Expression patterns of chicken Toll-like receptor mRNA in tissues, immune cell subsets and cell lines.
The Toll-like receptor (TLR) family of cell surface molecules represent a major component of the pattern recognition system, which enables both vertebrates and invertebrates to detect invading microorganisms and mount an anti-microbial response. The TLR repertoire of mouse and man has been intensively studied and in this manuscript we report the identification of ESTs with homology to chTLR5 and chTLR7, and independently confirm the identification of chTLR 1/6/10 and 3 in the EST databases. We have determined the mRNA expression patterns for seven chicken TLRs (chTLR) in a wide range of chicken tissues, isolated immune cell types and cultured cells. Some of the chTLR were expressed in most tissues (chTLR1/6/10, chTLR3, chTLR4 and chTLR5), whereas others exhibited more restricted expression patterns (chTLR2 type 1, type 2 and chTLR7). Similarly distinct patterns of chTLR expression were seen with innate and adaptive immune cell types isolated from peripheral blood or spleen and with cultured cells of somatic or immunological origin. An understanding of the TLR repertoire for different tissues, immune cell subsets and cultured cell types allows more refined interpretation of immune induction in response to chicken pathogens.


The role of recessive inheritance in early-onset epileptic encephalopathies: a combined whole-exome sequencing and copy number study.
Early-onset epileptic encephalopathy (EE) and combined developmental and epileptic encephalopathies (DEE) are clinically and genetically heterogeneous severely devastating conditions. Recent studies emphasized de novo variants as major underlying cause suggesting a generally low-recurrence risk. In order to better understand the full genetic landscape of EE and DEE, we performed high-resolution chromosomal microarray analysis in combination with whole-exome sequencing in 63 deeply phenotyped independent patients. After bioinformatic filtering for rare variants, diagnostic yield was improved for recessive disorders by manual data curation as well as molecular modeling of missense variants and untargeted plasma-metabolomics in selected patients. In total, we yielded a diagnosis in ∼42% of cases with causative copy number variants in 6 patients (∼10%) and causative sequence variants in 16 established disease genes in 20 patients (∼32%), including compound heterozygosity for causative sequence and copy number variants in one patient. In total, 38% of diagnosed cases were caused by recessive genes, of which two cases escaped automatic calling due to one allele occurring de novo. Notably, we found the recessive gene SPATA5 causative in as much as 3% of our cohort, indicating that it may have been underdiagnosed in previous studies. We further support candidacy for neurodevelopmental disorders of four previously described genes (PIK3AP1, GTF3C3, UFC1, and WRAP53), three of which also followed a recessive inheritance pattern. Our results therefore confirm the importance of de novo causative gene variants in EE/DEE, but additionally illustrate the major role of mostly compound heterozygous or hemizygous recessive inheritance and consequently high-recurrence risk.


Suppressor of Hairless is required for signal reception during lateral inhibition in the Drosophila pupal notum.
Suppressor of Hairless (Su(H)) activity is zygotically required in larval imaginal discs for the singling out of adult sense organ precursor (SOP) cells: loss of Su(H) function results in too many proneural cluster cells adopting the SOP fate, while overexpression of the Su(H) protein prevents SOP specification. Su(H) null mutant alleles are recessive lethal at the late larval and early pupal stages. The development of Su(H) mutant cells in pupae was therefore studied in somatic clones. Clonal analysis first showed that Su(H) is required for the regular spacing of microchaete precursor cells, as clusters of mutant SOPs were detected at positions where singled out sense organ cells are normally found. Second, Su(H) mutant SOPs produced neuron-like cells, consistent with a late defect in Notch (N) signalling. Third, a careful cell-by-cell analysis of clone borders showed that Su(H) mutant cells may adopt the SOP fate even when directly adjacent to wild-type cells. Finally, quantitative clone border analysis indicates that the relative level of Su(H) gene dosage appears to bias the selection of the future SOP: cells with a higher level of Su(H) activity are more likely to adopt the epidermal fate. These results show that notum cells strictly require Su(H) activity for receiving the lateral inhibitory signal. Thus, the DNA-binding protein encoded by the Su(H) gene may act downstream of the N receptor to implement the epidermal, non-SOP fate.


Ultra-Widefield Imaging for Pediatric Retinal Disease.
This article explores the development of retinal imaging, with particular emphasis on ultra-widefield imaging and the key concept of field of view. Two ultra-widefield imaging platforms are examined in detail-Optomap and Spectralis-noncontact imaging systems that include protocols for performing angiography in infants. Applications of ultra-widefield imaging are illustrated using case studies, including diagnosis, monitoring, and screening.


Determinants of infant feeding method in relation to risk factors for breast cancer.
Breastfeeding is considered to be an important factor for maternal and children's health. However, the epidemiological findings related to the effect of breastfeeding on women's health, especially with respect to breast cancer development, are inconsistent. Determinants of infant feeding method may contribute to the inconsistency. A total of 24,769 women aged 40-64 in Miyagi Prefecture, Japan, responded to a self-administered questionnaire survey in 1990. Using the data obtained from 22,085 parous women, we calculated odds ratios (ORs) for the choice of "breastfeeding only" during reproductive period. Late age at menarche (> or = 16 years, OR = 1.57) and high body mass index (BMI) at 20 years of age (> or = 24, OR = 1.31) were associated with the choice of breastfeeding only. Late age at birth of first child (> or = 28 years, OR = 0.29), history of breast cancer in mother (OR = 0.68), and high educational level (more than a high school education, OR = 0.53) reduced the possibility of choosing breastfeeding only. The results indicate that the choice of infant feeding method is associated with several breast cancer risk factors. Based on this finding, we should construct appropriate breast cancer risk models for parous women and investigate the changes in the effects of breastfeeding and other breast cancer risk factors among these risk models. Especially in a risk model controlling for breastfeeding, the effects of other breast cancer risk factors should be reevaluated. Through comparisons among different risk models, we may find the best-fitted risk model and identify the true effect of breastfeeding.


Aldose reductase and AGE-RAGE pathways: central roles in the pathogenesis of vascular dysfunction in aging rats.
Aging is inevitably accompanied by gradual and irreversible innate endothelial dysfunction. In this study, we tested the hypothesis that accentuation of glucose metabolism via the aldose reductase (AR) pathway contributes to age-related vascular dysfunction. AR protein and activity levels were significantly increased in aged vs. young aortic homogenates from Fischer 344 rats. Immunostaining revealed that the principal site of increased AR protein was the aortic endothelium as well as smooth muscle cells. Studies revealed that endothelial-dependent relaxation (EDR) in response to acetylcholine was impaired in aged rats compared to young rats and that treatment with the AR inhibitor (ARI) zopolrestat significantly improved EDR in aged rats. Methylglyoxal (MG), a key precursor of advanced glycation endproducts (AGEs), was significantly increased in the aortas of aged rats vs. young rats. Consistent with central roles for AR in generation of MG in aging, ARI treatment significantly reduced MG levels in aged rat aorta to those in young rats. Treatment of aged rats with soluble(s) RAGE, a soluble form of the chief signal transduction receptor for AGEs, RAGE, significantly improved EDR in aged rats, thus establishing the contribution of age-related increases in AGEs to endothelial dysfunction. These findings reveal that significant increases in AR expression and activity in aged rat vasculature linked to endothelial dysfunction may be mitigated, at least in part, via ARI and that aging-linked increased flux via AR generates AGEs; species which transduce endothelial injury consequent to their interaction with RAGE. These data demonstrate for the first time that AR mediates aging-related vascular dysfunction, at least in part, via RAGE.


Alterations in AdeS and AdeR regulatory proteins in 1-(1-naphthylmethyl)-piperazine responsive colistin resistance of Acinetobacter baumannii.
Colistin resistant Acinetobacter baumannii strains are of great concern worldwide. However, the role of efflux pumps in colistin resistance needs to be elucidated. We investigated the changes in colistin MICs of 29 colistin resistant A. baumannii isolates in response to resistance-nodulation-division (RND)-type efflux pump inhibitor (EPI) and the alterations in AdeR and AdeS two-component regulatory proteins previously associated with the overproduction of AdeAB. The EPI, 1-(1-naphthylmethyl)-piperazine (NMP), led to significant reductions in colistin MICs. At least one of the following amino acid substitutions was found in AdeS proteins from 18 of the isolates: L172P, A94V, V27I, V32I, G186V, and G164A. Besides, A136V and V120I alterations were identified in AdeR from five isolates. Therefore, EPI-responsive colistin resistance in our isolates is most likely due to the action of an RND-type efflux system. The underlying mechanism of resistance might be the result of certain AdeRS alterations, leading to AdeAB overexpression.


Gene expression profiling in human skeletal muscle during recovery from eccentric exercise.
We used cDNA microarrays to screen for differentially expressed genes during recovery from exercise-induced muscle damage in humans. Male subjects (n = 4) performed 300 maximal eccentric contractions, and skeletal muscle biopsy samples were analyzed at 3 h and 48 h after exercise. In total, 113 genes increased 3 h postexercise, and 34 decreased. At 48 h postexercise, 59 genes increased and 29 decreased. On the basis of these data, we chose 19 gene changes and conducted secondary analyses using real-time RT-PCR from muscle biopsy samples taken from 11 additional subjects who performed an identical bout of exercise. Real-time RT-PCR analyses confirmed that exercise-induced muscle damage led to a rapid (3 h) increase in sterol response element binding protein 2 (SREBP-2), followed by a delayed (48 h) increase in the SREBP-2 gene targets Acyl CoA:cholesterol acyltransferase (ACAT)-2 and insulin-induced gene 1 (insig-1). The expression of the IL-1 receptor, a known regulator of SREBP-2, was also elevated after exercise. Taken together, these expression changes suggest a transcriptional program for increasing cholesterol and lipid synthesis and/or modification. Additionally, damaging exercise induced the expression of protein kinase H11, capping protein Z alpha (capZalpha), and modulatory calcineurin-interacting protein 1 (MCIP1), as well as cardiac ankryin repeat protein 1 (CARP1), DNAJB2, c-myc, and junD, each of which are likely involved in skeletal muscle growth, remodeling, and stress management. In summary, using DNA microarrays and RT-PCR, we have identified novel genes that respond to skeletal muscle damage, which, given the known biological functions, are likely involved in recovery from and/or adaptation to damaging exercise.


