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Introduction {#sec1-1}
============
Infliximab (IFX), a chimeric anti-TNFα antibody, is effective in inducing and maintaining remission in a considerable proportion of IBD patients refractory to any other treatments \[[@ref1],[@ref2]\]. However, 8-12% of adult and/or pediatric patients fail to respond to the induction regimen (known as primary non responders) and approximately 40% of patients who respond initially and achieve clinical remission inevitably lose response over time\[[@ref3],[@ref7]\]. Lack of response to IFX is a stable trait and suggests that the differences in response might be in part genetically determined. Considering the high cost and safety profile of this drug, genetic targeting of patients responding to this therapy is certainly of great interest \[[@ref8]\]. So far, limited candidate gene association studies with response to IFX have been reported \[[@ref9]-[@ref11]\]. Recently, a genome-wide association study (GWAS) in paediatric IBD patients has revealed that the 21q22.2/BRWDI loci were associated with primary non response \[[@ref12]\]. Furthermore, although TNFa gene is of great interest as a candidate gene for pharmacogenetic approaches few studies have been performed to date and some have led to contradictory results \[[@ref10],[@ref11],[@ref13]-[@ref15]\].
All anti-TNF agents share an IgG1 Fc fragment, but the contribution of the Fc portion to the response to treatment among currently used TNF blockers remains unknown. Receptors for IgG-Fc portion (FcR) are important regulatory molecules of inflammatory responses. FcR polymorphisms alter receptor function by enhancing or diminishing the affinity for immunoglobulins \[[@ref16]\]. Three major classes of FcR that are capable of binding IgG antibodies are recognised: FcγRΙ (CD64), FcγRΙΙ (CD32), and FcγRΙΙΙ (CD16). FcγRΙΙ and FcγRΙΙΙ have multiple isoforms (FcγRΙΙΙA/C and B; FcγRΙΙΙA and B) \[[@ref16]\]. The most frequent polymorphism of *FcγRΙΙΙA* is a point mutation affecting amino acids in codon 158 in the extracellular domain. This results in either a valine (V158) or a phenylalanine (F158) at this position. Recently, it has been reported that CD patients with *FcγRΙΙΙA* -158V/V genotype had a better biological and possibly better clinical response to IFX \[[@ref17]\]. However, further studies did not confirm this observation \[[@ref18]\].
The aim of this study was to assess whether the *TNF* and/ or *FcγRΙΙΙA* gene polymorphisms are genetic predictors of response to IFX, in a cohort of Greek patients with adult or paediatric onset of CD.
Patients - Methods {#sec1-2}
==================
Patients {#sec2-1}
--------
We enrolled 106 consecutive patients with newly diagnosed CD attending the outpatient IBD Clinic at the 1^st^ Department of Gastroenterology, "Evangelismos" Hospital (79 adults) or the 1^st^ Department of Pediatrics, University Hospital of Athens "Aghia Sophia"(27 children). The diagnosis of CD was based on standard clinical, endoscopic, radiological, and histological criteria \[[@ref1],[@ref19]\]. Eligible patients should have inflammatory (luminal) disease and be naive to IFX.
IFX was administered intravenously at a dose of 5mg/kg at weeks 0, 2, 6 and then every 8 weeks. Clinical and serological responses were assessed using the Harvey-Bradshaw Index (HBI) \[[@ref20]\] and the serum levels of C-reactive protein (CRP), respectively, at baseline (before the 1st infusion of IFX), the day before each subsequent IFX infusion and after 12 weeks of treatment. Ileocolonoscopy was performed by a single endoscopist (GJM) at baseline and after 12-20 weeks of therapy to assess mucosal healing. Any changes in endoscopic appearance compared to baseline endoscopy were classified in four categories \[[@ref21],[@ref22]\] \[[Table 1](#T1){ref-type="table"}\]. Patients were classified in accordance to response to IFX therapy as shown in [table 2](#T2){ref-type="table"}. The ethical committee of the participating hospitals approved the study. Research was carried out according to Helsinki Convention (1975) and written inform consent was obtained in advance from each patient.
######
Grading of endoscopic mucosal lesions \[[@ref21],[@ref22]\]
######
Classification of the study population due to response to infliximab therapy
Genotyping {#sec2-2}
----------
Genomic DNA from whole blood containing EDTA was extracted using standard techniques (NucleoSpin Blood kit, Macherey-Nagel, Germany). All polymerase chain reactions (PCRs) were run under conditions previously described \[[@ref23]\]. Primer sequences for the gene polymorphism at --308 were forward 5′-GGG ACA CAC AAG CAT CAA GG-3′ and reverse 5′-GGG ACA CAC AAG CAT CAA GG-3′, for the polymorphism at −238 forward 5′-ATC TGG AGG AAG CGG TAG TG-3′ and reverse 5′-AGA AGA CCC CCC TCG GAA CC-3′. The PCR products were digested at 37 °C with NcoI to detect the SNP in the −308 gene allele and MspI to detect the polymorphism of the −238 nucleotide. The -857 C/T polymorphism was analyzed by allele-specific PCR method24 using the primers TNF857-C: 5′-aag gat aag ggc tca gag ag-3′, TNF857-N: 5′-cta cat ggc cct gtc ttc g-3′ and TNF857-M: 5′-t cta cat ggc cct gtc ttc a-3′. The --158V/F polymorphism of FcγRΙΙΙA gene was detected as described by Leppers-van de Straat et al \[[@ref25]\] using the primers 5′-CTG AAG ACA CAT TTT TACT CC CAA (A/C)-3′ and 5′-TCC AAA AGC CAC ACT CAA AGA C-3′. The PCR products were then subjected to 3% agarose-gel electrophoresis. "No target" controls were included in each PCR batch to ensure that reagents had not been contaminated.
Statistical Analysis {#sec2-3}
--------------------
Genotype frequencies were compared with the chi-square with Yate's correction using S-Plus (v. 6.2Insightful, Seattle, WA). Odds ratios (ORs) and 95 confidence intervals (CIs) were obtained with GraphPad (v. 3.00, GraphPad Software, San Diego, CA). The p values are all two-sided. Correction for multiple testing was not applied in this study. *P* values of \< 0.05 were considered to be significant.
Results {#sec1-3}
=======
Patient demographic and clinical characteristics are given in [Table 3](#T3){ref-type="table"}. There were 68 (64.15%) complete responders, 25 (23.58%) partial responders and 13 (12.26%) non responders to IFX in this study. There were no statistical differences in the mean age, gender, disease duration, location and behavior and smoking habits between complete or partial responders and primary non-responders. There was no disagreement between HBI scores and serum CRP levels. Although, the post-treatment CRP levels were significantly lower in complete responders compared to partial and non-responders, the decrease in CRP levels did not differ significantly between the three groups. Post-treatment CRP levels and mean HBI score were significantly lower in complete responders compared to pre-treatment values in contrast to partial and/or non-responders where the CRP levels and the mean HBI score did not differ significantly.
######
Demographic, clinical and biological characteristics of the study population
The -238 G/A, -308 G/A, and -857 C/T polymorphisms of the TNF gene and the -158 V/F polymorphism in the *FcγRΙΙΙA* gene were successfully determined in all subjects. The genotype distribution in complete, partial and non-responders were presented in [Table 4](#T4){ref-type="table"}. No significant difference was observed for the polymorphism tested. In addition, although there may be genetic differences in early (paediatric)-onset and late (adult)-onset CD we were unable to detect any such differences although the number of paediatric patients included in the current study did not allow firm conclusions.
######
Genotype frequency in complete responders, partial responders and non responders
In the present study, we could not correlate the decrease in serum CRP levels with the genotypes tested in any particular group of patients since in most of the cases serum CRP levels dropped by more than 25% after 12 weeks of treatment. However, no significant decrease in CRP was observed between the TNF genotypes tested. Regarding the -158 V/F polymorphism in the *FcγRΙΙΙA* gene, the relative decrease in serum CRP levels was greatest in VV
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INTRODUCTION {#s1}
============
Hepatitis B virus (HBV) is still a major global health problem, with an estimated 257 million people worldwide that are chronically infected with HBV ([@B1]). HBV, together with duck hepatitis B virus (DHBV) and several other related animal viruses, belongs to the *Hepadnaviridae* family ([@B2]). The HBV virion is comprised of an outer envelope and an inner icosahedral nucleocapsid (NC) assembled by 240 copies of core protein (HBc) and packaged with a 3.2-kb partially double-stranded circular DNA genome ([@B3][@B4][@B8]). In addition to DNA-containing virions, a large amount of incomplete viral particles, such as hepatitis B surface antigen (HBsAg) particles, empty virions, and naked capsids, can also be released from cells in the process of virus replication ([@B9]). Subviral HBsAg particles are spherical or rodlike and are present in vast excess over virions in sera of CHB patients ([@B2]). Empty virions share the same structure as DNA-containing virions but are devoid of nucleic acids ([@B10][@B11][@B14]). Naked capsids, which exit cells via a route different from that of virions ([@B15][@B16][@B17]), have the same structure as NCs but are either empty or filled with viral RNA and immature viral DNA ([@B7], [@B11], [@B18][@B19][@B20]).
In NC, pgRNA undergoes reverse transcription into minus-strand DNA, followed by plus-strand DNA synthesis ([@B2], [@B21][@B22][@B24]). Intracellular NCs can be packaged with viral nucleic acids at all levels of maturation, including pgRNA, nascent minus-strand DNA, minus-strand DNA-RNA hybrids, and relaxed circular DNA (RC DNA) or double-stranded linear DNA (DSL DNA) ([@B5], [@B7]). Only the NCs with relatively mature viral DNA (RC or DSL DNA) are enveloped and secreted as virions. HBV replicating cells can release empty core particles assembled from HBc proteins and NCs that contain various species of replicative intermediate nucleic acids into the culture supernatant. However, while free naked capsids could be readily detected *in vitro* ([@B7], [@B11], [@B18][@B19][@B20]), they are hardly found in the blood of HBV-infected patients ([@B17], [@B25], [@B26]).
Although extracellular HBV RNA was detected in both *in vitro* cell culture systems and in clinical serum samples, its origin and composition remain controversial. It was proposed that extracellular HBV RNA represents pgRNA localized in virions ([@B27]). However, HBV spliced RNA and HBx RNA were also detected in culture supernatant of HBV stably replicating cells as well as in sera of CHB patients ([@B28], [@B29]). In addition, extracellular HBV RNA was also suggested to originate from damaged liver cells ([@B30]), naked capsids, or exosomes ([@B11], [@B29]). Hence, these extracellular RNA molecules have never been conclusively characterized. Here, we demonstrate that extracellular HBV RNAs are heterogeneous in length, ranging from full-length pgRNA (3.5 kilonucleotides \[knt\]) to RNA fragments with merely several hundred nucleotides. These RNA molecules represent 3′ receding pgRNA fragments that have not been completely reverse transcribed to DNA and pgRNA fragments hydrolyzed by the RNase H domain of polymerase in the process of viral replication. More importantly, extracellular HBV RNAs are localized in naked capsids and in virions in culture supernatants of HBV replicating cells and also circulate as CACs and virions in blood of hepatitis B patients.
RESULTS {#s2}
=======
Extracellular HBV RNAs are heterogeneous in length and predominantly integral to naked capsids instead of virions in HepAD38 cell culture supernatant. {#s2.1}
------------------------------------------------------------------------------------------------------------------------------------------------------
To ascertain the origin of extracellular HBV RNA, we first examined viral particles prepared from culture medium of an *in vitro* HBV stably transduced cell line. A human hepatoma HepAD38 cell line was used in this study, as it sustains vigorous HBV replication under the control of a tetracycline-repressible cytomegalovirus (CMV) promoter ([@B31]). Total viral particles were concentrated and centrifuged over a 10% to 60% (wt/wt) sucrose gradient. Most of the subviral HBsAg particles, virions, and empty virions were detected between fractions 9 to 14 ([Fig. 1A](#F1){ref-type="fig"}, upper and middle). Naked capsids, detected only by anti-HBcAg and not by anti-HBsAg antibodies, settled in fractions 5 to 8 ([Fig. 1A](#F1){ref-type="fig"}, middle and lower). The majority of viral nucleic acids were detected in fractions between 4 and 11 ([Fig. 1B](#F1){ref-type="fig"}, upper), which coincided with the fractions containing virions (fractions 9 to 11), naked capsids (fractions 4 to 7), and the mixture of these particles (fraction 8). Consistent with previous observations, HBV virions are packed with mature viral DNA (RC or DSL DNA), while naked capsids contain both immature single-stranded DNA (SS DNA) and mature viral DNA ([Fig. 1B](#F1){ref-type="fig"}, upper). Moreover, Northern blot results showed that most of the HBV RNA was detected in the naked capsids ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 4 to 7), whereas only a very small amount was associated with virions ([Fig. 1B](#F1){ref-type="fig"}, lower, fractions 9 to 11). HBV RNA detected in naked capsids ranged from the full length of pgRNA down to a few hundred nucleotides (shorter than the HBx mRNA \[0.7 knt\]). Moreover, RNA molecules within virions were much shorter than those within naked capsids. We excluded the possibility of artifacts generated by the SDS-proteinase K extraction method, as a similar RNA blot pattern was obtained using a TRIzol reagent to extract both intracellular nucleocapsid-associated and extracellular HBV RNA (not shown). Furthermore, quantification of viral RNA extracted by either the SDS-proteinase K method or TRIzol reagent produced a very similar copy number, except that the TRIzol reagent is known to preferentially extract RNA rather than DNA (not shown). Moreover, the RNA signal detected by Northern blotting could not be attributed to DNA fragments generated by DNase I treatment, which would reduce DNA to below the detection limit of the hybridization method (not shown). Furthermore, the RNA signal could be completely removed by an additional RNase A treatment (not shown).
{#F1}
To confirm the above-described results and to better separate naked capsids from HBV virions, isopycnic CsCl gradient ultracentrifugation was employed. Naked capsids were observed mainly in fractions 5 to 7, with densities ranging from 1.33 to 1.34 g/cm^3^ ([Fig. 2A](#F2){ref-type="fig"}). The smearing bands of naked capsids were likely caused by high concentrations of CsCl salt, as fractionation of naked capsids in a 1.18-g/cm^3^ CsCl solution produced single bands. Virions, detected by both anti-HBcAg and anti-HBsAg antibodies ([Fig. 2A](#F2){ref-type="fig"}, upper and middle), were packaged with viral DNA ([Fig. 2A](#F2){ref-type="fig"}, lower) and settled in fractions 13 to 15, with densities ranging from 1.23 to 1.25 g/cm^3^. In agreement with the results shown in [Fig. 1](#F1){ref-type="fig"},
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Koolstra K, Beenakker J‐WM, Koken P, Webb A, Börnert P. Cartesian MR fingerprinting in the eye at 7T using compressed sensing and matrix completion‐based reconstructions. Magn Reson Med. 2019;81:2551--2565. 10.1002/mrm.27594 30421448
**Funding information**
This project was partially funded by the European Research Council Advanced Grant 670629 NOMA MRI.
1. INTRODUCTION {#mrm27594-sec-0005}
===============
Ophthalmologic disease diagnosis conventionally relies mainly on ultrasound and optical imaging techniques such as fundus photography and fluorescent angiography (FAG), MRI is increasingly being used in the radiological community.[1](#mrm27594-bib-0001){ref-type="ref"}, [2](#mrm27594-bib-0002){ref-type="ref"}, [3](#mrm27594-bib-0003){ref-type="ref"} One of the main advantages of MRI is its capability to assess nontransparent tissues such as ocular tumors or structures behind the globe such as the eye muscles. Currently, however, these applications are mainly based on qualitative MRI methods using the large number of tissue contrasts addressable by MR. As an example, in Graves' ophthalmopathy fat‐suppressed T~2~‐weighted MRI is the standard to detect inflammation in the eye muscles,[4](#mrm27594-bib-0004){ref-type="ref"}, [5](#mrm27594-bib-0005){ref-type="ref"} whereas in the diagnosis of retinoblastoma, a rare intraocular cancer in children, standard T~1~‐ and T~2~‐weighted MRI is often performed to confirm the presence of the tumor and to screen for potential optic nerve involvement.[2](#mrm27594-bib-0002){ref-type="ref"} In more recent ophthalmologic applications of MRI, such as uveal melanoma (the most common primary intraocular tumor), quantitative MRI techniques including DWI[6](#mrm27594-bib-0006){ref-type="ref"} and DCE imaging[7](#mrm27594-bib-0007){ref-type="ref"} have been shown, but currently diagnosis is still based on qualitative methods.[3](#mrm27594-bib-0003){ref-type="ref"}
To personalize treatment plans quantitative parameters of the tissues involved, as can be acquired invasively for example by performing biopsies,[8](#mrm27594-bib-0008){ref-type="ref"} are highly desirable. However, quantitative parameter mapping by means of MRI requires long examination times, which would result in significant eye‐motion artifacts, as well as patient discomfort.[9](#mrm27594-bib-0009){ref-type="ref"} MR fingerprinting (MRF) is a recently introduced method for rapid quantitation of tissue relaxation times and other MR‐related parameters.[10](#mrm27594-bib-0010){ref-type="ref"} It uses a flip angle sweep to induce a unique signal evolution for each tissue type. Incoherent undersampling can be applied during sampling of the MRF train, enabling acceleration of the MRF scans.[10](#mrm27594-bib-0010){ref-type="ref"} Together with its ability to measure simultaneously T~1~ and T~2~, MRF offers a solution to the problem of obtaining quantitative measures in an efficient manner and in relatively short scanning times.
One of the main challenges in ocular imaging is in‐plane and through‐plane eye motion, often associated with eye blinking.[11](#mrm27594-bib-0011){ref-type="ref"}, [12](#mrm27594-bib-0012){ref-type="ref"}, [13](#mrm27594-bib-0013){ref-type="ref"} The motion results in corrupted k‐space data that introduces artifacts and blurring throughout the entire image. Shortening the scans would reduce motion‐related artifacts, but standard acceleration techniques are not optimal for the current eye application due to the following 3 reasons. First, a cued‐blinking protocol is typically used to control and reduce the eye motion.[3](#mrm27594-bib-0003){ref-type="ref"}, [11](#mrm27594-bib-0011){ref-type="ref"} This requires an instruction screen placed at the end of the MR tunnel to be visible to the patient which complicates the use of small phased array receive coils in front of the eye, blocking the view. Instead, a custom‐built single‐element eye loop coil is used, which provides a high local SNR[3](#mrm27594-bib-0003){ref-type="ref"} and screen visibility, but which clearly excludes the possibility of scan acceleration by means of parallel imaging.[14](#mrm27594-bib-0014){ref-type="ref"} Second, the gel‐like vitreous body has an extremely long T~1~, particularly at high field.[15](#mrm27594-bib-0015){ref-type="ref"} Its value of 3 to 5 s requires a long duration of the MRF sequence to encode the MR parameters (T~1~,T~2~) sufficiently. Thus, using a flip angle train with a small number of RF pulses is not feasible, hindering scan time reduction. Finally, a time‐efficient spiral sampling scheme, usually applied in MRF,[10](#mrm27594-bib-0010){ref-type="ref"}, [16](#mrm27594-bib-0016){ref-type="ref"}, [17](#mrm27594-bib-0017){ref-type="ref"}, [18](#mrm27594-bib-0018){ref-type="ref"}, [19](#mrm27594-bib-0019){ref-type="ref"} introduces off‐resonance effects in each of the individual MRF images.[20](#mrm27594-bib-0020){ref-type="ref"} This occurs even when combined with unbalanced sequences such as fast imaging with steady state precession,[16](#mrm27594-bib-0016){ref-type="ref"} which are in themselves robust to off‐resonance effects.[21](#mrm27594-bib-0021){ref-type="ref"} The off‐resonance effects present in spiral sampling schemes are much stronger at high field, where they result in blurring,[22](#mrm27594-bib-0022){ref-type="ref"} caused by strong main field inhomogeneities (particularly in the eye region due to many air‐tissue‐bone interfaces), as well as the presence of significant amounts of off‐resonant orbital fat around the eye.
In this work, a Cartesian sampling scheme is used, which is more robust than spiral sampling to off‐resonance effects, but which is significantly less time‐efficient.[23](#mrm27594-bib-0023){ref-type="ref"} With such a Cartesian sampling scheme, undersampling artifacts have a more structured nature compared with spiral sampling, which increases the temporal coherence of the artifacts in the MRF image series.[10](#mrm27594-bib-0010){ref-type="ref"}, [20](#mrm27594-bib-0020){ref-type="ref"} In this case, direct matching of the measured MRF signal reconstructed by plain Fourier transformations, to the simulated dictionary elements is not sufficiently accurate for high undersampling factors.[24](#mrm27594-bib-0024){ref-type="ref"}, [25](#mrm27594-bib-0025){ref-type="ref"} Therefore, the quality of the reconstructed MRF data has to be improved before the matching process.
Compressed sensing (CS) has been introduced as a technique to reconstruct images from randomly undersampled data by enforcing signal sparsity (in the spatial dimension only or both in spatial and temporal dimensions),[26](#mrm27594-bib-0026){ref-type="ref"}, [27](#mrm27594-bib-0027){ref-type="ref"} allowing a scan time reduction in many applications.[28](#mrm27594-bib-0028){ref-type="ref"}, [29](#mrm27594-bib-0029){ref-type="ref"}, [30](#mrm27594-bib-0030){ref-type="ref"} The flexibility of MRF toward different sampling schemes and undersampling factors makes it possible to reconstruct the source images by means of CS.[27](#mrm27594-bib-0027){ref-type="ref"}, [31](#mrm27594-bib-0031){ref-type="ref"}, [32](#mrm27594-bib-0032){ref-type="ref"} Higher acceleration factors might be feasible if the correlation in the temporal dimension is better used.[33](#mrm27594-bib-0033){ref-type="ref"} Examples of such reconstructions specifically tailored to MRF are given in Davies et al, Pierre et al, and Zhao et al[34](#mrm27594-bib-0034){ref-type="ref"}, [35](#mrm27594-bib-0035){ref-type="ref"}, [36](#mrm27594-bib-0036){ref-type="ref"} which take into account the simulated dictionary atoms in the image reconstruction process.
Recent work has shown that the temporal correlation in the MRF data can be exploited even further by incorporating the low rank structure of the data into the cost function,[37](#mrm27594-bib-0037){ref-type="ref"} a technique which was introduced into MR in Liang[
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Historical conceptualizations of depression
===========================================
There is a long tradition in phenomenologlcal psychopathology that stresses basic bodily alterations as core features of depressive states. Thus, Wernicke used the term "vital feelings" to describe certain somatic symptoms occurring in affective psychoses.^[@ref1]^ Vital feelings refer to the close relationship of the body to the awareness of self. They determine the way we experience our body and the impression we assume our physical presence makes on other people. Vital feelings are somatic affects localized In different parts of the body. Whereas vital feelings constitute the bodily background of our normal experiences, they may move to the fore In a depressive mood. For example, depressed patients very often complain of a headache which is described not exactly as an ordinary pain, but more as an unbearable pressure "like a band around the head." Other disturbed vital feelings affect the chest or the abdomen, and mediate unpleasant sensations of weight, tension, heaviness, or Inhibition, totally absorbing the focus of attention. In quite a similar way Dupré speaks of "coenestopathic states" which mean a distressing, qualitative change of normal physical feeling In certain areas of the body during an episode of depressive mood. It Is a global loss of vitality In which all bodily parts and functions may be altered, and all their performances depressed.^[@ref2]^ Kurt Schneider considered these disturbances of vital feelings to be the core of cyclothymic depression. In his psychopathologlcal assessment they were of paramount diagnostic significance In depressive Illness, more or less equivalent to the first-rank symptoms In schizophrenia.^[@ref3]^ Huber discriminated between vital disturbances on the one hand and vegetative symptoms In depression on the other.^[@ref4]^ Vital disturbances refer to the vital feelings just mentioned. They comprise a loss of general vital tone of the body, a prevailing fatigue or exhaustibility, and various forms of somatic dysesthesia, typically of a static, more localized character affecting head, chest, heart region, or abdomen. All-pervasive sensations of anesthesia, stiffness, and alienation of the total body may characterize a somatopsychic depersonalization in depression which may appear as a Cotard\'s syndrome in its extreme form. If the vital disturbances take on a peculiar form that is difficult to describe in ordinary everyday words, Huber speaks of a "coenesthetic" depression which must be typologically differentiated from the bizarre states of coenesthetic schizophrenia. Vegetative symptoms are closely associated with these vital disturbances and coenesthesias in depression. Disturbances of sleep, appetite, and digestion are most frequent. However, there may be many other vegetative symptoms in depression such as disordered salivation, transpiration and lacrimation, cardiac arrhythmias and dyspnea, loss of libido and various sexual dysfunctions, dys- or amen? orrhea, loss of or increase in body weight, decreased turgor of the skin, loss of hair, decrease in body temperature, nausea, vomiting, meteorism, dizziness, sweating, or sensations of coldness. Both vital disturbances, coenesthesias and vegetative symptoms, are typically coexistent with the well-known affective, behavioral, and cognitive symptoms of depression. With respect to the different settings of medical care, however, these psychological symptoms of depression may be masked by a dominant reporting of somatic symptoms. M. Bleuler addressed the point in his book *Depressions in Primary Care,* in 1943: *"It is a common and frequent observation that depressive patients with single somatic complaints come to the consulting room of the general practitioner, internal specialist, and even the surgeon, gynecologist, ophthalmologist, urologist and other medical specialists, and spontaneously, they only speak of somatic phenomena while concealing their state of depressive mood. They report palpitations, tightness of the chest, loss of appetite, obstipation, pollakiuria, amenorrhea and many others. Only when one looks at their psychic state does one discover that they report numerous hypochondriac ideas also in other areas, that in addition they produce depressive ideas of impoverishment and sin, that beyond that their whole stream of thoughts is inhibited, that the depression manifests itself not only in the somatic complaints reported, but in various other bodily expressions."^[@ref5]^*
In spite of this long-standing psychopathological view on the somatic foundation of depressive mood, at least in moderate and severe clinical states, it is bewildering that the official psychiatric classification systems of the *Diagnostic and Statistical Manual of Mental Disorders,* 4th edition *(DSM-IV)* and the *ICD-10* *Classification of Mental and Behavioral Disorders. Clinical Descriptions and Diagnostic guidelines (ICD-10)* only marginally appreciate somatic symptoms as diagnostic criteria for depressive disorders while focussing on the psychological symptoms of affect and cognition. So, *DSM-IV* lists only three criteria of somatic symptoms for major depressive disorder: sleep disturbance, appetite disturbance, and fatigue or loss of energy. And correspondingly, in *ICD-10,* disturbances of sleep and appetite, loss of libido, and amenorrhea are the only somatic symptoms considered to be of diagnostic significance for major depression. Beyond this short list of predominantly vegetative symptoms, no painful physical symptoms are mentioned in either the *DSM-IV* or *ICD-10.* There seems to be a major shift In diagnostic practice, however; the second version of the *Diagnostic and Statistical Manual of Mental Disorders,* 4th edition, Text Revision *(DSM-IV TR)* now Includes new criteria referring to "excessive worry over physical health and complaints of pain (eg, headaches or joint, abdominal, or other pains)."^[@ref6]^ This supplement of diagnostic criteria Is Indicative of an againIncreasing awareness of the importance of somatic symptoms in depression.
What is meant by "somatic" in somatic symptoms of depression?
=============================================================
In the literature there are many terms used to describe somatic symptoms in depression: somatic, somatlzed, physical, bodily, somatoform, painful, psychosomatic, vegetative, medically unexplained, masked, etc.^[@ref7]^ These diverse terms refer to different theoretical or diagnostic concepts. For states of depressive mood the neutral term "somatic" is preferred, comprising various bodily sensations that a depressed individual perceives as unpleasant or worrisome. These dysesthesias are very often localized In certain body parts or organs, or may affect the whole body In Its vital condition, as In the case of fatigue or loss of energy. Several basic physical dysfunctions, such as those of sleep, appetite, or digestion, are also to be included in the term "somatic." In addition, It may be clinically relevant to differentiate between painful and nonpalnful somatic symptoms of depression. From a diagnostic perspective one has to keep in mind that somatic symptoms play a significant role both in primary psychiatric disorders, first and foremost depressive and anxiety disorders, and in somatoform disorders. And In differential diagnosis, somatic symptoms must be considered as possibly even Indicative of underlying somatic diseases. A diagnostic challenge may be seen In the well-known fact that depressive, anxiety, somatoform disorders, and medical conditions are frequently coexistent, or Interact In the Individual patient.^[@ref8]-[@ref10]^ Regarding the assessment of somatic symptoms, Kroenke correctly points out that diagnosis very often is more approximative than precise. Presented somatic symptoms may be either clearly attributed to a distinct medical disorder or be placed into one of the following heuristic categories: somatoform disorder, another primary psychiatric disorder (often depression and/or anxiety), functional somatic syndrome (eg, irritable bowel syndrome, fibromyalgia, chronic fatigue syndrome), "symptom-only" diagnosis (eg, low back pain, idiopathic dizziness) or only partially explained by a defined medical disorder (eg, many states of chronic pain).^[@ref11]^
Epidemiological studies may provide an illuminating survey of the prevalence of somatic symptoms in depressive disorders, especially those encountered in primary care, and the prognostic value of somatic symptoms regarding their development in the further course of illness.
Somatic symptoms of depressive disorders in inpatient care and primary care
===========================================================================
In a clinical study, Hamilton reported that somatic symptoms prevailed in a great majority of depressed patients.^[@ref12]^ Somatic symptoms, particularly somatic anxiety and fatigue, were documented in up to 80% of a sample of 260 women and 239 men suffering from major depression. These somatic symptoms very frequently had an underlying psychopathologically relevant hypochondriasis, both in women and men. This study confirmed earlier studies showing that depressive disorders with predominantly somatic presentation were likely to be the most common form of depression, both in inpatient and outpatient care.^[@ref13],[@ref14]^ Hagnell and Rorsman stressed the Indicative significance of somatic symptoms in depressed primary care patients regarding their risk of suicide.^[@ref15]^
Epidemiological studies designed to establish prevalence figures for depressive disorders In primary care during recent years have uniformly demonstrated that depressive disorders are highly prevalent at this level of medical care.^[@ref16]-[@ref19]^ For the great majority of depressed patients seeking professional help in the official health care system, general practitioners and internists are the decisive interface for diagnosis and treatment of depression.^[@ref20]^ Primary-care patients with depression very often present with somatic complaints. This seems to be more the rule than the exception worldwide.^[@ref21],[@ref22]^ Two of the three most common symptoms reported during a current depressive episode were somatic (tlred/no energy/listless: 73%, broken sleep/decreased sleep: 63%) as shown by the European Study Society study (DEPRES II).^[@ref23]^ This study, however, also underlined that 65% of the depressed primary care patients suffered from a concomitant medical condition pointing to some likely difficulties In differential diagnosis. The multlcenter International study (n =1146) conducted by
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Irinotecan (CPT-11, Campto®) -- a semisynthetic, water-soluble derivative of the plant alkaloid camptothecin -- is the standard of care in the treatment of advanced colorectal cancer when 5-fluorouracil (5-FU)-based therapy has failed ([Cunningham *et al*, 2001](#bib5){ref-type="other"}). Phase II trials have demonstrated objective response rates of 16--27% in pretreated patients, with stabilisation of disease in a further 40--60% of patients ([Rougier *et al*, 1997](#bib13){ref-type="other"}; [Van Cutsem *et al*, 1999](#bib17){ref-type="other"}). Median overall survival rates of up to 10 months are achievable when irinotecan is used in relapsed/refractory colorectal cancer ([Shimada *et al*, 1993](#bib15){ref-type="other"}; [Rothenberg *et al*, 1996](#bib12){ref-type="other"}, [1999](#bib11){ref-type="other"}; [Pitot *et al*, 1997](#bib10){ref-type="other"}; [Rougier *et al*, 1997](#bib13){ref-type="other"}; [Van Cutsem *et al*, 1999](#bib17){ref-type="other"}). Two European phase III trials investigating the efficacy and safety of irinotecan, following 5-FU failure in advanced colorectal cancer, have demonstrated significant improvements in survival compared with best supportive care and 5-FU ([Cunningham *et al*, 1998](#bib6){ref-type="other"}; [Rougier *et al*, 1998](#bib14){ref-type="other"}). The main adverse events accompanying treatment with irinotecan in these trials were diarrhoea, neutropenia, fatigue, nausea and vomiting.
Although 350 mg m^−2^ as an intravenous infusion every 3 weeks is the standard recommended dosage of irinotecan, pharmacokinetic parameters of irinotecan-lactone and the active metabolite SN-38-lactone vary between individuals ([Xie *et al*, 2002](#bib18){ref-type="other"}). This may be attributed to differences in the levels of the enzymes that metabolise irinotecan, notably carboxylesterase for SN-38. Furthermore, the variable interindividual patient exposure to SN-38 has been identified as an important determinant of toxicity ([Mathijssen *et al*, 2002](#bib8){ref-type="other"}).
At the same time, there is convincing evidence of a dose--response relationship, and therefore a rationale for increasing doses when possible. In a phase I trial by [Abigerges *et al* (1995)](#bib1){ref-type="other"}, there were two recommended doses: 350 mg m^−2^ without high-dose loperamide and 600 mg m^−2^ with high-dose loperamide. With the exception of one responder treated at 260 mg m^−2^, all objective responses were observed at dose levels above 350 mg m^−2^. [Merrouche *et al* (1997)](#bib9){ref-type="other"} provided further support for this from a phase I trial in which an increased tumour response was seen at an irinotecan dose level of 500 mg m^−2^.
Thus, these data suggest that a fixed-dose strategy for administration of irinotecan may not be optimal for all patients, thereby comprising treatment. The interindividual variability in pharmacokinetic parameters and dose--response relationship provided the rationale for investigating a dose optimisation strategy for irinotecan ([Chabot *et al*, 1995](#bib4){ref-type="other"}). The present study investigated different strategies, using doses of irinotecan up to 500 mg m^−2^, as single-agent therapy in the treatment of patients with metastatic colorectal cancer resistant to 5-FU.
METHODS
=======
Patients
--------
Eligibility criteria included metastatic, histologically proven adenocarcinoma of the colon or rectum progressing on 5-FU-based chemotherapy (adjuvant and/or palliative); administration of ⩽2 5-FU-based regimens in the adjuvant setting or ⩽1 in the palliative setting; World Health Organization (WHO) performance status (PS) of ⩽2; adequate haematological, renal and hepatic function. Exclusion criteria included prior treatment with topoisomerase-I inhibitors; evidence of central nervous system metastases; prior history of chronic diarrhoea; current infection; or any other serious illness or medical condition.
Study design and conduct
------------------------
This was a prospective, randomised, multicentre, open-label, phase II study. The study was conducted in accordance with the Declaration of Helsinki (Hong Kong revision, 1989) and with the approval of the Ethics Committee (Institutional Review Board) at each participating centre. Written informed consent was obtained from each patient prior to his or her enrolment into the trial. An independent Monitoring Committee regularly assessed the safety and efficacy issues and reviewed the conduct of the study if needed. An External Response Review Committee (ERRC) assessed tumour responses without knowledge of the randomisation arm. The aim of the study was to determine the optimal dosing strategy in terms of efficacy and safety of single-agent irinotecan (by individual dose optimisation based on patient tolerance to treatment, or optimisation based on specific baseline risk factors) in the treatment of 5-FU-resistant patients with metastatic colorectal cancer. The primary efficacy endpoint was the overall response rate.
### Dosing scenarios
Patients were randomised to one of three groups (A, B and C (outlined below)), each group receiving irinotecan as a 30 min intravenous infusion scheduled every 21 days. This dosing interval could be extended to a maximum of 35 days in the event of persistent toxicity to allow satisfactory recovery from the previous cycle. Doses \<250 mg m^−2^ or \>500 mg m^−2^ were not used in this study; patients who exhibited significant toxicity at 250 mg m^−2^ were withdrawn from the study.
Group A was the reference group in which a fixed dose of 350 mg m^−2^ of irinotecan was administered on Day 1. In subsequent cycles, the dose of irinotecan could be decreased (but not increased) according to the presence of significant toxicity at this dose.
Groups B and C investigated dosing scenarios to select patients for whom the higher dose of irinotecan (500 mg m^−2^) could be optimally used. Patients randomised to Group B received irinotecan at a starting dose of 250 mg m^−2^ followed by increasing doses (350 and 500 mg m^−2^) depending on the tolerance observed in the preceding cycle. In the event of significant toxicity, dose reductions were implemented.
In Group C, the irinotecan dose was based on protocol-defined toxicity risk factors identified at baseline: grade 3--4 neutropenia (bilirubin \>70% upper limit of normal (UNL), haemoglobin \<12 g dl^−1^, \>3 organs involved) and/or grade 3--4 diarrhoea (PS⩾1, creatinine \>70% UNL ([Freyer *et al*, 2000](#bib7){ref-type="other"})). Patients could be started at an irinotecan dose of 500 mg m^−2^ in the absence of toxicity risk factors. The starting dose of irinotecan was 350 mg m^−2^ in patients with one risk factor or one factor from each group, and 250 mg m^−2^ for patients with \>2 risk factors or two factors from the same group. The dose was not escalated, but could be reduced to 250 mg m^−2^ in the event of significant treatment-emergent toxicity.
Concomitant treatments and follow-up
------------------------------------
Antiemetic drugs were administered as premedication to irinotecan infusions. Atropine was permitted for acute anticholinergic symptoms and loperamide (or similar) for delayed diarrhoea. In addition, preventative oral antibiotic therapy (e.g. an oral fluoroquinolone) was administered to patients with persistent (\>48 h) grade 4 diarrhoea or for diarrhoea associated with grade 3--4 neutropenia or fever. No granulocyte-colony-stimulating factor (G-CSF) support was allowed. All patients were followed until disease progression, unacceptable toxicity or death occurred, or the patient chose to withdraw from the trial. In all cases, in each group where toxicity necessitated a dose reduction, delay or study treatment termination, the patient was followed up until the event had resolved.
Efficacy, safety and pharmacokinetic evaluations
------------------------------------------------
Tumour response rate, the primary efficacy end point, was measured according to WHO criteria and evaluated by the ERRC. Response was defined as complete (CR) plus partial (PR) response and as tumour growth control in terms of stabilisation of disease (PR plus no change/stable disease). Secondary efficacy variables were the duration of response and disease stabilisation, time to progression (TTP), time to treatment failure (TTF) and overall survival. The duration of response was measured from the first day of infusion of irinotecan to the first date that disease progression was noted or to the date of death for any reason. Time to progression was calculated from the date of randomisation to the first documented date of progression or the date of death for any reason. Time to treatment failure was the period between the date of randomisation and the date
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Introduction {#sec1}
============
Acute aortic dissection (AAD) is a relatively uncommon medical emergency with a high mortality after symptom onset. The mortality of acute type A aortic dissection increases by 1--2% per hour during the first 48 h if no treatment is received \[[@cit0001]\]. Meanwhile, other common causes of acute chest pain, such as acute myocardial infarction (AMI) and pulmonary embolism (PE), also require rapid differentiation from AAD due to their critical and lethal characteristics \[[@cit0002]\]. However, the misdiagnosis rate of AAD has been reported to be approximately 30% on initial evaluation \[[@cit0003], [@cit0004]\]. Currently, noninvasive imaging modalities, including enhanced computed tomography (CT), transesophageal echocardiography (TEE) and magnetic resonance imaging (MRI), have been developed to improve the diagnosis of AAD, but these imaging modalities are expensive, time-consuming and unavailable at the bedside. Therefore, a rapid, cheap, reliable and sensitive laboratory test is urgently needed to diagnose AAD.
D-dimer, the degradation product of cross linked fibrin, is significantly elevated in AAD patients \[[@cit0005]--[@cit0008]\] and has been suggested for use as a complementary marker to rule out AAD \[[@cit0005]--[@cit0007], [@cit0009]--[@cit0011]\]. However, in real-world clinical practice, AAD, PE and AMI are all thrombogenic diseases with high mortality, and whether the D-dimer level is helpful for differentiating these diseases remains to be elucidated. We therefore conducted a prospective cohort study to evaluate the validity and reliability of D-dimer level for differentiating AAD from other types of acute chest pain, including PE, AMI, unstable angina (UA), and other uncertain diagnoses of chest pain.
Material and methods {#sec2}
====================
Study population {#sec2.1}
----------------
A single-center, prospective cohort study was conducted in Fuwai Hospital (the National Center for Cardiovascular Diseases in China) from January 2009 to January 2010. A series of consecutive patients with acute chest pain who presented to the emergency department (ED) of Fuwai Hospital within 24 h of symptom onset were enrolled in a prospective manner. Baseline clinical characteristics such as sex, age, Stanford types of AAD, intervals from onset of symptoms to hospital admission, medical histories, baseline parameters of physical examinations and laboratory tests including C-reactive protein (CRP), imaging examinations, in-hospital managements, ED diagnosis and discharge diagnosis were recorded according to pre-designed case report forms. The study protocols were approved by the appropriate institutional review boards of Fuwai Hospital and complied with the Declaration of Helsinki. All subjects provided written informed consent.
D-dimer test and diagnosis {#sec2.2}
--------------------------
Plasma D-dimer levels were measured using a stago-evolution device (France) in patients with chest pain immediately following admission. The results collected are expressed in micrograms per milliliter. The effective detection range of the assay is 0.22--20 µg/ml. Diagnoses of AAD and PE were confirmed by aorta or pulmonary angiography with multi-detector CT scan. Acute myocardial infarction was confirmed by acute chest pain, elevated cardiac-enzyme levels (cardiac troponin I or T, or the MB fraction of creatine kinase exceeded the 99^th^ percentile upper reference limit), documented findings of a new ST segment elevation/depression or a new T wave inversion on electrocardiography, and/or with evidence of obstructive coronary artery on angiography. Unstable angina was confirmed by chest pain, ST segment depression or T wave changes with evidence of obstructive coronary artery on angiography, but without the elevation of cardiac enzymes.
Statistical analysis {#sec2.3}
--------------------
Continuous variables are presented as mean ± SD or median and interquartile range according to whether they follow Gaussian distributions. Categorical data are presented as numbers and proportions. Baseline characteristics between groups were compared using Student's *t* test or the nonparametric Mann-Whitney test for continuous data and the χ^2^ test for categorical data. Receiver-operating characteristic (ROC) curves were constructed to calculate the sensitivity for AAD. The area under the curve (AUC) was calculated. A *p-*value \< 0.05 was considered statistically significant. The statistical calculations were performed with SPSS 19.0 (SPSS Inc., Chicago, Illinois, USA).
Results {#sec3}
=======
A total of 790 patients were enrolled, including 202 AAD, 43 PE, 315 AMI, 136 UA, and 94 cases with other uncertain diagnoses. Of the 202 AAD patients confirmed by CT angiography, 119 (58.9%) were Stanford type A AAD cases and 83 (41.0%) were Stanford type B AAD cases.
Patient demographics and baseline characteristics are shown in [Table I](#t0001){ref-type="table"}. Compared to the patients with other causes of chest pain, AAD patients were more likely to be younger and male and tended to have concomitant hypertension but rarely have diabetes mellitus (all *p* \< 0.001).
######
Baseline characteristics of AAD patients and non-AAD (PE, UA, AMI, and uncertain diagnosis)
Parameter AAD (*n* = 202) Non-AAD *P*-value
------------------------------------ ----------------- ----------- ------------ ------------ ----------- ----------
Age \[years\] 51 ±12 55 ±17 61 ±12 60 ±12 54 ±17 \< 0.001
Male, *n* (%) 169 (83.7) 21 (48.8) 102 (75.0) 254 (80.6) 65 (69.1) \< 0.001
Systolic blood pressure \[mm Hg\] 141 ±31 129 ±21 138 ±23 128 ±23 133 ±23 \< 0.001
Diastolic blood pressure \[mm Hg\] 80 ±21 81 ±10 87 ±57 79 ±14 81 ±14 0.535
Heart rate \[beats per minute\] 81 ±19 87 ±17 72 ±13 76 ±18 80 ±28 \< 0.001
Body mass index \[kg/m^2^\] 24.6 ±3.2 25.7 ±3.7 26.7 ±4.2 25.5 ±3.4 26.2 ±4.9 0.450
Creatinine kinases \[U/l\] 269 ±544 85 ±61 97 ±84 497 ±688 109 ±105 \< 0.001
Fasting blood glucose \[mmol/l\] 7.5 ±1.9 6.3 ±1.6 7.4 ±3.1 8.4 ±3.4 7.1 ±2.7 \< 0.001
Hypertension, *n* (%) 133 (65.8) 13 (31.0) 86 (63.2) 161 (51.3) 42 (46.2) \< 0.001
Diabetes mellitus, *n* (%) 5 (2.5) 2 (4.8) 31 (22.8) 68 (21.7) 13 (14.3) \< 0.001
Hypercholesterolemia, *n* (%) 18 (8.9) 3 (7.1) 34 (25.0) 75 (24.0) 13 (14.3) \< 0.001
Stroke, *n* (%) 10 (5.0) 2 (4.8) 13 (9.6) 33 (10.5) 7 (7.7) 0.471
Smoker, n (%) 64 (31.7) 7 (16.7) 31 (22.8) 105 (33.5) 18 (19.8) 0.060
Drinker, *n* (%) 21 (10.4) 0 (0.0) 6 (4.4) 14 (4.5) 6 (6.6) 0.110
AAD -- acute aortic dissection, PE -- pulmonary embolism, UA -- unstable angina, AMI -- acute myocardial infarction.
The D-dimer level was elevated (\> 0.50 µg/ml) in 190 (94.1%) AAD patients. The D-dimer level in AAD patients was approximately 9-fold higher than that in non-AAD patients (median: 4.19 vs. 0.45 µg/ml, *p* \< 0.05). [Figure 1](#f0001){ref-type="fig"} shows the D-dimer level in patients with different causes of chest pain. The D-dimer level was significantly higher in patients with AAD than in patients with UA (median: 0.38 µg/ml, *p* \< 0.001), AMI (median: 0.45 µg/ml, *p* \< 0.001) and other uncertain diagnoses (median: 0.44 µg/ml, *p* \< 0.001), but it was comparable with that of PE patients (median: 2.72 µg/ml, *p* = 0.065). Similarly, the D-dimer level in PE patients was significantly higher than that in patients with UA, AMI, or other uncertain diagnoses (all *p* \< 0.001). Moreover, patients with type A AAD had higher D-dimer levels
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Background
==========
Polysaccharide-rich fungi and plants have been employed for centuries by cultures around the world for their dietary and medicinal benefits \[[@B1]-[@B5]\]. Often thought to merely support normal bowel function and blood glucose and lipid levels \[[@B6]-[@B8]\], certain polysaccharides have attracted growing scientific interest for their ability to exert marked effects on immune system function, inflammation and cancers \[[@B9]-[@B11]\]. Many of these chemically and structurally diverse, non- to poorly-digestible polysaccharides have been shown to beneficially affect one or more targeted cellular functions *in vitro*\[[@B11]-[@B16]\], but much of the *in vivo*literature consists of studies in which polysaccharides were injected \[[@B1],[@B2]\]. For clinicians and scientists interested in immunologic effects following dietary intake, the value of such studies is uncertain. Polysaccharides that elicit effects *in vitro*or by injection may be ineffective or have different effects when taken orally \[[@B17]\]. We thus decided to conduct a systematic review to evaluate the specific immunologic effects of dietary polysaccharide products on rodents and human subjects.
Methods
=======
Literature review
-----------------
Studies were identified by conducting electronic searches of PubMed and Google Scholar from their inception to the end of October 2009. The reference lists of the selected articles were checked for additional studies that were not originally found in the search.
Study selection and data extraction
-----------------------------------
The following search terms were combined with the term polysaccharide: dietary AND immune, or oral AND immune, or dietary AND inflammation, or oral AND inflammation. When specific polysaccharides or polysaccharide-rich plants and fungi were identified, further searches were conducted using their names with the same search terms. Studies were selected based on the following inclusion criteria:
1\. Rodent or human studies
2\. The presence of test group and control group (using either placebo, crossover, sham, or normal care)
3\. Studies reporting statistically significant immunomodulatory effects
4\. English language
5\. Studies published up to October 2009.
Two researchers (JER, EDN) reviewed the list of unique articles for studies that fit the inclusion criteria. Uncertainties over study inclusion were discussed between the researchers and resolved through consensus. Searches were then conducted to obtain specific polysaccharide product information: safety (using the search terms: toxicity, NOAEL, LD~50~), composition and structure, and disposition.
Quality assessment
------------------
Each study was assessed as to whether or not it reported a significant outcome measure for the polysaccharide intervention group.
Results
=======
A total of 62 rodent publications (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}) and 15 human publications (Table [4](#T4){ref-type="table"}) were deemed appropriate for inclusion in this review. Available structural and compositional information for these immunomodulatory polysaccharides are provided in Table [5](#T5){ref-type="table"} and safety information is provided in Table [6](#T6){ref-type="table"}. The majority of animal studies explored models in which animals were injected or implanted with cancer cells or tumors, were healthy, or were exposed to carcinogens. Other studies investigated immunodeficient, exercise-stressed, aged animals, or animals exposed to inflammatory agents, viruses, bacterial pathogens, pathogenic protozoa, radiation or mutagens. Human studies assessed immunomodulatory effects in healthy subjects, or patients with cancers, seasonal allergic rhinitis or aphthous stomatitis. Because of the limited number of human studies, we included some promising open-label controlled trials. Human study durations ranged from four days to seven years; daily doses ranging from 100-5,400 mg were reported to be well-tolerated.
######
Immunomodulatory Glucan Extracts: Oral Animal Studies
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Source Extract Animal Dose/day Duration of study Treatment Effects Reference
------------------------------------- ---------------------------------- --------------------------------------------------------------------------- ---------------------------------------------------------------------- ------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------
*Agaricus*\ α-1,6 and\ 8-week ♀ C3H/He mice (5/group) 100 mg/kg IG every 3 days 1 month Healthy animals ↑ \#s splenic T lymphocytes (Thy1.2, CD4+ and CD8+) \[[@B24]\]
(*A. blazei*) *subrufescens* α-1,4 glucans
Aqueous 7-9-week ♂ Balb/cByJ mice (40/group) 1 ml 0.45N, 0.6N, or 3N aqueous extract 2 months All doses ↑ serum IgG levels, CD3+ T cell populations and PML phagocytic activity \[[@B22]\]
7-9-week male Balb/cByJ mice (40/group) 1 ml 0.45N, 0.6N, or 3N aqueous extract 10 weeks IP injection of OVA at 4 weeks 0.6N and 3N ↑ levels of OVA-specific serum IgG 28 days post-immunization; all doses ↑ delayed-type hypersensitivity and TNF-α secreted from splenocytes at 10 weeks; 0.6N ↑ splenocyte proliferation at 10 weeks
5-6 -week ♀ BALB/cHsdOla mice (8/group × 2) One 200 μl extract day 1, orogastric intubation 1 week Injected IP fecal solution day 2 ↓ CFU in blood of mice with severe peritonitis & improved overall survival rate in all peritonitis groups \[[@B46]\]
6-week BALB/c nu/nu mice (7/group) 2.5 mg extract days 20-41, drinking water 41 days Injected SC Sp-2 myeloma cells day 1 ↓ tumor size & weight after 21 days treatment \[[@B65]\]
Aqueous, acid treated 6-week ♀ C57BL/6 mice (10/group) 20, 100 or 500 μg/ml, drinking water 9 days Injected IP human ovarian cancer cells day 1 500 μg/ml ↓ tumor weight \[[@B66]\]
20, 100 or 500 μg/ml, drinking water 3 weeks Injected IV murine lung cancer (3LL) cells 100 & 500 μg/ml ↓ \#s metastatic tumors
Aqueous, with 200 ng/day\ 6-week ♀ BALB/c mice (10/group) 200 ng days 5-21 3 weeks Injected Meth A tumor cells day 1 ↓ tumor size & weight \[[@B23]\]
β-glucan
2 weeks Injected Meth A tumor cells ↑ cytotoxic T lymphocyte activity & spleen cell IFN-α protein
300 mg 5 days Healthy animals ↑ splenic NK cell activity
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Introduction
============
Phytogenic feed additives (PFA) have attracted a lot of attention in recent years. When used in diets, these substances exhibit antioxidative properties and antimicrobial activity, improve nutrient absorption, and could ultimately improve animal performance ([@bib26]; [@bib30]; [@bib37]). PFA are less toxic, have fewer side effects, and are residue-free compared with synthetic feed additives, and are thought to be ideal feed additives in animal production ([@bib25]). Therefore, many phytogenic compounds have been recommended for use as feed additives.
Fenugreek (*Trigonella foenum-graecum* L) belongs to the Leguminosae family and is cultivated predominantly in India, West Asia, the Mediterranean, North Africa, and Canada ([@bib29]). The seeds are generally used for condiments in various food preparations, are regarded to have great nutritive value and restorative properties, and have been used in folk medicine for centuries for their hypoglycemic, anthelmintic, antibacterial, antiinflammatory, antipyretic, and antimicrobial properties ([@bib49]; [@bib7]; [@bib50]). The major active components of fenugreek seeds are 4-hydroxyisoleucine, trigonelline, galactomannan with flavonoids, carotenoids, coumarins, and saponins, which confer pharmacological activity and beneficial effects ([@bib46]; [@bib7]).
Recently, the modes of PFA action in vivo, including aromatic plants, plant extracts, their single active components, or their blended additives, have been investigated in several studies ([@bib54]; [@bib6]; [@bib37]; [@bib38]). However, in laying hens, a comparatively small number of studies have investigated the effects of fenugreek seed on production performance and egg quality. Therefore, in this study, an attempt was made to evaluate the fenugreek seed as natural growth promoter in laying hen diets. The evaluation included biochemical changes in egg production, egg quality, blood profiles, cecal microflora, and excreta noxious gas emission.
Materials and Methods
=====================
Ethical Considerations
----------------------
The experimental protocols describing the management and care of animals were reviewed and approved by the Animal Care and Use Committee of Dankook University.
Experimental Design, Animals, and Housing
-----------------------------------------
A total of 384, 26-week-old (Hyline-brown) laying hens were used in this 6-week trial. Laying hens were randomly assigned to one of three treatments with eight replicates (16 hens/replicate). The experimental treatments were: control, basal diet (FSE 0%); fenugreek seed extract (FSE) 0.05%, basal diet + 0.05% FSE; FSE 0.1%, basal diet + 0.1% FSE. The FSE used in our study was Nutrifen^®^ (Emerald Seed Products Ltd., Avonlea, Canada), which contains ≥ 15.0 mg/g saponin. Nutrifen^®^ was composed of 3584 kcal/kg ME, 10.4% moisture, 28.0% CP, 9.9% crude fiber, 10.4% crude fat, and 4.6% crude ash. It also contained macrominerals as follows; 0.34% calcium, 0.28% sulfur, 0.74% phosphorus, 0.26% magnesium, 36.2 mg/kg zinc, 21.4 mg/kg manganese, and 36.2 mg/kg iron. Laying hens were provided ad libitum access to water and feed. All the diets were formulated in mash form to meet or exceed the [@bib36] nutrition requirement ([Table 1](#T1){ref-type="table"}). Treatment additives were included in the diet by replacing the same amount of corn. Laying hens were allowed to adjust to the environment for 5 days prior to beginning the feeding trial, during which they were fed a basal diet. They were raised in an ambient-regulated house, in which the temperature was maintained below 23°C and the light regime was set on a 16:8-light: dark cycle throughout the entire experiment. Birds were individually reared in adjacent steel cages fitted with a nipple drinker, feeder, and an egg collecting plate.
###### Formula and chemical composition of basal diet (as-fed basis)
Item (%)
-------------------------------------------------------- -------------
Ingredients
Corn 50.40
Soybean meal (CP 46%) 18.70
Wheat grain 10.00
Corn gluten meal 2.00
Wheat bran 5.00
Animal fat 4.40
Limestone 7.50
Dicalcium phosphate (P 18%) 1.40
Salt 0.30
[dl]{.smallcaps}-Met (50%) 0.10
Vitamin premix[^1^](#tf1){ref-type="table-fn"} 0.10
Trace mineral premix[^2^](#tf2){ref-type="table-fn"} 0.10
Total 100
Calculated values
ME (kcal/kg) 2,904
CP (%) 15.02
Lys (%) 0.78
Met + Cys (%) 0.65
Ca (%) 3.25
P (%) 0.61
Provided per kilogram of premix: 125,000 IU vitamin A; 2,500 IU vitamin D~3~; 10 mg vitamin E; 2 mg vitamin K~3~; 1 mg vitamin B~1~; 5 mg vitamin B~2~; 1 mg vitamin B~6~; 15 mg vitamin B~12~; 500 mg folic acid; 35,000 mg niacin; 10,000 mg Ca-Pantothenate and 50 mg biotin.
Provided per Kg of diet: 8 mg Mn (as MnO~2~); 60 mg Zn (as ZnSO~4~); 5mg Cu (as CuSO~4~·5H~2~O); 40mg Fe (as FeSO~4~·7H~2~O); 0.3 mg Co (as CoSO~4~·5H~2~O); 1.5 mg I (as KI), and 0.15 mg Se (as Na~2~SeO~3~·5H~2~O).
Laying Production, Performance, and Egg Quality
-----------------------------------------------
The hen-day egg production and egg weights were recorded daily, while feed consumption was measured weekly. The feed conversion ratio was calculated as the feed consumption per hen divided by egg weight per day per hen. For each treatment, 40 normal eggs (five eggs/cage) were collected randomly at 32 weeks and used to determine the egg quality. Eggshell color scores were determined using an eggshell color fan on a 1--15 scale (1=light to 15=dark brown) by a single trained evaluator. Haugh units, albumen height, and yolk color were determined, using an egg multi tester (Touhoku Rhythm Co., Ltd., Tokyo, Japan). Eggshell breaking strength was evaluated, using an Eggshell force gauge, model II (Robotmation Co., Ltd., Tokyo, Japan), and eggshell thickness was measured, using a dial pipe gauge (Ozaki Mfg. Co., Ltd., Tokyo, Japan).
Blood Profile
-------------
At the end of the experiment, 16 laying hens were randomly selected from each treatment (two hens/replication) and blood samples were taken from the jugular vein by a sterilized syringe with needle. Then, the samples were transferred to either a vacuum or K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, FranklinLakes, NJ, USA). The blood samples were centrifuged at 2000×g at 4°C for 15 min to separate the serum. High-density lipoprotein (HDL), low-density lipoprotein (LDL), and total cholesterol, and immunoglobulin G (IgG) concentrations in the serum were then analyzed using an automatic biochemistry blood analyzer (HITACHI747, Tokyo, Japan). Whole blood samples from the K~3~EDTA vacuum tube were analyzed immediately to determine the white blood cells (WBC), red blood cells (RBC), and lymphocyte concentrations using an automatic blood analyzer (ADVIA 120, Bayer, Tarrytown, NY, USA).
Cecal Microflora
----------------
At the end of the experiment, samples of cecal contents were collected from 16 laying hens randomly selected from each treatment, then placed on ice for transportation to the laboratory, where analyses were immediately performed using the method described by [@bib51]. One-gram of pooled cecal content sample was diluted 1:9 (wt/vol) with phosphate buffer saline solution (PBS; 0.1M, pH 7.0). Then, 10-fold serial dilutions (10^−3^ to 10^−6^) of cecal content samples were generated with PBS and placed onto Mac-Conkey (Difco Laboratories, Detroit, MI, USA) and *Lactobacillus*-Rogosa agar plates (Difco Laboratories) to isolate the *Escherichia coli
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Updated to correct an editorial error in the element named in the deck.
The largest industrial application of olefin metathesis today is the synthesis of propylene from ethylene and butenes^[@ref1]^ employing WO~3~ on SiO~2~, a relatively long-lived and regenerable catalyst that operates at 350--400 °C. It is widely proposed that high temperatures are required because the percentage of metal sites actually involved in the metathesis reaction is extremely low, or the reaction that generates alkylidenes is not a high yield reaction, or both. A recent paper by Copéret, Mashima, and co-workers^[@ref2]^ tackles head-on the question concerning how in WO~3~/SiO~2~ catalysts the alkylidene is formed from an olefin alone. Hundreds of papers have attempted to answer this question, although one has to admit that there may not be a single answer for all supported oxide catalysts or all olefins.
Copéret and Mashima employ Me~4~BTDP to reduce four-coordinate (SurfO)~2~WO~2~ sites on silica in the absence of olefins to give 2,3,5,6-tetramethylpyrazine, hexamethyldisiloxane, and M(IV) sites ([eq [1](#eq1){ref-type="disp-formula"}](#eq1){ref-type="disp-formula"}). Analogously, five-coordinate (SurfO)~4~WO sites are also reduced to (SurfO)~4~W(IV) sites. When the purple solid containing a high percentage of W(IV) sites produced in this manner is then exposed to *cis*-4-nonene and heated to 70 °C, 1000 equiv of the alkene are metathesized in 6 h. When instead ethylene is added to the purple solid, solid-state NMR studies reveal that propene is formed along with unsubstituted square pyramidal metallacyclobutane and metallacyclopentane complexes. A variety of experiments led the authors to conclude that above 70 °C, metathesis activity can be ascribed to a relatively efficient contraction of a metallacyclopentane ring to a metallacyclobutane ring, from which loss of propylene generates an initial methylidene complex (eq 2). Ultimately, rearrangement of a metallacyclobutane complex to an olefin results in reduction to W(IV) and reformation of a metallacyclopentane and subsequently another methylidene. It is not yet known whether only TBP (SurfO)~2~W(O)(C~4~H~8~) sites undergo this "ring-contraction" to give a methylidene.
"Ring-contraction" was discovered in the process of exploring reactions between tantalum(III) olefin complexes and terminal olefins to give two dimers of the terminal olefins, not metathesis products. This reaction turned out to be a good model for nonmetathetical steps in alkylidene/metallacycle chemistry of Mo and W.^[@ref3],[@ref4]^ It was recognized at the time that "the MC~4~ to MC~3~ ring contraction is a straightforward and reasonable way of forming an alkylidene ligand from olefins---assuming that some MC~3~ complexes which form in this manner will cleave to give metathesis-type products instead of rearranging."^[@ref3]^ Although unsubstituted d^0^ metallacyclopentane (MC~4~) complexes of Mo and W (especially) have been observed as the end products of a decomposition "cascade" in the presence of ethylene,^[@ref5],[@ref6]^ there is little hard evidence in homogeneous systems that alkylidenes arise from M(IV) olefin complexes^[@ref7]^ through ring-contraction of metallacyclopentanes in homogeneous metathesis reactions at 22 °C. Virtually the only exception in Mo-based or W-based olefin metathesis systems is the catalytic homologation of vinyltributylstannane to allyltributylstannane in the presence of ethylene,^[@ref8]^ which can so far only be explained through a ring-contraction mechanism. An alternative to ring-contraction as a mechanism of forming an alkylidene is a mechanism in which an allyl hydride is formed through allylic CH activation in an olefin. Allyl hydrides are intermediates in rearrangement of a metallacyclobutane to an olefin and consequent reduction of a d^0^ complex to a d^2^ olefin complex with loss of metathesis activity, so formation (to some degree) of a metallacyclobutane from an alkenyl hydride also seems feasible.
The work by Copéret and Mashima may revolutionize the synthesis and use of inexpensive supported metathesis catalysts for hydrocarbons on an industrial scale by allowing the use of much lower temperatures than currently employed. It also may open up opportunities for regenerating catalysts in flow systems. However, it remains to be seen to what extent functional groups are tolerated as metathesis substrates or whether C=C bond isomerization^[@ref7]^ becomes a complication at the temperatures employed. Finally, it also must be noted that the level of selectivity found in homogeneous catalysts today^[@ref9]^ may be difficult to match in a heterogeneous catalyst since the latter are unlikely to contain true (100%) "single sites" that can be tuned with the high level of molecular precision as soluble catalysts.
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1. Introduction {#sec0005}
===============
Cytokines are a broad and loose group of small cell-signaling proteins that play important roles in cell growth, proliferation, differentiation, apoptosis, angiogenesis, immunity and inflammatory response. They include interferons (IFN), interleukins (IL), chemokines, lymphokines, and tumor necrosis factors (TNF). They are produced by a variety of cells, including macrophages, B and T lymphocytes, mast cells, endothelial cells, epithelial cells, fibroblasts, and various stromal cells. Cytokines that are produced at the site of infection stimulate and coordinate innate and adaptive immune responses against invading pathogens ([@bib0135], [@bib0185], [@bib0365]). They act through their matching receptors on the surface of target cells, followed by cascades of intracellular signaling. One such frequently activated intracellular signaling is the JAK-STAT pathway, which is indispensable and pivotal in many biological processes including immunity and inflammatory response ([@bib0275], [@bib0340]). Dysregulation of JAK-STAT signaling results in immunodeficiency and immune-mediated disorders ([@bib0270], [@bib0275]). Mutations in the components of JAK-STAT pathway cause immunodeficient and autoimmune disorders ([@bib0050], [@bib0190]). Due to the importance of JAK-STAT signaling in the host immune response, it is often targeted by pathogens, including PRRSV ([@bib0290], [@bib0400], [@bib0405]).
PRRSV causes a contagious disease that is characterized by reproductive failure in sows and respiratory disease of variable severity in pigs of all ages ([@bib0235]). PRRS has caused substantial economic losses to the swine industry and remains one of the most economically important diseases in pigs since it was first reported in 1987 ([@bib0165], [@bib0265]). A typical feature of the immune response to PRRSV infection in pigs is delayed production and low titer of virus neutralizing antibodies, and weak cell-mediated immune response ([@bib0210], [@bib0235], [@bib0435]). PRRSV infection is also characterized by prolonged viremia followed by the persistent presence in regional lymph nodes for as long as 250 days ([@bib0425]). One of the possible reasons for the weak protective immune response is that PRRSV interferes with innate immunity, including type I interferons (IFNs), and cytokine-mediated JAK-STAT signaling ([@bib0010], [@bib0045], [@bib0290], [@bib0350], [@bib0405], [@bib0395]).
1.1. JAKs and STATs {#sec0010}
-------------------
In mammals, there are four JAKs: JAK1, JAK2, JAK3, and Tyk2, ranging in size from 120 to 140 kDa ([@bib0140]). JAK1, JAK2, and Tyk2 are ubiquitously expressed, whereas JAK3 expression is restricted to cells of the hematopoietic system. The JAK protein is pre-associated with cytokine receptors in the cytoplasmic side and is an important determinant of their levels and signaling potential ([@bib0140]). Upon cytokine binding, the receptor chains are brought into close proximity, leading to the juxtaposition of two JAK kinase domains and consequent trans-phosphorylation. Once activated, JAKs phosphorylate STAT proteins via Src homology 2 (SH2) domain interaction ([@bib0155]). Even though responding to different cytokines, JAKs selected by different receptors activate specific STAT members for defined functions ([@bib0140]).
There are seven mammalian STAT proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, which range from 750 to 950 amino acids in polypeptide length and feature several conserved domains ([@bib0330], [@bib0340]). STATs are latent transcription factors located in the cytoplasm until activated. Each STAT member responds to a defined set of cytokines ([@bib0205], [@bib0270], [@bib0275]). The seven STATs go through similar activation processes and exhibit global conservation in function ([@bib0340]). In brief, ligand-mediated receptor multimerization leads to trans-phosphorylation of JAKs, which then create docking sites in the receptor for STATs and phosphorylate them. Phosphorylated STATs form homodimer or heterodimer complexes, followed by translocation into the nucleus by importins and binding to response element in DNA to activate or repress transcription of a defined set of genes ([@bib0340]).
1.2. STAT signaling and functions {#sec0015}
---------------------------------
Among the seven STATs, STAT1 and STAT2 mainly mediate the IFN-activated signaling ([@bib0270]). STAT1 is involved in signaling by type I, type II, and type III IFNs. In response to type I IFNs (IFN-α or IFN-β), STAT1 and STAT2 are phosphorylated, followed by heterodimer formation and then interaction with interferon regulatory factor 9 (IRF9) to form a heterotrimer known as interferon-stimulated gene factor 3 (ISGF3) ([@bib0090]) ([Fig. 1](#fig0005){ref-type="fig"} A). The ISGF3 is translocated into nucleus and binds to interferon-stimulated response element (ISRE) in DNA to activate the expression of interferon-stimulated genes (ISGs). Upon IFN-γ stimulation, activated STAT1 forms homodimers, followed by nuclear translocation and activation of gene expression via binding to interferon-gamma-activated-sequence (GAS) in DNA ([@bib0270]). Type III IFNs also activate STAT1 and STAT2 for ISRE transactivation like type I IFNs ([@bib0470]).Fig. 1PRRSV interference with type I IFN-activated JAK-STAT signaling. A. Canonical signaling. IFN-α/β binds to their receptors IFNAR-1 and IFNAR-2 on the cell membrane and activates the JAK-STAT1/STAT2 pathway. The phosphorylated STAT1 and STAT2 form heterodimer, followed by interaction with IRF9 to form interferon-stimulated gene factor 3 (ISGF3). Karyopherin α1 (KPNA1), an adaptor protein binding ISGF3, is essential to mediate the nuclear import of ISGF3 via interaction with karyopherin β1 (KPNB1). The ISGF3 binds to interferon-stimulated response element (ISRE) in DNA to activate transcription of interferon-stimulated genes (ISGs). "P" besides STATs indicates phosphorylation. PRRSV nsp1β inhibits ISGF3 nuclear translocation via inducing degradation of KPNA1. PRRSV N protein also inhibits ISGF3 nuclear translocation. PRRSV nsp2 reduces ISG15 production and conjugation via its deubiquitination activity. PRRSV induces elevation of miRNA miR-30c to downregulate JAK1 and SOCS1 to inhibit JAKs. PRRSV inhibits PKR during its early infection of pulmonary alveolar macrophages. B. STAT1-independent signaling. Type I IFNs activate alternative JAK-STAT2 signaling without STAT1. The ISGF3-like complex binds to ISRE and interferon-gamma-activated sequence (GAS) to activate alternative sets of ISGs. PRRSV reduces STAT2 protein to inhibit this pathway.Fig. 1
In addition to the canonical signaling described above, IFNα signaling occurs through alternative complexes containing STAT2 and IRF9 without STAT1 ([@bib0030], [@bib0110]) ([Fig. 1](#fig0005){ref-type="fig"}B). Moreover, STAT2 can form heterodimer with other STATs, like STAT3 and STAT6, followed by binding to diverse sequences, like GAS. Further studies demonstrate the existence of a STAT1-independent IFN signaling pathway, in which STAT2/IRF9 directs a prolonged antiviral activity ([@bib0025], [@bib0035]).
STAT3 is activated by many cytokines and had multiple functions including differentiation of T helper 17 (Th17) and generation of CD8^+^ T cell memory response ([@bib0055], [@bib0275], [@bib0380]). Numerous cytokines including IL-5, IL-6, IL-9, IL-10, IL-11, IL-12, IL-21, IL-22, IL-27, oncostatin M (OSM), IFN-γ, TNF-α and leukemia inhibitory factor (LIF), trigger STAT3 activation ([@bib0125], [@bib0205]). The IL-6 family of cytokines including IL-6, OSM, and LIF bind to the receptor complex containing the common glycoprotein 130 (gp130) and activate STAT3, known as gp130/JAK-STAT3 signaling ([Fig. 2](#fig0010){ref-type="fig"} ). STAT3 is needed for differentiation of follicular T helper and Th1 cells ([@bib0295]), as well as activation and maturation of dendritic cells (DCs) ([@bib0285]). Mutations in STAT3 cause autosomal dominant hyper-IgE syndrome, a rare multisystem primary immunodeficiency characterized by recurrent bacterial infections in skin and lung and with abnormally high levels of IgE ([@bib0160], [@bib0245]). STAT3 is indispensable for promoting host defense against virus infections. For instance, during Herpes simplex virus-1 (HSV-1) infection, STAT3 promotes the activation of CD8+ T cells response ([@bib0460]). It has been shown that gp130-STAT3 signaling is critical for the innate immune response against coxsackievirus B3 virus (CVB3) infection ([@bib0450]). In addition, STAT3 plays a protective role in regulating virus-induced proinflammatory response, as shown in STAT3 knock-out studies ([@bib0100], [@bib0195], [@bib0240]). Highly pathogenic avian influenza (HPA
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Introduction {#s1}
============
Evidence-based veterinary medicine (EVM) can be defined as 'the use of current best evidence in making clinical decisions' ([@R4]). Additionally, when making evidence-based decisions, the circumstances of the patient alongside the circumstances and values of the owner must also be taken into consideration ([@R3]). Although EVM was first mentioned in 1998 ([@R19]), it is less advanced than in the medical field in relation to the availability of synthesised evidence and the support available for the integration of evidence by clinicians into their practice ([@R10]). The first step in EVM is to identify relevant answerable questions ([@R29]), and veterinary clinicians have a crucial role in highlighting these ([@R25], [@R12]). By identifying what common species and conditions clinicians experience in practice, researchers can prioritise studies so that a large proportion of the profession will gain from future studies.
To our knowledge, few published studies describe the entire veterinary population (including both practising and non-practising members) and what species and conditions practitioners commonly encounter. A comprehensive survey of veterinarians in the UK was conducted by the Royal College of Veterinary Surgeons (RCVS) in 2010 where it was reported that the species veterinary clinicians mostly worked with were dogs, cats, horses, cattle and rabbits ([@R23]). Another study by [@R17] found that cats were more commonly seen than dogs in small and mixed animal practice in The Netherlands. Conditions seen in practice in the United States were investigated by [@R18] who found that the most common clinical finding was dental calculus followed by gingivitis from 120,000 consultations in cats and dogs. [@R16] found that the majority of clinical time in equine practice was spent on lameness and reproduction in The Netherlands.
The aim of this study was to describe the UK veterinary population, and what species and conditions veterinary clinicians think they commonly encounter in practice. A second aim was to gather data relating to how much information veterinary clinicians perceived was available for these species.
Materials and methods {#s2}
=====================
Population of interest {#s2a}
----------------------
The target population was all members of the veterinary profession within the UK. The sampling frame was the RCVS register of members. All veterinary surgeons legally practicing in the UK must be registered with the RCVS. This register incorporates individuals, including non-practicing and retired individuals, who have consented for their details to be made available to external organisations for research or marketing purposes. A questionnaire was used to collect data from individuals on this register. As a census of all individuals on the list was conducted, a sample size calculation was not carried out.
Questionnaire structure {#s2b}
-----------------------
Several methods were employed to increase response rates, including a mixed-mode survey design (utilising both paper-based and online methods) ([@R8], [@R26], [@R6]). The questionnaire was made up of 36 questions and had four main sections; a copy of the questionnaire is available on request. The questions in the first section concerned the collection of demographic information about respondents. The second section was made up of open questions requiring clinicians to nominate up to four species they most frequently encountered, and the three main conditions or complaints they thought they saw most commonly in those species with associated perceived information levels ([Fig 1](#VETREC2013101745F1){ref-type="fig"}). The other two sections are not discussed here and will appear in a separate manuscript. Questions were constructed using recommendations from several resources to optimise clarity, minimise ambiguity and to avoid leading terminology ([@R7], [@R13], [@R31], [@R14], [@R9], [@R28], [@R2], [@R6]).
{#VETREC2013101745F1}
Questionnaire development and distribution {#s2c}
------------------------------------------
Pretesting of the survey questions was carried out by researchers within the Centre for Evidence-based Veterinary Medicine (CEVM). Piloting of the survey was carried out three times (24 and 25 people, respectively, for paper version and once transferred to the online format, 8 people for online version) with a combination of private veterinarians, academic veterinarians, veterinary specialists and government veterinarians. Formatting of the questionnaire was carried out using TeleForm V.10.5.2 (Verity Inc. 2010), an automated content capture system. This programme enables scanning of completed questionnaires to facilitate entry of closed question data (open question data was manually entered) into a Microsoft Office Access V.14.0.6 (2010 Microsoft Corporation) database automatically. The software of Cvent (2011 Cvent Inc.), an online survey company, was used to construct the online version of the finalised paper questionnaire.
The questionnaires were printed on magnolia coloured paper to make them easily identifiable against white paper. White envelopes were printed with the CEVM logo and the words 'THIS IS A SCIENTIFIC RESEARCH STUDY. THIS IS NOT JUNK MAIL, AN APPEAL FOR DONATIONS OR MARKET RESEARCH' to make it distinguishable from marketing mailings. A pen, chocolate and a return postage paid envelope were included and a prize incentive was offered (£500 towards the continuing professional development course/s of choice). If participants filled in the online version, they had an extra chance of winning £50 worth of department store vouchers.
The RCVS mailing list was obtained in October 2010. An initial mailing was posted to all individuals on this list between 1st and 5th November 2010; a link to Cvent was included allowing participants to choose to complete either an online or paper version of the questionnaire. A first reminder was sent six weeks later to non-responders followed by a second copy of the questionnaire 10 weeks later for those still not responding.
Data entry {#s2d}
----------
Returned paper-based questionnaires were scanned using Teleform, with the system set to check 10 per cent of questionnaires to enable the detection of scanning errors. Questionnaires were accepted from respondents until scanning was completed (November 2011); coding of the common conditions and complaints was completed in May 2012. Responses received electronically were downloaded into a Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) document from Cvent and integrated into a Microsoft Access V.14.0.6 (2010 Microsoft Corporation) database with the paper responses.
Data coding {#s2e}
-----------
Data relating to the common conditions or complaints nominated by veterinary clinicians were classified according to species and type of condition. Classification definitions were primarily based on those created by N. J. [@R24], with some modifications for suitability across all species. Species were coded according to animal or production type (see online supplementary Appendix 1). The type of condition or complaint was coded according to the category it was most relevant to in relation to either body system (eg, musculoskeletal) or topic (eg, behaviour) (see online supplementary Appendix 2). This was further broken down to another level of classification which more specifically described the nature of the problem (see online supplementary Appendix 3), resulting in two levels of classification for each condition or complaint (eg, Musculoskeletal-ligament). Additionally, the condition or complaint was coded into a 'type' according to whether it was a disease, a clinical sign the animal might be presented for, or was deemed unclassifiable (see online supplementary Appendix 4).
One researcher (MLB) coded all conditions. If conditions were unknown to the coder or required clarification, the online resource Merck Veterinary Manual ([@R20]) was used. A second veterinary resource (eg, textbook, online veterinary resource, colleagues, Google 2012) was used if the condition was not found in the first resource. A Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) spreadsheet of coding was created to maintain consistency for the same complaints or conditions. At the end of the coding process, a second researcher (TDN) identified any discrepancies between similar conditions, and conferred with the first researcher (MLB).
Data management and analysis {#s2f}
----------------------------
The dataset was transferred to a Microsoft Excel V.14.0.6 (2010 Microsoft Corporation) document for data management. Frequency tables and graphs were generated in Excel and RStudio (R Core Team 2011). A posthoc sample size analysis was performed using Raosoft ([www.raosoft.com/samplesize.html](www.raosoft.com/samplesize.html)). There was a high degree of correlation between observations for perceived information level within clinician and species. In order to account for this clustering, the median perceived information level within species for each veterinarian was calculated. A χ^2^ test (excluding 'don\'t know' observations) was then used to determine if perceived information level was different between species. The level of statistical significance was set at P\<0.05. Some questions were left unanswered by participants, therefore, the number of responses per question could be less than the total number of respondents; the number of respondents per question is identified where appropriate.
This project received ethical approval from the ethics research committee at the School of Veterinary Medicine and Science at The University of Nottingham.
Results {#s3}
=======
Response rate {#s3a}
-------------
Of the 14,532 questionnaires distributed, 5407 (37 per cent) were returned. Of these: 259 were return to sender, 230 were retired veterinarians, 72 were returned blank, 3 stated that the veterinarian was deceased and 1 was blank except for one comment box. Therefore, 4842 responses (33%; CI 32% to 35%) could be used in the
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**Zhang X, Bao S, Lai D, Rapkins RW, Gillies MC. Intravitreal triamcinolone acetonide inhibits breakdown of the blood-retinal barrier through differential regulation of VEGF-A and its receptors in early diabetic rat retinas. Diabetes 2008;57:1026--1033**
In the print version of the article listed above, the second affiliation for Xinyuan Zhang is incorrect. The correct affiliation is as follows: Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China. The online version reflects these changes.
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1. Introduction {#s0005}
===============
Interactions of integral membrane proteins with their surrounding lipid environment play a key role in stabilising their structure and influencing their activity. To obtain insight into the nature and type of interactions occurring between lipids and integral membrane proteins a range of biophysical techniques including electron spin resonance [@bb0005; @bb0010] and fluorescence [@bb0145; @bb0020] spectroscopy have been used in numerous studies. This has provided a wealth of information on the specificity of proteins for particular classes of lipids and the affinity of these interactions [@bb0025; @bb0030]. These studies have revealed that in addition to the 'bulk' lipids whose dynamic properties remain largely unchanged by the presence of integral membrane proteins within the bilayer, there exist a population of lipids whose motional freedom is constrained through their interaction with integral membrane proteins. The motionally restricted population of lipids can be segregated into 'annular lipids' which exhibit low affinity interactions with the hydrophobic surface of membrane proteins and 'non-annular lipids' which exert high affinity interactions at sites located in clefts on the protein surface or at the interface between protein subunits [@bb0025; @bb0035; @bb0040; @bb0045]. The interactions at both sites play an important role in modulating the function of integral membrane proteins, but it has recently become apparent that occupation of the non-annular binding site can be particularly critical for function [@bb0050]. Despite the importance of these interactions a detailed atomic level description of the type and nature of the interplay between lipids and integral membrane proteins is limited as the lipids are often missing from high-resolution crystal structures of membrane proteins.
One of the best-studied ion channels is the potassium channel, KcsA from *Streptomyces lividans*. This potassium channel, in common with other members of this family, has an absolute requirement for anionic lipids such as phosphatidylglycerol, phosphatidylserine or cardiolipin for function; in their absence the channel exists in a non-conducting state [@bb0055; @bb0060]. The binding of lipids to KcsA has been extensively investigated by fluorescence quenching studies that clearly identify annular and non-annular populations of lipids [@bb0035; @bb0050]. Binding of lipids to the annular sites revealed marked differences between the inner and extracellular leaflets, with the extracellular side showing similar affinities for anionic and zwitterionic lipids. In contrast the intracellular side showed a twofold higher affinity for anionic lipids over phosphatidylcholine, presumably due to the clustering of charged residues on KcsA close to the bilayer surface. Fluorescence quenching studies have revealed that the non-annular binding sites show a high degree of selectivity of binding anionic lipids almost exclusively, albeit with moderate affinity.
The crystal structure of KcsA in detergent has provided valuable insights into the interaction of lipids with the non-annular binding site with electron density being seen at the interface between the protein subunits indicating the presence of a diacylglycerol-like moiety. The crystal structure revealed that the *sn*-1 chain of this lipid molecule was tightly buried in the groove between the pore helix and the M2 helix whilst the *sn*-2 chain was less intimately associated with the protein [@bb0055; @bb0065]. Subsequent studies have revealed the lipid present in the KcsA crystals to be phosphatidylglycerol which co-purifies with the KcsA [@bb0055] (pdb accession number 1K4C), suggesting that the phosphatidylglycerol headgroup exhibits significant motion or disorder within the crystal, resulting in the absence of electron density. The absence of electron density in the region corresponding to the lipid headgroup has precluded a detailed understanding of how the phosphatidylglycerol is recognised. The presence of two key arginine residues (R64 and R89) in close proximity to the proposed headgroup region suggested a putative role for electrostatic interactions between the sidechains and the anionic lipids within the binding site [@bb0055]. The proposed interactions between R64 and R89 have been investigated by molecular dynamics, revealing the formation of H-bonds between the headgroups of the anionic lipids phosphatidic acid and phosphatidylglycerol and the arginine residues [@bb0070]. Notably, these interactions were absent or reduced in bilayers containing the zwitterionic lipid phosphatidylethanolamine [@bb0070].
To demonstrate the feasibility of detecting interactions between non-annular binding sites and anionic lipids we have undertaken a 31P magic-angle spinning (MAS) NMR study of KcsA reconstituted into a model lipid bilayer composed of phosphatidylcholine and the negatively charged phosphatidylglycerol. The application of MAS permits the acquisition of ^31^P NMR spectra in which the individual lipid components are resolved on the basis of their chemical shift [@bb0075]. The chemical shift observed provides information on the local electrostatic environment of the phosphate moiety of the lipid headgroup and is thus an excellent reporter on the interaction of the lipid headgroup with the protein [@bb0080; @bb0085; @bb0090]. Typically the application of NMR to study lipid/protein interactions has proved challenging as exchange between the bulk lipid and annular sites occurs too rapidly on the NMR timescale to permit the observation of the bound lipids. Furthermore, interactions between the lipids and the proteins are typically hydrophobic in nature with little difference in electrostatic environment between the bound and free lipids resulting in only minor perturbations in chemical shift. In contrast, phosphatidylglycerol bound at the non-annular binding site is predicted to experience a significantly different electrostatic environment from the annular/bulk lipids with higher-affinity interactions resulting in longer residency times within the binding site. Below we demonstrate that this results in a spectroscopically distinct species that we resolve in the ^31^P MAS-NMR spectrum of KcsA reconstituted into lipid vesicles, providing insights into the interactions involved in lipid binding. Using site directed mutagenesis we have demonstrated that the perturbation in electrostatic environment arises through the interaction of the lipid head group with the positively charged sidechain of R64 and R89. Single-channel current recordings have been measured to ascertain the role that these residues play in determining the channel gating behaviour of KcsA.
2. Materials and methods {#s0010}
========================
2.1. Materials {#s0015}
--------------
Palmitoyloleoyl-phosphatidylcholine (POPC) and palmitoyloleoyl-phosphatidylglycerol (POPG) were purchased from Avanti Polar Lipids (Alabaster, AL). The pQE32 vector and M15\[PREP\] *Escherichia coli* strain were bought from Qiagen (UK). The detergent, dodecylmaltoside (DDM) was from Anatrace (UK). The other reagents for the purification were obtained from Sigma (UK).
2.2. Cloning and mutagenesis of KcsA {#s0030}
------------------------------------
The pQE32 vector containing the KcsA gene with a hexahistidine epitope at the N-terminus, kindly donated by Professor Lee (University of Southampton, UK), was expressed in M15 cells. Three KcsA mutants were generated by site-directed mutagenesis using the Quik-change protocol from Stratagene (La Jolla, CA). Three KcsA mutants were prepared replacing the arginine with the slightly smaller but uncharged leucine at residue 64 (R64L), 89 (R89L) or at both sites (R64,89L). The mutants were generated by PCR using synthetic oligonucleotide primers containing the desired mutations (Eurofins MWG, UK). Complementary oligonuceotides, 5′-AGCTGATCAC GTATCCGTTA GCGCTGTGGT GGTCC-3′ and 5′-GACCACCACA GCGCTAACGG ATACGTGATC AGCTG-3′ were used as forward and reverse primers respectively to create the R64L mutation. Similarly, R89L was produced using the synthetic oligonucleotides 5′-GTGACTCTGT GGGGCCTGC TCGTGGCCG TGGTGGTGA T-3′ and 5′-ATCACCACCA CGGCCACGA GCAGGCCCC ACAGAGTCA C-3′ as primers. Polymerase chain reaction was used to generate the mutants and the resultant PCR product was Dpn1- treated for 1 hr at 37 °C to digest any methylated parental DNA. The DNA was used to transform competent M15 \[PREP\] *E.coli* cells that were then plated onto agar plates supplemented with ampicillin. Mutations were confirmed by sequencing.
2.3. Over expression and purification of KcsA {#s0035}
---------------------------------------------
M15 *E.coli* cells transformed with KcsA or one of the KcsA mutants were used to inoculate 10 mL of Luria Broth (LB) medium containing 100 μg/mL of ampicillin. The overnight culture was then used to inoculate 1 L of LB containing 100 μg/mL of ampicillin and grown to an OD~600~ of 0.8 at 37 °C. Over-expression of KcsA wild type and mutant protein was induced by the addition of IPTG to a final concentration of 1 mM and the culture was grown for a further 4 h at 37 °C. The cells were harvested at 4 °C by centrifugation at 12,000 *g* for 20 min. The cell pellet was resuspended in buffer A (50 mM Tris, 150 mM NaCl, 150 mM KCl, pH 7.4) with 1 mM PMSF and sonicated on ice for 5 min: 15 s on; 20 s off at power level 7 (Misonix sonicator). The membrane fraction was clarified by ultracentrifugation at 420,000 *g* for 40 min at 5 °C. The membrane-containing pellet was homogenised and
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(J Am Heart Assoc. 2017;6:e007026 DOI: 10.1161/JAHA.117.007026.)29042422
Clinical PerspectiveWhat Is New?In this nonrandomized, retrospective, observational study of patients undergoing cardiac resynchronization therapy (CRT) with quadripolar (QUAD) and non‐QUAD left ventricular leads, programmed to biventricular, single‐site left ventricular pacing, QUAD was associated with a lower total mortality, cardiac mortality, and heart failure hospitalization.These benefits were observed after both CRT‐defibrillation and CRT‐pacing, after adjustment for heart failure etiology.Re‐interventions for left ventricular displacement or phrenic nerve stimulation, which were lower with QUAD, were associated with worse outcomes.What Are the Clinical Implications?The markedly better outcomes after CRT observed with QUAD supports their preferential use over non‐QUAD in clinical practice.The relative benefits of CRT‐defibrillation over CRT‐pacing requires further evaluation in the QUAD era.
Introduction {#jah32587-sec-0008}
============
Cardiac resynchronization therapy (CRT), with CRT‐defibrillation (CRT‐D) or without (CRT‐pacing \[CRT‐P\]) defibrillation, is a standard treatment for selected patients with heart failure (HF) with severe left ventricular (LV) dysfunction and a wide QRS complex.[1](#jah32587-bib-0001){ref-type="ref"} Since the first transvenous CRT implantations were undertaken in the 1990s,[2](#jah32587-bib-0002){ref-type="ref"}, [3](#jah32587-bib-0003){ref-type="ref"} improvements in delivery catheter and LV lead design, as well as implantation techniques using venoplasty and snaring, have helped to improve implantation success. Prominent among the challenges still encountered at implantation and thereafter is achieving acceptable LV pacing thresholds without phrenic nerve stimulation (PNS).[4](#jah32587-bib-0004){ref-type="ref"} Deactivation of the LV lead mainly occurs as a result of LV lead displacement which, in studies using unipolar and bipolar leads, occurs more frequently than with atrial or right ventricular leads.[5](#jah32587-bib-0005){ref-type="ref"}, [6](#jah32587-bib-0006){ref-type="ref"}, [7](#jah32587-bib-0007){ref-type="ref"}, [8](#jah32587-bib-0008){ref-type="ref"}, [9](#jah32587-bib-0009){ref-type="ref"}
Since their launch in 2010, quadripolar LV leads (QUAD) have been considered by implanters as a "game‐changer," even before robust clinical evidence emerged in their favor. Observational studies and a randomized, controlled trial[10](#jah32587-bib-0010){ref-type="ref"} have since shown that QUAD is associated with higher implant success rates and lower rates of re‐interventions for LV lead displacement or PNS.[11](#jah32587-bib-0011){ref-type="ref"}, [12](#jah32587-bib-0012){ref-type="ref"}
Some observational studies have suggested that CRT‐D using QUAD programmed to single‐site LV pacing also improves survival.[12](#jah32587-bib-0012){ref-type="ref"}, [13](#jah32587-bib-0013){ref-type="ref"}, [14](#jah32587-bib-0014){ref-type="ref"} These findings, however, are not consistent,[15](#jah32587-bib-0015){ref-type="ref"} and there is uncertainty as to whether they also apply to CRT‐P. Moreover, the possible influence of HF etiology and the effects of QUAD on HF hospitalization and mode of death remain largely unexplored.
