Control of the type 3 secretion system in Vibrio harveyi by quorum sensing through repression of ExsA.
The type 3 secretion system (T3SS) genes of Vibrio harveyi are activated at low cell density and repressed at high cell density by quorum sensing (QS). Repression requires LuxR, the master transcriptional regulator of QS-controlled genes. Here, we determine the mechanism underlying the LuxR repression of the T3SS system. Using a fluorescence-based cell sorting approach, we isolated V. harveyi mutants that are unable to express T3SS genes at low cell density and identified two mutations in the V. harveyi exsBA operon. While LuxR directly represses the expression of exsBA, complementation and epistasis analyses reveal that it is the repression of exsA expression, but not exsB expression, that is responsible for the QS-mediated repression of T3SS genes at high cell density. The present work further defines the genes in the V. harveyi QS regulon and elucidates a mechanism demonstrating how multiple regulators can be linked in series to direct the expression of QS target genes specifically at low or high cell density.


Improved tabletability after a polymorphic transition of delta-mannitol during twin screw granulation.
In most formulations processed via continuous twin screw granulation microcrystalline cellulose (MCC) and/or lactose are used as excipients, but mannitol is also a preferred excipient for wet granulation and tableting due to its non-hygroscopicity and inertness. Therefore, the aim of the current study was to investigate the influence of process parameters on critical quality attributes of granules (moisture content, solid state, morphology, size distribution, specific surface area, friability, flowability and hygroscopicity) and tablets (tensile strength and friability) after twin screw granulation of δ-mannitol. The δ-polymorph was selected since a moisture-induced transformation to β-mannitol was observed during batch wet granulation, which exhibited a unique morphology with a large surface area and improved tabletability. A full factorial experimental design was performed, varying screw speed (400-900rpm), granulation temperature (25-40°C), number of kneading elements (6 or 12) and liquid-to-solid (L/S) ratio, on the granulation unit of a ConsiGma™-25 line (a continuous powder-to-tablet manufacturing system). After tray drying the granules were milled and tableted. The results showed that the polymorphic transition from δ- to β-mannitol also occurred during twin screw granulation, although the residence time and L/S ratios were much lower in continuous twin screw granulation compared to batch processing. However, the polymorphic transition was not complete in all experiments and depended on the L/S ratio, screw speed and number of kneading elements. Nevertheless all granules exhibited the unique morphology linked to the polymorphic transition and had a superior tabletability compared to granules produced with β-mannitol as starting material. This was attributed to enhanced plastic deformation of the granules manufactured using δ-mannitol as starting material. In addition, it was concluded that mannitol was granulated via a different mechanism than other, less-soluble, excipients (e.g. lactose, microcrystalline cellulose) due to its high solubility and dissolution rate as the influence of process parameters on the mannitol granule characteristics was different.


Application of Capsid Integrity (RT-)qPCR to Assessing Occurrence of Intact Viruses in Surface Water and Tap Water in Japan.
Capsid integrity (RT-)qPCR has recently been developed to discriminate between intact forms from inactivated forms of viruses, but its applicability to identifying integrity of viruses in drinking water has remained limited. In this study, we investigated the application of capsid integrity (RT-)qPCR using cis-dichlorodiammineplatinum (CDDP) with sodium deoxycholate (SD) pretreatment (SD-CDDP-(RT-)qPCR) to detect intact viruses in surface water and tap water. A total of 63 water samples (surface water, n = 20; tap water, n = 43) were collected in the Kanto region in Japan and quantified by conventional (RT)-qPCR and SD-CDDP-(RT-)qPCR for pepper mild mottle virus (PMMoV) and seven other viruses pathogenic to humans (Aichivirus (AiV), noroviruses of genotypes I and II, enterovirus, adenovirus type 40 and 41, and JC and BK polyomaviruses). In surface water, PMMoV (100%) was more frequently detected than other human pathogenic viruses (30%-60%), as determined by conventional (RT-)qPCR. SD-CDDP-(RT-)qPCR also revealed that intact PMMoV (95%) was more common than intact human pathogenic viruses (20%-45%). In the tap water samples, most of the target viruses were not detected by conventional (RT-)qPCR, except for PMMoV (9%) and AiV (5%). PMMoV remained positive (5%), whereas no AiV was detected when tested by SD-CDDP-(RT-)qPCR, indicating that some PMMoV had an intact capsid, whereas AiV had damaged capsids. The presence of AiV in the absence of PMMoV in tap water produced from groundwater may demonstrate the limitation of PMMoV as a viral indicator in groundwater. In addition to being abundant in surface water, PMMoV was detected in tap water, including PMMoV with intact capsids. Thus, the absence of intact PMMoV may be used to guarantee the viral safety of tap water produced from surface water.


Therapeutic Approach to Cystic Neoplasms of the Pancreas.
Management of cystic neoplasms of the pancreas is challenging as it relies on radiologic and cyst fluid markers to discriminate between benign and pre-cancerous lesions, however their ability to predict malignancy is limited. While asymptomatic serous cystadenomas can be managed conservatively, mucinous cystic neoplasms and intraductal papillary mucinous neoplasms are more difficult to manage. A selective approach, based on the preoperative likelihood of high-grade dysplasia or invasive disease, is the standard of care. Research is focusing on the development of pre-operative markers for identifying high risk lesions, which will spare patients with low-risk or benign lesions the risks of pancreatectomy.


Correlation Between Diabetic Peripheral Neuropathy and Sarcopenia in Patients with Type 2 Diabetes Mellitus and Diabetic Foot Disease: A Cross-Sectional Study.
The present study was designed to determine the relationships between sarcopenia and diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes mellitus (T2DM) and diabetic foot disease (DFD) respectively. A total of 1104 patients with T2DM and 257 patients with DFD were included in the study, which was designed as a cross-sectional study. Body composition was assessed using dual-energy X-ray-absorptiometry (DXA). The diagnosis of sarcopenia was based on the Baumgartner criteria. DPN was assessed by Neuropathy symptom score (NSS) and Neuropathy disability score (NDS), and the severity of neuropathy was divided into non-neuropathy symptom (NS), Mild NS, Moderate NS and Severe NS according to NSS. Logistic regression analyses were carried out to determine the relations of sarcopenia and DPN in patients with T2DM and NSS in patients with DFD, respectively. The prevalence of DPN was 80.0% in T2DM patients with sarcopenia and 70.3% in non-sarcopenia patients (P=0.007). Logistic regression analyses showed DPN was one of the independent risk factors for sarcopenia in T2DM patients (OR 1.564 [95% CI: 1.004, 2.435], P=0.048). The prevalence of DPN had no statistical significance in DFD patients with or without sarcopenia. However, the NSS of DFD patients with sarcopenia was higher than that of non-sarcopenia patients. In the multivariate logistic regression analysis, NSS was determined to be associated with sarcopenia in DFD patients (OR 1.387[95% CI: 1.074, 1.789], P=0.012). The appendicular lean mass (ALM) of DFD patients without NS was higher than patients with mild, moderate and severe NS (20.71±2.73 vs 16.57±3.62 vs 17.99±3.54 vs 17.23±3.29 Kg, P=0.028). DPN is an independent risk factor for sarcopenia in patients with T2DM and NSS is also independently correlated with sarcopenia in patients with DFD, with the latter being more obvious with the aggravation of neurological symptoms in DFD patients.


Use of Alternative Medicine Among Norwegian Cancer Patients is Associated with Mental Distress - A Follow-up Study.
The purpose of this study was to investigate the relationship between the use of alternative medicine (AM) by cancer patients' and the level of self-perceived mental distress. One hundred and fifty-seven cancer patients were included in a longitudinal questionnaire-based study 12 months after their first contact with the Department of Oncology, University Hospital of Tromsø, during the period 1990 to 1991. Eligible patients received follow-up questionnaires after 24 and 60 months recording their use of AM and measuring their perceived mental distress (GHQ-20). A total of 64 (41%) of the 157 patients who completed at least one questionnaire reported using AM one or more times during follow-up (ever-use). Between 65% and 79% of patients who were eligible at each point of the study completed the GHQ questionnaire. After 60 months of follow-up, 104 patients were still alive and 54 patients (52% of the survivors) had completed all the questionnaires. At inclusion in the study, significantly more AM users reported mental distress (57%) than non-users (20%) (p=0.001). After 24 and 60 months, the corresponding figures were 42% versus 29% (p=0.18), and 30% versus 10% (p=0.02), respectively. When adjusted for sex, age and disease progression, use of AM was found to be an independent factor highly associated with mental distress at 12 months (OR=5.5, 95% CI 2.1-14.4). Later in the study, progression/relapse of cancer proved to be a more important predictor of mental distress. These findings suggest that, in North Norway, seeking alternative treatment is more common among mentally distressed cancer patients.


[Successive changes in the blood composition of experimental normal beagle dogs associated with age].
Eight hematologic parameter values, 16 serum biochemical constituents, serum protein fractions and albumin-globulin ratios were determined in blood samples obtained from 879 normal, healthy Beagle dogs of both sexes which had been reproduced and bred in our laboratories. The blood samples were collected from the Beagles that ranged in monthly ages from 1 to 12 and in monthly ages from 13 to 121, which were classified as the adult class. As a result, red blood cell counts, hemoglobin concentrations and packed cell volumes increased with growth. Red blood cell parameters of normal Beagles in our laboratories were rather higher than those in literatures presented by many other researchers. MCV decreased and MCHC increased gradually with age. Total serum protein concentrations increased with growth. alpha 1-1 and alpha 1-2 Globulin fractions descended, but beta 2 and gamma globulin fractions ascended in serum proteins. Alkaline phosphatase activities, inorganic phosphorus concentrations and glucose concentrations decreased conspicuously with growth. Leucine aminopeptidase activities and calcium concentrations decreased slightly. Serum cholinesterase and LDH activities showed a tendency to diminish similarly. Blood urea nitrogen and creatinine concentrations multiplied gradually. Hematologic parameters became almost steady in our 7-month-old dogs or older ones and serum biochemical constituents had a tendency to be stable in our 7- to 9-month-old dogs or older ones in the blood. White blood cell counts, alkaline phosphatase activities, inorganic phosphorus concentrations, glucose concentrations, leucine aminopeptidase activities and calcium concentrations were lowest in the adult class.


