentiation in the nervous system. It encodes a transcription factor related to the products of the Drosophila sexual regulator doublesex (dsx), which also regulates yolk protein transcription and male sense-organ differentiation. The similarities between mab-3 and dsx led us to suggest that some aspects of sex determination may be evolutionarily conserved. Here we find that mab-3 is also required for expression of male-specific genes in sensory neurons of the head and tail and for male interaction with hermaphrodites. These roles in male development and behavior suggest further functional similarity to dsx. In male sensory ray differentiation we find that MAB-3 acts synergistically with LIN-32, a neurogenic bHLH transcription factor. Expression of LIN-32 is spatially restricted by the combined action of the Hox gene mab-5 and the hairy homolog lin-22, while MAB-3 is expressed throughout the lateral hypodermis. Finally, we find that mab-3 transcription is directly regulated in the intestine by TRA-1A, providing a molecular link between the global regulatory pathway and terminal sexual differentiation.


Genome-wide DNA methylation and gene expression analyses of monozygotic twins discordant for intelligence levels.
Human intelligence, as measured by intelligence quotient (IQ) tests, demonstrates one of the highest heritabilities among human quantitative traits. Nevertheless, studies to identify quantitative trait loci responsible for intelligence face challenges because of the small effect sizes of individual genes. Phenotypically discordant monozygotic (MZ) twins provide a feasible way to minimize the effects of irrelevant genetic and environmental factors, and should yield more interpretable results by finding epigenetic or gene expression differences between twins. Here we conducted array-based genome-wide DNA methylation and gene expression analyses using 17 pairs of healthy MZ twins discordant intelligently. ARHGAP18, related to Rho GTPase, was identified in pair-wise methylation status analysis and validated via direct bisulfite sequencing and quantitative RT-PCR. To perform expression profile analysis, gene set enrichment analysis (GSEA) between the groups of twins with higher IQ and their co-twins revealed up-regulated expression of several ribosome-related genes and DNA replication-related genes in the group with higher IQ. To focus more on individual pairs, we conducted pair-wise GSEA and leading edge analysis, which indicated up-regulated expression of several ion channel-related genes in twins with lower IQ. Our findings implied that these groups of genes may be related to IQ and should shed light on the mechanism underlying human intelligence.


Comparison of admission random glucose, fasting glucose, and glycated hemoglobin in predicting the neurological outcome of acute ischemic stroke: a retrospective study.
Hyperglycemia is a known predictor of negative outcomes in stroke. Several glycemic measures, including admission random glucose, fasting glucose, and glycated hemoglobin (HbA1c), have been associated with bad neurological outcomes in acute ischemic stroke, particularly in nondiabetic patients. However, the predictive power of these glycemic measures is yet to be investigated. This retrospective study enrolled 484 patients with acute ischemic stroke from January 2009 to March 2013, and complete records of initial stroke severity, neurological outcomes at three months, and glycemic measures were evaluated. We examined the predictive power of admission random glucose, fasting glucose, and HbA1c for neurological outcomes in acute ischemic stroke. Furthermore, subgroup analyses of nondiabetic patients and patients with diabetes were performed separately. Receiver operating characteristic (ROC) analysis revealed that admission random glucose and fasting glucose were significant predictors of poor neurological outcomes, whereas HbA1c was not (areas under the ROC curve (AUCs): admission random glucose = 0.564, p = 0.026; fasting glucose = 0.598, p = 0.001; HbA1c = 0.510, p = 0.742). Subgroup analyses of nondiabetic patients and those with diabetes revealed that only fasting glucose predicts neurological outcomes in patients with diabetes, and the AUCs of these three glycemic measures did not differ between the two groups. A multivariate logistic regression analysis of the study patients indicated that only age, initial stroke severity, and fasting glucose were independent predictors of poor neurological outcomes, whereas admission random glucose and HbA1c were not (adjusted odds ratio: admission random glucose = 1.002, p = 0.228; fasting glucose = 1.005, p = 0.039; HbA1c = 1.160, p = 0.076). Furthermore, subgroup multivariate logistic regression analyses of nondiabetic patients and those with diabetes indicated that none of the three glycemic measures were associated with poor neurological outcomes. Fasting glucose is an independent predictor of poor neurological outcomes in patients with acute ischemic stroke and had greater predictive power than that of admission random glucose and HbA1c. The predictive power of glycemic measures for poor neurological outcomes did not differ significantly between the nondiabetic patients and those with diabetes.


