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+{"text": "Desmoid tumour is a benign, non metastasising neoplasm characterised by an elevated deposition of organic macromolecules in the extracellular matrix (ECM). The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases involved in the degradation of ECM macromolecules. The MMPs and their natural inhibitors (TIMPs) have been implicated in tumour growth, invasion and metastasis. In this study we provide evidence that the in vitro cultured cell line from desmoid tumour accumulates more collagen fibres in the ECM than healthy fibroblasts.3H-thymidine incorporation, MMP expression by substrate gel zymography and TIMP expression by Western blot analysis.We investigated collagen accumulation by Desmoid fibroblasts showed a reduction in MMP activity and an increase of type I and III collagen and TIMPs compared to normal fibroblasts.The increase in collagen in desmoid fibroblasts was due to inhibited collagen degradation (reduction of MMP activity) rather than to increased collagen synthesis. Adding toremifene, an anti-estrogen triphenylethylene derivate, to desmoid fibroblasts reduced collagen accumulation by decreasing mRNA expression and increasing collagen degradation. Confluent cultures were obtained after 48 h of in vitro maintenance. The cells were cultured for 12 h in MEM. The medium was then discarded to avoid serum factor contamination. Toremifene was dissolved in ethanol and all the cultures were maintained in MEM containing ethanol or MEM containing toremifne in ethanol and treated as described below.Fibroblast cell lines were obtained from patients with Gardner's syndrome and were provided by NIGMS . The GMO 6965 cell line was obtained from phenotypically healthy fibroblasts, and the GMO 6888 cell line was obtained from desmoid fibroblasts. All cell lines were cultured in Eagle's minimal essential medium (MEM)  supplemented with 20% fetal bovine serum (FBS) , 2% non-essential amino acids (GIBCO), 2 mM L-glutamine, 100 U/ml penicillin and 100 U/ml streptomycin in a humidified 5% CONormal (control) and desmoid fibroblasts were cultured for 24 h in MEM and ethanol or MEM containing 1 \u03bcM toremifene in ethanol . Then 50 \u03bcl of sterile 0.4% trypan blue solution  was added to each culture well; cultures were incubated at 37\u00b0C for 15 min. Viable cells (trypan blue negative) and dead cells (trypan blue positive) were counted by a Burker chamber.3H-proline  in the presence or absence of 1 \u03bcM toremifene. In a second set of experiments desmoid fibroblasts were cultured in MEM supplemented with L-ascorbic acid (50 \u03bcg/ml), \u03b2-aminopropionitrile fumarate (50 \u03bcg/ml) for 48 h with or without toremifene. 3H-proline was added for 48 h, for the last 24 and for the last 3 h. At the end of the labelling period collagen was extracted using the method of Webster and Harvey  dCTP (3000 Ci/mM) by random priming (Amersham RPN 1601). Hybridisation was performed at 65\u00b0C overnight using 106cpm/ml probe in the same buffer used for pre-hybridisation. After hybridisation, the nylon membrane was washed twice in 1 mM EDTA pH 8, 20 mM Na2HPO4 pH 7.2 and 5% SDS at 65\u00b0C (5 min each) and then washed twice with 1 mM EDTA pH 8, 20 mM Na2HPO4 pH 7.2 and 1% SDS at 65\u00b0C (5 min each). The filters were stripped and re-hybridised with a GAPDH probe to assess blot loading. For autoradiography the membranes were exposed to Kodak X-Omat film  at -80\u00b0C for 1 day. Autoradiographies were analysed by computerised scanning densitometry. Results are expressed as the ratio of procollagen \u03b11(I)/control GAPDH densitometry signals.Total RNA was isolated from confluent cultures of normal and desmoid fibroblasts maintained for 48 h in MEM alone or supplemented with 1 \u03bcM toremifene using the method of Chomczynski and Sacchi . For NorA 670 bp Eco RI-Hind III cDNA fragment from human pro-collagen \u03b11(I) and a 1.3 kb PstI cDNA fragment from rat glyceraldehyde-3-phosphate dehydrogenase were used as probes in hybridisation .2, 0.02% sodium azide, pH 7.4) and then dialysed overnight against the same buffer. The latent procollagenase was activated with trypsin (10 \u03bcg/ml) and then the trypsin was inactivated with soybean trypsin inhibitor (100 \u03bcg/ml). Acetic acid soluble type I collagen (25 \u03bcl of a solution 2 mg/ml) from bovine skin was incubated with the activated collagenase solution for 24 h. The products of the collagen digestion were separated by electrophoresis using 6% acrylamide gel containing SDS. The gels were stained with 0.25% Coomassie brilliant blue G-250 , destained appropriately  and fixed . The digestion products were quantified with a computerised scanner.Collagenase activity was determined using the method of Khorramizadeh et al., . Conflueg to remove cell debris, dialysed against bidistilled water for 24 h, lyophilised and used for zymography and Western blot analysis as described below.Confluent normal (GMO 6965) and desmoid (GMO 6888) fibroblasts were washed with 0.9% NaCl and cultured for 12 h in serum-free MEM. This medium was discarded to avoid contamination by serum factors and cells were cultured for the next 24 h in MEM and ethanol (control) or in MEM containing 1 \u03bcM toremifene in ethanol . Conditioned media (CM) were collected and centrifuged for 10 min at 350 2, 0.02% Brij-35 and 200 mM NaCl at 37\u00b0C for 24 h. Gels were stained with Coomassie brilliant blue G-250. Proteinase activity, observed as cleared (unstained) regions, was converted to dark regions to better observation of bands.CM were analysed for gelatinases and collagenases by zymography. Samples were separated under non reducing condition on 6% polyacrylamide gels containing 1 mg/ml of gelatin  or 1 mg/ml collagen (Sigma) . In one CM were analysed for type I and type III collagen, MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 by Western blotting using specific monoclonal antibodies. Aliquots of CM, containing 50 \u03bcg of proteins, were separated on SDS-10% polyacrylamide gels under reducing conditions and transferred on to a nitrocellulose membrane. The membrane was blocked with blocking solution  and incubated with the specific monoclonal antibody in antibody solution . Bound antibody was detected with a sheep anti-mouse peroxidase-conjugated antibody in antibody solution. Western analysis was performed using chemiluminescence reagents from Amersham Pharmacia Biotech.Protein concentrations were determined by the Lowry assay  of aliqut-test. Data are expressed as the means \u00b1 SD of four determinations. In other experiments, the results are reported as means \u00b1 SD of three separate experiments, each performed in quadruplicate. Statistical analysis was performed by Student's two-tailed t-test and by analysis of variance (ANOVA) followed by Sheffe F-test.In some experiments, statistical analysis was performed using Student's The amount of dead cells and viable cells in normal fibroblasts, desmoid fibroblasts and desmoid fibroblasts plus toremifene was evaluated after 24 h of in vitro maintenance in the presence of trypan blue Table . Granted3H-proline  mRNA level in normal and desmoid fibroblasts . Toremifene down regulated procollagen mRNA expression by 58% in desmoid cells.Northern blots were performed to analyse procollagen \u03b1Media from normal and desmoid fibroblasts with or without toremifene were analysed by Western blotting to evaluate the presence of type I and III collagen using specific monoclonal antibodies Fig. . Densito1 and \u03b12 chains of type I collagen into their corresponding 3/4 and 1/4 fragments than the collagenase in desmoid fibroblasts  gene expression, which showed mRNA levels were only lower in desmoid cells treated with toremifene. Normal and desmoid fibroblasts expressed different amounts of MMPs. Several studies suggest that MMPs are over-expressed in malignant tumour progression and facilitate both local tumour invasion and metastasis [Desmoid tumour is a benign non-invasive and non-metastasising neoplasm with an abnormal ECM macromolecule deposition which is stimulated by TGF\u03b21 ,23,24. T1 . There iroblasts , in the tastasis ,28. Difftastasis . They matastasis ,30. Nishtastasis  showed Mtastasis ,33. Thertastasis . In thistastasis . Moreovetastasis  which istastasis . Using Wtastasis ; hence, Upregulation in both inhibitors of MMPs may explain why the Desmoid tumour is characterised by an abundant deposition of ECM macromolecules and is neither malignant nor invasive. Toremifene addition to desmoid fibroblasts reduced the accumulation of collagen fibres but its mechanism of action remains unclear. Toremifene increased MMP-1 and MMP-2 activity by 8% and 25% respectively and decreased TIMP-1 by 18%. Despite these modest effects type I collagen degradation in 3/4 and 1/4 fragments increased almost 4-fold.1 was 6-fold higher in desmoid than in normal fibroblasts and that toremifene significantly reduced TGF\u03b21 activity and TGF\u03b21 membrane-receptors [1 because the growth factor enhances organic macromolecule accumulation in the ECM via a reduction in MMP-1 and MMP-2 [1 activity. Further studies on the regulation of MMP activities may clarify the role of toremifene on ECM degradation and provide important clues about pathogenesis of desmoid tumour.Our previous studies showed that TGF\u03b2eceptors . So the nd MMP-2  and an ind MMP-2 , so favoThe author(s) declare that they have no competing interests.CB carried out collagen synthesis, collagenase activity and drafted the manuscript.CL and GB participated in the design of the study and carried out Northern blot analysis.LM and GG carried out RT-PCR, zimography and oestrogen receptor assay.AB and LC carried out Western blot analysis and performed the statistical analysis.PL conceived of the study, and participated in its design and coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Injury to the peritoneum during surgery is followed by a healing process that frequently results in the attachment of adjacent organs by a fibrous mass, referred commonly as adhesions. Because injuries to the peritoneum during surgery are inevitable, it is imperative that we understand the mechanisms of adhesion formation to prevent its occurrence. This requires thorough understanding of the molecular sequence that results in the attachment of injured peritoneum and the development of fibrous tissue. Recent data show that fibroblasts from the injured peritoneum may play a critical role in the formation of adhesion tissues. Therefore, identifying changes in gene expression pattern in the peritoneal fibroblasts during the process may provide clues to the mechanisms by which adhesion develop.In this study, we compared expression patterns of larger number of genes in the fibroblasts isolated from adhesion and normal human peritoneum using gene filters. Contributions of TGF-beta1 and hypoxia in the altered expression of specific genes were also examined using a semiquantitative RT-PCR technique.Results show that several genes are differentially expressed between fibroblasts of normal and adhesion peritoneum and that the peritoneal fibroblast may acquire a different phenotype during adhesion formation. Genes that are differentially expressed between normal and adhesion fibroblasts encode molecules involved in cell adhesion, proliferation, differentiation, migration and factors regulating cytokines, transcription, translation and protein/vesicle trafficking.Our data substantiate that adhesion formation is a multigenic phenomenon and not all changes in gene expression pattern between normal and adhesion fibroblasts are the function of TGF-beta1 and hypoxia that are known to influence adhesion formation. Analysis of the gene expression data in the perspective of known functions of genes connote to additional targets that may be manipulated to inhibit adhesion development. Peritoneal adhesions resulting from surgical injury are often associated with pelvic pain, bowel obstruction and infertility . EpidemiParietal and visceral peritoneum that surfaces the intraperitoneal organs is covered by a layer of squamous epithelial cells, the mesothelium. The submesothelial layer consists of fibroblasts, macrophages and blood vessels. Surgical abrasion to the peritoneum releases mesothelial cells, macrophages, fibroblasts, and blood containing cytokines and several cell types at the site of injury. Coagulation of blood creates a fibrinous mass between injured surfaces. In some patients fibrinolysis of clot followed by proliferation of mesothelial cells covers the wound. In others, failure of fibrinolysis followed by proliferation and migration of fibroblasts into the proteinous mass generates fibrous tissues of adhesion. Consequently, the process of adhesion formation include inflammatory response, fibrin deposition, cell-proliferation, -differentiation, -migration, -death, angiogenesis, extra cellular matrix (ECM) turnover regulated by cytokines, hypoxia, genetic and epigenetic factors .Recent studies illustrate roles of peritoneal fibroblasts in adhesion development -10. It iTissues were collected at the initiation of surgery and after the entry into the abdominal cavity of female patients (25\u201350 years) undergoing laparatomy for pelvic pain as described earlier . All pat2) for 3 days in DMEM (Life Tech.) supplemented with 10% fetal bovine serum (Life Tech.) and antibiotics . The monolayer of cells were passaged in trypsin-EDTA solution (Life Tech.). Cells at 3\u20135 passages were cultured in serum free medium in 75 cm2 flasks  to 75% confluency prior to studies.The primary cultures were maintained in a humidified incubator . Radiolabeled cDNA was separated from the free nucleotides using Bio-Spin 6 chromatography column (Bio-Rad Laboratories). Gene filters were labeled as adhesion or normal fibroblasts and washed with 0.5% sodium dodecyl sulfate (SDS) prior to prehybridization. Individual membrane was transferred to separate roller tubes of the hybridization oven , each containing MicroHyb hybridization solution (Research Genetics) supplemented with Human Cot-1 DNA (Life Technology) and Poly dA (Research Genetics). The membranes were rotated at 10 RPM and at 42\u00b0C for 2 h. Radio-labeled cDNA prepared from adhesion and normal fibroblasts total RNA was denatured by heating in a boiling water bath for 3 min. The denatured probes were then injected into the prehybridization solution containing respective membrane. The membranes were hybridized with respective probe for 18 h at 42\u00b0C. The hybridization solution was then replaced with washing solution (2 \u00d7 SSC containing 1% SDS). The temperature of the oven was raised to 50\u00b0C and RPM of rotors was increased to 15. Membranes were washed for 20 minutes when washing solution was replaced with a batch of fresh and prewarmed (50\u00b0C) washing solution. Washing was continued for additional 20 min. A third wash was performed with 0.5 \u00d7 SSC solution containing 1% SDS at 55\u00b0C for 15 minutes. Membranes with cDNA spots facing up were covered with Saran wrap and exposed to phosphor screen (Kodak) for overnight. The screen was scanned with a Phosphor Imager . After acquisition of signal intensities from the normal and adhesion fibroblasts of one patient, filters were stripped according to protocol and subjected to gene filter experiments with the RNA samples from a second patient and images were scanned. Tiff images obtained from normal and adhesion fibroblasts of two patients were analyzed using Pathway 4 software (Research Genetics) for the identification of differentially expressed genes between the normal and adhesion fibroblasts of each patient.Total RNA was isolated from monolayer of fibroblasts at 12 h of culture in serum free medium using Trizol reagent (Invitrogen Inc.). Human Gene Filters  containing 4325 known human cDNA spots were used for the identification of differentially expressed genes between adhesion and normal fibroblasts from human peritoneum. Method suggested by the manufacturer was strictly followed. In brief, 10 \u03bcg of total RNA from monolayer cultures of fibroblasts were subjected to cDNA synthesis in presence of 10 \u03bcl 2, 0.2 mM dNTP, 0.5 U Taq Polymerase  and 1 \u03bcM each of sense and antisense primers. Primer sequences were determined using Primer3 software from the Internet . The control primers (sense 5'-ggaggttcgaagacgatcag-3' and antisense 5'-cgctgagccagtcagtgtag-3') were expected to provide an amplicon of 509 bp from human 18S ribosomal subunit cDNA (gi: 337376). Accession numbers of genes of interests are provided in Table Steady-state levels of mRNA of selected genes that are known to have a role in cellular adhesion, proliferation, migration, apoptosis and demonstrating different expression levels between adhesion and normal fibroblasts in the gene filter experiments were verified further by a previously described semiquantitative RT-PCR method . Total R. Band intensities were plotted to determine the linearity of PCR reactions for the amplification of target transcripts. Target cDNA were amplified by PCR from normal and peritoneal fibroblasts at specific PCR cycle within its linear range of amplification. Total RNA samples from normal and adhesion fibroblasts of 4 patients  were used for the RT-PCR experiments. Optical densities obtained from amplicons of 4 patients  were used to derive mean \u00b1 standard error of mean values representing relative levels of each mRNA species in normal and adhesion fibroblasts.Initially cDNA of interests were amplified from normal peritoneal fibroblasts at different (25 to 35) PCR cycles. PCR products were subjected to agarose gel electrophoresis. Molecular weight marker  were also loaded in adjacent lanes. DNA in the gel were stained with 1:10,000 dilution of SYBR Green I dye  and photographed using a DC 120 Kodak digital camera  for the verification of size and analysis of band intensity using Image J software 1  or hypoxia (2% Oxygen) for 24 h. Control plates were cultured for the same duration in absence of TGF-\u03b21 or hypoxia. Total RNA was isolated from the control, TGF-\u03b21 and hypoxia treated cells and subjected to RT-PCR reactions as described above to determine relative levels of 18S ribosomal subunit and gene specific transcripts in the control and treated cells. RT-PCR experiments were conducted twice with the normal peritoneal fibroblasts isolated from 3 patients to have six control, six TGF-\u03b21 treated and six hypoxia exposed amplicons. This included normal fibroblasts from one new patient and two patients that were used exclusively for RT-PCR experiments for the confirmation of gene array data. Optical densities of amplicons from six control or treated cells per mRNA species were used to derive the mean \u00b1 standard error of mean values for comparison.Effects of TGF-\u03b21 or hypoxic conditions on the steady state levels of specific gene transcripts in the normal peritoneal fibroblasts were also studied to examine the possibility of adhesion causing factors potentiating the gene expression pattern in the normal fibroblasts similar to adhesion fibroblasts. Normal peritoneal fibroblasts were cultured in six well culture plates (FALCON). When confluent, monolayer of cells in culture were exposed to 1 ng/ml TGF-\u03b2Band intensity value of each RT-PCR experiment  was used to derive Mean \u00b1 Standard error of Mean using Statview 4.5 software . Differences between Means were tested for significance by one-way analysis of variance with the specific post hoc test using the same software to compare differences in the steady state levels of different mRNA species.Hybridization intensities of radio labeled cDNA from normal or adhesion fibroblasts from both the patients were different when analyzed using Pathway software. Comparison of hybridization intensities from individual gene spots between normal and adhesion fibroblast RNA Figure  demonstrGene filter data from two patients showed similar expression pattern of collagen type 1 alpha 2), Collagen type III , fibronectin 1, Matrix metalloproteinase-1 (MMP-1), Transforming Growth Factor beta-1 (TGF-\u03b21), TGF-\u03b22 and tissue plasminogen activator as reported earlier using multiplex PCR technique  interacts with laminin1 P1, collagen I, collagen IV, perlecan and fibulin-2 in the extracellular space and stabilizes the basement membrane. It also interacts with \u03b16\u03b21 and \u03b13\u03b21 integrin receptors on cells . RelativTM4SF molecules (tetraspanins) play important roles in cell migration and in the generation of complexes with integrins functionally relevant for cell motility, tumor progression and wound healing . It is pVersicans (CSPG2) are also known to influence \u03b14\u03b21 and \u03b12\u03b21 integrin mediated invasion of melanoma cells . Higher Because MT-1e transcripts are detected in cell types that have undergone myoepithelial differentiation , signifiContrary to increase in the above mentioned mRNA species in the adhesion fibroblasts, steady state levels of IGFBP3 precursor transcript were found to be lower. Because IGFBP-3 is known to inhibit cell growth by sequestering IGF, its decreased level may enhance proliferation of adhesion fibroblasts . ReducedOur attempts to examine the regulatory roles of TGF-\u03b21 and hypoxia, factors known to promote adhesion development , on the 2 (PGE2) is shown to inhibit TGF-\u03b21 induced expression of \u03b1-SMA, production of Collagen I and the transformation of fibroblasts to myofibroblasts via EP2 signaling [P311 (PTZ17) and Interferon \u03b3 treatments, which inhibits TGF-\u03b21 induced myofibroblast transformation [It is known that a new phenotype of fibroblasts is induced during wound healing. These fibroblasts, termed- myofibroblasts, express higher levels of \u03b1-smooth muscle actin and vinculin-containing fibronexus adhesion complexes . Fibroblignaling . Additioormation ,47.During the course of normal wound healing, myofibroblasts disappear, possibly by apoptosis . In cont2, EP2 blockers, interferon \u03b3, P311 and applying measures to induce apoptosis in the peritoneal fibroblast at the site of injury. The obvious question \u2013 \"how to maintain apoptosis at a desired level for normal peritoneal healing?\" however, remains to be answered.It is evident from our study that steady state levels of several genes are different between adhesion and normal peritoneal fibroblasts in human and that adhesion development may be a function of several genes. Changes in the functional interdependence of these genes at the site of injury may transform normal peritoneal fibroblast into cell type/s with altered phenotype. These cells- designated as adhesion fibroblasts may mimic previously known myofibroblasts and are highly proliferative. These cells resist apoptosis and secrete ECM molecules to renovate basement membrane. With changed expression pattern of cell surface molecules these cells may respond to intracellular signaling for migration over the fibrin clot. This altered nature of adhesion fibroblasts therefore may play a major role in the formation of the fibrous mass of adhesion-tissue that bridges adjacent and injured peritoneum. Blocking changes in the expression or function of genes necessary for this transformation of normal peritoneal fibroblasts may curtail adhesion formation. This could be achieved by the application of PGECollagen type IV  chain (COL4A2), Nidogen 2 (NID2), Chondroitin sulfate proteoglycan 2 (CSPG2), S100 Calcium binding protein A10 (S100A10), Transmembrane 4 superfamily member 1 (TM4SF1), Metallothioneine (hMT-Ie), Insulin-like growth factor binding protein 3 precursor (IGFBP3), Transforming growth factor (TGF), Prostaglandin E2 (PGE2), Urokinase Plasminogen activator receptor (uPAR), Annexin 2 and S100A10 complex (AIIt), tissue Plasminogen Activator (tPA), Plasminogen Activator Inhibitor (PAI), Cyclooxigenase (COX), Matrix metalloproteinase (MMP), Tissue Inhibitor of Metalloproteinase (TIMP), Interferon \u03b3 (IFN-\u03b3), IL (Interleukin).GMS and MPD were responsible for the isolation of peritoneal fibroblasts from normal peritoneum and adhesion tissues as well as establishing hypoxia chambers. MPD provided patient information and valuable suggestions during writing the manuscript. UKR performed microarray and semiquantitative RT-PCR experiments, analyzed the data and wrote the manuscript."}
+{"text": "We have previously shown that the bronchodilatory effect of deep inspiration is attenuated in individuals with COPD. This study was designed to investigate whether the impairment in this effect is associated with loss of alveolar attachments.We measured deep inspiration (DI)-induced bronchodilation in 15 individuals with and without COPD  undergoing lobar resection for peripheral pulmonary nodule. Prior to surgery, we measured TLCO and determined the bronchodilatory effect of deep inspiration after constricting the airways with methacholine. The number of destroyed alveolar attachments, as well as airway wall area and airway smooth muscle area, were determined in tumor-free, peripheral lung tissue.The bronchodilatory effect of deep inspiration correlated inversely with the % destroyed attachments  and directly with the airway smooth muscle area , but not with the total wall area .We postulate that attenuation of airway stretch due to loss of alveolar attachments contributes to the loss of the bronchodilatory effect of lung inflation in COPD. We have recently demonstrated that the ability of deep inspirations to dilate constricted airways is impaired in subjects with COPD . We haveWhen a deep inspiration takes place, radial traction is applied to the outer airway walls by virtue of the forces of interdependence between the airways and the surrounding parenchyma , which aThe loss of the bronchodilatory effect of lung inflation in COPD may result from factors that unlink the parenchyma from the airways. COPD is accompanied by destructive changes of alveolar walls and consequent reduction in the number of alveolar attachments on the airways -7. We poIn order to obtain lung tissue for morphometric and correlative analyses, we enrolled subjects undergoing lobar resection for peripheral pulmonary nodule, with the assumption that a significant proportion would have been smokers and would presumably show evidence of reduced integrity of alveolar attachments . SubjectPrior to surgical lung resection, each subject underwent a clinical evaluation and a complete respiratory functional assessment that included spirometry, and determinations of lung volume and of the single-breath CO diffusing capacity.The clinical evaluation included a questionnaire that derives from the IUALTD  bronchial symptom questionnaire  and a phIn a series of subsequent visits, the bronchodilatory effect of deep inspirations was determined, as previously described ,11. Inha1 and FVC. At baseline, 3 acceptable combined partial/maximal forced expiratory maneuvers were performed, and the best was retained for analysis. Subjects were then instructed to abstain from taking deep breaths for a period of 20 minutes. Thereafter, the single dose of methacholine was administered as five tidal breaths followed, 3 minutes later, by a single partial/maximal combined spirometric measurement, as described above. If the targeted reduction in IVC (at least 15%) was not attained, another single dose challenge was performed. This was done during the same session (at one hour) if the reduction in IVC was within 5% of baseline, or postponed to the next day. Single dose provocations with increasing doses were conducted in this manner until the expected level of reduction in IVC was reached. The provocation in which the targeted reduction in IVC from baseline was attained was extended with 4 deep inspirations taken immediately after the post-methacholine IVC spirometry. Following these deep inspirations, another IVC maneuver was performed. The difference between the IVC obtained after the 4 deep inspirations and the post-methacholine IVC that preceded the 4 deep inspirations was used to calculate the bronchodilatory effect of deep inspirations. This was expressed as percent change from the post-methacholine IVC (% bronchodilation).To measure IVC, the subject forcefully expired from end-tidal volume to residual volume  and immediately inhaled to total lung capacity (IVC = TLC \u2013 RV). The maneuver continued with a forced expiration to RV. This forced expiration allowed us to also calculate FEV1, is not influenced by a lung inflation maneuver because RV is reached through a partial forced expiration. FEV1 and FVC data were utilized in secondary analyses.We have used IVC in studying the effects of deep inspiration because, assuming that TLC does not change , the priFor tissue morphometry, we applied the methodology previously described by Saetta and colleagues . Two to e), the internal perimeter along the subepithelial basement membrane (Pbm), the lumenal diameter (Dl), the external area (Ae), the internal area (Ai), and the muscle area (WAm) were evaluated. The thickness of the nonrespiratory bronchioles  was obtained by the difference between the external area and the internal areas (WA = Ae \u2013 Ai). Values of Dl, WAm and WAtot were normalized by dividing for Pbm [All airways with internal diameter \u2264 2 mm were retained for analysis. Non-respiratory bronchioles with incomplete walls at the edges of the sections or with a short/long diameter ratio < 1/3 were excluded. After this selection, each patient had at least four non-respiratory bronchioles suitable for morphometry. In each airway, the external perimeter  and the morphometric variables. Unpaired t-tests were used to assess differences between groups. In all analyses, two-tailed values of p = 0.05 were considered statistically significant.A total of fifteen subjects took part in the study . Seven of them had a diagnosis of COPD, confirmed by our clinical and functional evaluation. No subject received a diagnosis of asthma. Eleven out of the 15 subjects were smokers . None of the non-smokers received the diagnosis of COPD. Baseline lung function and lung tissue morphometric characteristics for each individual are presented in Table The median single methacholine dose required to induce the targeted reduction in IVC, in the absence of deep inspiration, was 25 mg/ml (range: 0.025\u201375 mg/ml). The % reduction in IVC in the protocol devoid of deep inspiration was 20 \u00b1 1.8% (mean \u00b1 SEM). The % bronchodilation by deep inspiration was 4.3 \u00b1 2.1% with a range of -13% to 18%.We found a significant inverse correlation between the bronchodilatory effect of deep inspiration and the percentage of destroyed alveolar attachments . Overall, bronchodilation was substantially reduced in this group (4.3 \u00b1 2.1%), compared to an average value of around 20% that we would have expected in healthy individuals of the same age, based on our previously published data . It is a8 to -13%The lack of correlation between the thickness of airway wall and the attenuation of the bronchodilatory effect of deep inspiration is also in agreement with the report by Corsico and colleagues , and indThe results of our study support the hypothesis that the attenuation of airway stretch due to loss of alveolar attachments represents an important cause for the impairment in the bronchodilatory effect of lung inflation in COPD. Whether the progressive impairment of the beneficial effect by deep inspiration has clinical and prognostic implications in these subjects needs to be addressed in future studies.Dr. Togias' participation in this work was supported by NIH grant RO1 HL61277.The authors declare that they have no competing interest.NS conceived and designed the study, performed the clinical and functional assessments, analyzed and interpreted the data, drafted the manuscript; AB carried the morphometric assessment and participated to the interpretation of the findings; RM carried the clinical and functional assessments and participated to the interpretation of the data; AMV participated to the design and the coordination of the study and the interpretation of the results; AT conceived and participated to the design of the study, the analysis of the data and the interpretation of the results, and contributed significantly to draft the manuscript; VB helped in the design and the organization of the study, as well as the interpretation of the results."}
+{"text": "Despite intensive studies, the clinical opportunities for patients with fibrosing liver diseases have not improved. This will be changed by increasing knowledge of new pathogenetic mechanisms, which complement the \"canonical principle\" of fibrogenesis. The latter is based on the activation of hepatic stellate cells and their transdifferentiation to myofibroblasts induced by hepatocellular injury and consecutive inflammatory mediators such as TGF-\u03b2. Stellate cells express a broad spectrum of matrix components. New mechanisms indicate that the heterogeneous pool of (myo-)fibroblasts can be supplemented by epithelial-mesenchymal transition (EMT) from cholangiocytes and potentially also from hepatocytes to fibroblasts, by influx of bone marrow-derived fibrocytes in the damaged liver tissue and by differentiation of a subgroup of monocytes to fibroblasts after homing in the damaged tissue. These processes are regulated by the cytokines TGF-\u03b2 and BMP-7, chemokines, colony-stimulating factors, metalloproteinases and numerous trapping proteins. They offer innovative diagnostic and therapeutic options. As an example, modulation of TGF-\u03b2/BMP-7 ratio changes the rate of EMT, and so the simultaneous determination of these parameters and of connective tissue growth factor (CTGF) in serum might provide information on fibrogenic activity. The extension of pathogenetic concepts of fibrosis will provide new therapeutic possibilities of interference with the fibrogenic mechanism in liver and other organs. Experimental and clinical studies of the past twenty years or so provide a detailed knowledge of structure and composition of extracellular matrix (ECM) in normal and fibrotic liver tissue ,2, of the.g., degree of hydroxylation of prolin and lysin), glycoproteins (variations of the carbohydrate structure) and proteoglycans (changes of the degree of sulfation of the glycosaminoglycan side chains)  are the most sensitive and fibrogenesis, e. g., in alcoholic liver injury, starts here first [1 (I), respectively) differentially indicating functional heterogeneity [Fibrosis is characterized by a severalfold increase of the extracellular matrix that comprises collagens, structural glycoproteins, sulphated proteoglycans and hyaluronan, by a histological redistribution with preferred initial matrix deposition in the subendothelial space of Disse leading to the formation of an incomplete subendothelial basement membrane creating additional diffusion barriers between hepatocytes and the liver sinusoid (\"capillarization of sinusoids\"), and by changes in the microstructure of collagens (ns) Fig. . It is kns) Fig. . As a cons) Fig. . HSC areat liver . The spiat liver . About 8at liver -21, as Cd repair , they ard repair ,24, in sd repair ,26 and gd repair , in the d repair , in regud repair , and in d repair -32. Somed repair , which id repair -38. The d repair , is reled repair . In the d repair , but alsd repair ,41-44, sd repair ,34,37 tod repair , the stid repair  phagocytd repair , and leud repair . In addid repair -52 and td repair  promote d repair -56. Addid repair  to the ed repair . The matd repair ,60 initid repair ,62, and d repair ,64. Extrlability . Antagonlability  or inhiblability  leads tolability . This ob process . The act process . The mod process . Accordiagonists , trichosferences . Thus, cferences . Due to ferences -76, the re first . The hetre first . As an ere first ,80. Usinogeneity . Taken togeneity .e.g., bone marrow stem cell therapy). It was recently extended for HSC and (myo-)fibroblasts under experimental and clinical conditions. By transplantation of genetically tagged bone marrow or of male bone marrow (Y-chromosome) to female mice, it was demonstrated that up to 30% of HSC in the liver originate from the bone marrow and acquire the MFB phenotype under injurious conditions [4-cirrhotic mice liver derive from the bone marrow [+ (haematopoietic origin), CD34+ (progenitor cell), and type I collagen+ (capability of matrix synthesis) [Several studies have pointed to the bone marrow as a source of immature, multipotent cells in various organs. Bone marrow cells have the capacity to differentiate to hepatocytes, cholangiocytes, sinusoidal endothelial cells, and Kupffer cells, if the adequate micro-environment of the liver is present ,84. Thisnditions . Anothere marrow . Even ine marrow . Anothere marrow . They arnthesis) , and exhnthesis) . Fibrocynthesis) . The mobnthesis) , which inthesis) . This menthesis) . Activatnthesis) . The homnthesis) .i.e., myocardial infarction). Investigations over the last years have proven that peripheral blood monocytes can be differentiated in vitro to hepatocyte-like cells if they are exposed with macrophage-colony stimulating factor (M-CSF) and specific interleukins (monocyte-derived neo-hepatocytes) [e.g., of the lung and kidney. The differentiation is positively influenced by G-CSF, M-CSF, monocyte chemotactic peptide 1 (MCP-1), and other chemokines and haematopoietic growth and differentiation factors, which are expressed and secreted by activated HSC [+ and CXCR4+ (a chemokine receptor) [Recent studies indicate a highly developed multi-differentiation potential of a subgroup of circulating blood monocytes, which can be recruited quickly for tissue repair processes . In additocytes) ,97. Althated HSC ,98-100 aated HSC . It is oated HSC  and, conated HSC . C-reacteceptor) . G-CSF aseptum transversum mesenchyme, from endoderm or from the mesothelial liver capsule [e.g., in kidney and lung [In vitro and in vivo observations made in blood vessels following sustained inflammation support a hypothesis that endothelial cell transformation to myofibroblast-like cells may explain the increase of matrix proteins and of MFB pathognomonic of fibrotic diseases [in vitro and of the mouse hepatocyte cell line AML12 [1(I) collagen mRNA expression and type I collagen deposition. Thus, hepatocytes are capable of EMT changes and type I collagen synthesis. A further source of EMT are cholangiocytes . In primary biliary cirrhosis (PBC) it was proven that bile duct epithelial cells express FSP-1 (S100A4) and vimentin as early markers of fibroblasts [In vitro studies with cultured human cholangiocytes have confirmed the clinical observations described. Thus, EMT proves to be a general pathogenetic principle of chronic cholestatic liver diseases [Beside activation and transdifferentiation of HSC, a cell type, which is developmentally most likely derived from the  capsule , an incrand lung . This prand lung  Fig. 7)septum trand lung . Similardiseases . Very rene AML12 . The EMTroblasts . The biddiseases . In addidiseases  turned odiseases ,113. Thidiseases .e.g., proteinkinase A [2-macroglobulin change the balance between TGF-\u03b2 and BMP-7 in favour of an anti-EMT effect due to binding a neutralization of TGF-\u03b2 [e.g., vascular endothelial cells to fibroblasts), and the fine tuning of the bioactive TGF-\u03b2/BMP-7 ratio and of their adaptor- and trapping proteins offer multiple regulatory possibilities of influencing fibrogenesis. These mechanisms are known in some detail for the kidney [The molecular inducers of EMT are TGF-\u03b2 , epidermkinase A  and epidkinase A . Thus, Ekinase A . It has kinase A . BMP-7 ikinase A , which tkinase A . In addiof TGF-\u03b2 . Similarof TGF-\u03b2 , which iof TGF-\u03b2 ,124 chanof TGF-\u03b2 . CTGF isof TGF-\u03b2 -128, whiof TGF-\u03b2 . CTGF inof TGF-\u03b2 . This isof TGF-\u03b2 ,131. Thue kidney , but neeNewly recognized pathogenetic mechanisms of fibrosis described above provide several innovative options for therapy of liver fibrogenesis and non-invasive diagnostic strategies Table . The detThe determination of CTGF in serum or plasma is suggested as a further innovative parameter of fibrogenesis, since this modulator protein is strongly up-regulated in the fibrotic liver, synthesized and secreted by parenchymal and non-parenchymal cells  and sinc+, CD45+, and collagen I positivities as identifying markers might be a way for evaluation of their diagnostic potential. Alternatively, these antigens might be detected by amplifying their mRNA using a quantitative PCR approach. In addition, a re-evaluation of the high concentrations of G-CSF, GM-CSF, and M-CSF in serum of cirrhotic patients published previously [The flowcytometric detection of circulating fibrocytes in blood or in buffy coat leucocytes by using CD34eviously  as mobileviously  might bei.e., on tumor development and progression, auto-immunopathy and degenerative diseases [Numerous publications discuss anti-fibrotic therapeutic strategies by inhibition of TGF-\u03b2 ,136-138,diseases . Therefodiseases , stimuladiseases , and inhdiseases . Similardiseases . Presentdiseases ,131 can diseases  \u2013 are indiseases ,131. In e.g., primary biliary cirrhosis might activate a pathogenetic pathway different from alcoholic fibrosis. These facts point to the important notion that results obtained with various models of experimental fibrogenesis cannot be generalized because different classes of (myo-)fibroblasts are generated by diverse pathways. Furthermore, HSC-activation in culture cannot be regarded any longer as the almost dogmatic paradigm of the liver fibrogenic mechanism as it was in the past. Since now detailed information on the molecular cascades of intracellular fibrogenic signaling is available, we have learned that several of them are modulated cell-type specifically. Therefore, it is conceivable that distinct subpopulations of fibroblasts and their transient precursor cell types respond differently to major fibrogenic cytokines, e.g., TGF-\u03b2. If this is the case, the complexity of the fibrogenic mechanisms will increase strongly in the future and the experimental conditions have to be described in detail. Taken together, studies on fibrogenesis in the liver (and other organs as well) are now pushed forward a lot, hopefully resulting in new impulses for therapy and diagnosis.The above described changing view on the pathogenetic mechanisms of liver fibrosis clearly suggests that one has to reconsider the exclusive role of HSC in the development of fibrosis. Although some of the newly proposed fibrogenic mechanisms have to be consolidated by additional experimental evidence in vitro and in situ, they indicate the presence of distinct subpopulations of myofibroblasts/fibroblasts in fibrosing liver, of which HSC-derived fibrogenic cells are only one of several sources. Most important, the composition of (myo-)fibroblasts may vary with the etiology of fibrosis, The author(s) declare that they have no competing interests.All the authors contributed equally to this work. All authors read and approved the final manuscript."}
+{"text": "It has been previously shown that cultured granulosa cells (GCs) derived from human ovarian preovulatory follicles contain choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine (ACh) synthesis. They also produce ACh and express functional muscarinic ACh receptors. ACh can act on GCs to increase proliferation, disrupt gap junctional communication, alter intracellular calcium levels, as well as expression of transcription factors, suggesting an unrecognized role of ACh in GC function. To gain further insights into the possible role of ACh in the ovary, we examined ChAT expression in the gland before and after birth, as well as in adults, and studied the regulation of ACh production by FSH.ChAT immunohistochemistry was performed using ovarian samples of different species and ages . ACh was measured by HPLC and/or a fluorescence based method in rat ovaries and in a FSH receptor-expressing cell line (rat GFSHR-17) cultured with or without FSH.In adult rat, as well as in all other species, ovarian ChAT immunoreactivity is associated with GCs of antral follicles, but not with other structures, indicating that GCs are the only ovarian source of ACh. Indeed ACh was clearly detected in adult rat ovaries by two methods. ChAT immunoreactivity is absent from embryonic and/or neonatal ovaries (mouse/rat and monkey) and ovarian development in embryonic mice null for ChAT appears normal, suggesting that ACh is not involved in ovarian or follicular formation. Since ChAT immunoreactivity is present in GCs of large follicles and since the degree of the ChAT immunoreactivity increases as antral follicles grow, we tested whether ACh production is stimulated by FSH. Rat GFSHR-17 cells that stably express the FSH receptor, respond to FSH with an increase in ACh production.ACh and ChAT are present in GCs of growing follicles and FSH, the major driving force of follicular growth, stimulates ACh production. Since ACh stimulates proliferation and differentiation processes in cultured GCs, we suggest that ACh may act in the growing ovarian follicle as a local mediator of some of the actions ascribed to FSH. Several publications have challenged the concept that acetycholine (ACh) in the ovary derives from a cholinergic innervation of the gland see -5]. Inst. Inst5]]Ca [Ca channels are necessary for hCG to exert its stimulatory action on steroid production [Furthermore, ChAT protein was detected in GCs of large antral follicles by immunohistochemisty ,7-9. TheCa ). Data foduction . It is toduction ,13. Releoduction ,14,15.Whether ACh production is restricted to preovulatory follicles and how the production of ACh by antral follicles is regulated, remain unknown . We haveParaffin-embedded, fixed human ovaries (n = 3) were from patients undergoing gynecological surgery. The procedures were approved by the local Ethics Committees and patients gave written consent to the use of tissues. Additional samples (n = 4) were from a tissue bank, as described . SectionMonkey ovarian samples (obtained at necropsy or during surgery for unrelated reasons at the ONPRC-OHSU) were from postnatal day 0, 1, and 3\u201317 years of age, total of 10 samples). The care and housing of rhesus macaces (Macaca mulatta) at the ONPRC was previously described ,17. AnimMouse and rat ovarian samples used  17, 18, 20, postnatal day 0, day 1 and adult) were derived from previous studies [ChAT knockout (-/-) mice were generously provided by Fred Gage and Kuo-Fen Lee . Mice weThe ABC method and DAB as a chromogen were used for immunohistochemistry, while FITC-labeled secondary antiserum was used for immunofluorescence. All procedures were performed as described ,11,20,22We studied whether ACh is present in adult rat ovaries. To this end ovaries from 11 adult rats, used in unrelated experiments, were examined. Animals were kept at the animal house of the Technical University of Munich and killed according to NIH/local animal care guidelines.Ovaries were rapidly removed, frozen on liquid nitrogen and ACh was either determined using the HPLC method, as described previously  or a 2O2 resulting from this reaction, interacts with Amplex Red (7-dihydroxyphenoxazine) in the presence of horseradish peroxidase to form the highly fluorescent resorufin. The samples were transferred into a 96-well plate and measured in a fluorescence plate reader  every 5 min for 40 min. All measurements were performed in triplicates. As this assay detects not only ACh, but also choline, we measured each sample in the absence of AChE, as well. Sensitivity of the assay was as low as 0.3 \u03bcM, with a range of detection from 0.3 \u03bcM to 100 \u03bcM ACh. For quantification purposes, ACh solutions in the range of 0 \u03bcM to 3 \u03bcM were measured, and a calibration curve was generated from the values obtained. The ratio of fluorescence intensities obtained in the presence of AChE divided by those in its absence was 1.2 \u00b1 0.1 . A value larger than one indicates that ACh was present in the samples. The choline detected by the assay can derive from endogenous sources and/or originate from non-enzymatic hydrolysis of ACh and/or from enzymatic hydrolysis of ACh by intrinsic esterase activity of the tissue. Thus, to semi-quantitatively assess ACh concentrations we subtracted the choline values obtained in absence of AChE from those in its presence.Briefly, in this assay ACh is hydrolyzed by ACh esterase (AChE), the choline formed is then oxidized by choline oxidase, and the HGFSHR-17 cells are derived from antral rat follicles, but are not able to produce estrogens . Yet, thChAT immunoreactivity, as expected, was observed in human, monkey and rat adult ovaries only in GCs of large antral follicles, but not in small follicles see Fig.  and 2. IFurthermore ovarian development in embryonic mutant mice null for ChAT was normal with respect to organ size and cellular composition, ruling out an involvement of ACh in the early ovarian development Fig. .Expression of ChAT in adult rat ovaries is associated with the presence of ACh (n = 6); the mean value was 2.47 \u00b1 0.90 pmol (mean \u00b1 SEM) as detected by a HPLC technique. The presence of ACh in rat ovaries was corroborated using a fluorescence technique (n = 5), and the values obtained ranged between 22 and 60 pmol (mean \u00b1 SEM: 43 \u00b1 7 pmol).Since follicular growth and GC development depend on FSH, we hypothesized that ACh production may be stimulated by the gonadotrophin. To test this possibility we determined the production of ACh in the rat GFSHR-17 cell line challenged with pFSH for 24 h. This cell line stably expresses the FSH receptor and is immunoreactive for ChAT Fig. .The present results demonstrate (1) the presence of ACh in adult rat ovary and (2) indicate that GCs of growing antral ovarian follicles in both rodents and primates are the only detectable structures, which are immunoreactive for ChAT, implying that GCs are the source of ovarian ACh. Furthermore (3) our results indicate that ChAT staining increases in GCs of growing antral ovarian follicles in both rodents and primates and (4) show that FSH stimulates production of ACh in cells derived from an antral follicle. Finally, (5) since ChAT is absent from embryonic and neonatal ovary and embryonic development in ChAT-/- mice appears normal, these data support the hypothesis that ACh is a signaling molecule important for the antral follicle, which is produced by non neuronal cells of the ovary under gonadotrophin control.A role of ACh in the ovary has only recently been suggested, based on studies with human GCs . These cWe confirmed that in rodents and primates only growing follicles express ChAT, and thus, are likely to produce ACh. The ChAT protein is not found in embryonic and neonatal ovary and is first detected in follicles that are beginning to develop an antrum, i.e. a stage supported by FSH, which drives subsequent follicular growth ,13,24. SThe increased ChAT immunoreactive signal detected in larger antral follicles, as compared with smaller antral follicles, suggested that ACh production may be regulated by FSH. This conclusion is supported by the results of our studies with immortalized GCs, which stably express functional FSH receptors , as thesWe clearly detected ACh itself by two techniques in homogenates from adult rat ovaries, in which ChAT immunoreactive GCs are present. Preliminary unpublished studies with immature rat ovarian homogenates (from ovaries of days 1\u20136) have sofar indicated that ACh in these samples is below detection limits. These results are consistent with the observed and documented lack of ChAT in the neonatal ovary (present study). More detailed studies, which are beyond the present report, are however required to examine ovarian ACh levels during sexual maturation and correlate them with ovarian and follicular development.When measuring ovarian ACh content in adults we did not differentiate between ovaries bearing differently sized follicles or corpora lutea and the results obtained reflect a thus expected heterogeneity. Furthermore the two different methods employed provided somewhat different absolute values. Several reasons may account for these differences, which have to do with the principles of the assays used (HPLC detection versus enzymatic assay). Furthermore, it is possible that the magnitude of the changes is, at least in part, determined by changes in ACh metabolism. ACh is a very labile molecule with a very short half life time. Our preliminary data from fluorescence ACh assays strongly suggests presence of esterases degrading ACh, and preliminary Western blot data implicates butyrylcholine esterase ,26 as onACh has only recently been recognized as a \"cyto-transmitter\" that, produced by non-neuronal cells, can act throughout the body in an autocrine and/or paracrine fashion (see ). It is ACh and ChAT are present in GCs of growing follicles and the hormone FSH, the major driving force of follicular growth, stimulates ACh production. Since ACh stimulates proliferation and differentiation processes in cultured GCs (see summary in -3), we sThe author(s) declare that they have no competing interests.AM conceived of the study, coordinated the work, analyzed the data, and drafted the manuscript. BP and ES were responsible for breeding and genotyping ChAT null and wild-type mice and dissecting of the ovaries. They also contributed to the writing of the paper. AA provided GFSHR-17 cells and contributed to the manuscript. GAD and SRO collected monkey ovaries and made major contributions to writing of the manuscript. AK was involved in cell culture experiments and together with LK developed and performed all fluorescence ACh measurements, and both were involved in drafting of the mansucript. IW performed all HPLC ACh measurements, helped in analysis of the data and writing of the paper. All authors read and approved of the manuscript."}
+{"text": "This study was designed to compare two different modalities of TBI which are currently used in clinical practice. The same dose of 750 cGy was given to CBA mice either in a single dose at a low dose rate (4 cGy min-1) (STBI) or in a fractionated regimen (six fractions of 125 cGy three times a day) at a higher dose rate (25 cGy min-1) (FTBI). After TBI completion we simultaneously studied the in vivo radiation response of bone marrow cells, two murine bone marrow clonogenic cells (CFU-S and GM-CFC) and peripheral blood lymphocytes and granulocytes for a period of 1 month. The percentage of spleen erythrocytic and granulocytic colonies was also determined. No significant differences were observed between the two groups in the first 48 hours after irradiation except in bone marrow cell numbers, probably due to differences in the overall treatment time between the two TBI schedules. After the first 48 hours the repopulation patterns of the different cells were very similar in both groups. These findings suggest that the different dose rates and fractionation used in this study caused similar radiation damage to the murine haemopoietic system. Moreover, no significant repopulation occurred during the longer overall treatment time of the fractionated regimen. These preliminary results must be corroborated with a larger range of doses before any firm conclusion can be drawn."}
+{"text": "P < 0.01 compared with vehicle). Treatment with IL-4 or interferon-\u03b3 was without effect, and there was no difference in 11\u03b2-HSD1 expression between fibroblasts (from any site) obtained from patients with rheumatoid arthritis or osteoarthritis. In the presence of 100 nmol/l cortisone, IL-6 production \u2013 a characteristic feature of synovial derived fibroblasts \u2013 was significantly reduced in synovial but not dermal or bone marrow fibroblasts. This was prevented by co-treatment with an 11\u03b2-HSD inhibitor, emphasizing the potential for autocrine activation of glucocorticoids in synovial fibroblasts. These data indicate that differences in fibroblast-derived glucocorticoid production (via the enzyme 11\u03b2-HSD1) between cells from distinct anatomical locations may play a key role in the predeliction of certain tissues to develop persistent inflammation.Stromal cells such as fibroblasts play an important role in defining tissue-specific responses during the resolution of inflammation. We hypothesized that this involves tissue-specific regulation of glucocorticoids, mediated via differential regulation of the enzyme 11\u03b2-hydroxysteroid dehydrogenase type 1 (11\u03b2-HSD1). Expression, activity and function of 11\u03b2-HSD1 was assessed in matched fibroblasts derived from various tissues  obtained from patients with rheumatoid arthritis or osteoarthritis. 11\u03b2-HSD1 was expressed in fibroblasts from all tissues but mRNA levels and enzyme activity were higher in synovial fibroblasts . Expression and activity of the enzyme increased in all fibroblasts following treatment with tumour necrosis factor-\u03b1 or IL-1\u03b2 (bone marrow: 8-fold and 37-fold, respectively, compared to vehicle; dermal fibroblasts: 4-fold and 14-fold; synovial fibroblasts: 7-fold and 31-fold; all  The profound effects of glucocorticoids on the immune system have underpinned their widespread use as therapeutic agents for inflammatory diseases . In addiExpression of 11\u03b2-HSD1 occurs primarily in GR-rich tissues, where the enzyme acts to increase local levels of active glucocorticoids, thereby providing a system for autocrine induction of GR-mediated responses ,6. Promin = 6) and osteoarthritis . Fibroblasts were isolated by mechanical digestion of tissue followed by dissociation in 5 mmol/l EDTA for 2 hours. Dissociated tissue was then washed and transferred to a culture flask. The fibroblasts were then cultured through to a maximum of seven passages in complete fibroblast medium consisting of RPMI-1640, 1% (vol/vol) non-essential amino acids, 1% penicillin/streptomycin, 1% sodium pyruvate, 2 mmol/l glutamine and 20% heat-inactivated foetal bovine serum  [All reagents used in cell culture were obtained from Sigma  unless otherwise stated. Fibroblasts were isolated from biopsies of matched skin, bone marrow and synovium removed during total knee arthroplasty  from consex, UK) . Fibrobl1 Alexa 633 antiserum ; and the UCHT-1 (CD3) antiserum  with secondary anti-mouse IgG2b TRITC . Immunohistochemical analyses and subsequent image visualization were carried out as described previously [Cryostat tissue sections of synovial tissue from patients with RA were analyzed using goat polyclonal antibodies to 11\u03b2-HSD1  with anti-sheep/goat biotin AB360 as secondary antibody (The Binding Site). Immunohistochemistry was also carried using polyclonal antisera to the following: the endothelial marker von Willebrand Factor  with a secondary anti-rabbit AMCA antiserum ; the fibroblast marker ASO2 (CD90) with a secondary anti-mouse IgGeviously .RNA was extracted from cultured fibroblasts using the single-step extraction method . Aliquots (1 \u03bcg) of RNA were then reverse transcribed using random hexamers in a 20 \u03bcl volume, as stated in the manufacturer's protocol  .\u0394\u0394Ct. Probe and primer sequences used are summarized in Table Expression of mRNA for 11\u03b2-HSD1, H6PDH, GR\u03b1, GR\u03b2, IL-6, CCAAT/enhancer binding protein (C/EBP)\u03b1 and C/EBP\u03b2 was assessed using real-time PCR in an ABI 7500 system . The reactions were performed in 25 \u03bcl aliquots on a 96 well optical reaction plate (Sigma). Primers for 18S were used in conjunction with our target primers as an internal reference. Reactions contained TaqMan universal PCR master mix (Applied Biosytems), 900 nmol primers, 100\u2013200 nmol TaqMan probe and 25\u201350 ng cDNA. Target genes probes were labelled with FAM and 18S probes were labelled with VIC. Reactions occurred as follows: 50\u00b0C for 2 minutes, 95\u00b0C for 10 minutes, 44 cycles of 95\u00b0C for 15 seconds and 60\u00b0C for 1 minutes. Data were obtained as Ct values  according to the manufacturer's guidelines (Applied Biosytems), and used to determine \u0394Ct values (Ct of target gene - Ct of housekeeping gene) as raw data for gene expression (high \u0394Ct = low gene expression). The fold change in gene expression was determined by subtracting \u0394Ct values for treated cells from their respective control samples. The resulting \u0394\u0394Ct values were then used to calculate fold change in gene expression according to the expression 2Confluent cells in 12-well plates were cultured with 1 ml complete fibroblast medium, containing 100 nmol/l cortisone (to measure reductase activity) or cortisol (dehydrogenase activity) along with tritiated tracer for 18 hours. Steroids were extracted from growth medium using dichloromethane (5\u201310 vol) and separated by thin-layer chromatography (TLC) using ethanol:chloroform (8:92) as the mobile phase. TLC plates were analyzed using a Bioscan imaging detector  and the fractional conversion of steroids was calculated. In each case total protein concentration was assessed using a 96-well plate assay kit . Results were expressed as pmol product/hour per mg protein. All experiments were carried out in triplicate.Cortisol levels in supernatants from cultured cells were measured using a commercially available sandwich ELISA in accordance with the manufacturer's instructions . Data were expressed as pg cortisol/mg protein. Soluble IL-6 in supernatents from cultured cells was measured using a commercially available sandwich ELISA in accordance with the manufacturer's instructions . Data were expressed as pg IL-6/mg protein.Data are reported as mean \u00b1 standard deviation (SD) of replicate mean values for separate patient cell cultures. However, because of interindividual variation, data in Figures in situ expressed 11\u03b2-HSD1. Confocal immunohistochemistry  with those from OA patients (n = 3) indicated that there was no significant difference in expression of 11\u03b2-HSD1, H6PDH, GR\u03b1, or GR\u03b2 mRNA in either vehicle or TNF-\u03b1-treated fibroblasts between the two disease types (data not shown).To investigate the significance of 11\u03b2-HSD1 expression in fibroblasts, further studies were carried out using primary cultures of fibroblasts isolated from matched synovium, bone marrow and skin. Analysis of mRNA from these cells indicated that expression of 11\u03b2-HSD1 was greatest in synovial fibroblasts (\u0394Ct = 20.5 \u00b1 2.2), followed by dermal (\u0394Ct = 23.3 \u00b1 1.8) and bone marrow fibroblasts , dermal (14-fold and 4-fold increase) and synovial (31-fold and 7-fold) fibroblasts Figure . By cont3H-cortisone and 3H-cortisol metabolism by scanning analysis of TLC indicated predominant reductase activity in all three cell types, being highest in synovial fibroblasts, followed by bone marrow fibroblasts, and lowest in dermal fibroblasts , bone marrow  and synovial fibroblasts , indicating that all three cells had a similar capacity for GR-mediated responsiveness  that arStromal cells play a pivotal role in normal, physiological inflammation and persistent inflammatory disease by expressing factors such as cytokines and chemokines that define local immune responses. We postulate that endogenous activation of glucocorticoids via the enzyme 11\u03b2-HSD1 is another important component of stromal cell function by providing an autocrine mechanism for the localized regulation of inflammation. Stromal cell 11\u03b2-HSD1 may also play a key role in responses to therapeutic glucocorticoids by increasing their concentration in a tissue-specific manner.C/EBP = CCAAT/enhancer binding protein; Ct = the cycle number at which logarithmic PCR plots cross a calculated threshold line; ELISA = enzyme-linked immunosorbent assay; GR = glucocorticoid receptor; 11\u03b2-HSD = 11\u03b2-hydroxysteroid dehydrogenase; H6PDH = hexose-6-phosphate dehydrogenase; IL= interleukin; NADPH = nicotinamide adenine dinucleotide phosphate, reduced form; OA = osteoarthritis; PCR = polymerase chain reaction; RA = rheumatoid arthritis; SD = standard deviation; TLC = thin-layer chromatography; TNF = tumour necrosis factor.The authors declare that they have no competing interests.RSH cultured cells, extracted and analyzed RNA, and carried out enzyme activity studies. AF derived initial primary cultures. MSC developed initial hypothesis and co-wrote. GP derived initial primary cultures of fibroblasts. KR helped in the generation of cell lines and analyzed data. DLH carried out immunohistochemical analyses. EHR organized ELISA analyses. PMS developed initial hypothesis. CDB developed initial hypothesis, supervised collection of tissue and generation of cells, and co-wrote. MH developed initial hypothesis, supervised all cell analyses and co-wrote. All authors read and approved the final manuscript."}