[Assessment of jugular vein hemodynamics to determine physiological activity during mental work].
Venous cerebral hemodynamics has been studied in 20 virtually sound military personnel. 120 instrumental tests were carried out. Blood flow rate in proximal and distal parts of internal jugular veins has been determined by means of ultrasonic doplerography. Blood flow rate in a distal part of internal jugular vein was shown increased considerably during small "ortostasis" conditions, that's why it is advised to determine blood flow rate in a distal part of jugular vein when we assess physiological activity of mental work, but for all that time averaging of max. blood flow in internal jugular vein should be taken as a control index.


Progress of fibrinolysis during tumor necrosis factor infusions in humans. Concomitant increase in tissue-type plasminogen activator, plasminogen activator inhibitor type-1, and fibrin(ogen) degradation products.
Several investigators have reported that tumor necrosis factor (TNF) can alter the production of plasminogen activator type-1 (PAI-1) and plasminogen activators (PAs) by endothelial cells in vitro. We have examined the in vivo effects of recombinant human TNF administration on fibrinolysis as assessed by parameters in plasma during a 24-hour period of continuous TNF infusion to 17 cancer patients with active disease. The plasma levels of PAI activity increased sevenfold after 3 and 24 hours of TNF infusion. This was the result of an increase of PAI-1 antigen; PAI-2 antigen was not detectable. Plasma concentrations of tissue-type PA (t-PA) antigen increased twofold to fivefold after 3 and 24 hours of TNF infusion, whereas urokinase-type PA antigen levels in plasma remained unaltered. After 3 hours of TNF infusion the plasma levels of alpha 2-antiplasmin were slightly decreased, 5% on average, suggesting that fibrinolysis continued. After 24 hours of TNF infusion a highly significant increase in fibrin- plus fibrinogen-degradation products, and separately of fibrin degradation products and fibrinogen degradation products, was found. This indicates that fibrinolysis persisted, at least partly, in the presence of high levels of PAI activity. Whereas PAI-1 production increased, t-PA production by human endothelial cells in vitro remains unaltered or even decreases on TNF addition. It has been shown previously that TNF infusion in our patients results in thrombin and fibrin generation. Therefore, it is possible that thrombin, not TNF, is the actual stimulus for t-PA production in our patients. We speculate that fibrin is formed during TNF infusions and that plasmin is generated by t-PA action immediately on the initial formation of (soluble) fibrin molecules. Such a process may explain the generation of degradation products of both fibrin and fibrinogen during infusion of TNF in patients.


[Methodological approaches to controlling the antibiotic content in disks for determining microorganism antibiotic sensitivity].
The content of antibiotics in the discs is the main requirement to their quality. There are 2 methods for determination of the antibiotic content in the discs: (1) a method with preliminary extraction of the antibiotic from the disc followed by determination of its content in the extract biologically and (2) a method using 3 or more standard discs containing different amounts of the antibiotic. A study was performed to show the advantages and limitations of the methods. The conditions for determination of the activity of 22 antibiotics contained in the discs with the method using the standard discs were developed. Comparison of the results obtained with the 2 methods showed that they did not differ significantly. However, it is considered advisable to use for the disc control the method based on direct diffusion of the antibiotic from the disc into agar, since in this case the antibiotic amount in the disc is determined under conditions as much as possible close to the conditions of the disc using for determination of the microbial antibiotic sensitivity.


Computed tomography-guided percutaneous biopsy of bone lesions: rate of diagnostic success and complications.
To determine the rates of diagnostic success and complications of computed tomography (CT)-guided percutaneous biopsy of bone lesions suspected for malignancy. Retrospective study including 186 cases of CT-guided percutaneous biopsies of bone lesions in the period from January, 2010 to December, 2012. All the specimens were obtained with 8-10 gauge needles. The following data were collected: demographics, previous history of malignancy, data related to the lesion, to the procedure, and to histological results. Most patients were women (57%), and the mean age was 53.0 ± 16.4 years. In 139 cases (74.6%), there was diagnostic suspicion of metastasis and the most common primary tumors were breast (32.1%) and prostate (11.8%). The bones most commonly involved were spine (36.0%), hip (32.8%) and long bones (18.3%). Complications occurred in only three cases (1.6%) including bone fracture, paresthesia with functional impairment, and needle breakage requiring surgical removal. The specimens collected from 183 lesions (98.4%) were considered appropriate for diagnosis. Malignant results were more frequently found in patients who had a suspected secondary lesion and history of known malignancy (p < 0.001), and in patients who underwent PET/CT-guided procedures (p = 0.011). CT-guided percutaneous biopsy is a safe and effective procedure for the diagnosis of suspicious bone lesions.


A 12 week longitudinal study of microbial translocation and systemic inflammation in undernourished HIV-infected Zambians initiating antiretroviral therapy.
Undernourished, HIV-infected adults in sub-Saharan Africa have high levels of systemic inflammation, which is a risk factor for mortality and other adverse health outcomes. We hypothesized that microbial translocation, due to the deleterious effects of HIV and poor nutrition on intestinal defenses and mucosal integrity, contributes to heightened systemic inflammation in this population, and reductions in inflammation on antiretroviral therapy (ART) accompany reductions in translocation. HIV-infected, Zambian adults with a body mass index <18.5 kg/m2 were recruited for a pilot study to assess the relationships between microbial translocation and systemic inflammation over the first 12 weeks of ART. To assess microbial translocation we measured serum lipopolysaccharide binding protein (LBP), endotoxin core IgG and IgM, and soluble CD14, and to assess intestinal permeability we measured the urinary excretion of an oral lactulose dose normalized to urinary creatinine (Lac/Cr ratio). Linear mixed models were used to assess within-patient changes in these markers relative to serum C-reactive protein (CRP), tumor necrosis factor-α receptor 1 (TNF-α R1), and soluble CD163 over 12 weeks, in addition to relationships between variables independent of time point and adjusted for age, sex, and CD4+ count. Thirty-three participants had data from recruitment and at 12 weeks: 55% were male, median age was 36 years, and median baseline CD4+ count was 224 cells/μl. Over the first 12 weeks of ART, there were significant decreases in serum levels of LBP (median change -8.7 μg/ml, p = 0.01), TNF-α receptor 1 (-0.31 ng/ml, p < 0.01), and CRP (-3.5 mg/l, p = 0.02). The change in soluble CD14 level over 12 weeks was positively associated with the change in CRP (p < 0.01) and soluble CD163 (p < 0.01). Pooling data at baseline and 12 weeks, serum LBP was positively associated with CRP (p = 0.01), while endotoxin core IgM was inversely associated with CRP (p = 0.01) and TNF-α receptor 1 (p = 0.04). The Lac/Cr ratio was not associated with any serum biomarkers. In undernourished HIV-infected adults in Zambia, biomarkers of increased microbial translocation are associated with high levels of systemic inflammation before and after initiation of ART, suggesting that impaired gut immune defenses contribute to innate immune activation in this population.


Invasive and non-invasive modalities of imaging carotid stenosis.
Despite recent therapeutic advances, acute ischemic complications of atherosclerosis remain the primary cause of morbidity and mortality in Western countries, with carotid atherosclerotic disease one of the major preventable causes of stroke. As the impact of this disease challenges our healthcare systems, we are becoming aware that factors influencing this disease are more complex than previously realized. In current clinical practice, risk stratification relies primarily on evaluation of the degree of luminal stenosis and patient symptomatology. Adequate investigation and optimal imaging are important factors that affect the quality of a carotid endarterectomy (CEA) service and are fundamental to patient selection. Digital subtraction angiography is still perceived as the most accurate imaging modality for carotid stenosis and historically has been the cornerstone of most of the major CEA trials but concerns regarding potential neurological complications have generated substantial interest in non-invasive modalities, such as contrast-enhanced magnetic resonance angiography. The purpose of this review is to give an overview to the vascular specialist of the current imaging modalities in clinical practice to identify patients with carotid stenosis. Advantages and disadvantages of each technique are outlined. Finally, limitations of assessing luminal stenosis in general are discussed. This article will not cover imaging of carotid atheroma morphology, function and other emerging imaging modalities of assessing plaque risk, which look beyond simple luminal measurements.


Stable or seral? Fire-driven alternative states in aspen forests of western North America.
As important centres for biological diversity, aspen forests are essential to the function and aesthetics of montane ecosystems in western North America. Aspen stands are maintained by a nuanced relationship with wildfire, although in recent decades aspen mortality has increased. The need to understand the baseline environmental conditions that favour aspen is clear; however, long-term fire history reconstructions are rare due to the scarcity of natural archives in dry montane settings. Here, we analyse a high-resolution lake sediment record from southwestern, Utah, USA to quantify the compositional and burning conditions that promote stable (or seral) aspen forests. Our results show that aspen presence is negatively correlated with subalpine fir and that severe fires tend to promote persistent and diverse aspen ecosystems over centennial timescales. This information improves our understanding of aspen disturbance ecology and identifies the circumstances where critical transitions in montane forests may occur.


Feeding problems in preterm infants of preeclamptic mothers.
Maternal disease can cause prematurity and neonatal complications, notably feeding problems. To determine the relationship between maternal disease and the nature and severity of neonatal feeding problems, we compared feeding profiles, time to demand feeding and length of hospital stay between preterm infants of preeclamptic mothers, mothers with amniotic infection and mothers with other disease causing prematurity. The retrospective study used labour ward data collected from 2002 to 2005 in a tertiary university centre to analyse three groups of singletons born at <32 completed gestational weeks to mothers with preeclampsia (n = 61), amniotic infection (n = 55) and non-preeclamptic non-amniotic infection controls (n = 55). The groups were similar in gestational age, birthweight and sex ratio; all infants received enteral feeding according to departmental guidelines. Feeding profiles and enteral/oral nutrition were compared. Feeding problems occurred in 46% of the preeclamptic group, 11% of the amniotic infection group and 13% of controls. Full oral demand feeding was established at 36 0/7 weeks postmenstrual age, 35 3/7 weeks (P = 0.03) and 35 2/7 weeks (P < 0.0001), respectively. Feeding problems were the main cause of delay (7-10 days) in hospital discharge in the preeclamptic group (P = 0.0002). Feeding problems are greater, and hospital stay longer, in preterm infants of preeclamptic mothers than in other preterm infants.