Methods {#jah32587-sec-0009}
-------
This is a nonrandomized, retrospective, observational study comparing clinical outcomes of patients undergoing CRT‐D and CRT‐P device implantation using unipolar, bipolar, and quadripolar leads in a single center (Queen Elizabeth Hospital, Birmingham, United Kingdom) from February 2010 to January 2017. The study was approved by the Clinical Audit Department at the Queen Elizabeth Hospital, which does not require informed consent for audit of clinical care delivery. The study conforms to the Declaration of Helsinki.
Implantation {#jah32587-sec-0010}
------------
Device implantation was undertaken using standard techniques with patients under local anesthesia and intravenous sedation. Access was gained via subclavian, axillary, and cephalic veins. The LV pacing site was chosen by the implanter on the basis of lead stability, absence of PNS, and adequate pacing parameters. An implant was considered a failure in the event of failure to deploy all desired leads and device at the index procedure. The first QUAD was implanted in February 2010. The following QUAD leads were used: Quartet 1458Q (St. Jude Medical, Sylmar, CA), Attain Performa (Medtronic Inc, Minneapolis, MN), and Acuity X4 (Boston Scientific, Marlborough, MA). The choice of vector was made at implantation and was made on the basis of presence or absence of PNS.
Follow‐Up {#jah32587-sec-0011}
---------
Patients were followed up in dedicated device therapy clinics. Before 2013, patients underwent systematic echocardiographic optimization. To this end, patients in sinus rhythm underwent transmitral Doppler‐directed optimization of atrioventricular delay using an iterative technique before discharge and at every scheduled visit thereafter. In patients with sinus rhythm, atrial pacing was set at 60 beats/min, and the pacing mode was set to DDDR with an interventricular delay of 0 to 4 ms, according to the manufacturer. In patients with permanent atrial fibrillation, right ventricular and LV leads were implanted and a CRT generator was used, plugging the atrial port and programming the generator to a ventricular triggered mode. In patients with uncontrolled atrial fibrillation despite medical therapy with suboptimal biventricular pacing capture (\<98%), atrioventricular junction ablation was undertaken, according to the individual clinician\'s decision. After 2013, echocardiographic optimization was only undertaken in symptomatic nonresponders.
End Points {#jah32587-sec-0012}
----------
The primary end point was total mortality, which included cardiac transplantation. Secondary end points included cardiac mortality and unplanned HF hospitalization. The first event was included in the analysis. With respect to mode of death, sudden cardiac death was defined as a "natural, unexpected death due to cardiac causes, heralded by an abrupt loss of consciousness within 1 hour of the onset of acute symptoms,"[16](#jah32587-bib-0016){ref-type="ref"} whereas death from pump failure was defined as "death after a period of clinical deterioration in signs and symptoms of heart failure despite medical treatment"[17](#jah32587-bib-0017){ref-type="ref"} or cardiac transplantation. Mortality data were collected through medical records every 3 months by investigators who were blinded to all other patient data. Mortality and event data were collected by separate investigators who were blinded to all other data, except patient identifiers.
Statistical Analysis {#jah32587-sec-0013}
--------------------
In preliminary analyses, no differences in outcomes emerged between unipolar and bipolar LV leads (data not shown). On this basis, the latter were classified as "non‐QUAD" in statistical analyses. Normality was tested using the Shapiro--Wilk test. Continuous variables are expressed as mean (±SD) and compared using the Student *t* test. Categorical variables were compared using the χ^2^ tests. Kaplan--Meier curves and the log‐rank tests were used to assess observed cumulative survival and to test for differences in survival, respectively. Cox proportional hazard models were used to compare hazard rates of subgroups. Variables reaching a *P*\<0.10 on univariable analyses were entered in multivariable models, and further backward elimination was applied for the final multivariable models. Confounders included in final models were the following: quadripolar lead, sex (male), age at implantation, New York Heart Association (NYHA) class, creatinine, QRS duration, and medication of angiotensin‐converting enzyme inhibitors/angiotensin receptor blockers. Proportionality hypotheses were verified by visual examination of log (survival) graphs to ensure parallel slopes, and by examining Schoenfeld residuals. Statistical analyses were undertaken using Stata 14 (StataCorp, Houston, TX). A 2‐sided *P*≤0.05 was considered statistically significant.
Results {#jah32587-sec-0014}
=======
Baseline Characteristics {#jah32587-sec-0015}
------------------------
Over the study period of 6.9 years, 847 patients underwent CRT (CRT‐D: 436 \[51.5%\]; CRT‐P: 411 \[48.5%\]), using QUAD (287 \[33.9%\]), unipolar (63 \[7.43%\]), or bipolar (497 \[58.7%\]) leads. Implantations using unipolar and bipolar leads were classified as non‐QUAD. As shown in Table [1](#jah32587-tbl-0001){ref-type="table"}, the groups were well matched for age, sex, cause of cardiomyopathy, comorbidities, proportion of upgrades from pacemaker, atrial rhythm (sinus rhythm or atrial fibrillation), QRS morphology, QRS duration, and left ventricular ejection fraction. Compared with the non‐QUAD group, QUAD were more likely to be in NYHA class I and II (*P
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Background
==========
Diamond holds a variety of extraordinary physical and chemical properties, facilitating its possible applications in novel functional devices \[[@B1]-[@B7]\]. As a semiconductor with a wide bandgap of 5.47 eV, it is a promising candidate for short-wavelength optoelectronic devices such as ultraviolet light-emitting diodes. The extreme mechanical hardness of diamond endows it with potential applications in nanomechanical devices. When doped with boron, it was found to display superconductivity around liquid helium temperature. To utilize the qualities of diamond, it is imperative to grow high-quality materials. Chemical vapor deposition is an efficient and versatile technique for the growth of diamond. A large body of experiments and theories are dedicated to understanding the growth process \[[@B8]\]. Graphitic-like surface reconstructions on stepped C(111) surfaces are predicated by first-principles calculations \[[@B9]\]. Surface graphitization of diamond nanoparticles is investigated from an experimental viewpoint \[[@B10]\]. A unique character of diamond growth is the existence of *sp*^2^-hybridized bonds in the graphitic-like layer of diamond surfaces, in contrast to other group IV element semiconductors (Si and Ge), which do not exhibit energetically favorable *sp*^2^ bonding configurations. This may account for different surface reconstructions on Si and diamond surfaces \[[@B11]\]. Besides low-index surfaces, high-index Si surfaces are extensively investigated to unveil their atomic and electronic structures \[[@B12],[@B13]\], whereas less attention has been paid to the study of high-index diamond surfaces. The graphite-like *sp*^2^ bonding is expected to give rise to the significant difference between high-index diamond and Si surfaces.
Graphene, a two-dimensional atomic crystal with graphite-like *sp*^2^ bonding, has attracted considerable interests due to its novel physical and chemical properties and its potential applications in nanoelectronics and optoelectronics \[[@B14]\]. Large-scale graphenes are grown on metal substrates \[[@B15]\]. Here, we explore the formation of graphene-like stripes on a reconstructed high-index diamond C(331) surface using first-principles density functional theory (DFT) calculations. During the structural relaxation of the bulk-terminated surface, the terrace C atoms in the first layer delaminate from the second layer, leading to local *sp*^3^ to *sp*^2^ rehybridization and the formation of graphene-like stripes on the surface. The driving force for the graphitic-like reconstruction is the presence of high-density dangling bonds on the surface, which gives rise to the rebonding of top-layer atoms. The comparison of the calculated absolute surface energies of C(331), C(111), and C(110) demonstrates the relative stability of the C(331) surface with the graphitic-like reconstruction. Local density of electronic states (LDOS) analysis reveals the occurrence of localized electronic states near the Fermi level (FL), which may play an essential role in determining the surface conductivity \[[@B16],[@B17]\].
Methods
=======
The calculations are conducted in the framework of the DFT method by DMol^3^ codes \[[@B18]\]. We use the Perdew-Burke-Ernzerhof generalized gradient approximation \[[@B19]\]. A double numeric basis set including *d*-polarization function, all electron treatment, and an 8 × 2 × 1 Monkhorst-Pack *k-*point mesh for the Brillouin zone sampling \[[@B20]\] are employed to carry out geometry optimization and electronic band structure calculations. Spin-unpolarized self-consistent field calculations are performed with a convergence criterion of 2.0 × 10^−5^ hartree (1 hartree = 27.2114 eV) for total energies. The maximum force tolerance is 0.004 hartree Å^−1^, and the maximum displacement tolerance is 0.005 Å.
The periodically repeated slabs separated by approximately 10 Å of vacuum are used to represent the surface structures. Each slab of C(331) surface is composed of 11 atomic layers with 40 C atoms and 6 H atoms per unit cell. The H atoms are used to passivate the surface C atoms at the bottom of the slabs to make the calculation more efficient. The dashed lines in Figure [1](#F1){ref-type="fig"}a and the dashed box in Figure [1](#F1){ref-type="fig"}b indicate the supercell used for the calculation. Each slab of H-passivated C(331) surface is composed of 12 atomic layers with 40 C atoms and 12 H atoms per unit cell. The dashed lines in Figure [2](#F2){ref-type="fig"} indicate the supercell used for the calculations.
![**Calculated atomic structure of diamond C(331) surface with graphene-like stripes.** (**a**) The dashed lines indicate the supercell viewed from the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The large circles denote the C atoms, and the small circles denote the H atoms. (**b**) The dashed box indicates the supercell viewed from the \[331\] direction, and the bottom is viewed from the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The large circles denote the C atoms of the graphitic layer, and the smaller circles indicate the *sp*^3^-bonded C atoms in the outmost surface. The other C and H atoms are represented by the smallest circles.](1556-276X-7-460-1){#F1}
{#F2}
Results and discussion
======================
Figure [1](#F1){ref-type="fig"} shows the atomic structure of the graphene-like stripes formed on the reconstructed diamond C(331) surface calculated after the structural relaxation of the bulk-terminated surface. We allow this surface to relax using a steepest descent algorithm. The top-layer C atoms exhibit the *sp*^2^ bonding configuration in the graphene-like structure, as shown in Figure [1](#F1){ref-type="fig"}b. Upon structural relaxation, the terrace C atoms (see 4 and 10 C atoms in Figure [3](#F3){ref-type="fig"}) delaminate from the subsurface diamond and form the graphene-like stripes along the $\left\lbrack {0\overline{1}1} \right\rbrack$ direction. The energetically favorable hexagonal rings are found to emerge in the graphitic layer on the reconstructed surface. The driving force for the graphitic-like reconstruction on the surface is the presence of high-density dangling bonds which have unpaired electrons. This situation is similar to the reconstruction of the C(111) surface, where the top-layer C atoms are rearranged to make the dangling bonds become the nearest neighbors and form the π bonding \[[@B21]\]. For the C(331) surface, the delamination of the terrace C atoms can lead to the formation of graphite-like *sp*^2^ bonds, thereby reducing the energetically unfavorable dangling bonds.
{#F3}
The representative C-C bond lengths for the graphitic-like reconstructed C(331) surface are shown in Figure [3](#F3){ref-type="fig"}. The distance between the delaminated C atom and the subsurface C atom increases to approximately 2.51 Å, much larger than the bond length of diamond (1.54 Å). The bond lengths for the C atoms in the graphitic structure decrease to 1.44 and 1.46 Å. These values are quite close to the bond length of graphite (1.42 Å), whereas much smaller than that of diamond. The C atoms with the unsatu-rated dangling bonds at the subsurface positions remain *sp*^3^-hybridized in character, although they have stretched by almost 34%. The C-C bonds are stretched to 1.62 and 1.57 Å for the outmost C atoms attached to the second-layer C atoms. The severe subsurface rebonding increases the elastic strain, which is energetically unfavorable. The competition between the favorable *sp*^2^ bonding in the graphitic layer and the unfavorable strain energy leads to the graphitic-like reconstruction of the C(331) surface.
The energetic stability of the C(331) surface is studied by comparing its absolute surface energy (ASE) with those of low-index diamond C(111) and C(110) surfaces \[[@B21]-[@B23]\]. In the centrosymmetric slab used for computing the ASE, the top and bottom surfaces are physically equivalent. After full structural relaxation, the same *n* × *m* surface reconstruction is observed to occur on both sides of the slab. Therefore, it allows calculating directly the ASE. For the slab with *N* atoms at the atomic configuration $\left\{ R_{i} \right\}$, the surface energy per 1 × 1 surface cell, $E_{\
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1. Introduction {#sec1}
===============
Nuclear magnetic resonance spectroscopy (NMR, or MRS) has enormous potential for the study of biochemical and physiological changes in cancer tissues, due to its noninvasive nature and the large quantity of specific molecular information it can generate. Despite the sensitivity limitations of the technique, the inherent complexity of the spectra, and inevitable presence of overlapping resonances, there have been several successful NMR-metabonomics studies of cell tissue culture and culture extracts. The focus has been on elucidating the physiopathology of tumors and tumor cells, their drug toxicology and drug resistance, often with a view to identifying diagnostic markers \[[@B1]--[@B8]\]. A further significant complication in such studies arises from variability in the metabolite profile from sample to sample. This reflects many factors \[[@B9]\] including minor variations in growing conditions, the biochemical heterogeneity of the growing cells, the effect of different batches of sera (if used), and variations in cell and sample preparation. These additional factors may mask the inherent metabolite distribution, which may be diagnostic of the pathophysiological state of interest.
Experimental complications and difficulties also compromise the extraction of critical information from *in vivo* MRS experiments. In this case, the problems arise from the use of different MR-protocols, which affect the quality of the water suppression, differences in echo time and in the baseline, and so forth. While the causes are different in origin, they have a similar effect on the application. For both forms of magnetic resonance, many of these issues can, in principle, be addressed by improved experimental design, however, it is common for additional sources of variance to be identifiable only after extensive experimentation. In addition to technical issues are the natural physiological variability and the individual treatment history of the subject. As a result, there is an ongoing requirement for the development of magnetic resonance-based diagnostics using advanced statistical-, or other data-, analysis techniques which can reduce or compensate for additional sources of variability.
^1^H NMR spectra of intact tissues or whole-cell samples are inherently complex due to the large number of contributing species which results in significantly overlapping resonance signals. Cell membranes also produce magnetic field inhomogeneity, further broadening the spectra \[[@B10]\]. In the case of cancer cells, a significant proportion of the lipids reside in a fluid environment and hence appear in the liquid-state ^1^H spectra as strong "mobile-lipid" resonances \[[@B7], [@B8], [@B11]\]. Although the identification of the major resonances in ^1^H NMR spectra can be used to characterise the metabolite profile, the complexity of the data sets usually necessitates the use of data reduction and pattern recognition techniques. These can provide information on the biochemical and physiological changes in cancer tissues, related to their physiopathology, drug toxicology, and drug resistance \[[@B12], [@B13]\]. Prominent amongst such techniques is principal component analysis (PCA), \[[@B14], [@B15]\] which involves diagonalisation of the spectral correlation or covariance matrix to identify independent sources of variance (principal components) across the set of spectra, and ranking of the components by their contribution to the overall variance. Thus, PCA is an unsupervised approach to data reprojection that can reveal the presence of classes, it has been applied to a variety of problems in biological science \[[@B16], [@B17]\].
Artificial Neural Networks (ANNs) belong to the so-called Artificial Intelligence group of methods, which were inspired by neurobiology and by the architecture of the human brain \[[@B18]\]. In recent times, these approaches have found applications in many branches of science. For example, they have been used in chemotaxonomy to classify limpets \[[@B19]\] from HPLC mass spectrometric data and in the identification of insect species from morphological measurements \[[@B20]\]. ANNs can be used to model data where the relations, or functions, are not known.
There have been some reports of the use of artificial intelligence and network methods in medical diagnostics which have involved analysis of magnetic resonance spectroscopic data. El-Deredy et al. \[[@B21]\] used ANNs to achieve reasonable prediction of the measured *in vitro* chemotherapeutic response from ^1^H NMR of glioma biopsy extracts. More recently, Suna et al. \[[@B22]\] demonstrated the diagnostic potential of unsupervised approaches to classification by successfully analysing simulated ^1^H NMR spectra using self-organising maps. This approach allowed the identification of stages along a metabolic pathway ranging from "normolipidaemic" to "metabolic syndrome". Tate and coworkers \[[@B23]\] reported the trial of an automated decision support system for classification of brain tumors from *in vivo* MRS, which showed a small but significant improvement in diagnostic accuracy over spectroscopy used and interpreted on its own.
In recent work \[[@B24]\], we reported PCA of ^1^H NMR spectra recorded for a group of human lung carcinoma cell lines in culture and ^1^H NMR analysis of extracts from the same samples. The samples studied were cells of lung tumor origin with differing chemotherapy drug resistance patterns. For whole-cell samples, it was found that the statistically significant causes of spectral variation were an increase in the choline and a decrease in the methylene and mobile lipid ^1^H resonance intensities, which were correlated with our knowledge of the level of resistance displayed by the different cell lines. In this paper, we investigate the use of artificial neural network (ANN), a supervised method, to classify lung carcinoma. Two sets of whole-cell ^1^H NMR spectra will be examined. These were recorded for two groups of human lung carcinoma cell lines, these were grown in culture and characterised over two different periods by two different groups of researchers (each consisting of a biologist and a spectroscopist), who both adhered to the same experimental protocol and used the same spectrometer. The cell lines studied include (i) the parent cell line DLKP, a human squamous nonsmall cell lung carcinoma; (ii) DLKP-A; (iii) DLKP-A5F, two resistant daughter lines; (iv) A549, a human lung adenocarcinoma cell line. The study also examines the capability of supervised techniques to compensate for experimental sources of variance, which may include operator bias and the cell culture growth process and in particular provide a test case for the application of ANN architectures in the identification and monitoring of resistance states in cancer tissue by MRS.
2. Experimental {#sec2}
===============
2.1. Cell Samples {#sec2.1}
-----------------
The cell lines DLKP \[[@B25], [@B26]\], DLKP-A \[[@B27]\], DLKP-A5F \[[@B28]\], and A549 were grown in culture to approximately 70--80% confluency in 175 cm^2^ tissue culture flasks. Culture conditions were as follows: DLKP, DLKP-A, and DLKP-A5F and were cultured in minimal essential medium/Hams F12 (1 : 1, v/v) supplemented with 5% fetal calf serum and 2 mM L-glutamine. A549 was cultured in Dulbecco\'s modified Eagle\'s medium/Hams F12 (1 : 1, v/v) supplemented with 5% fetal calf serum. Cells were cultured as monolayers in tissue culture flasks and incubated at 37°C. A cell count was performed and c. 5 × 10^7^ cells were separated and pelleted. These were then resuspended in deuterated PBS buffer and were kept in a container at 37°C before the start of the NMR measurements. The methods used were described in detail previously \[[@B24]\]. DLKP cells express a small amount of the multidrug resistance protein-1 (MRP-1) MDR drug efflux pump \[[@B25], [@B26]\]. DLKP-A \[[@B27]\] is a highly resistant clone of DLKP, which overexpresses the P-gp drug efflux pump. DLKP-A5F \[[@B28]\] was derived from DLKP by a different drug exposure profile, it is also highly drug resistant. A549 is an unrelated human lung adenocarcinoma cell line which was obtained from the American Type Culture Collection.
The first group of 13 samples, G1_13_21, were grown by a biologist during a six-month period, they were analysed by a first NMR spectroscopist. G1_13_21 contained 21 spectra and so was relatively sparse, it comprised DLKP \[4 samples, 6 spectra\], DLKP-A \[[@B4], [@B6]\], DLKP-A5F \[[@B3], [@B5]\], and A549 \[[@B2], [@B4]\]. The second group of 17 samples, G2_17_33, was grown independently, by a second biologist during a later six-month period and was analysed by a second spectroscopist \[[@B24]\]. G2_17_33 contained 33 spectra, it comprised DLKP \[[@B3], [@B6]\], DLKP-A \[[@B5], [@B10]\], DLKP-A5F \[[@B5], [@B9]\], and A549 \[[@B4], [@B8]\]. Thus for the integrated study presented here, a total of 30 samples were prepared and 54 ^1^H spectra was recorded. The same protocols and methods were used by all the researchers for cell growth and NMR spectroscopy. The biologist and spectroscopist who produced G1_13_21 will be collectively referred to as R1, and the biologist and spectroscopist who produced G2_17_33 will be referred to as R2. Due to the significant work involved in producing the large number of cells required for each spectrum, the number of samples in the study is inevitably somewhat limited. However, the total data set is larger than those usually reported in the analysis of NMR data by pattern recognition methods \[[@B16], [@B17], [@B
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Diet is believed to be the single most important contributor to colonic carcinogenesis ([Tomatis *et al*, 1990](#bib25){ref-type="other"}). Experimental data have shown that saturated fatty acids (SFAs) and n-6 polyunsaturated fatty acids (PUFAs) have tumour-enhancing properties in the colon ([Reddy and Maeura, 1984](#bib18){ref-type="other"}; [Zhao *et al*, 1991](#bib28){ref-type="other"}, [Woutersen *et al*, 1999](#bib26){ref-type="other"}). Epidemiological data suggest that increased consumption of all meat or red meat, which contains high levels of SFAs, is strongly associated with colorectal cancer ([Giovannucci and Goldin, 1997](#bib10){ref-type="other"}; [Sandhu *et al*, 2001](#bib19){ref-type="other"}), but there is only limited evidence on the role of dietary n-6 PUFAs ([Zock and Katan, 1998](#bib29){ref-type="other"}; [Flood *et al*, 2003](#bib9){ref-type="other"}).
The putative mechanism through which dietary n-6 PUFAs may enhance colonic carcinogenesis is the increased formation of prostaglandins, with the rate-limiting and committal step being mediated by the cyclooxygenase (COX)-2 enzyme ([Dubois *et al*, 1998](#bib7){ref-type="other"}). Prostaglandins possess a wide spectrum of procarcinogenic properties ([Handler *et al*, 1990](#bib11){ref-type="other"}; [Cowlen and Eling, 1993](#bib5){ref-type="other"}; [Coffey *et al*, 1997](#bib4){ref-type="other"}; [Dermott *et al*, 1999](#bib6){ref-type="other"}). We therefore hypothesised that functional *COX-2* gene polymorphisms may impact on the conversion of n-6 PUFAs into prostaglandins, with consequent change in level of cancer risk. A single nucleotide polymorphism (−*765G*\>*C)* in the promoter region of the *COX-2* gene was recently described ([Papafili *et al*, 2002](#bib16){ref-type="other"}). We therefore investigated whether this *COX-2* gene polymorphism was related to colorectal cancer risk within a population-based, prospective cohort of middle-aged and older Chinese men and women in Singapore.
MATERIALS AND METHODS
=====================
Study subjects
--------------
The study design and subject recruitment of the Singapore Chinese Health Study have been described ([Hankin *et al*, 2001](#bib12){ref-type="other"}). Briefly, 63 257 Chinese women and men aged 45--74 years belonging to the Hokkien or Cantonese dialect group were enrolled in the study between April 1993 and December 1998. At recruitment, information on lifestyle factors and usual diet over the last year was obtained through in-person interviews. The dietary component of the questionnaire was validated through a series of 24-h food recalls ([Hankin *et al*, 2001](#bib12){ref-type="other"}). Respondents were asked to choose from predefined frequency and portion size categories for each of the 165 listed food/beverage items that he/she consumed during the past 12 months. We used the Singapore Food Composition Table to estimate average daily intake of 96 nutrient and non-nutrient compounds for each study subject ([Hankin *et al*, 2001](#bib12){ref-type="other"}). The Institutional Review Boards at the University of Southern California and the National University of Singapore had approved this study.
We identified incident colorectal cancer cases through the population-based cancer registry in Singapore ([Chia *et al*, 2000](#bib3){ref-type="other"}). As of 30 April 2002, 592 colorectal cancer cases had occurred among cohort participants. All cases (including one carcinoid tumour and two *in situ* cancers) were histologically confirmed except three (ascertained by death records and clinical evidence). Details of the biospecimen collection, processing and storage procedures have been described ([Koh *et al*, 2003](#bib14){ref-type="other"}). Briefly, we attempted to collect blood and single-void urine specimens from a random 3% sample of cohort enrollees. If the subject refused to donate blood, he/she was asked to donate buccal cells. We collected blood/buccal cell samples from 1194 subjects during April 1994--July 1999. Of these subjects, 13 developed colorectal cancer by 30 April 2002, and the remaining 1181 subjects constituted the referent group for the present study. We also attempted to collect blood/buccal cell and urine samples from all incident colorectal cancer cases. Of the 592 colorectal cancer cases, 312 (53%) donated blood/buccal cell samples.
COX-2 genotyping
----------------
Genomic DNA was extracted from buffy coats (228 cases and 895 controls) and buccal cell samples (84 cases and 286 controls) using a QIAamp 96 DNA Blood Kit (Qiagen, Valencia, CA, USA). A TaqMan assay for the −*765G*\>*C COX-2* polymorphism was developed using a TaqMan PCR Core Reagent kit (Applied Biosystems Inc., Foster City, CA, USA). The oligonucleotide primers for amplification of the polymorphic region of *COX-2* were GC093 for (5′-CATTAACTATTTACAGGGTAACTGCTTAGG-3′) and GC093rev (5′-CCCCCTCCTTGTTTCTTGGA-3′). In addition, the fluorogenic oligonucleotide probes (TaqMan MGB Probes; ABI) used to detect each of the alleles were GC093F (5′-CTTTCCCGCCTCTCT-3′) labelled with 6-FAM to detect the *G* allele and GC093V (5′-CTTTCCCCCCTCTCT-3′) labelled with VIC to detect the *C* allele. Experimental samples were compared to 12 controls to identify the three genotypes at each locus (*GG, GC, CC*). All samples were processed without knowledge of their case/control status. Any samples that were outside the parameters defined by the controls were identified as noninformative and were retested. Four controls and two cases had noninformative *COX-2* genotypes and were excluded from the present analysis.
Statistical analysis
--------------------
Data were analysed by standard methods for unmatched case--control studies ([Breslow and Day, 1980](#bib1){ref-type="other"}). Unconditional logistic regression models were used to examine the associations between *COX-2* genotypes and risk of colorectal cancer, and their possible modification by n-6 PUFA intake. The associations were measured by odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) and *P*-values (two-sided). Limited by the very low frequency of the *CC* genotype (0.003), the *GC* and *CC* genotypes were combined when compared with the *GG* genotype. All ORs were adjusted for age (year) at recruitment, year of recruitment, gender, dialect group (Cantonese, Hokkien), level of education (no formal schooling, primary school, secondary school and higher), body mass index (\<20, 20 to \<24, 24 to \<28, 28+ kg m^−2^), smoking status (never, exsmoker, current smoker), frequency of alcohol consumption (nondrinker, monthly drinker, weekly drinker, daily drinker), and familial history of colorectal cancer (yes, no).
RESULTS
=======
Of the 592 incident colorectal cancer cases, 282 were excluded from the present analysis due to unavailable blood/buccal cell samples (*n*=280) or noninformative *COX-2* genotype (*n*=2). Cases included in the present study (*n*=310) were comparable to those excluded in terms of age (mean: 65.4 *vs* 66.1 years), but slightly different in gender (57 *vs* 49% male), dialect group (45 *vs* 37% Cantonese) and level of education (69 *vs* 60% attaining primary school education or higher).
In total, 180 (58%) cases had colon cancer, and the remaining cases had either rectal or rectosigmoid cancers. [Table 1](#tbl1){ref-type="table"} Table 1Selected characteristics of colorectal cancer cases and controls, the Singapore Chinese Health Study**CharacteristicsControls (*n*=1177)Cases (*n*=310)*P*-value^a^** Mean age±s.d.^b^ (years)56.5±8.161.3±7.5\<0.001 Number (%) *Sex* Males509 (43.2)178 (57.4)\<0.001 Females668 (56.8)132 (42.6) *Dialect group* Cantonese571 (48.5)138 (44.5)0.23 Hokkien606 (51.5)172 (55.5) *Level of education* No formal schooling318 (27.0)95 (30.6)0.01 Primary school504 (42.8)151 (48.7) Secondary school288 (24.5)54 (17.4) A level/university67 (5.7)10 (3.2) *Body mass index* (*kg* *m*^−*2*^
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1. Introduction {#sec1}
===============
Stroke is the second most common cause of death in developed countries and thus is a major health problem \[[@B1]\]. According to the 2009 Annual Public Health Report by the Korean National Statistical Office, cerebrovascular disease, or stroke, was the second-leading cause of disease-related deaths in Korea, after cancer \[[@B2]\]. In Korea, many stroke patients receive traditional medical care because the country has its own system of traditional alternative medicine called Traditional korean medicine (TKM), the role of which has been emphasized in stroke management \[[@B3]\].
The Korean medical diagnosis system has unique characteristics similar to the traditional Chinese medical diagnosis system. One such feature is pattern identification (PI), which is based on information obtained from four diagnostic processes including inspection, listening and smelling, inquiry, and palpation \[[@B4]\]. PI is a diagnostic system that entails a comprehensive analysis of symptoms and signs, with implications for determining the cause, nature, and location of the illness, the patient\'s physical condition, and the patient\'s treatment \[[@B3], [@B5]\]. The inspection process involves the examination of the patient\'s symptoms or disease by observing his or her shape, expression, and tongue \[[@B6]\], among others. Observation of the tongue, also known as tongue diagnosis, is an important procedure in diagnosis by inspection in TKM. The status of the tongue is an important indicator in the diagnosis of one\'s condition, including the physiological and clinicopathological changes of internal organs in the body \[[@B7]\]. A number of studies have shown that tongue diagnosis plays an important role in clinical prognosis and treatment \[[@B8]--[@B15]\], specifically in patients with a history of stroke. However, the clinical competence of tongue diagnosis was determined by the experience and knowledge of the clinicians who used tongue diagnosis. Environmental factors, such as differences between light sources and levels of brightness also had significant influences on clinicians and their diagnostic decisions using the tongue. Unfortunately, much of the experiences in traditional tongue diagnosis have not been verified scientifically or quantitatively. Therefore, it is necessary to build an objective diagnostic standard for tongue diagnosis \[[@B7]\]. We investigated the reliability of TKM tongue diagnosis in stroke patients by evaluating interobserver reliability regarding tongue indicators as achieved by TKM practitioners.
2. Methods {#sec2}
==========
2.1. Study Subjects {#sec2.1}
-------------------
The data for this analysis were collected as part of the project named the Fundamental Study for the Standardization and Objectification of Pattern Identification in TKM for Stroke (SOPI-Stroke). Stroke patients admitted to the following oriental medical university hospitals, Kyung Hee Oriental Medical Center (Seoul), Kyung Hee East-West Neo Medical Center (Seoul), Dong Guk International Hospital (Kyunggi-do), Kyung Won Oriental Medical Hospital (Incheon), Dae Jeon Oriental Medical Hospital (Daejeon), Dong Sin Oriental Medical Hospital (Gwangju), Won Kwang Oriental Medical Hospital (Jeollabuk-do), Dae Gu Hanny University Medical Center (Daegu), and Sang Ji Oriental Medical Hospital (Gangwon-do), participated in this study between February 2010 and December 2010 ([Figure 1](#fig1){ref-type="fig"}). All patients provided written informed consent under procedures approved by institutional review boards (IRB). Eligibility inclusion criteria were that participants had to be enrolled within 30 days of the onset of their symptoms as confirmed by imaging diagnosis, such as computerized tomography (CT) or magnetic resonance imaging (MRI). Exclusion criteria were traumatic stroke patients, such as those with subarachnoid, subdural, or epidural hemorrhage.
2.2. Data Processing and Analysis {#sec2.2}
---------------------------------
All patients were seen by two experts from the same department in each site, who were well trained in standard operation procedures (SOPs) \[Appendix\] and were subjected to an examination of the status of the tongue, tongue color (pale, red, and bluish purple), fur color (white fur, yellow fur), fur quality (thick fur, thin fur, moist fur, and dry fur), and special tongue appearance (teeth marked, enlarged, mirror, and spotted). The examination parameters were extracted from parts of a case report form (CRF) for the standardization of stroke diagnosis that had been developed by an expert committee organized by the Korean Institute of Oriental Medicine (KIOM). These assessments were given individually without discussions among the clinicians. Descriptions of the grading severity for each variable were scored as the following: 1 = very much so, 2 = Much so, and 3 = Not so much. In particular, the clinicians had to measure the stroke PI of each patient following the fire-heat pattern, the phlegm-dampness pattern, the blood stasis pattern, the qi deficiency pattern, and the Yin deficiency pattern, as suggested by the KIOM \[[@B3], [@B16]--[@B18]\].
Interobserver reliability was measured in three ways, using simple percentage agreements, Cohen\'s kappa coefficient and Gwet\'s AC~1~ statistic, as well as via the corresponding confidence intervals (CI). Kappa, the preferred measure of rater reliability for nominal data, measures the reliability of agreement between two or more independent raters using a rating scheme with mutually exclusive categories. In general, definitive kappa interpretations have been proposed \[[@B19]--[@B24]\]. For most purposes, however, values ≤0.40 represent poor agreement, values between 0.40 and 0.75 represent moderate to good agreement, and values ≥0.75 indicate excellent agreement \[[@B24]\]. The AC~1~ statistic is not vulnerable to the well-known paradoxes that make Kappa appear ineffective \[[@B25]--[@B27]\]. First, interobserver reliability for the tongue indicator among all subjects was calculated via simple percentage agreements, Cohen\'s kappa coefficient, and Gwet\'s AC~1~ statistic. Later, interobserver reliability regarding PI with same opinions between the raters was calculated in the same way. The blood stasis pattern was omitted because the sample size was too small (*n* = 1). The data were statistically analyzed with SAS software, version 9.1.3 (SAS Institute Inc., Cary, NC).
2.3. Ethical Approval {#sec2.3}
---------------------
This study was approved by institutional review board of the KIOM and by each of the oriental medical university hospitals.
3. Results {#sec3}
==========
A total of 658 stroke patients were enrolled in the study. Thirty patients were excluded from analysis due to PI omitted by any one of 2 TKM clinicians. The interobserver reliability results regarding tongue indicators for all subjects (*n* = 628) are shown in [Table 1](#tab1){ref-type="table"}. The kappa measure of agreement between the two experts was generally moderate to good for the tongue indicators, ranging from 0.42 to 0.69, except for moist fur (*κ* = 0.29) and spotted (*κ* = 0.37). Moreover, the AC~1~ measure of agreement between the two experts was generally high for the tongue indicators, ranging from "moderate" (AC~1~ = 0.43) to "excellent" (AC~1~ = 0.97). Agreement, as assessed by the kappa values, was considerably lower than the AC~1~ values in most cases.
The results of interobserver reliability for subjects of PI with the same opinion between the raters are shown in [Table 2](#tab2){ref-type="table"}. A total of 451 stroke patients received PI with the following same resulting opinions by the raters: Fire-Heat Pattern (*n* = 147), Phlegm-Dampness Pattern (*n* = 158), Yin Deficiency Pattern (*n* = 80), and Qi Deficiency Pattern (*n* = 66). The blood stasis pattern was excluded because the sample size was too small (*n* = 1).
The kappa measure of agreement for the subjects of PI was generally moderate to good for the tongue indicators, ranging from 0.40 to 0.72, except for moist fur (*κ* = 0.31). Moreover, the AC~1~ measure of agreement between the two experts was generally high for the tongue indicators, ranging from "moderate" (AC~1~ = 0.5) to "excellent" (AC~1~ = 0.98) ([Table 2](#tab2){ref-type="table"}).
4. Discussion {#sec4}
=============
Inspection of the tongue in TKM diagnosis, as well as in western medicine \[[@B28]\], is one of the most important approaches for obtaining significant evidence in diagnosing the patient\'s health conditions \[[@B7]\]. It is used to observe the color, coating, and body of the tongue, among other features, in rendering a disease diagnosis.
Also, as tongue diagnosis has played a prominent role in the diagnosis and subsequent treatment of stroke patients, it has attracted an increasing amount of attention in oriental medicine \[[@B8]--[@B15]\]. Park et al. \[[@B12]\] analyzed markers that classified tongue body color, fur, fur quality, dryness, and shape to standardize tongue diagnosis and PI for stroke patients. Choi et al. \[[@B14]\], to assess the usefulness of tongue diagnosis in evaluating PI, observed the coating of the tongue and compared it with PI in acute stroke stage patients within 72 hours from the onset of stroke. In his study, a red tongue was significantly related to the fire-heat pattern and the yin deficiency pattern, while a faint white tongue was related to the phlegm-dampness pattern. Thin fur was related to the Wind
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Introduction {#Sec1}
============
Small heat-shock proteins (sHSPs) are a diverse family of proteins that share a conserved ≈ 90-residue α-crystallin domain (ACD) that is flanked by variable N- and C-terminal regions (Basha et al. [@CR3]; Hilton et al. [@CR17]; McHaourab et al. [@CR28]). Although sHSPs are relatively small as monomers (12 to 42 kDa), the majority assemble into large oligomers. These range in size from 12 to \> 40 subunits, with some family members being monodisperse and others forming polydisperse ensembles (Basha et al. [@CR3]; Hilton et al. [@CR17]; McHaourab et al. [@CR28]). Found in all kingdoms of life, many sHSPs have been demonstrated in vitro to act as ATP-independent molecular chaperones with the ability to capture denaturing proteins in a partially unfolded form such that they can be reactivated by the cell's ATP-dependent chaperones. Recent reviews have described models for this canonical mechanism of sHSP chaperone action; however, details are derived primarily from in vitro studies with recombinant proteins and model interactors from non-homologous organisms (Haslbeck and Vierling [@CR16]; Treweek et al. [@CR40]). Thus, a major gap in our understanding of sHSP mechanism is the considerable lack of information about which substrates they protect in the cell.
In order to investigate the properties of proteins that are sHSP interactors, we identified HSP16.6 from the single-celled cyanobacterium *Synechocystis* sp. PCC 6803 (hereafter *Synechocystis*) as an ideal system to interrogate. HSP16.6 is the only sHSP in *Synechocystis* (Giese and Vierling [@CR12]; Lee et al. [@CR25]). It is strongly induced at high temperature, and cells deleted for HSP16.6 (Δ16.6) grow normally at optimal growth temperature but are sensitive to heat stress (Giese and Vierling [@CR12], [@CR13]). The temperature-sensitivity phenotype of Δ16.6 cells has enabled studies of sHSP properties required for activity in vivo in a homologous system. Crucially, point mutations in the N-terminal domain were found to decrease heat tolerance in vivo, but to have no effect on the efficiency of chaperone function in assays with model substrates in vitro (Giese et al. [@CR14]). This observation emphasizes the need to identify native interactors of sHSPs and renders *Synechocystis* an excellent system with which to do so.
We previously used immunoprecipitation and mass spectrometry (MS)-based proteomics to identify 13 proteins associated in vivo with HSP16.6 from *Synechocystis* cells that had been heat-stressed prior to cell lysis (Basha et al. [@CR2]). Notably, these 13 proteins were not detected in equivalent pull-downs from cells that had not been heat-stressed, or when recombinant HSP16.6 was added to heat-stressed Δ16.6 cells before lysis (to control for sHSP-protein interactions that might occur in the lysate, as opposed to during heat stress in vivo). Although these proteins were associated with the sHSP in the soluble cell fraction, they were also found in the insoluble cell fraction after heat stress (Basha et al. [@CR2]). All of these proteins, whose functions span a variety of cellular processes, including translation, transcription, secondary metabolism, and cell signaling, could be released from the immunoprecipitate by addition of DnaK, co-chaperones, and ATP (Basha et al. [@CR2]). In addition, one of these interactors, a serine esterase, when purified, was shown to be heat sensitive and to associate with HSP16.6 and thereby be protected from insolubilization (Basha et al. [@CR2]). While these data identified 13 proteins as potential interactors for canonical sHSP chaperone function, their relatively small number meant it was not possible to derive any common protein features that might dictate interaction with the sHSP.
Here, we have extended the identification of HSP16.6-interactors to a total of 83 proteins by performing an affinity pull-down from heat-stressed *Synechocystis*. By performing rigorous bioinformatic analyses, we provide new insights into the primary and secondary structural properties of proteins that interact with sHSPs in the soluble cell fraction during stress. We also catalogue the functions of the interactors and compare these to sHSP interactors previously identified in two other prokaryotes, *Escherichia coli* and *Deinococcus radiodurans* (Bepperling et al. [@CR4]; Fu et al. [@CR10]). Our combined results indicate that sHSPs protect a specific yet diverse set of proteins from aggregation in the cell.
Methods {#Sec2}
=======
Affinity isolation of HSP16.6-interacting proteins {#Sec3}
--------------------------------------------------
Isogenic *Synechocystis* strains were used in which the wild-type HSP16.6 gene had been replaced with a spectinomycin resistance gene (*aadA* gene) (ΔHSP16.6 strain) or with the spectinomycin gene and HSP16.6 carrying a Strep-tag II affinity tag (WSHPQFEK) on the C-terminus (HSP16.6-Strep strain) (Basha et al. [@CR2]). This HSP16.6-Strep strain had been shown previously to behave like wild type in assays of heat tolerance (Basha et al. [@CR2]), and recombinant HSP16.6-strep protein was equivalent to untagged protein in assays of chaperone activity in vitro (Friedrich et al. [@CR9]).
Cells were grown in 50-mL cultures at 30 °C as described previously to *A*~730~ ≈ 0.2 (Basha et al. [@CR2]) and then subjected to treatment at 42 °C for 2 h followed by 1 h recovery at 30 °C, to allow accumulation of HSP16.6-Strep protein. Control samples were prepared directly after this treatment, while heat-stressed samples were treated for an additional 30 min at 46 °C. To control for interaction of HSP16.6-Strep protein during sample processing, recombinant HSP16.6-Strep protein was added to heat-stressed samples of the ΔHSP16.6 strain directly after heat treatment at a concentration matching that in heat-stressed cells. Cells were harvested, suspended in 1.5 mL lysis buffer (25 mM HEPES-KOH, 0.2 M NaCl, 0.5% Triton X-100, 5 mM ϵ-aminocaproic acid, 1 mM benzamidine, 1 μg mL^−1^ leupeptin, and 1 mM EDTA, pH 7.5), and opened as described previously (Basha et al. [@CR2]). The soluble fraction was mixed with 30 μL of Strep-Tactin resin (Sigma) at 4 °C for 2 h. Resin was washed six times in lysis buffer, and bound proteins were eluted using either sample buffer (for SDS-PAGE) or isoelectric focusing (IEF) rehydration buffer (for 2D gels) (7.0 M urea, 2.5 M thiourea, 2% CHAPS, 2% IPG buffer pH 3--10 NL (Amersham Biotech), and 3 mg mL^−1^ dithiothreitol).
For 2D gel analysis, pH 3--10 NL first dimension strips (18 cm; Amersham Biotech) were rehydrated overnight at room temperature using 600 μL of sample in IEF rehydration buffer. IEF was carried out for 2 h at 150 V, 2 h at 300 V, 5 h at 500 V, and 7 h at 3500 V. The second dimension was separated by 11--17% SDS-PAGE for 30 min at 15 mA and then for 7 h at 25 mA. Samples were also separated by SDS-PAGE according to standard protocols, using 8% acrylamide gels in order to afford good separation of proteins above 100 kDa, which are typically not well resolved on the 2D system. Gels were silver stained according to a previous protocol (Rabilloud [@CR33])*.*
Protein identification by means of mass spectrometry {#Sec4}
----------------------------------------------------
Proteins unique to the heat-stressed HSP16.6-Strep sample were excised from 1D or 2D gels and digested with trypsin, and peptides were prepared for MS as described previously (Basha et al. [@CR2]). Peptide extracts were introduced onto a 100-μm I.D. × 5-cm C18 column using an autosampler and separated with a 25-min gradient of 2--100% acetonitrile in 0.5% formic acid. The column eluate was directed into a Thermo Finnigan LCQ Deca ion trap mass spectrometer. The mass range scanned was 400 to 1500 *m*/*z*, and data-dependent scanning was used to select the three most abundant ions in each parent scan for tandem MS. Peptides were searched using SEQUEST and allowed for static modification of Cys (57 Da; iodoacetamidation), and differential modification of Met (16
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Introduction {#Sec1}
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Alcohol use disorders (AUD) are common and associated with significant morbidity and mortality \[[@CR1]--[@CR3]\], but are substantially undertreated. In 2013, 16.6 million U.S. adults met diagnostic criteria for an AUD, but research suggests only 7.8% received any formal treatment \[[@CR4]\]. One of the major gaps in treatment for AUD is the significant under-utilization of medications that are effective for treating AUD \[[@CR1], [@CR5], [@CR6]\]. Three medications---*disulfiram*, *acamprosate*, and *naltrexone* (both oral and injectable)---have FDA approval specifically for the treatment of AUD, and *topiramate* has strong meta-analytic support \[[@CR7]\]. Efforts to increase treatment of AUD with medications is motivated in part because the modality may address many reported barriers to receiving any formal AUD treatment \[[@CR4], [@CR8]\]. For instance, psychosocial treatments are often offered in group settings, heightening stigma-related issues for some patients, whereas medications can be provided on an individual basis \[[@CR9]\]. In addition, patients may not be ready to abstain \[[@CR8], [@CR10]\]. Further, though this may be shifting over time \[[@CR11], [@CR12]\], many treatment programs view abstinence as the ultimate goal \[[@CR8]\], whereas abstinence is not required with all medications and reduced drinking can be a goal of medication treatment \[[@CR9]\]. Finally, AUD medications can be offered across healthcare settings, including primary care, which has been highlighted as an optimal setting for expansion of care for AUD \[[@CR8], [@CR13], [@CR14]\].
Despite the promise of medication treatment for addressing several known barriers to AUD treatment and national recommendations encouraging medications be made available to all patients with AUD \[[@CR15], [@CR16]\], rates of pharmacotherapy for AUD remain extremely low. Among patients with AUD, 4-12% are treated pharmacologically \[[@CR1], [@CR6], [@CR17]--[@CR21]\]. Among subsets of patients with AUD and co-occurring schizophrenic, bipolar, posttraumatic stress or major depressive disorder, receipt of medications for AUD ranged from 7 to 11%, whereas receipt of medications for the comorbid disorder ranged from 69 to 82% \[[@CR19]\]. This gap in the quality of AUD treatment is well known, and the substantial barriers to provision of AUD medications in diverse contexts have been described \[[@CR22]--[@CR27]\]. However, the optimal strategies for addressing these barriers and increasing use of medications for AUD treatment remain elusive.
In recent years, two related lines of research have contributed to knowledge regarding strategies to increase use of medications to treat AUD: evaluations of care delivery interventions and evaluations of implementation interventions. Care delivery interventions typically focus on improving patient-level clinical outcomes (e.g., reduction in heavy drinking days or abstinence from alcohol use), but often secondarily assess patient- or clinician-level process outcomes focused on treatment receipt (e.g., engagement in pharmacotherapy for AUD). Implementation interventions are typically designed to improve patient- or clinician-level process outcomes, but sometimes secondarily include patient-level clinical outcomes when the evidence for the effects of the underlying practice is weak (so called Hybrid I studies) \[[@CR28]\]. Other key differences exist between these types of research that may influence both clinical and process outcomes. Most importantly, care delivery interventions typically involve recruitment of patients who are willing to be randomized to the treatment arms contained within the new care delivery model. Thus, these trials may be restricted to patients who are at least open to, if not actively interested in, treatment for AUD. On the other hand, evaluations of implementation interventions typically recruit and intervene on clinical entities (e.g., providers, clinics, hospitals) who serve large groups of patients who likely have more variable interest in treatment. Further, evaluations of care delivery interventions are typically designed to establish the effectiveness (or lack thereof) of particular care delivery models. Thus, these studies generally put significant effort and resources into ensuring fidelity to the care delivery model. On the other hand, implementation evaluations are often trying to establish the effectiveness of bundles of strategies (interventions) to increase uptake of practices that do not depend on external research resources. Thus, evaluations of implementation interventions may measure fidelity as a process outcome but typically exert less direct control \[[@CR29]\].
Even though care delivery and implementation interventions differ in terms of methodology, patient inclusion criteria, and primary outcomes, they may evaluate the effectiveness of the same underlying implementation strategies, such as reorganizing, supplementing, or intervening on existing models of care \[[@CR29]\]. The fact that the same component implementation strategies (e.g., audit and feedback) have been evaluated by these different research designs with very different patient populations affords an opportunity to take stock of the effectiveness of these interventions, and to distill insights into which designs, contexts, and component strategies appear to drive outcomes. Therefore, our goal was to conduct a structured review of published evaluations of care delivery and implementation interventions that have either primarily or secondarily aimed to increase use of pharmacotherapy for patients with AUD, with the goal of identifying component strategies that may be effective in increasing pharmacologic treatment of AUD. Our review was guided by an existing taxonomy of implementation strategies and terms identified via a three-round modified-Delphi process \[[@CR30]\]. The purpose of our review was to learn which components have been tried most commonly and which strategies might be associated with larger effects. Also, due to the fact that evaluations of care delivery interventions exert greater efforts to ensure fidelity and include patients willing to be randomized, we hypothesized that higher adoption of medications for AUD will be observed in those contexts compared to implementation interventions, which typically aim to intervene on clinician and patient populations with greater variability in treatment motivation, knowledge, and preferences.
Methods {#Sec2}
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For this structured literature review, we sought to identify published evaluations of care delivery and implementation interventions reporting effects on receipt of medication treatments for patients with AUD. We reviewed literature through May 2018. Studies were identified via searching PubMed, Google Scholar, and PsychInfo with relevant search terms (e.g., pharmacotherapy, alcohol use disorder medications, AUD medications, naltrexone, Acamprosate, disulfiram, medication-assisted treatment). We also reviewed reference lists from identified studies to identify additional studies that may have been missed by our search. Finally, because we have personally conducted and/or served as co-investigators on related studies, additional studies were also identified via networking. Once identified, each individual article was coded for implementation strategies used, as guided by Powell et al.'s refined compilation of implementation strategies resulting from the Expert Recommendations for Implementing Change (ERIC) project \[[@CR30]\]. All articles were independently reviewed and coded by two investigators (EW and TM). When multiple articles and/or published protocols or commentaries were identified that described a single intervention and/or implementation effort, these articles were aggregated to the level of the intervention (e.g., three studies had adjoining published protocol papers, which were coded under the umbrella of a single study). Once coded, all authors met to review coding discrepancies, discuss interpretation of codes, arrive at consensus, and revise individual codes based on consensus.
After reaching internal consensus on coding, we reached out to the lead or senior author of each study to ask whether our codes aligned with their understanding/interpretation of their study and associated report. We shared Powell et al's description of strategies and asked them to review our coding to see if they thought we had missed or miscoded anything. Finally, process (e.g., rates of prescribed AUD pharmacotherapy) and alcohol use outcome data were extracted from each study and described. All authors reviewed the coding of implementation strategies against study outcomes data to qualitatively identify sets of implementation strategies that might have been be most effective for increasing provision of AUD medications and report whether interventions that increased AUD pharmacotherapy also improved alcohol use outcomes.
Results {#Sec3}
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Our literature review identified nine studies that evaluated interventions to primarily or secondarily increase utilization of pharmacotherapy for AUD. Four were randomized clinical trials of care delivery interventions designed to improve alcohol-related outcomes \[[@CR31]--[@CR38]\]. Four were quasi-experimental evaluations of large-scale implementation interventions designed to increase medication receipt \[[@CR39]--[@CR43]\], and one was a quasi-experimental evaluation of targeted implementation intervention in a single-site \[[@CR44]\]. Two additional studies were identified but not included. The first reported on a large-scale implementation intervention designed to increase screening and brief intervention for unhealthy alcohol use and secondarily assessed whether the implementation was associated with increased receipt of AUD medications among those who screened positive \[[@CR45]\]. However, it was not clear how many of the patients who screened positive met diagnostic criteria for AUD and thus would have been eligible for medication treatment, and, though findings regarding medication use were summarized, detailed data were not reported. The second report was a description of a demonstration project to implement extended release naltrexone in Los Angeles County, but no evaluation of the program's effect on receipt of medication treatment among patients with AUD was reported \[[@CR46]\].
Table [1](#Tab1){ref-type="table"} presents implementation strategies identified by our internal coding process across each identified study (labelled with X). All lead or senior authors of studies responded to our request for review of the codes and added additional codes (labelled with an O). Implementation strategies used were variable across the studies, and no strategy was used across all studies (Table [1](#Tab1){ref-type="table"}). The most frequently used strategies were assessing readiness and identifying barriers and facilitators, distributing educational materials, facilitating relay of clinical data to providers (audit and feedback), and providing ongoing consultation. Strategies less frequently used involved
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INTRODUCTION {#s1}
============
Hepatocellular carcinoma (HCC) is one of the most common malignant tumors and ranks the third highest cause of cancer-related deaths worldwide \[[@R1], [@R2]\]. The resection rate of HCC has increased over decades due to the improvements in early diagnostic methods and surgical techniques. However, the postoperative recurrence rate and overall survival (OS) are not optimistic due to limited response to various adjunctive therapies and aggressive behaviors in advanced stages of HCC \[[@R3]\]. Thus, an accurate understanding of the biological behavior of therioma is critical in predicting the prognosis of HCC patients. Traditional prognostic factors related to the clinicopathological characteristics of the neoplasm after hepatic resection such as tumor size, vascular invasion, tumor-node-metastasis(TNM) stage, functional liver reserve and Child-Pugh class have a limited clinical value for outcome prediction \[[@R4]\]. Therefore, a variety of other potential molecular predictive markers need to be further identified.
A sequential process, including escape from the primary tumor site, local invasion, systemic transport through vascular or lymphatic vessels, adhesion to distant organs, re-colonization, and expansion, is believed to be involved in hepatic carcinogenesis. Epithelial to mesenchymal transition (EMT) is known to play a pivotal role in the diffusion of cancer cells and the growth of tumors, in which epithelial cells lose their polarity and cell-cell contacts due to repressed expression of E-cadherin and up-regulated expression of mesenchymal markers such as N-cadherin, vimentin and α--smooth muscle actin (α--SMA) \[[@R5]\]. EMT could enhance not only the capacity of invasion and migration but resistance to apoptosis and chemoresistance in cancer. EMT may alter the gene expression of epithelial cells due to the activation of EMT-inducing transcription factors (EMT-TFs). In this meta-analysis, we focused on the most prominent inducers of EMT such as the zinc finger E-box binding homeobox (ZEB1 and ZEB2), the zinc-finger transcriptional repressor (Snail and Slug), and the basic helix-loop-helix transcription factor (Twist1) through searching the published literature \[[@R6], [@R7]\], knowing that EMT-TFs are directly or indirectly involved in metastasis of malignant cells through a series of signaling cascades, including the wingless-related integration site(Wnt), serine/threonine-specific protein kinase (Akt), mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) pathways \[[@R8], [@R9]\].
During the past decade, much research has begun noticing the correlation between the expression of EMT-TFs and the prognosis of HCC. However, the results are often unconvincing due to the limited sample sizes. Here, we sought to perform a meta-analysis to evaluate clinicopathological and prognostic significance of EMT-TFs overexpression in HCC patients, especially those with high incidences of recurrence after curative resection.
RESULTS {#s2}
=======
Study selection and patient characteristics {#s2_1}
-------------------------------------------
The initial search identified 418 potentially relevant studies. After screening, 10 published studies including 1,334 patients were selected for this pooled analysis \[[@R10]--[@R19]\]. A flowchart depicting the selection of the eligible literature is shown in Figure [1](#F1){ref-type="fig"}.
{#F1}
All the included studies were retrospectively analyzed, with the sample size ranging from 40 to 323 (median 133). The overexpression of EMT-TFs was reported in 662 (49.6%) of the 1,334 included patients. The highest positive expression rate was Twist1, accounting for 60.3%, followed by Snail (51.9%), ZEB2 (50.3%), ZEB1 (43.6%) and Slug (29.4%). These studies were published between 2007 and 2015. Among all cohorts, Asia was the only source region of the 10 included studies, including 9 studies from China \[[@R11]--[@R19]\] and one from Japan \[[@R10]\]. Newcastle-Ottawa Quality Assessment Scale was applied to assess these studies. The result showed that the quality scores ranged from 5 to 8 (median 6.5), indicating a relatively high study quality.
Characteristics of the included studies are listed in Table [1](#T1){ref-type="table"}. All the studies focused on OS, with a median follow-up period of at least 48 (48--80) months. The definition of EMT-TFs positive expression was based on immunohistochemistry (IHC) or western blot analysis (WB) evaluation in all the eligible articles, as expressed as the percentage of positive cells or/and staining intensity. Hazard ratios (HRs) and 95% confidence intervals (CIs) were directly recorded in 8 studies \[[@R10]--[@R12], [@R15]--[@R19]\] and could be inferred from two other studies using the Tierney\'s methods described above, among which one \[[@R14]\] were calculated by variance and *P* value, and the other \[[@R13]\] was estimated only by Kaplan-Meier survival curves.
###### Characteristics of the included studies
EMT-TFs Author Year Country Case EMT-TFs Positive(%) Treatment Antibody method Outcome MFu time (months) NOS score
--------- ---------- ------- --------- ----------- --------------------- ----------- ------------ ------------ ---------------------- ---------------------- ----------- ---- ---- ---
ZEB1 Motoyuki 2013 Japan 108 23 (21.3) Surgery goat polyclonal 1:100 SantaCruz, CA, USA IHC OS 60 8
Zhou 2011 China 110 72 (65.5) Surgery NA NA NA SantaCruz, CA, USA WB OS 60 7
ZEB2 Cai 2012 China 248 150 (60.5) Surgery rabbit polyclonal 1:100 Sigma, St.Louis, USA IHC OS 80 8
Yang 2015 China 92 21 (22.8) Surgery rabbit polyclonal 1:100 Abcam, Cambridge, UK IHC OS 60 5
Snail1 Zhou 2014 China 323 161 (49.8) Surgery NA NA NA Novus, USA IHC OS 60 6
Zhao 2012 China 97 57 (58.8) Surgery NA NA 1:250 SantaCruz, CA, USA IHC OS 60 7
Slug Zhang 2013 China 119 35 (29.4) Surgery NA NA NA Danvers, MA, USA IHC OS 60 8
Twist1 Zhang 2010 China 100 70 (70.0) Surgery rabbit polyclonal 1:50 SantaCruz, CA, USA IHC OS 76 7
Zhao 2011 China 97 51 (52.6) Surgery NA NA 1:100 SantaCruz, CA, USA IHC OS 60 7
Niu 2007 China 40 22 (55.0) Surgery rabbit monoclonal 1:50 SantaCruz, CA, USA IHC OS 48 6
Abbreviations: EMT-TFs = epithelial to mesenchymal transition-inducing transcription factors; NA = not available;
IHC = Immunohistochemistry; WB = Western Blot; MFu = median Follow-up; OS = overall survival; NOS = Newcastle-Ottawa Scale.
Evidence synthesis {#s2_2}
------------------
EMT-TFs and OS in HCC {#s2_3}
---------------------
The pooled HR for OS indicated that EMT-TF positive expression was associated with poor OS \[HR = 1.71; 95% CI: 1.40--2.08; *p* \< 0.00001\] in HCC with a statistically significant 71% increase in the risk for mortality (Figure [2](#F2){ref-type="fig"}). Seeing that the heterogeneity test showed a *P* value of 0.08 and an I^2^ statistic index of 41%, we considered that there may be relatively substantial heterogeneity between these studies, and therefore we used the random-effects model.
{#F2}
Figure [3](#F3){ref-type="fig"} shows the impact of various individual EMT-TFs on the survival of HCC patients. The significantly higher HRs for OS was Slug \[HR = 2.12; 95% CI: 1.16--3.86; *p* = 0.01\]. But as the transcription factor was reported in only one study, the result should be considered with caution. In addition to ZEB2 \[HR = 1.23; 95% CI: 0.59--2.57; *p* = 0.58\], HRs for Twist1 \[HR = 2.04; 95% CI: 1.50--2.78; *p* \< 0.00001
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Introduction
============
Better adjuvant therapy, improved metal implants, and innovative surgical techniques have led surgeons to consider limb salvage surgery as an alternative treatment for malignant bone tumour other than amputation. Orthopaedic oncology patients have a chance for an active, disease-free life after limb salvage surgery. In the first evidence-based study, Simon *et al.* had reported the benefits of limb-salvaging procedures for bone tumours.[@b1-rado-46-03-189] Their multicentre study reported the rates of local recurrence, metastasis and survival in 227 patients with osteosarcoma in the distal femur and suggested that the Kaplan-Meier curves of the patients without recurrence were not statistically different between limb-salvaging surgery and amputation patients during a 5.5-year follow-up. Limb-salvage surgery was considered as safe as an amputation in the management of patients with high-grade osteosarcoma.
The goal of limb-salvaging surgery is to preserve the function of limbs, prevent tumour recurrence, and enable the rapid administration of chemotherapy or radiotherapy.[@b2-rado-46-03-189] It can be reached with meticulous technique, detailed operative planning, and the use of endoprosthetic replacements and/or bone grafting. For a successful limb-salvage surgery in high-grade malignant tumour, such as sarcomas, a wide margin is necessary to obtain a local control.[@b3-rado-46-03-189]--[@b5-rado-46-03-189] Since marginal and intralesional margins are related to local recurrence, the reconstruction with limb-salvaging options should be carefully considered. The clinical outcome of the limb-salvage surgery with arthroplasty is closely related to the accuracy of the surgical procedure. To improve the final outcome, one must take into account the length of the osteotomy plane, as well as the alignment of the prosthesis with respect to the mechanical axis in order to keep the balance of the soft tissues. Furthermore, the parameters measured with the 3D imagine must be used during the individual manufacture of implant in order to reconstruct the skeletal structure accurately. Therefore, geometric data (such as length of leg, offset) and morphologic data are required.
Magnetic resonance imaging (MRI) was beneficial for tumour detection and consequently staging of musculoskeletal neoplasia. MRI became an ideal imaging modality for musculoskeletal neoplasia because of superior soft-tissue resolution and multiplanar imaging capabilities and had a significant impact on the ability to appropriately stage lesions and adequately plan for limb-salvage surgery.[@b6-rado-46-03-189],[@b7-rado-46-03-189] In contrast, multi-slice spiral computed tomography (CT) could provide super three-dimensional morphological delineation of the diseased bone. Theoretically, the complimentary use of these two imaging modalities could give the surgeon a more accurate way to implement preoperative planning than the conventional application of 2D images.
The purpose of this prospective study was to report our initial experience with limb salvage surgery for orthopaedic oncology patients by using both MR imaging and multi-slice spiral CT for preoperative planning.
Patients and methods
====================
Patients and preparation
------------------------
The study protocol has complied with all relevant national regulations and institutional policies and has been approved by the local institutional ethics committee. Informed consent was obtained from all patients before the procedure. Patients with malignant bone tumours of lower/upper limb were enrolled in the study.
Preoperative work-up consisted of history and clinical examination, routine laboratory tests and an aesthetic assessment, plain radiography of the limb, 64-slice spiral CT scan of the limb and chest, Technetium-99m bone scan, and, in all of the cases, MRI of the affected limb. Antibiotics were administrated before the surgery. Biopsy was performed for pathological examination. Chemotherapy was commenced 6 weeks before the surgery in those cases which were diagnosed as osteosarcoma and dedifferentiated chondrosarcoma. Patients were classified according to the Enneking staging system.[@b8-rado-46-03-189],[@b9-rado-46-03-189]
The patients received a detailed narrative of conventional, surgical and amputation options after the limb salvage surgery at their own request. Nine consecutive patients with lower/upper limb malignant tumour of bone (5 women, 4 man, mean age 28.6 years, range: 19--52 years) were treated with limb-salvaging procedures. Lesion size (longitudinal direction), location and histology are summarized in [Table 1](#t1-rado-46-03-189){ref-type="table"}.
MR imaging
----------
MR images were performed at a 1.5-T superconductive unit (Gyroscan Intera, Philips Medical Systems, Netherlands) and a synergy surface coil was used. The sequences included transverse, sagittal and coronal turbo spin echo T1- and fat-suppressed T2-weighted images. The parameters of these sequences were TR/TE=400 /20 ms for T1-weighted imaging, TR/TE=3500 /120 ms for T2-weighted imaging and a field of view of 480 mm×480 mm for sagittal imaging and 40 mm×40 mm for transverse imaging and 480 mm×480 mm for coronal imaging with a matrix of 512×512, 4--6 signals acquisition and a slice thickness/gap = 5/0.5 mm. Contrast enhanced sagittal, coronal and transverse T1-weighted imaging were obtained after the intravenous injection of gadopentetate dimeglumine (Magnevist, Schering, Berlin, Germany) with a dosage of 0.2 mmol/kg of body weight.
Multi-slice spiral CT
---------------------
CT scan was performed by using a 64-slice spiral CT (Sensation 64, Siemens Medical Systems, Germany). The raw data obtained using an axial collimation of 64×0.6 mm, a pitch of 1.0, a tubular voltage of 120 KV and a tubular current of 360 mAs, were reconstructed into contiguous 1-mm thick slices with an increment of 0.5 mm and a field of view of 376 mm × 376 mm and a matrix of 512 × 512 by using the standard soft tissue and bone algorithm. These thin-slice images were postprocessed by using the techniques of multiplanar reformation (MPR) and volume rendering (VR) to demonstrate the lesion details and perform related measurements.
Preoperative planning
---------------------
All preoperative radiographs were evaluated by one radiologist and two consultant orthopaedic surgeons, who were members of the surgical team performing the operations. First, the osteotomy plane was determined separately on CT and MRI. On orthogonal coronal enhanced MR images and CT MPR images, the bulk margin of the tumour in the medullary cavity was defined according to the different signal characteristics or attenuation of the tumour itself and the marrow oedema around the tumour. Then, the maximum distance from the top of the greater trochanter to this tumour margin was measured on orthogonal coronal T1-weigthted MRI images, if the tumour was located in the proximal part of femur. The maximum distance from the knee joint line to the tumour margin was measured for the tumours located in the distal part of femur. The maximum distance was defined as the intramedullary extension of the primary tumour and subsequently was used as a reference for the CT measurement. The osteotomy plane on CT MPR images was defined 30 mm distal from the margin of tumour. This distance was also used to determine the length of the extra medullary part of the prosthesis. After the osteotomy plane had been determined, the detailed shape of the medullary cavity of the preserved part of the femur was assessed using the orthogonal MPR technique for determining the diameter and length of the intra medullary part of prosthesis. Diameters of the medullary cavity at the level of the osteotomy plane and the level of the narrowest plane were measured to determine the diameter of the intra medullary stem of the prosthesis. The length of the intra- medullary stem of the prosthesis should be well matched to the length of medullary cavity of the preserved part of femur, which would be optimal if it had an equal length to the extra medullary part of prosthesis. Finally, the centre axis of the femoral shaft measured on CT was used as a reference. Offset, the distance from the central axis of the femoral shaft and the rotation centre of the femoral head, was the index used to determine the neck length of the prosthesis.
Surgery
-------
All patients underwent en bloc resection and customized prosthetic reconstruction. An anterolateral incision encircling the biopsy scar was used. Limb-salvage surgery consisted of intentional marginal excision, preserving important structures such as major neurovascular bundles, tendons, and ligaments. The osteotomy plane, 30 mm distal from the primary tumour was confirmed based on MRI for all patients. For patients with lesion in the proximal part of femur/humerus, the customized prosthesis was secured using methylmethacrylate cement after the resection. For patients with the tumour in the distal part of the femur, en bloc resection including the tibial plateau was performed and the customized prosthesis was secured using methylmethacrylate cement in both the tibia and femur after the resection. The extensor mechanism was reconstructed by reattachment of the patellar tendon to the slot on the tibial component. After surgery, functional rehabilitation and neoadjuvant chemotherapy were performed.
Postoperative measurement
-------------------------
After surgery, the patients were followed with a mean of 13 months (range, 9 to 20 months). The postoperative assessment of prosthesis was performed on plain radiography. The central axis of the femoral or humeral shaft and offset were defined. The vertical distance from the line between the top of bilateral ischial tuberosities to the femoral
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Building upon the pioneering work of Vicsek *et al.*[@b1], physicists, mathematicians and biologists have contemplated the self-organization of living-organism groups into flocks as an emergent process stemming from simple interaction rules at the individual level[@b2][@b3][@b4]. This idea has been supported by quantitative trajectory analysis in animal groups[@b5][@b6][@b7], together with a vast number of numerical and theoretical models[@b3][@b4], and more recently by the observations of flocking behaviour in ensembles of non-living motile particles such as shaken grains, active colloids, and mixtures of biofilaments and molecular motors[@b8][@b9][@b10][@b11][@b12]. From a physicist\'s perspective, these various systems are considered as different instances of polar active matter, which encompasses any ensemble of motile bodies endowed with local velocity--alignment interactions. The current paradigm for flocking physics is the following. Active particles are persistent random walkers, which when dilute form a homogeneous isotropic gas. Upon increasing density, collective motion emerges in the form of spatially localized swarms that may cruise in a sea of randomly moving particles; further increasing density, a homogeneous polar liquid forms and spontaneously flows along a well-defined direction[@b1][@b13][@b14]. This picture is the outcome of experiments, simulations and theories mostly performed in unbounded or periodic domains.
Beyond this picture, significant attention has been devoted over the last five years to confined active matter[@b3][@b12][@b15][@b16][@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24][@b25][@b26]. Confined active particles have consistently, yet not systematically, been reported to self-organize into vortex-like structures. However, unlike for our understanding of flocking, we are still lacking a unified picture to account for the emergence and structure of such vortex patterns. This situation is mostly due to the extreme diversity in the nature and symmetries of the interactions between the active particles that have been hitherto considered. Do active vortices exist only in finite-size systems as in the case of bacterial suspensions[@b17], which lose this beautiful order and display intermittent turbulent dynamics[@b27] when unconfined? What are the necessary interactions required to observe and/or engineer bona fide stationary swirling states of active matter?
In this paper, we answer these questions by considering the impact of geometrical boundaries on the collective behaviour of motile particles endowed with velocity--alignment interactions. Combining quantitative experiments on motile colloids, numerical simulations and analytical theory, we elucidate the phase behaviour of *polar* active matter restrained by geometrical boundaries. We use colloidal rollers, which, unlike most of the available biological self-propelled bodies, interact via well-established dynamical interactions[@b11]. We first exploit this unique model system to show that above a critical concentration populations of motile colloids undergo a non-equilibrium phase transition from an isotropic gaseous state to a novel ordered state where the entire population self-organizes into a single heterogeneous steadily rotating vortex. This self-organization is *not* the consequence of the finite system size. Rather, this emergent vortex is a genuine state of polar active matter lying on the verge of a macroscopic phase separation. This novel state is the only ordered phase found when unidirectional directed motion is hindered by convex isotropic boundaries. We then demonstrate theoretically that a competition between alignment, repulsive interactions and confinement is necessary to yield large-scale vortical motion in ensembles of motile particles interacting via alignment interactions, thereby extending the relevance of our findings to a broad class of active materials.
Results
=======
Experiments
-----------
The experimental setup is fully described in the *Methods* section and in [Fig. 1a,b](#f1){ref-type="fig"}. Briefly, we use colloidal rollers powered by the Quincke electrorotation mechanism as thoroughly explained in ref. [@b11]. An electric field **E**~**0**~ is applied to insulating colloidal beads immersed in a conducting fluid. Above a critical field amplitude *E*~Q~, the symmetry of the electric charge distribution at the bead surface is spontaneously broken. As a result, a net electric torque acts on the beads causing them to rotate at a constant rate around a random axis transverse to the electric field[@b28][@b29][@b30]. When the colloids sediment, or are electrophoretically driven, onto one of the two electrodes, rotation is converted into a net rolling motion along a random direction. Here, we use poly(methyl methacrylate) (PMMA) spheres of radius *a*=2.4 μm immersed in a hexadecane solution.
As sketched in [Fig. 1a](#f1){ref-type="fig"}, the colloids are handled and observed in a microfluidic device made of double-sided scotch tape and of two glass slides coated with an indium-tin-oxide layer. The ITO layers are used to apply a uniform DC field in the *z*-direction, with *E*~0~=1.6 V μm^−1^ (*E*~0~=1.1*E*~Q~). Importantly, the electric current is nonzero solely in a disc-shaped chamber at the centre of the main channel. As exemplified by the trajectories shown in [Fig. 1b](#f1){ref-type="fig"} and in [Supplementary Movie 1](#S1){ref-type="supplementary-material"}, Quincke rotation is hence restrained to this circular region in which the rollers are trapped. We henceforth characterize the collective dynamics of the roller population for increasing values of the colloid packing fraction *φ*~0~.
Individual self-propulsion
--------------------------
For area fractions smaller than , the ensemble of rollers uniformly explores the circular confinement as illustrated by the flat profile of the local packing fraction averaged along the azimuthal direction *φ*(*r*) in [Fig. 2a](#f2){ref-type="fig"}. The rollers undergo uncorrelated persistent random walks as demonstrated in [Fig. 2b,c](#f2){ref-type="fig"}. The probability distribution of the roller velocities is isotropic and sharply peaked on the typical speed *v*~0~=493±17 μm s^−1^. In addition, the velocity autocorrelation function decays exponentially at short time as expected from a simple model of self-propelled particles having a constant speed *v*~0~ and undergoing rotational diffusion with a rotational diffusivity *D*^−1^=0.31±0.02 s that hardly depends on the area fraction (see [Supplementary Note 1](#S1){ref-type="supplementary-material"}). These quantities correspond to a persistence length of that is about a decade smaller than the confinement radius *R*~c~ used in our experiments: 0.9 mm\<*R*~c~\<1.8 mm.
At long time, because of the collisions on the disc boundary, the velocity autocorrelation function sharply drops to 0 as seen in [Fig. 2c](#f2){ref-type="fig"}. Unlike swimming cells[@b26][@b31], self-propelled grains[@b8][@b22][@b23] or autophoretic colloids[@b32], dilute ensembles of rollers do not accumulate at the boundary. Instead, they bounce off the walls of this virtual box as shown in a close-up of a typical roller trajectory in [Fig. 2d](#f2){ref-type="fig"}, and in the [Supplementary Movie 1](#S1){ref-type="supplementary-material"}. As a result, the outer region of the circular chamber is depleted, and the local packing fraction vanishes as *r* goes to *R*~c~, [Fig. 2a](#f2){ref-type="fig"}. The repulsion from the edges of the circular hole in the microchannel stems from another electrohydrodynamic phenomenon[@b33]. When an electric field is applied, a toroidal flow sketched in [Fig. 1a](#f1){ref-type="fig"} is osmotically induced by the transport of the electric charges at the surface of the insulating adhesive films. Consequently, a net inward flow sets in at the vicinity of the bottom electrode. As the colloidal rollers are prone to reorient in the direction of the local fluid velocity[@b11], this vortical flow repels the rollers at a distance typically set by the channel height *H* while leaving unchanged the colloid trajectories in the centre of the disc. This electrokinetic flow will be thoroughly characterized elsewhere.
Collective motion in confinement
--------------------------------
As the area fraction is increased above , collective motion emerges spontaneously at the entire population level. When the electric field is applied, large groups of rollers akin to the band-shaped swarms reported in[@b11] form and collide. However, unlike what was observed in periodic geometries, the colloidal swarms are merely transient and ultimately self-organize into a single vortex pattern spanning the entire confining disc as shown in [Fig. 3a](#f3){ref-type="fig"} and [Supplementary Movie 2](#S1){ref-type="supplementary-material"}. Once formed, the vortex is very robust, rotates steadily and retains an axisymmetric shape. To go beyond this qualitative picture, we measured the local colloid velocity field **v**(**r**, *t*) and use it to define the polarization field **Π**(**r**, *t*)≡**v**/*v*~0~, which quantifies local orientational ordering. The spatial average of **Π** vanishes when a coherent vortex forms, therefore we use its projection along the azimuthal direction as a macroscopic order parameter to probe the transition from an isotropic gas to a polar-v
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1. Introduction {#sec1}
===============
Health care is changing dynamically in the 2010s. The economic recession and problems with recruiting professionals \[[@B1], [@B2]\], staff retention \[[@B3]\], creating healthy work environments \[[@B4], [@B5]\], and a growing demand for customer orientation \[[@B6]\] pose challenges for nurse managers\' work. More expertise in management is needed to respond to these issues.
One essential area of nurse manager\'s management skills is the use of different leadership styles \[[@B7]\]. Leadership styles can be seen as different combinations of tasks and transaction behaviours that influence people in achieving goals \[[@B8]\]. Earlier studies indicate that nurse manager\'s effective leadership style is affiliated to staff retention \[[@B5]\], work unit climate \[[@B4]\], nurses\' job satisfaction \[[@B9], [@B10]\], nurses\' commitment \[[@B11]\], and patient satisfaction \[[@B12]\].
Transformational leadership style \[[@B5], [@B6], [@B13], [@B14]\] and transactional leadership \[[@B7]\] help to respond to these issues. Transformational leadership refers to the leader\'s skills to influence others towards achieving goals by changing the followers\' beliefs, values, and needs \[[@B7]\]. Transactional leadership complements and enhances the effects of transformational leadership outcomes \[[@B15]\].
There are certain skills required from nurse managers so as to be able to use these effective leadership styles. The skills include the ability to create an organization culture that combines high-quality health care and patient/employee safety and highly developed collaborative and team-building skills \[[@B1]\]. Nurse managers also need to have the readiness to observe their own behaviour \[[@B16]\] and its effects on the work unit; as a result, employees can adjust to a better leadership style. These kinds of skills are related to manager\'s emotional intelligence (EI).
EI is an ability to lead ourselves and our relationships effectively \[[@B17]\]. It has been defined as the ability to observe one\'s own and others\' feelings and emotions, to discriminate among them and to use this information to direct one\'s thinking and actions \[[@B18]\]. EI is composed of personal competence and social competence. Self-awareness and self-management are reflections of personal competence, influencing the way the leader manages him/herself. Social awareness and relationship management reflect social competence, which affects how the leader manages relationships with others \[[@B17]\].
Nurse managers with that skill can easily form relationships with others, read employees\' feelings and responses accurately, and lead successfully \[[@B19]--[@B21]\]. Emotionally intelligent leaders\' behaviour also stimulates the creativity of their employees \[[@B22]\]. Goleman et al. \[[@B23]\] have identified visionary, coaching, affiliate, and democratic styles as resonant, and pacesetting and commanding styles as dissonant leadership styles. Most leaders use both resonant and dissonant leadership styles. The leadership styles of Goleman et al. are applied as the basis of this study because earlier studies refer to the significance of these styles, especially that of EI in manager\'s work. In addition, these leadership styles are one way of aiming to carry out transformational leadership. Especially visionary, coaching, affiliate, and democratic styles include elements that promote transformational leadership. Such elements are for example the leader being visionary and empowering staff \[[@B4]\].
This paper focuses on Finnish nurse managers\' leadership styles. The Finnish health care system is a strong institution where health care services are offered to all citizens and funded by taxes \[[@B24]\]. It has widely recognized that health care services in Finland are of high-quality Despite recent concerns about equity issues, Finns are in general very satisfied with their health care services. \[[@B25]\]. Consequently it is important to explore nurse managers\' leadership styles especially in this context.
2. Materials and Methods {#sec2}
========================
2.1. Aim of the Study {#sec2.1}
---------------------
The intention of this study was to explore nurses\' and supervisors\' perceptions of nurse leaders\' leadership styles. The research questions were as follows: what kind of leadership styles do nurse managers use and what are the factors affected by their leadership styles.
2.2. Participants {#sec2.2}
-----------------
To achieve the aim of this study data were collected through open interviews. The majority of Finnish nurse managers, nurses, and supervisors work in hospitals or long-term facilities. Selection of participants was performed in convenience sampling \[[@B26]\]. Participants were selected paying attention to the fact they were of different ages, working in different wards and units (e.g., psychiatry, internal diseases, gerontology) in either hospitals or long-term facilities, and had worked with more than one nurse manager.
The researcher contacted the participants and asked whether they were interested in taking part in the study. The participants were informed about the aim of the study. Participation was voluntary. Prior to the interviews each participant signed a form where they gave their consent to participate in the study. A total of 11 nurses and 10 supervisors, 20 women and one man, from eight Finnish hospitals and five long-term care facilities participated in the study. The age of the nurses varied between 30 and 53 and their experience in health care between 7 and 25 years. The age of the supervisors varied between 38 and 59 and their experience as supervisors between 5 and 21 years. Both nurses and supervisors had worked with many nurse leaders and they were interviewed about nurse managers in general. They thus had experience of different nurse managers on different wards and they were able to describe leadership styles from various aspects.
2.3. Data Collection and Analysis {#sec2.3}
---------------------------------
Semistructured interviews were used to gather data on the perceptions of nurse managers\' leadership styles and factors affected by leadership styles. Interviews were usually carried out in the office in the participants\' workplace. All interviews were recorded with individual consent. Participants were initially asked to describe their work and earlier study and work history. They were subsequently asked about their perception of leadership styles and asked to describe the leadership styles used by their nurse managers. After that they were asked about factors affected by leadership styles. Each interview was approached individually, guided by participants\' responses. The interview sessions lasted between 30 and 85 minutes. Every interview was transcribed word for word from the recordings. Interviewing was continued until saturation of the data was achieved \[[@B27]\].
Because nurses and supervisors might have differed in their perceptions of leadership styles, the data were first analysed separately in two separate groups, following the same process for each group. Content analysis was chosen because it is a research method for making valid inferences from data to the contexts of their use \[[@B28]\].
The interview texts were read through multiple times, based on the author\'s empirical and theoretical preunderstanding of the professional area of the participating nurses and nurse managers. A structured categorization matrix of leadership styles was developed based on the primal leadership model \[[@B23]\] and research of Vesterinen et al. \[[@B29]\]. When using a structured matrix of analysis, an item of the data that does not fit the categorization frame is used to create its own concept, based on the principles of inductive content analysis \[[@B30]\]. When both the data of nurses and superiors were analysed, the results were compared. The categories and subthemes were congruent and therefore the results are presented together, albeit paying attention to differences and similarities of the perceptions of nurses and superiors.
The data analysis of the factors affected by leadership styles was inductive. All the data of nurses and supervisors were analysed together. This process included open coding, creating categories, and abstraction. A classification framework of the factors was formed inductively by defining categories and sub-themes. The criteria for allocating a unit to a category were formed by asking questions if the unit was suitable to the category. The sub-themes were named using descriptive concepts and classified as "belonging" to a particular category. After that, the categories were given names \[[@B31]\].
2.4. Trustworthiness {#sec2.4}
--------------------
The trustworthiness of this study has been ensured by confirming truth value, consistency, neutrality, and transferability of this study \[[@B32]\].
When considering this study from the viewpoint of trustworthiness, there are some threats that should be taken into consideration. The researcher collected the data and performed the analysis alone and the interpretation could have been affected by her professional history \[[@B33]\]. With interviews there is a risk that respondents try to please the interviewer by reporting things they assume s/he wants to hear. The researcher confirmed the truth value of the study by selecting participants in convenience sampling. The respondents\' age distribution was wide and they worked in different units. Their perspectives and descriptions were broad and gave a diverse picture.
The truth value of this study was also confirmed by analysing data as they emerged based on the interviews. To ensure the trustworthiness of the study quotes from interviews are included in the results.
In view of consistency, the research process is described so that it can be repeated if necessary. This gives a possibility to understand the limitations of the process of data collection and analysis. To ensure neutrality in this study, interpretations were based on original data. This is confirmed by citations from the interview data.
In this study the sample was small, consisting of Finnish nurses and supervisors, and the results only reflect their perceptions of leadership styles. As a result, transferability of results is limited. However, when considering the main objective in this study, it was not transferability of research results, but it was to enhance understanding of leadership styles and use it for future studies.
2.5. Ethical Considerations {#sec2.5}
---------------------------
The data for this study were collected following approval from the administrations of the organizations. All participants were informed of the
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1. Introduction {#sec1}
===============
Soil organic C (SOC) and total N (TN) are very important C and N pools in the terrestrial ecosystems \[[@B1], [@B2]\]. As the components of labile C and N pools in soils, dissolved organic C (DOC) and N (DON) and soil ammonium and nitrate N (NH~4~ ^+^-N and NO~3~ ^−^-N) play crucial roles in the biogeochemistry of C and N and in the nutrient transformation \[[@B3]--[@B5]\]. With the context of climatic warming, how SOC, TN, DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N respond is vital to global C and N cycling \[[@B1], [@B2]\]. However, inconsistent results on the responses of these C and N pools to climatic warming have been observed with respect to vegetation types and initial soil characteristics \[[@B2], [@B3], [@B6]--[@B14]\]. For example, He et al. \[[@B2]\] demonstrated that six-year warming (\~1.4°C increase of 10 cm soil temperature) significantly decreased soil C by 129.3 g m^−2^ in a temperate steppe of Inner Mongolia. In contrast, Li et al. \[[@B7]\] found that two-year warming significantly increased SOC in an alpine meadow (\~2.1°C increase of air temperature) but significantly reduced TN in an alpine swamp meadow (\~2.3°C increase of air temperature) on the Tibetan Plateau. Hagedorn et al. \[[@B13]\] indicated that one-growing-season warming (\~4°C increase of 5 cm soil temperature) did not significantly influence DOC. Song et al. \[[@B1]\] pointed out that six-year warming (\~1.2°C increase of 10 cm soil temperature) significantly reduced DOC in a temperate steppe in Inner Mongolia. Biasi et al. \[[@B15]\] indicated that two-year warming (\~0.9°C increase of 5 cm soil temperature) did not have obvious effects on DON, NH~4~ ^+^-N, NO~3~ ^−^-N, and N~min~ in a lichen-rich dwarf shrub tundra in Siberia. Bai et al. \[[@B14]\] stated that experimental warming (\~0.6--6.7°C in soil temperature) had a significant positive effect on N~min~ but not on TN across all biomes. Therefore, how climatic warming acts on C and N cycling still remains unclear.
More than 70% of the Tibetan Plateau is covered with grasslands \[[@B16]\]. The alpine grasslands of this Plateau are one of the systems most sensitive to global change \[[@B17], [@B18]\]. In alpine grasslands, understanding the responses of SOC, DOC, TN, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N to climatic warming are crucial for predicting future changes in soil fertility and C sequestration. The alpine meadow is one of the most typical grasslands types on the Tibetan Plateau being subjected to climatic warming \[[@B19]\]. Information on how these C and N pools along an elevation gradient respond to climatic warming is scarce on the Tibetan Plateau. Here we set up a warming experiment in an alpine meadow at three elevations (i.e., 4313 m, 4513 m, and 4693 m) on the Northern Tibetan Plateau.
The main objective was to investigate the effects of short-term experimental warming on SOC, TN, DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N. Our previous study indicated that short-term experimental warming could not affect soil microbial biomass \[[@B20]\] and soil microbial activity regulated the balances of soil C and N pools in the alpine meadow \[[@B21]\]. We hypothesized that experimental warming may not affect these C and N pools in this study.
2. Materials and Methods {#sec2}
========================
2.1. Study Area, Experimental Design, and Soil Sampling {#sec2.1}
-------------------------------------------------------
A detailed description of the study area, the warming experimental design, the measurements of microclimate factors (including soil temperature and soil moisture), and the soil sampling are given in Fu et al. \[[@B20], [@B22]\].
Briefly, three alpine meadow sites were established at three elevations (i.e., a low (30°30′N, 91°04′E, and 4313 m), mid- (30°31′N, 91°04′E, and 4513 m), and high (30°32′N, 91°03′E, and 4693 m) elevation) at Damxung Grassland Observation Station of Tibet Autonomous Region in China in May 2010.
Annual mean air temperature and precipitation is 1.3°C and \~476.8 mm, respectively \[[@B20], [@B21]\]. The vegetation is*Kobresia*-dominated alpine meadow and roots are mainly concentrated in the topsoil layer (0--20 cm) \[[@B21], [@B22]\]. The soil is classified as sandy loam, with pH of 6.0--6.7, organic matter of 0.3--11.2%, and total N of 0.03--0.49% \[[@B20], [@B22]\].
Open top chambers (OTCs, 3 mm thick polycarbonate) were used to enhance temperature \[[@B22], [@B23]\]. The bottom and top diameters and the height of OTCs were 1.45 m and 1.00 m and 0.40 m, respectively \[[@B20], [@B22]\]. For each site, four OTCs and their paired control plots (1 m × 1 m) were randomly established in May 2010. There was \~3 m distance between plots.
Daily mean soil temperature (*T* ~*s*~) during the study period of July-September in 2011 inside the OTCs increased by 1.26°C, 0.98°C, and 1.37°C at the low, mid-, and high elevation, respectively, compared to control plots \[[@B20]\]. In contrast, experimental warming decreased daily mean soil moisture (SM) by 0.04 m^3^ m^−3^ in all sites \[[@B20]\]. Daily mean *T* ~*s*~ decreased with increasing elevation from the low to high elevation \[[@B20]\].
We collected topsoil samples (0--20 cm depth) inside each plot using a probe 3.0 cm in diameter on July 7, August 9, and September 10, 2011 \[[@B20]\]. Five soil subsamples were randomly sampled and composited into one soil sample for each plot \[[@B20]\]. Subsamples of the fresh soil were used to measure DOC, DON, NH~4~ ^+^-N, and NO~3~ ^−^-N and other subsamples of the fresh soil were air-dried for the measurements of SOC and TN.
2.2. Soil Analysis {#sec2.2}
------------------
A more detailed description of measurements of soil inorganic N (N~min~, i.e., sum of NH~4~ ^+^-N and NO~3~ ^−^-N), DON, and DOC can be found in Fu et al. \[[@B21]\]. Briefly, soil inorganic N in 20 g fresh soil sample was extracted with 100 mL K~2~SO~4~, filtered through 0.45 *μ*m membrane, and analyzed on a LACHAT Quikchem Automated Ion Analyzer. Dissolved organic C and TN (DTN) in another 20 g fresh soil sample was extracted with 100 mL ultrapure water and filtered through 0.45 membrane. The extractable SOC and TN concentrations in the ultrapure water extracts were measured using a Liqui TOC II elementar analyzer (Elementar Liqui TOC, Elementar Co., Hanau, Germany) and a UV-1700 PharmaSpec visible spectrophotometer (220 nm and 275 nm), respectively. We also analyzed dissolved inorganic N (DIN) in the ultrapure water extracts on a LACHAT Quikchem Automated Ion Analyzer. Then DON was calculated as the difference between DTN and DIN. The potassium dichromate method was used to determine SOC \[[@B24]\]. Soil TN was measured on a CN analyzer (Elementar Variomax CN). Soil microbial biomass (MBC) and N (MBN) data were obtained from Fu et al. \[[@B20]\].