Activated proliferation of B-cell lymphomas/leukemias with the SHP1 gene silencing by aberrant CpG methylation.
Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2'-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes.


Language, social behavior, and the quality of friendships in adolescents with and without a history of specific language impairment.
Language is drawn on extensively in friendships but has received scant attention in the developmental literature. This study compared friendship quality in 16-year-old adolescents with and without specific language impairment (SLI), testing the extent it is predicted by individual differences in social behaviors and language ability. Participants were 120 adolescents with SLI and 118 typically developing (TD) adolescents. After considering the effects of nonverbal IQ and prosocial and difficult behavior, language measures were found to be associated with friendship quality. The TD participants enjoyed normal friendships, whereas the participants with SLI were more likely to exhibit poorer quality (although 60% experienced good quality of friendships). Longitudinal analyses identified early language difficulties as predictive of poorer friendship quality in adolescence.


Noise-based volume rendering for the visualization of multivariate volumetric data.
Analysis of multivariate data is of great importance in many scientific disciplines. However, visualization of 3D spatially-fixed multivariate volumetric data is a very challenging task. In this paper we present a method that allows simultaneous real-time visualization of multivariate data. We redistribute the opacity within a voxel to improve the readability of the color defined by a regular transfer function, and to maintain the see-through capabilities of volume rendering. We use predictable procedural noise--random-phase Gabor noise--to generate a high-frequency redistribution pattern and construct an opacity mapping function, which allows to partition the available space among the displayed data attributes. This mapping function is appropriately filtered to avoid aliasing, while maintaining transparent regions. We show the usefulness of our approach on various data sets and with different example applications. Furthermore, we evaluate our method by comparing it to other visualization techniques in a controlled user study. Overall, the results of our study indicate that users are much more accurate in determining exact data values with our novel 3D volume visualization method. Significantly lower error rates for reading data values and high subjective ranking of our method imply that it has a high chance of being adopted for the purpose of visualization of multivariate 3D data.


KatG-Mediated Oxidation Leading to Reduced Susceptibility of Bacteria to Kanamycin.
Resistance to antibiotics has become a serious problem for society, and there are increasing efforts to understand the reasons for and sources of resistance. Bacterial-encoded enzymes and transport systems, both innate and acquired, are the most frequent culprits for the development of resistance, although in Mycobacterium tuberculosis, the catalase-peroxidase, KatG, has been linked to the activation of the antitubercular drug isoniazid. While investigating a possible link between aminoglycoside antibiotics and the induction of oxidative bursts, we observed that KatG reduces susceptibility to aminoglycosides. Investigation revealed that kanamycin served as an electron donor for the peroxidase reaction, reducing the oxidized ferryl intermediates of KatG to the resting state. Loss of electrons from kanamycin was accompanied by the addition of a single oxygen atom to the aminoglycoside. The oxidized form of kanamycin proved to be less effective as an antibiotic. Kanamycin inhibited the crystallization of KatG, but the smaller, structurally related glycoside maltose did cocrystallize with KatG, providing a suggestion as to the possible binding site of kanamycin.


Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells.
A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications.


Parathyroid hormone-like biological activity in urine.
1. The parathyroid hormone-like biological activity of concentrated urine was measured by the increase of plasma calcium concentration after intravenous injection of the sample into chickens. 2. Urine was tested in hypoparathyroid patients, normal volunteer subjects, primary hyperparathyroid patients before and after surgery and patients with secondary hyperparathyroidism. 3. In primary and secondary hyperparathyroidism the biological activity was significantly higher than in urine from normal subjects, which was in turn significantly higher than the activity in the urine of hypoparathyroid patients. This bioactivity diminished after surgical removal of a hyperparathyroid adenoma. 4. Decreased activity after trypsinization indicated the peptidic nature of the hypercalcaemic substance.


An intelligent controller for automated operation of sequencing batch reactors.
In this paper the results are presented of original research into the automatic and "intelligent" detection of breakpoints in Dissolved Oxygen (DO) profiles. The research has been based on a large body of data collected from laboratory SBRs operating on synthetic wastewater. Two different approaches were followed to identify the endpoints. The paper analyses and evaluates the results of automatic detection on the basis of geometric features in the DO profiles. This was followed by classification of the detected breakpoints using different soft computing techniques based on Neural Network (NN), Fuzzy Neural Network (FuNN) and Evolving Fuzzy Neural Network (EfuNN) software systems for breakpoint classification. A high rate of successful detection and classification was obtained with up to 96% of the decisions made correctly. In order to overcome the limitations of this system to adapt to dynamically changing process conditions, an intelligent control model was developed by a combination between an Evolving Fuzzy Neural Net (EfuNN) combined with a logic decision unit. This system has the ability to "learn on-the-fly" and adjust its response pattern in order to maintain a high rate of successful breakpoint detection under varying changing process conditions. This software system has been sucessfully embedded on a small programmable controller for integration into larger process control systems for the operation of SBR plants.


Olive oil consumption, plasma oleic acid, and stroke incidence: the Three-City Study.
To determine whether high olive oil consumption, and high plasma oleic acid as an indirect biological marker of olive oil intake, are associated with lower incidence of stroke in older subjects. Among participants from the Three-City Study with no history of stroke at baseline, we examined the association between olive oil consumption (main sample, n = 7,625) or plasma oleic acid (secondary sample, n = 1,245) and incidence of stroke (median follow-up 5.25 years), ascertained according to a diagnosis validated by an expert committee. In the main sample, 148 incident strokes occurred. After adjustment for sociodemographic and dietary variables, physical activity, body mass index, and risk factors for stroke, a lower incidence for stroke with higher olive oil use was observed (p for trend = 0.02). Compared to those who never used olive oil, those with intensive use had a 41%(95% confidence interval 6%-63%, p = 0.03) lower risk of stroke. In the secondary sample, 27 incident strokes occurred. After full adjustment, higher plasma oleic acid was associated with lower stroke incidence (p for trend = 0.03). Compared to those in the first tertile, participants in the third tertile of plasma oleic acid had a 73% (95% confidence interval 10%-92%, p = 0.03) reduction of stroke risk. These results suggest a protective role for high olive oil consumption on the risk of stroke in older subjects.


Intraoperative Consultation and Macroscopic Handling: The International Society of Urological Pathology (ISUP) Testicular Cancer Consultation Conference Recommendations.
The International Society of Urological Pathology held a conference on issues in testicular and penile pathology in Boston in March 2015, which included a presentation by the testis macroscopic features working group. The presentation focused on current published guidance for macroscopic handling of testicular tumors and retroperitoneal lymph node dissections with a summary of results from an online survey of members preceding the conference. The survey results were used to initiate discussions, but decisions on practice were made by expert consensus rather than voting. The importance of comprehensive assessment at the time of gross dissection with confirmation of findings by microscopic assessment was underscored. For example, the anatomic landmarks denoting the distinction of hilar soft tissue invasion (pT2) from spermatic cord invasion (pT3 category) can only be determined by careful macroscopic assessment in many cases. Other recommendations were to routinely sample epididymis, rete testis, hilar soft tissue, and tunica vaginalis in order to confirm macroscopic invasion of these structures or if not macroscopically evident, to exclude subtle microscopic invasion. Tumors 2 cm or less in greatest dimension should be completely embedded. If the tumor is >2 cm in greatest dimension, 10 blocks or a minimum of 1 to 2 additional blocks per centimeter should be taken (whichever is greater).


Roles and responsibilities of the interpreter in interactions with speech-language pathologists, parents, and students.
The Individuals with Disabilities Education Act Amendments of 1997 (IDEA '97) specify that students must be assessed in their primary language. With the ever-increasing number of diverse languages spoken by students, it is very unlikely that even an English-bilingual speech-language pathologist's other language will match that of a student who is referred for a speech-language assessment. Knowing how to work effectively with an interpreter is, therefore, essential for all speech-language pathologists. This article reviews the process of interpretation during parent conferences and student assessments. It describes the specific roles and responsibilities of the speech-language pathologist-interpreter team, with a focus on the linguistic, cultural, and professional skills that the interpreter should have to ensure a successful outcome. Data obtained from bilingual Spanish/English-speaking speech-language pathologists are provided to confirm and describe some important features desired in an interpreter who collaborates with a speech-language pathologist. Suggestions for training and certifying future interpreters are outlined. Finally, models are presented for successful interactions with interpreters.


Risk of suicide in households threatened with eviction: the role of banks and social support.
One of the greatest effects of the financial crisis in Spain has been the enormous increase in the number of evictions. Several studies have shown the association of evictions with different aspects of the physical and mental health. Furthermore, evictions have been associated with an increased risk of suicide. Our objective was to evaluate the risk of suicide among victims of eviction and investigate whether it is associated with specific characteristics of households and interviewees, the eviction process and social support, and health needs. A total of 205 participants from households threatened with eviction in Granada, Spain, and 673 being the total number of members of these households, were interviewed in one-on-one sessions between April 2013 and May 2014. Through a questionnaire, information was obtained on physical and mental health, characteristics of their eviction process and support networks, and the use of health services. Almost half of the sample (46.7%) were at low (11.8%), moderate (16.9%), or high suicide risk (17.9%). Household and interviewee features had a limited association with suicide risk. On the contrary, the risk of suicide is greater with a longer exposure to the eviction process. In addition, threatening phone calls from banks increased significantly the risk of suicide, especially among men. Suicide risk was also associated with low social support, especially among women. Interviewees at risk of suicide received more help from nongovernmental organizations than those who were not at risk. In interviewees at risk, the main unmet needs were emotional and psychological help, especially in men. A high percentage of those at risk of suicide declare having large unmeet health needs. Finally, there was a tendency among the evicted at risk of suicide to visit emergency room and primary care more often than those not at risk, especially among women. To our knowledge, this is the first study showing that when banks adopt a threatening attitude, suicide risk increases among the evicted. As hypothesized, when the evicted felt socially supported, suicide risk decreased. Emotional help was the main mediator of suicide risk and the main unmet need, especially among men.