Breast Cancer, Version 4.2017, NCCN Clinical Practice Guidelines in Oncology.
Ductal carcinoma in situ (DCIS) of the breast represents a heterogeneous group of neoplastic lesions in the breast ducts. The goal for management of DCIS is to prevent the development of invasive breast cancer. This manuscript focuses on the NCCN Guidelines Panel recommendations for the workup, primary treatment, risk reduction strategies, and surveillance specific to DCIS.


An update on the pathobiological relevance of nuclear receptors for cancers of the head and neck.
Cancers of the head and neck are among the most common neoplasms worldwide, characterized by local tumor aggressiveness, high rate of early recurrence, development of metastasis and second primary tumors. Although disease management of head and neck cancer has improved significantly, overall survival-rates remained largely unchanged over the last decades. Thus, in addition to modern chemo-radiation treatment strategies combined with sophisticated surgery, there is still a need for molecular markers and key regulatory factors exploitable for chemoprevention and targeted therapies. A critical event in carcinogenesis is the uncontrolled modulation of genetic programs, mediated by deregulated signaling cascades, together with downstream transcriptional modulators. Hence, nuclear receptors, belonging to a superfamily of transcription factors implicated in a broad spectrum of physiological and pathophysiological processes, have also been associated with HNC. Enhanced expression of several nuclear receptors has been shown in head and neck cancer cells, and strategies targeting these molecules have been developed and tested in the clinics. In particular, the effects of retinoids targeting nuclear receptors of the thyroid hormone receptor-like receptor subfamily have been vigorously examined in large clinical chemoprevention trials. This review seeks to provide a general overview of nuclear receptors' molecular functions and summarizes their prognostic/therapeutic relevance, as well as the (pre)clinical studies targeting nuclear receptors in HNC.


Making sense of microplastics? Public understandings of plastic pollution.
This paper explores people's knowledge and understandings of microplastics; the role of media in framing perceptions and socio-cultural dimensions to popular solutions to reduce single-use plastics. We conducted 6 focus groups (2016-17) involving participants with no obvious knowledge of microplastics and some with special interest. Most people were unaware of microplastics though environmentally conscious participants had heard of microbeads due to media reporting concerning regulation. Few made connections between their personal use of plastics and ocean pollution. Plastic pollution was associated with macro-plastic 'islands' in the Great Pacific Garbage Patch and powerful media images of charismatic wildlife entanglement remote from participants lives. The scale of microplastics (not easily detected), poor understanding of the science behind microplastics and cultural ideas about healthy and appropriate behaviour presents barriers to change. Science communicators, NGOs, industry and policy makers must take account of media representations and the culturally embedded nature of plastics in society.


The relationship between hypertension and health-related quality of life: adjusted by chronic pain, chronic diseases, and life habits in the general middle-aged population in Japan.
The aim of this study was to examine the relationship between hypertension and health-related quality of life (HRQoL) adjusted by chronic pain, other chronic diseases, and life habits in the general middle-aged population in Japan. This study is a population-based cross-sectional study. In this study, 1117 participants aged 40-65 years and living in Shika Town completed a self-administered questionnaire including Short Form-36 (SF-36). The scores of SF-36 among hypertensives were compared with those of normotensives. The independent association of hypertension with each SF-36 subscale was analyzed using a multiple linear regression model adjusted by age, BMI, chronic pain, chronic diseases, sleep, exercise, and occupational status. We analyzed two groups; Group 1 which contained 846 participants completed the questionnaire without coronary heart disease and cerebral vascular disease, Group 2 which contained 686 participants without coronary heart disease, cerebral vascular disease, or diseases accompanied by chronic pain (gastroduodenal ulcer, fracture, osteoarthritis, osteoporosis, rheumatoid arthritis, and disc herniation). In Group 2, hypertensive women had a lower general health perception than normotensive women [unstandardized coefficients; B = -8.84, 95 % confidence interval (95 % CI) = -13.3 to -4.34, standardized coefficients; β = -0.200, p < 0.001], whereas hypertensive men had higher social functioning than normotensive men (B = 5.66, 95 % CI = 1.30-10.0, β = 0.149, p < 0.05) after adjusting by chronic pain and life habits. These results may be due to the sex difference in the light of the perception for health.


Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA.
The crystal structure of the DNA complex of a STAT-1 homodimer has been determined at 2.9 A resolution. STAT-1 utilizes a DNA-binding domain with an immunoglobulin fold, similar to that of NFkappaB and the p53 tumor suppressor protein. The STAT-1 dimer forms a contiguous C-shaped clamp around DNA that is stabilized by reciprocal and highly specific interactions between the SH2 domain of one monomer and the C-terminal segment, phosphorylated on tyrosine, of the other. The phosphotyrosine-binding site of the SH2 domain in each monomer is coupled structurally to the DNA-binding domain, suggesting a potential role for the SH2-phosphotyrosine interaction in the stabilization of DNA interacting elements.


A requirement of Polo-like kinase 1 in murine embryonic myogenesis and adult muscle regeneration.
Muscle development and regeneration require delicate cell cycle regulation of embryonic myoblasts and adult muscle satellite cells (MuSCs). Through analysis of the Polo-like kinase (Plk) family cell-cycle regulators in mice, we show that Plk1's expression closely mirrors myoblast dynamics during embryonic and postnatal myogenesis. Cell-specific deletion of Plk1 in embryonic myoblasts leads to depletion of myoblasts, developmental failure and prenatal lethality. Postnatal deletion of Plk1 in MuSCs does not perturb their quiescence but depletes activated MuSCs as they enter the cell cycle, leading to regenerative failure. The Plk1-null MuSCs are arrested at the M-phase, accumulate DNA damage, and apoptose. Mechanistically, Plk1 deletion upregulates p53, and inhibition of p53 promotes survival of the Plk1-null myoblasts. Pharmacological inhibition of Plk1 similarly inhibits proliferation but promotes differentiation of myoblasts in vitro, and blocks muscle regeneration in vivo. These results reveal for the first time an indispensable role of Plk1 in developmental and regenerative myogenesis.


A lentivirus-based system for Cas9/gRNA expression and subsequent removal by Cre-mediated recombination.
A major concern of CRISPR and related genome engineering technologies is off-target mutagenesis from prolonged exposure to Cas9 and related editing enzymes. To help mitigate this concern we added a loxP site to the 3'-LTR of an HIV-based lentiviral vector capable of expressing Cas9/gRNA complexes in a wide variety of mammalian cell types. Transduction of susceptible target cells yields an integrated provirus that expresses the desired Cas9/gRNA complex. The reverse transcription process also results in duplication of the 3'-LTR such that the integrated provirus becomes flanked by loxP sites (floxed). Subsequent expression of Cre recombinase results in loxP-to-loxP site-specific recombination that deletes the Cas9/gRNA payload and effectively prevents additional Cas9-mediated mutations. This construct also expresses a gRNA with a single transcription termination sequence, which results in higher expression levels and more efficient genome engineering as evidenced by disruption of the SAMHD1 gene. This hit-and-run CRISPR approach was validated by recreating a natural APOBEC3B deletion and by disrupting the mismatch repair gene MSH2. This hit-and-run strategy may have broad utility in many areas and especially those where cell types are difficult to engineer by transient delivery of ribonucleoprotein complexes.


Selective binding of glutathione conjugates of fatty acid derivatives by plant glutathione transferases.
Proteomic studies with Arabidopsis thaliana have revealed that the plant-specific Tau (U) class glutathione transferases (GSTs) are selectively retained by S-hexylglutathione affinity supports. Overexpression of members of the Arabidopsis GST superfamily in Escherichia coli showed that 25 of the complement of 28 GSTUs caused the aberrant accumulation of acylated glutathione thioesters in vivo, a perturbation that was not observed with other GST classes. Each GSTU caused a specific group of fatty acyl derivatives to accumulate, which varied in chain length (C(6) to C(18)), additional oxygen content (0 or 1), and desaturation (0 or 1). Thioesters bound tightly to recombinant GSTs (K(d) approximately 1 microm), explaining their accumulation. Transient expression of GSTUs in Nicotiana benthamiana followed by recovery by Strep-tag affinity chromatography allowed the respective plant ligands to be extracted and characterized. Again, each GST showed a distinct profile of recovered metabolites, notably glutathionylated oxophytodienoic acid and related oxygenated fatty acids. Similarly, the expression of the major Tau protein GSTU19 in the endogenous host Arabidopsis led to the selective binding of the glutathionylated oxophytodienoic acid-glutathione conjugate, with the enzyme able to catalyze the conjugation reaction. Additional ligands identified in planta included other fatty acid derivatives including divinyl ethers and glutathionylated chlorogenic acid. The strong and specific retention of various oxygenated fatty acids by each GSTU and the conservation in binding observed in the different hosts suggest that these proteins have selective roles in binding and conjugating these unstable metabolites in vivo.


Nonsurgical mouse model of endometriosis-associated pain that responds to clinically active drugs.
Endometriosis is an estrogen-dependent inflammatory disease that affects approximately 10% of women. Debilitating pelvic or abdominal pain is one of its major clinical features. Current animal models of endometriosis-associated pain require surgery either to implant tissue or to remove the ovaries. Moreover, existing models do not induce spontaneous pain, which is the primary symptom of patients with chronic pain, including endometriosis. A lack of models that accurately recapitulate the disease phenotype must contribute to the high failure rate of clinical trials for analgesic drugs directed at chronic pain, including those for endometriosis. We set out to establish a murine model of endometriosis-associated pain. Endometriosis was induced nonsurgically by injecting a dissociated uterine horn into a recipient mouse. The induced lesions exhibited histological features that resemble human lesions along with an increase in proinflammatory cytokines and recruitment of immune cells. We also observed the presence of calcitonin gene-related peptide-, TRPA1-, and TRPV1-expressing nerve fibers in the lesions. This model induced mechanical allodynia, spontaneous abdominal pain, and changes in thermal selection behavior that indicate discomfort. These behavioral changes were reduced by drugs used clinically for endometriosis, specifically letrozole (aromatase inhibitor) and danazol (androgen). Endometriosis also induced neuronal changes as evidenced by activation of the NF-κB signaling pathway in TRPA1- and TRPV1-expressing dorsal root ganglion neurons. In conclusion, we have established a model of endometriosis-associated pain that responds to clinically active drugs and can, therefore, be used to identify novel therapies.