2.3. Statistical Analysis {#sec2.3}
-------------------------
In order to examine the elevation effect, repeated-measures ANOVA with experimental warming and elevation as the between subject factors and with sampling date as the within subject factor was performed for a specific soil property (i.e., SOC, TN, DOC, DON, ratio of DOC to DON (DOC/DON), NH~4~ ^+^-N, NO~3~ ^−^-N, ratio of NH~4~ ^+^-N to NO~3~ ^−^-N(NH~4~ ^+^-N/NO~3~ ^−^-N), and N~min~). At each site, repeated-measures ANOVA with experimental warming (i.e., OTCs versus control) as the between subject factor and with sampling date as the within subject factor was conducted for each soil property. Single factor linear reg
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The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.
Introduction {#s1}
============
Mechanical ventilation (MV) has been used in critical care patients for decades. In spite of its life-saving potential, it has several shortcomings. A number of experimental studies have shown that mechanical ventilation may result in the appearance of inflammatory mediators in the lung [@pone.0114247-Uhlig1] and subsequently in oedema. [@pone.0114247-Dreyfuss1] Ventilator-Induced Lung Injury (VILI) causes macro and microscopic unspecific changes[@pone.0114247-Katzenstein1] similar to those found in patients with Acute Respiratory Distress Syndrome (ARDS). As it happens with ARDS, VILI is basically the result of important changes in the permeability of the alveolar-capillary membrane. [@pone.0114247-Dreyfuss2] The potential of mechanical ventilation for triggering or worsening pulmonary damages has been shown in animal models where the application of non-physiological ventilatory parameters (mostly very high tidal volumes) aggravated the condition of animals with a previously injured lung [@pone.0114247-Corbridge1], and even caused an injury in those without a previous pulmonary pathology. [@pone.0114247-Dreyfuss1] The use of low tidal volumes has proved to be a better approach in ARDS patients, survival being improved in strategies based on its usage. [@pone.0114247-Amato1]--[@pone.0114247-Villar1] Interestingly, recent experimental and clinical work has demonstrated that MV with low tidal volume can induce similar pulmonary changes to those noticed for VILI [@pone.0114247-Cobelens1]--[@pone.0114247-Wolthuis1] and that its appearance may be related to MV exposure time. [@pone.0114247-Hegeman1]
Aquaporins are a family of small transmembrane proteins that help water to move fast, selectively and bi-directionally through lipid bi-layers. [@pone.0114247-Kozono1], [@pone.0114247-Ma1] 13 different types have been identified in mammals, [@pone.0114247-Verkman1] from which the lung is known to express four: AQP-1, in the pulmonary capillary endothelium (especially alveolar), and the visceral pleura; AQP-3, in the tracheal epithelium; AQP-4, in the tracheal and bronchial epithelium; and AQP-5, on type I pneumocyte cells of the alveoli, on the membrane adjoining to the alveolar lumen. [@pone.0114247-King1] Their role in the development and resolution of pulmonary oedema gives rise to controversy, although it does seem to play a part in VILI. [@pone.0114247-Hales1]
This research aimed to verify if MV with low or moderately high tidal volumes (10 ml/Kg) sustained over time results in lung injury, subsequently altering pulmonary water content and microvascular permeability, as observed in VILI, and to objectivize what happens with AQP 1 and 5 expression, both types mainly involved in the formation of lung oedema, under the same ventilation conditions.
Material and Methods {#s2}
====================
1. Ethics statement {#s2a}
-------------------
The project was carried out after approval from the Ethics Committee for Animal Experimentation and Wellbeing of the Research Foundation of Valencia\'s Hospital Clínico Universitario.
2. Animal model and monitoring {#s2b}
------------------------------
A total of 30 rats were anaesthetised by intraperitoneal injection of ketamine 80 mg/kg and xylazine 5 mg/kg. 5 rats (group C or controls) were sacrificed by intravenous injection of 100 mg/Kg thiopental. The rest of the animals (n = 25) were performed a surgical tracheostomy, using a teflon cannula (Surflo, 16G). Rats were randomly allocated into two groups. 12 rats were ventilated for 2 hours (group 2H) with a Harvard Rodent Ventilator, model 683 (Harvard Apparatus) with a tidal volume of 10 ml/kg and a respiratory rate of 90 breaths/minute. 13 rats were ventilated with exactly the same parameters for 4 hours (group 4H). The cervical vascular bundle was dissected, and the right internal jugular vein and the right carotid artery were catheterized to continuously monitor heart rate (HR) and mean arterial pressure (MAP). Peak inspiratory pressure and respiratory system compliance were continuously recorded.
Anaesthesia was maintained by continuous intravenous infusion of ketamine and cisatracurium using dosis of 100 mcg/Kg/min and 2--3 mcg/Kg/min, respectively (20 ml of ketamine 5%, 10 ml of cisatracurium 0,2% and 20 ml of saline solution 0,9% at a rate of approximately 0,1 ml/h in the internal jugular vein). Anesthesia was supplemented, in cases in which it was necessary, by administration of an intravenous bolus of 0.1 ml of the mixture. Gasometric samples were taken in all animals in groups 2H and 4H at the beginning of MV and 30 minutes before the end of MV.
Rats were sacrificed by intravenous injection of sodium thiopental. The left lung was used for the determination of lung water content. The right lung of 6 rats from groups 2H and 4H was used for determining AQP 1 and AQP 5 expression. Lungs were either frozen in liquid nitrogen or paraffin-embedded for immunohistochemical sectioning and marking. 2 rats in group C and 4 rats from groups 2H and 4H were used to establish pulmonary macrovascular permeability.
3. Measuring lung oedema {#s2c}
------------------------
Lungs were dried with filter paper and placed on a Petri dish with known weight to obtain lung wet weight (LWW). They were then placed in a drying chamber at 80°C for 96 hours and their dry weight (LDW) was determined.
Two indicators of the amount of oedema were obtained: Lung WW/DW ratio and the proportion of pulmonary water, expressed in percentages (%~water~). The latter parameter was estimated using this formula: % ~water~ = (LWW - LDW)/LWW \* 100
4. Measuring microvascular permeability {#s2d}
---------------------------------------
Microvascular permeability was quantified using Evans Blue Dye. 0.5 ml Evans Blue was injected intravenously (30 mg/Kg) 30 minutes before sacrificing the animal. Rats were sacrificed by exsanguination from the carotid artery, but saline was simultaneously infused via the jugular vein in the same amount as that of the blood extracted.
After death, the right lung was separated and immersed in formamide (5 ml) and homogenised for 2 min. The resulting suspension was incubated at 37°C/18 h and then centrifuged at 5000xg/30 minutes, and the supernatant was measured. Concentration of Evans Blue in the supernatant was spectrophotometrically determined.
5. Study of aquaporin expression {#s2e}
--------------------------------
### 5.1 Western blot {#s2e1}
Proteins were extracted from previously frozen lungs. A Compartmental Protein Extraction Kit (Chemicon International, Temecula CA) was used. 200--400 mg tissue was homogenised in cold buffer C (1 ml/g tissue) and Ultra Turrax (KA, Staufen, Germany). Two protein fractions were obtained for each sample: cytoplasm and membrane, and they were quantified. Proteins in each fraction (100 mg) were separately run on a Tris-HCl/SDS gel, 8% acrylamide, and they were transferred onto a nitrocellulose membrane (Hybond-ECL, Amersham). After washing the membrane with distilled water, it was blocked with a PBS/Tween solution, 0.2%, with 5% skimmed milk. It was then incubated with the primary antibody during 2 hours at room temperature. The antibodies used were Anti-Rat AQP1 (Alpha Diagnostic, San Antonio, TX) and Anti-Rat AQP5 (Alpha Diagnostic, San Antonio, TX), both with a 2 mg/ml concentration. After several washes with PBS/Tween 0.2%, it was incubated with the secondary antibody Anti-Rabbit IgG (DAKO, Glostrup, Denmark) in 1∶2000 dilution. β-actin expression was detected as an internal control, and relative protein content was analysed using the enhanced chemoluminiscence method.
### 5.2 Real-time polymerase chain reaction with reverse transcriptase {#s2e2}
Lungs were cut with a microtome, three sections being obtained for each sample for total RNA extraction with TRIZOL (Reagent InvitrogenTM Life Technologies) as in the phenol extraction method described by Chomczynsky. [@pone.0114247-Chomczynski1] Microsections were added 1 ml TRIZOL and homogenized (Polytron PT 1200, Kinematica AG) and centrifuged at 10.000 rpm/10 min at 4°C. The supernatant was removed and RNA was precipitated by adding 0.1 volumes of sodium acetate 3 M, 2.5 volumes cold ethanol and 0.5 µl glycogen (20 mg/ml). RNA was centrifuged again, air-dried and resuspended in 20 µl Tris/EDTA buffer. RNA was reversely transcribed to cDNA with Superscript II (Invitrogen), by incubation
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Introduction {#s1}
============
Usher syndrome (USH) is an autosomal recessive disorder characterized by hearing loss (HL), retinitis pigmentosa (RP) and vestibular dysfunction. Three clinical subtypes can be distinguished. USH type 1 (USH1) is the most severe among them because of profound HL, absent vestibular responses, and prepubertal onset RP. USH type 2 (USH2) is characterized by congenital moderate to severe HL, with a high-frequency sloping configuration. The vestibular function is normal and onset of RP is in the first or second decade. The onset of the visual symptoms such as night blindness in USH usually occurs several years later than in USH1. USH type 3 (USH3) is characterized by variable onset of progressive HL, variable onset of RP, and variable impairment of vestibular function (normal to absent) [@pone.0090688-Kimberling1], [@pone.0090688-Yan1].
To date, nine genetic loci for USH1(*USH1B-H*, *J*, and *K*) have been mapped to chromosomes 11q13.5, 11p15.1, 10q22.1, 21q21, 10q21-q22, 17q24-q25, 15q22-q23 (*USH1H* and *J*), and 10p11.21--q21.1 [@pone.0090688-Yan1], [@pone.0090688-Jaworek1], [@pone.0090688-Riazuddin1]. Six of the corresponding genes have been identified: the actin-based motor protein myosin VIIa (*MYO7A*, USH1B) [@pone.0090688-Weil1]; two cadherin-related proteins, cadherin 23 (*CDH23*, USH1D) [@pone.0090688-Bork1] and protocadherin 15 (*PCDH15*, USH1F) [@pone.0090688-Ahmed1]; and two scaffold proteins, harmonin (*USH1C*) [@pone.0090688-Verpy1] and sans (*USH1G*) [@pone.0090688-Mustapha1]; the Ca^2+^- and integrin-binding protein (*CIB2*, USH1J) [@pone.0090688-Riazuddin1]. In Caucasian USH1 patients, previous studies showed that mutations in *MYO7A*, *USH1C*, *CDH23*, *PCDH15*, and *USH1G*, were found in 39--55%, 7--14%, 7--35%, 7--11%, and 0--7%, respectively (the frequency of *CIB2* is still unknown) [@pone.0090688-Ouyang1], [@pone.0090688-Bonnet1], [@pone.0090688-LeQuesneStabej1]. In Japanese, Nakanishi et al. showed that *MYO7A* and *CDH23* mutations are present in USH1 patients [@pone.0090688-Nakanishi1], however, the frequency is not yet known. In addition, mutations in three corresponding genes (usherin *USH2A* [@pone.0090688-Eudy1], G protein-coupled receptor 98; *GPR98* [@pone.0090688-Weston1], and deafness, autosomal recessive 31; *DFNB31* [@pone.0090688-Aller1]) have been reported so far in USH2, and USH3 is caused by mutations in the clarin 1 (*CLRN1*) [@pone.0090688-Joensuu1] gene.
Comprehensive molecular diagnosis of USH has been hampered both by genetic heterogeneity and the large number of exons for most of the USH genes. The six USH1 genes collectively contain 180 coding exons [@pone.0090688-Riazuddin1], [@pone.0090688-Mustapha1], [@pone.0090688-Ouyang1] the three USH2 genes comprise 175 coding exons [@pone.0090688-Weston1], [@pone.0090688-Aller1], [@pone.0090688-Nakanishi2], and the USH3 gene has five coding exons [@pone.0090688-Joensuu1]. In addition some of these genes are alternatively spliced ([@pone.0090688-Riazuddin1], [@pone.0090688-Ahmed1], [@pone.0090688-Verpy1], [@pone.0090688-Aller1], [@pone.0090688-Joensuu1] and NCBI database: <http://www.ncbi.nlm.nih.gov/nuccore/>). Thus far, large-scale mutation screening has been performed using direct sequence analysis, but that is both time-consuming and expensive. We thought that targeted exon sequencing of selected genes using the Massively Parallel DNA Sequencing (MPS) technology would enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis.
Therefore, in this study, we have conducted genetic analysis using MPS-based genetic screening to find mutations in nine causative USH genes (except *CIB2*) in Japanese USH1 patients.
Results {#s2}
=======
Mutation analysis of the nine USH genes in 17 unrelated USH1 patients revealed 19 different probable pathogenic variants, of which 14 were novel ([Table 1](#pone-0090688-t001){ref-type="table"}).
10.1371/journal.pone.0090688.t001
###### Possible pathogenic variants found in this study.
{#pone-0090688-t001-1}
Gene Mutation type Nucleotide change Amino acid change exon/intron number Domain control (in 384 alleles) SIFT Score PolyPhen Score Reference
---------- --------------- ------------------- ------------------- -------------------- -------------- -------------------------- ------------ ---------------- --------------------------------
*MYO7A* Frameshift c.1623dup p.Lys542GlnfsX5 Exon 14 \- N/A \- \- Le Quesne Stabej et al. (2012)
c.4482_4483insTG p.Trp1495CysfsX55 Exon 34 \- N/A \- \- This study
c.6205_6206delAT p.Ile2069ProfsX6 Exon 45 \- N/A \- \- This study
Nonsense c.1477C\>T p.Gln493X Exon 13 \- N/A \- \- This study
c.1708C\>T p.Arg570X Exon 15 \- N/A \- \- This study
c.2115C\>A p.Cys705X Exon 18 \- N/A \- \- This study
c.6321G\>A p.Trp2107X Exon 46 \- N/A \- \- This study
Missense c.2074G\>A p.Val692Met Exon 17 Motor domain 0 0.09 0.982 This study
c.2311G\>T p.Ala771Ser Exon 20 IQ 2 0.0026 0.01 0.825 Nakanishi et al. (2010)
c.6028G\>A p.Asp2010Asn Exon 44 FERM 2 0 0 0.925 Jacobson et al. (2009)
*CDH23* Frameshift c.3567delG p.Arg1189ArgfsX5 Exon 30 \- N/A \- \- This study
c.5780_5781delCT p.Ser1927Cysfs16 Exon 44 \- N/A \- \- This study
Splicing c.5821-2A\>G ? Intron 44 \- N/A \- \- This study
Nonsense c.6319C\>T p.Arg2107X Exon 48 \- N/A \- \- Nakanishi et al. (2010)
*PCDH15* Splicing c.158-1G\>A ? Intron 3 \- N/A \- \- This study
Nonsense c.1006C\>T p.Arg336X Exon 10 \- N/A \- \- This study
c.2971C\>T p.Arg991X Exon 22 \- N/A \- \- Roux et al. (2006)
c.3337G\>T p.Glu1113X Exon 25 \- N/A \- \- This study
Missense c.3724G\>A p.Val1242Met Exon 28 Cadherin 11 0
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1. Introduction {#sec1-polymers-08-00159}
===============
Phenol-formaldehyde (PF) is a high-performance resin that is synthesized by the copolymerization of phenol with formaldehyde. It is widely applied for industrial uses, including adhesives, impregnating resins, and plastics. The excellent properties of PF resin include high mechanical, thermal, and weather stability \[[@B1-polymers-08-00159]\]. However, the lower curing rate and required higher curing temperature compared to other thermosetting adhesives limit the application of PF resins for use in impregnating resins or adhesives \[[@B2-polymers-08-00159],[@B3-polymers-08-00159]\]. Many attempts have been made to accelerate the curing rate or lower the curing temperature, including testing of various catalysts or additives to alter the reaction kinetics, such as carboxylic acid esters \[[@B4-polymers-08-00159],[@B5-polymers-08-00159]\], anhydrides \[[@B6-polymers-08-00159]\], amides \[[@B7-polymers-08-00159]\], carbonate \[[@B8-polymers-08-00159]\], and metallic ions \[[@B9-polymers-08-00159]\]. Additionally, the effects of the condensation condition on the PF resin structure and properties have been well studied by conventional analytical techniques. For example, various mechanisms of PF resin hardening accelerated by catalysts or additives have been reported \[[@B10-polymers-08-00159]\]. Some additives, such as sodium carbonate, act solely to accelerate the curing reaction, but other additives, such as propylene carbonate, both accelerate the reaction and also increase the average functionality of the PF reaction system to allow a tighter final network \[[@B10-polymers-08-00159]\]. The properties of basic catalysts, such as the valence and ionic radius of hydrated cations, affected the mechanisms and kinetics of PF resin condensation and, thus, the composition of the final products \[[@B9-polymers-08-00159]\]. Some studies also reported that an alkaline catalyst promoted the formation of dimethylene ethers in the polymerization reaction, and that *ortho* to *ortho* (*o*,*o*′) ethers were more stable \[[@B11-polymers-08-00159]\]. However, there has been no comprehensive study about the action of catalysts to increase or decrease the ratio of *ortho*/*para* reaction position or analysis of the corresponding physicochemical properties of the accelerated PF resins.
The aromatic ring of phenol has *ortho* and *para* positions capable of reaction with formaldehyde under certain conditions, but the *para* position has higher reactivity than the *ortho* position. The presence of two *ortho* positions and one *para* position in an aromatic ring generally could lead to a PF resin containing mostly *ortho* hydroxymethyl groups \[[@B2-polymers-08-00159]\]. However, in the process of PF resin synthesis, some catalysts could make more formaldehyde or methylol toward phenol *ortho* positions to increase the ratio of *ortho*/*para* substituted positions \[[@B12-polymers-08-00159]\], leading to more reactive functional groups or more unreacted *para* positions at the curing stage, which may shorten the curing time and increase the cross-linking degree of cured PF resin.
Since metal ions can accelerate the curing of PF resins, we tested the ability of barium hydroxide (Ba(OH)~2~), sodium carbonate (Na~2~CO~3~), lithium hydroxide (LiOH), and zinc acetate ((CH~3~COO)~2~Zn) to decrease curing temperature and accelerate the curing rate of PF resins. To elucidate the chemical structure of the cure-accelerated PF resins, we performed quantitative liquid ^13^C NMR to analyze the structural features. Finally, possible synthesis mechanism of metal-mediated polymerization of PF resins was proposed based on the chemical structure analysis and thermogravimetric (DTG) curve.
2. Materials and Methods {#sec2-polymers-08-00159}
========================
2.1. Materials {#sec2dot1-polymers-08-00159}
--------------
Phenol, formaldehyde (37%), Ba(OH)~2~, Na~2~CO~3~, LiOH, and (CH~3~COO)~2~Zn were obtained from Zhong'an Chemical Industries, Beijing, China and were used directly without further purification, and all other chemicals were AR grade and obtained from Beijing Chemical Industries, Beijing, China.
2.2. Preparation of PF Resins {#sec2dot2-polymers-08-00159}
-----------------------------
The catalyst-accelerated PF resin was synthesized by batch polymerization with phenol and formaldehyde at a molar ratio of 1:2.2, and the additive amount of catalyst was 6% based on the total mass of PF resin. In the first step, phenol was mixed in a flask with one third of the formaldehyde and one third of the catalyst. The mixture was quickly heated to 70 °C, and then the heater was turned off. The temperature of the mixture increased to 90 °C due to the heat produced by polymerization reaction and remained at 93--95 °C for 1 h. In the second step, the remaining formaldehyde and two thirds of the catalyst were added to the flask and the mixture was heated to 90 °C and kept at that temperature for 0.5 h. Finally, the mixture was cooled to 40 °C to yield PF resin. PF resins with different catalysts were synthesized with the same procedure.
2.3. Preparation of Plywood {#sec2dot3-polymers-08-00159}
---------------------------
Three-layer plywood (400 mm × 400 mm × 4.8 mm) was prepared with a single poplar veneer in the middle and two poplar veneers on the top and bottom simulating actual industrial parameters. The middle poplar veneer was coated with 125--150 g/m^2^ resin on each side. Four pieces of three-layer plywood for each catalyst-accelerated resin (including control) were hot-pressed under 1.2 MPa at 100, 110, 120, and 130 °C, respectively. The hot-press time was 7 min, including the first one minute and the last one minute to load and unload the pressure, respectively.
2.4. Characterization of PF Resins {#sec2dot4-polymers-08-00159}
----------------------------------
The solid (non-volatile) content of resol resin was determined in accordance with ASTM standard D4426-01. The viscosity of resin was measured using a Brookfield DV-II viscometer (AMETEK-BROOKFIELD Corporation, Middleboro, MA, USA) using 61\# rotor with spinning rate of 100 rpm. Gel time was defined as the time period from the immersion of the test tube into the oil bath (135 °C) to the beginning of the resin gelation (resin forming a string when a glass rod was lifted from the resin).
2.5. Characterization of the Plywood {#sec2dot5-polymers-08-00159}
------------------------------------
The shear strength was measured as per ASTM D906-98.
2.6. FT-IR Analysis of PF Resins {#sec2dot6-polymers-08-00159}
--------------------------------
The resins were placed in 0.01 MPa vacuum at 60 °C for 4 h to dry to non-volatility. FT-IR spectra of vacuum-dried PF resins were performed in a Nicolet IS10 instrument (Thermo Fisher Scientific Corporation, Waltham, MA, USA). Each spectrum was recorded with 32 scans in a frequency range of 600--4000 cm^−1^ at a spectral resolution of 4 cm^−1^.
2.7. Contact Angle Measurement {#sec2dot7-polymers-08-00159}
------------------------------
The contact angle measurements of the PF resins were performed on the tangential surfaces of wood samples with an optical contact angle apparatus (OCA 20 DataPhysics Instruments GmnH, Filderstadt, Germany). Sessile droplets (3 μL, measured with a microsyringe) of liquid resin were placed on the wood surface. The right and left angles of the drops on the surface were collected at intervals of 0.1 s for a total duration of 60 s, and the average angle was calculated.
2.8. Quantitative Liquid ^13^C NMR Measurement {#sec2dot8-polymers-08-00159}
----------------------------------------------
All of the resins were characterized by quantitative ^13^C NMR spectroscopy with a VARIAN INOUR-300 (JEOL Corporation, Tokyo, Japan) spectrometer with a frequency of 75.51 MHz using the inverse-gated decoupling method. All of the spectra were recorded at room temperature with a delay time of 8 s, a 13 h acquisition time and a 15.4 μs pulse width (90°). About 8000 scans were accumulated to obtain spectra for each spectrum. The chemical shifts of each spectrum were accurate to 0.1 ppm and all the resin samples were directly used for ^13^C NMR measurement.
2.9. Thermogravimetric Analysis (TG) of Resins {#sec2dot9-polymers-08-00159}
----------------------------------------------
Samples were dried at 120 °C for 2 h to evaporate the moisture and then TG was performed in a nitrogen atmosphere within a temperature range from room temperature to 700 °C, with a heating rate of 10 °C/min.
3. Results and Discussion {#sec3-polymers-08-00159}
=========================
3.1. Performance of the Catalyst-Accelerated PF Resin {#sec3dot1-
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Introduction {#Sec1}
============
Headache is the commonest neurological disorder in the community with variable intensity, ranging from a trivial nuisance to a severe, disabling, acute or chronic disorder, and may impose a substantial burden on sufferers and on society \[[@CR1], [@CR2]\]. It is one of the commonest reasons for visiting the neurology clinics worldwide \[[@CR3]--[@CR5]\], exerting significant burden on its sufferers and impairing daily function especially when accompanied by other symptoms, hence adversely affecting quality of life \[[@CR6]\]. According to the World Health Organisation (WHO), 1.7 -- 4% of the adult population of the world have headaches on 15 or more days every month \[[@CR7]\] and a lifetime prevalence of more than 90% has been attributed to headache disorders in most populations of the world \[[@CR8]\].
It is known that Africans have a higher threshold for pain and may not present to the clinic just for an 'ordinary headache' \[[@CR9]\]. Local experiences show that patients suffering from other chronic neurological disorders present very late to doctors and sometimes never do so \[[@CR9]\]. Chronic headaches produce individual and societal burdens, the former referring to its effect on family, social and recreational activities and the latter referring to effects on healthcare cost (direct costs) and work and function (indirect costs), including absenteeism and reduced effectiveness \[[@CR10]\].
There is limited data for headache prevalence in Africa. In 2004, the 1-year prevalence of headache from a door-to-door survey of rural south Tanzania was 23.1% (18.8% males and 26.4% females) \[[@CR11]\]. Getahu and colleagues in Ethiopia found a 1-year prevalence rate of 73.2% \[[@CR12]\]. A 1992 study from Ibadan, South West Nigeria, found the crude life-time prevalence for at least one episode of headache to be 51% \[[@CR13]\].
In Nigeria, there is a paucity of data on the national prevalence and burden of chronic headaches \[[@CR14]\] despite the fact that it is the commonest presenting neurological disorder in the authors' environment \[[@CR1], [@CR3]\], and therefore the possibility that a big headache problem exists in Nigeria. There are also no known studies of the prevalence and characterization of headache among Nigerian healthcare workers or healthcare workers in South East Nigeria hence the relevance of this study.
Aim of the study {#Sec2}
----------------
The aim of this preliminary study was to determine the frequency and pattern of headaches among a population of healthcare workers in a tertiary health institution located in South East Nigeria.
Methods {#Sec3}
=======
This was an epidemiological sampling-based study (Figure [1](#Fig1){ref-type="fig"}) using a semi-structured questionnaire. The questionnaire was pre-tested in another health facility at Nsukka (a local government area similar to the study area) for content validity. English language was used to reduce cross- cultural misinterpretations and wrong understanding of terms.Figure 1**Flow chart of research activities.**
The questionnaire was self- administered to all various cadres of health workers in medical unit of University of Nigeria Teaching Hospital, a tertiary health institution located in Enugu, South- East Nigeria, over a 3- month period from September -- November 2013, selected by simple random method out of the various units in the hospital e.g. surgical, medical, laboratory, physiotherapy, nutrition, administrative, laundry, transport, security, and medical record. Within these are various cadres of hospital staff: physicians, nurses, pharmacists and cleaners. Out of a total of 141 only 133 gave consent and hence were studied, giving a response rate of 94.3%. To ascertain the overall prevalence of headache, subjects were asked if they have ever had a headache within the previous six months and to note any association. They were to rate the severity of headache based on a scale of mild, moderate and severe. The impact of these severe headaches on the daily activity and the number of days they occur in a month were recorded. The character of the pain, location, duration, and the total numbers of times in the 6 months preceding the date of administering the questionnaire were also noted.
Statistical Package for Social Sciences version 16 was used in statistical analysis. Comparison of multiplex groups was carried out with One Way ANOVA test. On the other hand comparison of two distinct groups was carried out with student t test. Chi-square test (and/or Fisher's exact test) was used in analysis of categorical variables. The results were revealed as mean ± SD. P value \<0.05 was interpreted as statistically meaningful. Ethical approval was obtained from the hospital ethics committee.
Results {#Sec4}
=======
Of the 2,450 hospital employees (450 medical doctors, 630 nurses, 50 pharmacists, and 1320 laboratory and administrative staff), 141 were selected using simple random method from the employment register and eventually only 133 health workers (71 males and 62 females) gave informed consent and were studied (response rate 94.3%). More of the respondents were males (53.4%) and most were within the 25 - 34 years age group (46.6%). Most of the workers had worked for only ≤5 years (72.9%). Table [1](#Tab1){ref-type="table"} illustrates.Table 1**Demographic distribution and work experience of health workers**VariableFrequencyPercent***Sex***Male7153.4Female6246.6Total133100.0***Age Group***15 -- 24139.825 -- 346246.635 -- 443627.145 -- 541511.355 -- 6464.565 and above10.7Total133100.0***Number of years worked***1 -- 59772.96 -- 101813.511 -- 1575.316 -- 2043.021 -- 2553.826 -- 3021.5Total133100.0
The prevalence of headache in the past 6 months was 88.0% (among males the prevalence was 87.3% while in females it was 88.7%). There was no significant difference observed between the sexes (p = 0.806). In both sexes, primary headaches were more prevalent (71.0% in males and 76.4% in females). There was also no significant difference in the prevalence of the primary headaches among the sexes (p = 0.509). See Table [2](#Tab2){ref-type="table"}.Table 2**Prevalence of headache among the health workers**General prevalence of headache in the past 6 monthsVariablesFrequencyPercentHeadache present11788.0Headache absent1612.0**Sex prevalence of headache**Male (%)Female (%)Headache present62 (87.3)55 (88.7)Headache absent9 (12.7)7 (11.3)Total71 (100.0)62 (100.0)χ^2^ = 0.060; P value = 0.806Type of headachePrimary44 (71.0)42 (76.4)\*Secondary18 (29.0)13 (23.6)Total62 (100.0)55 (100.0)χ^2^ = 0.436; P value = 0.509\*Secondary headache is headache with a definitive and identifiable cause found for it i.e. those with pre-existing conditions that may cause the headache e.g. hypertension, cervical spondylosis, refractive error, sleep apnoea, malaria and other febrile conditions \[[@CR15]\].
Most respondents reported ≤5 episodes of headache in the last 6 months (74.4%) and these were typically of short-lasting durations, \<60 minutes (44.4%). There was no observed periodicity to the headaches in 57.3% of cases (see Table [3](#Tab3){ref-type="table"}). Most of the headaches were not located in any particular part of the head or side-locked (71.7%); were described as mildly severe in 59.8% of cases while 88.0% of respondents did not suffer any sleep disruption. The headaches were often not significantly disabling (73.4%) and in 93.2% of respondents did not lead to absenteeism or affect productivity at work (Table [4](#Tab4){ref-type="table"}).Table 3**Characterization of the headaches**Variable/CharacteristicsFrequency (N =117)Percent***Number of episodes in past 6 months***1 -- 58774.46 -- 102521.411 -- 1532.616 -- 2021.6**Usual duration of headaches**Seconds1916.2Minutes5244.4Hours3630.9Days108.5***Usual time of day of the headache***Morning1815.4Afternoon1512.8Night1311.1Continuous43.4No particular time6757.3***Is the headache becoming stronger, last longer or occur more frequent?***Yes2218.8No9581.2**What is the commonest nature of the headache?**Throbbing/exploding4336.8Sharp43.4Tightness54.3Dull65.1Aching2420.5Pressure in head3227.3Grinding32.6Table 4**Usual location and severity of the headache*Usual Location of headache***FrequencyPercentLeft side32.6Forehead97.7Around the head/ill-defined119.4Right side21.7Both Temples21.7Top of the head10.9Neck21.7Back of head32.6No particular side8471.7***Severity of headache***Mild7059.8Moderate4538.5severe21.7***Is the headache strong enough to wake you from sleep?***
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INTRODUCTION
============
Silicon (Si), in the form of dissolved silicic acid---often referred to as silicate and hereafter abbreviated as DSi---is an inorganic nutrient instrumental to ocean functioning. Its availability modulates processes as relevant as ocean primary productivity ([@R1]) and the exchange of CO~2~ with the atmosphere ([@R2]). Regional patterns of DSi availability largely result from the consumption of this nutrient by marine organisms (silicifiers) to build their skeletons of biogenic silica (BSi), with diatom utilization among the best known and quantified ([@R1], [@R3]). However, sponges, radiolarians, silicoflagellates, choanoflagellates, testate amoebae, and chrysophyceans, among others, also consume DSi in the ocean ([@R4]), but their activity remains poorly quantified and little understood from a physiological and molecular perspective. In the diatoms, the kinetics of DSi uptake have been investigated in a large variety of species, all of which were reported initially to follow a saturable Michaelis-Menten model. It is also that those saturable kinetics shift into nonsaturable uptake when DSi availability drastically increases and/or under particular physiological conditions ([@R5], [@R6]). Likewise, membrane silicon transporters (SITs) incorporating actively ambient DSi into the diatom cell have long been described and, although passive transporters have not been identified yet, a diffusion-based uptake has been described for at least some diatoms at high DSi availability \[reviewed in ([@R7])\]. In contrast, little is known about these physiological and molecular processes in other groups of marine silicifiers. Because diatoms are restricted to the photic zone of the ocean, a major gap in knowledge relative to DSi utilization in the dark ocean by nonphototrophic silicifiers persists. The discoveries that extensive aggregations of highly silicified sponges are common in the deep sea ([@R8]), that they can accumulate substantial amounts of BSi at the regional scale ([@R9], [@R10]), and that they trigger significant losses of BSi from the ocean ([@R11]) have raised considerable interest in deciphering how Si is processed in these singular, sponge-dominated, deep-sea systems. The lack of knowledge regarding these processes in sponges currently hinders the ability to understand the Si utilization in the dark ocean and makes it difficult to model adequately the role of the biological component in the marine biogeochemical cycle of silicon ([@R3], [@R11]).
Information on the physiology of DSi consumption by sponges is sparse and derived exclusively from shallow-water species in the class Demospongiae. These studies indicate Michaelis-Menten kinetics but with optimal DSi consumption attained at environmental DSi concentrations of \>100 μM ([@R12]--[@R16]). Because DSi concentrations higher than 100 µM are virtually never reached in the shallow waters of the modern ocean ([@R17]), the skeletal growth of all shallow-water demosponges investigated to date is therefore chronically limited by Si availability ([@R15], [@R16], [@R18]--[@R20]). Whether this kinetic limitation also applies to sponges in the class Hexactinellida---deep-sea specialists characterized by impressive siliceous skeletons---remains unknown. The physiology of DSi consumption and the molecular pathways of DSi uptake remain largely uninvestigated in hexactinellids, hampered by impediments to conducting in situ and laboratory experimentation with such relatively large and delicate deep-sea animals. Nevertheless, the interest is enormous. Major differences in the kinetics of DSi utilization between the two major lineages of siliceous sponges (i.e., Demospongiae and Hexactinellida) cannot be discarded, as silicification in demosponges revolves around the activity of the nonsoluble silicatein enzyme ([@R21]), while the process in hexactinellids appears to be governed by a phylogenetically unrelated, soluble enzyme, glassin ([@R22]). In addition, because hexactinellids have essentially syncytial organization while demosponges have a conventional cellular organization, the membrane transporters involved in Si utilization may not be shared but may be lineage specific instead. Very little is known on molecular Si transport in demosponges ([@R23], [@R24]) and nothing in hexactinellids. This lack of knowledge obscures the understanding of the evolution of the biosilicification process in the animal kingdom and its relationships to that in other organisms.
Here, we characterized experimentally the kinetics of DSi consumption in the hexactinellid sponge *Vazella pourtalesii* (Schmidt, 1870), a rosellid distributed from \~100 to 935 m in the northwest Atlantic that forms extensive monospecific aggregations on the deep continental shelf off Nova Scotia ([Fig. 1A](#F1){ref-type="fig"} and fig. S1), eastern Canada ([@R25]). We tested the laboratory-based kinetic model by comparing its predictions to both in situ determinations of DSi consumption rates using incubation chambers ([Fig. 1, B to D](#F1){ref-type="fig"}, and movies S1 and S2) and rates of BSi production derived from individuals of a known age grown in the wild on an artificial substrate ([Fig. 1, E and F](#F1){ref-type="fig"}). In combination with those physiological experiments, we conducted a quantitative large-scale assessment of gene expression as a function of DSi availability. The results of this study offer a mechanistic explanation for the kinetics of DSi utilization in hexactinellids and provide fresh insights into the molecular systems of Si transport and their evolution within sponges and across other silicifying organisms.
{#F1}
RESULTS
=======
Modeling and testing the physiology of DSi consumption
------------------------------------------------------
Live sponges were collected using the remotely operated vehicle (ROV) Remotely Operated Platform for Ocean Sciences (ROPOS) and taken to the laboratory for incubation in progressively increasing DSi concentrations (12, 30, 60, 100, 150, 200, and 250 μM DSi; see Materials and Methods). Initially, all 11 assayed individuals increased their DSi consumption rate in response to the progressive increase of DSi availability in the seawater ([Fig. 2, A and B](#F2){ref-type="fig"}, and tables S1 and S2). As also known for demosponges, the DSi consumption rate notably varied among individuals, with an average maximum consumption of 0.106 ± 0.050 μmol Si per milliliter of sponge tissue and per hour (hereafter given as μmol Si ml^−1^ hour^−1^) at an average DSi concentration of 150.9 ± 69.3 μM. Over that concentration threshold, the consumption rate of most individuals did not increase with increasing DSi availability, revealing that the Si transport system reaches the maximum speed (i.e., optimal utilization) at about 150 μM DSi and saturates at higher concentrations.
{#F2}
Unlike in all demosponges studied to date, the model best fitting the average DSi consumption rate of *V. pourtalesii* in response to DSi availability did not follow Michaelis-Menten kinetics (*r*^2^ = 0.841, *P* = 0.004; [Fig. 2A](#F2){ref-type="fig"}). An exponential rise to a maximum (ERM) model showed the best fit (*r*^2^ = 0.898, *P* = 0.002; [Fig. 2, A and B](#F2){ref-type="fig"}) to the empirical data on DSi consumption as a function of DSi availability, "consumption rate = *a* (1 − *b*^\[DSi\]^)". Although the difference in statistical fit between the "Michaelis-Menten" and "ERM" models was apparently small, the ERM model was built on parameters with higher statistical significance (*a* = 0.093 ± 0.008, *P* \< 0.001
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When Landauer argued in 1961 that any physical realisation of erasure of information has a fundamental thermodynamic work cost he irrevocably linked thermodynamics and information theory[@b1][@b2][@b3][@b4][@b5][@b6][@b7][@b8][@b9]. A practical consequence of this insight is that all computers must dissipate a minimal amount of heat in each irreversible computing step, a threshold that is becoming a concern with future computer chips entering atomic scales. The treatment of general *quantum* information processing tasks within the wider framework of quantum thermodynamics has only recently begun[@b13]. Quantum mechanics differs from classical mechanics in at least three central aspects: the special nature of measurement, the possibility of a quantum system to be in a superposition and the existence of quantum correlations. The thermodynamic energy needed to perform a (selective) measurement has been investigated[@b10] and the total work for a closed thermodynamic measurement cycle explored[@b11]. The catalytic role of quantum superposition states when used in thermal operations has been uncovered[@b12] and it has been shown that work can be drawn from quantum correlations[@b13][@b14] in a thermodynamic setting, see [Fig. 1](#f1){ref-type="fig"}. In particular, del Rio *et al.*[@b14] showed that contrary to Landauer's principle, it is possible to *extract* work while performing erasure of a system's state when the system is correlated to a memory. This can occur if and only if the initial correlations imply a negative conditional entropy, a uniquely quantum feature. The thermodynamic process does however now require operation on degrees of freedom external to the system, i.e. the memory's.
Results
=======
Projections and the optimal work value of removing coherences
-------------------------------------------------------------
Our motivation is here to shed light on the implications of performing a measurement on a quantum state that has coherences. We will consider this task in the thermodynamic setting of Landauer's erasure, involving a heat bath at fixed temperature *T* and operation on *N* → ∞ uncorrelated and identically prepared copies of the system (i.i.d. limit). This is of interest in the context of the quantum Jarzynski equality, for example, and will also be central for experiments testing quantum thermodynamic predictions in the future. To tackle this question we define the information-theoretic "projection" for a given initial quantum state *ρ* and a complete set of mutually orthogonal projectors . Such state transformation can be seen as analogous to the state transfer of erasure, , to a blank state . Physically, this projection can be interpreted as the result of an unread, or unselective[@b15], measurement of an observable that has eigenvector projectors . In an unselective measurement the individual measurement outcomes are not recorded and only the statistics of outcomes is known. In the literature the implementation of unselective measurements is often not specified, although it is typically thought of as measuring individual outcomes, e.g. with a Stern-Gerlach experiment, see [Fig. 2a](#f2){ref-type="fig"}, followed by mixing. The crux is that the information-theoretic projection can be implemented in many physical ways. The associated thermodynamic heat and work will differ depending on *how* the projection was done and we will refer to the various realisations as "thermodynamic projection processes". One possibility is decohering[@b16] the state in the so-called pointer basis, , a thermodynamic process where an environment removes coherences in an uncontrolled manner resulting in no associated work. In general it is possible to implement the state transfer in a finely controlled fashion achieving optimal thermodynamic heat and work values.
Of particular importance in thermodynamics is the projection of the system's initial state *ρ* onto the set of energy eigenstates of the system's Hamiltonian with *E*~*k*~ the energy eigenvalues. Here the state's off-diagonals with respect to the energy eigenbasis are removed - a state transformation that is frequently employed in quantum thermodynamic derivations and referred to as "dephasing" or "measuring the energy". Our key observation is that there exists a thermodynamic projection process realising this transformation and allowing to draw from the quantum system a non-trivial *optimal average work* of
Here *T* is the temperature of the heat bath with which the system is allowed to interact, see illustration [Fig. 1](#f1){ref-type="fig"}, *k*~*B*~ is the Boltzmann constant and *S* is the von Neumann entropy. Crucially, this work is strictly positive for quantum states with coherences. Extending the key observation to general projections one finds that optimal thermodynamic projection processes can be implemented that allow to draw an average work of
where an additional internal energy change term appears.
Physical interpretation and assumptions made to derive the optimal work
-----------------------------------------------------------------------
The optimal work values stated in Eqs. [(1](#eq12){ref-type="disp-formula"}) and ([2](#eq14){ref-type="disp-formula"}) are valid for processes applied to classical and quantum states alike. While for a classical ensemble the entropy change, , will be zero this is not so in the general quantum situation, where initial non-diagonal quantum states result in a strictly positive entropy change[@b17]. We note that while the optimal work values are in principle attainable, practical implementations may be suboptimal resulting in a reduced work gain or a higher work cost.
The physical meaning of can be grasped by considering a lower bound[@b18] on it, , see Supplement. Here *d* is the dimension of the system and denotes the Hilbert-Schmidt norm. The first factor quantifies the distance of the initial state from the fully mixed state, while the second factor, , quantifies the angle between the diagonal basis of *ρ* and the projection basis . These terms correspond to incoherent and coherent mixing contributions. The entropy change is non-trivially bounded only if the initial state is not an incoherent mixture with respect to that basis. The entropy bound is the largest for pure initial states whose basis is mutually unbiased with respect to . In this case the optimal entropy change is .
One may wonder where the work has gone to. There are two equivalent approaches to the accounting of work. In the present analysis the focus is on the work that the system exchanges, as done in statistical physics[@b5][@b19][@b20][@b21][@b22]. In this approach it is often not explicitly mentioned where the work goes to, but the only place work can go to are the externally controlled energy sources. Similarly, the heat, i.e. the energy change minus the work, is established implicitly. For example, in the experimental realisation of classical Landauer erasure with a colloidal silica bead trapped in an optical tweezer[@b21], the dissipated heat of erasure was calculated by knowing the applied tilting forces and integrating over the bead's dynamics. The second approach is to collect work in a separate work storage system[@b23], as illustrated by the weight in [Fig. 1](#f1){ref-type="fig"} and detailed in the Supplement. Both the implicit and the explicit treatment of work are equivalent in the sense that the results obtained in one approach can be translated into the other.
The thermodynamic assumptions made to prove Eq. [(2)](#eq14){ref-type="disp-formula"} are congruent with current literature[@b9][@b23][@b24][@b25]; specifically they are: (T0) an isolated system is a system that only exchanges work and not heat; (T1) the validity of the *first law* relating the internal energy change, Δ*U*, of the system during a process to its average heat absorbed and work drawn, ; (T2) the validity of the *second law* relating the system's entropy change to its average absorbed heat, , when interacting with a bath at temperature *T*, with equality attainable by an optimal process; (T3) the thermodynamic entropy to be equal to the von Neumann entropy in equilibrium as well as out-of-equilibrium, . In addition we make the following standard quantum mechanics assumptions: (Q0) an isolated system evolves unitarily; (Q1) control of a quantum system includes its coherences. Details of the proof are in the Methods Summary. We note that in the single-shot setting whole families of second laws apply[@b7][@b8] that differ from (T2) stated above. However, in the limit of infinitely many independent and identically prepared copies of the system these collapse to the standard second law, (T2), on the basis of which Eq. [(2)](#eq14){ref-type="disp-formula"} is derived.
From the information-theory point of view the projections considered here constitute just one example of the larger class of trace-preserving completely positive (TPCP) maps characterising quantum dynamics. Of course, all TPCP maps can be interpreted thermodynamically with the assumptions stated above, resulting in an optimal average work given by a free energy difference. Erasure is another such map whose study forged the link between information theory and thermodynamics. The benefit of discussing "projections" here lies in the insight that this focus provides: it uncovers that coherences offer the potential to draw work making it a genuine and testable quantum thermodynamic feature. This work is non-trivial even when the thermodynamic process is operated on the system alone, not involving any side-information[@b14] stored in other degrees of freedom.
Qubit example for drawing optimal work
--------------------------------------
To gain a detailed understanding of thermodynamic projection processes that give the optimal work stated in Eq. [(1)](#eq12){ref-type="disp-
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Background {#Sec1}
==========
Congenital cataracts are diagnosed within the first year of life. These cataracts are one of the leading causes of blindness in children and are estimated to occur with a prevalence of 3--6 per 10,000 live births \[[@CR1]\]. Congenital cataracts may appear either in isolation or in association with other ocular or systemic anomalies. Up to 25 % of congenital cataracts are thought to be caused by genetic defects \[[@CR2]\]. The genetic landscape of mutations causing congenital cataract is extremely diverse; more than 40 genes and additional loci have been associated with nonsyndromic cataract \[[@CR2]--[@CR6]\].
*GCNT2* (glucosaminyl (N-acetyl) transferase 2, I-branching enzyme) was first identified in 2001 as the gene encoding for the glycosyltransferase responsible for the human blood group I antigen. Recessive mutations in *GCNT2* result in an adult i blood group phenotype, which is also associated with congenital cataracts in some cases \[[@CR7]\]. Alternative splicing of the *GCNT2* gene produces three transcripts (A, B, and C). The three transcripts share a common second and third coding exon with a unique first exon for each isoform; differing expression profiles were identified for the transcripts with only the *GCNT2B* isoform expressed in lens epithelial cells and only the *GCNT2C* isoform expressed in reticulocytes \[[@CR8]\]. To date, seven missense mutations, one nonsense mutation, and two large deletions have been reported; mutations in exon 1C, affecting only the *GCNT2C* isoform, cause the adult i blood group without cataracts while mutations/deletions affecting exons 2 and 3, shared by all isoforms, result in the adult i blood group along with congenital cataract \[[@CR7]--[@CR11]\].
Case presentation {#Sec2}
=================
Patient 1(individual II:1) is an 18-month old Pakistani female affected with bilateral dense central congenital cataract (Fig. [1a,](#Fig1){ref-type="fig"} Table [1](#Tab1){ref-type="table"}) which were visually significant and required extraction at 2 months of age, mild asymmetry of the palpebral fissures, and left nasolacrimal duct obstruction; her development is normal and growth parameters are generally normal with the exception of borderline microcephaly (length 83.8 cm, 75--90th centile; weight 10.2 kg, 25--50th centile; and head circumference 44 cm (3rd centile)). Physical exam at 4 months of age identified hypotelorism (familial) and mildly widely-spaced nipples. Her younger brother, age 6 months, was similarly affected with visually significant bilateral dense central congenital cataracts requiring extraction around 2 months of age; his length (67.5 cm, 25--50th centile), weight (6.8 cm, 5--10th centile), and head circumference (42.5 cm, 10--25th centile) are all within the normal range (Table [1](#Tab1){ref-type="table"}). Family history shows unaffected second-cousin parents with additional endogamous mating within the family. A double second-cousin to the proband is affected with bilateral non-syndromic anophthalmia/ microphthalmia with no additional details available.Fig. 1Patient photographs and pedigree. **a** Photograph of Patient 1's eyes at 2 months of age showing bilateral cataract. **b** Pedigree showing both affected siblings with a homozygous deletion of 6p24.3 while the unaffected parents are heterozygous carriers. WT: wild type; black arrow indicates probandTable 1Phenotype and genotype information of the affected patientsPatientLens phenotypeOther featuresDevelopmentDeletionGenes involvedPatient 1Bilateral dense central congenital cataracts; extraction at \~2 months of ageBorderline microcephaly (3rd centile), mild asymmetry of the palpebral fissures, left nasolacrimal duct obstruction, hypotelorism, somewhat widely-spaced nipplesWNL97.9 kb homozygous deletion of 6p24.3The first coding exons of *GCNT2A* and *GCNT2B,* two 5'noncoding exons of *GCNT2A*, and a part of the region upstream of *TFAP2A*Patient 2Bilateral dense central congenital cataracts; extraction at \~2 months of ageNoneWNL97.9 kb homozygous deletion of 6p24.3The first coding exons of *GCNT2A* and *GCNT2B,* two 5'noncoding exons of *GCNT2A*, and a part of the region upstream of *TFAP2A*
Materials and methods {#Sec3}
=====================
Whole exome sequencing was performed by Macrogen (previously Axeq) and analyzed as previously described \[[@CR12]\]; briefly, exome data from the proband was analyzed using the SNP & Variation Suite (SVS; Golden Helix, Bozeman, MT, USA) to identify/exclude mutations in the coding and splicing regions of 40 known nonsyndromic cataract genes and 7 additional crystallins \[[@CR3]--[@CR6]\]; synonymous variants and variants with a frequency of \>1 % in the general population ([http://exac.broadinstitute.org](http://exac.broadinstitute.org/), <http://evs.gs.washington.edu/EVS/>, <http://www.1000genomes.org/>) were considered to be benign variants. Copy number variation analysis was completed by screening exome sequencing data using the Copy Number Inference From Exome Reads (CoNIFER) v0.2.2 software package as previously outlined \[[@CR13]\]; regions of interest were further verified by independent quantitative PCR reactions using DNA samples from the proband and other available familial samples with SYBR Green PCR Master Mix (Applied Biosystems/Life Technologies, Carlsbad, CA, USA). qPCR reactions utilized three region-specific probes (Additional file [1](#MOESM1){ref-type="media"}: Table S1) and were performed as follows: primers located within regions of interest were designed using Primer3Plus software (<http://sourceforge.net/projects/primer3/>) using qPCR settings. Each reaction was comprised of five nanograms of DNA in a total reaction volume of 12uL. Each primer set was run three times in triplicate using patient, parental or control DNA on a Bio-Rad CFX Connect Real-Time PCR machine (Bio-Rad, Hercules, CA, USA). A primer set for the housekeeping gene *RPPH1* (ribonuclease P RNA component H1) was used to normalize all data. A probe located in *NDP* (Norrie disease (pseudoglioma)), located on the X-chromosome, was used as a copy-loss control. All experiments included a no-template control and an unaffected human DNA sample with presumably normal copy number at each region for comparison. Copy number changes were calculated using the 2^-ΔΔCt^ method as previously described \[[@CR14]\]. Following qPCR confirmation, the size and exact breakpoints of the deletion were determined using a series of regular PCR reactions that utilized primers located on both ends of the region (as defined by CoNIFER and qPCR analysis) and standard conditions (Additional file [1](#MOESM1){ref-type="media"}: Table S1). Since the patients were apparently homozygous for the deletion, no amplification product indicated that the primer(s) are located inside of the deleted region while the presence of a PCR product indicated primers outside of the deletion. Once sequences bordering the deleted region on the centromeric and telomeric sides were determined, the corresponding primers were used to amplify a 1.5 kb region across the breakpoints. The resultant product was cloned into pCRII-TOPO® (Life Technologies, Carlsbad, CA, USA) vector using the manufacturer's protocols and sequenced bidirectionally with M13 forward and reverse primers using Big Dye Terminator v3 chemistry and an ABI 3730XL sequencer (Applied Biosystems/Life Technologies, Carlsbad, CA, USA); the obtained sequences were compared with the corresponding reference sequence using BLAST (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>).
Results and discussion {#Sec4}
======================
Review of the whole exome sequencing (WES) data from Patient 1 did not identify any potentially pathogenic variants (with only two synonymous variants) in known nonsyndromic cataract genes. The WES data was then analyzed for copy number variation which revealed a potential 208-kb deletion (6p24.3 chr6: 10,412,788-10,621,660) affecting *TFAP2A* and *GCNT2*. The deletion was verified using qPCR probes located in the first coding exon of *GCNT2* isoform A and the first exon of *TFAP2A*; the qPCR confirmed deletion of the *GCNT2* sequence in both unaffected parents (haploid, heterozygous) and affected children (complete loss, homozygous) while diploid copy of the *TFAP2A* sequence was identified in all family members. Further analysis of the region by a series of regular PCR reactions using affected DNA identified the centromeric breakpoint between chr6:10472330--10472606 (set 7; diploid) and chr6:10474759--10474901 (set 8; complete loss) and the telomeric breakpoint between chr6:10570580--10570905 (set 13, complete loss) and chr6:
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Introduction
============
Diabetic cardiomyopathy contributes to increased cardiovascular mortality in diabetes mellitus (DM) patients and is characterized by a progressive alteration of left ventricular (LV) function. At a preclinical stage, a decrease in systolic myocardial strain has been suggested in echocardiographic studies.
MRI techniques remain the gold standard for quantification of myocardial deformation but only a single study suggested systolic abnormalities in type 2 DM patients with evidence of diastolic dysfunction.
MR-tagging is the most common technique for strain calculation using CMR but is intrinsically limited in measuring transmural variations. Cine-Displacement ENcoding Imaging with Stimulated Echoes(DENSE) has been recently proposed as an alternative that benefits from an increased spatial resolution.
Purpose
=======
To evaluate whether cine-DENSE and MR-tagging confirm the existence of a sub-clinical myocardial dysfunction in a population of type 2 DM patients with no sign or history of heart disease and normal conventional echo and MRI parameters.
Methods
=======
37 patients with type 2 DM (50.6±5.6 years, 8 females, HbA1c 7.6±1.2%) and 21 age-matched controls (49.7±8.0 years, 11 females) underwent a CMR study on a 1.5T scanner. Subjects were excluded if standard echocardiography showed significant abnormality. After a standard CMR study for conventional LV function assessment, two-dimensional cine-DENSE pulse sequence with short-echo train echo-planar imaging readout and cine-tagging with complementary spatial modulation of magnetization(CSPAMM) were acquired in short axis views at the same basal, mid and apical levels. LV volumes and ejection fraction were measured on cine-MRI images. Regional circumferential maximal systolic strain(ε~c~) was calculated from cine-DENSE and MR-tagging acquisitions on 16 LV segments. Average maximal systolic strain in each slice and a whole heart mean value(ε~c~mean) for each patient were calculated. Post-processing of cine-DENSE acquisitions included adaptive phase-unwrapping and spatial filtering. CSPAMM images were processed using *InTag* post-processing toolbox (Creatis, Lyon, France) implemented in OsiriX software (Geneva, Switzerland) with motion estimation based on the *Sine Wave Modeling* approach.
Results
=======
Standard cine-MRI LV function parameters were normal and comparable between groups (table [1](#T1){ref-type="table"}). Whereas LV ejection fraction was similar in the 2 groups, cine-DENSE showed a significant decrease in ε~c~ at basal, mid and apical LV level and in ε~c~mean in the DM group as compared to controls. MR-tagging confirmed a decrease in ε~c~ at the 3 LV levels and in ε~c~mean in DM patients as compared with controls.
######
Left ventricular function in type 2 diabetes mellitus patients and controls.
DM patients Controls P
----------------------- ---------------- ---------------- ---------
LVEDV (mL) 120 ± 26 129 ± 28 0.26
LVESV (mL) 41 ± 12 41 ± 12 0.90
LVEF (%) 66 ± 6 68 ± 6 0.30
ε~c~ base MR-tagging -0.173 ± 0.040 -0.200 ± 0.028 0.004
ε~c~ mid MR-tagging -0.177 ± 0.045 -0.220 ± 0.035 \<0.001
ε~c~ apex MR-tagging -0.189 ± 0.056 -0.232 ± 0.025 \<0.001
ε~c~ mean MR-tagging -0.179 ± 0.045 -0.216 ± 0.025 \<0.001
ε~c~ base cine-DENSE -0.134 ± 0.019 -0.155 ± 0.019 \<0.001
ε~c~ mid cine-DENSE -0.150 ± 0.021 -0.174 ± 0.020 \<0.001
ε~c~ apex cine- DENSE -0.153 ± 0.022 -0.193 ± 0.018 \<0.001
ε~c~ mean cine-DENSE -0.144 ± 0.016 -0.171 ± 0.016 \<0.001
LVEDV= left ventricular end-diastolic volume; LVESV= left ventricular end-systolic volume; LVEF= left ventricular ejection fraction; ε~c~ =Régional circumferential maximal systolic strain.
Conclusions
===========
Cine-DENSE and MR-tagging confirm subclinical myocardial dysfunction in asymptomatic patients with Type II DM.
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Background {#Sec1}
==========
Hemangioblastomas (HB) are highly vascular tumors, which account for approximately 3 % of all tumors of the central nervous system (CNS) \[[@CR1]\]. It occurs in a subset of CNS locations, including the cerebellum (37 %), brainstem (10 %), and spinal cord (50 %) \[[@CR1]\]. They are classed as grade one tumors under the World Health Organization\'s classification system. While most of these tumors are low grade and benign, some hemangioblastomas can present aggressive and occasionally malignant behavior. Hemangioblastomas occur as sporadic tumors (75 %) or as a manifestation of an autosomal dominantly inherited disorder, von Hippel-Lindau (VHL) disease (25 %) \[[@CR2]\].
VHL hemangioblastomas are most commonly caused by germline exon deletions or truncating mutations \[[@CR3]\] of the Von Hippel-Lindau *(VHL*) tumor-suppressor gene. The VHL protein, which is the critical part of a ubiquitin ligase protein complex that binds to the hypoxia-inducing factors HIF-1 and HIF-2 transcription factors and targets them for ubiquitination and proteosomal degradation. Dysregulation of this VHL-associated function causes increased expression of a variety of growth factors, including erythropoietin, PDGF, VEGF and TGF. Upregulation of these factors may lead to angiogenesis and tumorigenesis. Additional mechanisms of tumorigenesis have been described outside of the HIF pathway, including alterations in microtubule binding and stabilization, abnormal extracellular matrix composition as well as apoptosis and transcription regulation \[[@CR4]\]. For most VHL disease related hemangioblastomas, the inactivation or loss of both alleles of the VHL gene is required. In addition to the phenotypic variability associated with allelic heterogeneity, genetic modifiers may influence the phenotypic expression of VHL disease. Allelic variants in the *CCND1, MMP1* and *MMP3* genes have been reported to influence hemangioblastoma development \[[@CR5]\]. This reiterates the need for elucidating other genetic alterations specific for hemangioblastoma beside the hits of *VHL* gene. Moreover, in a subset of tumors including mostly sporadic hemangioblastomas, the genetic pathways involved in tumorigenesis have not been defined yet \[[@CR6]\].
Copy number variants (CNVs) are alterations of DNA sections in result of genomic deletions (fewer than the normal number) or duplications (more than the normal number) on certain chromosomes and are common to many human cancers. Comparative genomic hybridization (CGH) by single nucleotide polymorphism (SNP) arrays is a cutting edge technology that allows characterization of CNVs. SNP array karyotyping provides genome-wide assessment of copy number and loss of heterozygosity (LOH) in one assay. SNP array platforms, such as Affymetrix SNP 6.0 (Affymetrix, Santa Clara, CA, USA), often identify amplifications/deletions at a single gene level, which could not have been accomplished by previous methods. Thus, modern SNP arrays offer a powerful method for the discovery of oncogene and tumor suppressor gene involvement in tumors, as well as for improved cancer classification \[[@CR7]\].
In contrast to surveillance of genome wide alterations by CGH arrays it is possible to directly quantify the absolute copy number of specific DNA loci by Droplet Digital PCR (ddPCR). In ddPCR, target sequences are amplified by PCR and the reaction products are partitioned into droplets and amplified to endpoint with TaqMan probes as in qPCR, then their concentrations are determined based on the number of fluorescently positive and negative droplets in a sample well. The absolute number of target and reference DNA molecules is calculated and provides the target copy number variation (CNV) \[[@CR8]\].
In the present study we used high-resolution SNP arrays for the first time to genome wide analysis of aberrations in hemangioblastomas aiming at the identification of novel pathogenetic mechanisms and possible targets for rational therapy. We validated the main reoccurring genetic changes by ddPCR highly precise quantification.
Methods {#Sec2}
=======
Study population {#Sec3}
----------------
A total of 44 hemangioblastoma samples were used for the present study. Thirteen frozen samples obtained from The Sourasky Medical Center, Tel Aviv, Israel were used for the CGH analysis. Additional 32 formalin fixed paraffin embedded (FFPE) samples from Sheba Medical Center, Tel Hashomer, Israel were used as validation group. The study was approved by the ethical review boards of both Sheba and Tel Aviv Sourasky Medical Centers and was consistent with the declaration of Helsinki including informed consents. Clinical parameters, such as sex, age at diagnosis, and pathologic classification were collected from patient records. Clinical information of the patient's cohort is outlined in Table [1](#Tab1){ref-type="table"}.Table 1Cohort characteristicsCharacteristicFrozenParaffinAverage age48.553.5Median age5153Spinal samples43Brain samples829Total number1232
CGH analysis {#Sec4}
------------
DNA was purified from frozen tissues using DNeasy (Qiagen Inc., Valencia, CA). One sample of pooled normal genomic DNA, provided by Affymetrix, was used as experimental positive control. 250 ng of genomic DNA was digested with *Nsp*I (New England Biolabs, Inc) and then ligated to Nsp adaptors. The adaptor-ligated DNA fragments were amplified, fragmented using DNase I, end labelled with a biotinylated nucleotide, and hybridized to a human cytoscan HD array (Affymetrix) at 50 °C for 17 h. After hybridization, the arrays were washed, stained, and finally scanned with a GeneChip scanner 3000 (Affymetrix). All procedures were performed according to the manufacturer's protocols. Array experiments were performed using the high-resolution Affymetrix CytoScan HD microarray (Affymetrix, Inc, Santa Clara, CA) containing 2,696,550 markers of which 1,953,246 are non-polymorphic markers and 750,000 SNPs with over 99 % accuracy to detect accurate breakpoint estimation as well as loss of heterozygosity (LOH) determination. This chip covers 340 International Standards for Cytogenomic Arrays (ISCA) constitutional genes, 526 cancer genes (99.6 %) and 36,121 RefSeq genes. The chip uses marker intervals of 25 markers / 100 kb. Analysis of CEL files from the Affymetrix CytoScan HD Array or Cytogenetics Whole-Genome 2.7 M Array was done with the Chromosome Analysis Suite (ChAS) software for cytogenetic analysis. Signal processing was done by Signal Covariate Adjustment, Fragment Correction, Dual Quantile Normalization and PLIER signal summarization. Dual Quantile Normalization was done to equalize each array's intensity distribution copy number and SNP probes separately. For SNP markers, multiple probes for each allele were summarized to single values. Copy number (CN) was calculated by hidden Markov model copy number segments after log2 calculation, high pass filter image correction, log2 ratio covariate adjustment and systematic residual variables removal. The baseline for CN = 2 (normal autosomal copy number state) was established and used by the analysis software by Affymetrix company using a set of 380 phenotypically normal individuals named as reference. The reference sample includes 186 females and 194 male. For chromosome X, only females were used and for chromosome Y only males were used. Log2 ratios for each marker are calculated relative to the reference signal profile. Results of the summarized Data (CYCHP files) were viewed as chromosomal aberrations in table and graphical formats. We also added visual inspection of probe performance for altered segments. Reference intensity intended to represent the copy normal state (typically 2). Log ratios above 0 mean CN gain, log ratios below 0 mean CN loss and log ratios around 0 represent no change. Abnormal DNA copy numbers are identified automatically using 25 markers for loss/50 markers for gains.
VHL sequencing {#Sec5}
--------------
To screen the *VHL* gene for mutations in our cohort, we performed direct sequencing of the coding region. Exons 1, 2 and 3 of the *VHL* gene and their immediately flanking sequences were amplified by PCR as described previously \[[@CR9]\]. The PCR amplification products were purified by using the QIAquick PCR Purification Kit (Qiagen), according to the manufacturer's instructions. The amplification primers were used as primers in the sequencing reactions, except for exon 1, for which we designed a new cycle sequencing primer (5′CGAAGATACGGAGGTCGA3′). Cycle sequencing was performed using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems, Foster City, CA, USA), followed by isopropanol precipitation. The fragments were sequenced by automated sequencing analysis on an ABI Prism 377 sequencer (Applied Biosystems).
Droplet digital PCR {#Sec6}
-------------------
Copy Number validation was done on all samples, frozen and paraffin embedded, hemangioblastoma biopsies. Genomic DNA was purified using QIAamp DNA mini (Qiagen). Copy number variation (CNV) test was performed by droplet digital PCR (ddPCR) as previously described \[[@CR10]\]. In short, 16 ng of genomic DNA samples were added to 2xddPCR supermix (Bio-Rad) with final concentration of 500nM of each primer and 250nM probe in duplex of the tested gene and RNaseP. RNaseP served as a CNV = 2 reference gene. Probes for the tested genes contained a FAM reporter and RNaseP contained HEX. The genomic DNA and PCR reaction mixtures were partitioned into an emulsion of approximately 20,000 droplets using the QX100 droplet generator (Bio-Rad, USA). The
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The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.
Introduction
============
Pulmonary sequestration was first described by Pryce in 1946 in the Journal of Pathology and Bacteriology \[[@REF1]\]. Pulmonary sequestration is a rare congenital malformation of the respiratory tract. It constitutes approximately 0.15%-6.4% of all congenital pulmonary malformations \[[@REF2]\]. It is defined as a non-functioning mass of parenchymal lung tissue that lacks communication with the tracheobronchial tree and is supplied by an anomalous systemic artery. Anatomically, it is classified as intralobar sequestration, where it is located within a normal lobe without its own visceral pleura, or extralobar sequestration, which is outside the normal lung with its own visceral pleura. Here, we present a case of a 45-year-old female who had a diagnosis of intrapulmonary sequestration \[[@REF3]\].
Case presentation
=================
A 45-year-old Caucasian female presented with a left-sided breast mass. An excisional biopsy showed a high-grade phyllodes tumor, which was treated by resection. Preoperatively, a chest radiograph was obtained, which revealed a left lower lobe shadow suspicious of consolidation. At the time, this was considered to be a community-acquired pneumonia though she had no symptoms. She was hemodynamically stable. Laboratory investigations were within normal limits. She was subsequently treated with a seven-day course of antibiotics. Follow-up chest radiograph showed persistence of the infiltrate which led to a non-contrast computed tomography (CT) scan of the chest. It revealed a moderate consolidation of the left lower lobe and hilar lymphadenopathy (Figure [1](#FIG1){ref-type="fig"}).
{#FIG1}
She, again, denied any signs of infection including sputum production, hemoptysis, shortness of breath or cough. She also denied a history of frequent pulmonary infections. She was a former smoker and had a 20-pack year history. A fiberoptic bronchoscopy was conducted for her unresolving left lower lobe infiltrate. Brushing of the area of infiltration was obtained along with the lymph node biopsy, which turned out to be non-malignant. At this juncture, a chest CT scan with contrast was obtained, which showed an artery from descending aorta feeding the area of infiltration highly suggestive of pulmonary sequestration (Figures [2](#FIG2){ref-type="fig"}, [3](#FIG3){ref-type="fig"}).
{#FIG2}
{#FIG3}
After discussion with the patient, it was decided to pursue a resection. She was referred to a cardiothoracic surgeon for a video-assisted thoracoscopic surgery (VATS) lobectomy procedure. The patient underwent left lower lobectomy. Pathology report after lobectomy confirmed the features consistent with an intralobar sequestration.
Discussion
==========
Pulmonary sequestration is a rare congenital malformation of dysplastic lung tissue. The sequestered lung does not communicate with the tracheobronchial tree and is supplied by an anomalous systemic arterial source, most commonly from the aorta \[[@REF4]\]. Intralobar sequestration is four times more common than the extralobar type \[[@REF5]\]. Diagnosis during adulthood is relatively uncommon as 60% of cases are diagnosed in the first decade of life and is a rarity in adults greater than 40 years of age \[[@REF6]\].
Most patients develop symptoms in infancy or early childhood. Symptoms may be non-specific, including cough, chest pain and shortness of breath. Some patients develop recurrent pneumonias and bronchiectasis \[[@REF7]\]. Approximately 15% of patients remain asymptomatic \[[@REF8]\]. Our patient was asymptomatic and had denied previous pulmonary infections.
As in our patient, sequestration occurs mostly in the left hemithorax, in the posterior basal segment of the left lower lobe \[[@REF8],[@REF9]\]. In 75% of the patients, the supply to the intralobar sequestration is from the descending thoracic aorta \[[@REF9]\].
CT angiography scan is the imaging test of choice as it can show the anomalous artery feeding into the pulmonary sequestration \[[@REF4],[@REF9]\]. Non-contrasted CT imaging is sometimes adequate to aid in the diagnosis of sequestration. However, in our case, the diagnosis was not clear until CT angiography was performed. Pulmonary angiography is considered to be the gold standard; however, it is not commonly used as the diagnosis is mostly clinched on CT scan imaging.
On cut section, the intralobar pulmonary sequestration represents mucus filled airways and small cysts which may be filled with purulent material. On histological examination, there is mucus stasis in the airways and a systemic artery accompanies the airways \[[@REF10]\].
Surgical resection is considered the treatment of choice for intralobar sequestration especially in symptomatic patients \[[@REF8],[@REF11],[@REF12]\]. Even in asymptomatic patients, surgical resection is often recommended due to the risk of serious future complications, including infections, massive hemoptysis and malignant transformation \[[@REF2],[@REF13]\]. The usual surgery is lobectomy either via VATS or standard thoracotomy. Our patient had a VATS lobectomy performed.
Conclusions
===========
Pulmonary sequestration is an uncommon finding especially in the adult population. Imaging modalities, especially contrast-enhanced CT scan, can aid in diagnosis by localizing the aberrant arterial blood supply to the sequestered lung parenchyma. Contrasted CT scan is also important for preoperative evaluation in these patients.
The authors have declared that no competing interests exist.
Consent was obtained by all participants in this study
We would like to acknowledge that we presented a poster presentation discussing the same case under the same heading at the Chest Conference 2019.
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Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor with a median survival of 12 months. Conventional therapeutic strategies of radiotherapy and chemotherapy are inadequate to eradicate GBMs because of the diffuse nature of GBM, acquired or innate resistance to therapy and the presence of blood--brain--barrier (BBB).^[@bib1],\ [@bib2]^ To alter the *status quo* that has remained unchanged over 25 years, novel targets and therapeutic strategies need to be developed.
Evasion of apoptosis is a key hallmark of cancers that exhibit resistance against therapeutics,^[@bib3]^ making the reactivation of dormant apoptotic programs a favorable approach in treatment. Activating extrinsic apoptosis using death ligands, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), is a promising strategy because of its tumor specificity.^[@bib4]^ Several TRAIL-based therapies such as recombinant human TRAIL and death receptor agonists have been developed and have shown success in preclinical models.^[@bib5]^ Similarly, activating intrinsic apoptosis by inhibiting the antiapoptotic Bcl-2 proteins, Bcl-2 and Bcl-XL, with BH3 peptides has shown success in preclinical tumor models.^[@bib6],\ [@bib7]^ However, the major obstacle in proapoptotic therapies is innate or acquired resistance of tumor cells to the proapoptotic agents.^[@bib8]^
Aberrant regulation of the apoptosis pathway components could be responsible for the failure of desired response to the proapoptotic agents.^[@bib3]^ One well-characterized misregulation is the epigenetic silencing of proapoptotic genes, such as death receptor 4 (*DR4*).^[@bib9]^ However, epigenetic regulation of chemotherapy response of cancer cells has been demonstrated to be dynamic and reversible.^[@bib10]^ Numerous studies have shown that overcoming TRAIL resistance is possible by using secondary agents, such as the histone deacetylase inhibitors,^[@bib11],\ [@bib12],\ [@bib13]^ corroborating the idea that the resistance can be reversed by epigenetic reprogramming.
The epigenome of cells are maintained by dynamic histone and DNA modifications throughout the chromatin by a group of chromatin-modifying enzymes (CMEs) called 'writers' such as histone acetyltransferases, histone methyltransferases (HMTs) and DNA methyltransferases (DNMTs).^[@bib14]^ These modifications can be removed by 'erasers' such as histone deacetylases (HDACs) and histone demethylases (HDMs), or can be recognized by 'readers' such as bromodomain-containing proteins.^[@bib15]^ Although the molecular mechanisms leading to aberrant cancer epigenomes are becoming better understood,^[@bib16],\ [@bib17],\ [@bib18]^ most studies in GBM merely focus on DNA hyper/hypomethylation.^[@bib19]^ Therefore, an unbiased and comprehensive assessment of roles of CMEs in apoptosis resistance is needed.
In this study, we aimed to interrogate the function of CMEs regulating apoptotic response in GBM and undertook a loss-of-function approach using short-hairpin RNAs (shRNAs) that were designed against a select but diverse set of CMEs and associated proteins. We have shown that loss of KDM2B -- a H3K36-specific histone demethylase -- primed GBM cells for apoptosis through the induction of proapoptotic genes and the suppression of antiapoptotic genes. Our results suggest KDM2B as a novel central epigenetic regulator of GBM cell apoptosis and identify it as a potential proapoptotic target for future of GBM treatment.
Results
=======
Interrogation of the CMEs reveals KDM2B as a modulator of TRAIL-induced apoptosis in GBM cells
----------------------------------------------------------------------------------------------
To identify the key CMEs regulating the apoptotic response of GBM cells, we applied a shRNA-based loss-of-function screen in U87MG cell line ([Figure 1a](#fig1){ref-type="fig"}). We used a library of 60 shRNAs published before,^[@bib20]^ and expanded it to 125 shRNAs targeting 48 different CME genes with two or three separate shRNAs. The targeted CMEs included DNMTs, HMTs, HDMs, methylated DNA-binding proteins, polycomb-group proteins (PRCs) and a few transcription factors ([Figure 1b](#fig1){ref-type="fig"}). We first assessed the effects of shRNAs alone on U87MG cell viability, to identify shRNAs displaying intrinsic toxicity before TRAIL treatment. Two of the 125 shRNAs were eliminated from further analysis in the screen as they caused \~100% cell death after puromycin selection, possibly due to problems with viral packaging. Accordingly, 6 out of 125 shRNAs reduced cell viability more than 2 S.D. compared with shControl ([Figure 1c](#fig1){ref-type="fig"}). These were shRNAs targeting KDM5C (68±1%), KDM4C (71±1%), KDM4A (75±1%), KDM4A (75±1%), Set1A (77±1%) and KDM3B (77±1%). Five out of 125 shRNAs targeting MeCP2 (115±1%), MBD1 (115±1%), AHCY (117±5%), TCF3 (117±1%) and EZH1 (121±2%) caused minor increases in viability. However, the majority of the shRNAs did not significantly alter cell viability ([Figure 1c](#fig1){ref-type="fig"}). To identify shRNAs that modulated TRAIL sensitivity, we assessed the percent viability changes after TRAIL treatment. Accordingly, we categorized CMEs into two groups based on the phenotype observed upon their silencing compared with shControl cells: (1) suppressors of apoptosis, if their knockdown sensitized the cells to TRAIL, (2) enhancers of apoptosis, if their knockdown conferred cells more TRAIL resistant ([Figure 1d](#fig1){ref-type="fig"}). While TRAIL caused 25±4% reduction in the viability of shControl cells, there were seven shRNAs that sensitized cells to TRAIL. These were targeting five genes, namely *Suv39H2* (41±2%), *G9A* (42±2%), *NR2F2* (45±4%), *RING1A* (48±2 or 50±3%) and *KDM2B* (41±2 or 44±2%) ([Figure 1e](#fig1){ref-type="fig"}). We then focused on the genes *RING1A* and *KDM2B*, whose knockdown led to a phenotype with two independent shRNAs ([Figure 1e](#fig1){ref-type="fig"}). Taken together, our screen identified KDM2B and RING1, an H3K36-specific demethylase and an E3 ubiquitin-protein ligase of H2AK119, respectively, as novel regulators of TRAIL response.
Loss of KDM2B cooperates with TRAIL to reduce GBM cell viability
----------------------------------------------------------------
To assess the function of the novel apoptosis-modulating CMEs we identified in GBMs, we first checked the knockdown efficiency of the shRNAs and observed silencing down to 17--40% ([Supplementary Figure 1](#sup1){ref-type="supplementary-material"}). We then focused on KDM2B, as its protumorigenic functions have been demonstrated in various solid and hematological malignancies;^[@bib21],\ [@bib22]^ however, its function in GBMs was not defined. Quantitative RT-PCR (qRT-PCR) analysis of *KDM2B* levels revealed that both shRNAs reduced *KDM2B* mRNA levels down to \~50% ([Figure 2a](#fig2){ref-type="fig"}). However, shKDM2B-2 led to a more robust reduction at protein levels ([Figure 2b](#fig2){ref-type="fig"}). To assess whether the shRNAs targeting KDM2B is specific, we checked the levels of other KDM family members upon KDM2B knockdown and observed no major alterations in their levels ([Supplementary Figure 2](#sup1){ref-type="supplementary-material"}).
To further validate the screen results, we conducted ATP-based cell viability analysis of cells transduced with both shKDM2B vectors and verified that cells with reduced KDM2B exhibit cell death significantly more than the controls ([Figure 2c](#fig2){ref-type="fig"}). To examine these differences in cell death further, we used an assay that measures cell growth in real time, where cells' electrical impedance in a well is measured and transformed into a cell index. Accordingly, silencing of KDM2B not only augmented TRAIL response but also accelerated the process of cell death ([Figure 2d](#fig2){ref-type="fig"}). There was no observable difference between untreated cells, showing that KDM2B knockdown has no significant effects on short-term (24 h) proliferation dynamics. This phenotype was also validated by live-cell imaging, where the morphology of individual cells, as well as cell death processes, was observed in real time ([Figure 2e](#fig2){ref-type="fig"} and [Supplementary Videos 1--4](#sup1){ref-type="supplementary-material"}). As shown by automated quantification of cellular blebs that were indicative of apoptotic bodies, the process of apoptosis was accelerated in shKDM2B cells compared with controls ([Figure 2f](#fig2){ref-type="fig"}). The number of apoptotic bodies per frame reached its maxima within 6 h of TRAIL treatment in shKDM2B cells, earlier and significantly in higher numbers than shControl cells. To examine whether KDM2B effects can be recapitulated in additional GBM cell lines, we used a more TRAIL-sensitive line, T98G in parallel. There, KDM2B
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1. Introduction {#sec1-ijerph-17-04838}
===============
Recent studies show a rise in overweight and obesity among children and adolescents to 18% worldwide in 2016 \[[@B1-ijerph-17-04838]\], with one in five school-aged children in Europe being overweight or obese \[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838]\]. Also in The Netherlands, childhood obesity rates are high. In 2019, 12% of children aged 4 to 12 were overweight or obese \[[@B4-ijerph-17-04838]\]. Overweight or obesity in childhood is especially problematic, since this often continues into adolescence and adulthood and is related to an increased risk of negative health consequences, such as type II diabetes, hypertension, respiratory disease and various types of cancer \[[@B2-ijerph-17-04838],[@B3-ijerph-17-04838],[@B5-ijerph-17-04838]\]. Overweight and obesity are preventable and treatable \[[@B6-ijerph-17-04838]\], especially at a young age \[[@B5-ijerph-17-04838],[@B7-ijerph-17-04838]\], by improving healthy nutrition behaviours and increasing physical activity (PA) (also called energy balance-related behaviours (EBRBs)) \[[@B8-ijerph-17-04838],[@B9-ijerph-17-04838],[@B10-ijerph-17-04838]\]. To target children's EBRBs \[[@B8-ijerph-17-04838],[@B11-ijerph-17-04838]\], multiple settings \[[@B12-ijerph-17-04838]\], such as the school \[[@B13-ijerph-17-04838]\] and home environment should be involved in interventions \[[@B8-ijerph-17-04838],[@B13-ijerph-17-04838]\].
Since children spend a large proportion of their weekdays at school, and schools reach many children, schools have been a popular intervention setting for decades \[[@B13-ijerph-17-04838],[@B14-ijerph-17-04838],[@B15-ijerph-17-04838]\]. However, the influence of the home setting on children's EBRBs is profound, especially for young children, since parents determine the food availability at home, and influence children's nutrition and PA behaviour by practices like modelling and rule setting \[[@B16-ijerph-17-04838],[@B17-ijerph-17-04838],[@B18-ijerph-17-04838],[@B19-ijerph-17-04838]\]. Therefore it is important to also focus on the home setting when implementing school-based energy balance-related interventions \[[@B15-ijerph-17-04838],[@B17-ijerph-17-04838]\].
Most effective changes in the home setting are accomplished through interventions with direct parental involvement (e.g., parents attending educational sessions, or counselling sessions) \[[@B13-ijerph-17-04838],[@B16-ijerph-17-04838],[@B19-ijerph-17-04838],[@B20-ijerph-17-04838],[@B21-ijerph-17-04838]\]. However, directly involving parents is often resource- and labour-intensive, making these types of interventions less feasible \[[@B20-ijerph-17-04838]\]. Also, the recruitment \[[@B22-ijerph-17-04838]\] and prolonged engagement of parents in these types of interventions have proven to be challenging \[[@B3-ijerph-17-04838],[@B22-ijerph-17-04838],[@B23-ijerph-17-04838]\]. Only about one-third of invited families participate in any intervention activity \[[@B3-ijerph-17-04838],[@B24-ijerph-17-04838]\], 40 to 60% of whom drop out \[[@B24-ijerph-17-04838]\]. In addition, the participants of parental involvement interventions tend to be mostly high socioeconomic status (SES) parents \[[@B25-ijerph-17-04838]\], and it is particularly challenging to engage parents with a low SES \[[@B3-ijerph-17-04838],[@B25-ijerph-17-04838]\], a lower educational level, single parents and those of ethnic minority groups \[[@B24-ijerph-17-04838]\]. This is discouraging, since these parents/families are most in need of interventions. For example, research from a large Dutch cohort study has shown that there are socioeconomic and ethnic inequalities in child health \[[@B26-ijerph-17-04838]\]. At a young age, non-Western children were more likely to be overweight compared to Dutch children, with mother's educational level being one of the contributing factors in explaining this higher prevalence in overweight in these children \[[@B27-ijerph-17-04838]\]. Beyond socioeconomic health disparities, child characteristics and the supportiveness of the home environment to perform healthy behaviours are also important predictors of children's PA and nutrition behaviour. For instance, children's nutrition behaviour is determined by their preference for healthy and/or unhealthy foods, and their PA and sedentary behaviour is associated with their activity preferences \[[@B28-ijerph-17-04838]\]. Also, children raised in an environment in which healthy nutrition \[[@B29-ijerph-17-04838]\] and sufficient PA \[[@B29-ijerph-17-04838],[@B30-ijerph-17-04838]\] are less valued are more at risk for developing unhealthy EBRBs. Therefore, vulnerability of the population is not restricted to well-known socioeconomic differences, but also includes specific child preferences and the health climate regarding EBRBs in the home environment.
To involve more parents, and particularly those of vulnerable populations, the strategy of indirect parental involvement is an alternative. Even though indirect parental involvement is assumed to be less effective in changing health behaviours compared to direct parental involvement, it can lead to greater adoption and implementation rates \[[@B21-ijerph-17-04838]\]. In indirect parental involvement, parents are engaged in a way that the intervention implementers do not communicate or engage directly (i.e., face to face, or personally) with them. Instead, parents are informed via school media, or children function as the messenger. Examples of indirect parental involvement are the provision of information (newsletters, tip sheets), invitations to participate in or attend activities (events, educational sessions) and prompts or assignments directed at the child and/or parent with the aim of involving parents \[[@B19-ijerph-17-04838]\]. Previous research has shown that some of these strategies are more promising than others, for example prompting children to engage in intervention activities together with their parent(s) seems more promising than providing newsletters or invitations for optional intervention activities \[[@B17-ijerph-17-04838],[@B19-ijerph-17-04838]\]. The difference between these strategies could be described as passive (not requiring a specific action or response on the part of the parents) indirect involvement versus active (performing activities) indirect involvement.
To our knowledge, there is little to no research on the participation rates of the strategy of the latter type of indirect parental involvement in school-based interventions, while this information is crucial to be able to draw conclusions on potential strategies to involve parents in (school-based) interventions \[[@B21-ijerph-17-04838]\]. Given the fact that children of vulnerable populations are less active, more sedentary and have unhealthier diets \[[@B31-ijerph-17-04838],[@B32-ijerph-17-04838],[@B33-ijerph-17-04838]\], it is possible that these children and their parents are less interested in energy balance-related interventions. Therefore, gaining insight in participation rates of children and parents in interventions using an indirect parental involvement strategy is needed. Moreover, empirical evidence is lacking on who is engaged in this strategy and therefore, it is warranted to know whether vulnerable children and parents engage in these interventions using this strategy.
In the current study, we evaluated the potential of Challenge Me to engage children and parents, especially those of vulnerable populations. Challenge Me was a parental involvement intervention in which children are challenged to perform PA and nutrition-related activities by themselves and with their parents. The main aim of the current study is to examine whether conducting challenges is a feasible intervention strategy to engage children and parents in school-based energy balance-related interventions. With engagement, we refer to participation in the intervention, i.e., performing the intervention activities, and not enrolment. Additionally, a second aim is to gain insight in whether children in need for improvements, i.e., vulnerable populations, are engaged in the intervention using this indirect-involvement strategy. For this aim, we focussed on child demographics, child characteristics (preferences) and the climate towards health behaviours in the home environment.
2. Materials and Methods {#sec2-ijerph-17-04838}
========================
2.1. Design {#sec2dot1-ijerph-17-04838}
-----------
An exploratory cross-sectional study design was conducted. The Medical Ethics Committee of the Maastricht University Medical Centre and Maastricht University provided ethical approval for this study (METC163027, national number: NL58554.068.16).
2.2. Challenge Me Intervention {#sec2dot2-ijerph-17-04838}
------------------------------
### 2.2.1. Intervention Development {#sec2dot2dot1-ijerph-17-04838}
The intervention, called Challenge Me, challenged children to perform PA and nutrition-related challenges together with their parent(s) or guardian(s). Challenge Me has been developed as part of a larger evaluation \[[@B34-ijerph-17-04838]\], and is specifically focused on improving parental involvement. In the larger evaluation study, eight primary schools, located in low SES neighbourhoods (based on scoring of The Netherlands Institute for
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All relevant data are within the paper and its Supporting Information files.
Introduction {#sec001}
============
Ecosystem services are defined as the benefits that nature provides to society \[[@pone.0235320.ref001]\]. Ecosystems such as forests, grasslands, croplands, coastal zones, and urban areas offer different services to society. These include provisioning services (food, water, wood, and fibers), regulation services (which affect climate, flooding, disease, waste, and water quantity and quality), cultural services (recreational opportunities, aesthetic, and spiritual values), and support services (soil formation, photosynthesis, and nutrient cycling) \[[@pone.0235320.ref001], [@pone.0235320.ref002]\]. Regulating services are obtained directly from ecosystems without any transformational process \[[@pone.0235320.ref001]\]. Water regulation is one of such services, which has great importance to society by providing adequate quality water and maintaining the water cycle \[[@pone.0235320.ref003], [@pone.0235320.ref004]\].
Water regulation in forest ecosystems involves the processes that take place after precipitation. These include interception, evapotranspiration, surface and subsurface flow, infiltration, soil erosion control, water quality, and groundwater replenishing, among others \[[@pone.0235320.ref001]\]. These water and soil movement-related processes are affected by various climatic and topographic factors, as well as by soil and vegetation cover types. In a forest system, a portion of the rain is intercepted by the top and other layers of the canopy. The intercepted rain evaporates and returns to the atmosphere, while the non-intercepted portion reaches the ground and deep parts of the soil \[[@pone.0235320.ref005]\]. Forest cover plays an important role in intercepting, capturing, and channeling rainfall. In addition, it is one factor that can be directly manipulated by resource managers using silvicultural practices that consist of different harvesting techniques of various intensities.
For the purpose of this study, we focused on the processes that regulate water movement, starting from the canopy, to the forest floor, and into the stream channels. This includes the processes of throughfall, stemflow, and surface runoff. Throughfall refers to the amount of water that passes directly through the forest canopy or drips from branches and leaves of trees \[[@pone.0235320.ref006]\]. It accounts for 60 to 90% of rainfall \[[@pone.0235320.ref007]\]. Stemflow is the fraction of the water that comes in contact with the forest canopy and runs down the trunks of trees and bushes, before being deposited on the ground \[[@pone.0235320.ref008], [@pone.0235320.ref009]\]. It is often ignored in rainfall studies because it is thought to be insignificant and expensive to measure, particularly when forests are composed by rough-barked trees \[[@pone.0235320.ref010]--[@pone.0235320.ref012]\]. Stemflow values represent between 1 to 4% of total rainfall, although some studies reported values up to 20% for certain forest types \[[@pone.0235320.ref007], [@pone.0235320.ref011], [@pone.0235320.ref012]\]. In many areas, particularly semi-arid ones, stemflow creates important islands of soil moisture and nutrients around the stem and contributes to streamflow and groundwater generation \[[@pone.0235320.ref007]\].
Surface runoff refers to the rainfall that flows over the surface of the soil directly into nearby channels and bodies of water \[[@pone.0235320.ref011]\]. It is often referred to as sheet flow, e.g., the water that resembles a braiding pattern of threads, without forming channels larger than rills and gullies \[[@pone.0235320.ref013]\]. In addition to vegetation and other surface obstructions, the rate of flow is dependent upon soil characteristics. There are numerous studies that describe the effect of soil infiltration capacity on surface runoff \[[@pone.0235320.ref014]--[@pone.0235320.ref017]\]. However, there are only a few studies that have addressed the impacts of vegetation cover on surface runoff in Mexico. Furthermore, there are only a limited number of studies in Mexico that have assessed the relationship between surface runoff and forest density, which, as we said above, can be manipulated through direct silvicultural treatments.
Silvicultural treatments affect hydrological fluxes. Intensive silvicultural treatments (e.g. stand thinning from above or clear-cutting) change forest density \[[@pone.0235320.ref018]\], eventually modifying throughfall and stemflow at both stand level and individual tree level \[[@pone.0235320.ref019]\], while increasing surface runoff \[[@pone.0235320.ref020]\]. The potential impact of the increased water from the surface flow may eventually affect site productivity and the provision or regulation of other ecosystem services (e.g., plant diversity, soil erosion control, carbon sequestration, etc.) \[[@pone.0235320.ref021], [@pone.0235320.ref022]\]. Varying levels of tree density affect water cycle components (namely interception, evapotranspiration, infiltration, and surface runoff), causing variations in water soil movement and groundwater reserves \[[@pone.0235320.ref021], [@pone.0235320.ref023]\]. For example, heavy rainfall occurrences, following highly intensive vegetation cover treatments (such as clear-cuts), result in increased surface runoff causing soil erosion, flooding, and water turbidity \[[@pone.0235320.ref023]\]. For short periods, large water and soil movements can modify the quality, quantity, and distribution of water resources \[[@pone.0235320.ref024]\].