+{"text": "The mechanism of bowel obstruction in colorectal cancer is likely to involve interactions between tumour cells, host fibroblasts and the extracellular matrix. The role of fibroblast-mediated matrix reorganisation in malignant structures of the large bowel was examined in an in vitro collagen matrix model in which tumour cells and fibroblasts were cultured under serum-free conditions. Colon cancer cells secreted a factor(s) which enhanced the ability of colon fibroblasts to contrast a collagen matrix without an associated mitogenic response by the fibroblasts. Within uncontracted collagen gels marked elongation of fibroblast cell processes was observed in the presence of the tumour-derived factor(s). We propose that matrix reorganisation by host fibroblasts in the wall of the human colon is responsible, at least in part, for malignant large bowel obstruction."}
+{"text": "Antineoplastic drugs, given in the perioperative period, are thought to be a hazard to wound repair. Since fibroblast collagen synthesis is crucial to healing, we examined the effects of bleomycin, cisplatin and 5-fluorouracil on collagen synthesis in confluent cultures of fibroblasts from human colon and skin. The drugs were added in final concentrations between 0.1 and 50 microM. Bleomycin did not affect collagen synthesis in colon fibroblasts but inhibited synthesis in skin fibroblasts. Collagen synthesis in colon fibroblasts was strongly, and specifically, inhibited by cisplatin while synthesis in skin fibroblasts was affected only slightly. 5-Fluorouracil had no effect whatsoever on the collagen synthetic capacity in either colon or skin fibroblasts. If skin fibroblasts were cultured in the presence of transforming growth factor beta (TGF beta), the antineoplastic agents inhibited the TGF beta-stimulated collagen synthesis at far lower concentrations than those needed to suppress non-stimulated synthesis. This effect was not observed in fibroblasts from colon. The possible implications of these observations, as pertain to the use of perioperative chemotherapy, are discussed. Since 5-fluorouracil did not directly affect collagen synthesis in colon fibroblasts under any of the conditions tested it is suggested that the data support the contention that this drug is relatively harmless for intestinal healing."}
+{"text": "Tumor-stroma reaction is associated with activation of fibroblasts. Nemosis is a novel type of fibroblast activation. It leads to an increased production of growth factors and proinflammatory and proteolytic proteins, while at the same time cytoskeletal proteins are degraded. Here we used paired normal skin fibroblasts and cancer-associated fibroblasts (CAF) and primary and recurrent oral squamous cell carcinoma (SCC) cells to study the nemosis response.Fibroblast nemosis was analyzed by protein and gene expression and the paracrine regulation with colony formation assay. One of the normal fibroblast strains, FB-43, upregulated COX-2 in nemosis, but FB-74 cells did not. In contrast, CAF-74 spheroids expressed COX-2 but CAF-43 cells did not. Alpha-SMA protein was expressed in both CAF strains and in FB-74 cells, but not in FB-43 fibroblasts. Its mRNA levels were downregulated in nemosis, but the CAFs started to regain the expression. FSP1 mRNA was downregulated in normal fibroblasts and CAF-74 cells, but not in CAF-43 fibroblasts. Serine protease FAP was upregulated in all fibroblasts, more so in nemotic CAFs. VEGF, HGF/SF and FGF7 mRNA levels were upregulated to variable degree in nemosis. CAFs increased the colony formation of primary tumor cell lines UT-SCC-43A and UT-SCC-74A, but normal fibroblasts inhibited the anchorage-independent growth of recurrent UT-SCC-43B and UT-SCC-74B cells.Nemosis response, as observed by COX-2 and growth factor induction, and expression of CAF markers \u03b1-SMA, FSP1 and FAP, varies between fibroblast populations. The expression of CAF markers differs between normal fibroblasts and CAFs in nemosis. These results emphasize the heterogeneity of fibroblasts and the evolving tumor-promoting properties of CAFs. Tumor microenvironment plays a major role in cancer progression and fibroblasts are known to be key components of the tumor stroma. Recently it has been suggested that stromal fibroblasts initially inhibit early stages of carcinogenesis and later under the paracrine influence of the transformed epithelia become activated leading to promotion of cancer growth. The dependence of carcinomas on stromal fibroblasts decreases as the cancer progresses, partly through a switch in epithelial cells from paracrine to autocrine regulation Nemosis, a phenomenon of fibroblast activation  is the sixth most common malignancy worldwide and the overall patient survival is poor. This is mainly due to high rates of cancer recurrence and local invasion, partly caused by p53 gene mutations, which can be found in more than 70% of HNSCCs Based on the previous results that under the influence of malignant cells normal nemotic fibroblasts start to resemble CAFs, the objective of this work was to study the nemosis response of autologous skin and cancer-associated fibroblasts, to compare the expression of CAF markers between these fibroblasts strains and their fate in nemosis and to investigate how these different fibroblast populations influence the patient-matched oral SCC cells. Our study shows that both normal and cancer-associated fibroblasts show variation between individuals, seen as varying basal CAF expression levels and different growth factor responses in nemosis, and have a differential impact on the SCC cells. The behaviour of the studied CAF markers in nemosis followed the general nemosis response: cytoskeletal \u03b1-SMA and FSP1 were downregulated and proteolytic FAP was upregulated. The only exception was one of the CAF strains that upregulated FSP1 in nemosis. Major systematic differences between normal and cancer-associated fibroblasts were the decreased basal levels of growth factors in CAFs and the capability of nemotic CAFs to start to regain the \u03b1-SMA expression and the increased FAP expression in nemosis compared to their normal counterparts.First we wanted to investigate the expression of the previously used nemosis marker COX-2 in the four fibroblast populations. There was no basal expression of COX-2 in any of the fibroblast strains, and it was not induced in monolayer culture. However, when cultured as spheroids the normal fibroblasts FB-43 started to express COX-2 after 48 hours , but thiin vitro are considered to be in a state resembling wound healing. Both CAF strains expressed \u03b1-SMA, CAF-74 slightly more than CAF-43. Interestingly, also the normal FB-74 cells expressed \u03b1-SMA, but no protein expression was detected in the other normal fibroblast cell strain FB-43. Time-dependent downregulation of \u03b1-SMA was seen in spheroids but not in the monolayer cultures, caused by the degradation of cytoskeleton in these fibroblasts going through nemosis. This is in line with previous results by Bizik et al. We also looked at the protein levels of vimentin and \u03b1-SMA in these cells. All four fibroblast populations expressed vimentin in equal amounts, as expected, since fibroblasts Since the protein levels of \u03b1-SMA varied between different fibroblast populations, we decided to investigate also the expression of other widely used CAF markers FSP1 and FAP. Gene expression pattern of these three genes in the fibroblasts grown as spheroids for 0, 24, 48 and 72 hours was analyzed using quantitative real-time PCR. Q-PCR was chosen as the method over immunoblotting because of its higher sensitivity. GAPDH was used as a reference gene that the expression of target genes was normalized to, after which relative fold expression ratios were calculated. The basal expression level of \u03b1-SMA, FSP1 and FAP was significantly lower (P<0.01) in CAF-43 cells than in normal FB-43 fibroblasts . HoweverNemosis response of the CAF markers between these fibroblast populations showed also variation. The \u03b1-SMA level was drastically downregulated in spheroids, reflecting the protein levels and indicating the decomposition of cytoskeleton in these spheroids. This was also true for FB-43 cells, for which we could not detect protein expression. Differing from the normal fibroblasts, the CAFs started to regain the \u03b1-SMA expression at 72 hours; when compared to normal fibroblasts the increase was statistically significant (P<0.05) . FSP1 mRThe other hallmark of nemosis is the increased production of several growth factors, including VEGF, HGF/SF and FGF7 (KGF). Therefore we used Q-PCR to study the expression levels of these genes in the four fibroblast populations. Both CAF strains had lower basal expression levels of VEGF, HGF/SF (P<0.05) and FGF7 (P<0.01) mRNA compared to the paired normal fibroblasts . When grAnchorage-independent growth of UT-SCC carcinoma cell lines was tested using the soft-agarose assay. During the three-week observation period all four carcinoma cell lines formed colonies, but clear difference between primary and recurrent tumor cell lines was seen . When cuThe UT-SCC colony formation results were in line between cells obtained from the two individuals; however, there was variation between individuals when observing closely the underlying monolayer fibroblast cultures. Spontaneous spheroid formation was seen in the underlying monolayer culture of FB-43 and CAF-43 fibroblasts when co-cultured with both 43A and 43B SCC cells . FB-74 aTo elucidate the reason for the different behavior of the fibroblast strains on monolayer cultures, and particularly the slow growth rate of CAF-74 cells, we performed senescence-associated beta-galactosidase  staining. SA-\u03b2-gal activity is the most commonly used marker for cellular senescence. Premature stress-induced senescence is caused by oxidative stress, DNA damage and oncogene activation Primary carcinomas are considered to be unorganized organs that are composed of various cell types, including cancer cells, fibroblasts and other mesenchymal cells, and cells related to immunity and vasculature. The tumor-stroma microenvironment leads to fibroblast activation and paracrine signaling between fibroblasts and cancer cells The objective of this work was to investigate the nemosis response of patient-matched normal and cancer-associated fibroblasts, and to study the expression pattern of CAF markers and their behaviour in nemosis. Only one of the normal fibroblast strains (FB-43) and one of the CAFs (FB-74) induced COX-2 in nemosis. This is in contrast with previously published results, where COX-2 induction has been considered a hallmark feature of nemosis. However, in those studies fibroblasts have been from neonatal origin and here we have used fibroblasts obtained from adults. This conflicting result therefore indicates that COX-2 should not be solely used to measure nemosis response, but other markers, such as the profile of secreted proteins, should be investigated as well.The other key feature of nemosis is the time-dependent degradation of cytoskeleton. On protein level three of the fibroblast strains expressed \u03b1-SMA, surprisingly also the normal skin fibroblasts FB-74. The other normal fibroblast strain FB-43 did not express \u03b1-SMA at the protein level. However, when measuring the mRNA levels with the more sensitive Q-PCR method, all four fibroblast populations showed \u03b1-SMA expression and this was downregulated in nemosis. The time-dependent downregulation on both protein and mRNA level is in line with previous results on nemosis, indicating the decomposition of the cytoskeleton. Interestingly the CAFs started to regain the \u03b1-SMA mRNA expression at 72 h, the difference was significant when compared to their normal counterparts. In contrast to our results, a study by Shannon et al. We investigated also the mRNA levels of two other CAF markers, FSP1 and FAP. In nemosis FSP1 levels decreased in FB-43, FB-74 and CAF-74 spheroids, but increased in CAF-43 cells. The third investigated CAF marker FAP was upregulated in nemosis, more in CAFs than in normal fibroblasts, the difference was significant with the 43 fibroblast strains. With all three CAF markers the nemosis response followed the pattern of decreased expression of cytoskeletal genes (\u03b1-SMA and FSP1) and increase in proteolytic gene expression (FAP). Clearly different response was seen with CAF-43 cells where, instead of downregulation of FSP1, the levels increased in nemosis.The heterogeneity of fibroblasts becomes evident when looking at the basal levels of the CAF marker expression; CAF-43 cells had lower levels of all three markers, CAF-74 had less \u03b1-SMA, slightly more FSP1 and over 10-fold more FAP. These results also emphasize that \u03b1-SMA, the most commonly used CAF / myofibroblast marker, should not be used solely to define activated fibroblasts.Another hallmark of nemosis is the induction of growth factors. It has been shown that oral fibroblasts produce significantly more FGF7 and HGF/SF when compared to skin fibroblasts Based on these results it seems that the capability of normal and cancer-associated fibroblasts to produce these growth factors in nemosis is somewhat related to the extent they are needed in cancer progression. The dependence of tumors on stromal fibroblasts, and particularly on the growth factors they produce, decreases in the course of tumor progression. Epithelial cells require FGF7 to break the epithelial polarization. FGF7 is only expressed by stromal cells and its receptor FGFR2IIb only by epithelial cells, indicating the role FGF7 in the beginning of tumor progression It has been well established that CAFs, but not normal fibroblasts, are capable to promote tumor progression The observed spontaneous spheroid formation of FB-43 and CAF-43 in monolayer cultures is in line with the results from Kankuri et al. in vitro model of fibroblast activation, may have its in vivo counterpart in cancer-associated fibroblasts and is a valuable tool in studying the variations between fibroblasts obtained from different individuals. Nemosis response, particularly of the CAF markers \u03b1-SMA and FAP, could therefore be used as a prognostic marker to predict the stromal reaction of tumors.In conclusion, this study clearly demonstrates that fibroblasts obtained from different individuals vary in gene expression and behavior and that the expression of CAF markers differs between normal fibroblasts and CAFs in nemosis. Both normal and cancer-associated fibroblasts modulate tumor cells, normal fibroblasts by inhibiting the growth of invasive SCC cells and CAFs by further enhancing the growth of primary SCC cells. Nemosis, an All used cell strains had been previously established 2 atmosphere in Dulbecco's modified Eagle's medium (DMEM)  and supplemented with 5% fetal calf serum (FCS) (Invitrogen), 0.3 mg/ml glutamine, 100 \u00b5g/ml streptomycin and 100 U/ml penicillin. Fibroblast spheroids were formed as described previously 5 cells/ml) were plated on agarose-coated U-bottom 96-well plates . Monolayer cultures were plated at the same density either on flat-bottomed 96-well plates (for immunoblotting) or on 6-cm dishes (for Q-PCR) . Cells were harvested at 24 h, 48 h and 72 h. As zero-hour time point the single cell suspension at the time of seeding was used. Fibroblasts were used till passage number 20 and UT-SCC cell lines till passage number 55.All cell populations were cultured at +37\u00b0C in 5% COThe samples were harvested in 2\u00d7 sample buffer (125 mM Tris (pH 6.8), 4% sodium dodecyl sulfate (SDS), 0.01% bromophenol blue, 10% \u03b2-mercaptoethanol, 10% glycerol) and equal amounts of protein from each sample were resolved by 10% SDS-PAGE. Proteins were transferred to nitrocellulose membrane  and blocked with 2.5% non-fat powdered milk in TBS .The following primary antibodies were used: rabbit polyclonal anti-COX-2 , rabbit polyclonal anti-GAPDH , mouse monoclonal anti-\u03b1-SMA , mouse monoclonal anti-vimentin 65EE3;  and mousThe samples for Q-PCR were harvested in RNAprotect Cell Reagent and total RNA was extracted according to the manufacturer's instructions using RNeasy Plus Mini kit . Using the SuperScript VILO cDNA synthesis kit (Invitrogen) 500 ng of RNA from each sample were reverse-transcribed according to the manufacturer's instructions. Real-time quantitative PCR was done using DyNAmo Capillary SYBR Green Quantitative PCR kit  with a LightCycler Instrument . Primer sequences are listed in 4 fibroblasts / ml or 5\u00d7104 SCC cells / ml. In order to determine the role of paracrine signaling between fibroblasts and carcinoma cells the assay was modified so that fibroblasts (2.5\u00d7104 fibroblasts / ml) were first plated in wells as a monolayer and incubated for 24 hours. Medium was aspirated and bottom agarose was laid on top of semi-confluent cells and allowed to solidify, after which the top agarose with or without SCC cells (5\u00d7104 SCC cells / ml) was overlaid. The plates were cultured at +37\u00b0C in 5%-CO2 incubator for 3 weeks without further feeding. The formed colonies were scored by calculating number of colonies in ten random views of 10\u00d7 magnification in duplicate using an inverted microscope (Olympus CKX41) and photographed (Olympus DP12).The colony formation assay was based on method described by Zheng et al. 2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 in 40 mM phosphate buffer, pH 6) over night. Images of random views were captured at 4\u00d7 magnification and the blue cells, indicating senescence, were calculated.Senescence-associated beta-galactosidase  activity was stained as described by Dimri et al. t-test.All experiments were done in duplicates and repeated three times. The mean and SEM of all three experiments are shown. GraphPad Prism software was used to calculate statistical significance that was determined by unpaired Student's"}
+{"text": "F) of the Spanish Habsburg kings from an extended pedigree up to 16 generations in depth and involving more than 3,000 individuals. The inbreeding coefficient of the Spanish Habsburg kings increased strongly along generations from 0.025 for king Philip I, the founder of the dynasty, to 0.254 for Charles II and several members of the dynasty had inbreeding coefficients higher than 0.20. In addition to inbreeding due to unions between close relatives, ancestral inbreeding from multiple remote ancestors makes a substantial contribution to the inbreeding coefficient of most kings. A statistically significant inbreeding depression for survival to 10 years is detected in the progenies of the Spanish Habsburg kings. The results indicate that inbreeding at the level of first cousin (F\u200a=\u200a0.0625) exerted an adverse effect on survival of 17.8%\u00b112.3. It is speculated that the simultaneous occurrence in Charles II (F\u200a=\u200a0.254) of two different genetic disorders: combined pituitary hormone deficiency and distal renal tubular acidosis, determined by recessive alleles at two unlinked loci, could explain most of the complex clinical profile of this king, including his impotence/infertility which in last instance led to the extinction of the dynasty.The kings of the Spanish Habsburg dynasty (1516\u20131700) frequently married close relatives in such a way that uncle-niece, first cousins and other consanguineous unions were prevalent in that dynasty. In the historical literature, it has been suggested that inbreeding was a major cause responsible for the extinction of the dynasty when the king Charles II, physically and mentally disabled, died in 1700 and no children were born from his two marriages, but this hypothesis has not been examined from a genetic perspective. In this article, this hypothesis is checked by computing the inbreeding coefficient ( The Habsburg dynasty  was one of the sovereign dynasties of Europe. The Spanish branch of this dynasty ruled over the Spanish kingdoms from 1516 to 1700. Under Habsburg rule, Spain reached the zenith of its influence and power in Europe and the world-wide Spanish Empire reached its apogee. The last king of the Spanish Habsburg dynasty was Charles II. He was physically disabled, mentally retarded and disfigured. He proved impotent since no children were born from his two marriages. When Charles II died in 1700 the line of the Spanish Habsburgs died with him and a new dynasty- the French Bourbons- was installed in Spain. In the historical literature it is frequently speculated that strong preference for consanguineous marriages within the Spanish Habsburg line contributed to its extinction. In order to keep their heritage in their own hands, the Spanish Habsburgs began to intermarry more and more frequently among themselves and the result, in a few generations, was a fatal inbreeding that brought the male line of the Spanish Habsburgs to extinction Three lines of evidence support the inbreeding hypothesis. First of all, the incidence of consanguineous marriages in the Spanish Habsburgs was remarkable . The SpaF  of the Spanish Habsburgs by means of the FSpeed computer programme Genealogical information of the Spanish Habsburg kings obtained from several sources In order to evaluate the impact of inbreeding on survival in the Spanish Habsburg dynasty, mortality data of progenies of eight royal families were obtained from several sources F) of the Spanish Habsburg kings must be calculated. Inbreeding coefficients of the Spanish Habsburgs were computed from an extended pedigree traced back as far as 16 ancestral generations from the last Spanish Habsburg king and involving more than 3,000 individuals  and very close to the expected value in an incestuous union as parent-child or brother-sister (F\u200a=\u200a0.250 in both cases). The development of the inbreeding coefficients with increasing pedigree depth shows that the inbreeding levels for most Spanish Habsburg kings are not definitely established until pedigrees of at least 10 generations depth are analyzed (F\u200a=\u200a0.008), and Philip IV and his first wife Elizabeth of Bourbon (F\u200a=\u200a0.050). Both marriages did not give place to a king since, in the first case, only daughters were born and, in the second, the heir to the throne, the prince Baltasar Charles, died at early age. These historical contingencies were relevant for the Spanish Habsburg dynasty since when queens Elizabeth of Valois and Elizabeth of Bourbon died, Philip II and Philip IV married their nieces, Anna and Mariana, respectively, and consequently an important increase in the inbreeding coefficient of the resulting progenies was produced.In order to assess the possible effects of inbreeding on the Spanish Habsburg dynasty the inbreeding coefficient  based on a large data set of first cousins The effect of inbreeding on survival in the Spanish Habsburg dynasty is investigated from mortality data of eight royal families . Two difEl Hechizado (\u201cThe Hexed\u201d). According to contemporary writings, he was often described as \u201cbig headed\u201d and \u201cweak breast-fed baby\u201d. He was unable to speak until the age of 4, and could not walk until the age of 8. He was short, weak and quite lean and thin. He was described as a person showing very little interest on his surroundings . He first marries at 18 and later at 29, leaving no descendency. His first wife talks of his premature ejaculation, while his second spouse complaints about his impotency. He suffers from sporadic hematuria and intestinal problems (frequent diarrhea and vomits). He looked like an old person when he was only 30 years old, suffering from edemas on his feet, legs, abdomen and face. During the last years of his life he barely can stand up, and suffers from hallucinations and convulsive episodes. His health worsens until his premature death when he was 39, after an episode of fever, abdominal pain, hard breathing and comma. In the light of the knowledge of the current clinical genetics and on information gathered by the historians on the health of Charles II we might speculate on the origin of his illness. Although we recognize that it is highly speculative, some of the health problems suffered by Charles II could have been caused by the action of detrimental recessive genes given his high inbreeding coefficient (F\u200a=\u200a0.254) with 25.4% of his autosomal genome expected to be homozygous. In this sense, the simultaneous occurrence in Charles II of two genetic disorders determined by recessive alleles, combined pituitary hormone deficiency  and distal renal tubular acidosis , could explain an important part of his complex clinical profile. The most frequent genetic cause of hereditary CPHD is mutations in PROP1 (5q) that cause a multiple endocrine deficit of pituitary hormones ATP6V0A4 (7q) or ATP6V1B1 (2q) genes As already mentioned, Charles II, the last king of the Spanish Habsburg dynasty, presented important physical and mental disabilities suffering from a number of different diseases during his life"}
+{"text": "The authors explain how the international framework of human rights can be better used to help reduce child poverty and improve child survival rates. The purpose of this short paper is to explain how the international framework of human rights can be better used to help reduce child poverty and improve child survival rates.It is estimated that over 10 million children in developing countries die each year, mainly from preventable causes. In approximately half of these deaths, malnutrition is a contributory cause . HoweverWhile medical interventions can, in principle, prevent most young children from dying early, they cannot remove the underlying causes of poor health, which are linked directly to the severely deprived or absolutely poor living conditions suffered by 30% of the world's children ,6. For eThe Lancet [In recent years, the importance of the link between child rights and child survival has been contested. In 2004, an editorial in e Lancet  argued te Lancet .We argue that a rights-based strategy will increase child survival, in part by reducing child poverty, but only if some rights are prioritised over others. UNICEF, under Bellamy, adopted a position in which all the rights in the UN Convention on the Rights of the Child (UNCRC) were regarded as of equal importance, and both developed and developing countries were urged to realise these rights progressively  ,10. ThisThere is a clear need to prioritise the realisation of rights in policy so that action can be divided into successive stages according to degree of severity of transgression and available resources. Ensuring child survival provides a good basis for this prioritisation, but to be effective, actions need to tackle both the symptoms and the underlying causes. The UNCRC see  establisThe right to life, survival, and developmentNon-discriminationDevotion to the best interests of the childRespect for the views of the childThe right to an adequate standard of living and social securityArticle 24 (1) of the UNCRC states that:\u201cStates Parties recognize the right of the child to the enjoyment of the highest attainable standard of health and to facilities for the treatment of illness and rehabilitation of health. States Parties shall strive to ensure that no child is deprived of his or her right of access to such health care services\u201d.Similarly, Article 24 (2) of the UNCRC continues:\u201cStates Parties shall pursue full implementation of this right and, in particular, shall take appropriate measures:(a) To diminish infant and child mortality;(b) To ensure the provision of necessary medical assistance and health care to all children with emphasis on the development of primary health care;(c) To combat disease and malnutrition, including within the framework of primary health care, through, inter alia, the application of readily available technology and through the provision of adequate nutritious foods and clean drinking-water, taking into consideration the dangers and risks of environmental pollution;(d) To ensure appropriate pre-natal and post-natal health care for mothers;(e) To ensure that all segments of society, in particular parents and children, are informed, have access to education and are supported in the use of basic knowledge of child health and nutrition, the advantages of breastfeeding, hygiene and environmental sanitation and the prevention of accidents;(f) To develop preventive health care, guidance for parents and family planning education and services.\u201dIf these rights were to be fulfilled, child survival rates would rapidly improve.A human rights approach offers the possibility for progressive interventions into child poverty and child survival in three ways. First, conventions like the UNCRC have been signed by most countries in the world and thus can be considered to embody universal values and aspirations. Second, human rights conventions place a legal obligation upon states, a view endorsed by Mary Robinson (former UN High Commissioner for Human Rights) in her speech to the 2002 World Summit on Sustainable Development in Johannesburg, South Africa: \u201c\u2026a human rights approach adds value because it provides a normative framework of obligations that has the legal power to render governments accountable\u201d .Any comprehensive understanding of the root causes of poverty and the 10 million annual premature child deaths cannot ignore the legal and institutional structures that create and perpetuate income and wealth imbalances within society. Thus, human rights provide a challenge to these structures .Third, rights-based language can help to direct policy. It shifts the focus of debate from the personal failures of the \u201cpoor\u201d to the failure of macro-economic structures and policies implemented by nation states and international bodies  to eradicate poverty. Hence, child poverty in this context is no longer described as a \u201csocial problem\u201d but a \u201cviolation of rights\u201d .There are objections to the human rights approach. One question is whether human rights, as formally expressed in human rights conventions, are genuinely universal . CritiquA second question is whether economic, social, and cultural rights  have been subjugated to civil and political rights, despite the insistence of human rights advocates on the \u201cindivisibility\u201d of these rights  can be considered to be on a continuum ranging from \u201cgood health\u201d to \u201cpoor health/death\u201d . SimilarRegrettably, there is little international case law at present that identifies the location of this \u201cjudicial\u201d threshold with respect to many social, economic, and cultural rights, such as the right to health care. Social scientists therefore have a responsibility to help identify such \u201cjudicial\u201d thresholds\u2014a methodological issue we have sought to address in previous research .The international framework of child rights is a useful theoretical and political tool in taking action to reduce child poverty and improve child health . A rightall nation states, be it within their jurisdiction or through international cooperation and action, requires widespread reinforcement and support. This places special obligations upon those who operate in the interests of the powerful nation states at the supranational level to ensure that child survival rates are improved by the fulfilment of children's human rights, particularly their economic and social rights. Solely concentrating on medical interventions that increase child survival, while ignoring other violations of children's human rights, is unlikely to ensure the health and well being of children in the long term.Child rights fulfilment by states can only be properly assessed within the global context of poverty and an equal appraisal of developed and developing countries. Thus, the guidance given by the Committee on the Rights of the Child  , which s"}
+{"text": "Airway remodeling in COPD includes reorganization of the extracellular matrix. Proteoglycans play a crucial role in this process as regulators of the integrity of the extracellular matrix. Altered proteoglycan immunostaining has been demonstrated in COPD lungs and this has been suggested to contribute to the pathogenesis. The major cell type responsible for production and maintenance of ECM constituents, such as proteoglycans, are fibroblasts. Interestingly, it has been proposed that central airways and alveolar lung parenchyma contain distinct fibroblast populations. This study explores the hypothesis that altered depositions of proteoglycans in COPD lungs, and in particular versican and perlecan, is a result of dysregulated fibroblast proteoglycan production.1 were examined in vitro in centrally and distally derived fibroblasts isolated from COPD patients (GOLD stage IV) and from control subjects.Proliferation, proteoglycan production and the response to TGF-\u03b21 triggered similar increases in proteoglycan production in distally derived fibroblasts from COPD patients and control subjects. In contrast, centrally derived fibroblasts from COPD patients were less responsive to TGF-\u03b21 than those from control subjects.Phenotypically different fibroblast populations were identified in central airways and in the lung parenchyma. Versican production was higher in distal fibroblasts from COPD patients than from control subjects (p < 0.01). In addition, perlecan production was lower in centrally derived fibroblasts from COPD patients than from control subjects (p < 0.01). TGF-\u03b2The results show that fibroblasts from COPD patients have alterations in proteoglycan production that may contribute to disease development. Distally derived fibroblasts from COPD patients have enhanced production of versican that may have a negative influence on the elastic recoil. In addition, a lower perlecan production in centrally derived fibroblasts from COPD patients may indicate alterations in bronchial basement membrane integrity in severe COPD. Chronic obstructive pulmonary disease (COPD) is a progressive disease characterized by a reduction in respiratory airflow that is not possible to normalize . The redInterestingly, in terms of the turnover of ECM, opposing pathological processes occur in the COPD lung as the ECM is degraded in alveoli and there is excessive deposition of ECM (fibrosis) in bronchi and in bronchioles . The maj1 in vitro.In this study, we hypothesized that altered levels of proteoglycans in COPD lungs may be dependent on dysregulated proteoglycan production in fibroblasts and hence that there are alterations in fibroblast phenotypes in COPD patients compared to control subjects. In particular, we wanted to determine whether enhanced alveolar versican deposition is due to higher versican production by distal fibroblasts. We also wanted to examine whether perlecan production was altered in centrally derived fibroblasts. Thus, we isolated centrally and distally derived fibroblasts from lung explants from COPD patients and from biopsies from healthy control subjects in order to assess proteoglycan production, proliferative potential, and responsiveness to TGF-\u03b22-agonist salbutamol (400 \u03bcg) and did not respond to methacholine test doses ), or other lung diseases were included as control subjects. This study was approved by the Swedish Research Ethical Committee in Lund (FEK 91/2006 and FEK 213/2005).Patients (n = 8) suffering from very severe COPD (GOLD stage IV) who were undergoing lung transplantation at Lund University Hospital were included in the study. The patients had quitted smoking at least 6 months before the lung transplantation. Non-smokers (n = 12) with no clinical history of asthma, . Proliferation was defined as absorbance at 48 h divided by the absorbance after 6 h. With this method the amount of adsorbed dye has been shown to be proportional to the cell number recorded on a Coulter counter -sulfate incorporation into glycosoaminoglycan side-chains measured on a scintillation counter . Individual proteoglycans were separated by SDS-PAGE and quantified using densitometry. The contribution of individual proteoglycans to the total proteoglycan content was calculated as the value for each proteoglycan divided by the sum of all the measured proteoglycans  by densitometry.Proteoglycan production in fibroblasts was determined as previously described . Briefly1-stimulated proteoglycan production was different from basal levels. Differences were considered significant at p < 0.05. All analyses were performed using GraphPad Prism software version 4.00 .Data are expressed as mean \u00b1 SEM. Statistical differences between groups were determined by multiple comparisons using Kruskal-Wallis test. The Mann-Whitney test was used to compare statistical differences between groups. The Wilcoxon signed rank test was used to determine whether TGF-\u03b21 was 19.7% (14-24%); (mean and range) in COPD patients. The corresponding numbers were 106.7% (95-116%) for control subjects. Mean age was 62 years (53-66) in the COPD group. For control subjects the mean age was 30 years (24-41). All the COPD patients were ex-smokers whereas none of the controls had a history of smoking. Distal fibroblast cultures were obtained from all patients and control subjects, while central fibroblasts were obtained from 6 of 8 COPD patients and 7 of 12 control subjects.Characteristics of COPD patients and control subjects are shown in Table The localization of versican, perlecan, biglycan, and decorin staining from one representative COPD subject is presented in Figure Isolated fibroblasts from COPD patients and control subjects were characterized using antibodies to mesenchymal markers Figure . Both ceThe proliferative potential of isolated fibroblasts was quantified using the crystal violet assay Figure . In contThe basal proteoglycan production was investigated, as shown in Figure 1 for 24 hours and the proteoglycan production was quantified and compared to basal levels  and biglycan (3.6-fold). In distal fibroblasts from control subjects TGF-\u03b21 induced significant increases of versican (1.8-fold), perlecan (1.4-fold) and biglycan (3.1-fold). However, TGF-\u03b21 induced a significant decrease of decorin (1.4-fold) but no change in the production of the other proteoglycans in central fibroblasts from COPD patients. In distal fibroblasts from COPD patients, TGF-\u03b21 induced significant increases in the production of versican (2.1-fold), perlecan (1.5-fold) and biglycan (2.9-fold). Distal fibroblasts from COPD patients had a higher TGF-\u03b21-response in the production of versican (p < 0.05), perlecan (p < 0.05), biglycan (p < 0.01), and decorin (p < 0.01) than central fibroblasts from COPD patients. No such difference was observed between distal and central fibroblasts from control subjects.Fibroblasts were stimulated with TGF-\u03b2s Figure . In centWe next examined the contribution of each proteoglycan to the total proteoglycan production defined as the sum of versican, perlecan, biglycan and decorin Figure . The con1.Our data indicate that fibroblast in central airways are phenotypically different from those present in the alveolar parenchyma. In COPD patients the phenotypes of these fibroblast populations are altered which may have implications for disease development. Enhanced alveolar versican deposition has been reported in tissue sections from COPD patients, and this may have a negative influence on the elastic recoil. We show that this may be due to dysregulated versican production in fibroblasts. Furthermore, data indicates that there may be structural changes linked to altered molecular composition of basement membranes in cartilaginous airways in severe COPD as the perlecan production was lower and perlecan is important for the basement membrane integrity. Surprisingly, central fibroblasts from COPD patients were poor responders to the pro-fibrotic mediator TGF-\u03b2et al. reported morphological differences between central fibroblasts, which were larger and had more projections, and distal fibroblasts, which were thinner and more spindle-shaped [Our data support the idea that there are unique and phenotypically different fibroblast populations in central airways and in the lung parenchyma ,7. Kotare-shaped . We obsee-shaped . Furthere-shaped -19. In o1 did not differ between central and distal fibroblasts from control subjects. However, the production of all the investigated proteoglycans was higher in distal fibroblasts than in central fibroblasts from COPD patients following TGF-\u03b21-stimulation. It cannot be explained with a dysregulated response in distal fibroblasts as it was similar in fibroblasts from COPD patients and control subjects. A more plausible explanation is that central fibroblasts from COPD patients are poor TGF-\u03b21-responders. This is intriguing as bronchial remodeling in COPD often includes deposition of ECM and TGF-\u03b21 is believed to be one of the most prominent inducer of matrix production in fibroblasts. Several studies have reported increased expression of TGF-\u03b21 in the airway epithelium and submucosa of patients with COPD and this was proposed to be a product of cigarette smoke as control smokers had similar staining pattern [1 and have as a consequence become less responsive. In the present study TGF-\u03b21 induced an increase in proteoglycan production in distal fibroblasts from COPD patients. TGF-\u03b21-induced transcription of versican, perlecan, and biglycan requires a functional downstream SMAD signalling pathway [et al. have smokers with similar smoking history as the COPD patients. Furthermore, it cannot be excluded that the use of biopsy material as the source of control fibroblasts in the present study also may contribute to the difference.Our data show that the production of biglycan was higher in distal fibroblasts than in central fibroblasts from control subjects. This difference was not observed in COPD patients. Interestingly, biglycan has previously been reported to promote cytoskeletal changes resulting in a more migratory phenotype which may be important in wound healing processes . Further pattern -23. One  pathway ,25. This pathway . The def1. Previous results from the same group showed that cellular modulations that caused a decrease in versican content or the chondroitin-sulfate side chain content of versican enhanced protoelastin synthesis and elastic fiber assembly in smooth muscle cells [To the best of our knowledge, this is the first study to examine the proteoglycan production in both centrally and distally derived fibroblasts from COPD patients. In our study, an alteration in proteoglycan production was observed in fibroblasts from COPD patients. Versican production was higher in distal fibroblasts from COPD patients than in distal fibroblasts from control subjects. This is consistent with a study that reported increased versican mRNA-expression in distal fibroblasts from COPD patients (GOLD stages II and IV) compared to smoking controls . It is ale cells ,29. ThesStructural changes in the small airway wall, including basement membranes, have been suggested to contribute to airflow obstruction in COPD . Less isAn interesting observation in the present study was the shift in the relative production of individual proteoglycans. Distally derived fibroblasts from COPD patients had higher production of versican and decorin and a lower production of perlecan and biglycan than distal fibroblasts from control subjects relative to the total amount of proteoglycan production. Surprisingly, these data are consistent with a rat model investigating bleomycin-induced pulmonary fibrosis, which showed that biglycan production was increased early in the fibrotic process and declined over time, while decorin production was enhanced later in this process . In a suThe present study was based on fibroblasts isolated from lung explants from COPD patients undergoing lung transplantation and from bronchial and transbronchial biopsies from control subjects. As lung tissue from the two groups is acquired from different sources it may be a potential caveat. Transbronchial biopsies are primarily composed of alveolar tissue but there is a risk of contamination with small airway structures. It was reported that distal lung sampling via transbronchial biopsies contains small airways in one in four to eight biopsies . We haveIn summary, the present study supports the idea of the existence of phenotypically distinct fibroblast populations in central airways and in the lung parenchyma. In addition, we show that fibroblasts isolated from COPD patients have alterations in proteoglycan production that may have implications for the pathogenesis. Distally derived fibroblasts from COPD patients had higher versican production, which may have a negative influence on the elastic recoil. Centrally derived fibroblasts from COPD patients had lower production of the basement membrane-stabilizing proteoglycan perlecan, and immunostaining of lung sections showed altered deposition of biglycan and decorin in the basement lamina reticularis. These abnormalities in airway basement membrane composition in COPD are likely to have functional consequences and may thus be regarded as a pathogenic feature in the disease.OH has received two research grants from AstraZeneca 2006 and 2008. JE serves as consultant to MedImmune Inc. and received research grants from MedImmune and GlaxoSmithKline. CGL has served on advising boards and given paid lectures organized by various pharmaceutical firms  and has received institutional grants from AstraZeneca and GlaxoSmithKline. LE and MD are employed by AstraZeneca. LB, KN and GWT have no competing interests.OH and KN performed studies on proliferation and proteoglycan production on primary fibroblasts. OH performed the immunostainings, interpreted the data and drafted the manuscript. MD, LB, LE, JE and CGL contributed to the design of the study and to the writing process. GWT initialized the study and had overall responsibility for the design and writing of the study. All authors read and approved the final manuscript."}
+{"text": "Alteromonas espejiana, BAL 31 catalyzes the degradation of single-stranded and linear duplex DNA to 5\u2032-mononucleotides, cleaves negatively supercoiled DNA to the linear duplex form, and cleaves duplex DNA in response to the presence of apurinic sites.The extracellular nuclease from per se impairs the enzyme's functionality, possibly due to the tract's bulky nature and preventing efficient progression through the active site.In this work we demonstrate that BAL 31 activity is affected by the presence of guanine in single-stranded DNA oligomers. Specifically, nuclease activity is shown to be affected by guanine's presence in minimal homopolymeric tracts in the middle of short oligomer substrates and also by its presence at the 3\u2032 end of ten and twenty base oligomers. G\u2022C rich regions in dsDNA are known to cause a decrease in the enzyme's nuclease activity which has been attributed to the increased thermal stability of these regions, thus making it more difficult to unwind the strands required for enzyme access. Our results indicate that an additional phenomenon could be wholly or partly responsible for the loss of activity in these G\u2022C rich regions. Thus the presence of a guanine tract This study has revealed that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA (ssDNA) oligomers. Specifically, BAL 31 nuclease activity was found to be affected by the presence of guanine in ssDNA oligomers. Alteromonas espejiana, BAL 31 comprises several nuclease activities designated as \u201cslow\u201d (S) and \u201cfast\u201d (F) depending upon the relative rates with which they catalyze the terminally directed hydrolysis of duplex DNA The extracellular nuclease from Although BAL 31 activity with duplex DNA is relatively well characterized 10 homopolymers was carried out for varying periods  at 37\u00b0C and with two different enzyme concentrations (0.5 and 1.0 U). Samples were prepared in triplicate. Oligomer hydrolysis was monitored quantitatively by the separation and detection of dNMPs by HPLC .BAL 31 hydrolysis of the four dN by HPLC . Of the 10 could be an artifact due to guanine homoploymer self aggregation to form higher order structures 2+ and Ca2+ for enzymatic activity. Since the enzyme requires a large deactivation temperature (\u223c85\u00b0C) and presumably is still active at elevated temperatures, the enzyme reaction temperature was increased from 37\u00b0C to 45\u00b0C, 50\u00b0C, and 55\u00b0C and the dG10 oligomers were incubated over a twenty-four hour period with 0.5 U enzyme. DT10 oligomers were incubated under the same conditions as a control. Samples were prepared in quintuplet. The dT10 samples produced 82.6\u00b19.9%, 88.2\u00b15.9%, and 98.4\u00b18.4% dTMP at 45\u00b0C, 50\u00b0C, and 55\u00b0C respectively. The dG10 samples produced 0\u00b10%, 1.31\u00b10.94%, and 0.78\u00b10.46% dGMP at 45\u00b0C, 50\u00b0C, and 55\u00b0C respectively  contained two guanines that \u2018capped\u2019 both the 5\u2032 and 3\u2032 ends (G2-CAP), (ii) contained a homopolymeric stretch of four guanines in the middle of the decamer (G4-MID), (iii) contained no guanine residues (G-NO), (iv) contained a homopolymeric stretch of four guanines at the 5\u2032 end of the decamer (G4-5\u2032CAP), and (v) contained a homopolymeric stretch of four guanines at the 3\u2032 end of the decamer (G4-3\u2032CAP). A series of 20-mers were also used that (i) contained a homopolymeric stretch of four guanines in the middle of the 20-mer (G4-MID-L), (ii) contained three guanines that \u2018capped\u2019 both the 5\u2032 and 3\u2032 ends (G3-CAP-L), and (iii) contained four guanines that \u2018capped\u2019 both the 5\u2032 and 3\u2032 ends (G4-CAP-L).To further delineate the sequence length and position requirements for the dG2-CAP, and G4-MID were incubated at 37\u00b0C with 0.5 U enzyme for a twenty-four hour period (in quintuplet) and, as before, their hydrolysis efficiency was measured by the recovery of the constituent nucleotides. An ANOVA analysis was conducted to determine whether the variation in recovery rates were significant. The G2-CAP oligomer produced nucleotides at recovery rates similar to those seen with the non-G containing homopolymeric oligomers and with the G-NO oligomer  indicating that the refractory nature of guanine tracts to BAL 31 digestion was an inherent property of the enzyme. Moreover, the USB Corporation purified the enzyme using SDS-PAGE and reported only seeing two bands corresponding to the \u2018fast\u2019 and \u2018slow\u2019 forms of the enzyme. In order for the activity seen to be due to a contaminant, the contaminating species would have had to have been of similar size as one of the species comprising the BAL 31 enzyme and co-elute as such.The above experiments used oligomers at a concentration of 300 \u00b5M and the hydrolysis proceeded at 37\u00b0C. In order to preclude the possibility that self aggregation of the G4-5\u2032CAP, G4-3\u2032CAP, G4-MID-L, G3-CAP-L and G4-CAP-L to see if further insight could be obtained into the mechanism of action of BAL 31. For all five oligomers the mean recovery of each nucleotide after BAL 31 hydrolysis is shown in 3-CAP-L was the same as from the other four oligomers . G4-5\u2032CAP was statistically the same as G4-3\u2032CAP and G3-CAP-L, where G4-3\u2032-CAP was additionally similar to G4-MID-L. G4-CAP-L, G3-CAP-L, and G4-MID-L had the same amount of dCMP recovery according to statistical analysis. Recovery of dAMP from G4-CAP-L, G4-5\u2032CAP, G3-CAP-L, and G4-MID-L did not differ significantly and recovery from G4-3\u2032CAP and G4-MID-L did not differ significantly. In addition G4-MID-L did not differ from G4-3\u2032CAP. G3-CAP-L and G4-MID-L do differ significantly. The recoveries of dTMP from G4-CAP-L, G4-MID-L, and G4-5\u2032CAP were statistically indistinguishable, and recovery from G4-5\u2032CAP was similar to G3-CAP-L. Recovery from G4-3\u2032CAP differed from the other four oligonucleotides. Most importantly, recovery of dGMP differed significantly between all oligomers except G4-CAP-L and G3-CAP-L.Further studies were carried out using the oligomers G4-5\u2032CAP) was refractory to G tract hydrolysis whereas, in contrast, almost complete recovery of dGMP occurred with the decamer with four guanines at the 3\u2032 end (G4-3\u2032CAP). Surprisingly, the hydrolysis of the other non-G nucleotides was more efficient with G4-5\u2032CAP than with G4-3\u2032CAP. This data supports the notion that, independent of any endonuclease activity it may possess, the BAL 31 acts as a ssDNA 3\u2032\u21925\u2032 exonuclease particularly when the substrate is of minimal length (10 mer) as previously reported with single stranded viral \u03a6X174 (wild type) DNA4-MID-L) as opposed to being capped at both ends (G3-CAP-L and G4-CAP-L). While ostensibly mimicking its shorter counterpart G4-MID, the G4-MID-L 20-mer, in contradistinction to its shorter counterpart G4-MID, was not more refractory to hydrolysis than its capped end homologs . Thus an increase in the length of the polynucleotide chain from 10 to 20 appeared to overcome the G-tract mediated inhibition of hydrolysis when the G tract was in the middle of the polynucleotide chain.The decamer that has four guanines at the 5\u2032 end  compared with the single functional group on the other purine nucleotide, adenine. Thus one hypothesis is that the active site of the enzyme is spatially constrained in such a manner that the bulky guanine tracts are bound and catalyzed more inefficiently than the other nucleotides. The resulting loss of \u2018processive momentum\u2019 could explain the activities noted in this study.Future studies could include the use of nucleotide analog substrates containing a variety of different exocyclic architectures to permit further testing of the postulated hypothesis of enzyme inhibition by bulky nucleotides. Such studies would also provide a more precise delineation of the steric impediments to efficient catalysis. Further characterization of the noted ssDNA nuclease activity would include determining, after chromatographic fractionation, whether the activity was present in the F and/or S isoforms.10, dC10, dA10, dG10, 5\u2032-GGATCATCGG (G2-CAP), 5\u2032-ATCGGGGATC (G4-MID), 5\u2032-ATATCATCAC (G-NO), 5\u2032-GGGATCTCATCTCATCTGGG (G3-CAP-L), 5\u2032-GGGGTCTCATCTCATCGGGG (G4-CAP-L), 5\u2032-GGGGATCTCA (G4-5\u2032CAP), 5\u2032-ATCTCAGGGG (G4-3\u2032CAP) and 5\u2032-ATCTCATCGGGGTCATCTCA (G4-MID-L)) were cartridge purified . The deoxycytidine 5\u2032-monophosphate (dCMP), thymidine 5\u2032-monophosphate (dTMP), deoxyadenosine 5\u2032-monophosphate (dAMP), and deoxyguanosine 5\u2032-monophosphate (dGMP) standards were obtained from Sigma Aldrich . The nuclease BAL 31 was purified from the culture medium of Alteromonas espejiana BAL 31 containing a mixture of \u201cfast\u201d and \u201cslow\u201d species . Quality control assays and double stranded endonuclease activity were checked for by the manufacturer.Single stranded oligomers  into 1.5 ml microcentrifuge tubes and dehydrating in a vacuum centrifuge. Reactions were carried out in a 20 \u00b5l reaction volume containing 300 \u00b5M ssDNA oligomer, 20 mM Tris-HCl (pH 8.1 at 25\u00b0C), 600 mM NaCl, 12 mM CaCl18 column with a 10\u00d72.1 mm guard column . The ion-pairing technique was employed using buffers described by Tavazzi et al 2PO4, 0.125% methanol, 12 mM tetrabutyl ammonium hydroxide, pH 7.0), and buffer B  were used in a 50\u223650 (v\u2236v) isocratic combination unless otherwise stated. A flow rate of 1.0 ml/min was maintained constant throughout the analysis and the analysis was conducted at ambient temperature (\u223c22\u00b0C). The use of HPLC to detect mononucleotides released by BAL 31 has been used previously with an alkaline salt gradient Samples were analyzed using ion-pairing HPLC. The HPLC apparatus consisted of a SpectraSystem P2000 pump and a UV6000LP diode array detector , equipped with a 5 cm light-path flow cell and data was collected between 200 and 300 nm. Data were acquired and analyzed by a PC using the XCalibur\u00ae software package provided by the HPLC manufacturer. Separation of the nucleotides was carried out using a Pinnacle II 250\u00d74.6 mm, 5 \u00b5m particle size CMolecular species identification was determined by matching retention times and absorption spectra to prepared standards. The peak areas of hydrolysis products obtained from HPLC-UV absorption measurements were quantified using the program XCalibur\u00ae applying an Avalon algorithm of peak detection.The recovery percentage was calculated by dividing the number of moles of nucleotide recovered by the maximum number of nucleotide moles possible and multiplying by 100%. For example, 6 nmol of a 10-oligomer will theoretically yield a possible 60 nmol of nucleotides. Statistical analysis of data was carried out using ANOVA analysis where the between sample and within sample variances were compared using a one-sided F-test"}