Spatial aspects of tree mortality strongly differ between young and old-growth forests.
Rates and spatial patterns of tree mortality are predicted to change during forest structural development. In young forests, mortality should be primarily density dependent due to competition for light, leading to an increasingly spatially uniform pattern of surviving trees. In contrast, mortality in old-growth forests should be primarily caused by contagious and spatially autocorrelated agents (e.g., insects, wind), causing spatial aggregation of surviving trees to increase through time. We tested these predictions by contrasting a three-decade record of tree mortality from replicated mapped permanent plots located in young (< 60-year-old) and old-growth (> 300-year-old) Abies amabilis forests. Trees in young forests died at a rate of 4.42% per year, whereas trees in old-growth forests died at 0.60% per year. Tree mortality in young forests was significantly aggregated, strongly density dependent, and caused live tree patterns to become more uniform through time. Mortality in old-growth forests was spatially aggregated, but was density independent and did not change the spatial pattern of surviving trees. These results extend current theory by demonstrating that density-dependent competitive mortality leading to increasingly uniform tree spacing in young forests ultimately transitions late in succession to a more diverse tree mortality regime that maintains spatial heterogeneity through time.


Assessment of anticoagulation intensity and management of bleeding with old and new oral anticoagulants.
Warfarin is effective for the prevention and treatment of thromboembolism but produces variable anticoagulant effects and requires routine monitoring of the international normalized ratio (INR) to optimize the balance between efficacy and safety. The new oral anticoagulants (NOACs) have a more predictable anticoagulant effect and were recently demonstrated to be at least as efficacious and safe as warfarin despite being administered in fixed doses without routine coagulation monitoring. Specific laboratory tests have been developed to measure the anticoagulant effect of the NOACs but are not yet widely available, and the relation between drug levels and both coagulation test results and outcomes is uncertain. It remains to be demonstrated whether adjustment of the dose of NOACs, according to the results of laboratory testing, may lead to even greater efficacy and safety. The principles of bleeding management in patients treated with NOACs compared with patients receiving warfarin are similar. Most patients can be safely managed by interrupting drug treatment, performing local measures to stem the bleeding, and providing transfusion support as required. In patients with major or life-threatening bleeding and those requiring surgery, the anticoagulant effects of warfarin can be reversed using oral or intravenous vitamin K, fresh frozen plasma (FFP), and prothrombin complex concentrates (PCCs). Specific antidotes are under development for the NOACs but are not yet approved for clinical use. PCCs and recombinant factor VIIa may improve hemostasis in patients in whom bleeding develops during treatment with a NOAC, but their efficacy is unproven.


Interactions of human galectins with Trypanosoma cruzi: binding profile correlate with genetic clustering of lineages.
We report here the specific interaction between several members of the human galectin family with the three developmental stages of several genetic lineages of the protozoan parasite Trypanosoma cruzi. We provide data of specific and differential binding of human galectin (gal)-1, -3, -4, -7 and -8 to 14 strains of T. cruzi that belong to the six genetic lineages representing the genetic diversity of the parasite. It is shown that galectins preferentially bind forms present in the host, trypomastigotes and amastigotes, compared with the non-infective epimastigote present on the intestinal tract of the vector, reflecting the changes on glycosylation that occur during the metacyclogenesis and amastigogenesis process. Also, it is evidenced that galectin binding to the parasites promotes binding to the host cells and higher infection rates. In addition, evidence is provided indicating that the intracellular amastigotes may take over the cytosolic pool of some galectins when released to the extracellular medium. Finally, by applying unweighted pair group method analysis to the galectin-binding profile to either cell-derived trypomastigotes or amastigotes, we show that the differential-binding profile by the host galectins to the six lineages resembles the clustering based in genetic data. Therefore, the differential-binding profile for the six lineages could have implications in the immunopathology of Chagas' disease, affecting the complex network of immune responses on which galectins mediate, thus providing linkage clues to the notion that different lineages may be related to different clinical forms of the disease.


Hypertensive Disorders of Pregnancy - A Life-Long Risk?!
Background: Arterial hypertension is one of the most important causes of cardiovascular diseases, and the latter are responsible for almost half of the deaths in the industrialised nations. Hypertensive disorders of pregnancy constitute one of the most frequent causes of feto-maternal morbidity and mortality; on the other hand the occurrence of a hypertensive disorder of pregnancy represents a risk for the later development of hypertension and the cardiovascular risks resulting therefrom. The aim of this article is to demonstrate the association of hypertensive disorders of pregnancy with consecutive cardiovascular diseases. Materials and Methods: Specific selective literature research. Results: After the occurrence of a hypertensive disorder of pregnancy the relative risks for hypertension are 3.7 (2.70-5.05), for ischaemic heart disease 2.2 (1.86-2.52), for cerebral insult 1.8 (1.45-2.27) and for mortality resulting from cardiovascular causes 1.5 (1.05-2.14) and are thus significant. According to a recent study 56 % of internal specialists and 23 % of gynecologists do not know about the association of preeclampsia with ischemic heart disease, 48 % and 38 % respectively are not aware of the link with stroke and 79 % and 77 % respectively are not aware of the association with a reduced life expectancy after preeclampsia. The presence of hypertension is not known by many of the patients, merely 28-38 % receive an appropriate therapy. Conclusion: Adequate follow-up after hypertensive disorders of pregnancy and the early recognition of and therapy for hypertension represent the cornerstones in the prevention of late cardiovascular sequelae. General practitioners, specialist for internal medicine and gynaecologists have a special responsibility with regard to the reduction of later complications.


Repression and activation domains of RME1p structurally overlap, but differ in genetic requirements.
Rme1p, a repressor of meiosis in the yeast Saccharomyces cerevisiae, acts as both a transcriptional repressor and activator. Rme1p is a zinc-finger protein with no other homology to any protein of known function. The C-terminal DNA binding domain of Rme1p is essential for function. We find that mutations and progressive deletions in all three zinc fingers can be rescued by fusion of RME1 to the DNA binding domain of another protein. Thus, structural integrity of the zinc fingers is not required for the Rme1p-mediated effects on transcription. Using a series of mutant Rme1 proteins, we have characterized domains responsible for repression and activation. We find that the minimal transcriptional repression and activation domains completely overlap and lie in an 88-amino-acid N-terminal segment (aa 61-148). An additional transcriptional effector determinant lies in the first 31 amino acids of the protein. Notwithstanding the complete overlap between repression and activation domains of Rme1p, we demonstrated a functional difference between repression and activation: Rgr1p and Sin4p are absolutely required for repression but dispensable for activation.


Phase III randomized multicenter study of a humanized anti-CD33 monoclonal antibody, lintuzumab, in combination with chemotherapy, versus chemotherapy alone in patients with refractory or first-relapsed acute myeloid leukemia.
Lintuzumab (HuM195) is an unconjugated humanized murine monoclonal antibody directed against the cell surface myelomonocytic differentiation antigen CD33. In this study, the efficacy of lintuzumab in combination with induction chemotherapy was compared with chemotherapy alone in adults with first relapsed or primary refractory acute myeloid leukemia (AML). Patients with relapsed or primary resistant AML (duration of first response, zero to 12 months) were randomly assigned to receive either mitoxantrone 8 mg/m(2), etoposide 80 mg/m(2), and cytarabine 1 g/m(2) daily for 6 days (MEC) in combination with lintuzumab 12 mg/m(2), or MEC alone. Overall response, defined as the rate of complete remission (CR) and CR with incomplete platelet recovery (CRp), was the primary end point of the study, with additional analyses of survival time and toxicity. A total of 191 patients were randomly assigned from November 1999 to April 2001. The percent CR plus CRp with MEC plus lintuzumab was 36% v 28% in patients treated with MEC alone (P = .28). The overall median survival was 156 days and was not different in the two arms of the study. Apart from mild antibody infusion-related toxicities (fever, chills, and hypotension), no differences in chemotherapy-related adverse effects, including hepatic and cardiac dysfunction, were observed with the addition of lintuzumab to induction chemotherapy. The addition of lintuzumab to salvage induction chemotherapy was safe, but did not result in a statistically significant improvement in response rate or survival in patients with refractory/relapsed AML.


Progression of insulin resistance in individuals with type 1 diabetes: A retrospective longitudinal study on individuals from Thailand.
To investigate temporal changes in glycaemic control and weight contributing to insulin resistance (IR), in Thai individuals with type 1 diabetes (T1D). Longitudinal data of 69 individuals with T1D were retrospectively collected over a median follow-up of 7.2 years. The estimated glucose disposal rate (eGDR), a marker of IR, was calculated using an established formula. Individuals were assigned as insulin-sensitive T1D (the latest eGDR≥8 mg/kg/min), or insulin-resistant T1D/double diabetes (the latest eGDR<8 mg/kg/min). Generalised linear mixed model was employed to compare the temporal patterns of HbA1c, BMI, and eGDR between the two groups. 26 insulin-resistant T1D had a gradual decline in eGDR, corresponding with increased weight and HbA1c. In contrast, 43 insulin-sensitive T1D had stable insulin sensitivity with an improvement in HbA1c over time, associated with a modest weight gain. Fluctuations of glucose levels were observed during the early diabetes course leading to unstable eGDR, thus limiting the use of eGDR to classify insulin-resistant T1D. T1D individuals who eventually develop IR are likely to experience early increasing IR over time. In contrast, those who ultimately do not have IR, maintain their insulin sensitivity throughout their course at least in the medium term.