Functional neuroanatomy of grief: an FMRI study.
In this study the authors examined the functional neuroanatomy of grief, which to their knowledge has not been studied previously in functional neuroimaging research. Grief was elicited in eight bereaved women through photographs of the deceased versus a stranger, combined with words specific to the death event versus neutral words. Use of both pictures and words resulted in a 2x2 factorial design. Three brain regions were independently activated by the picture and word factors: posterior cingulate cortex, medial/superior frontal gyrus, and cerebellum. The two factors also activated distinct regions: for the picture factor, they were the cuneus, superior lingual gyrus, insula, dorsal anterior cingulate cortex, inferior temporal gyrus, and fusiform gyrus; and for the word factor, they were the precuneus, precentral gyrus, midbrain, and vermis. The interaction of the two factors showed significant activation in the cerebellar vermis. Grief is mediated by a distributed neural network that subserves affect processing, mentalizing, episodic memory retrieval, processing of familiar faces, visual imagery, autonomic regulation, and modulation/coordination of these functions. This neural network may account for the unique, subjective quality of grief and provide new leads in understanding the health consequences of grief and the neurobiology of attachment.


Identification of an immunoreactive Brucella abortus HtrA stress response protein homolog.
An 11-kb fragment of Brucella abortus genomic DNA cloned into the BamHI site of pUC9 expressed a 60-kDa protein in Escherichia coli DH5-alpha. Antibodies reactive with this 60-kDa protein were detected by Western blot (immunoblot) analysis in sera from mice, cattle, and goats experimentally infected with B. abortus, in sera from mice experimentally infected with Brucella melitensis, and in serum from a dog experimentally infected with Brucella canis. Similar results were seen with sera obtained from cattle and dogs with naturally acquired brucellosis. The gene encoding the 60-kDa Brucella protein was localized to a 2-kb EcoRI fragment which was also reactive in Southern blots with genomic DNA from other strains of B. abortus as well as with genomic DNA from B. melitensis and B. canis. Nucleotide sequence analysis of the cloned EcoRI fragment revealed an open reading frame encoding a protein with a predicted molecular mass of 51,847 Da and an isoelectric point of 5.15. Comparison of the deduced amino acid sequence of the immunoreactive Brucella protein with the SWISS-PROT protein sequence data base revealed that it shares > 40% amino acid sequence identity with the E. coli and Salmonella typhimurium HtrA stress response proteins. Computer-assisted analysis of this amino acid sequence also predicted that the putative Brucella HtrA homolog contains an export signal sequence and a serine protease active site, two structural features characteristic of previously described HtrA proteins. A potential sigma E type heat shock promoter sequence was detected upstream of the cloned Brucella htrA gene, and Northern (RNA) blot analysis demonstrated that exposure of B. abortus 2308 to heat shock conditions resulted in a transient elevation of htrA transcription. These results strongly suggest that the immunoreactive 60-kDa Brucella protein is a member of the HtrA class of stress response proteins.