This study used hydrological models to analyze throughfall, stemflow, and surface runoff in a managed pine-oak forest in northern Mexico. Hydrological models, which relate the flow of water to some stand variables, enable an evaluation of the impact of changes in forest cover on the water resources within a watershed \[[@pone.0235320.ref025]\]. The models can help determine the best forest management scenarios in places where regulation services are combined with provisioning ecosystem services. The objectives of this study were to evaluate the effects that forests, in terms of some forest tree and stand variables, have on throughfall, stemflow, and surface runoff in a temperate area of northern Mexico. The working hypotheses are that throughfall and stemflow are different depending on tree size and genus, and that stand density affects surface runoff.
Materials and methods {#sec002}
=====================
The study area is located in the mountainous region of the Sierra Madre Occidental, within the municipality of Durango, which lies in the southern part of the state of Durango. The experimental site is located in a private property known as Molinillos ([Fig 1](#pone.0235320.g001){ref-type="fig"}). The owners of this 2,866-hectare property have played a leading role in promoting a healthy silvicultural management, biodiversity conservation, and ecotourism in the region \[[@pone.0235320.ref026]\]. They allowed us to conduct research and field measurements on their property. The current management plan includes the application of non-intensive tree regeneration methods (selective harvesting) as well as intensive methods (seed tree retention or clear-cuts) in different parts of the property \[[@pone.0235320.ref018], [@pone.0235320.ref026]\]. Of the total area, about 2,050 ha are under timber management, with the following treatment distribution: clear-cuts 3.5%, tree retention 14%, thinning 27%, and individual selection 55.5% \[[@pone.0235320.ref026]\].
{#pone.0235320.g001}
The climate in the region is temperate and sub-humid, with moderate levels of rainfall in the summer and parts of December and January. In the coldest month (January), daily temperatures can reach anywhere between -3 °C and 18 °C. In the warmest month (June), daily temperatures vary between 15 °C and 35 °C. Historical records show that the mean annual temperature varies from 8 °C to 26 °C, while the annual average is 13.3 °C. The annual rainfall varies from 443 to 1450 mm, with an average of 917 mm \[[@pone.0235320.ref027]\].
Regional elevation ranges from 1,500 to 3,000 meters above sea level. However, the elevation in the plots is closer between 2,360 and 2,630 meters. The typical slope ranges between 20% and 60%. Runoff water flows toward the hydrographic system of the Acaponeta River basin and eventually into the Pacific Ocean. The natural, pine-oak forests include mixtures of *Pinus strobiformis*, *P*. *cooperii*, *P*. *durangensis*, *P*. *engelmannii*, *P*. *teocote*, *P*. *leiophylla*, *Quercus coccolobifolia*, *Q*. *ruogosa*, *Q*. *sideroxilla*, *Q*. *obtusata*, and *Arbutus* spp. The type of soils are Regosol, Litosol, Eutric Cambisol, and Luvisol cromico types \[[@pone.0235320.ref026]\].
Tree and stand variables {#sec003}
------------------------
The information of the tree and
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Introduction {#Sec1}
============
Psoriasis is a chronic, immune-mediated, inflammatory skin disorder that is currently incurable. Consequently, the majority of people with psoriasis require long-term treatment to maintain disease control. Traditional immunosuppressive systemic treatments, such as acitretin, methotrexate, cyclosporine, hydroxyurea, and thioguanine, may be effective in controlling psoriasis in some patients but significant toxicity and the need to closely monitor patients limit the viability of these treatments for long-term, continuous use \[[@CR23]\]. Recently developed systemic therapies that selectively target specific pathways in the inflammatory cascade of psoriasis generally have a much improved safety profile compared with traditional therapies \[[@CR26]\].
Efalizumab (anti-CD11a; Raptiva^®^) is a recombinant humanized monoclonal IgG~1~ antibody that has been approved for the treatment of moderate-to-severe chronic plaque psoriasis. It interferes with the pathogenesis of psoriasis via multiple mechanisms, including inhibition of T-lymphocyte trafficking and T-lymphocyte activation and reactivation \[[@CR1], [@CR10], [@CR11], [@CR21], [@CR25]\]. The safety and efficacy profile of efalizumab has been established in numerous clinical trials, in which more than 3,500 patients were enrolled and treatment was assessed for up to 3 years \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\].
Although psoriasis can be associated with the co-morbidity of psoriatic arthritis, a minority of patients with psoriasis (7--30%) will develop this joint disease \[[@CR27]\]. Nevertheless, psoriatic arthritis constitutes a major consideration in patients who are receiving long-term treatment for their psoriasis. A Nordic study of more than 5,000 patients with psoriasis showed that patients with arthritis exhibited greater impairment of psoriasis-related quality of life (QoL), longer disease duration, and greater self-reported disease severity, compared with patients who had psoriasis but no co-morbid arthritis \[[@CR27]\].
A low incidence of arthropathy adverse events (AEs; any form of joint disease) associated with efalizumab treatment has been reported in both clinical studies and routine clinical practice \[[@CR8], [@CR12]\]. However, anecdotal reports of arthropathy in routine clinical practice have expressed concern that efalizumab may be associated with exacerbation of arthropathy \[[@CR8]\]. To address this concern, we conducted a large-scale pooled analysis of safety data from five Phase III clinical trials (including open-label extensions of two of these studies) and two Phase III open-label clinical trials of efalizumab to explore whether arthropathy AEs were associated with efalizumab treatment in patients with psoriasis.
Methods {#Sec2}
=======
The primary objective of this pooled safety analysis was to assess the incidence of arthropathy AEs in patients who had received either efalizumab or placebo. Safety data were pooled from five randomized, double-blind, placebo-controlled clinical trials (including data from two open-label extension studies of two of these trials) and two open-label clinical trials of efalizumab \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\]. Patients included in these Phase III studies were aged ≥18 years and had moderate-to-severe chronic plaque psoriasis, a psoriasis area and severity index (PASI) score of ≥12 at screening, and plaque psoriasis covering ≥10% of body surface area. All patients were candidates for either systemic anti-psoriatic therapy or had received systemic anti-psoriatic therapy. Patients included in these trials received subcutaneous injections with efalizumab, 1--4 mg/kg once weekly or 2 mg/kg once-every-other week, or placebo. Details of individual study methodologies are described in other publications \[[@CR4]--[@CR6], [@CR12]--[@CR17], [@CR22]\].
Arthropathy AEs were defined according to the Coding Symbols for Thesaurus of Adverse Reaction Terms (COSTART) \[[@CR3]\] preferred terms 'arthritis' and 'arthrosis', or the Medical Dictionary for Regulatory Activities (MedDRA, <http://www.meddramsso.com/NewWeb2003/index.htm>) preferred terms 'arthritis not otherwise specified (NOS)', 'psoriatic arthropathy', 'arthropathy NOS', 'monoarthritis', 'polyarthritis', and 'osteoarthritis NOS'.
Treatment groups analyzed {#Sec3}
-------------------------
Due to the variety of study designs, five analyses were considered: 'first-treatment phase', 'first exposure phase', 'extended treatment phase', 're-treatment phase', and 'long-term treatment' (see Table [1](#Tab1){ref-type="table"}). Table 1Summary of the Phase III data from five placebo-controlled clinical trials (including data from two open-label extension studies of two of these trials) and two open-label clinical trials of efalizumab included in the pooled safety analysisPublication (protocol number)Study designNumber of patients in each analysisFirst treatment (0--12 weeks)Efalizumab sc 1--4 mg/kg qw or 2 mg/kg qowFirst exposure\
Extended treatment (13--24 weeks)Long-term treatment^a^(≤36 months)Re-treatmentPlaceboEfalizumab 1 mg/kgEfalizumab 2 mg/kgLeonardi \[[@CR13]\] (ACD2058g)Randomized, double-blind, parallel-group, placebo-controlled170162166462123--55Lebwohl \[[@CR12]\] (ACD2059g)Randomized, double-blind, parallel-group, placebo-controlled122232243579289----Gordon \[[@CR4]\] (ACD2390g)Randomized, double-blind, parallel-group, placebo-controlled187368--368------Papp \[[@CR17]\] (ACD2600g)Randomized, double-blind, parallel-group, placebo-controlled236449--449--449--Sterry \[[@CR22]\] (IMP24011)Randomized, double-blind, parallel-group, placebo-controlled264529--772308--145Papp \[[@CR16]\] (ACD2062g)Open-label------34137--365Gottlieb \[[@CR5], [@CR6]\] (ACD2243g)Open-label------339^b^290339--Menter \[[@CR14]\] (ACD2391g)Open-label extension^c^of study ACD2390g \[[@CR4]\]------174342----Menter \[[@CR15]\] (ACD2601g)Open-label extension^c^of study ACD2600 g \[[@CR17]\]------217622635^d^--Pooled analysis97917404093,3942,111n.a.^e^565*qow* once-every-other week, *qw* once weekly, *sc* subcutaneous^a^Number of patients at start^b^Patients received combined therapy with fluocinolone acetate (*n* = 169) or petrolatum (*n* = 170) for weeks 9--12; for months 3--15, the dose of efalizumab could be escalated to 4 mg/kg per week for up to 4 weeks if clinically indicated^c^Some patients are included in the analyses more than once because patients in the open-label extension studies are also included in analyses of the parent studies^d^Included patients who received either efalizumab or placebo in the parent study \[[@CR17]\]^e^Not applicable because data are analyzed and reported separately for the study by Gottlieb et al. \[[@CR6]\] and the study published by Papp \[[@CR17]\] and Menter \[[@CR15]\]
It is worth noting that most of the studies included in this pooled analysis were designed and conducted before efalizumab had received regulatory approval and before it was known that doses of more than 1 mg/kg once weekly (the approved dose) did not confer additional treatment benefit (EMEA, Raptiva Summary of Product Characteristics; FDA US, FDA Prescribing Information for Raptiva). For this reason, only the efalizumab 1 mg/kg once-weekly dose data are reported for the 'first treatment phase' of the analysis. Due to the wide variety of study designs included in the pooled analysis, data for patients receiving any dose of efalizumab are combined for all other treatment phases analyzed.
The 'first treatment phase' analysis included 0--12-week data from patients in the five placebo-controlled studies who received either efalizumab 1 mg/kg once weekly or placebo. This analysis allows a comparison between the efalizumab and placebo treatment groups.
The 'first exposure phase' included 12-week data from all studies in patients who had their first exposure to any dose of efalizumab and, thus, did not include placebo data. This analysis was conducted to include the maximum number of patients who received efalizumab for their first 12 weeks of treatment (i.e., it included those patients who first received efalizumab treatment after crossing over from a placebo group, as well as the patients who first received efalizumab during weeks 0--12).
The 'extended treatment phase' analysis included 13--24-week data in patients given any dose of efalizumab who had already received efalizumab during the first treatment phase.
The 'long-term treatment phase' analysis included all patients who received continuous long-term treatment (up to 36 months) with any dose of efalizumab. Data were analyzed in 12-week segments to assess change in the incidence of arthropathy
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Background {#Sec1}
==========
Hypoxia is common after stroke, and associated with poor outcomes. In this article, we have reviewed the physiology of oxygen transport, the cerebrovascular response to hypoxia and pathophysiology, incidence and aetiology behind hypoxia in stroke and its subsequent clinical consequences. We have then reviewed all randomised clinical trials looking at the use of supplemental oxygen therapy in acute stroke and made conclusions regarding current evidence and recommendations for clinical practice.
Oxygen physiology {#Sec2}
=================
The normal adult range of arterial oxygen pressure (PaO2) is 11.0--14.4 kPa and the normal range for arterial oxygen saturation (SaO2) is 95--98% \[[@CR1]\]. The term hypoxia refers to oxygen levels below normal. It includes both tissue (e.g. brain, myocardium) hypoxia and hypoxia in the blood (hypoxaemia). Tissue hypoxia is defined by the concentration of oxygen in blood and also tissue perfusion, whilst hypoxaemia is defined by the concentration of oxygen in inspired air and its transfer into the blood \[[@CR2]\].
Following inhalation oxygen is taken up in the lung capillaries via diffusion down an oxygen concentration gradient across the alveoli \[[@CR3], [@CR4]\]. Oxygen binds to the haemoglobin molecule, which can carry four oxygen molecules; each binding and changing the shape of the haemoglobin molecule and increasing its affinity for oxygen \[[@CR5]\]. A small amount of oxygen is also dissolved in plasma. This proportion increases in hyperoxia, when all haemoglobin is saturated \[[@CR6]\]. Oxygen dissociates from the haemoglobin molecule in the tissues owing to the relatively hypercapnic and acidic environment (the Bohr effect) \[[@CR3]\].
Oxygen is a vital substrate that supports virtually all metabolic processes. 90% of oxygen intake is engaged in the cytochrome C oxidase system in the mitochondria \[[@CR7]\] generating adenosine triphosphate (ATP), which acts as the main energy substrate within cells. A continuous supply of oxygen is required to secure a continuous supply of ATP maintaining sufficient energy for cerebral neuronal and cellular activity. This facilitates an efficient energy producing process making 38 molecules of ATP during aerobic respiration, equivalent to 1270 joules (J) energy, in comparison to two molecules of ATP (67 J of energy) during anaerobic respiration \[[@CR7], [@CR8]\].
The anoxic brain {#Sec3}
================
20% of all human oxygen consumption is utilised by the brain \[[@CR9]\]. The brain has no oxygen or glucose (the other important substrate in the ATP producing equation) stores. Thus complete disruption of cerebral blood flow very rapidly results in an anoxic, hypoglycaemic state, which via a variety of mechanisms ultimately leads to cell death. Excitatory neurotransmitters, such as glutamate, bind to a variety of receptors and allow for an influx of calcium ions that help formulate the chemical signal for depolarisation \[[@CR10], [@CR11]\]. Normally the re-uptake of glutamate is an active energy-driven process. In the absence of ATP this process fails, resulting in an extracellular accumulation of glutamate, which continually stimulates receptors leading to a persistent influx of calcium ions \[[@CR12]\]. Furthermore, the Na+/Ca2+ ATP driven pump normally used to eliminate calcium fails, also due to a lack of ATP \[[@CR13]\]. The resultant high intracellular calcium triggers multiple cascades that ultimately lead to mitochondrial dysfunction and cell death. Furthermore, instead of producing ATP, glial cells have been shown to release ATP extracellularly \[[@CR11]\]. Aside from rendering this unusable by mitochondria, ATP also stimulates the P2X7 receptor, which again leads to significant calcium influx and ultimately cell death \[[@CR13]\]. The other major mechanism of cellular demise is via the formation of free radicals facilitated by the reduction of iron from its ferric (Fe3+) to its ferrous (Fe2+) form and the initiation of inflammatory cascades \[[@CR12]\].
Cerebral blood flow in hypoxia {#Sec4}
==============================
In normoxic states, cerebral blood flow is very tightly controlled by the partial pressure of carbon dioxide (PaCO~2~). Any hypocapnic state will result in vasoconstriction and reduction in regional cerebral blood flow and a hypercapnic state leads to the reverse with vasodilatation and an increase in cerebral blood flow. Cerebral blood flow is somewhat less responsive to changes in PaO~2~, which has the opposite effect to carbon dioxide; a hypoxic state causing cerebral vasodilatation with the aim of improving oxygen delivery and a hyperoxic state causing vasoconstriction \[[@CR14], [@CR15]\].
In a hypoxic state, whilst the vasodilatory response improves flow, the detection of hypoxia by peripheral chemoreceptors will in turn lead to an increase in respiratory drive, increasing arterial oxygen content. However, the consequence of this is also an increase in the clearance of carbon dioxide, which would theoretically cause vasoconstriction and reduced cerebral blood flow \[[@CR9]\]. It appears there is a threshold to which the hypoxic response predominates (and the carbon dioxide one attenuated) at a PaO~2~ of around 50--60 mmHg \[[@CR9], [@CR14]\]. Whilst the carbon dioxide mediated vascular response is mediated via a direct change in vessel wall pH \[[@CR15]\], the oxygen response appears to be mediated by the deoxygenated erythrocyte via a number of mechanisms; which include release of ATP and the subsequent actions of endothelial nitric oxide synthase on the vessel wall, reduction of nitrite to nitric oxide and the activity of S-nitrosohaemoglobin \[[@CR9]\].
The cerebral vascular response to hypoxia is not uniform. A study found that in an induced isocapnic hypoxic state increases in cerebral blood flow were most prominent in basal ganglia nuclei, the putamen, thalamus, nucleus accumbens and pallidum \[[@CR16]\]. Studies of blood flow in individual vessels have found that flow in the internal carotid artery is maintained during hypoxia and that vertebral artery flow is increased \[[@CR17]\]. This had led to the hypothesis that blood flow is increased in this region to preserve vital brainstem structures, or that possibly the posterior circulation vasculature is less susceptible to the effects of carbon dioxide for similar reasons.
Neurological effects of hypoxia {#Sec5}
===============================
The neurological consequences of hypoxia are dependent upon the speed of onset, the severity of hypoxia, and the level of tissue perfusion. Rapid decreases in PaO~2~, as in a cardiorespiratory arrest, can lead to permanent neurological damage within minutes. However, lower, less abrupt changes, can be tolerated if the decrease in oxygen occurs in a gradual manner, such as ascending at altitude, where individuals can acclimatise and develop tolerance to lower oxygen partial pressures or, (to a lesser degree), in chronic smokers. Initial clinical features include altered judgement, difficulty in completing complex tasks, and impairment in short term memory \[[@CR18], [@CR19]\], but in the longer term deficits can be more widespread and span physical and neuropsychological domains. Seizures occur in up to a third of individuals within a day of exposure to hypoxia, and are commonly partial complex or myoclonic in nature. Intractable forms of either of these types of seizure are associated with a poor prognosis \[[@CR20]\]. Cognitive impairment domains include amnesia, visuospatial deficits, frontal lobe symptoms, impairment of executive function, and impairments in language \[[@CR21]\]. These are covered in more detailed reviews on the subject \[[@CR22], [@CR23]\]. Involvement of the basal ganglia, a region particularly susceptible to hypoxic injury, can result in delayed Parkinsonism in older subjects, dystonia mainly in younger people, choreo-athetosis, and tremors \[[@CR20]\]. Varying degrees of unilateral or bilateral motor impairment may be observed depending on both the anatomical level and extent of corticospinal tract involvement. In very rare cases, the syndrome of delayed post-hypoxic leukoencephalopathy may occur weeks after a seemingly rapid recovery from the original insult. This condition is characterised by rapid deterioration in cognition, emergence of extra-pyramidal signs, and loss of executive function as a results of severe demyelination \[[@CR24]\]. The severity of leukoencephalopathy can be assessed by magnetic resonance imaging (MRI) \[[@CR20]\]. Electroencephalography, somatosensory evoked potentials and MRI can provide valuable information about the severity of hypoxic injury, but also aid in prognostication together with overall clinical state \[[@CR25]\].
Hypoxia in the context of a stroke {#Sec6}
==================================
There is no specific definition as to what constitutes hypoxia in an acute stroke, and it is therefore reasonable to assume that normal values for the general population apply.
Sulter and colleagues \[[@CR26]\] monitored 49 consecutive patients who presented with an acute stroke within 12 h duration using pulse oximetry for 48 h. Patients were considered hypoxic and treated with supplemental oxygen if saturations were below 96% for more than 5 min. This occurred in 63% \[[@CR31]\] of patients, with 28 of those returning to 'normal' oxygen saturations following administration of up to 5 L/min of oxygen. The remaining three required much higher concentrations. Factors associated with hypoxia in this group were stroke severity, presence of dysphagia, and older age. Roffe et al. \[[@CR27]\] recruited 118 patients (100 of whom had adequate measurements by pulse oximetry) and found that the mean daytime awake SO~2~ was 94.5 ± 1.7% in stroke patients and 95.8 ± 1.7% in healthy controls. Nocturnal saturations were reduced to 93.5 ± 1.9% in the stroke group and 94.3 ± 1.9% in controls. In the stroke group the average 4% oxygen desaturation index (ODI) (number of times per
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Raw deep sequencing cannot be provided because these data are derived from patient samples. Because of the Minimum Necessary Requirement of the HIPAA Privacy Rule these data may not be deposited into a public repository. A Supporting Information File accompanying the submission contains the de-identified results and interpretations of all clinical NGS tests included in our analysis next to the results of single-gene testing of the same specimen.
Introduction {#sec001}
============
The advance of next-generation sequencing (NGS) is a cornerstone of a recent development in molecular pathology, variably referred to as "personalized," "precision," or "individualized" medicine. Much of the focus of clinical NGS has been on oncology, as there are clear diagnostic, prognostic, and therapeutic implications for a multitude of genomic mutations in both solid and liquid malignancies. For example, in non-small cell lung cancers, activating mutations of the *EGFR* gene predict therapeutic response to tyrosine kinase inhibitors (TKI) such as erlotinib, gefitinib, and afatinib \[[@pone.0152851.ref001]--[@pone.0152851.ref003]\]. The KRAS protein acts downstream of EGFR, and thus mutations of the *KRAS* gene predict resistance to TKI \[[@pone.0152851.ref002],[@pone.0152851.ref004]--[@pone.0152851.ref006]\]. In metastatic melanoma, *BRAF* V600 mutations predict response to dabrafenib, vemurafenib, and trametinib \[[@pone.0152851.ref007]\]. Mutations of the *FLT3* and *NPM1* genes affect the prognosis of karyotypically normal acute myeloid leukemia and aid in the decision whether or not to pursue hematopoietic stem cell transplantation \[[@pone.0152851.ref008]\]. In addition, activating mutations of *JAK2*, which encodes a tyrosine kinase essential for cytokine and growth factor signaling, are found in a large proportion of patients with myeloproliferative neoplasms \[[@pone.0152851.ref009]\]. Ruxolitinib is a JAK1/JAK2 inhibitor approved for the treatment of myelofibrosis \[[@pone.0152851.ref010]\], and additional JAK2 inhibitors are in clinical development \[[@pone.0152851.ref011]\].
Detection of mutations in *EGFR*, *KRAS*, *BRAF*, *FLT3*, *NPM1*, and *JAK2* is most commonly accomplished by targeted tests that are designed to detect one or at most a small number of mutations in a single gene. However, NGS is gaining momentum as a complementary test for a number of reasons. Firstly, clinical trials for targeted cancer therapies rely on detection of mutations that are frequently not covered by existing targeted tests. In contrast to the laborious and lengthy process of validating and implementing a new molecular assay testing for one or a few mutations, NGS greatly simplifies the task of providing coverage of one of more additional mutations of interest. Secondly, targeted tests can provide misleading results. For example, the widely used FDA-approved cobas® *EGFR* Mutation Test only detects exon 19 deletion and L858R mutations, which together only comprise the mutations found in 85% of *EGFR*-mutated lung cancers \[[@pone.0152851.ref012]\]. In a significant proportion of cases, this test fails to identify therapeutically targetable mutations. Thirdly, targeted tests may fail to detect the very mutation they are designed to detect. Our group has recently reported a striking failure of two separate single-gene tests for the *BRAF* gene to detect a V600E mutation in a melanoma specimen \[[@pone.0152851.ref013]\]. The mutation was clearly demonstrated by concurrent NGS analysis. Finally, as has recently become apparent, tumors frequently harbor mutations that are therapeutically targetable but are not typically seen in that tumor type. Due to its massively parallel nature, NGS is very well-suited for detecting mutations in unexpected genes. One study reported a three-fold increased yield of clinically actionable mutations with NGS as compared to traditional molecular approaches targeting mutation hotspots \[[@pone.0152851.ref014]\].
Multiple recent studies have investigated the potential utility of NGS for detection of clinically actionable cancer mutations with encouraging results \[[@pone.0152851.ref014]--[@pone.0152851.ref025]\]. With the exception of one study \[[@pone.0152851.ref021]\], all demonstrated excellent performance of NGS on various platforms as measured by detection of point mutations and small insertions and deletions (indels). In many cases, additional potentially important variants were uncovered by NGS. All of the aforementioned studies were designed to validate clinical NGS pipelines that were not yet in clinical practice. As a consequence, they enrolled selected samples from previously examined specimens. With the exception of one commercially-sponsored study \[[@pone.0152851.ref014]\], in all cases a very limited number of samples was re-examined (range 13--61), often from only a single tissue type. While these important contributions confirm the potential usefulness of clinical NGS, they do not address the important question whether a well-validated NGS pipeline performs at an acceptable level in day-to-day clinical practice. Here we present a summative analysis of mutation results and quality control metrics obtained during the first year (March 2013 through March 2014) of clinical solid and liquid malignancy NGS carried out at the Center for Personalized Diagnostics at the University of Pennsylvania Health System. More than 900 specimens were submitted and processed during this time frame. We report that using our validated molecular and bioinformatics pipeline \[[@pone.0152851.ref026]\] with pre-determined tumor percentage and DNA quality cutoffs, we achieved excellent NGS data quality as determined by virtually perfect concordance between NGS and targeted single-gene tests for various genes in a large number of solid and liquid malignancy specimens.
Materials and Methods {#sec002}
=====================
Specimen Characteristics and Processing {#sec003}
---------------------------------------
Over the course of the study duration, 938 liquid and solid tumor specimens were submitted to the Center for Personalized Diagnostics ([Table 1](#pone.0152851.t001){ref-type="table"}). Specimens were eligible for NGS if they passed the tumor percentage, DNA quality, and DNA quantity thresholds that had been determined at the time of the validation of the NGS assay, which preceded the study period. Briefly, specimens with \<10% tumor were not eligible for NGS, because sequencing of samples with lower tumor percentages frequently yielded changes that were represented in fewer than five unique reads, making it difficult to distinguish true variants from sequencing artifacts. For similar reasons, DNA quality and quantity were judged to be insufficient, and the specimen was ineligible for NGS, if the DNA concentration was \<1 ng/μL; the DNA concentration was \<5 ng/μL with \>20% DNA degraded; the DNA concentration was \<50 ng/μL with \>45% DNA degraded; or DNA degradation was \>60%. Degraded DNA was defined as the proportion of DNA under 1000 bp in length.
10.1371/journal.pone.0152851.t001
###### Characteristics of Specimens by Tumor Site.
{#pone.0152851.t001g}
Tumor site Number of specimens submitted for NGS Specimens with DNA quality or quantity inadequate for NGS analysis Number of "shared" specimens (i.e., results are available from both NGS and targeted tests)
------------------ --------------------------------------- -------------------------------------------------------------------- ---------------------------------------------------------------------------------------------
Lung 196 13 (6.6%) 101 (51.5%)
Brain 174 9 (5.2%) 6 (3.4%)
Bone Marrow 161 0 95 (59.0%)
Lymph Node 70 8 (11.4%) 32 (45.7%)
Peripheral blood 56 0 29 (51.8%)
Liver 36 4 (11.1%) 5 (13.9%)
Skin 23 2 (8.7%) 5 (21.7%)
Other 222 24 (10.8%) 31 (14.0%)
Tumor site is not necessarily tissue of origin. Please note that in contrast to solid tumor specimens, all liquid specimens (bone marrow and peripheral blood) were adequate for NGS processing.
To determine the tumor percentage and volume of solid tumors, hematoxylin- and eosin-stained tissue specimens were evaluated by an anatomic pathologist, and the region with the highest tumor burden was marked. Genomic DNA was extracted from fresh bone marrow or peripheral blood using the Gentra Puregene Cell Kit (Qiagen, Netherlands). For formalin-fixed, paraffin-embedded (FFPE) specimens, tissues were macro-dissected from 5 μM or 10μM slides. Scrapings were dewaxed with Qiagen Deparaffinization Solution and purified with Gentra Puregene Tissue reagents following the manufacturer's protocol (Qiagen, Netherlands). DNA quantification was performed using the Qubit Broad Range assay following manufacturer's protocols (Life Technologies, CA). Agilent Genomic TapeScreens were used following manufacturer's protocols to assess the degree of DNA degradation (Agilent, CA).
Targeted Molecular Testing {#sec004}
--------------------------
### *EGFR*, *KRAS*, and *BRAF* Assays {#sec005}
The mutational status of *EGFR* exons 19 and 21 was determined using a laboratory-developed test (LDT) as previously described \[[@pone.0152851
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Introduction {#s1}
============
All organisms live in environments that vary through time and such environmental heterogeneity can impose highly variable selection pressures on populations. In this situation, an allele may be beneficial during one environmental regime and subsequently deleterious during another. Such an allele would be subject to short bursts of directional selection, alternately being favored and disfavored. When this situation occurs in diploids, the heterozygote can have a higher geometric mean fitness than either homozygote and allelic variation at this locus could be maintained for long periods despite being subject to directional selection at any given time [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1]. This situation is referred to as marginal overdominance and is a form of balancing selection.
There is substantial evidence for the maintenance of phenotypic and genetic variation by temporally variable selection in a variety of organisms. For instance, evolutionary response to rapid changes in selection pressures has been demonstrated for morphological and life-history traits in mammals [@pgen.1004775-Gershenson1], [@pgen.1004775-Grant1], birds [@pgen.1004775-Tarwater1]--[@pgen.1004775-Wall1], plants [@pgen.1004775-Brakefield1], invertebrates [@pgen.1004775-Hairston1]--[@pgen.1004775-RodriguezTrelles1], and others (reviewed in [@pgen.1004775-Bell1], [@pgen.1004775-Siepielski1]). Chromosomal inversions and allozyme alleles in a variety of drosophilids vary among seasons [@pgen.1004775-Dobzhansky1]--[@pgen.1004775-Ananina1] suggesting that these polymorphisms confer differential fitness in alternating seasons. Further, in some species of drosophilids, life-history [@pgen.1004775-Bouletreaumerle1], [@pgen.1004775-Schmidt1], morphological [@pgen.1004775-Stalker1], [@pgen.1004775-Tantawy1] and stress tolerance traits [@pgen.1004775-Miyo1], [@pgen.1004775-Dev1] also fluctuate seasonally suggesting that these traits respond to seasonal shifts in selection pressures.
Although theoretical models suggest that temporal variation in selection pressures can maintain fitness-related genetic variation in populations [@pgen.1004775-Gillespie1]--[@pgen.1004775-Hedrick1] and empirical evidence from a variety of species [@pgen.1004775-Gershenson1]--[@pgen.1004775-Dev1] demonstrates that variation in selection pressures over short time periods does alter phenotypes and allele frequencies, we still lack a basic understanding of many fundamental questions about the genetics and evolutionary history of alleles that undergo rapid adaptation in response to temporal variation in selection pressures. Specifically, we do not know how many loci respond to temporally variable selection within a population, the strength of selection at each locus, nor the effects of such strong selection on neutral genetic differentiation through time. We do not know whether adaptation at loci that respond to temporally variable selection is predictable nor do we know the relationship between loci that respond to temporally variable selection and spatially varying selection. Finally, it is unclear whether rapid adaptation to temporally variable selection pressures is primarily fueled by young alleles that constantly enter the population but cannot be maintained for long periods of time or, rather, by old alleles that have possibly been maintained by variable selection associated with environmental heterogeneity despite short bursts of strong directional selection.
To address these questions, we estimated allele frequencies genome-wide from samples of *D. melanogaster* individuals collected along a broad latitudinal cline in North America and in the spring and fall over three consecutive years in a single temperate orchard. We demonstrate that samples of flies collected in a single Pennsylvania orchard over the course of several years are as differentiated as populations separated by 5--10° latitude. We identify hundreds of polymorphisms that are subject to strong, temporally varying selection and argue that genetic draft [@pgen.1004775-Gillespie3] in the wake of rapid, multilocus adaptation is sufficient to explain the high degree of genetic turnover that we observe in this population over several years. We examine the genome-wide relationship between spatial and temporal variation in allele frequencies and find that spatial genetic differentiation, but not clinality *per se*, in allele frequency is a good predictor of temporal variation in allele frequency. Moreover, at SNPs subject to seasonal fluctuations in selection pressures, northern populations are more similar to spring populations than southern ones are. Next, we show that allele frequencies at SNPs subject to seasonal fluctuations in selection pressures become more 'spring-like' (i.e., they move towards the average spring frequency) immediately following a hard frost event and that seasonally variably SNPs tend to be associated with two seasonally variable phenotypes, chill coma recovery time and starvation tolerance. Finally, we demonstrate that some of the loci that respond to temporal variation in selection pressures are likely ancient, balanced polymorphisms that predate the split of *D. melanogaster* from its sister species, *D. simulans*. Taken together, our results are consistent with a model in which temporally variable selection maintains fitness-related genetic variation at hundreds of loci throughout the genome for millions of generations if not millions of years.
Results/Discussion {#s2}
==================
Genomic differentiation through time and space {#s2a}
----------------------------------------------
To test for the genomic signatures of balancing selection caused by seasonal fluctuations in selection pressures, we performed whole genome, pooled resequencing of samples of male flies collected in the spring and fall over three consecutive years (2009--2011) in a temperate, Pennsylvanian orchard. We contrast changes in allele frequencies through time with estimates of allele frequencies we made from five additional populations spanning Florida to Maine along the east coast of North America over a number of years (2003--2010) largely during periods of peak abundance of *D. melanogaster* ([Fig. 1A](#pgen-1004775-g001){ref-type="fig"}, [Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). From each population and time point, we sampled approximately 50--100 flies and resequenced each sample to average read depth of 20--200× coverage ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}, and see [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"}). Estimates of allele frequency using this sampling design have been shown to be highly accurate [@pgen.1004775-Gillespie3].
![Experimental design and genomic turnover through time and space.\
(A) Map of sampling locations in North America used in this study. Grey boxes represent individual samples from each locale. Genome-wide differentiation among spatially (B) and temporally (C) separated samples, measured as genome-wide average *F~ST~* (y-axis). Lines represent the predicted value of *F~ST~* based on the linear (A; y = a+bx) and non-linear (B; y = ab^X^) regression. Note: Pennsylvanian samples are not represented in (B) and the negative *F~ST~* in (B) results from the conservative correction of heterozygosity [@pgen.1004775-Rashkovetsky1], [@pgen.1004775-Turelli1]. In addition, please note that there are four estimates of pairwise *F~ST~* between the two replicate Maine and Florida samples (corresponding to a difference in latitude of 20°) and that there are two estimates of *F~ST~* between each of the remaining clinal populations and each Maine and Florida replicate sample. Error bars represent 95% confidence intervals based on 500 blocked bootstrap samples of ∼2000 SNPs.](pgen.1004775.g001){#pgen-1004775-g001}
As a point of departure and to provide context for understanding the magnitude of genetic variation through the seasons, we first examined genetic differentiation along the cline ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"}, [Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}). We calculated genome-wide average *F~ST~* among pairs of populations (excluding Pennsylvanian populations; hereafter 'spatial *F~ST~*') as well as the proportion of SNPs where average spatial *F~ST~* between a pair of populations is greater than expected by chance conditional on our sampling design and assuming panmixia using allele frequency estimates of 500,000 common polymorphisms ([Table S1](#pgen.1004775.s008){ref-type="supplementary-material"}). Genome-wide average spatial *F~ST~* ([Fig. 1B](#pgen-1004775-g001){ref-type="fig"}) as well as the proportion of SNPs where spatial *F~ST~* is greater than expected by chance ([Fig. S1A](#pgen.1004775.s001){ref-type="supplementary-material"}) is positively correlated with geographic distance (*r* = 0.75; *p* = 7e-5), a pattern consistent with isolation by distance [@pgen.1004775-Wright1]. Pooled resequencing did identify polymorphisms in or near genes previously shown to be clinal in North American populations (see [Text S1](#pgen.1004775.s011){ref-type="supplementary-material"}) demonstrating that clines are stable over multiple years. This suggests that populations sampled along the cline represent resident
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###### Strengths and limitations of this study
- The study data were obtained through a questionnaire carried out by face-to-face interviews. The youth were trained to obtain dietary diet from their parent and grandparent. They were accompanied by the research assistant during data collection.
- This study provides important insights of differences in food types consumed between the younger and older Pacific generation.
- Exploratory factor analyses was used to examine dietary data between groups, providing robust findings.
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- A major limitation is the small sample size of the study; however, this is a feasibility study, and the results are limited to the study participants.
- The use of the dietary diversity questionnaire is based on a limited sampling frame of food group diversity over a 7-day period; therefore, it may not be completely representative of all food items and food groups that may have been consumed by the different age generations.
Introduction {#s1}
============
Pacific peoples in New Zealand (NZ) are at greater risk of developing long-term conditions such as prediabetes, diabetes[@R1] and cardiovascular disease[@R4] due to obesity, defined as having a body mass index (BMI) \>30 kg/m^2^. The prevalence of obesity (67% in adults aged 15+ years) in Pacific peoples is twice that in the general population (33%).[@R5] Obesity has been viewed as a result of the changing westernised environmental pressures,[@R6] leading to an energy expenditure/energy storage mismatch that operates through dietary behaviours.[@R6] Often modernised dietary patterns, characterised by high energy dense and palatable food,[@R8] and eating habits, such as having more access to less healthy snacks and food,[@R9] explain the weight gain.[@R11] The findings from the NZ national nutritional survey on Pacific peoples reported similar patterns of energy, micronutrient intake, dietary supplement use and dietary habits (eg, consumption of breakfast and other food types, fruit and vegetable intake and salt intake), compared with the general population.[@R12]
Obesity rates continue to rise for all young people aged 15--24 years, with the highest increase among young Pacific peoples (up by 50% between 2007 and 2012).[@R13] Previous NZ research has documented the impact of family meals on the dietary quality of young people[@R14] and found that eating family meals together had an overall positive effect on the home food environment[@R15] and that it encouraged the availability of more healthy food.[@R10] Results from the Youth'12 Survey highlighted similar findings that shared family meals were associated with positive outcomes for young people, which were accentuated for those living in the lower deprivation neighbourhoods (66%), compared with those in the higher deprivation areas (58%).[@R16] However, the survey results do not provide any clues as to the obesity-related mechanisms to further understanding about why young people continue to eat unhealthily. It has also been shown that young people experiencing poverty, irrespective of living in either low deprivation or more affluent neighbourhoods, does not explain overweight and obesity issues for young people.[@R17] Little research has focused on understanding the Pacific concept of socialisation and food as inter-related activities, and there is increasing recognition of the importance of this if obesity prevention strategies are to effective.[@R18]
In 2014, we investigated obesity-related health issues among 30 Pacific youth from the Wellington and Auckland regions of NZ in the pilot study, '*Chewing the facts on fat! What does that say about me?*'. The methodology and scope of the study has been published elsewhere.[@R19] As part of that study, we also examined Pacific youths' diet and eating habits as it relates to obesity development. In particular, we explored dietary diversity as a form of investigating diet quality, which could be useful in identifying dietary component needs and provide insights to guide development of intervention strategies to improve diet quality and health outcomes for young Pacific peoples. The aim of the current paper is to examine the intergenerational dietary patterns among 30 young Pacific adults aged 15--24 years, and 34 parents and grandparents, from Wellington and Auckland regions.
Method {#s2}
======
From a pilot study of 30 young Pacific adults aged 15--24 years in Wellington and Auckland, NZ, we investigated the social cultural determinants of the obesogenic environment. The study was conducted in two phases, and the original study methodology has been previously published,[@R19] which described the recruitment, questionnaire data and the social demography of the participants, from phase 1. This paper presents data mainly from phase 2, obtained from young Pacific peoples who were trained to interview and obtain information from one Pacific parent and one Pacific grandparent to examine and compare older generation's dietary habits with those of the Pacific youth group (phase 1) using the Pasifika dietary diversity questionnaire (described below). As Pacific peoples are not a homogenous group, we will refer to them as Pasifika, defined as a collective group of people representing the different Pacific Island nations and their respective languages, social cultural realities and protocols.
Patient involvement {#s2a}
-------------------
Patients were not involved in the planning or design of the study.
Demography {#s2b}
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Basic descriptive data were obtained from the parent and grandparent as per the protocol (face to face) described in phase 1 of the study,[@R19] including measured weight and height, from which BMI was determined. We used the international standard cut-offs in defining obesity.[@R20] BMI was analysed as a continuous variable, with a BMI of ≥30 kg/m^2^ and 25--29.9 kg/m^2^ defined as being obese and overweight, respectively.[@R21] Waist-to-hip circumference was also measured, from which the waist--hip ratio (WHR) was determined to provide a measure of central adiposity to indicate associated risk of incident cardiovascular events.[@R22] The waist-to-height ratio (WtHR) was also calculated, as an adjunct measure of central obesity, which is less prone to measurement error than WHR.[@R23] Deprivation was assessed using the NZDep2013 measure,[@R25] a small area-based measure of deprivation derived from the 2013 Census, which uses nine variables (benefit income, employment, household income, communication, transport, support, qualifications, living space and home ownership) from the Census to place small area blocks on a deprivation scale from 1 to 10 with 10 representing the most deprived 10% of NZ areas, while 1 represents the 10% least deprived areas. For analyses, deprivation was categorised into quintiles combining deciles 1--2, 3--4, 5--6, 7--8 and 9--10. Ethnicity was defined according to standard NZ Census data[@R26] and self-identified by each participant; however, for participation in the project, parents and grandparents needed to be self-identifiable as being of Pacific ethnicity. Where possible, the participants were also invited to record any physician-diagnosed conditions to highlight the presence of morbidity.
Dietary diversity {#s2c}
-----------------
The Pacific dietary diversity questionnaire was compiled by the research team to assess the scope of individual foods and food groups in Pacific peoples' diets. The dietary diversity questionnaire aims to capture the 'range of food' that people consume over a 7-day period and not to measure quantity of food items like the 'food frequency questionnaire'. This type of assessment has been used among other indigenous groups successfully, and it has been proven to be effective than a quantitative assessment of describing the quality of the diet.[@R27] This questionnaire was pretested among an independent community group of Pasifika youth (n=30) from Wellington, and it was adapted to include common food that would be consumed by Pasifika people in NZ (eg, povi masima \[salted meat\]).
Data on different individual foods and food groups that were consumed over a 7-day period (reference period) were collected and recorded (dichotomised: yes/no) by the Pacific youth who were participants in phase 1 and were trained by the research team at a single day workshop. The training involved familiarising and understanding the questionnaire and prompts. Following the training day, each youth arranged and organised a face-to face interview (accompanied by a research assistant), with a parent and grandparent. The questionnaire included both nutritious and discretionary food and food groups to encapsulate the diversity of food groups and of food items. The questionnaire data produced a total of 26 food groups (15 nutritious and 11 discretionary) specifically consumed by the study participants. However, using exploratory factor analyses (EFA) (see below) in order to create meaningful summary patterns that describe types of diet, we had refined the food groups down to 13, as determined by the total percentage of items consumed within a food grouping, per person. The groupings are: group 1: meats, poultry and fish diversity; group 2: dairy products diversity; group 3: bread, cereals and starchy vegetable diversity; group 4: legumes and nut diversity; group 5: fruit diversity; group 6: vegetable diversity; group 7: oil and fat diversity; group 8: drinks diversity; group 9: alcohol diversity; group 10: sauces, spreads and flavouring diversity; group 11: sweets and sweet snacks diversity; group 12: savoury snacks diversity; and group 13: take way food diversity.
Eating habits and meal patterns, food choices and related cultural and social influences were also investigated, but the results are not presented here.
We also included a measure of acculturation using a tool developed by coauthor JK and his colleagues[@R33
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1. Introduction {#sec1-ijerph-17-00783}
===============
The decline in youth's healthy behaviors and related consequences \[[@B1-ijerph-17-00783]\] is of concern for public health in general and for national defense in particular, which requires a sufficient number of physically fit and mentally healthy military personnel. In Lithuania, there is evidence that young people do not partake in adequate physical activity; that is, only around 30% of students aged 18 years comply with the recommendation to be active ≥1 h on at least 5 days a week \[[@B2-ijerph-17-00783]\]. The trends for health-related physical fitness among adolescents have deteriorated in the past 20 years, and the indicators of cardiorespiratory fitness have decreased by nearly 50% during this period \[[@B3-ijerph-17-00783]\]. Only 13--14% of high school students comply with recommendations for healthy nutrition \[[@B4-ijerph-17-00783],[@B5-ijerph-17-00783]\], 11% of young people consume alcohol \[[@B6-ijerph-17-00783]\]. Moreover, 22% experience psychological distress \[[@B7-ijerph-17-00783]\]. Multiple health behaviors, psychological distress, and their associated risk factors, such as obesity and poor physical fitness, are associated with poor mental health outcomes, the risk of cardiovascular disease, type 2 diabetes, certain types of cancer \[[@B8-ijerph-17-00783],[@B9-ijerph-17-00783]\], depression \[[@B10-ijerph-17-00783],[@B11-ijerph-17-00783]\], and anxiety \[[@B12-ijerph-17-00783]\]. Unhealthy behaviors also impose an economic burden; for example, one study estimated that physical inactivity alone costs USD 53.8 billion in 2013 for healthcare worldwide \[[@B13-ijerph-17-00783]\].
Health is the main criterion for accepting or rejecting young men into military service (MS) and is strongly related to the ability to perform military duties. Unsatisfactory results of youth recruitment to Lithuanian MS have been presented: on average, 58.6% of Lithuanian young men proceed through the full procedure of military enlistment. Among those who do not pass these procedures, 33%--37% experience psychological problems, 29%--33% have cardiovascular diseases, and 13% have musculoskeletal problems \[[@B14-ijerph-17-00783]\]. Other countries face similar problems of rejection from MS. For instance, analysis of the reasons for rejection from MS in the USA found that about 22% of rejections were because of problems with bones or joints, flat feet, or hernias, 15% because of organ defects, 13% because of defects of the cardiovascular system, 12% because of nervous system or mental problems, and 10% because of communicable diseases \[[@B15-ijerph-17-00783]\]. In the USA, Hispanic men appear to have a better health profile than their white and black peers, except for the prevalence of overweight, which is higher in Hispanic men \[[@B16-ijerph-17-00783]\].
Although health behaviors alone are not criteria for enlistment into MS, they might explain the reasons for some instances of rejection. Given that some health behaviors are risk factors for the occurrence of many lifestyle-related diseases \[[@B17-ijerph-17-00783]\], it is critical to identify whether and how health behaviors differ between young men who are deemed eligible and those who are ineligible for MS and to take appropriate actions to prevent adverse health behaviors from their onset.
Soldiers must be both physically and mentally healthy. However, mental health issues are among the main factors for rejection from MS \[[@B14-ijerph-17-00783],[@B15-ijerph-17-00783]\]. Identifying psychological distress along with health behaviors might help to provide a more complete understanding of health indicators in conscripts as psychological distress is an indicator of mental health \[[@B18-ijerph-17-00783]\].
Given the associations between many health-related behaviors, the complex analysis of a set of risk behaviors instead of evaluation of individual associations may help to reduce the risk of missing potential confounders for enlistment into MS \[[@B19-ijerph-17-00783]\].
Several studies have examined psychological distress, health behaviors \[[@B20-ijerph-17-00783],[@B21-ijerph-17-00783]\], and changes in health behavior \[[@B22-ijerph-17-00783]\] during MS. Enlistment is based on a medical examination of draftees, which may reject unhealthy individuals. As a result, these studies have evaluated health behaviors in relatively healthy youth but have not examined whether health behavior is related to the rejection of military recruits.
The aim of this study is to identify and compare health behaviors and psychological distress between male conscripts rejected for and enlisted into MS. We expect to find that healthier behaviors and low distress level would be related to a higher rate of enlistment into MS.
2. Materials and Methods {#sec2-ijerph-17-00783}
========================
2.1. Study Design and Procedure {#sec2dot1-ijerph-17-00783}
-------------------------------
This nationally representative cross-sectional study was performed among Lithuanian conscripts. In Lithuania, 9-month-long MS is compulsory. In accordance with the conscription procedure of the Lithuanian Armed Forces, a list of potential draftees is created by an automatic electronic selection system each year and includes male Lithuanian citizens of compulsory MS-eligible age (19--26 years). Other male and female young adults can express their wish to be conscripted on a priority basis.
A stratified random sampling was used. There are four centers for military recruitment in Lithuania that recruit conscripts during the whole year. The data were gathered in all four centers for 4 months from June to October 2018. Each conscript in these centers was approached during this period and asked to sign a consent form to participate in the study and to complete the study questionnaire. There was an equal chance (probability) that participants included in the study would be enlisted or rejected for MS.
The decision to accept a recruit for enlistment into MS is made by medical experts and is based on an individual medical examination and previous medical reports. The criteria for rejection from MS are defined in Order V-1142/V-1139, which provides a list of disorders along with their severity, and is signed by the Ministers of State Defense and Health Care. These criteria also indicate the minimum height requirement: 160 and 155 cm for men and women, respectively. Obesity without comorbid illness is not a reason for rejection from MS \[[@B23-ijerph-17-00783]\]. Enlistment in and rejection from MS were identified from the medical records for each study participant.
2.2. Participants {#sec2dot2-ijerph-17-00783}
-----------------
In 2018, there was a list of 10,340 conscripts, 209 of them female. This study included 1427 conscripts (which represented 13.80% of the total population), of whom 1296 returned their completed questionnaire along with their consent to participate in the study. The response rate was 90.82%. Female recruits were invited to participate in the study, but because only 53 agreed to participate, they were later excluded because of the small sample size. Finally, 1243 young male potential conscripts were included in the analysis. The participants were aged 19--26 years and their mean age was 22.50 ± 2.43 years.
The Ministry of National Defense of the Republic of Lithuania approved the research. Ethics approval (No SMTEK-28, 2018) was obtained from the Ethics Committee of Lithuanian Sports University. The investigations were carried out following the rules of the Declaration of Helsinki of 1975, revised in 2013. Participants were informed of the tasks in the study before data collection, and all participants gave their informed consent for inclusion before they participated in the study.
2.3. Measurements {#sec2dot3-ijerph-17-00783}
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### 2.3.1. Physical Activity {#sec2dot3dot1-ijerph-17-00783}
The World Health Organization (WHO) defines moderate physical activity as activity that noticeably accelerates the heart rate and includes activities equivalent in intensity to brisk walking or bicycling. Vigorous physical activity causes rapid breathing and substantially increases heart rate, and includes activities such as jogging, aerobic dance, and bicycling uphill \[[@B24-ijerph-17-00783]\]. To assess physical activity in the participants in this study, we used the 2005 US Department of Defense Survey of Health Related Behaviors Among Active Duty Military Personnel \[[@B21-ijerph-17-00783]\]. The study participants were asked, "During the past 7 days, for leisure-time physical activity, how often did you usually do each of the following?". Participants were also asked, "During the past 7 days, when you did leisure-time physical activity, how long did you usually do each of the following?" For both questions, detailed descriptions and examples of what constitutes moderate and vigorous physical activity were presented.
In this study, we assessed physical activity during the preceding 7 days instead of the 30 days in the original questionnaire. Also, instead of using categorical answers (such as "5 or 6 days" and "at least 20 minutes") for each question, we provided the opportunity to write the exact numbers of days, hours, and/or minutes per day. The number of minutes spent in was totaled. Participants whose MVPA was \<2.5 h/week were coded as not meeting health-related physical activity requirements and those whose MVPA was ≥2.5 h/week were coded as meeting health-related physical activity requirements \[[@B25
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Background {#Sec1}
==========
The prevalence of obesity in the general population has increased dramatically over the last 30 years and it seems likely that the environmental changes that have provoked these increases have also affected people with severe mental illness (SMI); in fact, the rates of overweight and obesity have increased even more rapidly in this cohort \[[@CR1]\]. Obesity adversely affects the physical health and psychological well-being of people with SMI and if weight gain is attributed to treatment, this can lead to non-adherence and risk of relapse.
Schizophrenia is a major psychiatric disorder that alters the individual's perception, thoughts, affect and behaviour and may involve a loss of insight and has a lifetime prevalence of approximately 1% \[[@CR2]\]. Schizoaffective disorder is recognised as a separate condition to schizophrenia and is more likely to occur in women at a later age. This disorder affects an individual's thoughts and emotions \[[@CR3]\]. Although individuals with first-episode psychosis do not fulfil the diagnostic criteria for schizophrenia or schizoaffective disorder, 90--95% of people presenting with a non-affective psychotic episode (i.e. not mania and not depressive psychosis) will still meet the criteria for a schizophrenia spectrum disorder 2 years later. Mortality rates are increased two to three fold in people with SMI and life expectancy is reduced by 10--20 years. Approximately 75% of all deaths in people with schizophrenia are caused by physical illness with cardiovascular disease being the commonest cause \[[@CR4]\]. Overweight and obesity contribute to this excess morbidity and mortality. Recent studies indicate that obesity is two to three times more common among people with SMI \[[@CR5]\]. Obesity occurs early in the natural history of schizophrenia with a significant proportion of people with first-episode psychosis being overweight prior to any treatment. Substantial weight gain (\> 7%) often occurs rapidly within 6--8 weeks after antipsychotic-treatment initiation \[[@CR6]\]. While most weight gain occurs early in treatment, longer-term observational studies suggest that weight gain continues for at least 4 years albeit at a slower rate \[[@CR7]\].
Individuals with schizophrenia are more likely to consume a diet that is rich in fat and refined carbohydrates while containing less fibre, fruit and vegetables than the general population \[[@CR8]\]. Although there are fewer studies, people with first-episode psychosis also have poor diets \[[@CR9]\]. Physical inactivity and the social and urban deprivation experienced by those with schizophrenia may contribute further to the increased obesity rates \[[@CR8], [@CR10]\]. There may be disease-specific effects of schizophrenia, such as genetic susceptibility, that have additive or synergistic actions to increase body weight further \[[@CR5]\]. However, the most important factor related to weight gain in people with SMI is the use of antipsychotic medications, which are among the most obesogenic drugs. Weight gain is the commonest side effect of second-generation antipsychotic medication, affecting between 15 and 72% of patients \[[@CR11]\]. Other psychotropic drugs are often prescribed to people with schizophrenia and include some antidepressants and mood-stabilising drugs, such as lithium and sodium valproate; these may also induce significant weight gain \[[@CR12]\].
Both lifestyle and pharmacological interventions lead to significant reductions in body weight in the general population. Not only are the interventions clinically effective, they are also cost-effective because of the benefits of long-term improved health outcomes, including decreased mortality \[[@CR13]\]. It is likely that similarly effective interventions for people with schizophrenia will also lead to improvements in health and would be a major step towards reducing the health inequalities experienced by people with schizophrenia. As the weight gain associated with antipsychotic medication result in some people discontinuing their medication, we hypothesis that effective weight-management strategies may also lead to improved adherence to antipsychotic medication and reduced relapse and hospitalisation rates.
Some studies have suggested that short-term lifestyle interventions could support weight reduction in people with SMI. A meta-analysis of non-pharmacological interventions in people with SMI \[[@CR14]\] reported a mean reduction in weight of 3.12 kg over a period of 8--24 weeks. However, the results of longer-term studies are more mixed. A recent meta-analysis found significant weight loss in only two of six studies with interventions lasting longer than a year \[[@CR15]\]. Most studies have included a mixed population of people with SMI and two large studies, which included only people with schizophrenia, found no effect of a lifestyle intervention on body weight \[[@CR16], [@CR17]\]. These latter studies suggest the weight management in people with schizophrenia may require a different approach from other SMIs such as bipolar disorder.
Given the challenges of implementing lifestyle change in people with schizophrenia and the lack of long-term effectiveness, alternative approaches are needed to manage overweight and obesity. A wide variety of treatments have been subject to clinical studies but currently no drug treatments are licensed for the treatment of antipsychotic-medication-associated weight gain or obesity in people with SMI with the exception of orlistat \[[@CR18]\]. The long-term use of the latter, however, is extremely limited by high discontinuation rates, making it of little value in routine clinical practice \[[@CR19]\].
To date, there have also been three completed trials of glucagon-like peptide 1 (GLP-1)-receptor agonists in people with SMI, two of which used exenatide and one used liraglutide (maximum dosage 1.8 mg) \[[@CR20]--[@CR22]\]. Liraglutide is a GLP-1-receptor agonist, with 97% homology to human GLP-1, which induces weight loss in humans mainly by reducing appetite and caloric intake, rather than increasing energy expenditure. There were contrasting results in the exenatide studies with one showing no difference between groups after 12 weeks of treatment \[[@CR20]\] but in the other the exenatide arm had greater mean weight loss (− 5.29 vs − 1.12 kg; *P* = 0.015), and reduced glycosylated haemoglobin (HbA~1c~) levels (− 0.21% vs 0.03%; *P* = 0.004) \[[@CR21]\]. In the liraglutide (maximum dosage 1.8 mg) study, glucose tolerance improved in the liraglutide group and body weight decreased compared with placebo (− 5.3 kg; 95% confidence interval (CI) − 7.0 to − 3.7 kg) \[[@CR22]\].
Liraglutide is approved for the management of obesity at a dosage of 3.0 mg daily, which is higher than the dosage used to treat diabetes \[[@CR23]\]. In a 56-week, double-blind trial involving 3731 participants without type-2 diabetes, 63.2% of the intervention arm compared with 27.1% in the placebo arm group lost at least 5% of their body weight, and 33.1% and 10.6%, respectively, lost more than 10% of their body weight \[[@CR23]\]. We have, therefore, chosen to use liraglutide (maximum dosage 3.0 mg) as we postulate that a higher dosage of liraglutide may offer even greater weight loss in people with SMI than the 1.8-mg dosage employed in the previous study or other currently available GLP-1-receptor agonists.
Aims and objectives {#Sec2}
-------------------
The aim of this pilot study is to undertake a double-blind, randomised controlled trial (RCT) of the use of liraglutide (maximum dosage 3.0 mg daily) in comparison to placebo in 60 obese or overweight people with schizophrenia, schizoaffective disorder or first-episode psychosis to assess the feasibility and acceptability of delivering a full-scale trial evaluating treatment with liraglutide in people with schizophrenia, schizoaffective disorder and first-episode psychosis.
Methods {#Sec3}
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Design {#Sec4}
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This study is a double-blind, randomised pilot study of the use of liraglutide (maximum dosage 3.0 mg daily) in comparison to placebo (Fig. [1](#Fig1){ref-type="fig"}). It is important to include a double-blind placebo for two main reasons; there is evidence that people would be less likely to consent to a trial that includes a placebo arm because of the risk of not receiving an active treatment. As our ability to recruit to the trial was one of our key aims, it is important to assess whether the inclusion of a placebo prevented us from recruiting to the trial. Previous experience from weight-management trials that include pharmaceuticals have been plagued by high dropout rates in the placebo arm as the participants are able to assess the effectiveness of treatment. To adequately power a full RCT of liraglutide, we will need to know the likely dropout rate in the placebo group in this patient population. Fig. 1Consolidated Standards of Reporting Trials (CONSORT) study diagram
Setting {#Sec5}
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The study will be take place in a variety of community and inpatient mental health locations in the Southern Health NHS Foundation Trust. Public and Patient Involvement was actively included in the development of the trial and will continue throughout the trial.
Ethics approval and consent to participate {#Sec6}
------------------------------------------
South Central -- Hampshire B Research Ethics Committee (REC) approved the study on 17 April 2018 with REC reference: 18/SC/0085. Only those who agree to provide written informed consent will be included in the study. The study will be conducted in keeping with Good Clinical Practice (GCP) and the International Conference of Harmonisation (ICH) standards. The Trial Steering
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Introduction {#s1}
============
Traditionally, ecological and eco-evolutionary epidemiological models describe the dynamics of infectious diseases by considering susceptible, infected and recovered hosts with host-to-host, or host-environment-host transmission [@pone.0071621-Kermack1]--[@pone.0071621-Hudson1]. A number of modifications--such as seasonality [@pone.0071621-Altizer1], or within-host dynamics [@pone.0071621-Mideo1]--have been introduced to the SI- and SIR-models in various attempts to explain recurrent outbreak disease dynamics. However, natural epidemics often show a variety of dynamics that do not correspond to the predictions made by the classical models. One reason for this is that the underlying assumptions on disease transmission are unrealistic for pathogens that spend a considerable amount, or even the most part of their life cycle, in the outside-host environment. A large proportion of opportunist pathogen species also grow actively in the outside-host environment. These environmental pathogens form an increasing problem for human health [@pone.0071621-Cangelosi1], and thus a better theoretical understanding of their epidemiology is required. Currently, most models for environmental transmission allow only decay of pathogens in the outside-host environment [@pone.0071621-Codeco1], [@pone.0071621-Day1], but see [@pone.0071621-Merikanto1]. In addition, the outside-host environment is riddled with other microbes which frequently interact with the pathogen. This means that while active growth as well as ecological interactions in the environment are likely to be profoundly important, they are yet poorly understood factors in disease dynamics.
The role of environmental transmission in disease dynamics and the evolution of virulence has attracted increasing interest [@pone.0071621-Day1], [@pone.0071621-Roche1], both due to human pathogen outbreaks such as cholera [@pone.0071621-Colwell1] and emergent animal diseases, e.g. columnaris disease [@pone.0071621-Pulkkinen1]. Worldwide, there is an ongoing battle against opportunistic infections, which are often persistent due to the pathogens' ability to grow outside hosts. Broad-spectrum antibiotics and disinfectants are used *en masse* to prevent environmental infections to humans and cultivated animals. This is likely to cause changes in the composition of environmental communities and have an impact on ecosystem functioning, health and disease [@pone.0071621-Ding1]. Theoretically, an environmentally transmitted pathogen can be highly lethal as the trade-offs between transmission and virulence associated with obligate pathogens are reduced [@pone.0071621-Walther1]. This is because by killing a host--which is not required to be alive for pathogen transmission--the pathogen gains access to an enormously rich resource for saprotrophic growth, which can lead to a positive transmission-virulence relationship [@pone.0071621-Kunttu1]. In most studies, however, the environment represents simply a reservoir into which the pathogen particles are shed from infected hosts and from which the surviving pathogen individuals may re-enter susceptible hosts, without explicit description of the outside-host dynamics other than the decay rate of the pathogen.
Interspecific interactions, such as competition, mutualism, predation, and parasitism constitute the core of ecological research. An important implication of these interactions is that the dynamics and stability of individual populations within ecological networks (e.g., communities or food webs) can strongly depend on the composition of these networks and the details of between-species interactions [@pone.0071621-DeRuiter1]--[@pone.0071621-Pimm1]. In general, similar co-occurring species compete for limiting resources [@pone.0071621-Hibbing1] and are attacked by parasites and predators [@pone.0071621-Abedon1]. All of these different ecological interactions can affect the density of pathogens and other interacting species in the community, thereby affecting the probabilities of infection outbreaks. Therefore, understanding the role of ecological interactions in the outside-host environment is likely to be of great importance for uncovering mechanisms behind the dynamics of many environmentally transmitted diseases such as *Vibrio cholera* [@pone.0071621-deMagny1], group A *streptococci* [@pone.0071621-Beres1], *Staphylococcus aureus* [@pone.0071621-Eveillard1], and *Flavobacterium columnare* [@pone.0071621-Pulkkinen1].
We explore the dynamics of a model that combines environmental opportunist pathogen--host dynamics to community dynamics outside the host. By the term environmental opportunist pathogen we mean an organism that is both (i) able to grow in the environment in the absence of hosts and (ii) infect susceptible hosts. Whether the pathogen can infect one or several host species is not important in this simple model in which we consider all (susceptible) hosts similar from the pathogens perspective. The model contains susceptible and infected hosts as in a classical SI-model and a competitive community in the outside-host environment. One of the competitors is a pathogen that can return from a dead host to the environment and thus the host does not represent an ecological or evolutionary dead end for the pathogen. This is opposite to the common assumption of the theory of "co-incidental virulence" [@pone.0071621-Brown1]. Further, we assume that there is a trade-off between virulence and environmental competitive ability. This assumption also differs from the expectation under co-incidental virulence theory [@pone.0071621-Brown1], where resource acquisition or fighting against natural enemies outside the host are positively linked to a pathogens ability to cause infections. Life-history trade-offs can reduce virulence because the machineries for resource acquisition and defence in the outside- vs. inside-host environments require specialisation [@pone.0071621-Gower1], [@pone.0071621-Mikonranta1]. The choice of the functional form of the infectivity response can crucially affect the model dynamics [@pone.0071621-Boldin1], [@pone.0071621-McCallum1]. We explore both linear and sigmoidal infection rate in response to pathogen density, and assume that the infected hosts can either recover back to the susceptible class or die from the disease. The sigmoidal infectivity response incorporates dose-dependence, i.e., exposure to pathogen densities below a certain level is unlikely to cause an infection, as observed in many laboratory experiments [@pone.0071621-Aaby1]--[@pone.0071621-McLean1]. The model behavior is explored in a parameter range that is likely to cover typical environmentally growing opportunist micro-parasites (e.g., bacteria, protozoa, or fungi) that infect multicellular hosts ranging from taxa with fast growth rates (e.g., nematodes and insects) to taxa with slow growth rates (e.g., vertebrates).
A striking feature of the model is that reducing competitor species richness in the outside-host environment can lead to an abrupt emergence of disease outbreaks. If the infectivity response of the pathogen is a sigmoidal function of pathogen density in the environment, epidemiological dynamics are sensitive to the intensity of competitive suppression by the outside-host community. With linear infectivity response and pathogen growth in the environment no disease outbreaks are observed, i.e. population dynamics remain stable, and pathogen and host densities are relatively insensitive to manipulation of diversity. Under sigmoidal infectivity, reduction of competitive pressure on the pathogen, either due to loss of diversity from the outside-host community (e.g., due to use of disinfectants), or increased loss rate of all species in the outside-host community (e.g., due to application of non-specific antibiotics) can lead to catastrophic disease outbreaks.
Methods {#s2}
=======
The dynamical model is a combination of the classical SI-disease dynamics [@pone.0071621-Kermack1] for hosts and the Lotka--Volterra competition model for an outside-host community. The susceptible and infected hosts at time *t* are denoted by *S*(*t*) and *I*(*t*), respectively. The pathogen, *P*(*t*), has *n* competitors, with densities denoted by *B~i~*(*t*), i.e., community size is *N* = *n* +1. The population densities vary according to the following differential equations:
In the absence of an infection, susceptible hosts follow a logistic growth model with a growth rate *r~h~* and a carrying capacity *K~h~* (eqn. 1.1). In the presence of an infective pathogen infected host individuals are formed. The infected individuals compete for resources with the susceptible individuals, but do not contribute to host reproduction (this assumption has no qualitative effect on the dynamics). Susceptible hosts are infected with a rate *βSf*(*P*) depending on the infectivity response *f*(*P*). Two alternative infectivity response functions were explored. Most previous theoretical work has assumed a linear infectivity response. However, we argue that a more realistic assumption for many circumstances is a sigmoidal dose-dependent response that is supported by empirical data [@pone.0071621-Aaby1]--[@pone.0071621-McLean1] as well as theoretical analysis [@pone.0071621-Pujol1]. The sigmoidal infectivity function has the following mechanistic interpretation. With low pathogen densities the immune system can effectively overcome most pathogen invasions and therefore the probability of an infection per unit time must increase nonlinearly at low densities. With high pathogen densities the effect of increasing pathogen density on the probability of infection per time unit must saturate since the
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All coordinates and structure factor files are available from the PDB database (accession numbers: 6KR2 and 6KR3).
Introduction {#sec001}
============
The flaviviruses are a large group of positive-strand RNA viruses, including dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), tick-borne encephalitis virus (TBEV), and Omsk hemorrhagic fever virus (OHFV). The majority of flaviviruses are mosquito-borne or tick-borne, sometimes causing human encephalitis or hemorrhagic diseases. The recent ZIKV outbreaks in South and North America, and more recently in Southeast Asia, have intensified the global threats of flaviviruses, in part due to the capabilities of the virus to cause birth defects through maternal-fetal transmission \[[@ppat.1008484.ref001]\]. The flavivirus RNA genome is 10--11 kilo-bases in length, bearing a type 1 cap and lacking a poly-adenine tail. It encodes a large polyprotein that is further processed by viral and host proteases, yielding three structural proteins C/prM/E, and seven nonstructural proteins NS1/NS2A/NS2B/NS3/NS4A/NS4B/NS5 \[[@ppat.1008484.ref002]\]. Being a unique natural fusion of an N-terminal methyltransferase (MTase) and a C-terminal RNA-dependent RNA polymerase (RdRP), the NS5 is the largest and most conserved protein encoded by flaviviruses. The NS5 MTase catalyzes the guanylyltransfer and both the guanine N7 and nucleoside 2′-O methylation steps in the capping process, and is a single-domain module adopting a common S-adenosyl-L-methionine (SAM)-dependent MTase fold \[[@ppat.1008484.ref003],[@ppat.1008484.ref004]\]. The K-D-K-E catalytic tetrad sits in the center of the MTase catalytic cleft, with the methyl donor SAM binding site and the cap binding site residing on the opposite sides. The RdRP module is the central molecular machine governing the viral genome replication, and has an encircled human right hand architecture with palm, fingers, and thumb domains surrounding the active site \[[@ppat.1008484.ref005],[@ppat.1008484.ref006]\]. The fingers domain can be further divided into index, middle, ring, and pinky subdomains according to nomenclatures first used in describing the poliovirus (PV) RdRP ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}) \[[@ppat.1008484.ref007],[@ppat.1008484.ref008]\]. Among the seven viral RdRP catalytic motifs, A/B/C/D/E are within the most conserved palm, and F/G are part of the ring and pinky fingers, respectively. Motifs A/B/C/F contain amino acids highly conserved in all viral RdRPs, and these conserved residues have highly analogous spatial arrangements around the polymerase active site \[[@ppat.1008484.ref009],[@ppat.1008484.ref010]\]. Being an entity bearing two active sites and multiple essential viral enzymatic activities, NS5 has become a very attractive system in flavivirus research, and understanding the interplay between its MTase and RdRP modules is undoubtedly critical.
{#ppat.1008484.g001}
Among the evidence related to MTase-RdRP crosstalk, high-resolution structures of full-length NS5 are essential in providing direct and informative readout of the interactions between the two modules. To date, two types of global conformations have been observed in full-length NS5 structures \[[@ppat.1008484.ref011]\]. The conformation revealed by the JEV NS5 structure (named JEV-mode hereinafter) features a medium size interface (\~1540 Å^2^, for all interface area values presented in this study, the total buried solvent accessible surface from both side of the interface was accounted) with a conserved hydrophobic core \[[@ppat.1008484.ref008],[@ppat.1008484.ref012]\], and is also observed in recently reported ZIKV, yellow fever virus (YFV), and DENV serotype 2 (DENV2) full-length NS5 crystal structures \[[@ppat.1008484.ref013]--[@ppat.1008484.ref018]\]. In such a conformation, the MTase approaches the RdRP from its backside and interacts with the RdRP middle finger, ring finger, and an index finger helix bearing part of a nuclear localization signal (NLS-helix) \[[@ppat.1008484.ref019]\] ([Fig 1A](#ppat.1008484.g001){ref-type="fig"}). The second conformation was observed in two different crystal forms of DENV serotype 3 (DENV3) NS5 (named DENV3-mode hereinafter) \[[@ppat.1008484.ref020],[@ppat.1008484.ref021]\] ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). In this case, the MTase also approaches the RdRP from the backside, but it is related to the JEV conformation by an approximately 110° rotation around an axis passing near its center of mass with less than 6 Å translation. The RdRP index and middle fingers are still involved in the interactions in a slightly larger interface (\~1650--1780 Å^2^), and the nature of the interactions is instead primarily polar. Notably, the NTP binding ring finger (motif F) contacts with the MTase are absent in the DENV3 structures and the ring finger itself and the adjacent motif G residues in the pinky finger are largely disordered ([Fig 1B](#ppat.1008484.g001){ref-type="fig"}). Motif G participates in RNA template binding and has been proposed to participate in the translocation step after every phosphoryl transfer reaction \[[@ppat.1008484.ref022],[@ppat.1008484.ref023]\]. Hence, the JEV-mode conformation likely represents a state more suitable for polymerase synthesis from the structural perspective.
With two monomer conformation modes and eight crystal forms identified, more than 10 NS5 dimer interfaces can be recognized in the aforementioned NS5 crystal structures with no obvious conservative features. A couple of studies did focus on some of these dimer interface interactions, even though the primary NS5 solution state is monomer \[[@ppat.1008484.ref016],[@ppat.1008484.ref021]\]. Either by probing the inter-molecular interactions, deleting the MTase domain, or mutating the MTase-RdRP linker, multiple *in vitro* polymerase assay-based studies together suggest that the MTase regulates the RdRP catalytic activities, albeit to overall moderate extents \[[@ppat.1008484.ref012],[@ppat.1008484.ref014],[@ppat.1008484.ref016],[@ppat.1008484.ref018],[@ppat.1008484.ref024],[@ppat.1008484.ref025]\]. However, the RdRP assays established in all these studies, no matter in primer-dependent or *de novo* (including dinucleotide driven) format, did not demonstrate the formation of a processive RdRP elongation complex (EC), at least for the majority of the polymerase molecules, and all these assays require the manganese ion (Mn^2+^) for catalysis, albeit in combination with the magnesium ion (Mg^2+^) in some cases. In other words, the mutation-derived effect on RdRP synthesis observed in these studies may only reflect overall changes in non-processive RdRP synthesis activity, while specific alteration of either RdRP initiation or elongation cannot be clearly judged. Furthermore, none of these studies characterized both the JEV- and DENV3-mode monomer conformations to distinguish their differences in RdRP synthesis, except for our previous JEV NS5 study that only probed the JEV-mode conformation \[[@ppat.1008484.ref012]\]. Therefore, the precise mechanism of the regulation and the explicit contribution of either NS5 conformation remain to be clarified.
In this work, we report two crystal forms of DENV2 NS5 that reveal two conformational states bearing clear analogies to those observed in the JEV-mode and DENV3-mode NS5 structures, respectively. Virological data further support the conservation and the functional importance of both conformation modes. NS5 constructs bearing mutations specifically probing two modes of MTase-RdRP intra-molecular interfaces were tested in *in vitro* polymerase assays, and only the JEV-mode interface related mutants inhibited polymerase initiation primarily through a three-fold reduction in the Michaelis constant of the initiating NTP (*K*~*M*,*NTP*~), while polymerase EC properties were not much affected by
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1. Introduction
===============
The synthesis of metal sulfide nanocrystals has attracted much interest for both fundamental research and technological applications in the past decades \[[@B1-nanomaterials-02-00113],[@B2-nanomaterials-02-00113],[@B3-nanomaterials-02-00113],[@B4-nanomaterials-02-00113],[@B5-nanomaterials-02-00113],[@B6-nanomaterials-02-00113]\]. Metal sulfide nanocrystals have been prepared by a wide range of synthetic methods, one of which involves the direct decomposition of molecular precursors \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B12-nanomaterials-02-00113],[@B13-nanomaterials-02-00113],[@B14-nanomaterials-02-00113]\]. Molecular precursor approach has recently been developed as an efficient route to prepare monodispersed semiconductor nanocrystals \[[@B7-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B15-nanomaterials-02-00113]\] and, in some cases, unique shape-control has been achieved \[[@B9-nanomaterials-02-00113],[@B11-nanomaterials-02-00113],[@B16-nanomaterials-02-00113]\].
One of the earliest precursors used for preparing metal sulfides in the literature is *N*,*N*'-dialkyl dithiocarbamate. In this preparation, the precursor was injected into hot coordination solvents such as trioctylphosphine oxide (TOPO) under nitrogen atmosphere at high temperatures \[[@B7-nanomaterials-02-00113],[@B8-nanomaterials-02-00113],[@B9-nanomaterials-02-00113],[@B10-nanomaterials-02-00113],[@B11-nanomaterials-02-00113]\]. Metal bis(benzylthiolates) \[[@B12-nanomaterials-02-00113]\] and metal salts of alkylxanthate \[[@B17-nanomaterials-02-00113],[@B18-nanomaterials-02-00113],[@B19-nanomaterials-02-00113]\] have also been used as precursors to prepare nanocrystalline sulfides of zinc, cadmium or lead via pyrolysis at 150--400 °C. It was found that Lewis base such as hexydecylamine (HDA), trioctylphosphine (TOP) or tributylphosphine (TBP) could lower the reaction temperature for the alkylxanthate precursors. Recently, there are also several reports on the preparation of ternary and metal sulfide nanocrystals via the decomposition of thiocarboxylate precursors \[[@B20-nanomaterials-02-00113],[@B21-nanomaterials-02-00113],[@B22-nanomaterials-02-00113],[@B23-nanomaterials-02-00113],[@B24-nanomaterials-02-00113]\]. Most of these syntheses, nevertheless, require elevated or refluxing temperatures.
In this paper, we report a generalized precursor method operating at room temperature for the synthesis of various metal sulfide nanocrystals. The precursors we use are metal thiobenzoates \[M~x~(SCOC~6~H~5~)~y~ or simply MTB\]. These MTB precursors are air-stable and could be readily prepared from thiobenzoic acid and the corresponding metal salts following the known literature method \[[@B25-nanomaterials-02-00113]\]. We illustrate the generality of this MTB method by preparing four types of semiconductors from both transition and main group metals: Ag~2~S, Cu~2−x~S, In~2~S~3~ and CdS. We discuss in this report the basis of our approach and also attempt to understand the reaction mechanism through theoretical Density Functional Theory (DFT) calculations. With this insightful knowledge, we demonstrate how to manipulate the stability of the precursor, and thus the reaction kinetics, to generate different nanostructures.
2. Experimental Section
=======================
2.1. Synthesis of Precursors and Metal Sulfide Nanocrystals
-----------------------------------------------------------
Commercially available compounds such as thiobenzoic acid (Fluka), ether, ethanol, chloroform (all from J. T. Baker), sodium bicarbonate (Dumont), silver nitrate (Merck), indium chloride (Fluka), acetonitrile, cadmium acetate, copper chloride, 2,2'-bipyridine, octylamine, dodecylamine and oleylamine (all from Aldrich) were used as received.
All of the MTB precursors used in this paper (M = Ag, Cu, In, Cd; TB = thiobenzoate) were prepared according to literature methods \[[@B25-nanomaterials-02-00113]\]. The products obtained were washed with ethanol, dried, and recrystallized from chloroform or ether. Purity of the crystals was checked with microanalysis and their decomposition profiles were investigated by thermogravimetric analysis (TGA).
For the synthesis of silver sulfide nanoparticles, AgTB (3 mmol) was stirred in toluene (5 mL) at room temperature, and then added with 1.8 mmol of an amine. A homogeneous clear brown solution formed quickly and the solution was stirred for a further 3 hours. After that, 10 mL of ethanol was added to induce turbidity in the mixture. The brown or dark brown nanoparticles are isolated by centrifugation, washed several times with ethanol and acetone, and then dried under vacuum. The resulting powder can be easily re-dispersed in toluene, hexane, chloroform and other non-polar solvents.
The experimental procedure is similar for the synthesis of copper sulfide nanoparticles, except that the reaction mixture turned blue and was stirred overnight. Dark brown or black copper sulfide nanoparticles were isolated, which can be re-dispersed in non-polar solvents such as toluene, hexane and chloroform.
The synthesis of cadmium sulfide nanoparticles is similar to silver sulfide and copper sulfide, except that the reaction mixture turned yellow and was stirred for 4 hours at room temperature. Pale yellow product was separated by centrifugation after adding 10 mL of ethanol. The product isolated, after washing with ethanol and acetone, can be re-dispersed in chloroform, hexane and toluene.
For the synthesis of indium sulfide nanoparticles, InTB (0.3 mmol) was stirred in toluene (5 mL) at room temperature, and then 1.2 mmol of octylamine (OA) was injected to give a yellow solution. After stirring for 6 hours, 10 mL of ethanol was added to induce the formation of turbidity. The particles were purified similarly to the previous procedure. For the preparation of oleylamine-capped In~2~S~3~ nanoparticles, it was found necessary to further add 40 µL of propylamine to speed up the reaction.
2.2. Characterizations
----------------------
TGA was recorded on a SDT 2960 Simultaneous DTA-TGA by heating approximately 10 mg of the precursor under inert N~2~ flow (flow rate 90 mL/min) at a heating rate of 10 °C/min. X-ray diffraction (XRD) analysis was carried out on a Siemens D5005 X-ray powder diffractometer with Cu Kα radiation (40 kV, 40 mA). The powdered sample was mounted on a sample holder and scanned with a step size of 2θ = 0.05° in the range of 20° to 90°. UV-Visible spectra were recorded using a Shimadzu UV-2550 UV/Vis spectrophotometer. FT-IR spectra were recorded using a FTS 165 Bio-Rad FTIR spectrophotometer in the range of 4000--400 cm^−1^ on KBr or nujol mulls. Transmission electron microscopy (TEM) images were obtained on a 100 kV JEM-100CXII TEM and 200 kV JEOL 2010F microscope. Samples were prepared by placing a drop of the dispersed nanoparticles onto a copper grid with carbon film, and were allowed to dry in a desiccator.
2.3. Computational Methodology
------------------------------
Calculations were performed using the hybrid density functional B3LYP \[[@B26-nanomaterials-02-00113],[@B27-nanomaterials-02-00113]\] method with the effective core potential LanL2DZ \[[@B28-nanomaterials-02-00113],[@B29-nanomaterials-02-00113],[@B30-nanomaterials-02-00113],[@B31-nanomaterials-02-00113]\] basis set. All reported energies include zero point energy corrections. All calculations were performed using the Gaussian 98 \[[@B32-nanomaterials-02-00113]\] suite of programs. Optimization was performed without any constraints and the optimized structures were verified to be equilibrium structures or transition states from frequency calculations. An equilibrium structure is characterized by all real frequencies while a transition state has one and only one imaginary frequency.
3. Results and Discussion
=========================
3.1. Thermal Behavior of the MTB Precursors
-------------------------------------------
The decomposition profiles of the four MTB precursors were investigated and their TGA curves are presented in [Figure 1](#nanomaterials-02-00113-f001){ref-type="fig"} and [Table 1](#nanomaterials-02-00113-t001){ref-type="table"}. The analysis indicated a clear onset of decomposition at \~ 200--300 °C in each case. The residual weight after each complete decomposition was found to be close to the expected remaining weight of the corresponding metal sulfide. The slightly higher measured values are
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Introduction
============
Porcine reproductive and respiratory syndrome (PRRS), first reported in 1987 in the USA, is a global infectious disease that has resulted in widespread economic loss in the swine industry. PRRS is characterized by reproductive failure in pregnant sows (*e.g.* abortions and stillbirths), respiratory distress in pigs of different ages, and severe immune suppression \[[@B13],[@B16]\]. The PRRS virus (PRRSV) is the recognized causative agent of this syndrome. Two different genotypes of PRRSV have been described: European or type 1, and North American or type 2 \[[@B6],[@B21]\]. In China, most isolated strains are of type 2 \[[@B3]\].
PRRSV is an enveloped, positive, single-stranded RNA virus in the genus *Arterivirus*, which belongs to the family Arteriviridae within the order Nidovirales \[14\].The PRRSV genome is 15 kb in length and contains at least 10 open reading frames (ORFs). Of these, ORF1a and ORF1b represent nearly 75% of the viral genome and encode two large polyproteins (pp), pp1a and pp1ab, respectively. These pps can be hydrolyzed into at least 16 small nonstructural proteins (Nsps). Of these 16 Nsps, at least 14 are involved in viral genome replication and transcription \[[@B10]\]. The Nsp1, Nsp2, and Nsp7 proteins elicit a strong immune response in pigs. Fourteen days after pigs are infected with PRRSV, anti-PRRSV Nsp7 antibodies can be detected, and high levels of antibodies in pigs can last for up to 202 days \[[@B2]\].
A diagnosis of PRRSV is important for its prevention and control. The laboratory diagnostic tests commonly used at present include reverse transcriptase polymerase chain reaction (PCR), quantitative real-time PCR \[[@B19],[@B22]\], and four serological detection methods \[[@B1],[@B4],[@B5],[@B20]\]: the indirect fluorescence assay (IFA), the immunoperoxidase monolayer assay (IPMA), the serum neutralization test (SN), and the enzyme-linked immunosorbent assay (ELISA). In recent years, by using the PRRSV N protein as an antigen, the commercial IDEXX ELISA kit (IDEXX Laboratories, USA) has been widely used for the detection of PRRSV antibodies. Recently, an ELISA kit that uses recombinant Nsp7 protein as the antigen has been introduced, and the concurrence rate between that kit and the IDEXX ELISA assay has been reported to be up to 97.6% \[[@B2]\]. Although traditional laboratory tests offer good sensitivity and specificity, these detection methods require professional and technical personnel and specialized equipment. In addition, there are many methodological aspects that must be constantly improved and complemented.
In developing countries, vaccination is an important strategy for preventing and controlling PRRSV. To monitor the titer of anti-PRRSV antibodies after vaccination in order to confirm the effectiveness of the vaccination, quick and easy techniques suitable for field testing are needed. Compared with the traditional detection methods, an immunochromatographic strip method has the advantages of being easy to use, providing an answer rapidly and at a low cost, and not requiring specialized equipment or technical personnel, thus such a method is suitable for field testing for antigens or antibodies. In this study, we developed an immunochromatographic test strip for detecting anti-PRRSV antibodies and conducted a preliminary field study of the strip.
Materials and Methods
=====================
Serum samples
-------------
Pigs were experimentally inoculated with either of two strains of PRRSV having differing levels of virulence, HN07-1 and BJ-4, in order to provide positive test serum samples. HN07-1 strain was isolated during an atypical PRRSV outbreak in Henan Province, China in 2007 \[[@B17]\]. The BJ-4 strain was a typical North American (VR2332)-like PRRSV isolated in 1996 in China \[[@B17]\]. The field sera for antibody testing were collected from several swine herds (all sampled pigs were more than 2-weeks-old) within Henan Province, China. Positive serum samples for porcine circovirus-2 (PCV-2), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine parvovirus (PPV), and foot-and-mouth disease virus (FMDV) were collected and stored in the Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences.
Preparation of recombinant Nsp7 protein of PRRSV
------------------------------------------------
For the expression of a recombinant Nsp7 proteins in *Escherichia* (*E.*) *coli*, reverse transcriptase PCR was performed on viral genomic RNA from PRRSV isolate BJ-4, obtained from Dr. Hanchun Yang of China Agricultural University. To amplify the Nsp7 gene the following oligonucleotide primers were used: forward primer, 5′-CGC**GGATCC**TCTCTGACTGGTGCCCTCGCTATG-3′ and reverse primer, 5′-CCG**CTCGAG**TTCCCATTGAACTCTTCCAT-3′ (restriction sites in bold font). The amplified gene was then ligated into the pET-28a vector. The recombinant plasmid was transformed to *E. coli* BL21-competent cells. Single colonies were obtained and tested by PCR and sequencing, and a positive clone was grown at 37℃ in LB broth supplemented with 100 µg/mL ampicillin to an optical density of 0.8 at 600 nm. Expression of the recombinant protein was induced by 100 mM isopropyl-β-D-thiogalactopyranoside (IPTG, TAKARA Bio, China) for 8 h at 37℃. Cells were then harvested by centrifugation (7000 × g for 30 min).
Purification of recombinant Nsp7 protein
----------------------------------------
The recombinant Nsp7 protein was purified by immobilized-metal affinity chromatography (IMAC) using a polyhistidine tag and further purified by a gel filtration column Superdex200 (GE Healthcare, Sweden). The cell pellet was suspended and lysed by sonication on ice. The lysate was centrifuged at 16,000 × g for 30 min, and the supernatant was collected and transferred to a Ni-NTA His Band Resin column pre-equilibrated with binding buffer (500 mM NaCl, 20 mM Tris, 5 mM imidazole). More than five column-volumes of washing buffer (500 mM NaCl, 20 mM Tris, 20 mM imidazole) was added to remove the nonspecific binding proteins. The target protein was eluted with elution buffer (500 mM NaCl, 20 mM Tris, 400 mM imidazole). The purity and relative concentration of the recombinant Nsp7 was determined by SDS-PAGE. The protein was further fractionated by gel filtration on a column of Superdex200 in a buffer of 50 mM Tris, 150 mM NaCl by using the Bio-Rad BioLogic system (Bio-Rad Laboratories, USA). The protein of interest was collected in different fractions according to its different states of aggregation. The final protein products were examined by SDS-PAGE before storing at −80℃.
Western blot
------------
For western blot analysis, 4 µg purified recombinant Nsp7 protein were subjected to 15% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membrane was washed with phosphate-buffered saline-Tween20 (PBST) and blocked with 5% skimmed milk. After washing three times with PBST, the membranes were reacted with PRRSV-positive sera; a PRRSV-negative serum were used as a negative control. After incubating at 37℃ for 1 h, the resulting blot was treated with secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG (Abbkine; WuHan AmyJet Scientific, China) for 1 h. As the substrate for color development, 3-amino-9-ethylcarbazole (AEC) was used.
The antigenicity of the separated protein fractions compared by ELISA
---------------------------------------------------------------------
The antibody binding capability of the monomer, dimer, and larger aggregate of the recombinant Nsp7, which were separated by Superdex200 gel filtration column, were compared by indirect ELISA assay. The separated proteins were diluted to the appropriate concentration in 50 mM sodium carbonate bicarbonate buffer (pH 9.6). After incubation for 14 h at 4℃, antigen-coated plates were washed five times with phosphate-buffered saline (PBS) containing 0.05% Tween 80 then blocked with 5% skimmed milk powder dissolved by PBST for 1 h at 37℃. Then, appropriate dilutions in PBST of PRRSV-positive HN07-1, PRRSV-positive BJ-4, and PRRSV-negative pig sera were incubated in the antigen-coated wells at 37℃ for 30 min. Secondary antibody horseradish peroxidase-conjugated rabbit-anti-pig IgG was added at a final dilution of 1:2,000, and the mixture incubated for a further 30 min at 37℃. Finally, 3′,3′,5′,5′-tetramethylbenzidine was added as a substrate. Color development was stopped with 2 M H~2~SO~4~, and the OD value at 450 nm was read on a spectrophotometer.
Conjugation of antigen with colloidal gold
------------------------------------------
Colloidal gold with an average particle diameter of approximately 20 to 25 nm was obtained by reduction of a HAuCl4 solution with 1% trisodium citrate. Three milliliters of 1% trisodium citrate (w/v) was added to 100 mL of 0.01% HAuCl~4~·3H~2
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"pile_set_name": "PubMed Central"
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Background
==========
Human immunodeficiency virus type-1 (HIV-1) is characterized by extensive genetic heterogeneity. Molecular epidemiologic studies have demonstrated that globally, the most prevalent forms of HIV-1 are subtypes (clades) C, B and A \[[@B1]-[@B3]\]. Subtype C, which accounts for almost 50% of all HIV-1 infections globally, predominates in sub-Saharan Africa and India \[[@B1]-[@B3]\]. Subtype B is the main genetic form in the Americas, Australia and western Europe; subtype A predominates in areas of central and eastern Africa (Kenya, Uganda, Tanzania and Rwanda) and in eastern Europe \[[@B1]-[@B3]\]; and subtype D is distributed mainly in east Africa, including Uganda \[[@B1]\]. HIV-1 subtypes differ by as much as 20-25% at the genetic level \[[@B2]\], and have varying biological characteristics, including differences in disease progression, pathogenicity, transmissibility and co-receptor usage \[[@B1],[@B2],[@B4]-[@B7]\].