Applying pattern recognition methods plus quantum and physico-chemical molecular descriptors to analyze the anabolic activity of structurally diverse steroids.
The great cost associated with the development of new anabolic-androgenic steroid (AASs) makes necessary the development of computational methods that shorten the drug discovery pipeline. Toward this end, quantum, and physicochemical molecular descriptors, plus linear discriminant analysis (LDA) were used to analyze the anabolic/androgenic activity of structurally diverse steroids and to discover novel AASs, as well as also to give a structural interpretation of their anabolic-androgenic ratio (AAR). The obtained models are able to correctly classify 91.67% (86.27%) of the AASs in the training (test) sets, respectively. The results of predictions on the 10% full-out cross-validation test also evidence the robustness of the obtained model. Moreover, these classification functions are applied to an "in house" library of chemicals, to find novel AASs. Two new AASs are synthesized and tested for in vivo activity. Although both AASs are less active than some commercially AASs, this result leaves a door open to a virtual variational study of the structure of the two compounds, to improve their biological activity. The LDA-assisted QSAR models presented here, could significantly reduce the number of synthesized and tested AASs, as well as could increase the chance of finding new chemical entities with higher AAR.


Efficacy of umbilical cord-derived mesenchymal stem cells in the treatment of type 2 diabetes assessed by retrospective continuous glucose monitoring.
Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have been proved a promising clinical strategy for the treatment of diabetes, and time in range (TIR) has been demonstrated a new metric of glycemic control links to diabetes complications. To further assess the therapeutic effect of UC-MSCs on TIR, a phase II study investigating the efficacy of UC-MSCs in Chinese adults with type 2 diabetes (T2D) assessed by retrospective continuous glucose monitoring (CGM) was conducted. In this randomized and placebo-controlled trial, a total of 73 patients were randomly assigned to receive intravenous infusion of UC-MSCs (n = 37) or placebo (n = 36) 3 times at 4-week intervals and followed up for 48 weeks. The primary endpoint was the changes in TIR and glycosylated hemoglobin (HbA1c). TIR and HbA1c were both significantly improved in UC-MSCs and placebo groups after 48 weeks of therapy compared with baseline. Compared with placebo group, UC-MSCs group exhibited more pronounced changes at 9 and 48 weeks from baseline in TIR (26.54 vs. 15.84 and 21.36 vs. 6.32) and HbA1c (-1.79 vs. -0.96 and -1.36 vs. -0.51). More patients in UC-MSCs group achieved the glycemic control target of TIR ≥ 70% and HbA1c < 7% at 9 and 48 weeks than in placebo group (59.5% vs. 27.8% and 43.2% vs. 11.1%). The C-peptide area under the curve (AUCC-pep) was an independent risk factor associated with efficacy in T2D undergoing UC-MSCs intervention. These results illustrate that UC-MSCs administration via intravenous infusion is an effective approach for ameliorating TIR.


Role of inorganic nitrogen oxides and tumor necrosis factor alpha in killing Leishmania donovani amastigotes in gamma interferon-lipopolysaccharide-activated macrophages from Lshs and Lshr congenic mouse strains.
The capacity of mature bone-marrow-derived macrophages and resident peritoneal macrophages from Lshr versus Lshs congenic mice to kill intracellular Leishmania donovani amastigotes when activated by recombinant gamma interferon-lipopolysaccharide (rIFN-gamma-LPS) was examined. IFN-gamma alone in doses up to 100 U/ml was unable to activate macrophages to kill L. donovani amastigotes in vitro; LPS was a necessary secondary stimulus. Similarly, LPS alone in doses up to 100 ng/ml produced no leishmanicidal activity. In bone marrow macrophages, a dose-dependent increase in leishmanicidal activity was observed as increasing rIFN-gamma-LPS dose combinations were introduced, with Lshr macrophages maintaining a significant but not dramatic advantage within any particular dose combination. For peritoneal macrophages, the reverse was true, with macrophages from Lshs mice being more efficient at killing for doses of LPS up to 10 ng/ml with doses of rIFN-gamma in the range of 11 to 33 U/ml. The degree of killing in both bone marrow and peritoneal macrophages correlated well with the levels of nitrites measured in the supernatants at 72 h, and a highly significant correlation was observed between 4-, 24-, or 72-h tumor necrosis factor alpha (TNF-alpha) release and nitrite production measured at 72 h. Inclusion of 200 microM NG-monomethyl-L-arginine, a competitive inhibitor of the L-arginine-dependent pathway for the synthesis of inorganic nitrogen oxides, inhibited the killing, as did the addition of neutralizing anti-TNF-alpha antibody. These results are consistent with previous data showing an important autocrine role for TNF-alpha in enhancing production of inorganic nitrogen oxides by primed or activated macrophages. In addition, our results suggest that production of TNF-alpha and nitrites after priming or activation signals may be under a different regulatory control in mature bone marrow macrophages than in the resident peritoneal macrophage population.


DNA methylation-based age prediction using massively parallel sequencing data and multiple machine learning models.
The field of DNA intelligence focuses on retrieving information from DNA evidence that can help narrow down large groups of suspects or define target groups of interest. With recent breakthroughs on the estimation of geographical ancestry and physical appearance, the estimation of chronological age comes to complete this circle of information. Recent studies have identified methylation sites in the human genome that correlate strongly with age and can be used for the development of age-estimation algorithms. In this study, 110 whole blood samples from individuals aged 11-93 years were analysed using a DNA methylation quantification assay based on bisulphite conversion and massively parallel sequencing (Illumina MiSeq) of 12 CpG sites. Using this data, 17 different statistical modelling approaches were compared based on root mean square error (RMSE) and a Support Vector Machine with polynomial function (SVMp) model was selected for further testing. For the selected model (RMSE = 4.9 years) the mean average error (MAE) of the blind test (n = 33) was calculated at 4.1 years, with 52% of the samples predicting with less than 4 years of error and 86% with less than 7 years. Furthermore, the sensitivity of the method was assessed both in terms of methylation quantification accuracy and prediction accuracy in the first validation of this kind. The described method retained its accuracy down to 10 ng of initial DNA input or ∼2 ng bisulphite PCR input. Finally, 34 saliva samples were analysed and following basic normalisation, the chronological age of the donors was predicted with less than 4 years of error for 50% of the samples and with less than 7 years of error for 70%.


Management and patients perspective regarding a common peroneal nerve schwannoma: a rare cause of lower leg pain in a young individual.
The differential diagnosis of exercise-induced lower leg pain in young individuals is extensive and includes entities such as chronic exertional compartment syndrome, popliteal arterial entrapment syndrome, cystic adventitial disease, medial tibial stress syndrome and tibial stress fractures. Peripheral nerve-related lower leg pain syndromes are unusual. We present a 41-year-old woman who was diagnosed with an ancient schwannoma of the right common peroneal nerve (CPN) as a rare cause of lower leg pain. This case report provides simple diagnostic bedside tools that may be used to diagnose CPN abnormalities. Altered lateral lower leg skin sensation was found using a simple cotton swab, whereas a nodule was palpated at the fibular head. A positive Tinel sign indicated CPN dysfunction. MRI confirmed the diagnosis. Surgical resection is simple and curative.


Accumulation of xylem transported protein at pit membranes and associated reductions in hydraulic conductance.
Proteins and traces of polysaccharide are the only polymeric colloids consistently transported in the xylem sap of plants. The hypothesis that such proteins could have physical inhibitory effects on xylem water transport was investigated. Ovalbumin, with a molecular weight of 45 kDa and a molecular diameter of 5.4 nm, is an inert, water-soluble protein that is midway along the size range of endogenous xylem sap proteins. Solutions of ovalbumin conjugated to a fluorescent marker and supplied to transpiring shoot explants of tobacco (Nicotiana tabacum L.) and olive (Olea europaea L.) were shown by confocal laser scanning microscopy to accumulate specifically at wall-based pit membranes that connect neighbouring xylem conduits. In addition, pressure-induced perfusion of micro-filtered ovalbumin solutions, at concentrations similar to those of endogenous xylem sap proteins, through the xylem of tobacco stem or olive twig segments resulted in the retention of c. 40% of the ovalbumin and reductions in the axial hydraulic conductance of the xylem. Smaller molecules such as Texas Red 3000 (MW 3 kDa) and Alexafluor 488-cadaverin conjugates (MW 0.64 kDa) did not show similar characteristics. The partial reduction in xylem hydraulic conductance appeared to be related to the accumulation of ovalbumin at xylem pit membranes and the consequent fouling of trans-membrane water-conducting pores with smaller diameters than those of the ovalbumin molecules. Potential implications of these novel findings for whole-plant water relations are considered.


Azathioprine in treatment of bullous pemphigoid.
Azathioprine was given to 11 patients with pemphigoid who had been on long-term maintenance therapy with prednisone or prednisolone. In nine of these prednisone therapy was withdrawn and all were maintained symptom-free on azathioprine alone, while in two the dose of prednisone was considerably reduced. One patient who had never received corticosteroids was controlled by azathioprine alone during the initial acute phase of the illness. Since azathioprine acts slowly, it is recommended that corticosteroids should be used together with azathioprine during the acute stage. Thus azathioprine is valuable in long-term management of pemphigoid, particularly in patients showing corticosteroid toxicity or in whom the minimum maintenance dose is dangerously high.


Protein profiling in brain tumors using mass spectrometry: feasibility of a new technique for the analysis of protein expression.
The purpose of this research was to perform a preliminary assessment of protein patterns in primary brain tumors using a direct-tissue mass spectrometric technique to profile and map biomolecules. We examined 20 prospectively collected, snap-frozen normal brain and brain tumor specimens using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS), and compared peptide and protein expression in primary brain tumor and nontumor brain tissues. MS can be used to identify protein expression patterns in human brain tissue and tumor specimens. The mass spectral patterns can reliably identify glial neoplasms of similar histological grade and differentiate them from tumors of different histological grades as well as from nontumor brain tissues. Initial bioinformatics cluster analysis algorithms classified tumor and nontumor tissues into similar groups comparable with their histological grade. We describe a novel tool for the analysis of protein expression patterns in human glial neoplasms. Initial results demonstrate that MALDI-MS technology can significantly aid in the process of unraveling and understanding the molecular complexities of gliomas. MALDI-MS accurately and reliably identified normal and neoplastic tissues, and could be used to discriminate between tumors of increasing grades.