+{"text": "Finally, we will discuss and resolve some of the objections to our position including conflicts that may arise when more than one individual considers an avatar to be part of the self.There is an astounding silence in the peer-reviewed literature regarding what rights a person ought to expect to retain when being represented by an avatar rather than a biological body. Before one can have meaningful ethical discussions about informed consent in virtual worlds, avatar bodily integrity, and so on, the status of avatars vis-\u00e0-vis the self must first be decided. We argue that as another manifestation of the individual, an individual\u2019s avatar should have rights analogous to those of a biological body. Our strategy will be to show that (1) possessing a physical body is not a necessary condition for possessing rights; (2) rights are already extended to representations of a person to which no biological consciousness is attached; and (3) when imbued with intentionality, some prostheses become \u201cself.\u201d We will then argue that avatars meet all of the conditions necessary to be protected by rights similar to those enjoyed by a biological body. The structure of our argument will take the form of a conditional. We will argue that  Medical ethics is a fluid field that is constantly evolving in response to new technological developments. One of the recent innovations to impact medical ethics is the Internet and the development of virtual worlds and interactions. Virtual interactions can be as mundane as a virtual business meeting or as provocative as the Red Light Center (www.redlightcenter.com) dedicated to sex. Some virtual worlds such as Second Life (secondlife.com) allow for a range of interactions between individuals similar to those in the nonvirtual world. Among these interactions are those relevant to bioethics, particularly the treatment of various psychological disorders including group therapy, patient education, medical education, research, and so on -6.An individual navigates many of these virtual worlds as a construct known as an \u201davatar,\u201d named after the worldly incarnations of the Hindu gods. Avatars may or may not share physical similarities with the person they represent. Depending on the world, avatars can be individualized by \u201cvirtual plastic surgery\u201d to emphasize the idealized characteristics of an individual.If virtual sex is available, can virtual rape be far behind? In the first six months of 2003, the South Korean police received 22,000 reports of crimes committed by characters in online games . In 2007Accusations of virtual crime have forced governments to consider the legal status of virtual property. Given that virtual property often has value in the nonvirtual world, it comes as no surprise that theft of virtual property has been prosecuted as if it were theft of material property. What does come as a surprise is the astounding silence in the peer-reviewed ethics literature regarding what rights a person should expect to retain when being represented by an avatar rather than a biological body. This lack of peer-reviewed literature is particularly surprising given the claim in the existing literature that \u201c[i]t appears likely that [online] gaming and its associated notions of play may become a metaphor for a range of human social interactions, with the potential for new freedoms and creativity as well as new oppressions and inequality\u201d . Before nothing but an avatar, requires an examination of the paradigm of what it means to be \u201cself,\u201d or, to use the terminology of the philosophy literature, what it is that constitutes one\u2019s personal identity ypical language used in these definitions [of emotional abuse] is injury to the psychological capacity or emotional stability of a child\u201d . In VirgIf the previous example is not convincing, a will provides more direct evidence that having a physical body is not a necessary condition for possessing rights. A will provides instructions regarding a deceased individual\u2019s property. Having died, the author of the will no longer has a physical body nor any kind of mental activity. Even so, a person\u2019s wishes in regard to his or her property are binding; that is to say, individuals retain some amount of postmortem property rights.In summary, a body is not a necessary condition to possess rights. And some rights continue even when a consciousness is no longer present. This does not imply that all inanimate objects qualify for rights. But in the proper circumstances, which we will discuss below, an avatar, which has neither a corporeal existence in the nonvirtual world nor inherent consciousness, can be a candidate for rights based on being part of the \u201cself.\u201d. Some moral/legal change occurs when the prosthetic limbs are attached to the victim of the assault. Imbuing the limbs with intentionality changes the moral status of the limbs from that of inanimate objects to extensions of the individual to whom they are attached.We already afford rights to some representations of an individual that are not connected in a biological sense to the individual\u2019s consciousness. Consider the case of an individual with prosthetic limbs. If these limbs are stolen from a house and destroyed, they are considered property: a burglary and destruction of property have occurred. Now consider two cases in which an individual is attacked. In the first attack (A1), the assailant misses the intended target entirely. This is unpleasant because of awareness of potential danger, but there is no bodily harm: the victim sustained no physical damage. In the second attack (A2), assume that the only damage that occurs during the assault is to the victim\u2019s prosthetic limbs while they are attached to the individual. Most readers will intuit that A2 reaches a different level of harm than A1 even though the prosthetic limbs are a nonbiological manifestation of the individualPerhaps a more intuitive example comes from the transplantation of a biological \u201cprosthesis\u201d from one person to another. Take for example, a biological face, hand, or arm graft. These are prostheses, albeit of a sophisticated nature. Is it the case that these cannot be a part of the person because they are prostheses and do not contain the same DNA? Does being part of the \u201cself\u201d require a prosthesis to contain the same DNA as the user? We do not believe this to be the case. Rather, the person receiving the transplanted part gives it intentionality. Otherwise the part would simply be an inanimate object, unattached, and a candidate for burial or cremation.We would argue that a biological prosthesis does not differ fundamentally from a sophisticated mechanical arm or a functional prosthetic mechanical eye. The difference between a biological prosthetic arm and a nonbiological prosthetic arm is one of degree and not of kind. Both are prostheses and devoid of rights (as a person) unless given intentionality. As future nonbiological prostheses become more sophisticated and integrated into the nervous system, this will become more self-evident. ,17.It does not appear that biological identity is necessary for something to be given intentionality and thus to become self. This does not imply that every object can be considered self. However, it allows for, although does not prove, that avatars can be \u201cself.\u201d Avatars are analogous to prostheses in that they allow the user to manipulate the environment even though they have no direct biological connection to the consciousness of the individual. The individual thinks, moves a muscle, and the prosthesis moves. This is the same path taken by an avatar. The individual thinks, moves muscles (in this case controlling a keyboard or a mouse), and the avatar carries out an action. If, as we argue, we are going to assign \u201cself\u201d to one biological or nonbiological corporeal manifestation of an individual (prosthetic limbs) there should be no barrier to assigning similar rights to another nonbiological manifestations of an individual (the avatar).within the representation. This is the case with rights afforded those in a permanent vegetative state. There are legal protections against assaulting and otherwise violating those in a persistent vegetative state. For our purposes, the reason for these rights is immaterial. Whether it is because there was once a consciousness present or because society finds violation of a biologically live body abhorrent is irrelevant. The point remains that consciousness need not be biologically present for the representation to have rights as a person. This is analogous to an avatar. It is a representation of an individual, and we will argue below that it is part of the self, even though there is no biological consciousness present within the representation.Likewise, we already assign rights to the corporeal representation of an individual whether or not a biological consciousness is present The fact that having a physical body is not a necessary condition for an entity to possess rights does not show that avatars have rights. A young child\u2019s imaginary play friend does not have a physical body. It would be absurd to argue that it had rights as a person.1. In this world, people are directly and permanently connected to a virtual world via brain-scanning hardware. The person thinks and their avatar in the virtual world moves. The nonvirtual world and virtual worlds are phenomenologically indistinguishable. All representations of the \u201cself\u201d in this world are via an avatar. What happens to the avatar is fed back into the brain scan system causing pain, pleasure, and other sensations. Higher emotions such as fear, greed, and love are also affected by the virtual world. It is unquestionably true that residents of this Matrix-like world can make legitimate rights claims on each other, the right to be free from torture, for example. Any rights claimed would be assigned to the avatar since it is the only manifestation of self that can act that exists in this world.Our first argument showing that avatars are appropriate candidates for rights is a thought experiment. Let us imagine a \u201cMatrix-like\u201d world. Let us call this world MW1 can log in and out of the virtual world at will. Let us call this world MW2. Does the ability to log in and out change legitimacy of rights claims in the virtual world? Nothing of moral  importance has changed about an individual\u2019s relationship to the virtual world except that there are now two worlds in which an individual can make rights claims: a virtual world and a nonvirtual world.Now let us suppose that instead of being permanently hardwired into the virtual world, the residents of MW2 in all ways except that brain-scan technology has yet to be invented. People can only interact with the virtual world via a computer monitor and keyboard . There is an important difference between MW2 and W. The behavior of, and toward, avatars can no longer cause certain phenomenological states such as physical pain or the sensations of touch, taste, and smell, for example . Other phenomenological states will still be affected by events in the virtual world such as joy, despair, anxiety, and annoyance.Let us examine a third world, W. It is similar to MW1 and W is one of degree and not of kind. If the difference is only in degree, people can still make legitimate rights claims for their avatars; all that changes is the degree of strength of said claims [The difference between MWd claims . The use2 will cause no such pain for a user in W, some behavior that is not permissible in MW2 will be permissible in W. Just as one has a right not to be assaulted, even when the assault causes no physical harm, avatars in W will have certain rights as part of the self. These may include bodily integrity and freedom from fear. The closer the identification between the avatar and the individual, the more complete the rights of the person-as-avatar. In the \u201cMatrix-like\u201d world, where phenomenologically the prosthetic is the person, the rights of the avatar are identical to those of the personGiven that an action that would cause physical pain to the user in MW1, we made modifications that made MW1 come to resemble W, which is in all relevant aspects similar to our own world. We further argued that each modification was only a change of degree, and not a change in kind. Given that avatars in MW1 have rights, it follows that avatars in W also have rights.Starting with a paradigm that made the case for legitimate rights claims in MW. For example, while my avatar is engaged in an online community I find myself facing unwelcome sexual advances. Things quickly escalate out of control. I find myself feeling extremely uncomfortable. Depending on the situation, my only recourse may be to disconnect my avatar. This looks like a violation of right to free access. One may now argue that \u201cAha, didn\u2019t they just say the rights of an avatar are dependent on the proxy rights of the individual and not inherent to the avatar?\u201d To this we would answer an emphatic yes. It shows the degree of identity between an avatar and the \u201dperson.\u201d Essentially, they are phenomenologically one and the same at this point. Changing the rights of the avatar changes the rights of the person. My avatar has certain rights and violating those rights is also a violation of my rights.Our final argument in favor of the rights of avatars is related to the previous argument, that is, avatars have rights by proxy of the rights of their users\u201d change as the domain changes. What counts as a normal interaction in the nonvirtual world is not a paradigm of normal interaction in the virtual world. Additionally, what count as \u201cnormal\u201d interactions in the virtual world can and do change as technology and programming evolve, allowing ever more phenomenologically complex interactions to occur. By basing the standard for rights possession by nonbiological extensions on \u201cnormal\u201d interactions, necessary embeddedness captures the social nature of rights and manages to apply them to inanimate objects. For example, persons who are visually impaired may use haptic devices to provide tactile feedback in lieu of visual cues. In the case of the vitual world, haptic devices can substitute for vision allowing people who are visually impaired to locate other online individuals, navigate passageways, and so on [The ability of a person to have interactions in more than one social milieu  requires a redefinition of the \u201cself.\u201d One way we propose to redefine self is by using a principle we call \u201cnecessary embeddedness.\u201d An object is \u201cnecessarily embedded\u201d and has rights as part of the self to the extent that the object is necessary for \u201cnormal\u201d interaction within some specific domain. Note that \u201cnormal interactionsnd so on . Withoutnd so on . Thus, w1 have rights. We posit that because avatars in MW1 are more strongly \u201cnecessarily embedded\u201d than those in W, our intuitions recognize the legitimacy of rights claims in MW1 while they do not for avatars in W.We would also argue that applying the principle of necessary embeddedness may correct our mistaken intuitions on rights. In our Matrix thought experiment, it is not intuitively clear that avatars in W have rights; however, it is clear that avatars in MWBy this point the observant reader will have noticed a somewhat disconcerting trend with our argumentation. Any number of our arguments seem to lead to a question with an unacceptable answer: If an individual decides that a doll (or other object) is his representation in the nonvirtual world, should it have rights as \u201cself?\u201d By the argumentation expressed so far, it would appear that we must also accept the conclusion that a doll is an appropriate candidate for rights if an individual deems it so.We have two responses to this conclusion. First, we intuitively assign rights to prosthetic limbs. As demonstrated above, we recognize on some level that prostheses are part of \u201cself\u201d when attached to an individual. Prostheses in the future will be more integrated with \u201cself\u201d as prostheses that interface directly with the brain and the biological body are developed ,18. Seco) with the world, they should be recognized as an extension of one\u2019s self. Thus, they have a legitimate right to protection while in use; interfering with their active use would constitute assault and not a property crime. An analogy would be plucking out a functional prosthetic eye or a cochlear implant. As in the Matrix scenario, there is a difference in degree between interfering with someone\u2019s white cane and their prosthetic eye but not a difference in kind.This still begs the point about assigning \u201cself\u201d to a doll or a pair of glasses. We would suggest that if someone posits that \u201ca doll is my representation in the world and part of my \u201cself,\u201d\u201d it has to meet the \u201ctest of destruction,\u201d that is, would destruction of this doll hinder my ability to function in the world in a fundamental sense (not just the depression suffered from the loss of an object)? Does the doll meet the test of necessary embeddedness? If the answer is no, it is not considered \u201cself.\u201d It is clearly separate from self in that its destruction does not fundamentally change the individual\u2019s ability to function in the world. Similarly, one can raise the question about glasses, a wheelchair, or the white cane carried by visually impaired persons. We would argue that to the extent that individuals imbue these prostheses with intentionality and they are needed for \u201cnormal\u201d interactions  in which an individual does assign \u201cself\u201d to a doll, and the doll\u2019s destruction hinders or prevents the individual from interacting in the world . In this case, we would argue that the doll does have a right to bodily integrity as part of the self of the individual; attacking the doll has a fundamental consequence for the individual. However, this would apply to a very limited subset of individuals. Another caveat is that one obviously cannot define another individual as \u201cself\u201d even if the other \u201cself\u201d is being used as a prosthesis . Nor can one claim as \u201cself\u201d a prosthesis to which someone already has a right. This is discussed further below.As we note above, rights require a social milieu. Thus, conflicts can arise which require rules. An interesting case occurs when more than one person claims a prosthesis as part of the self. For example, imagine a team of scientists who work with the Mars Rover. The Mars Rover is necessarily embedded for the scientists; it is the only way for them to interact with the surface of Mars. Some particular scientist (perhaps a bit deluded) comes to think about the Mars Rover as a part of himself. The Mars Rover then fulfills the two criteria for rights that we laid out previously: it is necessarily embedded and it is viewed as part of the self. Now imagine that not one but two (slightly deluded) scientists claim the Mars Rover as an extension of the self. Is the Mars Rover an extension of both scientists\u2019 selves? There are two caveats to add to the theory of necessary embeddedness. First, if an agent, A, is aware that some object, O, is possessed by another individual, A cannot extend rights to O. A\u2019s knowledge of the preexisting rights claim by some other person precludes A\u2019s making it a legitimate extension of the self. In the case of the Mars Rover, the scientists cannot extend their selves to the Rover because the Rover is known to be public property. Second, if two individuals make two competing, seemingly equal legitimate rights claims on an object at the same time, property law would come into play to resolve the conflict. If the claims are indeed equal, the claims would cancel each other out and neither scientist would be able to claim the object as self.1; call it MW1* . In this world, two individuals share the same avatar. When one person goes to sleep, the other person wakes up and controls the avatar. In this case there does not seem to be any ownership claims that might undermine one individual\u2019s rights claims to the avatar as self. Nor is it the case that the rights claims overlap temporally; each individual makes his or her rights claim in turn and with the agreement of the other.                A more interesting scenario occurs when \u201cborrowing\u201d a prosthesis. Imagine a world similar to MWThe concept of something serially being the \u201cself\u201d of two individuals seems at first counterintuitive. However, a real-world example would be a transplanted heart. The heart is serially part of the \u201cself\u201d of two individuals. In fact, if you remove it, the \u201cself\u201d of each individual will cease to exist . And, there is no inherent barrier to the heart being transplanted serially to others (once immunologic barriers are removed). Thus, the concept of something being the \u201cself\u201d of two individuals serially does not require a paradigm shift. In the case of the avatar, if there is an intention to deceive other people (for example identify theft) on the part of one party using the avatar, laws about personal identity come into play.If the two individuals do not know each other and are not aware they are sharing an avatar, then there is a rights conflict . This would be similar to someone living in my vacation home without my knowledge. The fact that I am unaware of the occurrence does not mean that I do not have rights to my property.one of the most fundamental rights. Thus, it should be afforded to avatars in that they can be considered part of \u201cself.\u201d The degree of rights assigned to an avatar as part of the self will depend on the degree of identity between the avatar and the person. In MW1 (the \u201cMatrix-like\u201d world), the identity between the avatar and the self is complete. Thus, the avatar, as the only social and phenomenological manifestation of self, will be entitled to the full rights enjoyed by a person .The right to bodily integrity is current virtual interactions. Thus, assault or other violation of the self-as-avatar will have consequences in the nonvirtual world. The likelihood of this will increase in the future as more complex tactile feedback is given to the user .We believe it is obvious a priori that the right to bodily integrity is one of the most fundamental rights. Before one can even contemplate higher rights , one needs to be free from fear of assault, rape (a particularly heinous type of assault), torture, murder, and so on. As noted above, certain states\u2014such as joy, despair, anxiety, annoyance, and love\u2014may be felt as direct consequences of That avatars are candidates for rights also raises the question of informed consent within virtual worlds. We would argue that some type of informed consent should be required for virtual world research. Even a research questionnaire will have an impact on the individual whether the questionnaire is administered in the virtual or nonvirtual world. The virtual world inherently affords a degree of anonymity that may or may not exist in the nonvirtual world depending on the study design . However, there will be some emotional impact on the individual in the study. Thus, consent, perhaps in a modified form, should be obtained in the virtual world as it is in the nonvirtual world.We have outlined a case showing that avatars, as extensions of the self, are candidates for rights. The subset of rights one\u2019s avatar should enjoy will be dependent on the degree of phenomenological identity between the individual and his or her avatar. Drawing this line is difficult. Some people will have a stronger emotional reaction to a violation of their avatar than others. And, the situation in which the avatar is participating will obviously modify these rights. One would expect a greater degree of bodily integrity within virtual group therapy than in a combat simulation.Areas of future research could focus on expanding the discussion of necessary embeddedness and it\u2019s application to virtual and nonvirtual worlds as well as further discussion of what elements of informed consent are necessary in a virtual world. A discussion of what might constitute the \u201cinalienable\u201d rights of an avatar should also be undertaken."}
+{"text": "In a Phase I study, melphalan 140 mg/m2 was administered to 8 patients with disseminated malignant melanoma. Marrow was removed from the patients immediately before melphalan administration and returned i.v. 8 h later. Studies on marrow culture and melphalan pharmacokinetics predicted that this was a safe time to administer non-cryopreserved marrow. Four patients received lower doses of i.v. melphalan without autologous marrow. In the group receiving autologous marrow the time for recovery of peripheral-blood granulocytes to 800/mm2 or greater was significantly less (P = 0.01) than in those not receiving marrow. In 7 patients the tumour showed evidence of response to the drug and there was 1 complete remission. This treatment deserves investigation in patients with tumours more sensitive to drugs than melanoma."}
+{"text": "Knowledge of the appearances of normal bone marrow, metastases involving marrow,            and therapy-related marrow changes shown by MR imaging is necessary in order to avoid            misdiagnosis. This article reviews MR imaging techniques and the findings that allow            distinction of normal yellow (fatty) marrow and red marrow from tumor in marrow,            as well as the identification of marrow changes resulting from radiation therapy or            treatment with marrow-stimulating drugs in patients with musculoskeletal tumors."}
+{"text": "Tracheobronchopathia osteochondroplastica (TO) is a relativelyrare benign disease, around 300 and 130 cases have been reportedin English and Japanese literature, respectively. Most of thecases have been diagnosed incidentally at autopsy. Due to thewidespread use of fiberoptic bronchoscopy (FOB), the number ofcases diagnosed upon examination is increasing. Here, we report acase of a 72 year-old man with a history of crown aspiration, whowas diagnosed as TO upon removal of a foreign body using FOB. Thediagnosis of TO and the removal of an aspirated crown by FOB arediscussed."}
+{"text": "Chronic Obstructive Pulmonary Disease (COPD) is characterized by defective extracellular matrix (ECM) turnover as a result of prolonged cigarette smoking. Fibroblasts have a central role in ECM turnover. The TGF\u03b2 induced Smad pathway provides intracellular signals to regulate ECM production. We address the following hypothesis: fibroblasts have abnormal expression of genes in the Smad pathway in COPD, resulting in abnormal proteoglycan modulation, the ground substance of ECM.We compared gene expression of the Smad pathway at different time points after stimulation with TGF\u03b2, TNF or cigarette smoke extract (CSE) in pulmonary fibroblasts of GOLD stage II and IV COPD patients, and controls.Without stimulation, all genes were similarly expressed in control and COPD fibroblasts. TGF\u03b2 stimulation: downregulation of Smad3 and upregulation of Smad7 occurred in COPD and control fibroblasts, indicating a negative feedback loop upon TGF\u03b2 stimulation. CSE hardly influenced gene expression of the TGF\u03b2-Smad pathway in control fibroblasts, whereas it reduced Smad3 and enhanced Smad7 gene expression in COPD fibroblasts. Furthermore, decorin gene expression decreased by all stimulations in COPD but not in control fibroblasts.Fibroblasts of COPD patients and controls differ in their regulation of the Smad pathway, the contrast being most pronounced under CSE exposure. This aberrant responsiveness of COPD fibroblasts to CSE might result in an impaired tissue repair capability and is likely important with regard to the question why only a subset of smokers demonstrates an excess ECM destruction under influence of cigarette smoking. Chronic Obstructive Pulmonary Disease (COPD) is a severe, slowly progressive and disabling disease associated with accelerated lung function decline. COPD consists of emphysema, small airways disease, and chronic bronchitis, which may be present alone or in combination of different intensities. Emphysema is due to an extensive extracellular matrix (ECM) destruction of lung parenchyma -5. In adPulmonary fibroblasts are essential cells in tissue repair processes since they are key producers of ECM constituents . TGF\u03b2 isIdeally, fibroblasts should be capable of repairing cigarette smoke induced lung damage. This repair process appears inadequate in a subset of smokers, leading to gradually increasing parenchymal ECM destruction and emphysematous changes . We addr1-antitrypsin deficient. Emphysema was assessed by routine histological examination of lung tissue, performed by an experienced pulmonary pathologist (WT). The clinical characteristics of the groups are presented in table ).To answer the question whether genes of the Smad pathway are differentially expressed in COPD and controls, we stimulated primary fibroblasts of patients with stage II and stage IV COPD and controls with TGF\u03b2, TNF and cigarette smoke extract (CSE) for 1, and 24 hours. Primary fibroblasts were cultured from lung tissue, obtained from 23 individuals. Classification of COPD severity was based on the 2003 Global initiative for chronic obstructive lung disease (GOLD) criteria . Three g1 73% of predicted) was derived from non-involved lung tissue from patients undergoing resective surgery for pulmonary carcinoma. Tissue was always taken as far as possible from the tumor, or from a non-involved lobe. Histopathologically emphysematous lesions were present, yet of limited but varying severity. The moderate forms can be histopathologically demonstrated by the finding of isolated or free-lying segments of viable alveolar septal tissue or isolated cross sections of pulmonary vessels [Tissue of GOLD stage II COPD patients  was obtained from COPD patients undergoing surgery for lung transplantation or lung volume reduction. All individuals had quitted smoking for at least 1 year before surgery. The resected tissue showed both macroscopically and microscopically severe emphysematous lesions, often accompanied by bullae. Sub-pleural fibrous areas were avoided.Tissue of GOLD stage IV COPD patients (median FEV1 86% of predicted) was derived from non-involved lung tissue from patients undergoing resective surgery for pulmonary carcinoma. Patients had no airway obstruction, nor chronic airway symptoms like cough and sputum production. Material was always taken as far as possible from the tumor, or from a non-involved lobe. No histopathological abnormalities were present.Tissue of the control group . Isolated cells were characterized as fibroblasts by morphological appearance and expression pattern of specific proteins as described previously . FibroblFibroblast cultures cultured in complete culture medium ). Experiments were performed on fibroblasts of passage 5/6 with confluent growth. After reaching confluence, fibroblasts were cultured for 24 h on 0.5% FBS culture medium before the stimulations started. Cells were washed with Ham's culture medium without FBS and incubated with the appropriate stimulus, diluted in complete culture medium. TGF\u03b2  was used in a concentration of 100 u/ml, TNF (R&D systems) in a concentration of 1000 u/ml and CSE was used in a concentration of 2.5%. CSE was prepared according to a standardized protocol by bubbling the puffs of 4 cigarettes (Kentucky University research cigarettes) through 50 ml of Ham's F12 (Cambrex). The medium was filter sterilized with a 22 um filter. After the designated duration of the stimulation (1 and 24 hours) cells were harvested by trypsinization  followed by two wash steps. Non-stimulated fibroblasts were also harvested at 1 h and 24 h for comparison of effect of stimulation. Cell pellets were lysed using lysis buffer of the RNeasy mini kit for RNA isolation . Optimal concentrations and durations of the stimulation were determined in previous experiments (data not shown). As a check for an adequate effect of TGF\u03b2 stimulation, expression of plasminogen activator inhibitor (PAI)-1, being a classical TGF\u03b2 regulated gene, was analyzed, similar to the other genes. PAI-1 was significantly upregulated by TGF\u03b2 in all fibroblast samples tested.-delta-delta-Ct was taken for each stimulated fibroblast sample, which was compared to its unstimulated counterpart and presented as a percentage of this basal value; each basal values was set to 100%. Several housekeeping genes were tested for the influence of the experimental procedure on the expression. Ribosomal protein S9 was chosen as most optimal household gene because gene expression was most stable under basal as well as stimulation conditions.Fibroblast total RNA was isolated using the RNeasy mini kit (Qiagen). RNA quantity and quality (OD 260/280) were determined by optical density measurements on the Nanodrop. Total RNA was treated with DNase I during RNA isolation and run over the column to remove genomic DNA (Qiagen RNA-se free DNAse set). Three ng mRNA was transcribed into cDNA by reverse transcriptase II . Real time PCR was performed on an ABI7900 HT sequencer with \"Assay on Demands\" from Applied Biosystems , according to the manufacturers' instructions. Expression of the following genes was analyzed: Smad2, 3, 4, 7, versican, biglycan, and decorin. Data were analyzed by the delta-delta-Ct method . In brieDifferences in subject characteristics and real time data between the gene expression at basal levels were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test. Differences between gene expression after stimulation were analyzed using the Wilcoxon signed rank test. To analyze differences between COPD- and control fibroblasts, we calculated the percent change from the stimulated value as compared to the basal value. Significant differences in this percent change between COPD fibroblasts and controls were also analyzed using the Kruskal-Wallis test followed by a Mann-Whitney U test. The level of significance used was < 0.05, all reported P-values are two-sided.Downregulation of Smad3 gene expression occurred at 24-hour stimulation, while Smad2, Smad4, biglycan and versican gene expression were upregulated figures . Smad7 gUpregulation of Smad2 and 7 gene expression occurred at 24-hour stimulation. Smad4 was upregulated at both 1-hour and 24-hour stimulation. Biglycan was downregulated after 1 hour. Versican gene expression was downregulated at 24-hour stimulation figures .No effect on Smad genes occurred. The genes for biglycan, decorin, and versican were downregulated after 1 hour of exposure, without an effect at 24 hours.Whereas Smad3 gene expression in COPD stage II fibroblasts was significantly downregulated at 1- and 24-hour stimulation  stimulation. We provide suggestive evidence that this is indeed the case. Since the Smad pathway coordinates a delicate balance between fibrosis and excess ECM destruction, alterations in this pathway can contribute to the development of COPD. All investigated genes were similarly expressed in control and COPD fibroblasts without stimulation. In contrast, we observed a stimulus dependent gene expression of the Smad pathway with differential expression in COPD and control fibroblasts. In particular Smad3 and Smad7 were aberrantly regulated in COPD fibroblasts under influence of TNF and CSE figure . MoreoveLittle is known about the regulation of Smad and ECM gene expression in lung tissue fibroblasts of COPD patients by TGF-\u03b2, TNF, and CSE, factors that are of prime importance in COPD development. We therefore first investigated the regulation of the Smad pathway in control fibroblasts and observed that TGF\u03b2 stimulation in general resulted in downregulation of Smad3 gene expression while Smad2, 4, 7, biglycan and versican gene expressions were upregulated. This downregulation of Smad3 gene expression by TGF\u03b2, together with the upregulation of Smad7 supports an inhibitory feedback mechanism because a reduced presence of Smad3 mRNA will result in reduced levels of Smad3 protein, as suggested previously . SurprisAn unexpected finding was that TNF induced upregulation of Smad2, 4 and 7 mRNA in control fibroblasts after 24-hrs, similar to TGF\u03b2. Furthermore, TNF induced an early downregulation of biglycan and later downregulation of versican. TNF has been demonstrated to be able to inhibit ECM production by fibroblasts . This isThe second research question we addressed is whether the Smad pathway is differentially expressed under basal conditions in fibroblasts of COPD patients and control subjects. We observed no significant differences between the groups suggesting that the altered modulation of the ECM as observed in COPD is not due to intrinsic differences in basal expression levels of the Smad pathway genes in fibroblasts. We can not rule out that the basal gene expression is normalized due to culturing of the fibroblasts for several passages. However, our data show that these fibroblasts are still capable of responding to stimuli and this particularly uncovers the differential fibroblast response in COPD patients and controls.The third question we addressed is whether the differences in ECM modulation as observed in COPD can be ascribed to a differential modulation of the Smad pathway under influence of COPD relevant cytokines and cigarette smoke, the most important etiologic risk factor of COPD. Stage II and IV COPD fibroblasts displayed up- and downregulation of Smad genes under influence of TGF\u03b2 and TNF in the same direction as the control fibroblasts. As CSE exposure mainly induced inhibition of expression, it seems likely that a general effect may play a role, although of course some of the presumed > 5000 cigarette smoke compounds also may have a specific role affecting certain receptors or downstream mediators. Importantly, CSE exposure induced differential effects on Smad4 and 7, and biglycan and decorin gene expression of COPD fibroblasts, findings that were not or only transiently observed in control fibroblasts. Apparently, fibroblasts of COPD patients are more reactive to components of cigarette smoke extract. Of importance to the pathogenesis of COPD, CSE blocked the repair effect of COPD pulmonary fibroblasts, as represented by a decreased Smad3 gene expression at 1 hour and Smad 4 expression at 24 hour, together with the elevated Smad7 gene expression at 24 hours. It has been demonstrated that CSE is capable to upregulate the expression of GADD34, a cell cycle related protein . The inhIn contrast to the increased Smad7gene expression in our lung tissue fibroblasts, we previously demonstrated a reduced expression of Smad7 protein in bronchial epithelial cells of COPD patients compared to control subjects . In addiLung fibroblasts from COPD patients as well as controls showed an upregulation of biglycan and versican gene expression after 24-hour TGF\u03b2 stimulation. This indicates that lung fibroblasts of COPD patients are capable of upregulating ECM genes on gene expression level, and the results depend on the effects of the local cytokine microenvironment and the presence of cigarette smoke. Of interest, and compatible with our previous observations, decorin gene expression was mainly downregulated in COPD fibroblasts, a finding that occurred with all stimuli, and only transiently at 1 hour after CSE in control fibroblasts. This highlights the putative important role of decorin in COPD.Obviously our data are a first set of experiments that now need expansion at the protein level and including additional kinetic data. There are some limitations to our study since our experiments were focused on intracellular regulation, and hence it is not always clear whether the direction of the modulation is a direct result of the applied stimulus or the result of a cellular counteraction, as a response to upregulation of other cellular factors by this applied stimulus. Therefore, our data is conclusive as to our hypothesis that there is an aberrant regulation of the Smad pathway, but it is only supportive as to any conclusion of the exact underlying nature of this aberrant regulation.In conclusion, our study in control and COPD fibroblasts shows similar regulation of the Smad pathway in COPD and controls without stimulation, but differential effects of particularly cigarette smoke on fibroblast expression of the Smad-genes, an intracellular pathway that is involved in regulation of ECM gene expression. TGF\u03b2, TNF, and CSE cause differential downregulation of decorin gene expression in COPD patients, at least partially via the Smad pathway. Our findings may explain why only a subset of smokers demonstrates an excess parenchymal ECM destruction under influence of cigarette smoking. Smad3, 4 and 7 have to be considered as important factors in the defective repair process of COPD fibroblasts, since smoke exposure affects expression of these genes in COPD but not in control fibroblasts. Because of the chronicity of COPD in combination with its slow progression, even subtle differences in this pathway can have a great impact in the outcome of the disease.ECM: Extracellular matrix; COPD: Chronic Obstructive Pulmonary Disease; TGF\u03b2: Transforming Growth Factor \u03b2; TNF: Tumor Necrosis Factor; CSE: Cigarette Smoke Extract; GOLD: Global initiative for Chronic Obstructive Lung Disease; FEV1: Forced Expiratory Volume in 1 second; Ct: Concentration in TimeThe authors declare that they have no competing interests.AZ carried out the data analysis and drafted the manuscript. DP and WT participated in the design of the original study, were responsible for clinical and histological patient data and contributed substantially to the manuscript. MRJ and AZ carried out real-time PCR. MRJ, JAN and JTWMV carried out the fibroblast isolations and culture and stimulation experiments and contributed to the manuscript.Results of real-time PCR analysis 1 and 24 h, GOLD stage II and IV and control. The data provided represent results of real-time PCR analysis at 1 and 24 h, presented for control and disease stage, and per gene of interest and per stimulation. Values are based on 2-delta-delta-Ct values.Click here for file"}
+{"text": "The activation involved the processing of proMMP-2 by a membrane-bound metalloproteinase. We propose that, in the ovarian tumour microenvironment, interaction between tumour cells and fibroblasts may enhance fibroblast production of the proMMP-2 and TIMP-2. Collagen I, also present in the ovarian tumours, then induces these fibroblasts to activate proMMP-2 even in the presence of TIMP-2. This active MMP-2 can associate with the cell surface of tumour cells and fibroblasts and is used in the processes of tissue remodelling and invasion. \u00a9 1999 Cancer Research CampaignThe matrix metalloproteinase MMP-2 is up-regulated in epithelial cancers and its mRNA localizes to stromal fibroblasts. In this paper we show that co-culture of ovarian carcinoma cells with fibroblasts resulted in an enhanced release of proMMP-2 and TIMP-2 into the culture medium. Cell\u2013cell interaction was a major factor in this response and carcinoma cells stimulated proMMP-2 release from fibroblasts but not vice versa. Collagen I, in a dose-dependent fashion, induced activation of proMMP-2 by tumour-derived, but not normal, fibroblasts. Antibody to \u03b2"}
+{"text": "Human bone marrow obtained from patients with neutropenia contains a cell population which is absent or diminished in normal marrow. The abnormal population is composed of cells of volume 200-300 mum3 which sediment at 5-5 to 8-5 mm/h. Normal marrow contains one cell class giving rise to increased numbers of grnaulocyte colonies after mass culture, while marrow obtained from neutropenic patients, or from patients with marrow regeneration, shows two such populations; one of these cell classes corresponds to the abnormally large cells demonstrated on velocity sedimentation analysis. This population of large cells may represent a group of either self-renewing cells related to the committed granulocyte progenitors or the pluripotent stem cell."}
+{"text": "It would be useful to have psychometrically-sound measures of dental fear for Hispanics, who comprise the largest ethnic minority in the United States. We report on the psychometric properties of Spanish-language versions of two common adult measures of dental fear , as well as a measure of fear of dental injections .Spanish versions of the measures were administered to 213 adults attending Hispanic cultural festivals, 31 students , and 100 patients at a dental clinic. We also administered the questionnaire to 136 English-speaking adults at the Hispanic festivals and 58 English-speaking students at the same college where we recruited the Spanish-speaking students, to compare the performance of the English and Spanish measures in the same populations.The internal reliabilities of the Spanish MDAS ranged from 0.80 to 0.85. Values for the DFS ranged from 0.92 to 0.96, and values for the NS ranged from 0.92 to 0.94. The test-retest reliabilities (intra-class correlations) for the three measures were 0.69, 0.86, and 0.94 for the MDAS, DFS, and NS, respectively. The three measures showed moderate correlations with one another in all three samples, providing evidence for construct validity. Patients with higher scores on the measures were rated as being more anxious during dental procedures. Similar internal reliabilities and correlations were found in the English-version analyses. The test-retest values were also similar in the English students for the DFS and NS; however, the English test-retest value for the MDAS was better than that found in the Spanish students.We found evidence for the internal reliability, construct validity, and criterion validity for the Spanish versions of the three measures, and evidence for the test-retest reliability of the Spanish versions of the DFS and NS. According to the U.S. Bureau of Census statistics estimate for July 1, 2005, Hispanics comprise the largest ethnic minority in the United States . Most HiTo date, little is known about dental fear in Hispanics. One study found that dental fear was not a significant factor in explaining why Hispanics in the Southwest did not utilize dental services . AnotherThe report of a 2004 conference which focused on Hispanic oral health highlighted the need for the development of reliable and valid measures for the Hispanic population . MeasureThe two most frequently used measures of overall dental fear in adults are Corah's Dental Anxiety Scale (DAS)  and KleiThe Modified Dental Anxiety Scale (MDAS)  was deveThe original DFS was a 27-item questionnaire . The iteOne group has developed a Spanish-language version of the 27-item DFS for use in Spain . A seconA third English-language questionnaire has been developed to measure dental injection phobia in greater detail  . The NS In summary, while there are valid measures of dental fear in English, there is a dearth of Spanish-language versions for use with Hispanics. Therefore, we translated the MDAS, DFS and NS into Spanish and assessed their psychometric properties in three samples of Hispanics: Spanish-speaking adults attending Hispanic community festivals, college students in an area whose population is heavily Hispanic, and Hispanic patients receiving care in a dental clinic. Since English-speaking persons also attended the community festivals and college, we also elected to obtain data on the English versions of these measures in these two populations, which permitted us to compare the performance of the Spanish and English versions in the same populations.Ethical review boards at the University of Washington and Heritage University approved the studies.Data were collected from three groups of participants. For an overall examination of the Spanish-language versions of the fear questionnaires, we recruited participants from two community festivals which celebrated Hispanic culture. One festival took place in Seattle, an urban city, and the second festival took place in a largely-Hispanic area in rural Eastern Washington. These participants gave oral consent. In order to gather test-retest data, a second group of participants included Spanish- and English-speaking college students from Heritage University, a university in a largely-Hispanic area of Eastern Washington. While the language of instruction at the University is English, many students speak Spanish. These participants gave oral consent. The third group of participants included Hispanic patients at a dental clinic in rural Eastern Washington in order to examine the criterion validity of the Spanish-language measures. The adult patients gave written consent. For patients younger than 18, parents provided written consent and the minor patients provided written assent. Summary information about the English- and Spanish-speaking participants is shown in Table We compiled two questionnaires, each of which included the MDAS, DFS, NS, and demographic questions in either English or Spanish. The English language questionnaires were translated into Spanish by a professional translator and back-translated. Since we were focusing our data collection in rural Eastern Washington, an area with a large number of Hispanic residents, a bilingual individual from that area made minor changes to make the Spanish more consistent with local usage. During the first administration of the Spanish questionnaire, some participants found the translated version of one DFS item  to be too abstract. After consultation with bilingual experts, we replaced the original Spanish word for \"I perspire\" with a simpler word (\"Sudo\"). Subsequent participants did not express any difficulty with the simpler word. The Spanish versions of the MDAS and NS are included in the appendix. For copyright reasons, the Spanish version of the DFS is not included here. Interested persons may contact the author for more information.At the community festivals, we recruited both Spanish and English speaking participants to provide comparisons between languages. Since our primary interest was in gathering Spanish data, if a potential participant was bilingual, he/she was asked to complete the Spanish language questionnaire. A small number of potential Spanish participants, primarily older, were turned away because of literacy concerns. A total of 136 participants completed the English questionnaire, while 213 completed the Spanish questionnaire. Participants received a gift card for a supermarket as an incentive.In the college student sample, we first recruited English-speaking students to complete the English questionnaire twice in English, about 1 to 3 weeks apart. Fifty eight students completed the questionnaire in English. Thirty eight of them completed the questionnaire both times, 17 only completed the first questionnaire, and 3 only completed the second questionnaire. In a different academic year, we recruited Spanish-speaking students to complete the Spanish version twice, about 1 to 3 weeks apart. Thirty one students completed the Spanish version of the questionnaire. One student only completed the first questionnaire, and the remaining 30 completed the questionnaire at both administrations. Students were offered the chance of winning a gift certificate at the University bookstore as an incentive.In the third sample, patients who had dental appointments in a community clinic on the days when the research assistant was present, and who were able to read the Spanish questionnaire, were invited to participate. Recruitment continued until 100 patients agreed to participate. Adolescent and adult patients completed the Spanish version of the questionnaire and indicated what kind of dental treatment they had received. Due to low numbers for some of the specific dental treatments, the patients were categorized according to whether they had received invasive treatment requiring dental injection  or not . Thirty-six percent stated that they had received one of the invasive treatments, 41% stated that they had only received one or more of the non-invasive treatments, and the remaining 23% did not indicate what their dental treatment had been. The research assistant rated the patients' observed anxiety using Corah's Interval Scale of Anxiety Response (ISAR) . PatientQuestionnaire data were entered into an Excel data base and checked for accuracy. If a participant gave two answers to an item, the mean value was substituted. No other changes were made to the original data. Only completed questionnaires were included in each analysis. Analyses were done in SPSS Version 14.0 . In the student samples, scores from the first questionnaire completed were used for summary statistics. Cronbach's alpha was used to determine internal reliability. Intra-class correlations (two-way mixed) were used to compute test-retest reliability. Construct validity was examined by inter-correlating the three measures, since they were presumed to be tapping similar underlying constructs related to dental fear . CriteriTable Tables Criterion validity was assessed in the sample of dental patients. Behavioral ratings of anxiety (ISAR) were made for 97 of the 100 patients. In these ratings, higher scores indicated greater observable anxiety. The correlations between the ISAR and the two general dental fear measures were r = 0.37 for the MDAS (p < 0.001) and r = 0.39 for the DFS (p = 0.001). The correlation between the ISAR and the NS was r = 0.45 (p = 0.012) for patients who had received a dental injection as part of their dental treatment.This study is the first to examine the psychometrics of Spanish versions of the MDAS and the NS. We also developed and assessed a new translation of the 20-item DFS for Hispanics.We found very good evidence for the internal reliabilities of all three Spanish versions of the fear measures, with coefficients ranging from 0.80 to 0.96. We also found good evidence for the test-retest reliability of the DFS and NS. The one anomalous finding was that the test-retest reliability for the Spanish version of the MDAS was lower. This was surprising, since the summary statistics for all three measures were similar for the English and Spanish students, and the test-retest for the English version of the MDAS was acceptable. Although the item analysis of the Spanish MDAS indicated that all five items were measuring the same construct, the comparison of each item across both administrations indicated that one item  was not answered consistently, contributing to the lower test-retest value. As a result of these analyses, we subsequently had three additional bilingual translators look at this item, and there is disagreement across all six of our translators over whether the Spanish translation for \"drilled\" is accurate.The significant correlations found between the three fear measures in the Spanish samples provide evidence of their construct validity. In addition, the similarities in magnitude seen in the Spanish correlations when compared with the English results in both the community and student samples provide evidence that the Spanish versions appear to be behaving similarly to their English counterparts. Given that the English versions have established validity, these similarities across language versions provide additional evidence for the construct validity of the Spanish language versions.In each sample, the correlations between the MDAS and DFS were higher than those between the NS and either the MDAS or the DFS. While many fearful patients express anxiety about dental injections, it is important to keep in mind that dental fear presents heterogeneously, and not all fearful patients are anxious about injections . It is lIn terms of criterion validity, the relationships between the participants' self-report and the observer rating were significant for both of the general fears measures, as well as for the NS for those patients who experienced a dental injection. According to Cohen , the magThere are several reservations about our study. For practical purposes, we used samples of convenience in this study, and it would be desirable to replicate the results using other samples of Hispanics. As described above, the test-retest value found for the Spanish MDAS was lower than expected, perhaps due to the translation of one item. In addition, this study used a written questionnaire to measure dental fear. We found that some Hispanics could not read and therefore could not participate in the study. It is possible that the Hispanics whom we turned away might have different attitudes towards dental fear. Other studies of Hispanics have developed alternatives to written questionnaires when literacy rates are lower, such as those described by Boiko and colleagues . It woulIn sum, we found evidence for the internal reliability, construct validity, and criterion validity for the Spanish versions of the MDAS, DFS, and NS. We also found evidence for the test-retest reliability of the DFS and NS. On the other hand, the test-retest reliability of the Spanish MDAS was lower than expected.The authors declare that they have no competing interests.TC and SEC were the primary contributors to the design of the study. TC, MAC, LJG and SEC collected data. All authors contributed to the data analyses. TC drafted the manuscript. All authors read and approved the final manuscript.Spanish MDAS Items:1. \u00bf'C\u00f3mo se sentir\u00eda si tuviera que ir a su dentista para un tratamiento ma\u00f1ana?2. \u00bf'C\u00f3mo se sentir\u00eda si estuviera sentado/a en la sala de espera (esperando por el tratamiento)?3. \u00bf'C\u00f3mo se sentir\u00eda si estuvieran a punto de agujerarle un diente?4. \u00bf'C\u00f3mo se sentir\u00eda si estuvieran a punto de quitarle el sarro de los dientes y pul\u00edrselos?5. \u00bf'C\u00f3mo se sentir\u00eda si estuvieran a punto de ponerle una inyecci\u00f3n de anestesia local en su enc\u00eda, sobre uno de los dientes de arriba de la parte de atr\u00e1s de su boca?Spanish NS Items:Con respecto a las inyecciones dentales, yo creo que:1. La aguja anest\u00e9sica puede golpear un nervio o algo y herirme.2. Nada es tan doloroso como una aguja en mi boca.3. Soy muy dif\u00edcil de anestesiar.4. Si mi garganta se adormece por la inyecci\u00f3n, no podr\u00e9 respirar.5. Si mi garganta se adormece por la inyecci\u00f3n, no podr\u00e9 ingerir.6. Ver la aguja es aterrorizante.7. Soy sensible a la Novoca\u00edna y puede hacerme da\u00f1o.8. El adormecimiento no desaparecer\u00e1.9. Sentir que la aguja pica la piel es la peor parte.10. El dentista puede resbalarse y lastimarme.11. Solo la idea de una aguja penetrando mi cuerpo me causa terror.12. No se por que las agujas me aterrorizan.13. Inyectar qu\u00edmicos desconocidos en mi cuerpo me causa miedo.14. Me da miedo ver la aguja acercarse a mi boca.15. La aguja no esta limpia y puedo contraer una enfermedad.16. La aguja no esta limpia y puedo contraer una infecci\u00f3n.17. Es posible que me mueva y haga que el dentista me lastime.18. Pueda que sangre como resultado de la inyecci\u00f3n.The pre-publication history for this paper can be accessed here:"}
+{"text": "We have investigated, by immunocytochemical means, the value of an antiserum raised to milk-fat-globule membranes in detecting metastatic deposits of breast carcinoma in conventional histological sections of liver, lymph nodes and marrow. The antiseum recognizes a membrane component, which we have called Epithelial Membrane Antigen, and which is confined to but widely distributed in epithelial tissues and tumours derived from them. In the sections examined, a greater extent of tumour infiltration was usually found, largely due to the identification of single malignant cells which normally go unrecognized with conventional stains. The number of positive samples was only increased, however, in sections of marrow aspirates, and the reasons for this are discussed. We suggest that further increases in detection rates could be attained by using the antiserum on cytological smears of marrow or even in cell suspensions."}