Studies of HIV-1 co-receptor tropism, which have been conducted primarily in populations where subtype B infections predominate, have demonstrated a relationship between HIV-1 co-receptor use and disease stage. In general, early stages of infection and disease are characterized by greater prevalence of only C-C chemokine type 5 (CCR5)-tropic (R5) HIV-1, which has been associated with slower progression to AIDS \[[@B8]-[@B12]\]. The emergence of C-X-C chemokine receptor type 4 (CXCR4)-using virus (X4) has been associated with greater treatment experience and higher risk of death, and coincides with more rapid CD4^+^T-cell depletion and disease progression \[[@B6],[@B8],[@B9],[@B12],[@B13]\]. Some variants of HIV-1 can use either co-receptor (dual/mixed-tropic \[DM\] HIV-1); these can be found in all stages of infection, but are more common in infections of longer duration, with lower CD4^+^cell counts and higher viral loads \[[@B12]-[@B14]\]. Despite the emergence of X4-using variants in some patients, only R5 infection typically persists in the majority of patients. Nearly 50% of patients who die of HIV-1 disease have only R5 HIV-1 detectable at the time of their death, indicating that CCR5 remains a critical co-receptor throughout the course of HIV infection \[[@B12],[@B15]\].
Although HIV-1 co-receptor usage and its relationship to disease stage have been studied in the developed world, where subtype B predominates, such relationships are less well understood for subtypes A, C and D. The R5 phenotype is predominant in subtype C HIV-1 infections, whereas X4-using virus has been reported infrequently, even in advanced disease. R5-using virus is more common in subtype A than subtype D HIV-1 infections, and a high proportion of subtype D infections shows D/M tropism throughout the course of disease \[[@B16]-[@B24]\]. However, some of these previous studies have been limited by small sample sizes.
The introduction of the CCR5 antagonist, maraviroc, for HIV-1 therapy \[[@B25]\] has increased interest in the epidemiology of tropism and relationships with HIV-1 subtype. A greater understanding of the tropism of non-B subtype HIV-1 is key for the optimal use of CCR5 antagonists in the treatment of these infections in the developing world, and HIV-1 prevention strategies, such as topical microbicides and systemic pre- or post-exposure prophylaxis. In addition, this information will be important for management of clinic populations in the developed world that include individuals with non-B subtype infections who have migrated from endemic countries \[[@B26]\]. HIV-1 tropism can be determined by genotypic and phenotypic methods. While genotypic assays may have lower specificity and sensitivity, retrospective analyses have found that they are comparable to phenotypic tropism assays for prediction of response to treatment with CCR5 antagonists, in populations pre-screened with a phenotypic assay \[[@B27],[@B28]\].
The clinical development programme for maraviroc, the first-in-class CCR5 antagonist, used the Trofile^®^phenotypic assay (Monogram Biosciences, South San Francisco, California) \[[@B29]-[@B31]\], which determines tropism via the expression of full-length *env*genes of multiple viruses isolated from patient plasma and can detect 10% of X4 variants with 100% sensitivity. More recently, a Trofile^®^assay with enhanced sensitivity to improve detection of low-level X4-using variants has been developed that can detect 0.3% of these variants with 100% sensitivity. Otherwise, assay validation performance characteristics are equivalent between the original and enhanced Trofile^®^assays \[[@B31]\]. The enhanced Trofile^®^assay has been validated in a number of studies by re-testing the co-receptor tropism of clinical samples that were initially determined using the original assay \[[@B31]-[@B34]\]: in a re-analysis of samples from the Phase 3 MERIT study, which evaluated the efficacy of maraviroc in treatment-naïve patients with CCR5-tropic virus, 15% of enrolled patients (n = 106/721) were reclassified as having D/M virus with the enhanced assay \[[@B34]\].
The aim of this study was to estimate the prevalence of R5-, D/M-, and X4-tropic HIV-1 among isolates obtained from patients with HIV-1 subtype C infection from India and South Africa, and with subtype A/A1 and D infection from Uganda, and to explore the demographic and clinical characteristics associated with R5 infection. In addition, the study examined the ability of the Trofile^®^assay to determine tropism of non-B subtypes of HIV-1, which previously had not been explored in a large study.
Methods
=======
Study design
------------
HIV-1-infected, antiretroviral therapy (ART) treatment-naïve (TN) and treatment-experienced (TE) viremic patients were recruited into this prospective, cross-sectional, epidemiologic study from HIV clinics in South Africa (four sites), Uganda (one site), and India (seven sites) in 2007 and 2008. Sites were selected if they had considerable experience with both HIV management and HIV research. The study protocol was approved by the institutional review board at each site.
Both TN and TE adults (aged 18 years or older) were eligible for enrolment in India and Uganda. In South Africa, where the MERIT study had previously been conducted in TN patients \[[@B35]\], only TE adults were eligible for inclusion in the present study. No patients were recruited from any other study.
Patients who had received less than 10 days of ART were considered to be TN; those who had experienced failure of at least one three-drug ART regimen were considered to be TE. To maximize the external validity and generalizability of the study, only one member of a known infection cluster (i.e., only one member of a family affected by HIV) was eligible for enrolment.
Study procedure
---------------
At a single visit, the study was explained to patients, and verbal and written informed consent were obtained prior to conduct of any study procedures. Demographic, and HIV clinical history and treatment data were collected; blood was drawn and analyzed for CD4^+^and CD8^+^T cells using BD FACS™ CAP (Becton Dickinson and Company, Franklin Lakes, New Jersey), and for HIV-1 RNA levels using Amplicor HIV-1 Monitor™ UltraSensitive Assay (Roche Diagnostics, Indianapolis, Indiana).
For individuals with viral loads exceeding 500 copies/mL, HIV-1 subtype was determined based on reverse transcriptase and protease gene sequence (Monogram Biosciences). *Pol*subtyping was performed by generating three distinct sets of partial-length nucleotide sequences from patient-derived reverse transcriptase and protease genes. Genetic sequence comparison was determined by the Basic Local Alignment Search Tool algorithm, and subtype was resolved by a customized software package that was validated against the publically available tool on the National Center for Biotechnology Information web site at the National Institutes of Health. Samples determined to be HIV-1 subtypes of interest (A/A1 and D in Uganda; C in South Africa and India) were further tested for viral tropism.
HIV-1 co-receptor tropism was determined using the original Trofile^®^assay \[[@B29],[@B30]\] for samples collected from patients in South Africa and Uganda. The Indian cohort was enrolled after the South African and Ugandan cohorts, thus Indian patients were tested using the newly available enhanced Trofile^®^assay, which had replaced the original assay. Samples were prepared for both the original and enhanced assays in the same way: 1 mL plasma samples underwent centrifugation and viral RNA was isolated, purified and subjected to polymerase chain reaction (PCR) amplification of the entire HIV envelope gene (*env*). Co-transfection of HIV *env*expression vectors and HIV-1 genomic vectors produced pseudoviruses containing full length *env*genes derived from patient virus populations. Tropism was determined by measuring the ability of the pseudovirus population to efficiently infect target cells co-expressing CD4 with either the CXCR4 or CCR5 co-receptor \[[@B29],[@B31]\].
For commercial Trofile^®^testing, up to 3 mL of plasma and up to three attempts at RNA PCR amplification are performed for each patient specimen. For this study of non-subtype B specimens, however, only a single attempt at amplification was made with 1 mL samples and primers that were optimized on subtype B specimens (hereafter referred to as \"standard primers\"). In the event that samples yielded a non-reportable tropism result, re-testing was performed using modified primers that were optimized for subtypes A, C and
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Introduction
============
Myopia is the main cause of preventive blindness worldwide, especially in adolescents ([@B1], [@B2]). Thus, it is one of the main priorities among the five projects under the 'Vision 2020 Action' launched by WHO ([@B3]). In recent years, the incidence of myopia has increased rapidly worldwide ([@B4]), especially among adolescents in East and Southeast Asia ([@B5], [@B6]). The prevalence of myopia among adolescents is at 96.5% ([@B7]) in South Korea, 81.6% ([@B8]) in Singapore and 95.5% ([@B9]) in Shanghai.
Myopia not only affects adolescents' school performance and future career choice ([@B10]) but also causes glaucoma, cataract and other serious complications ([@B11]). Thus, many researchers have devoted themselves to gaining a more in-depth understanding of the prevention and control of adolescent myopia ([@B12], [@B13]).
Collaborative research networks can help other researchers expand their field of research or join groups conducting related studies. Bibliometric studies of scientific collaboration have been conducted in various fields ([@B14], [@B15]), providing different levels of cooperation frequency in research practice. One of the methods used to study such collaboration is the co-authorship network analysis, which focuses on finding patterns of contacts or interactions between social actors. Author, country, and institution are the subjects of cooccurrence relationship; thus, analyzing their cooccurrence relationship can better reflect the truth of scientific research and academic communication, because the cooperation of authors, institutions and countries can measure the cooperation at different levels ([@B15]).
However, to date, no bibliometric analysis of scientific literature in myopia prevention and control had been carried out and published. As such, this study aimed to describe the diversity of cooperation among authors, institutions, and countries in the study of adolescent myopia prevention and control. Specifically, for adolescent myopia prevention and control research, our main goal is to explore the following content: firstly, analysing the overall status of collaborative research among authors, institutions and countries; secondly, determining the institutions and authors at the core of the cooperative research network; and thirdly, identifying countries that have a strong cooperative relationship.
Materials and Methods
=====================
The search for papers to be included in the analysis was conducted in one day (Sep 25, 2017) to avoid bias resulting from daily updating in the database. The Web of Science Core Collection annually collects a large number of journals and records each publication, including bibliographic information (i.e., author, institution and country or region), which we used to locate publications. All papers published within the period of 1997--2016 were evaluated. Search terms included combinations of terms, such as 'adolescent', 'children', 'student', 'myopia', 'myopic', 'prevention', 'control' and 'management'. Literature types, such as meeting abstracts, letters, correction, news item, book chapter, retracted publication, editorial material, non-English literature and repeated articles, were excluded. To ensure reliability, profile information of each included article was extracted by two independent reviewers, resulting in a reliability check of 100% of the selected abstracts. A search query that was used for data extraction from Web of Science database looked like this: TS= ((adolescent myopia OR children myopia OR student myopia OR adolescent myopic OR children myopic OR student myopic) AND (prevention OR control OR management)).
Social network analysis (SNA) is a method of structural analysis applied in many research fields. It focuses on relationship research and is mainly used to describe and measure relationships and information between individuals ([@B16]). SNA has been proven to be effective in studies on scientific collaboration network ([@B17], [@B18]). The same method is used in the current study.
To analyze and identify critical issues, we used SATI (Statistical Analysis Toolkit for Informetrics) (ver. 3.2) to build the co-occurrence matrix ([@B19]) and transformed the data format with Ucinet 6.0 ([@B20]) to finally obtain co-occurrence mapping. VOS viewer (Visualisation of Similarities viewer) software (ver. 1.6.6) was employed to draw the co-country (region) maps by using literature title packets ([@B21]). Excel 2016 (Microsoft, Redmond, DC, USA) and Netdraw (ver. 2.118) were also used in the research. In addition, some measures of our network, including degree centrality, betweenness centrality, closeness centrality, density, and diameter, were evaluated ([@B22]). Degree centrality refers to the number of neighbors to a node in the network ([@B15]). In this case, the greater its connection to other nodes in the network, the more important is the node.
Betweenness centrality refers to the number of the shortest paths passing through a given node ([@B23]). The higher the betweenness centrality of the node, the greater the ability to control the information passed between the other nodes. The closeness centrality is used to measure the distance of one node to other nodes in a network. Nodes with high closeness centrality obtain information better than other nodes or tend to have a more direct influence on other nodes ([@B16]). Density is calculated through the actually observed ties divided by all possible ties whose value is between 0 and 1 ([@B24]). Density values tend to reach 0 in sparse networks, and close to one in tightly connected networks ([@B24]). The diameter represents the longest measuring distance in a connected network; it shows the number of steps required from one side of the network to the other ([@B16]).
Ethical considerations
----------------------
This study did not require any ethical consideration as it does not include any human or animal to be the object of study.
Results
=======
A systematic search for publications on adolescent myopia prevention and control retrieved 624 articles in Web of Science Core Collection, excluding one duplicate. After further screening of titles and abstracts, 9 editorial materials, 4 letters and a meeting abstract were removed, leaving 610 eligible papers.
The scale and overall trend of collaborative research
-----------------------------------------------------
[Figure 1](#F1){ref-type="fig"} shows the number of publications issued annually and the number of papers published through collaboration with authors, institutional cooperation and country (region) cooperation. The number of papers, co-authors, co-institutions and country (region) cooperative papers has increased significantly from 1997 to 2016, particularly after 2011. In general, the total number of published articles since 1997 has increased more than six-fold, from 11 in 1997 to 79 in 2016; the institutional cooperation increased more than five-fold, the author cooperation increased by twelve-fold and the country (region) cooperation increased by fifteen-fold.
{#F1}
[Fig. 2](#F2){ref-type="fig"} reveals the average number of authors, institutions and countries per article from 1997 to 2016. The average number shows a gradually increasing trend.
{#F2}
The increase in number of authors was from 3.91 to 4.34, from 1.36 to 2.51 for institutions and from 0.82 to 1.25 for countries per paper. Overall, the rates of cooperation among authors, institutions and countries were 93%, 57.9% and 21.5%, respectively.
In general, the number of SCI journal papers produced by institutional cooperation is the largest (accounting for 56.6%), followed by papers generated through intra-institutional collaboration (accounting for 36.1%) and papers produced without collaboration (accounting for only 7.4%). [Figure 3](#F3){ref-type="fig"} shows the percentage of papers studied in each of the different institution collaboration types and their changes over time. The percentages of single-author papers have decreased by 26.7% from 1997 to 2016, whereas that of institution-collaborated papers increased by 24.4%. The percentage of papers produced through single authorship has always been higher than that of institutional collaboration from 1997 to 2000 but decreased after 2006.
{#F3}
Authors' collaborative research
-------------------------------
Results of scientific research are published in the form of papers, and the status of co-authorship in papers reflects the collaboration among authors. Researchers who study the growth of co-authorship articles produced by multiple authors regard co-authorship of papers as a significant scientometric indicator of researching on cooperation among authors ([@B25]).
More important researchers were expected to have published more articles, thus scholars who published more than four articles were included in the co-authorship networks. Overall, 75 researchers with 371 co-authored experiments meet this condition. Five authors not cooperated with other authors were excluded. The research collaboration network between authors is shown in [Fig. 4](#F4){ref-type="fig"}.
{#F4}
Each node of the figure represents an author, and the connections among the nodes represent the collaboration relationships among authors. The weight of a link indicates the number of publications co-authored by two scholars. In this author's collaboration network, the highest degree centrality of Allen, Peter M. and O\'Leary, Daniel J. was 5.83, indicating that they had 5.83 collaborators and that they played a pivotal role in the co
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Introduction
============
Resistin is a cysteine-rich protein, which is mainly secreted from monocytes and macrophages in humans ([@b1-or-40-06-3392]--[@b3-or-40-06-3392]). It is associated with inflammation and malignant neoplasms ([@b4-or-40-06-3392]--[@b5-or-40-06-3392]). Blood resistin levels are demonstrated to be increased in certain cancer patients compared with healthy controls, including esophageal squamous cancer, gastric, colorectal, breast and endometrial cancer, and malignant lymphoma ([@b6-or-40-06-3392]--[@b12-or-40-06-3392]). Resistin is considered to be a risk factor for breast cancer ([@b9-or-40-06-3392],[@b13-or-40-06-3392]) and biomarker of disease progression of esophageal squamous cancer, gastric and colorectal cancer ([@b6-or-40-06-3392]--[@b8-or-40-06-3392]). It is an independent prognostic factor of pancreatic ductal adenocarcinoma ([@b5-or-40-06-3392]). Resistin can promote prostate cancer cell proliferation through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B signaling pathway in human prostate cancer cell lines PC-3 and DU-145 ([@b14-or-40-06-3392]).
However, most of the reported studies only demonstrated the association between serum or plasma resistin and malignancy. Few reports measured the level of resistin expression in cancer tissues, even though it is less well studied and controversial in lung cancer. Certain reports demonstrated a higher concentration of resistin in the blood was demonstrated in non-small cell lung cancer (NSCLC) patients compared with the controls ([@b15-or-40-06-3392]--[@b17-or-40-06-3392]). One of the reports assessed resistin expression in the marginal area of lung cancer tissue and non-cancer region by immunofluorescence staining in 10 cases ([@b17-or-40-06-3392]). Another revealed that blood resistin levels were similar between cancer group and non-cancer group ([@b18-or-40-06-3392]). Furthermore, the clinical significance and biological function remain largely unknown.
However, lung cancer is one of the most common malignancies worldwide, with higher morbidity and poorer prognosis ([@b19-or-40-06-3392]--[@b20-or-40-06-3392]). The most common form of lung cancer is NSCLC, which includes lung adenocarcinoma and squamous carcinoma. At present, lung adenocarcinoma replaces squamous carcinoma as the dominating type of NSCLC. The aim of the present study is to determine the resistin expression in lung adenocarcinoma tissues, clinical significance and biological function *in vitro* and *in vivo*.
Materials and methods
=====================
### Patients and tissue samples
A total of 70 consecutive cases of newly diagnosed lung adenocarcinoma patients at Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) from January to December 2008, with complete clinical and pathological data, were selected retrospectively in the present study and followed up for at least five years. Paired cancer and adjacent non-cancerous tissue samples, which were located more than 1 cm away from the tumor, were obtained through open surgeries. The paraffin-embedded tissue samples were stained with hematoxylin-eosin and confirmed lung as adenocarcinoma again.
The clinicopathological characteristics of the patients were recorded. The tumor staging of NSCLC was defined according to the tumor, node and metastasis system. The study was comprised of 70 cases of lung adenocarcinoma (38 male cases, 32 female cases), with an average age of 61 years old (36--77 years old). All patients received treatments (including operation, chemotherapy or radiotherapy), which conformed to the guidelines of NSCLC.
### Immunohistochemistry
For immunohistochemical staining, 5-µm paraffin-embedded tissue sections were heated for 1 h at 70°C, deparaffinized with a xylene soak, followed by rehydration via the addition of alcohol at decreasing concentrations (100, 95, 85 and 75%) for 5 min/step. A 96°C water-bath was used for antigen retrieval in 0.01 mol/l sodium citrate buffer (10 mM, pH 6.0) for 20 min. Next, endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide for 15 min at room temperature. Subsequently, slides were blocked with goat serum (cat. no. ZLI-9022; OriGene Technologies, Inc., Beijing, China; 1:1) at 37°C in a wet box for 30 min and then incubated by the primary antibody (rabbit polyclonal antibody against human resistin; cat. no. BS7730; Bioworld Technology, Inc., St. Louis Park, MN, USA; 1:100) overnight at 4°C in moist chambers. Following washing with 0.01 mol/L PBS (pH 7.2) three times, slides were incubated with a biotinylated secondary rabbit anti-mouse antibody (cat. no. PV-6000; OriGene Technologies, Inc.; 1:1) for 25 min at 37°C. Following incubating in horseradish peroxidase marked streptomycin avidin working fluid at 37°C for 30 min, slides were treated with avidin biotin-peroxidase complex using 3,3′-diaminobenzidine as a chromogen, and then counterstained with hematoxylin for 30 sec at room temperature and examined by light microscopy (Olympus Corporation, Tokyo, Japan). PBS was used as a negative control instead of a primary antibody.
### Evaluation of immunohistochemical staining
Existing tan or brown particles in the nucleus or cytoplasm indicated positive cells, which must conform to the following conditions: i) The cellular structure was clear; ii) the location of positive granules was accurate; iii) staining was significantly increased compared with the background.
In a 400× high power filed, randomly selected from 10 different cancer cell fields of view, the percentage of (a) positively stained cells was calculated as follows: 0--5% positive cells, score 0; 6--25% positive cells, score 1; 26--50% positive cells, score 2; 51--75% positive cells, score 3; 76--100% positive cells, score 4. Then, the (b) staining intensity was evaluated: Colorless, score 0; light yellow, score 1; deep yellow and tan, score 2; brown, score 3.
The expression of resistin was based on the product of (a) × (b): Score 0, negative (−); score 1--4, weakly positive (+); score 5--8, positive (++); score 9--12, strongly positive (+++). (−) and (+) were regarded as low expression group, (++) and (+++) were categorized as the high expression group ([Fig. 1A](#f1-or-40-06-3392){ref-type="fig"}). The result of each specimen was independently evaluated by two qualified and expert pathologists, blinded to the patients\' clinical data. The few cases with discordant results were reevaluated and final scores were consensual.
### Cell culture
Human lung adenocarcinoma cell lines A549 and H1975 were obtained from the Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere of 95% air and 5% CO~2~. Cells were divided according to transfection of overexpression resistin plasmid for the resistin group and empty vector for the control group.
### Transfection and isolation of stable transfectants
Lipofectamine™ 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.), endo-free maxiplasmid kit (Tiangen Biotech Co., Ltd., Beijing, China); pcDNA3.1-(+)/resistin plasmids were established by OriGene Technologies, Inc. (cat. no. RC210942) and the primers were as follows: Forward primer, 5′-CCCACCGAGAGGGATGAAAG-3′ and reverse primer, 5′-CAGTGACATGTGGTCTCGGC-3′; forward primer, 5′-CAGCTCACCATGGATGATGATATC-3′ and reverse primer, 5′-AAGCCGGCCTTGCACAT-3′ (β-actin).
A fragment of the rat resistin cDNA fragment (285 bp) was inserted at the unique *Eco*RI site in the anti-sense orientation as determined by sequencing. The final concentration of resistin was 100 nM. A total of 2×10^6^ A549 and H1975 cells grown in 60 mm Petri dishes were transfected with 10 µg of the recombinant plasmid using lipofectamine, as described by the supplier (Gibco; Thermo Fisher Scientific, Inc.). A total of 24 h later, fresh RPMI-1640 media containing 10% FBS was added and replaced 48 h later. Monoclonal cells were selected with NeoR. Then the cells were cultured for 24 h following transfection.
### Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from the cells by TRIzol (cat. no. 15596026; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer\'s protocol. Prime Script RT reagent kit (DRR037A; Takara Bio, Inc., Otsu, Japan) were used for cDNA generation (42°C for 30--60 min, 70°C for 15 min). RT-qPCR was performed
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INTRODUCTION {#sec1-1}
============
The main goal of periodontal treatment is to control the inflammation in periodontal tissues and to regenerate the lost tissues predictably. To meet this goal it is critical to guide the tissues capable of regeneration.\[[@ref1][@ref2][@ref3]\] Guided tissue regeneration is an accepted method for enhancement of lost periodontal tissue. In this technique a barrier membrane is used to prevent epithelial cell migration and stabilization of the clot into the defect. This prevention results in the migration of periodontal ligament cells and osteoblasts into defect and these cells are known to be responsible for tissue regeneration.\[[@ref4]\] Different types of barrier membranes are introduced that had shown favorable results due to different studies.\[[@ref5]\] These membranes are different in composition and structure, but all of them prevent the migration of epithelial and gingival connective tissue cells into the defect and ideally, a barrier membrane should enhance the cell attachment and migration of the progenitor cells.\[[@ref5][@ref6][@ref7][@ref8][@ref9][@ref10]\] Wound healing is a complex process which includes cell migration, cell attachment to various extracellular matrix components, and cell proliferation.\[[@ref11][@ref12]\] Cell attachment process is a four-step sequence which includes adsorption of glycoproteins to the substrate surface, cell contact, attachment, and spreading.\[[@ref9][@ref10]\] Cell proliferation begins after these events.\[[@ref5]\] Tissue integration property ensures the stabilization of the wound and inhibits the migration of epithelial cells, which results in better gain of clinical attachment levels.\[[@ref13][@ref14][@ref15]\]
According to their degradation characteristics, barrier membranes are divided into two groups of resorbable and non-resorbable membranes. Collagen is the most common material used as resorbable membranes.\[[@ref5]\] It facilitates hemostasis and wound stability by promotion of platelet aggregation along with fibroblast migration which accelerates wound closure,\[[@ref16][@ref17]\] but collagenous membranes are not stiff enough to resist soft tissue pressure during healing.\[[@ref16][@ref18]\]
Polytetrafluroethylene (PTFE) is the main composition of non-resorbable membranes.\[[@ref19]\] Although their biocompatibility and positive effect on bone regeneration was shown, but a second surgery is required for their removal which may traumatize the newly formed immature periodontal tissue and causes patient discomfort and increases the treatment time and cost.\[[@ref20]\] Also, the membrane stiffness may result in tissue dehiscence which is the main reason of treatment failure 3 weeks after membrane placement and exposes the membrane which leads to bacterial infection and decrease in the levels of gained clinical attachment.\[[@ref21][@ref22][@ref23][@ref24]\] An alternative to an expanded PTFE membrane is a high-density polytetrafluroethylene (d-PTFE) membrane which is commercially available as TXT-200 and GBR-200. High-density polytetrafluroethylene membranes have small porosities, so bacterial contamination is eliminated and therefore there is no need of primary closure when they are being used and they can be left exposed to the oral cavity.\[[@ref25][@ref26][@ref27][@ref28]\]
The acellular dermal matrix (Alloderm) was originally introduced in medicine for reconstructive plastic surgeries but is also used in dentistry in various periodontal procedures like root coverage and keratinized tissue augmentation around teeth and implants.\[[@ref29][@ref30][@ref31]\] It has many advantages, but the absence of cells and vessels makes tissue incorporation slower, therefore, attempts of culturing fibroblasts on Alloderm were performed to achieve early wound healing and decrease wound contraction in periodontium.\[[@ref32][@ref33][@ref34][@ref35]\]
Fibroblasts play an important role in the healing process. It has been shown that the key factor in the success of regenerative treatment is the recruitment or delivery of cells to the defect site and the production of suitable extracellular matrix along with the periodontal tissues.\[[@ref36][@ref37]\]
Introduction of specific cell adhesion molecules to the membrane surfaces may lead to specific tissue responses. Different growth factors and proteins have been introduced and one of them is enamel matrix derivatives. A commercially available product of enamel matrix derivatives is called Emdogain^®^ (EMD). It is an acidic extract of low molecular weight procine enamel proteins mainly amelogenin and a propylene glycol alginate vehicle.\[[@ref38][@ref39]\] Different studies showed that EMD enhances the adhesion, proliferation, and matrix production of periodontal ligament fibroblasts, stimulates cell growth, and production of insulin growth factor-1 and transforming growth factor-β1 in periodontal ligament cells although it has no appreciable effect on osteoblastic differentiation and has no effect on epithelial cells.\[[@ref37][@ref38]\] All of the described characteristics of EMD make it a suitable functional material for regenerative treatments. Therefore, its effects on cell adhesion to different materials were investigated in the present study.
There was also no available study that had compared the fibroblast adhesion among TXT-200, GBR-200, Alloderm, and collagenous membrane (RTM Collagen, Cytoplast^®^) or the effect of EMD on fibroblast attachment to these common barrier membranes. The present study was performed to compare cell adhesion among the prementioned membranes and also to investigate the effect of EMD on gingival fibroblast attachment.
MATERIALS AND METHODS {#sec1-2}
=====================
For this experimental *in vitro* study, gingival fibroblast cells (NCBI Codece C165) were provided by Pasteur Institute of Iran. Cells were cultured in a culture flask and cultured in the presence of Dulbecco\'s modified Eagle medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) containing 10% Fetal Calf Serum and 100 μg/ml of penicillin, streptomycin, and amphotericin B. The flask was kept in 37°C in a 5% CO~2~ atmosphere in an incubator with humidity. The medium was changed twice a week. Cells were cultured for 3 weeks and passaged for five times.
Four different barrier membranes were used in this study. Two non-resorbable dense polytetrafluoroethylene membranes GBR-200 (GBR1224, LOT: 2541) (Cytoplast®, Osteogenic Biomedical, Lubbock, TX, USA), TXT-200 (TXT1224, LOT: 3688) (Cytoplast®), RTM Collagen (RTM2030, LOT:C2030263) (Cytoplast®) and acellular dermal matrix (ADM, 302111, LOT: B42234) (Alloderm, Biohorizons, Birmingham, AL, USA).
Each membrane was cut into two 6×6-mm pieces and washed with sterile saline solution according to the supplier\'s instructions. In RTM Collagen and ADM groups, membranes were washed with sterile saline solution until the protect paper was floating. A 48 wells culture plate was used in this experiment. Five groups of four close wells were selected. Four groups were used for membranes (each group containing four wells for each membrane). All of the membranes were adapted at the bottom of the selected group of wells. No membrane was added to the fifth group and it served as a control group to check the growth of seeded cells. 10 μg/mL of EMD (LOT: C2822, Emdogain®, Straumann, Malmö, Sweden) was added to two wells of each group (EMD+) and two wells were left without any EMD (EMD-). Cells were seeded at a density of 100,000 cell/well on the membranes. Plate was placed in a 37°C incubator with humidity and 5% CO~2~ atmosphere for 24 hours. The growth of seeded cells in the fifth group was evaluated by means of a light microscope.
Then cells were washed four times with phosphate buffer saline (PBS) to remove non-adherent cells. The membranes were fixed in 2.5% glutaraldehyde for 2 hours, washed five times with distilled water for 20 minutes, treated with 1% osmium tetroxide for 1 hour, washed again five times with distilled water for 20 minutes and finally dehydrated through a series of graded ethanol solutions and left for 24 hours in room temperature to dry. To finish the process, they were coated with gold and analyzed with Field Emission Scanning Electron Microscope (Hittachi s4160, Stanford, CA, USA). An operator not aware of the experimental set up analyzed the membranes with SEM. Each membrane was divided into four intellectual parts under SEM with ×300 magnifications and one image was taken from each part. Another two observers totally unaware of the experiment counted the cells on each image and if there was a difference, the least cell count was recorded.
Data was analyzed by independent t-test, one-way ANOVA, two-way ANOVA, and *post hoc* LSD test with SPSS18 (version 18;SPSS Inc, Chicago, IL, USA). *P* \< 0.05 in independent t-test analysis and *P* \< 0.001 in one-way ANOVA, two-way ANOVA, and *post hoc* LSD analysis was considered statistically significant.
RESULTS {#sec1-3}
=======
Figures [1](#F1){ref-type="fig"}--[4](#F4){ref-type="fig"} illustrates the membranes in EMD- and EMD+ groups under SEM with ×300 magnifications and [Table 1](#T1){ref-type="table"} shows the gained data after cell counting process by two observes.
{#F1}
![SEM illustration of TXT-200 membrane, a- EMD- group
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Background {#Sec1}
==========
Bronchopleural fistula (BPF) is a relatively infrequent but potentially fatal complication of pulmonary resection. BPF can be divided into peripheral or central, based on the location of the leakage, and BPF occurs in about 1.5 to 28 % of pneumonectomy cases, and is associated with high death rate \[[@CR9], [@CR30]\]. It is estimated that incidence of BPF after pneumonectomy and lobectomy for lung cancer is 4.5--20 % and 0.5 %, respectively, and the incidence of BPF is highest after right pulmonary resection and right lower lobectomy \[[@CR31]\]. The etiology of BPF includes incomplete tumor resection, use of steroids, intraoperative infection and prolonged postoperative mechanical ventilation as major risk factors of BPF \[[@CR31]\]. The clinical manifestations of BPF can be frequently classified as acute, subacute, and chronic. An acute BPF presents as tension pneumothorax, with pleural cavity communicating abnormally with the airways, and is associated with purulent sputum expectoration, dyspnea, and reduction in established pleural effusion \[[@CR22]\]. The presentations of subacute and chronic BPF are commonly related to a pleural space with infection, manifesting as a more invisible form with fever, dry cough, and malaise with different levels of respiratory disorder \[[@CR33]\]. Traditional treatments of BPF include thoracotomy after drainage and primary repair, which is based on vascularized muscular flaps and omental grafts tissues \[[@CR20]\]. Amplatzer vascular plug, which was originally designed for the transcatheter closure of vascular structures, has also been reported as a safe and effective method to treat small postoperative BPF \[[@CR9]\]. Fruchter et al. also found that the technique of Amplatzer double-disk occluder implantation may be suitable for both large and small BPFs which originate from the main bronchi and lobar bronchi, respectively \[[@CR8]\]. Additionally, endoscopic approaches and bronchoscopy are common methods of treating BPF to avoid thoracotomy \[[@CR27], [@CR36]\].
Bronchofiberscope (BFS) is a precision instrument employed to diagnose bronchial diseases using of the light guide composed by the fine fibers formed by tens of thousands of high transmittance glass or acrylic resin \[[@CR12], [@CR16]\]. BFS is designed to offer advantageous features such as easy operation method, clear vision, mild trauma, tolerance of surgery by patients, and high safety profile, which reduces or avoids complications associated with tracheotomy and prevents local infection \[[@CR25], [@CR32]\]. Clinically, BFS has multiple uses, including removing foreign bodies, eliminating secretions, treating nasopharyngeal carcinoma, central lung cancer, alveolar cell carcinoma, esophageal fistula, hemoptysis, obstruction, assisting endotracheal intubation treatment and placing gastric tube \[[@CR1], [@CR11], [@CR21]\]. Previous studies have revealed that BFS is also an excellent diagnostic tool for early detection of various intrabronchial injuries, and the attached biopsy sampling feature is helpful in the identification of early lesions, and to carry out poly excision surgery for the studies on bronchus and lung diseases \[[@CR15], [@CR18], [@CR19]\]. Previous studies reported various treatment methods for BPF using BFS, and the methods include gelfoam, shot put plugs, and tissue adhesives. However, these methods have significant deficiencies, evident from the fact that treatment fistula under 3 mm was efficient using these methods, but they show poor efficacy in treatment of BPF beyond 3 mm, particularly those beyond 10 mm \[[@CR6], [@CR28], [@CR35], [@CR37]\]. Phenol, also named carbolic acid, is a sweet-smelling colorless liquid used to prepare resins, preservatives, fungicides, drugs (e.g., aspirin), and also is used to disinfect surgical instruments \[[@CR4], [@CR24], [@CR38]\]. 88 % carbolic acid was found to be efficacious with all alopecia areata patients and can be considered as a treatment of choice for stable alopecia areata \[[@CR3]\]. Moreover, spot peel with 88 % phenol can be a cost-effective procedure for idiopathic guttate hypomelanosis, which can be combined with other medical therapies \[[@CR26]\].
There are no studies using carbolic acid to treat BPF with the help of BFS at present. Therefore, we investigated the efficiency of carbolic acid treatment of BPF in post-pulmonectomy patients, by instilled 100 % carbolic acid with the aid of BFS.
Methods {#Sec2}
=======
Ethics statement {#Sec3}
----------------
This study was conducted with the approval of the Institutional Review Board of Liaoning Tumor Hospital, Shenyang. The informed written consent was collected from each eligible patient and the whole study was performed based on the Declaration of Helsinki \[[@CR14]\].
Study population {#Sec4}
----------------
A total of 12 patients with post-pulmonectomy BPF were enrolled at the Department of Thoracic Surgery, Liaoning Tumor Hospital, Shenyang between February 2009 and March 2012. Orificium fistulae were confirmed by bronchoscope and the average diameter was 4.5 mm. The eligible patients included eight males and three females, with an average age of 56 years (range, 45 \~ 71 years). Three patients had BPF after the right pneumonectomy, six after the left pneumonectomy, one after the right middle and low lobectomy and two after left upper lobectomy.
Preoperotive preparation {#Sec5}
------------------------
Electrocardiogram, routine blood tests and biochemical examination were performed in all the patients. Patients were fasted for 4 \~ 6 h in preparation for surgery and received 10 mg diazepam and 1 mg atropine via intramuscular injection about 30 min before operation. In addition, 1 % lidocaine was used for nasopharyngeal anesthesia by nebulizer.
Intraoperative methods {#Sec6}
----------------------
All patients were instructed to take supine position except 2 patients with short breath in sitting position. The BFS (Olympus BF1T40) was inserted into the trachea through nasal cavity. Heart rate, blood pressure and SpO2 was monitored. Patients received local nasopharyngeal anesthesia with 2 % lidocaine to alleviate irritant reaction. The bronchus around the suture was bubbling when the patient breathed deeply. The fistula was observed via BFS. After the drainage of secretion, hematocele or pus around the BPF, a bronchoscopy biopsy forceps was used for removing necrotic tissues and a 1.8 mm flexible tube was guided through the biopsy hole. The distal end of BFS was brought out and fixed 0.3 cm above the fistula. With breath holding, 100 % carbolic acid solution (0.5--1.0 ml) was instilled to bronchial mucosa through the BFS. The bronchial mucosa became pale after treatment and finally the flexible tube and bronchoscope were removed.
Postoperative and histological observation {#Sec7}
------------------------------------------
After the surgery, the patients were treated with closed drainage of thoracic cavity, anti-inflammatory, symptomatic and supportive treatments. Gas discharge in thoracic drainage tube was observed, and fistula healing were measured via BFS. The treatments were repeated if there was gas discharge from thoracic drainage tube, or further observations were made. Patients could leave hospital after blood routine test showing no evidence of dyspnea, fever, positive culture of fluid drainage (3 times). Paraffin sections (4 \~ 6 μm) of bronchial stump were stained by hematoxylin and eosin (HE) to observe the irritation of bronchial stump after instilled with carbolic acid solution.
Results {#Sec8}
=======
Outcome characteristics of BPF {#Sec9}
------------------------------
In the 12 patients with BPF, the median diameter of the BPF orifice was 4.5 mm, according to the intraoperative observation. Specifically, 3 patients showed a fistula diameter of 3 mm or smaller, 6 patients showed a fistula diameter of 3 \~ 5 mm, and 3 patients exhibited a fistula diameter of 5 mm or larger, 1 of whom had a fistula diameter of 7 mm (Table [1](#Tab1){ref-type="table"}). Serious complications, such as haemorrhage, severe dyspnea and SpO2 declines, did not occur in all the 12 patients during bronchoscopic therapy. Of note, BPF orifices in 5 patients closed after 5 treatments with carbolic acid, 1 patient through 2 treatments, 1 patient through 3 treatments, 2 patients through 4 treatments and 3 patients through 7 treatments (Fig. [1](#Fig1){ref-type="fig"}). Follow-up was conducted for six months after bronchoscopy. Based on the data collected, the average treatment time of the 12 patients was calculated as 20 min and the average time of fistula closure was 30 days. Importantly, the cure rate was 100 %.Table 1Characteristics and outcomes of BPF patientsAge\
(y)GenderInitial symptomSurgical methodBronchopleural fistulaeSize (mm)Treatment timesCure time (d)Follow up148maleLow feverRight PNY3535alive256maleHigh feverRight PNY3.5535alive350maleBlood sputumLeft PNY1321alive471maleIrritating coughLeft upper LBY1.5428alive557femaleLow feverRight upper LBY4535alive665maleFever/air bubbleLeft PNY5749unknown764maleCough/feverLeft PNY7749alive859male
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The content published in Cureus is the result of clinical experience and/or research by independent individuals or organizations. Cureus is not responsible for the scientific accuracy or reliability of data or conclusions published herein. All content published within Cureus is intended only for educational, research and reference purposes. Additionally, articles published within Cureus should not be deemed a suitable substitute for the advice of a qualified health care professional. Do not disregard or avoid professional medical advice due to content published within Cureus.
Introduction
============
Ganglion cysts are soft tissue masses that arise from the joint capsule and tendons which are commonly seen on the dorsal side of the hand and wrist \[[@REF1]\]. One of the treatment options is surgical excision \[[@REF2]\]. Surgery is usually performed with local anesthesia infiltration in sedated-unsedated patients or under general anesthesia.
In recent years, ultrasound (US) guidance has made distal peripheral nerve blocks of the upper extremity technically safe and feasible options to achieve anesthesia for hand and wrist surgery \[[@REF3]\]. Nowadays, US-guided regional blocks should be the first choice for such surgical procedures where the possible risks of general anesthesia and the necessity of clear airways are considered \[[@REF4]\]. US-guided nerve blocks have many advantages, such as the avoidance of nerve damage, with a clear definition of nerves from the surrounding structures, control of the distribution of local anesthetics, visualization of needle position, faster block onset time, improved block qualities, and a reduction in the volumes of local anesthetics \[[@REF5]-[@REF6]\]. The success rate of regional anesthesia has increased when performed under ultrasound guidance. Ultrasound identifies nerves easily, which cannot be defined by surface anatomic landmarks and alternative sites can be determined by scanning the nerve along its route \[[@REF6]-[@REF7]\].
Forearm blocks may especially be useful for minor surgeries of the hand. However, background data are limited regarding the performance of a peripheral nerve blockade at the level of the upper arm for hand surgery. There is also a lack of reports about the optimal sites for needle insertion regarding US-guided radial nerve blocks. Most studies about median, ulnar, and radial nerves present these blocks as rescue techniques for a failed or incomplete proximal (infraclavicular, axillary, interscalene, or supraclavicular) upper extremity block \[[@REF4],[@REF8]-[@REF11]\].
The mid-humeral region enables anesthesiologists to selectively administer local anesthetics to different nerves and block the four main nerves of the upper extremity separately \[[@REF12]\]. This technique may have advantages over proximally performed approaches, such as the avoidance of needle trauma to central structures, decreased motor blockade, and smaller amounts of local anesthetic drugs used to achieve anesthesia for a narrower area \[[@REF4]\].
In this case report, we present three cases of US-guided radial nerve blocks performed at the mid-humeral region. Efficient surgical anesthesia was achieved for ganglion cyst excision at the hand dorsum using the minimal dose of local anesthesia. We discuss the potential indications and advantages of a US-guided radial nerve block at the mid-humeral region for patients undergoing hand surgery.
Case presentation
=================
Written informed consent has been obtained from all patients for the anesthetic/surgical procedures and for the publication of this case report. All patients underwent the excision of ganglion cysts at the dorsum of the hand (Figure [1](#FIG1){ref-type="fig"}).
{#FIG1}
Standard monitorization (electrocardiogram, blood pressure, pulse oximetry) was applied at the block room. An intravenous cannula was placed and infusion of 0.9% NaCl solution was started. The patients were sedated by administering 0.03 mg/kg of midazolam and 0.5 µg/kg of fentanyl intravenously. After sedation, the position of the arm was set at the 90 degrees abduction of the arm and 90 degrees of the forearm on the surgery table. The ultrasound probe was placed transversally at the mid-humeral region, on the posterolateral aspect of the arm. The needle was entered from the lateral side of the arm in the medial direction within the plane of the ultrasound beam (Figure [2](#FIG2){ref-type="fig"}).
{#FIG2}
The block area was sterilized with iodine solution and the US transducer was covered with a sterile cap. We performed needle insertion at the mid-humeral region, which is located midway between the anterior process of the acromion and the lateral epicondyle of the humerus, as described by Foxall et al. \[[@REF6]\]. The radial nerve could be visualized easily by locating the ultrasound probe on the posterolateral aspect of the arm at this level. A 13 MHz linear transducer was used (LOGIQ P5®, General Electric, USA). The radial nerve was visualized as an oval, heterogeneous structure that consisted of hypoechoic and hyperechoic structures, which represent the nerve fascicles and connective tissue. Using the in-plane technique, a 22 Ga 8 cm echogenic needle (Stimuplex® Ultra 360® Braun, Melsungen, Germany) was introduced into the mid-humeral region, aiming to enter the fascial plane next to the radial nerve. After the localization of the needle tip on the radial nerve, 0.15 ml/kg of 0.5% bupivacaine was injected. The distribution of the local anesthetic drug and the tip of the needle was visualized in real time during the procedure, aiming to spread around the nerve (Figure [3](#FIG3){ref-type="fig"}).
{#FIG3}
We did not use a nerve stimulator, as the nerve was clearly identified under ultrasound guidance. The patients were followed up with pinprick tests after the blocks for loss of sensation. The incisions were within the sensory dermatomal innervation area of the radial nerve (Figure [4](#FIG4){ref-type="fig"}).
{#FIG4}
Case 1
A 33-year-old female patient presented with complaints of swelling and pain in the dorsum of the right hand. She had a 2x2 cm mass and a soft cystic lesion, which was diagnosed as a ganglion cyst at the dorsum of the hand (Figure [1](#FIG1){ref-type="fig"}). Past medical history was unremarkable with American Society Anesthesiology (ASA) classification I. Surgical history examination revealed two cesarian-section operations. Ten milliliters of local anesthetic (bupivacaine 0.5%) was administered to encircle the radial nerve without entering the humeral side. Surgical anesthesia was achieved at the 25th minute after local anesthetic administration. A mild motor block was observed, as the patient could move her hand parallel to gravity. The patient was cooperative and reported minor discomfort during the excision of the cyst from its base where it originated, but there was no need for additional analgesic drugs during the surgery. There were no symptoms of cardiovascular, respiratory, or central nervous system side-effects. The surgery proceeded uneventfully and lasted about 20 minutes. The block was considered successful without the necessitation of conversion to general anesthesia.
Case 2
A 28-year-old female patient with complaints of swelling in the wrist dorsum of the right hand. The patient\'s medical and surgical history was unremarkable and evaluated as ASA I class. Fifteen milliliters of local anesthesia (10 ml bupivacaine 0.5 % and 5 ml lidocaine 2%) was administered around the radial nerve under ultrasound guidance. The block procedure was uneventful. The patient was cooperative during the operation and did not report pain at the beginning of the surgery. During the excision of the cyst from its base, the patient complained of discomfort. Fentanyl 50 µg intravenous was administered and 3 milliliters of 2% prilocaine was infiltrated to the surgical area. The surgery lasted 30 minutes, uneventfully. The block was considered successful without the need for conversion to general anesthesia.
Case 3
A 24-year-old male patient with a complaint of swelling at the wrist dorsum of the right hand was diagnosed with a ganglion cyst. The patient was evaluated as ASA I class with no remarkable medical and surgical history. After identifying the radial nerve under ultrasound guidance, 10 milliliters of 0.5% bupivacaine was administered. There were no symptoms of side effects during the block procedure. The patient reported minor discomfort, which was resolved with the administration of 50 µg intravenous fentanyl and infiltration of 3 milliliters 2% prilocaine into the surgical area. The surgical procedure was completed in 30 minutes without any complications. The block was considered successful, with no need of conversion to general anesthesia.
Discussion
==========
In this case report, a US-guided radial nerve block was applied from the mid-humeral level, which allowed sufficient surgical anesthesia for the excision of the gang
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All relevant data are within the the paper and its Supporting Information file.
Introduction {#sec001}
============
There is strong evidence that insufficient physical activity (PA) is associated with increased risk of developing lifestyle-related diseases and premature death \[[@pone.0175190.ref001]\]. Metabolic syndrome (MetS) is not consistently defined, but includes: overweight, abdominal obesity, insulin resistance, dyslipidemia, and hypertension in various combinations \[[@pone.0175190.ref002]\]. The presence of MetS carries a high risk for developing cardiovascular disease and type 2 diabetes \[[@pone.0175190.ref003]\]. Importantly, MetS is also associated with physical inactivity, further aggravating the risk of cardiovascular events \[[@pone.0175190.ref004]\].
The definition of PA is "any bodily movement produced by skeletal muscles that results in energy expenditure" and can be categorized as e.g. a household, occupational, leisure time, and sporting activity \[[@pone.0175190.ref005]\]. Exercise is PA with the objective to improve or maintain physical fitness components and is categorized in terms of the type, frequency, duration, intensity, and purpose \[[@pone.0175190.ref006]\]. The internationally recommended minimum level of PA \[[@pone.0175190.ref007]\] is moderate-intensity aerobic PA 150 min per week or, alternatively, vigorous-intensity aerobic PA 75 min per week, which has been associated with a clinically relevant risk reduction. Additional health benefits can be achieved by increased PA, above the national recommendation levels \[[@pone.0175190.ref008]\].
Despite the evidence-based positive effects of regular PA on health, implementing PA as an integrated method of treatment in health care remains a major challenge \[[@pone.0175190.ref009]\]. The Swedish National Board of Health's guidelines for disease prevention methods recommend the use of individual-based dialogue, written information, training diaries, a pedometer, and structured follow-up when the patient's PA level is insufficient \[[@pone.0175190.ref010]\]. An example of such a treatment strategy is physical activity on prescription (PAP), which is individually tailored for each patient and prescribed for preventive and therapeutic purposes as a first-line treatment.
Meta-analyses of international PAP studies show varying results, with small to medium positive intervention effects when comparing increased PA levels with usual care. However, there is uncertainty due to the lack of high quality studies and further research is needed with more homogenized, comparable PAP interventions, longer follow-up, and objective measures of outcome \[[@pone.0175190.ref011], [@pone.0175190.ref012]\]. Swedish lifestyle interventions, including PAP, has shown to be cost-effective \[[@pone.0175190.ref013], [@pone.0175190.ref014]\] and the Swedish PAP intervention method had positive effects on PA levels, body composition, cardio metabolic risk factors, and health related quality of life (HRQOL) \[[@pone.0175190.ref015], [@pone.0175190.ref016]\]. Although scientific evidence has resulted in clinical treatment guidelines \[[@pone.0175190.ref010]\] and there are some evaluated Swedish PAP studies \[[@pone.0175190.ref015]--[@pone.0175190.ref018]\], PA is still underutilized as a treatment strategy in Swedish health care \[[@pone.0175190.ref019], [@pone.0175190.ref020]\]. There is still a lack of knowledge about PAP interventions suitable for different patient groups to improve their PA level and health outcomes. Further studies are needed evaluating clinical feasible PAP strategies on a large sample \[[@pone.0175190.ref021]--[@pone.0175190.ref023]\].
In primary care in the city of Gothenburg, health care centers have implemented PAP-treatment, individualized for patients with metabolic risk factors, with the purpose of increasing the PA level and health benefits. This specific model of PAP-treatment in daily clinical work has not been evaluated and may add new insights on how the extent of the intervention affects the PA level and health status.
The aim of this observational study was to explore the association between PAP-treatment and the PA level of patients with metabolic risk factors and the relationship between changes in the PA level and health outcomes, including metabolic risk factors and HRQOL at the 6-month follow-up.
Methods {#sec002}
=======
Study design {#sec003}
------------
This is a prospective, longitudinal observational study with a 6-month follow-up of PAP-treatment in a daily clinical primary care practice. The present study is part of an ongoing study with a 5 year follow-up.
Study population {#sec004}
----------------
The study population included 444 patients, aged 27--85 years. The patients were selected as a convenience sample from 15 primary health care centers in Gothenburg center/west. The patients agreed with their health care provider to participate in the study before they were prescribed PA and were included prospectively from 2010 to 2014. The population of central/western Gothenburg is 220 000 and has a higher socio-economic status compared with Gothenburg overall \[[@pone.0175190.ref024]\]. The inclusion criteria were: physically inactive, having at least one component of MetS present, and receiving PAP-treatment. The patients also had to understand the Swedish language to fill in the questionnaires.
Intervention {#sec005}
------------
The patient was informed of the possibility to receive treatment with PAP by written information in the waiting room and orally by their caregiver. All authorized personnel were educated on the effects of PA according to the *Physical activity in the prevention and treatment of disease* (FYSS) \[[@pone.0175190.ref025]\] and the concept of the Swedish PAP model. Authorized personnel, mainly nurses, at the health care centers prescribed PA to the patients. The PAP included a dialogue with the patient, based on the principles of motivational interviewing (MI) \[[@pone.0175190.ref026]\]. Each patient's previous and current level of PA and their preferences for different kinds of PAs were elucidated. Furthermore, the patient's motivation, self-efficacy, and readiness to change PA behaviour were evaluated. This information served as the basis for the selection of the type and volume of the PA. The volume of the chosen PA was determined using the FYSS reference book, and the most suitable activity was prescribed at the appropriate relative intensity using the Borg's rate of perceived exertion scale \[[@pone.0175190.ref027]\] as well as duration and frequency. To help the patient to choose a suitable PA, a registry of the local supply of PA's was presented. It was possible to recommend two different types of PA in the PAP. This resulted, for each patient, in an individually tailored PA recommendation planned in dialogue with the patient and followed by a structured follow-up. The patients were offered individually adjusted support during the 6 month intervention period, either by revisits or telephone contacts.
Measurements {#sec006}
------------
The measurements described below were conducted when PA was first prescribed as well as at the 6-month follow-up.
### PA level {#sec007}
The PA level was the primary outcome and four questionnaires were used due to the known complexity of PA assessments. 1. Self-assessment was according to the American College of Sports Medicine (ACSM) and American Heart Association (AHA) public health recommendations. The patient responded to two PA questions (ACSM/AHA questionnaire), where 30 min of moderate-intensity PA per day resulted in 1 point and 20 min of more vigorous-intensity PA per day resulted in 1.7 point during each specific day of the week. A value of \<5 points indicated an inadequate PA level \[[@pone.0175190.ref028]\]. 2. The International Physical Activity Questionnaire (IPAQ) assessed the level of PA during the last 7 days. This instrument is extensively tested and translated into Swedish and can assess vigorous- and moderate-intensity PA, walking, and sitting time \[[@pone.0175190.ref029], [@pone.0175190.ref030]\]. 3. The Saltin-Grimby Physical Activity Level Scale (SGPALS) assessed leisure time PA during the past year at four different levels, from sedentary/physically inactive to vigorous physically active \[[@pone.0175190.ref031]\]. The levels have been validated against e.g. metabolic risk factors \[[@pone.0175190.ref032], [@pone.0175190.ref033]\] and the SGPALS has been published in an updated Swedish form \[[@pone.0175190.ref034]\]. 4. A six-grade PA scale, which is a further development of the SGPALS (Frändin/Grimby), was used and includes household activities \[[@pone.0175190.ref035]\]. This scale correlates with physical performance and self-assessed fitness and is used to classify PA among the elderly \[[@pone.0175190.ref036]\].
### Anthropometrics {#sec008}
Body weight was measured with light clothing and without shoes to the nearest 0.1 kg using an electric scale (Carl Lidén AFW D300, Jönköping, Sweden). Body height was measured in an upright position without shoes to the nearest 0.5 cm using a scale fixed to the wall (Personmått PEM 136, Hultafors, Sweden), and the body mass index (BMI) was calculated. Waist circumference (WC), to the nearest 0.5 cm, was measured in a standing exhaled position, with a measuring-tape (Kirchner Wilhelm, Aspberg, Germany) placed on the patient's skin between the lower rib and the iliac crest.
### Syst
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1. Introduction {#s0005}
===============
Human saliva is the complex fluid secreted by major and minor salivary glands. Saliva secretion is under the control of the autonomic nervous system. The three major salivary glands are parotid, sublingual and submandibular. Human saliva consists of water, glycoproteins, enzymes, antimicrobial substances and electrolytes. Glycoproteins in saliva are responsible for the viscoelastic characteristics giving a lubricative film, which enables the free movement of oral tissues. The mucin and electrolytes in saliva maintain the oral mucosa in its hydrated state and thus providing mucosal integrity ([@b0140]). The major functions of human saliva are lubrication, antimicrobial and cleansing activity, remineralisation of enamel with calcium and phosphate and facilitating eating and speech. Salivary gland dysfunction such as xerostomia (subjective sensation of dry mouth) and hyposalivation (diminished salivary flow) ([@b0055]) are relatively common problems that can give difficulties in speech, problems with eating, mucosal infections, denture intolerance, increased dental caries, periodontal disease and loss of life quality. The usual therapies for xerostomia and hyposalivation are drinking large quantities of water, using chewing gum, candies and artificial saliva. The aims of using artificial saliva are to ensure lubrication of oral tissues, relieve the sensation of dry mouth, and protect the tooth tissues from decay. There are several approaches to produce artificial saliva, including the imitation of natural saliva which is quite complex. Usually, the commercially available artificial saliva formulation composes of carboxymethylcellulose (CMC), sodium carboxymethylcellulose (SCMC) and hydroxyethylcellulose as thickening and lubricating agents. In fact, mucilage can be found in various parts of many plants. These mucilages can be used as thickening, moisturizing and lubricating agents in artificial saliva formulations. Beside mucilages in these plants, there are also several bioactive compounds which are advantageous for the development as artificial saliva, e.g. antioxidant and anti-adherent activity. Antioxidants can enhance the immune system and prevent cancer in oral cavity, while the anti-adherent activity is one of the important properties to prevent the adhesion of microorganisms on the teeth.
Mucilage is water soluble polysaccharide found in a widespread number of plants and also in some microorganisms. There are many plants which contain mucilage such as *Basella alba* Linn., *Hibiscus esculentus* Linn., *Litsea glutinosa* (Lour.) C.B. Robinson, *Ocimum canum* Sims., *Plantago ovate* Forssk., *Scaphium scaphigerum* G. Don. and *Trigonella foenum-graecum* Linn. ([@b0125]). *Basella alba* Linn. (called in common name as Ceylon Spinach or Phak Plung in Thai), family Basellaceae is a wildly cultivated, cool season vegetable (plants that have adapted to cool climates. They prefer temperatures between 55° and 75° F, which are late winter, early spring, late summer, autumn and early winter) with climbing growth habit ([Fig. 1](#f0005){ref-type="fig"}). Ceylon Spinach is an edible vegetable and has long been used as thickening agents in soups and stews. It is rich in vitamins A and C, as well as iron, calcium and soluble fiber. In addition, mucilage from this plant has also been used as topical Thai traditional medicines for the treatment of irritant, bruise, ringworm and laboring. Its stem and leaves are used as mild laxative, diuretic and antipyretic. The Ayurvedic treatment in India has used its leaves and stems for anticancer such as melanoma, leukemia and oral cancer ([@b0005]). Its mucilage is composed of mainly polysaccharides with the pH ranging between 5.3 and 5.4, containing D-galactose as a major monosaccharide and exhibiting slow swelling capacity ([@b0020]). Our previous study has demonstrated that the mucilages of *B. alba* Linn. extracted by distilled water at pH 11 using microwave for 3 min gave the highest DPPH radical scavenging and metal chelating activity of 1.01 and 11.14 folds of vitamin C and EDTA, respectively ([@b0105]). Therefore, the objective of this present study was to develop a biological active artificial saliva formulation containing mucilage from Ceylon Spinach by evaluating the physicochemical properties (viscosity, rheology wetting time) and biological activities including anti-oxidant activity and anti-adherent activity.Fig. 1The whole plant of Ceylon Spinach (*Basella alba* Linn.) showing its flowers, Family Basellaceae.
2. Materials and methods {#s0010}
========================
2.1. Materials {#s0015}
--------------
Ceylon Spinach was purchased from the local fresh market in Chiang Mai province, Thailand during April-May 2012 and identified by a botanist of the botanical garden at Faculty of Pharmacy, Chiang Mai University in Thailand. A voucher specimen (BAL-258) was deposited in the herbarium of Chiang Mai University (CMU) herbarium and flora database, Department of Biology, Faculty of Science, Chiang Mai University, Thailand. Vitamin C (L-(+)-ascorbic acid), 2, 2-diphenyl-1-picryhydrazyl (DPPH), sulforhodamine B (SRB) and resazurin sodium were from Sigma Chemical Co. (St. Louis, MO, USA). Calcium chloride was purchased from Merck, Germany. Potassium chloride and sodium fluoride from Ajax Finechem Pty Ltd., Australia were used. Tris (hydroxymethyl) methylamine was purchased from Fisher Scientific UK Limited, UK. All other chemicals and reagents were of analytical grade.
2.2. Mucilage extraction {#s0020}
------------------------
The fresh flowers of Ceylon Spinach (100 g) were cut into small pieces, macerated with 700 ml distilled water for 24 h and microwaved at 600 W intensity for 5 min. The mixture was then pressed through a muslin cloth. The filtrate containing the mucilage was centrifuged at 4,660g (centrifuge machine, Fisher Scientific Inc., New York, U.S.A) 25 °C for 30 min. The supernatant was collected, mixed with 95% ethanol (3 folds in volume) to precipitate the mucilage and re-centrifuged at 4,660g for 15 min. The precipitate was collected and the remaining ethanol in the precipitate was removed by a rotary evaporator (Buchi, Flawil, Switzerland) (50 ± 2 °C) until all ethanol was evaporated and lyophilized by a lyophilizer (Christ FOC-1 Model K-40 equipment, Balzers-Pfeiffer GmbH, Asslar, Germany) at −50 ± 2 °C. The dried lyophilized powder of the mucilage was kept at room temperature (25 ± 2 °C) until use.
2.3. Phytochemical assays {#s0025}
-------------------------
The mucilage was analyzed for phytochemical constituents (anthraquinones, glycosides, tannin, carotenoids, flavonoids and alkaloids) using the standard methods ([@b0100]). For anthraquinone, 0.05 g of the mucilage was put into a dry test tube, added with 2 ml of chloroform and shaken for 5 min. The mucilage was filtered. The filtrate was mixed with an equal volume of 10% ammonia solution and shaken. A pink violet or red color in the ammoniacal layer (lower layer) indicated the presence of anthraquinone. The qualitative assay of reducing sugars was performed by TLC method. The mucilage dissolved in water was spotted on the silica gel plate in comparing to the standard reducing sugars (glucose, fructose and sucrose). The filtrate was resolved on the TLC plate coated with silica gel 60. The mobile phase was butanol/acetic acid/diethyl ether/water (9:6:1:3). The spot on the plate was sprayed with 10% H~2~SO~4~ and heated. Sucrose, glucose and fructose were used as the standards. For tannins, 0.05 g of the mucilage was mixed with 2 ml of 15% FeCl~3~ solution. The blue-black precipitate indicated the presence of tannins. For carotenoid, each mucilage sample was extracted with chloroform in a test tube with vigorous shaking. The resulting mixture was filtered and 0.1 ml of H~2~SO~4~ was added. The blue color at the interface showed the presence of carotenoids. For the presence of flavonoid, 2 ml of the mucilage solution mixed with 1 ml of the concentrated HCl and magnesium ribbon gave the pink tomato-red color. For alkaloids, an amount of 0.05 g of the mucilage in 2 ml of 1.5%v/v HCl was boiled on a water bath and 6 drops of the Dragendorff's reagent were added. The orange precipitate indicated the presence of alkaloids.
2.4. The development of artificial saliva formulations containing mucilage from Ceylon Spinach {#s0030}
----------------------------------------------------------------------------------------------
### 2.4.1. Preparation of the artificial saliva formulations {#s0035}
Five developed artificial saliva formulations were prepared ([Table 1](#t0005){ref-type="table"}). The compositions of the formulations were mucilage from Ceylon Spin
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{
"pile_set_name": "PubMed Central"
}
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1.. INTRODUCTION
================
The pattern of stresses transferred to the bone have great influence on the success or failure of an orthopedic implant. The evaluation of bone stresses is so complex that it cannot be accomplished analytically, necessitating the application of FEA (Finite Element Analysis) techniques; three types of FE model, axisymmetric, bi-dimensional and three-dimensional, are considered in the literature \[[@R1]-[@R5]\]. Two-dimensional models are flawed in that stresses outside the plane of analysis are disregarded, while 3D models require a large number of elements and, consequently, long calculation times. The axisymmetric model, where the only simplification is that the screw thread is modeled as a disk, can be considered a good compromise between 2D and 3D models.
With regard to the constraints, the condition of osseointegration is usually simulated, and the post-operative condition it is rarely considered in the literature \[[@R3],[@R4],[@R6]\]. However, these two conditions produce significantly different results and can be considered the limit conditions under which orthopedic screws operate, so that the analysis of both cases can provide useful information.
Two boundary conditions (pull out test; alternative condition) are simulated in the literature \[[@R4],[@R7]\], but the relation between their respective results has not been analyzed. This relation is relevant because the pull out test is the standard test for orthopedic screws, while the screws, once implanted, are subjected to different loading conditions.
Regarding bone material, most models in the literature consider bone an isotropic, elastic and homogenous material \[[@R1]-[@R4],[@R7]-[@R9]\], while in reality bone is anisotropic because of its trabecular structure. In this study, bone isotropy was assumed in order to obtain more general results, while bone structure was defined by a single parameter, its volumetric density.
In the literature, the geometric and mechanical parameters of the screw generally considered are: pitch \[[@R10],[@R11]\], length, flank angle, and material \[[@R2]-[@R5]\]. Few authors have considered the fillet radius \[[@R4],[@R7]\], while many models have sharp edges \[[@R1],[@R3],[@R12]\]. Little emphasis has been given to screw performance in relation to bone density \[[@R5],[@R13]\] even though it is well known that the density of the bone determines its mechanical properties \[[@R14]-[@R18]\].
The present study evidences that it is not possible to select the appropriate screw without considering bone density. Different models were developed, considering the geometry and mechanical properties of commercial screws, and a parametric analysis was undertaken to assess how the strength of the bone-screw system varies for different values of thread pitch and bone density. Actually, this paper introduces a methodology where a multi-parametric structural numerical analysis is integrated with a multi-factorial analysis in order to be able to summarize a great amount of results and to build predictive analytic models. Overall, it was possible to determine some criteria for system optimization.
2.. MATERIALS AND METHODOLOGY
=============================
Stress analysis required the construction of apposite FE models, which were then validated by means of experimental tests.
2.1.. Finite Element Model
--------------------------
The model shown in Fig. (**[1](#F1){ref-type="fig"}**) was developed using MSC MARC^®^ 2003 software.
The bone consists of cortical bone (E =11 GPa, ν=0.33, 5 mm in thickness) and trabecular bone. Two different trabecular bone densities were simulated (Table **[1](#T1){ref-type="table"}**), and their respective mechanical properties were obtained from the relations \[[@R14], [@R16]\]:
$$E = 2015 \cdot \mathit{\rho}^{25}$$
$$\mathit{\sigma}_{U} = 0.0042 \cdot E - 0.039$$
where:
ρ is bone density (g/cm^3^);
E is Young's modulus (MPa);
σ~U~ is the ultimate tensile stress (MPa).
The bone density values were chosen to simulate recurrent clinical situations (adult patient with mild bone resorbing) and significantly different Young's modulus (1:2 ratio).
With regard to the screws, four different constitutive materials were simulated in order to study both currently used materials such as stainless steel (screw models n. 9-10) and titanium Ti6Al4V (screw models n. 3-8), and more innovative solutions such as low stiffness titanium (Ti12Mo5Ta, screw model n.2) and PMMA reinforced by an inner Ti12Mo5T cylinder (screw model n.1); the last two materials have been chosen in order to better approach trabecular bone's Young modulus.
The general geometry of the simulated screws and the mesh details are shown in Fig. (**[2](#F2){ref-type="fig"}**) (symbols as in Table **[2](#T2){ref-type="table"}**); they were generated from an effectively produced geometry (screw model n. 9), varying all parameters, one by one.
All the screw threads were simulated with symmetrical flanks, fillet radii were all equal, and the pitch was constant along the screw axis. The axisymmetric model entails shorter calculation times.
The constraints and boundary conditions are shown in Fig. (**[1](#F1){ref-type="fig"}**): they correspond to the pull out test (Fig. **[1a](#F1){ref-type="fig"}**) or to an alternative condition, with a different position of the constraint (Fig. **[1b](#F1){ref-type="fig"}**); this alternative condition should represent a more realistic working condition. According to Saint Venant's, the second condition is equivalent to the first one, however the small displacement hypothesis cannot be assumed a priori in this case, due to the low stiffness of trabecular bone. For both configurations, the immediate post-operative condition and that of complete osseointegration were simulated: the first condition was created through a 'touch' contact between the screw and trabecular bone (whatever displacement is allowed with the exception of penetration), while the second was obtained through a 'glue' contact (the two components are completely bounded to each other).
The mean number of elements per model was 7500.
2.2.. Maximum Allowed Force Evaluation
--------------------------------------
The maximum allowed force was calculated by iteration (non-linear case), specifying that the maximum stress on the bone must not exceed its ultimate tensile stress. This hypothesis is quite restrictive: actually bone is not a fragile material, tension peaks produce localized plastic deformations and stresses are redistributed over a wider area. However bone is likely to be stressed by dynamic loads and fatigue damage may occur when stress peaks produce a crack which progressively propagates during loading, until structural failure occurs.
2.3.. Experimental Tests
------------------------
The finite element model with 'touch' boundary condition was validated by means of experimental tests. All tests were performed on an hydraulic INSTRON 8872 test machine, equipped with a ±5 kN load cell.
Pull out tests \[[@R19]\] were conducted replacing bone by polyurethane foam, as reported in the literature \[[@R20]\].
The mechanical properties of polyurethane foam were determined by ten compression and ten tensile static tests, where a linearly increasing displacement was applied. The nominal stress was calculated from the force, divided by the initial specimen section; the nominal strain was calculated from the current displacement, divided by the initial specimen height.
Young's modulus was determined through five dynamic tests, applying a pulsating sinusoidal load (Fig. **[3](#F3){ref-type="fig"}**, left).
Young's modulus was calculated as 6 MPa (s.d. = ± 1.21 MPa), while ultimate tensile stress was 0.42 MPa (s.d. = ± 0.084 MPa); these data were input into the numeric model to be validated. Compression tests produced a progressive packing of the foam (reaching up to 60% height reduction) which does not break but shows buckling.
The pull out tests required specific equipment: two holes drilled in the opposite sides of a rectangular metal profile (obtained from an extruded bar) and a polyurethane block was placed within the box profile (Fig. **[4](#F4){ref-type="fig"}**).
A bolt screwed into the lower hole was then held by the lower jaw of the machine (the bolt was left free to move in the cross-sectional plane, in order to avoid bending moments acting on the screw). A screw was inserted through the upper hole and inserted into the polyurethane foam. The metal profile simulated the cortical layer of the FE model, and prevented the polyurethane foam deforming when the screw was pulled. A preparatory hole (6.5 mm in diameter) was drilled in the foam before inserting the screw, which was always set at the same height. The tests were conducted on a standard metrical screw (M10, UNI 4536) whose geometry was known in detail, allowing an accurate FE model to be constructed. The lower end of the shaft was threaded with 17 threads.
The screw was pulled at a speed of 2 mm/min. The pull out test was repeated 23 times and the mean force determined was 120 N (s.d. = ±14 N). A typical force versus displacement curve is shown in Fig. (**[4](#F4){ref-type="fig"}**).
2.4.. Analysis of FE Model Results
----------------------------------
Ten different screw models were realized (Table **[2](#T2){ref-type="table
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Introduction {#s1}
============
Iron (Fe) deficiency is among the most prevalent micronutrient deficiencies in humans. Since plants constitute the primary source of nutrients for a large part of the world's population, the improvement of plants in terms of nutrient bioavailability is considered a priority [@pone.0099234-deBenoist1]. Micronutrients like Fe are often present in an un-soluble form in the soil. Plants are able to mobilize such nutrients for uptake into the roots. Plants can also mobilize Fe from internal stores. Understanding the regulation of Fe acquisition and internal Fe utilization is of high importance for precision breeding of crops that are improved to either tolerate growth on alkaline and calcareous soils with poor Fe bio-availability or to accumulate a higher content of this micronutrient in bio-available form in the edible plant parts.
Genetic traits have been associated with micronutrient content and usage in plants, for example [@pone.0099234-Uauy1], [@pone.0099234-Baxter1]. Another trait was found in soybean as being linked to transcription factor genes encoding the soybean homologs of *BHLH38* and *BHLH39* [@pone.0099234-Peiffer1]. The potential importance of these two transcription factor genes for Fe mobilization had previously been uncovered in studies on the plant model *Arabidopsis thaliana*. *BHLH38* and *BHLH39* belong to the so-called subgroup Ib(2) *BHLH* genes [@pone.0099234-Pires1] and they are functionally redundant [@pone.0099234-Wang1]--[@pone.0099234-Wang2]. In fact, *BHLH38* and *BHLH39* are tandem duplicates on the chromosome, and they share similarity with two other *BHLH* genes, namely *BHLH100* and *BHLH101* [@pone.0099234-Wang1], [@pone.0099234-Heim1]--[@pone.0099234-ToledoOrtiz1]. All these four subgroup Ib(2) *BHLH* genes are highly induced by low Fe supply in roots and leaves while they are not usually found expressed under sufficient Fe supply [@pone.0099234-Wang1]. Expression of *BHLH39* and *BHLH101* in response to iron can be followed using the public microarray data in Arabidopsis [@pone.0099234-Bauer1]--[@pone.0099234-Yang1] and it was found that they occur in a co-expression network along with several Fe homeostasis genes like *FERRIC REDUCTASE OXIDASE3* (*FRO3*), *NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN4* (*NRAMP4*) and *NICOTIANAMINE SYNTHASE4* (*NAS4*) [@pone.0099234-Ivanov1]. From the co-expression with Fe homeostasis genes it can be concluded that the subgroup Ib(2) *BHLH* transcription factor genes likely perform regulatory functions in the context of Fe homeostasis and internal Fe mobilization. The bHLH protein POPEYE (PYE, belonging to another bHLH subgroup) is also induced by Fe deficiency within this co-expression network and it acts as a negative regulator of *FRO3*, *NRAMP4* and *NAS4,* presumably to avoid over-activation of Fe mobilization [@pone.0099234-Long1]. PYE is regulated by BRUTUS (BTS) that is also found in this co-expression network [@pone.0099234-Ivanov1], [@pone.0099234-Long1]. bHLH subgroup Ib(2) can physically interact with the bHLH FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) [@pone.0099234-Wang2], [@pone.0099234-Yuan1]. FIT is expressed specifically in roots and has been shown to be essential for Fe uptake [@pone.0099234-Bauer2]--[@pone.0099234-Yuan2] by regulating the expression of the genes encoding ARABIDOPSIS H^+^-ATPASE2 (AHA2) [@pone.0099234-Ivanov1], Fe reductase FERRIC OXIDASE2 (FRO2) [@pone.0099234-Jakoby1], [@pone.0099234-Robinson1] and the IRON-REGULATED TRANSPORTER1 (IRT1) [@pone.0099234-Jakoby1], [@pone.0099234-Eide1]. From ectopic FIT expression experiments along with yeast promoter activation assays and inducible FIT activation in plants, it can be concluded that FIT targets *FRO2* and *IRT1* gene promoters [@pone.0099234-Jakoby1], [@pone.0099234-Yuan2], [@pone.0099234-Meiser1], [@pone.0099234-Sivitz2]. However, FIT induces *IRT1* and *FRO2* only upon Fe deficiency even when overexpressed [@pone.0099234-Jakoby1], [@pone.0099234-Meiser1]. The activation of FIT at low Fe can be explained with the presence of bHLH subgroup Ib(2) factors. Indeed, the double overexpression of FIT together with either bHLH subgroup Ib(2) protein leads to an increase of Fe acquisition responses under sufficient Fe supply conditions, and it was therefore proposed that the function of bHLH subgroup Ib(2) might be to induce Fe deficiency responses in conjunction with FIT [@pone.0099234-Wang2], [@pone.0099234-Yuan1]. However, the occurrence of *BHLH* subgroup Ib(2) genes in the *PYE* coexpression network, their non-expression upon sufficient Fe (where *FIT* and *IRT1* are active although at low level) and their high induction upon Fe deficiency not only in roots but also in leaves (in contrast to Fe acquisition genes) renders this hypothesis questionable. Moreover, contradictory results have been published with regard to the function of bHLH subgroup Ib(2) proteins. In one report, double *bhlh100 bhlh101* knockout mutants were demonstrated to develop a more severe leaf chlorosis than the wild type upon Fe deficiency, while no phenotype was apparent upon Fe sufficiency. Although some Fe homeostasis genes appeared mis-expressed, the gene knockouts did not affect the plants' abilities for Fe uptake and the regulation of *FRO2* and *IRT1* upon sufficient or deficient Fe supply [@pone.0099234-Sivitz1]. In contrast to that, in another report, bHLH subgroup Ib(2) knockouts including *bhlh100 bhlh101* and a triple knockout *bhlh39 bhlh100 bhlh101* were demonstrated to affect Fe acquisition responses and to have low *FRO2* and *IRT1* expression upon sufficient or deficient Fe supply [@pone.0099234-Wang3]. This latter finding was rather puzzling, and it was not further explained how this finding fits to the observation that the *BHLH* genes are not normally expressed upon sufficient Fe supply, when Fe also needs to be acquired via FRO2 and IRT1 [@pone.0099234-Jakoby1], [@pone.0099234-Vert1]. Thus, the function of the bHLH subgroup Ib(2) transcription factors in Fe uptake is still open for debate.
Very interestingly, it has been shown that *BHLH38* and *BHLH39* were induced after application of salicylic acid ( = SA) by the SA-inducible Dof ( = DNA binding with one finger) transcription factor OBF BINDING PROTEIN3 (OBP3) [@pone.0099234-Kang1]. Binding of OBP3 to promoter elements in *BHLH38* and *BHLH39* genes and their subsequent activation was demonstrated (in these studies *BHLH38* and *BHLH39* were named *OBP3 RESPONSIVE GENE2*, *ORG2*, and *OBP3 RESPONSIVE GENE3*, *ORG3*) [@pone.0099234-Kang1]. Jasmonic acid negatively affects the onset of Fe mobilization and the induction of *FRO2* and *IRT1* [@pone.0099234-Maurer1], while ethylene enhances the responses [@pone.0099234-Garca1]--[@pone.0099234-Lingam1]. Since SA, jasmonic acid and ethylene act in stress response networks, the possibility exists that perhaps, there is a link between SA and the up-regulation of Fe deficiency responses.
Here, we made use of the triple knockout mutant *bhlh39 bhlh100 bhlh101* (*3xbhlh*) that we constructed to investigate the functions of these *BHLH* genes in the Fe deficiency response and to further shed light on the question whether SA is involved in mediating the onset of Fe uptake via the induction of *BHLH* subgroup Ib(2) genes. We discuss that *BHLH39*, *BHLH100* and *BHLH101* are essential for a subset of Fe deficiency responses but not including up-regulation of *IRT1* and *FRO2*. We suggest that these transcription
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{
"pile_set_name": "PubMed Central"
}
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Introduction
============
RNA interference (RNAi) is a highly efficient gene-silencing mechanism in which a small interfering RNA (siRNA) binds a target mRNA, guiding mRNA cleavage via an RNA-induced silencing complex (RISC).^[@bib1],[@bib2]^ This biological phenomenon is widely used as a genetic tool in biomedical research. Advances in RNA chemistry have expanded siRNA applications toward therapeutic development, with robust efficacy seen in phase 2 clinical trials for liver diseases (*e.g.*, transthyretin amyloidosis).^[@bib3],[@bib4],[@bib5]^
Despite its prevalence in biomedical research, the use of RNAi in neurodegenerative research has been limited.^[@bib6]^ There is a significant unmet need for simple, effective, and nontoxic siRNA delivery methods to modulate gene expression in primary neurons and brain. A range of approaches has been evaluated,^[@bib7]^ including AAV viruses,^[@bib8],[@bib9]^ peptide conjugates,^[@bib10]^ oligonucleotide formulations,^[@bib11]^ infusion of naked or slightly modified siRNAs,^[@bib12],[@bib13]^ ultrasound,^[@bib14]^ and convection-enhanced based delivery.^[@bib15]^ None of these approaches has received wide acceptance due to toxicity, a requirement for extensive repetitive dosing, and/or limited spatial distribution. Lipofection and electroporation of siRNAs are challenging in primary neurons due to low transfection efficiencies and their extreme sensitivity to external manipulation.^[@bib16]^ Delivery of siRNA precursors (Lentiviruses and AAV) has been used successfully, but viral transduction cannot readily be turned off and requires extensive formulation and experimental optimization to achieve reproducible, nontoxic silencing in neuronal cells.^[@bib17],[@bib18],[@bib19],[@bib20],[@bib21],[@bib22]^
In this study, we describe the delivery, distribution, and silencing capacity of hydrophobically modified siRNAs (hsiRNAs) in primary neurons and in mouse brain. hsiRNAs are siRNA-antisense hybrids containing numerous chemical modifications (see **[Figure 1](#fig1){ref-type="fig"}** and **Supplementary Table S1** for exact chemical composition of compounds used) designed to promote biodistribution and stability while minimizing immunogenicity. As a model for our studies, we silenced the huntingtin (*Htt*) gene, the causative gene in Huntington\'s disease (HD). HD is an autosomal-dominant neurodegenerative disorder caused by a toxic expansion in the CAG repeat region of the huntingtin gene leading to a variety of molecular and cellular consequences. Tetrabenazine, the only FDA-approved therapy for HD, seeks to alleviate disease symptoms but does not treat the actual problem: the gain of toxic function caused by mutant *Htt*. Recent studies suggest that transient neuronal knockdown of *Htt* mRNA can reverse disease progression without compromising normal cellular function *in vivo*.^[@bib23]^ At present, RNA interference via siRNA or antisense oligonucleotide is one of the most promising therapeutic approaches for transient *Htt* mRNA silencing.
We performed a screen of hsiRNAs targeting *Htt* mRNA and identified multiple functional compounds. We showed that primary neurons internalize hsiRNA added directly to the culture medium, with membrane saturation occurring by 1 hour. Direct uptake in neurons induces potent and long-lasting silencing of *Htt* mRNA for up to 3 weeks *in vitro* without major detectable effects on neuronal viability. Additionally, a single injection of unformulated (without cationic lipid or AAV formulation) *Htt* hsiRNA into mouse brain silences *Htt* mRNA with minimal neuronal toxicity.
Efficient gene silencing in primary neurons and *in vivo* upon direct administration of unformulated hsiRNA represents a significant technical advance in the application of RNAi to neuroscience research, enabling technically achievable genetic manipulation in a native, biological context.
Results
=======
hsiRNAs are efficiently internalized by primary neurons
-------------------------------------------------------
hsiRNA is an asymmetric compound composed of a 15-nucleotide modified RNA duplex with a single-stranded 3′ extension on the guide strand (**[Figure 1a](#fig1){ref-type="fig"}** and **Supplementary Table S1**).^[@bib24],[@bib25]^ Pyrimidines in the hsiRNA are modified with 2′-O-methyl (passenger strand) or 2′-fluoro (guide strand) to promote stability, and the 3′ end of the passenger strand is conjugated to a hydrophobic teg-Chol (tetraethylene glycol cholesterol) to promote membrane binding and association.^[@bib26]^ The single-stranded tail contains hydrophobic phosphorothioate linkages and promotes cellular uptake by a mechanism similar to that of antisense oligonucleotides.^[@bib27]^ The presence of phosphorothioates, ribose modifications, and a cholesterol conjugate contribute to overall hydrophobicity and are essential for compound stabilization and efficient cellular internalization.
Previous studies have shown that hydrophobically modified siRNAs bind to a wide range of cells and is readily internalized without the requirement for a transfection reagent.^[@bib26],[@bib28],[@bib29]^ Here, we evaluated whether asymmetric hydrophobically modified siRNAs are efficiently internalized by primary neurons. We found that, when added to the culture medium, Cy3-labeled hsiRNAs rapidly associated with primary cortical neurons (**[Figure 1b](#fig1){ref-type="fig"}**). These Cy3-labeled hsiRNAs were observed in every cell in the culture, demonstrating efficient and uniform uptake. Initially, hsiRNAs mainly associate with neurites and, over time, accumulate in the cell bodies. Treatment of primary neurons with a previously identified hsiRNA targeting *Ppib*^[@bib26],[@bib28]^ (encodes cyclophilin B) reduced target mRNA levels by 90%, further supporting that the observed compound internalization results in potent gene silencing (**[Figure 1c](#fig1){ref-type="fig"}**).
Identification of hsiRNAs that silence huntingtin mRNA
------------------------------------------------------
Robust uptake and efficacy observed with hsiRNAs in primary cortical neurons encouraged us to identify functional compounds that target *Htt* mRNA, the single gene responsible for the development of Huntington\'s disease. The hsiRNA\'s extensive chemical scaffold^[@bib26],[@bib28]^ is essential for stability, minimization of innate immune response,^[@bib30],[@bib31]^ and cellular internalization but imposes significant restrictions on sequence space by potentially interfering with the compound\'s RISC-entering ability. To maximize the likelihood of identifying functional *Htt* hsiRNAs and to evaluate the hit rate for this type of chemistry, we designed (using conventional criteria described in Materials and Methods) and synthesized hsiRNAs targeting 94 sites across the human *Htt* mRNA (**Supplementary Table S1**). The panel of hsiRNAs was initially screened for efficacy in HeLa cells by adding hsiRNA directly to the culture medium (without lipofection or electroporation) to a final concentration of 1.5 µmol/l and evaluating impact on levels of *Htt* and housekeeping (*Ppib*) gene mRNA expression using the QuantiGene (Affymetrix, Santa Clara, CA) assay. At this concentration, 24 hsiRNAs reduced *Htt* mRNA levels to less than 50% of control levels, including 7 hsiRNAs that reduced *Htt* mRNA levels below 30% of control (**[Figure 2a](#fig2){ref-type="fig"}**). Unlike unmodified siRNA libraries, creating a library with extensive 2\'-O-methyl and 2\'-fluoro modifications introduces additional constraints on sequence selection. As a result, hit rates for modified siRNA screens are lower than that seen for conventional unmodified siRNA.^[@bib32],[@bib33],[@bib34],[@bib35]^ Functional hsiRNAs targeted sites distributed throughout the mRNA, except the distal end of the 3′ UTR, which later was shown to be part of the alternative *Htt* gene isoform^[@bib36]^ not expressed in HeLa cells (data not shown). Discounting the \~32 hsiRNAs targeting long 3′ UTR sites absent from the *Htt* isoform in HeLa cells, almost 40% of hsiRNAs showed some level of activity at 1.5 µmol/l, demonstrating that the evaluated chemical scaffold is well tolerated by the RNAi machinery and a functional compound can be easily identified against a wide range of targets.
Half-maximal inhibitory concentrations (IC~50~) for passive uptake of hsiRNAs ranged from 82 to 766 nmol/l (**Supplementary Table S1 and Figure S1**). In lipid-mediated delivery, eight of the most active hsiRNAs had IC~50~ values ranging from 4 to 91 pmol/l (**Supplementary Table S1**). The best clinically active siRNAs are usually characterized by IC~50~ values in the low pmol/l range.^[@bib37]^ An ability to identify highly potent compounds with low picomolar IC~50~ values suggests that the hsiRNA chemical scaffold does not interfere with siRNA biological activity in selected compounds. The most potent hsiRNA targeting position, 10150 (HTT10150), and an unmodified conventional siRNA version of HTT10150 showed similar IC~50~ values in lipid-mediated delivery (4 and 13 pmol/l respectively, **[Figure 2c](#fig2){ref-type="fig"}**), further confirming that the hsiRNA chemical scaffold does not interfere with RISC loading or function. Only the fully modified hsiRNA, and not the unmodified version, silenced *Htt* mRNA by passive uptake (**[Figure 2b](#fig2){ref-type="fig"}**). Thus, the chemical scaffold described here does not interfere with RISC assembly and is sufficient to support unformulated compound uptake and efficacy. HTT10150 was used for subsequent studies.
Potent and specific silencing with unformulated hsiRNAs in primary neurons
--------------------------------------------------------------------------
HTT10150 induced a concentration-dependent silencing at 72 hours and
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{
"pile_set_name": "PubMed Central"
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Introduction {#s1}
============
Immunity is essential for survival, yet energetically expensive and potentially self-damaging. The immune system needs to be tightly regulated and highly responsive to changes in external and internal environments, adapting to developmental stage [@ppat.1003720-Kollmann1], nutritional state [@ppat.1003720-Becker1], and stress [@ppat.1003720-Cohen1]. Hormonal control of the immune system is not well-understood, and lies at the heart of a diverse range of clinically relevant phenomena including immune circadian rhythm [@ppat.1003720-Silver1], [@ppat.1003720-Stone1], obesity induced inflammation [@ppat.1003720-Fantuzzi1] and age- and gender-differences in immune function [@ppat.1003720-Kollmann1], [@ppat.1003720-Gilliver1].
*Drosophila* has proven to be a fruitful model of innate immunity [@ppat.1003720-Lemaitre1]. The innate immune system in the fly comprises humoral and cellular responses [@ppat.1003720-Lemaitre1]. The humoral response is best characterized by the secretion of antimicrobial peptides (AMPs) locally by epithelia such as the gut, or systemically by the fat-body, a functional analogue of the mammalian liver. This response to bacterial or fungal infection is mainly regulated by the Toll and Imd signaling pathways. Cellular immunity is performed by hemocytes, phagocytic circulating immune cells. *Drosophila* has become a powerful system to study phagocytosis due to high conservation of the processes involved [@ppat.1003720-Stuart1], [@ppat.1003720-Stuart2]. Cellular immunity is required to survive various types of bacterial infections, complementing the humoral response [@ppat.1003720-AvetRochex1]--[@ppat.1003720-Ulvila1] and is involved in the response to wasp parasitoid infestation [@ppat.1003720-Krzemien1]. In addition, in the larvae, hemocytes play a role in inter-organ communication, as they are required for the fat-body to mount a full humoral antimicrobial response after an intestinal infection [@ppat.1003720-Charroux1], [@ppat.1003720-Basset1], [@ppat.1003720-Wu1]. Hemocytes are also recruited to wounds in embryo and larvae [@ppat.1003720-Babcock1]--[@ppat.1003720-Wood1], potentially clearing damaged tissue and preventing further dispersal of microorganisms from unsterile wound sites. In parallel to their role in immunity, hemocytes perform developmental and homeostatic functions such as the phagocytosis of apoptotic cells and extracellular matrix secretion. These functions are essential at embryonic stages, for example, for central nervous system and renal tubule development [@ppat.1003720-Bunt1], [@ppat.1003720-Olofsson1]. The crosstalk between immunity and nutritional state or stress [@ppat.1003720-Becker1], [@ppat.1003720-DiAngelo1]--[@ppat.1003720-Storelli1] in *Drosophila* is beginning to be unravelled, however, developmental regulation of the immune system remains enigmatic.
Ecdysone is a steroid hormone, similar to mammalian estrogens and androgens; peaks in ecdysone titer regulate the major developmental transitions in the fly, including metamorphosis. The pupal stage lasts 4 days from the end of the 3^rd^ larval instar, after which adult flies eclose [@ppat.1003720-Thummel1]. The biologically active form of ecdysone, 20-hydroxyecdysterone (20-E, hereafter referred to as 'ecdysone') coordinates tissue remodelling at metamorphosis [@ppat.1003720-Thummel1]. This hormone activates a nuclear receptor, the Ecdysone Receptor (EcR), which acts as a heterodimer with its partner Ultraspiracle (USP), a homologue of the mammalian Retinoid X Receptor. Together, they activate the transcription of primary response genes, which in turn activate the transcription of a battery of late response genes [@ppat.1003720-Thummel1]. This transcriptional cascade ultimately leads to the induction of both cell death in larval tissues and differentiation and proliferation of the imaginal discs into adult tissues [@ppat.1003720-Thummel1]. In addition, several lines of evidence indicate that ecdysone regulates some aspects of hemocyte behaviour. In the larva, while the majority of hemocytes are in circulation, approximately one third of the total population interacts with tissues, attaching in repeated patches to the dorsal epithelium along the longitudinal axis [@ppat.1003720-Lanot1]. Despite the fact that hemocytes from these patches are mostly immotile at larval stages, it has been noticed that they disperse at metamorphosis [@ppat.1003720-Lanot1]. This observation correlates with the recent finding that *ex vivo*, hemocytes activate motility and morphological changes after metamorphosis [@ppat.1003720-Sampson1]. Cell shape changes can be prematurely triggered in larvae by ecdysone injection [@ppat.1003720-Lanot1]. Last, it is long known that ecdysone treatment is important to potentiate AMP gene expression and phagocytosis after an immune challenge in hemocyte-derived cell culture lines [@ppat.1003720-Dimarcq1]. Although these results suggest an ecdysone-dependent regulation of hemocyte function, *in vivo* evidence of a direct effect of ecdysone signaling on hemocyte behaviour, and its functional relevance, is lacking.
Here, we explore the hormonal regulation of *Drosophila* hemocytes at metamorphosis and its impact on *Drosophila* immunity using an *in vivo* approach. We demonstrate that direct activation of ecdysone signaling in hemocytes is necessary to increase their developmental and immune activities at metamorphosis, including phagocytosis. We show that this activation is essential to respond efficiently to and survive pathogenic challenge.