Ex vivo preparations of the intact vomeronasal organ and accessory olfactory bulb.
The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm(3)) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.


Central Macular Thickness Monitoring after a Taxane-Based Therapy in Visually Asymptomatic Patients.
Taxanes are drugs used in various chemotherapeutical protocols to treat solid tumors. They have multiple systemic adverse effects, such as bone marrow suppression, alopecia, nausea, and vomiting, and may rarely cause ocular symptoms. In the past decade, a few reported cases have shown the occurrence of a cystoid macular edema with significant visual loss after the use of a taxane-based chemotherapy. The aim of this study was to compare the central macular thickness (CMT) before and after the initiation of a taxane-based therapy in visually asymptomatic patients and to elucidate the possible impact of these drugs on the vision of cancer patients. Patients with a confirmed diagnosis of a solid tumor were screened for any ophthalmic disease before inclusion and had a baseline macular spectral domain optical coherence tomography (OCT; RTVue-100; Optovue Inc., Fremont, CA, USA) before the initiation of a taxane-based chemotherapy according to different protocols, such as 4EC-4T, 3FEC/3T, or 4TC. OCT was repeated after 4 cycles (or 3 months) of treatment, and CMT was compared to baseline. Patients presenting diabetic retinopathy, age-related macular degeneration or any condition that causes macular edema confirmed by ophthalmic examination were excluded. Fifty eyes of 25 patients were included; 92% of the subjects were female with a mean age of 48.52 years, 88% were diagnosed with breast cancer, 8% with esophageal cancer, and 4% with ovarian cancer. Docetaxel was the taxane administered to 92% of the patients. The received dose of docetaxel ranged between 110 and 160 mg. The other patients had paclitaxel in their protocols. No significant macular edema or drop in visual acuity were noted in any patient. Nevertheless, the mean CMT was found to be increased, particularly in the parafoveal and perifoveal areas (mean difference of +2.22 μm; p = 0.001). Taxane-based chemotherapy regimens seem to increase macular thickness, with a relative sparing of the fovea, in patients without significant macular edema. Further research is required to better explain the pathophysiology and possible impact of this phenomenon.


Use of the Beck Depression Inventory-II with African American primary care patients.
The Beck Depression Inventory-II (BDI-II) is one of the most common self-report instruments used for depression screening. However, there is a lack of research examining the effectiveness of this instrument in detecting depression in an African American primary care sample. The current study included 220 African American primary care patients who completed the BDI-II and were administered a diagnostic interview to establish depressive diagnoses. Results indicated that depressed patients demonstrated significantly greater BDI-II total scores compared to non-depressed patients. The recommended cutoff score of 14 for screening for depression appears to be appropriate for African American patients in the primary care setting. This cutoff yielded sensitivity of 87.7% and specificity of 83.9%. Positive predictive value of the BDI-II was .70, and negative predictive value was .94. Similar to findings with predominantly Caucasian samples, current results suggest the BDI-II is an appropriate and accurate instrument to use for depression screening among African American primary care patients.


Properties of separable covariance matrices and their associated gaussian random processes.
A number of properties of separable covariance matrices are summarized. Expressions for the divergence of the corresponding two-dimensional Gaussian random processes are given in terms of row and column covariance matrices, and in terms of linear prediction parameters and maximum likelihood spectral estimates. Such time and frequency domain expressions are not widely known, even for one-dimensional random processes.


Developmental pathways of childhood dark traits.
The dark triad of personality has traditionally been defined by 3 interrelated constructs, defined as Narcissism, Machiavellianism, and Psychopathy. Although the content of each of these constructs is clearly represented in childhood maladaptive trait measures, no studies have jointly addressed the prospective developmental course of this core set of maladaptive characteristics throughout childhood and adolescence. The current study uses latent growth modeling to explore how early dark traits develop over time, relying on a selected set of 6 childhood maladaptive traits that conceptually cover the adult dark triad. Across a 5-wave multi-informant design spanning 10 years of childhood, adolescence, and emerging adulthood (Nwave 1 = 717, 54.4% girls, age range T1 = 8-14.7 years, mean age = 10.73), results indicate that childhood dark traits show to some extent shared growth across time, although notable unique growth variance was also observed. Early dark traits further demonstrate significant association patterns with an adult dark triad measure across informants and are increasingly able to discriminate among more and less prototypical profiles of adult dark triad scores. Findings are discussed from a developmental psychopathology framework, underscoring that the proposed set of childhood dark traits represents a meaningful developmental precursor of the adult dark triad. (PsycINFO Database Record


Regulation of cockroach fat-body metabolism by the corpus cardiacum in vitro.
Incubation in vitro of young, adult, male Leucophaea maderae fat body with extracts of corpora cardiaca or intact corpora cardiaca results in stimulation of oxygen consumption but reduction in carbon dioxide evolved from carbohydrate. The carbohydrate is preferentially used for trehalose synthesis, and the endogenous metabolism of the fat body appears to be supported by increased lipid utilization. A hormone from the corpus cardiacum is most likely responsible for these effects and may act at two points, at least, in the glycolytic pathway.


Impact of Minimally Invasive Approach on Attainment of a Textbook Oncologic Outcome Following Gastrectomy for Gastric Cancer: A Review of the National Cancer Database.
Textbook oncologic outcome (TOO) is a composite outcome measure realized when all desired short-term quality metrics are met following an oncologic operation. This study examined whether minimally invasive gastrectomy (MIG) is associated with increased likelihood of TOO attainment. The 2010-2016 National Cancer Database was queried for patients with gastric cancer who underwent gastrectomy. Surgical approach was described as open (OG), laparoscopic (LG), or robotic (RG). TOO was defined as having met five metrics: R0 resection, AJCC compliant lymph node evaluation (n ≥ 15), no prolonged length of stay (< 75th percentile by year), no 30-day readmission, and receipt of guideline-accordant systemic therapy. Of 21,015 patients identified, 5708 (27.2%) underwent MIG (LG = 21.9%, RG = 5.3%). Patients who underwent RG were more likely to have met all TOO criteria, and consequently TOO. Logistic regression models revealed that patients undergoing MIG were significantly more likely to attain TOO. MIG was associated with a higher likelihood of adequate LAD, no prolonged LOS, and concordant chemotherapy. Patients who underwent LG and achieved TOO had the highest median OS (86.7 months), while the OG non-TOO cohort experienced the lowest (34.6 months). The median OS for the RG TOO group was not estimable; however, the mortality rate (.7%) was the lowest of the six cohorts. RG resulted in a significantly increased likelihood of TOO attainment. Although TOO is associated with increased OS across all surgical approaches, attainment of TOO following MIG is associated with a statistically significantly higher median OS.


A novel immunotherapy targeting MMP-14 limits hypoxia, immune suppression and metastasis in triple-negative breast cancer models.
Matrix metalloproteinase-14 (MMP-14) is a clinically relevant target in metastatic cancers due to its role in tumor progression and metastasis. Since active MMP-14 is localized on the cell surface, it is amenable to antibody-mediated blockade in cancer, and here we describe our efforts to develop novel inhibitory anti-MMP-14 antibodies. A phage-displayed synthetic humanized Fab library was screened against the extracellular domain of MMP-14 and a panel of MMP14-specific Fabs were identified. A lead antibody that inhibits the catalytic domain of MMP-14 (Fab 3369) was identified and treatment of MDA-MB-231 breast cancer cells with Fab 3369 led to significant loss of extracellular matrix degradation and cell invasion abilities. In mammary orthotopic tumor xenograft assays, MMP-14 blockade by IgG 3369 limited tumor growth and metastasis. Analysis of tumor tissue sections revealed that MMP-14 blockade limited tumor neoangiogenesis and hypoxia. Similar effects of MMP-14 blockade in syngeneic 4T1 mammary tumors were observed, along with increased detection of cytotoxic immune cell markers. In conclusion, we show that immunotherapies targeting MMP-14 can limit immune suppression, tumor progression, and metastasis in triple-negative breast cancer.


An accountability evaluation for the International Food & Beverage Alliance's Global Policy on Marketing Communications to Children to reduce obesity: A narrative review to inform policy.
United Nations (UN) organizations have urged governments to restrict the marketing of unhealthy food and nonalcoholic beverage products to children. This study conducted an accountability evaluation for the International Food & Beverage Alliance's (IFBA's) Global Policy on Marketing Communications to Children (Global Policy) compared with UN and other best-practice recommendations. We used the National Academy of Medicine's LEAD (ie, locate, evaluate, assemble evidence to inform decisions) framework to identify evidence (January 2004 to October 2018). We assigned a progress score (ie, none, limited, some, extensive) for five accountability steps. No progress was made to appoint an empowered body to evaluate IFBA's Global Policy. IFBA and the Access to Nutrition Foundation made some progress to take and share the account. Diverse actors made no progress to hold IFBA to account for nonadherence or to strengthen accountability structures for future compliance. IFBA could strengthen its Global Policy to align with best practices. UN organizations and other stakeholders should encourage IFBA firms to restrict the marketing of unhealthy foods and beverages to reduce children's obesity risk. This evaluation is relevant to all firms and industry associations that market products to children that undermine their diet and health.


Tai Chi for cancer survivors: A systematic review toward consensus-based guidelines.
To manage acute, long-term, and late effects of cancer, current guidelines recommend moderate-to-vigorous intensity aerobic and resistance exercise. Unfortunately, not all cancer survivors are able or willing to perform higher intensity exercise during difficult cancer treatments or because of other existing health conditions. Tai Chi is an equipment-free, multicomponent mind-body exercise performed at light-to-moderate intensity that may provide a more feasible alternative to traditional exercise programs for some cancer survivors. This systematic review evaluated the therapeutic efficacy of Tai Chi across the cancer care continuum. We searched MEDLINE/PubMed, Embase, SCOPUS, and CINAHL databases for interventional studies from inception to 18 September 2020. Controlled trials of the effects of Tai Chi training on patient-reported and objectively measured outcomes in cancer survivors were included. Study quality was determined by the RoB 2 tool, and effect estimates were evaluated using the Best Evidence Synthesis approach. Twenty-six reports from 14 trials (one non-randomized controlled trial) conducted during (n = 5) and after treatment (after surgery: n = 2; after other treatments: n = 7) were included. Low-level evidence emerged to support the benefits of 40-60 min of thrice-weekly supervised Tai Chi for 8-12 weeks to improve fatigue and sleep quality in cancer survivors. These findings need to be confirmed in larger trials and tested for scaling-up potential. Insufficient evidence was available to evaluate the effects of Tai Chi on other cancer-related outcomes. Future research should examine whether Tai Chi training can improve a broader range of cancer outcomes including during the pre-treatment and end of life phases.


