The role of U2AF35 and U2AF65 in enhancer-dependent splicing.
Splicing enhancers are RNA sequence elements that promote the splicing of nearby introns. The mechanism by which these elements act is still unclear. Some experiments support a model in which serine-arginine (SR)-rich proteins function as splicing activators by binding to enhancers and recruiting the splicing factor U2AF to an adjacent weak 3' splice site. In this model, recruitment requires interactions between the SR proteins and the 35-kDa subunit of U2AF (U2AF35). However, more recent experiments have not supported the U2AF recruitment model. Here we provide additional evidence for the recruitment model. First, we confirm that base substitutions that convert weak 3' splice sites to a consensus sequence, and therefore increase U2AF binding, relieve the requirement for a splicing activator. Second, we confirm that splicing activators are required for the formation of early spliceosomal complexes on substrates containing weak 3' splice sites. Most importantly, we find that splicing activators promote the binding of both U2AF65 and U2AF35 to weak 3' splice sites under splicing conditions. Finally, we show that U2AF35 is required for maximum levels of activator-dependent splicing. We conclude that a critical function of splicing activators is to recruit U2AF to the weak 3' splice sites of enhancer-dependent introns, and that efficient enhancer-dependent splicing requires U2AF35.


Quercetin enhances the antitumor effect of trichostatin A and suppresses muscle wasting in tumor-bearing mice.
Quercetin, a flavonol, displays anti-inflammatory and anti-cancer properties. This study aimed to investigate whether a diet containing 0.1% or 1% quercetin (LQ and HQ, respectively) enhances the anti-tumor effects of trichostatin A (TSA) and prevents muscle wasting induced by TSA. The positive control group received quercetin intraperitoneally (IQ). Three weeks after injecting A549 cells, nude mice were given TSA alone or in combination with quercetin administered orally or intraperitoneally for 16 weeks. Tumor volumes as well as body, muscle and epididymal fat weights were determined during or after the experiment. Quercetin given as a diet supplement dose-dependently enhanced the anti-tumor potency of TSA (p < 0.05). The enhancing effect of HQ was similar to that of IQ. HQ also significantly increased the expression of p53, a tumor suppressor, in tumor tissues compared with the TSA alone group. In addition, TSA-induced loss of gastrocnemius muscle weight was inhibited by oral quercetin in a dose dependent manner; the efficiencies of LQ and HQ were similar to or better than IQ. Moreover, both LQ and HQ decreased TSA-induced activation of Forkhead box O1 (FOXO1), a crucial transcription factor that regulates muscle wasting associated genes. Consistently, LQ and HQ suppressed muscle wasting associated proteins atrophy gene-1 and muscle ring-finger protein-1 expression as well as increased the myosin heavy chain level in the gastrocnemius muscles. Besides, quercetin attenuated TSA-increased oxidative damage and proinflammatory cytokines (p < 0.05). These findings demonstrate that a diet containing 0.1% or 1% quercetin enhances the antitumor effect of TSA and prevents TSA-induced muscle wasting.


Resveratrol-induced remodelling of myocellular lipid stores: A study in metabolically compromised humans.
In non-athletes, insulin sensitivity correlates negatively with intramyocellular lipid (IMCL) content. In athletes, however, a pattern of benign IMCL storage exists, which is characterized by lipid storage in type I muscle fibres, in small and numerous lipid droplets (LDs) preferable coated with PLIN5, without affecting insulin sensitivity. Administration of resveratrol has been promoted for its beneficial effects on glucose homeostasis. We observed that 30 days of oral resveratrol administration (150 mg/day) in metabolically compromised individuals showed a 33% increase in IMCL (placebo vs. resveratrol; 0.86 ± 0.090 AU vs. 1.14 ± 0.11 AU, p = 0.003) without impeding insulin sensitivity. Thus, the aim of the present study was to examine if a resveratrol-mediated increase in IMCL content, in metabolically compromised individuals, changes the LD phenotype towards the phenotype we previously observed in athletes. For this, we studied IMCL, LD number, LD size, subcellular distribution and PLIN5 coating in different fibre types using high-resolution confocal microscopy. As proof of concept, we observed a 2.3-fold increase (p = 0.038) in lipid accumulation after 48 h of resveratrol incubation in cultured human primary muscle cells. In vivo analysis showed that resveratrol-induced increase in IMCL is predominantly in type I muscle fibres (placebo vs. resveratrol; 0.97 ± 0.16% vs. 1.26 ± 0.09%; p = 0.030) in both the subsarcolemmal (p = 0.016) and intermyofibrillar region (p = 0.026) and particularly in PLIN5-coated LDs (p = 0.024). These data indicate that administration of resveratrol augments IMCL content in metabolically compromised individuals towards a LD phenotype that mimics an 'athlete like phenotype'.


Stem cells as vectors to deliver HSV/tk gene therapy for malignant gliomas.
The prognosis of patients diagnosed with malignant gliomas including glioblastoma multiforme (GBM) is poor and there is an urgent need to develop and translate novel therapies into the clinic. Neural stem cells display remarkable tropism toward GBMs and thus may provide a platform to deliver oncolytic agents to improve survival. First we provide a brief review of clinical trials that have used intra-tumoral herpes simplex virus thymidine kinase (HSV/tk) gene therapy to treat brain tumors. Then, we review recent evidence that neural stem cells can be used to deliver HSV/tk to GBMs in animal models. While previous clinical trials used viruses or non-migratory vector-producing cells to deliver HSV/tk, the latter approaches were not effective in humans, primarily because of satellite tumor cells that escaped surgical resection and survived due to low efficiency delivery of HSV/tk. To enhance delivery of HSV/tk to kill gliomas cells, recent animal studies have focused on the ability of neural stem cells, transduced with HSV/tk, to migrate efficiently and selectively to regions occupied by GBM cells. This approach holds the promise of targeting GBM cells that have infiltrated the brain well beyond the original site of the tumor epicenter.


TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-kappaB dependent induction of cFLIPL.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a "death ligand"-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-kappaB and JNK signalling pathways. To determine the role of TGF-beta-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-kappaB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-kappaB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-kappaB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-kappaB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1-/- MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-kappaB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.


Exploring cellular memory molecules marking competent and active transcriptions.
Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation. We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription. Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.


Early detection of bipolar disorder: a pilot familial high-risk study of parents with bipolar disorder and their adolescent children.
Disturbances in cognition, affect, sleep and activity have been identified in bipolar disorder (BD) but little is known about the possible role of these factors in the development of the condition. We studied these variables in a familial high-risk sample. Twenty-five children (13-19 years) of bipolar parents were compared with 22 similar aged children of age- and sex-matched healthy controls. Participants were assessed using Schedule for Affective Disorders and Schizophrenia-Lifetime version (SADS-L) and completed self-report measures of dysfunctional attitudes, behavioural inhibition/activation, social rhythms, coping styles and subjective experience of sleep. Children completed a 7-day recording of actigraphy (sleep and activity) and a 7-day diary measuring self-esteem, positive and negative affect and reactions to positive and negative events. Fifty-six per cent of children of bipolar parents (CBP) reported mood symptoms compared to 9% of children of control parents (CC). The CBP group had coping styles and instability of self-esteem consistent with abnormal strategies for regulating affect. Both groups also differed on sleep measures. The majority of differences observed were between CBP with a current or past mood diagnosis and CC. BD parents reported dysfunctional coping styles and (to a lesser extent) disrupted activity patterns. A familial high-risk strategy for studying the role of psychological factors in BD is feasible and informative. This pilot study indicates that abnormal coping styles, instability of self-esteem and dysregulation of sleep may be early markers of bipolar illness. However, current findings need to be explored further in longitudinal studies to clarify which potential markers are truly predictive of BD.


Malaria immunization in Rhesus monkeys. A vaccine effective against both the sexual and asexual stages of Plasmodium knowlesi.
Rhesus monkeys were immunized with a preparation of Plasmodium knowlesi parasites containing principally microgametes with lesser numbers of macrogametes and asexual trophozoites. The antigen mixture was emulsified in Freund's complete adjuvant (FCA) and administered intramuscularly. After one or two inoculations of from 10(5) to 10(7) microgametes in FCA, monkeys showed high levels of circulating anti-gamete antibodies as demonstrated by various in vitro microgamete immobilization or transmission blocking tests. After challenge with P. knowlesi, immunized monkeys developed low level asexual parasitemias and were not infectious to feeding mosquitoes as measured by growth of the parasite on the mosquito gut. Control monkeys developed rapidly rising, usually fatal infections and were highly infectious to mosquitoes. Anti-gamete antibodies appear to neutralize the sexual parasites and prevent mosquito infection within the gut of the recently fed mosquito vector. Suppression of asexual parasitemia in immunized monkeys may be due to the presence of asexual trophozoites in the antigen mixture or to antigens common to both sexual and asexual stages of the parasite. A vaccine effective as a single injection capable of interrupting malaria transmission from man to man whereas reducing the severity of the disease in infected individuals offers a new approach to the control of one of the major diseases affecting man.


Do activities of cytochrome P450 (CYP)3A, CYP2D6 and P-glycoprotein differ between healthy volunteers and HIV-infected patients?
In inflammation and infection, cytochrome P450 (CYP) enzyme activities are down-regulated. Information on possible discrepancies in activities of CYP enzymes and drug transporters between HIV-infected patients and healthy people is limited. We used midazolam, dextromethorphan and digoxin as in vivo phenotyping probes for CYP3A (CYP3A4/5), CYP2D6 and P-glycoprotein activities, respectively, and compared these activities between 12 healthy Caucasian volunteers and 30 treatment-naive HIV-infected patients. Among the HIV-infected patients, the overall CYP3A activity (apparent oral midazolam clearance) was approximately 50% of the activity observed in healthy volunteers (point estimate 0.490, 90% confidence interval [CI] 0.377-0.638). The CYP2D6 activity (plasma ratio area under the curve [AUC]; AUC(dextromethorphan)/AUC(dextrorphan)) was essentially unchanged (point estimate 1.289, 90% CI 0.778-2.136). P-glycoprotein activity was slightly lower in patients (digoxin maximum concentration point estimate 1.304, 90% CI 1.034-1.644). The overall CYP3A activity was approximately 50% lower in HIV-infected patients than in healthy volunteers. The CYP2D6 activity was highly variable, but, on average was not different between groups, whereas a marginally lower P-glycoprotein activity was observed in treatment-naive HIV-infected patients.