Results {#s2}
=======
1. The Ecdysone Receptor is required in the hemocytes for their activation at metamorphosis {#s2a}
-------------------------------------------------------------------------------------------
To test whether ecdysone signaling cell-autonomously regulates hemocyte shape changes at metamorphosis, we used the *Hml-(Hemolectin-)ΔGal4* driver [@ppat.1003720-Sinenko1] to specifically express green fluorescent protein (GFP) and dominant-negative constructs of the three known EcR isoforms under the control of the UAS sequence. We imaged hemocytes *ex vivo* by bleeding larvae or pupae at precise time points after puparium formation (APF) ([Fig. 1A--B](#ppat-1003720-g001){ref-type="fig"}) and *in vivo* through the dorsal epidermis ([Fig. 1C--D](#ppat-1003720-g001){ref-type="fig"}). The first hour of the 12h 'prepupal' period is characterized by a translucent pupal case (which then darkens). Control hemocytes (from *HmlΔGal4, UAS-GFP/+* pupae, here after referred to as *HmlΔ*\>GFP) displayed a clear change of morphology over metamorphosis: they became gradually more polarized, with many cytoplasmic protrusions and a higher number of vacuoles, increasing in size dramatically, likely due to their greater spread and many phagocytic vesicles ([Fig. 1A and C](#ppat-1003720-g001){ref-type="fig"}). In striking contrast, hemocytes expressing a dominant negative (DN) form of the EcRB1 isoform (from *HmlΔGal4, UAS-GFP/UAS-EcRB1DN* pupae, here after referred to as *HmlΔ\>EcRB1DN*) did not show any obvious change of size or morphology ([Fig. 1B and D](#ppat-1003720-g001){ref-type="fig"}). Similar results were obtained when we analysed by flow cytometry the properties of the hemocyte population at the onset of pupariation. Control hemocytes displayed a clear shift both in Forward Scatter (FSC) and Side Scatter (SSC), indicating an increase in cell size and granularity, respectively ([Fig. 1E](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}). In contrast, the FSC and SSC of hemocytes expressing *EcRB1DN* remained stable over metamorphosis and similar to the parameters observed for control hemocytes in late 3^rd^ instar larvae (L3 wandering; L3W; [Fig. 1F](#ppat-1003720-g001){ref-type="fig"} and [S1](#ppat.1003720.s001){ref-type="supplementary-material"}).
![Ecdysone signaling is required for hemocyte activation at metamorphosis.\
(A--D) Analysis of the morphology of control hemocytes (A, C) and hemocytes expressing a DN form of the EcR receptor (EcRB1DN; B, D) at precise time points before and after puparium formation (APF). (A, B) *Ex vivo* analysis of bleeds; (C, D) *in vivo* analysis of cells visualized under the dorsal epithelium. Green, endogenous GFP. Blue, DAPI. Red, phalloidin.
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Introduction {#s1}
============
Tuberculosis (TB) is caused by the pathogenic species, *Mycobacterium tuberculosis (Mtb)*; together with human immunodeficiency virus (HIV/AIDS) infection, TB is among the most prevalent and severe of the infectious diseases worldwide. In 2019, an estimated 10 million people developed active tuberculosis in association with 1.6 million deaths ([@B1]). Infection with *Mtb* triggers an immune response, however *Mtb* can survive and grow by circumventing the host immune detection. One of the pathological characteristics of the successful infection with *Mtb* is the formation of granulome, which are organized cellular structures that include a variety of innate and adaptive immune cells that surround the *Mtb*-infected phagocytes ([@B2]--[@B5]). During the formation of granulome, intricate host-*Mtb* interactions occur at the infectious site and this pathogen can escape various host immune responses, which ultimately prevent *Mtb* elimination by these systems. Once *Mtb* enters the host, its cell wall components and proteins are detected by Toll-like receptors (TLRs), primarily by TLR2 and TLR4. *Mtb* is engulfed by professional phagocytic cells such as a macrophage, dendritic cell (DC), or neutrophil, and becomes incorporated into the subcellular organelle formed by the fusion of the phagosome and lysosome to create the phagolysosome, however *Mtb* is able to manipulate the endocytic pathway by suppressing fusion of the phagosome containing the bacteria with lysosomes. Infected macrophages synthesize and release both inflammatory and antimicrobial genes and molecules, including interleukin (IL)-1β, IL-6, IL-12, tumor necrosis factor (TNF), inducible nitric oxide synthase/nitric oxide synthase 2 (iNOS/NOS2), and chemokines which activate both the innate and adaptive immune systems. Activated immune cells secrete protective molecules to the extracellular space to promote recruitment of other immune cells to form a granuloma ([@B4], [@B6]). Interestingly, endogenous proteins expressed by *Mtb* serve to perturb the formation of phagolysosome, the permitting its survival and proliferation within macrophages. For preventing excessive lung damage during *Mtb* infection, *Mtb* also elicits the production of protective factors that promote its survival including anti-inflammatory mediators such as IL-4, IL-10, IL-13, and transforming growth factor β (TGF-β) ([@B7]--[@B9]) and several human TB studies show that these factors has been shown to be increased in the active TB patients ([@B10], [@B11]). These immunosuppressive factors play key roles in limits effective the immune defense to *Mtb* ([@B12], [@B13]). *Mtb* will persist and exacerbate pathophysiological manifestations within the granulome; this will ultimately result in progression of disease and dissemination to the other hosts ([@B5], [@B14]). As a major focus of this disease process, mycobacterial granulome have been the subject of intense scrutiny mainly focused on mechanisms of formation, function, maintenance, and evolution.
Recently, there has been an increasing appreciation of the important relationship that exists between essential metabolism and immune cell function. Metabolic reprogramming in immune cells, a phenomenon known as immunometabolism, focuses on unique cellular functions that are essential for the immune response. During TB infection, host cells undergo profound metabolic change, which results in differential control of various cytokines and chemokines associated with inflammation, clearance, inhibition, and progression of *Mtb* infection ([@B15], [@B16]). Specifically, a shift in the use of pathways promoting glucose and lipid metabolism can be an important feature for directing host cell function to promote mycobacterial survival with the granulome ([@B17]). At homeostasis, cells in "resting" condition utilize oxidative phosphorylation (OXPHOS) to produce ATP from NADH and FADH2 by facilitating transfer of protons and electrons. Cells typically switch from OXPHOS to glycolysis in order to generate ATP under oxygen-depleted or hypoxic conditions ([@B18]). Similarly, glycolysis is main form of metabolism in immune cells that promote the inflammatory response in the immune system. This observation--that immune cells utilize glycolysis even in the presence of adequate concentrations of oxygen (i.e., aerobic glycolysis)-- is known as the "Warburg effect." To date, the Warburg effect has been explored primarily with respect to cancer metabolism. Although aerobic glycolysis generates fewer ATP molecules per cycle than does OXPHOS, this pathway is capable of rapid generation of ATP required by immune cells. Additionally, aerobic glycolysis requires a number of specific precursors, including nucleotides, amino acids, and lipids ([@B19]). Because metabolic reprogramming is essential for immune cell function, studies that explore this phenomenon in also provide new insight into the relationship between host immune cells and infection with *Mtb*. Furthermore, predisposing factors for TB, including diabetes, and HIV also related to immunometabolism against TB pathogenicity. Diabetes mellitus (DM) is a mainly risky factor for occurring active TB ([@B20]--[@B22]). In DM, innate immune cells undergo activation for releasing cytokines, recruiting neutrophils, upregulate T cell activation and antigen recognition ([@B23], [@B24]). Metabolism of DM is characterized by increasing glucose production and impairing glucose uptake. Expression of glucose transporter and glycolytic enzymes is elevated in DM ([@B25]). In DM, High glucose level increased IL-10 production, impaired macrophage phagocytic ability for promoting better milieu for survival and proliferation of TB ([@B26], [@B27]). Additionally, HIV is also other pathogen to be associated with pathogenicity of TB ([@B28]--[@B30]). In HIV-1-infected primary CD4^+^ T cells, glycolytic metabolism is induced with high pro-inflammatory response and increased production of virus ([@B31], [@B32]). Interestingly, glycolytic metabolism is regulated by HIV-1 infection in macrophage alleviated Warburg effects ([@B33]). These factors promote the activation of TB by reprogramming the metabolism.
A variety of antibiotics have been introduced for promoting eradication of *Mtb* infection, including 6--9 months courses of isoniazid, rifampicin, ethambutol, and pyrazinamide. However, the emergence of multidrug-resistant TB (MDR-TB) or extensively drug-resistant TB (XDR-TB) has become a major challenge toward designing effective treatments and for eradication of this disease ([@B34], [@B35]). Among the approaches to this challenge, host-directed therapy (HDT) has been introduced as a means to potentiate and to amplify the effectiveness of current treatments used for TB ([@B36]). A clear understanding of the molecular interactions between host cell metabolism and accommodations made to *Mtb* may provide new strategies to combat infection. Here we review the current understanding of the metabolic relationship between the host and the *Mtb* pathogen. We also suggest several new strategies that may enhance host metabolic pathways and thereby promote protective antimicrobial functions in the setting of TB infection.
Metabolic Reprogramming in TB {#s2}
=============================
Warburg Effect in Immune Cells
------------------------------
Immune cells provide critical protection and maintain homeostasis in the mammalian host. There are currently many studies that suggest that the functions of immune cells are largely reliant on specific aspects of host metabolism. These studies, which have generated a field known as immunometabolism, have provided us for a new focus for understanding how and why immune cells exist or persist in a specific metabolic state in order to support or direct functional changes. Several recent reports suggest that different metabolic signatures have a direct impact on specific effector functions characteristic of the innate and adaptive immune systems ([@B37]). As such, among the primary functions of immune cells, there are those that generate an inflammatory response, actions typically undertaken by M1-polarized macrophages, DCs, neutrophils, and effector T cells, and those that promote an anti- inflammatory response, which include M2-polarized macrophages, as well as regulatory and memory T cells. The basic metabolic profiles of these cells differ significantly from one another. Inflammatory immune cells generate energy in the form of ATP mainly via glycolytic metabolism; by contrast, immune cells that promote anti- inflammatory activities generate ATP via oxidative phosphorylation and fatty acid oxidation ([@B38]--[@B43]). These observations have been best characterized for polarized macrophages. The predominant phenotypes of macrophages are known as M1 and M2 ([@B44], [@B45]). M1 macrophages, activated by lipopolysaccharide (LPS) and IFN-γ, promote pro-inflammatory and antibacterial functions in immune system, and they produce nitric oxide (NO) and reactive oxygen species (ROS) which are fundamental components of the pathways used to eradicate bacteria. The main metabolic pathway used by these cells is glycolysis, which results in rapid production of ATP via inhibition of the trichloroacetic acid (TCA) cycle and OXPHOS in mitochondria; this is a critical factor due to the fact that M1 macrophages require rapid generation of ATP to activate inflammation. By contrast, M2 macrophages promote anti-inflammatory responses and tissue repair; these cells mainly utilize OXPHOS and fatty acid oxidation in order to generate ATP; this takes place via efficient pathways localized in the mitochondria ([@B46]--[@B51]). In T cells, metabolic state is reprogrammed according to T cell subsets. Naïve T cells mainly use OXPHOS for generating energy. Upon TCR stimulation, glycolytic metabolism is upregulated for differentiating into activated T cell. Th1, TH2, and Th17 effector cells mainly depend on aerobic glycolysis. While, regulatory and memory T cells use fatty acid oxidation and OXPHOS for differentiation and functions ([@B52], [@B53]). Mammalian target of rapamycin (mTOR) and AKT signaling is essential for regulating metabolism of T cells and cytokine responses ([@B54]). Recently, cyclophililn D (
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There are errors in the Funding section. The correct funding information is as follows: This study was supported by the National Cancer Institute of the National Institutes of Health under award number K08CA155035 and the Melanoma Research Alliance. The authors are also grateful to Timothy Dattels for his generous support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Introduction
============
Diabetes mellitus is a common endocrine metabolic disease estimated to affect 629 million individuals in 2045 according to the International Diabetes Federation. T2DM is the extremely prevalent form of diabetes that accounts for 90% of diagnosed cases and is associated with insulin resistance and chronic hyperglycemia ([@B13]). Many clinical studies reported a broad spectrum of lower urinary tract symptoms in diabetic patients ([@B16]), accounting for 90--95% of all diabetes cases ([@B17]). DBD is a major lower urinary tract complication of diabetes and was first described by [@B26]. Such complication is traditionally described as a triad of increased capacity, decreased sensation, and poor emptying ([@B11]) and has affected over 50% of diabetic patients ([@B12]; [@B28]). DBD development is divided into two phases: the compensated phase, which occurs in the early phase and is characterized by an OAB; and the decompensated phase, which occurs in the late phase and is characterized by an atonic bladder ([@B36]; [@B24]).
The pathogenesis of DBD is multifactorial and accompanied by the structural and functional impairments of the bladder ([@B37]). The bladder structural remodeling of DBD, such as the increase in bladder capacity, total BWT, and smooth muscle content, was observed in STZ-induced diabetic mice. Such remodeling may be a physical alteration to increase the urine volume ([@B22]). The two major functions of bladder are urine storage and urine disposal, and the uncoordinated contraction in the OAB of a diabetic greatly affects the urine storage ability of this organ ([@B10]). Bladder contraction is mainly mediated by purinergic and cholinergic pathways ([@B23]). In particular, ATP is the purinergic messenger released from varicosities or bulbous nerve endings of neurons, and the contractile responses mediated by ATP play a key role in DBD ([@B44]). Solute carrier family 17 member 9 (SLC17A9) is a member of the solute-carrier protein family that plays an indispensable role in the vesicular storage of ATP ([@B31]; [@B20]). The translocation of neurotransmitter-filled vesicles to the varicose terminal is the first step in the release of vesicular neurotransmitters, followed by the merger of vesicles with the membrane of the varicose terminal and the precise and rapid release of their contents into the synaptic cleft ([@B33]). In addition, the motor of vesicles for transportation to the varicose membrane in the cells is mainly provided by myosin motors, particularly myosin Va ([@B2]). Several studies found that the purinergic inhibitory neurotransmission was impaired in myosin Va-deficient mice ([@B6]). Such finding suggested that myosin Va played an important role in purinergic neurotransmission ([@B3]).
Diabetic bladder dysfunction, particularly OAB, is not life threatening to humans. However, this dysfunction seriously affects the quality of the life of patients ([@B24]). The treatment methods for DBD changed when the phase progresses. Anticholinergic drugs, such as tolterodine and solifenacin, are the main treatment options for DBD patients with OAB. However, many side effects, including dry mouth, dry eyes, and memory loss, occurs after the treatment with anticholinergic drugs, thus rendering poor life quality for the patients. In the late phase, surgical intervention was the only therapeutic method for patients who did not benefit from pharmacological and behavioral treatments ([@B45]). However, pharmacological and surgical interventions were largely ineffective in clinics ([@B12]; [@B40]). Therefore, new effective treatments for DBD are urgently needed.
In the treatment of diabetic OAB, traditional Chinese medicine and natural plant components have recently attracted increasing attention due to their safeness, few side effects, and excellent activity ([@B41]). SQW is a traditional Chinese herbal formula that was first recorded on *Fu Ren Liang Fang* in the Southern Song Dynasty (between 1127 and 1279 CE). This medicine is a mixture of three Chinese medicines: *roots of Lindera aggregata (Sims) Kosterm. (Lauraceae), roots of Alpinia oxyphylla Miq., (Zingiberaceae), and rhizomes of Dioscorea oppositifolia L. (Dioscoreaceae)* at a 1:1:1 ratio ([@B14]). SQW has been used to treat lower urinary tract symptoms, such as nocturia, urgency, and child bedwetting for hundreds of years ([@B4]). We have recently reported that SQW had therapeutic effects on the OAB of bladder outlet obstruction rat models by modulating the TRPV1 expression ([@B18]). In China, SQW is often used in the clinical treatment of diabetic OAB. However, its mechanism remains unclear, and its therapeutic effect has not been investigated in animal studies. Therefore, we designed experiments to explore the effects and therapeutic mechanisms of SQW in diabetic OAB mouse model.
Materials and Methods {#s1}
=====================
Reagents and Materials
----------------------
Suo Quan Wan was purchased from Hunan Hansen Pharmaceutical Co., Ltd. (China), and the quality control was provided by the company based on Chinese Pharmacopeia employing by high performance liquid chromatography (HPLC) technology from SQW samples ([@B9]). Three Chinese herbals were ground and mixed evenly at a 1:1:1 ratio and appropriate volumes of distilled water were used to make these powders to SQW compound. The doses were adopted according to the Experimental Methodology of Pharmacology, based on clinical usage, the Bios method ([@B39]). SQW H was 2.208 g/kg, SQW M was 1.104 g/kg, and SQW L was 0.552 g/kg. The tolterodine dose for the positive group was 0.82 mg/kg.
Streptozotocin was purchased from TOKU-E Co., Ltd. (Japan). HFD (45% fat) and control diet were purchased from Guangdong Medical Laboratory Animal Center (China). Tolterodine was purchased from Chengdu Dikang Pharmaceutical Co., Ltd. (China). Roche dynamic Bg meter was purchased from Hoffmann-La Roche Inc. (Switzerland), and carbachol was obtained from Shandong Bausch & Lomb Freda Pharmaceutical Co., Ltd. (China). α,β-methylene ATP was purchased from Tocris Bio-Techne Ltd. (United Kingdom). FastQuant RT Kit (with gDNAse) and Talent qPCR PreMix (SYBR Green) were purchased from TIANGEN Biotech (Beijing) Co., Ltd. (China). TRIzol reagent was purchased from Thermo Fisher Scientific (United States). RIPA lysis buffer and protease inhibitor cocktail (100×) were obtained from CoWin Biosciences (China). All other reagents used were of analytical grade.
Preparation and HPLC Conditions of SQW
--------------------------------------
Suo Quan Wan samples were weighted 0.3 g and extracted with 25 mL of methanol-hydrochloric acid solution using heating reflux method and then cool the solution. Finally, the solution was filtered through 0.45 μm nylon membranes before injection.
According to the Chinese pharmacopoeia 2015, the content of norisoboldine should be more than 0.4 mg/0.3 g, and the content of allantoin is more than 0.48 mg/0.3 g. The HPLC conditions and gradient elution were shown as [Tables 1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}.
######
Chromatographic condition for norisoboldine.
Column C18 (25°C)
------------ ----------------------------------------- -------
Solvent A Acetonitrile
Solvent B 0.5% formic acid and 0.1% triethylamine
Flow rate 1.0 mL/min
Wavelength 280 nm
Time (min) A (%) B (%)
0 10 90
13 22 78
30 22 78
######
Chromatographic condition for allantoin.
Column C18 (25°C)
------------ ------------ -------
Solvent A Methanol
Solvent B H~2~O
Flow rate 1.0 mL/min
Wavelength 191 nm
Time (min) A (%) B (%)
0 8 92
10 10 90
20 10 90
Animal Model and Treatment
--------------------------
All experimental protocols and animal procedures complied with the ethical principle guidelines of the National Research Council. A total of 100 male C57BL/6J mice (18--22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in the Experimental Animal Center of Guangzhou University of Chinese Medicine (No. S2017051, Guangzhou, China) under room temperature and exposed to a 12 h/12 h light--dark cycle, with free access to food and water. The animals were fed with normal diet for 3 days and then divided into two groups, namely, diabetic (*n* = 85) and control (*n* = 15) groups. The mice in the diabetic group were fed with HFD, whereas those in the control group received normal diet. After 4-week feeding, the mice in the diabetic group were injected with STZ at 100 mg/kg dissolved in citrate buffer for four times (0.05 M, pH 4.3--4.5). The mice in the control group were treated with an equal volume of vehicle (0.05 M citric acid
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Introduction {#sec1}
============
In contrast to bulk silver, nanometric silver materials exhibit many extraordinary properties such as a high surface-to-volume ratio,^[@ref1],[@ref2]^ quantum tunneling effects,^[@ref3],[@ref4]^ an abundance of free electrons,^[@ref5]^ surface plasmon resonance,^[@ref2],[@ref6]−[@ref8]^ and antibacterial behaviors.^[@ref9],[@ref10]^ Because of these unique properties, noble silver nanomaterials are widely applied in diverse areas, including thermotherapy,^[@ref5],[@ref11]^ medicine,^[@ref2],[@ref5],[@ref12]^ sensors,^[@ref13]−[@ref16]^ surface-enhanced spectroscopy,^[@ref6],[@ref17]−[@ref20]^ biology,^[@ref2]^ catalysis,^[@ref21]−[@ref28]^ and electronics.^[@ref29]−[@ref31]^ Among these many applications, the catalysis of the reduction of nitroarenes to aromatic amines is increasingly attracting attention because of pharmaceutical needs and the importance of this industry.^[@ref25],[@ref26],[@ref32]−[@ref38]^ Various strategies have been proposed to reduce nitroarenes more efficiently and more rapidly using silver nanomaterials. These strategies include depositing silver nanoparticles (AgNPs) on supports used as heterogeneous catalysts,^[@ref24],[@ref26],[@ref39]−[@ref42]^ combining AgNPs with reduced graphene oxide or graphene oxide as catalysts,^[@ref43]−[@ref47]^ and using silver nanocolloids as a quasi-homogeneous nanocatalyst.^[@ref25],[@ref33],[@ref48]−[@ref54]^ All of the aforementioned catalytic approaches are efficient and selective. However, the reaction rate for heterogeneous catalysis is rather low and quasi-homogeneous catalysis suffers from possible aggregation of the nanocatalyst. Furthermore, the procedures for preparing such nanocatalysts are somewhat complex and time-consuming.
Applications of magnetic nanoparticles have also been extensively investigated in recent years because they feature many notable characteristics, such as a high surface-to-volume ratio, easy attraction and redispersion, and paramagnetism. Because of these crucial properties and advantages, the combination of AgNPs and magnetic nanoparticles has become one of the most favorable approaches for the catalytic reduction of nitroarenes.^[@ref55]−[@ref60]^ In this study, a simple but facile method was applied in a single step to prepare silver-doped magnetic nanoparticles (AgMNPs) for the catalytic reduction of nitroarenes through spontaneous oxidation--reduction and coprecipitation. When mixing Fe^2+^ with Ag^+^, a spontaneous reaction is caused by the difference in standard reduction potential between the ionic species. When Ag^+^ is reduced to Ag^0^, an equivalent number of moles of Fe^2+^ ions are simultaneously oxidized to Fe^3+^. After the addition of precipitation agents, AgNPs were coprecipitated with iron oxide magnetic nanoparticles, which led to the formation of AgMNPs. The proposed preparation can be achieved in a single step, and the prepared AgMNPs can subsequently be utilized as nanocatalysts for the reduction of *o*-nitroaniline (*o*-NA). The parameters (pH, temperature, and amount of nanocatalyst) that affect the morphology and composition of the prepared AgMNPs and efficiencies of the catalytic reduction were systematically studied to gain a greater understanding of the characteristics of the AgMNPs prepared using the method proposed in this study. Additionally, the catalytic activity of the AgMNPs prepared for the reduction of other nitroarenes and their recyclability were investigated to fully evaluate their potential for practical applications.
Results and Discussion {#sec2}
======================
Effect of Oxidation--Reduction Time on AgMNP Preparation {#sec2.1}
--------------------------------------------------------
[Figure S1](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the typical measured hysteresis loops of the prepared AgMNPs, which confirmed that the prepared AgMNPs were paramagnetic and usable for further applications. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} depicts the transmission electron microscopy (TEM) images of AgMNPs obtained using various reaction times. From the images, dark-sphere-like Ag nanoparticles were mixed with light-colored Fe~3~O~4~ NPs because Ag has a higher electron density that allows fewer electrons to transmit.^[@ref61],[@ref62]^ The AgNPs formed after a 10 min reaction time were larger than those formed after a 2 min reaction time. Notably, the size of the Fe~3~O~4~ NPs was mostly unaffected by the reaction time.
![TEM images of prepared AgMNPs with oxidation--reduction times of (a) 2 min, (b) 8 min, and (c)--(f) 10 min, where the \[Fe^2+^\]~0~ to \[Ag^+^\]~0~ ratios are (a)--(c) 3:1, (d) 2:1, (e) 4:1, and (f) 6:1. \[Fe^2+^\]~0~ are all 12 mM, and the magnifications of the images are all 100 000×. The yellow arrows indicate the examples of AgNPs for each sample.](ao-2017-019876_0008){#fig1}
[Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}a shows the evolution of the UV--vis spectra for the reduction of *o*-NA catalyzed by AgMNPs over time. The absorbance peak at 412 nm, which corresponds to the characteristic *o*-NA peak,^[@ref63]^ decreased as the reaction proceeded. The variations of the spectra indicated that *o*-NA was reduced to 1,2-phenylenediamine (1,2-PPD).^[@ref41],[@ref64]^ The relative concentration (*C*~t~/*C*~0~) of *o*-NA was obtained by dividing the absorbance recorded at 412 nm at the specified time (*C*~t~) by the absorbance at 412 nm before the addition of AgMNPs (*C*~0~). The results in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b were plotted using various AgMNPs prepared using various reduction durations. In [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, the catalytic efficiency of the AgMNPs shows no significant difference between when the reaction time was 4--10 min and when more than 95% of *o*-NA was reduced within 240 s. This finding suggests remarkable catalytic activity. By contrast, achieving the same conversion percentage required more than 500 s when the reaction time to prepare the AgMNPs was 2 or 12 min. Because the catalytic efficiency of nanocatalysts depends on their size and the amount of catalyst loaded,^[@ref26]^ we concluded that the reaction time to achieve the optimal morphology and catalyst loading was 10 min. For comparison, the results of an experiment conducted in parallel, where AgMNPs were replaced with Fe~3~O~4~ NPs, are also plotted in [Figure [2](#fig2){ref-type="fig"}](#fig2){ref-type="fig"}b, showing that the reduction of *o*-NA can proceed only when AgNPs are doped. In the absence of AgNPs, the reduction of *o*-NA is suspended.
{ref-type="fig"}](#fig1){ref-type="fig"}c. (b) *C*~t~/*C*~0~ of 1 mM *o*-NA (412 nm) versus the catalytic reduction time in the presence of 30 mM NaBH~4~ and AgMNPs prepared with (■) 2 min, (●) 4 min, (▲) 6 min, (▼) 8 min, (◆) 10 min, and (★) 12 min of oxidation--reduction time during the preparation, where the other conditions are the same as described in (a). Parallel experiment (□) uses 20 mg of Fe~3~O~4~ NPs as nanocatalysts, where the other conditions are the same as described as (a).](ao-2017-019876_0001){#fig2}
Effect of Fe^2+^/Ag^+^ on AgMNP Preparation {#sec2.2}
-------------------------------------------
As described in the previous section, catalytic efficiency is related to the size and amount of doped AgNPs. In addition, we expected the morphology and amount of doped AgNPs to be affected by the ratio of initial concentration of Fe^2+^ to Ag^+^ because AgNO~3~ acts as the oxidation agent in the formation of AgMNPs. [Figure [1](#fig1){ref-type="fig"}](#fig1){ref-type="fig"} shows the TEM images of the AgMNPs with different ratios of \[Fe^2+^\]~0~ to \[Ag^+^\]~0~. The images reveal that the morphologies of the AgNPs are similar. [Figure S2](http://pubs.acs.org/doi/suppl/10.1021/acsomega.7b01987/suppl_file/ao7b01987_si_001.pdf) shows the typical X-ray diffraction (XRD) spectra of the prepared AgMNPs and
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INTRODUCTION {#s1}
============
Whilst oncogenesis is driven by a multitude of complex, non-programmed molecular events, there are a number of key features of this process, not least of which is the aberrant activation of genes that would normally be silenced in a given tissue context \[[@R1]\]. The so called cancer/testis (CT) or cancer germline (CG) genes are one such group of genes that are frequently activated in a range of different human cancer types \[[@R2]-[@R4]\]. These genes have expression normally restricted to the human germline, many being testis-specific \[[@R2]-[@R4]\]. They have come under intense scrutiny since their original identification as the immunological privilege of their normal germline setting means that the proteins they encode can elicit an immunological response when aberrantly produced in cancers and so have exceptional potential in immunotherapeutics \[[@R5]\]; for example, the *NY-ESO-1* gene product has been successfully targeted in an adoptive therapeutic approach to melanoma therapy \[[@R6]\].
Despite this interest, remarkably little is known about the normal germline function of most CT genes. Moreover, it has been demonstrated that germline genes in *Drosophila melanogaster* are required for the oncogenic process and that the human orthologues of these *Drosophila* genes have up-regulated expression in a range of human cancers, although the functional implications for oncogenesis of this up-regulation remains unclear \[[@R7],[@R8]\]. Interestingly, down-regulation of a number of CT genes in human cancer cells results in perturbation of cellular proliferative potential \[for example, see [@R9],[@R10]\]. These findings open up the exciting possibility that CT genes might encode functions that are required for tumour homeostasis and it has recently been proposed that tumours become 'addicted' to these germline factors \[[@R11],[@R12]\], and recently, meiotic factors have been shown to contribute to telomere maintenance in cancer cells via the ALT pathway \[[@R13], [@R14]\]. The full extent of germline gene requirement is unclear, but these findings expose a new therapeutic opportunity by directly targeting the tumour-associated function of the CT gene products. Additionally, a number of studies have revealed another clinically important feature of CT genes; their expression appears to drive drug resistance as depletion of the gene products results in enhanced sensitization to anti-cancer drugs \[for example, see [@R15]\] expanding the therapeutic potential of this important class of cancer genes.
Germline gene expression profiling has also recently been demonstrated to have applications in prognostics and patient stratification. In a seminal study, Rousseaux and co-workers demonstrated that expression of a sub-set of germ line genes in some lung cancers delineated patients with aggressive, metastasis prone tumours with poor prognosis \[[@R16]\]; they extended this by indicating that this cohort of patients might benefit from a drug therapeutic regime that had previously been dismissed for more general use in lung cancer patients, indicating that profiling patients for expression of a small sub-set of germline genes could be used in therapeutic decision making. Understanding germline gene expression is also critical as drug-induced augmentation of expression has also been postulated to be a potential enhancer of immunotherapeutics, the rationale being that further up-regulation of a tumour-specific antigen will result in enhanced immunological targeting of the tumour \[for example, see [@R17]\].
Taking all these factors together reveals the importance of understanding the regulatory mechanisms for somatic germline gene silencing and their aberrant activation in tumours. To date, the regulation of a number of CT genes has been studied and it has been demonstrated that DNA methylation of regulatory elements, such as promoter-associated CpG islands plays a fundamental role in the somatic silencing of these genes and the hypomethylation of these regulatory DNA regions in cancers is linked to gene activation \[for example, see [@R18]-[@R23]\], whereas gene body hypomethylation has been linked to gene down regulation in cancers \[[@R24]\]. Expression of these genes also becomes activated or further up-regulated upon enforced hypomethylation by the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR), and to date, all CT genes studied have up-regulated expression in response to this chemotherapeutic agent, indicating a commonality in the mechanistic pathway for somatic CT gene silencing \[for example, see [@R18]-[@R23]\].
To date, most of the CT genes whose expression has been studied are located on the X chromosome (X-CT genes) and belong to large paralogous gene families \[[@R2]-[@R4]\]. Recently, a computational pipeline combining expressed sequence tag and microarray meta-analyses of the human orthologues of mouse spermatocyte-specific genes revealed a large cohort of new CT genes that were expressed in a broad spectrum of cancer types \[[@R25]-[@R29]\]. Unlike the X-CT genes, the majority of these genes are autosomally encoded and are single copy. To date, the clinical potential of these genes remains largely unexplored. In this current study, analysis of the expression of a small sub-set of these genes reveals a novel feature of CT genes, which indicates that some have a unique mechanism for somatic transcriptional silencing. This is a significant finding as these genes and their associated gene products have an increased prominence in clinical applications and hence the sub-classification of CT genes will play an important role in diagnostics, stratification and therapeutics.
RESULTS {#s2}
=======
All CT genes studied to date (mostly X-CT genes) require hypermethylation of regulatory DNA sequences for somatic silencing and are activated by the hypomethylating agent 5-aza-CdR. Given the clinical potential of enhanced up-regulation of immunogenic CT antigens, we set out to explore whether a similar DNA hypermethylation silencing mechanism was operating in the recently identified autosomally encoded CT genes \[[@R25],[@R27]\]. To do this, we selected a small sub-group of these genes that remained transcriptionally silenced in the colorectal cancer cell lines HCT116 and SW480 (*ARRDC5, C4orf17, C20orf201, DDX4, NT5C1B, STRA8, TDRD12*). We also selected two previously characterized CT genes (both X-CT genes) that remained transcriptionally silenced in these two cell lines to serve as exemplar controls for hypermethylation regulated CT genes, *SSX2* and *GAGE1*. To determine whether the novel CT genes are silenced via hypermethylation mediated mechanisms, similar to the characterized X-CT genes, we treated the two cell lines with the DNA methyltransferase inhibitor 5-aza-CdR to determine whether inhibition of DNA methyltransferase activity can activate these genes. Following 5-aza-CdR treatment of HCT116 and SW480 we made cDNA and carried out RT-PCR and agarose gel electrophoresis analysis of the products. The two X-CT genes were activated from the silent state with relatively low levels of 5-aza-CdR (0.1 μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). Some of the novel, autosomally encoded CT genes were similarly activated (*C20orf201, DDX4, STRA8, TDRD12*), although *C20orf201* and *DDX4* required a slightly higher 5-aza-CdR concentration for activation (0.5 μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). Additionally, activation of *STRA8* requires slightly higher concentrations of 5-aza-CdR in SW480 (Figure [2](#F2){ref-type="fig"}) than HCT116 (Figure [1](#F1){ref-type="fig"}), which indicates subtle regulatory differences between tumour cell types. However, surprisingly, three genes (*ARRDC5, C4orf17, NT5C1B*) remained tightly transcriptionally silenced, even at high concentrations of 5-aza-CdR in both cell lines (15.0 μM; Figure [1](#F1){ref-type="fig"}; Figure [2](#F2){ref-type="fig"}). This unexpected result reveals an important distinction in the way CT gene silencing is epigenetically regulated, revealing a hypermethylation-independent pathway. Interestingly, the X-CT genes (*GAGE1, SSX2*) remained activated for a prolonged period following removal of the hypomethylating agent, as did the autosomally encoded CT genes that were activated with the lowest concentration of 5-aza-CdR (*STRA8, TDRD12*) (Figure [3](#F3){ref-type="fig"}); however, the other two autosomally encoded CT genes, *C20orf201* and *DDX4*, which required slightly higher concentrations of 5-aza-CdR for activation, reverted to the silent state relatively soon after removal of the hypomethylating agent (Figure [2](#F2){ref-type="fig"}). This indicates a much greater transcriptional elasticity to the methylation-dependent silencing mechanisms for some CT genes.
![A sub-group of germline genes remain refractory to activation by epigenetic modulating agents\
RT-PCR was used to analyse activation of a group of germline genes that are normally silenced in the cancer cell line HCT116 (an additional colorectal cell line gives similar results \[see Supplementary Figure S1)\]. Whilst a cohort of known and newly identified germline genes become activated at low doses of the demethylating agent 5-aza-CdR (*GAGE1, SSX2, STRA8, TDRD12*) and others become activated with slightly higher levels of 5-aza-CdR (*C20orf201, DDX4*), some remain tightly silenced, even at high concentrations of 5-aza-CdR (*ARRDC5, C4orf17, NT5C1
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Introduction
============
Gingival reactive lesions like pyogenic granuloma have frequent occurrence around natural dentition, however, their association with dental implants is not common. The causes of pyogenic granuloma (PG) in relation to dental implants are not clear mainly due to few published cases ([@B1]-[@B8]).
Tooth-related PG is a result of tissue response to minor injury or chronic low-grade irritation ([@B9]-[@B16]). Clinically, oral PG is characterized as a soft mass of smooth or lobulated appearance that could be sessile or pedunculated and frequently presents ulceration. The lesion grows rapidly for a few weeks and the colour ranges from pink to red purple and haemorrhage may occur either spontaneously or after minor trauma ([@B8]). Its incidence is relatively common and accounts for 3.81-7% of all biopsies harvested from the oral cavity ([@B13]-[@B16]).
Microscopically, the lesion is characterized by prominent capillary growth in hyperplastic granulation tissue, which suggests a strong activity of angiogenesis. The blood vessels often show a clustered or medullary pattern separated by less vascular fibrotic septa, leading some authorities to consider PG as a polypoid form of capillary hemangioma ([@B17]).
The lesions of PG may be found in the oral cavity or extraorally. The most frequent intraoral localization is the gingiva (about 60-70%), but lesions can occur on the lips (14%), tongue (9%), buccal mucosa (7%) and palate (2%) ([@B18]-[@B24]). Possible treatment methods are excision, curettage, cryotherapy, sclerotherapy, chemical and electrical cauterization, cryotherapy and the use of lasers with the carbon dioxide (CO2) or argon ([@B25]-[@B29]). Conservative local excision is the preferred form of treatment and recurrence rates after excision range from 0% to 16% ([@B29]).
However, to the best of the authors' knowledge, only 5 cases of pyogenic granuloma in association with a dental implant have been reported in the international literature ([@B1]-[@B3],[@B7],[@B8]). Within the context of the scarce information available on these lesions, the aim of the present study was to report 10 novel clinical cases of pyogenic granuloma in association with titanium dental implants and to elucidate potential risk factors. Finally, the presence of marginal bone loss was evaluated.
Material and Methods
====================
Patients charts at the service of oral medicine of Anitua's Dental Clinic (Alava, Spain) were revised from 1991 to 2011. Patients selection was based on the following inclusion criteria:
• Treatment of pyogenic granuloma.
• The presence of histopathological diagnosis.
• Lesion in relation to dental implants.
All patients who did not fulfill all inclusion criteria were excluded from the study.
Data were collected to report on patient age, gender, patient´s disease, lesion site, type of dental implant (surface and morphology), predisposing factors (trauma, prosthesis type, poor oral hygiene), clinical and radiographic features, diagnosis, treatment and recurrence. Orthopantomography (OPG) of all lesions were examined to compare the presence or absence bone resorption around dental implants.
A descriptive statistical analysis of all variables were performed. Then the relationship between PG and marginal bone loss was analyzed by nonparametric Spearman correlation. The effect of surface type on marginal bone loss was also analyzed with one-way ANOVA and Levene post hoc test. The statistical significance was set at *p*-value \< 0.05. All the statistical analyses were performed using the SPSS v15.0 for Windows statistical software package (SPSS Inc., Chicago, IL, USA).
Results
=======
Ten patients with pyogenic granuloma in relation to dental implants had been identified. They were 2 males and 8 females. Patients' age ranged from 21 to 92 years and all were non-smokers. Five of the ten patients (50%) had systemic disorders: cardiac arrhythmia (1 patient), hypertension (2 patients), atrial fibrillation (2 patients), Type II diabetes mellitus (2 patients), hepatitis C (1 patient ), hypothyroidism (1 patient). Within the group of patients with systemic disease, 3 of them were using 1 to 2 drugs daily, whereas the remaining patient took more than 2 drugs.
With regard to oral hygiene habits, 20% of patients reported to brush once a day, 50% did twice daily and 30% brushed three times a day. A 90% of the patients received professional prophylaxis twice a year and the other 10% once a year. In the use of hygiene products the obtained results were as follows: a) use of mouthwash: only was used by 3 patients (37.5%), b) use of dental floss: only one patient (12.5%), and c) interproximal brushes: 3 patients (37.5%).
The distribution of PG lesions was even between maxilla and mandible (50% for each region), and the most common oral site affected by PG was the area of tooth 41 (2 cases).
The development of PG was related to only accumulation of dental plaque (one patient), bad prosthetic design (one patient), and both factors (one patient). In 4 patients, there had been a combination of tissue pressure by the prosthesis and poor oral hygiene. However, no etiological factor could be related to the development of PG in 3 patients. The clinical size of the lesions ranged from 1.1 x 0.6 mm to 36 x 19 mm. The mean diameter was 7.2 mm. All the lesions were excised and sent for histological examination. The defects were covered with a autologous fibrin membrane (Anitua's protocol). During the first week after the operation, all patients were given analgesic and 0.2% chlorhexidine gluconate mouthwash. During the follow-up period (range two months to 10 years), there were no recurrences.
The histopathological reports indicated the diagnosis of PG and the description of highly vascular proliferation that resembles granulation tissue (Fig. [1](#F1){ref-type="fig"}).
Figure 1Histological images of the pyogenic granuloma showing an appearance similar to granulation tissue. The histological type of the pyogenic granuloma is non-lobular capillary hemangioma. Arrow heads label blood vessels surrounded by connective tissue.
The surfaces of the implants associated with the lesion were smooth (2 implants), machined (3 implants) and rough (5 implants). In no case there was a natural tooth adjacent to the implants related to the lesion. The characteristics of diameters and lengths of the implants studied can be seen in figure [2](#F2){ref-type="fig"}. The average load time of the implants studied was 115 months (SD = 67.5), ranging from a range of 9 to 184 months. Oral rehabilitation was performed with complete prosthesis in 9 patients. The mean mesial bone loss was 2.14 mm (range 0 to 6.50 mm, SD = 2.07) and the mean of distal bone was 1.66 mm (range 0 to 3.75 mm, SD = 1.21.
Figure 2Diameter and length of dental implants related to the pyogenic granuloma.
There were no statistically significant association between the PG area and the marginal bone loss. However the smooth implant surface showed a significant influence on bone loss (Anova: *p* = 0.001) (Fig. [3](#F3){ref-type="fig"}).
Figure 3Peri-implant bone loss grouped by type of surface. The bone loss was the highest for implants with smooth surface.
Discussion
==========
The clinical and histopathological findings have confirmed the diagnosis of pyogenic granuloma in 10 patients. The present study is the one with the highest number of implant-related PG lesion that are available until now in the scientific literature. These PG lesions have been diagnosed as non-lobular capillary hemangioma. There are two histological types of PG. The first type is characterized by proliferating blood vessels that are organized in lobular aggregates. This histological type of PG was called lobular capillary hemangioma (LCH type). The second type (non-LCH type) consist of highly vascular proliferation that resembles granulation tissue ([@B1],[@B4],[@B11]).
Literature data indicated that PG is rarely associated with dental implants, as there are only five cases reported ([@B1]-[@B3],[@B7],[@B8]). However, other reactive lesions such as gingival hyperplasia caused by phenytoin, allergy to titanium abutments or peripheral giant cell granulomas have been reported in the international literature. Causes of conventional oral pyogenic granulomas are not clear, although it has been shown that different stimuli irritants that can trigger them, such as repeated trauma, poor oral hygiene and hormonal problems ([@B1]-[@B20]). About 30-50% of patients with PG have a history of local trauma ([@B9]).
Considering PG, in the case reported by Dojcinovic *et al.* ([@B1]), the inappropriate healing cap has resulted in dental plaque accumulation and chronic inflammation of the peri-implant tissues, triggering the development of a PG. However this was not the cause for PG in the case reported by Olmedo *et al.* ([@B2]). The authors have pointed out to the presence of "metal-like" particles and have postulated that these particles could be the result electrochemical phenomena, corrosion, friction, or a synergistic combination of these events ([@B4],[@B5]). Once released, these particles may trigger an inflammatory response mediated by cytokines and macrophages ([@B5]). This inflammatory reaction could perpetuate the pseudo-periodontal pocket that generates the lesion around the implant ([@B5]).
In the case reported by Etöz *et al.* ([@B
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Introduction {#sec1}
============
In total hip arthroplasty (THA), optimum component position is critical for long-term success of the operation by decreasing rates of wear, aseptic loosening, and dislocation \[[@bib1], [@bib2], [@bib3], [@bib4]\]. Recognizing the importance of acetabular component position, Lewinnek et al published a "safe zone" of 5° to 25° for anteversion and 30° to 50° for acetabular abduction based on their experience with dislocations after posterior THA \[[@bib5]\]. This still serves as the standard for ideal acetabular component position but has been called into question given the importance of the spinopelvic relationship \[[@bib6],[@bib7]\]. In addition to acetabular component position, emphasis has also been placed on femoral component positioning.
Historically, surgeons have identified the importance of keeping the femoral component out of varus because of increased rates of failure with varus cemented femoral stems \[[@bib8],[@bib9]\]. With the use of cementless femoral fixation, varus positioning of the femoral stem has not been shown to lead to the same increased failures \[[@bib10]\]. However, as compared with cemented femoral stems, many cementless femoral stems provide less ability to adjust the version of the component as a stable press-fit requires the stem to adapt to the proximal geometry of the native femur. Consequently, recent attention has been given to the combined version (CV) of the acetabular and femoral components, with the goal of improving impingement-free range of motion and decreasing instability \[[@bib11], [@bib12], [@bib13]\]. The concept of CV was originally introduced by Ranawat, and he described the use of the "Ranawat sign" to determine CV intraoperatively when using the posterior approach \[[@bib14]\]. While no optimum femoral version has been described, Dorr proposed a CV safe zone of 25°-50° based on previous anatomical studies and his experience with decreased instability in this range \[[@bib15]\]. More recent studies have attempted to quantify a combined anteversion that minimizes impingement \[[@bib16],[@bib17]\].
Native femoral anteversion can vary a great deal, and intraoperative judgment of femoral component version can be difficult. Using preoperative computerized tomography (CT) scans of a group of 46 patients scheduled for primary THA, Bargar et al \[[@bib18]\] found a large range of native femoral version from 6° of retroversion to 33° of anteversion. Dorr et al compared the surgeon's estimate of femoral component anteversion in the posterior approach with the postoperative CT measurement of version and found a poor precision of the surgeon\'s estimate with a correlation coefficient of only 0.688 \[[@bib19]\]. In addition, this study found that only 45% of the femoral stems landed within the desired range of 10°-20° of anteversion.
In direct anterior total hip arthroplasty (DA-THA), femoral component broaching and insertion occurs while the patient is positioned supine with the leg fully extended, and the leg below the knee is often draped from the surgeon\'s view. Despite published results of comparable patient outcomes from the DA-THA with other THA approaches, some have questioned the ability to appropriately orient the femoral component with respect to femoral anteversion, via this approach \[[@bib20], [@bib21], [@bib22], [@bib23]\]. Previous studies have reported on improved acetabular component positioning in DA-THA \[[@bib24],[@bib25]\]. However, no prior study, to our knowledge, has examined the combined anteversion of the femoral and acetabular components in DA-THA. This study aims to analyze the combined femoral and acetabular anteversion with cross-sectional imaging and quantify this relative to the CV "safe zone" described by Dorr.
Materials and methods {#sec2}
=====================
After obtaining institutional review board approval, patients were approached for enrollment in the study. An patient who was undergoing a primary DA-THA from the senior author (JBM) was a candidate for enrollment. Patients with femoral or acetabular hardware were excluded from this study. Thirty consecutive patients were enrolled in the study. Four blinded observers independently recorded the measurements (2 fellowship-trained arthroplasty surgeons, one hip and knee fellow, and one orthopaedic resident). All implants were positioned using intraoperative fluoroscopy based on preoperative templating. A CORAIL femoral stem (DePuy, Warsaw, IN) and a PINNACLE acetabular cup (DePuy, Warsaw, IN) were used for all the cases. The senior author standardized intraoperative images by matching the anteroposterior (AP) pelvis fluoroscopic view with the preoperative AP pelvis standing radiograph.
One month after surgery, all patients had a standing AP pelvis and a cross-table lateral radiograph taken, which were used for acetabular component position measurement. Abduction and anteversion measurements of the acetabulum were made from the digital radiograph using the TraumaCad (Voyant Health, Columbia, MD) hip abduction measurement tool. Femoral component position measurements were taken from limited supine CT scan of the hip and knee with 2.5-mm cuts (General Electric BrightSpeed, Fairfield, CT). CT was not selected for acetabular component position to minimize patient radiation exposure. Angular measurements were calculated using the axis of the top of neck of the femoral stem relative to both the posterior condylar axis (PCA) and the transepicondylar axis (TEA).
The CV was then calculated for the TEA and the PCA by adding the femoral anteversion calculated from the CT scan with the anteversion measured from the standing AP pelvis radiograph.
Statistical analysis {#sec2.1}
--------------------
Measurements from the 4 observers were combined, and the mean and standard deviation were calculated. The Pearson correlation coefficient was also measured for each observer, with the kappa values reported, and compared with the group for all measurements. The mean for each measurement was used to determine the number of components placed in the "safe zone." Statistical analysis was performed with the use of SAS software (SAS Institute, Raleigh, NC).
Results {#sec3}
=======
Of the 30 enrolled patients, 29 had an appropriate CT scan obtained. One patient had a CT scan performed without adherence to the protocol precluding reference of femoral version to the axes of the knee and was excluded from the results.
The mean acetabular abduction and anteversion were 39.3° (standard deviation \[SD\] = 4.2°) and 27.2° (SD = 4.7°), respectively. The mean stem anteversion was 17.5° (SD = 10.8°) from the TEA and 21.7° (SD = 11.3°) from the PCA. Ten of the 30 cups were placed inside of the "safe zone" of Lewinnek for acetabular anteversion, but all cups were within the "safe zone" for abduction ([Fig. 1](#fig1){ref-type="fig"}).Figure 1The Lewinnek "safe zone." Ten of the 30 cups were placed inside of the "safe zone" of Lewinnek for acetabular anteversion, but all cups were within the "safe zone" for abduction.
Combined femoral and acetabular component anteversion from the TEA resulted in 79% (23 of 29) of patients within the "safe zone" of 25°-50° with accurately oriented components ([Fig. 2](#fig2){ref-type="fig"}).Figure 2Combined anteversion. Combined femoral and acetabular component anteversion.
Pearson correlation coefficients were high for both stem anteversion from the TEA (R = 0.96) and the PCA (R = 0.98); however, the kappa coefficient for interobserver reliability for combined component anteversion was greater for the TEA (kappa = 0.83 vs 0.65).
Discussion {#sec4}
==========
Component positioning has been recognized as an important factor in the long-term survival of THA \[[@bib5],[@bib12],[@bib26]\]. Muller et al. \[[@bib27]\] suggested a cup anteversion of 10°-15° and femoral anteversion of 10° to be ideal. Lewinnek et al. \[[@bib5]\] followed with their study that found a lower dislocation rate when the acetabular components were positioned in a safe zone of 30°-50° of inclination and 5°-25° of anteversion. A study by Biedermann et al. \[[@bib28]\] found the lowest dislocation rates with acetabular components positioned at 45° of inclination and 15° of anteversion. More recently, Dorr proposed a CV safe zone of 25°-50° based on previous anatomical studies and his experience with decreased instability in this range \[[@bib15]\].
Many authors have begun to appreciate the importance of the combined femoral and acetabular anteversion on dislocation rates and impingement \[[@bib3],[@bib11],[@bib12],[@bib29]\]. Ranawat and Maynard \[[@bib6]\] suggested the importance of the combination of femoral and acetabular anteversion and recommended 45° for women and between 20° and 30° for men. Jolles et al. \[[@bib12]\] found that when the combined anteversion was outside of a range of 40°-60°, the patient's dislocation was 6.9 times higher. Hisatome and Doi \[[@bib29]\] examined combined anteversion in a mathematical model to find the optimum positions to avoid neck impingement with different sized components. They recommended an ideal position, while not accounting for patient's pelvic inclination, of 45° of cup ab
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1. Historical Origins {#sec1-viruses-12-00132}
=====================
At a meeting of the Fellows of the National Institute of Agricultural Botany in Cambridge, UK, on 14 November 1924, Dr. Redcliffe Salaman gave a lecture entitled "Degeneration of the Potato---An Urgent Problem" \[[@B1-viruses-12-00132]\]. He reported that "potato degeneration", namely the decrease in yield when potatoes were grown year after year from tubers, rather than from true seed, cost the UK between five and ten million pounds sterling each year. He noted that the condition was first reported in 1778 at a meeting in Manchester, and called "potato curl". It was worse in lowland crops and in the Southern UK than in crops grown on higher ground and in the north, and although some thought it was caused by disease, perhaps insect-borne, others believed it was a form of senility resulting from repeated vegetative reproduction! Salaman concluded that that degeneration was caused by a complex of tuber-borne pathogens.
Salaman's talk was successful, as it induced the Ministry of Agriculture to found the Potato Virus Research Station in Cambridge and appoint him as director, and in turn he appointed Kenneth Smith as entomologist, who soon separated some of the components of potato curl and identified the viruses he called potato virus X and potato virus Y (PVY) \[[@B2-viruses-12-00132]\].
Other viruses similar to PVY were soon reported, for example, henbane mosaic virus \[[@B3-viruses-12-00132]\], which was like PVY in causing mosaic symptoms, being transmitted by sap, although relatively unstable in it, and also by being transmitted by aphids in short feeds. These viruses, which became known as potyviruses, short for "potato virus Y group viruses" \[[@B4-viruses-12-00132]\], were among those included in early attempts to devise biological taxonomies of plant viruses \[[@B5-viruses-12-00132]\] based on the length of their filamentous particles \[[@B6-viruses-12-00132]\]. They were also distinguished from other plant viruses by having serologically distinct virions and, biologically, by having distinct host ranges and causing distinct symptoms, and by their properties in infective sap, such as dilution end point, thermal inactivation point, and longevity in vitro. Sixteen different potyviruses had been described in 1959. Subsequently, in this pre-sequencing era, a combination of techniques, including sucrose density gradient centrifugation, analytical ultracentrifugation, ultraviolet spectrophotometry, and polyacrylamide gel electrophoresis were also included to establish the sedimentation coefficients and buoyant densities of virions, and the molecular weights of protein subunits and % nucleic acid contents, as all these properties provided additional distinguishing characteristics when novel viruses were being described \[[@B7-viruses-12-00132]\].
Virus identification and taxonomy were transformed later, when methods for sequencing genes were invented in the 1970s and applied to plant viruses \[[@B8-viruses-12-00132],[@B9-viruses-12-00132]\], and it was established that hierarchical groupings based on viral protein and gene sequences, including those of potyviruses, confirmed and extended those that had been devised previously by using phenotypic characters, serological tests, etc. As a result, 57 potyviruses had been identified by 1991 \[[@B10-viruses-12-00132]\], using sequences of the "part NIb-CP" region of their genomes, as this was bracketed by convenient primer sites \[[@B11-viruses-12-00132],[@B12-viruses-12-00132]\]. By 2000, over 1000 potyvirus sequences were recorded in the GenBank database, and there are now more than 26,000. The potyviruses now form a family, the *Potyviridae* \[[@B13-viruses-12-00132]\], containing at least eight genera of which the aphid-transmitted potyviruses, including the first described, PVY, make up the largest genus, *Potyvirus*. This large plant virus genus is one of the most important economically because of the yield and quality losses it causes in a wide range of crops worldwide. Moreover, some of its members currently endanger food security in developing countries by causing devastating diseases in tropical and subtropical food crops \[[@B14-viruses-12-00132]\].
2. The Origins of the Potyviridae {#sec2-viruses-12-00132}
=================================
The potyvirids are distinguished from other viruses by specific molecular differences, together with a combination of phenotypic properties \[[@B13-viruses-12-00132],[@B15-viruses-12-00132]\]. All potyviruses infect plants; most are transmitted in nature by arthropods---mostly by aphids---though bymoviruses are transmitted by root-infecting plasmodiophorids, which are cercozoan amoebae. Some potyviruses are seed-borne \[[@B16-viruses-12-00132]\]. Potyvirid virions are flexuous filaments, 680--900 nm long and 11--20 nm in diameter. Each is helically constructed from 1400 to 2140 subunits of a coat protein (CP), and a positive-sense, single-stranded RNA genome (usually monopartite, but bipartite in the genus *Bymovirus*) of 8--11 kb in total, which is wound into a groove within the CP subunits.
The ancestry and origins of the potyvirids is being revealed by studies of the structure and sequences of their genes and proteins. These show that their genomes are polyphyletic in origin, as there are significant similarities between three of their genes and those of viruses in three otherwise-unrelated virus genera; two detected by the protein sequence similarity of the helicase region of the CI protein and the RdRp region of the NIb protein, respectively, and the third by the structure of the CP \[[@B17-viruses-12-00132],[@B18-viruses-12-00132]\]. A BLASTp search of the GenBank protein sequence database (Sept 2019), using the eight main motif regions of the polyprotein of PVY (NC_001616), and excluding matches with sequences from the *Potyviridae*, found only significant matches between the "DEAD helicase-helicase C" region of the CI protein and that of classical swine fever and hog cholera pestiviruses (chance probability, 1e-12\_-16; 30% identity, 13% indels), and no others. Likewise, there were significant similarities between the PVY RdRp region and that of astroviruses (see below). Structural studies have only been reported for the CP protein of one potyvirus, watermelon mosaic virus (WMV), and reveal a structure that is closely similar to those of other viruses with flexuous filamentous virions, including two potexviruses, and also the enveloped flexuous nucleoproteins of orthomyxoviruses and bunyavirids, which include tomato spotted wilt tospovirus \[[@B19-viruses-12-00132],[@B20-viruses-12-00132],[@B21-viruses-12-00132]\]; all these CPs have a core domain rich in alpha helices. Each CP subunit interacts with five nucleotides (nts), and has 8.8 subunits per turn in a left-handed helix, with a pitch of 34.5--35 Å. Its N-terminus is external to the virion, and its C-terminus internal. The terminal regions interact with adjacent subunits and provide flexibility to the virion. In serological studies, the N-terminus is dominant and, in potyviruses, is also involved with aphid interactions \[[@B22-viruses-12-00132],[@B23-viruses-12-00132]\].
The sequences of their RNA-dependent RNA polymerases (RdRps) place the potyvirids in the "Picornavirus Supergroup" ([Figure 1](#viruses-12-00132-f001){ref-type="fig"}) \[[@B19-viruses-12-00132],[@B24-viruses-12-00132]\], where the potyvirids are outsiders, as most of the others have icosahedral virions made of eight-stranded antiparallel beta-barrel proteins, the so-called 'jelly roll' proteins. In the Wolf et al. \[[@B24-viruses-12-00132]\] taxonomy of RdRps ([Figure 2](#viruses-12-00132-f002){ref-type="fig"}), the potyvirid RdRps form a cluster that is sister to an RdRp found in a metagenome, bufivirus UC1-gp2, isolated from "wastewater" collected in San Francisco. The sister clade to the potyvirid/bufivirus clade of RdRps are mostly those of the astroviruses, a group of gut-infecting viruses that are found in a wide range of animals, mostly mammals or birds. They have 28--35 nm diameter isometric virions (<https://en.wikipedia.org/wiki/Astrovirus> (accessed July 2019)). Sister to the RdRp clade of potyviruses/bufivirus/astroviruses are the RdRps of hypoviruses, amalgaviruses, partitiviruses, and picobirnaviruses, many of them metagenomes, including one from *Phytophthora infestans* \[[@B25-viruses-12-00132]\] and two from leeches \[[@B26-viruses-12-00132]\]. None of the motifs identified in potyvirus proteins, other than the RdRp, match those encoded by
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All relevant data are within the paper and its Supporting Information files.
Introduction {#sec005}
============
Globally, an estimated 225 million women have unmet need for family planning \[[@pone.0153907.ref001]\]. Increasing acceptance and use of family planning requires more than increasing access to health services: truly effective family planning programs must address the social and gender norms that present critical barriers to sexual and reproductive health. Use of family planning is powerfully shaped by social norms, including perceived acceptability of family planning, social pressure for large families, and perceived opposition to family planning by religious and community leaders and spouses \[[@pone.0153907.ref002]\].
Rigid gender roles and unequal power between men and women inhibit couple communication and joint decision-making about family planning: power dynamics in couples have been found to influence the use of family planning and other health services \[[@pone.0153907.ref003]--[@pone.0153907.ref005]\]. Fear of and experience with intimate partner violence or gender-based violence are barriers to contraceptive use \[[@pone.0153907.ref006]--[@pone.0153907.ref009]\]. Women who report intimate partner violence are at higher risk for unintended pregnancy than women who do not report violence \[[@pone.0153907.ref008]\]. Analyses of data from Demographic and Health Surveys (DHS) in a number of African countries suggest that key dimensions of gender equity and women's empowerment---including equitable beliefs about gender roles, women's ability to negotiate sexual activity, and women's control over household economic decision-making---are associated with higher contraceptive use \[[@pone.0153907.ref010]\]. Several studies have also found a positive association between spousal communication and the use of contraception \[[@pone.0153907.ref011]--[@pone.0153907.ref014]\].
Despite evidence demonstrating the important relationships between gender, women's empowerment, and family planning, there is little consensus about how to best measure these complex social constructs. Researchers have attempted to measure specific domains of women's empowerment, and several scales have been developed including measures of mobility \[[@pone.0153907.ref015]\], power in relationships \[[@pone.0153907.ref003]\], gender norms and decision-making autonomy \[[@pone.0153907.ref016]\], control over assets \[[@pone.0153907.ref017]\], and social capital \[[@pone.0153907.ref018],[@pone.0153907.ref019]\]. The lack of a standard set of widely used measures for women's empowerment, however, hampers our ability to compare program effects across studies, populations, and cultural contexts.
The objective of the research described in this article was to evaluate the impact of a CARE intervention that catalyzed community-level dialogue about gender, sexuality, and family planning on household-level gender dynamics and reported use of family planning among men and women in Kenya. For this evaluation, we used several scales from WE-MEASR (Women's Empowerment-Multidimensional Evaluation of Agency, Social Capital, and Relations) (see [S1 File](#pone.0153907.s001){ref-type="supplementary-material"}), a new tool that CARE developed following multi-year research into the effects and impact of our women's empowerment programming \[[@pone.0153907.ref020]\].
Methodology {#sec006}
===========
Study Setting {#sec007}
-------------
According to the 2008--09 Kenya DHS, approximately one in four women has an unmet need for family planning \[[@pone.0153907.ref021]\]. A 2013 analysis of DHS data found that Kenyan women who do not use family planning report method-related concerns, especially fear of harmful side effects, as the most common reason for non-use (43%); opposition to family planning (both the perceived religious prohibition of family planning and opposition by husbands/partners) is the second most commonly cited reason (16%) \[[@pone.0153907.ref022]\].
CARE conducted the research described here as part of the Family Planning Results Initiative (FPRI), which we implemented in Siaya County, Nyanza province, Kenya from July 2009 through December 2012. The majority of the county's population of 842,304 is under 30 years old, and 46.1% are between 0 and 14 years of age \[[@pone.0153907.ref023]\]. Over 90% of Siaya County is rural and, like the rest of Nyanza, falls behind the national average on various indicators of development, gender inequality, poverty, education, and health, including infant and under-five mortality, antenatal care coverage, and HIV prevalence \[[@pone.0153907.ref021]\]. The contraceptive prevalence rate among married women in Nyanza province is among the lowest in Kenya: 37.3% of women use any method (compared to 45.5% nationally), and only 32.9% use a modern method (39.4% nationally) \[[@pone.0153907.ref021]\].
The Intervention {#sec008}
----------------
CARE used its *Social Analysis and Action* approach \[[@pone.0153907.ref024]\] to shape FPRI's central intervention about 150 community-based facilitators were trained and held ongoing community dialogues about gender, sexuality, and family planning over three and a half years (July 2009-December 2012). At any one time, an average of 65 facilitators were actively convening dialogues. Monthly planning meetings were held to plan activities and ensure adequate location coverage and topic variation over the entire study area. On a quarterly basis, CARE observed and provided feedback on dialogues and completed progress reviews with facilitators. Based on our monitoring data, CARE organized 759 dialogues between July 2009 and December 2012. Dialogues were held in a variety of venues and were promoted by community leaders or organized around pre-scheduled community meetings. Based on proximity to a respective venue, they were attended by participants from several villages, and, many times, were included in villages' development plans.
The dialogues were designed to normalize communication about sensitive topics like gender norms and FP, and to catalyze participants' critical analysis of how gender norms and dynamics restrict family planning acceptability and use. During dialogues, community leaders and satisfied family planning users acted as role models and overtly expressed support for family planning, equitable gender norms, open communication and shared decision-making regarding family planning between spouses.
Trained, community-based facilitators convened dialogues in an array of settings, such as markets, churches, women's groups and village meetings. Local theatre groups often performed during the dialogues. Facilitators included community health care workers, religious leaders, local government officials, and teachers. CARE provided ongoing support to facilitators in the form of training and supervision, and also provided transport reimbursement and lunch/refreshments on days when dialogues or trainings were conducted; no incentives were provided for participation in intervention activities to any other community members.
All of CARE's activities were coordinated with the District Health Medical Team (DHMT.) During the intervention period, the DHMT coordinated several interventions designed to increase availability and access to contraceptives, including in-service training for providers, strengthening of systems for ordering contraceptives, and some community outreach activities (FP counseling and method provision). To our knowledge, no other similar FP dialogue interventions or activities were implemented by other organizations during the intervention period.
Theory of Change {#sec009}
----------------
The intervention aimed to challenge and shift key community norms about gender dynamics and family planning, with the ultimate goal of creating a social environment that is more supportive of equitable gender relations and the use of family planning. Our research goals were to determine whether and how the ongoing dialogues shifted social norms, and whether and how these shifts at the community level influenced communication, decision-making, and family planning use at the couple or household level. Our theorized pathway of change is shown in [Fig 1](#pone.0153907.g001){ref-type="fig"}.
{#pone.0153907.g001}
Evaluation Design {#sec010}
-----------------
We used both qualitative and quantitative methods to evaluate the intervention. At baseline (February 2009) and again at endline (December 2012), we conducted household surveys to collect data about men and women's socio-demographic characteristics, pregnancy intentions, family planning knowledge, beliefs, attitudes, and use. At endline only, we measured key gender-related beliefs and behaviors and exposure to the intervention, which enabled us to determine whether these were important predictors of family planning use. Also at endline, we conducted in-depth, qualitative interviews with purposively selected couples from the intervention area (described below) to explore changes in communication, decision-making, and gender roles over the previous four years and to identify specific enablers and barriers to family planning use.
Ethics Statement {#sec011}
----------------
CARE obtained approval to conduct this research from the Institutional Research and Ethics Committee at Moi University in Eldoret, Kenya.
Quantitative Data Collection {#sec012}
----------------------------
Through independent, cross-sectional household surveys, married men (20--49 years) and women (18--45 years) were interviewed by trained interviewers using standardized questionnaires at baseline and endline. Although the two samples were entirely independent (no attempt was made at endline to include or exclude respondents who participated at baseline), both were drawn from
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Background
==========
The prevalence of type 2 diabetes rapidly rose over the past three decades. However its burden is not homogeneously distributed: racial and ethnic minorities usually experience higher prevalence than their non-minority counterparts \[[@B1]\] and are at higher risk of developing diabetes-related complications such as blindness, kidney damage, or depression, impacting both quality of life and mortality rates \[[@B2],[@B3]\].
Self-management, defined as the patient's ability to manage not only the symptoms inherent to a chronic condition but also its treatment and associated lifestyle changes \[[@B4]\], has become increasingly important in the treatment of type 2 diabetes \[[@B5],[@B6]\]. However, because adequate diabetes self-management (DSM) may require considerable lifestyle changes to several domains, namely having a healthy diet, exercising, or glucose monitoring, not all the patients are able to properly follow the self-management plans agreed with their healthcare professionals or advised by clinical guidelines. Racial and ethnic minorities are less likely to engage in DSM behaviors than other population groups, which partially explains observed disparities in health outcomes \[[@B7]\]. Some of the barriers faced by these groups for achieving an adequate DSM are related to characteristics of the groups (such as health literacy or health beliefs) and also of the healthcare system (namely accessibility of culturally sensitive information) \[[@B8],[@B9]\].
Several review studies have assessed the effect of DSM educational programs on the general population \[[@B6],[@B10]-[@B17]\]. Those studies have established that DSM educational programs can improve glycemic control \[[@B11]-[@B16]\], and identified the key characteristics for improving glycemic control, including face-to-face delivery, teaching methods based on cognitive reframing \[[@B11]\], and higher contact time between participant and educator \[[@B16]\]. A smaller number of studies have reviewed the evidence of the effect of these programs on racial/ethnic minorities \[[@B18]-[@B20]\]. Only one study \[[@B18]\] examined their impact on glycemic control, observing a reduction on glycated hemoglobin of 0.32%. There were however some limitations underlying that study, as some of the included interventions combined educational programs with other types of quality improvement strategies, making it difficult to disentangle the individual effect of the educational components. More importantly, no previous meta-regression study has identified the key common characteristics of successful educational programs targeted to racial/ethnic minority groups. This represents a considerable gap in the literature, as programs for racial/ethnic minority groups should have different components from those targeting other population groups, namely addressing the cultural idiosyncrasies associated with each group. Therefore it is not reasonable to assume that the same type of successful program will be equally successful when applied to racial/ethnic minority groups. Additionally, in the past years several clinical trials examining the impact of educational programs to improve diabetes self-management on racial/ethnic minorities have been published, making now possible a more detailed review and analysis of the available evidence regarding the effectiveness of these interventions.
In this work we systematically reviewed DSM educational interventions specifically targeted to racial/ethnic minority groups. We studied the characteristics and costs of the interventions, and analyzed their impact on diabetes knowledge, self-management behaviors, and clinical outcomes. Whenever data were available, we performed meta-analyses to examine the short and long-term effects of the interventions, and meta-regressions to identify common characteristics of the interventions associated with better results.
Methods
=======
The review and its procedures were planned, conducted, and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines \[[@B21]\].
Data sources and searches
-------------------------
A comprehensive core search strategy was developed for Medline through Ovid (combining MeSH terms and keywords) and then adapted and implemented in EMBASE and CINAHL (search strategy available in Additional file [1](#S1){ref-type="supplementary-material"}: Table S1). Gray literature and additional articles were searched in twelve more bibliographic sources (Additional file [2](#S2){ref-type="supplementary-material"}: Table S2). The search was not restricted by language or publication date. For all the references selected to be included in the review, backward and forward citation searches were performed in ISI Web of Knowledge. All searches were conducted in October 2012. A bibliographical database was created using EndNote X6, and used to store and manage the retrieved references.
Study selection
---------------
We included studies analyzing the effectiveness of DSM educational programs targeted to racial/ethnic minority groups with type 2 diabetes. We only included those studies in which at least 90% of the participants pertained to a racial/ethnic minority group considered to be at a higher risk for diabetes complications than the majority population group. Racial/ethnic minority group was defined as a population group with a race or ethnicity different from that of the majority population group of the host country. Groups at higher risk of diabetes complications were identified based on available literature. Interventions had to be exclusively educational, without including any other component such us financial incentives, clinician education or case management. In order to avoid possible comparisons between programs carried out in very heterogeneous settings, with very different health systems and population needs, we restricted this review to those interventions conducted in countries that were members of the OECD \[[@B22]\], when study selection was conducted (November 2012).
Eligible designs were randomized controlled trials (RCTs), including cluster randomized controlled trials; controlled trials, including quasi-randomized trials; controlled before-after studies; and non-controlled before-after studies. Studies including a control group were only eligible in case the intervention was compared with care as usual.
Titles and abstracts were screened for eligibility, and those fulfilling the inclusion criteria were included in the next stage, where the full texts of the selected articles were retrieved and assessed. Those that met the inclusion criteria were included for data extraction. Two reviewers independently screened citations, and any disagreements were solved by consensus with a third reviewer.
Data extraction and quality assessment
--------------------------------------
We designed and used structured forms to extract information of interventions' characteristics and their effectiveness. We used a previously developed taxonomy of DSM educational programs to characterize the interventions \[[@B23]\]. The following information was extracted: setting, ethnic group, administration formats, teaching methods, educational contents, educators' background, use of peer educators, and duration. Information of interventions' cost and cost-effectiveness was also extracted.
We critically appraised the studies using the Quality Assessment Tool for Quantitative Studies \[[@B24]\], which enables the assessment of both internal and external validity, classifying them into three categories (good, fair or poor) depending on six aspects: selection bias, study design, confounders, blinding, data collection and withdrawals/ dropouts. Two reviewers independently extracted all the information and critically appraised the studies. Disagreements were solved through discussion with a third reviewer. When necessary we contacted the authors of the studies to request additional information.
Data synthesis and analysis
---------------------------
The effectiveness of the interventions was assessed in terms of their impact on 1) diabetes knowledge, 2) diabetes self-management behavior, and 3) clinical outcomes. Diabetes knowledge was ascertained by measures reflecting the theoretical knowledge the patients had about their condition. Diabetes self-management behavior measured the performance of specific activities related to adequate DSM (diet, exercise, glucose control, foot self-examination, etc.). Clinical outcomes included hemoglobin A1c (HbA1c), body mass index (BMI), or blood pressure, amongst others. All outcomes in all the studies were examined and classified as measuring one of these three domains. Variables that measured other domains were not included in the analysis.
Additionally, we conducted independent meta-analyses to analyze short and long-term (six months post-intervention) effect of the interventions on glycemic control. Eligibility criteria for the meta-analyses included randomized controlled trials comparing the interventions with usual care. The mean (standard deviation) of HbA1c levels in each study were extracted. This information was transformed into weighted mean difference and 95% confidence intervals (CI) were calculated and combined using random-effects models. We imputed unreported standard deviations by use of established methods \[[@B25]\]. Heterogeneity was quantified by the I^2^ statistic, where I^2^ ≥ 50% was considered evidence of substantial heterogeneity \[[@B26]\]. Sources of heterogeneity were investigated by a Galbraith plot. Publication bias was quantitatively assessed with Egger test.
We used bivariate meta-regression to explore relationships between effect size (ES) and interventions characteristics. The number of included studies was insufficient to perform a multivariate regression analysis. We conducted a sensitivity analysis, excluding the studies with higher risk of bias. All analyses were conducted with Stata, version 12.0 (StataCorp, College Station, Texas). For all the analyses, statistical significance was accepted at *p* \< 0.05.
Results
=======
Article identification
----------------------
Figure [1](#F1){ref-type="fig"} reports the screening process. A total of 1,988 unique references were retrieved. 1,386 references were excluded based on title and abstract, resulting in 602 references being included in the next stage. Full text articles were retrieved and assessed, with 24 studies meeting the eligibility criteria. Backward and forward search of these 24 articles identified thirteen additional studies, resulting in thirty seven articles being included in the review \[[@B27]-[@B63]\]. Thirty five of them reported one single intervention, whereas two articles reported two distinct interventions per article. Overall, thirty seven articles were identified, which analyzed the effectiveness of thirty nine different interventions.
{#F1}
Characteristics of the studies and interventions
------------------------------------------------
Table [1](#T1){ref-
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1.. Introduction
================
Heart rate has been an important factor in indicating patients' health for quite a long time. As people are paying more and more attention on their health, long-term heart rate monitoring is of more importance to everyone as it's a valuable indicator for early diagnosis of dozens of diseases. Furthermore, long-term heart rate monitoring is also essential for sports enthusiasts and professional athletes. According to these requirements, it is necessary to design and fabricate a device which is suitable for long-term heart rate monitoring and free from external disturbance. The wearing comfort, the reliability and the cost become three key issues for long-term heart rate monitoring for daily use.
Several conventional methods have already been developed to measure heart rate, most of which are based on electrocardiograms (ECGs) \[[@b1-sensors-15-03224]\], photoplethysmography (PPG) \[[@b2-sensors-15-03224]\], and the piezoelectric effect \[[@b3-sensors-15-03224]--[@b5-sensors-15-03224]\]. Other principles in heart rate measurement have also been verified by researchers \[[@b6-sensors-15-03224]--[@b8-sensors-15-03224]\]. Among these methods for heart rate monitoring, ECG is the most widely used. It is the standard diagnosis technique for heart disease in hospitals but the specialized equipment needed is clearly not suitable for household applications. Household heart rate monitors based on ECG technique have also been commercialized, but chest strap transmitters are usually needed, which is not appropriate for long-term use. Strapless wrist band ECG heart rate monitors have been developed these days. However, the contradiction between reliable electric connection and the comfort of wearing still has not been settled in a satisfactory way. Techniques for PPG require hard photoelectric modules which are tightly attached to the tissue with a certain penetration depth, for instance, the fingers. For the reasons above, these two kinds of heard rate monitor system are not appropriate for long-term ordinary daily use because of the inconvenience in user experience, especially at night. Monitors based on piezoelectric pressure sensor also have the similar limits.
2.. System Design
=================
In this work, a comfort, reliable heart rate monitor for long-term household use is proposed. Unlike the previous ECG, PPG and piezoelectric devices, a flexible pressure sensor with the capability of measuring heart rate by sensing the pulsation of the artery is used in the system. This solution is adopted based on the following considerations.
Since the sensor of the monitoring system is the only component which needs to inevitably be in contact with the human skin, trying to have a flexible sensor is crucial in realizing a comfortable, simple heart rate monitor. In this work, a robust, low-cost novel flexible pressure sensor has been proposed and fabricated. The polymer-based flexible sensor has no hard components which it can ensure its comfort and fitness for wearing all day in any situation.
Furthermore, to distinguish the tiny pressure changes caused by the artery pulse, the flexible pressure sensor's sensitivity needs to be improved. Based on previous works in this area, a highly sensitive structure is proposed. The conventional structure with one piezoresistive layer is replaced by a novel structure with two piezoresistive layers. The contact interface of the two layers is modified with micro structures. These microstructures are realized with modern soft lithography technology and are treated as the key features for the sensor's high sensitivity. In addition to that, for the specific morphology of the surface microstructures utilized, the pressure sensor device also has a perfect linearity performance.
However, due to the high sensitivity of the pressure sensor, noises may be induced by the wearer's muscle movements. This inevitable daily movement can cause pressure changes between the wearer's skin and the device. Such noises can easily make the monitor fail when heart rate counting. In order to solve this problem while maintaining a relatively low cost in signal processing, an Analog Signal Processing (ASP) system is proposed to process the signal and extract the heart rate information from it. The whole system is composed of a sensor subsystem and the ASP subsystem. The sensor subsystem is an elastic belt with the flexible pressure sensor attached on it. The tightness of the belt can be tuned in order to provide a proper pressure for measurement. The system also includes an ASP subsystem to process the pressure signals, a counter, an indicator, and a timing circuit which is used to stop the measurement.
2.1.. Flexible Pressure Sensor
------------------------------
To realize the characteristic of flexibility in addition to the pressure sensing capability, a flexible piezoresistive carbon black/silicone rubber nanocomposite is adopted as the functional material \[[@b9-sensors-15-03224]--[@b12-sensors-15-03224]\]. For its stretchable, flexible properties and piezoresistive effect, this nanocomposite material has already been used in tactile sensors for robots to provide contact or grasping force feedback \[[@b13-sensors-15-03224],[@b14-sensors-15-03224]\]. They are also some other sensors for mechanics that adopt the material for their piezoresistive properties and energy absorbing capabilities in preventing mechanical collisions \[[@b15-sensors-15-03224],[@b16-sensors-15-03224]\]. In addition to that, all other materials used in device are flexible. [Figure 1](#f1-sensors-15-03224){ref-type="fig"} shows a simple schematic diagram demonstrating the assembly and packaging steps used in fabricating the device.
Two layers of surface-modified piezoresistive polymer nanocomposite films are placed face-to face inside the device as the pressure sensing layer. These polymer films are packaged by another two layer of patterned Flexible Copper Clad Laminate (FCCL) films which act either as the package material or as the electrode of the device. To isolate this device from external moisture and contamination, another layer of PI bond-ply is used as the adhesion layer to ensure these four layers of material a good firm bond. These bond-ply layers have also been previously patterned by a laser cutting machine to save the space for the pressure sensing layer. The scale of the device's sensing part is 15 × 30 mm^2^.
Since the human artery pulse in the wrist is weak and the excited pressure variation is not easy to sense, the flexible device' sensitivity needs to be further improved. Several previous efforts have been made to promote the devices' sensitivity in this research area \[[@b17-sensors-15-03224]--[@b23-sensors-15-03224]\]. A pressure sensor with record sensitivity with highly sensitive material with hollow polymer sphere in it has been proposed by Bao's group \[[@b17-sensors-15-03224]\]. Another type of research mainly utilizea tiny features' very sensitive contact state to realize high sensitivity \[[@b18-sensors-15-03224],[@b19-sensors-15-03224]\]. They usually have polymer-based micrometer scale pyramid structures to make the device electrically sensitive to external pressure loading. Devices of this type usually have a very sensitive electric response at the beginning when an array of unique pyramids gets contact to the opponent electrode. However, as the pressure goes higher, device's response become dull as the pyramidal surface get saturated, as shown in [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}.
A certain degree of pre-pressure needs to be maintained between the sensor and the human skin in order to ensure effective contact state and signal acquisition and the wrist artery pulse induced pressure varies (range \< 3 kPa) based on this pre-pressure bias. Furthermore, the pre-pressure also changes with the differences between users when they fasten on the wrist belt. Consequently, the real effective span for the heart rate measurements will be around 8--18 kPa. Several previous works on flexible pressure sensors have reported sensitivity high enough for heart rate measurement. However, these high sensitivities only exist within a limited range around 0 Pa. As shown in [Figure 2b](#f2-sensors-15-03224){ref-type="fig"}, the slope of the device response decreases as pressure goes higher, like the sensitivity. When the pressure reaches the effective range, devices becomes so dull that it can't respond to the artery pulse. As to the proposed novel device with full-scale high sensitivity ([Figure 2a](#f2-sensors-15-03224){ref-type="fig"}), artery pulse-induced pressure variation will make it respond intensively compared with previous ones.
To have this full-scale sensitive pressure response, a new flexible pressure sensing device have been proposed in this work. The surface of the nanocomposite film has been modified with micrometer scale bump structures randomly distributed on surface with certain variation in height and lateral size. [Figure 2c](#f2-sensors-15-03224){ref-type="fig"}--d shows the SEM photos of the microbumps from the top view and side view, respectively. Unlike the typical pyramid structures of the same size, the distributed tiny structures' size makes them gradually touch with the opponent conductive surface along the whole pressure loading process so as to have a full scale high sensitivity. By surface profile measurement, the overall surface obeys the Gaussian Random distribution. By numerical simulation with MATLAB, the device response is expected to be linear.
Furthermore, the micrometer scale piezoresistive bumps on the surface are very sensitive to external pressure loading as the pressures are concentrated on these tiny structures. Large deformations of these piezoresistive structures lead to dramatic conductance variations of the device. Besides that, for the distributed bump size, the conducting paths are established gradually. The capability in conductance variation is even higher. Based on above statement, the device sensitivity is expected to be higher.
The devices' sensing capabilities were tested using a device test platform composed of a micropositioner, force gauge and the electric measurement
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Introduction {#s1}
============
The intestinal mucosa is a critical effector site for elimination of enteric pathogens. *Toxoplasma gondii*, a ubiquitous protozoan parasite, is a prime example of such a pathogen. Mammals are infected with *T. gondii* primarily by the ingestion of tissue cysts from undercooked meat or oocysts excreted in the feces of felines, which are the sole definitive hosts. Upon infection, the parasite induces a potent Th1 immune response that is characterized by high levels of IL-12 and IFN-γ [@ppat.1003706-Denkers1], [@ppat.1003706-Dupont1]. Initial IL-12 production is largely the result of MyD88-dependent Toll-like receptor (TLR) signaling in dendritic cells, and the parasite profilin molecule has been identified as a ligand for TLR11 and TLR12 [@ppat.1003706-Scanga1]--[@ppat.1003706-Sukhumavasi1]. IL-12 activates natural killer (NK) cells to initiate IFN-γ production and promotes T-cell differentiation towards a Th1 program. Ultimately IFN-γ is the critical cytokine involved in controlling *Toxoplasma*. While *in vitro* experiments suggest that macrophages activated by this cytokine acquire anti-*Toxoplasma* activity through upregulation of immunity-related GTPase (IRG) molecules that mediate destruction of the parasitophorous vacuole [@ppat.1003706-Zhao1]--[@ppat.1003706-Khaminets1], the *in vivo* function of IFN-γ is less clear.
Inflammatory monocytes are an important component of defense against microbial pathogens, including *Toxoplasma* [@ppat.1003706-Dunay1]. These cells express high levels of Ly6C/G (Gr-1) and are recruited from the bone marrow via chemokine (C-C motif) receptor 2 (CCR2) [@ppat.1003706-Geissmann1]. During *Listeria monocytogenes* infection, inflammatory monocytes are recruited from the bone marrow to the spleen and liver where they differentiate into TNF-α- and nitric oxide (NO)-producing DCs (Tip-DCs). There they are essential for bacterial clearance and mouse survival [@ppat.1003706-Shi1], [@ppat.1003706-Serbina1]. Likewise, CCR2-dependent inflammatory monocytes are recruited to the lung during *Mycobacteria tuberculosis* infection where they protect mice from disease by recruiting and activating T cells and by producing NO [@ppat.1003706-Peters1], [@ppat.1003706-Peters2]. Mucosal defense against *T. gondii* has also recently been shown to require CCR2-dependent inflammatory monocytes [@ppat.1003706-Dunay1]. Upon recruitment to the small intestine, these cells control the parasite either indirectly by production of IL-12 and TNF-α or directly through production of NO and IRG proteins [@ppat.1003706-Yarovinsky1]--[@ppat.1003706-Andrade1], [@ppat.1003706-Zhao1], [@ppat.1003706-Taylor1], [@ppat.1003706-Dunay1], [@ppat.1003706-Ling1]. While CCR2 enables recruitment of inflammatory monocytes to sites of infection, the factors that coordinate their activation and acquisition of effector function are not known.
CXCR3 is a Th1-associated chemokine receptor, and cells expressing this receptor respond to the IFN-γ-inducible chemokines CXCL9, 10, and 11 [@ppat.1003706-Groom1]. The receptor is expressed predominantly by T cells and NK cells and is rapidly upregulated upon cell activation. There is evidence that CXCR3 expression enables T-cell entry into sites of infection, although the outcome of recruitment varies among pathogens. In the case of *Leishmania major*, recruitment is protective as CXCR3-expressing T cells are required for the resolution of cutaneous lesions [@ppat.1003706-Rosas1]. However, in the case of *Plasmodium berghei* ANKA, CXCR3 is pathogenic because it allows entry of proinflammatory cells into the CNS, resulting in cerebral malaria [@ppat.1003706-Campanella1].
Here we determined the role of CXCR3 in the intestinal immune response to *Toxoplasma*. We found that loss of CXCR3 negatively affected host survival against oral infection. This was associated with diminished recruitment of CD4^+^ T cells to the lamina propria (LP), decreased T cell IFN-γ secretion, impaired inflammatory monocyte effector function, and inability to control the parasite in the intestinal mucosa. Reconstitution with CXCR3-competent CD4^+^ T cells restored inflammatory monocyte function, resulting in improved survival against the parasite. Protective effects of adoptively transferred CD4^+^ T lymphocytes depended upon their ability to produce IFN-γ, but occurred independently of CD4 expression of CD40L. Our data show that CXCR3 enables Th1 recruitment to the intestinal LP, where these cells instruct activation of CCR2-dependent inflammatory monocytes, in turn controlling infection. These results establish CXCR3 as a major determinant orchestrating communication between effectors of innate and adaptive immunity, enabling effective host defense against infection.
Results {#s2}
=======
CXCR3 and its ligands CXCL9 and CXCL10 are upregulated during acute toxoplasmosis {#s2a}
---------------------------------------------------------------------------------
Because CXCR3 and its chemokine ligands are strongly associated with Th1 responses, we asked whether this proinflammatory axis was induced during *Toxoplasma* infection in the intestinal mucosa. Accordingly, mice were orally inoculated with cysts, and relative levels of CXCR3, CXCL9 and CXCL10 mRNA expression were measured over the course of acute infection. We found strong upregulation of CXCR3 and its specific chemokine ligands as early as Day 4 post-infection in both the ileum and mesenteric lymph nodes (MLN) ([Fig. 1A](#ppat-1003706-g001){ref-type="fig"}). Overall, peak CXCR3 mRNA levels were attained by Day 6 post-inoculation.
{#ppat-1003706-g001}
In order to examine CXCR3 expression in more detail, we utilized *Cxcr3* eGFP reporter (CIBER) mice, a bicistronic reporter strain in which cells expressing CXCR3 also express eGFP [@ppat.1003706-Oghumu1]. We found a large increase in CXCR3 populations of both CD4^+^ and CD8^+^ T lymphocytes in MLN, spleen (SPL) and LP compartments following infection ([Fig. 1B](#ppat-1003706-g001){ref-type="fig"}). In general, CXCR3 upregulation was most pronounced in the CD4^+^ population. For example, in the MLN there was a 6-fold increase in CD4^+^CXCR3^+^ cells but only a 2-fold increase in CD8^+^CXCR3^+^ lymphocytes. NK cells are also known to express CXCR3 and are an important source of early IFN-γ during *T. gondii* infection [@ppat.1003706-Qin1], [@ppat.1003706-Sher1]. However, while CXCR3-GFP expression was relatively high on naïve NK cells, the GFP expression was in fact reduced during infection, suggesting lack of a role for CXCR3^+^ NK cells during intestinal infection ([Fig. S1](#ppat.1003706.s001){ref-type="supplementary-material"}). We next examined expression of the activation marker CD27 on GFP^+^ and GFP^−^ T lymphocytes in infected mice. CD27 was significantly lower in CXCR3^−^GFP^−^ cells, suggesting an altered maturation state of the CXCR3^−^ T cells ([Fig. S2A and B](#ppat.1003706.s002){ref-type="supplementary-material"}). Likewise, there was a lower percentage of CD27^+^ cells amongst CXCR3-GFP^−^ CD8^+^ T lymphocytes, although the decreases in expression were not as striking as with the CD4^+^ lymphocytes ([Fig. S2C and D](#ppat.1003706.s002){ref-type="supplementary-material"}).
*Cxcr3^−/−^* mice are increased in susceptibility and are prone to severe intestinal damage following *T. gondii infection* {#s2b}
---------------------------------------------------------------------------------------------------------------------------
To further examine the role of CXCR3 during *T. gondii* infection, mice deficient in CXCR3 were orally inoculated with low virulence ME49 cysts, and the outcome of infection was monitored. While all wild-type (WT) mice survived acute infection with 30 cysts, *Cxcr3^−/−^* animals displayed increased susceptibility with nearly 75% of mice dying by 2 weeks post-infection ([Fig. 2A](#ppat-1003706-g002){ref-type="fig"}). When the cyst dose was increased to 50, all
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Our individual data points can not be publicly deposited as supporting information due to ethical restrictions imposed by Nagoya University International Bioethics Committee, since the data contains the sensitive personal information. Information regarding our data can be requested at the following e-mail address: Kazunori Hashimoto (e-mail: <khashim@med.nagoya-u.ac.jp>).
Introduction {#sec001}
============
Exposure to arsenic (As) via drinking water is a health risk for humans \[[@pone.0198743.ref001]--[@pone.0198743.ref003]\]. Previous studies showed that exposure of humans to As was associated with cancer of the bladder, kidney, skin, prostate, lung and liver \[[@pone.0198743.ref004]\] and with cardiovascular disease \[[@pone.0198743.ref005]\]. It was also shown that exposure to As was associated with neurological diseases including cognitive impairment in children \[[@pone.0198743.ref006], [@pone.0198743.ref007]\] and adults \[[@pone.0198743.ref008]\] and with diabetes mellitus \[[@pone.0198743.ref009]--[@pone.0198743.ref012]\].