Kinetic mechanistic studies of Cdk5/p25-catalyzed H1P phosphorylation: metal effect and solvent kinetic isotope effect.
Cdk5/p25 is a member of the cyclin-dependent, Ser/Thr kinase family and has been identified as one of the principle Alzheimer's disease-associated kinases that promote the formation of hyperphosphorylated tau, the major component of neurofibrillary tangles. We and others have been developing inhibitors of cdk5/p25 as possible therapeutic agents for Alzheimer's disease (AD). In support of these efforts, we examine the metal effect and solvent kinetic isotope effect on cdk5/p25-catalyzed H1P (a histone H-1-derived peptide) phosphorylation. Here, we report that a second Mg(2+) in addition to the one forming the MgATP complex is required to bind to cdk5/p25 for its catalytic activity. It activates cdk5/p25 by demonstrating an increase in k(cat) and induces a conformational change that favors ATP binding but has no effect on the binding affinity for the H1P peptide substrate. The binding of the second Mg(2+) does not change the binding order of substrates. The reaction follows the same rapid equilibrium random mechanism in the presence or absence of the second Mg(2+) as evidenced by initial velocity analysis and substrate analogue and product inhibition studies. A linear proton inventory with a normal SKIE of 2.0 +/- 0.1 in the presence of the second Mg(2+) was revealed and suggested a single proton transfer in the rate-limiting phosphoryl transfer step. The pH profile revealed a residue with a pK(a) of 6.5 that is most likely the general acid-base catalyst facilitating the proton transfer.


PARENTS, SONS, AND GLOBALIZATION IN TANZANIA: IMPLICATIONS FOR ADOLESCENT HEALTH.
As the global youth population grows exponentially across Africa, there is increasing recognition of the risky health behaviors impeding boys' healthy transitions through puberty. This study in Tanzania sought to capture boys' voiced experiences of transitioning through adolescence, and the masculinity norms shaping boys' engagement in risky behaviors. A critical finding was the gap in parent-son communication around pubertal body changes and avoidance of risk behaviors. Findings also suggest influences from globalization and modernization are changing boys' pubertal experiences and introducing new challenges for parents attempting to provide guidance. Given evidence from high-income countries indicating parents can serve as protective factors for young people during the transition through adolescence, additional research is needed to understand current parent-son dynamics and potential interventions.


Dose-mass inverse optimization for minimally moving thoracic lesions.
In the past decade, several different radiotherapy treatment plan evaluation and optimization schemes have been proposed as viable approaches, aiming for dose escalation or an increase of healthy tissue sparing. In particular, it has been argued that dose-mass plan evaluation and treatment plan optimization might be viable alternatives to the standard of care, which is realized through dose-volume evaluation and optimization. The purpose of this investigation is to apply dose-mass optimization to a cohort of lung cancer patients and compare the achievable healthy tissue sparing to that one achievable through dose-volume optimization. Fourteen non-small cell lung cancer (NSCLC) patient plans were studied retrospectively. The range of tumor motion was less than 0.5 cm and motion management in the treatment planning process was not considered. For each case, dose-volume (DV)-based and dose-mass (DM)-based optimization was performed. Nine-field step-and-shoot IMRT was used, with all of the optimization parameters kept the same between DV and DM optimizations. Commonly used dosimetric indices (DIs) such as dose to 1% the spinal cord volume, dose to 50% of the esophageal volume, and doses to 20 and 30% of healthy lung volumes were used for cross-comparison. Similarly, mass-based indices (MIs), such as doses to 20 and 30% of healthy lung masses, 1% of spinal cord mass, and 33% of heart mass, were also tallied. Statistical equivalence tests were performed to quantify the findings for the entire patient cohort. Both DV and DM plans for each case were normalized such that 95% of the planning target volume received the prescribed dose. DM optimization resulted in more organs at risk (OAR) sparing than DV optimization. The average sparing of cord, heart, and esophagus was 23, 4, and 6%, respectively. For the majority of the DIs, DM optimization resulted in lower lung doses. On average, the doses to 20 and 30% of healthy lung were lower by approximately 3 and 4%, whereas lung volumes receiving 2000 and 3000 cGy were lower by 3 and 2%, respectively. The behavior of MIs was very similar. The statistical analyses of the results again indicated better healthy anatomical structure sparing with DM optimization. The presented findings indicate that dose-mass-based optimization results in statistically significant OAR sparing as compared to dose-volume-based optimization for NSCLC. However, the sparing is case-dependent and it is not observed for all tallied dosimetric endpoints.


The profits of organized crime: the illicit drug trade in Canada.
Massive funds that are produced from street-level crime are laundered upwards into the criminal organizations for the benefit of their top-level members. The laundering systems used to distribute the proceeds of crime are designed to conceal the size of the organization as well as the identity of its members. Crime syndicates are attracted to criminal activity that produces the highest profits with the lowest risks. Although organized crime syndicates derive profits from a wide variety of criminal activity, their most sought after ventures principally involve the consensual type of crimes, such as drug trafficking, where no one individual can be readily identified as a legal victim for the purpose of recovering the proceeds of a criminal act. A recent survey indicated that illicit drug trafficking accounted for 87 per cent of the cash flow generated from organized crime. When the proceeds are disbursed, the criminals are free of the usual civil liability respecting the ownership of the proceeds. Canada currently has several laws which enhance the ability of law-enforcement authorities to trace the proceeds of crime and prosecute those who possess the assets. This legislation has some limitations with respect to seizure and forfeiture of certain types of proceeds. For example, forfeiture of the illegally acquired property is difficult when there is no original legal owner to initiate legal action, and even more difficult if the assets are located abroad.


S1P3 receptor influences key physiological properties of fast-twitch extensor digitorum longus muscle.
To examine the role of sphingosine 1-phosphate (S1P) receptor 3 (S1P3) in modulating muscle properties, we utilized transgenic mice depleted of the receptor. Morphological analyses of extensor digitorum longus (EDL) muscle did not show evident differences between wild-type and S1P3-null mice. The body weight of 3-mo-old S1P3-null mice and the mean cross-sectional area of transgenic EDL muscle fibers were similar to those of wild-type. S1P3 deficiency enhanced the expression level of S1P1 and S1P2 receptors mRNA in S1P3-null EDL muscle. The contractile properties of S1P3-null EDL diverge from those of wild-type, largely more fatigable and less able to recover. The absence of S1P3 appears responsible for a lower availability of calcium during fatigue. S1P supplementation, expected to stimulate residual S1P receptors and signaling, reduced fatigue development of S1P3-null muscle. Moreover, in the absence of S1P3, denervated EDL atrophies less than wild-type. The analysis of atrophy-related proteins in S1P3-null EDL evidences high levels of the endogenous regulator of mitochondria biogenesis peroxisome proliferative-activated receptor-γ coactivator 1α (PGC-1α); preserving mitochondria could protect the muscle from disuse atrophy. In conclusion, the absence of S1P3 makes the muscle more sensitive to fatigue and slows down atrophy development after denervation, indicating that S1P3 is involved in the modulation of key physiological properties of the fast-twitch EDL muscle.


Investigation of the role of FcγR and FcRn in mAb distribution to the brain.
To evaluate the role of Fc receptors (FcR) on IgG distribution to the brain, the disposition of 8C2, a murine monoclonal IgG1 antibody, was investigated after intravenous administration in FcRn α-chain knockout mice, FcγRIIb knockout mice, FcγRI/RIII knockout mice, and C57BL/6 control mice. (125)I-8C2 was co-administered with (51)Cr-labeled red blood cells to allow accurate assessment of residual blood content in brain samples. Blood and brain tissues were harvested from subgroups of three mice at several time-points up to 10 days, and radioactivity was counted. The blood and brain areas under 8C2 concentration vs time curves (AUCs) were calculated using the linear trapezoidal rule, and the associated standard deviations (SD) were assessed using a modified Bailer method. Concentration data were also analyzed with a semiphysiological population pharmacokinetic model. The brain/blood AUC ratios were comparable across all strains of mice (ratios ± SD): 0.00774 ± 0.000452, 0.00841 ± 0.000535, 0.00636 ± 0.000548, and 0.00917 ± 0.000478 for C57BL/6 control mice, FcγRI/RIII knockouts, FcγRIIb knockouts, and FcRn α-chain knockout mice (p > 0.05). Statistically significant improvement in model fitting of the data was shown with incorporation of a strain-specific parameter for antibody clearance for FcRn knockout mice; however, no significant improvements in model fitting were found for strain effects on any other parameter, including the brain uptake clearance or efflux clearances for 8C2. The predicted 8C2 brain efflux clearance was found to be ∼135-fold faster than the brain uptake clearance, consistent with the observed low ratio of brain-blood exposure. The experimental results and modeling results indicate that, in mice, FcRn and FcγR do not contribute to the "blood-brain barrier" that limits mAb uptake into the brain.


The voluntary food intake by growing pigs of diets containing 'treated' rapeseed meals or extracts of rapeseed meal.
The intake by growing pigs of a diet containing British rapeseed meal (Brsm) extracted once with water was greater than that of a similar diet containing untreated Brsm, and a diet containing Brsm extracted twice with water was eaten more readily than one containing Brsm extracted once with water. Treatment of Brsm with slaked lime increased slightly its acceptability by growing pigs. The addition of a water extract of Brsm to a soya-bean meal (Sbm) diet reduced voluntary intake compared with the voluntary intake of the same Sbm diet with water added. Addition to the diet of a water extract passed through an anion exchange column increased food intake but if a cation exchange column was used there was no effect on intake. Treated rapeseed meals and extracts were analysed for glucosinolates, sinapine and tannins; the results indicated that glucosinolates rather than sinapine or tannin were associated with low feed intake.