+{"text": "By in vivo covisualization of oskar mRNA with Staufen, its putative trafficking protein, we find oskar mRNA to be present in particles distinct from Staufen for part of its transport. oskar mRNA stably associated with Staufen near the posterior pole. We observe oskar mRNA to oligomerize as hundreds of copies forming large particles which are necessary for its long range transport and localization. We show the formation of these particles occurs in the nurse cell nucleus in an Hrp48-dependent manner. We present a more refined model of oskar mRNA transport in the Drosophila oocyte.Efficient mRNA transport in eukaryotes requires highly orchestrated relationships between nuclear and cytoplasmic proteins. For  Drosophila melanogaster oocyte offers an ideal model system to study mRNP interactions with proteins involved in transport oskar mRNA (osk), which specifies the inchoate pole plasm as well as abdominal and germline determinants osk during its transport have also been identified. Many act to aggregate oskar transcripts during transport, a process that is likely conserved for many other mRNAs across species osk than for any other localized transcript The transport and localization of mRNAs to particular sites within cells is of broad biological importance and is essential in diverse processes such as growth and differentiation, asymmetric cell division, long-term memory formation, axon guidance and the establishment of the basic body axes osk transport, and the first identified affecting its posterior localization is Staufen Drosophila and mammals, the best studied of these being the Drosophila oocyte osk either during or just after the transcript exits from the nurse cell nucleus, and believed to escort osk to the posterior pole of the oocyte osk in vivo though the precise spatial location within the oocyte of their interaction during the localization process remains unknown osk is impaired in Staufen mutants osk in vitro, two lines of evidence are commonly used to suggest that it does so in vivo: mutations in Staufen abrogate the localization of oskosk at the oocyte posterior implying a functional link between the two osk to Oskar protein A protein central to osk transport is poorly understood. Understanding the spatio-temporal relationship between osk and these trans-acting proteins necessitates approaches permitting the direct observation of such dynamic events in vivotrans-acting factors with individual mRNP particles in real time, such as those introduced by Zimyanin et al, permit the precise description of transport events and how they are influenced by these factors osk particle during its transport.How Staufen and other proteins contribute to osk during localization in real-time. We covisualize native individual osk particles and Staufen in vivo. We show osk does not require Staufen to reach the posterior region of the oocyte, strongly suggesting Staufen is necessary for only a terminal step in the localization of osk. We demonstrate and directly observe for the first time what has only been hypothesized, that osk is transported and anchored in large, higher order RNP assemblies that are constantly reshaped in their form during transport. We find that the nurse cell nuclear protein Hrp48 plays a key role in the formation of these structures. We confirm recent studies that osk transport in the nucleus can be modeled by what we term diffusive transport whilst in the nurse cell and the oocyte it occurs via super-diffusive transport. Thus we build upon contemporaneous studies to develop a more refined model of long-range transport of osk.Here we begin to answer these questions by relating for the first time the spatial position where Staufen begins to escort oskar mRNA (osk) using molecular beacons. Molecular beacons are internally quenched hairpin shaped oligonucleotides probes that become fluorescent upon hybridization with their target sequence osk while ignoring other contents of the oocyte osk localization is disrupted, the posterior signal is lost; when the 3\u2032 UTR of bicoid mRNA  is transplanted on the osk coding sequence, the signal emanates from the anterior of the oocyte; and when two molecular beacons designed to bind side-by-side on oskar mRNA as a FRET (Fluorescence Resonance Energy Transfer) pair are introduced in oocytes, a clear posterior localization is observed, indicating specific hybridization and detection of osk with localization unperturbed. Furthermore, the target specificity of molecular beacons has been confirmed in various other cellular contexts We visualized endogenous osk coding sequence. Using carefully controlled concentrations resulted in higher fluorescence intensity and lower background signals, thus permitting high speed imaging in 3-D  Single particle analysis was used to characterize the mobility of mRNP particles in different compartments. The relationship between mean square displacement (MSD) and time interval can be used to classify the motion of a particle as \u2018classical diffusion,\u2019 \u2018confined diffusion\u2019 or \u2018active transport\u2019 osk motion had an \u03b1\u200a=\u200a1.4465, in oocytes \u03b1\u200a=\u200a1.166, and in the nucleus we determined the diffusion coefficient of osk to be 0.0124 \u00b5m2/s. Modeling of MSD to these specific types of non-Brownian motion is further described in the Supporting Information section. The behavior of osk in the nucleus was similar to the mobility of other mRNP complexes in the nucleus of cultured cells osk in the oocyte and nurse cell cytoplasm is attributable to active transport, which could either occur via molecular motors or movement under the influence of cytoplasmic flows.The exponent alpha, extracted from the power-law fitting the MSD curves, reflects the motion dynamics (\u03b1\u200a=\u200a1 corresponds to Brownian motion). Values of \u03b1 greater than 1 (termed \u201csuper-diffusion\u201d) are indicative of directed motion and those less than 1 relate a motion that is freely diffusive and undirected oskar mRNA  factors loaded co-transcriptionally (i.e. hnRNPA/B family member Hrp48 and Bruno), and during splicing of the first intron, such as the exon junction complex members (EJC) (i.e. Y14 and Mago nashi), (2) factors loaded in the cytoplasm which may connect osk to active transport pathways (i.e. Staufen), and finally (3) those factors loaded when osk reaches the posterior compartment to spatially control translation (as yet unidentified).A high degree of interplay between nuclear and cytoplasmic proteins is apparent in the correct transport and localization of osk to the posterior tip of the oocyte osk to the anterior end, the middle of the oocyte and with slight posterior localization oskar mRNA are notably absent in the literature. Therefore, in order to investigate the role of Staufen protein in the transport of osk, we co-visualized the two molecules in live oocytes and studied their mobility during oocyte development. To visualize Staufen protein in live oocytes, we utilized flies in which the natural Staufen gene was replaced with a Staufen-GFP gene . Further the biophysical characteristics of osk motion in the Stau (When stage 7\u201310 oocytes mutant for Staufen are probed rse cell . We obse compare . Furtherthe Stau ) retaineosk transcripts they continued to form the large particles that were similar to those observed in wild-type oocytes. We also observed that when we calculated the absolute displacement of osk particles, we found their displacement to be extensive, and the particles were able to occupy large areas of the oocyte chamber including the posterior of the oocyte . Around the ring canal, several osk particles underwent frequent collisions with Staufen particles, though they did not result in permanent associations. Those osk particles that seemed unable to cross the ring canal despite remaining in its vicinity for an extended period of time (up to 45 min) did so while remaining mobile. The particles repositioned themselves frequently prior to each attempt at crossing the ring canal  we did not find a single example when osk persisted as a cocmplex with Staufen-GFP, while entering and exiting the ring canal  and glycine-rich domains abrogating this localization osk 3\u2032-UTR termed the Bruno response elements (BREs) oskosk 3\u2032- UTR, as well as to a translational activation element near the 5\u2032 end of the mRNA, we examined its role in the in vivo oligomerization of osk.In order to understand the processes that lead to formation of large aggregates of osk in the nurse cell and oocyte cytoplasm, with no accumulation whatsoever of osk at the posterior pole (data not shown). Interestingly, in the nurse cell nuclei, we saw the accumulation of osk particles that appeared to be relatively immobile , we ruled out a direct relationship between osk particle formation and Staufen particle formation in Hrp48 mutants. Hrp48 is one of the three most abundant hnRNPs in Drosophila and is thought to bind most, if not all, transcripts in the nucleus We sought to shed light on the role Hrp48 could play in the reshaping/oligomerization process and whether the formation of oligomers was an essential precursor to localization. To this end, we imaged Hrp48 mutant egg chambers in a Staufen-GFP background . We obseimmobile  as compaimmobile . Furtherckground . The lacin vivo dynamics of the osk particles in the nurse cell nuclei of these Hrp48 mutants. We used QUIA to analyze and track single osk particles and reconstructed their motion in 3D , aubergine, is required for osk translation, and the microRNA mir280 has been computationally predicted to bind oskar mRNA osk near the posterior, we propose that Staufen is the trans-acting factor that is able to stably associate with osk and spatially restricts the translation of Oskar protein to the posterior of the oocyte.The Macdonald group first posited Drosophila Staufen dsRBD3, which is essential for Staufen function and osk localization, shares common structural features with the N-terminal domain of bacterial ribosomal RNA-binding protein S5 osk, but rather to another component of the mRNP, as it is speculated to do in neurons. Bolstering this observation is the finding that Staufen 1 and 2 in mammals are recruited to the polysomes and not to newly synthesized cytoplasmic mRNA osk to polysomes osk to polysomes for translation this would also explain the observed phenotype of increased Staufen accumulation at the posterior due to increased osk expression Studies in neurons have also questioned the role Staufen plays in the direct trafficking of mRNPs osk is transported. Our findings are reinforced by the recent work of Zimyanin et al. describing the motion of osk to be \u201cbiased random walk\u201d. Our biophysical data, and especially our highly refined spatial data describing specific velocities in specific regions is the first of its kind for osk. It largely concurs with the velocities given by Zimyanin et al., 0.04 \u00b5m/s\u00b10.02 versus our largest class of osk particles, which is 0.022 \u00b5m/s. We find this transport to be super-diffusive or active transport in the oocyte, while in other compartments it occur at lesser speeds. Experiments we conducted in Hrp48 mutant oocytes strongly suggest that the nuclear and cytoplasmic aggregation of osk and its assembly into higher order RNPs, is an essential step in its transport during stage 7 to 10. It has been observed that the action of molecular motors, kinesin I and dynein, generate cytoplasmic flows which serve to traffic large particles such as oskosk to the plus ends of the microtubule in the oocyte. Once osk reaches the posterior pole, it is \u2018captured\u2019 and the anchoring phase is initiated. Oskar protein acts to trap osk mRNA at the posterior, spatially controlling its translation, and creates a key feedback loop in the accumulation of osk. This may occur due to the prepositioning of ribosomes at the posterior, thus making synthesis of Oskar protein possible only there. This is true in neurons where ribosomes positioned at the post-synaptic, density to facilitate spatiotemporal control of translation of localized mRNAs.Our data in Staufen mutants begs the question of how osk translational and localization control that have been hypothesized in Drosophilaosk interactions, and those described by Zimyanin et al. The first would occur prior to, and independent of, posterior localization, and is characterized by the trans-acting factors, loaded in the nucleus and in the cytoplasm, which repress osk translation. The release from translational repression would be the second level. It would also require posterior localization of osk and as Gunkel et al., 1998, determined, a locally restricted interaction with a trans-acting factor would occur at the posterior of the oocyte to permit translation. Finally, the third level of translational control would occur as a translational activation event independent of both the initial repression and localization. This latter level of control is suggested by the phenotype of Staufen mutants. Under normal circumstances, posterior localization is a pre-requisite for osk translation. However, Staufen mutants still lack osk activity when the first level of control is abolished, i.e. translational repression mediated by nuclear factors such as Bruno. This implicates a requirement for Staufen in translation, independent of localization. Prior to our data, it was difficult to reconcile the ability of Staufen to be a key translational activator and be the major protein involved in the transport of osk. Some models of transport had suggested Oskar protein is actively degraded as osk is transported until it reaches the posterior where the degradation mechanism is silenced osk and Staufen near the posterior, is able to reconcile the role of Staufen with its demonstrated role as a translational activator.Long-range transport of mRNA depends on nuclear factors overwhelmingly acting to repress translation during transport and spatial control of translation of these mRNAs. This may be coupled to three different levels of osk interactions from our data are only stable at the mid-posterior, which still suggests an as of yet unidentified mechanism by which their stable interaction is spatially restricted to this region. A possible mechanism is the microRNA dependent recruitment of RISC to osk. Cook et al., 2004 suggested that mir280 binds to osk would recruit the RISC components armitage (armi) and aubergine (aub). Like Staufen, these proteins are required for the timely translation of osk. Evidence that may link Staufen to RISC comes from the recent finding showing a high degree of conservation between the RBD of Staufen and the Drosophila C virus (DCV) protein Drosophila to destroy viral RNA, thus enabling DCV to more successfully infect Drosophila. This high degree of conservation in the RBDs suggests Staufen may act through RISC to control osk translation. In addition, armi, an SDE3 homolog, contains eight motifs characteristic of the Upf1p family of ATP-dependent RNA helicases. In mammals, Staufen 1 recruits Upf1 to specific 3'UTRs where it acts in mRNA decay oskar mRNA decay at the posterior pole during later stages of oogenesis.Staufen-osk to be in a higher order RNP assembly that, if disrupted, leads to an inability to localize at the posterior or associate with Staufen there. Our results reveal the dynamic nature of osk particle assembly and movement through the various compartments of the oocyte and illuminate the dynamics of what was, until now, an opaque process. Our highly specific detection technique for mRNAs enabled for the first time, the continuous real-time, simultaneous covisualization of a native mRNA and a protein. Engaging rapid acquisition microscopy, we observed the dynamic behavior of osk and Staufen from a nurse cell through the ring canal and into the oocyte. We observed the formation of large mRNP particles undergoing extensive reshaping prior to and upon exiting the ring canal, all in a Staufen independent manner.We determine that the transport process from the nurse cell nucleus to the oocyte posterior requires osk, are sequestered in large complexes \u2018masking\u2019 them from translation and maintaining their stability. Chekulaeva and coworkers have proposed that Bruno binds to recognition sites in the 5\u2032- and 3\u2032- UTRs of osk, acts to promote the oligomerization of osk and represses translation. Our results are the first visual confirmation of osk transported in such oligomers of many osk transcripts. Through an elegant set of experiments, Spirin proposed that in unfertilized eggs, all stored  mRNAs exist in a large \u2018informosome,\u2019 defined as the \u2018masked\u2019 form of mRNA, found in equilibrium with inactive oligoribosomes, or mRNAs loaded with ribosomes. We found these \u2018informosomes\u2019 to be absent in Hrp48 mutants, and believe that this would also hold true in Bruno mutant flies. Thus, we show in vivo confirmation that high order assemblies of mRNPs, through nuclear factors, play a key role in osk transport. Such aggregates may possess hundreds of transcripts (Supporting Information), which remain efficiently \u2018masked\u2019 for transport over long distances. Given that Bruno and Hrp48 are thought to form homodimers that permit such oligomerization The formation of such oligomers has been recently proposed osk to be transported by cytoplasmic motors and, flows as implicated in studies by Zimyanin et al. Such large RNA oligomers/granules serving in transport have been strongly speculated to exist in several biological systems. The study of the biophysical processes that oskar oligomers and Staufen undergo, are a crucial beginning in understanding how osk RNA and subsequently Oskar protein are spatially positioned within the developing oocyte. Combining powerful detection methods, fluorescent proteins and image analysis algorithms, it will be possible to offer novel approaches to deciphering interactions between mRNAs, their trans-acting factors, cytoskeletal structures and molecular motors in vivo.Overall, the oligomerization of mRNPs is essential for D3/TM3 D3cnsP{GFP-stau7N}/Cyo linhaK02647 Hrp48K02814, Hrp48K16203We used the following fly stocks: OreR, stauDrosophila oocytes or into most posterior nurse cells, coming from females of various genetic backgrounds.Molecular beacons were designed by D. Bratu and synthesized by M. Mhlanga & C. Gouyette at the Plateforme de Synth\u00e8se d'Oligonucl\u00e9otides Longs \u00e0 Haut Debit (Institut Pasteur) as described in osk and Staufen-GFP were performed using QUIA software (Institut Pasteur) as described in the Supplementary Data. All microinjection and microscopy experiments were done at room temperature.The oocytes were teased in Halocarbon oil 700 (Fisher) on a glass cover slip. Microinjections were performed using an Eppendorf manipulator and Femtojet microinjector, delivering femtoliters of the molecular beacon solution. Live microscopy was performed with a Perkin-Elmer UltraView RS Nipkow-disk confocal system controlled with PE-viewer software (version 1.0.0.9) on a Zeiss Axiovert 200 inverted microscope with 40\u00d7 objective  acquired by a Hamamatsu Orca II ER cooled CCD camera or an Andor EMCCD camera. Chromatic aberration was verified before image capture by alignment of 15 \u00b5m Focal Check Fluorescent Microspheres (Molecular Probes). With this objective, pixel size is 164.83 nm. All analysis was done in 3D with a minimum of 12 Z-stacks and 1 \u00b5m Z-step used. Exposure time was 200 ms at excitation wavelength 488 nm and 568 nm/647 nm taken in continuous acquisition mode (i.e. no pause between Z stacks). Acquired images were converted from RAW format to 16-bit TIFF using a custom made ImageJ javascript written by M. Marchand, E.Glory & M. Mhlanga. Image analysis on these TIFF files such as 3D detection and tracking of Data S1Supplemental data information(0.24 MB DOC)Click here for additional data file.Figure S1Distribution of oskar molecular beacon in a stage 9 wild type oocyte. Single optical section of oocyte chamber, microinjected at the anterior with an oskar-specific molecular beacon labeled with TMR. Upon microinjection, the probe diffuses rapidly throughout the cytoplasm. Images were acquired every 10 sec. for 30 min. Within 100 sec., the fluorescent signal appears brightly at the posterior pole, indicating the presence of already localized oskar transcript. We quantified the fluorescent level emitted by the molecular beacon over a region encompassing the entire length of the oocyte . The anterior signal decreases upon diffusion of the probe from the injection site, and increases almost spontaneously to maximum levels, reflecting a thorough detection of the osk population at the posterior. N\u200a=\u200a5. Posterior is oriented to the right. Scale bar is 25 \u00b5m.(2.82 MB EPS)Click here for additional data file.Figure S2Particle reshaping. Oskar mRNP imaged at the ring canal as it transverses into the oocyte. A Z-projection of 1 \u00b5m optical slices is displayed as a time sequence. The montage shows the accumulation of particles into a large RNP aggregate, which changes in shape as it funnels through the ring canal. The aggregate breaks down and dissipates as it progresses into the oocyte cytoplasm. See Video 8. Scale bar is 10 \u00b5m.(30.32 MB EPS)Click here for additional data file.Figure S3Barentsz and oskar. To visualize clearly the association between osk and Barentsz the red and green channels were de-noised. Cancellations of the background and noise effects make the superposition of the two channels particularly demonstrative of the colocalization at a given time. This process involves first a multi scales B3 wavelet transform which is especially adapted to isotropic signals such as RNA particles, and then a statistical thresholding in each band. Here six tracks of oskar-Barentsz-GFP complexes are shown with their movements in the cytoplasm of the nurse cell. Barentsz is present at the nuclear envelope (most likely the outer envelope) and loads oskar mRNPs as they exit the nucleus (see movie 12). The Barentsz GFP transgenic fly also expresses endogenous (unlabeled) Barentsz protein, therefore particles of oskar mRNP without Barentsz GFP are visible.(1.48 MB DOC)Click here for additional data file.Figure S43D plus time tracking of oskar mRNA. (A) The velocity and MSD of osk were calculated in a wild-type oocyte injected with molecular beacons. The resulting tracks were superimposed on the final timepoint image. Using an advanced version of our standard algorithm QUIA, three regions of the oocyte were selected to analyze the velocity of osk. Osk motion is heterogenous with many molecules engaged in slow motion as well as a small sub-population engaged in rapid motion at a given time. Three regions within the oocyte are arbitrarily selected, and depicted in Green, Red and Yellow boxes. (B) The tracks within each of the Green, Red and Yellow regions were analyzed for their velocity. The number of displacements refers to the number of tracks. The tracking algorithm is able to give important information as to how subpopulations of osk particles behave in differing regions of the oocyte. This is especially important since the movement is not uniform throughout the entire oocyte chamber. Posterior is oriented to the right. Scale bar is 25 \u00b5m.(1.00 MB PDF)Click here for additional data file.Movie S1Nuclear export of oskar mRNPs from nucleus of a nurse cell at stage 8 of oogenesis. Molecular beacons were injected in a nurse cell cytoplasm. As the probes diffused into a neighboring nurse cell via a ring canal, they became sequestered into its nucleus. Native oskar mRNPs were visualized as they formed and exited that nucleus into the cytoplasm. Red arrowheads at time frame 440 sec mark some of the particles. Still frames with tracks are portrayed in (0.96 MB MOV)Click here for additional data file.Movie S2Oskar mRNP traffic in stage 9 oocytes. Oskar mRNP particles form in the nurse cell and are transported into oocyte via the ring canals. Arrowheads indicate several particles during an early time-point frame (60 sec). Fluorescent signal accumulates in a crest at the posterior of the oocyte indicating the localization of endogenous oskar mRNP indicated by another arrowhead that appears during a few late time-point frames of the movie (1400 sec). Time is noted on the top right corner. More details are found in legend of (0.93 MB MOV)Click here for additional data file.Movie S3Multidimensional tracking of oskar mRNPs of the sequence in Movie 1 using QUIA . The oskar mRNPs are indicated by different color balls with the respective tracks. This representation of the 4D reconstruction enables visualization of several particles moving between the nucleus and the cytoplasm.(1.11 MB MOV)Click here for additional data file.Movie S4In vivo dynamics of oskar mRNPs over the entire egg chamber. Several red arrowheads indicate the formation of oskar mRNP particles in the nurse cells and as they are transported into the oocyte. White arrows close to the dorsal and ventral cortexes and towards the posterior indicate the direction of mRNP movement within the oocyte. A red arrow throughout the movie marks the posterior location where oskar mRNP arrests in transport prior to reaching the posterior as has been observed in Bratu et al., 2003.(1.06 MB MOV)Click here for additional data file.Movie S5Covisualization of oskar mRNP and Staufen-GFP. Z-projections of 12\u00d71.2 \u00b5m optical slices played over 90 min are represented in a red (oskar mRNP), a green (Staufen-GFP) and merged-colors time-lapse movies. The merged representation highlights the temporal colocalization of oskar mRNP with Staufen-GFP throughout the egg chamber. A zoomed view of the merged movie enables the colocalization Staufen and oskar mRNP to be observed only near the posterior of the oocyte, contrary to expectations. The dynamic movement of red and green particles persists throughout the egg chamber during the time of the acquisition.(3.13 MB MOV)Click here for additional data file.Movie S6In vivo dynamics of oskar mRNP is Staufen mutant oocytes. The oskar mRNP is able to reach all parts of the oocyte. QUIA generates tracking data for each detected particle that meets minimum displacement and speed criteria predetermined by the user. Yellow tracks are shown only for those detected oskar mRNPs that are displaced at least 2.5 \u00b5m in distance. (1.40 MB MOV)Click here for additional data file.Movie S7Oskar mRNP transport through the ring canal. The passage of oskar mRNP as it remodels to cross the ring canal from the nurse cell into the oocyte. Many particles are detected across the ring canal and 4 are numbered to correspond to the analysis data in (1.97 MB MOV)Click here for additional data file.Movie S8oskar mRNP and Staufen behavior at the ring canal. Z-projections of 12\u00d71.2 \u00b5m optical slices played over 30 min; Staufen (green) and oskar mRNP (red) are shown to undergo numerous collision that do not result in stable associations once they exit the ring canal and enter the oocyte. Extensive reshaping of oskar mRNP is also observed as a big particle funnels through the canal and breaks down into smaller particles within the oocytes.(1.32 MB MOV)Click here for additional data file.Movie S9Oskar mRNP dynamics in Hrp48 mutant oocytes. Oskar mRNPs are seen as small particles in the nurse cell nucleus. The particles undergo movements within the nucleus but are not seen exiting as in Movie 1. Arrowheads indicate oskar mRNPs. Colored tracks of individual particles are shown in parallel; the circle represents the last time point on the track.(2.24 MB MOV)Click here for additional data file.Movie S10Models of colocalization estimation. Movie 10 has a model of a synthetic sequence with two colored \u201cblobs\u201d that cross each other several times. Movie 11 is the identical sequence with noise and background signals added to the simulation. This is further described in the Supporting information section.(0.15 MB MOV)Click here for additional data file.Movie S11Models of colocalization estimation. Movie 10 has a model of a synthetic sequence with two colored \u201cblobs\u201d that cross each other several times. Movie 11 is the identical sequence with noise and background signals added to the simulation. This is further described in the Supporting information section.(3.78 MB MOV)Click here for additional data file.Movie S124D representation of Barentsz-GFP and oskar mRNA in stage 8 egg chamber. Barentsz protein (green) localizes at the nuclear envelope in the nurse cells. It is seen loading on oskar mRNA (red) and maintaining a stable association with oskar mRNA in time.(2.37 MB MOV)Click here for additional data file."}
+{"text": "P=0.02, CI: 1.0\u20131.2) during cycle 1 and over the entire treatment period . Pretreatment homocysteine concentrations did not predict for toxicity. In conclusion, fixed-dose capecitabine appears to have similar efficacy and safety compared to the currently recommended dose schedule based on body surface area and simplifies drug administration. A high pretreatment folate may be predictive of increased toxicity from capecitabine.The purpose of this study was to evaluate the safety and activity of fixed-dose capecitabine in patients with advanced colorectal cancer and to correlate pretreatment plasma concentrations of homocysteine and serum and red cell folate with toxicity. Patients received capecitabine 2000\u2009mg (4 \u00d7 500\u2009mg tablets) twice daily on days 1\u201314 every 3 weeks. They were reviewed weekly during the first cycle and then three weekly for safety assessment. Eligibility criteria were advanced/metastatic colorectal cancer, \u2a7d2 prior chemotherapy regimens, ECOG performance status 0\u20132 and life expectancy >12 weeks. A total of 60 patients were enrolled and 55 were evaluable for efficacy. The median age was 72 years and 63% of patients had a performance status of 1 or 2. Confirmed tumour responses were reported in 15 patients , 15.7\u201340.3%). The median time to disease progression was 4.9 months and median overall survival was 11.2 months. The median ratio of fixed dose to body surface area (BSA)-calculated dose was 88% (range 65\u2013108%). Significant myelosuppression was not observed. Grade 2/3 treatment-related adverse events were diarrhoea (34%), fatigue (27%), stomatitis (15%) and hand\u2013foot syndrome (22%). Dose reduction due to adverse events was required in 16 patients (29%) and multiple reductions in five patients (9%). There was no grade 3/4 haematological toxicity, any grade 4 adverse events or treatment-related deaths. Patients with higher pretreatment levels of serum folate experienced significantly greater toxicity ( As a single agent, 5-FU improves median survival by approximately 6 months . AttemptCapecitabine is an oral fluoropyrimidine that preferentially delivers 5-FU to the tumour via a three-step enzymatic conversion, the final step being catalysed by thymidine phosphorylase, which has a higher activity within the tumour compared with healthy tissue  inhibitors or folate antagonists . Previou9/l), platelets (<100 \u00d7 109/l), serum creatinine or serum bilirubin , and ALT, AST or alkaline phosphatase .Patients were eligible for entry into the study if they had locally advanced or metastatic colorectal cancer with measurable or evaluable disease. Patients could have previously received up to two prior chemotherapy regimens for metastatic disease. If administered, adjuvant therapy should have been completed \u2a7e6 months prior to study entry. Histological or cytological confirmation of colorectal adenocarcinoma was required. Patients had to be at least 18 years of age and have a life expectancy >12 weeks. ECOG performance status (PS) was required to be between 0 and 2. Patients were not included if they had active or extensive brain metastases, active systemic infection, inflammatory bowel disease, unstable cardiac disease or untreated vitamin B12 deficiency. Patients who were pregnant or actively lactating were excluded. Patients had to be able to be changed from warfarin to enoxaparin sodium  version 2.0 , biochemistry, liver function tests (LFTs), vitamin B12, carcinoembryonic antigen (CEA), serum and red cell folate and plasma homocysteine. Serum and red cell folate were measured in a central laboratory using National Committee for Clinical Laboratory Standards (NCCLS) procedures. Serum and red cell folate were measured using a competitive binding receptor assay . Samples were not batched, but analysed as patients were enrolled.Baseline CT imaging of the chest, abdomen and pelvis was obtained within 3 weeks of treatment commencement. At the start of each treatment cycle, history and physical examination were repeated and blood samples were taken for FBC, biochemistry, LFTs and CEA. Patients were reviewed weekly during cycle one and then every 3 weeks for safety assessment. All safety evaluations were graded according to the NCI-CTC except hand\u2013foot syndrome (HFS). Hand\u2013foot syndrome was classified as grade 1 , grade 2 (painful erythema with swelling or affecting daily living activities) or grade 3   both during cycle 1 and for the whole course of treatment and the following parameters was determined using logistic regression: gender, PS, number of sites of metastases, prior pelvic radiotherapy, creatinine clearance, homocysteine, serum folate, red cell folate, serum vitamin B12 and serum albumin. The odds ratio and its 95% confidence interval (95% CI) were used to quantify the extent of any association. Kaplan\u2013Meier survival curves were used to illustrate the distributions of the time to progression and overall survival.A total of 60 patients were enrolled from three centres in Australia between January 2002 and August 2003. Four patients were excluded from analysis as they were not dosed according to protocol and one patient withdrew consent. Therefore, the efficacy and safety analyses included 55 patients.The demographic and clinical characteristics of the patients are summarised in At the time of analysis, 48 patients had died. The cause of death in all patients was disease progression. The best tumour response over the entire treatment period for all treated patients is shown in 2. Assuming the normal recommended dose of capecitabine (1250\u2009mg\u2009m\u22122 twice daily on days 1\u201314 every 3 weeks) had been used, the total dose calculated on BSA should have been 63\u2009350\u2009mg during the first cycle of therapy. The median total flat dose received by patients in cycle 1 was 56\u2009000\u2009mg. Therefore, the median ratio of fixed-dose to BSA-calculated dose was 88% (range 65\u2013108%). Thus, patients received a median dose 12% lower than the BSA-calculated dose.At the time of analysis, five patients were still receiving capecitabine. The median duration of therapy with capecitabine was 97 days and the median number of cycles received was 6.5. The median dose intensity received was 100% (range 25\u2013114%). The median BSA value of the enrolled patient cohort was 1.8\u2009mThe most common treatment-related adverse events are reported in P=0.8), CEA (P=0.5), homocysteine (P=0.2), red cell folate (P=0.7), serum vitamin B12 (P=0.3) and albumin (P=0.9). A significant association was observed between high pretreatment serum folate and worse toxicity . On further analysis, elevated levels of pretreatment serum folate were associated with a higher incidence of grade 2/3 diarrhoea (P=0.001) and grade 2/3 nausea and vomiting (P=0.032) compared to grade 0/1 toxicity. For serum folate levels less than 17.96\u2009nmol\u2009l\u22121, no significant toxicity was seen. When assessing for potential predictors of toxicity over the entire treatment period, serum folate remains significant .Baseline laboratory test results were not available for all patients . The relIn the current study, we demonstrated that fixed dose capecitabine can be safely and effectively used as an alternative to conventional BSA-corrected dosing strategy in the treatment of metastatic colorectal cancer. Furthermore, we have shown that a raised serum folate level may be predictive of worse toxicity, in particular diarrhoea, nausea and vomiting.vs 64 years, respectively). Furthermore, our results concur with the efficacy results of the BSA-calculated dose schedule of capecitabine in a similarly elderly population (median 75 years). In a study reported by With respect to efficacy, the overall response rate was 28%, time to disease progression was 4.9 months and median overall survival was 11.2 months. These results are similar to the combined results of phase III studies of the currently recommended BSA-calculated dosage schedule for capecitabine, which recruited over 1200 patients: overall response rate of 26%, time to disease progression of 4.6 months and overall survival of 12.9 months . In compOverall fixed-dose capecitabine was well tolerated. As anticipated, diarrhoea and HFS were the predominant adverse effects. The majority of adverse events resolved with treatment delay, while a single dose reduction was required in 29% of patients and multiple reductions in only 9%. In total, 15% of patients ceased treatment because of adverse events and this was mainly due to gastrointestinal events. There were no treatment-related grade 4 adverse events or deaths.vs 9%), HFS (17 vs 11%), nausea and vomiting (2 vs 2%) and stomatitis (2 vs 0%). Therefore, toxicity appeared to be comparable in those receiving fixed-dose capecitabine.The toxicity data reported in this study are comparable to that of the combined safety analysis of the two-phase III studies of BSA-calculated dose schedule of capecitabine  in our study of fixed-dose capecitabine (P=0.02). This association remained significant for the entire treatment period (P=0.04). In particular, a higher incidence of grade 2/3 diarrhoea and nausea and vomiting was seen during cycle 1. These results are contrary to studies in the antimetabolites, in which low levels of serum folate, as indicated by a raised serum homocysteine level, were associated with worse toxicity  can be simply made up using the 500-mg tablet. This provides convenience for the patient and avoids potential confusion that might lead to miss-dosing by confusion between 500 and 150-mg tablets. The current results also suggest that a high pretreatment concentration of serum folate may be predictive of increased toxicity from capecitabine. This observation requires further investigation."}
+{"text": "Thong style flip-flops are a popular form of footwear for children. Health professionals relate the wearing of thongs to foot pathology and deformity despite the lack of quantitative evidence to support or refute the benefits or disadvantages of children wearing thongs. The purpose of this study was to compare the effect of thong footwear on children\u2019s barefoot three dimensional foot kinematics during walking and jogging.Thirteen healthy children (age 10.3\u2009\u00b1\u20091.6 SD years) were recruited from the metropolitan area of Sydney Australia following a national press release. Kinematic data were recorded at 200 Hz using a 14 camera motion analysis system  and simultaneous ground reaction force were measured using a force platform . A three-segment foot model was used to describe three dimensional ankle, midfoot and one dimensional hallux kinematics during the stance sub-phases of contact, midstance and propulsion.p; = 0.005 walk and by 8.1\u00b0, p; = 0.005 jog); increased midfoot plantarflexion during midstance  and propulsion ; increased midfoot inversion during contact  and reduced hallux dorsiflexion during walking 10% prior to heel strike  at heel strike  and 10% post toe-off .Thongs resulted in increased ankle dorsiflexion during contact (by 10.9\u00b0, Ankle dorsiflexion during the contact phase of walking and jogging, combined with reduced hallux dorsiflexion during walking, suggests a mechanism to retain the thong during weight acceptance. Greater midfoot plantarflexion throughout midstance while walking and throughout midstance and propulsion while jogging may indicate a gripping action to sustain the thong during stance. While these compensations exist, the overall findings suggest that foot motion whilst wearing thongs may be more replicable of barefoot motion than originally thought. Thongs  are a common footwear choice for Australian children. They arDespite the possible advantage of thongs compared to other footwear options for children, there is no evidence that they are beneficial. Indeed, there are concerns that thongs may be harmful. In a recent survey of 272 parents of children, thongs were implicated by the parents as contributing to 15% of forefoot and 22% of rearfoot complaints. HoweverTo our knowledge, no quantitative evidence to support or refute the benefits or disadvantages of children wearing thongs has been reported in the literature. The aim of this paper is to compare the kinematic effects of wearing thongs on children\u2019s feet with a barefoot control condition during walking and jogging using three dimensional motion analyses. It is hypothesised that, compared to barefoot, wearing thongs will see reduced hallux motion, greater midfoot dorsiflexion and ankle eversion.Study participants were thirteen children (8 girls and 5 boys) between 8 and 13 years of age (mean age 10.3\u2009\u00b1\u20091.6 SD years) from the metropolitan area of Sydney Australia who volunteered in response to publicly displayed posters, press release and fourteen radio interviews. Power analysis using data from a previous footwear study protocolInclusion criteria stipulated healthy children free of known foot deformity, and not requiring medical consultation for foot or leg pathology in the preceding six months, Beighton Score less than 5/9 to exclude hypermobile children and a foShank and rearfoot segments were defined using 3, 12 mm diameter, non-collinear reflective markers per segment  were considered as a single universal joint with its centre located at the midpoint between the markers on the medial and lateral malleolus. The forefoot segment X and Y axes had their origin in line with the navicular marker and the Z axis in line with the rearfoot joint centre. The axis system origin for the shank segment was midway between the medial and lateral femoral condyles. All segment X axes were initially aligned with the laboratory -Z axis (down), segment Y axis pointing anteriorly (X axis of laboratory) and the segment Z axis pointing to the right of the participant (-Y axis of the laboratory). For the shank segment the X axis was subsequently aligned with the rearfoot joint centre.The three degrees of freedom ankle joint angle was described using the joint coordinate system according to International Society of Biomechanics (ISB) recommendations. The midStudy participant characteristics were recorded Table\u00a0 and reflVideo data were recorded at 200 Hz using a 14 camera motion analysis system . The initial right foot ground reaction force was measured using a force platform . Calibration of all fourteen cameras was completed prior to each session of data collection. Residual error for the motion analysis system, representing the accuracy with which the system could reconstruct marker location within the captured volume, was <0.5mm across all testing sessions.All trials were truncated at 20% prior to heel-strike of the right foot and at 20% after the right foot toe-off and time normalised to the right foot\u2019s stance phase. In accordance with previous literature, the kinematic data were smoothed at 5\u2009Hz for walkFour events were used to define the three stance sub-phases: foot contact (heel contact to foot flat), mid-stance (foot flat to heel rise) and propulsion (heel rise to toe off). Foot flat and heel rise events were defined within stance phase using the minimum of the posteriorly directed and the zero-crossing of the anterior-posterior ground reaction force respectively.p\u2009<\u20090.05 was set to determine the significance of range of motion value and mean difference.For the primary discrete variable of the footwear condition thong to barefoot, a two by five nested repeated measures analysis of variance was used . Bonferoni adjustments to condition and gait were applied to test significant differences between footwear conditions and gaits walking and jogging over five trials. The threshold of . During jogging, barefoot and in thong conditions, foot-flat occurred at 20 and 22 percent of stance and heel-rise at 44 and 44 percent of stance respectively. Participant barefoot and thong ankle, midfoot and hallux range of motion (ROM) and walking and jogging velocity are shown in Table\u00a0Mean age, height, mass, Beighton Score, FPI, dominant leg and thong size for the participants are presented in Table\u00a0p; = 0.010, 95% CI ) more dorsiflexed in the thong condition when compared to barefoot  throughout the contact phase . Ankle frontal and transverse plane motion in the thong condition closely followed that of barefoot throughout the stance phase.At heel strike the ankle was 10.4\u00b0,   than when barefoot. During mid-stance, the midfoot was more plantarflexed by 6.6\u00b0,  when thongs were worn. The midfoot was more plantarflexed when thongs were worn during the propulsive phase by 6.7\u00b0,    at heel strike by 4.9\u00b0,  and at 110% of stance by 10.7\u00b0,   and following toe-off at 110% of stance by 5.8\u00b0,    and propulsion by 5.4\u00b0,   over the entire stance phase when thongs were worn but not significantly so  . Midfoot transverse plane motion in the thong condition showed no difference to barefoot throughout the stance phase  (TableHallux sagittal plane motion was unaffected by thongs while jogging.The purpose of this study was to examine the effects of wearing thongs on selected foot kinematics while children were walking and jogging using the barefoot condition as a baseline. Children adapted to wearing thongs with altered ankle kinematics during the contact phase while walking and jogging and midfoot adaptations during midstance while jogging. Hallux adaptations were observed while walking prior to and during weight acceptance and after toe off. Overall ankle, midfoot and hallux range of motion was unaffected while wearing thongs compared to barefoot.Self-selected barefoot walking velocity Table\u00a0 in the pOnly small differences were seen when children wore thongs compared to barefoot for the tested foot model. The overall pattern and range of joint angle motion for ankle, midfoot and hallux kinematics were comparable between barefoot and thong conditions during both walking and jogging Table\u00a0. BarefooAnkle and midfoot adaptations occurred during the contact and midstance phases while wearing thongs compared to barefoot. Significant ankle dorsiflexion during walking Figure\u00a0 and joggAn action to grip thongs may be present during midstance and in particular during propulsion with greater midfoot plantarflexion while walking Figure\u00a0 and to aAnecdotally, clinicians have believed it necessary to claw one\u2019s toes to maintain thongs. This popular belief has been found lacking with hallux plantar pressure measures reduced when wearing thongs. The preReduced hallux dorsiflexion immediately prior-to and at heel strike while walking may indicate an action to grip and lever the thong to make contact with the heel in preparation for weight acceptance at heel strike Figure\u00a0. This adThere were a number of limitations to the current study. Firstly, the inclusion criterion was restrictive. This limits the extent to which the findings can be generalized, and cannot be applied to those children with excessively flat or highly arched feet. Further research is required to substantiate the current findings. Our study considered the influence of thongs on children\u2019s kinematics in the immediate time after the thongs were put on and prior wear of thongs was not controlled for. Children were not separated into groups of habitual or infrequent wearers of thongs which may have an effect on condition familiarity and the individual methods to secure the thong. Future studies should include side-stepping tasks and should examine the inverse dynamics during prolonged wearing of thongs to better understand pathological implications of the processes necessary to maintain thongs and their effect.Thongs had a minimal effect on walking and jogging at self-selected speed. The adaptations seen in this study may be necessary to maintain contact between the thong and the foot. In particular, increased contact phase ankle dorsiflexion, during walking and jogging with reduced hallux dorsiflexion during walking suggests a need to retain the thong during weight acceptance. Greater midfoot plantarflexion during midstance while jogging and propulsion while walking and jogging suggests a gripping action to retain the thong during stance. Reduced hallux dorsiflexion after toe-off during walking indicates a gripping action may be necessary during early swing. These adaptations may result in muscle overuse syndromes for rearfoot dorsiflexors and midfoot plantarflexors with prolonged thong wear, however further evidence is required to explore these areas. While differences were statistically significant, clinical importance is yet to be determined and so, overall, foot motion whilst wearing thongs may be more replicable of barefoot motion than originally thought.The authors declare no competing interests.AC carried out participant recruitment, data collection and analysis, statistical analysis, interpretation of results and manuscript draft. AG assisted data collection, statistical interpretation and editing the manuscript. AH and BV assisted in editing the manuscript. RS conceived the study, constructed the methodology, procedure for statistical analysis and interpretation and study overall coordination and helped to edit the manuscript. All authors read and approved the final manuscript."}
+{"text": "Nondirective meditation techniques are practiced with a relaxed focus of attention that permits spontaneously occurring thoughts, images, sensations, memories, and emotions to emerge and pass freely, without any expectation that mind wandering should abate. These techniques are thought to facilitate mental processing of emotional experiences, thereby contributing to wellness and stress management. The present study assessed brain activity by functional magnetic resonance imaging (fMRI) in 14 experienced practitioners of Acem meditation in two experimental conditions. In the first, nondirective meditation was compared to rest. Significantly increased activity was detected in areas associated with attention, mind wandering, retrieval of episodic memories, and emotional processing. In the second condition, participants carried out concentrative practicing of the same meditation technique, actively trying to avoid mind wandering. The contrast nondirective meditation > concentrative practicing was characterized by higher activity in the right medial temporal lobe . In conclusion, the present results support the notion that nondirective meditation, which permits mind wandering, involves more extensive activation of brain areas associated with episodic memories and emotional processing, than during concentrative practicing or regular rest. Many types of meditation used for stress management and health can be described as a cycle of volitional and spontaneous cognitive processes  are described as paying attention to whatever comes into ones awareness - whether it is a thought, emotion, or body sensation - just following it until something else emerges without trying to hold onto it or change it in any way , \u201cloving-kindness meditation\u201d , and \u201cchoiceless awareness\u201d (open monitoring of mind wandering) compared to inexperienced controls , including frontal, parietal regions, lateral occipital cortex, and insula  with different types of attentional focus would affect the subjective experience and the pattern of brain activation during meditation assessed by fMRI.The National Committee for Medical Research Ethics in Norway approved the study. Informed written consent was obtained from all participants before inclusion.Twenty-seven experienced practitioners of Acem meditation (18 men and 9 women) were recruited. All participants were regular practitioners (2 \u00d7 30 min daily) and had extensive experience with longer meditation periods, including participation in at least one 3-week long retreat. Twenty-four were right handed, ascertained by the Edinburg Handedness Inventory , presented in randomized order. In each run the practitioners performed a sequence of four meditation blocks lasting 3, 5, 4, and 3 min respectively, interspersed with five resting blocks lasting 1 min each. Block length was varied in order to avoid \u201cfalse\u201d fMRI activation induced by expectation. All subjects were scanned with eyes closed. Concentrative practicing and rest were used as contrasts for nondirective meditation. This would minimize the possible effect of underlying traits in the subjects, each subject serving as his or her own control. Immediately following each scanning run, all participants were asked to complete a questionnaire assessing their meditation experiences: extent of mind wandering compared to regular home practice, whether they became drowsy or briefly fell asleep, and to what extent the sound from the MRI scanner was disturbing. They also confirmed whether they had been able to carry out the meditation tasks.TR = 3000 ms; flip angle = 90\u00b0; TE = 35 ms; FOV = 230 mm; slice thickness = 2.5 mm; matrix = 64 \u00d7 64 giving an in-plane resolution of 3.6 \u00d7 3.6 mm2. Also a high-resolution T1-weighted image series was collected using a three-dimensional magnetization-prepared rapid gradient echo sequence (MP-RAGE) consisting of 182 contiguous sagittal slices of 1.2-mm thickness with an in-plane resolution of 1 \u00d7 1 mm. For analysis, all images were reconstructed to 1 mm3.Structural and functional scanning was performed using a 3T Philips Intera scanner  with an 8-channel SENSitivity Encoding (SENSE) head-coil . Using BOLD-sensitive imaging, a total of 400 volumes was acquired for each run with a gradient-echo echo-planar-imaging pulse sequence. Each volume consisted of 44 contiguous axial slices, with the following scan parameters: SENSE-reduction factor = 2.2, www.fmrib.ox.ac.uk/fsl/). First, non-brain tissue was removed from the T1-weighted anatomical image-series using the Brain Extraction Tool . For the contrast nondirective meditation > concentrative practicing it was set to p < 0.05 and cluster forming z > 2.3 (p < 0.0214). To increase sensitivity, the threshold was set less stringently for the latter comparison, because the expected difference between two similar conditions is usually smaller and the variability greater than for respective comparisons with rest. For all three contrasts, correlation analysis with years of experience as independent variable was performed in FEAT using FLAME-1 algorithm. Years of experience was defined as an extra environmental variable for all three contrasts. Brain areas were identified by FSL atlases and other relevant sources for functional data as referenced.The two-level random effects statistical analysis of the fMRI data was carried out using Bayesian estimation techniques with FEAT : (1) How disturbing was the scanner sound in the background: 0 = not at all, 1 = some, 2 = much. (2) What was the extent of mind wandering compared to regular meditation outside the scanner: 0 = less, 1 = similar, 2 = more. (3) Did you become drowsy or fall asleep: 0 = wakeful, 1 = drowsy, 2 = fell asleep. The questionnaire data were analyzed in Microsoft Excel . Fisher's exact test was performed to assess whether mind wandering, drowsiness and disturbance by scanner depended on the mode of practicing (nondirective vs. concentrative) in 2 \u00d7 2 tables, excluding table lines with zero-cells. As described below, participants who fell asleep during scanning, were excluded from further analyses.Data from a brief questionnaire administered immediately after each fMRI recording indicated a trend for less mind wandering with concentrative practicing compared to regular meditation. Even though the meditation blocks were short and the number of participants small, a larger number experienced less mind wandering during concentrative practicing than during nondirective meditation, whereas the numbers of participants who were wakeful/drowsy and disturbed some/much by noise were similar during nondirective and concentrative practicing, respectively Table . A majorThe fMRI assessments showed that nondirective meditation activated several regions of the cerebral cortex as well as subcortical structures significantly more than during resting. However, compared to nondirective meditation, during concentrative practicing fewer areas were activated more than at rest. Some regions in the right temporal lobe were activated significantly stronger during nondirective meditation than concentrative practicing. The activated areas for each contrast are detailed below. There was no correlation between activation and years of meditation experience.nondirective meditation > rest was found in several regions, including orbitofrontal, motor, somatosensory, visual, association, and limbic areas  and the temporal lobe  were activated more than at rest.Increased signal for the contrast s Figure ; Table 2primary and supplementary motor areas of the left hemisphere, extending into Broca's area.Large clusters were also detected in the occipital lobe covering vision areas in the middle occipital gyrus and striate cortex. In the posterior part of the frontal lobe, activation occurred in sensorimotor and secondary sensory regions including part of the precuneus were activated. There was no change in Wernicke's receptive speech area.In the left parietal lobe, temporal lobe, three clusters were found: the fusiform cortex/inferior temporal gyrus/parahippocampal gyrus including the visual processing and facial areas, the hippocampus, and the amygdala.In the right cingulate cortex, separate clusters in the right and left anterior regions were activated, as well as in the right posterior regions. Activated clusters were also seen in two non-cortical regions: In the left basal ganglia , and in a right and a left cerebellar region.In the nondirective meditation < rest, showed no positive activation.The opposite contrast, concentrative practicing > rest revealed significant activation in three regions  showed stronger activation of the anterior cingulate cortex and the dorsal medial prefrontal cortex than control subjects  and in an anterior cingulate cluster during nondirective meditation Figure , Table 2Hippocampus activation has been associated with mind wandering by detailed temporal analysis of meditation with focused attention on breath . As emphasized in a recent source of mindfulness-based cognitive therapy , performed with an effort to reduce mind wandering, reduced the extent of default mode network activation compared to nondirective meditation, but not below the level of resting.Altogether, our findings support the notion that nondirective meditation is conducive for default mode network activation. They also indicate that this activation is related to the relaxed focus of attention, which allows spontaneous thoughts, images, sensations, memories, and emotions to emerge and pass freely, accepting them as part of the meditation process. Since the relaxed focus of attention is a core component of several practices, we speculate that mental activities associated with default mode network activation, may be essential for state and trait effects. Further research is needed to determine whether this activation is associated with retrieval of episodic memories and emotional processing during nondirective meditation.Svend Davanger, \u00d8yvind Ellingsen, and Are Holen perform voluntary work for Acem School of Meditation, an international not for profit organization. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Implementation of user-friendly, real-time, electronic medical records for patient management may lead to improved adherence to clinical guidelines and improved quality of patient care. We detail the systematic, iterative process that implementation partners, Lighthouse clinic and Baobab Health Trust, employed to develop and implement a point-of-care electronic medical records system in an integrated, public clinic in Malawi that serves HIV-infected and tuberculosis (TB) patients.Baobab Health Trust, the system developers, conducted a series of technical and clinical meetings with Lighthouse and Ministry of Health to determine specifications. Multiple pre-testing sessions assessed patient flow, question clarity, information sequencing, and verified compliance to national guidelines. Final components of the TB/HIV electronic medical records system include: patient demographics; anthropometric measurements; laboratory samples and results; HIV testing; WHO clinical staging; TB diagnosis; family planning; clinical review; and drug dispensing.Our experience suggests that an electronic medical records system can improve patient management, enhance integration of TB/HIV services, and improve provider decision-making. However, despite sufficient funding and motivation, several challenges delayed system launch including: expansion of system components to include of HIV testing and counseling services; changes in the national antiretroviral treatment guidelines that required system revision; and low confidence to use the system among new healthcare workers. To ensure a more robust and agile system that met all stakeholder and user needs, our electronic medical records launch was delayed more than a year. Open communication with stakeholders, careful consideration of ongoing provider input, and a well-functioning, backup, paper-based TB registry helped ensure successful implementation and sustainability of the system. Additional, on-site, technical support provided reassurance and swift problem-solving during the extended launch period.Even when system users are closely involved in the design and development of an electronic medical record system, it is critical to allow sufficient time for software development, solicitation of detailed feedback from both users and stakeholders, and iterative system revisions to successfully transition from paper to point-of-care electronic medical records. For those in low-resource settings, electronic medical records for integrated care is a possible and positive innovation.The online version of this article (doi:10.1186/s13104-016-1943-4) contains supplementary material, which is available to authorized users. As the numbers of patients co-infected with human immunodeficiency virus (HIV) and tuberculosis (TB) increase in sub-Saharan Africa, reliance on paper-based registers may result in poor record management and, ultimately, suboptimal clinical care. One innovative approach to improve data quality and strengthen the continuum of care is the implementation of user-friendly, real-time electronic medical record (EMR) system. EMR systems may be well received by clinicians , improveIn 2005, Lighthouse Trust, the largest public provider of antiretroviral therapy (ART) and TB services in Malawi\u2019s central region, in partnership with Baobab Health Trust, a local non-governmental organisation that leads the development of EMR system in the country, developed and launched an ART-focused EMR system. By 2006, both of Lighthouse Trust\u2019s clinics, Lighthouse and Martin Preuss centre (MPC), successfully employed the real-time, touchscreen-based EMR system to manage ART patients. Data management for more than 36,000 ART patients at both clinics is now entirely based on the EMR system resulting in near completeness of patient level data. Due to the EMR system success in improving both patient management and reliable record keeping, described previously [Although the current ART EMR system in Malawi is successful, one significant limitation is its singular programmatic focus. Nationally, almost 60\u00a0% of TB patients are co-infected with HIV . At MPC In 2010, Lighthouse Trust, with its partners Baobab Health Trust, the Malawi National TB Program (NTP), and the IeDEA Collaboration at the Institute of Social and Preventive Medicine at University of Bern in Switzerland, received a one-year innovation development grant from US President\u2019s Emergency Plan for AIDS Relief (PEPFAR) to develop and implement a real-time, touchscreen-based EMR system for TB and TB/HIV integrated care. The TB/HIV EMR system is expected to significantly increase service quality and facilitate program monitoring at MPC. If proven beneficial, the system will expand from MPC to other clinics.This paper shares insights and lessons learned from our three-phased approach to planning, developing, and implementing the point-of-care TB/HIV EMR system for TB and TB/HIV co-infected patient management implemented at MPC clinic. We first detail the steps taken in conjunction with developers, system users and local stakeholders during system planning. Then, we note the system components designed to aid patient care followed by our experiences in system deployment. Then, we discuss the delays and unexpected challenges we overcame during implementation. These experiences and lessons may assist providers, clinics and organizations in other resource limited settings that are considering developing and deploying TB/HIV EMR systems.The Malawi national Health Science Research Committee (NHSRC) provided a general blanket approval for the use of routine programmatic data for monitoring and evaluation every year; and as such, a separate formal ethics approval was not required from NHSRC.https://github.com/baobabhealthtrust/TB-ART.The TB/HIV EMR system and its components are Open Source. The code and system components are available for non-commercial, scientific use at While the technical processes were being discussed with management and technical staff at Lighthouse and Baobab, a series of concurrent meetings were conducted by Lighthouse staff with Baobab system developers, DHO TB staff, NTP, and MoH\u2019s HIV department. Due to the rapid nature of the award and expected results, these initial, highly-participative meetings were held over the course of several weeks, helping ensure buy-in, create ownership, and discuss perceived barriers to TB/HIV EMR implementation , pharmacy, and exit. Unlike the simpler flow for ART patients who largely follow the same visit flow, most TB suspects and patients fell under one of five, distinct, patient-provider encounter types depending on HIV status, TB suspects, TB patients , follow-up TB patients already in care with unknown HIV status or not yet on ART, follow-up patients on ART at MPC, and transfer-in TB patients  are moved to archive areas; the process of identifying the longest dormant file is automated in the TB/HIV EMR system. As patient numbers continue to increase, filing space will run out, leaving the potential for missing or misplaced files. To address this challenge, the TB/HIV EMR system reduces the need for complete paper files; however, the system does retain capacity for generating printed summary files for patient who transfer. As more ART facilities adopt TB/HIV EMR technology for ART and TB/HIV services, we expect the use of paper-based records to stop.Compliance with guidelines can present challenges especially with complicated patients. The TB/HIV EMR system is designed to enforce compliance to national TB and ART/PMTCT guidelines by requiring clinicians to complete key tasks such as ascertainment of HIV status and ART status, as needed. The user interface software was designed to reduce system complexity by presenting only one question and possible answers on one screen page, helping to reduce user error and ensure data completeness. The questions are simple and concise. Each question requires an answer to progress. The system prompts healthcare providers to consider additional prevention and treatment tasks during reviews, including ordering lab samples , consider drug contraindications, and counsel on drug adherence.Drug dispensing with multiple regimens, including those for TB and HIV can become difficult and riddled with errors in stock management. In the TB/HIV EMR system, the drug dispensing process uses the same barcode scanning technology employed for patient IDs. Unique standard barcodes for different drug regimens and quantities are pre-printed on labels and placed in the pharmacy. For each patient receiving TB and/or ART drugs, pharmacy technicians scan the corresponding barcode for the prescribed drugs and quantities Fig.\u00a0. This teDetermination of next appointment date requires consideration of each patient\u2019s drug requirements, adherence, and clinical review schedule\u2014a process that is hard to do correctly by hand. In the TB/HIV EMR system, the system automatically calculates and presents the next appointment date in a calendar-format for provider confirmation based on the drug quantities dispensed and adherence profile, allowing the provider to change the date based on clinical or patient need Fig.\u00a0. At the The primary TB/HIV EMR system users are TB clinical officers, nurses, clinic clerks and health surveillance assistants. Access rights for specific tasks are assigned according to cadre and system management level. For instance, the clinic clerk\u00a0cadre has access to registration and lab information modules only and cannot access tasks assigned to clinicians. A TB clinician trained in ART has access to all tasks through TB/HIV patient clinical review and prescription while a pharmacy technician is only allowed to dispense drugs. Super-users  and system developers have access to edit patient data including amending current and previous patients\u2019 visit records. System usage logs record usernames of all users who enter or change patient data records for quality assurance and supervision purposes.As components of the system were being developed, a series of provider-focused pre-testing sessions were organized with mixed groups of up to 10, total, clinicians, clerks, and data staff to assess clarity and order of questions, and to verify compliance to national guidelines. We also tested various patient flow scenarios to ensure skip patterns were followed, e.g. family planning questions were not asked to young children or assessment of ART eligibility in confirmed HIV-negative patients. Also, pre-test sessions ensured that system alerts designed to remind clinicians to conduct HIV tests, order follow-up lab tests, consider drug contraindications, etc., worked as expected. Early pre-testing of TB/HIV EMR question flow and content helped system users understand the questions, identify missing or misplaced content, and avoid errors in misinterpretation.After successfully pre-testing the system off-site, an intensive, two-day practical training was provided to approximately 20 healthcare providers and support staff at MPC\u2014those with and without previous EMR experience. This training session was held over a weekend so that all staff could attend without affecting patients. Service providers first practiced specific tasks  according to their cadre  using touchscreen computers. Subsequent TB/HIV EMR practice activities mimicked diverse patient visit types requiring system users to \u201cmove\u201d patients from registration through various clinic review services to drug dispensing. As system developers also attended the training, many issues in patient management and flow identified by users on Saturday could be immediately addressed by code fixes for further review and testing on Sunday. More complicated coding issues required additional days to correct and follow-up test with a smaller group of staff. Subsequently, through the initial on-site testing processes, system users identified various additional bottlenecks and system inconsistencies that required the developers to further refine system before a full clinic-based launch. After each system revision, additional, brief hands-on trainings were held on site with users to gain confidence and provide further feedback. When the system was completed, another weekend training was held with all clinic staff to demonstrate the final system and allow users to gain confidence in the system.As an indication of the volume of data that can now be more easily accessed for monitoring and evaluation, in only our first 4\u00a0months of full system implementation, from April and July 2013, 787 individuals were registered in the TB/HIV EMR system: 369 (45\u00a0%) TB suspects and 418 (55\u00a0%) TB patients  system intranet and terminals were tested and (2) a system dry-run from clinic registration to drug dispensing was conducted using diverse imaginary patients to identify and rectify additional issues before live deployment. Still, we encountered problems that required software developers to provide additional on-site assistance to avoid challenges in data quality while providers gained TB/HIV EMR skills and confidence.EMR systems require strict data security protocols to ensure both patient confidentiality and data privacy. The TB/HIV EMR system has several built-in security features: (1) each user  has a unique username and password for logging in; (2) each user or cadre is restricted to their specific domain (e.g. service area); (3) only select \u201csuper users\u201d may edit or revise patient entries; (4) patient information accessed by providers is restricted to the current patient (through entering patient\u2019s unique ID or scanning its barcode); (5) data are stored in a password-protected, database and are accessible only by highly-trained internal staff; (6) the TB/HIV EMR system keeps an audit trail by tracking users, locations and timestamps for all data changes; and (7) data is encrypted during off-site data back up using secure encryption protocols.Securing start-up and sustainable funding are critical for system initiation and maintenance. Financial requirements were categorized into two groups; start-up and ongoing costs. Start-up (one-time) costs included purchasing hardware and developing software (Additional fileAlthough implementation of large TB/HIV EMR system is complex, our three-phase approach to planning, design, and implementation allowed us to successfully launch the TB/HIV system in MPC clinic due to several key factors. First, an extensive, highly-participatory planning period with stakeholders ranging from service providers to MoH officials before and during the system development process, resulting in generating provider buy-in and ownership by the NTP who plan to adapt the system for national use. Second, alerts to order lab samples encourage high quality clinical practices while maintaining user-end simplicity, ensuing that providers were reminded to include all components of patient care even when faced with complicated patient management. Features such as prompts to order lab sample have the potential to decrease disparities in patient care because system-decision support is given to all providers. Third, working to ensure a consistent power-supply with a mix of electrical power and reliable backup system helped avoid unnecessary system failures and lost data. Fourth, a well-functioning paper-based TB registry greatly aided adoption of the TB/HIV EMR system because the clinic and all service providers already understood the importance of accurate and quality data in patient management. Lastly, adequate time should be allocated to mapping patient flows, assessing clarity and order of questions, development of software development and pre-test sessions to avoid unplanned delays and maintain user confidence.Overall, the TB/HIV EMR system demonstrates the potential to enhance integration of TB/HIV services and facilitate improved decision-making among providers. We plan to further evaluate the data collected to inform service integration, strengthen monitoring and evaluation, and, ultimately, improve patient treatment outcomes. In the meantime, the system will be scaled up to other high-burden TB/HIV centers in Malawi for additional testing and evaluation at the national level. We believe the additional benefits of the TB/HIV EMR system towards improving patient care will become apparent as the system is expanded to other clinics and clinicians over time. Despite the personnel, financial, and technical investments required for success, other clinics, organizations, and public healthcare service providers should consider TB/HIV EMR systems as a beneficial investment in patient care."}