Levels of toxic elements in noninvasive biological samples including nails, hair and urine have been determined to investigate associations with hearing loss in humans \[[@pone.0198743.ref013], [@pone.0198743.ref014]\]. A univariate analysis showed a significant correlation of As levels in fingernails with amplitude of distortion product otoacoustic emission (DPOAE) at 2 kHz, which reflects activity levels of outer hair cells, in 30 subjects aged 9--78 years residing in the gold mining community of Bonanza, Nicaragua \[[@pone.0198743.ref015]\]. Thus, it is possible that As in nails is a reliable index for As-mediated hearing loss in humans. However, there is no direct evidence showing correlations between As levels in toenails and hearing levels.
In our previous study using pure tone audiometry (PTA), an association of oral exposure to As via drinking water with hearing loss in young people was shown by multivariate analysis with adjustments for confounders including age, smoking, sex and BMI \[[@pone.0198743.ref016]\]. However, multivariate analysis has not been performed to demonstrate the association of As levels in nails with hearing loss in humans, although age and smoking are known to be strong factors affecting hearing levels in humans \[[@pone.0198743.ref014], [@pone.0198743.ref017]--[@pone.0198743.ref019]\].
In experimental studies, oral exposure to As has been shown to cause several health risks including carcinogenesis \[[@pone.0198743.ref020]\] and cardiovascular diseases \[[@pone.0198743.ref021]\]. Levels of toxic elements in inner ears, which are known to be the sensory organ for hearing, have been determined to demonstrate the correlation with hearing loss \[[@pone.0198743.ref022], [@pone.0198743.ref023]\]. Exposure of guniea pigs to As at 200 mg/L via intraperitoneal injection for 2 months resulted in morphological impairments of the inner ear \[[@pone.0198743.ref024]\]. Our previous study has showed that exposure of young mice to As via drinking water caused accumulation of As in inner ears, resulting in hearing loss \[[@pone.0198743.ref016]\]. However, it is not clear whether there is a correlation between As levels in inner ears and nails in mice.
In this study, we epidemiologically and experimentally investigated whether As levels in nails are reliable as non-invasive indexes for As-mediated hearing loss in humans and whether As levels in nails are associated with As levels in inner ears in mice.
Materials and methods {#sec002}
=====================
Epidemiological study {#sec003}
---------------------
We obtained information on age, sex, smoking history, weight and height in 145 healthy subjects aged from 12 to 55 years by self-reported questionnaires in Bangladesh as previously described \[[@pone.0198743.ref014]\]. We obtained informed consent in written form from all of the subjects. For subjects aged 12 to 18 years, we obtained consent in written form from their parents. The subjects were only Bangladeshi people who lived mainly in rural areas. The subjects living in one area drink tap water and the rest of the subjects living in another area drink tube well water contaminated with As. The subjects included students, housewives and businessmen. The subjects did not have portable music players with earphones and had no occupational exposure to wielding fumes. None of the subjects had a habit of drinking alcohol. We excluded only subjects who had a history of ear diseases or illness at the time of the survey. We calculated body mass index (BMI) by diving weight (kg) by the square of height (m^2^) and used definitions of underweight (\< 18.5 kg/m^2^), normal weight (18.5--24.9 kg/m^2^) and overweight (≥ 25 kg/m^2^) set by the WHO \[[@pone.0198743.ref025]\]. We set the mean value of age (29.6 years) of the subjects in this study as the cut-off value for age. We collected 0.1--2 cm samples of toenails from each subject by using a ceramic nail clipper. We also collected urine samples from each subject and stored the samples in 50 ml sterile tubes at -80°C until measurements. We determined the hearing levels at 1, 4 and 8 kHz in addition to 12 kHz of the participants by PTA, since hearing level at 12 kHz was shown to be sensitive to environmental stresses \[[@pone.0198743.ref014], [@pone.0198743.ref017], [@pone.0198743.ref026]\]. We used an iPod with earphone-type headphones (Panasonic RP-HJE150) in a soundproof room as described previously \[[@pone.0198743.ref027], [@pone.0198743.ref028]\]. We output pure tones at each frequency to a subject until the subject recognized the sound. We stood behind the subject and provided the subject with an initial stimulus of 5 decibels (dB) and then increased the sound level by 5 dB step-by-step. The subject raised their hand when they recognized the sound. We evaluated the sound level recognized by each subjects as hearing threshold. Pure tones at each frequency from the earphones in the soundproof room were verified by using a noise level meter (Type 6224 with an FFT analyzer, ACO CO., LTD, Japan). The epidemiological study was approved by Nagoya University International Bioethics Committee following the regulations of the Japanese government (approval number: 2013--0070) and the Faculty of Biological Science, University of Dhaka (Ref. no. 5509/Bio.Sc).
Experimental study {#sec004}
------------------
Hairless mice having the C57BL/6J background (1 month old, female, body weight of 10--15 g) were procured from Hoshino Laboratory Animal, Inc. Three or four mice were housed in a cage under super pathogen-free (SPF) conditions with a standard temperature of 23 ± 2°C and a 12-h light/dark cycle. The mice were fed a standard rodent chow (Clea Rodent Diet CE-2). Neither randomization nor blinding investigation were used in this animal study. In brief, we exposed mice (n = 7) to 22.5 mg/L of sodium arsenite (NaAsO~2~, Sigma-Aldrich) dissolved in distilled water for 2 months via drinking water and changed the drinking water every week as previously described \[[@pone.0198743.ref016]\]. The exposure dose was based on the As exposure for mice at 10 and 100 ppm via drinking water in a previous study \[[@pone.0198743.ref029]\]. The control group (n = 6) was given just distilled water. After exposure for 2 months, mice were anesthetized with isoflurane and sacrificed by decapitation. Nails and inner ears were collected and kept in a 1.5 ml tube. For the collection of inner ears, we first identified temporal bones at the bottom of the skull and then carefully removed the cranial nerves and tissues using standard forceps. The inner ears were dislodged by pushing down on the posterior semicircular canal with the thumb of a hand and fixing the tip region of the otic bone capsule with standard forceps. After carefully removing extra tissues adhered to the inner ears, we used the inner ears for ashing. The experimental study was approved by the Institutional Animal Care and Use Committee in Nagoya University (approval number: 28251) and followed the Japanese Government Regulations for Animal Experiments.
Measurement of As levels in biological samples {#sec005}
----------------------------------------------
As levels in biological samples including toenails and urine were measured by inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7500cx) as described previously \[[@pone.0198743.ref014], [@pone.0198743.ref022], [@pone.0198743.ref023]\]. In brief, toenails were washed with detergent water. One or two drops of acetone were added and then the samples were kept at room temperature until starting the ashing. Samples were ashed by incubation in 3 ml of HNO~3~ at room temperature overnight followed by incubation at 80°C for 3 hours. Samples were further incubated in 1--1.5 ml of H~2~O~2~ at 80°C for 3 hours. After cooling, Milli-Q water was added to the samples to adjust the final volume to 5 ml. In the case of urine, 4 ml of urine was incubated in 1 ml of HNO~3~ at room temperature overnight followed by incubation
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{#sp1 .67}
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Introduction {#s1}
============
Some of the most diverse and bizarre organelle genomes of all eukaryotes come from the Volvocales, which is a large order of predominantly freshwater green algae, belonging to chlorophycean class of the Chlorophyta. Volvocalean mitochondrial and plastid DNAs (mtDNAs and ptDNAs) show an impressive array of architectures, nucleotide landscapes, and coding compositions ([Table 1](#pone-0057177-t001){ref-type="table"})-- and see Leliaert et al. [@pone.0057177-Leliaert1] and Lee and Hua [@pone.0057177-Lee1] for additional compilations. Moreover, certain volvocalean species, particularly those within the "*Reinhardtinia* clade" *sensu* Nakada et al. [@pone.0057177-Nakada1], have proven to be excellent systems for testing contemporary hypotheses on the evolution of organelle genome expansion and linearization [@pone.0057177-Smith1], [@pone.0057177-Michaelis1], [@pone.0057177-Smith2].
10.1371/journal.pone.0057177.t001
###### Completely sequenced organelle genomes from volvocalean green algae.
{#pone-0057177-t001-1}
Species Clade (lineage) Organelle genome architecture
-------------------------------- ---------------------------- ------------------------------- -------- ---- -------- ---- ------ -------------------
MITOCHONDRIAL DNA
* Chlamydomonas* *reinhardtii* *Reinhardtinia*(volvocine) Linear 16--19 55 67--82 7 0--3 EU306617--23
* Chlamydomonas* *moewusii* *Xenovolvoxa* Circular 23 65 54 7 9 AF008237
* Chlorogonium* *elongatum* *Caudivolvoxa* Circular 23 62 53 7 6 Y13643--4,Y07814
* Dunaliella salina* *Caudivolvoxa* Circular 28 66 42 7 18 GQ250045
* Gonium pectorale* *Reinhardtinia*(volvocine) Circular 16 61 73 7 1 AP012493
* Polytomella capuana* *Reinhardtinia* Linear 13 43 82 7 0 EF645804
* Polytomella parva* *Reinhardtinia* Linear 16 59 66 7 0 AY062933-4
* Polytomella* sp.SAG 63--10 *Reinhardtinia* Linear 16 58 66 7 0 GU108480-1
* Volvox carteri* *Reinhardtinia*(volvocine) Circular 35 66 \<40 7 3 EU760701,GU084821
PLASTID DNA
* Chlamydomonas* *reinhardtii* *Reinhardtinia*(volvocine) Circular 204 66 44 66 7 FJ423446
* Dunaliella salina* *Caudivolvoxa* Circular 269 68 35 66 \>35 GQ250046
* Gonium pectorale* *Reinhardtinia*(volvocine) Circular 223 70 44 66 3 AP012494
* Volvox carteri* *Reinhardtinia*(volvocine) Circular 525 57 \<20 66 9 GU084820
Note: Values rounded to whole numbers. Clade names are based on Nakada et al. [@pone.0057177-Nakada1]. Percent coding includes all annotated protein-, rRNA-, and tRNA-coding regions as well as non-standard ORFs, such as the *rtl* gene in the *C. reinhardtii* mtDNA. Gene number includes standard protein-coding genes, but does not include intronic or nonstandard ORFs, like *rtl*. Duplicate genes and introns were counted only once. Genome statistics for *P. parva* and *P. piriformis* are based on the concatenation of the two mitochondrial chromosomes; those for *V. carteri* should be considered as approximations as the mtDNA and ptDNA contain assembly gaps due to unresolved repeats. For *C. reinhardtii*, the mitochondrial genome size, intron number, and coding content can vary because of optional introns.
Most of our understanding of volvocalean mitochondrial and plastid genomes is limited to unicellular species, such as the model organisms *Chlamydomonas reinhardtii* and *C. globosa* (previously misidentified as *C. incerta*; see Nakada et al. [@pone.0057177-Nakada2]) [@pone.0057177-Michaelis1], [@pone.0057177-Popescu1], the colorless and wall-less *Polytomella capuana*, *P. parva*, and *P. piriformis* [@pone.0057177-Smith2], [@pone.0057177-Fan1], [@pone.0057177-Smith3], and the halotolerant β-carotene-rich *Dunaliella salina* [@pone.0057177-Smith4]. Surprisingly little is known about the organelle genomes of colonial and multicellular volvocaleans, which are found within the volvocine lineage of the *Reinhardtinia* clade ([Figure S1](#pone.0057177.s001){ref-type="supplementary-material"}). Volvocine algae are preeminent models for studying the evolution of multicellularity [@pone.0057177-Kirk1], [@pone.0057177-Sachs1], and span the gamut of cellular complexity, from simple 4-celled species (e.g., *Tetrabaena*), to 8-64-celled colonial forms (e.g., *Gonium*), all the way to highly complex spheroidal taxa, with more than 500 cells (e.g., *Volvox*) [@pone.0057177-Nozaki1], [@pone.0057177-Herron1]. It is estimated that multicellular volvocine species last shared a common unicellular ancestor ∼200 million years ago [@pone.0057177-Herron1].
Of the 8 different volvocalean algae for which complete mtDNA and/or ptDNA sequences are available [@pone.0057177-Smith4], only one is multicellular: *Volvox carteri*, which is comprised of ∼4,000 cells. The organelle genomes of this species are distended with repetitive noncoding DNA, and similar palindromic repeats are located in both the mitochondrial and plastid compartments [@pone.0057177-Smith5]. Moreover, the *V. carteri* ptDNA, at ∼525 kb, is among the largest plastomes ever observed (from any eukaryote) [@pone.0057177-Smith1], dwarfing that of *C. reinhardtii*, which is 204 kb [@pone.0057177-Maul1]. Although smaller than its plastid counterpart, the ∼35 kb mtDNA of *V. carteri* is still larger than any the other completely sequenced volvocalean mitochondrial genome. It is hypothesized that the expanded organelle genomes of *V. carteri* are a consequence of a low organelle mutation rate and/or a small effective population size [@pone.0057177-Smith1].
The *V. carteri* mtDNA assembles as a circular molecule, contrasting the linear (or linear fragmented) architectures of all other well-studied *Reinhardtinia*-clade mitochondrial genomes, including those of *C. reinhardtii*, *Polytomella* spp., and the multicellular *Pandorina morum* [@pone.0057177-Michaelis1], [@pone.0057177-Smith4], [@pone.0057177-Moore1]. These linear mtDNAs have evolved complex terminal structures [@pone.0057177-Michaelis1], [@pone.0057177-Smith3], called mitochondrial telomeres, which form long palindromic repeats at the genome ends. The origin and number of times that linear mitochondrial chromosomes have evolved within the *Reinhardtinia* is unknown, but it has been argued that they arose only once [@pone.0057177-Smith1]. If true, this would imply that in a recent ancestor of *V. carteri*, the mtDNA reverted from a linear to a circular form.
To learn about organelle genome architecture within multicellular volvocine algae and to gain insight into ptDNA expansion and the origin of linear mtDNAs, we sequenced the mitochondrial and plastid genomes
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Introduction {#s1}
============
Mitochondria perform a dual role in the life and death of the cardiomyocyte. When functioning normally they generate the energy required for normal cellular processes and survival. However, in situations of cellular stress such as during acute myocardial ischemia-reperfusion injury (IRI), they can become dysfunctional and be the arbitrators of cardiomyocyte death. Therefore, new treatment strategies which are capable of preventing mitochondrial dysfunction during acute IRI may reduce myocardial injury, preserve cardiac function and improve clinical outcomes in patients with ischemic heart disease.
In this regard, the mitochondrial serine-threonine protein kinase, PTEN (phosphatase and tensin homologue on chromosome 10)-induced kinase 1 (PINK1), may provide a novel therapeutic target for cardioprotection [@pone.0062400-Siddall1]. Mutations in the PINK1 gene are responsible for the autosomal recessive PARK6 inherited form of early onset Parkinson disease, a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra [@pone.0062400-Valente1]. Genetic ablation of PINK1 in neurons results in mitochondrial dysfunction characterized by: mitochondrial membrane depolarization [@pone.0062400-WoodKaczmar1], [@pone.0062400-Gandhi1], reduced mitochondrial respiration and ATP levels [@pone.0062400-Park1], increased oxidative stress [@pone.0062400-WoodKaczmar1], [@pone.0062400-Gandhi1], [@pone.0062400-Clark1]--[@pone.0062400-Dagda1], mitochondrial calcium overload [@pone.0062400-Gandhi1], and enhanced susceptibility to mitochondrial permeability transition pore (MPTP) opening [@pone.0062400-Gandhi1]. In contrast, wild-type PINK1 has been reported to protect neurons from mitochondrial dysfunction [@pone.0062400-Valente1], reduce mitochondrial cytochrome C release and caspase 3 and 9 activation [@pone.0062400-Petit1], [@pone.0062400-Wang2], and attenuate apoptotic cell death [@pone.0062400-Valente1], [@pone.0062400-Petit1].
Interestingly, PINK1 protein is highly expressed in the myocardium [@pone.0062400-Unoki1] but its role in the heart, is not clear [@pone.0062400-Siddall1], [@pone.0062400-Siddall2]. Given its beneficial effects on mitochondrial function and neuroprotective properties, we investigated whether PINK1 could also protect the heart against acute IRI. We find that the loss of PINK1 increases the heart\'s vulnerability to ischemia-reperfusion injury and this may be by worsening mitochondrial function.
Materials and Methods {#s2}
=====================
Animal experiments were conducted in strict accordance with the *Animals* (*Scientific Procedures*) *Act 1986* published by the UK Home Office and the *Guide for the Care and Use of Laboratory Animals* published by the US National Institutes of Health (NIH Publication No. 85--23, revised 1996). Approval has been granted by a local ethics review board at University College London. All efforts were made to minimize suffering.
HL-1 Cell Culture and PINK1 Over-expression {#s2a}
-------------------------------------------
HL-1 cells are an adherent murine atrial cell line that spontaneously beat in culture (the cells were obtained from Claycomb) [@pone.0062400-Claycomb1]. Cells were cultured in tissue culture flasks pre-coated for 2--3 hrs with 10 µg/ml fibronectin (diluted in 0.02% gelatin). Growth medium (Claycomb media supplemented with 10% FBS, 2 mM L glutamine (Invitrogen, Gibco), 0.1 mM norepinephrine (prepared in 30 mM ascorbic acid), 500 IU penicillin and 500 µg streptomycin (PAA Laboratories)) was changed every 1--2 days and cells were maintained at 37°C in 95%O~2~/5%CO~2~ with 90% humidity.
A similar vector expressing PINK1 under the control of the CMV promoter (Addgene plasmid 13315: pcDNA-DEST53 PINK1) from Addgene Inc., Cambridge, MA was used to over-express PINK1. HL-1 cells were seeded onto fibronectin coated glass cover slips and upon reaching 50--60% confluence were transfected for 24 hours using Fugene6 ® (Roche, UK) according to manufacturer's instructions. The pEGFP expression plasmid (Clontech) was included for identification of successfully transfected cells, at a ratio of 1∶2. The vector control group was designated as cells transfected with an empty plasmid expression vector (RcCMV). A similar vector expressing PINK1 under the control of the CMV promoter (Addgene plasmid 13315: pcDNA-DEST53 PINK1) from Addgene Inc., Cambridge, MA was used to over-express PINK1 in a separate set of cells. The pEGFP expression plasmid (Clontech) was included for identification of successfully transfected cells, at a ratio of 1∶2. The transfection efficacy was 60--70% of cells. Culture media containing transfection components was replaced with fresh growth medium and cells were incubated overnight. Unfortunately due to a lack of a specific commercially available PINK1 antibody were not able to demonstrate PINK1 protein expression or localization.
Simulated Ischemia-reperfusion Injury in HL-1 Cells Over-expressing PINK1 {#s2b}
-------------------------------------------------------------------------
In order to determine the effect of PINK1 over-expression on the susceptibility to simulated ischemia-reperfusion injury (SIRI), HL-1 cells were subjected to a sustained episode of lethal hypoxia and reoxygenation [@pone.0062400-Lim1], [@pone.0062400-Smith1]. Culture medium was removed and replaced with hypoxic buffer (comprising in mM: KH~2~PO~4~ 1.0, NaHCO~3~ 10.0, MgCl~2~.6H~2~O 1.2, NaHEPES 25.0, NaCl 74.0, KCl 16, CaCl~2~ 1.2 and NaLactate 20 at pH 6.2, bubbled with 100% nitrogen) and then placed in an airtight custom-built hypoxic chamber kept at 37°C for 12 hours to simulate ischemia. Following the period of simulated ischemia, the cells were removed from the hypoxic chamber and placed in normoxic Claycomb medium (containing 3 µM propidium iodide) and returned to a tissue culture incubator, to simulate reperfusion. Following 1 hour simulated reperfusion at 37°C, the percentage of GFP-transfected cells stained with propidium iodide was determined using a Nikon Eclipse TE200 fluorescent microscope in order to calculate the percentage cell death in each treatment group. For each treatment group 80 cells were counted, taken from four randomly-selected fields of view. This experiment was repeated on at least four separate occasions giving a total of 320 cells per treatment group. For a time-matched normoxic control group, HL-1 cells were placed in normoxic buffer (comprising in mM: KH~2~PO~4~ 1.0, NaHCO~3~ 10.0, MgCl~2~.6H~2~O 1.2, NaHEPES 25.0, NaCl 98.0, KCl 3, CaCl~2~ 1.2, d-glucose 10.0, Na pyruvate 2.0 at pH 7.4, bubbled with 5% CO~2~/95% O~2~) for the total 13 hours duration of the experiment and the percentage cell death was determined.
ROS-induced MPTP Opening in HL-1 Cells Over-expressing PINK1 {#s2c}
------------------------------------------------------------
To determine the effect of PINK1 over-expression on the susceptibility to MPTP opening, a previously validated cell model of MPTP opening was utilized [@pone.0062400-Zorov1]. Confocal laser-stimulation of the fluorophore tetra methyl rhodamine methyl (TMRM), which accumulates into mitochondria, generates reactive oxygen species (ROS) within mitochondria. This cell model can be used to simulate the events occurring at reperfusion, in which the production of ROS results in MPTP opening and the collapse of the mitochondrial membrane potential [@pone.0062400-Zorov1], [@pone.0062400-Davidson1]. The collapse of mitochondrial membrane potential in this cell model has previously been verified as indicating MPTP opening as it coincides with the redistribution of calcein from the mitochondria to the cytosol [@pone.0062400-Hausenloy1].
Culture medium was removed and replaced with Krebs imaging buffer. Cells were then loaded with the 3 µM TMRM for 15 min at 37°C and washed with Krebs imaging buffer. The time taken to induce mitochondrial membrane depolarization is recorded as a measure of susceptibility to MPTP opening. This was defined as the time taken to reach half the maximum TMRM fluorescence intensity. Twenty transfected cells were randomly selected for the induction and detection of MPTP opening from each treatment group, and this was repeated in four independent experiments giving a total of 80 cells per treatment group. As a positive control and in order to confirm that mitochondrial membrane depolarization was indicative of MPTP opening, following TMRM loading, a group of cells were pre-treated for 10 minutes with the MPTP inhibitor, cyclosporin A (0.2 µM) [@pone.0062400-Lim1], [@pone.0062400-Hausenloy2], [@pone.006
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Introduction {#tca13044-sec-0001}
============
Nivolumab, a PD‐1 antibody, is an immune checkpoint inhibitor (ICI) that has been proven to be active in patients with several different tumor types. Nivolumab has been shown to have an overall response rate (ORR) of approximately 20% in patients with previously treated non‐small cell lung cancer (NSCLC).[1](#tca13044-bib-0001){ref-type="ref"}, [2](#tca13044-bib-0002){ref-type="ref"} However, nivolumab is not effective in more than 40% of NSCLC patients who experience disease progression, despite nivolumab treatment. To improve the efficacy of this promising immunotherapy, additional modalities such as chemotherapy, radiotherapy (RT), or other ICIs may also be administered to patients in whom ICIs have not been completely effective. Interestingly, RT stimulates a systemic immune response and causes the release of tumor‐related antigens.[3](#tca13044-bib-0003){ref-type="ref"} Recent preclinical studies have demonstrated a synergistic tumor response with RT and the blockade of PD‐1.[4](#tca13044-bib-0004){ref-type="ref"}, [5](#tca13044-bib-0005){ref-type="ref"}, [6](#tca13044-bib-0006){ref-type="ref"} It is possible that tumor‐specific immunity is induced by radiation. Although RT plays an important role in the local control and elimination of tumors, it also contributes to the induction of antitumor immune responses, and the immunosuppressive and immunostimulatory effects of RT.[6](#tca13044-bib-0006){ref-type="ref"} Radiation‐induced cell death causes a release of danger signals such as HMGB1, ATP, and HSP70, and the dendritic cells can stimulate activated CD8 T cells and tumor‐specific T cells.[6](#tca13044-bib-0006){ref-type="ref"} In addition to immune activation, RT induces transforming growth factor‐β (TGF‐β), an immunosuppressive cytokine. To reduce the immunosuppressive functions, a combination of RT and TGF‐β inhibitors has been identified as a valuable option in preclinical settings.[6](#tca13044-bib-0006){ref-type="ref"} A recent experimental study demonstrated that fractionated RT increases PD‐L1 surface expression on tumor cells, suggesting a key rationale for the combination of RT with ICIs.[7](#tca13044-bib-0007){ref-type="ref"}
Of the 98 patients registered in the KEYNOTE‐001 phase I trial, Shaverdian *et al.* reported that the duration of progression‐free survival (PFS) with pembrolizumab was significantly longer in patients previously administered RT than in those not treated with RT.[8](#tca13044-bib-0008){ref-type="ref"} Fiorica *et al.* also reported that nivolumab treatment after hypofractionated RT improved the outcome in 35 patients with pretreated or metastatic NSCLC.[9](#tca13044-bib-0009){ref-type="ref"} These results suggest that previous RT clinically improves tumor response and immune reaction to ICIs, such as nivolumab or pembrolizumab. However, the synergistic effect of ICIs and previous RT was not fully elucidated in these studies. As the former study was a phase I trial, pembrolizumab was administered at different doses and treatment deliveries.[8](#tca13044-bib-0008){ref-type="ref"} The latter study was limited by a small sample of only 35 patients.[9](#tca13044-bib-0009){ref-type="ref"} Analysis of these studies shows that immunotherapy after previous RT prolongs survival;[8](#tca13044-bib-0008){ref-type="ref"}, [9](#tca13044-bib-0009){ref-type="ref"} however, neither the ORRs of ICIs after previous RT nor the populations that might benefit from ICI treatment were included. Little detailed clinical data of the effect of ICI administration after previous RT has been reported; therefore, we attempted to elucidate the potential synergistic antitumor effect of nivolumab after RT in patients with previously treated NSCLC.
Methods {#tca13044-sec-0002}
=======
Patient eligibility and data collection {#tca13044-sec-0003}
---------------------------------------
The eligibility criteria for our retrospective analysis were: histologically or cytologically proven advanced NSCLC with stage III or IV disease or recurrence after surgical resection; age \> 20 years; patients with disease progression after at least one prior cytotoxic chemotherapy treated with nivolumab; *EGFR* mutation‐positive patients administered EGFR‐tyrosine kinase inhibitors prior to any cytotoxic chemotherapy; and patients with available ORR data of nivolumab according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Patients were excluded if they had: a concomitant serious illness, such as myocardial infarction in the previous three months; uncontrolled angina pectoris, heart failure, uncontrolled diabetes mellitus, uncontrolled hypertension, interstitial pneumonia, or lung disease; an infection or other disease contraindicating chemotherapy; or were pregnant or breastfeeding. This study was approved by the institutional ethics committee of the Saitama Medical University International Medical Center.
Treatment and efficacy evaluation {#tca13044-sec-0004}
---------------------------------
Nivolumab was intravenously administered at 3 mg/kg every two weeks. A complete blood cell count, differential count, routine chemistry measurements, physical examination, and toxicity assessment were performed on a weekly basis. Acute toxicity was graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. The tumor response was evaluated according to RECIST version 1.1.[10](#tca13044-bib-0010){ref-type="ref"}
Statistical analysis {#tca13044-sec-0005}
--------------------
*P* \< 0.05 indicated statistical significance. Fisher\'s exact tests were conducted to examine the association between the categorical variables. The Kaplan--Meier method was used to estimate survival as a function of time, and survival differences were analyzed by log‐rank tests. PFS was defined as the time from the initiation of nivolumab therapy to tumor recurrence or death from any cause, while overall survival (OS) was defined as the time from the initiation of nivolumab therapy to death from any cause. Statistical analyses were performed using GraphPad Prism 4 and JMP 8.0.
Results {#tca13044-sec-0006}
=======
Patient demographics {#tca13044-sec-0007}
--------------------
From February 2016 to December 2017, 152 patients with pretreated NSCLC were administered nivolumab. Twenty‐eight patients were excluded because of inadequate medical information or the absence of an evaluable target lesion. Thus, a total of 124 patients (*n* ~males~ = 93, *n* ~females~ = 31; median age: 69 years; range: 31--85 years) were eligible for analysis. Patient characteristics are listed in Table [1](#tca13044-tbl-0001){ref-type="table"}. A total of 99 patients had a smoking history. Clinical staging indicated that 27 patients had stage III disease, 77 had stage IV disease, and 20 patients developed recurrence after surgical resection. The patients were divided into RT and non‐RT groups.
######
Comparison of demographics in patients treated with or without RT before nivolumab
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Variables All patients\ Patients administered any RT before Nivo (*n* = 66) Patients not administered RT before Nivo (*n* = 58) *P*
(*n* = 124)
---------------------------------------------------------- --------------- ----------------------------------------------------- ----------------------------------------------------- ----------
Age
≦ 69/\> 69 65/59 36/30 29/29 0.71
Gender
Male/female 93/31 51/15 42/16 0.54
Smoking
Yes/no 99/25 52/14 47/11 0.82
ECOG PS
0/1--3 59/65 31/35 18/40 0.09
Stage
III/IV 27/97 20/46 7/51 0.71
T factor
T1--2/T3--4 68/56 35/31 33/25 0.58
N factor
N0/N1‐3 20/104 12/54 8/50 0.62
Histology
Adeno/non‐adeno 65/59 31/35 34/24 0.21
*EGFR* mutation status
Mutant/wild 14/104 10/51 4/53 0.15
Nivo response
CR or PR/SD or PD
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Cedell[@bib1] described talus fracture in 4 young active sportsmen in 1974. These injuries are rare, and from then until now, only a few cases have been reported. The patient may arrive in the emergency department with pain and tenderness in the posteromedial region of the talus. However, most of the time, the injury\'s similarity to an ankle sprain on normal anteroposterior and lateral ankle radiographic views may contribute to misdiagnosis or delayed diagnosis.[@bib2]
Different treatments of this injury are supported in the literature: conservative treatment of nondisplaced or minimally displaced fractures, open reduction--internal fixation for displaced fractures, or fracture excision for malunion of displaced fractures that cause posteromedial ankle impingement.[@bib2], [@bib3]
Technique {#sec1}
=========
The diagnosis can be made using anteroposterior and lateral ankle radiographs ([Fig 1](#fig1){ref-type="fig"}) and computed tomography ([Fig 2](#fig2){ref-type="fig"}).Fig 1Preoperative anteroposterior and lateral radiographs of a right ankle showing a posteromedial talus fracture (arrow).Fig 2Preoperative 3-dimensional right ankle reconstruction showing a displaced posteromedial talus fracture with malunion (arrow).
Preoperative Setup {#sec1.1}
------------------
The patient is placed in the prone position with application of a thigh tourniquet to provide a bloodless surgical field. All bony prominences are padded. A small support is situated under the lower leg, making it possible to move the ankle freely. The ankle should be kept in a plantar-flexed position to relax the neurovascular bundle. The operative leg is prepared and draped in standard fashion. No traction is required.
Portal Placement {#sec1.2}
----------------
According to the recommendations of Van Dijk et al,[@bib4] the posterolateral portal is created through an incision at the level of or slightly above the tip of the lateral malleolus, just lateral to the Achilles tendon. Careful palpation of the Achilles tendon before portal insertion reduces the risk of damaging the tendon. Blunt dissection to the level of the joint is carried out with a small mosquito clamp directed anteriorly, pointing in the direction of the interdigital web space between the first and second toes. It is exchanged with a 4.5-mm arthroscope shaft (Arthrex, Naples, FL) with a blunt trocar.
The posteromedial portal is made just medial to the Achilles tendon, at the same level as the posterolateral portal. A mosquito clamp is introduced and directed toward the arthroscope shaft at 90° to touch this shaft; it should pass the neurovascular bundle without a problem. A 4.0-mm 30° arthroscope (Arthrex) is used through the posterolateral portal. The posterolateral portal is used as the viewing portal, and the posteromedial portal serves as the working portal ([Fig 3](#fig3){ref-type="fig"}). If there is any difficulty in determining whether the arthroscope is inserted correctly, confirmation of its position by fluoroscopy is recommended.Fig 3The patient is in the prone position. Portal placement is shown for the right ankle. Placement of the posterolateral portal is performed just lateral to the Achilles tendon at the level of the tip of the malleolus. A 4-mm 30° arthroscope is in position. The posteromedial portal is made just medial to the Achilles tendon, at the same level as the posterolateral portal. A shaver is introduced and directed toward the arthroscope shaft.
Surgical Correction of Posterior Ankle Impingement {#sec1.3}
--------------------------------------------------
Before the surgeon addresses the pathology, it is paramount to identify the flexor hallucis longus (FHL) and confirm that it moves with passive motion of the hallux. A base loop is passed around the tendon. The FHL is used as the medial border of the working area, which helps prevent injury to the neurovascular bundle. Then, debridement starts with an arthroscopic shaver (Arthrex) and radiofrequency device (Arthrex) inserted through the posteromedial portal.[@bib4]
When soft-tissue debridement is complete, the fracture malunion is clearly defined ([Fig 4](#fig4){ref-type="fig"}). Then, it is time to perform resection. This may include partial or total resection to correct the impingement ([Fig 5](#fig5){ref-type="fig"}). We perform partial resection of the fragment with a motorized 4.0-mm burr (Arthrex) to restore the normal shape of the posterior talus. We check, under arthroscopic control, that range of ankle motion is completely restored and posterior ankle impingement is corrected ([Fig 6](#fig6){ref-type="fig"}).Fig 4Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. The fracture malunion (yellow arrow) and posterior ankle impingement are visualized. A shaver is introduced through the posteromedial portal. The flexor hallucis longus is on the medial side with a base loop around it (green arrow).Fig 5Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Partial resection of the fracture malunion (arrow) is performed. A burr is introduced through the posteromedial portal.Fig 6Arthroscopic view of the posteromedial aspect of the right ankle from the posterolateral portal. Final reshaping of the posterior talus is performed, and posterior ankle impingement (red arrow) is corrected. The flexor hallucis longus is on the medial side with a base loop around it (green arrow).
Complications {#sec1.4}
-------------
Although we have not encountered any complications with this procedure, the major complication is injury to the tibial neurovascular bundle. It is recommended to keep the ankle in plantar flexion during the procedure and keep the instruments lateral to the FHL.
Postoperative Rehabilitation Protocol {#sec1.5}
-------------------------------------
The patient is discharged on the day after surgery using crutches. Weight bearing is allowed. A rehabilitation program to gain full mobility and strength is followed. A control computed tomography scan is ordered 1 month after surgery ([Fig 7](#fig7){ref-type="fig"}). Return to sports is gradually permitted, based on functional demands, with the most demanding activities being avoided until 3 months after ankle arthroscopy.Fig 7Three-dimensional reconstruction 1 month after arthroscopic resection of a malunion of a posteromedial talus fracture showing correction of posterior ankle impingement (arrow).
A step-by-step summary of our technique is provided in [Table 1](#tbl1){ref-type="table"}. Pearls and pitfalls are presented in [Table 2](#tbl2){ref-type="table"}, and advantages and disadvantages are listed in [Table 3](#tbl3){ref-type="table"}. Key steps of the procedure are shown in [Video 1](#appsec1){ref-type="sec"}.Table 1Step-by-Step Summary of Arthroscopic Treatment of Malunion of Posteromedial Talus Fracture1.Position the patient in the prone position with application of a thigh tourniquet to provide a bloodless surgical field.2.Keep the ankle in a plantar-flexed position to relax the neurovascular bundle.3.Establish the posterolateral and posteromedial portals.4.Use a 4-mm 30° arthroscope inserted through the posterolateral portal.5.Identify the FHL and confirm that it moves with passive motion of the hallux.6.Start debridement with an arthroscopic shaver and radiofrequency device inserted through the posteromedial portal.7.Identify the fracture malunion.8.Perform partial resection of the fragment with a motorized 4.0-mm burr.9.Check, under arthroscopic control, that range of ankle motion is completely restored and posterior ankle impingement is corrected.[^1]Table 2Pearls and PitfallsPearlsPitfallsUse the prone position; pad all bony prominences.Pressure sores and lateral femoral cutaneous nerve neuropathyNote that no traction is required.No free movement of ankleKeep the ankle in a plantar-flexed position.Neurovascular bundle injuryPerform careful palpation of the Achilles tendon.Achilles tendon injuryIdentify the tip of the lateral malleolus for correct portal placement.Incorrect portal placementUse the arthroscope shaft as a guide to place the posteromedial portal.Neurovascular bundle injuryUse fluoroscopy to confirm the position and direction of the arthroscope if necessary.Incorrect portal placementIdentify the FHL by passive motion of the great toe and pass a base loop around the tendon.Neurovascular bundle injuryPerform a detailed examination of posterior ankle impingement.Incorrect portal placementIdentify the fracture malunion.No identification of fracture malunionReshape the posterior talus.No impingement correctionAvoid excessive bone resection.Excessive bone resection leading to ankle instabilityCheck complete ankle motion and correction of posterior ankle impingement.No improvement in clinical resultsTable 3Advantages and DisadvantagesAdvantages The procedure allows excellent access to the posterior ankle compartment. The procedure is minimally invasive. The procedure allows posterior ankle impingement to be corrected. The recovery time is shorter.Disadvantages The technique is challenging. Neurovascular bundle injury can occur. An
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Introduction {#Sec1}
============
Organic light-emitting diodes (OLEDs) are being widely applied in displays for mobiles and televisions owing to their self-emitting characteristics, excellent colour gamut, high-speed operation, and applicability in flexible or stretchable devices^[@CR1]^. Practical OLEDs function based on phosphorescence owing to their nearly 100% internal phosphorescence efficiency^[@CR2],[@CR3]^. However, the external quantum efficiency of OLEDs is \~20% because the surface plasmon polariton modes at the metal-organic interface, the waveguide mode in the indium tin oxide (ITO)/organic layer, and the substrate mode in the glass substrate produce light loss^[@CR4]--[@CR8]^. Generally, light loss in the substrate mode is larger than that in the waveguide mode due to small differences in refractive index between the ITO/glass interface and the glass/air interface^[@CR9]^. In addition, microcavity top-emitting organic light-emitting diodes (TEOLED) without the loss of the substrate mode and having the advantage of improved colour purity and aperture ratio have been applied to mobile displays. However, microcavity TEOLED originally had a problem associated with viewing angles due to the blue shift that occurred with the variation of the sight angle, even though the light efficiency along the normal direction was amplified. Therefore, researchers have attempted to find solutions for improving the light extraction and viewing angle of OLEDs^[@CR10]^.
Different approaches have been adopted with the aim of optimizing light extraction and viewing angle, including micro lens array (MLA)^[@CR5],[@CR11]--[@CR16]^, dielectric Bragg gratings^[@CR17]--[@CR19]^, surface plasmons^[@CR20]^, buckling patterns^[@CR21]^, periodic corrugation^[@CR8]^, low-index grids^[@CR22]^, and photonic crystals^[@CR23]^. Methods that modify the internal or external surfaces of the substrate of OLEDs minimize the total internal reflection. Such surface shapes are produced by different technologies which traditionally involve photo-lithography or printing, moulding, and embossing methods^[@CR24]--[@CR28]^. Therefore, these technologies employ at least the photo-lithographic step or utilize lithographic templates once. They generally end up being considerably complex or expensive for mass production. On the other hand, other researchers have reported a simple scattering layer synthesized by competitive and scalable methods. In these methods, particles or voids having different refractive indices were embedded in a polymer matrix and utilized for the scattering layer^[@CR29]--[@CR36]^. However, because the particles or voids are micro-sized or the pitch between the nearest particles or voids is micro-sized, these techniques reveal a small effect of diffraction that is difficult to control. In addition, to prevent the loss of the substrate mode, the most representative MLA has larger visibility than a nano-sized array because of its size. Alignment is a problem in the case of high-resolution displays with very small pixels.
In this paper, we propose a simple method to fabricate random nanoscale rods (RNRs) as scattering layers for enhancement of light extraction and improvement of viewing angle characteristics in OLEDs. In our device, there is virtually no spectral distortion or shift, which are issues observed in 2D photonic crystal-based OLEDs, and the spectral shift according to the change in viewing angle is reduced. This layer is fabricated by a cost-effective and scalable procedure that consists of coating and etching the polymer at low temperatures (below 100 °C) without photomasks or templates. Additionally, the RNRs can control the diffraction as well as scattering effects in the visible wavelength range owing to the distance between closest rods and are compatible with high-resolution displays for virtual reality because the process of alignment between the individual pixels in the panel and the scattering layer is unnecessary. The RNRs can also be applied in flexible displays and lighting owing to the use of etched polymers. OLEDs equipped with RNRs exhibit superior optical properties such as high external quantum efficiency (EQE), high luminance efficiency (LE), and colour variation with changes in viewing angle, to control OLEDs.
Results and Discussion {#Sec2}
======================
To investigate the optical effect of RNRs on glass, the electrical field distribution was simulated by the finite-difference time domain (FDTD) method. The boundary conditions for the simulation were set for a perfect optically matched layer to avoid the reflection of electromagnetic waves at the edges of the structure on all the sides, except for the metal cathode layer. The simulated structure consisted of an aluminium cathode, *N*,*N*'-bis(naphthalen-1-yl)-*N*,*N*'-bis(phenyl)-benzidine (NPB; refractive index n = 1.81), tris(8-hydroxy-quinolinato)aluminium (Alq~**3**~; n = 1.72), ITO (n = 1.9), glass (n = 1.52), SU-8 polymer, and RNRs (n = 1.59). A dipole source of visible wavelength was generated in the Alq~**3**~ layer, and one transverse electric (TE) and two transverse magnetic (TM) modes were used^[@CR37]^. To reflect the randomness of RNRs in the simulation, their height was varied from 1000 nm to 1200 nm and width from 50 nm to 150 nm (Supplementary Information Fig. [S1](#MOESM1){ref-type="media"}). In the reference structure, most of the light incident at an angle above the critical angle became a waveguide because of the difference in refractive index between glass and air (Fig. [1a](#Fig1){ref-type="fig"}). In order to confirm the optical effect of the SU-8 polymer, a simulation using SU-8 without the pattern was performed (Fig. [1b](#Fig1){ref-type="fig"}). As a result, the optical path was almost unchanged, and the difference between the refractive indices of glass and air slightly increased; this difference seemed to be slightly adversely affected for external light extraction. In contrast, in the RNRs present on the glass structure, some portions of the waveguide light were allowed to escape from the glass substrate, and light extraction increased below the critical angle (Fig. [1c](#Fig1){ref-type="fig"}). Optical phenomena such as refraction, diffraction, and interference were produced by the RNRs because the differences in refractive index at the interface between glass and air changed and the random periodic rods revealed distances between each other that were similar to the visible wavelength. Figure [2e](#Fig2){ref-type="fig"} illustrates the light path in OLEDs. The photons trapped (rays 2 and 3) within the glass are scattered by the RNRs, increasing the probability of them being emitted from the structure. Furthermore, as a result of simulation, in which scattering occurs several times in one rod, it can be inferred that scattering occurs more frequently when the height of the rod is greater.Figure 1FDTD simulation of E-field distribution induced by transverse magnetic and electric dipoles: (**a**) Reference (w/o RNRs), (**b**) unpatterned SU-8 on a glass structure, and (**c**) RNRs on the glass structure.Figure 2Cross-sectional scanning electron microscopy images of random nanoscale rods (RNRs) obtained under different plasma etching conditions: (**a**) RNRs 1, (**b**) RNRs 2, (**c**) RNRs 3, and (**d**) RNRs 4, and (**e**) schematic illustration of OLED outcoupling enhancement by the RNR scattering layer.
It is desirable to fabricate a nanostructure of proper height and periodicity that is comparable to the wavelength of the visible range in order to enhance light extraction. For manufacturing RNRs, SU-8 polymer was chosen because it is highly transparent to visible light and has a higher refractive index than glass. Figure [2](#Fig2){ref-type="fig"} shows the cross-sectional scanning electron microscopy images of the RNRs obtained under different plasma etching conditions. Obtained by using only an oxygen plasma for a duration of 9 min (denoted as RNRs 1), the structure was random columnar-like with high aspect ratio and density^[@CR38]^. The height and density of RNRs 1 were about 1200 nm and 9.7 ea/µm^2^, respectively (Fig. [2a](#Fig2){ref-type="fig"}, Supplementary Information Fig. [S2a](#MOESM1){ref-type="media"}). The width of the individual rods was observed to be approximately 50 nm. To control the height and density, we applied an additional argon plasma treatment at higher powers and lower process pressures than the oxygen plasma. The low pressure led to an increase in the mean free path of argon ions and reduced the energy loss of these ions as they approached the RNRs. In general, the etching process using argon plasma carried out in a reactive ion etcher resulted in anisotropic and directional features due to the utilization of the physical bombardment energy^[@CR38]^. As a result of these features, the height and density were adjusted as much as a target to the little changed width of the RNRs. The three treated RNRs shown in Fig. [2b--d](#Fig2){ref-type="fig"} corresponded to argon plasma treatment durations of 3, 6, and 9 min (denoted as RNRs 2, RNRs 3, and RNRs 4, respectively), while the conditions of the oxygen plasma process remained unchanged. As the duration of the argon plasma treatment increased, the
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Introduction {#ss1}
============
Throughout Europe birth-rates have declined over the last decades, and in most countries they are well below the replacement level ([@CIT0001]). In Sweden, the number of children being born per woman has fluctuated greatly since the mid-1970s but has now been predicted to stabilize at a level around 1.8 ([@CIT0002]). The declining birth-rate can partly be explained by an increasing age at first birth ([@CIT0003]); furthermore, today there is clear empirical evidence of the postponement of the first child in several countries ([@CIT0004; @CIT0005; @CIT0006]).
Higher education has been associated with delayed childbearing ([@CIT0007]), and the association between female education and the age of becoming a first-time parent has been well documented ([@CIT0008; @CIT0009]), showing that highly educated women are more likely to pursue careers and postpone having children ([@CIT0005]). Women\'s labour force participation has been associated with postponement largely due to the incompatibility of caring for children and participation in the paid labour force. However, it has been possible to combine female employment and childbearing when the reduction in work--family conflict was facilitated by state or policy interventions, such as in some Scandinavian countries ([@CIT0010; @CIT0011]).
It seems, however, that postponed childbearing is a result of several different reasons. For example, efficient and reliable oral contraception has had an impact on family planning in many modern societies ([@CIT0012; @CIT0013]). A US study found that multiple partnerships before marriage, higher levels of non-marital cohabiting, and difficulty in finding a suitable partner might contribute to later births ([@CIT0014]). It has also been reported that young adults delay childbearing until they are financially secure, so they can afford to support children ([@CIT0015]). In addition, the emergence and development of assisted reproductive technologies (ARTs) have been suggested to promote perceptions that childbearing can be resumed at a later and more convenient phase of life ([@CIT0016]).
Surveys concerning attitudes towards future parenthood and awareness about fertility among undergraduate and postgraduate students in Sweden have shown that these young women and men had largely positive attitudes towards having children, but they were not sufficiently aware of the limitations associated with ageing ([@CIT0017; @CIT0018; @CIT0019]). Similar results have been reported in a recent study among Finnish university students, in which one-third of the women and more than half of the men believed that a marked decline in female fertility begins after the age of 45 ([@CIT0020]). In a Canadian survey among female undergraduate students, it was found that women were aware that fertility declines with age, but they overestimated the chances of pregnancy at any age and were not aware of the steep rate of fertility decline with age ([@CIT0021]). However, knowledge about the influence of female age on childbearing success was not predictive of their childbearing intentions.
The adverse effects of ageing on reproduction are complex and multifactorial. It is known that women\'s ability to conceive starts to decline in their late 20s and rapidly falls by the mid-30s, mainly due to reduced quantity and quality of ovarian follicles ([@CIT0003; @CIT0022]). In addition, after the age of 35 an increase in pregnancy complications and prenatal maternal morbidity, as well as impaired prenatal and postnatal outcome of the child has been reported ([@CIT0023; @CIT0024; @CIT0025]). There is also increasing evidence that paternal age, at least over the age of 40, is associated with lower fertility-related outcome and anomalies in the offspring ([@CIT0026; @CIT0027]).
Societal attitude towards family and children also forms the context in which the subjective preferences and assessments regarding family formation are made. The tendency towards individualism, the endeavour for self-fulfilment, increasing freedom of choice, and greater tolerance towards new lifestyles and reproductive behaviours have also been suggested to influence the decline in births and the postponement of childbearing ([@CIT0028]).
Most research concerning postponed childbearing has focused on women and has mostly been based on standardized questionnaires or register data ([@CIT0029]); however, the information gathered from the viewpoint of highly educated young people themselves is more random. Thus, the aim of the present study was to gain a more comprehensive understanding of how young, highly educated women and men without children, who had started their professional career, reflect on fertility and postponed parenthood.
Materials and methods {#ss2}
=====================
We performed individual interviews with 22 women and 18 men between 24 and 38 years of age. The criteria for inclusion were 4 years or more of university education, having started a professional career, and not yet having children. The participants were recruited using advertisements placed in different work-places where the majority of staff had a higher education. Those who were interested in participating were requested to phone or send an e-mail to the project leaders for further information and arrangement of a suitable time for the interview. The interviews took place in a convenient room offered by the work-place, or outside the office. The interviews were audiotaped and lasted for about half an hour. The interview guide covered two main areas: 1) personal attitudes towards having children in the future and 2) reflections on fertility and postponement of parenthood. The findings related to personal attitudes towards future parenthood have previously been published ([@CIT0030]). All interviews were transcribed verbatim and analysed according to qualitative content analysis ([@CIT0031]).
The transcripts were read several times to gain a sense of the whole. Constellations of words, sentences, or paragraphs related to the study aim were identified and condensed. Thereafter, codes were created, and finally the codes were sorted into seven subcategories and two categories according to their content and meaning. The process of developing categories is illustrated in [Tables I](#T1){ref-type="table"} and [II](#T2){ref-type="table"}.
######
Examples of condensed meaning units, codes, subcategories, and categories.
Meaning units Codes Subcategory Category
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------ --------------------------------------------------------- ------------------------------------------------------------------
'In a larger city one has so many other things to keep you busy anyway, so children come significantly later. Yes, but yeah, maybe it\'s time, I thought a little like that, that it\'s about time\' City life offers many alternatives to family formation A consequence of contemporary lifestyles Postponed parenthood---a rational adaptation to societal changes
'To delay having children is probably due to the increased fear of growing up, to seriously be an adult because it\'s so nice to have to worry only about yourself\' A sign of extended immaturity, or egocentricity
'Today it is expected to not only get an education but also to travel, learn a new language, live overseas and much more, and parenthood doesn\'t fit so well in the story\' Expectations of further learning and striving for experiences and adventures A consequence of competing priorities
'There are more women who study at universities, more women than men that graduate, and maybe the birth of a child is viewed as an obstacle to one\'s career, that must be the explanation\' Women\'s increased investment in higher education
'To have a child before 25 is not exactly something that is encouraged and maybe also not having children after 40 either\' Socially mediated perceptions about the timing of childbearing A consequence of prevailing discourses about parenthood
'There is such an incredibly negative view about having a child when you are young, and among my friends there are many that see it as completely abnormal to have a child before 30\' Early parenthood is unfavourable
######
Women\'s and men\'s reflections on fertility and postponed parenthood.
Subcategories Categories
--------------------------------------------------------- ------------------------------------------------------------------
Unconsidered and taken for granted Fertility---an imperceptive and retrievable capacity
Unpredictable and different for women and men
Restorable by medical technology
Replaceable by alternatives to a biological child
A consequence of contemporary lifestyles Postponed parenthood---a rational adaptation to societal changes
A consequence of competing priorities
A consequence of prevailing discourses about parenthood
Results {#ss3}
=======
In the following section, the two categories 'Fertility---an imperceptible and retrievable capacity\' and 'Postponed childbearing---a rational adaptation to societal changes\' are presented. Quotations from the interviews are used for illustration.
Fertility---an imperceptible and retrievable capacity {#ss4}
-----------------------------------------------------
The category 'Fertility---an imperceptible and retrievable capacity\' consisted of four subcategories: 'Unconsidered and taken for granted\', 'Unpredictable and different for women and men\', 'Restorable by medical technology\', and 'Replaceable by alternatives to a biological child\'.
### Unconsidered and taken for granted {#ss5}
This subcategory consisted of statements showing that even though most informants wanted to have children in the future, some had never reflected on their own reproductive capacity. It also included comments that revealed perceptions about fertility as something natural. Some referred to a healthy body, while others put confidence in their genetic heritage. In addition, some held the opinion that there is no point in reflecting too much about one\'s fertility as it might cause unfounded worries.
> My own fertility, nah, I have not thought about that; actually, I have never done that. (w12)
> I presume that I can deliver what\'s required when it comes to
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Introduction {#Sec1}
============
Pre-diabetes (pre-DM), typically defined as blood glucose levels above normal but below diabetes thresholds, has been increasing globally and has a high chance of developing diabetes mellitus (DM)^[@CR1]^. It is estimated that there were 318 million adults suffering from impaired glucose tolerance in the world by 2015^[@CR2]^. As to the Chinese adult population, a cross-sectional survey in 2010 reported that the estimated prevalence of pre-DM was 50.1%^[@CR3]^. Previous studies^[@CR4]^ have shown that about 5--10% of pre-DM progressed to DM every year, and persistent hyperglycemia leads to the complications that are the major source of morbidity, mortality, and cost. Nowadays, There is a common conception that this natural history is not inevitable^[@CR5]^. Randomized controlled trials showed that lifestyle intervention or glucose-lowering medications could delay and even reverse the natural course of pre-DM^[@CR6]--[@CR8]^. The American Diabetes Association (ADA) recommends at least annual screening via testing fasting plasma glucose (FPG), 2-hr 75 g oral glucose tolerance test (OGTT), or hemoglobin A1c (HbA1c) in those with pre-DM^[@CR9]^. However, clinicians are far from satisfied with the presently used method of monitoring glycemic level because of its hysteresis. FPG, OGTT and HbA1c are used more as a diagnostic than a predictive marker. Interestingly, multiple cross-sectional and prospective cohort studies have revealed the metabolism of impaired branched-chain amino acid (BCAA), aromatic amino acid (AAA), free fatty acid (FFA), acylcarnitines and glycerophospholipid are associated with insulin resistance, and many metabolites were considered as biomarkers for the prediction of pre-DM and DM^[@CR10]--[@CR13]^. However, little is known about the potential metabolic biomarkers of different glycemic prognoses among subjects with pre-DM. More importantly, building a metabolic model for predicting the transition from pre-DM to NGR or DM would be helpful for the early prevention and treatment among individuals with pre-DM.
Metabolomics provides a snapshot of the metabolic dynamics that reflects the response of living systems to pathophysiological stimuli and/or genetic modifications and surrounding environment. Furthermore, in many ways, tanscriptomic, genomic, and proteomic changes are upstream of the final physiology of cells, whereas the metabolic profile is likely closer in response to the disease process^[@CR14]^. Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) of biofluids can easily detect hundreds of individual species in a single clinical sample, reflecting the biochemical fingerprint of the organism^[@CR15]^. Characterized by sensitivity and high mass accuracy, the technique has been employed in identifying novel biomarkers for cancers^[@CR16],[@CR17]^, metabolic disorders^[@CR18]--[@CR20]^, drug toxicity and function^[@CR21],[@CR22]^, and so on.
With the current study, we characterized the metabolic profiles of fasting plasma samples among the 108 pre-DM at baseline with different outcomes ten years later utilizing untargeted UPLC-QTOF-MS analysis. 23 and 22 metabolites were identified as biomarkers for transition to NGR and DM from pre-DM, respectively. And the underlying biochemical pathways leading to different prognoses were investigated.
Results {#Sec2}
=======
Demographic and Clinical Characteristics {#Sec3}
----------------------------------------
108 participants with pre-DM from a longitudinal cohort study were followed up for ten years and were divided into 3 groups according to different glycemic outcomes. 20 participants progressed to DM, 20 regressed to NGR, and 68 remained at pre-DM, respectively.
At baseline, there were no significant differences in ages, gender, body mass index, blood glucose, lipid profile, blood pressure as well as general health conditions among these 3 groups (Supplemental Table [S1](#MOESM1){ref-type="media"}). At the end-point of the study, no significant differences in the biochemical characteristics were found among the three groups, except for fasting glucose, 2-h glucose and HbA1c (Table [1](#Tab1){ref-type="table"}).Table 1Characteristics of the study participants at end-point.NGR (n = 20)Pre-DM (n = 68)DM (n = 20)Age, years57.4 ± 8.860.4 ± 8.957.9 ± 10.1Gender, N, male/female7/1325/437/13Body mass index, kg/m^2^24.0 ± 3.124.8 ± 2.926.1 ± 3.9Waist circumference, cm83.3 ± 7.587.1 ± 8.488.3 ± 11.9Waist-hip ratio0.88 ± 0.040.90 ± 0.060.92 ± 0.08Hypertension, %45.057.470.0Family history of DM, %35.033.845.0Current smoker, %5.022.115.0Current drinker, %15.013.20.0**Physical activity**Inactive, %5.011.810.0Medium, %70.073.575.0Active, %25.014.715.0SBP, mmHg132.5 ± 19.4138.9 ± 14.8138.7 ± 17.5DBP, mmHg77.4 ± 8.880.5 ± 9.580.4 ± 10.4Fasting glucose, mmol/L5.2 ± 0.2^\*^5.6 ± 0.58.0 ± 2.9^\*\*\#^2-h glucose, mmol/L6.0 ± 0.8^\*\*^7.6 ± 1.813.1 ± 4.7^\*\*\#^HbA1c, %5.3 ± 0.5^\*\*^5.8 ± 0.37.2 ± 1.4^\*\*\#^Fasting insulin, pmol/mL8.4 ± 3.79.2 ± 5.011.3 ± 3.8^\*a^2-h insulin, pmol/mL38.9 ± 23.571.2 ± 65.458.7 ± 36.8HDL, mmol/L1.4 ± 0.31.5 ± 0.41.5 ± 0.4LDL, mmol/L3.1 ± 0.73.0 ± 0.93.5 ± 1.1TC, mmol/L5.2 ± 1.25.5 ± 1.86.2 ± 1.3^b^TG, mmol/L1.4 ± 0.72.0 ± 2.62.8 ± 2.3^\*\#^BUA, umol/L311.9 ± 69.7338.7 ± 88.5321.3 ± 79.4CR, umol/L81.1 ± 18.672.8 ± 29.062.6 ± 25.7ALT, U/L20.8 ± 9.023.0 ± 16.833.1 ± 32.7AST, U/L24.5 ± 6.527.3 ± 17.828.1 ± 16.8γGT, U/L24.0 ± 21.431.8 ± 30.531.8 ± 17.2Values are mean ± SD or %. \*p \< 0.01, \*\*p \< 0.001 compared to pre-DM and ^\#^p \< 0.001 compared to NGR. ^a^Exact significance (2-tailed), p = 0.015 compared to NGR; ^b^p = 0.017 compared to pre-DM. SBP: systolic blood pressure; DBP: diastolic blood pressure; HbA1c: hemoglobin A1c; HDL: high density lipoprotein; LDL: low density lipoprotein TC: total cholesterol; TG: triglyceride; BUA: blood uric acid; CR: creatinine; ALT: alanine transaminase; AST: aspartate transaminase; γGT: γ glutamyl transferase.
Quality control {#Sec4}
---------------
The robustness and stability of the method was assessed by repeat analysis of a representative pooled quality control (QC) sample during sample runs. The overlapped total ion current chromatograms of the QC sample demonstrated the repeatability of our UPLC-QTOF-MS system (Supplemental Fig. [S1A](#MOESM1){ref-type="media"}). The principal component analysis (PCA) performed on QC and other groups revealed that QC samples were clustered in the PCA scores plot (Supplemental Fig. [S2A](#MOESM1){ref-type="media"}). The percentage coefficient of variation (CV%) of peak intensity was estimated as 4.1--18.6%. These results collectively indicated good repeatability, reliability, and stability of this method for metabolite analysis.
Plasma metabolite profile and makers for NGR and DM {#Sec5}
---------------------------------------------------
Representative base peak intensity (BPI) chromatograms of plasma samples indicated that the sample metabolites attained suitable separation. Typical
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1. Introduction {#sec1-pharmaceutics-10-00021}
===============
The discovery of vascular endothelial growth factor (VEGF) \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and the subsequent recognition of its critical role in the pathogenesis of several chorioretinal vascular conditions constitute the most important advances in ophthalmology over the past 30 years. Strong evidence correlates the development of both neovascularization and macular edema in the two most common causes of blindness in industrialized nations---neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR)---with the upregulation of VEGF \[[@B3-pharmaceutics-10-00021]\]. Furthermore, disease severity frequently correlates with intraocular VEGF concentrations, thereby making VEGF a logical target for therapeutic intervention.
Soon after VEGF was discovered and sequenced, the production of inhibitory molecules began \[[@B4-pharmaceutics-10-00021]\]. Thus far, five VEGF-neutralizing molecules (pegaptanib, Macugen^®^, Bausch & Lomb, Bridgewater, NJ, USA; ranibizumab, Lucentis^®^, Genentech, S. San Francisco, CA, USA/Roche, Basel, Switzerland; aflibercept, Eylea^®^, Regeneron, Tarrytown, NY, USA; conbercept, Chengdu Kanghong Pharmaceutical Group, Chengdu, China; and bevacizumab, Avastin^®^, Genentech, S. San Francisco, CA, USA/Roche, Basel, Switzerland) have been used to treat ophthalmologic conditions, though only the first three have received United States Food and Drug Administration (US FDA) approval for intraocular use. Intravitreal therapy usually begins with monthly injections (in accordance with package labeling) but most physicians will attempt to extend the time between injections as much as possible with either monthly *pro re nata* (PRN) or treat and extend strategies \[[@B5-pharmaceutics-10-00021]\]. Treatment intervals for many patients cannot be extended beyond eight weeks \[[@B6-pharmaceutics-10-00021]\], resulting in a large group of patients who require frequent injections for long periods of time. This large number of intravitreal injections burdens physicians and their staffs, and challenges patients' compliance. Therefore, new, longer acting anti-VEGF medications and drug delivery systems are needed to improve outcomes, optimize compliance, and reduce the total cost of care.
This manuscript discusses extended duration anti-VEGF therapies that have been recently introduced, as well as those that are in various stages of development.
2. Vascular Endothelial Growth Factor (VEGF) Physiology and Pharmacokinetics {#sec2-pharmaceutics-10-00021}
============================================================================
VEGF was discovered independently by two research groups in 1989 \[[@B1-pharmaceutics-10-00021],[@B2-pharmaceutics-10-00021]\] and its important role in both physiologic angiogenesis and pathological neovascularization was realized almost immediately. VEGF is actually a group of molecules that segregate into seven closely related families: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PlGF) \[[@B7-pharmaceutics-10-00021]\]. Each of the families is characterized by common, critical binding sequences, and most families contain multiple isoforms that share similar binding properties and biological actions.
VEGF-A synthesis is upregulated in eyes with chorioretinal vascular conditions, including nAMD, diabetic macular edema (DME), and retinal vein occlusion (RVO) \[[@B3-pharmaceutics-10-00021]\], and is believed to play a central role in the development of these conditions. Several in vivo models show that VEGF-A promotes the growth of choroidal neovascular membranes \[[@B8-pharmaceutics-10-00021]\] and produces retinal vascular lesions that resemble DR \[[@B9-pharmaceutics-10-00021]\]. Evidence suggests that VEGF~165~ may be the most biologically active isoform because of its high tissue concentrations and 10-fold potentiation of activity through its interaction with the transmembrane co-receptor neuropilin-1 \[[@B10-pharmaceutics-10-00021]\]. Most VEGF inhibitory molecules block the receptor binding region (amino acids 81--92) of VEGF-A isoforms, whereas pegaptanib interacts with the heparin binding region (amino acids 110--165) of VEGF~165~. Research suggests that VEGF-B, VEGF-C, VEGF-D, and PlGF may also contribute to pathologic ocular angiogenesis in humans but their relative contribution is not known \[[@B11-pharmaceutics-10-00021],[@B12-pharmaceutics-10-00021]\].
Increased VEGF synthesis by vascular endothelial cells, glia, pericytes, Müller cells, retinal pigment epithelium (RPE) cells, and invading leukocytes \[[@B13-pharmaceutics-10-00021],[@B14-pharmaceutics-10-00021]\] results from tissue ischemia and inflammation \[[@B15-pharmaceutics-10-00021],[@B16-pharmaceutics-10-00021]\]. Cells throughout the retina and choroid respond to increased VEGF concentrations but the primary targets are retinal and choroidal vascular endothelial cells \[[@B17-pharmaceutics-10-00021]\].
VEGF-A has a short half-life of 30 min in the eye and serum, and homeostatic concentrations are generally low (approximately 9 ng/mL) \[[@B18-pharmaceutics-10-00021]\]. Some systemic conditions increase serum VEGF concentrations but chorioretinal vascular conditions produce insufficient VEGF to meaningfully change serum levels.
3. Currently Available Therapies {#sec3-pharmaceutics-10-00021}
================================
Several anti-VEGF drugs have been developed exclusively for ocular use or, in the case of bevacizumab, are used off-label for chorioretinal vascular conditions. Peak clinical efficacies of these drugs (except for pegaptanib) are similar and though product labels describe different injection intervals (monthly or every two months) the differences in their duration of action are on the order of only days. Currently available drugs, recently failed therapies, and drugs and systems under development are listed in [Table 1](#pharmaceutics-10-00021-t001){ref-type="table"}.
3.1. Pegaptanib {#sec3dot1-pharmaceutics-10-00021}
---------------
Pegaptanib (molecular weight (MW) of 50 kDa), an aptamer to VEGF, was the first ocular drug approved for the intravitreal treatment of neovascular age-related macular degeneration (nAMD). Clinicians hoped that q6week treatment with pegaptanib would improve best corrected visual acuity (BCVA) but in most eyes it only decreased the rate of vision loss by approximately one half \[[@B19-pharmaceutics-10-00021]\]. Its use dropped significantly when more potent anti-VEGF drugs were introduced and pegaptanib is rarely used today.
3.2. Bevacizumab {#sec3dot2-pharmaceutics-10-00021}
----------------
Bevacizumab is a full-length, recombinant, humanized, monoclonal antibody (MW of 149 kDa) that binds all isoforms of VEGF-A. It was developed and approved for the intravenous treatment of several advanced solid tumors (colorectal carcinoma, non-small cell lung carcinoma, renal cell carcinoma, glioblastoma, and breast cancer, though this approval was rescinded in 2011) \[[@B20-pharmaceutics-10-00021]\].
Single injections of bevacizumab were first given to patients with nAMD and macular edema due to a central retinal vein occlusion (CRVO) in 2005 \[[@B39-pharmaceutics-10-00021],[@B40-pharmaceutics-10-00021]\], and within six months off-label use of bevacizumab had become the accepted standard-of-care treatment of chorioretinal vascular conditions. Hundreds of ocular disease studies have established bevacizumab's efficacy and safety, though the best evidence comes from the Comparison of Age-related Macular Degeneration Treatment Trials (CATT) for nAMD and the Diabetic Retinopathy Clinical Research Network Protocol T trial for DME \[[@B6-pharmaceutics-10-00021],[@B21-pharmaceutics-10-00021]\].
The use of bevacizumab varies among countries due to regulatory restrictions, reimbursement policies, and availability of safely compounded drug. Because physicians have accumulated extensive clinical experience with bevacizumab and are able to acquire it inexpensively, bevacizumab remains the most commonly used anti-VEGF drug in the United States.
3.3. Ranibizumab {#sec3dot3-pharmaceutics-10-00021}
----------------
Ranibizumab is a recombinant, humanized, monoclonal antibody fragment (Fab with MW of 48 kDa) that binds all isoforms of VEGF-A \[[@B4-pharmaceutics-10-00021]\]. It has been approved by the United States Food and Drug Administration (USFDA) for the treatment of nAMD (2006), DME, DR, macular edema due to vein occlusions, and choroidal neovascular membranes (CNVM) associated with high myopia \[[@B22-pharmaceutics-10-00021]\].
Following completion of the phase III MARINA and ANCHOR trials, ranibizumab was approved for the monthly treatment of nAMD and subsequently for PRN treatment. The CATT trial reported that PRN treatment is non-inferior to monthly treatment for nAMD \[[@B6-pharmaceutics-10-00021]\] though pooled data from CATT and IVAN suggest that PRN is inferior to monthly injections. Ran
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1. Introduction {#sec1-materials-13-03111}
===============
The efficiency, stability, and lifetime of any device is strongly dependent on the stability of compounds and alloys that a given device is made of. The attempts to understand the particular state of matter are crucial points of understanding its implications for application in advanced electronic devices. This gain in importance when exotic states of matter are considered. Certain exotic behavior, namely the insulating character of the bulk and metallic character of the surface, was observed in a large group of materials, which were later classified as topological insulators \[[@B1-materials-13-03111],[@B2-materials-13-03111]\]. Among classified topological insulators the Bi~2~Te~3~ compound, considered as good material for thermoelectric \[[@B3-materials-13-03111],[@B4-materials-13-03111]\] and optoelectronic \[[@B5-materials-13-03111],[@B6-materials-13-03111]\] applications, was selected as the subject of this study. Topological insulators (TIs) are a novel type of quantum materials of which the bulk part is a band insulator whereas the surface always exhibits the presence of electronic states carrying electric current \[[@B2-materials-13-03111],[@B7-materials-13-03111]\]. This phase shows a clear bulk energy gap separation like any ordinary insulator or semiconductor, but the difference is in the presence of the gapless edge states (2D) or surface states (3D) which are protected by time-reversal symmetry. The unusual nature of the TI surface states resulted in the widespread interest in the fabrication and characterization of this class of materials. The nature of the TI class of materials resulting from its unique spin nature is gaining in importance when one realizes that TIs properties may entail generation of quasiparticles and electronic states which are not accessible in classic condensed-matter systems. Not surprisingly the topological insulators are considered as promising materials for multiple applications in next generation electronic or spintronic devices \[[@B8-materials-13-03111],[@B9-materials-13-03111]\] as well as for applications in energy conversion such as thermoelectrics \[[@B10-materials-13-03111],[@B11-materials-13-03111]\].
It is well known that defects, strain, and doping influence are important features of the TIs. Coulomb, magnetic, or disorder perturbations can modify the surface states \[[@B12-materials-13-03111],[@B13-materials-13-03111],[@B14-materials-13-03111]\]. Elemental doping and alloying in the TI materials allows for the control of the Fermi level via population with the n-type or p-type carriers which is also a crucial step towards the future junctions of topological insulators that associate two doped TI layers \[[@B15-materials-13-03111],[@B16-materials-13-03111]\]. Among doped TIs, special interest is placed on magnetic dopants, mostly due to the possible interplay between topological magnetic orders, which may lead to the implementation of exotic quantum phenomena, such as the magnetoelectric effect \[[@B17-materials-13-03111]\] and quantum anomalous Hall effect (QAHE) \[[@B18-materials-13-03111],[@B19-materials-13-03111]\], and later on the exploration of its potential device applications. Recent research is focused mostly on TI's electronic structure and their thermoelectric properties and include structures containing transition metals dopants like V, Mn, Cr, Fe, and Cu \[[@B20-materials-13-03111],[@B21-materials-13-03111],[@B22-materials-13-03111],[@B23-materials-13-03111],[@B24-materials-13-03111],[@B25-materials-13-03111]\], and rare earth dopants Gd, Ce, Y, Sm, Dy, and Eu \[[@B8-materials-13-03111],[@B9-materials-13-03111],[@B10-materials-13-03111]\]. Mn and Fe doping of the Bi~2~Te~3~ causes the opening of a small gap at the Dirac point in the surface Dirac cone \[[@B22-materials-13-03111]\]. Further, with the formation of ferromagnetic state only at the surface, a state which leads to opening the gap, has been confirmed for the Mn doped Bi~2~(Te,Se)~3~ as well as for the Cr-doped (Bi,Sb)~2~Te~3~ thin films \[[@B26-materials-13-03111]\]. Bi~2~Te~3~ doped with rare earth is discussed mostly from the point of view of modifying the thermoelectric properties \[[@B27-materials-13-03111],[@B28-materials-13-03111],[@B29-materials-13-03111],[@B30-materials-13-03111],[@B31-materials-13-03111],[@B32-materials-13-03111],[@B33-materials-13-03111]\]. Controlling the distribution of magnetic dopants within the TI's matrix resulting from sample preparation procedures and chemical potentials of the constituent atoms is still challenging and requires in-depth and careful characterizations at the atomic level. This is especially the case when considering nanoscale systems, where already a controlled fabrication, quality control, as well as subsequent preservation of the completed structure or even device requires specific attention.
In our previous paper \[[@B34-materials-13-03111]\], we have studied the multilayered structures of Bi~2~Te~3~ and europium and shown that the interface between the layers is not stable and the reaction that took place at room temperature led to the decomposition of the originally layered structure and formation of new phases. This observation led us to focus on the electronic and crystallographic structures of a simplified structure where europium is doped in the Bi~2~Te~3~ thin film. Europium by itself is quite chemically active, and possesses interesting magnetic properties related to its valence state. It is worth noting that it is reasonable, at a first glance, to assume that europium atoms will partially substitute the bismuth atoms in the Bi~2~Te~3~ matrix as it happens for the Bi~2~Te~3~ doped with Gd \[[@B35-materials-13-03111],[@B36-materials-13-03111]\]. Recently, the homogeneous incorporation of Eu^2+^ into the Bi sites of the Bi~2~Te~3~ lattice was observed \[[@B37-materials-13-03111]\]. Our point of interest was strongly aimed at the electronic structure of the obtained film, especially on the valence state of europium. Europium in alloys and compounds occurs in 2+ and 3+ valence states. Eu^3+^ is non-magnetic (J = 0) while the Eu^2+^, observed for pure Eu and europium in intermetallic alloys and compounds \[[@B38-materials-13-03111],[@B39-materials-13-03111]\], has a large pure spin moment (J = 7/2). A mixed valence state has also been found in many intermetallic compounds. In the presented studies, the response of thin film of Bi~2~Te~3~ to the disorder introduced by Eu dopant is discussed. The studies were performed on thin films with an unprecedented combination of techniques such as photoemission spectroscopy, TOF-SIMS, RHEED, femtosecond spectroscopy, and LC-AFM.
2. Materials and Methods {#sec2-materials-13-03111}
========================
2.1. Materials {#sec2dot1-materials-13-03111}
--------------
The molecular beam epitaxy system was used for the growth of two thin films of Bi~2~Te~3~. The films were grown in accordance with the procedure we developed earlier \[[@B40-materials-13-03111]\] for the Bi~2~Te~3~ monocrystalline films. Freshly cleaved (110) 0.15--0.17 mm thick Muscovite mica substrates (Ted Pella, Inc., Mica grade V1) was used as a substrate for the Bi~2~Te~3~ films. In order to prevent the problem of the sample surface charging resulting from the used substrate, the mica surface was ground using silver paste. The mica was outgassed at 300 °C for 1.5 h under UVH conditions (5 × 10^−9^ mbar) and kept at 120 °C during the deposition of the Bi~2~Te~3~ films. High-purity Bi (99.999% Aldrich Chem. Co., Milwaukee, WI, USA), spectrographically standardized Te ingot (Johnson Matthey Chemicals, London, UK), and europium were evaporated by conventional effusion Knudsen cells (K-cell). The growth rate of each element, controlled by quartz microbalance, was customized to obtain assumed stoichiometry. The total growth rate of the films was about 0.2 QL min^−1^. In order to obtain a uniform distribution of deposited elements, the films were grown in the co-deposition mode. For the europium doped Bi~2~Te~3~ film, the growth rates of elements were adjusted as if Eu atoms were to substitute for bismuth in the Bi~2~Te~3~ crystal lattice. Assumed level of europium dopants was set at the level of about 2--3%. After the deposition process the films were annealed at 100 °C for 18 h. During the deposition process, the vacuum was of about 4 × 10^−9^ mbar for the undoped film and about 9 × 10^−9^ mbar for Bi~
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Introduction {#Sec1}
============
Rhinosinusitis (RS) is a condition that arises from inflammation of the nose and the paranasal sinuses resulting in two or more symptoms including nasal blockage, obstruction, congestion, and nasal discharge \[[@CR1]••, [@CR2]••\]. Additional symptoms may include facial pain and pressure, reduction or loss of smell. During diagnosis, endoscopic signs of polyps, mucopurulent discharge, and mucosal obstruction in the middle meatus and computerized tomography (CT) changes within the ostiomeatal complex may be observed \[[@CR2]••\]. RS can be classified into two major types based on the duration and nature of the disease. Acute rhinosinusitis (ARS) involves the presentation of two or more symptoms for the duration of less than 12 weeks, with the damage to the nasal passage being often reversible. On the other hand, chronic rhinosinusitis (CRS) involves remodeling of the nasal passage due to long-standing inflammation causing persistent symptoms for more than 12 weeks \[[@CR2]••, [@CR3]\]. Regardless of duration, both ARS and CRS can be caused by infection by pathogens or allergies against environmental allergens. However, in most cases, the key trigger of RS is often a viral infection that either initiates or exacerbates the symptoms \[[@CR1]••, [@CR2]••, [@CR4]••\].
Epidemiology of ARS and CRS {#Sec2}
===========================
RS is a very common condition encountered in primary care, accounting for about 15% of the population in Western countries \[[@CR4]••\]. The prevalence of ARS is relatively high with an estimated prevalence rate of 6--15% worldwide according to various epidemiological studies \[[@CR2]••, [@CR5]\]. On the other hand, studies on the epidemiology and prevalence of CRS are less comprehensive compared to ARS. This is due to the fact that CRS encompasses many forms of presentation which confound the study parameters when accounting for their prevalence. A prevalence of 5--15% prevalence is estimated both in the USA and Europe based on compilation of available studies \[[@CR2]••, [@CR6]--[@CR9]\].
Pathophysiology and Mechanism of Action of Viral Infection in ARS and CRS {#Sec3}
=========================================================================
The nature of the disease of RS is highly dependent on the predisposing conditions such as allergic rhinitis, nasal deformity, immune deficiency, and other environmental factors. These underlying conditions can be exacerbated following primary damage caused by viral infections and the secondary damage caused by the host response against the virus, resulting in the pathology of RS and its symptoms. In ARS, the primary and secondary damage does not result in permanent changes to the nasal passage, and the ARS symptoms arising from the acute infection typically subside within 12 weeks. On the other hand, when the host responses triggered by the viral infection cause permanent alteration of the anatomy of the nasal passage such as remodeling of the epithelial layer, long-term effects may present in the form of CRS \[[@CR2]••, [@CR3], [@CR10]\].
As the main cause of ARS is viral, the host immune response that leads to the pathophysiology of RS is mainly antiviral in nature. The antiviral immune response involves nonspecific and specific components that require the coordination between different cell types including neutrophils, macrophages, eosinophils, dendritic cells, epithelial cells, mast cells, natural killer cells, and lymphocytes \[[@CR11]••, [@CR12]•\]. An ideal scenario is when the elicited immune response is timely and culminates in early viral elimination and minimal damage to the host. However, the cascade of inflammation initiated by the epithelial cells normally lead to damage by the infiltrating cells, causing edema, engorgement, fluid extravasation, mucus production, and sinus obstruction in the process, eventually leading to ARS or exacerbating ARS \[[@CR1]••, [@CR2]••\].
The exact roles and mechanisms of respiratory viruses in ARS exacerbation are still under debate. It is often speculated that the T-helper 1 (Th1) response is initiated from the epithelial innate immune response via toll-like receptors 3, 7, and 9 (TLR 3, TLR7, and TLR9) due to the virus infection \[[@CR13], [@CR14]••\]. Depending on the type of viral pathogen, the pathogen-sensing molecules in turn activate the production and secretion of nuclear factor-κB (NF-κB), interferon-β (IFN-β), tumor necrosis factor-α (TNF-α), and interleukins-1β, 6, and 8 (IL-1 β, IL-6, and IL-8), which are potent inducers or recruiters of neutrophils and macrophages \[[@CR4]••, [@CR12]•\]. The initial action of neutrophils against virus-infected cells usually contributes to the early symptoms of an acute respiratory virus infection. Following this, the further secretion of TNF-α and interferon-γ (IFN-γ) increases the recruitment of Th1 cells and cytotoxic T-cells which leads to the clearance of the viral pathogens and viral-infected cells. The process of clearing the virus generates dead epithelial and infiltrating cells which contribute to the pathology of ARS. It also creates an environment suitable for secondary bacterial infections (such as *Staphylococcus aureus* and *Streptococcus pneumoniae*), which represent another factor that exacerbates ARS symptoms initiated by a viral infection \[[@CR11]••, [@CR12]•, [@CR15]\].
On the other hand, CRS pathology is not as straightforward as ARS as the former is associated with multiple factors and disease presentations. However, virus infection is known to contribute to and exacerbate CRS. People suffering from CRS often have predisposing factors that contribute to the inflammatory condition in the nasal passage. One factor is the remodeling of the nasal epithelium due to the extended period of inflammation. During viral infection, the similar inflammatory process causes exacerbation as with ARS. In addition, the destruction of the epithelial layer and macrophage recruitment also induce the production of matrix metalloproteinase-9 (MMP-9), a key remodeling protein that mediates repair of the epithelial and extracellular matrix \[[@CR16]\]. The remodeling of the nasal passage in turn exacerbates the symptoms of CRS even further, as normal epithelial function is further diminished due to possible hyperplasia and replacement of epithelial cells with fibroblasts following respiratory viral infection.
Key Viruses Causing the Pathophysiology of ARS and CRS Exacerbation {#Sec4}
===================================================================
Viruses account for at least 80 to 90% of the ARS occurrence, with rhinovirus (RV), respiratory syncytial virus (RSV), influenza virus, coronavirus (CorV), parainfluenza virus (PIV), adenovirus (AdeV), and enterovirus (EV) playing a major role in ARS exacerbation \[[@CR2]••, [@CR4]••, [@CR9]\]. These viruses induce strong Th1 responses which lead to the pathology of ARS exacerbation. In addition, RV and PIV may contribute further to the exacerbation as their infection upregulates ICAM-1, viral receptor for major type of RV \[[@CR17]--[@CR19]\] (Tan et al., unpublished observation). ICAM-1 is also a signaling protein that activates the migration and infiltration of immune cells to contribute further to the exacerbation and pathophysiology of ARS \[[@CR20]\]. RV and CorV are the most common viruses isolated from adult ARS, accounting for approximately 50% of ARS diagnosis. Geographically, there are also other viruses isolated from patients with ARS, e.g., human bocavirus is frequently isolated from ARS cases in Taiwan \[[@CR21]\].
On the other hand, most of the viruses that exacerbate ARS will also cause similar exacerbation in CRS patients. There is a high rate of viral detection from the nasal wash of CRS patients including viruses commonly found in ARS patients, with RV also being the most prevalent virus in CRS exacerbation \[[@CR22]\]. Human bocavirus and metapneumovirus (hMPV) are also found in the nasal washes of CRS patients during flare ups. Moreover, there are also certain viruses (herpes virus, Epstein-Barr virus, and human cytomegalovirus) found in CRS patients with nasal polyps, and it is currently unclear whether they contribute to exacerbation of CRS \[[@CR23]\]. Due to the multifactorial nature of CRS exacerbation, it is relatively harder to identify key viruses that exacerbate CRS compared to ARS, and thus, a direct association between CRS and viral exacerbation is still unclear. The putative underlying mechanism of viral ARS and CRS exacerbation is summarized in Fig. [1](#Fig1){ref-type="fig"} based on existing literature \[[@CR1]••, [@CR2]••, [@CR11]••, [@CR12]•, [@CR14]••\].Fig. 1Putative underlying mechanisms of viral-induced ARS and CRS exacerbation. The figure shows the chain of events leading to ARS and CRS exacerbation from common respiratory virus infections (rhinovirus and influenza virus). When a respiratory virus infects its respective host cells in the nasal epithelium, the intracellular pathogen sensors TLRs 3, 7, and 9 are activated to initiate an antiviral cellular response via the activation of STAT1/2 and NF-κB transcription factors. These activated transcription factors then induce the expression of interferons (IFNs), interferon-stimulated genes (ISGs), chemokines, and cytokines against the virus. The cascade of intracellular events leads to the recruitment of innate responders such as neutrophils, macrophages, and
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Introduction
============
The emergence of stem cells as a therapeutic for many diseases, disorders and injuries has brought excitement among scientists, clinicians and patients alike regarding the potential treatment of previously untreatable conditions. However, the implementation of many stem cell therapies in patients may still be years away. When considering translating these therapies into patients, there are two principal concerns that must be resolved: I. Can the stem cells efficiently produce the desired therapeutic outcome, albeit tissue replacement or repair, in vitro?; and II. Can the in vitro studies be replicated in vivo, both short- and long-term, with increased confidence? Much of the past research has concentrated on question one, or more appropriately, the philosophy of can we apply the method? However, to recognize stem cells as key factors in the treatment of various ailments, we need to rest assured that we can also answer question two -- Is this a viable treatment approach? These questions are aside from the ethical implications surrounding the field, which ask should we do it.
In any case, stem cells will continue to be researched as a potential treatment for a multitude of diseases and disorders. Considerable progress has been made in addressing the first question stated above -- can we do it. A vast number of tissue types have been generated from both embryonic (ES cells) and adult stem cells. ES cells are pluripotent cells derived from the inner cell mass of the blastocyst, which hold tremendous potential in generating specified tissue types ([@b25-btt-2-699]). However, the potential for immune rejection, together with the possibility of tumor formation has caused their application in humans to proceed with caution ([@b25-btt-2-699]). Adult stem cells tend to be tissue-specific cells with limited differentiation potential compared with ES cells. Adult stem cells are clinically attractive therapies due to their reduced risk of tumorigenesis and ability to expand with relative ease ([@b8-btt-2-699]).
Among the many types of adult stem cells, those resident to the bone marrow (BM), particularly mesenchymal stem cells (MSCs), have gained extensive interest among scientists and clinicians ([@b12-btt-2-699]). MSCs are mesodermal cells primarily resident to the adult BM, which undergo lineage-specific differentiation to generate bone, fat, and cartilage among other tissue types ([@b3-btt-2-699]). MSCs have also been reported to transdifferentiate into defined ectodermal and endodermal tissues in vitro, thus alluding to their inherent plasticity (Choi and Panayi 2001; [@b9-btt-2-699]; [@b14-btt-2-699]; [@b27-btt-2-699]; [@b19-btt-2-699]; [@b21-btt-2-699]; [@b23-btt-2-699]; [@b34-btt-2-699]). MSCs are available for autologous therapies, have a unique ability to bypass immune rejection and are inherently migratory ([@b31-btt-2-699]). These properties of MSCs make them particularly well suited when considering the second question posed earlier -- can in vitro findings be accurately recapitulated in vivo? Whereas tissues derived from ES cells or other types of stem cells may be rejected when transplanted, MSCs offer the potential for allogeneic transplantation and a readily available source of "off-the-shelf" stem cells for personalized therapies.
However, the unique immune properties of MSCs do not guarantee that the cells will produce the desired therapeutic outcome or even that they will not be rejected. In vitro, a MSC's growth conditions can be closely monitored to favor stem cell growth and/or differentiation. In vivo, the transplanted MSCs are exposed to local immune cells and soluble mediators that could influence the cells' behavior, either positively or negatively regarding the desired outcome. This concept of the tissue microenvironment has become a growing concern among researchers, and may be the ultimate factor in deciding whether a stem cell therapy succeeds or fails ([@b18-btt-2-699]; [@b37-btt-2-699]; [@b17-btt-2-699]; [@b29-btt-2-699]).
A prototypical example of a tissue microenvironment affecting stem cell behavior is observed among hematopoietic stem cells (HSCs) and their niche within the BM. HSCs are relatively quiescent cells located close to the BM endosteum at relatively low oxygen concentration ([@b18-btt-2-699]). As HSCs differentiate, the maturing immune cells migrate towards the central sinus of the BM under progressively higher oxygen concentrations ([@b18-btt-2-699]). The change in oxygen is a key determinant in the maturation of the immune cells before they leave the BM and migrate into the peripheral circulation ([@b18-btt-2-699]). In contrast, MSCs are located close to trabecular bone near the central sinus of the BM ([@b3-btt-2-699]). As MSCs migrate towards the endosteum under progressively lower concentrations of oxygen, the stem cells differentiate into stromal fibroblasts, which form the principal support structure for immune cell maturation ([@b3-btt-2-699]).
This example demonstrates that local microenvironmental changes in variables such as oxygen concentration can drastically affect the behavior of MSCs. Since MSCs have been shown to generate a vast number of tissues, they have clinical implications in a wide array of diseases and disorders. Among possible transplantation sites are tissues such as cardiac, neural, pancreatic and bone. Each tissue provides a unique local microenvironment that can affect the success of the therapy. The problem facing researchers is accurately developing in vitro models to recapitulate the tissue microenvironment so that cellular behavior can be observed prior to transplantation. This is no easy task considering the dynamic nature of the microenvironment.
Transplantation of MSCs alone will generate a local immune response and disrupt homeostasis within the tissue milieu by causing release of inflammatory mediators, such as cytokines. The anatomy of the BM is such that MSCs are in direct interaction with immune cells and form synapse-like structures with innervating nerve fibers ([@b3-btt-2-699]). MSCs express receptors for many cytokines and neurotransmitters, thus demonstrating their potential to respond to local microenvironmental changes ([@b20-btt-2-699]). Excessive cytokine release within the transplantation site could lead to the production of other soluble factors by the MSCs themselves. If these factors are immunoreactive, then other immune cells could infiltrate the tissue and cause an exacerbated immune response, rejection of the transplant or differentiation of the MSCs ([Figure 1A](#f1-btt-2-699){ref-type="fig"}). On the other hand, MSCs have been shown to be a potent source of trophic factors ([@b29-btt-2-699]). These findings indicate that MSCs could also be used to aid normal tissue repair, perhaps even more so than in cell replacement. Whether transplanted MSCs cause an immune insult or help repair injured tissues may be difficult to determine unless appropriate models are developed to better predict the outcome. However, if MSCs are found to negatively impact the host microenvironment through exposure to soluble mediators, there are still potential methods to develop effective therapeutics. When considering the example presented in [Figure 1A](#f1-btt-2-699){ref-type="fig"}, inclusion of specific cytokine receptor antagonists or inhibitors could suppress the untoward effects of the host microenvironment on the transplanted MSCs, thus leading to defined therapeutic outcomes ([Figure 1B](#f1-btt-2-699){ref-type="fig"}). Throughout the remainder of this review, we will address the feasibility of using similar pharmacologic approaches in MSC transplants, while focusing on three ubiquitous microenvironmental factors: IL-1α/β, TNFα, and SDF-1α. Specifically, we will examine how receptor antagonists or inhibitors to these factors, whether federally approved or in development, may limit the potential negative influences of the tissue microenvironment.
Interleukin-1α/β
================
IL-1α and IL-1β are members of the IL-1 superfamily of cytokines. These pro-inflammatory mediators are primarily synthesized by macrophages, monocytes and dendritic cells, and are responsible for immune defense against infection ([Table 1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). IL-1α and IL-1β are also key regulators of hematopoesis and the inflammatory process ([Table 1](#t1-btt-2-699){ref-type="table"}) ([@b13-btt-2-699]). Both cytokines are found throughout the body, thus they are expected to be present within the local microenvironment of most tissues.