Phenotypes developed in secretin receptor-null mice indicated a role for secretin in regulating renal water reabsorption.
Aquaporin 2 (AQP2) is responsible for regulating the concentration of urine in the collecting tubules of the kidney under the control of vasopressin (Vp). Studies using Vp-deficient Brattleboro rats, however, indicated the existence of substantial Vp-independent mechanisms for membrane insertion, as well as transcriptional regulation, of this water channel. The Vp-independent mechanism(s) is clinically relevant to patients with X-linked nephrogenic diabetes insipidus (NDI) by therapeutically bypassing the dysfunctional Vp receptor. On the basis of studies with secretin receptor-null (SCTR(-/-)) mice, we report here for the first time that mutation of the SCTR gene could lead to mild polydipsia and polyuria. Additionally, SCTR(-/-) mice were shown to have reduced renal expression of AQP2 and AQP4, as well as altered glomerular and tubular morphology, suggesting possible disturbances in the filtration and/or water reabsorption process in these animals. By using SCTR(-/-) mice as controls and comparing them with wild-type animals, we performed both in vivo and in vitro studies that demonstrated a role for secretin in stimulating (i) AQP2 translocation from intracellular vesicles to the plasma membrane in renal medullary tubules and (ii) expression of this water channel under hyperosmotic conditions. The present study therefore provides information for at least one of the Vp-independent mechanisms that modulate the process of renal water reabsorption. Future investigations in this direction should be important in developing therapeutic means for treating NDI patients.


Proving the value of simulation in laparoscopic surgery.
To assess the McGill Inanimate System for Training and Evaluation of Laparoscopic Skills (MISTELS) physical laparoscopic simulator for construct and predictive validity and for its educational utility. MISTELS is the physical simulator incorporated by the Society of American Gastrointestinal and Endoscopic Surgeons (SAGES) in their Fundamentals of Laparoscopic Surgery (FLS) program. MISTELS' metrics have been shown to have high interrater and test-retest reliability and to correlate with skill in animal surgery. Over 200 surgeons and trainees from 5 countries were assessed using MISTELS in a series of experiments to assess the validity of the system and to evaluate whether practicing MISTELS basic skills (transferring) would result in skill acquisition transferable to complex laparoscopic tasks (suturing). Face validity was confirmed through questioning 44 experienced laparoscopic surgeons using global rating scales. MISTELS scores increased progressively with increasing laparoscopic experience (n = 215, P < 0.0001), and residents followed over time improved their scores (n = 24, P < 0.0001), evidence of construct validity. Results in the host institution did not differ from 5 beta sites (n = 215, external validity). MISTELS scores correlated with a highly reliable validated intraoperative rating of technical skill during laparoscopic cholecystectomy (n = 19, r = 0.81, P < 0.0004; concurrent validity). Novice laparoscopists were randomized to practice/no practice of the transfer drill for 4 weeks. Improvement in intracorporeal suturing skill was significantly related to practice but not to baseline ability, career goals, or gender (P < 0.001). MISTELS is a practical and inexpensive inanimate system developed to teach and measure technical skills in laparoscopy. This system is reliable, valid, and a useful educational tool.


Vindel: a simple pipeline for checking indel redundancy.
With the advance of next generation sequencing (NGS) technologies, a large number of insertion and deletion (indel) variants have been identified in human populations. Despite much research into variant calling, it has been found that a non-negligible proportion of the identified indel variants might be false positives due to sequencing errors, artifacts caused by ambiguous alignments, and annotation errors. In this paper, we examine indel redundancy in dbSNP, one of the central databases for indel variants, and develop a standalone computational pipeline, dubbed Vindel, to detect redundant indels. The pipeline first applies indel position information to form candidate redundant groups, then performs indel mutations to the reference genome to generate corresponding indel variant substrings. Finally the indel variant substrings in the same candidate redundant groups are compared in a pairwise fashion to identify redundant indels. We applied our pipeline to check for redundancy in the human indels in dbSNP. Our pipeline identified approximately 8% redundancy in insertion type indels, 12% in deletion type indels, and overall 10% for insertions and deletions combined. These numbers are largely consistent across all human autosomes. We also investigated indel size distribution and adjacent indel distance distribution for a better understanding of the mechanisms generating indel variants. Vindel, a simple yet effective computational pipeline, can be used to check whether a set of indels are redundant with respect to those already in the database of interest such as NCBI's dbSNP. Of the approximately 5.9 million indels we examined, nearly 0.6 million are redundant, revealing a serious limitation in the current indel annotation. Statistics results prove the consistency of the pipeline on indel redundancy detection for all 22 chromosomes. Apart from the standalone Vindel pipeline, the indel redundancy check algorithm is also implemented in the web server http://bioinformatics.cs.vt.edu/zhanglab/indelRedundant.php .


The oncoprotein EVI1 and the DNA methyltransferase Dnmt3 co-operate in binding and de novo methylation of target DNA.
EVI1 has pleiotropic functions during murine embryogenesis and its targeted disruption leads to prenatal death by severely affecting the development of virtually all embryonic organs. However, its functions in adult tissues are still unclear. When inappropriately expressed, EVI1 becomes one of the most aggressive oncogenes associated with human hematopoietic and solid cancers. The mechanisms by which EVI1 transforms normal cells are unknown, but we showed recently that EVI1 indirectly upregulates self-renewal and cell-cycling genes by inappropriate methylation of CpG dinucleotides in the regulatory regions of microRNA-124-3 (miR-124-3), leading to the repression of this small gene that controls normal differentiation and cell cycling of somatic cells. We used the regulatory regions of miR-124-3 as a read-out system to investigate how EVI1 induces de novo methylation of DNA. Here we show that EVI1 physically interacts with DNA methyltransferases 3a and 3b (Dnmt3a/b), which are the only de novo DNA methyltransferases identified to date in mouse and man, and that it forms an enzymatically active protein complex that induces de novo DNA methylation in vitro. This protein complex targets and binds to a precise region of miR-124-3 that is necessary for repression of a reporter gene by EVI1. Based on our findings, we propose that in cooperation with Dnmt3a/b EVI1 regulates the methylation of DNA as a sequence-specific mediator of de novo DNA methylation and that inappropriate EVI1 expression contributes to carcinogenesis through improper DNA methylation.


Genetics of male sterility in gynodioecious Plantago coronopus. II. Nuclear genetic variation.
Inheritance of male sterility was studied in the gynodioecious species Plantago coronopus using five plants and their descendants from an area of approximately 50 m2 from each of four locations. In each location, crosses between these five plants yielded the entire array of possible sex phenotypes. Both nuclear and cytoplasmic genes were involved. Emphasis is placed on the nuclear (restorer) genetics of two cytoplasmic types. For both types, multiple interacting nuclear genes were demonstrated. These genes carried either dominant or recessive restorer alleles. The exact number of genes involved could not be determined, because different genetic models could be proposed for each location and no common genetic solution could be given. At least five genes, three with dominant and two with recessive restorer allele action, were involved with both cytoplasmic types. Segregation patterns of partially male sterile plants suggested that they are due to incomplete dominance at restorer loci. Restorer genes interact in different ways. In most instances models with independent restorer gene action were sufficient to explain the crossing results. However, for one case we propose a model with epistatic restorer gene action. There was a consistent difference in the segregation of male sterility between plants from the two cytoplasmic types. Hermaphrodites of cytoplasmic type 2 hardly segregated male steriles, in contrast to plants with cytoplasmic type 1.


Hadrosauroid Dinosaurs from the Late Cretaceous of the Sultanate of Oman.
Fragmentary post-cranial remains (femora, tibia, vertebrae) of ornithischian dinosaurs from the Late Cretaceous of the Sultanate of Oman are described and referred to hadrosauroids. The specimens come from the Al-Khod Conglomerate, of latest Campanian to Maastrichtian age, in the north-eastern part of the country. Although the fragmentary condition of the fossils precludes a precise identification, various characters, including the shape of the fourth trochanter of the femur and the morphology of its distal end, support an attribution to hadrosauroids. With the possible exception of a possible phalanx from Angola, this group of ornithopod dinosaurs, which apparently originated in Laurasia, was hitherto unreported from the Afro-Arabian plate. From a paleobiogeographical point of view, the presence of hadrosauroids in Oman in all likelihood is a result of trans-Tethys dispersal from Asia or Europe, probably by way of islands in the Tethys shown on all recent paleogeographical maps of that area. Whether hadrosauroids were widespread on the Afro-Arabian landmass in the latest Cretaceous, or where restricted to the « Oman island » shown on some paleogeographical maps, remains to be determined.


Possible Molecular Targets of Novel Ruthenium Complexes in Antiplatelet Therapy.
In oncotherapy, ruthenium (Ru) complexes are reflected as potential alternatives for platinum compounds and have been proved as encouraging anticancer drugs with high efficacy and low side effects. Cardiovascular diseases (CVDs) are mutually considered as the number one killer globally, and thrombosis is liable for the majority of CVD-related deaths. Platelets, an anuclear and small circulating blood cell, play key roles in hemostasis by inhibiting unnecessary blood loss of vascular damage by making blood clot. Platelet activation also plays a role in cancer metastasis and progression. Nevertheless, abnormal activation of platelets results in thrombosis under pathological settings such as the rupture of atherosclerotic plaques. Thrombosis diminishes the blood supply to the heart and brain resulting in heart attacks and strokes, respectively. While currently used anti-platelet drugs such as aspirin and clopidogrel demonstrate efficacy in many patients, they exert undesirable side effects. Therefore, the development of effective therapeutic strategies for the prevention and treatment of thrombotic diseases is a demanding priority. Recently, precious metal drugs have conquered the subject of metal-based drugs, and several investigators have motivated their attention on the synthesis of various ruthenium (Ru) complexes due to their prospective therapeutic values. Similarly, our recent studies established that novel ruthenium-based compounds suppressed platelet aggregation via inhibiting several signaling cascades. Our study also described the structure antiplatelet-activity relationship (SAR) of three newly synthesized ruthenium-based compounds. This review summarizes the antiplatelet activity of newly synthesized ruthenium-based compounds with their potential molecular mechanisms.