+{"text": "MIT suppressed TNBC cell survival, at least partially, by transcriptionally down-regulating KLF5, an oncogenic transcription factor specifically expressed in basal TNBC. Finally, MIT suppressed TNBC cell growth in a xenograft mouse model. Taken together, our findings suggested that MIT inhibits basal TNBC via the Sp1/KLF5 axis and that MIT may be used for TNBC treatment.As the most malignant breast cancer subtype, triple-negative breast cancer (TNBC) does not have effective targeted therapies clinically to date. As a selective Sp1 inhibitor, Mithramycin A (MIT) has been reported to have anti-tumor activities in multiple cancers. However, the efficacy and the mechanism of MIT in breast cancer, especially TNBC, have not been studied. In this study, we demonstrated that MIT suppressed breast cancer cell survival in a dosage-dependent manner. Interestingly, TNBC cells were more sensitive to MIT than non-TNBC cells. MIT inhibited TNBC cell proliferation and promoted apoptosis  Breast cancer is highly heterogeneous and is clinically classified into several subtypes based on the expression of the estrogen receptor (ER\u03b1), PR (progesterone receptor), and human epidermal growth factor 2 (HER2): the luminal A subtype (ER\u03b1+ and/or PR+ with low levels of Ki-67), the luminal B subtype (ER\u03b1+ and/or PR+ with high levels of Ki-67), the HER2 subtype (HER2+), and triple-negative subtype 3. Compared to other subtypes, TNBC is associated with higher cancer grades, higher metastasis rates, and poorer prognosis5. Furthermore, TNBC with high Ki-67 levels and BRCA gene mutations have been reported to have poorer prognosis8. For women with metastatic TNBC, the 5-year survival rate is less than 30%4. Furthermore, there are no effective targeted therapies for TNBC, and adjuvant chemotherapy is currently the only treatment option clinically. Although some molecules, such as poly-ADP ribose polymerase (PARP)9, epidermal growth factor recptor (EGFR)10, phosphoinositide 3-kinase (PI3K)/AKT11, etc. have been revealed to be promising molecular targets for TNBC treatment, and their inhibitors have now entered clinical trials, no targeted therapy has been approved for TNBC patients currently. Therefore, there is an urgent need to develop effective treatments for this aggressive subtype of breast cancer. TNBC is also heterogeneous and may be further classified into 7 subtypes based on gene expression profiling12.Breast cancer is the most commonly diagnosed malignancy among women in China, accounting for approximately 15% of new cancers in women16. High KLF5 expression is an unfavorable prognostic marker correlated with shorter survival for breast cancer patients18. We previously found that KLF5 is highly and specifically expressed in basal TNBC cell lines19, and depletion of KLF5 significantly suppresses basal TNBC cell proliferation, survival, and tumor growth22. Pharmacological inhibition of KLF5 suppressed TNBC stemness and growth23. Taken together, KLF5 could serve as a promising therapeutic target for basal TNBC.As a member of the Kr\u00fcppel-like transcription factor family, human Kr\u00fcppel-like factor 5 (KLF5) has been implicated in promoting breast cell proliferation, survival, migration, stemness maintenance, and tumorigenesis26, via binding to GC-rich sequences of the target genes\u2019 promoter regions27. Inhibition of Sp1 has been found to suppress cell survival in various cancer cell models29. We previously reported that Sp1 is essential for basic transcription of KLF530. As a selective Sp1 inhibitor, Mithramycin A (MIT) inhibits Sp1 binding to DNA. It has been reported that MIT has anti-tumor activities in multiple cancers, including prostate cancer31, cervical cancer32, lung and esophageal cancer33, etc. Furthermore, MIT has been clinically used to treat several types of cancer, including testicular cancer34 and chronic myeloid leukemia35. However, whether MIT has anti-tumor effects on breast cancer, especially TNBC, has not been elucidated.Specificity protein 1 (Sp1), a member of the Sp/Kr\u00fcppel-like factor transcription factor family, regulates multiple cellular functions and promotes tumor progression by regulating cell cycle, apoptosis, and metastasisKLF5 transcription in time and dosage-dependent manners via inhibiting Sp1 binding to the KLF5 promoter. Ectopic overexpression of KLF5 partially rescued MIT-induced loss of cell viability. Our data suggest that MIT could serve as a candidate therapeutic drug for basal TNBC.In the present study, we demonstrated that MIT suppressed breast cancer cell survival, especially TNBC cell survival. MIT suppressed 50 ranging from 12.9\u2009nM to more than 100\u2009nM , ER+ breast cancer cell lines (MCF7 and T47D), HER2+ breast cancer cell lines (SKBR-3 and BT474) and TNBC cell lines , with MIT and measured cell viability. As shown in Fig.\u00a0nM Table\u00a0.Figure 1Since MIT significantly decreased cell viability Fig.\u00a0, we firs20, and Sp1 is a key transcription factor for KLF5 transcription30. MIT is well known to inhibit Sp1 recruitment to its target gene promoters37. We hypothesize that MIT suppresses basal TNBC through down-regulating the KLF5 expression by blocking the recruitment of Sp1 to the KLF5 gene promoter. To test this hypothesis, we treated HCC1806 cells with MIT and detected KLF5 expression. As shown in Fig.\u00a0FGF-BP expression in a time- and dosage-dependent manner. Similar results were observed in HCC1937 and MCF10A cell lines  and reconstituted in DMSO to a final concentration of 100\u2009mM for storage.All cell lines were purchased from the American Type Culture Collection (ATCC). The immortalized breast epithelial cell line MCF10A was maintained in DMEM/Ham\u2019s F-1250/50 medium supplemented with 5% horse serum, 0.5\u2009\u03bcg/ml hydrocortisone, 10\u2009\u03bcg/ml insulin, 20 ng/ml epidermal growth factor, 0.1\u2009\u03bcg/ml cholera enterotoxin, and 2 mM L-glutamine. TNBC cell lines HCC1806 and HCC1937 were cultured in RPMI-1640  containing 5% fetal bovine serum (FBS), 1.5\u2009g/L sodium bicarbonate, and 1\u2009mM sodium pyruvate. These cells were maintained in a humidified atmosphere with 5% CO49. Briefly, 40\u2009\u03bcg of each protein sample was subjected to SDS-PAGE and blotted onto polyvinylidene fluoride (PVDF) membranes. After incubating with specific primary antibodies at 4\u2009\u00b0C overnight, membranes were incubated with horseradish peroxidase (HRP) conjugated secondary antibodies . The images were taken using an ImageQuant LAS4000 Biomolecular imager , and were processed using Adobe Photoshop Elements 2.0 .Western blot analysis was performed as described previously49.The anti-PARP antibody was purchased from Cell Signaling Technology . The anti-cleaved Caspase3 antibody was from Imagenex . The anti-\u03b2-actin antibody was from Sigma . The anti-ER and anti-HER2 antibodies were from Santa Cruz . The anti-PR antibody was from Invitrogen . The anti-KLF5 rabbit polyclonal antibody has been described previouslyCell proliferation of HCC1806 and MCF10A cells was measured using the Click-iT EdU Alexa Fluor 647/488 Imaging Kit (Invitrogen), following the manufacturer\u2019s instructions.For cell cycle analysis, cells were plated in 6-well plates and treated with either MIT or DMSO control at an indicated dosage for 24\u2009h before cell cycle analysis. Briefly, the cells were trypsinized and fixed with 75% ethanol at 4\u2009\u00b0C overnight. The fixed cells were stained with propidium iodide (PI) buffer  in the dark for 30\u2009min at room temperature. Cell cycle was analyzed on an Accuri C6 flow cytometer  within 4\u2009h.4 to 4\u2009\u00d7\u2009104 cells/well. One day after plating, the cells were treated with either MIT or DMSO vehicle control at indicated dosage for another 48\u2009h. The cells were then fixed with 10% trichloroacetic acid (TCA) followed by staining with 0.4% (W/V) SRB for 30\u2009min at room temperature, and extra dye was washed with 1% acetic acid. Finally, 10\u2009mM Tris base was added to dissolve the dye and the optical densities at 530\u2009nm were measured using a spectrophotometric plate reader .Cell viability was measured using a Sulforhodamine B assay . Briefly, breast cells were plated in 48-well plates at densities varying from 1\u2009\u00d7\u20091016. The vector control and KLF5 expression lentiviruses were prepared according to published protocols50. HCC1806 cells were plated in either 12-well plates at a density of 1\u2009\u00d7\u2009105/well or in 48-well plates at a density of 2\u2009\u00d7\u2009104/well. Sixteen hours after plating, the cells were infected with prepared viruses. Cell culture medium was changed after 24\u2009h. The cells were fixed for the SRB assay after treating with 25\u2009nM MIT for 48\u2009h.The lentiviral FUCGW-KLF5 expression vector was constructed as described previously6 HCC1806 cells/point were implanted into mammary fat pads of the mice. Tumor sizes were measured using Vernier calipers once tumors became palpable. Tumor volumes were calculated using the following equation: tumor volume (cm3)\u2009=\u2009\u043b(length\u2009\u00d7\u2009width2)/6. When the tumor size reached 100 mm3, the mice were randomly and equally distributed into two groups, which were treated daily with either a saline control or MIT (0.3\u2009mg/kg). Tumor size was monitored twice per week. All mice were sacrificed and tumors were collected for analysis when the tumors reached approximately 1\u2009cm in the largest diameter.Animal care and experimental procedures were, in accordance with the Institutional Guidelines, reviewed and approved by the Animal Ethics Committee of Kunming Institute of Zoology, the Chinese Academy of Sciences, and all experiments were performed in accordance with relevant guidelines and regulations. Twelve 6-week-old female Balb/c nude mice were purchased from SJA Laboratory Animal Co., Ltd . 1\u2009\u00d7\u200910P values less than 0.05 were considered as significant.All experiments were repeated at least three times. When appropriate, the data were pooled and expressed as the mean\u2009\u00b1\u2009standard deviation and analyzed by Student\u2019s t-test. The datasets generated during the current study are available from the corresponding author on reasonable request.Supplementary data"}
+{"text": "The aim of this study is to investigate the prevalence and severity of gingival inflammation and associated risk indicators in South American adults.Multi-stage samples totaling 1,650 adults from Porto Alegre (Brazil), Tucum\u00e1n (Argentina), and Santiago (Chile) were assessed. The sampling procedure consisted of a 4-stage process. Examinations were performed in mobile dental units by calibrated examiners. A multivariable logistic regression model was utilized for associating variables as indicators of gingival inflammation (GI) . Statistical significance was set at 0.05.A total of 96.5% of the adults have GI. Regarding the severity of GI, 22.5% of participants examined have mild GI, 74.0% have moderate GI, and 3.6% have severe GI. The multivariate analyses identify the main risk indicators for GI as adults with higher mean of Calculus Index (OR=18.59); with a Visible Plaque Index \u226530% (OR=14.56); living in Santiago (OR=7.17); having \u226412 years of schooling (OR=2.18), and females (OR=1.93).This study shows a high prevalence and severity of gingival inflammation, being the first one performed in adult populations in three cities of South America. It is characterized by swelling, redness, and bleeding at the gums and it is described as an inflammatory reaction upon the pro-inflammatory cytokines that modulate the balance between humoral and cell-associated immune responses28. This clinical feature is characteristic of both gingivitis and periodontitis. GI is considered to be one major class of periodontal conditions, and is recognized to result from the increase in supragingival plaque and the ensuing interactions between the microbiota of biofilm and host response26. Consequently, the prevention of plaque accumulation and early treatment of GI reduce the risks associated with the development of the more destructive periodontal disease5, which has also been associated with systemic conditions8.Gingival inflammation (GI) is a common clinical feature detected in children and adults29. In recent decades, cross-sectional and longitudinal epidemiological studies on periodontitis in adults were performed in Chile12 and Brazil14. Moreover, analytical approaches designed to identify associated factors that could be risk indicators for gingival inflammation are nonexistent. Our main objective in this multi-center, population-based, cross-sectional and epidemiological study is to investigate the prevalence, severity, and risk indicators for gingival inflammation in representative samples of the adult populations of Porto Alegre (Brazil), Tucum\u00e1n (Argentina), and Santiago (Chile).Understanding the epidemiologic pattern of GI is essential for planning appropriate public-health services. It has been clearly demonstrated that plaque-induced GI is prevalent at all ages of the dentate populationThe present cross-sectional, representative study utilized stratified, multistage probability samples of the civilian, noninstitutionalized adult populations in three South American cities. Data were collected between January and July of 2014.21  with a precision rate of 95% and a 2% error, we determined that a sample size of 550 adults would be appropriate for each of the three cities in the study. To do so, the formula to estimate the prevalence of a population (n=Z21-\u03b1/2 P(1-P)/e2) was used.Our sampling approach considered various age subgroups . Considering previously published information that estimated a prevalence of gingivitis of 93.9%The study participants were selected using a multi-staged probability sampling process. Age groups were formed according to a proportional approach to the base population registries in the total of urban administrative regions in Porto Alegre, Tucum\u00e1n, and Santiago, according to the last census in each city, and considering differences in gender and age.st stage); Tract census (2nd stage); Blocks (3rd stage); and Individuals within the age group (4th stage). The cities were chosen according to logistics and interests in the three countries. Using maps of each city, primary census sectors were randomly chosen. The number of sectors in each city was determined according to the city size and census distribution. If the access to a primary census sector was not possible, the next available census sector was chosen. In each census sector, the blocks were randomly chosen. On each block, households were consecutively approached according to the sector starting point, until the number of participants expected for each sector was reached. Places such as nursing homes and commercial establishments were not included. When no potential participants were available for examination in a household, the next household was visited.The sampling process consisted of four stages: City . Intra- and inter-examiner kappa coefficients for the Visible Plaque Index, Calculus Index, and Gingival Index were above 0.7. In addition, the structured questionnaire was standardized for each of the three study locations.Each team consisted of one clinical examiner, totaling three dental examiners  and each conducted exams using a manual periodontal probe (UNC-15) and mobile dental units. Good clinical practice standards were used and warranted. Periodontal clinical parameters were evaluated in all teeth, excluding third molars. The parameters evaluated were Visible Plaque Index (VPI), Calculus Index (CI), and Gingival Index (G-Index). Visual plaque assessment was determined using absence (0) or presence (1) of dental plaque according to Ainamo & Bay. The L\u00f6e modification of the L\u00f6e-Silness index was used to evaluate gingival health. Each tooth was divided into six surfaces, three facial and three lingual, as follows: 1) mesio-facial; 2) mid-facial; 3) disto-facial; 4) mesio-lingual; 5) mid-lingual; and 6) disto-lingual. Third molars and those teeth with cervical restorations or prosthetic crowns were excluded from the scoring procedure. Absence (0) or presence (1) of calculus was scored in lower anterior teeth (CI). Each tooth was divided into three lingual surfaces, as follows: disto-lingual, medio-lingual, and mesio-lingual. At the end of clinical examinations, those participants who were diagnosed with periodontal pathologic conditions were provided a written report of their condition and advised to seek an oral health consultation.per day), moderate (5\u201310 per day), and heavy (over 10 per day). The educational level of the study participants was also categorized as \u226412 or >12 years of school. The G-Index, VPI, GBI, and CI indices are represented in the data as mean values.The prevalence of gingival inflammation was defined as the percentage of study participants with a mean Gingival Index (G-Index) \u22650.5. Gingival Bleeding Index (GBI) was defined as the percentage of sites with G-Index \u22652. When appropriate, estimates were isolated for interproximal sites. Severity of gingival inflammation was defined as G-Index 0.5\u20131.0 for mild, 1.1\u20132.0 for moderate, and >2.0 for severe gingival inflammation. Study participants were classified by their smoking habits as either nonsmokers or smokers. Smokers were further classified as light . A multi-variable logistic regression model was built to assess the contribution of each variable . The occurrence of gingival inflammation was considered if G-Index \u22650.5. In addition, variables were analyzed in the model and allowed if the coefficient was modified by at least 10%. VPI was dichotomized at 30%. Odds ratios were calculated with 95% confidence intervals, and statistical significance was defined as p<0.05.A total of 1650 adults from Porto Alegre, Tucum\u00e1n, and Santiago participated in this study. Females represent 52.5% of the overall study participants, while one third of the participants are in the >50 years age group.Considering the total sample of the present study, no statistically significant difference is observed between genders . In the Participants who smoke present a higher prevalence of gingival inflammation as compared with nonsmokers, but the difference was not statistically significant (p=0.194). Those participants with self-reported diabetes and hypertension also present a significantly higher prevalence of gingival inflammation as compared with those not reporting these conditions. Within cities, only in Tucum\u00e1n there is a statistically significant difference in gingival inflammation prevalence between those who self-report hypertension and those who do not . Adults with a visible plaque index \u226530% present a significantly higher prevalence of gingival inflammation than those with visible plaque index <30%  .The multivariate logistic regression model, which was designed to assess indicators that could be related to gingival inflammation prevalence (G-Index\u22650.5) in the adult samples, demonstrates that subjects with higher CI mean (OR=18.59); with a VPI \u226530% (OR= 14.56); living in Santiago (OR=7.17); having \u226412 years of schooling (OR=2.18), and females (OR=1.93) are more likely to present gingival inflammation. This model was adjusted for age, presence of diabetes, and self-reported hypertension and smoking .30. Comparing our results with that from previous studies is somewhat hindered by the use of different nomenclature and diagnostic criteria across studies. The Community Periodontal Index (CPI) was used to report the occurrence of probing pocket depth, calculus, and gingival inflammation in the Hungarian adult population, and gingival bleeding (CPI=1) was observed in 8% of the population15. The National Health and Nutrition Examination Survey III (NHANES III) conducted in the USA between 1988\u20131994 demonstrated that 50% of the adult USA population had gingival inflammation, using gingival bleeding as the criterion3. A study conducted in Italy, using bleeding on probing (BoP) as the criterion, determined that the prevalence of individuals showing at least one site positive for BoP was 99%11. All the aforementioned studies demonstrate that the occurrence of gingival inflammation is almost universally correlated with poor gingival conditions. In addition, there is a large discussion in the literature regarding how to define disease criteria. It would be interesting to develop guidelines to suggest which criteria should be standardized for use in epidemiological studies for gingival conditions16. Concerning the severity of gingival inflammation, the present study shows that 22.5% of adults have mild gingival inflammation, 74.0% have moderate gingival inflammation, and 3.6% have severe gingival inflammation. In contrast, representative studies conducted in Europe and Australia demonstrated that the prevalence of severe gingival inflammation is comparably much higher  in these populations17. The multivariate logistic regression models that we employed show that a female with \u226412 years of schooling, a higher dental calculus index, \u226530% of VPI, and living in Santiago has an increased likelihood of presenting gingival inflammation, when adjusting for age, presence of diabetes, and self-reported hypertension and smoking. In this study, to live in Santiago was a risk indicator for presenting GI. It could be because these subjects had lower oral hygiene habits compared with those from the other cities. Indeed, subjects with presence of GI belonging to Santiago had a higher average percentage of VPI, compared with subjects with GI of Tucum\u00e1n and Porto Alegre. In fact, the plaque index has been associated with increased presence of gingival inflammation and worst level of oral hygiene in other populations9. There are limited studies at the Latin American level on the prevalence of periodontal conditions27, which allow contrast with the findings in this study. Although subjects belonging to Santiago present greater gingival inflammation and increased risk of it, is based with those reported for that country, where Chilean adults had a high prevalence of periodontal destruction and indicators of gingival inflammation12.The population examined in the present study is comprised of a random sample of individuals aging 18 years or older from Porto Alegre (Brazil), Tucum\u00e1n (Argentina), and Santiago (Chile). To our knowledge, this is the first study conducted to assess the prevalence of gingival inflammation in a representative sample of adult populations from three Latin American cities. The sampling strategy that we employed was successful in achieving a representative and balanced sample of participants, since the individuals examined in each age group for each of the three cities are in the same proportion as in the whole study population for the three cities combined. In this study, 95.6% of the 1650 adults examined from three South American cities present a G-Index >0.5. This information corroborates the data reported for adults from Jordan, China, and the United States of America, which demonstrated that adults with a G-Index >0.5 comprise 75.8%, 97.9%, and 93.9% of their respective populations4. In this regard, different explanations have been raised in the literature to explain such findings. For example, those in lower socioeconomic situations are also more likely to smoke, and smoking has been found as a significant risk factor for periodontitis10. On the other hand, individuals with higher socioeconomic status are likely to have more positive attitudes regarding oral hygiene and self-care, and better access to available health care options. Therefore, low incomes and low levels of education seem to be variables with good predictability for periodontal diseases7. The prevalence of gingival inflammation in females found in the present study contradicts results from Greece, where women have been shown to have better oral hygiene and gingival status than men22. Perhaps this difference is more about culture than gender. It should be noted that only when adjusting the multivariate model by confounding variables, female gender appears as a possible risk indicator for the presence of gingival inflammation. On the other hand, the longitudinal recovery of bleeding sites was positively influenced in females25.The present study also demonstrates a socioeconomic gradient in the oral health of the populations examined. Participants from lower socioeconomic groups present significantly poorer gingival health as compared with participants in higher socioeconomic groups20.Regarding the self-reported diabetes and hypertension among our participants, 7.4% have diabetes and 19.3% have hypertension. Participants with self-reported diabetes and hypertension present a significantly higher prevalence of gingival inflammation than those not reporting these conditions. Previous studies have reported comparable results regarding this association between type 2 diabetes and periodontal disease25 (2002), in which study participants (soldiers of the German Armed Forces serving between December 1999 and May 2000) who smoked were found to have more prevalent bleeding on probing and more calculus than nonsmokers. Smoking was first identified as a risk factor in periodontal diseases from an analysis of data by Ismail, et al.18 from data collected from 1971 to 1975. An analysis by the National Health and Nutrition Examination Survey in the U.S. (NHANES I) was able to demonstrate an association between smoking and periodontal diseases that was independent of oral hygiene, age, or other probable risk factors. Since then, there has been enough evidence to identify smoking as a risk factor for periodontitis13. Studies performed in randomly chosen groups of patients demonstrated that tobacco use and oral hygiene are risk indicators for periodontitis; in particular, smokers were invariably shown to have a higher prevalence and progression of destructive periodontal diseases24.We find that current smokers comprise 28.8% of our total study participants, and these smokers present a slightly higher prevalence of gingival inflammation than nonsmokers in our study, but this finding was not statistically significant. These results contrast with the findings of Muller, et al.23. These findings highlight the challenge in responding to the diversity of urgent oral health needs worldwide, particularly in developing communities.There is a strong need in Latin America to focus on more effective intervention programs to prevent and control periodontal diseases at national levels. It should be emphasized that since periodontitis begins as gingivitis, it is reasonable to conclude that the control of gingival inflammation can be beneficial to the population as a whole to prevent both the onset and the progression of periodontal damage caused by periodontitis. While the disability-adjusted life-years (DALYs) due to severe periodontitis and untreated caries have increased since 1990, those due to severe tooth loss have decreased. Oral conditions are all ranked among the top 100 detailed causes of DALYs6. Poor awareness of the importance of periodontal health and the consequences of the disease among the public and even among some general dental practitioners is one of the most common reasons for failure to control and treat periodontal diseases effectively on a population basis19. We recommend that effective intervention programs for the prevention and control of periodontal diseases should be implemented at national levels, and the need for such implementation seems to be extremely important in the three countries we studied here. We believe that there is a strong need across Latin America to improve the population's self-awareness about oral health through better oral health education that promotes good oral hygiene and regular dental care.Gingival inflammation, which could be considered a reversible and easily controlled disease in stage of gingivitis, is found to be highly prevalent among adult study participants from the three cities in Latin America of this study. In addition, these individuals are also more likely to attend preventive or follow-up visits because socioeconomic characteristics (such as income and level of education) influence the pattern and type of dental services usedThis study was aimed to detect gingival inflammation instead of establishing the diagnosis of either gingivitis or periodontitis in order to determine the risk of gingivitis onset or periodontitis progression in the study population.Considering our findings together, it is possible to conclude that gingival inflammation is highly prevalent in the three Latin American cities studied. Overall, 95.6% of the participants aging 18 years or older had gingival inflammation, considering that more than two-thirds have moderate gingival inflammation and 3.6% have severe gingival inflammation. In general terms, the presence of gingival inflammation is positively associated with risk indicators such as gender, socioeconomic variables, and the presence of plaque. The present investigation serves as the basis for a longitudinal analysis of oral health in populations of South American adults, and for the development of strategies to improve the health care systems that serve them."}
+{"text": "In the UK, eating disorders affect upward of 725,000 people per year, and early assessment and treatment are important for patient outcomes. Around a third of adult outpatients in the UK who are referred to specialist eating disorder services do not attend, which could be related to patient factors related to ambivalence, fear, and a lack of confidence about change. This lack of engagement has a negative impact on the quality of life of patients and has implications for service costs.To describe the development of a Web-based program (\u201cMotivATE\u201d) designed for delivery at the point of referral to an eating disorder service, with the aim of increasing service attendance.We used intervention mapping and a person-based approach to design the MotivATE program and conducted a needs assessment to determine the current impact of service nonattendance on patients  and services (through a service provision survey to understand current issues in UK services). Following the needs assessment, we followed the five steps of program development outlined by Bartholomew et al (1998): (1) creating a matrix of proximal program objectives; (2) selecting theory-based intervention methods and strategies; (3) designing and organizing the program; (4) specifying adoption and implementation plans; and (5) generating program evaluation plans.The needs assessment identified current nonattendance rates of 10%-32%. We defined the objective of MotivATE as increasing attendance rates at an eating disorder service and considered four key determinants of poor attendance: patient ambivalence about change, low patient self-efficacy, recognition of the need to change, and expectations about assessment. We chose aspects of motivational interviewing, self-determination theory, and the use of patient stories as the most appropriate ways to enable change. Think-aloud piloting with people with lived experience of an eating disorder resulted in positive feedback on the MotivATE program. Participants related well to the stories used. Nonetheless, because of feedback, we further modified the program in line with patients\u2019 stage of change and addressed issues with the language used. A consultation with service staff meant that we could make clear implementation plans. Finally, a randomized controlled trial is currently underway to evaluate the MotivATE program.Using intervention mapping, we have developed a novel pretreatment Web-based program that is acceptable to people with eating disorders. To our knowledge, this is the first such program. The model of development described here could be a useful template for designing further programs for other difficult-to-engage populations. Eating disorders  have a prevalence rate of 1 in 600,000\u2013725,000 people in the UK, affecting up to 6.4% of adults at any time ,4. EatinAfter a person in the UK initially seeks help for his or her eating disorder , he or she is usually referred to a specialist eating disorder service for an initial assessment appointment. However, research highlights that up to a third of people referred for specialist psychological treatment do not access the services and that 16.4% of these people simply do not attend their first scheduled assessment appointment . ReasonsWe developed the MotivATE program using intervention mapping, which recognizes three phases of program development: needs assessment, program development, and evaluation . As recoThe aims of the needs assessment were to understand the problem of nonattendance at adult eating disorder services and to consider both patients\u2019 personal barriers and service-level barriers that the program would need to address. In line with the person-based approach, our first step was to review our own qualitative research with people who described themselves as not wanting to receive treatment for an eating disorder  and thosBartholomew et al  outline The needs assessment, team discussions, and consultations with eating disorder-focused charities enabled us to determine (a) the objectives of the program, (b) the behaviors that potential users needed to engage in, (c) the factors (or determinants) influencing these behaviors, and (d) the target population (and any subgroups) who would use the program.We reviewed the current theory related to increasing motivation to change, including the current evidence relating to eating disorders, and then considered practical strategies for implementing this theory. As per the person-based approach, this enabled us to develop guiding principles for the design of the program .The team designed the structure and content of the intervention during team meetings, and we used open-source LifeGuide software to create the MotivATE platform . We starPeople with experience of an eating disorder then qualitatively evaluated our initial prototype of the program via think-aloud interviews to understand beliefs about the relevance, acceptability, and usability of the program . We recrThis phase involved making plans for the delivery of the program: two team members demonstrated the program to staff at a local eating disorder service that was typical of outpatient services across the UK and invited feedback about implementation.The final stage of the intervention mapping described by Bartholomew et al  is to geThe idea to develop a program to address attendance at psychological services for eating disorders originated from previous qualitative research conducted by the authors ,14-15. QWaller et al  reportedThe main objective of the program is to \u201cincrease attendance at an eating disorder service\u201d. In contrast to the opt-in process detailed above, which simply asks patients to make an appointment at a convenient time, the MotivATE approach aims to increase attendance rates by fostering personal intrinsic motivation and increased self-efficacy to attend while addressing negative beliefs and expectations about the service. The target population for the program was all adults who have been referred to an outpatient eating disorder service for an assessment appointment. People in the UK are usually referred to an eating disorder service by their general practitioner, although there are other routes to treatment, depending on the structure of the local service model. Some services allow referral by any health or social care professional, and others welcome the person to refer himself or herself. Clinicians at the service review the referral letter to determine the next steps, guided by the urgency of referral, risks, etc. When the person is suitable for outpatient treatment , the service will contact the person to invite him or her for an assessment appointment . The wait times for the assessment appointment are different across the UK, ranging from 4 weeks to 6 months. It is when inviting the person for assessment that we intend to include the invitation to use MotivATE.People who have been referred to the service may be in one of three of the five stages of change . First, The needs assessment and the identification of the target group enabled us to ascertain four main determinants that the program needed to address: (1) patient ambivalence about change, (2) low patient self-efficacy, (3) recognition of the need to change, and (4) expectations about assessment. This resulted in the final matrix of proximal program objectives outlined in The main objective of the program is to increase motivation to attend an assessment appointment. Motivational therapies are advocated in the treatment of eating disorders  and are We also drew on self-determination theory (SDT) for the intervention, which is in keeping with the person-based approach. SDT recognizes that human behavior centers on three innate needs: autonomy, competence, and relatedness . People The program objectives highlighted in the previous stage and the examination of theory and practical strategies allowed us to develop guiding principles, outlined by the person-based approach, for the program .We aimed to present a professional-looking, gender- and age-neutral program. The color scheme is blue and white, and images are based on nature, with the intention of depicting positive well-being see . No imagThe final MotivATE program consists of four 15-minute Web-based modules , and it was clear from talking to participants that the website should not be overtly about \u201crecovery\u201d; instead, it should \u201csow the seed\u201d (Sam) and focus on how to prepare for the assessment appointment. Early versions of the program also discussed the concepts of recovery and psychological treatment, but based on the following feedback, the final program only considered the first step of the assessment appointment:I freaked a little bit then. Thinking about change in the future. I\u2019m going to move onto something else (chooses quiz to distract from message on page) because that\u2019s what I would do.Sally, eating disorder not otherwise specifiedscales and motivational exercises, but these have connotations related to body weight, so we removed these references.Some participants (particularly those with a history of anorexia nervosa) believed that some modules were too long, and we therefore decided to shorten the modules to no longer than 10 minutes, focusing on essential information. Participants also commented on the terminology. For example, we referred to question We used the results from the think-aloud interviews to modify the program, and further consultations with participants as well as consultations with charity directors and others with experience of eating disorders confirmed that the program was acceptable to users.We demonstrated the MotivATE program to clinical and administrative staff at one eating disorder service before we asked for feedback about implementing MotivATE in their practice. Two key points arose: (1) it should not impact existing service resources and staff time, and (2) the modules needed to be short enough to optimize engagement of patients. MotivATE would be a free, stand-alone program that would not require training for service staff or patients. Invitation to MotivATE would occur via the same materials as the standard invitation to attend an assessment and would therefore not impact staff time and resources. We are planning a process evaluation with program users and service staff to further explore implementation.We have already evaluated the acceptability and usability of the intervention as part of the program development described here. A randomized controlled trial (RCT) is also currently underway to evaluate the impact of the MotivATE program on improving attendance at an eating disorder service .This paper described the development of a pretreatment Web-based program designed to support adults with eating disorders as they prepare for an assessment appointment at a specialist outpatient eating disorder service. We have yet to establish the program\u2019s value for and impact on service attendance rates, although the RCT will enable us to determine this. Nonetheless, we believe the program will improve attendance at assessment appointments based on comments from service users and service staff.The combined approach of using intervention mapping and a person-based approach worked well, as it allowed us to structure our development process in a logical manner by identifying areas of need, setting out key objectives from the outset, and highlighting key theory and strategies that could be used while always ensuring that each of these components would work for the target user. Our person-based approach is a key strength of the development process. Specifically, this program needs to be acceptable, motivating, and empowering for users who already find it difficult to engage with services. Interviews with service users and service staff also revealed important shortcomings of the first version of the intervention, developed based on existing literature and theory, which we have been able to address and tailor more to service user needs . The use of existing qualitative literature and theory was also fundamental to our intervention design since they not only ensured an evidence-based approach to program development, but they also enabled the inclusion of a variety of processes designed to increase motivation, and allowed us to consider the characteristics of populations that can be difficult to engage. Our needs assessment has also demonstrated the potential usefulness of the MotivATE program across services in the UK, although one weakness of our service provision survey is that for practical reasons (resources and time demands making it difficult for services to engage), we included only four services. Nonetheless, the study still provides a more generalized view than existing studies, which have focused only on one area of the UK .One difficulty in the design phase was the production of a Web-based intervention that utilized the principles of motivational interviewing and SDT, which rely heavily on face-to-face interaction. Use of a person-based language style, which enabled us to provide autonomy support , and optDespite positive feedback regarding the acceptability of the MotivATE program, a key unknown is whether people will register for and engage with it, particularly as Web-based interventions are known to have low rates of engagement . Our RCTTo our knowledge, the MotivATE program is the first intervention designed for delivery to eating disorder patients prior to formal contact with an eating disorder service with the aim of enhancing engagement with face-to-face services. If the program is effective, it could act as a template for the design of similar pretreatment Web-based programs for other patient groups in which ambivalent attitudes and/or a lack of engagement with services may be an issue."}
+{"text": "A LEMON method which consists of following assessments; Look-Evaluate-Mallampati-Obstruction-Neck mobility is a fast and easy technique to evaluate patients\u2019 airways in the emergency situation. And a modified LEMON method, which excludes the Mallampati classification from the original LEMON score, also can be used clinically. We investigated the relationship between modified LEMON score and intubation difficulty score in adult trauma patients undergoing emergency surgery.Prediction of difficult airway is critical in the airway management of trauma patients.We retrospectively reviewed electronic medical records of 114 adult trauma patients who underwent emergency surgery under general anesthesia. All patients\u2019 airways were evaluated according to the modified LEMON method before anesthesia induction and after tracheal intubation; the intubating doctor self-reported the intubation difficulty scale (IDS) score. A difficult intubation group was defined as patients who had IDS scores >\u20095P\u2009<\u20090.001). The difficult intubation group showed higher modified LEMON score than the non-difficult intubation group . Limited neck mobility was the only independent predictor of intubation difficulty .The modified LEMON score was significantly correlated with the IDS score (The modified LEMON score is correlated with difficult intubation in adult trauma patients undergoing emergency surgery. Successful airway securement by an experienced physician is crucial in the management of trauma patient . HoweverConventional tools for predicting difficult airway, such as the Mallampati score, have a limited application in trauma patients . The LEMIn the present study, we retrospectively investigated the ability of the LEON score to predict intubation difficulty by assessing the correlation between the LEON score and intubation difficulty score in adult trauma patients undergoing emergency surgery.After the approval of the institutional review board , electronic medical records of adult trauma patients who underwent emergency surgery under general anesthesia in an operating theater between March 2016 and August 2016 were reviewed retrospectively. Patients who were already intubated before anesthesia induction or underwent surgical procedures under regional anesthesia were excluded.1, the number of supplementary intubation attempts; N2, the number of supplementary operators; N3, the number of alternative intubation techniques used; N4, glottic exposure as defined by the Cormack and Lehane grade ; N5, the lifting force applied during laryngoscopy (N5 = 1 if a subjectively increased lifting force was required); N6, external laryngeal pressure to improve glottic exposure ; and N7, position of the vocal cords at intubation . The IDS score is the sum of N1 through N7. An IDS score between 1 and 5 represents slight difficulty, and IDS score >\u20095 represents moderate to major difficulty. In the present study, patients were divided into the difficult intubation group (group D) and non-difficult intubation group (group ND) according to whether their IDS score was >\u20095 or \u2264\u20095.All patients\u2019 airways were evaluated by the well-trained residents or attending members of staff of the anesthesia department before anesthesia induction. Patient\u2019s airway assessment was performed according to the LEON method Fig.\u00a0, left ast test or Mann-Whitney U test. Categorical variables were analyzed using a chi-square test, and the Cochran-Armitage test was used for trend analysis. Correlation between the LEON score and IDS was calculated using the Spearman\u2019s rank correlation. To determine the relationship between one dependent factor and one or more independent factors, a logistic regression analysis was performed. Data are expressed as means\u2009\u00b1\u2009standard deviations, medians [interquartile ranges], or number (%). P\u2009<\u20090.05 was considered to be statistically significant.All analyses were performed using MedCalc\u00ae 16.2 . Continuous variables were compared using the Student\u2019s During study period, a total of 114 cases were reviewed. Patients\u2019 characteristics are shown in Table\u00a0P\u2009<\u20090.001). The IDS score was 6 [P\u2009<\u20090.001). The Cormack-Lehane grade was significantly higher in group D than in group ND (3 [P\u2009<\u20090.001). The number of intubation attempts was higher in group D than in group ND (2 [P\u2009<\u20090.001). The median intubation time was also longer in group D than in group ND .The LEON score was significantly correlated with the IDS score  . The logistic regression analysis showed that the LEON score showed a significant correlation with intubation difficulty . Among the variables in the LEON score, limited neck mobility was the only independent predictor of intubation difficulty  (Table\u00a0The LEON score was higher in group D than in group ND (3 \u20135 vs. 2 In the present study, we found that the LEON score correlated with the intubation difficulty and a LEON score of \u22653 could predict intubation difficulty in trauma patients.Airway management is a challenging issue in adult trauma patients. Trauma patients may present with a variety of airway difficulties, ranging from promptly recognized to unanticipated difficult airways . MoreoveIt has been previously well-validated that the LEON score can effectively predict difficult intubation in the emergency department . HoweverThis study has several limitations. First, the study was conducted with a retrospective design via analysis of electronic medical records. Although the intubating doctor recorded the intubation information right after the induction of anesthesia, the data may have been unreliable because they were self-reported data. Moreover, because the initial intubation device can be selected by intubator\u2019s choice, a selection bias may affect the result. A relatively small population for a retrospective study also contributes to the result. Second, the results of the present study do not reflect patients with very severe and complex traumatic injuries that require immediate airway access. Because we evaluated patients undergoing emergency surgery in the operating theater, patients who underwent immediate tracheal intubation in the emergency department were not included. Third, we used NMBA to facilitate tracheal intubation in the present study. Since NMBA can provide the relaxation of soft tissues and comfortable condition without patient movements, the IDS score can be differed in clinical situations of tracheal intubation without NMBA.The LEON score may be used as one of the evidence predicting difficult airway, thereby being helpful to increase safety in the airway management of adult trauma patients undergoing emergency surgery. A patient with LEON score \u2265\u20093 may have the possibility of difficult intubation, and even in using video laryngoscopy, the limited neck mobility may contribute to the intubation difficulty."}