Functional testing of an inhalable nanoparticle based influenza vaccine using a human precision cut lung slice technique.
Annual outbreaks of influenza infections, caused by new influenza virus subtypes and high incidences of zoonosis, make seasonal influenza one of the most unpredictable and serious health threats worldwide. Currently available vaccines, though the main prevention strategy, can neither efficiently be adapted to new circulating virus subtypes nor provide high amounts to meet the global demand fast enough. New influenza vaccines quickly adapted to current virus strains are needed. In the present study we investigated the local toxicity and capacity of a new inhalable influenza vaccine to induce an antigen-specific recall response at the site of virus entry in human precision-cut lung slices (PCLS). This new vaccine combines recombinant H1N1 influenza hemagglutinin (HAC1), produced in tobacco plants, and a silica nanoparticle (NP)-based drug delivery system. We found no local cellular toxicity of the vaccine within applicable concentrations. However higher concentrations of NP (≥10(3) µg/ml) dose-dependently decreased viability of human PCLS. Furthermore NP, not the protein, provoked a dose-dependent induction of TNF-α and IL-1β, indicating adjuvant properties of silica. In contrast, we found an antigen-specific induction of the T cell proliferation and differentiation cytokine, IL-2, compared to baseline level (152±49 pg/mg vs. 22±5 pg/mg), which could not be seen for the NP alone. Additionally, treatment with 10 µg/ml HAC1 caused a 6-times higher secretion of IFN-γ compared to baseline (602±307 pg/mg vs. 97±51 pg/mg). This antigen-induced IFN-γ secretion was further boosted by the adjuvant effect of silica NP for the formulated vaccine to a 12-fold increase (97±51 pg/mg vs. 1226±535 pg/mg). Thus we were able to show that the plant-produced vaccine induced an adequate innate immune response and re-activated an established antigen-specific T cell response within a non-toxic range in human PCLS at the site of virus entry.


Brain Information Sharing During Visual Short-Term Memory Binding Yields a Memory Biomarker for Familial Alzheimer's Disease.
Alzheimer's disease (AD) as a disconnection syndrome which disrupts both brain information sharing and memory binding functions. The extent to which these two phenotypic expressions share pathophysiological mechanisms remains unknown. To unveil the electrophysiological correlates of integrative memory impairments in AD towards new memory biomarkers for its prodromal stages. Patients with 100% risk of familial AD (FAD) and healthy controls underwent assessment with the Visual Short-Term Memory binding test (VSTMBT) while we recorded their EEG. We applied a novel brain connectivity method (Weighted Symbolic Mutual Information) to EEG data. Patients showed significant deficits during the VSTMBT. A reduction of brain connectivity was observed during resting as well as during correct VSTM binding, particularly over frontal and posterior regions. An increase of connectivity was found during VSTM binding performance over central regions. While decreased connectivity was found in cases in more advanced stages of FAD, increased brain connectivity appeared in cases in earlier stages. Such altered patterns of task-related connectivity were found in 89% of the assessed patients. VSTM binding in the prodromal stages of FAD are associated to altered patterns of brain connectivity thus confirming the link between integrative memory deficits and impaired brain information sharing in prodromal FAD. While significant loss of brain connectivity seems to be a feature of the advanced stages of FAD increased brain connectivity characterizes its earlier stages. These findings are discussed in the light of recent proposals about the earliest pathophysiological mechanisms of AD and their clinical expression.


The influence of sex chromosome aneuploidy on brain asymmetry.
The cognitive deficits present in individuals with sex chromosome aneuploidies suggest that hemispheric differentiation of function is determined by an X-Y homologous gene [Crow (1993); Lancet 342:594-598]. In particular, females with Turner's syndrome (TS) who have only one X-chromosome exhibit deficits of spatial ability whereas males with Klinefelter's syndrome (KS) who possess a supernumerary X-chromosome are delayed in acquiring words. Since spatial and verbal abilities are generally associated with right and left hemispheric function, such deficits may relate to anomalies of cerebral asymmetry. We therefore applied a novel image analysis technique to investigate the relationship between sex chromosome dosage and structural brain asymmetry. Specifically, we tested Crow's prediction that the magnitude of the brain torque (i.e., a combination of rightward frontal and leftward occipital asymmetry) would, as a function of sex chromosome dosage, be respectively decreased in TS women and increased in KS men, relative to genotypically normal controls. We found that brain torque was not significantly different in TS women and KS men, in comparison to controls. However, TS women exhibited significantly increased leftward brain asymmetry, restricted to the posterior of the brain and focused on the superior temporal and parietal-occipital association cortex, while KS men showed a trend for decreased brain asymmetry throughout the frontal lobes. The findings suggest that the number of sex chromosomes influences the development of brain asymmetry not simply to modify the torque but in a complex pattern along the antero-posterior axis.