A systematic performance analysis of the simultaneous algebraic reconstruction technique (SART) for limited angle tomography.
The design of limited angle tomography systems requires the optimization of various imaging parameters in order to achieve useful as well as reliable results. Algebraic reconstruction techniques, specifically the SART algorithm, have given excellent results in CT and are being actively considered for limited angle commercial applications such as tomosynthesis. In this study, we simulate a range of limited angle scenarios by systematically varying a number of key imaging parameters, and examine the performance of the SART algorithm under these variations. The phantoms used are basic ellipsoids in 2D, yielding analytical projections, and an MR-derived breast phantom in 3D.


Antigen Targeting to Human HLA Class II Molecules Increases Efficacy of DNA Vaccination.
It has been difficult to translate promising results from DNA vaccination in mice to larger animals and humans. Previously, DNA vaccines encoding proteins that target Ag to MHC class II (MHC-II) molecules on APCs have been shown to induce rapid, enhanced, and long-lasting Ag-specific Ab titers in mice. In this study, we describe two novel DNA vaccines that as proteins target HLA class II (HLA-II) molecules. These vaccine proteins cross-react with MHC-II molecules in several species of larger mammals. When tested in ferrets and pigs, a single DNA delivery with low doses of the HLA-II-targeted vaccines resulted in rapid and increased Ab responses. Importantly, painless intradermal jet delivery of DNA was as effective as delivery by needle injection followed by electroporation. As an indication that the vaccines could also be useful for human application, HLA-II-targeted vaccine proteins were found to increase human CD4+ T cell responses by a factor of ×103 in vitro. Thus, targeting of Ag to MHC-II molecules may represent an attractive strategy for increasing efficacy of DNA vaccines in larger animals and humans.


Local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen improves western blot sensitivity.
This study evaluates the use of local Borrelia burgdorferi sensu stricto and Borrelia afzelii strains in a single mixed antigen for in-house IgG western blots in the routine diagnostic setting by comparing it with the current in-house protocol. Sera from 233 patients from areas of Scotland with low and high prevalence for Lyme borreliosis were tested by western blots prepared from reference strain antigen (B burgdorferi sensu stricto) and mixed antigen (B burgdorferi sensu stricto and B afzelii). Results were scored using original and revised criteria, and results were compared. The mixed antigen produced significantly more bands than the reference antigen. Using the original interpretation criteria the mixed antigen produced more positive results than the reference antigen (90 versus 85). When the revised criteria were applied to the mixed antigen there were 14 more patients with positive results than with the reference antigen (99 versus 85); this difference was significant. Although 22 patients were positive with the mixed antigen and revised criteria, but negative/equivocal with the reference antigen, eight patients who were positive with reference antigen remained negative with the mixed antigen. The positive predictive value of the two antigen preparations was the same (96%). The negative predictive value of the mixed antigen with revised criteria was higher than the reference antigen (96% versus 88%), but the specificity was similar (97% versus 98%). The mixed antigen and revised interpretation criteria have been successfully incorporated into the routine diagnostic testing service, increasing the sensitivity of the in-house IgG western blot test for Scottish patients.


The I148M PNPLA3 polymorphism influences serum adiponectin in patients with fatty liver and healthy controls.
Reduced adiponectin is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH), and the I148M Patatin-like phospholipase domain-containing 3 (PNPLA3) polymorphism predisposes to NAFLD and liver damage progression in NASH and chronic hepatitis C (CHC) by still undefined mechanisms, possibly involving regulation of adipose tissue function. Aim of this study was to evaluate whether the I148M PNPLA3 polymorphism influences serum adiponectin in liver diseases and healthy controls. To this end, we considered 144 consecutive Italian patients with NAFLD, 261 with CHC, 35 severely obese subjects, and 257 healthy controls with very low probability of steatosis, all with complete clinical and genetic characterization, including adiponectin (ADIPOQ) genotype. PNPLA3 rs738409 (I148M) and ADIPOQ genotypes were evaluated by Taqman assays, serum adiponectin by ELISA. Adiponectin mRNA levels were evaluated by quantitative real-time PCR in the visceral adipose tissue (VAT) of 35 obese subjects undergoing bariatric surgery. Adiponectin levels were independently associated with the risk of NAFLD and with the histological severity of the disease. Adiponectin levels decreased with the number of 148 M PNPLA3 alleles at risk of NASH both in patients with NAFLD (p = 0.03), and in healthy subjects (p = 0.04). At multivariate analysis, PNPLA3 148 M alleles were associated with low adiponectin levels (<6 mg/ml, median value) independently of NAFLD diagnosis, age, gender, BMI, and ADIPOQ genotype (OR 1.67, 95% c.i. 1.07-2.1 for each 148 M allele). The p.148 M PNPLA3 variant was associated with decreased adiponectin mRNA levels in the VAT of obese patients (p < 0.05) even in the absence of NASH. In contrast, in CHC, characterized by adiponectin resistance, low adiponectin was associated with male gender and steatosis, but not with PNPLA3 and ADIPOQ genotypes and viral features. The I148M PNPLA3 variant is associated with adiponectin levels in patients with NAFLD and in healthy subjects, but in the presence of adiponectin resistance not in CHC patients. The I148M PNPLA3 genotype may represent a genetic determinant of serum adiponectin levels. Modulation of serum adiponectin might be involved in mediating the susceptibility to steatosis, NASH, and hepatocellular carcinoma in carriers of the 148 M PNPLA3 variant without CHC, with potential therapeutic implications.


Child Passenger Safety.
Despite significant reductions in the number of children killed in motor vehicle crashes over the past decade, crashes continue to be the leading cause of death to children 4 years and older. Therefore, the American Academy of Pediatrics continues to recommend the inclusion of child passenger safety anticipatory guidance at every health supervision visit. This technical report provides a summary of the evidence in support of 5 recommendations for best practices to optimize safety in passenger vehicles for children from birth through adolescence that all pediatricians should know and promote in their routine practice. These recommendations are presented in the revised policy statement on child passenger safety in the form of an algorithm that is intended to facilitate their implementation by pediatricians with their patients and families. The algorithm is designed to cover the majority of situations that pediatricians will encounter in practice. In addition, a summary of evidence on a number of additional issues affecting the safety of children in motor vehicles, including the proper use and installation of child restraints, exposure to air bags, travel in pickup trucks, children left in or around vehicles, and the importance of restraint laws, is provided. Finally, this technical report provides pediatricians with a number of resources for additional information to use when providing anticipatory guidance to families.


Evolutionary history of the sequestrate genus Rossbeevera (Boletaceae) reveals a new genus Turmalinea and highlights the utility of ITS minisatellite-like insertions for molecular identification.
The sequestrate (truffle-like) basidiomycete genera Rossbeevera, Chamonixia, and Octaviania are closely related to the epigeous mushroom genera Leccinum and Leccinellum. In order to elucidate the properties and placement of several undescribed sequestrate taxa in the group and to reveal the evolutionary history of Rossbeevera and its allies, we conducted phylogenetic analyses based on three nuclear (ITS, nLSU, EF-1α) and two mitochondrial DNA loci (ATP6 and mtSSU) as well as precise morphological observations. Phylogenetic analyses of three nuclear loci suggest a complex evolutionary history with sequestrate fruiting bodies present in several clades, including a previously unrecognized sister clade to Rossbeevera. Here we propose a new sequestrate genus, Turmalinea, with four new species and one new subspecies as well as two new species of Rossbeevera. The three-locus nuclear phylogeny resolves species-level divergence within the Rossbeevera-Turmalinea lineage, whereas a separate phylogeny based on two mitochondrial genes corresponds to geographic distance within each species-level lineage and suggests incomplete lineage sorting (ILS) and gene introgression within several intraspecific lineages of Rossbeevera. Furthermore, topological incongruence among the three nuclear single-locus phylogenies suggests that ancient speciation within Rossbeevera probably involved considerable ILS. We also found an unusually long, minisatellite-like insertion within the ITS2 in all Rossbeevera and Turmalinea species. A barcode gap analysis demonstrates that the insertion is more informative for discrimination at various taxonomic levels than the rest of the ITS region and could therefore serve as a unique molecular barcode for these genera.


Human Immunology through the Lens of Evolutionary Genetics.
Pathogen-imposed selection pressures have been paramount during human evolution. Detecting such selection signatures in ancient and modern human genomes can thus help us to identify genes of temporal and spatial immunological relevance. Admixture with ancient hominins and between human populations has been a source of genetic diversity open to selection by infections. Furthermore, cultural transitions, such as the advent of agriculture, have exposed humans to new microbial threats, with impacts on host defense mechanisms. The integration of population genetics and systems immunology holds great promise for the increased understanding of the factors driving immune response variation between individuals and populations.


Ubiquitous parasites drive a 33% increase in methane yield from livestock.
Of anthropogenic methane emissions, 40% can be attributed to agriculture, the majority of which are from enteric fermentation in livestock. With international commitments to tackle drivers of climate change, there is a need to lower global methane emissions from livestock production. Gastrointestinal helminths (parasitic worms) are globally ubiquitous and represent one of the most pervasive challenges to the health and productivity of grazing livestock. These parasites influence a number of factors affecting methane emissions including feed efficiency, nutrient use, and production traits. However, their effects on methane emissions are unknown. This is to our knowledge the first study that empirically demonstrates disease-driven increases in methane (CH4) yield in livestock (grams of CH4 per kg of dry matter intake). We do this by measuring methane emissions (in respiration chambers), dry matter intake, and production parameters for parasitised and parasite-free lambs. This study shows that parasite infections in lambs can lead to a 33% increase in methane yield (g CH4/kg DMI). This knowledge will facilitate more accurate calculations of the true environmental costs of parasitism in livestock, and reveals the potential benefits of mitigating emission through controlling parasite burdens.