Gut microbiome and chronic prostatitis/chronic pelvic pain syndrome.
Analysis of the human microbiome continues to reveal new and previously unrealized associations between microbial dysbiosis and disease. Novel approaches to bacterial identification using culture-independent methods allow practitioners to discern the presence of alterations in the taxa and diversity of the microbiome and identify correlations with disease processes. While some of these diseases that have been extensively studied are well-defined in their etiology and treatment methods (colorectal cancer), others have provided much more significant challenges in both diagnosis and treatment. One such condition, chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), has several etiological and potentiating contributions from infection, inflammation, central nervous system (CNS) changes, stress, and central sensitization-all factors that play important roles in the crosstalk between the human body and its microbiome. No singular cause of CP/CPPS has been identified and it is most likely a syndrome with multifactorial causes. This heterogeneity and ambiguity are sources of significant frustration for patients and providers alike. Despite multiple attempts, treatment of chronic prostatitis with monotherapy has seen limited success, which is thought to be due to its heterogeneous nature. Phenotypic approaches to both classify the disease and direct treatment for CP/CPPS have proven beneficial in these patients, but questions still remain regarding etiology. Newer microbiome research has found correlations between symptom scores and disease severity and the degree of dysbiosis in urine and gut (stool) microbiomes in these patients as compared to un-afflicted controls. These findings present potential new diagnostic and therapeutic targets in CP/CPPS patients.


A phase II trial of neoadjuvant erlotinib in patients with muscle-invasive bladder cancer undergoing radical cystectomy: clinical and pathological results.
To evaluate the clinicopathological efficacy of neoadjuvant erlotinib (an epidermal growth factor receptor, EGFR, inhibitor) for invasive bladder cancer in patients undergoing radical cystectomy (RC) as despite definitive surgical therapy, only half of patients undergoing RC will have long-term disease-free survival, and effective adjunctive therapies, especially using agents with lower toxicity, would be a significant advance in the treatment of invasive bladder cancer. The primary endpoint of this phase II trial is to determine the effect of neoadjuvant erlotinib (150 mg once daily for 4 weeks) before RC on the pathological complete response rate (pT0 rate) in RC specimens. In addition, the safety of therapy with erlotinib was also evaluated. Patients selected for study included those with histologically confirmed muscle-invasive bladder cancer who had undergone initial transurethral resection. In all, 20 patients with clinical stage T2 disease had neoadjuvant erlotinib therapy followed by RC. On surgical pathology, five patients (25%) were pT0; in addition, seven (35%) were clinically down-staged (< or = pT1) and 15 (75%) had organ-confined disease at surgical pathology. At a mean follow-up of 24.8 months, 10 patients remain alive and with no evidence of disease, four with organ-confined disease had progression and nine died, including six from disease and three from other causes. Erlotinib was tolerated in all patients, with drug rash being the most common side-effect, in 15 patients (75%). Interestingly, all pT0 and pTis/T1 patients had a rash. The EGFR inhibitor erlotinib, when administered in the neoadjuvant setting, can have beneficial effects in terms of surgical pathology and short-term clinical outcomes in patients undergoing RC for invasive bladder cancer. Analyses are underway to examine the molecular correlates of the apparent clinical effect of neoadjuvant therapy in these patients.


Interventions to Improve Antiretroviral Therapy Adherence Among Adolescents and Youth in Low- and Middle-Income Countries: A Systematic Review 2015-2019.
Adolescents and youth living with HIV have poorer antiretroviral treatment (ART) adherence and viral suppression outcomes than all other age groups. Effective interventions promoting adherence are urgently needed. We reviewed and synthesized recent literature on interventions to improve ART adherence among this vulnerable population. We focus on studies conducted in low- and middle-income countries (LMIC) where the adolescent and youth HIV burden is greatest. Articles published between September 2015 and January 2019 were identified through PubMed. Inclusion criteria were: [1] included participants ages 10-24 years; [2] assessed the efficacy of an intervention to improve ART adherence; [3] reported an ART adherence measurement or viral load; [4] conducted in a LMIC. Articles were reviewed for study population characteristics, intervention type, study design, outcomes measured, and intervention effect. Strength of each study's evidence was evaluated according to an adapted World Health Organization GRADE system. Articles meeting all inclusion criteria except being conducted in an LMIC were reviewed for results and potential transportability to a LMIC setting. Of 108 articles identified, 7 met criteria for inclusion. Three evaluated patient-level interventions and four evaluated health services interventions. Of the patient-level interventions, two were experimental designs and one was a retrospective cohort study. None of these interventions improved ART adherence or viral suppression. Of the four health services interventions, two targeted stable patients and reduced the amount of time spent in the clinic or grouped patients together for bi-monthly meetings, and two targeted patients newly diagnosed with HIV or not yet deemed clinically stable and augmented clinical care with home-based case-management. The two studies targeting stable patients used retrospective cohort designs and found that adolescents and youth were less likely to maintain viral suppression than children or adults. The two studies targeting patients not yet deemed clinically stable included one experimental and one retrospective cohort design and showed improved ART adherence and viral suppression outcomes. ART adherence and viral suppression outcomes remain a major challenge among adolescents and youth. Intensive home-based case management models of care hold promise for improving outcomes in this population and warrant further research.


Functional and clinical outcome of ileal (Studer) neo-bladder reconstruction: single centre experience from Pakistan.
To determine the medium and long-term outcome of orthotopic continent urinary diversion with ileal (Studer) neo-bladder following radical cystectomy. Case series. Department of Surgery, The Aga Khan University Hospital, Karachi, from January 1998 to September 2010. Thirty eight patients underwent radical cystectomy for invasive bladder tumor with ileal neo-bladder (Studer type) reconstruction. Peri-operative and late complications, functional outcome of neo-bladder, urinary continence, metabolic and upper urinary tract status and overall survival were evaluated in all patients. A total of 29 patients (23 males and 6 females) with mean age of 59 ± 12 years were included for the final analysis. The mean duration of surgery (both radical cystectomy and urinary diversion) was 520 ± 70 minutes. Perioperative complication rate was 24% (n=7) with surgical site infection in 4 patients, sepsis in 1 patient and 2 had ureteroileal leak. At 6 months follow-up, 22 patients were fully continent while 7 patients had minimal stress / nocturnal incontinence. The continence rate was 93% (n=27) at one year follow-up. The mean capacity of neo-bladder at 6 months was 384 ± 66 mLs. The late complication rate was 17% (n=5). Three patients developed anastomotic stricture requiring transurethral incision of neo bladder neck, one formed stone in neo-bladder and one developed incision hernia. All patients had preserved renal functions on follow-up. The survival rate was 80% (n=23) at a median follow-up of 66.4 ± 36 months. Two patients developed local recurrence and four developed distant metastasis. Studer ileal neo-bladder is a safe and effective option for urinary diversion in select patients with good oncological and functional outcomes comparable to contemporary literature, even in a low volume center.


Uniparental disomy screen of Irish rare disorder cohort unmasks homozygous variants of clinical significance in the TMCO1 and PRKRA genes.
A uniparental disomy (UPD) screen using whole genome sequencing (WGS) data from 164 trios with rare disorders in the Irish population was performed to identify large runs of homozygosity of uniparental origin that may harbour deleterious recessive variants. Three instances of whole chromosome uniparental isodisomy (UPiD) were identified: one case of maternal isodisomy of chromosome 1 and two cases of paternal isodisomy of chromosome 2. We identified deleterious homozygous variants on isodisomic chromosomes in two probands: a novel p (Glu59ValfsTer20) variant in TMCO1, and a p (Pro222Leu) variant in PRKRA, respectively. The overall prevalence of whole chromosome UPiD in our cohort was 1 in 55 births, compared to 1 in ∼7,500 births in the general population, suggesting a higher frequency of UPiD in rare disease cohorts. As a distinct mechanism underlying homozygosity compared to biallelic inheritance, the identification of UPiD has important implications for family planning and cascade testing. Our study demonstrates that UPD screening may improve diagnostic yields by prioritising UPiD chromosomes during WGS analysis.


Predictors of outcome in World Federation of Neurologic Surgeons grade V aneurysmal subarachnoid hemorrhage patients.
Only a small percentage of World Federation of Neurologic Surgeons grade V aneurysmal subarachnoid hemorrhage patients have a favorable outcome. The influence of clinical parameters on outcome was assessed. Retrospective evaluation of consecutive patients admitted from 2000-2007 with grade V subarachnoid hemorrhage at two institutions by evaluating, over time, the motor value of the Glasgow Coma Scale, effects of external ventricular drainage and rebleeding on outcome. Six-month outcome was assessed with the extended Glasgow Outcome Scale; favorable outcome was defined as good recovery or moderately disabled. Of 126 patients, 28 had absent brainstem reflexes, without improvement after external ventricular drainage. Rebleeding occurred in 26 patients, resulting in treatment withdrawal in 14. Only one patient had a favorable outcome after rebleeding. Of the 84 remaining patients, 61 improved at day 2 after subarachnoid hemorrhage to Glasgow Coma Scale motor value ≥4; 24 of these (39%) had a favorable outcome. All 23 patients with a Glasgow Coma Scale motor value ≤3 had an unfavorable outcome or died. Patients younger than 65 yrs of age had a better outcome (p < .03). Hydrocephalus was present in 71 of 84 patients. Favorable outcome was similar for patients with a positive external ventricular drainage response (8 of 28) as compared to no response to external ventricular drainage (12 of 43). The high rebleeding rate and subsequent poor outcome in World Federation of Neurologic Surgeons grade V patients warrants early treatment to secure the ruptured aneurysm. Favorable outcome was seen in 39% of patients with a Glasgow Coma Scale motor value ≥4 at day 2. In this study, patients with Glasgow Coma Scale motor value ≤3 at day 2 all had a very poor prognosis.