Our laboratory has previously demonstrated that MSCs express IL-1RI, which is the principal receptor for both IL-1α and IL-1β ([@b20-btt-2-699]). Interestingly, membrane expression of the receptor was maintained throughout the course of transdifferentiation of MSCs into functional neurons ([@b20-btt-2-699]). If IL-1α or IL-1β were found to have negative effects on MSCs, then these effects may also be seen on transplantable tissues differentiated from MSCs. These results have implications regarding the ideal stage of stem cell implantation, whether undifferentiated, partly differentiated or fully differentiated.
IL-1α was found to alter the behavior of undifferentiated MSCs and neurons differentiated from MSCs ([@b20-btt-2-699]). Specifically, stimulation of MSCs with IL-1α caused production of the neurotransmitter, substance P (SP), by undifferentiated and differentiated cells ([@b20-btt-2-699]). Similar effects were not observed in cells stimulated with IL-1β ([@b20-btt-2-699]). SP has involvement in various physiological functions, such as the perception of pain
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INTRODUCTION {#sec1-1}
============
Hospital staffs are the most important group of health care providers. As a result of their continuous hard work, they are frequently faced with physical and psychological health problems. If these problems linger on, disappointment and boredom will settle, which in turn will lower the quality of health care services. Health care quality is highly dependent on clinical and professional ethical principles, which are under the influence of religious beliefs. An influential factor helping preservation of psychological stability is to believe in and to rely on God; closeness to God and performing religious duties brings about peace. Spirituality is a human dimension that combines our existence with concepts such as human nature, sacred mental experience, tendency to know more, elevation toward Good, and finding a meaning to life. It has also been defined as a number of values, attitudes, and hopes related to the superior existence and thus guiding us throughout life.\[[@ref1]\]
The relationship between the mental health and spirituality has received much attention recently. Research shows spirituality is highly influential in physical and mental health preservation. Many researchers have concluded that spirituality has an immense effect on mental health.\[[@ref2][@ref3]\]
According to WHO definition, health is total physical, mental, and social existential well-being, and mental health is the ability to establish harmonious communication with others, modify surroundings, and resolve conflicts.\[[@ref4]\] Behaviors such as reliance on God, praying, and going on pilgrimage can promote solace and mental health through peace and developing hope and positive thinking. Religious beliefs increase resistance against calamities, and thus help preserve physical and mental health, prevent infliction of diseases, and finally promote hopefulness.\[[@ref5][@ref6][@ref7][@ref8][@ref9]\]
According to the cognitive-emotional-religious theory, man cannot comprehend the meaning of life without religious beliefs. This theory discusses religious beliefs and God, existence, and man, and proposes that these beliefs can be applied to treat psychological problems.\[[@ref10]\] From a clinical viewpoint, lack of appraisal of religious spiritual dimensions sets obstacles in the way of (1) acknowledging significance of these dimensions for health and (2) learning about the mechanisms involved.\[[@ref11]\] Islam as an ideology presents an impeccable and health-preserving lifestyle; its orders cover a wide range of life aspects, including personal and social ethics, interpersonal relations, and physical and mental health.\[[@ref11]\] Some studies indicate a correlation between reliance on God and high self-esteem,\[[@ref12]\] lower levels of depression, more mature social behavior,\[[@ref13][@ref14]\] and better mental predisposition.\[[@ref15]\]
Kendler *et al*.\[[@ref16]\] studied a number of spiritual/religious themes and identified those factors which had a significant inverse relationship with lack of mental health. Hill *et al*. found that true religious belief acts as a motivational drive and is closely related to physical and mental health.\[[@ref17]\] Koenig *et al*. and Smith *et al*. also showed that health is a causal result of the relationship between higher levels of spirituality and reliance on God and trust in God enables people to tolerate hardships of life much better since they believe in God and entrust their lives into his hands.\[[@ref18][@ref19][@ref20]\] Also, James states that the foundation of religions, that is based on spiritual relation with God, meaningfulness of the whole universe, and meaningfulness of life is the essence of spirituality.\[[@ref21]\]
Modern analyses have reported that there is a significant positive relationship between religious duties and concerns and health and life time.\[[@ref22][@ref23]\] Koenig *et al*. also found that spiritual/religious beliefs are important predictors of youth depression.\[[@ref18]\] Bahrami and Tashak\[[@ref24]\] reported a significant positive relationship between religious orientation and mental health promotion and decrease in psychological disorders. The study done by Omran-Nasab\[[@ref25]\] also indicated that there was a significant correlation between religious beliefs and mental health. Cohen, Yoon, and Johnston in a study on 168 patients with various physical disorders showed that there is generally a positive relationship between positive spiritual tolerance and mental health and a negative relationship between negative spiritual tolerance and mental health, but there was no relationship between more personal religious duties such as praying and mental health.\[[@ref26]\] Cohen and Hall also studied 1,000 aged people and found that there was a relationship between some religious beliefs (fear of God, fear of death, and belief in life after death) and feeling of well-being and it is more significant in Protestants than in Catholics and Jews.\[[@ref27]\] Krause also suggested that the relationship between calamities of life and depression symptoms in aged people reduces when they believe God knows better when to respond to their prayers.\[[@ref28]\] Keyes and Reitzes\[[@ref29]\] emphasize the importance of the impact of religious identity on dignity and depression symptoms in retired workers. Mazidi and Ostovar\[[@ref30]\] concluded that both Islam and Christianity affect mental health of Iranian youth positively.
The findings of Jang *et al*., Hills *et al*., and Doolittle *et al*.\[[@ref2][@ref3][@ref31]\] showed that spirituality is beneficial to physical and mental health. Some researcher found a close relationship between religiousness and elevation of soul and health.\[[@ref32][@ref33]\] In other words, positive tendencies such as feeling of happiness, joyousness, and hope for future are related to spirituality.\[[@ref23][@ref34][@ref35]\]
The favorable effect of positive constructs such as optimism and hope for future on physical and mental health has been confirmed by various studies.\[[@ref36]\] Snyder\'s theory of hope made researcher devote a lot of research to the relationship between hope and health.\[[@ref37][@ref38]\] Snyder believes hope is not a passive drive emerging only in dark moments of life, but a cognitive process through which one activity pursues goals. He states that hope is (1) a goal-determining process through which people (2) build up strategies to achieve goals and (3) are motivated enough to apply those strategies. These three components are known as goals, pathway thinking, and agency thinking.\[[@ref39]\] Thus, those staffs who are hopeful develops enough motivation to initiate and perform hard tasks, and emphasizing their capabilities, move toward their grand goals. Snyder also believes there is a positive relationship between positive emotions and purposefulness in life.\[[@ref40]\] Other studies have also indicated the relationship between hope and positive emotions is a positive relationship, and it has a negative relation with depression, anxiety, and negative emotions in general.\[[@ref40]\]
Similarly, Hezarjaribi *et al*.,\[[@ref41]\] believe that there is a maximum significant relationship between hope for future and joyousness, and with the increase of the feeling of joy, hope for future is also enhanced. Many researchers such as Bandloo *et al*. believe important factors, including personality traits, cognitive components, and religious attitudes pave the way for feeling of joyousness.\[[@ref30]\] They have also reported a direct relationship between genetic, personality, cognitive, and religious factors, and anxiety on one hand and joyousness and hope for future on the other.\[[@ref40]\] In other words, religious attitudes and practices definitely help people find a meaning to life. Once hospital staff finds a meaningful life, hope for God\'s support, receive social and religious support, and feel belonging to a holy and graceful existence, they can survive hardships and calamities of life and maintain their mental health successfully; these people can work more fruitfully and provide better services.\[[@ref3]\]
Thus, considering how crucial the job is, and also regarding the lack of a research on spiritual variables in Iran, this study was designed to answer this research question: Is there a relationship between religious/spiritual dimensions of one\'s character and his/her mental health and his/her hope for future?
MATERIALS AND METHODS {#sec1-2}
=====================
This was a correlational study. The statistical population included all state hospital\'s staffs in Shiraz-who were selected randomly using the sample size formula for correlational studies and matched with Cohen\'s table of sample size.\[[@ref42]\] Out of the 250 selected participants, 212 questionnaires were returned (85.2%). Females (156 people) formed 73.6% and males (56 people) formed 26.4% of the sample size. In six hospitals in the study, 95 staff (44.8%) were single, 112 staff (52.8%) were married, and 5 (2.4%) did not report their marital status. Regarding the educational level, 1 (0.5%) had not finished high school, 12 (5.7%) had high school diplomas, 19 (0.9%) had associate degrees, 160 (75.5%) had bachelor degrees, and 13 (6.1%) abstained from reporting. Ethical considerations were observed by reassuring subjects that their personal information would be regarded strictly confidential and by offering them to feel free to or not to answer the questionnaires.
Data collection was done through using three questionnaires:
Mental health questionnaire: the 12-item Goldberg and William\'s mental health questionnaire was one of the tools used in this study. The 4-point Likert scale (0 = always to 3 = Never) was applied. The total score was 36. The highest score indicated the lowest level of mental health. Goldberg *et al*.,\[[@ref43]\] have reported that the reliability of their test is 0.78 and Pearson correlation coefficient for 3 factors, and the total score is 0.57 if *P* \< 0.000. Ebadi *et al*.,\[[@ref44]\] applying the tool on a research sample of 18-25-year old Iranians, reported an internal consistency of 0.81 for the tool. They also confirmed the validity
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"pile_set_name": "PubMed Central"
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INTRODUCTION
============
Patient expect for improved esthetics has driven the advancement of ceramic for use with fixed partial prostheses.[@B1] Many clinical studies demonstrate excellent long-term success of ceramic restorations. In recent year, strong ceramic cores unioning esthetic veneering porcelains have become popular as all ceramic restorations which have compensated the brittleness of porcelain and unesthetic metal substructure. The clinical success of all ceramic prosthesis depends on a number of factors, such as composition of the ceramic material and the cementation procedure.[@B2] Hence, bonding to ceramic requires strict attention to detail for optimal clinical outcomes.[@B3]
A vital importance is due to the adhesive strength and durability of the complex formed between the three different components: the resin cement, the ceramic surface and the tooth surface especially in anterior laminate veneers.[@B4][@B5] In some cases, significant amounts of exposed dentin is usually unavoidable during the preparation of anterior teeth,[@B1][@B6] the protection is required during the period between preparation and cementation for prevention of post-operative sensitivity and bacterial invasion.[@B2] They suggested the application of the dentin bonding agent immediately after tooth preparation. This new technique of dentin bonding agent application prevents the bacterial invasion and dentin sensitivity during the provisional states, and the technique is concerned with increased bond strength *in vitro*.[@B7] Magne (2005) also recommended application of dentin adhesive to the freshly cut dentin when a significant area of dentin has been exposed during preparation for indirect restoration. The dentin could be sealed immediately after tooth preparation with IDS prior to impression taking.[@B3]
IDS is the application of dentin bonding agent to freshly cut dentin when it is exposed during tooth preparation for indirect restorations. IDS protects the dentin against bacterial leakage and tooth sensitivity before cementation of final prosthesis. An advanced protocol, IDS is devised to address the challenges of preparation, provisionalization, and the final cementation of indirect restorative procedures. The general protocol of IDS includes the use of filled three-step total etch systems, two-step total-etch systems, and two-step self-etch systems incorporating low elastic liners.[@B8]
Especially prosthodontic patients, complex inlay, onlay and veneer situations may require longer periods with provisional restoration until the final ceramic restoration is delivered.[@B9] The provisional restoration must protect the pulp from thermal changes as well as from invasive microorganisms in the mouth. However, it is difficult to takestable and sealed provisionals as they detached easily during temporary states, allowing microleakage of bacteria and sensitivity.[@B10] In addition, the bond strength between resin composite and a pre-treated ceramic restoration has been described to be negatively affected by external factors such as thermocycling, fatigue and water sorption.[@B11] Also, cement breakdown can bring about results as microleakage, marginal discoloration, debonding, secondary caries, pulpal irritation, and decreased fracture load.[@B12]
We pose the clinical assumption that the dentin surface after IDS procedure is exposed to the oral environment and factors that could result in fatigue may influence the physical and mechanical properties of dentin bond strength. There are no studies available concerning the dentin bond strength on exposed sealed dentin with a long delay under thermocycling.
The aim of this study was to evaluate the effect of IDS on dentin bond strength of lithium disilicate ceramic (IPS Empress II, Ivoclar) under various thermocycling periods of 1, 2, 7, 14 days.
MATERIALS AND METHODS
=====================
For tooth preparation, freshly extracted sound human mandibular third molars stored in solution saturated with thymol were used. The midcoronal dentin surfaces were created after the removal of the occlusal half of the crown using a low-speed diamond saw (Isomet: Buehler Ltd., Lake Bluff, IL, USA). Each specimen was individually secured to a silicon mold (diameter: 25 mm, height: 15 mm) and self-curing polyester resin (CH-304, Aekyung Chemical Co., Ltd., Seoul, Korea) was poured to create a resin-embedded specimen block. The surface was wet polished to create hybrid layer with 320, 400 and 600 grit SiC abrasive papers. Total 50 specimens were prepared. The experimental groups were divided into four groups (10 specimens per group) according to the thermocycling period (1, 4, 7, 14 days). The control group consisted of 10 specimens without thermocycling.
A total of 50 ceramic discs (diameter: 4 mm, height: 2 mm) of lithium disilicate ceramic (IPS Empress II ingot, shade A1, Ivoclar Vivadent AG, Schaan, Liechtenstein) were fabricated. The surfaces were etched with 9.5% hydrofluoric acid (HF) (Porcelain etchant, Bisco Inc., Schaumburg, IL, USA) for 90 seconds and one layer of silane coupling agent (Porcelain primer, Bisco Inc., Schaumburg, IL, USA) was applied and allowed to air dry for thirty seconds at room temperature. The surface treatments of the ceramic discs were according to the manufacturer\'s instruction. The materials used in this study are shown in [Table 1](#T1){ref-type="table"}.
For immediate dentin sealing procedure, the dentin surface was etched with 32% phosphoric acid (H~3~PO~4~) for 15 seconds, followed by rinsing with distilled water and air drying for 5 seconds. Then, five coat of dentin bonding primer (3-step etch-rinse adhesive system; All Bond II, Bisco Inc., Shaumburg, IL, USA) with a light brushing motion for 30 seconds was applied to the surface and air thinning for 3 seconds. After one coat of Pre-bond resin, the layer was light polymerized (VIP Junior light curing unit, Bisco Inc., Schaumburg, IL, USA) for 20 seconds at 600mW/cm^2^. Ceramic disc was attached with dual-cured resin cement (Duo-link, Bisco Inc., Shaumburg, IL, USA) on IDS treated dentin surface.
After then, the specimens of experimental groups were submitted to 1500 thermal cycles between 5℃ and 55℃ (dwell time of 30 seconds) in a thermal cycling machine (Thermocycling testing machine, CDM-127, CDM, Korea) for 1, 2, 7, 14 day (group 1d, 2d, 7d, 14d) ([Table 2](#T2){ref-type="table"}).[@B13][@B14] Control group (0d) was not thermocycled and the specimens were stored for 14 days in deionized water at room temperature.
Ceramic discs after surface treatment with ceramic etchant and silane coupling agent were cemented to the surfaces of specimens with or without thermocycling with dual-cured resin cement (Duo-link, Bisco Inc., Shaumburg, IL, USA) ([Fig. 1](#F1){ref-type="fig"}). The excess cement was removed with a disposable microbrush, followed by light curing (VIP Junior light curing unit, Bisco Inc., Schaumburg, IL, USA) with a light intensity of 600 mW/cm^2^. The light was applied for 100 seconds (20 seconds each from occusal, buccal, lingual, mesial and distal aspects).
The extrusion shear bond strength test (SBST)[@B15][@B16] represents a confinement situation for the composite, and the resulting interface would be more likely to present defects that resemble clinical conditions. After the cementation procedures, the specimens of control group and thermocycled groups were stored in distilled water for 24 hours at 37℃. The SBST was conducted in a universal testing machine (Instron, Shimadzu, Japan) at a crosshead speed of 1.0 mm/min.
For the scanning electron microscopy (SEM) analysis, the dentin surfaces of specimens from each group were air dried and gold coated with a sputter coater (IB-3 ION coater, Elko Co., Tokyo, Japan) and examined under scanning electron microscope (Scanning Electron Microscopy, S-2300, Hitachi, Co., Ltd., Tokyo, Japan). The specimens were vertically sectioned using a low-speed diamond saw under water lubrication to observe the interface of dentinresin-ceramic.
Bond strength values were analyzed using one-way analysis of variance and followed by Tukey\'s HSD multiple comparison tests. Statistical analysis was conducted using SAS software version 9.1 for windows (SAS Institute Inc., Cary, NC, USA). The significance level for all statistical tests was set at 0.05.
RESULTS
=======
The values of the shear bond strength recorded in debonding force (DF) for the specimens in all groups. The mean values and standard deviation of shear bond strength are shown in [Fig. 2](#F2){ref-type="fig"}. Two thermocycled groups had lower mean values than the control group. The analysis of variance indicated no statistically significant difference in shear bond strength between all groups, however the mean value started to decrease in group 7d, and group 14d showed the lowest mean bond strength in all groups.
SEM micrograph of fractured surface of dentin side after SBST (original magnification ×100) are shown in [Fig. 3](#F3){ref-type="fig"}. SEM analysis of fractured surface in the control group, thermocycled 1d and
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INTRODUCTION
============
For decades, high ligation and saphenous vein stripping with phlebectomy of varices were the gold standard procedures in the treatment of the incompetence of the great saphenous vein (GSV) \[[@b1-vsi-30-102],[@b2-vsi-30-102]\]. An alternative method was cryostripping which is less invasive than traditional stripping \[[@b3-vsi-30-102]\]. Because of the need for minimal invasiveness, the endovenous procedures spread worldwide in the treatment of the incompetence of the saphenous trunks in the last decade \[[@b4-vsi-30-102],[@b5-vsi-30-102]\].
At the end of the last century, Milleret and Le Pivert \[[@b6-vsi-30-102]\] described cryosclerosis as a safe, efficient and feasible endovenous cryoablation technique to treat the incompetence of the GSV. The segmental freezing of the saphenous trunk was performed with a flexible cryoprobe by inguinal approach. In 1987, Le Pivert \[[@b7-vsi-30-102]\] published the experiences of 350 patients treated by this method. In 1994, Garde \[[@b8-vsi-30-102]\] concluded that the technique was safe and efficient based on the results of a randomized controlled trial on 800 patients. We have completed a prospective non-randomized trial to compare cryosclerosis and classical stripping, and regarding the short term results, this endovenous procedure seems to be effective. The electron microscopic examination proved that immediate ultrastructural changes were formed in the GSV \[[@b9-vsi-30-102]\].
CASE
====
We present the histological changes of the GSV at 2 years after cryoablation. The examined vein piece was harvested from the proximal femoral part of the GSV from a 31-year-old female patient who underwent cryosclerosis 23 months ago. This procedure was performed by inguinal approach, after high ligation a cryoprobe was introduced into the GSV and segmental freezing of the vessel wall was carried out. The patient was admitted again to our department of surgery because of recurrent varicosity that caused pain and limb swelling at the end of the day. Ultrasonography showed the recanalization of the GSV. Saphenofemoral reflux and an incompetent posteromedial perforating vein were detected as possible reasons. High ligation and cryostripping with phlebectomy of varices were performed under spinal anesthesia. The GSV was very crusted and segmental strictures were found when the cryoprobe was introduced into the vessel lumen. A small vein piece was harvested from the proximal femoral part of the GSV for histological examination. The patient healed without any postoperative complications.
Hematoxyllin-eosin, Picro-sirius Red, van Gieson and immunohistochemical stains (CD34, smooth muscle actin) were performed. The anatomical structure of the vein couldn't be recognized microscopically. The wall was collagenized and padded by endothelium that was caused by progressive fibrosis ([Fig. 1](#f1-vsi-30-102){ref-type="fig"}). The patient consented to this trial and agreed to apply her records.
DISCUSSION
==========
Generally, the role of the endovenous ablation techniques (endovenous laser ablation \[EVLA\], radiofrequency ablation \[RFA\], ultrasound guided foam sclerotherapy, mechanochemical ablation, steam ablation) has increased in the treatment of the incompetence of the GSV. These are widely studied, safe and efficient procedures. The most frequently used methods are EVLA and RFA. The long term results are favorable, but the recanalization of the GSV could be observed some years after the treatment that may cause recurrent varicosity with symptoms \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b5-vsi-30-102]\].
In this short report, the presented method is cryosclerosis, which is the endovenous cryoablation of the GSV. This procedure is not widely known. The reported patient had recurrent varicosity with symptoms. Cryosclerosis was ineffective 2 years after the treatment; however, segmental stenoses of the GSV were observed during the operation. The incompetence of the saphenofemoral junction and the posteromedial perforating vein could cause the recurrent disease. The role of tumescent fluids during cryosclerosis is not clear and it is not routinely used, but it might be useful since by decreasing the volume of the intraluminal blood, vein wall destruction can be achieved more efficiently \[[@b10-vsi-30-102],[@b11-vsi-30-102]\]. A prospective trial is necessary to assess the effect of injecting warming liquid around the GSV before cryosclerosis.
The histomorphological effects of the endovenous ablation methods have been previously investigated, mostly from animal experiments \[[@b12-vsi-30-102]--[@b14-vsi-30-102]\]. Their long term effects have not been described yet. It's typical for all procedures that the vein wall is destroyed by thermal, mechanical or chemical effects \[[@b1-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102],[@b9-vsi-30-102],[@b10-vsi-30-102]\]. Heger et al. \[[@b12-vsi-30-102]\] summarized in their review on EVLA that the heat induced thrombosis and the thermal damage in the vein wall results in inflammation and activation of the immune system (fibroblast migration). Finally, the vein wall becomes collagenized and results in occlusion or stenosis. The presented histomorphological examination showed that the whole vein treated by cryosclerosis underwent typical remodeling, thus the anatomical structure couldn't be recognized. The wall became crusted because of collagen deposition by fibroblast activity. However, the GSV wasn't occluded yet segmental strictures were observed ([Fig. 1](#f1-vsi-30-102){ref-type="fig"}).
Cryosclerosis seems to have the same effect as the other familiar endovenous ablation techniques \[[@b1-vsi-30-102],[@b2-vsi-30-102],[@b4-vsi-30-102],[@b6-vsi-30-102]--[@b9-vsi-30-102]\]. The reason for recanalization of the GSV is not clear, with recurrent incompetence of the saphenofemoral junction and the posteromedial perforating vein being one of the reasons, but it is also possible that the remodeling process of the GSV was not effective enough. The pathogenesis of recanalization after the endovenous procedures and recurrent varicosity has been studied, but more evidence is needed \[[@b5-vsi-30-102]\].
Considering that this article is only a case report, it has serious limitations. More cases are necessary to assess the real effect of cryosclerosis on the GSV, but the procedure seems to be efficient in obliterative remodeling of the vein.
Conflict of interest: None.
{#f1-vsi-30-102}
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Introduction {#S1}
============
Porcine circovirus has long harmed the sound development of pig husbandry, and especially the PCV3 that has appeared in recent years has caused tremendous economic losses for pig husbandry ([@B17]; [@B21]). Current research indicates that PCV is mainly divided into three genotypes ([@B8]). PCV1 is generally considered non-pathogenic ([@B27]). PCV2 is widely prevalent worldwide, previous studies have shown that PCV2 is the main pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) and swine dermatitis and nephrotic syndrome (PDNS) ([@B1]; [@B30]). However, in recent years, some researchers have detected PCV3 from PDNS piglets ([@B22]; [@B13]). Studies have shown that PCV3 and PCV2 are mixed infections and have become popular in many countries. It has been reported that PCV3 may cause reproduction disorder in sows and PDNS in adult pigs ([@B22]). Similar to PCV2, PCV3 is often mixed with PRRSV, PCV2 ([@B2]). However, unlike PCV2, there are currently commercial vaccines to prevent PCV2 infection ([@B23]), and there is still a shortage of vaccines and related drugs to prevent PCV3.
Obtaining standard virus strains is the basis for the development of PCV3 vaccines and related biological products. However, no experimental report on the successful isolation of PCV3 has been reported. With the development of genetic engineering technology, a variety of virus strains have been constructed through reverse genetics ([@B34]; [@B10]). With the continuous analysis of the genome structure and function of PCV2 and PCV3, researchers have obtained infectious clones by constructing eukaryotic expression vectors of PCV2 and PCV3 ([@B28]; [@B11]). According to the genome structure of PCV, the porcine circovirus genome is a single-stranded negative-stranded DNA, the virions are only 14--17 nm, and there is no capsule on the surface of the virus capsid ([@B18]; [@B32]). The size of the PCV1 and PCV2 genome research surface is 1767--1768 bp and contains 11 open reading frames (ORFs), of which ORF1 is a virus replication--related protein (Rep) and is a necessary element of virus replication ([@B3]). ORF2 encodes the viral capsid protein (Cap) ([@B20]) and is commonly used in the study of subunit vaccines and diagnostic reagents ([@B16]). Unlike the genomes of PCV1 and PCV2, the full length of the PCV3 genome is 2000 bp ([@B24]). Some researchers have predicted the PCV3 genomic DNA, and currently they have predicted a total of three ORFs ([@B22]). One Rep protein composed of 297 amino acids was encoded by ORF1 ([@B33]). Besides, another Cap protein covering 214 amino acids by ORF2 was replicated in the opposite direction and a protein with an unknown function and containing 231 amino acids by ORF3 ([@B5]). Whether PCV3 has other open reading frames and their functions still needs further study.
The above research is to construct infectious clones under the premise of grasping the genome structure of the virus. However, for some newly discovered circular DNA viruses, the genome structure and function are undefined yet, so it is hard to construct infectious clones with the application of the eukaryotic expression vector. This study intends to use the biological characteristics of PCV3 circular DNA to construct PCV3 infectious clones and construct a Kunming mouse infection model without the help of exogenous expression vectors. The results showed that the PCV3 strain can infect the myocardium and lung of mice. This study will provide a method for the construction of infectious clones of other circular DNA viruses and lay a foundation for the study of the pathogenic mechanism of PCV3.
Materials and Methods {#S2}
=====================
Cells and Cultures {#S2.SS1}
------------------
The 3D4/21 cell line (iCell Bioscience Inc., Shanghai, China) was cultivated in Dulbecco's minimum essential medium (MEM, Gibco) supplemented by 10% fetal bovine serum at 37°C in a humidified 5% CO~2~ incubator. The cloning and construction of recombinant expression plasmids was carried out in *E. coli* strain DH5α cells (Takara Bio, Dalian, China). The prokaryotic expression vector pET-32a (+) and *E. coli* BL21 (DE3) cells were harvested from the stocks of our laboratory. The SP2/0 cells were also acquired from the stocks.
Viral Gene and Primer Synthesis {#S2.SS2}
-------------------------------
The PCV3 gene sequence (GenBank accession number: [MH107162.1](MH107162.1)) was taken to form a loop on Snap-Gene software, and the position of the unique restriction site was adopted to open the sequence, termed as the rearranged linear PCV3 gene sequence. Since the PCV3 is circular, the cyclization of the original linear DNA sequence was considered, the *Hin*dIII restriction site was recruited as the reopening site. When the opening process was completed, the *Hin*dIII restriction site was added to the end of the linear sequence. The newly rearranged sequence was synthesized by Sangon (Shanghai, China). [Figure 1](#F1){ref-type="fig"} illustrates the gene pattern of the novel PCV3 and presents the design pattern of the circular PCV3 amplification primer and detection primer. Primers listed in [Table 1](#T1){ref-type="table"} were designed in accordance with the rearranged PCV3 gene sequence and PCV3 Cap gene sequence; the designed primers were synthesized by Sangon (Shanghai, China).
######
Primers used for the construction and identification of the recombinant virus.
Primer Sequence
---------------------------------------------------------------------
PCV3 M1: 5′-CCC[AAGCTT]{.ul}GTGCGGATGCGGCTGCGCG-3′;
PCV3 L2: 5′- CCC[AAGCTT]{.ul}CCCGCGTTTTCCCACAACC-3′;
PCV3 L3: 5′- GGTTGTGGGAAAACGCGGG-3′;
PCV3 M2: 5′-CGCGCAGCCGCATCCGCACAAGCTT-3′;
PCV3 Cap *Bam*HI: 5′-CGC[GGATCC]{.ul}ATGAGACACAGAGCTATATTCAGAA-3′;
PCV3 Cap *Hin*dIII: 5′-CCC[AAGCTT]{.ul}TTAGAGAACGGACTTGTAACGAATC-3′
The underlined sequences represent restriction enzyme sites introduced. PCV3 M1, PCV3 L2: PCV3 complete genome amplification primers; PCV3 L3 is a reverse complementary sequence of PCV3 L2; PCV3 M2 is the nucleotide sequence next to the primer binding site of PCV3 L2; PCV3 Cap BamHI, PCV3 Cap HindIII: The design of the primer is the reverse complement of the negative chain of PCV3 with the Cap gene sequence, and specific primers were then designed according to the reverse complementary DNA sequence.
{#F1}
Amplification and Cyclization of the PCV3 Genome {#S2.SS3}
------------------------------------------------
To amplify the newly synthesized rearranged PCV3 gene, PCV3 M1, PCV3 L2 primers, and KFX-101 high fidelity enzymes (TOYOBO, Japan) were adopted. The final volume of reaction fluid reached 100 μL. The PCR cycling conditions included: pre-denaturation at 94°C for 2 min, 30 cycles at 98°C for 10 s, 58°C for 30 s, and 68°C for 2 min, as well as the final extension for 10 min at 68°C. PCR products were preserved at 16°C for subsequent experiments.
The amplified PCR products of rearranged PCV3 gene were purified following the instructions of the DNA Purification Kit (Takara Bio, Dalian, China). Subsequently, the purified PCR product was digested by *Hin*dIII restriction endonuclease (NEB, Beijing, China) and then incubated in a water bath at 37°C for 5 h. Then, the digested products of *Hin*dIII restriction endonuclease were purified following the manufacturer's instructions (Takara Bio, Dalian, China). Next, the purified DNA fragments were connected based on T4 DNA ligase instructions (NEB, Beijing, China) at 16°C for 12 h. Last, the cyclized PCV3 DNA was harvested.
Transfection of Cyclized PCV3 DNA {#S2.SS4}
---------------------------------
The cyclized PCV3 DNA was transfected into 3D4/21 cells (60% confluency)
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Background {#Sec1}
==========
The development of resistance to cytotoxic agents represents a major concern in cancer chemotherapy. Multi-drug resistance (MDR) is associated with over-expression of transmembrane glycoprotein (P-gp) which functions as a drug efflux pump, reducing the intracellular levels of cytotoxic drugs (Juranka et al. [@CR29]). P-gp belongs to the ATP-binding cassette (ABC) transport proteins, which also include the multi-drug resistance associated protein 1 (MRP1) (Shen et al. [@CR63]; Biedler and Spengler [@CR4]; Efferth et al. [@CR15]), or the breast cancer resistance protein (BCRP/ABCG2) (Shen et al. [@CR63]). The oncogene epidermal growth factor receptor (EGFR) (Biedler and Spengler [@CR4]; Efferth et al. [@CR15], [@CR16]) and the deletions or inactivation of tumor suppressor gene p53 (el-Deiry [@CR17]) have also been involved in MDR mechanism of cancer cells. Overcoming this resistance requires a permanent search of new antineoplastic agents. In the past, natural products from plant kingdom have revealed a high potential as cytotoxic drug reservoir (Kuete and Efferth [@CR33]). According to the World Health Organization, about 80 % of the population of developing countries relies on traditional medicines, mostly plant drugs, for their primary health care needs (FAO [@CR20]). It has also been reported that modern pharmacopoeia still contain at least 25 % drugs derived from plants and many others which are synthetic analogues (FAO [@CR20]). Therefore, fighting cancers with botanicals represents a very promising alternative, especially regarding the diversity of plant's secondary metabolites. African flora has previously been found to be very fruitful in the search of antiproliferative molecules. Many compounds including xanthones: 8-hydroxycudraxanthone G, morusignin I, cudraxanthone I (Kuete et al. [@CR38]), and xanthone V1 (Kuete et al. [@CR35]), benzophenones: guttiferone E and isogarcinol (Kuete et al. [@CR39]), quinone: 2-acetylfuro-1,4-naphthoquinone (Kuete et al. [@CR35]), flavonoids: 4-hydroxylonchocarpin, 6,8-diprenyleriodictyol (Kuete et al. [@CR36]), 2′,4′-dihydroxy-3′,6′-dimethoxychalcone and 4′-hydroxy-2′,6′-dimethoxychalcone (Kuete et al. [@CR41]; Dzoyem et al. [@CR13]) and alkaloids: isotetrandrine (Kuete et al. [@CR44]) and montrofoline (Kuete et al. [@CR45]) displayed good antiproliferative effects against various cancer cell lines. In a collaborative research programme between the Council for Scientific and Industrial Research (CSIR) in South Africa and the National Cancer Institute (NCI) in the USA, several South African plant extracts exhibited anticancer activity against a panel of three human cell lines (breast MCF7, renal TK10 and melanoma UACC62) (Fouche et al. [@CR21], [@CR22]). African medicinal plants such as *Fagara heitzii* (Dzoyem et al. [@CR14]), *Echinops giganteus*, *Xylopia aethiopica*, *Piper capense*, *Imperata cylindrica* (Kuete et al. [@CR37]), *Beilschmiedia acuta*, *Clausena anisata* (Kuete et al. [@CR40]) also displayed good cytotoxicity towards drug-sensitive and drug-resistant cancer cell lines. In our ongoing search of anticancer products from African medicinal flora, we designed the present study to investigate the cytotoxicity of 11 plants traditionally used to manage cancer or disease states bearing relevance to cancer or cancer-like symptoms, such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases (Kuete et al. [@CR44]). The study was extended to the evaluation of the ability of the three most active extracts from two medicinal plants, *Annona muricata* Lin. (Annonaceae) and *Passiflora edulis* Sims (Passifloraceae) to alter the cell cycle distribution, caspases activity, mitochondrial membrane potential (MMP) and to increase the production of reactive oxygen species (ROS) in leukemia CCRF--CEM cells.
Methods {#Sec2}
=======
Plant material and extraction {#Sec3}
-----------------------------
All medicinal plants tested are traditionally used in the management of cancer or disease states with symptoms related to cancer. Plants were collected in different regions of Cameroon in January 2012. They included *Pachypodanthium staudtii*, *Alchornea floribunda*, *Annona muricata*, *Canarium schweinfurthii*, *Hibiscus esculentus*, *Colocasia esculenta*, *Moringa oleifera*, *Triumphetta pentandra*, *Xanthosoma mafaffa*, *Euphorbia prostata* and *Passiflora edulis*. The plant parts investigated are shown in Table [1](#Tab1){ref-type="table"}. The plants were identified at the National Herbarium (Yaoundé, Cameroon), where voucher specimens were deposited under the reference numbers shown in Table [1](#Tab1){ref-type="table"}. The air-dried and powdered plant material (50 g) was soaked in methanol (200 mL) for 48 h, at room temperature. The methanol extract was concentrated in vacuum under reduced pressure at 68 °C to give the crude extract. This extract was completely dried at room temperature, then conserved at 4 °C until further use.Table 1General information and reports on evidence of biological activities and chemistry of the studied plantsSpecies (family); voucher number^a^Traditional usesParts used (%yield)^b^Bioactive or potentially bioactive componentsBioactivity of crude extract*Alchornea floribunda* Müll. Arg. (Euphorbiaceae)\
4595/HNCTreatment of [bacterial]{.ul} and parasitic infections, painful urination in children (Adjanohoun et al. [@CR2]; Jiofack et al. [@CR28]), urinary, respiratory and intestinal problems, pains in the heart, diarrhoea, ovarian problems, stomach ailments and intestinal disorders (Siwe Noundou et al. [@CR66]), [trypanosomiasis]{.ul}, urinary, respiratory and intestinal disorders (Musuyu Muganza et al. [@CR49]; Mesia et al. [@CR47]), [inflammation]{.ul} (Okoye et al. [@CR58])Bark (18.91 %) and leaves (4.56 %)Eugenol, cadinol, nanocosaine, ethyl iso-allocholate, 3-acetoxy-7,8-epoxylanostan-1-ol (Okoye et al. [@CR58])Antibacterial activities of crude against *Bc*, *Ef*, *Ec*, *Sa*, *Kp*, *Mc*, *Pm*, *Ss* (Siwe Noundou et al. [@CR66]); topical anti-inflammatory effects (Okoye et al. [@CR58])*Annona muricata* Lin. (Annonaceae); 18681/SRF/CamTreatment of wounds and insomnia; [antiparasitic]{.ul}, insecticidal (Rajeswari [@CR60])Leaves (4.50 %), seeds (9.15 %), pericarp (5.17 %)Epomuricenins-A and B, montecristin, cohibins-A and B, muridienins-1 and 2, muridienins-3 and 4, muricadienin and chatenaytrienins-1, 2 and 3 and sabadelin, murihexol, donhexocin, annonacin A and Annonacin B (Rajeswari [@CR60])Antimicrobial activities of aqueous, ethanol and methanol extracts against *Sa*, *Vc*, *Ec*, *Se*, *Lv* and *On* (Vieira et al. [@CR68]) and *Pv*, *Sp*, *Bs*, *St*, *Kp*, *Ea* (Rajeswari [@CR60]), *Lb*, *Lp*, *Hv* (Rajeswari [@CR60]), *Ec*, *Ea*, *Kp*, *Ps* (Dzotam et al. [@CR11])*Canarium schweinfurthii* Engl. (Burceraceae); 16929/SRF/CamTreatment of [malaria]{.ul}, constipation, diarrhea, rheumatism and [sexually transmitted diseases]{.ul} (Koudou et al. [@CR32])Fruits (0.78 %)Saponins, cardiac glycosides, tannins, flavonoids and steroids (Ngbede et al. [@CR52])Antimicrobial activities of EO against *Bc*, *Ef*, *Ec*, *Li*, *Se*, *Sd*, *Sa*, *Pm*, *Sc*
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Introduction {#section1-2050313X19827737}
============
Congenital diaphragmatic eventration is an abnormal diaphragmatic elevation caused by insufficient or absent muscularization of the pleuroperitoneal membrane.^[@bibr1-2050313X19827737]^ It is difficult to assess the exact incidence of this abnormality because it is rare and generally diagnosed incidentally on chest radiography. Symptoms of diaphragmatic eventration vary from asymptomatic, mild gastrointestinal disease to life-threatening diaphragm rupture.^[@bibr2-2050313X19827737]^ Both induction and emergence from anesthesia should be smooth to avoid abdominal pressure increase, as this may cause diaphragmatic rupture.^[@bibr3-2050313X19827737]^ If the diaphragm ruptures, it should be rapidly diagnosed and treated. Lung ultrasonography can be used to monitor diaphragmatic movement on a real-time basis.^[@bibr4-2050313X19827737]^ This case report describes the anesthetic management of a pediatric patient with congenital diaphragmatic eventration and perioperative observation of diaphragmatic motion using lung ultrasonography.
Case report {#section2-2050313X19827737}
===========
A 28-month-old male (height: 87.8 cm, weight: 11.3 kg) was scheduled for excisional biopsy of osteochondroma on the right distal ulna. The patient had a family history of osteochondroma, and the surgery was planned to prevent further deformity of the right arm. The boy was born at a gestational age of 29 weeks and 2 days with a birth weight of 1400 g. He was transferred to the neonatal intensive care unit (NICU) after birth and remained there for 54 days. Haziness was noted in the right lower lung on chest radiography 30 days after the infant's admission to the NICU ([Figure 1(a)](#fig1-2050313X19827737){ref-type="fig"}). Based on this finding, fluoroscopy was performed and the patient was diagnosed with eventration, which persisted until his discharge. At the time of discharge, no abnormal diaphragmatic movement was observed on fluoroscopy despite right hemidiaphragm elevation. After the patient was discharged from the hospital, his clinical course was uneventful and similar to that of other babies of the same age. As part of the pre-operative evaluation, chest radiography showed an abnormal finding indicating eventration of the right diaphragm ([Figure 1(b)](#fig1-2050313X19827737){ref-type="fig"}). Since no pulmonary symptoms were present, the possibility of atelectasis or pneumonic consolidation was ruled out. The patient was already diagnosed with diaphragmatic eventration by fluoroscopy and ultrasonography. Blood test and electrocardiogram results were all within normal ranges.
{#fig1-2050313X19827737}
Because the patient was non-cooperative as a result of his young age, anesthesia was induced with ketamine 1.5 mg/kg before surgery. With the ventilator, spontaneous tidal volume was about 100 mL before intubation. After the administration of 0.8 mg/kg rocuronium as a muscle relaxant, bag-valve-mask ventilation was initiated with pressure less than 15 cmH~2~O and tidal volume under 100 mL. Intubation was performed with a cuffed endotracheal tube (size 4.0). Lung sounds were clear on the upper and lower left side. However, on the right side, lung sounds were auscultated only on the upper side, but not on the lower side. Anesthesia was maintained with sevoflurane and remifentanil. Bronchoscopy (3.1 mm diameter, Olympus America, Brooklyn Park, MN, USA) was used to identify three openings on the right bronchus. Initially, volume-controlled ventilation was set at tidal volume 100 mL, respiratory rate 25, fraction of inspired oxygen (FiO~2~) 50%, and flow 3 L. With these initial ventilator settings, peak pressure was 25 cmH~2~O. Before the initiation of surgery, the zone of apposition was examined using ultrasonography. To prevent an increase in abdominal pressure, the probe was placed on the lateral chest wall instead of the abdomen.^[@bibr5-2050313X19827737]^ Lung sliding and pleural edge were observed in the 8th through the 10th intercostal spaces on the left side and in the 5th through the 7th intercostal spaces on the right side.
At about 15 min after the initiation of surgery, the peak pressure suddenly rose to 28--30 mmHg. Lung ultrasonography was repeated to determine whether the rise in pressure was due to single lung ventilation and diaphragmatic rupture. In both lung fields, the level of lung sliding motion remained the same as that observed during the initial examination. The diaphragm demonstrated good movement. The sevoflurane concentration was increased to 2.5% to heighten the depth of anesthesia. To prevent diaphragmatic rupture, the tidal volume was lowered to 90 mL. Consequently, the peak pressure dropped to 22 cmH~2~O while maintaining end-tidal CO~2~ (ETCO~2~) at about 38 mmHg. Following surgery, the motion of the diaphragm was examined with real-time lung ultrasonographic imaging in the post-anesthesia care unit. It was difficult to observe slow movement of lung sliding because the child was crying. However, the pleural edge moved rapidly in the caudal direction in the zone of apposition when the patient cried, creating effects similar to a sniffing test (see [Supplemental Video](https://journals.sagepub.com/doi/suppl/10.1177/2050313X19827737)). Thus, the possibility of diaphragmatic rupture was excluded.
Discussion {#section3-2050313X19827737}
==========
Diaphragmatic eventration is so rare that it is difficult to measure the exact incidence. Although there are some reports of the anesthetic management of adult patients with this condition, cases of pediatric patients are limited.^[@bibr6-2050313X19827737]^ The reported incidence of pediatric congenital diaphragmatic eventration is 1/1400,^[@bibr7-2050313X19827737]^ and only one case of spontaneous rupture of a congenital diaphragmatic eventration in an infant has been reported.^[@bibr8-2050313X19827737]^ However, the exact incidence of the condition is unknown in the general population.
Partial diaphragm elevation has generally been found on the anteromedial right hemidiaphragm, while complete elevation has been observed on the left hemidiaphragm.^[@bibr9-2050313X19827737]^ While hypoplastic lung-related diaphragmatic eventration is diagnosed using radiography, fluoroscopy, and computed tomography (CT),^[@bibr7-2050313X19827737]^ it is difficult to count the number of lung lobes on CT images. However, three lobe openings on the right lung were confirmed on bronchoscopy in the present case during general anesthesia. In addition, diaphragmatic motion was noted on fluoroscopy, although congenital eventration of the diaphragm is normally associated with inadequate development of muscles or absence of the phrenic nerve.
Extra precautions are required when administering general anesthesia in patients with diaphragmatic eventration. A sudden increase in intra-abdominal pressure may cause diaphragmatic rupture, especially in patients with an abnormal diaphragm. Therefore, the prevention of severe coughing and bucking in patients should be ensured. If diaphragmatic rupture occurs, cardiac output will decrease with the migration of intra-abdominal organs from the intra-abdominal space to the intra-thoracic space, resulting in compression of the heart, aorta, and vena cava. Thus, it is important to maintain sufficient anesthetic depth during induction and emergence. In addition, low-volume bag-valve-mask ventilation is necessary to prevent peak pressure increase. Moreover, total intravenous anesthesia is preferred to balanced anesthesia with inhalation as the latter causes hypoxic pulmonary vasoconstriction. Peak pressure may rise upon single lung ventilation or migration of abdominal organs to the thoracic area after diaphragmatic rupture. When the peak pressure does rise, it is important to determine its cause. In addition, lung ultrasonography can be useful to evaluate diaphragm function, although there is no consensus on the sensitivity of ultrasound in this assessment.^[@bibr10-2050313X19827737]^
Abnormal diaphragmatic motion during breathing can be examined in M-mode ultrasonography.^[@bibr4-2050313X19827737]^ Normal diaphragmatic movement during inspiration shows movement toward the transducer when the transducer is on the right below the normal diaphragm position. When the diaphragm ruptures, the diaphragm may appear to be floating or invisible, or a subphrenic fluid collection may appear on ultrasonography.^[@bibr11-2050313X19827737]^ Ultrasonography may show herniation of the solid abdominal contents, such as the liver, omentum, or a bowel segment with peristaltic activity. If the liver sliding that is hepatic parenchymal movement is shown on the right upper trunk instead of the lung parenchyma, it may indicate liver herniation.^[@bibr12-2050313X19827737]^ Comparison of the amplitude of diaphragmatic movement and the alteration of changes in diaphragm thickness with the contralateral side is also important. In this case, liver and lung diaphragm were found at the locations expected from the results of pre-operative chest posteroanterior (PA) imaging of the right side: five or six rib levels higher than on the left side. When the patient cried, normal diaphragmatic excursion was observed. The
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Introduction {#s1}
============
Diabetic dyslipidaemia is characterised by hypertriglyceridaemia, low high-density lipoprotein (HDL) cholesterol (c) and normal low-density lipoprotein-cholesterol (LDLc) but preponderance of small-dense, highly atherogenic particles. The increase in free fatty acids (FFAs) as degradation products of triglycerides (TGs) is associated with the development of insulin resistance [@pone.0027208-Wilding1].
Cholesteryl Ester Transfer Protein (CETP) and Hepatic Lipase (HL) are central enzymes in the metabolism of HDL particles and reverse cholesterol transport. CETP is responsible for an exchange of cholesteryl ester (CE) for triglycerides (TGs) between LDL and HDL and TG rich-lipoprotein particles [@pone.0027208-Morton1]. The result is an enrichment of HDL and LDL particles in TGs, which makes them good substrates for HL [@pone.0027208-Thuren1]. The latter catalyses the hydrolysis of the TGs and phospholipids present in several lipoprotein subclasses, leading to changes in the size and density of lipoproteins [@pone.0027208-Thuren1]. The increased activity of either enzyme results in lower HDLc levels and a predominance of small, dense HDL and LDL particles [@pone.0027208-Lagrost1], [@pone.0027208-Zambon1].
The variations in the *CETP* gene, which lead to changes in enzyme function, have consequences on lipoprotein composition. *CETP* deficiency in humans is characterized by increases in HDLc, whereas increases in its activity are associated with an enrichment of HDL particles in TGs and a decrease in HDLc levels [@pone.0027208-Ikewaki1]. The most extensively studied polymorphism in *CETP* is Taq1B (rs708272) [@pone.0027208-Kondo1]. The G allele, also called B1, is associated with higher enzymatic activity, higher CETP mass and lower HDLc levels [@pone.0027208-Noone1], [@pone.0027208-Boekholdt1]. It has been estimated that this polymorphism is responsible for 5.8% of the variation in HDLc levels [@pone.0027208-Corella1].
Studies in transgenic mice demonstrate that the over-expression of the gene encoding HL, *Lipc*, leads to a marked decrease in plasma HDLc levels[@pone.0027208-Isaacs1] , an observation supported by human studies showing an inverse correlation between HL activity and HDLc concentrations [@pone.0027208-Blades1]. The -G250A *LIPC* polymorphism (rs2070895) [@pone.0027208-Todorova1], located in the promoter region of the gene, has been extensively studied in relation to enzyme activity and lipid metabolism. The minor allele (A) is associated with a reduction of transcriptional activity *in vitro* [@pone.0027208-Deeb1] and a 15--45% reduction in enzymatic activity [@pone.0027208-Tahvanainen1]. In humans, the minor allele has also been associated with an increased HDLc concentration and more buoyant LDL particles [@pone.0027208-Tahvanainen1], [@pone.0027208-Zambon2]. Studies assessing the association of this variant with T2D show conflicting results [@pone.0027208-Zacharova1].
Diabetes is often preceded and even predicted, by the presence of dyslipidemia [@pone.0027208-Todorova1]. Thus, mechanisms involved in the development of diabetic dyslipidemia may also play a role in the pathogenesis of T2D. The effects of the mentioned polymorphisms in *CETP* and *LIPC* on HDLc concentrations are well established, but their relation with the risk of T2D is less known. Therefore, the aim of our study was to analyze the relationship between polymorphisms in these two genes and the presence of diabetes and insulin resistance in a Canarian population.
Methods {#s2}
=======
Study population {#s2a}
----------------
The Telde study is a cross-sectional population-based study on the prevalence of diabetes and cardiovascular risk factors in Telde, a city located on the island of Gran Canaria, Spain. The study population and design of this survey has been previously described [@pone.0027208-Boronat1]. An oral glucose tolerance test (OGTT) was performed and the subjects were classified (using ADA 1997 criteria) as diabetic (n = 115) and pre-diabetic (n = 116) if they had impaired fasting glucose, impaired glucose tolerance or both. A total of 226 subjects with a normal OGTT were selected, after matching for gender and age with the other two groups. All participants gave their written informed consent for participation in the study, which was carried out according to the declaration of Helsinki and approved by the local ethics committee.
Genetic analyses {#s2b}
----------------
The biochemical analyses and insulin resistance parameters have been described previously [@pone.0027208-Novoa1]. Genomic DNA was extracted from whole blood (n = 457) using a salting-out method. The Taq1B *CETP* polymorphism was amplified by PCR-RFLP as described by June Hsieh Wu [@pone.0027208-Wu1] and the *G-250A LIPC* polymorphism was analyzed by AMRS-PCR (Amplification Refractory Mutation System-Polymerase Chain Reaction) [@pone.0027208-Ye1]. Two pairs of primers were used, one which amplifies a fragment of 366 bp, common to both alleles (outer primers: 5′-CTT TTC TTT TTC TTT GGG CTT AGG CT-3′ and 5′-AAG ACT GCC CAT TAA TAA TTA ACC TCT CAA-3′) and another pair specific for the SNP (inner primers): 5′-CAA GGT CAG AGT TCC AAA TTA ATC CAC-3′ for the G allele and 5′-TTC CAA ACA CAA CAC AGT AGC TTT CAA-3′ for the A allele. The primers were designed *"in silico"* in a free access web (<http://cedar.genetics.soton.ac.uk>, accessed in August 2007) and then checked for specificity (<http://blast.ncbi.nlm.nih.gov/Blast.cgi>, accessed in August 2007). The PCR reaction was carried out in a total volume of 25 µl containing 1∶5 ratio of outer to inner primer concentration. The annealing temperatures were 70°C during 15 sec for the outer primers and 58°C during 25 sec for the inner primers with a 30 sec extension at 72°C. PCR products were mixed with 2 µl of loading buffer and run on 2.5% agarose gel stained with Ethidium Bromide. This resulted in 3 DNA fragments: one of 366 bp, one of 234 bp for the A allele and one of 185 bp for the G allele ([figure 1](#pone-0027208-g001){ref-type="fig"}).
{#pone-0027208-g001}
Statistical analysis {#s2c}
--------------------
Statistical analyses were performed with SPSS for WINDOWS, version 13 (SPSS Inc., Chicago, IL). The quantitative variables are described as mean ± standard deviation (S.D). Before further analyses, variable distribution was checked with the Kolmogorov--Smirnov test. A logarithmic transformation was performed for variables not following a Gaussian distribution. Differences between groups were analyzed using either analysis of variance or analysis of covariance, both with the Bonferroni post hoc correction test, after adjusting for age, gender, Body Mass Index (BMI) and waist. The categorical variables were compared using Fisher\'s exact test for 2×2 tables and chi-squared or the Mantel-Haenszel test for linear association. The independent contribution of each polymorphism to DM2 risk was analyzed by a multinomial logistic regression model, which included age, gender, BMI and waist. All tests were considered significant if p was \<0.05.
Regarding the effect of the interaction of both polymorphisms on the risk of diabetes, the reference category was defined by the non-B1B1 genotype, regardless of the -250G/A *LIPC* genotypes (nonB1B1*CETP* genotype). A second group included B1B1 and non-GG genotypes (B1B1CETP/non-GGLIPC) and a third, B1B1 CETP and *LIPC* GG genotypes (B1B1CETP/GGLIPC).
Results {#s3}
=======
Patient description {#s3a}
-------------------
The anthropometric, clinical and genetic characteristics of the whole population and their classification according to the OGTT are shown in [table 1](#pone-0027208-t001){ref-type="table"}. The frequencies of the B1B1, B1B2 and B2B2 genotypes of the
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Background
==========
Diatoms are important primary producers in the ocean \[[@B1]\], contributing approximately 40% to global marine productivity. Although diatoms often dominate phytoplankton communities in nutrient-rich ecosystems, members of this diverse group are also adapted to survive and persist in nutrient-limited conditions. The development of large diatom blooms upon nutrient resupply demonstrates the metabolic plasticity inherent to their ability to recover rapidly from nutrient limitation.
Iron is an essential nutrient for all organisms and in particular for photoautotrophic organisms. It functions as a powerful electron carrier in iron-sulfur- and heme-containing proteins and as such is a required component of the photosynthetic apparatus. Solubility of iron in seawater is low and phytoplankton growth in marine habitats is often limited by iron availability. This is best illustrated in high-nitrate low-chlorophyll (HNLC) regions, remote oceanic areas that lack any form of regular iron supply and suffer from a persistent shortage of this micronutrient. Here, although other commonly limiting nutrients like nitrate or phosphate are present at high concentrations, primary productivity - and biomass as a whole - is low \[[@B2]\].
Numerous large-scale iron fertilization experiments have confirmed that iron is the limiting nutrient in HNLC regions \[[@B3]\]. Phytoplankton blooms induced by iron fertilization were dominated by diatoms and carbon export to the deep-sea floor could be observed in some cases. The strong response of diatoms to the input of iron in HNLC regions has been a motivation for exploring large-scale iron fertilization as a possible bioengineering strategy to sequester CO~2~into the ocean in HNLC regions, which are otherwise rich in macronutrients.
Genome projects on the model organisms *Thalassiosira pseudonana*\[[@B4]\] and *Phaeodactylum tricornutum*\[[@B5]\] have already generated a wealth of insights into the metabolic complexity of diatoms \[[@B6]\], a consequence of the secondary endosymbiosis event that gave rise to this group \[[@B7]\]. This secondary endosymbiosis brought together the benefits of a heterotrophic host and the \'red\'-type photosynthesis of red alga cells, which already have an elemental composition low in iron \[[@B8]\].
The impact of iron availability on phytoplankton growth has led to the evolution of strategies to counteract iron limitation. Well established parts of the low-iron response found in diverse phytoplankton species are the reduction of the chloroplast system, the corresponding development of a chlorotic phenotype, compensation mechanisms (replacement of iron-rich elements with iron-poor substitutes) and the activation of high-affinity iron-uptake systems \[[@B9]\]. The substitution of ferredoxin by flavodoxin \[[@B10]\], the use of plastocyanin instead of cytochrome c~6~\[[@B11]\] and a variant stoichiometry of photosynthetic complexes \[[@B12]\] are notable adaptive strategies to facilitate diatom growth in low-iron conditions.
Oceanic and neritic phytoplankton species can be distinguished from each other by their growth characteristics and their tolerance to nutrient limitation \[[@B13]\]. Unlike many other *Thalassiosira*species that are predominantly found in coastal waters, *Thalassiosiraoceanica*is adapted to oligotrophic conditions and is highly tolerant to iron limitation in particular. Therefore, we chose *T. oceanica*CCMP1005 as a model for a comprehensive analysis of its low-iron response in the context of genomic information.
Here, we explore the complex cellular response of *T. oceanica*to low-iron conditions with genomics, transcriptomics and proteomics approaches complemented by reverse transcription-quantitative PCR (RT-qPCR) analyses. We present a metabolic reconstruction of the iron limitation response based on the transcriptomics data from cells grown under iron-limited versus iron-replete conditions. A metabolic isotope labeling approach using ^14^N/^15^N was established for *T. oceanica*and showed the response to iron limitation at the protein expression level in a marine diatom for the first time. General characteristics of the \'diatom\' low-iron response and its ecological implications are discussed, as well as the constraints for species-specific adaptations to low-iron environments.
Results
=======
Characteristics of the *T. oceanica*genome
------------------------------------------
The genome of the centric diatom *T. oceanica*CCMP1005 (Figure [1](#F1){ref-type="fig"}) was *de novo*assembled from 725 Mb of Roche 454 sequence read information, generated using nuclear genomic DNA (gDNA) of an axenic clonal culture as substrate \[[@B14]\]. The current assembly version comprises 51,656 contigs of total size 92.15 Mb at N50 = 3,623 (that is, 50% of the genomic sequence information is present as contigs ≥3,623 bases). From a median 8.7-fold coverage of long contigs (≥10 kb) we estimated a true haploid nuclear genome size of 81.6 Mb, suggesting some redundancy in the current assembly. This estimate is in good agreement with the 159 Mb measured by van Dassow *et al*. \[[@B15]\] for the diploid G1 DNA content. The gene finder tool AUGUSTUS \[[@B16]\] predicts 37,921 protein gene models that cluster into a non-redundant set of 29,306 models including pseudogenes and short ORFs; 10,109 models have BLAST hits to the National Center for Biotechnology Information (NCBI) nr protein database at a conservative E-value cutoff of 1.0E-30 and thus are more indicative of the expected true protein-coding gene number (that is, expressed genes excluding pseudogenes and short ORFs). Best BLAST hits are listed in Additional file [1](#S1){ref-type="supplementary-material"}. In Table [1](#T1){ref-type="table"} we present an overview of the most abundant Clusters of Orthologous Groups (COG) domains in *T. oceanica*. The abundances of diverse groups of ATPases were overall very similar to those for other diatoms. A group of 19 chitinases is shared between the two centric *Thalassiosira*species.
{#F1}
######
Most abundant protein domains in diatom genomes
COG To Tp Pt Fc
-------------------------- ------------------------------------------------------------------------------------------------------------------------- ----- ----- ----- -----
ATPases
COG0515 SPS1, serine/threonine protein kinase 115 132 119 137
COG0464 SpoVK, ATPases of the AAA+ class 90 43 38 44
COG1132 MdlB, ABC-type multidrug transport system, ATPase and permease components 54 44 47 51
COG1222 RPT1, ATP-dependent 26S proteasome regulatory subunit 50 41 37 42
COG0465 HflB, ATP-dependent Zn proteases 49 37 35 39
COG1223 Predicted ATPase (AAA+ superfamily) 44 39 34 41
COG3899 Predicted ATPase 42 48 11 2
COG2274 SunT, ABC-type bacteriocin/lantibiotic exporters, contain an amino-terminal double-glycine peptidase domain 42 52 50 61
COG5265 ATM1, ABC-type transport system involved in Fe-S cluster assembly, permease and ATPase components 40 33 30 33
COG4618 ArpD, ABC-type protease/lipase transport system, ATPase and permease components 39 41 42 43
COG4987 CydC, ABC-type transport system involved in cytochrome bd biosynthesis, fused ATPase and permease components 31 50 46 56
COG4988 CydD, ABC-type transport system involved in cytochrome bd biosynthesis, ATPase and permease components 29 52 49 60
COG0488 Uup, ATPase components of ABC transporters with duplicated ATPase domains 29 50 46 53
COG1131 CcmA, ABC-type multidrug transport system, ATPase component
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Handled by Yu Xue
Introduction {#s0005}
============
Cancer somatic mutations and viral oncogenes can generate tumor-specific protein sequences that are entirely absent from normal human cells. These novel proteins may result in the formation of tumor-specific antigens (TSAs) [@b0005]. As an important type of TSAs, neoantigens are generated by tumor-specific proteins, and presented by major histocompatibility complexes (MHCs) on cell surfaces through antigen presentation, where they can be recognized by T-cell receptors (TCRs) [@b0010], [@b0015]. Recently, neoantigens have attracted a large amount of attention, because they are potential biomarkers to distinguish tumor cells from normal cells. Neoantigens are of critical importance for cancer immunotherapy in the following two aspects. First, the neoantigen burden and quality can be used to predict therapeutic effects for immune checkpoint blockade therapy, such as blockage of programmed death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) [@b0020], [@b0025], [@b0030]. Second, neoantigens can be used as potential targets for cancer immunotherapy, such as personalized cancer vaccines [@b0035], [@b0040] and adoptive cell therapy (ACT) [@b0045]. Therefore, there is an urgent need to identify neoantigens accurately for cancer patients.
With the progress of cancer immunogenomics, several kinds of integrated software have been developed for tumor-specific neoantigen detection, such as TSNAD [@b0050] and pVAC-seq [@b0055]. The most critical function of such software is to predict the binding affinities between mutant peptides and human leukocyte antigen (HLA) alleles. To achieve this, a lot of well-acknowledged and popular tools, such as NetMHC [@b0060], NetMHCpan [@b0065], sNebula [@b0070], and HLA-CNN [@b0075], can be used. In addition, several databases can provide necessary information for the development of tools to predict the affinities between peptides and HLA alleles. For example, the Immune Epitope Database (IEDB) is an important immune-related database, providing a large amount of valuable and experimentally-validated information of immune epitopes [@b0080]. The International Immunogenetics Information System (IMGT) offers information about antibodies, TCRs, MHCs, and so on [@b0085]. Taking advantage of existing neoantigen prediction software, several neoantigen-related databases have been built. For example, TRON Cell Line Portal (TCLP) presents potential neoantigens of 1082 cancer cell lines [@b0090]. The Cancer Immunome Atlas (TCIA) presents the relationship between tumor genotypes and immunophenotypes based on 20 solid cancers, and provides a quantitative index for immunotherapy response [@b0095]. With the rapid growth of cancer genomics data, researchers are able to discover potential shared neoantigens across tumor patient populations [@b0100], [@b0105].
In this study, we developed a tumor-specific neoantigen database (TSNAdb v1.0) from pan-cancer immunogenomic analyses. Based on the 7748 tumor samples of 16 tumor types from The Cancer Genome Atlas (TCGA), we predicted the binding affinities between mutant/wild-type peptides and HLA class I molecules. Datasets we used include somatic mutation data of tumor samples from TCGA and the corresponding HLA allele data from TCIA. Two different versions of NetMHCpan, v2.8 [@b0065], and v4.0 [@b0110], were used for prediction. Furthermore, we also conducted extensive analyses and presented detailed information of potential neoantigens generated by somatic mutations, utilizing the related filtering tools embedded in TSNAD [@b0050]. In addition, we employed the recurrent missense mutations in combination with the highly frequent HLA alleles to predict and analyze potential shared neoantigens. Our study would provide a platform to discover putative targets for neoantigen-based cancer immunotherapy.
Database content and usage {#s0010}
==========================
Data source {#s0015}
-----------
We collected somatic mutations and HLA alleles of 7748 tumor samples across 16 tumor types from TCGA (Release7.0, <https://portal.gdc.cancer.gov>) and TCIA (<https://tcia.at/home>), respectively. These tumor samples carry 972,187 missense mutations, among which 18,897 were found recurrently (at least three occurrences in all tumor samples). We selected the top 100 HLA alleles (frequency \>0.5%) of 7748 tumor samples and combined them with the recurrent missense mutations to predict potential shared neoantigens. Moreover, we also extracted 13,459 recurrent missense mutations from 9155 samples derived from the International Cancer Genome Consortium (ICGC) (Release20, <https://icgc.org/>) and 16 highly frequent HLA alleles (frequencies \>5%) from the 1000 Genome Project [@b0115] for the prediction of potential shared neoantigens.
Neoantigen prediction {#s0020}
---------------------
We took the information on somatic mutations and HLA alleles of each tumor sample and employed NetMHCpan v2.8 [@b0065] and NetMHCpan v4.0 [@b0110] for neoantigen prediction, using the filtering tools embedded in our previously-developed software TSNAD [@b0050]. All the peptides with 8--11 amino acids that contain missense mutations were extracted as mutant peptides, and the corresponding wild-type peptides were extracted as references. We collected the mutant peptides and HLA alleles with binding affinity IC~50~ \< 500 nM (including strong binding with IC~50~ \< 150 nM and weak binding with 150 nM \< IC~50~ \< 500 nM), without consideration of the binding level between their corresponding wild-type peptides and HLA alleles. We then clustered prediction results based on tumor types and calculated the frequencies of shared neoantigens. Compared with NetMHCpan v2.8, NetMHCpan v4.0 is trained based on both binding affinity data and mass spectrometry data, thus adopting stricter criteria for binding prediction. Consequently, 3,707,562 and 1,146,961 neoantigens were predicted by NetMHCpan v2.8 and v4.0, respectively, among which, 716,876 neoantigens were found in both predictions. The potential shared neoantigens based on recurrent mutations and highly frequent HLA alleles were predicted in the similar way.
Web interface {#s0025}
-------------
To facilitate the utilization of TSNAdb, we have established a web interface to browse and analyze neoantigens. The web interface comprises five main pages ([Figure 1](#f0005){ref-type="fig"}A): (i) Home, (ii) Browse, (iii) Search, (iv) Validation, and (v) Download. In the following context, we exemplify the usage of TSNAdb with the results predicted by NetMHCpan v2.8.Figure 1**An overview of the TSNAdb web interfaceA.** TSNAdb comprises five main components: (i) Home; (ii) Browse; (iii) Search; (iv) Validation, and (v) Download. The distribution of HLA alleles (**B)** and somatic missense mutations (**C)** extracted from TCGA and TCIA are listed. **D.** The average neoantigen loads across different tissues. **E.** Top 20 genes with predicted neoantigens in 7748 samples. **F.** The result of 'Search' page under the selection of "NetMHCpan2.8, *KRAS*, and TCGA". **G.** The result of 'Search' page under the selection of "NetMHCpan2.8, *KRAS*, and ICGC". **H.** Example of validation data from IEDB in 'Validation' page (top) and partial information on 'Download' page (bottom). The predicted binding level 'Strong' indicates strong binding with IC~50~ \< 150 nM. TCGA, The Cancer Genome Atlas; TCIA, The Cancer Immunome Atlas; HLA, human leukocyte antigen; TSNAD, tumor-specific neoantigen detector; ICGC: International Cancer Genome Consortium; IEDB, Immune Epitope Database; WT, wild type.
In the 'Home' page, TSNAdb provides a statistical table about the database, including the number of projects, samples, HLA alleles ([Figure 1](#f0005){ref-type="fig"}B), mutations ([Figure 1](#f0005){ref-type="fig"}C), and predicted neoantigens of each tumor type. The table presents 3,707,562 potential neoantigens from the 7748 tumor samples across 16 tumor types. The average predicted neoantigen loads vary across different tumor types ([Figure 1](#f0005){ref-type="fig"}D). For instance, the average predicted neoantigen load for uterus cancer is 1957, which is 21 for thyroid cancer. Besides, users can get the detailed information about the predicted neoantigens of each tumor type by clicking on the tissue name in the table. The content of each tumor type includes top 20 HLA alleles and top 20 genes with predicted neoantigens in this tumor type, which are shown as two figures. All predicted neoantigens in this tumor type would be displayed below figures.
To further understand the distribution of neoantigens at gene level, users can retrieve neoantigens by feeding in a gene name in the 'Browse' page ([Figure 1](#f0005){ref-type="fig"}E). The retrieval results provide more functions than that indicated in each tumor type
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{
"pile_set_name": "PubMed Central"
}
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INTRODUCTION {#s1}
============
Pediatric glioblastoma (GBM) is a relatively rare primary brain tumor in children \[[@R1]\]. Maximum surgical resection is considered the key and prognosis-determining factor in the treatment, followed by radiotherapy \[[@R1], [@R2]\]. Unfortunately, pediatric GBM is poorly studied at the molecular and genomic levels. Radiotherapy plays a critical role in eradicating the post-surgical residual microtumor \[[@R3]\]. Yet the molecular and genomic changes post-radiation in pediatric GBM have not been well examined. We have recently identified many radiation-responsive genes in adult radioresistant GBM cells that explain the radioresistance and increased malignant features of recurrent GBM \[[@R4]\]. However, adult and pediatric GBMs are distinct from each other at both molecular and genetic levels \[[@R2]\]. We are presenting a full RNA sequencing profile of both the radiation-naïve pediatric GBM (SJ-GBM2) cell line and stable radioresistant pediatric GBM (SJ-GBM2-10gy) cell line that we recently developed \[[@R5], [@R6]\]. Our data demonstrated that radiation perturbed the expression of many genes related to many different known pathways in cancer biology. The irradiated cells exhibited an enhanced growth rate, overexpressed protease cathepsin B, and both subunits of the rate-limiting enzyme of DNA synthesis ribonucleotide reductase (RR). In this study, we shed light on the irradiation responsive mRNA changes that transform the tumor cells toward a more aggressive and resistant form, for which treatment choices are limited. This study opens the door to further examining the possibility of targeting these modified pathways as a therapeutic strategy to block GBM tumor recurrence and progression.
RESULTS {#s2}
=======
Radioresistant irradiated pediatric GBM cells exhibited higher growth rate than control cells {#s2_1}
---------------------------------------------------------------------------------------------
The *in-vitro* growth rate of the SJ-GBM2 and SJ-GBM2-10gy cells was evaluated using an MTT growth assay over a period of 10 days. SJ-GBM2-10gy cells showed a superior divergent growth starting from day 3, having the difference in growth maximized on day 7 when the control cells significantly slowed down their proliferation rate (Figure [1](#F1){ref-type="fig"}). The control cells SJ-GBM2 reached about 7.4 growth fold in 10 days, while the irradiated cells reached a 10.5 fold from their baseline. This represents an estimated 30% increase in growth (Figure [1A](#F1){ref-type="fig"}).
{#F1}
Irradiated radioresistant pediatric GBM overexpresses ribonucleotide reductase {#s2_2}
------------------------------------------------------------------------------
To probe for a mechanism promoting the superior growth rate in irradiated cells, the expression of both ribonucleotide reductase (RR) subunits was measured. The RR enzyme, specifically the RRM2 subunit, has been reported to be essential for proliferation and invasion of GBM cells \[[@R7]\]. Immunoblotting of control and irradiated cells revealed an increase in the expression of RRM1 subunit by 2-fold, and an increase in the RRM2 subunit by 3.5-fold in irradiated cells relative to control cells (Figure [1B](#F1){ref-type="fig"}). This increase in cellular expression of RRM2 was confirmed by immunofluorescence probing of intact cells, demonstrating the distribution of RRM2 in the cytoplasm of the irradiated cells and was greater than the control cells (Figure [1B, 1C](#F1){ref-type="fig"}).
Irradiated radioresistant pediatric GBM overexpresses pro-cathepsin B {#s2_3}
---------------------------------------------------------------------
We were interested in evaluating whether protease may play of role in promoting invasion and progression of irradiated radioresistant GBM. Cathepsin B, a cysteine protease, has been shown to play a role in tumor growth and invasion \[[@R8], [@R9]\]. We probed for the differential expression level of pro-cathepsin B in control and irradiated cells. Western blot of cell lysates and immunofluorescence of intact cells revealed 3-fold overexpression of pro-cathepsin B in irradiated cells over control cells (Figure [1B, 1C](#F1){ref-type="fig"}). In addition to being localized to the cytoplasm, pro-cathepsin B was present in the processes of the irradiated cells, a feature that may be important in the invasiveness of GBM into the surrounding tissue (Figure [1C](#F1){ref-type="fig"}).
Irradiation of pediatric GBM cells induces differential expression of 1192 radiation-responsive genes {#s2_4}
-----------------------------------------------------------------------------------------------------
Total mRNAs from SJGBM2 and SJGBM2-10gy cells were harvested and subjected to further analysis. To screen for global mRNA changes following irradiation, we profiled transcriptomes of the control SJ-GBM2 cell line and its derivative radioresistant irradiated SJ-GBM2-10gy cells by RNA sequencing (Table [1](#T1){ref-type="table"} and [Supplementary Table 1](#SD2){ref-type="supplementary-material"}). The criteria for differentially expressed genes were 2-fold or greater than statistically significant values (*P* \< 0.05). We identified 1192 radiation responsive genes. Among these 1192 radiation responsive genes, 584 were upregulated and 608 were downregulated ([Supplementary Tables 2](#SD3){ref-type="supplementary-material"} and [3](#SD4){ref-type="supplementary-material"}).
###### Enriched gene ontology categories of differentially expressed genes following irradiation based on sets of statistically significant (more than 2-fold) upregulated and downregulated genes (*P* \< 0.05)
Differentially expressed Category *P*-value
-------------------------------------------------------- --------------------------------------- -----------
**Upregulated** GO:0048863: Stem cell differentiation 6.60E-03
GO:0010628: Positive regulation of gene expression 7.70E-03
GO:0002040: Sprouting angiogenesis 3.20E-02
GO:0008284: Positive regulation of cells proliferation 2.40E-02
GO:0070848: Response to growth factor 7.40E-02
GO:0001558: Regulation of cell growth 7.60E-02
GO:0055114: Oxidation reduction process 3.00E-02
GO:0071356: Cellular response to tumor necrosis factor 3.60E-02
GO:0006954: Inflammatory response 4.10E-02
GO:0016055: Wnt signaling pathway 8.40E-02
GO:0043066: Negative regulation of apoptotic process 9.40E-02
GO:0004222: Metalloendopeptidase activity 2.40E-02
**Downregulated** GO:0007155: Cell adhesion 4.90E-03
GO:0043065: Positive regulation of apoptosis 1.30E-04
GO:008285: Negative regulation of cell proliferation 4.10E-02
GO:0002020: Protease binding 1.20E-03
Experiments were performed in triplicate.
Upregulation of genes promoting tumor growth and aggressiveness following irradiation {#s2_5}
-------------------------------------------------------------------------------------
Gene ontology analysis of the mRNA data was utilized to categorize genes into functional groups. It revealed that upregulated genes were enriched in positive regulation of stem cells differentiation, angiogenesis, cell proliferation, cell growth, inflammatory response, positive regulation of the Wnt signaling pathway, response to hypoxia, metalloendopeptidase activity, cellular response to tumor necrosis factor and negative regulation of apoptotic process (Table [1](#T1){ref-type="table"} and [Supplementary Table 2](#SD3){ref-type="supplementary-material"}, and [Supplementary Table 4](#SD5){ref-type="supplementary-material"}). The upregulated genes were enriched in positive regulation of gene expression and tumor cell proliferation such as KIT, connective tissue growth factor CTGF and ID1, ID2, and TLE1-FOXG1 transcriptional factors that have been reported to enhance growth and proliferation of GBM cells \[[@R10]--[@R13]\]. G protein-coupled receptor kinase 5 (GRK5) plays an important role in tumor cells' proliferation \[[@R14]\]. Fibroblast growth factor 4 (FGF4) also has been correlated with a greater malignancy profile in high-grade gliomas \[[@R15]\].The radioresistant cells had upregulated expression of many anti-apoptotic genes, including BCL2, CD74, and WT1, which regulates GBM cells proliferation and apoptosis \[[@R16]\]. A significant upregulation (10-fold) of the AIM2 gene, a tumor-associated antigen, was found. This gene upregulation was observed in GBM patients and
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{
"pile_set_name": "PubMed Central"
}
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1. Introduction
===============
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive hematologic malignancy derived from precursors of plasmacytoid dendritic cells. This disease entity was recognized in the 2008 World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues, where it was separately included in the group of acute myeloid leukemia (AML) and related precursor neoplasm.^\[[@R1]\]^ This disease almost always presents with cutaneous involvement as the 1st manifestation, with subsequent or concurrent spread to bone marrow and peripheral blood.^\[[@R2]--[@R4]\]^ Although it is extremely rare, a minority but significant proportion of patients present without skin lesions. Furthermore, BPDCN present at other sites has not been yet reported. To date, nasal cavity lesion as the 1st manifestation in BPDCN has not been reported yet. Here we report 2 cases of BPDCN preseting as masses of nasal cavity and nasopharynx with leukemic manifestation without skin lesion in adolescent patients. In addition, we briefly reviewed previous cases of BPDCN without skin manifestation.
2. Case reports
===============
2.1. Case 1
-----------
The 1st patient was a 16-year-old girl who presented with recurrent epistaxis. She had no significant medical history or family history of cancer or known genetic disorders. On sinonasal computed tomography (CT), a 2.9-cm sized, polypoid mass was noted in the nasal cavity. Cutaneous examination was unremarkable. Biopsy of this mass was performed. Histologically, the nasal mucosa diffusely expanded. It was infiltrated by atypical lymphoid infiltrates. Infiltrative tumor cells were diffuse, monomorphic medium-sized cells with fine chromatin, irregular nuclei, and scanty cytoplasm, showing blastic morphology. Mucosal glands often became widely spaced and lost. An angiocentric and angiodestructive growth pattern were not identified. Mucosal ulceration and necrosis were not identified either (Fig. [1](#F1){ref-type="fig"}A, B). Immunohistochemically, atypical lymphoid cells were positive for CD2, CD4, CD56, and CD123 with focal weak staining for TCL-1, but negative for CD20, CD3, TdT, MPO, and EBV-encoded small RNA (Fig. [1](#F1){ref-type="fig"}C--F). No clonal TCRG or IgH gene rearrangement was detected. Peripheral blood work-up revealed pancytopenia while bone marrow biopsy revealed involvement of neoplastic cells, similar to histology and immunohistochemical findings of nasal cavity mass.
{#F1}
The patient was treated with induction chemotherapy with Berlin-Frankfurt-Münster regimen used for acute lymphoblastic leukemia. She achieved complete remission. After the 1st remission, she received allogenic peripheral blood stem-cell transplant (PBSCT). No relapse was observed at 14 months after transplantation. Interestingly, she had no skin lesions at initial diagnosis or during the course of their illness.
2.2. Case 2
-----------
The 2nd patient was a previous healthy 17-year-old female who presented with nasal obstruction and voice change for a month. CT scans revealed a large enhancing nasopharyngeal mass involving adenoid and several small indeterminate lymph nodes at the neck. Biopsy of the nasopharyngeal mass was performed. Microscopically, the nasopharyngeal mucosa was entirely replaced by diffuse atypical lymphoid cells with blastoid morphology (Fig. [2](#F2){ref-type="fig"}A, B). Immunohistochemically, these atypical lymphoid cells were positive for CD4, weak CD56, CD123, TCL1, and TdT, but negative for CD20, CD3, CD8, and CD1a (Fig. [2](#F2){ref-type="fig"}C--F). Peripheral blood count results were as follows: WBC, 4890/μL; Hb, 11 g/dL; and platelet, 127/μL. Blast was measured 13% of WBCs. Bone marrow biopsy showed infiltration of blastic tumor cells and demonstrated increase of CD4+, CD56+, TdT+, CD10−, and CD34− blasts up to 95% of total nucleated cells. Abdominal scan revealed mild hepatosplenomegaly while PET scan suggested hypermetabolism at nasopharynx, systemic lymph nodes, breast, liver, spleen, and bone marrow. Based on these findings, the diagnosis was most consistent with BPDCN for the 2 cases.
{#F2}
The patient was treated with AraC/Idarubicin (AId) induction chemotherapy. However, persistent blasts (32.5% of total nucleated cells) were observed in bone marrow biopsy. She is now taking Cladribin/Ara-C/G-CSF (CLAG) reinduction chemotherapy. After the remission, she received allogenic PBSCT. No relapse was observed at 11 months after transplantation. Interestingly, she also had no skin lesions at initial diagnosis or during the course of their illness.
3. Discussion
=============
In this study, we report 2 cases of BPDCN in adolescent patients who had unusual extracutaneous manifestation without skin lesion. Clinicopathologically, the differential diagnosis of our cases included extranodal NK/T-cell lymphoma, acute leukemia of ambiguous lineage, and NK lymphoblastic leukemia/lymphoma. Nasal cavity lesion as the 1st clinical manifestation and CD56-positive tumor cells raised the possibility of extranodal NK/T-cell lymphoma. However, absence of angioinvasion, no expression of cytoplasmic CD3, and cytotoxic granule proteins such as granzyme B and no association with EBV ruled out the diagnosis of extranodal NK/T-cell lymphoma. According to the 2017 WHO criteria, tumors that express some immunophenotypic features of BPDCN but not all immunohistochemical markers may be better classified as "acute leukemia of ambiguous lineage."^\[[@R5]\]^ At present, NK lymphoblastic leukemia/lymphoma is considered a provisional entity. It should be diagnosed after ruling out BPDCN. Blastic cells expressing CD56 and CD2 raised the possibility of NK lymphoblastic leukemia/lymphoma. However, CD4 positivity made it doubtful for such diagnosis. In such cases, immunohistochemical analysis including the most characteristic and reliable marker is essential for the diagnosis of BPDCN. BPDCN was initially characterized by the expression of CD4, CD56, and the lack of B cells, T cells, myeloid or monocytic cells, and NK cell markers. More specific plasmacytoid dendritic cell markers (CD123, CD303, and TCL1) have been recently used to diagnose BPDCN.^\[[@R6],[@R7]\]^ Since they are concomitantly expressed in only 46% of patients, it has been proposed that diagnosis of BPDCN can be made when 4 of these 5 markers (CD4, CD56, CD123, CD303, and TCL1) are expressed.^\[[@R8]\]^ Although tumor cells of the 1st case showed focal positive for TCL1, both of 2 cases showed all 5 markers except CD303 which was not performed in our institution. Therefore, our 2 cases were histologically diagnosed with BPDCN.
The BPDCN without cutaneous lesion is exceedingly rare to diagnose. Patients without cutaneous involvement have been described in the literature. Table [1](#T1){ref-type="table"} presents a summary of 39 published cases of BPDN without skin involvement. Bone marrow involvement was observed in the majority of patients at diagnosis. Through hematopathology consultation service at the National Institutes of Health, Jegalian et al^\[[@R6]\]^ have evaluated 55 BPDCN cases. Among them, 9 (16%) patients lacked cutaneous disease at presentation. A retrospective multicenter study of 43 patients (the GIMEMA study) presenting with leukemic manifestation was reported in 2012.^\[[@R14]\]^ Among 43 patients, 8 (19%) cases had no cutaneous manifestations.^\[[@R14]\]^ In these patients lacking skin involvement, other extracutaneous and extramedullary sites in lymph node, spleen, and liver are most commonly observed. Rauh et al^\[[@R13]\]^ have demonstrated that patients with BPDN without skin involvement and leukemic presentation show adverse prognosis than those with skin involvement. Interestingly, no case of BPDCN presenting with nasal cavity mass has been reported. It is of note that we identified nasal cavity as the unusual site of BPDCN.
######
Summary of 39 published cases of BPDCN without skin involvement in the literatures.
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{
"pile_set_name": "PubMed Central"
}
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1. Introduction
===============
*Mycobacterium tuberculosis* is counted with justification among the most dangerous and successful microorganisms in today's world, especially in developing countries which have become the reservoir of resistant strains (the most burdened countries are in South Africa and East Asia) \[[@B1-molecules-19-00651]\]. These strains are causing the biggest number of problems connected to tuberculosis (TB) treatment \[[@B2-molecules-19-00651]\]. The main problem is resistance. It can be divided into three groups. The first one is called multidrug-resistant TB (MDR-TB) and these microbes are resistant to all first-line antituberculotic drugs (pyrazinamide - PZA, isoniazid - INH, rifampicin - RIF, ethambutol - ETH, streptomycin - STR). The second group is more treacherous. This type of resistance is called extensively or extremely drug-resistant TB (XDR-TB) with the resistance to the first-line anti-TB agents isoniazid and rifampicin together with the resistance to any fluoroquinolone used in the therapy and to at least one of three injectable second-line antituberculotic drugs (amikacin, kanamycin or capreomycin) \[[@B3-molecules-19-00651]\]. The last and the latest category was the most dreaded and was called totally drug-resistant TB (TDR-TB) and the first case was recorded in India \[[@B4-molecules-19-00651]\]. These mycobacterial strains were resistant to all current known therapy. Nowadays, this group has disappeared with the approval of bedaquiline for therapy of resistant forms of tuberculosis. Another problem is connected with the HIV pandemic. These two infectious diseases are influencing each other in a synergic way so this has resulted in efforts to develop new anti-tubercular agents.
This work deals with a microwave-assisted synthesis of pyrazinamide analogues with potential antimycobacterial activity. It is caused by the fact that pyrazinamide is counted among the first-line anti-tuberculosis drugs used in current therapy. Its unique ability to kill the dormant forms of *Mycobacterium tuberculosis* is crucial in shortening the time needed for the treatment, so PZA has sterilizing activity especially in combination with rifampicin \[[@B5-molecules-19-00651]\].
PZA itself has multiple mechanisms of action. The first described was the activation of this prodrug *via* the enzyme pyrazinamidase (EC 3.5.1.19) to form pyrazinoic acid (POA). This metabolite causes a lowering of the inner compartment pH in mycobacterial cells. This leads to inhibition of membrane transport and then to cellular death \[[@B6-molecules-19-00651],[@B7-molecules-19-00651],[@B8-molecules-19-00651]\]. The gene encoding this enzyme is called *pncA* gene and its mutation is responsible for the origin of mycobacterial resistance to PZA \[[@B9-molecules-19-00651]\].
The second mechanism of action is connected with fatty acid synthase I (FAS I) (EC 2.3.1.85). It is suggested that the disruption of metabolism can be caused by inhibition of the cell membrane synthesis, which is essential for the survival of *Mycobacteria*, but this mechanism was mainly rejected for PZA itself by Boshoff because there is only low inhibition \[[@B10-molecules-19-00651]\]. On the other hand, the PZA analogues such as 5‑chloropyrazinamide, esters of pyrazinoic acid and esters of 5‑chloropyrazinoic acid were proven to act in this way \[[@B11-molecules-19-00651],[@B12-molecules-19-00651],[@B13-molecules-19-00651]\].
Recent research has suggested a novel mechanism of action of PZA - inhibition of *trans*-translation. This process is vital for survival and virulence of *Mycobacteria* and its inhibition leads to blockage of the proteosynthetic apparatus in ribosomes and to cellular death. These assumptions were proven by Zhang *et al.* \[[@B14-molecules-19-00651]\].
Although PZA is the first-line antituberculotic drug, it was found that this molecule has exhibited other interesting biological activities such as antifungal, antibacterial, antiviral and antineoplastic effects \[[@B15-molecules-19-00651],[@B16-molecules-19-00651],[@B17-molecules-19-00651],[@B18-molecules-19-00651],[@B19-molecules-19-00651],[@B20-molecules-19-00651]\].
There is another application of PZA derivatives that can be used in agriculture. The most successful pyrazine derivative diquat-dibromide (6,7-dihydrodipyrido\[1,2-a:2\',1\'-c\]pyrazinediium-dibromide), a non-selective, contact herbicide, which has been used to control many submerged and floating aquatic macrophytes, was found to interfere with the photosynthetic process by releasing strong oxidizers that rapidly disrupt and inactivate cells and cellular functions (at present banned in many EU countries) \[[@B21-molecules-19-00651]\]. Many structural variations of pyrazine compounds with herbicidal properties can be found in the patent literature \[[@B22-molecules-19-00651],[@B23-molecules-19-00651],[@B24-molecules-19-00651],[@B25-molecules-19-00651]\]. However, several pyrazine derivatives were also described as inhibitors of Hill reaction which inhibit photosynthetic electron transport (PET) in photosystem (PS) 2 \[[@B18-molecules-19-00651],[@B26-molecules-19-00651]\]. The site of action of these PET inhibitors in the photosynthetic apparatus was situated predominantly on the donor side of PS2, in the section between oxygen evolving complex and intermediate D^·^, *i.e.*, tyrosine radical (Tyr~D~•) occurring on the 161^st^ position in D~2~ protein. Consequently, these compounds can be considered as PS2 herbicides which could have ultimately adverse effect on plant growth. In general, the PET-inhibiting effectiveness of pyrazine derivatives depends on compound lipophilicity and σ Hammett constants of individual substituents. Hosseini *et al.* studied the electronic and structural descriptors, which are the main factors for the cytotoxicity in the series of substituted *N*-phenylpyrazine-2-carboxamides \[[@B27-molecules-19-00651]\].
This study is focused on preparation of *N*-substituted structural and functional derivative of PZA (5-chloro-6-methylpyrazine-2,3-dicarbonitrile) that was treated with ring-substituted benzylamines using the advantages of a microwave reactor. It should be stressed that this type of syntheses has become popular due to its higher yields, shorter reaction times or solvent savings in comparison with conventional organic syntheses \[[@B28-molecules-19-00651]\]. One of the main advantages is the heating. It is uniform through the volume of the sample and the microwaves usually interact with molecules themselves not vessel sides. Another benefit is connected with the temperature reached by the solvent used. The final temperature is usually far higher than the standard boiling point of the solvent when using over- pressurized systems. It is reached and bypassed in seconds. Improved heating usually leads to higher yields and shorter reaction times. There is one limitation for choosing the conditions. It is the polarity of the solvent when the non-polar solvents cannot be used in the way the polar ones can be. If the polar solvent is used in the reaction, there is a direct coupling of microwaves with molecules. More polar solvents have greater ability to interact with microwave radiation. Using the solvents with low polarity (low absorbers) leads to longer times of heating and reaction. On the contrary, if the reagents themselves are polar it could lower the disadvantages of non-polar solvents. Finally there are new approaches to microwave accelerated methods using ionic liquids or solid phase reactions (adsorption on mineral oxides, phase transfer catalysis, neat reactions) \[[@B29-molecules-19-00651]\]. Microwave assisted condensation in polar solvent is used in this work to accelerate the aminodehalogenation reaction. The conditions for the synthesis were proven experimentally. Antimycobacterial activity of the all prepared compounds was determined and compounds were evaluated also in relation to inhibition of photosynthetic electron transport (PET) in spinach (*Spinacia oleracea* L.) chloroplasts. The structure-activity relationships between the chemical structure and *in vitro* biological activities of evaluated compounds are discussed.
2. Results and Discussion
=========================
2.1. Chemistry
--------------
The starting compound 5-chloro-6-methylpyrazine-2,3-dicarbonitrile and the final compounds **1**--**15** were synthesized according to the general procedure shown in [Scheme 1](#molecules-19-00651-f006){ref-type="scheme"}. The aminodehalogenation reaction of this starting compound and ring-substituted benzylamines yielded a series of 15 secondary amines of which 14 were novel. 5-(Benzylamino)-6-methylpyrazine-2,3-dicarbonitrile (**6**) was previously synthesised by Takematsu *et al.* and the reported melting point was 118--119 °C \[[@B30-molecules-19-00651]\]. The compound we obtained melted at 128.7--130.7 °C. This difference can be caused by the mode of crystallization. All reactions were
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{
"pile_set_name": "PubMed Central"
}
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