+{"text": "Type 1 and type 2 diabetes differ with respect to pathophysiological factors such as beta cell function, insulin resistance and phenotypic appearance, but there may be overlap between the two forms of diabetes. However, there are relatively few prospective studies that have characterised the relationship between autoimmunity and incident diabetes. We investigated associations of antibodies against the 65 kDa isoform of GAD (GAD65) with type 1 diabetes and type 2 diabetes genetic risk scores and incident diabetes in adults in European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct, a case-cohort study nested in the EPIC cohort.n = 15,802) and in incident diabetes cases . Type 1 diabetes and type 2 diabetes genetic risk scores were calculated. Associations between GAD65 antibodies and incident diabetes were estimated using Prentice-weighted Cox regression.GAD65 antibodies were analysed in EPIC participants (over 40 years of age and free of known diabetes at baseline) by radioligand binding assay in a random subcohort  after adjustment for sex, centre, physical activity, smoking status and education. The genetic risk score for type 1 diabetes but not type 2 diabetes was associated with GAD65 antibody positivity in both the subcohort  and incident cases  after adjusting for age and sex. The risk of incident diabetes in those in the top tertile of the type 1 diabetes genetic risk score who were also GAD65 antibody positive was 3.23  compared with all other individuals, suggesting that 1.8% of incident diabetes in adults was attributable to this combination of risk factors.Our study indicates that incident diabetes in adults has an element of autoimmune aetiology. Thus, there might be a reason to re-evaluate the present subclassification of diabetes in adulthood.The online version of this article (10.1007/s00125-019-05016-3) contains peer-reviwed but unedited supplementary material, which is available to authorised users. Diabetes mellitus has classically been thought to have two major aetiological subtypes: type 1 diabetes, which is characterised by autoimmune destruction of beta cells with subsequent insulin deficiency, and type 2 diabetes, which is associated with insulin resistance and a relative insulin secretory deficit . AssociaSusceptibility for development of GAD65 antibody and type 1 diabetes is at least in part explained by risk alleles located within the HLA region on chromosome 6 \u201323. We hThus, we aimed to investigate the association between GAD65 antibody levels measured using a competition assay, which increases the precision of the assessment of autoantibody positivity, and incident adult-onset diabetes in a large, multi-centre, population-based prospective study in eight European countries in people who were over 40 years of age and free of known diabetes at baseline. In addition, we investigated whether genetic risk scores (GRSs) for type 1 diabetes and type 2 diabetes are associated with GAD65 antibody positivity, and the potential for either of these risk scores to modify the association of GAD65 antibodies with incident adult-onset diabetes.The design and methods of the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct case-cohort study have previously been described . A totalAll study participants gave informed consent, and the investigation has been carried out in accordance with the Declaration of Helsinki as revised in 2008.Blood samples were drawn at the time of participation in EPIC, at which time all participants were free of known diabetes. Blood plasma was prepared and stored at \u2212196\u00b0C in liquid nitrogen at the coordinating centre at the International Agency for Research into Cancer (IARC) in Lyon, France, or in liquid nitrogen in local biorepositories except for Ume\u00e5, where \u221280\u00b0C freezers were used .[35]S-GAD65 was produced in an in vitro coupled transcription and translation system with SP6 RNA polymerase and nuclease-treated rabbit reticulocyte lysate  as previously described [The samples had been subject to at least two freeze-thaw cycles before being analysed for GAD65 antibody. Recombinant escribed . The WHOescribed  was incl50. Given the low sample volume, we chose to use the competitor at the optimal concentration (200 ng/ml) found in previous experiments, in which we titrated the amount of rhGAD65 necessary to give maximal competition. In a variation of the traditional receiver operating characteristic (ROC) analysis, we plotted GAD65 antibody levels of samples that were competed by \u226550% against GAD65 antibody levels of samples that were competed by <50%. The area under the ROC curve was 0.97, indicating excellent predictive ability of the GAD65 antibody measurement. At a cut-off level of \u226565 U/ml, the measurement had 99% specificity and 85% sensitivity  and incident cases  were analysed for GAD65 antibodies in a radiobinding assay (RBA) as previously described [To determine the cut-off for GAD65 antibody positivity, we used a competition assay employing recombinant human GAD65 (rhGAD65)  as previously described . A totalescribed .2). Waist circumference was measured either at the narrowest circumference of the torso or at the midpoint between the lower ribs and the iliac crest. Hip circumference was measured horizontally at the level of the largest lateral extension of the hips or over the buttocks. Anthropometric data were mostly self-reported in the Oxford centre, and waist and hip circumferences were not measured in the Ume\u00e5 centre (n = 1845).Weight, height, and waist and hip circumferences were measured with participants not wearing shoes and in light clothing or underwear, as described previously . BMI wasStandardised information on highest educational level  and smoking status  was collected by questionnaire at baseline . PhysicaSamples were processed for array-based genotyping if they had sufficient DNA that could be successfully genotyped on TaqMan  or Sequenom  platforms and had sex chromosome genotypes concordant with self-reported sex. Samples that failed one genotyping round for reasons that did not relate to sample quality  were repeated. Samples were genotyped on the Illumina 660 W-Quad BeadChip, the Illumina HumanCoreExome-12 or the Illumina HumanCoreExome-24 . Samples genotyped on the Illumina 660 W-Quad BeadChip were randomly selected from the available samples with the number of individuals selected per centre being proportional to the percentage of total cases in that centre. The Danish samples were not available for genotyping at this stage. Genotyping was carried out at the Wellcome Trust Sanger Institute. Most of the remaining non-Danish samples were genotyped on the Illumina HumanCoreExome-12 at Cambridge Genomic Services in the University of Cambridge Department of Pathology. Finally, the Danish samples and repeat samples due to poor genotyping were genotyped on the Illumina HumanCoreExome-24 also at Cambridge Genomic Services. Sample quality control criteria varied slightly by array but included call rate , X chromosome heterozygosity concordance with self-reported sex, outliers for heterozygosity and concordance with previous genotyping results.From the genome-wide array data, we calculated a type 1 diabetes GRS as a weighted average of 33 SNPs, including five HLA variants. The relevant SNPs and their individual associations with GAD65 antibody positivity are described in ESM Table Baseline characteristics of the analysis sample were summarised by GAD65 antibody status (negative/positive), separately within the subcohort and incident diabetes cases, using means and standard deviations for continuous variables  and percentages for categorical variables.The association between GAD65 antibody status (positive/negative) and incident diabetes was estimated using Prentice-weighted Cox regression, which is appropriate for estimating association in a case-cohort study. We fitted models within each country and the estimated HRs were combined across countries using random effects meta-analysis. We fitted three models including the following covariates: Model 1\u2014age , sex and centre; Model 2\u2014also including physical activity, smoking status and education; Model 3\u2014also including family history of diabetes. In order to study the effects of high vs low GAD65 antibody levels, GAD65 antibody-positive individuals were further subdivided into those with GAD65 antibody equal to or above, and those with GAD65 antibody below, 167.5 U/ml, which is the median antibody level in the GAD65 antibody-positive group in this study. Prentice-weighted Cox regression was also used to test possible multiplicative interactions of GAD65 antibody status with: (1) sex; (2) BMI category; (3) waist/hip ratio (WHR) category (sex-specific tertiles); and (4) type 1 diabetes GRS tertile. The interactions with anthropometry measures were tested because of prior studies suggesting that adiposity could moderate the association of autoimmunity with diabetes , 10. In The associations of the type 1 diabetes GRS and the type 2 diabetes GRS with GAD65 antibody status (positive/negative) were estimated separately in the subcohort and the incident diabetes cases, using logistic regression adjusted for age and sex, since the prevalence of autoimmunity is associated with age and sex. Models were fitted within each country and estimated odds ratios combined across countries using random effects meta-analysis. ORs (and 95% CIs) per risk allele of each of the individual SNPs contributing to the type 1 diabetes GRS were also calculated in the subcohort using the same method.The association between the type 1 diabetes GRS and incident diabetes, by GAD65 antibody status, was estimated using Prentice-weighted Cox regression as described above.Three hundred and sixteen (2.0%) individuals in the subcohort and 413 (3.4%) incident cases were GAD65 antibody positive as defined by having a level of \u226565 U/ml Table .Table 1Bp = 0.099).GAD65 antibody positivity was associated with a higher incidence of diabetes Table  consisten = 10,893), the HR for incident diabetes in those positive for GAD65 antibodies compared with those individuals who were negative was 2.22  without adjustment and 2.13  with adjustment for a five HLA haplotype risk score. There was no association between any individual type 2 diabetes-associated SNP and GAD65 antibody level , WHR category (p <0.001) and type 1 diabetes GRS tertile (p <0.001), but no interaction of GAD65 antibody status with sex, on the hazard of diabetes. The associations between GAD65 antibody positivity and incident diabetes were strongest in the lowest categories of BMI and WHR, and in the highest tertile of type 1 diabetes GRS  when adjusted for age, sex, physical activity, smoking status, education and BMI. However, there were significant interactions of GAD65 antibody status with BMI category . There was no evidence of an association among GAD65 antibody-negative individuals . In order to contextualise these interactions, we also analysed the interaction in terms of tertiles of the type 1 diabetes GRS rather than standard deviations. Among the 26,693 individuals with relevant data, 552 (2.1%) were GAD65 antibody positive and 284 (1.1%) were GAD65 antibody positive and also in the top tertile of the type 1 diabetes GRS. Of these 284 individuals, 196 developed incident diabetes and 88 were non-cases. The HR for incident diabetes in the GAD65 antibody-positive individuals comparing the top tertile of the type 1 diabetes GRS with the combination of the other two tertiles was 4.83 . When the exposed group was defined as those who were GAD65 antibody positive and in the top tertile of the type 1 diabetes GRS, the HR for incident diabetes compared with all other individuals was 3.23 . From this it follows that the population attributable fraction, the theoretical fraction of all cases attributable to having GAD65 antibodies and high type 1 diabetes genetic risk, was 1.8%. Baseline characteristics of the subcohort stratified by both GAD65 antibody status and tertile of the type 1 diabetes GRS (high vs middle/low) are summarised in ESM Table This large, prospective, population-based European study found a significant association between GAD65 antibody positivity and development of incident diabetes after the age of 40 years in individuals free of known diabetes at baseline in eight European countries. A GRS for type 1 diabetes, but not type 2 diabetes, was associated with GAD65 antibody positivity. There was no overall association of the type 1 diabetes GRS with incident diabetes, but, in the subgroup of individuals who were positive for GAD65 antibodies, the type 1 diabetes GRS was strongly associated with incident diabetes. From these data we estimate that just under 2% of all cases of adult-onset diabetes are attributable to the combination of having a high genetic risk for type 1 diabetes and being positive for GAD65 antibodies.Our study provides definitive evidence of the association between GAD65 antibodies and incident diabetes in European populations, by virtue of the size of the population and extensive follow-up (nearly 4 million person-years), as well as the use of a specific method for defining antibody positivity. An association between higher GAD65 antibody levels and incident diabetes in adults has been suggested previously , 16. HowOur finding that type 1 diabetes GRS was associated with GAD65 antibody positivity in the subcohort is in line with the study by Mishra et al, which was based on prevalent latent autoimmune diabetes in adults (LADA) cases and non-diabetic control individuals . The HLAThis study indicates that autoimmunity plays a role in the aetiology of adult-onset diabetes, irrespective of how the people were classified clinically. We estimate that 1.8% of incident diabetes cases in adulthood are attributable to the combination of having a high genetic risk for type 1 diabetes and having GAD65 antibodies. Our interpretation is that there is an overlap in the aetiology in adult diabetes. It has been shown that presence of GAD65 antibodies predicts future beta cell dysfunction which could be the result of an autoimmune attack on the beta cells (reviewed in ), even iTaken together, there might be a reason to re-evaluate the present subclassification of diabetes in adulthood . This reOur study has limitations. The presence of GAD65 antibody at baseline suggests an autoimmune component in the aetiology of adult-onset diabetes over the age of 40. We deliberately sought to use this descriptive term to avoid forcing individuals into predetermined diagnostic categories, since this has the potential to create circular arguments , 39. We In conclusion, in this large, population-based prospective study, we found that GAD65 antibody positivity was associated with the incidence of diabetes after the age of 40 years. A GRS for type 1 diabetes but not type 2 diabetes was associated with GAD65 antibody positivity and with the incidence of diabetes in those who were GAD65 antibody positive. Our data suggest that incident diabetes in adults includes an element of autoimmune aetiology, which warrants further prospective studies into the risk factors associated. Moreover, future studies with a different design should investigate the implications of this finding for subclassification of incident diabetes in adults, selection of primary treatment modalities and frequency of risk factor assessment to enhance prognosis.ESM(PDF 230 kb)"}
+{"text": "Previous studies have shown that hoarding behavior usually starts at a subclinical level in early adolescence and gradually worsens; however, a limited number of studies have examined the prevalence of hoarding behavior and its association with developmental disorders in young adults. The aims of this study were to estimate the prevalence of hoarding behavior and to identify correlations between hoarding behavior and developmental disorder traits in university students.The study participants included 801 university students  who completed questionnaires .r\u2009=\u20090.283; CIR composite vs ASRS H/I, r\u2009=\u20090.147).Among 801 participants, 27 (3.4%) exceeded the CIR cut-off score. Moreover, the participants with hoarding behavior had a significantly higher percentage of ADHD traits compared to participants without hoarding behavior (HB(+) vs HB(\u2212), 40.7% vs 21.7%). In addition, 7.4% of HB(+) participants had autism spectrum disorder (ASD) traits, compared to 4.1% of HB(\u2212) participants. A correlation analysis revealed that the CIR composite score had a stronger correlation with the ASRS inattentive score than with the hyperactivity/impulsivity score (CIR composite vs ASRS IA, The results showed a high prevalence of ADHD traits in the university students with hoarding behavior. Moreover, we found that the hoarding behavior was more strongly correlated with inattentive symptoms rather than with hyperactivity/impulsivity symptoms. Our results support the concept of a common pathophysiology behind hoarding behavior and ADHD in young adults. Hoarding is defined as the acquisition of and failure to discard possessions of little use or value . Hoardingomi-yashiki,\u201d literally meaning rubbish (gomi) house (yashiki). Hoarding behavior increases the risk of falls, injuries, fires, and infection, thus threatening the safety and health not only of the hoarder but also of the community. Overall, hoarding behavior is a growing social problem in advanced countries.The prevalence of hoarding behavior in the general population has been reported to be 2\u20136%, suggesting that hoarding behavior is not rare , 7, 8. IThe characteristics of hoarding behavior have been examined from the viewpoints of comorbidity and trajectory. For example, major depression and anxiety disorders  showed high comorbidity with hoarding behavior in a large-scale study . AnotherTherefore, to understand the pathogenesis and progression of hoarding symptoms, it is useful to examine hoarding behavior from the viewpoint of developmental disorders. Indeed, there has been accumulating evidence that suggests an association between hoarding and developmental disorders. For example, Hacker et al. reported a high prevalence of hoarding in children with attention deficit hyperactivity disorder (ADHD) . In addiThis observational study was conducted at Kyushu University from April 2016 to April 2018. All participants were students who volunteered to participate and were recruited through the University\u2019s Interdisciplinary Graduate School of Engineering Sciences and Faculty of Arts and Science. We explained the study outline and asked for volunteers from students enrolled in psychology or health and safety courses. We excluded participants who had previously completed the same questionnaires in the authors\u2019 class to avoid duplicate participants. The Ethics Committee of the Faculty of Arts and Science, Kyushu University, Japan approved this study.The ASRS is a self-reported questionnaire designed to screen for adult ADHD , and it The AQ (Autism-Spectrum Quotient), developed by Baron-Cohen et al. , is a 50The clutter image rating  is a thrU test. Data are shown as means (\u00b1 SD) in Table\u00a0p\u2009<\u20090.05.Data were analyzed using IBM SPSS Statistics version 23.0 . Chi-square or Fisher\u2019s exact tests were used to compare the categorical variables. The Shapiro-Wilk test was employed to evaluate whether each set of measures was a normally distributed trait. Comparisons between men and women were performed using the Mann-Whitney p\u2009=\u20090.166). Table In total, 801 students  completed all of the questionnaires. Figure\u00a0p\u2009=\u20090.020). Next, we examined the proportion of participants with hoarding behavior and ASD traits (Table p\u2009=\u20090.320).Tables\u00a0ts Table . In the r\u2009=\u20090.663; bedroom vs kitchen, r\u2009=\u20090.422; living room vs kitchen, r\u2009=\u20090.539). The intercorrelation between the two subscales of the ASRS was moderate . The correlation among CIR composite, ASRS total, and AQ16 total scores was weak or moderate . The correlations between the CIR composite score and the ASRS subscales were weak or very weak .Next, we examined correlations among all the study variables. The results are shown in Table\u00a0r\u00a0=\u20090.25; CIR vs AQ16, r\u00a0=\u20090.194; ASRS vs AQ16, r\u00a0=\u20090.413). Finally, we found that the sub-scales of ASRS (inattentive symptoms and hyperactivity/impulsivity symptoms) have a statistically significant correlation with hoarding behavior .This study is the first large-scale survey to identify the relationships between hoarding behavior and developmental disorders in Japanese young adults. Our results gave a 3.4% prevalence rate for hoarding behavior among these university students. Moreover, we found that the participants with hoarding behavior had a significantly higher percentage of ADHD traits than participants without hoarding behavior (HB(+) vs HB(\u2212), 40.7% vs 21.7%). Furthermore, we found statistically significant correlations among CIR, ASRS, and AQ16  (AQ16\u2009\u2265\u200912) was lower than in the ADHD (+) group . In addition, correlation analyses indicated that there was a statistically significant but weak correlation between AQ16 score and CIR scores  . Since tIn conclusion, our study demonstrated that there is a high prevalence of ADHD traits in university students with hoarding behavior. Moreover, we found that hoarding behavior was more strongly correlated with inattentive symptoms than with hyperactivity/impulsivity. Our results may help elucidate the common pathophysiology underlying hoarding behavior and ADHD in young adults. Further studies are needed to develop effective interventions for young adults with hoarding behavior from the standpoint of developmental disorders."}
+{"text": "Previous studies modelling human neural crest differentiation from stem cells have resulted in a low yield of sympathetic neurons. Our aim was to optimise a method for the differentiation of human embryonic stem cells (hESCs) to sympathetic neuron-like cells (SN) to model normal human SNS development. SNAIL and SOX-9 neural crest specifier genes and the neural marker peripherin (PRPH) increased. Expression of the pluripotency marker OCT 4 decreased, whereas TP53 and LIN28B expression remained high at levels similar to SHSY5Y and IMR32 neuroblastoma cell lines. A 5-fold increase in the expression of the catecholaminergic marker tyrosine hydroxylase (TH) and the noradrenergic marker dopamine betahydroxylase (DBH) was observed by day 7 of differentiation. Fluorescence-activated cell sorting for the neural crest marker p75, enriched for cells expressing p75, DBH, TH, and PRPH, was more specific than p75 neural crest stem cell (NCSC) microbeads. On day 28 post p75 sorting, dual immunofluorescence identified sympathetic neurons by PRPH and TH copositivity cells in 20% of the cell population. Noradrenergic sympathetic neurons, identified by copositivity for both PHOX2B and DBH, were present in 9.4%\u2009\u00b1\u20095.5% of cells.  Using stromal-derived inducing activity (SDIA) of PA6 cells plus BMP4 and B27 supplements, the H9 hESC line was differentiated to neural crest stem-like cells and SN-like cells. After 7 days of PA6 cell coculture, mRNA expression of  We have optimised a method for noradrenergic SNS development using the H9 hESC line to improve our understanding of normal human SNS development and, in a future work, the pathogenesis of neuroblastoma. The neural crest is a transient embryonic cell population which undergoes extensive migration and differentiation to give rise to a diverse range of cell populations in the embryo, ranging from the peripheral nervous system ) to the craniofacial skeleton and pigment cells (reviewed by ). Neural\u03b2 superfamily, are secreted in the dorsal aorta and the gut . PR. PR26]. PHOX2B have been identified in hereditary neuroblastoma [DBH mRNA expression and immunostaining for DBH alone and DBH/PHOX2B copositivity.PHOX2B regulates the expression of PHOX2A and heart and neural crest-derived expressed protein 2 (HAND2). Hand2 is induced by BMPs and is first observed after the onset of Phox2B and Asc-1 expression. Overexpression of Hand2 has been shown to induce the generation of catecholaminergic neurons from neural precursor cells both in vitro and in vivo , 5. Furtblastoma , 29. DBHblastoma ; it catablastoma . In the Time-lapse analysis of p75+ ve and p75\u2212 ve cells showed that p75+ ve cells had increased migration compared with p75\u2212 ve cells, consistent with migratory properties of neural crest cells . These rOCT4 and LIN28B in pluripotent H9 cells decreasing with the onset of neural differentiation as expected [QRT-PCR gene expression analysis showed high expression of the pluripotency markers expected .TP53 expression was observed throughout differentiation at levels similar to those in neuroblastoma cell lines consistent with evidence that p53 regulates the proliferation and differentiation of neural progenitor cells independently of its role in the induction of apoptosis. In vivo studies using transgenic mouse models have demonstrated a fundamental role for p53 during neural stem cell self-renewal and differentiation [ntiation , 37.MYCN transformation of primary neural crest cells derived from day 9.5 mouse embryos [MYCN and common ALK mutations exert a role in neuroblastoma tumour initiation using neural crest progenitor cell lines MONC-1 and JoMa1 [MYCN to form neuroblastoma in vivo [Previous studies have used murine neural crest systems to investigate neuroblastoma development by  embryos . Furthernd JoMa1 , 40. Ver in vivo . The useIn conclusion, our study describes advancement in the generation of noradrenergic sympathetic neuron-like cells from hESC to improve our understanding of the normal development of the human SNS and abnormalities thereof including neural crest-derived malignancies such as neuroblastoma. This model could later be perturbed by oncogenic transformation of these cells with genes known to be important in the development of neuroblastoma including MYCN and/or ALK as has been reported for NCDSC, to better understand events leading to the development of neuroblastoma, its cell of origin, and new potential treatment targets."}
+{"text": "Induction therapy of multiple myeloma patients prior to autologous stem cell transplantation has changed from conventional chemotherapy to treatment based on proteasome inhibitors or immunomodulatory drugs. We used flow cytometry to analyze total monocyte and monocyte subset  peripheral blood levels before and following auto-transplantation for a consecutive group of myeloma patients who had received the presently used induction therapy.+ as well as classical and non-classical monocytes, whereas intermediate monocytes were not affected. Thus, melphalan has a non-random effect on monocyte subsets. Melphalan had a stronger effect on total and classical monocyte concentrations for those patients who had received induction therapy including immunomodulatory drugs. Total monocytes and monocyte subset concentrations decreased during the period of pancytopenia, but monocyte reconstitution occurred before hematopoietic reconstitution. However, the fractions of various monocyte subsets varied considerably between patients.The patients showed normal total monocyte concentrations after induction/stem cell mobilization, but the concentrations of classical monocytes were increased compared with healthy controls. Melphalan conditioning reduced the levels of total CD14The total level of circulating monocytes is normalized early after auto-transplantation for multiple myeloma, but pre- and post-transplant levels of various monocyte subsets show considerable variation between patients. Autoll months . The posl months , 8, espebright CD16negative), intermediate (CD14bright CD16dim) and non-classical (CD14dim CD16bright) monocytes [Monocytes constitute up to 10% of total circulating peripheral blood leukocytes in healthy individuals ; they caonocytes \u201315. Clasonocytes , 15, 16.Monocytes seem to be involved in the development of myeloma bone disease \u201320 throup\u2009=\u20090.004), and patients receiving their first and second auto-transplantation showed a similar decrease. The decreased leukocyte counts were seen with both analytical methods , and the levels measured by these two methods were significantly correlated . The total leukocyte levels prior to melphalan conditioning showed no association with age, induction treatment , response to induction treatment, circulating CD34+ cell level at the day of harvesting or duration of posttransplant neutropenia/cytopenia (data not shown).We first compared the total leukocyte counts in peripheral blood for myeloma patients Table\u00a0 followed+ monocytes did not differ between the 17 myeloma patients (Table p\u2009=\u20090.01) whereas we could not detect any significant differences between patients and controls for intermediate and non-classical monocytes. The three patients admitted for their second auto-transplantation showed total monocyte and monocyte subset concentrations within the range for the patients admitted for their first transplantation . The same was true for classical, intermediate and non-classical monocytes except that pre-harvesting CD34+ cell levels showed significant correlations to absolute and relative levels of intermediate  and non-classical monocytes , response to induction treatment, circulating CD34+ monocytes  and the various monocyte subsets  for 18 newly diagnosed myeloma patients .We first compared the relative levels of circulating total CD14+ monocytes did not differ between patients and healthy controls  both for the healthy controls and for all except five patients. We also observed wide variation ranges for the intermediate and non-classical monocyte subsets; exceptional patients showed intermediate monocyte levels exceeding 15% and non-classical monocytes levels up to 40% of the total CD14+ monocytes .The preconditioning percentages of the classical, intermediate and non-classical monocyte subsets among CD14+ monocytes before the conditioning therapy (day \u2212\u20092) and 2 days later immediately before the autologous stem cell reinfusion (day 0). Ten patients were available for this paired comparison , whereas lymphocyte  and thrombocyte levels  were significantly decreased similar to the monocyte levels.We finally compared the levels of circulating neutrophils, total lymphocytes and thrombocytes  immediately before and 2 days after melphalan conditioning (Table p\u2009=\u20090.002) and non-classical subsets (p\u2009=\u20090.0039). Thus, melphalan conditioning has a nonrandom early effect on circulating monocyte subsets. However, we observed a wide variation in the percentage of the various monocyte subsets among total CD14+ monocytes in preconditioning samples  and day 0 (i.e. before stem cell transplantation) for 10 patients  and classical monocytes  compared with patients receiving induction treatment without these drugs). Furthermore, the absolute and relative levels of total monocytes and the various monocyte subsets 2\u2009days after the conditioning therapy  showed no association with age, response to induction treatment or levels of circulating CD34+ cells at the first day of harvesting (data not shown). Finally, the day 0 pretransplant levels of circulating neutrophils, total lymphocytes and thrombocytes did not differ between patients that had received induction therapy with and without IMiDs (data not shown). Thus, the IMiDs seem to have a non-random effect on the various monocyte subsets that becomes detectable after the melphalan infusion.IMiDs can alter the monocyte phenotype , and we + monocytes) varied during pancytopenia when tested 6\u20138\u2009days after stem cell reinfusion. All patients showed <\u20095% intermediate monocytes, whereas classical monocyte levels varied between 8 and 92% (median 62%) and non-classical monocytes also showed a considerable variation .We investigated the peripheral blood concentrations of the three monocyte subsets during the period of severe neutropenia for 8 myeloma patients Table . In cont9/L. The median time from first day of neutropenia (i.e. first day with neutrophils \u22640.2\u2009\u00d7\u2009109/L) until neutrophil reconstitution was 4\u2009days (range 2\u20139\u2009days). Furthermore, thrombocyte reconstitution was defined as the first out of three consecutive days with thrombocyte counts above 20\u2009\u00d7\u2009109/L in transfusion-independent patients. The median duration of thrombocytopenia (i.e. thrombocyte levels below 20\u2009\u00d7\u2009109/L) was 4\u2009days (range 1\u20136\u2009days). Finally, time to neutrophil/thrombocyte reconstitution did not differ between patients receiving their first or second auto-transplantation and showed no significant associations with preconditioning (i.e. day \u2212\u20092) or pre-transplant (i.e. day 0) total monocyte levels.The levels of circulating total leukocytes, neutrophils and thrombocytes were measured by clinical hematology instrument) for all our patients. Neutrophil reconstitution was defined as the first of 3 days with neutrophils above 0.2\u2009\u00d7\u2009109/L) was 10\u2009days; the median monocyte level was then 0.23\u2009\u00d7\u2009109/L (range 0.05\u20130.78\u2009\u00d7\u2009109/L). The neutrophil levels at the first day of monocyte normalization were generally below the lower normal limit , i.e. for 18 patients the neutrophil levels were still below the lower normal limit. All patients still had severe thrombocytopenia  at the first day of monocyte normalization. Finally, there was no significant association between preconditioning or pre-transplant total monocyte levels and time to normalized circulating monocyte levels, and monocyte normalization did not differ for patients receiving induction treatment with or without IMiDs (data not shown).The absolute levels of total monocytes were followed daily during the period of early hematological reconstitution for 24 consecutive patients. The median time from transplantation until the monocyte levels exceeded the lower normal limit . The thrombocyte counts for all patients  were also below the lower normal limit. However, even at this early time point only 10\u201312\u2009days post-transplant most patients showed normalized absolute (concentration) and relative (percentage) levels of total CD14+ monocytes as well as the three monocyte subsets within the pre-transplant variation range.We compared the absolute and relative levels of various monocyte subsets at day +\u200910/+\u200912 posttransplant with the corresponding preconditioning levels (day \u2212\u20092); paired samples were then available only for eight patients  was tested early after neutrophil and platelet reconstitution, and at this time point  nine patients showed normal and six patients showed increased counts . The total monocyte counts tested at a later time point  showed increased levels for a minority of four patients .One of our collaborating local hospitals only investigated peripheral blood neutrophil but not monocyte counts at the out-patient evaluations; for this reason peripheral blood monocyte counts were only available at later time points for 15 patients. The total monocyte count  or pretransplant (n\u2009=\u200918) total monocyte or monocyte subset levels or with posttransplant time to normalized total monocyte levels (n\u2009=\u200924) (data not shown).The posttransplant observation time for patients receiving their first auto-transplantation was 27\u201336\u2009months (median 32\u2009months). Five patients had disease progression less than 2\u2009years post-transplant, one patient was lost from follow-up and all other patients remained in plateau phase during follow-up. Progression-free survival less than 2\u2009years showed no association with precondition  were decreased compared with the preconditioning levels. However, the levels of CD14+ monocytes were lower than the monocyte levels estimated in the alternative assays, and this difference is probably due to a random loss of cells during the washing steps.We analyzed the total number of monocytes by two different methodological approaches, i.e. by using a hemocytometer and by using flow cytometry to estimate the levels of CD14Our studies included all except one patient from a defined geographic area and during a defined time period; for this reason it should be regarded as a population-based study. We could not investigate all patients at every time point during the treatment. However, we would emphasize that this was due to practical reasons such as transfer of patients to their local hospital or long traveling distance from their home to the transplantation center; it was not because of the disease, the treatment or development of complications. Leukocyte levels show diurnal variations \u201336, and + and CD14+CD16low/negative classical monocytes are then associated with prolonged progression-free survival after auto-transplantation. Thus, taken together these studies show that monocyte reconstitution occurs early (according to our study very early) after auto-transplantation, but despite this normalization there is still a relatively wide variation between patients and this heterogeneity in monocyte (subset) levels seems to persist until day 100 posttransplant and may even have a prognostic impact. The antimyeloma effect of posttransplant monocyte targeting may therefore vary between patients and depend on the monocyte subset profile. IL6 is regarded as a possible target in multiple myeloma [Our present study showed that the preconditioning patient levels did not differ from healthy controls, i.e. the myeloma disease itself, the induction treatment and the stem cell mobilization by cyclophosphamide plus G-CSF have only minor effects on monocytes except for a slight increase of classical monocytes. In contrast, the melphalan conditioning seemed to have a nonrandom effect of the monocyte subsets before an early reconstitution of all three subsets was observed. However, it should be emphasized that there is a wide variation between patients with regard to the effects of the conditioning therapy. A short duration of this monocytopenia is also suggested by previous studies , 8, but  myeloma ; monocyt myeloma . IL6 is Most of our patients received only 3 or 4 induction cycles before stem cell transplantation, whereas 6\u2009cycles are now often recommended, especially for patients who have not received a complete remission , 41, 42.+ cell mobilization) was associated with the levels of intermediate and non-classical monocytes before conditioning therapy.Our comparison of induction treatments with and without IMiDs suggests that the post-conditioning monocyte concentrations are influenced by the previous use of immunomodulatory drugs in the induction therapy, whereas the capacity of stem cell mobilization and response to the induction therapy are less important. However, G-CSF responsiveness  for our patients that received their first auto-transplantation and a majority of them were still in a plateau phase. We could not detect any associations between time to progression (i.e. progression before 2 years posttransplant) and monocyte subset levels/reconstitution. However, these data should be interpreted with great care because the patient cohort is relatively small for such analyses and the observation time is short and patients with early relapse are few.Although our study is relatively small, we observed that the total level of circulating monocytes is normalized early after auto-transplantation for multiple myeloma. However, the levels of various monocyte subsets show considerable variation between patients. Clinical studies including larger number of patients and a longer observation time are needed to clarify whether these differences are associated with overall survival, time to relapse and/or frequencies of severe infections.Proteasomal inhibitors and IMiDs are now commonly used in induction treatment of young and fit myeloma patients prior to stem cell harvesting and auto-transplantation. These drugs may thereby influence the pretransplant immunological status of the patients and the immunocompetent cells in the stem cell graft. The aim of our present study was therefore to investigate the preconditioning status of the monocyte system in myeloma patients treated with induction chemotherapy based on IMiDs or proteasomal inhibitors, and to characterize the monocyte subset levels in autotransplanted patients during the early posttransplant period until hematological reconstitution.Our hospital is the only center for stem cell transplantation in a defined geographical area of Norway , and our patients represent all myeloma patients except one receiving autologous stem cell transplantation in this area during an 8 months period. Our study should therefore be regarded as a population-based study of unselected patients.6 CD34+ cells per kilo body weight, and the median time from start of induction to transplantation was 16\u2009weeks (range 13\u201328\u2009weeks). For patients receiving their first auto-transplantation the grafts had been stored for 3\u20135\u2009weeks, whereas for those patients receiving their second transplantation the grafts were stored for at least 2\u2009years. All patients received conditioning therapy with melphalan , 200\u2009mg/m2 administered as an intravenous infusion 2\u2009days before stem cell reinfusion. They received G-CSF 5\u2009\u03bcg/kg from day +\u20094 posttransplant until stable neutrophil recovery, i.e. peripheral blood neutrophil levels above 0.2\u2009\u00d7\u2009109/L for three consecutive days or exceeding 10\u2009\u00d7\u2009109/L.The diagnosis of multiple myeloma was based on generally accepted criteria , 45, andThe normal controls were healthy blood donors; in accordance with the approved routines at the Blood Bank, Haukeland University Hospital peripheral venous blood samples for medical research were donated after written informed consent.9/L) and thrombocytopenia. The last sample was collected on the first or second day with peripheral blood neutrophil counts exceeding 0.2\u2009\u00d7\u2009109/L (10\u201312\u2009days posttransplant). The control samples were derived from 17 healthy blood donors . All samples were processed at room temperature within 120\u2009min. All samples were collected between 08:00 and 10:00\u2009am. It was not possible to get samples from all patients at all four time points; this was due to either transfer to their local hospitals after stem cell reinfusion or the patient was not available for sampling at the indicated time in the morning.Blood samples were drawn in ACD-A  blood sampling vacuum tubes. We collected the first patient sample immediately before melphalan conditioning (day \u2212\u20092). The second sample was collected 2 days later immediately before reinfusion of the peripheral blood stem cell graft (day 0). The third sample was collected on day +\u20096 posttransplant when patients had severe neutropenia  anticoagulated whole blood and 46\u2009mL of lysing buffer  were mixed and incubated for 15\u2009min at room temperature. Subsequently, leukocytes were collected by centrifugation  and thereafter washed in phosphate-buffered saline with 1% Bovine Serum Albumin ,. The cells were reconstituted in 200\u2009\u03bcL 1% BSA/PBS with 10% immunoglobulin solution . The following mouse anti-human antibodies were included in the antibody panel : CD14 Alexa 488 (Clone M5E2), CD56 Alexa 647 (Clone B159), CD16 PerCpCy\u21225\u20135 (Clone 3G8), CD45 V500 (Clone HI30), CD11b V540 (Clone ICRF44 (44)) and HLA-DR PE (Clone G46\u20136). The staining procedure and gating strategy for identification of monocytes and monocyte subsets has been described in detail in a previous methodological article .All samples were analyzed by a 10-parameter BD FACS Verse flow cytometer equipped with 404, 488 and 640\u2009nm lasers. We used BD FACSuite\u2122 CS&T Research Beads  for regular quality control of the instrument, single-stained compensation bead samples  for compensation and unstained samples as gating controls. At least 5000 monocytes were analyzed for each sample (based on SSC/FSC properties). We used counting beads  when estimating the concentrations of monocytes/monocyte subsets. FlowJo software  was used for analysis of the results.Analyses of peripheral blood levels of total leukocytes and total monocytes were performed by using accredited clinical\u00a0hematology instrument .p-values were below 0.05.We applied IBM SSP statistics 23 for all statistical analyses. The Wilcoxon\u2019s rank sum test and the Wilcoxon\u2019s test for paired samples were used for comparison of different groups and for comparison of paired observations, respectively. The Pearson\u2019s test was used for correlation analyses. Differences were regarded as statistically significant when"}
+{"text": "Introduction: Urinary tract infections (UTIs) are one of the most common infections worldwide. Under special circumstances, clinicians must rely on laboratory findings, which might have a weak predicting value, misguiding the practitioners and leading to incorrect diagnosis and overuse of antibiotics. Therefore, there is an urgent need for reliable biomarkers in UTIs.Methods: We performed a literature search for biomarkers used in UTIs from January 1999 until May 2020. We used \u201curinary tract infection\u201d and \u201cbiomarker\u201d as the main key words in the PubMed, Medline and Cochrane databases. After peer review, we excluded the duplicates and identified the suitable articles, from which we collected the data and divided the available biomarkers into 5 groups: i) conventional markers; ii) promising, thoroughly studied biomarkers; iii) promising biomarkers that need further studies; iv) biomarkers of unknown significance; v) controversial, not useful markers.Results: We found 131 articles, mostly from the paediatric population. Neutrophil gelatinase-associated lipocalin (NGAL) and interleukins (IL) have a leading role in diagnosing and differentiating UTIs based on a lot of observational, comparative trials. Heparin Binding Protein (HBP), Lactoferrin (LF), Heat-Shock Protein-70 (HSP-70), Human Defensin-5 (HD-5), Lipopolysaccharide Binding Protein (LBP) and mass spectrometry studies are promising, but confirming data are lacking. The measurable components of the innate immune system and local host cell response could be appropriate biomarkers, but their significance is currently unknown.Conclusions: Conventional biomarkers for UTIs have low specificity. The use of urinary NGAL and interleukins could improve the sensitivity and specificity of laboratory diagnosis of UTIs. Conventional biomarkers  have weak accuracy in general for diagnosis and differential diagnosis for UTIs.The urothelium is an interactive immunological structure that produces an enormous quantity of molecules during the host-pathogene interaction. All of these molecules can form a subject for further research as a potential biomarker for UTIs.Most data is collected on NGAL and interleukins, but their role is not exclusive in diagnostics considering the limiting factors and the lack of well-designed, confirming studies.There are studies with at least 50 or more other biomarkers with either promising or controversial outcomes on their clinical usefulness, which we present briefly.Not only for diagnosis but also for etiologic diagnosis there is a novel technology (NMR-spectrometry) with convincing, but few studies.None of the presented biomarkers can render an exceptional or exclusive role in the field of diagnostics, thus further research is needed to identify suitable, reliable and easily measurable biomarkers. Urinary tract infections (UTIs) are amongst the most common infections worldwide. The diagnosis is based on symptoms, laboratory and imaging findings depending on the clinical manifestation. In this article, we will use the terminology as defined by the European Urological Association (EAU) in their guideline. For details, see Table 1 Non-febrile, symptomatic lower UTIs react with local mucosal responses; febrile UTIs (f-UTIs)/acute pyelonephritis (APN) have an added significance to systemic host response; patients with asymptomatic bacteriuria (ABU) normally have no or only a discrete host response. Although these approaches provide an appropriate diagnosis in the majority of cases, their diagnostic accuracy or clinical usability can be limited in some situations, including UTIs in infants and in patients with neurogenic conditions or continuous catheterization.Unfortunately, the widely used conventional biomarkers, including C-reactive protein (CRP), urine nitrite, leukocyte esterase, pyuria, and proteinuria, have a low sensitivity and specificity for predicting or differentiating UTIs, leading to over- or undertreatment. According to a retrospective pediatric study \u2013 analyzing 1,186 subjects, using these conventional diagnostic tools, 43% of the patients are overtreated, and 13% are undertreated, leading to recurrent infections, antibiotic resistance or kidney damage . It. It9]. IBased on the results, urinary NGAL can be a suitable marker for diagnosing UTIs in children and in adults. Although serum NGAL elevates during UTIs as well, referring to the listed data, urinary NGAL seems to be a more sensitive marker for predicting UTIs. For differential diagnosis of upper from lower UTIs, NGAL also could be a useful marker. Although the difference between the two groups was not significant in every study, a remarkable disparity could be observed between these groups . Urine NDuring the host cell-pathogen interaction, a cytokine burst occurs in order to recruit the cells of the innate immune system and enhance the defense against pathogens. In UTIs, cytokines are mainly produced locally in the uroepithelial cell lining of the bladder and secreted into the urine. Generally, creatinine correlation of the cytokine content to compensate for different dilutions of the urine is not considered necessary . Mostly,IL-6 and IL-8 are expressed rapidly after getting into contact with pathogens. IL-6 not only recruits immune cells, but also initiates gene cascades in order to produce antimicrobial peptides . Due to IL-8 has a central role in all inflammatory processes. Although its elevated concentration is observed in UTIs and can be a predictor of acute pyelonephritis, its specificity is low. It raises in every kind of congenital urinary anomaly, except antenatal renal pelvic dilatation. Thus, IL-8 is not suitable for diagnosing UTIs when an anatomical disorder is present .Escherichia coli infection. The opposite findings  could indicate the presence of UTIs caused by ESBL producing Klebsiella pneumoniae. Increased NLR and IL-8 not only refers to the etiology, but also indicates the occurrence of renal damage. Among the aforementioned markers, NLR had the strongest predictive value for diagnosing UTIs caused by ESBL producing E. coli with 88% sensitivity, 73% specificity, 93% PPV and 24% NPV , , , 39], , .Controversial data on their usefulness can be found for the following biomarkers: Beta-2-microglobulin, pentraxin-3, prosaposin, Tamm-Horsfall Protein, Arginin-Vasopressin , 41], , .UTIs are common infections in every age group. Under special circumstances, timely diagnosis might be challenging. In these situations, clinicians must decide on laboratory findings, which have a weak accuracy in general. As seen in the details, none of the aforementioned biomarkers can predict the presence of a UTI exclusively. Thus, the interest turns to novel markers, specifically in special patients populations, for example children, who cannot describe their symptoms reliably, or among patients with constant catheterization, who have asymptomatic bacteriuria with or without specific symptoms referring to infection. In these cases, measuring the severity of the immune response could be a suitable alternative to decide about the need of antibiotic treatment. In addition, the diagnostic accuracy of biomarkers can probably only be determined when combined with a detailed and standardized scoring of subjective symptoms along severity indexing and individual risk ratification. For this reason, using validated questionnaires, for example the Acute Cystitis Symptom Score questionnaire , 44], c, c44], cThere are specific difficulties in each patient population in which UTI biomarkers are needed, but unfortunately, the patient groups with the highest need for a biomarker are also the ones that are most difficult to study. Not only symptom scoring is difficult; when ABU is frequent, the host response differentiation is obstructed. Furthermore, the comparator, ideally intraindividual comparison before and after a UTI episode, is commonly replaced by matched controls. In our literature search, we found that approximately one third of the articles were on NGAL or on cytokines. Considering that cytokines are key components of the local host-cell response, these are promising biomarkers for differentiating upper from lower UTIs \u2013 especially IL-6 and IL-8 , 21]. F. F21]. FNGAL also has a potential in the diagnosis of UTIs with high sensitivity and specificity. However, an important limitation is that it is a common marker of kidney injury, thus acute or chronic disease and other potential conditions that are yet unknown can elevate its level along with an infectious disease. In this field, further observations are needed .Most of the published data on biomarkers focus on children. There were examples when the context were proven for adults as well, thus we believe that further findings should be valid for adults too. However, due to a lack of studies, the probative value is still missing . AnotherFinding suitable biomarkers for UTIs has limitations: The immune system is constantly influenced by varying conditions  which may influence the expression of biomarkers. Further research could include a search for bacterial metabolites or volatile organic compounds in urine , 44]. T. T44]. TEach of the biomarkers discussed above needs to be investigated and validated further in well designed clinical studies to determine their clinical usefulness for diagnosis and treatment outcome in patients with different categories of UTI, including uncomplicated and complicated UTI and urosepsis.Since the diagnostic accuracy and clinical usability of traditional biomarkers for UTIs in certain clinical settings are limited, there is a need for novel, accurate and widely available biomarkers. Urinary NGAL and interleukins are the most suitable biomarkers that are currently available for clinicians with a potential to improve the sensitivity and specificity of laboratory diagnosis of UTIs.6-OHNA: 6\u2019-hydroxy nicotinic acid 8-oxodG: 8\u2019-hydroxy-2\u2019-deoxyguanine ABU: Asymptomatic bacteriuria APN: Acute pyelonephritis ATP: Adenosine triphosphate BMP-2: Bone morphogenic protein-2 CA19-9: Carboanhydrate antigene 19-9 CAP37: Cationic antimicrobial protein of 37 kd (synonyme for HPB) CD14: Cluster of differentiation 14 CD44: Cluster of differentiation 44 CIC: Clean intermittent catheterisation CRN: Creatinine CRP: C-reactive protein CysC: Cystatin C ESBL: Extended spectrum beta lactamase H1-NMR: H1-nuclear magnetic resonance HBP: Heparin binding protein HD-5: Human defensin-5 HNP-1: Human neutrophil peptide-1 HNP-3: Human neutrophil peptide-3 HSP-70: Heat shock protein-70 IL: Interleukin IL-1B: Interleukin-1 beta IL-6: Interleukin-6 IL-8: Interleukin-8 LBP: Lipopolisaccharide binding protein LF: Lactoferrinl-UTI: Lower urinary tract infectionNGAL: Neutrophil gelatinase-associated lipocalinNLR: Neutrophil-lymphocyte RatioNPV: Negative predictive valueMCP-1: Monocyte chemotactic protein-1MMP-9: Matrix metalloprotease-9PCT: ProcalcitoninPGE2: Prostaglandin-E2PPV: Positive predictive valuesIL-1B: Serum interleukin 1 betasIL-6: Serum interleukin-6sIL-8: Serum interleukin-8s-NGAL: Serum neutrophil gelatinase-associated lipocalins-UTI: Symptomatic urinary tract infectionTAC: Total antioxidant capacityTMA: TrimethylamineTLR: Toll-like receptorTREM-1: Triggering receptor expressed on myeloid cells-1uIL-1B: Urinary interleukin-1 betauIL-6: Urinary interleukin-6uIL-8: Urinary IL-8u-NGAL: Urinary neutrophil gelatinase-associated lipocalinUPJO: Uretero-pelvic junction obstructionUTI: Urinary tract infectionu-UTI: Upper urinary tract infectionYKL-40: Tyrosine (Y) \u2013 lysine (K) \u2013 leucin (L) of 40 kDaVUR: Vesico-ureteral refluxThis article is also to be published as a chapter of the Living Handbook \u201eUrogenital Infections and Inflammations\u201c .The authors declare that they have no competing interests."}
+{"text": "Coronavirus disease (COVID-19), the infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported on December 31, 2019. Because it has only been studied for just over three months, our understanding of this disease is still incomplete, particularly regarding its sequelae and long-term outcomes. Moreover, very little has been written about the rehabilitation needs of patients with COVID-19 after discharge from acute care. The objective of this report is to answer the question \u201cWhat rehabilitation services do survivors of COVID-19 require?\u201d The question was asked within the context of a subacute hospital delivering geriatric inpatient and outpatient rehabilitation services.Three areas relevant to rehabilitation after COVID-19 were identified. First, details of how patients may present have been summarized, including comorbidities, complications from an intensive care unit stay with or without intubation, and the effects of the virus on multiple body systems, including those pertaining to cardiac, neurological, cognitive, and mental health. Second, I have suggested procedures regarding the design of inpatient rehabilitation units for COVID-19 survivors, staffing issues, and considerations for outpatient rehabilitation. Third, guidelines for rehabilitation  following COVID-19 have been proposed with respect to recovery of the respiratory system as well as recovery of mobility and function. A thorough assessment and an individualized, progressive treatment plan which focuses on function, disability, and return to participation in society will help each patient to maximize their function and quality of life. Careful consideration of the rehabilitation environment will ensure that all patients recover as completely as possible. Coronavirus disease (COVID-19), the infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported on December 31, 2019. Because it has only been studied for just over three months, our understanding of this disease is still incomplete, particularly its sequelae and long-term outcomes. Knowledge about COVID-19, including its presentation and treatment, is changing very rapidly, and guidelines are quickly being created and updated. Therefore, it is important to remain current by engaging in frequent reviews of new research.The objective of this report was to answer the question \u201cWhat rehabilitation services do survivors of COVID-19 require?\u201d The question was asked within the context of a subacute hospital delivering geriatric inpatient and outpatient rehabilitation services. As of April 14, 2020, very little has been written about the rehabilitation needs or outcomes for patients with COVID-19 after discharge from acute care. Upon thoughtful consideration of the question, it appears that the topics of greatest importance to allied health professionals treating patients with COVID-19 are the physical, cognitive, and psychosocial presentation of survivors, the procedures that would be required within a rehabilitation department, and the treatment that should be provided. These three topics are discussed in order below.Much of what has been published is based on expert opinion but not on direct observation of the actual trajectories of patients with COVID-19. Many of the early papers came from China and Italy , the locations that had the earliest experience with COVID-19; these can potentially provide insight into longer-term outcomes and ongoing patient needs. Organizations such as the World Health Organization (WHO) and physiotherapy organizations have also written acute-care clinical practice guidelines for patients with COVID-19 . Some auComorbidities, direct lung damage from COVID-19, and concurrent injuries to other organs and systems due to COVID-19 are all important considerations when creating a rehabilitation treatment plan for patients recovering from COVID-19. The information below presents several comorbidities and features of COVID-19; however, this knowledge continues to evolve.The leading comorbid conditions of patients with COVID-19 are hypertension (55%), coronary artery disease and stroke (32%), and diabetes (31%) . PatientThe most likely early complications are acute respiratory distress syndrome (ARDS) and sepsis/septic shock, multi-organ failure, acute kidney injury, and cardiac injury ,10,13. TCritical illness polyneuropathy (CIP) is a mixed sensorimotor neuropathy that leads to axonal degeneration; it may occur after COVID-19 ,14-16. ICritical illness myopathy (CIM), which presents in 48%-96% of ICU patients with ARDS, is a non-necrotizing diffuse myopathy with fatty degeneration, fiber atrophy, and fibrosis ,15,16. IPost\u2013intensive care syndrome is described separately from CIP and CIM; it is associated with reduced pulmonary function (restrictive pattern), reduced inspiratory muscle strength, poor knee extension, poor upper extremity and grip strength, and low functional capacity . ImprovePatients with COVID-19 who have physically recovered and have tested negative for the virus twice are deemed to be cured and noninfectious. However, there are reports of such patients subsequently testing positive 5-13 days later using a different manufacturer\u2019s test kit . The virIn one study , 20% of Acutely, 36.4% of patients with COVID-19 develop neurological symptoms, including headaches, disturbed consciousness, seizures, absence of smell and taste, and paresthesia ,21. PostSARS can induce neurological diseases such as polyneuropathy, viral encephalitis, and aortic ischemic stroke . In MERSPatients severely affected by COVID-19 are more likely to have acute kidney injury as well as secondary infection ,11. SurvOsteoporosis and avascular necrosis have been reported as sequelae of SARS . These cIn one study of patients with respiratory failure or shock, after ICU admission , median global cognition scores  were an average of 1.5 SD below the age-adjusted population mean and similar to those of patients with mild cognitive impairment . Among tIn research regarding ICU admissions for ARDS, adverse psychological impacts have been reported . Even afAfter discharge from acute care, some patients who have recovered from the acute respiratory effects of COVID-19 will need further rehabilitation. How many of these patients may need postacute care? In one study, 30% of patients hospitalized with sepsis  required facility-based care; another 20% required home health care .These suggestions regarding the design of an inpatient rehabilitation unit in this time of COVID-19, and the procedures to be followed, are mostly based on the experiences of China and Italy, who are ahead of Canada on the COVID-19 trajectory ,21,29-31A separate unit or area is suggested for the rehabilitation of patients post\u2013COVID-19 and other patients arriving on the unit.Depending on need, it has been suggested that dedicated facilities should be used to treat patients post\u2013COVID-19; examples may include underutilized rural hospitals or retrofitted unused buildings, such as university dormitories.It may be necessary to receive patients from acute care earlier than is generally done.Patients should stay in their rooms.Group therapy and therapy in rehabilitation gyms should be prohibited; therapy should be provided one-on-one in patients\u2019 rooms.Patients may be discharged to home sooner than usual (as soon as the family is able to take care of the patient) to free space.It may be difficult to discharge some patients because long-term care facilities and retirement homes may not be accepting new residents.Shared equipment must be decontaminated between patients; single-use equipment should be used where possible . Particular attention should be paid to electrode sponges, hydrocollator heat packs, gels, topical lotions, items for training manual dexterity, etc.Plan therapeutic activities to minimize the number of personnel involved when possible .Minimize the number of personnel entering a patient\u2019s room. Have a single staff member perform most  of the care and duties for a particular patient .Walking practice should be done in parts of the hospital that are not commonly used.Surgical masks should be worn by the patients and the therapists.Patients should be kept at least 2 meters apart and avoid talking or eating while facing each other.Several suggestions for how allied health care professionals can adapt to working with COVID-19 rehabilitation patients are provided here. These suggestions have been informed by early COVID-19 reports and adapted from acute care guidelines ,21,30,31Health checks for personnel should be done frequently.There may be personnel shortages due to staff illness, staff in isolation, or redeployment.There may be changes in staff/patient ratios due to the increased number of one-on-one treatments (due to patients not being seen in the rehabilitation gyms).Continuous staff training will be required due to changing protocols/guidelines.Time should be taken to train and retrain personnel in the use of personal protective equipment (PPE).Physiotherapists and speech-language pathologists should wear higher levels of PPE if they may be exposed to aerosols from post\u2013COVID-19 patients .It is important to seek ongoing input from front line staff to inform others. One group of rehabilitation professionals in Italy has been holding weekly webinars to stay up-to-date with the changing needs of rehabilitation during this time. These are available for an international audience.All nonrequired therapies and services should be cancelled, or telecommunication should be used to deliver them.The time taken to don PPE and perform infection control measures may decrease work efficiency.Allied health professionals should wear scrubs and a T-shirt at work and shower and change into street clothes before going home.Rehabilitation staff may be divided into two teams who work independently of each other. If several members of one team become ill, the other team can take over.Meetings should be held virtually when possible.If patients can be managed at home, this may be a good option, even for patients who might have been admitted to inpatient rehabilitation in the past ,32. IsolThe importance of rehabilitation after COVID-19 has been emphasized according to the framework of the International Classification of Functioning, Disability and Health ,35. The Recommendations from both China and Italy state that to avoid aggravating respiratory distress or dispersing the virus unnecessarily, respiratory rehabilitation should not begin too early ,37,38. IIn inpatient rehabilitation, respiratory assessment should include dyspnea, thoracic activity, diaphragmatic activity and amplitude, respiratory muscle strength , respiratory pattern, and frequency ,39. Card2 (<95%), blood pressure <90/60 or >140/90, heart rate >100 beats per minute, temperature >37.2 \u00baC, excessive fatigue, chest pain, severe cough, blurred vision, dizziness, heart palpitations, sweating, loss of balance, and headache [In the postacute phase, inspiratory muscle training should be included if inspiratory muscles are weak. Deep, slow breathing, thoracic expansion (with shoulder elevation), diaphragmatic breathing, mobilization of respiratory muscles, airway clearance techniques (as needed), and positive expiratory pressure devices can be added based on assessed needs ,39. Careheadache .Functional assessment should include muscle joint range of motion, strength testing, and balance  ,7,38. ExPhysiotherapy should begin in the acute inpatient setting and continue after transfer to inpatient rehabilitation ,38. EarlOccupational therapy should focus on ADL and instrumental ADL guidance as well as targeted interventions to facilitate functional independence and prepare patients for discharge . Speech-Rehabilitation after COVID-19 is similar to that provided for many patients in geriatric rehabilitation units who have been affected by illness or injury. Some may present with a variety of sequelae associated with the viral illness and with a prolonged stay in the ICU, possibly including mechanical ventilation. Many will have preexisting comorbidities. A thorough assessment and an individualized, progressive treatment plan which focuses on function, disability, and return to participation in society will help each patient to maximize their function and quality of life."}