Tumor Sequencing and Patient-Derived Xenografts in the Neoadjuvant Treatment of Breast Cancer.
Breast cancer patients with residual disease after neoadjuvant chemotherapy (NAC) have increased recurrence risk. Molecular characterization, knowledge of NAC response, and simultaneous generation of patient-derived xenografts (PDXs) may accelerate drug development. However, the feasibility of this approach is unknown. We conducted a prospective study of 140 breast cancer patients treated with NAC and performed tumor and germline sequencing and generated patient-derived xenografts (PDXs) using core needle biopsies. Chemotherapy response was assessed at surgery. Recurrent "targetable" alterations were not enriched in patients without pathologic complete response (pCR); however, upregulation of steroid receptor signaling and lower pCR rates (16.7%, 1/6) were observed in triple-negative breast cancer (TNBC) patients with luminal androgen receptor (LAR) vs basal subtypes (60.0%, 21/35). Within TNBC, TP53 mutation frequency (75.6%, 31/41) did not differ comparing basal (74.3%, 26/35) and LAR (83.3%, 5/6); however, TP53 stop-gain mutations were more common in basal (22.9%, 8/35) vs LAR (0.0%, 0/6), which was confirmed in The Cancer Genome Atlas and British Columbia data sets. In luminal B tumors, Ki-67 responses were observed in tumors that harbored mutations conferring endocrine resistance ( p53, AKT, and IKBKE ). PDX take rate (27.4%, 31/113) varied according to tumor subtype, and in a patient with progression on NAC, sequencing data informed drug selection (olaparib) with in vivo antitumor activity observed in the primary and resistant (postchemotherapy) PDXs. In this study, we demonstrate the feasibility of tumor sequencing and PDX generation in the NAC setting. "Targetable" alterations were not enriched in chemotherapy-resistant tumors; however, prioritization of drug testing based on sequence data may accelerate drug development.


Pharmaceutical crystallization in surface-modified nanocellulose organogels.
A significant research focus in the pharmaceutical industry is on methods to improve drug uptake into the body by increased dissolution of poorly water soluble active pharmaceutical ingredients (APIs) or sustained drug release behavior, which results in higher overall uptake. Production of higher energy, higher solubility polymorphs is one approach to address this problem. Here we utilize natural materials, cellulose nanocrystals (CNCs), that have a high surface area covered with readily-modified hydroxyl groups to form organogels that promote API crystallization into polymorphs that differ from the as-received materials. We form the gels by oxidizing the CNCs and mixing them with an amine-containing surfactant, octadecylamine (ODA) in dimethylsulfoxide (DMSO) and we optimize the composition and preparation conditions for these gels. The APIs, sulfamethoxazole, sulfapyridine, and sulfamerazine, are added to the mixture prior to the gelation step and are expected to localize in the solvophobic regions of the physical gel and crystallize. We found that sulfamethoxazle recovered from the gels is in the amorphous state, while sulfapyridine crystallizes into a mixture of forms I, III and IV, and sulfamerazine crystallizes into forms I and II, which are different from the as-received materials. This system shows promise for rational design of nanocellulose organogel supports for heterogeneous crystallization of pharmaceutical materials with desired polymorphs.


MRI features of intracerebral hemorrhage within 2 hours from symptom onset.
MRI has been increasingly used in the evaluation of acute stroke patients. However, MRI must be able to detect early hemorrhage to be the only imaging screen used before treatment such as thrombolysis. Susceptibility-weighted imaging, an echo-planar T2* sequence, can show intracerebral hemorrhage (ICH) in patients imaged between 2.5 and 5 hours from symptom onset. It is unknown whether MRI can detect ICH earlier than 2.5 hours. We describe 5 patients with ICH who had MRI between 23 and 120 minutes from symptom onset and propose diagnostic patterns of evolution of hyperacute ICH on MRI. As part of our acute imaging protocol, all patients with acute stroke within 24 hours from symptom onset were imaged with a set of sequences that included susceptibility-weighted imaging, diffusion- and perfusion-weighted imaging, T1- and T2-weighted imaging, fluid-attenuated inversion recovery (FLAIR), and MR angiography using echo-planar techniques. Five patients with ICH had MRI between 23 and 120 minutes from the onset of symptoms. ICH was identified in all patients. Distinctive patterns of hyperacute ICH and absence of signs of ischemic stroke were the hallmark features of this diagnosis. The hyperacute hematoma appears to be composed of 3 distinct areas: (1) center: isointense to hyperintense heterogeneous signal on susceptibility-weighted and T2-weighted imaging; (2) periphery: hypointense (susceptibility effect) on susceptibility-weighted and T2-weighted imaging; and (3) rim: hypointense on T1-weighted imaging and hyperintense on T2-weighted imaging, representing vasogenic edema encasing the hematoma. MRI is able to detect hyperacute ICH and show a pattern of evolution of the hematoma within 2 hours from the onset of symptoms.


In vivo and in vitro vasoactive reactions of coronary arteriolar microvessels to nitroglycerin.
The actions of nitroglycerin on the coronary microcirculation are controversial, with some laboratories reporting that coronary arterioles dilate to the drug and others reporting that they do not. Our goal was to reconcile these disparate observations. Specifically, we hypothesized that dilation of coronary arterioles by nitroglycerin is overwhelmed by intrinsic autoregulatory escape mechanisms. Accordingly, we projected that coronary arterioles would show transient, but not sustained, dilation to nitroglycerin in vivo. Furthermore, we hypothesized that isolated coronary arterioles would show sustained dilation to the drug, because intrinsic escape mechanisms would be absent under these conditions. To test these hypotheses, we measured diameter changes of canine coronary microvessels in vivo during continuous nitroglycerin administration (intracoronary infusion or epicardial suffusion) using intravital fluorescent microscopy (n = 17 dogs) at two time points: early (1-3 min), when coronary artery blood flow velocity was increased, and late (15-20 min), after blood flow velocity returned to control. Tb study responses of coronary arterioles in the absence of autoregulatory influences, we measured the diameter of isolated canine coronary arterioles to varying doses of nitroglycerin (n = 8 vessels, maximal diameter 81 +/- 4 microns). During the early phase of nitroglycerin infusion (1,3, and 10 micrograms.kg-1.min-1), coronary arterioles dilated by 4 +/- 1, 7 +/- 2, and 13 +/- 2% (all P < 0.05), whereas small arteries dilated by 1 +/- 2, 3 +/- 1, and 4 +/- 1%, respectively (P < 0.05 for the higher doses). Coronary artery blood velocity measured increased by 45 +/- 15% (3 micrograms.kg-1.min-1, P < 0.05). Suffusion of nitroglycerin (10(-5) M) dilated coronary arterioles, but not small arteries, by 17 +/- 5% (P < 0.05) between 1 and 3 min. After 15-20 min of nitroglycerin (3 micrograms.kg-1.min-1 by intracoronary infusion), diameters of coronary arterioles and coronary artery blood velocity returned to control, whereas dilation of small arteries remained significant at 4 +/- 1%. Coronary arteriolar dilation by epicardial suffusion of nitroglycerin also waned to control values by 15-20 min, whereas dilation of small arteries was observed: 5 +/- 2% (P < 0.05). In vitro, nitroglycerin caused dose-dependent dilation of coronary arterioles to their maximal diameter, which was sustained for 20 min. Thus nitroglycerin dilates coronary arterioles and small arteries. The dilation in vivo is transient for arterioles but sustained for arteries. In vitro, the dilation is sustained. Because microvessels in vitro are capable of sustaining dilation for 20 min, we conclude that the waning of arteriolar dilation in vivo is related to autoregulatory escape from dilation by nitroglycerin.


Thyroid Dysfunction, Vitamin B12, and Folic Acid Deficiencies Are Not Associated With Cognitive Impairment in Older Adults in Lima, Peru.
Background: Reversible etiologies of cognitive impairment are common and treatable, yet the majority of mild cognitive impairment (MCI) and dementia research in Latin America has focused on irreversible, neurodegenerative etiologies. Objective: We sought to determine if thyroid dysfunction and vitamin B12 and folate deficiencies are associated with cognitive disorders among older adults with memory complaints in Lima, Peru. Methods: This was a retrospective review of patients who presented for cognitive evaluations to a multidisciplinary neurology clinic in Lima, Peru from January 2014 to February 2020. We included individuals aged ≥60 years, native Spanish-speakers, with at least a primary school educational level and a complete clinical assessment. Patients had either subjective cognitive decline (SCD), MCI, or dementia. One-way ANOVA and multiple logistic regression analyses were performed. Results: We included 720 patients (330 SCD, 154 MCI, and 236 dementia); the dementia group was significantly older [mean age SCD 69.7 ± 4.1, dementia 72.4 ± 3.7 (p = 0.000)] and had lower folate levels than SCD patients. The MCI group had higher free T3 levels compared with SCD patients. Those with lower TSH had greater dementia risk (OR = 2.91, 95%CI: 1.15-6.86) but not MCI risk in unadjusted models. B12 deficiency or borderline B12 deficiency was present in 34% of the dementia group, yet no clear correlation was seen between neuropsychological test results and B12 levels in our study. There was no association between MCI or dementia and thyroid hormone, B12 nor folate levels in adjusted models. Conclusion: Our findings do not support an association between metabolic and endocrine disorders and cognitive impairment in older Peruvians from Lima despite a high prevalence of B12 deficiency. Future work may determine if cognitive decline is associated with metabolic or endocrine changes in Latin America.