Urban behavioural adaptation.
A large and growing proportion of the world is impacted directly by human activities; among the most extreme of these is the spread of urban environments. Environmental change associated with urbanization represents a potentially potent source of selection. While urban environments generally have lowered biodiversity, some clades seem to thrive in urban settings. For example, many members of the bird family Turdidae, known as the 'truethrushes' and the blackbird Turdus merula (Fig. 1) in particular, are familiar urban species. Indeed, the colonization of urban environments by blackbirds has become a textbook case study for our understanding of the many ways a wild species can deal with urbanization. In this issue, Mueller et al. (Molecular Ecology, 00, 2013, 00) add to that story by beginning to address the genetic nature of behavioural adaptation of blackbirds colonizing urban areas. They do this by testing for divergence between paired urban and rural samples at a suite of candidate genes with hypothesized effects on behaviours thought to be important for the colonization of urban environments.They find evidence for consistent patterns of divergence at an exonic microsatellite associated with the SERT gene. SERT has a number of hypothesized behavioural effects, including harm avoidance, which may be associated with tolerating the hustle and bustle of urban environments. This is among the first evidence that behavioural differences between urban and rural environments have a genetic basis and this work suggests that urban environments can in some cases exert homogeneous selection pressures.


Shigella effector IpaB-induced cholesterol relocation disrupts the Golgi complex and recycling network to inhibit host cell secretion.
Shigella infection causes destruction of the human colonic epithelial barrier. The Golgi network and recycling endosomes are essential for maintaining epithelial barrier function. Here we show that Shigella epithelial invasion induces fragmentation of the Golgi complex with consequent inhibition of both secretion and retrograde transport in the infected host cell. Shigella induces tubulation of the Rab11-positive compartment, thereby affecting cell surface receptor recycling. The molecular process underlying the observed damage to the Golgi complex and receptor recycling is a massive redistribution of plasma membrane cholesterol to the sites of Shigella entry. IpaB, a virulence factor of Shigella that is known to bind cholesterol, is necessary and sufficient to induce Golgi fragmentation and reorganization of the recycling compartment. Shigella infection-induced Golgi disorganization was also observed in vivo, suggesting that this mechanism affecting the sorting of cell surface molecules likely contributes to host epithelial barrier disruption associated with Shigella pathogenesis.


Staurosporine inhibits invasion of erythrocytes by malarial merozoites.
Staurosporine, a protein kinase inhibitor, inhibits the invasion of rhesus by Plasmodium knowlesi merozoites with an IC50 of 250 nM. The drug exerts its effects primarily on the merozoite, with little or no effect on the erythrocyte. Okadaic acid, an inhibitor of protein phosphatases, can partially abrogate the inhibitory effects of staurosporine. Staurosporine arrests invasion at a step which is ultrastructurally similar to the arrest caused by cytochalasins B and D: the merozoite attaches, apically reorients, and forms a junction with the erythrocyte, but it does not internalize. These results suggest that protein phosphorylation within the merozoite plays an important role in the internalization step of invasion.


Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle.
Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×10(10) -1 × 10(11) particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 10(9) AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.Molecular Therapy - Nucleic Acids (2013) 2, e86; doi:10.1038/mtna.2013.16; published online 16 April 2013.


Infections and coinfections of questing Ixodes ricinus ticks by emerging zoonotic pathogens in Western Switzerland.
In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and "Candidatus Neoehrlichia mikurensis" were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia- and A. phagocytophilum-infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii, Borrelia lusitaniae, and Rickettsia monacensis, were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica, were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.