+{"text": "Streptococcus genus, with 98.04, 98.11, and 97.34% similarities to Streptococcus australis ATCC 700641T, Streptococcus rubneri LMG 27207T and Streptococcus parasanguinis ATCC 15912T, respectively. A sodA gene comparison exhibited a sequence identity of 92.6% with the closest strain Streptococcus australis ATCC 700641T. In silico DNA-DNA hybridization showed a highest DNA similarity value of 52% with Streptococcus australis ATCC 700641T. Comparing 18 biochemical traits, the similarities of the Streptococcus strain E24 were 72% with Streptococcus rubneri LMG 27207T, 78% with Streptococcus australis ATCC 700641T and 44% with Streptococcus parasanguinis ATCC 15912T. We suggest that based on the genotypic and phenotypic results that the strain E24 is a novel species of the Streptococcus genus and propose the name Streptococcus xiaochunlingii sp. nov. E24.A Gram-positive, \u03b1-hemolytic, catalase-negative, facultative anaerobic and non-motile coccus was isolated form throat swabs taken from the oropharynx of healthy children. The genome was shown to be 1.950,659 bp long and contained 42.03 mol% G + C bases with 1,942 protein-coding and 53 RNA coding genes. The results of 16S rRNA gene sequencing strongly suggested that the strain is a member of the  Streptococcus viridans-predominated bacterial community, followed by niche differentiation to Staphylococcus aureus and other bacteriae frequently leading to a composition of Moraxella spp., Dolosigranulum spp., and Streptococcus spp.  . During streptococci isolation a single, circular, unpigmented and \u03b1-hemolytic colony of 0.5\u20131.0 mm diameter emerged, which was purified by repeated streaking and finally stored at \u221280\u00b0C. The study was conducted in accordance with the \u201cDeclaration of Helsinki\u201d guidelines and approved by the Ethics Committee of Shenyang Medical College . Written informed consent was obtained from the legal representatives of the participating children.Streptococcus species from the NCBI database  and the 16S rRNA universal primers 27F/1492R . The sequence obtained by sequencing was aligned using Basic Local Alignment Search Tool (BLAST) of NCBI. We collected 16S rRNA gene sequences of all known database . Phylogedatabase  and maxidatabase  by closedatabase  and the database . Additiodatabase  with the2 and at 4, 15, 22, 30, 35, 37 and 42\u00b0C using nutrient agar containing sheep blood   . The nut, China) . The sus, China) .Cell sizes were calculated from images captured on a scanning electron microscope. Bacteria were fixed in glutaraldehyde phosphate (2.5%) buffer overnight at 4\u00b0C. They were then washed twice with phosphate buffer (PBS), fixed for 30 min in a 1% osmium tetroxide solution  and then washed 3 times with PBS before being dehydrated with 50, 70, 80, 90, 95 (twice), and 100% ethanol (three times), respectively (5 min per ethanol concentration). After natural drying, each sample was subjected to ion sputtering and then the gold was sprayed onto the surface and then viewed on a Hitachi 3400N scanning electron microscope .Streptococcus strain E24 solutions (concentration: 1 \u00d7 106 cfu/mL) divided into two groups, from which one group was heated to 80\u201390\u00b0C for 20 min and the other served as control. Then 50 \u03bcl solutions of each group were applied on nutrient agar plates containing 5% sheep blood. The strains were incubated under normal conditions at 37\u00b0C overnight to observe the bacterial colony formation. No colony growth was observed on the plate of the heat treatment group on the next day.Sporulation was tested with Isolates were characterized with Rapid ID32 Strep, API ZYM and API 50CH systems  as well as catalase  and oxidase  assays were carried out separately.Streptococcus strain E24 was determined using disk diffusion (The antibiotic susceptibility of the strain iffusion  on MuellFatty acid methyl esters (FAMEs) were analyzed using a gas chromatography/mass spectrometry [see  and Sas]. FAMEs Genomic DNA was extracted using the SDS methodology . AgaroseAfter preprocessing, appropriate data were gathered using SOAP (version 2.04) , 2010, SStreptococcus strain E24 located strains were selected based on BLAST alignment and the phylogenetic tree constructed from 16S rRNA gene results data.Average nucleotide identity (ANI) was used to assess genomic similarity and to define species with values of 95%. The ANI analysis was performed with OAT  and DNA-Streptococcus species retrieved from GenBank. Strain Streptococcus strain E24 shared 98.04, 98.11, and 97.34 % 16S rRNA gene sequence similarity with S. australis ATCC 700641T, S. rubneri LMG 27207T and S. parasanguinis ATCC 15912T, respectively. The housekeeping gene sodA .The newly determined 16S rRNA  isolate ene sodA  of Strepvide supra) for 24 h, when the bacteria became punctate, gray and formed \u03b1-hemolytic colonies having edges that were undulated. Gram staining was performed using the classical Gram stain procedure  and other members of the Streptococcus genus are shown in Catalase and oxidase tested negative and Streptococcus strain E24 revealed susceptibility to vancomycin, cefepime, Rina thiazole amine, penicillin, chloramphenicol and clindamycin, but resistance to ceftriaxone, ampicillin, and cefotaxime.Antibiotic susceptibility tests of Streptococcus strain E24 revealed that it was comprised of 1,950,659 bp with a 42.03% G + C content (VFSG00000000).A draft of the genome of  content . It is cStreptococcus strain E24  was less than S. pseudopneumoniae ATCC BAA-960T , S. tigurinus AZ 3aT , S. pneumoniae NCTC 7465T , S. parasanguinis ATCC 15912T  and S. sanguinis ATCC 10556T , but greater than that of S. dentisani CECT 7747T , S. infantis ATCC 700779T , S. mitis NCTC 12261T  and S. oralis ATCC 35037T . The G + C contents of Streptococcus strain E24 (42.03%) was greater than that of S. pseudopneumoniae ATCC BAA-960T (39.8%), S. dentisani CECT 7747T (41.1%), S. infantis ATCC 700779T (38.9%), S. mitis NCTC 12261T (41.1%), S. oralis (41.4%), S. pneumoniae NCTC 7465T (39.7%), S. parasanguinis ATCC 15912T (41.7%) and S. tigurinus AZ 3aT (40.3%), but less than that of S. sanguinis ATCC 10556T (43.2%).The genome sequence length of Streptococcus strain E24 was matched with 11 comparison strains .Streptococcus strain E24 is a non-motile, non-spore-forming, facultative anaerobic and Gram-positive coccus, isolated from the oropharynx of healthy children. Growth is achieved under aerobic, microaerophilic and anaerobic atmospheres and had a temperature growth range of 22 to 42\u00b0C, with the optimum temperature being 37\u00b0C. After 48 h of aerobic incubation on 5% sheep\u2019s blood\u2013enriched nutrition agar, colonies are pinpoint, grayish and \u03b1-hemolytic, with undulated edges and with a diameter of 0.5 to 1 mm. Cells are roughly round with a 0.5 \u03bcm diameter. Cells did not hydrolyse arginine, aesculin or starch except hippurate and were negative in tests for esterase, leucin arylamidase, valin arylamidase and \u03b2-D-galactosidase. Positive for \u03b2-D-glucuronidase, \u03b2-D-glucosidase and alkaline phosphatase. Does not produce acid from glycerol, erythritol, d- or l-arabinose, d-ribose, d- or l-xylose, d-adonitol, methyl \u03b2-D-xyloside, d-fructose-l-sorbose, l-rhamnose, dulcitol, d-mannitol, methyl \u03b1-D-mannopyranoside, methyl \u03b1-D-glucopyranoside, amygdalin, arbutin, aesculin, salicin, cellobiose, d-maltose, trehalose, inulin, melezitose, starch, glycogen, xylitol, gentiobiose, d-lyxose, d-raffinose, d-tagatose, d- or l-fucose, d- or l-arabitol, d-sorbitol, d-melibiose or 2- or 5-ketogluconate. Utilization of d-lactose is variable. They were capable of producing acid from D-galactose, D-glucose, d-mannose, inositol, N-acetylglucosamine and sucrose. Catalase and oxidase tested negative. The genome was shown to be 1.950,659 bp long and contained 42.03 mol% G + C bases with 1,942 protein-coding and 53 RNA coding genes. The results of 16S rRNA gene sequencing strongly suggested that the strain was a member of the Streptococcus genus, with 98.04, 98.11, and 97.34% similarities to Streptococcus australis ATCC 700641T, Streptococcus rubneri LMG 27207T and Streptococcus parasanguinis ATCC 15912T, respectively. A sodA gene comparison exhibited a sequence identity of 92.6% with the closest strain Streptococcus australis ATCC 700641T. In silico DNA\u2013DNA hybridization showed a closest DNA similarity value of 52% with Streptococcus australis ATCC 700641T. Streptococcus strain E24 has been deposited in the Korean Agricultural Culture Collection (deposition no. KACC 21425) and Guangdong Microbial Culture Collection Center (deposition no. GDMCC 1.1634). The genome and 16S rRNA gene sequences are recorded in the NCBI database .https://www.ncbi.nlm.nih.gov/, VFSG00000000; https://www.ncbi.nlm.nih.gov/, MN592637.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession numbers can be found below: The studies involving human participants were reviewed and approved by Ethics Committee of Shenyang Medical College . Written informed consent to participate in this study was provided by the participants\u2019 legal guardian/next of kin.XL and YZ were responsible for the conception and design of the study, drafted, commented, and revised the manuscript. XL, YZ, YS, HQ, and DL were in charge of statistical analysis. All authors were responsible for data acquisition and analysis, read and approved the final version of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3\u2032 end of the JSRV  Parvoviridae family -1-propanesulfonate (CHAPS) was added to each cell pellet before pipetting up and down to suspend the cells. Cells were allowed to lyse for 15 min on ice. Cell debris was removed by centrifuging the sample at 14,000 rpm and the supernatant was recovered for use in subsequent assays. A Bradford assay was performed on cell lysates according to the method of Sambrook and Russell to determine total protein concentration [After 48 h, cells were washed with 10 mL of ice cold PBS and scraped off the plate using a rubber policeman into 1 mL of PBS. Cells and PBS were spun down at 4000 rpm for 2 min to recover cells. 500 \u00b5L of TMNC lysis buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgClntration .2+ solution containing 0.1 M MgCl2 and 4.5 M \u03b2-mercaptoethanol, 1\u00d7 ONPG solution containing 4 mg/mL o-nitrophenyl-\u03b2-D-Galactoside (ONPG) dissolved in 0.1 M dibasic sodium phosphate buffer pH (7.5). 3 \u00b5L of 100\u00d7 Mg2+, 66 \u00b5L of 1\u00d7 ONPG, 30 \u00b5L of cell lysate, and 201 \u00b5L of 0.1 M sodium phosphate were mixed together to initiate the reaction. Reactions were incubated at 37 \u00b0C until a faint yellow color developed. Reactions were stopped by adding 500 \u00b5L of 1 M Na2CO3. To determine beta-galactosidase activity, absorbance was read at 420 nm using a BioTeK Powerwave XS2 plate reader.Beta-galactosidase assays were performed by the method of J. Miller . The fol10 vector genomes of AAV vectors was injected in a 100 \u00b5L volume into the tail vein. For intraperitoneal, 8 \u00d7 1010 vector genomes were injected into the intraperitoneal space in a 500 \u00b5L total volume containing the AAV vector plus PBS. For intranasal delivery, 1 \u00d7 1010 vector genomes were delivered in two aliquots of 40 \u00b5L each in order to maximize the chances that tissues deep in the lung would be transduced. A modified method of intranasal delivery was used so as to ensure vector delivery to the distal lung [Mouse experiments were performed in accordance with the guidelines set forth by the Canadian Council on Animal Care (CCAC). Eight-week old C57BL6/J mice were obtained from Charles River Laboratories . AAV vectors were produced by cotransfection of HEK 293 cells with genome and packaging plasmids as described previously . AAV vectal lung . Mice we2, 100 mM NaCl, 4% (wt/vol) CHAPS) using a Precellys 24 homogenizer , with ~200 \u00b5L of TMNC buffer in a FastPrep\u2122 Lysing Matrix A tube . Tissue homogenates were placed in a water bath at 65 \u00b0C for 1 h to inactivate endogenous heat-labile AP activity and subsequently clarified by centrifugation at 17,900\u00d7 g for 15 min at 4 \u00b0C to remove cell debris. The protein content of each sample was determined by the method of Bradford, and the AP activity in tissue lysates was determined, in triplicate, by a fluorometric assay using the 4-methylumbelliferyl phosphate (MUP)  substrate, as described previously [For in vitro studies, cell lysates were heated at 65 \u00b0C for 1 h to inactivate endogenous alkaline phosphatases. For 208F cells, 30 \u00b5g of total protein as determined by Bradford assay was loaded into each well of a 96 well plate for each 10 cm dish. For HTX, HEK 293, and HEK 293T cells, 100 \u00b5g of total protein as determined by Bradford assay were loaded into each well of a 96 well plate. Mouse tissues were harvested 4 weeks after vector administration, snap-frozen in liquid nitrogen and stored at \u221280 \u00b0C until assayed. A small piece  of the tissue to be analyzed was homogenized in TMNC lysis buffer  on their own demonstrated little or no expression when transfected into human embryonic kidney (HEK 293), human fibrosarcoma (HTX), or rat fibroblast (208F) cells . SimilarInclusion of the chicken beta actin promoter (CBA) markedly increased expression from constructs bearing the JE in all cell lines tested . CompariFor another series of constructs, the putative JE sequences were placed in an inverted orientation relative to their orientation in the JSRV provirus. Inverted JE sequences corresponding to short, medium and long lengths placed in front of the chicken beta actin promoter  all demonstrated a similar improvement as their non-inverted counterparts in 293, HTX, 293T and 208F cells, while constructs containing the inverted JE  of varying lengths but lacking the chicken beta actin promoter conferred low expression in all cell lines but 293T. The relatively low activity of the wild-type JSRV promoter is dwarfed by the high activity brought about by the combination of the JE, the U3 and R regions of the JSRV LTR placed upstream of the chicken beta actin promoter (pAJE71-U3R-CBA-AP) in 293, 293T, 208F, and HTX cells. In fact, expression was so high in 293T and HTX cells that it matched or exceeded expression by the CAG promoter (pACAGAP), currently one of the best promoters for constitutive expression from a variety of cell lines . A vectoConstructs possessing components of the JE or JSRV LTR but lacking the chicken beta actin promoter demonstrated low activity in 293, 293T, 208F and HTX cell lines . pAU3-APExpression of the JSRV promoter is primarily limited to the respiratory tract  and liver when mice are transduced via an AAV vector . InclusiIn histological sections, strong AP expression could be observed in lung alveolar cells and liver hepatocytes of mice transduced with a vector that bore the short JE and the CBA (AJE71-CBA-AP), similar to that of ACAGAP . HistoloQuantification of hPLAP enzymatic activity in tissues harvested from the vector transduced mice revealed that in all tissues evaluated, expression from AJE71-U3-CBA-AP was reduced relative to ACAG-AP, but was much higher than ACBA-AP. AJE71-CBA-AP was marginally higher than ACBA-AP but far lower than AJE71-U3-CBA-AP, demonstrating the utility of the JSRV U3 region .The MLV env intron (581 bp) was exchanged for a shorter sequence based on an optimized SV40 intron (93 bp) modified to include consensus splice sites. Transfection of HEK 293 cells with the SV40 intron containing construct compared to the parental plasmid containing the MLV intron demonstrated no visible difference in hPLAP expression .In its native context, within an integrated JSRV provirus, JE may function to enhance expression from the 5\u2019 LTR sequence to drive viral gene expression, or may participate in the process of tumor development by dysregulating the expression of cellular proto-oncogenes.A lack of expression was observed in constructs bearing only the JE, compared to constructs that possessed the JE and a functional heterologous promoter, the chicken beta actin promoter. Furthermore, inclusion of the JE in any of its varying lengths described here and in either forward or reverse orientations proved to show an increase in protein expression when combined with the chicken beta actin promoter. These characteristics strongly suggest that the JE is able to function as an enhancer sequence . ExtensiLow levels of expression observed for the constructs containing JE alone, in either forward or inverted orientation  indicate that JE itself is not able to function effectively as a promoter. This rules out the possibility of JE functioning as a promoter for a transactivating non coding RNA, as is the case in Moloney murine leukemia virus and feline leukemia virus, which both express noncoding RNAs from their 3\u2032 LTRs that are able to transactivate signaling pathways involved in cancer ,20.Evidence for a possible expression enhancing region or putative enhancer sequence located in regions flanking the JSRV env gene came to light in a paper by Sinn et al., wherein greatly increased JSRV envelope pseudotyped lentiviral vector titers were observed when env flanking sequences were included in the envelope expression cassette . PreviouThe identity of the transcription factor that binds to the JE has yet to be elucidated; however, one promising candidate is AP-2, for which there are four predicted sites within the JE71 sequence . A wide A high level of expression could be observed for AJE71-U3-CBA-AP in vivo, nearly matching ACAGAP for some individual mice . HoweverIn summary, we demonstrate the presence of an intragenic transcriptional enhancer element, found within the 3\u2032 end of the env gene. This JSRV-based enhancer element demonstrated strong expression in a variety of tissues, particularly respiratory and hepatic, and promoter/enhancer/intron cassettes derived from these elements can be pared down to a sufficiently small size (<630 bp in length), suitable for genome constraints imposed by AAV vector systems."}
+{"text": "OCA2) is a gene of great interest because of genetic variation affecting normal pigmentation variation in humans. The diverse geographic patterns for variant frequencies at OCA2 have been evident but have not been systematically investigated, especially outside of Europe. Here we examine population genetic variation in and near the OCA2 gene from a worldwide perspective. The very different patterns of genetic variation found across world regions suggest strong selection effects may have been at work over time. For example, analyses involving the variants that affect pigmentation of the iris argue that the derived allele of the rs1800407 single nucleotide polymorphism, which produces a hypomorphic protein, may have contributed to the previously demonstrated positive selection in Europe for the enhancer variant responsible for light eye color. More study is needed on the relationships of the genetic variation at OCA2 to variation in pigmentation in areas beyond Europe.Oculocutaneous Albinism type 2 ( OCA2) is a gene of interest for several reasons, not the least of which is its role in oculocutaneous albinism with about 30% of worldwide cases accounted for by 154 mutations in the OCA2 gene1. Two amino acid substitutions in the coding sequence were shown by Sviderskaya et al.2 to be associated with decreased expression of the OCA2 protein but not full ocular albinism. OCA2 was subsequently studied for its association with eye color but common variants are associated not just with variation in eye color but also with variation in skin color5. Different polymorphisms in the regulatory and coding regions are primarily associated with different eye, hair, and skin pigmentation phenotypes and show large frequency differences among populations from different parts of the world.Oculocutaneous Albinism type 2 , rs74653330 (Ala481Thr), rs1800407 (Arg419Gln), and rs1800401 (Arg305Trp). The Ala481Thr (rs74653330) and Val443Ile (rs121918166) variants were shown2 to be hypomorphic but not pathogenic in their studies of ocular albinism. The Val443Ile missense variant (rs121918166) has been reported at\u2009<\u20091% in Scandinavian populations10. These missense SNPs are distributed across 63\u00a0kb of the gene (Table Single nucleotide polymorphisms (SNPs) in the molecular region of OCA2 missense SNPs  have been studied in conjunction with pigmentation phenotypes, primarily in European and East Asian populations where the variants are most common. Walsh et al.13 found that including the genotype at rs1800407 in a regression equation improved the ability to predict eye color in their samples. Edwards et al.14 and Yuasa et al.17 found that rs1800414 was associated with skin color variation among individuals of East Asian ancestry. Eaton et al.18 studied both rs1800414 and rs74653330 on East Asians and found them to be independently associated with skin color. Rawofi et al.19 confirmed the association of rs1800414 with skin color and found it significantly associated with iris color. Lee et al.20 identified the derived allele at rs74653330 at a frequency of about 1% in Europeans. This hypomorphic OCA2*481Thr (rs74653330) allele was later found to be moderately frequent in many East Asian populations21.Three of the 8. The skin color effects of rs1800414 have been considered an example of parallel evolution for light skin color14. We are interested in these and other aspects of the population genetics of the OCA2 variants. To that end we have tested  project website23 in those populations and assembled the published data on population frequencies. The derived alleles show very distinct biogeographic variation. That global pattern of variation is the focus of this paper.Evidence of recent selection for the derived allele of rs12913832 at the enhancer is clearly documented in European populations as is selection for the derived allele at rs1800414 in East AsiaOCA2, rs1800414, rs74653330, and rs1800407 and the OCA2 enhancer SNP, rs12913832, in an intron of HERC2, that are the focus of this study. Data on all four of these SNPs come primarily from our genotyping studies (76 populations), from a collaboration with co-author Longli Kang (7 populations), and from the 22 relatively unadmixed populations of the 1\u00a0KG project (Phase 3)23. Additional individual SNP frequencies were obtained primarily from the published literature and were entered into the ALFRED database  before it became static. A fourth amino acid substitution, rs1800401 (Arg305Trp), has been typed in the 1\u00a0KG samples but is not included here because it has been otherwise studied largely in samples defined by pigmentation phenotypes  in a few populations25. The rare amino acid change  at SNP rs121918166 has only been studied on a small number of European populations10 and studied for its effect on eye, hair, and skin color. Only three of the 1\u00a0KG populations, all European, have the variant allele at rare frequencies ranging from 0.5% to 0.9%. All of the samples were collected with informed consent for population genetic studies such as this. Because all samples are completely anonymous, the allele frequency collection in this study is not considered human research.Table 26.Various methods were used to type the SNPs and are described in the multiple sources of the data. The source of data for each population sample is listed in Table https://www.goldensoftware.com). The haplotype frequencies were estimated using Phase version 2.1.128. Each population was phased separately.As these SNPs are simple co-dominant genetic systems allele frequencies were estimated by simple gene counting. The density plots were produced by Surfer (version 12.8) software  Figs.  and S2 hThe derived allele at rs1800407 Figs.  and S3 oThe rs12913832 SNP Figs.  and S4 iSNPs rs1800401 and rs121918166 have not been studied in as many populations as any of the four other SNPs and we have not considered them in this study. The variant at rs121918166 has only been observed at rare frequencies in Scandinavians. Based on the populations in the 1\u00a0KG the derived allele at rs1800401 occurs most frequently, 10% to 20%, in African and South Asian populations and is absent to\u2009<\u200912% in East Asia and Europe.24. Each of the four variants has a distinct geographic distribution but overlaps exist. In East Asia the hypomorphic rs74653330 allele overlaps somewhat with the rs1800414 variant but they appear to occur on separate haplotypes in the population. However, both the enhancer variant at rs12913832 and the amino acid substitution at rs1800407 occur frequently in Europe and surrounding areas and occur on the same chromosome at some unclear frequency.Evidence argues that the variant alleles at the four common SNPs depicted in Figs. 11 for nearly a decade and was suggested by Sturm et al.7 as functioning to increase the penetrance of the enhancer variant. Duffy et al.29 notes that heterozygosity for the derived allele at rs1800407 decreases the probability of green eyes on the homozygous derived rs12913832 background but increases it on a heterozygous rs12913832 background. Several studies have referred to the relationship of rs1800407, especially the 419Gln allele, and the enhancer variant as an example of epistasis32. However, if we consider the functional unit as production of a protein we necessarily include the rate of production of mRNA and the coding content of that mRNA. The term epistasis seems inappropriate because these two DNA variants are not functionally independent loci. The haplotype is the functional unit and the locus can be considered as a four-allele locus, at least with respect to the enhancer and rs1800407  of a hypomorphic OCA2 protein . Selection operated on some trait to increase the frequency of the enhancer variant; this cis combination of the two variants with a presumably hypomorphic protein might have been more strongly affected.If the doubly-derived chromosome for rs1800407 and rs12913832 results in \u201chigher penetrance\u201d for light eye color, the derived allele at rs1800407 must have a functional difference. While it was not studied by Sviderskaya et al.On a background of homozygosity for the enhancer (rs12913832) variant, the frequency of heterozygotes of the amino acid substitution (rs1800407) is 246/ or 7.5% in Duffy\u2019s largely British origin population sample. Those genotypes involve one chromosome that is doubly-derived  for the two variants and one that has only the enhancer variant. On a heterozygous enhancer background genotype, however, the amino acid substitution heterozygotes occur at a higher frequency of 529/ or 29.8%. Those nearly 30% of individuals are composed of both cis and trans genotypes for the two functional variants. The evidence is consistent with those two genotypes having different phenotypes as would be predicted by considering the functional context: the cis genotype has one fully normal protein at normal amounts and one variant protein produced at reduced amounts; the trans genotype has a normal protein at reduced amounts and a variant protein at normal amounts.29 are not necessarily in HW proportions depending on how they were ascertained, which is not specified. In fact, the ratio of the enhancer homozygotes to heterozygotes is 1.849 which is compatible with an enhancer variant frequency of about 0.79, essentially the same as in our summary  as heterozygous with a fully ancestral chromosome is largely absent; its expected frequency is quite small. Even assuming the haplotypes affect phenotype additively, to estimate the three different fitness parameters associated with the three derived chromosomes seems beyond the power of the existing data. We leave such estimation to others.OCA2 and SNPs at other genes to infer the iris, hair, and skin color of an individual from that individual\u2019s DNA. Those efforts are most recently integrated into the HIrisplex-S web site (https://hirisplex.erasmusmc.nl/). Such phenotype inference from a DNA sample can be very useful as an investigative lead in criminal forensics. Our data summaries demonstrate that two of the SNPs, rs12913832 and rs1800414, have common variants with strikingly different geographic patterns that makes them relevant to inference of biogeographic ancestry in some parts of the world. Indeed, rs12913832, the enhancer SNP, was incorporated in the Kidd Lab panel of 55 ancestry informative SNPs26 and rs1800414 is part of the 74 SNPs in a panel by Li et al.33.Many studies have reported on use of the genotypes at these sites at The population distribution of the chromosome with the derived enhancer variant (rs12913832) and the derived amino acid variant (419Gln for rs1800407) in cis is seen almost exclusively in northern Europe. Elsewhere, the rs1800407 variant (419Gln) occurs on a chromosome with the ancestral allele at the enhancer. The common occurrence of the doubly-derived (cis) chromosome, primarily in the populations with the strongest evidence of selection for the enhancer variant, strongly suggests selection on this chromosome in northern Europe. The north Eurasia distribution of the hypomorphic allele\u2013481Thr at rs74653330\u2013suggests parallel evolution for this variant as well.OCA2-HERC2 gene region are more extensive and suggest that more empirical studies are needed from more world regions so that we can refine and improve our knowledge. The studies supporting strong selection effects done thus far also support the view that more studies are important. Other genetic loci are known to influence pigmentation phenotypes. Their relative roles and the magnitude of their effects during development as well as the evolutionary impact of non-genetic factors will be more clearly understood when we have more worldwide data on the OCA2-HERC2 gene region.Our understanding of the role of the known functional and enhancer variants in human pigmentation phenotypes has grown markedly in recent decades but, thus far, the relationships have only been studied simultaneously and in relatively large samples in a subset of populations of European and East Asian ancestry. The very strong geographical frequency patterns shown by the existing patchwork of genetic data in the All subjects gave permission for collection of samples and use in population studies such as this. All samples are anonymous.Supplementary Tables.Supplementary Figures."}
+{"text": "As the 2019 novel coronavirus disease (COVID-19) pandemic rages globally, its impact has been felt in the stock markets around the world. Amidst the gloomy economic outlook, certain sectors seem to have survived better than others. This paper aims to investigate the sectors that have performed better even as market sentiment is affected by the pandemic. The daily closing stock prices of a total usable sample of 1,567 firms from 37 sectors are first analyzed using a combination of hierarchical clustering and shape-based distance (SBD) measures. Market sentiment is modeled from Google Trends on the COVID-19 pandemic. This is then analyzed against the time series of daily closing stock prices using augmented vector autoregression (VAR). The empirical results indicate that market sentiment towards the pandemic has significant effects on the stock prices of the sectors. Particularly, the stock price performance across sectors is differentiated by the level of the digital transformation of sectors, with those that are most digitally transformed, showing resilience towards negative market sentiment on the pandemic. This study contributes to the existing literature by incorporating search trends to analyze market sentiment, and by showing that digital transformation moderated the stock market resilience of firms against concern over the COVID-19 outbreak. The onslaught of the 2019 novel coronavirus disease (COVID-19) pandemic has impacted stock markets worldwide, with the stock prices of many firms seeing unprecedented fall. Many governments around the world imposed a shutdown on their cities in attempts to implement social distancing practices and slow down the spread of the life-threatening virus. Such shutdowns have brought what is classified as non-essential corporeal economic activities close to complete standstill. This has resulted in well-known brands, such as Hertz, JC Penny, and J Crew, filing for bankruptcy, and sparking concerns of large-scale economic recession or even economic collapse  Industry Digitalization Framework  have influenced a large number of economies worldwide. All of these epidemics have resulted in severe damage to the economy and loss of lives, e.g., 5% gross domestic product (GDP) of the US was reduced owing to the Influenza Palese ; as resuHowever, the new digital world has made the COVID-19 pandemic\u2019s impact on the global economy different from that of previous epidemics. The development of digital technologies in the past two decades has changed the way people and firms interact with each other. With the fast-growing Internet users worldwide, online shopping has become one of the most popular online activities, and the emergence of e-business platforms such as Amazon and Alibaba contribute to the rapid growth of online sales. In 2019, e-retail sales accounted for 14.1% of all retail sales worldwide, expected to reach 22% in 2023  to validate the groups of firms identified in the MGI Industry Digitalization Framework. It followed similar approaches employed by Sard\u00e1-Espinosa  and PapaPhase 2 modeled Google search trends and stock price changes jointly in an augmented VAR. In this phase, the analysis compared and contrasted the stock performance of firms across the three levels of digital transformation, i.e., low, medium, and high, based on the MGI Industry Digitalization Framework. The analysis in the two phases built on each other, supporting the notion that market sentiment towards the COVID-19 pandemic as reflected in Google search trends affects the stock prices.Using data from FactSet, we selected nearly 2000 firms listed on the NASDAQ based on their sectoral information in 2020. NASDAQ is one of the world\u2019s largest stock exchange based on market capitalization. Among the scholarly community, NASDAQ represents one of the most widely investigated stock exchange databases . The average market capitalization for the sampled stocks is USD8,587 million. The sample period  is chosen because it allows a reasonable amount of time lag for the COVID-19 outbreak to generate public attention and stir market sentiment. Additionally, the current research focuses on how sectoral digital transformation mitigates the effect of negative events on the stock market. We are more interested in the period from the outbreak of the COVID-19 to the eventual stock market crash (the affected period). There might be other potential confounding factors coming into play during the recovery period. For example, the U.S. coronavirus relief funds, established under the Coronavirus Aid, Relief, and Economic Security (CARES) Act, were announced in early April 2020. The stock market is likely to respond to such information on economic support. The chosen sample period ends when the CARES Act was announced, minimizing the potential confounding effect of government support and intervention on the stock market. For every entry, three data fields are obtained, namely, the daily price change, trading volume, and market cap. These data are used in the models in both Phase 1 and Phase 2. Table\u00a0In Phase 2, the search variable is operationalized as an \u201cinterest over time,\u201d measured on a relative scale of 0\u2013100, with peak popularity coinciding with a value of 100. While Google\u2019s indicator of interest, over time, has been used as a source of data in numerous studies   on March 11,\u00a02020. Thus, this cluster is named as the Resilient Cluster.By analyzing the extracted centroid, the times series patterns of the two clusters are then examined. For firms in Cluster 1, it is observed that a steeper declining trend of market capitalization occurs amid the COVID-19 outbreak. This is thus termed as the Sensitive Cluster and the Resilient Cluster, with a high, medium, and low level of digital transformation following the MGI Industry Digitalization Framework, to answer the research question and to validate the sectoring. To further simplify the research model and to enable in-depth investigation in Phase 2, Categories 2 to 5 in the original MGI Industry Digitalization Framework, which represent sectors with the potential to transform digitally further, are merged into one group of firms with a medium level of digital transformation. Firms under the most digital sectors (Category 1) and the most lagging sectors (Category 6) are left unchanged. It is then analyzed to assess if the level of digital transformation is associated with the two identified clusters. A descriptive summary of the sensitive and resilient clusters can be found in Table\u00a0The firms are cross-tabulated with percentages of their market capitalization decline under the Resilient Cluste. The majority of the firms in the lowest rung (60%), including agriculture, hotels, and healthcare, fall under the Sensitive Cluster. Firms with mid-level of sectoral digital transformation fall in between, with about half of the firms (48%) being in the Resilient Cluster. Additionally, Pearson \u03c72 statistic is used to test the two-way associations with frequencies in the cells (\u03c72 (1) =16.667, p\u2009<\u20090.05). The results provide some directional support to the relative impact of digital transformation. It also validates the relevance of the level of digital transformation  as a grouping variable to be used in the analysis in Phase 2.Consistent with expectation, more than two-thirds of the firms (68%) within the most digitally transformed sectors, such as telecommunications equipment, personal services, financial conglomerates, and advertising/marketing services, fall under the Google trends index represents the search volume of keywords by Google users, and it serves to indicate market sentiment and the public\u2019s attention to an event or incident , a negatively correlated movement of the two variables for the three groups of firms is observed. Stock prices move downwards when the search for coronavirus increased. We do not test correlation, given that it does not necessarily reflect causality, and Granger causality is used to test the leading relationship between these two variables. However, the typical Granger causality test cannot be relied on when one or both time series are non-stationary, which could lead to spurious causality ,,3), Yt r for Eq.  is H0: bX, Y, and Z removes the unit root. Thus, the maximum order of integration is set to be 1, denoted by I (1). As a next step, the VAR model is set up using the levels of the data without differencing and determining the appropriate lag length for the variables X and Y. Based on the information criteria of Akaike Information Criterion Hannan Quinn, Schwarz Criterion, and Final Prediction Error, it is decided that six lags are used in the analyses that followed. As noted by Toda and Yamamoto ,,3), giveX in Eq.  and the Table As the focus is on the impact that Google searches impose on stock price changes and also to differentiate the impact across the different groups, a detailed investigation into the results of Eq.  is then The results of Eq.  examine This study aims to investigate the sectors that have performed better even as market sentiment is affected by the COVID-19 pandemic. Market sentiment on the COVID-19 is modeled with Google search trends. The sectors are organized according to the MGI Industry Digitalization Framework. Performance is quantified by the stock prices of firms in the sectors. The analysis is carried out over two phases.In the first phase, a three-group model on how stock prices adjust to market sentiment towards the sudden emergence of the COVID-19 pandemic is established. The stock prices of a majority of firms across sectors with a higher level of digital transformation are found to have remained resilient to the impact of market sentiment, while sectors that lag across most digital transformation dimensions are among the most negatively affected.In the second phase , market sentiment on the COVID-19 pandemic, as reflected in Google search trends, is affirmed to be a predictor of stock performance. There is also further evidence to show that the digital transformation of firms mitigates the negative impact of market sentiment induced by large-scale unanticipated incidents. As the stock price of firms dips amid the coronavirus pandemic, firms with mid to high level of digital transformation have out-performed others. Analysis in the second phase also suggests that a rise in stock price can affect market sentiment.In answering \u201cwhat are the sectors that have performed better even as market sentiment is affected by the pandemic,\u201d the analysis reveals that sectors with a higher degree of digital transformation have performed better. Thus, during the COVID-19 pandemic, the market has reacted more favorably towards firms and sectors with a higher level of digital transformation and more negatively towards the laggards. An explanation for this is thus offered.The spread of the COVID-19 virus has led governments around the world to shut down their cities to slow down the rate and magnitude at which the pandemic is developing. Even as such shutdowns have brought many corporeal economic activities to a near-complete standstill, consumer purchases and even trade not only continue online, but they have increased considerably. Demand for Internet bandwidth as well as portable computing devices such as laptops and tablets also intensify as many people switch to working and learning online. Many are also predicting the emergence of a post-COVID-19 period, where digital transformation will be prominent. Given these, the markets may have given eminence and greater confidence to firms and sectors with a higher level of digital transformation as they are positioned better to sustain operations not only amid the pandemic, but also to recover faster in the post-COVID-19 period. Thus, sectors with a higher level of digital transformation have been more resilient in their performance and also performed better relative to sectors with lower levels of digital transformation.Further, this study contributes to understanding the role of digital transformation in the firms\u2019 stock market performance, particularly due to large-scale unanticipated incidents such as a pandemic. While previous research has investigated whether the use of technology can improve organizational performance, this research is among the first to demonstrate how digital transformation may give rise to stock market performance. This suggests that digital transformation is an important factor for investors.The findings from this study also suggest that investors consider recent stock prices when conducting further online search about COVID-19, and such search behaviors will drive changes in stock prices. However, the direction, magnitude, and timing of such effects depend on the level of digital transformation.Regarding the research method, this study demonstrates the feasibility of using a search trend index to model market sentiment, especially when investigating how large-scale anticipated incidents can affect the stock prices for a large number of firms or across sectors. This presents researchers with another approach to assess market sentiment and expands the research arsenal to include search indexes such as Google trends, which are readily available for use.This paper has limitations that offer opportunities for future research. First, our study examines how sectoral digital transformation mitigates the effect of market sentiment on negative events  on the stock market, focusing on the period from the outbreak of the COVID-19 to the eventual stock market crash. Since early April 2020, much of the stock markets\u2019 main indices have regained much of their lost territory for the year. The aggressive stimulus packages roll out by central banks and governments to boost growth, such as the U.S. coronavirus relief funds announced around early April 2020, have fueled recovery. Such confounding effects of government support and intervention on the stock market are difficult to account for in the current research model. Future research can look into how the intervention of governments and support of central banks restore investors\u2019 confidence and accelerate the recovery phase of the market cycle.The reopening of businesses and economies is also underpinning market optimism. It is plausible that sectoral digital transformation can also influence how firms perform during the recovery period. Therefore, a possible extension of our research is to investigate how digital transformation moderates the speed and extent of the stock market rebound, as it is of interest to both firms and investors. Firms in the most digitally advanced sectors are likely to be better positioned for the recovery and the post-pandemic period.Second, the literature on the state of digital transformation in sectors suggests a large and growing gap between companies within the sectors (Manyika et al. Lastly, this paper does not investigate the causes behind the growing gap between sectors in digital transformation. Future research may try to uncover how the different conditions lead to a widening gap between the \u201chaves\u201d and the \u201chave-mores\u201d: companies and sectors that are using their digital capabilities far more than others to innovate and transform how they operate."}
+{"text": "Chirality being a phenomenon of describing the ability of any object to exist as a pair of non-superimposable mirror images has aroused extensive attention as it is directly correlated with numerous biological systems . Specific to all living organisms, the mentioned microscopic self-assembled nanostructures exhibiting the supramolecular chirogenesis are spontaneously arranged from chiral building blocks through non-covalent interactions or from achiral components being influenced by an external chiral field  spectroscopy. Furthermore, Gaeta et al. disclosed that chiral porphyrin hetero-aggregates can act as templates to generate the chiral structures during the oxidative conversion of amino acid.Porphyrins refer to be prominent host molecules, which can be commonly used to study chiral supramolecular systems for their unique planar structures and abundant chemical property. In this respect, Fujiki et al. demonstrated that the signal of CPL (circularly polarized luminescence) and CD appeared through the intermolecular chirality transfer upon lanthanide (EuIII and TbIII) tris(\u03b2-diketonate) exposed to poly- and monosaccharide alkyl esters and \u03b1-pinene. Also, Nakakoji et al. investigated the ability of chiral discrimination between copper(II)-chiral tetradentate ligand and a chiral amino acid with the isotopically labeled/unlabeled enantiomer method. As revealed from this study, the matching steric interaction accounts for the observed chiral recognition. Trapp described that a transient stereodynamic catalyst is capable of efficiently amplifying the Soai's asymmetric autocatalysis. The coordination bond provides a good platform to study the property of materials  ion achieved the \u03c0-electronic communications and exciton coupling in the absorption spectrum.Besides the chirogenic porphyrin chemistry, Okada et al. summarized the synthesis and property of the optically-active phthalocyanines, as well as their related azamacrocycles from 2010 to 2020, which are of high significance for chemists to design highly desirable chiral macrocycles from achiral molecules. As summarized by Qiu et al., the C3 symmetrical cages were employed as efficient scaffolds to study chiral dynamics. With the use of non-linear chiroptical techniques, the chirality at surfaces can be clarified from novel perspectives. Gogoi et al. reviewed that chemists adopted the linear and non-linear optical methods to quantify the surface chirality, as an attempt to solve vital issues in surface biochemistry.Numerous fields generated the interest in macrocycles exhibiting the optically-active characteristics (Aav and Mishra, Kaasik and Kanger demonstrated the use of halogen bonding in catalytic stereoselective processes. Also, Kananovich et al. highlighted recent achievements in the catalytic enantioselective oxidations utilizing molecular oxygen, which placed the stress on the mechanisms of dioxygen activation and chirogenesis in the mentioned chemical transformations.Catalytic asymmetric chemistry is another field of supramolecular chirogenesis. In this respect, halogen bonding, with its high directionality, has been recognized as an attractive interaction for new molecular assemblies and its use in chiral systems just started to emerge. In this respect, Siligardi et al. outlooked that researchers used the CD imaging at high spatial resolution at Diamond B23 beamline to determine the homogeneity of the supramolecular structures of thin films deposited on fused quartz substrates.In related development In summary, this Research Topic collected various aspects of the supramolecular chirogenesis in chemical and related sciences. To be specific, the topic illustrates the emergence and characterization of chirogenesis and its potential applications. It is expected that this topic will guide the design and functionality of various chiral structures. The field of supramolecular chirogenesis and relevant sciences are significantly promising in understanding the origins of life in nature, as well as in developing novel medicines and smart materials.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The analysis of sentence lengths in the inaugural speeches of US presidents and the annual speeches of UK party leaders is carried out. Transcripts of the speeches are used, rather than the oral production. It is discovered that the average sentence length in these speeches decreases linearly with time, with the slope of 0.13 \u00b1 0.03 words/year. It is shown that among the analyzed distributions  the Weibull is the best distribution for describing sentence length. These two results can be considered a consequence of the principle of least effort. The connection of this principle with the well-known principles of maximum and minimum entropy production is discussed. The study of natural languages is extremely important not only for the human and social sciences, but also for the sciences that study the development patterns of complex systems . An important section of language science is quantitative linguistics, which uses mathematical methods to establish language laws . At pThe most important element of language is the sentence\u2014the object of this study. According to the Cambridge dictionary, a sentence is a group of words, usually containing a verb, that expresses a thought in the form of a statement, question, instruction, or exclamation. Sentences have semantic completeness; they express a particular thought of a person and serve to communicate it with other people. Based on the above sentence qualities, the study of these structural units is essential for cognitive science, which is of great interest. A metaphor from atomic physics would be very appropriate to illustrate this, especially for representatives of the natural sciences. Many properties of an atom are estimated by radiation (spontaneous and stimulated) that an atom emits and/or absorbs. A person (human brain) also \u201cemits\u201d and perceives elementary flows of thought in the form of sentences and the characteristics of this \u201chuman radiation\u201d can reveal a lot about both the person and their environment.An important quantitative characteristic of a sentence is its length, which can be measured in various ways . The study of sentence lengths does not require special linguistic training and can be easily processed by computer. As a result, this value has been studied for a long time and is used to determine the authorship of a work, the genre of the text, the cognitive development of the author or reader (listener), the level of language proficiency, etc. ,12,13,14The first regularity is a decrease in the average sentence length over time. The decrease may vary depending on the genre and language of the text ,16,17,18At the same time, the so-called principle of least effort  has exisL), among the variety of those that are similar in content {1, L2, \u2026, LnL}, i.e., L = min{1, L2, \u2026, LnL}. It is well known from mathematical statistics [L = min{1, L2, \u2026, LnL}, n\u2192\u221e and, 1, L2, \u2026, LnL being identically distributed random variables equal to zero or larger, L will obey the Weibull distribution function [Starting with G. Zipf, the discussed principle of least effort is used to explain the different frequencies of words of various lengths, the origins of scaling in human language, etc. . Howatistics ,31 that function ,31). ThuThe purpose of this work is to check the applicability of the Weibull distribution to the distribution of sentence lengths and to discover the law of the sentences length decrease over time. The results can provide additional justification for the applicability of the principle of least effort to elementary units of human speech that carry a particular thought.The object of this research was to study the public speeches of politicians. Previously, this has beencarried out several times . We analyzed text transcripts of the 59 inaugural speeches of US presidents from 1789 to 2021 and 224 texts of speeches of UK Party leaders from 1895 to 2018 . Note that the selected unit of measure for sentence length is not exclusive. Words were selected as a unit of measure for sentence length primarily because of the simplicity and the great prevalence of this approach. It is necessary to note that according to , sentencDespite the fact that the studied speeches belonged to a long period of time, the total number of words in the speeches did not change reliably . The aveStatistical analysis was performed using the well-known and widespread professional commercial product Statistica 12.0 (TIBCO Software). The data  are in open access . The parameters characterizing sentence length were calculated. They are listed below.The average sentence length. To calculate this parameter, the total number of words in a speech was divided by the number of sentences. The change in this parameter over time is shown in 2.The median is known to be a stable characteristic of the distribution, it is almost unaffected by outliers. According to 3.The decrease in sentence length over time is also demonstrated in The final results of this section are summarized in To analyze the sentence length distribution law, a number of speeches of the US presidents were excluded from the initial data. First, small speech texts, containing less than 40 sentences were excluded . Second, since it is the texts of public oral speeches that are analyzed, the texts of 1953, 1961, 1973, and 1981 were excluded because these speeches were not spoken, but were only written. Third, speech texts of 1801, 1805, 1837, 1877, 1881, 1893, 1941, 1965, and 1969 were not processed, since these speech texts have a multimodal distribution . Thus, the analysis of the distribution law was carried out at 31 inaugural speeches of US presidents. All 224 speeches of the UK party leaders were analyzed. However, 31 texts were excluded due to the low significance level (<0.05) of the results obtained in relation to all tested distribution laws. Single outliers were excluded from the datasets before data analysis.p-level value, the better this distribution describes the empirical data and, accordingly, the higher its place in comparison with others. Six distributions with no more than two parameters, such as log-normal, Weibull, folded normal, half normal , generalized Pareto, Rayleigh were analyzed in order to find the best theoretical distribution that describes the studied empirical distributions. The ranking of these distributions by the quality of data description was carried out according to the Kolmogorov\u2013Smirnov criterion: the larger the k), where \u03bb and k are the scale and shape parameters respectively. Examples of the experimental data description using the Weibull distribution are presented in Thus, according to the performed statistical analysis, the Weibull distribution is the most preferable for describing the studied speeches. The Weibull distribution (cumulative distribution function) has the form 1 \u2212 exp(\u2212 (x/\u03bb)The behavior of the parameters of the Weibull distribution over time is shown in Thus, the time behavior of the parameters of the Weibull distribution allows us to conclude that, over the past two hundred years, sentence length distribution has become less fuzzy, the width of the peak decreases, and its abscissa is slightly shifted to the left. This is demonstrated in Based on the calculation of sentence lengths in the text transcripts of the inaugural speeches of the US presidents for 228 years and the annual speeches of the UK party leaders for 123 years, two main results were obtained:1. The average sentence length for both US and UK speeches decreases linearly with time with the slope of 0.13 \u00b1 0.03 words/year and, on average, from 1900 to 2000, sentence length decreased with time from 30 to 16 words.2. Sentence length distribution for both US and UK speeches is better described by the Weibull distribution . The scale parameter of this distribution reliably decreases over time from 35 to 15. The shape parameter for US speeches does not change over time and is equal to 1.9 \u00b1 0.1, and the shape parameter for U\u041a speeches slightly changes over time from 1.5 to 1.8.These two results are in agreement with the principle of least effort: the speaker, attempting to minimize both their efforts and the listeners\u2019 effort, tends to choose the shortest possible sentence length from a potential set of sentences of approximately the same content. As a result, on the one hand, sentence length distribution begins to correspond to the distribution of minimum values\u2014the Weibull distribution, and on the other hand, at time intervals significantly longer than the speech preparation time, the average sentence length decreases. The detected change over time in the scale parameter of the Weibull distribution and in information entropy indicates that sentence length in public speeches is gradually becoming less diverse; it is being unified and standardized. Here we highlight the following idea. When establishing the distribution type for empirical data, most important are not statistical tests, but rather the theoretical justification. If we accept the principle of least effort, then the Weibull distribution clearly follows from it. If one assumes that the principle of least effort is not suitable here, then obviously, they must propose some other theoretical justification\u2014their principle\u2014and theoretically derive, for example, gamma or lognormal distributions from it. Currently, we do not see such attempts. The G. Zipf\u2019s principle, in our opinion, is very profound and productive, and many interesting consequences can be obtained from it. It has great potential, which has not yet been fully embraced by modern linguists. Our work and a number of works  \u201cemitted\u201d by the brain, corresponding to the Weibull distribution, is also able to stimulate the development of brain sciences. One of the possible directions related to brain biophysics may be the study of the energetic basis of the origin and development of thought and language. There are a number of works in this direction, in particular ,51,52,53"}