Convergence analysis of three classes of split-complex gradient algorithms for complex-valued recurrent neural networks.
This letter presents a unified convergence analysis of the split-complex nonlinear gradient descent (SCNGD) learning algorithms for complex-valued recurrent neural networks, covering three classes of SCNGD algorithms: standard SCNGD, normalized SCNGD, and adaptive normalized SCNGD. We prove that if the activation functions are of split-complex type and some conditions are satisfied, the error function is monotonically decreasing during the training iteration process, and the gradients of the error function with respect to the real and imaginary parts of the weights converge to zero. A strong convergence result is also obtained under the assumption that the error function has only a finite number of stationary points. The simulation results are given to support the theoretical analysis.


Overview of Histone Deacetylase Inhibitors in Haematological Malignancies.
Histone deacetylase inhibitors (HDACi) can induce hyperacetylation of both histone and non-histone target resulting in epigenetic reprogramming and altered activity, stability and localisation of non-histone proteins to ultimately mediate diverse biological effects on cancer cells and their microenvironment. Clinical trials have demonstrated single agent HDACi to have activity in hematological malignancies, in particular T-cell lymphoma and Hodgkin lymphoma. Combination strategies with standard therapies based on pre-clinical data are being employed with significant success due to their excellent side effect profile. Correlative studies will provide valuable information on the sub-groups of patients more likely to respond or be resistant to HDACi therapy, while long-term monitoring for toxicities is also needed.


[MOG encephalomyelitis: international recommendations on diagnosis and antibody testing].
Over the past few years, new-generation cell-based assays have demonstrated a robust association of autoantibodies to full-length human myelin oligodendrocyte glycoprotein (MOG-IgG) with (mostly recurrent) optic neuritis, myelitis and brainstem encephalitis, as well as with acute disseminated encephalomyelitis (ADEM)-like presentations. Most experts now consider MOG-IgG-associated encephalomyelitis (MOG-EM) a disease entity in its own right, immunopathogenetically distinct from both classic multiple sclerosis (MS) and aquaporin-4 (AQP4)-IgG-positive neuromyelitis optica spectrum disorders (NMOSD). Owing to a substantial overlap in clinicoradiological presentation, MOG-EM was often unwittingly misdiagnosed as MS in the past. Accordingly, increasing numbers of patients with suspected or established MS are currently being tested for MOG-IgG. However, screening of large unselected cohorts for rare biomarkers can significantly reduce the positive predictive value of a test. To lessen the hazard of overdiagnosing MOG-EM, which may lead to inappropriate treatment, more selective criteria for MOG-IgG testing are urgently needed. In this paper, we propose indications for MOG-IgG testing based on expert consensus. In addition, we give a list of conditions atypical for MOG-EM ("red flags") that should prompt physicians to challenge a positive MOG-IgG test result. Finally, we provide recommendations regarding assay methodology, specimen sampling and data interpretation, and propose for the first time diagnostic criteria for MOG-EM.


A step by step guide to phylogeny reconstruction.
The aim of this paper is to enable those who have never reconstructed a phylogeny to do so from scratch. The paper does not attempt to be a comprehensive theoretical guide, but describes one rigorous way of obtaining phylogenetic trees. Those who follow the methods outlined should be able to understand the basic ideas behind the steps taken, the meaning of the phylogenetic trees obtained and the scope of questions that can be answered with phylogenetic methods. The protocols have been successfully tested by volunteers with no phylogenetic experience.


Comparative immunogenicity and efficacy of equivalent outer membrane vesicle and glycoconjugate vaccines against nontyphoidal Salmonella.
Nontyphoidal Salmonellae cause a devastating burden of invasive disease in sub-Saharan Africa with high levels of antimicrobial resistance. Vaccination has potential for a major global health impact, but no licensed vaccine is available. The lack of commercial incentive makes simple, affordable technologies the preferred route for vaccine development. Here we compare equivalent Generalized Modules for Membrane Antigens (GMMA) outer membrane vesicles and O-antigen-CRM197 glycoconjugates to deliver lipopolysaccharide O-antigen in bivalent Salmonella Typhimurium and Enteritidis vaccines. Salmonella strains were chosen and tolR deleted to induce GMMA production. O-antigens were extracted from wild-type bacteria and conjugated to CRM197 Purified GMMA and glycoconjugates were characterized and tested in mice for immunogenicity and ability to reduce Salmonella infection. GMMA and glycoconjugate O-antigen had similar structural characteristics, O-acetylation, and glucosylation levels. Immunization with GMMA induced higher anti-O-antigen IgG than glycoconjugate administered without Alhydrogel adjuvant. With Alhydrogel, antibody levels were similar. GMMA induced a diverse antibody isotype profile with greater serum bactericidal activity than glycoconjugate, which induced almost exclusively IgG1. Immunization reduced bacterial colonization of mice subsequently infected with SalmonellaS Typhimurium numbers were lower in tissues of mice vaccinated with GMMA compared with glycoconjugate. S. Enteritidis burden in the tissues was similar in mice immunized with either vaccine. With favorable immunogenicity, low cost, and ability to induce functional antibodies and reduce bacterial burden, GMMA offer a promising strategy for the development of a nontyphoidal Salmonella vaccine compared with established glycoconjugates. GMMA technology is potentially attractive for development of vaccines against other bacteria of global health significance.


Domino double Michael-Claisen cyclizations: a powerful general tool for introducing quaternary stereocenters at C4 of cyclohexane-1,3-diones and total synthesis of diverse families of sterically congested alkaloids.
Reactions of substituted acetone derivatives with acrylic acid esters (>200 mol %) in the presence of t-BuOK (200 mol %) in t-BuOH-THF (1:1 by volume) turned out to proceed as a cascade process consisting of the first Michael addition, the second Michael addition, and the last Claisen reaction to afford 4,4-disubstituted cyclohexane-1,3-diones. Only more substituted enolates play the role of a Michael donor in this cascade process, and therefore the ketone took up two alkoxycarbonylethyl groups on the same carbon bearing more substituents. Such intermediates were followed by intramolecular Claisen reactions leading to cyclohexane-1,3-diones bearing quaternary stereogenic centers at C(4), which bears an alkoxycarbonylethyl group and the substituent of the starting acetone derivatives. Thus-obtained 4,4-disubstituted cyclohexane-1,3-diones were successfully employed for total syntheses of intricate alkaloids of biological interest such as (+)-aspidospermidine, (+/-)-galanthamine, (+/-)-lycoramine, and (+/-)-mesembrine, all featuring quaternary stereogenic centers. DFT calculations provided us with clear-cut explanations for the observed chemoselectivity of the cascade process involving ketone-based enolates under thermodynamically controlled conditions.


The correlation of selected physical examination findings and the efficacy of physiotherapy for chronic lumbosacral pain.
Lumbosacral pain is a significant social problem. Effective treatment of this ailment should be based on thorough diagnostic work-up. The goal of the present study was to determine which physical examination findings can help predict a positive outcome of physiotherapy, including kinesitherapy and physical therapy, aimed at symptomatic improvement. The study group consisted of 50 patients with lumbosacral pain syndromes presenting as lumbalgia and ischialgia. Statistically significant correlations were ascertained between certain physical examination findings before the therapy and after the therapy. The crossed Lasegue sign was the only component of the physical examination that correlated with the dynamic efficacy of the rehabilitation treatment.


Deltoid ligament. Functional analysis of the medial collateral ligamentous apparatus of the ankle joint.
On 34 osteoligamentous ankle preparations the function of the various components of the deltoid ligament has been elucidated by tracing mobility patterns after successive transection of the components in varying sequence. The anterior and posterior talofibular ligaments were included in the study to investigate the interaction between these structures and the deltoid ligament. The tibiocalcaneal and the intermediate tibiotalar ligaments control abduction of the talus. The anterior tibiotalar and talofibular ligaments control plantar flexion, while dorsiflexion is inhibited by the posterior tibiotalar and talofibular ligaments, and partly by the anterior talofibular ligament as well. In combination, the anterior and intermediate tibiotalar ligaments control external rotation, while the intermediate and posterior tibiotalar ligaments control both external and, together with the anterior talofibular ligament, internal rotation of the talus. Isolated, neither the anterior nor the posterior tibiotalar ligament appears to play any major role in ankle stability.


CD99 is a novel prognostic stromal marker in non-small cell lung cancer.
The complex interaction between cancer cells and the microenvironment plays an essential role in all stages of tumourigenesis. Despite the significance of this interplay, alterations in protein composition underlying tumour-stroma interactions are largely unknown. The aim of this study was to identify stromal proteins with clinical relevance in non-small cell lung cancer (NSCLC). A list encompassing 203 stromal candidate genes was compiled based on gene expression array data and available literature. The protein expression of these genes in human NSCLC was screened using the Human Protein Atlas. Twelve proteins were selected that showed a differential stromal staining pattern (BGN, CD99, DCN, EMILIN1, FBN1, PDGFRB, PDLIM5, POSTN, SPARC, TAGLN, TNC and VCAN). The corresponding antibodies were applied on tissue microarrays, including 190 NSCLC samples, and stromal staining was correlated with clinical parameters. Higher stromal expression of CD99 was associated with better prognosis in the univariate (p = 0.037) and multivariate (p = 0.039) analysis. The association was independent from the proportion of tumour stroma, the fraction of inflammatory cells and clinical and pathological parameters like stage, performance status and tumour histology. The prognostic impact of stromal CD99 protein expression was confirmed in an independent cohort of 240 NSCLC patients (p = 0.008). Furthermore, double-staining confocal fluorescence microscopy showed that CD99 was expressed in stromal lymphocytes as well as in cancer-associated fibroblasts. Based on a comprehensive screening strategy the membrane protein CD99 was identified as a novel stromal factor with clinical relevance. The results support the concept that stromal properties have an important impact on tumour progression.