Association between low uric acid levels and acute graft-versus-host disease.
Endogenous danger signals are increasingly recognized in the pathogenesis of graft-versus-host disease (GVHD). Uric acid (UA) is a known danger signal and is released from injured cells during conditioning for allogeneic hematopoietic stem cell transplantation (HSCT), but its role in GVHD is unclear. Here, we retrospectively analyze 228 consecutive patients with malignant diseases undergoing HSCT from 10/10-HLA-matched donors. Low UA levels at the time of HSCT (day 0) were significantly associated with acute GVHD grades II-IV in univariate (HR 0.836, p = 0.0072) and multivariate analyses (HR 0.815, p = 0.0047). There was no significant association between UA levels and overall survival, non-relapse mortality, and relapse. This is the first report demonstrating a negative association between UA levels and acute GVHD. Due to the easy assessment and established pharmaceutical modification of UA, our findings are potentially clinically relevant. Confirmation in independent cohorts and further investigations into underlying mechanisms, such as reduced antioxidative capacity in hypouricemia, are warranted.


Ectopic TBX1 suppresses thymic epithelial cell differentiation and proliferation during thymus organogenesis.
The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.


miRNA-135a promotes breast cancer cell migration and invasion by targeting HOXA10.
miRNAs are a group of small RNA molecules regulating target genes by inducing mRNA degradation or translational repression. Aberrant expression of miRNAs correlates with various cancers. Although miR-135a has been implicated in several other cancers, its role in breast cancer is unknown. HOXA10 however, is associated with multiple cancer types and was recently shown to induce p53 expression in breast cancer cells and reduce their invasive ability. Because HOXA10 is a confirmed miR-135a target in more than one tissue, we examined miR-135a levels in relation to breast cancer phenotypes to determine if miR-135a plays role in this cancer type. Expression levels of miR-135a in tissues and cells were determined by poly (A)-RT PCR. The effect of miR-135a on proliferation was evaluated by CCK8 assay, cell migration and invasion were evaluated by transwell migration and invasion assays, and target protein expression was determined by western blotting. GFP and luciferase reporter plasmids were constructed to confirm the action of miR-135a on downstream target genes including HOXA10. Results are reported as means ± S.D. and differences were tested for significance using 2-sided Student"s t-test. Here we report that miR-135a was highly expressed in metastatic breast tumors. We found that the expression of miR-135a was required for the migration and invasion of breast cancer cells, but not their proliferation. HOXA10, which encodes a transcription factor required for embryonic development and is a metastasis suppressor in breast cancer, was shown to be a direct target of miR-135a in breast cancer cells. Our analysis showed that miR-135a suppressed the expression of HOXA10 both at the mRNA and protein level, and its ability to promote cellular migration and invasion was partially reversed by overexpression of HOXA10. In summary, our results indicate that miR-135a is an onco-miRNA that can promote breast cancer cell migration and invasion. HOXA10 is a target gene for miR-135a in breast cancer cells and overexpression of HOXA10 can partially reverse the miR-135a invasive phenotype.


Relationship between renal injury and the antagonistic roles of angiotensin-converting enzyme (ACE) and ACE2.
Angiotensin-converting enzyme 2 (ACE2), a newly discovered carboxypeptidase in the renin-angiotensin system (RAS), antagonizes ACE activity and plays an active role during tissue injury. Yet the mechanism of its action is not well known. Using a streptozotocin (STZ)-induced renal injury rat model, we investigated the relationship between renal injury and the antagonism between ACE and ACE2. We assayed the levels of urea nitrogen, urine glucose, creatinine, and protein, Ace2, Ace, angiotensin II type 1 receptor (At1) and Mas receptor mRNA, and renal and plasma angiotensin II (Ang II) in STZ-treated and untreated rats. We also used histology and immunohistochemistry to assess glomerular injury and ACE2 glomerular and cortical expression. The amounts of urea nitrogen, urine glucose, creatinine, and protein were significantly higher in STZ-treated rats than in control rats (P < 0.01). There were significant pathological changes in the kidney upon STZ-treatment. Ace2 and Ace mRNA levels were significantly higher in STZ-treated rats than in control rats (P < 0.05 and P = 0.05, respectively). There was no significant difference in the Mas receptor and At1 mRNA levels in the 2 groups, although At1 levels showed an increase upon STZ-treatment. The Ang II level in the renal cortical tissue and plasma of STZ-treated rats was higher than that of control rats (P < 0.05). The increase in Ace mRNA levels was higher than that of Ace2 mRNA levels, leading to an elevated Ace/Ace2 ratio. Together, these data suggest that the ACE-Ang II-AT1 axis is the dominant axis in severe kidney injury.


Congenital Hypothyroidism due to a Low Level of Maternal Thyrotropin Receptor-Blocking Antibodies.
Maternal TSH receptor antibodies (TRAbs) can cross the placenta and affect fetal and neonatal thyroid function. Maternal TSH receptor-blocking antibodies (TBAbs) are a rare cause of congenital hypothyroidism. Following the discovery of a highly elevated TSH on her neonatal screening test, a 10-day-old girl with no familial history of thyroid disorder was referred to the pediatric endocrinology unit. Hypothyroidism was confirmed with a highly elevated TSH (817 mIU/L, reference range 0.4-3.1) and very low levels of FT4 (1.8 pmol/L, reference range 12-22). Anti-TPO antibodies were at 81 IU/mL (reference range <34), TRAbs at 1.7 IU/L (reference range <1.75), and thyroglobulin at 9.4 µg/L (reference range 3.5-77). The thyroid appeared normal on ultrasonography, and no radioiodine uptake was seen on the scintigraphy after the perchlorate discharge test. Concomitantly, a severe maternal hypothyroidism was discovered (TSH 224 mIU/L). The maternal ultrasound appeared normal, anti-TPO antibodies were moderately elevated, and TRAbs were at 3.2 IU/L. TBAbs activity was measured in the mother and her daughter, and a very high and similar blocking activity was observed in both patients (TBAbs 89%, reference range <10%). L-thyroxine treatment was introduced in the newborn and was successfully discontinued at 6.5 months of age, as the TBAbs activity decreased. We report herein a case of transient congenital hypothyroidism with a normal neonatal TRAbs level. In case of maternal TBAbs, similar activity of maternal TBAbs must be expected in the neonate, independently of the neonatal level of TRAbs.


Cobalt phosphide nanowire array as an effective electrocatalyst for non-enzymatic glucose sensing.
It is of significant importance for the design of electrodes to construct enhanced electrochemical sensing platforms, and a nanoarray offers an ideal architecture for the detection of molecules. In this communication, we demonstrate that cobalt phosphide nanowire array grown in situ on titanium mesh (CoP NA/TM) exhibits high catalytic activity towards electrooxidation of glucose. As a non-enzymatic electrochemical glucose sensor, this CoP NA/TM catalytic electrode possesses superior analytical performance with a short response time of less than 5 s, a wide linear range of 0.0005-1.5 mM, a low detection limit of 0.1 μM (S/N = 3), a high sensitivity of 5168.6 μA mM-1 cm-2, and a remarkable selectivity and long-term stability for glucose detection. We further demonstrate the successful use of such glucose biosensors in human blood serum and fruit juice.


Chondroitin sulfate B exerts its inhibitory effect on secondary lymphoid tissue chemokine (SLC) by binding to the C-terminus of SLC.
We previously reported that certain glycosaminoglycans (GAGs) bind secondary lymphoid tissue chemokine (SLC, CCL21) and that the SLC-binding GAGs, including chondroitin sulfate B (CS B), negatively modulate the function of SLC, although the mechanism remains unknown [J. Biol. Chem. 276 (2001) 5228]. To gain insight into the mechanism of inhibition, we used a C-terminally truncated SLC (SLC-T) that lacked clusters of basic amino acid residues that have been implicated in GAG binding. While SLC-T failed to bind any GAGs, it induced prominent intracellular Ca(2+) mobilization in CC chemokine receptor (CCR) 7-expressing cells, as did wild-type SLC. However, the SLC-T-induced Ca(2+) influx was not inhibited by CS B, unlike the SLC-induced Ca(2+) influx. These results demonstrate the requirement of the C-terminus of SLC for the inhibition of chemokine responses by CS B; that is, CS B exerts its inhibitory effect by binding to the C-terminus of SLC, thus defining the mode of action of CS B on certain chemokines.


Outcome of gallbladder preservation in surgical management of primary bile duct stones.
To evaluate the methods and outcome of gallbladder preservation in surgical treatment of primary bile duct stones. Thirty-five patients with primary bile duct stones and intact gallbladders received stone extraction by two operative approaches, 23 done through the intrahepatic duct stump (RBD-IDS, the RBD-IDS group) after partial hepatectomy and 12 through the hepatic parenchyma by retrograde puncture (RBD-RP, the RBD-RP group). The gallbladders were preserved and the common bile duct (CBD) incisions were primarily closed. The patients were examined postoperatively by direct cholangiography and followed up by ultrasonography once every six months. In the RBD-IDS group, residual bile duct stones were found in three patients, which were cleared by a combination of fibrocholedochoscopic extraction and lithotripsy through the drainage tracts. The tubes were removed on postoperative day 22 (range: 16-42 days). In the RBD-RP group, one patient developed hemobilia and was cured by conservative therapy. The tubes were removed on postoperative day 8 (range: 7-11 days). Postoperative cholangiography showed that all the gallbladders were well opacified, contractile and smooth. During 54 (range: 6-120 months) months of follow-up, six patients had mildly thickened cholecystic walls without related symptoms and further changes, two underwent laparotomies because of adhesive intestinal obstruction and gastric cancer respectively, three died of cardiopulmonary diseases. No stones were found in all the preserved gallbladders. The intact gallbladders preserved after surgical extraction of primary bile duct stones will not develop gallstones. Retrograde biliary drainage is an optimal approach for gallbladder preservation.