Whole Body Magnetic Resonance Imaging Features in Diffuse Idiopathic Skeletal Hyperostosis in Conjunction with Clinical Variables to Whole Body MRI and Clinical Variables in Ankylosing Spondylitis.
Discrimination of diffuse idiopathic skeletal hyperostosis (DISH) and ankylosing spondylitis (AS) can be challenging. Usefulness of whole-body magnetic resonance imaging (WB-MRI) in diagnosing spondyloarthritis has been recently proved. We assessed the value of clinical variables alone and in combination with WB-MRI to distinguish between DISH and AS. Diagnostic case-control study: 33 patients with AS and 15 patients with DISH were included. All patients underwent 1.5 Tesla WB-MRI scanning. MR scans were read by a blinded radiologist using the Canadian-Danish Working Group's recommendation. Imaging and clinical variables were identified using the bootstrap. The most important variables from MR and clinical history were assessed in a multivariate fashion resulting in 3 diagnostic models (MRI, clinical, and combined). The discriminative capacity was quantified using the area under the receiver-operating characteristic (ROC) curve. The strength of diagnostic variables was quantified with OR. Forty-eight patients provided 1545 positive findings (193 DISH/1352 AS). The final MR model contained upper anterior corner fat infiltration (32 DISH/181 AS), ankylosis on the vertebral endplate (4 DISH/60 AS), facet joint ankylosis (4 DISH/49 AS), sacroiliac joint edema (11 DISH/91 AS), sacroiliac joint fat infiltration (2 DISH/114 AS), sacroiliac joint ankylosis (2 DISH/119 AS); area under the ROC curve was 0.71, 95% CI 0.64-0.78. The final clinical model contained patient's age and body mass index (area under the ROC curve 0.90, 95% CI 0.89-0.91). The full diagnostic model containing clinical and MR information had an area under the ROC curve of 0.93 (95% CI 0.92-0.95). WB-MRI features can contribute to the correct diagnosis after a thorough conventional workup of patients with DISH and AS.


Environmental Contaminants and Congenital Heart Defects: A Re-Evaluation of the Evidence.
Congenital heart defects (CHDs) are a common birth defect of largely unknown etiology, with high fetal and neonatal mortality. A review of CHDs and environmental contaminant exposure found that meta-analyses showed only modest associations for smoking, vehicle exhaust components, disinfectant by-products and proximity to incinerators, with stronger results from the newer, larger and better quality studies masked by the typical absence of effect in older studies. Recent studies of exposure to agricultural pesticides, solvents, metals and landfill sites also showed associations. Certain contaminants have been associated with certain CHDs, with septal defects being the most common. Frequent methodological problems include failure to account for potential confounders or maternal/paternal preconception exposure, differences in diagnosing, defining and classifying CHDs, grouping of defects to increase power, grouping of contaminants with dissimilar mechanisms, exclusion of pregnancies that result in death or later life diagnosis, and the assumption that maternal residence at birth is the same as at conception. Furthermore, most studies use measurement estimates of one exposure, ignoring the many additional contaminant exposures in daily life. All these problems can distort and underestimate the true associations. Impaired methylation is a common mechanism, suggesting that supplementary folate may be protective for any birth defect.


A molecular rationale for the how, when and why of colorectal cancer screening.
Colorectal cancer remains a leading cause of cancer-related mortality in the United States. Recently, colorectal cancer screening and colorectal cancer prevention have gained national attention. In response, the American Gastroenterological Association, the American College of Gastroenterology and the Agency for Healthcare Policy and Research have published recommendations for colorectal cancer screening and surveillance in patients with sporadic as well as hereditary forms of colorectal cancer. This review will focus on the basic molecular differences underlying the formation of carcinoma in patients with sporadic colorectal cancer, and the heritable syndromes of familial adenomatous polyposis (FAP), hereditary non-polyposis colorectal cancer (HNPCC), and juvenile polyposis (JPS). By appreciating the molecular mechanisms underlying these four types of polyp cancer syndromes, the differences in clinical time course for progression from polyp to carcinoma and in current screening recommendations for patients with sporadic adenomas, FAP, HNPCC and JPS can be better understood.


Molecular assays for the diagnosis of sepsis in neonates.
Microbial cultures for diagnosis of neonatal sepsis have low sensitivity and reporting delay. Advances in molecular microbiology have fostered new molecular assays that are rapid and may improve neonatal outcomes. To assess the diagnostic accuracy of various molecular methods for the diagnosis of culture-positive bacterial and fungal sepsis in neonates and to explore heterogeneity among studies by analyzing subgroups classified by gestational age and type of sepsis onset and compare molecular tests with one another. We performed the systematic review as recommended by the Cochrane Diagnostic Test Accuracy Working Group. On 19 January 2016, we searched electronic bibliographic databases (the Cochrane Library, PubMed (from 1966), Embase (from 1982), and CINAHL (from 1982)), conference proceedings of the Pediatric Academic Societies annual conference (from 1990), clinical trial registries (ClinicalTrials.gov, International Standard Randomised Controlled Trial Number (ISRCTN) registry, and World Health Organization (WHO) International Clinical Trials Platform (ICTRP) Search portal), and Science Citation Index. We contacted experts in the field for studies. We included studies that were prospective or retrospective, cohort or cross-sectional design, which evaluated molecular assays (index test) in neonates with suspected sepsis (participants) in comparison with microbial cultures (reference standard). Two review authors independently assessed the methodologic quality of the studies and extracted data. We performed meta-analyses using the bivariate and hierarchical summary receiver operating characteristic (HSROC) models and entered data into Review Manager 5. Thirty-five studies were eligible for inclusion and the summary estimate of sensitivity was 0.90 (95% confidence interval (CI) 0.82 to 0.95) and of specificity was 0.93 (95% CI 0.89 to 0.96) (moderate quality evidence). We explored heterogeneity by subgroup analyses of type of test, gestational age, type of sepsis onset, and prevalence of sepsis and we did not find sufficient explanations for the heterogeneity (moderate to very low quality evidence). Sensitivity analyses by including studies that analyzed blood samples and by good methodology revealed similar results (moderate quality evidence). Molecular assays have the advantage of producing rapid results and may perform well as 'add-on' tests.


Endogenous IRAK-M attenuates postinfarction remodeling through effects on macrophages and fibroblasts.
Effective postinfarction repair requires timely suppression of innate immune signals to prevent the catastrophic consequences of uncontrolled inflammation on cardiac geometry and function. In macrophages, interleukin-1 receptor-associated kinase (IRAK)-M acts as a functional decoy preventing uncontrolled toll-like receptor /interleukin-1-mediated responses. Our study investigates the role of IRAK-M as a negative regulator of the postinfarction inflammatory response and as a modulator of cardiac remodeling. In wild-type mouse infarcts IRAK-M was upregulated in infiltrating macrophages and fibroblasts exhibiting a biphasic response. When compared with wild-type animals, infarcted IRAK-M(-/-) mice had enhanced adverse remodeling and worse systolic dysfunction; however, acute infarct size was comparable between groups. Adverse remodeling in IRAK-M(-/-) animals was associated with enhanced myocardial inflammation and protease activation. The protective actions of IRAK-M involved phenotypic modulation of macrophages and fibroblasts. IRAK-M(-/-) infarcts showed increased infiltration with proinflammatory CD11b+/Ly6C(hi) monocytes; leukocytes harvested from IRAK-M-null infarcts exhibited accentuated cytokine expression. In vitro, IRAK-M expression was upregulated in cytokine-stimulated murine cardiac fibroblasts and suppressed their matrix-degrading properties without affecting their inflammatory activity. Endogenous IRAK-M attenuates adverse postinfarction remodeling suppressing leukocyte inflammatory activity, while inhibiting fibroblast-mediated matrix degradation.


Ethics approval: a challenge for public health researchers in India.
There is increasing impetus, interest and opportunity for people working in public health programmes in India to carry out operational research (OR) around relevant programme issues and then publish that in peer-reviewed publications. These published researches are valuable in analysing, documenting and advocating for locally generated evidence to inform policy and practice. Ethics review and approval is an essential step in the process of OR but is often viewed as a barrier rather than a prerequisite of good practice in OR. Journals and peer reviewers are also increasingly requiring approvals from local institutional ethics committees (IECs).


Three-dimensional experiments and individual based simulations show that cell proliferation drives melanoma nest formation in human skin tissue.
Melanoma can be diagnosed by identifying nests of cells on the skin surface. Understanding the processes that drive nest formation is important as these processes could be potential targets for new cancer drugs. Cell proliferation and cell migration are two potential mechanisms that could conceivably drive melanoma nest formation. However, it is unclear which one of these two putative mechanisms plays a dominant role in driving nest formation. We use a suite of three-dimensional (3D) experiments in human skin tissue and a parallel series of 3D individual-based simulations to explore whether cell migration or cell proliferation plays a dominant role in nest formation. In the experiments we measure nest formation in populations of irradiated (non-proliferative) and non-irradiated (proliferative) melanoma cells, cultured together with primary keratinocyte and fibroblast cells on a 3D experimental human skin model. Results show that nest size depends on initial cell number and is driven primarily by cell proliferation rather than cell migration. Nest size depends on cell number, and is driven primarily by cell proliferation rather than cell migration. All experimental results are consistent with simulation data from a 3D individual based model (IBM) of cell migration and cell proliferation.