A pilot study of heart rate variability biofeedback therapy in the treatment of perinatal depression on a specialized perinatal psychiatry inpatient unit.
Heart rate variability biofeedback (HRVB) therapy may be useful in treating the prominent anxiety features of perinatal depression. We investigated the use of this non-pharmacologic therapy among women hospitalized with severe perinatal depression. Three questionnaires, the State Trait Anxiety Inventory (STAI), Warwick-Edinburgh Mental Well-Being Scale, and Linear Analog Self Assessment, were administered to 15 women in a specialized inpatient perinatal psychiatry unit. Participants were also contacted by telephone after discharge to assess continued use of HRVB techniques. The use of HRVB was associated with an improvement in all three scales. The greatest improvement (-13.867, p < 0.001 and -11.533, p < 0.001) was among STAI scores. A majority (81.9 %, n = 9) of women surveyed by telephone also reported continued frequent use at least once per week, and over half (54.6 %, n = 6) described the use of HRVB techniques as very or extremely beneficial. The use of HRVB was associated with statistically significant improvement on all instrument scores, the greatest of which was STAI scores, and most women reported frequent continued use of HRVB techniques after discharge. These results suggest that HRVB may be particularly beneficial in the treatment of the prominent anxiety features of perinatal depression, both in inpatient and outpatient settings.


Activation state of polymorphonuclear leukocytes in surgical patients: characterization of surface receptor expression.
We studied the expression of chemotactic, opsonic, and adherent receptors on the membranes of polymorphonuclear neutrophils (PMNs) from surgical patients because modulation of these receptors has been suggested in the control of critical PMN functions that directly or indirectly influence patient outcome. Healthy subjects who had PMN adherence, chemotaxis, and receptor measurements were compared with preoperative surgical patients and those who were within 48 hours of an admission to an intensive care unit (surgical intensive care unit) because of an acute illness. The following receptor/cell pattern was found in control subjects, preoperative patients, and patients in the surgical intensive care unit, respectively: formyl peptide (13,000 vs 18,000 vs 22,000), CR3 (59,000 vs 105,000 vs 121,000), fibronectin (21,000 vs 20,000 vs 35,000), FcII gamma (9,000 vs 20,000 vs 25,000), C5a (347 vs 265 vs 250 mean channel number), and FcIIIR (64,000 vs 75,000 vs 26,000). This receptor pattern was partly correlated directly or indirectly with the acute-phase response, neutrophil adherence, and PMN chemotaxis from these subjects. The data suggest that there are alterations in the expression and modulation of PMN surface membrane receptors in patients with a "stable" disease process compared with those with an acute illness, which may affect critical PMN functions needed to combat bacterial infections.


Continuous solid-phase extraction and preconcentration of bisphenol A in aqueous samples using molecularly imprinted columns.
A bisphenol A (BPA) molecularly imprinted polymer, the composition of which was optimised using a chemometric approach, has been applied to the selective preconcentration of the template from aqueous samples. The selectivity of the polymer toward BPA and related compounds was evaluated chromatographically. The BPA-imprinted polymer was packed in a column and used for continuous on-column solid-phase extraction (MISPE) of aqueous samples followed by subsequent analysis by HPLC with fluorescence detection of the eluted fractions. The composition of the washing solvent applied in the MISPE procedure was optimised to favour the specific interactions of the MIP with BPA and to remove the non-selectively bound matrix components. The MISPE method has proven to be effective for selective preconcentration of BPA in aqueous samples (recoveries >84% obtained in the eluate for 10-100 mL sample volumes) enabling detection and quantification limits of 1.0 and 3.3 ng mL(-1), respectively (based on 25 mL sample size). Analytical recoveries were between 92 and 101% for river water samples spiked with known amounts of BPA (30, 60, and 80 ng mL(-1)); relative standard deviations (RSD) were lower than 5.0%.


Genesis and mechanisms controlling tornillo seismo-volcanic events in volcanic areas.
Volcanic activity is often preceded or accompanied by different types of seismo-volcanic signals. Among these signals, the so-called tornillo (Spanish for "screw") events are considered to belong to a unique class of volcano-seismicity characterised by a long-duration coda, amplitude modulation and high-quality factor. These data constitute important evidence for the gas fraction inside magmatic fluids. However, the mechanism behind this unique signal remains not fully understood. Here we report new laboratory evidence showing that two different processes have either scale-invariant or scale-dependent effects in generating tornillo-like events. These processes are respectively the gas pressure gradient, which triggers the event and regulates the slow decaying coda, and the fluid resonance into small scale structures which, in turn, control the frequency content of the signal. Considering that the gas pressure gradient is proportional to the fluid flow, these new findings, as applied to volcanoes, provide new information to better quantify both gas rate and volume, and the dimension of the resonator.


Observations on vitamin K deficiency in the fetus and newborn: has nature made a mistake?
The microsomal mixed function oxidase system metabolizes xenobiotics (Phase I) to products that, if not activated and conjugated for excretion (Phase II), are capable of forming conjugates with cellular macromolecules, including DNA, resulting in toxic, mutagenic, or carcinogenic events. Benzo(a)pyrene (BP), a polycyclic aromatic hydrocarbon, is a model carcinogen for this system. Vitamin K1 (phylloquinone) is a regulator of BP metabolism. These studies demonstrate that K1 is capable of increasing Phase I metabolism and decreasing glutathione transferase activity (Phase II) in chick embryo liver; that deprivation of K1 reduces BP/DNA adducts in mouse liver and reduces tumor formation in mice given intraperitoneal BP; and that K1 supplementation increases BP induced tumor formation in mice. However, epidemiologic studies indicate that children of mothers who smoke during pregnancy may not be at increased risk of cancer. It is known that the placentas from these pregnancies exhibit markedly increased levels of arylhydrocarbon hydroxylase induced by the polycyclic aromatic hydrocarbons in tobacco smoke, but there is no corresponding increase in this enzyme activity in the fetus in such pregnancies. We suggest that the low vitamin K level is a secondary protective mechanism for xenobiotics, such as BP, that may escape the primary placental screen. The recently described role of vitamin K-dependent Gla protein as ligands for receptor tyrosine kinases, also establishes K as a link in cell growth and transformation. It is proposed that the small total body pool of K1 in the adult, which is sufficient only to meet continuing needs, and the even smaller pool in the fetus are protective. This protective effect of low K1 levels is particularly important in the presence of the high mitotic rates and rapid cell turnover in the avian embryo and mammalian fetus.


Ligand binding pocket of a novel Allatostatin receptor type C of stick insect, Carausius morosus.
Allatostatins (AST) are neuropeptides with variable function ranging from regulation of developmental processes to the feeding behavior in insects. They exert their effects by binding to cognate GPCRs, called Allatostatin receptors (AlstR), which emerge as promising targets for pesticide design. However, AlstRs are rarely studied. This study is the first reported structural study on AlstR-AST interaction. In this work, the first C type AlstR from the stick insect Carausius morosus (CamAlstR-C) was identified and its interaction with type C AST peptide was shown to be physically consistent with the experimental results. The proposed structure of CamAlstR-C revealed a conserved motif within the third extracellular loop, which, together with the N-terminus is essential for ligand binding. In this work, computational studies were combined with molecular and nano-scale approaches in order to introduce an unknown GPCR-ligand system. Consequently, the data obtained provided a reliable target region for future agonist/inverse agonist studies on AlstRs.


Structure-function analysis of cf-9, a receptor-like protein with extracytoplasmic leucine-rich repeats.
The tomato (Lycopersicon pimpinellifolium) resistance protein Cf-9 belongs to a large class of plant proteins with extracytoplasmic Leu-rich repeats (eLRRs). eLRR proteins play key roles in plant defense and development, mainly as receptor-like proteins or receptor-like kinases, conferring recognition of various pathogen molecules and plant hormones. We report here a large-scale structure-function analysis of an eLRR protein. A total of 66 site-directed mutants of Cf-9 were analyzed for activity in Avr9 recognition and for protein stability and the results interpreted with the help of a homology model of the Cf-9 structure. Conserved Trp and Cys pairs in the N-terminal LRR-flanking domain appear to be important for Cf-9 activity and are probably exposed at the putative concave inner surface of the Cf-9 protein, where recognition specificity also resides. Removal of each of the 22 putative N-linked glycosylation sites (PGS) revealed that many PGSs contribute to Cf-9 activity and that the PGSs in the putative alpha-helices of the LRR modules are essential. Immunoblot analysis and mass spectrometry showed that all but one of the PGSs are N-glycosylated. Introduction of glycosylation at the putative concave beta-sheet surface blocks Cf-9 activity, in some cases probably by disturbing specific recognition, and in another case by steric hindrance with existing N-glycans. The glycosylation pattern and several other features are conserved in other eLRR proteins, where similar mutations show similar phenotypes.


Coenzyme Q10 and male infertility.
We had previously demonstrated that Coenzyme Q10 [(CoQ10) also commonly called ubiquinone] is present in well-measurable levels in human seminal fluid, where it probably exerts important metabolic and antioxidant functions; seminal CoQ10 concentrations show a direct correlation with seminal parameters (count and motility). Alterations of CoQ10 content were also shown in conditions associated with male infertility, such as asthenozoospermia and varicocele (VAR). The physiological role of this molecule was further clarified by inquiring into its variations in concentrations induced by different medical or surgical procedures used in male infertility treatment. We therefore evaluated CoQ10 concentration and distribution between seminal plasma and spermatozoa in VAR, before and after surgical treatment, and in infertile patients after recombinant human FSH therapy. The effect of CoQ10 on sperm motility and function had been addressed only through some in vitro experiments. In two distinct studies conducted by our group, 22 and 60 patients affected by idiopathic asthenozoospermia were enrolled, respectively. CoQ10 and its reduced form, ubiquinol, increased significantly both in seminal plasma and sperm cells after treatment, as well as spermatozoa motility. A weak linear dependence among the relative variations, at baseline and after treatment, of seminal plasma or intracellular CoQ10, ubiquinol levels and kinetic parameters was found in the treated group. Patients with lower baseline value of motility and CoQ10 levels had a statistically significant higher probability to be responders to the treatment. In conclusion, the exogenous administration of CoQ10 increases both ubiquinone and ubiquinol levels in semen and can be effective in improving sperm kinetic features in patients affected by idiopathic asthenozoospermia.


