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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}
============
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}
=======
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}
=======
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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