Survey of human group C rotaviruses in Japan during the winter of 1992 to 1993.
Fecal specimens from patients with acute diarrhea were collected from 10 prefectures in Japan over a 6-month period (November 1992 to April 1993), and the specimens that were negative for human group A rotaviruses were screened for the presence of human group C rotaviruses (CHRVs) by the reverse passive hemagglutination test. Of 784 specimens examined, 53 samples (6.8%) that were collected in 7 of 10 prefectures were positive for CHRV, indicating that CHRVs are widely distributed across Japan. Most of the CHRV isolates were detected in March and April, and CHRVs mainly prevailed in children ages 3 to 8 years. The genome electropherotypes of eight strains isolated in five individual prefectures were surprisingly similar to each other and were different from those of CHRV strains isolated to date. The outer capsid glycoprotein (VP7) gene homologies of the isolates retrieved in 1993 were subsequently analyzed by the dot blot hybridization method. As a result, the VP7 genes of the isolates revealed very high levels of homology not only with each other but also with the VP7 gene of the OK118 strain isolated in 1988. These results suggest that a large-scale outbreak of CHRV occurred during the winter of 1992 and 1993 in Japan.


Recent trends in smoking and the role of public policies: results from the SimSmoke tobacco control policy simulation model.
After a period of steady decline in smoking prevalence, smoking rates leveled off in the United States between 1990 and 1997, but began falling between 1997 and 2003. Trends in smoking prevalence, and the role of tobacco control policies in affecting those rates, are examined. A computer simulation model is used in which smoking prevalence evolves through initiation and cessation, which are in turn influenced by tobacco control policies. The results of the model are compared to smoking prevalence measures from the US National Health Interview Survey between 1993 and 2003. We also consider the role of tax/price, clean air laws, media campaigns and youth access policies in influencing these rates. Both the SimSmoke model and data for recent years indicate that adult smoking prevalence changed little between 1993 and 1997, and even increased among youth. Between 1997 and 2003, smoking prevalence has been declining. Most age, gender and racial-ethnic groups show patterns similar to that of the entire population, with differences for those aged 18-24 years. The predominant trends were explained mainly by changes in price, with some residual effect of clean air laws, media campaigns and youth access laws. Among public tobacco control policies, price had the dominant effect on smoking prevalence between 1993 and 2003, because few states implemented other policies to the degree necessary to affect much change. Through continued tax increases, stronger clean air laws, extensive media campaigns and broader cessation treatment programs, there is the potential to have much larger reductions in smoking prevalence.


Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs.
Combination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases. We show that helper-dependent adenoviral vectors (HDAdVs) provide a highly efficient and safe method for correcting mutations in large genomic regions in human induced pluripotent stem cells and can also be effective in adult human mesenchymal stem cells. This type of approach could be used to generate genotype-matched cell lines for disease modeling and drug discovery and potentially also in therapeutics.


Availability, safety and quality of blood for transfusion in the Americas.
This article has two objectives: (1) to present for countries and territories of the Region of the Americas data on the number of blood donations, proportion of voluntary blood donors versus remunerated blood donors, coverage of screening for infectious agents, and separation of donated blood into its components and (2) to explore the relationships of those characteristics with economic and organisational factors in the countries and territories. We carried out comparative analyses using population and health information gathered annually by the Pan American Health Organization (PAHO) from national health officials from the countries in the Americas, as well as economic information (gross national product (GNP) per capita) obtained from publications of the World Bank. There is a direct correlation between the availability of blood for transfusion and GNP per capita. Seven countries with a GNP per capita above US dollar 10,000 per year account for 38% of the Regional population but 68% of the Regional blood donations. Voluntary blood donation is more common in the countries with better blood availability. There is no association between GNP per capita and coverage of screening for infectious agents. Nevertheless, of the six countries with a GNP per capita below US dollar 1,000, only one of the six screens all units for human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B surface antigen (HBsAg). Countries with a higher proportion of voluntary blood donors tend to have lower prevalence rates of infectious markers. Separation of blood into its components is also more common in countries with higher blood donation rates. The availability, safety and quality of blood for transfusion in the Americas needs to be improved. As part of that effort, national policies and strategies must be put into place so that the resources already allocated for blood services are better utilised.


Socio-economic, environmental and nutritional characteristics of urban and rural South Indian women in early pregnancy: findings from the South Asian Birth Cohort (START).
High frequency of low birth weight (LBW) is observed in rural compared with urban Indian women. Since maternal BMI is known to be associated with pregnancy outcomes, the present study aimed to investigate factors associated with BMI in early pregnancy of urban and rural South Indian women. Prospective observational cohort. A hospital-based study conducted at an urban and a rural health centre in Karnataka State. Pregnant women (n 843) aged 18-40 years recruited in early pregnancy from whom detailed sociodemographic, environmental, anthropometric and dietary intake information was collected. A high proportion of low BMI (32 v. 26 %, P<0·000) and anaemia (48 v. 23 %, P<0·000) was observed in the rural v. the urban cohort. Rural women were younger, had lower body weight, tended to be shorter and less educated. They lived in poor housing conditions, had less access to piped water and good sanitation, used unrefined fuel for cooking and had lower standard of living score. The age (β=0·21, 95 % CI 0·14, 0·29), education level of their spouse (β=1·36, 95 % CI 0·71, 2·71) and fat intake (β=1·24, 95 % CI 0·20, 2·28) were positively associated with BMI in urban women. Our findings indicate that risk factors associated with BMI in early pregnancy are different in rural and urban settings. It is important to study population-specific risk factors in relation to perinatal health.


Prognostic Value of Plasma Fibrinogen in Lung Cancer Patients: A Meta-Analysis.
Background: The prognostic role of plasma fibrinogen in lung cancer remains controversial. The aim of this meta-analysis was to assess the prognostic value of plasma fibrinogen in lung cancer. Methods: We performed a systematic literature search to identify eligible studies in PubMed, Embase and the Cochrane Library database. The hazard ratios (HR) and their 95% confidence intervals (CI) were collected from these eligible studies and were used to assess the relationship between plasma fibrinogen and lung cancer. Results: A total of 16 studies including 6,881 patients were selected in this meta-analysis. The results showed that elevated plasma fibrinogen in lung cancer patients was correlated with poor overall survival (OS) (HR = 1.38, 95% CI: 1.22-1.55, P < 0.001) and disease-free survival (DFS) / progress-free survival (PFS). (HR = 1.29, 95% CI: 1.01-1.65, P = 0.042). When stratified by cut-off value for OS and DFS/PFS, there was no significant heterogeneity. And the results of "cut-off value ≥ 400mg/dl" group showed that the high level of fibrinogen in serum was associated with worse OS and DFS/PFS of lung cancer. In further subgroup analysis by tumor histology, high plasma fibrinogen was also associated with worse OS in non-small cell lung cancer (NSCLC) (HR = 1.32, 95% CI: 1.14-1.53, P < 0.001). However, there was no significant association between high plasma fibrinogen and poor DFS in NSCLC patients (HR = 1.24, 95% CI: 0.97-1.57, P = 0.08). The Egger's regression test indicated evidence of publication bias for DFS/PFS. Conclusions: Elevated plasma fibrinogen, particularly defined as a plasma fibrinogen concentration of ≥ 400mg/dl, could be a promising indicator for worse OS in lung cancer patients, including NSCLC.


Antiretroviral drug resistance mutations in naïve and experienced patients in Shiraz, Iran, 2014.
Resistance to antiretroviral agents is a significant concern in the clinical management of HIV-infected individuals, particularly in areas of the world where treatment options are limited. In this study, we aimed to identify HIV drug-resistance-associated mutations in 40 drug-naïve patients and 62 patients under antiretroviral therapy (ART) referred to the Shiraz HIV/AIDS Research Center - the first such data available for the south of Iran. HIV reverse transcriptase and protease genes were amplified and sequenced to determine subtypes and antiretroviral- resistance-associated mutations (RAMs). Subtype CRF35-AD recombinant was the most prevalent in all patients (98 of 102, 96 %), followed by subtype A1, and subtype B (one each, 2 %). Among the 40 ART-naïve patients, two mutations associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance (two with Y115F and T215I) and three associated with non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance (two with G190S and Y181C, four with V179T) were found. Among ART-experienced patients, four mutations associated with resistance to NRTI, four with NNRTI, and five with protease inhibitors (PI) were found. Twenty patients with high levels of resistance were already on second-line therapy. We document for the first time in this region of Iran high levels of ART resistance to multiple drugs. Our findings call for more vigilant systematic ART resistance surveillance, increased resistance testing, careful management of patients with existing regimens, and strong advocacy for expansion of available drugs in Iran.


Genetic strategies for dissecting complex traits in biomass willows (Salix spp.).
Willows are highly diverse catkin-bearing trees and shrubs of the genus Salix. They occur in many growth forms, from tall trees to creeping alpines, and successfully occupy a wide variety of ecological niches. Shrubby willows (sub-genus Vetrix) have many characteristics that render them suited to cultivation in much faster growth cycles than conventional forestry. They respond well to coppicing, can be propagated vegetatively as cuttings and achieve rapid growth with low fertilizer inputs. As a result, willows grown as short rotation coppice are now among the leading commercially grown biomass crops in temperate regions. However, although willows have a long history of cultivation for traditional uses, their industrial use is relatively recent and, compared with major arable crops, they are largely undomesticated. Breeding programmes initiated to improve willow as a biomass crop achieved a doubling of yields within a period of <15 years. These advances were made by selecting for stem characteristics (height and diameter) and coppicing response (shoot number and shoot vigour), as well as resistance to pests, diseases and environmental stress, with little or no knowledge of the genetic basis of these traits. Genetics and genomics, combined with extensive phenotyping, have substantially improved our understanding of the basis of biomass traits in willow for more targeted breeding via marker-assisted selection. Here, we present the strategy we have adopted in which a genetic-based approach was used to dissect complex traits into more defined components for molecular breeding and gene discovery.