Illuminating the Reaction Pathways of Viromimetic Assembly.
The coassembly of well-defined biological nanostructures relies on a delicate balance between attractive and repulsive interactions between biomolecular building blocks. Viral capsids are a prototypical example, where coat proteins exhibit not only self-interactions but also interact with the cargo they encapsulate. In nature, the balance between antagonistic and synergistic interactions has evolved to avoid kinetic trapping and polymorphism. To date, it has remained a major challenge to experimentally disentangle the complex kinetic reaction pathways that underlie successful coassembly of biomolecular building blocks in a noninvasive approach with high temporal resolution. Here we show how macromolecular force sensors, acting as a genome proxy, allow us to probe the pathways through which a viromimetic protein forms capsids. We uncover the complex multistage process of capsid assembly, which involves recruitment and complexation, followed by allosteric growth of the proteinaceous coat. Under certain conditions, the single-genome particles condense into capsids containing multiple copies of the template. Finally, we derive a theoretical model that quantitatively describes the kinetics of recruitment and growth. These results shed new light on the origins of the pathway complexity in biomolecular coassembly.


Prospective change in health-related quality of life and subsequent mortality among middle-aged and older women.
We sought to determine prospective changes in health-related quality of life (HRQoL) measures and subsequent mortality in middle-aged and older women. We obtained data from 40 337 healthy women from the Nurses' Health Study aged 46 to 71 years in 1992. We used Cox proportional hazards regression to evaluate associations of changes in self-assessed physical and mental component summary (PCS and MCS) scores from the Short Form 36 Health Survey between 1992 and 1996 and between 1996 and 2000, with all-cause mortality through 2004. Women with low HRQoL (PCS and MCS scores) and the greatest HRQoL declines had higher mortality than did women with stable scores. Change in PCS score predicted mortality across the range of 4-year change: severe decline (relative risk [RR] = 3.32; 95% confidence interval [CI] = 2.45, 4.50), moderate decline (RR = 1.44; 95% CI = 1.16, 1.79), slight decline (RR = 1.35; 95% CI = 1.12, 1.63), no change (reference category), improvement (RR = 0.72; 95% CI = 0.56, 0.91; continuous P < .001). MCS score results were similar. Score increases were associated with lifestyle improvements, especially increased physical activity. Observed associations demonstrate the predictive validity of changes in self-assessed HRQoL for subsequent mortality in healthy populations. Future research should examine determinants of patterns of change.


Plasmodium falciparum, anaemia and cognitive and educational performance among school children in an area of moderate malaria transmission: baseline results of a cluster randomized trial on the coast of Kenya.
Studies have typically investigated health and educational consequences of malaria among school-aged children in areas of high malaria transmission, but few have investigated these issues in moderate transmission settings. This study investigates the patterns of and risks for Plasmodium falciparum and anaemia and their association with cognitive and education outcomes on the Kenyan coast, an area of moderate malaria transmission. As part of a cluster randomised trial, a baseline cross-sectional survey assessed the prevalence of and risk factors for P. falciparum infection and anaemia and the associations between health status and measures of cognition and educational achievement. Results are presented for 2400 randomly selected children who were enrolled in the 51 intervention schools. The overall prevalence of P. falciparum infection and anaemia was 13.0% and 45.5%, respectively. There was marked heterogeneity in the prevalence of P. falciparum infection by school. In multivariable analysis, being male, younger age, not sleeping under a mosquito net and household crowding were adjusted risk factors for P. falciparum infection, whilst P. falciparum infection, being male and indicators of poor nutritional intake were risk factors for anaemia. No association was observed between either P. falciparum or anaemia and performance on tests of sustained attention, cognition, literacy or numeracy. The results indicate that in this moderate malaria transmission setting, P. falciparum is strongly associated with anaemia, but there is no clear association between health status and education. Intervention studies are underway to investigate whether removing the burden of chronic asymptomatic P. falciparum and related anaemia can improve education outcomes.


Application of a multiplex PCR for the detection of protozoan pathogens of the eastern oyster Crassostrea virginica in field samples.
Populations of eastern oysters Crassostrea virginica along the east coast of North America have repeatedly experienced epizootic mass mortality due to infections by protozoan parasites, and molecular diagnostic methodologies are fast becoming more widely available for the diagnosis of protozoan diseases of oysters. In this study we applied a modified version of an existing multiplex polymerase chain reaction (PCR) for detection of the eastern oyster parasites Haplosporidium nelsoni, H. costale and Perkinsus marinus from field-collected samples. We incorporated primers for DNA quality control based on the large subunit ribosomal RNA (LSU rRNA) gene of C. virginica. The multiplex PCR (MPCR) simultaneously amplified genomic DNA of C. virginica, and cloned DNA of H. nelsoni, P. marinus and H. costale. In field trial applications, we compared the performance of the MPCR to that of the conventional diagnostic techniques of histopathological tissue examination and the Ray/Mackin fluid thioglycollate medium (RMFT) assay. A total of 530 oysters were sampled from 18 sites at 12 locations along the east coast of the United States from the Gulf of Mexico to southern New England. The modified MPCR detected 21% oysters with H. nelsoni, 2% oysters with H. costale, and 40% oysters with P. marinus infections. In comparison, histopathological examination detected H. nelsoni and H. costale infections in 6 and 0.8% oysters, respectively, and the RMFT assay detected P. marinus infection in 31% oysters. The MPCR is a more sensitive diagnostic assay for detection of H. nelsoni, H. costale, and P. marinus, and incorporation of an oyster quality control product limits false negative results.


Belt-MRF for large aperture mirrors.
With high-determinacy and no subsurface damage, Magnetorheological Finishing (MRF) has become an important tool in fabricating high-precision optics. But for large mirrors, the application of MRF is restricted by its small removal function and low material removal rate. In order to improve the material removal rate, shorten the processing cycle, we proposed a new MRF concept, named Belt-MRF to expand the application of MRF to large mirrors and made a prototype with a large remove function, using a belt instead of a very large polishing wheel to expand the polishing length. A series of experimental results on Silicon carbide (SiC) and BK 7 specimens and fabrication simulation verified that the Belt-MRF has high material removal rates, stable removal function and high convergence efficiency which makes it a promising technology for processing large aperture optical elements.


The evolution of mutualism in gut microbiota via host epithelial selection.
The human gut harbours a large and genetically diverse population of symbiotic microbes that both feed and protect the host. Evolutionary theory, however, predicts that such genetic diversity can destabilise mutualistic partnerships. How then can the mutualism of the human microbiota be explained? Here we develop an individual-based model of host-associated microbial communities. We first demonstrate the fundamental problem faced by a host: The presence of a genetically diverse microbiota leads to the dominance of the fastest growing microbes instead of the microbes that are most beneficial to the host. We next investigate the potential for host secretions to influence the microbiota. This reveals that the epithelium-microbiota interface acts as a selectivity amplifier: Modest amounts of moderately selective epithelial secretions cause a complete shift in the strains growing at the epithelial surface. This occurs because of the physical structure of the epithelium-microbiota interface: Epithelial secretions have effects that permeate upwards through the whole microbial community, while lumen compounds preferentially affect cells that are soon to slough off. Finally, our model predicts that while antimicrobial secretion can promote host epithelial selection, epithelial nutrient secretion will often be key to host selection. Our findings are consistent with a growing number of empirical papers that indicate an influence of host factors upon microbiota, including growth-promoting glycoconjugates. We argue that host selection is likely to be a key mechanism in the stabilisation of the mutualism between a host and its microbiota.


System analysis of cross-talk between nuclear receptors reveals an opposite regulation of the cell cycle by LXR and FXR in human HepaRG liver cells.
Transcriptional regulations exert a critical control of metabolic homeostasis. In particular, the nuclear receptors (NRs) are involved in regulating numerous pathways of the intermediate metabolism. The purpose of the present study was to explore in liver cells the interconnectedness between three of them, LXR, FXR, and PPARα, all three known to act on lipid and glucose metabolism, and also on inflammation. The human cell line HepaRG was selected for its best proximity to human primary hepatocytes. Global gene expression of differentiated HepaRG cells was assessed after 4 hours and 24 hours of exposure to GW3965 (LXR agonist), GW7647 (PPARα agonist), and GW4064 and CDCA (FXR synthetic and natural agonist, respectively). Our work revealed that, contrary to our expectations, NR specificity is largely present at the level of target genes, with a smaller than expected overlap of the set of genes targeted by the different NRs. It also highlighted the much broader activity of the synthetic FXR ligand compared to CDCA. More importantly, our results revealed that activation of FXR has a pro-proliferative effect and decreases the number of tetraploid (or binucleated) hepatocytes, while LXR inhibits the cell cycle progression, inducing hepatocyte differentiation and an increase in tetraploidism. Conclusion: these results highlight the importance of analyzing the different NR activities in a context allowing a direct confrontation of each receptor outcome, and reveals the opposite role of FXR and LXR in hepatocyte cells division and maturation.


Do elder emergency department patients and their informants agree about the elder's functioning?
To compare elder patients' and their informants' ratings of the elder's physical and mental function measured by a standard instrument, the Medical Outcomes Study Short Form 12 (SF-12). This was a randomized, cross-sectional study conducted at a university-affiliated community teaching hospital emergency department (census 65,000/year). Patients >69 years old, arriving on weekdays between 10 AM and 7 PM, able to engage in English conversation, and consenting to participate were eligible. Patients too ill to participate were excluded. Informants were people who accompanied and knew the patient. Elder patients were randomized 1:1 to receive an interview or questionnaire version of the SF-12. The questionnaire was read to people unable to read. Two trained medical students administered the instrument. The SF-12 algorithm was used to calculate physical (PCS) and mental (MCS) component scores. Oral and written versions were compared using analysis of variance. The PCS and MCS scores between patient-informant pairs were compared with a matched t-test. Alpha was 0.05. One hundred six patients and 55 informants were enrolled. The patients' average (+/-SD) age was 77 +/- 5 years; 59 (56%; 95% CI = 46% to 65%) were women. There was no significant difference for mode of administration in PCS (p = 0.53) or MCS (p = 0.14) scores. Patients rated themselves higher on physical function than did their proxies. There was a 4.1 (95% CI = 99 to 7.2) point difference between patients' and their proxies' physical component scores (p = 0.01). Scores on the mental component were quite similar. The mean difference between patients and proxies was 0.49 (95% CI = 3.17 to 4.16). The half point higher rating by patients was not statistically significant (p = 0.79). Elders' self-ratings of physical function were higher than those of proxies who knew them. There was no difference in mental function ratings between patients and their proxies. Switching from informants' to patients' reports in evaluating elders' physical function in longitudinal studies may introduce error.


Treating cachexia using soluble ACVR2B improves survival, alters mTOR localization, and attenuates liver and spleen responses.
Cancer cachexia increases morbidity and mortality, and blocking of activin receptor ligands has improved survival in experimental cancer. However, the underlying mechanisms have not yet been fully uncovered. The effects of blocking activin receptor type 2 (ACVR2) ligands on both muscle and non-muscle tissues were investigated in a preclinical model of cancer cachexia using a recombinant soluble ACVR2B (sACVR2B-Fc). Treatment with sACVR2B-Fc was applied either only before the tumour formation or with continued treatment both before and after tumour formation. The potential roles of muscle and non-muscle tissues in cancer cachexia were investigated in order to understand the possible mechanisms of improved survival mediated by ACVR2 ligand blocking. Blocking of ACVR2 ligands improved survival in tumour-bearing mice only when the mice were treated both before and after the tumour formation. This occurred without effects on tumour growth, production of pro-inflammatory cytokines or the level of physical activity. ACVR2 ligand blocking was associated with increased muscle (limb and diaphragm) mass and attenuation of both hepatic protein synthesis and splenomegaly. Especially, the effects on the liver and the spleen were observed independent of the treatment protocol. The prevention of splenomegaly by sACVR2B-Fc was not explained by decreased markers of myeloid-derived suppressor cells. Decreased tibialis anterior, diaphragm, and heart protein synthesis were observed in cachectic mice. This was associated with decreased mechanistic target of rapamycin (mTOR) colocalization with late-endosomes/lysosomes, which correlated with cachexia and reduced muscle protein synthesis. The prolonged survival with continued ACVR2 ligand blocking could potentially be attributed in part to the maintenance of limb and respiratory muscle mass, but many observed non-muscle effects suggest that the effect may be more complex than previously thought. Our novel finding showing decreased mTOR localization in skeletal muscle with lysosomes/late-endosomes in cancer opens up new research questions and possible treatment options for cachexia.


Effects of ethanol administration on hepatocellular ultrastructure of regenerating liver induced by partial hepatectomy.
Acute ethanol administration partially inhibits DNA and protein syntheses during liver regeneration (LR) induced by partial hepatectomy (PH) in rats. Previous findings that the magnitude of ethanol's deleterious effects on LR are related to the route and timing of its administration led us to perform studies at the ultrastructural level, comparing ethanol effects on PH-induced LR, as a consequence of its administration route. PH promoted alterations on the endoplasmic reticulum and mitochondria, accompanied by decreased glycogen and increased lipid content in cytoplasm. Structural nuclear and nucleolar activities were also evident. Intragastric ethanol administration practically abolished the adaptative changes found in PH-promoted regenerating hepatocytes, whereas its administration through the intraperitoneal route induced later ultrastructural modifications, indicating cellular proliferation. These results suggest that ethanol, under certain conditions, could stimulate liver proliferation triggered by PH. The mechanism underlying this surprising effect of ethanol on LR remains to be elucidated. However, it is suggested that an altered ethanol metabolism by rats subjected to PH could be involved.


Conditional Pten knock-out mice: a model for metastatic phaeochromocytoma.
Phaeochromocytomas (PCCs) are neuro-endocrine tumours of the adrenal medulla that are usually benign, but approximately 10% of patients develop metastases. Malignant PCCs can only be diagnosed with certainty if metastases are present. Here we describe adrenal tumours generated in a Pten conditional knock-out (KO) mouse model. We characterized the molecular alterations in these tumours and compared them with human PCC. Thirty-two of 41 (78%) male Psa-Cre;Pten-loxP/loxP mice presented adrenal tumours that were shown to be PCC by histology and by immunohistochemical staining for enzymes in the catecholamine biosynthetic pathway. In 6 of 17 investigated mice, histological and immunohistochemical evidence was obtained for the presence of PCC lung metastases. Array comparative genomic hybridization (CGH) analysis of the primary tumours showed loss of chromosomes 6 and 19, which are syntenic to human 3p and 11q. Another frequent alteration found was gain of chromosome 15, which is syntenic to human chromosome 5. The molecular aberrations in the mouse model corresponded to the alterations found in a subtype of human PCC, suggesting that the PCC of the Pten KO mice might be representative of human PCC. The mouse model should allow further studies into the pathogenesis of human malignant PCCs and into therapeutic strategies for these tumours.


P2Y(2) receptor agonist INS37217 enhances functional recovery after detachment caused by subretinal injection in normal and rds mice.
To evaluate the effects of INS37217 on the recovery of retinal function after experimental retinal detachment induced by subretinal injection. Subretinal injections of 1 micro L of fluorescent microbeads, saline, or INS37217 (1-200 micro M) were made by the transvitreal method in normal (C57BL/6) mice and in mice heterozygous for the retinal degeneration slow (rds) gene. Control, mock-injected animals underwent corneal puncture without injection. Histologic and ERG evaluations were made at 0 to 1 and 8 hours, and 1, 3, 7, 10, 14, and 60 days post injection (PI). DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL). A single subretinal injection of saline solution containing fluorescent beads caused a histologically evident retinal detachment and distributed the microbeads to almost all the subretinal space. Spontaneous reattachment occurred within 24 hours after injection and was accompanied by evident retinal folding that appeared largely resolved by 6 days later. Relative to controls, injection of saline resulted in approximately 40% recovery of dark-adapted a-wave amplitude at 24 hours PI and gradually improved to approximately 90% of controls at 2 months PI. Subretinal injection of saline containing INS37217 (10 micro M) significantly increased rod and cone ERG of normal and rds(+/-) mice at 1 and 10 days PI, when compared with injection of saline alone. Additionally, INS37217 reduced the number of TUNEL-positive photoreceptors and the enhanced rate of reattachment. Enhancement of ERG recovery by INS37217 is likely due to reduced retinal folding and cell death associated with detachment. These results support the use of INS37217 to help restore function after therapies that involve subretinal administration of drugs in animal models of retinal diseases.


Eosinophil hyporesponse of jirds induced by microfilariae of Brugia pahangi.
Male jirds (Meriones unguiculatus) were inoculated sc with 100 infective larvae of Brugia pahangi. After 16 weeks, the animals were reinoculated with a comparable number of organisms. Blood eosinophil responses during the 5 weeks subsequent to this attempt to reinfect were much lower than those of comparable naive animals, while the response to a heterologous infection (Toxocara canis) was comparable to that of controls. Mebendazole was given to infected animals for 2 weeks beginning 5 weeks (prepatent) or 16 weeks (patent) after infection. At comparable intervals after drug administration, the animals were reinoculated with infective larvae and the blood eosinophil response was measured over a 5 week period. The response in the animals treated during the prepatent period was higher than the untreated infected controls. Treatment during the patent period had no demonstrable effect. Jirds made artificially microfilaremic by intravenous inoculation of viable filaria before and after the standard infecting dose had a low eosinophil response to infective larvae. A primary experience of jirds with the microfilariae of B. pahangi evokes an eosinophil response. Subsequent inoculation of larvae did not produce a comparable response.


Identification of Mycobacterial RplJ/L10 and RpsA/S1 Proteins as Novel Targets for CD4+ T Cells.
Tuberculosis (TB) due to Mycobacterium tuberculosis remains a major global infectious disease problem, and a more efficacious vaccine is urgently needed for the control and prevention of disease caused by this organism. We previously reported that a genetically modified strain of Mycobacterium smegmatis called IKEPLUS is a promising TB vaccine candidate. Since protective immunity induced by IKEPLUS is dependent on antigen-specific CD4+ T cell memory, we hypothesized that the specificity of the CD4+ T cell response was a critical feature of this protection. Using in vitro assays of interferon gamma production (enzyme-linked immunosorbent spot [ELISPOT] assays) by splenocytes from IKEPLUS-immunized C57BL/6J mice, we identified an immunogenic peptide within the mycobacterial ribosomal large subunit protein RplJ, encoded by the Rv0651 gene. In a complementary approach, we generated major histocompatibility complex (MHC) class II-restricted T cell hybridomas from IKEPLUS-immunized mice. Screening of these T cell hybridomas against IKEPLUS and ribosomes enriched from IKEPLUS suggested that the CD4+ T cell response in IKEPLUS-immunized mice was dominated by the recognition of multiple components of the mycobacterial ribosome. Importantly, CD4+ T cells specific for mycobacterial ribosomes accumulate to significant levels in the lungs of IKEPLUS-immunized mice following aerosol challenge with virulent M. tuberculosis, consistent with a role for these T cells in protective host immunity in TB. The identification of CD4+ T cell responses to defined ribosomal protein epitopes expands the range of antigenic targets for adaptive immune responses to M. tuberculosis and may help to inform the design of more effective vaccines against tuberculosis.


Quantification of penicillin G during labor and delivery by capillary electrophoresis.
In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( approximately 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.