The prognostic significance of faecal calprotectin in patients with inactive inflammatory bowel disease.
Faecal calprotectin, an established biomarker used to assess mucosal inflammation, has been shown to correlate with endoscopic activity in inflammatory bowel disease (IBD). Longitudinal monitoring of faecal calprotectin, however, has rarely been employed beyond assessment of therapy response and post hoc analyses of clinical trials. To study whether consecutive measurements of faecal calprotectin every third month are useful for monitoring patients with IBD in clinical remission. Patients aged 18 years or older, with a known diagnosis of IBD in clinical remission, were prospectively studied. Patients provided faecal samples every third month and were prospectively followed until the first clinical relapse or the end of the 2-year follow-up period. Measurements (EK-CAL, Bühlmann Lab. AG, Switzerland) were done at the end of the study. A Cox model with time-dependent covariates was used for analysis. Among 104 patients, Crohn's disease (n = 49) and ulcerative colitis (n = 55), 37 had a relapse. A doubling of faecal calprotectin level between two consecutively collected samples was associated with a 101% increased risk of relapse (HR: 2.01; 95% CI: 1.53-2.65; P < 0.001). The relative risk of relapse attenuated with time (HR: 0.80; 95% CI: 0.75-0.86; P < 0.001), by a 20% decrease in risk of relapse per 3-month period since the sample was obtained. By consecutively measuring faecal calprotectin every third month, we quantified the risk of relapse related to faecal calprotectin change and observed attenuation of the risk across time. Our data suggest that longitudinal monitoring of faecal calprotectin is informative in predicting relapse in IBD.


Measurement of the third component of complement bound to red blood cells in patients with the cold agglutinin syndrome.
The amount of the third component of complement (C3) bound to red cells of patients with the cold agglutinin syndrome was determined by a quantitative assay, measuring the fixation of the first component of complement by anti-C3. Abrupt reduction in the serum concentration of cold agglutinin by plasmapheresis markedly decreased the hemolytic rate, but the amount of C3 bound to circulating cells did not change appreciably. When this patient was transfused with normal cells. C3 accumulated on the transfused cells within 48 h to the level present on his own cells, but selective destruction of the transfused cells did not occur. When patients were subjected to acute cold stress, cell-bound C3 rose abruptly and intravascular hemolysis occurred. These studies suggest that most of the C3 detected on the circulating red cells of cold agglutinin patients is in an inactive form, and that the rate of attachment of C3 to the membrane is important in determining hemolysis.


A 39-year-old woman with hypercholesterolemia.
Ms T, a 39-year-old woman, has a total cholesterol level of 277 mg/dL (7.17 mmol/L) and well-controlled hypertension; her brother had a stroke in his 30s. She is primarily concerned with her mother's history of breast cancer, but she would like to know if she can take a dietary supplement or if she needs to take cholesterol-lowering medication, and if so, whether she will need to continue it as well as adhere to her diet for the rest of her life. Her estimated 10-year risk of developing coronary heart disease (CHD) is 1% to 2% using the Framingham Risk Score, but that may underestimate her true risk as an African American woman with a family history of CHD. Recommendations for her, her longer-term risk for CHD, and evidence for lipid-lowering therapies are discussed.


Community-based wastewater treatment systems and water quality of an Indonesian village.
This paper examines the impact of community-based water treatment systems on water quality in a peri-urban village in Yogyakarta, Indonesia. Water samples were taken from the wastewater treatment plants (WWTPs), irrigation canals, paddy fields and wells during the dry and wet seasons. The samples were tested for biological and chemical oxygen demand, nutrients (ammonia, nitrate, total nitrogen and total phosphorus) and Escherichia coli. Water quality in this village is affected by the presence of active septic tanks, WWTP effluent discharge, small-scale tempe industries and external sources. We found that the WWTPs remove oxygen-demanding wastes effectively but discharged nutrients, such as nitrate and ammonia, into irrigation canals. Irrigation canals had high levels of E. coli as well as oxygen-demanding wastes. Well samples had high E. coli, nitrate and total nitrogen levels. Rainfall tended to increase concentrations of biological and chemical oxygen demand and some nutrients. All our samples fell within the drinking water standards for nitrate but failed the international and Indonesian standards for E. coli. Water quality in this village can be improved by improving the WWTP treatment of nutrients, encouraging more villagers to be connected to WWTPs and controlling hotspot contamination areas in the village.


Fish oil supplementation with and without added vitamin E differentially modulates plasma antioxidant concentrations in healthy women.
The purpose of this study was to assess the effect of fish oil with or without vitamin E on plasma vitamin antioxidants. Thirty-three apparently healthy women aged 18-28 yr were recruited from the university environs, and 30 completed the double-blinded, parallel design supplementation trial. Blood samples were collected at baseline (week 0) and following 28 d of supplementation with three capsules/d (0.8 g x 3) of either fish oil (FO) or FO with vitamin E (3 IU/g) (FOE). An additional blood sample was taken at day 91 (washout). Plasma antioxidant vitamins, fatty acid composition, and lipid peroxides were measured. Plasma alpha-tocopherol concentrations were increased significantly in both groups postsupplementation FO (P = 0.018) and FOE (P = 0.003) compared with baseline and washout values. Plasma retinol concentration was significantly increased (P = 0.034) compared with baseline and washout values following supplementation with FOE but not FO, while plasma beta-carotene was significantly increased (P = 0.036), compared with baseline and washout values, following supplementation with FO but not FOE. There was a trend (P = 0.059) toward decreased plasma ascorbic acid following FO supplementation compared with baseline and washout. Plasma lipid peroxides did not change following either supplementation. Results suggest that low-dose FO feeding with and without vitamin E differentially modulates plasma antioxidant vitamins but has no significant effect on lipid peroxidation.


A bacterial endo-β-1,4-glucuronan lyase, CUL-I from Brevundimonas sp. SH203, belonging to a novel polysaccharide lyase family.
Cellouronate is a (1,4)-β-D-glucuronan prepared by TEMPO-mediated oxidation from regenerated cellulose. We have previously isolated a cellouronate-degrading bacterial strain, Brevundimonas sp. SH203, that produces a cellouronate lyase (β-1,4-glucuronan lyase, CUL-I). In this study, the gene encoding CUL-I was cloned, and the recombinant enzyme was heterologously expressed in Escherichia coli. The predicted CUL-I protein is composed of 426 amino acid residues and includes a putative 21-amino acid signal peptide. The recombinant CUL-I specifically depolymerized β-1,4-glycoside linkages of cellouronate, and its mode of action was endo-type, like the native CUL-I. Sequence analysis showed CUL-I has no similarity to previously known polysaccharide lyases (PLs), indicating that CUL-I should be classified into a novel PL family.


Communicating effectively with vulnerable populations during water contamination events.
Water contamination events are a public health concern worldwide with significant potential to impact the global community. When communicating with the public during these crisis situations, it is vital to consider the multiple audiences who receive the messages. Before developing or delivering messages to a particular community, it is essential to be familiar with the community's characteristics, needs, concerns, and who is considered credible to that community. Vulnerable populations are those with difficulties in comprehension or accessibility that may limit their full understanding of risks and may mitigate the effectiveness of public health strategies. Vulnerable populations include, but are not limited to, the urban/rural poor, those who are mentally ill, intellectually disabled, medically vulnerable, at the extremes of age (children and the elderly), racial/ethnic minorities, and those with low literacy or limited English proficiency.A water contamination event poses a unique opportunity to work with diverse populations to effectively convey important health messages. Each population needs to receive appropriate public health messages. Becoming familiar with vulnerable populations and their needs prior to a water contamination event will help in identifying barriers and developing and refining effective messages in such a crisis. In water contamination crises, our publics' health depends on effective, targeted crisis communication.


What is driving the black-white difference in low birthweight in the US?
This is a first effort to quantify the contribution of different factors in explaining racial difference in low birthweight rate (LBW). Mother's health, child characteristics, prenatal care, socioeconomic status (SES), and the socioeconomic and healthcare environment of mother's community are important inputs into the birthweight production function, and a vast literature has delved into obtaining causal estimates of their effect on infant health. What is unknown is how much of the racial gap in LBW is explained by all these inputs together. We apply a nonlinear extension of the Oaxaca-Blinder method proposed by Fairlie to decompose this gap into the portion explained by differences in observed characteristics and the portion that remains unexplained. Data are obtained from several sources in order to capture as many observables as possible, although the primary data source is the Natality Detail Files. Results show that of the 6.8 percentage point racial gap in LBW, only 0.9-1.9 points are explained by white-black differences in endowments across those measures, and of those endowments, most of the gap in LBW is explained by the differences in SES. The unexplained difference is attributed to racial differences in the returns to or the marginal product of investments in infant health.


Risk of serious adverse cardiovascular events associated with varenicline: a systematic review and meta-analysis.
There have been postmarketing reports of adverse cardiovascular events associated with the use of varenicline, a widely used smoking cessation drug. We conducted a systematic review and meta-analysis of randomized controlled trials to ascertain the serious adverse cardiovascular effects of varenicline compared with placebo among tobacco users. We searched MEDLINE, EMBASE, the Cochrane Database of Systematic Reviews, websites of regulatory authorities and registries of clinical trials, with no date or language restrictions, through September 2010 (updated March 2011) for published and unpublished studies. We selected double-blind randomized controlled trials of at least one week's duration involving smokers or people who used smokeless tobacco that reported on cardiovascular events (ischemia, arrhythmia, congestive heart failure, sudden death or cardiovascular-related death) as serious adverse events asociated with the use of varenicline. We analyzed data from 14 double-blind randomized controlled trials involving 8216 participants. The trials ranged in duration from 7 to 52 weeks. Varenicline was associated with a significantly increased risk of serious adverse cardiovascular events compared with placebo (1.06% [52/4908] in varenicline group v. 0.82% [27/3308] in placebo group; Peto odds ratio [OR] 1.72, 95% confidence interval [CI] 1.09-2.71; I(2) = 0%). The results of various sensitivity analyses were consistent with those of the main analysis, and a funnel plot showed no publication bias. There were too few deaths to allow meaningful comparisons of mortality. Our meta-analysis raises safety concerns about the potential for an increased risk of serious adverse cardiovascular events associated with the use of varenicline among tobacco users.


In vivo functional analysis of the human mitochondrial DNA polymerase POLG expressed in cultured human cells.
The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.


Lifestyle behaviours associated with 5-year weight gain in a prospective cohort of Australian adults aged 26-36 years at baseline.
Whether not meeting common guidelines for lifestyle behaviours is associated with weight gain is uncertain. This study examined whether 5-year weight gain was predicted by not meeting guidelines for: breakfast consumption (eating between 6 and 9 am), takeaway food consumption (<2 times/week), television viewing (<2 h/day) and daily steps (≥10,000 steps/day). One thousand one hundred and fifty-five Australian participants (43% men, 26-36 years) completed questionnaires and wore a pedometer at baseline (2004-06) and follow-up (2009-11). Weight was measured or self-reported, with a correction factor applied. For each behaviour, participants were classified according to whether they met the guideline: consistently met at baseline and follow-up (reference group); not met at baseline but met at follow-up; met at baseline but not met at follow-up; consistently not met at baseline and follow-up. For each behaviour, weight gain was calculated using linear regression. Weight gain by number of guidelines met was also examined. Mean 5-year weight gain was 2.0 kg (SD:6.3). Compared to the reference group, additional weight (mean, 95% CI) was gained among those who did not meet the guideline at follow-up, or consistently did not meet the guideline, for breakfast (1.8 kg, 0.7-2.9; 1.5 kg, 0.1-2.8); takeaway food (2.2 kg, 0.7-3.6; 1.9 kg, 0.7-3.1); watching television (1.9 kg, 0.9-2.9; 1.4 kg, 0.4-2.3); and daily steps (2.6 kg, 1.1-4.04; 1.6 kg, 0.5-2.7). Those who met ≤1 guideline at follow-up gained 3.8 kg (95% CI 2.3-5.3) more than those meeting all guidelines. Individuals who adopted healthier behaviours between baseline and follow-up had similar weight gain to those who met the guidelines at both time points. Encouraging young adults to meet these simple guidelines may reduce weight gain.


Next generation sequencing uncovers unexpected bacterial pathogens in ticks in western Europe.
Ticks are highly susceptible to global environmental and socio-economical changes. Several tick-borne pathogens have been reported in new geographical regions while new species, strains or genetic variants of tick-borne microorganisms are continually being detected. However, tick-borne pathogens are still poorly understood, and it is estimated that half of all human tick-borne disease has an unknown origin. Therefore in order to prevent these diseases, more effort is required to identify unknown or unexpected tick-borne pathogens. Ixodes ricinus is the vector for a broad range of bacterial pathogens and the most prevalent tick in Europe. The aim of the present study was to evaluate the capability of Next Generation Sequencing (NGS) to extend the inventory of pathogenic bacteria carried by this species of tick in France. RNA and DNA were extracted from 1450 I. ricinus questing nymphs collected by flagging in Alsace, France. RNA was pooled and used for NGS. Following de novo assembly, bacterial contigs were assigned to the closest known taxonomy. DNA was used for real time PCR to confirm taxonomic species assignment of NGS-derived contigs for the doubtful cases, and for determination of prevalence. We have generated a global in-depth picture of tick-borne bacteria. We identified RNA from the main pathogenic bacterial species known to be transmitted by I. ricinus. In addition we also identified unanticipated bacterial species for which we have estimated the prevalence within those ticks inhabiting the studied areas. The data obtained from this study has proven that NGS has an enormous potential to detect the unexpected and provides the means to monitor pathogen occurrence.


Stable dispersions of azide functionalized ferromagnetic metal nanoparticles.
Ferromagnetic nanoparticles are covalently modified in order to enhance the dispersion stability as well as the antifouling properties. Insertion of an azide moiety allows "click"-reaction of a relevant tag molecule. This and the high saturation magnetization of the presented nanocomposite offer a promising platform for magnetic biosensors.


Electrochemical water oxidation using a copper complex.
Herein, we report the application of the mononuclear copper complex 1, [CuII(L)]2+, in electrochemical water oxidation catalysis (L = 1,3-bis(((1-methyl-1H-imidazol-2-yl)methyl)amino)propan-2-ol). The complex exhibits a N4 donor set consisting of two amine and two imidazole units and a dangling OH unit in close proximity to the copper ion. 1 exhibits a moderate apparent rate constant kcat of 0.12 s-1 in catalysis and operates at an overpotential of 0.83 V. Detailed investigations allowed us to derive a mechanism for water oxidation. The catalysis proceeds only under basic conditions, where [CuII(L)(OH)]+, 1H-1, is the main solution species, which indicates that a negatively charged ligand is necessary to drive the catalysis. Initial oxidation of 1H-1 is coupled to proton loss forming a copper(iii) species and further oxidation initiates oxygen evolution. Initial oxidation of 1 under neutral, i.e. non-catalytic, conditions is pH independent, highlighting the importance of PCET steps during catalysis. We collected reasonable evidence that catalysis proceeds via a water nucleophilic attack mechanism. The electrolyte presumably acts as a proton acceptor in catalysis as the onset potential depends on the buffer employed.


Down-regulation of cyclic nucleotide phosphodiesterase PDE1A is the key event of p73 and UHRF1 deregulation in thymoquinone-induced acute lymphoblastic leukemia cell apoptosis.
Thymoquinone (TQ), the active principle of Nigella sativa black seeds, has anti-proliferative properties on numerous cancer cell types. Others and we have previously reported that TQ acts as agent that triggers cell cycle arrest and apoptosis through either a p53- or p73-dependent pathway. However, the immediate targets recruited upon TQ-induced cytotoxicity have not yet been clearly identified. We therefore asked whether cyclic nucleotide phosphodiesterases (PDEs) could be involved in TQ-triggered pro-apoptotic reactivity; PDEs are regulators of intracellular levels of cyclic nucleotides and therefore can modulate cAMP and cGMP-dependent cell death pathways. Our results showed that TQ specifically repressed PDE1A expression in the acute lymphoblastic leukemia Jurkat cell line. This effect is concomitant with the previously described sequential deregulation of the expression of the tumor suppressor protein p73 and the epigenetic integrator UHRF1 (Ubiquitin-like, PHD Ring Finger 1). Interestingly, RNA-interference knock-down of PDE1A expression as well as decreased PDE1A expression induced growth inhibition of Jurkat cells, cell cycle arrest and apoptosis through an activation of p73 and a repression of UHRF1. Conversely, PDE1A re-expression counteracted the cellular pro-apoptotic effects of TQ in association with a p73 repression and UHRF1 re-expression. Altogether, our results show that TQ induced an initial down-regulation of PDE1A with a subsequent down-regulation of UHRF1 via a p73-dependent mechanism. This study further proposes that PDE1A might be involved in the epigenetic code inheritance by regulating, via p73, the epigenetic integrator UHRF1. Our findings also suggest that a forced inhibition of PDE1A expression might be a new therapeutic strategy for the management of acute lymphoblastic leukemia.


Data Analytics for Smart Parking Applications.
We consider real-life smart parking systems where parking lot occupancy data are collected from field sensor devices and sent to backend servers for further processing and usage for applications. Our objective is to make these data useful to end users, such as parking managers, and, ultimately, to citizens. To this end, we concoct and validate an automated classification algorithm having two objectives: (1) outlier detection: to detect sensors with anomalous behavioral patterns, i.e., outliers; and (2) clustering: to group the parking sensors exhibiting similar patterns into distinct clusters. We first analyze the statistics of real parking data, obtaining suitable simulation models for parking traces. We then consider a simple classification algorithm based on the empirical complementary distribution function of occupancy times and show its limitations. Hence, we design a more sophisticated algorithm exploiting unsupervised learning techniques (self-organizing maps). These are tuned following a supervised approach using our trace generator and are compared against other clustering schemes, namely expectation maximization, k-means clustering and DBSCAN, considering six months of data from a real sensor deployment. Our approach is found to be superior in terms of classification accuracy, while also being capable of identifying all of the outliers in the dataset.


Volunteer Conservation Action Data Reveals Large-Scale and Long-Term Negative Population Trends of a Widespread Amphibian, the Common Toad (Bufo bufo).
Rare and threatened species are the most frequent focus of conservation science and action. With the ongoing shift from single-species conservation towards the preservation of ecosystem services, there is a greater need to understand abundance trends of common species because declines in common species can disproportionately impact ecosystems function. We used volunteer-collected data in two European countries, the United Kingdom (UK) and Switzerland, since the 1970s to assess national and regional trends for one of Europe's most abundant amphibian species, the common toad (Bufo bufo). Millions of toads were moved by volunteers across roads during this period in an effort to protect them from road traffic. For Switzerland, we additionally estimated trends for the common frog (Rana temporaria), a similarly widespread and common amphibian species. We used state-space models to account for variability in detection and effort and included only populations with at least 5 years of data; 153 populations for the UK and 141 for Switzerland. Common toads declined continuously in each decade in both countries since the 1980s. Given the declines, this common species almost qualifies for International Union for the Conservation of Nature (IUCN) red-listing over this period despite volunteer conservation efforts. Reasons for the declines and wider impacts remain unknown. By contrast, common frog populations were stable or increasing in Switzerland, although there was evidence of declines after 2003. "Toads on Roads" schemes are vital citizen conservation action projects, and the data from such projects can be used for large scale trend estimations of widespread amphibians. We highlight the need for increased research into the status of common amphibian species in addition to conservation efforts focusing on rare and threatened species.


Histological, ultrastructural, and in situ hybridization study on enlarged cells in grouper Epinephelus hybrids infected by grouper iridovirus in Taiwan (TGIV).
Grouper iridovirus in Taiwan (TGIV) infection in the Epinephelus hybrid is a major problem in the grouper industry. ATPase gene sequences indicate that this virus is closely related to cell hypertrophy iridoviruses. Histologically, the appearance of basophilic or eosinophilic enlarged cells in internal organs is the most characteristic feature of this disease. These cells are acid-phosphatase positive and are able to phagocytose injected carbon particles. In our study, TGIV infection inhibited normal phagocytic ability in these cells in vivo after 4 d post-infection (p.i.) but not before 2 d p.i. Their staining properties and phagocytic ability suggested a monocyte origin of enlarged cells, which appeared in high numbers in the trunk kidney, head kidney, spleen and gill. After infection, the enlarged cells first appeared in the spleen, with an abundance peak at 64 h p.i. (Peak 1); at 120 h p.i., a second peak (Peak 2) occurred in the spleen, head kidney, trunk kidney and gill. Lower numbers of enlarged cells were observed in the liver, muscle, heart, eye, intestine, but no enlarged cells were found in the brain. A TGIV-specific DNA probe labeled most of the basophilic but not eosinophilic enlarged cells. Nuclei of infected cells were labeled during an early stage of the infection; at later stages, both nuclei and cytoplasms were labeled. Ultrastructurally, heterochromatins of the infected cells were marginated or aggregated to one side of the nuclei during the early stages of infection. Damage and rupture of the nuclear membrane started before formation of the viromatrix. Capsids were assembled in ring-shaped or disc-shaped structures. Bullet-shaped electron-dense material was present near the incomplete virus particles, and is speculated to be inserted into the capsids later.


Heterotopic auxiliary liver in an isolated and vascularized segment of the small intestine in rats.
Development of an auxiliary liver is of interest for treating several conditions. To examine whether an isolated intestinal segment will support development of a heterotopic auxiliary liver, we studied the fate of liver microfragments in rats. Small intestinal segments with intact circulation were created, and the small intestinal mucosa was removed. The intestinal segments were filled with autologous liver microfragments, and animals were studied for various periods. Initially, liver microfragments were engulfed by a serosanguineous exudate enriched in polymorphonuclear leukocytes, suggesting an early granulation-type response. Transplanted liver fragments were subsequently reorganized and showed morphologic integrity with typical hepatic lobular organization. Transplanted tissue contained healthy hepatocytes with abundant glycogen content. Transplanted liver remained intact in the small intestine for up to 40 days, although at later times portal fibrosis and bile ductular proliferation were apparent, despite the absence of cholestasis or hepatocellular abnormalities. In contrast, instillation of liver microfragments in the peritoneal cavity led to rapid loss of tissue integrity and phagocytotic clearance of transplanted tissue. Small intestinal segments denuded of the mucosal layer can support heterotopically transplanted liver. Further development of this auxiliary liver system will provide insights into mechanisms concerning neo-organogenesis and into potential therapeutic applications of heterotopic liver in specific diseases.


Inspection of three-dimensional structures of proteins with dynamical information from the normal mode analysis.
In this paper it is demonstrated that, to analyze structural data of proteins obtained from X-ray crystallography, the normal mode analysis in dihedral angle space can serve to supplement X-ray data as a useful system for gaining information on their dynamical as well as static structures. Especially, the following two subjects are discussed; first, the breakdown of the motions of a limited region in a polypeptide chain (e.g., an alpha-helix, a beta-strand or a loop) into internal and external motions reveals whether the region is flexible, or it is rigid but mobile when it has large fluctuations. Second, the correlation map between atomic motions serves to provide information for dividing the chain into segments, such as domains or modules, from a dynamical rather than from a geometrical point of view. It is shown that the modules proposed by M. Go appear distinctly in the correlation map as the regions in which clusters of atoms with positive correlation coefficients of their movements to each other exist. Furthermore, the modules are characterized by the negative correlation coefficients of the movements of the atoms in the clusters in a particular module to such movements in a different module.


Metabolite profiling identifies anandamide as a biomarker of nonalcoholic steatohepatitis.
The discovery of metabolite-phenotype associations may highlight candidate biomarkers and metabolic pathways altered in disease states. We sought to identify novel metabolites associated with obesity and one of its major complications, nonalcoholic fatty liver disease (NAFLD), using a liquid chromatography-tandem mass spectrometry method. In 997 individuals in Framingham Heart Study Generation 3 (FHS Gen 3), we identified an association between anandamide (AEA) and BMI. Further examination revealed that AEA was associated with radiographic hepatic steatosis. In a histologically defined NAFLD cohort, AEA was associated with NAFLD severity, the presence of nonalcoholic steatohepatitis, and fibrosis. These data highlight AEA as a marker linking cardiometabolic disease and NAFLD severity.


Utility of the Leptospermum scoparium Compound Lepteridine as a Chemical Marker for Manuka Honey Authenticity.
Manuka honey is a premium food product with unique antimicrobial bioactivity. Concerns with mislabeled manuka honey require robust assays to determine authenticity. Lepteridine is a Leptospermum-specific fluorescent molecule with potential as an authenticity marker. We describe a mass spectrometry-based assay to measure lepteridine based on an isotopically labeled lepteridine standard. Using this assay, lepteridine concentrations in manuka honey samples strongly correlated with concentrations quantitated by either high-performance liquid chromatography-ultraviolet (HPLC-UV) or fluorescence. A derived minimum lepteridine threshold concentration was compared with the New Zealand regulatory definition for manuka honey to determine "manuka honey" authenticity on a set of commercial samples. Both methods effectively distinguished manuka honey from non-manuka honeys. The regulatory definition excludes lepteridine but otherwise includes the quantification of multiple floral markers together with pollen analysis. Our findings suggest that the quantification of lepteridine alone or in combination with leptosperin could be implemented as an effective screening method to identify manuka honey, likely to achieve an outcome similar to the regulatory definition.


Mechanisms responsible for decline in transthoracic impedance after DC shocks.
To test the hypothesis that tissue hyperemia and edema in the current pathway cause a decrease in transthoracic impedance (TTI) following direct current (DC) shock, the thoracic-skin and skeletal blood flow and blood volume were measured in anesthetized dogs after three 100-J shocks. TTI declined 11% (P less than 0.01) after DC shocks. Blood flow increased 10-fold in skeletal muscle from 0.9 +/- 0.2 to 11.3 +/- 2.6 (SE) ml.100 g-1.min-1 (P less than 0.05). Blood flow did not change outside the current pathway. Blood volume increased in skin in the current pathway from 7.6 +/- 1.5 ml/100 g preshock to 17.5 +/- 2.0 ml/100 g (P less than 0.01) at 60 min after shock; skeletal muscle blood volume did not change. We also determined postshock tissue weight before and after 72 h of drying at 70 degrees C. The percentage decline from wet weight after drying was 68.4 +/- 3.4% in the current pathway vs. 64 +/- 3.8% outside the pathway (P less than 0.05), indicating the tissue in the current pathway was more edematous. Finally, hindlimb edema was induced by deliberate overperfusion. As hindlimb circumference (edema) increased, impedance declined. We conclude that increases in tissue blood flow or tissue blood volume or tissue edema contribute to the decline in transthoracic impedance after DC shock.


Pulmonary arterial hypertension in adults born with a heart septal defect: the Euro Heart Survey on adult congenital heart disease.
To investigate the role of pulmonary arterial hypertension (PAH) in adult patients born with a cardiac septal defect, by assessing its prevalence and its relation with patient characteristics and outcome. From the database of the Euro Heart Survey on adult congenital heart disease (a retrospective cohort study with a 5-year follow-up), the relevant data on all 1877 patients with an atrial septal defect (ASD), a ventricular septal defect (VSD), or a cyanotic defect were analysed. Most patients (83%) attended a specialised centre. There were 896 patients with an ASD (377 closed, 504 open without and 15 with Eisenmenger's syndrome), 710 with a VSD (275, 352 and 83, respectively), 133 with Eisenmenger's syndrome owing to another defect and 138 remaining patients with cyanosis. PAH was present in 531 (28%) patients, or in 34% of patients with an open ASD and 28% of patients with an open VSD, and 12% and 13% of patients with a closed defect, respectively. Mortality was highest in patients with Eisenmenger's syndrome (20.6%). In case of an open defect, PAH entailed an eightfold increased probability of functional limitations (New York Heart Association class >1), with a further sixfold increase when Eisenmenger's syndrome was present. Also, in patients with persisting PAH despite defect closure, functional limitations were more common. In patients with ASD, the prevalence of right ventricular dysfunction increased with systolic pulmonary artery pressure (OR = 1.073 per mm Hg; p<0.001). Major bleeding events were more prevalent in patients with cyanosis with than without Eisenmenger's syndrome (17% vs 3%; p<0.001). In this selected population of adults with congenital heart disease, PAH was common and predisposed to more symptoms and further clinical deterioration, even among patients with previous defect closure and patients who had not developed Eisenmenger's physiology.


Introduction of 2009 pandemic influenza A virus subtype H1N1 into South Africa: clinical presentation, epidemiology, and transmissibility of the first 100 cases.
We documented the introduction of 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) into South Africa and describe its clinical presentation, epidemiology, and transmissibility. We conducted a prospective descriptive study of the first 100 laboratory-confirmed cases of A(H1N1)pdm09 infections identified through active case finding and surveillance. Infected patients and the attending clinicians were interviewed, and close contacts were followed up to investigate household transmission. The first case was confirmed on 14 June 2009, and by 15 July 2009, 100 cases were diagnosed. Forty-two percent of patients reported international travel within 7 days prior to onset of illness. Patients ranged in age from 4 to 70 years (median age, 21.5 years). Seventeen percent of household contacts developed influenza-like illness, and 10% of household contacts had laboratory-confirmed A(H1N1)pdm09 infection. We found a mean serial interval (± SD) of 2.3 ± 1.3 days (range, 1-5 days) between successive laboratory-confirmed cases in the transmission chain. A(H1N1)pdm09 established itself rapidly in South Africa. Transmissibility of the virus was comparable to observations from outside of Africa and to seasonal influenza virus strains.