+{"text": "Decision analytic models are often used in economic evaluations to estimate long-term costs and effects of treatment which span beyond the time-frame of a clinical trial, therefore providing a better understanding of the long-term implications of decisions that conventional trial-based economic evaluations fail to provide. This is particularly relevant for considering oral health interventions in children as treatments may affect adult oral health. However, in the field of child oral health there has not been an evaluation of the quality and scope of decision analytical models which extend into adulthood. The aim of this review is to examine the scope and quality of decision modelling studies, with horizons extending into adulthood, within the field of child oral health.The following databases were searched: NHS Economic Evaluation Database (CRD York), MEDLINE, EMBASE, CINAHL, Web of Science, Scopus, the Cochrane Library and Econlit. Full economic evaluations, in the field of child oral health, published after 1997 which included a decision model with a horizon that extended beyond the age of 18\u00a0years old were included. Included studies were appraised against the Drummond checklist and the Consolidated Health Economic Evaluation Reporting Standards by calibrated reviewers.Four hundred studies were identified, of which nine met the inclusion criteria. Of the nine, eight were cost-effectiveness models. The majority focussed on the prevention or management of dental caries. The mean percentage of applicable Drummond checklist criteria met by the studies in this review was 82% . Discounting of costs and performing an incremental analysis were noted as key methodological weaknesses. The mean percentage of applicable CHEERS criteria met by each study was 82% . Justifying the type of model, analytical methods used, and sources of funding were most commonly unreported.There is a paucity of decision analytical models in the field of child oral health. Most of those that are available are of high methodological and reporting quality.The online version contains supplementary material available at 10.1186/s12903-021-01680-3. Dental disease is prevalent and carries a high burden amongst children worldwide. Untreated dental caries  and molaFor any given dental disease, a range of interventions can be offered to manage the problem and reduce the impact the disease has on the child. Often different treatment options have different costs and effects associated with them and choosing which to provide can be determined by efficiency (i.e. which intervention offers the most benefit for the least cost or the most favourable cost:benefit ratio). This can be assessed using an economic evaluation, the results of which can support decision makers to maximising benefits from limited healthcare resources , 17. EcoPrevious systematic reviews of economic evaluations in dentistry have either focused on specific areas of dental disease \u201323 or ecThe study protocol was registered with the Prospective Register of Systematic Reviews (PROSPERO\u2014CRD42020166717), and it followed the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement for reporting.A search strategy was developed  in the following electronic databases: NHS Economic Evaluation Database (CRD York), MEDLINE, EMBASE, CINAHL, Web of Science, Scopus, the Cochrane Library and Econlit. Searches covered the period from commencement of each database system until the 1st October 2020.The reference lists of included articles were examined to assess if additional relevant studies, that were not found during the database searches, should be included. Efforts were made to identify relevant unpublished \u2018grey\u2019 literature and conference proceedings through appropriate websites and databases such as OpenGrey. The EThOS database was also searched to identify relevant published UK theses.Full economic evaluation in the field of child oral healthInvolved a decision model with a horizon that extended beyond the age of 18\u00a0years oldPublished after 1997No language restrictions were placed. It should be noted that cost-minimisation studies may not be universally considered as full economic evaluations, however, it was agreed by the research team to include them in this review. Similarly, it was agreed by the authors that only studies published after 1997 should be included in this review. This was due to the Drummond checklist [For studies to be included in this review, they had to meet the following inclusion criteria:hecklist , which wStudies which used the lifetime of the tooth as a horizon were excluded. The reason for excluding these studies is that choosing such a horizon would not reflect the key differences in costs and consequences between interventions, e.g. if an evaluation compares filling and extraction of teeth, and the lifetime of the tooth was chosen as the horizon, once the tooth has been extracted the evaluation would cease yet there will still be benefits from the filling option that extend beyond this. Therefore, in these evaluations, comparing benefits obtained during childhood and adulthood from interventions carried in children cannot be fully assessed.Search results were organised using Endnote\u2122 X9. Duplicate articles were removed. Title and abstract screening against the eligibility criteria, was carried out independently by two reviewers (GDT & KC), with any disagreement resolved by consensus. If necessary, any unresolved differences were resolved by a third reviewer (CRV).Full texts were obtained for all titles that met these criteria. Two reviewers (GDT & KC) assessed the full texts against the inclusion/exclusion criteria independently, with any disagreement resolved by consensus. If necessary, any unresolved differences were resolved by a third reviewer (CRV).Two reviewers (GDT & HJR) extracted data and assessed both the methodological quality, independently. A calibration exercise (using 3 papers) was conducted with all reviewers prior to commencement of data extraction and methodological/reporting quality appraisal. Any disagreement was resolved by consensus, and where needed, any unresolved differences were resolved by a third reviewer (KC).Publication details publication year; author; countryStudy Characteristics oral health condition studied; nature of interventionEconomic evaluation characteristics type of economic evaluation; perspective of the model; costing data sources; data source (primary/secondary data); horizon; model design; model input and parameter details; model outcomeFor each selected article, the following data, split into three sections, were extracted:An assessment of methodological quality and reporting quality was undertaken. The most widely used checklist, the Drummond 10 item, 13 criteria checklist   24-item checklist was used . PublishDrummond Criteria: High\u2009>\u200950%; Moderate 32\u201350%; Low\u2009<\u200932% CHEERS criteria: High\u2009>\u200983%; Moderate 63\u201383%; Low\u2009<\u200963% It was agreed by the research team to use the same cut-offs for this review to allow a meaningful and direct comparison between the two studies.Rogers et al.  reportedSimple descriptive statistics, including inter- and intra-rater reliability scores, were conducted on the extracted data and quality appraisal results using IBM\u00ae SPSS\u00ae Statistics v25. Meta-analyses were inappropriate given the diversity of interventions covered in addition to the aim of the review being to identify good practice and appraise quality.Cohen\u2019s kappa (\u03ba) was calculated at 0.75 (93.2% agreement) for overall inter-rater agreement for the Drummond checklist, and at 0.77 (91% agreement) for the CHEERS checklist. These figures suggest substantial strength of agreement . To deteThe search resulted in 440 articles and after the removal of duplicates, 400 articles were identified for title and abstract screening. Twenty-three papers were included for review of the full text. Fourteen were excluded because they did not meet the inclusion criteria. Nine articles were included and underwent data extraction. Reference lists of included articles did not produce any further articles for inclusion. A summary of article selection is presented as a flowchart, based on PRISMA guidelines Fig.\u00a0. The pubAll nine studies included in the final analysis were written in the English language. All but one study undertook cost-effectiveness analyses . The remaining study reported a cost-cost analysis . The vast majority of studies were carried out in Germany , with six being carried out by the same first author (66%).n\u2009=\u20097, 78%), with one study (11%) relating to MIH and one relating to the caries increment as a result of a 20% tax on sugar-sweetened beverages (11%). Just over half of the studies undertook an individual patient-level microsimulation Markov model , one was a cohort simulation Markov model (11%), one system dynamics model (11%) and two did not clearly state their model design (22%). Justification for the choice of model was not always provided. Two thirds of the studies used a lifetime horizon . One study used nine years (11%), one ten years (11%) and whilst the remaining paper used 63.5\u00a0years, detailed as the remaining life expectancy of a 15-year old male simulated in their model (11%).Most studies focused on the prevention or management of dental caries  and for the CHEERS checklist was 82% . Applicability scores and an assessment overall quality, based on pre-determined cut-offs , of for An overall breakdown of whether studies met each criterion of the Drummond and CHEERS checklists are shown in Tables To the author\u2019s knowledge, this is the first systematic review that has assessed the methodological and reporting quality of DAM studies, with horizons extending beyond childhood, within the field of child oral health. Limited examples of using DAMs exist in the field of child oral health; however, those that do exist are of relatively high methodological and reporting quality.Using the high-, moderate- and low-level cut-offs of quality, as defined by Rogers et al. , the oveDiscounting of costs and performing an incremental analysis of the costs and outcomes were the most omitted criterion from studies included in this review. Future costs need to be discounted to reflect the amount spent or saved in the future should not weigh as heavily in decisions as those spent or saved today , 32. ThiAnother criterion that was frequently absent was an incremental analysis of the outcomes and costs of alternatives. Omitting this analysis fails to provide the stakeholders, for whom the results from any modelling study will be essential to guide clinical and policy decisions, as to whether the additional costs generated by one alternative over another is compared to the additional benefits generated. It could be argued that an incremental analysis is a fundamental output of any given economic evaluation .The overall reporting quality of economic evaluations included in this review was relatively high, with seven out of nine studies being in the high category. A median score of 87% against the CHEERS checklist was noted, which was similar to the median score of 83% reported by Rogers et al. . InteresThe worst performing criteria, in relation to the CHEERS checklist, were reporting the choice and justification of the chosen model, analytical methods used and sources of funding. Describing, but more importantly, justifying the choice of model, for example Markov-model or decision tree, is an essential piece of information needed for any modelling study . The typThe majority of the modelling studies in this review focussed on the prevention and/or management of dental caries. Similarly, Rogers et al.  noted thOnly one study in this review was not related to dental caries. Other dental conditions observed in children would benefit from modelling studies, for example management options for non-vital teeth with immature apices, management of anterior opacities due to MIH or the management of traumatic dental injuries. Although these conditions may not be as prevalent, these are just as impactful, and therefore this evidence found in this review supports the argument set out in Rogers et al.  that othAll but one of the modelling studies included in this review conducted a cost-effectiveness analysis. Most of the studies either used tooth retention years or caries increment as their outcome measure, which are appropriate when being applied to modelling studies, as they are both able to capture long-term outcomes associated with the interventions. This predominance of cost-effectiveness analyses is consistent with previous systematic reviews addressing oral health interventions , 26. HowThis is the first systematic review that has addressed the scope and quality of economic evaluations that involve decision analytical models extending into adulthood in the field of child oral health. A strength of this review was the involvement of both paediatric dental and health economics expertise at each stage of the review. Furthermore, a calibration exercise was undertaken for data extraction and quality assessment, against both checklists, which was beneficial as shown by the intra- and inter-rater agreements scores. In addition, this review complemented the recent review by Rogers et al. , by overOne acknowledged limitation was adopting the same cut-offs that were defined by Rogers et al.  These cuThere is a paucity of decision analytical models in the field of child oral health which span beyond the age of 18, however the few that are available are of relatively high methodological and reporting quality. Decision analytical models can help, and should be used to, understand the implications of dental decisions made in children whereby the outcomes extend beyond 18\u00a0years old, and further work using this methodology in child oral health would be valuable.Additional file 1. Search Strategy."}
+{"text": "When testing on n = 692 healthy people, we found a median (across participant) mean absolute error (within participant) of 9.5 years. Performance was more accurate (MAE around 7 years) in the prefrontal cortex and periventricular areas. We also introduce a new voxelwise method to reduce the age-bias when predicting local brain-age \u201cgaps.\u201d To validate local brain-age predictions, we tested the model in people with mild cognitive impairment or dementia using data from OASIS3 (n = 267). Different local brain-age patterns were evident between healthy controls and people with mild cognitive impairment or dementia, particularly in subcortical regions such as the accumbens, putamen, pallidum, hippocampus, and amygdala. Comparing groups based on mean local brain-age over regions-of-interest resulted in large effects sizes, with Cohen's d values >1.5, for example when comparing people with stable and progressive mild cognitive impairment. Our local brain-age framework has the potential to provide spatial information leading to a more mechanistic understanding of individual differences in patterns of brain ageing in health and disease.We propose a new framework for estimating neuroimaging-derived \u201cbrain-age\u201d at a local level within the brain, using deep learning. The local approach, contrary to existing global methods, provides spatial information on anatomical patterns of brain ageing. We trained a U-Net model using brain MRI scans from  Brain ageing is associated with cognitive decline and an increased risk of neurodegenerative disease, though these effects vary greatly between individuals. Brain atrophy, often measured using structural MRI, is commonly seen in many neurological diseases . This approach of using a separate linear regression model for each voxel is limited as it does not incorporate contextual information from neighbouring voxels, and is insensitive to non-linear relationships. Other studies have provided local or regional information by training separate models per region e.g., Kaufmann et al., Limited prior work on local predictions of brain-age are available. Of note, is the early work of Cherubini et al. , who use3 3D block, reporting MAE values between 2.16 and 4.19 depending on block origin. While these approaches are promising, the relatively large size of the patch limits spatial resolution which results in less insightful inference in clinical settings. For example, semantic dementia is associated with a relatively localised spatial pattern of atrophy, often the left anterior and middle temporal lobe  from participants aged 18\u201388 years, with a mean age of 54.29 \u00b1 18.59 and a male/female ratio of 322/330. More information can be found at https://www.cam-can.org/.This dataset is part of larger project which is trying to use epidemiological, behavioural and neuroimaging data to understand how individuals can best retain cognitive abilities into old age. All participants were scanned with 3 T Siemens TIM Trio scanner with a 32-channel head coil. The dataset consists of n = 494; age range = 19\u201380 years; mean age 45.18\u00b117.44; male/female ratio of 187/307) undergoing a multi-modal  neuroimaging. Only T1-weighted 3D MPRAGE  were used here. The goals of the SALD are to give researchers the opportunity to map the structural and functional changes the human brain undergoes throughout adulthood and to replicate previous findings. More information can be found at http://fcon_1000.projects.nitrc.org/indi/retro/sald.html.This comprises a large cross-sectional sample  with a 3D T1-weighted MPRAGE sequence . More information can be found at http://fcon_1000.projects.nitrc.org/indi/retro/wayne_11.html.The Wayne State longitudinal data set for the Brain Aging in Detroit Longitudinal Study, comprises 200 healthy individuals, with Here we used data from the Imperial College London project, STudy Of Reliability of MRI (STORM). The study comprises of 20 participants with a male-female ratio of 12/8, with a mean age at the first scan undertaken of 34.05 \u00b1 8.71. The participants were scanned for the second time at an average distance of 28.35 \u00b1 1.09 days. All participants were free from any neurological or psychiatric disorders. MPRAGE were acquired using a Siemens Verio 3T scanner .This study included 11 participants scanned in two different centres, mean age at first scan of 30.88 \u00b1 6.16 and with a male/female ration of 7/4. The two scanning sites were at Imperial College London, where a Siemens Verio 3T scanner was used to acquire MPRAGE , whereas a Philips Ingenia 3T scanner was used at the Academic Medical Center Amsterdam to acquire sagittal Turbo Field Echo . The mean interval between scans was 68.17 \u00b1 92.23 days.n = 609 cognitively normal adults and n=489 individuals at with MCI or dementia ranging in age from 42 to 95 years. Using Clinical Dementia Rating scale (CDR) scores, we classified participants as healthy control (HC), stable MCI, progressive MCI or AD, as detailed in https://www.oasis-brains.org/.This is a retrospective compilation of data for >1,000 participants that were collected across several ongoing projects through the WUSTL Knight ADRC over the course of 30 years. Participants include https://aibl.csiro.au/). The dataset contained n = 198 participants with Clinical Dementia Rating scale scores, detailed in This study is a study to discover which biomarkers, cognitive characteristics, and health and lifestyle factors determine subsequent development of symptomatic Alzheimer's Disease (AD) (https://www.fil.ion.ucl.ac.uk/spm/software/spm12/). This entailed tissue segmentation into grey matter (GM) and white matter (WM), followed by a non-linear registration procedure using the DARTEL algorithm  software package  is used to test the reproducibility of a certain quantitative measurement made by a specified number of observers which rate the same participant. The original formula is given as follows:where Here, we used ICC as defined by Shrout and Fleiss . The intIn this subsection, we summarise the design of our experiments and which datasets are used in each step.BAHC, CamCan, Dallas and SALD were used as training and validation sets (standard 80/20 split) for training the local brain-age U-net model.All Wayne State participants and healthy participants from OASIS3 and AIBL were used at testing time. We calculated mean absolute error (MAE) values globally (by averaging across voxel-level brain-predicted age for each participant) and at voxel level, alongside the Pearson's correlation coefficient between chronological age brain-predicted age at both global and voxel level.Within-scanner reliability and Scanner calibration datasets were used as test sets to compute voxel-level ICC values to assess the reliability of local brain-age when the same participant is scanned in two different scanners, respectively one the same scanner with short time interval.3) for each ROI, alongside ROI-level brain-PAD values which were computed by first averaging voxel-level \u201cbrain-predicted age\u201d inside an ROI, then subtracting the participant's chronological age. We calculated a Pearson's correlation coefficient for each ROI.Using subcortical and cortical ROIs from the Harvard-Oxford structural brain atlas, we obtained brain tissues volumes , again computing Cohen's d effect sizes. For all experiments in this part we have selected subjects above 60 years old from the healthy controls so as to have a similar chronological age distribution in relation to the groups with varying degrees of cognitive impairment.OASIS3 was used at testing time to assess the sensitivity of local brain-age to differences in brain structure between groups. For each participant we computed an mean across voxels global brain-PAD (adjusted for age bias using method described in section 2.6), which we use then to perform Welch's A visual overview of the study design is portrayed in 3 voxels. The convolutional layers in our network used an isotropic 3x3x3 filter, convolved over the input image after which element-wise multiplication with the filter weights and subsequent summation was performed at each location. Subsequently, to allow for non-linear modelling, we passed the obtained values through an \u201cactivation function\u201d; we used leaky rectified linear units with alpha = 0.2, more precisely max+min, thus allowing a small, non-zero gradient when the unit is not active.We used a fully convolutional neural network (CNN) inspired by the U-Net architecture introduced in Ronneberger et al. . Our netThe convolution operation is also controlled by its stride, which is how many pixels/voxels are skipped after every element-wise weight multiplication and summation. We set the stride equal to 1.Downsampling increases the effective field of view or \u201creceptive field\u201d of layers higher in the hierarchy. For the downsampling part of the U-Net we used at each scale two consecutive 3D 3 \u00d7 3 \u00d7 3 filter kernels with an initial number of channels = 64, which get multiplied by 2 as we progress down the downsampling path. For downsampling we used 2 \u00d7 2 \u00d7 2 average pooling.3 voxels blocks.For the upsampling part of the U-Net we inverted the downsampling architecture, with the downsampling layers being replaced by 2 \u00d7 2 \u00d7 2 upsampling layers. At each convolution we used a squeeze-and-excite unit. Squeeze & Excite networks were introduced in Hu et al.  and can Besides the voxel-level mean absolute error cost function on the output layer we introduced two additional cost functions at the two other scales of the architecture. We applied global average pooling followed by a dense layer to predict brain-age at block-level. The loss function can be expressed as follows:1 and \u03b12 are progressively decreased so that the gradients will exclusively flow from the voxel-level predictions after 50,000 training iterations. We used Adam  or brain-age \u201cdelta.\u201d A so-called \u201cregression dilution\u201d has been commonly observed in brain-age prediction algorithms, caused by noise in the neuroimaging features leading to a greater under- or over-estimate of age, the further away a sample is from the training set mean age. In other words, this effect results in the systematic under-estimation of brain-predicted age for older participants and over-estimation for younger participants, which increases as model performance decreases.Broadly speaking, two approaches to account for this effect have been reported:Age representing chronological age.where \u0394 is the brain-age delta of a group of participants from an external dataset that is used specifically for adjusting the bias. \u03b1 and \u03b2 are the parameters of a linear regression with the covariate Then, to obtain the bias-adjusted age we have the following equation:Another approach involves using the brain-predicted age in the linear regression, more specifically:where \u1ef9 denotes the brain-predicted age. de Lange and Cole  showed tn = 200) of participants randomly selected from the healthy participant datasets , who were not included in the training or validation set. We obtained testing time predictions for these participants and calculated their voxel-level brain-age delta \u0394i, v, where i indicates the i-th participant and v the v-th voxel. We then binned these participants based on their chronological age . Then, for each bin b we calculated the average voxel-level brain-age delta for that respective bin, which we denote as \u0394b,v. This value will represents the average brain-age delta for that voxel given the chronological age interval. Subsequently, to de-bias the voxel-level brain-age delta for a new participant , \u0394j,v we used the following formula:Here, we used a separate small batch , AIBL (n = 83) and Wayne State (n = 200) datasets. When looking at voxel-level MAE (unadjusted) values across the brain mask (r = 0.47), for Wayne State 8.09\u00b16.08 years , respectively for OASIS3 we get an average of 8.08\u00b16.40 years . We tested the local brain-age model on healthy participants combined from the OASIS3  and Scanner calibration (between-scanner) datasets, ICC was calculated per voxel. Test-retest reliability was very high with the vast majority of voxels having ICC <0.90  . This inWe examined patterns of local and global brain-age in people with MCI and dementia patients using cross-sectional data from OASIS3. Firstly, we investigated if the global-level  brain-predicted age (adjusted) corresponds to previously reported differences from models that directly predict global brain age. We averaged voxel-level brain-age (adjusted) across voxels per individual to generate an adjusted global-level brain age and then calculate global-level brain-PAD. Global-level brain-PAD (adjusted) mean (\u00b1 standard deviation) values were: \u22120.65 \u00b1 7.46 (median = 0.95) years for healthy controls, 3.07 \u00b14.29 (median = 2.83) years for stable MCI (sMCI), 5.77 \u00b1 5.41 (median = 4.94) years for progressive MCI (pMCI) and 4.34 \u00b1 6.78 (median = 4.63) years for AD patients.t-tests, finding significant differences between cognitively impaired groups and healthy controls in all cases . Comparisons between stable and progressive MCI patients and with AD patients were not significant: sMCI-pMCI p = 0.161, t = \u22121.44, df = 26.44, Cohen's d = \u22120.54, AD-sMCI t = 0.83, p = 0.414, df = 20.64, Cohen's d = 0.19, AD-pMCI t = \u22120.90, p = 0.3714, df = 28.51, Cohen's d = \u22120.21.We then assessed the significance of group differences using global-level brain-PAD values by performing independent two-sample Welch's t-test. Likewise, differences between participants MCI or dementia and healthy controls were significant . In contrast to the global-level results .Next, we examined local brain-PAD, summarising across all voxels within group. The mean voxel-level brain-PAD (adjusted) values were: healthy controls = \u22120.39\u00b10.85 (median = \u22120.44) years, sMCI = 3.07\u00b11.67 (median = 3.266) years, pMCI = 5.45\u00b11.74 (median = 5.663) years for pMCI, AD patients = 4.01\u00b11.71 (median = 4.229) years . We thend scores both for separating AD patients from healthy controls and stable from progressive MCI  is below what has been reported with purely global  including the hippocampus, and the amygdala, entorhinal, and parahippocampal cortices  and amygdala (d = 0.45), when comparing stable and progressive MCI patients. Here, our local brain-age framework resulted in d = 2.18 for the hippocampus and d = 1.5 for the bilateral amygdala in the same context. This suggests that use of the brain-age paradigm to capture local age-related changes, relative to a healthy ageing model, could increase statistical power in experimental research and clinical trials, relative to conventional volumetric imaging biomarkers. Potentially, the ROI-based brain-PAD values could even be used in a classification framework to distinguish between people with stable or progressive MCI.As biomarker of brain health, brain-age models may have clinical utility, either prognostically or in the context of clinical trials of neuroprotective treatments. While previous studies have reported standardised effect sizes from global brain-age, we used atlas ROIs to summarise regional values of local brain-PAD and generated Cohen's n et al.  reportedr et al.  generateOut proposed U-Net local brain-age framework has some strengths and weaknesses. Our model was assessed on a large multi-site testing set with a flat distribution of chronological age across the adult lifespan  was calculated starting from the first convolutional layer where the FOV is 33 voxels, which gets increased by 23 voxels per convolutional operation in the downstream part of the U-Net. The average pooling layers increase the FOV by 13 voxels, since their stride is set to 1, while the upsampling layers do not increase the FOV as they merely repeat existing information. Lastly, the first convolutional layer in the upstream layers only adds 13 voxels  whereas the second adds 33 voxels . While this means that our resolution is not necessarily at the voxel-level, 233 voxels is still substantially higher resolution compared to existing models in literature. In the 3D block approach of Bintsi et al. , in order to encourage the network to emphasise the context encoded in its lower layers. As the individual voxel location we are aiming to obtain a prediction for is not necessarily related to the imposed ground truth output, the U-Net architecture is biased toward using the context information. Hence, in the worst case scenario where no voxel-level relationship is learned, the true resolution of our voxel-level predictions is actually blocks of 23i et al. , blocks One drawback of this study is the categorisation of subjects into sMCI vs. pMCI vs. AD. We attempted to closely follow the ADNI clinical procedure, respectively to classify a subject as AD if they had a mini-mental state examination (MMSE) score between 24 and 26, CDR of 0.5 or 1.0. Subjects with an MMSE score between 24 and 30, CDR of 0.5 alongside memory complains, objective memory loss measured by education adjusted scores and absence of significant levels of impairment in other cognitive domains are classified as MCI. Unfortunately, for OASIS3 we only had access to CDR scores, hence there is the possibility of misclassifications occurring in our data curation.de facto method to quantitatively assess differences between groups at voxel-level , genotype or polygenic risk score, cognitive measures . Such an approach could be used as an alternative to techniques like VBM, to provide mechanistic insights into the relationship between local brain regions and individual deviations from healthy/normal levels of a given outcome measure. While VBM is the One potential direction to take local brain-age further is in disease subtyping. Local brain-PAD maps could be used as input to clustering algorithms with the goal of identifying subgroups of patients that have spatially similar patterns of brain ageing. The putative subgroups may undergoing distinct pathological processes that effect different regions of the brain and may have different trajectories of disease progression or may respond differently to treatments. Such approaches have been applied to volumetric brain maps before  under the European Union's Horizon 2020 research and innovation programme . DS was supported by the NIHR Biomedical Research Centre at Imperial College Healthcare NHS Trust and the UK Dementia Research Institute (DRI) Care Research and Technology Centre. JC acknowledges funding from UKRI/MRC Innovation Fellowship (MR/R024790/2).BG has received grants from European Commission and UK Research and Innovation Engineering and Physical Sciences Research Council, during the conduct of this study and is Scientific Advisor for Kheiron Medical Technologies, Advisor and Scientific Lead of the HeartFlow-Imperial Research Team, and Visiting Researcher at Microsoft Research. JC is a shareholder in and Scientific Advisor to BrainKey and Claritas HealthTech, both medical image analysis software companies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Obesity and diabetes are the most demanding health problems today, and their prevalence, as well as comorbidities, is on the rise all over the world. As time goes on, both are becoming big issues that have a big impact on people\u2019s lives. Diabetes is a metabolic and endocrine illness set apart by hyperglycemia and glucose narrow-mindedness because of insulin opposition. Heftiness is a typical, complex, and developing overall wellbeing worry that has for quite some time been connected to significant medical issues in individuals, all things considered. Because of the wide variety and low adverse effects, herbal products are an important hotspot for drug development. Synthetic compounds are not structurally diverse and lack drug-likeness properties. Thus, it is basic to keep on exploring herbal products as possible wellsprings of novel drugs. We conducted this review of the literature by searching Scopus, Science Direct, Elsevier, PubMed, and Web of Science databases. From 1990 until October 2021, research reports, review articles, and original research articles in English are presented. It provides top to bottom data and an examination of plant-inferred compounds that might be utilized against heftiness or potentially hostile to diabetes treatments. Our expanded comprehension of the systems of activity of phytogenic compounds, as an extra examination, could prompt the advancement of remedial methodologies for metabolic diseases. In clinical trials, a huge number of these food kinds or restorative plants, as well as their bioactive compounds, have been shown to be beneficial in the treatment of obesity. Diabetes mellitus (DM) is a metabolic disorder that may be caused by hereditary or environmental causes, and it increases the risk of various diseases in patients. Currently, available therapies are effective for some individuals, but not all, and there are currently no effective medicines available to combat this illness. As a result, there is a high need for novel antidiabetic medications ,2. On thMany conventional medications are used to treat obesity in today\u2019s world. The availability of these medications, as well as their potentially hazardous side effects, limits their use. As a result, developing safe, effective, and cost-effective entities with easy accessibility is critical. Plant-derived medications are thought to be the first line of defense in keeping people healthy by preventing diseases and their complications ,7. By acDiabetes has been known since old times, and the principal side effects were inordinate thirst, ceaseless peeing, and sleepiness. Heftiness is a critical danger factor for a scope of ongoing diseases, including T2DM, which is related to more noteworthy clinical consideration costs and a more restricted future. Free fatty acids (FFA) have a strong link to obesity, discomfort, and insulin resistance; therefore, lowering prolonged plasma levels is critical. In the therapy of obesity and T2DM, FFA should be a critical healing aim. As indicated by the World Health Organization (WHO), 35% of individuals possessed around 20 pounds of overabundant weight in 2008, with 11% being corpulent. Besides, the pervasiveness of T2DM has expanded from under 10% in 1980 to over 30% in recent years ,17. GlucIn this review, we focused on the relation between diabetes and obesity, which is a burning issue of the current world, and the medications of diabetes and obesity from plant-derived herbal products concentrating on various therapeutic targets.The most basic cause of obesity is either increased hunger or decreased calorie consumption due to controlling cellular functioning, physical activity, and other aspects of a person\u2019s life . The accThe secretion of fatty acids and triglycerides in the circulation results in the formation of fat cells all over the body, followed by atherosclerosis. These metabolic disorders may also arise as a result of this phenomenon. For the management of obesity, we must reduce circulation and stored fat levels. Additionally, as oxidative stress is a frequent element in some pathological disorders, such as obesity and other conditions, reducing oxidative stress can help to mitigate the susceptible repercussions of obesity and other difficulties. In addition to secreting the three substances, adipocytes secrete an adipocytokine, also known as lectin, adiponectin, and visfatin. Autophagy can be induced by adiponectin in the breast, colon, prostate, and female-specific carcinogenesis ,27,28. TDopamine regulates fat and helps the pancreas and the digestive tract to release their respective hormones. Hormones that sustain hunger, satiety, and body fat are under homeostatic control, but hormone abnormalities can lead to obesity. This means that in the development of novel anti-obesity drugs, these elements and their impact are extremely significant ,9,29.Obesity and diabetes, despite their differences in symptoms, share certain common characteristics. Insulin resistance has an impact on a huge number of persons . Insulin2 [2 is viewed as typical, while a weight record of 25 to 30 kg/m2 is viewed as overweight. Hypertension, T2DM, hyperlipidemia, and coronary supply route illness are a couple of the drawn-out impacts of abundant weight. The following four are the most well-known reasons for corpulence: food/drink admittance, activity (workout), inherited elements [Obesity is becoming all the more a worldwide issue, especially in developed nations. The WHO characterizes heftiness as a body mass index (BMI) of an excess of 30 kg/m2 . A weighelements , and clielements . Food anelements . Accordielements . Thus, pObesity can be addressed in a variety of ways. Smothering hunger is one method for reducing food intake . A varieGalega officinalis. Metformin has various incidental effects, including sickness, swelling, and flatulating. Other diabetes medications have been developed, yet none are as successful or convincing as metformin [Hyperglycemia, which is brought about by an absence of insulin discharge as insulin advancement, is an indication of diabetes . Cardiovetformin . Adjustietformin . This prDM is expected to become a pandemic worldwide, accompanied by metabolic and endocrine diseases, according to epidemiological studies. T2DM affects the majority of diabetic individuals, causing insulin resistance and insulin secretion problems. Dietary control, moderate exercise, and hypoglycemic and lipid-lowering medications are the most common treatments for T2DM. Despite the therapeutic benefits of most medicines for the treatment of T2DM, the majority of them might cause unwanted side effects. Herbal products have emerged as important sources of bioactive molecules for anti-T2DM medication development, given the pathophysiology of T2DM . RecentlMany studies have tracked down a significant connection between stoutness and the advancement of diabetes. Individuals who are overweight, especially around the stomach, are more insulin safe ,46,47,48Obesity and diabetes are instances of multifactorial sicknesses that arise because of the interchange of different hereditary and ecological factors. There is evidence that builds up the hereditary connection between fat and diabetes. Forty qualities have been connected to T2DM by a genome-wide association study (GWAS) and up-and-comer quality methodologies and a comparative number of qualities have been connected to heftiness by GWAS and up-and-comer quality methodologies. Most T2DM qualities give off an impression of being connected to a B-cell glitch, with far fewer occupied with insulin obstruction pathways random to fat ,58,59,60Phytogenic compounds have been utilized to treat an extent of illnesses for a significantly long time ,13. For Panax ginseng (Asian ginseng) and Panax quinquefolius (American ginseng). On the glucose and fat processing frameworks, ginsenoside Rb1 has a scope of constructive outcomes. Ginsenoside Rb1 (10 mg/kg) was implanted intraperitoneally into a run-of-the-mill eating routine dealt with mice and high-fat unhealthy-eating supported mice packs for three weeks, and it reduced body weight, fat substance, serum leptin, and serum nitric oxide back to normally observed levels or lower than the normal group. Additionally, the paraventricular focus of the operational hub appeared to diminish orexigenic neuropeptide Y (NPY) expression while boosting anorexigenic CCK expression when the high-fat diet (HFD) pack was given ginsenoside Rb1. P. ginseng contains various ginsenosides, all of which help to diminish pancreatic lipase creation [Ginsenoside is a foe of fat substances isolated from ginseng. Ginsenosides are copious in creation .P. ginseng are essentially higher in the root [The proportions of ginsenosides Re, Rb2, and Rd in the result of the root . Ginsenothe root . In heavthe root .Hoodia gordonii is a medicinal plant that has a significant ability to treat T2DM. P57AS3, an oxypregnane steroidal glycoside, is conveyed by H. gordonii and H. pilifera. P57AS3 was overseen into the third ventricle and diminished 24 h sustenance affirmations by 40\u201360% [H. gordonii coordinates dietary affirmation and centrality balance. Food affirmation and weight were lessened at all estimations when glycosides 1 and 2, segregated from dried H. Gordonii stems, were offered orally to rodents in segments from 6.25 to 50 mg/kg for eight days, appearing differently from the control group [H. gordonii fuse mitochondrial carnitine palmitoyltransferase-1 (CPT-1), thyroid synthetic substances, NPY, and affront-like progression factor-1 (IGF-1) [H.gordonii orally. Thyroid synthetic substances such as triiodothyronine (T3) and thyroxine (T4), similarly to CPT-1, were extended. CPT-1 is an oil destructive oxidation atom associated with the lipid processing framework, and its expanded levels infer better greasy destructive oxidation. As the carbohydrate processing framework advances, T3 and T4 stature mirror an expansion in energy utilization and glucose homeostasis. In this sense, H. gordonii might be useful in diminishing hunger, working on fast processing, and further developing starch absorption [y 40\u201360% . P57AS3,ol group . Various (IGF-1) . Food cosorption .Berries have long been known for their antioxidant effects. Blueberries have been demonstrated to have antioxidant, anti-obesity, and antidiabetic characteristics, as well as improving cardiovascular health ,74. AnthEnergy utilization includes thermogenesis, genuine development, and head centrality use . The twoNelumbo nucifera has been used to cure a range of diseases for millennia. It has a wide range of medicinal qualities, including relief from fever, burning skin problems, and clutter removal [N. nucifera shoots were fed to HFD-fed mice for five weeks, alpha-amylase and lipase activity were lowered but fat digestion and UCP3 mRNA expression was increased in C2C12 myotubes [N. nucifera, which contains eleven flavonoids, including the sesquiterpene eudesmane; thirteen megastigmanes; and eight alkaloids [N. ginseng is a root vegetable, such as P. ginseng. Nucifera has been shown to promote vitality consumption [ removal . The elimyotubes . The uprlkaloids ,84,85. Fsumption .Capsicum annuum, sometimes known as crimson chili peppers, is capsaicin. In Southeast Asia, China, and Latin America, it is commonly used as a flavoring [The active element of lavoring . Capsaiclavoring . In vitrlavoring . By uncolavoring . The mRNlavoring . Capsiatlavoring . Capsiatlavoring . Accordilavoring , capsaicC. sinensis is epigallocatechin gallate (EGCG) [One of the main catechins in e (EGCG) . At the e (EGCG) . EGCG lie (EGCG) , EGCG dee (EGCG) , EGCG deWild blueberries contain anthocyanin, an antioxidant polyphenol. When stout and skinny Zucker rats were fed a wild blueberry-enriched diet for eight weeks, the corpulent Zucker rats demonstrated an increase in dyslipidemia and changes in lipid digesting system features . The stoCinnamomum tree\u2019s bark. Cinnamon extract enacts PPAR, which increments fat assimilation and opposition [Cinnamon is made from the position . PPAR taposition . Water-dposition . Although several studies suggest a link between carbohydrate digestion and weight gain, the glucose digestive system is often disregarded when it comes to weight increase. The Atkins tally calories group seemed to lose more weight for a half year in a clinical exploration looking at every day tally calories  and Atkins thin down  groups . Diminis2 levels were measured to decide starch retention. Volunteers were offered the decision of black (0.1 g), green (0.1 g), or mulberry (0.1 g) teas at each stage. Individuals ate lipid-and starch-rich dinners, just as lipid-just and carb-free dinners. Increased breath-hydrogen concentration was observed after suppers including fat, carbohydrate, and tea, indicating carbohydrate malabsorption. The carbohydrate-free dinners made with or without tea extract, on the other hand, showed no differences. This suggests that the compounds in tea extract may reduce carbohydrate retention [By inhibiting alpha-amylase , alpha-getention .Soy has an assortment of effects against stoutness and is hostile to diabetic impacts . Food upS. vaccinia [Insulin resistance is improved by eating blueberries. Mice were fed freeze-dried whole blueberry powder supplemented with HFD for 8 weeks . Bluebervaccinia . KKAy mivaccinia . The PPAvaccinia . Furthervaccinia . This covaccinia . Anothervaccinia .Glycyrrhiza glabra extracts include amorfrutins, which can be used to treat diabetes. HFD-induced obese mice were given chemically produced amorfrutins [rfrutins . When amrfrutins .Glycyrrhiza orally [Glycyrrhiza extract binds to PPAR according to the results of a binding affinity assay [Glycyrrhiza extract can help with insulin sensitivity and hyperglycemia [KKAy mice were given an ethanol extract of a orally . Blood gty assay ,116. PPAty assay ,118. Thiglycemia .Trigonella foenumgraecum) are used as a food supplement and have a long history of therapeutic use for inducing labor, aiding digestion, and overall health [Fenugreek seeds , as compared with the same patients prior to treatment with Gymnema. During the same 21-day period, the patients\u2019 body weight increased by 0.9 kg, which was deemed to be non-significant. There was no control group in this study [A case series of eight T2DM patients given 10 mg of dried is study .In 1992, the same author presented a randomized controlled experiment with three research aims: For a total of 21 days, 16 healthy volunteers and 43 T2DM patients were given Gymnema or tolbutamide . The patients were not randomly assigned to groups, and neither the investigators nor the patients were blinded. The non-diabetic patients experienced a significant reduction in fasting blood sugar  on day 7. The diabetic patients in the Gymnema-treated group experienced significant reductions in both FBS (152 mg/dL to 133 mg/dL) and post-prandial blood sugar  at 21 days. On day 7, the diabetic patients in the tolbutamide therapy group had significantly lower FBS and PPBS, but not on day 14. Decreases also were reported in total cholesterol for both diabetic and non-diabetic patients . P-values were not provided for this short, non-randomized study .p < 0.001 level. Except for the Gymnema, 23 percent of the patients in the GS4 therapy group were able to cease taking all hypoglycemic medicines. There are a few caveats to be aware of when interpreting this study. The patients were not assigned to treatment groups at random, and there were disparities in fasting glucose levels at the start. They were, however, fairly well matched in terms of weight and BMI [For 20 months, 47 T2DM patients were followed in a lengthier controlled clinical trial. Each patient was already taking oral hypoglycemics and was maintaining a consistent treatment regimen with only partial control. The normal treatment was continued for half of the patients. For the other half, 400 mg of GS4, a Gymnema extract, was added to their daily routine. The average HBA1C in the GS4 group fell from 12% to 8.5%; FBS decreased as well, from 174 mg/dL to 124 mg/dL; total cholesterol and triglycerides improved from 260 mg/dL to 231 mg/dL and from 170 mg/dL to 140 mg/dL, respectively. All results were significant at the  and BMI .p < 0.05) and T2DM  patients. Significant reductions were also demonstrated in 30 min PPBS  and two-hour PPBS  in the Type 2 group [Ten healthy volunteers and six T2DM patients were given 2 g of an aqueous decoction of the shade-dried leaves of Gymnema three times per day in a modest, short-term trial. Significant reductions in both FBS and PPBS were demonstrated. After 10 days, significant reductions were seen in the FBS of both normal . Thirty-seven patients received their usual and customary care, whereas 27 insulin-dependent patients received 400 mg of Gymnema daily for eight months in addition to their usual insulin regimen. In the supplemented group, the average insulin need dropped from 60 uNPH/d to 45 uNPH/d. At least one episode of hypoglycemia occurred in all Gymnema patients, prompting insulin dosage reductions. Unfortunately, 40% of the patients withdrew before the completion of the trial due to one patient\u2019s severe hypoglycemia and practical difficulties with follow-up for the rest [Only one study evaluated the effectiveness of Gymnema in T1DM -induced diabetic rats were given conophylline orally, their blood glucose levels were lowered [Conophylline is a substance derived from the  lowered . Pancrea lowered . Accordi lowered . Further lowered .Anoectochilus roxburghii. In STZ-induced hyperglycemic rats, the oral treatment of kinsenoside lowered blood glucose levels [Kinsenoside is a chemical found in the jewel orchid e levels . Plasma e levels .Nymphaea stellata. This chemical was found to induce the partial production of pancreatic islet cells in one study [Initially, Nymphayol was used to isolate from ne study . In diabne study .Capsicum frutescens enhanced serum insulin concentration in streptozotocin-induced T2DM rats fed a high-fat (HF) diet. In the experimental procedures, the data from this study imply that 2% dietary Capsicum frutescens is insulinotropic rather than hypoglycemic [After 4 weeks of treatment, glycemic ,87.Momordica charantia fruit juice showed a significant drop in blood glucose levels and an increase in plasma insulin concentration. The result was attributed to a higher number of beta cells in treated mice compared to untreated animals. Momordicin, charantin, and a few other substances isolated from other portions of this plant, such as galactose-binding lectin and insulin-like protein, have been demonstrated to have insulin-mimetic activity [Diabetic rats treated with activity ,146.M. charantia fruits has also been demonstrated to partially induce insulin release from the isolated beta-cells of obese-hyperglycemic mice, implying that the insulin-releasing effect is caused by membrane processes being disrupted [M. charantia enhances pancreatic insulin secretion by increasing the renewal of partial cells in the pancreas or allowing the recovery of partially damaged cells [The aqueous extract of unripe isrupted . M. chared cells .M. charantia juice or a vehicle. Patients who took M. charantia juice had lower blood glucose levels than the vehicle control group. These findings suggest that M. charantia can lower blood glucose levels in diabetic mice and patients [A human clinical investigation was undertaken with 18 mature-onset diabetes patients . Thirty patients .Vitis vinifera [Resveratrol is a polyphenol that is found in the extract of vinifera . It possvinifera ,150,151.vinifera ,153 and vinifera ,158,159.vinifera ,161. It vinifera  and STZ-vinifera .Furthermore, resveratrol increased glucose-mediated insulin production in cells by activating SIRT1 , one of V. vinifera extract decreases glycogen phosphorylase b activity [V. vinifera extract can help T2DM patients with high blood and liver glucose levels [In a HepG2 cell line, activity . Glycogeactivity . So, inhe levels .Obesity and diabetes share certain basic characteristics and are intimately linked. In the 1970s, the term \u201cdiabesity\u201d was coined to stress the close link between obesity and diabetes. Some diabetes medications, on the other hand, may contribute to obesity. Sulfonylureas, for example, can cause weight gain as a side effect . ObesityIn female mice, genistein reduces food intake, body weight, and fat pad weight while increasing the apoptosis of adipose tissue in several animal investigations ,170. A lGenistein improved glucose and lipid metabolisms, raised insulin levels, and maintained pancreatic b cells in studies on C57BL/KsJ-db/db mice  and non-Vaccicum spp. [V. angustifolium (wild blueberry) ameliorate dyslipidemia via modulating the genes involved in lipid metabolism [Insulin resistance is improved ,110, thecum spp. . Bluebercum spp. . Furthertabolism .Glycyrrhiza extract. Hyperglycemia can be improved with this phytogenic substance [Glycyrrhiza glabra boosted oxidation while decreasing acetyl-CoA production [Glycyrrhiza extract may also help to reduce body fat, according to clinical investigations [Obesity and diabetes are affected by ubstance . The extoduction . Glycyrrigations ,178,179.Capsicum activates BAT, which causes thermogenesis ,180. WhiM. charantia extract. Adipocyte hypertrophy is suppressed by M. charantia extract, and lipogenic gene expression, such as FAS, ACC-1, and LPL, is reduced [M. charantia may act as both a lipogenesis inhibitor and a lipolysis stimulator. Hyperlipidemia and hyperglycemia may be reduced by M. charantia extract [Hyperglycemia  and hype reduced . As a re extract .Cinnamomum extract can help to reduce blood sugar levels [Cinnamomum extract appears to have a fat-burning effect [r levels ,131. Cing effect ,100. As Herbal products are the most abundant source of leads for developing new pharmacological entities, with a wide range of therapeutic indications and chemical structures . Blood gThe DPP-4 enzyme, also known as adenosine deaminase binding protein or CD26, inactivates oligopeptides such as glucagon-like peptide-1 (GLP-1) by eliminating N-terminal dipeptides. It is distributed throughout the body and is prevalent in endothelial cells ,188,189.The protein tyrosine phosphatase 1B (PTP1B) enzyme regulates tyrosine phosphorylation in cells and has an essential function in negatively regulating insulin signal transmission. Its inhibition substantially reduces triglyceride accumulation in adipose tissues in the context of excess nutrition. PTP1B is thus a possible target for T2DM ,191. TheThe \u03b1-glucosidase enzymes hydrolyze starch to simple sugars that help the body to digest dietary carbohydrates and starch and thus increase the levels of blood glucose for intestinal absorption . InhibitNuclear factor erythroid 2 (Nrf2) is a protein that combats oxidative stress and comprises seven functional domains. These are ARE and sMAF (Neh1), Keap1 (Neh2), CHD6-binding (Neh3), trans-activating, and CBP-binding areas (Neh4 and Neh5), \u00df-transducin repeat binding protein (\u00df-TrcP) (Neh6) and RXR\u03b1 domain binding (Neh7). Under stress, Nrf2 leaks out of the proteasomal degradation machine to accumulate and move from the cytosol to the nucleus. Insulin resistance is caused by oxidative stress, and nitrosative stress and elevated phosphorylation levels of extracellular signal-related kinase may suppress cardiac Nrf2 activity. The activation of Nrf2 may aid in the prevention of diabetic nephropathy. Nrf2 departs from Keap1 and enters the nucleus, where it binds to genes that code for antioxidant enzymes ,198,199.Insulin is an anti-hyperglycemic hormone produced by beta cells in the pancreas that helps to keep blood glucose levels in check . As a coIn hyperglycemia, aldose reductase (AR) is an NADPH-dependent Oxidoreductase and a major enzyme in the polyol pathway, converting glucose to sorbitol. Sorbitol cannot diffuse readily through the cell membrane, resulting in diabetic problems and a variety of cellular functional impairments. However, under normal physiological conditions, the hexokinase enzyme converts glucose to glucose-6-phosphate ,201,202.Autophagy is a lysosome-dependent homeostatic mechanism that contributes to the preservation of homeostasis in the cells and tissues. As a consequence, faulty autophagy contributes considerably to the development of metabolic disorders of carbohydrates, lipids, and proteins, such as T1DM and T2DM. People with diabetes who do not have enough insulin or cannot respond to it will have hyperglycemia and impaired autophagy. Therefore, autophagy offers a novel therapeutic target for diabetes, as activating autophagy has a range of benefits. AMPK and mTOR work together to act as essential regulators that monitor and control the amount of cell energy. It is generally assumed that inhibiting mTOR or activating AMPK is an efficient way to induce autophagy. The main anticipated herbal products that can trigger autophagy include resveratrol, berberine, quercetin, dihydromyricetin, and epigallocatechingallate  Table 1.Obesity is a complicated and multifaceted illness, and it is prevalent in the world\u2019s population, which is shifting away from the prior prevalence of infectious diseases and malnutrition. Obesity is a consequence of genetic predisposition, as well as a large number of high-energy meals being available, and a reduction in the physical activity required due to technological advancements. The idea that obesity is just a cosmetic issue for certain people is no longer valid since it is a worldwide pandemic ,210. The50 = 3.2 \u00b5M), chlorophyll a (IC50 = 5.0 \u00b5M), chlorophyll b (IC50 = 5.9 \u00b5M), and (-)-Epigallocatechin gallate (IC50 = 1.7 \u00b5M) from green tea leaves protect against dioxin toxicity through the suppression of AhR transformation [The aryl hydrocarbon receptor (AhR) is an evolutionarily fundamental protein that functions as a ligand-activated transcription factor that detects the presence of external chemicals such as Persistent organic pollutants (POPs) and activates the cytochrome P450 enzymes necessary for their elimination from the body ,212. AhRormation ,216.Silybum marianum, Citrus aurantium, Taraxacum officinale, resveratrol, Curcuma longa, caffeine, etc. reduce differentiation and increase lipolysis and apoptosis [Adipocytes grow from fibroblast-like preadipocytes during adipogenesis. Many transcription factors, including the CCAAT/enhancer-binding protein (C/EBP) gene family and peroxisome proliferator-activated receptor (PPAR), must be activated sequentially during adipogenesis. These cells must go through two stages to mature: adipocyte determination and adipocyte differentiation. Several variables have been found. Some stimulators include PPAR \u03b3, insulin-like growth factor I (IGF-1), macrophage colony-stimulating factor, fatty acids, prostaglandins, and glucocorticoids. Glycoproteins, transforming growth factor-\u03b2 (TGF-\u03b2), inflammatory cytokines, and growth hormones are all inhibitors. Aside from these, age, gender, and lifestyle may all influence the process in some manner. Obesity is caused by an increase in the number and size of adipocytes. Adipogenesis may cause central obesity  or peripheral obesity (subcutaneous tissue) . Herbal poptosis .Silybum marianum), lycopene from tomato, watermelon, papaya, orange, grapefruit, nobiletin from citrus fruit, baicalein from Scutellaria baicalensis, and quercetin from broccoli or onion [The melanocortin 4 receptor (MC4R) is a seven-transmembrane G-protein-coupled receptor (GPCR) that is encoded by a single exon gene on chromosome 18q22. MC4R is expressed in the hypothalamic paraventricular nucleus, a region of the brain that is intricately engaged in appetite control, and its activation leads to decreased food intake . Mc4r hoor onion .Radix astragali yield two types of saponins, astragaloside II and isoastragaloside I, which, when delivered to primary adipocytes, stimulate adiponectin secretion without impacting the release of other adipokines, offering a promising avenue for treating obesity-related illnesses [Adiponectin is a collagen-like plasma protein with a molecular weight of 30 kDa that is produced by adipocytes but mainly circulates in hexameric, oligomeric, and, to a lesser degree, trimeric forms ,229. Adillnesses .Phytogenic substances that alter obesity and diabetes are discussed in this review. While there has been some research toward a combined treatment for obesity and diabetes, there is currently no such treatment. Inflammation and insulin resistance are common features of both obesity and diabetes. As a result of the correlations between obesity and diabetes and the interesting properties of phytogenic synthetic substances, some phytogenic mixtures might be utilized to create medicines for the two issues. Herbal remedies, for instance, can assist with hyperglycemia and muscle versus fat synthesis. More examination into the potential outcomes of herbal products would be valuable. Plants can have perilous or inadequate impacts and unexplained results in regular item research, yet the choices are interminable. We characterize dynamic parts and normal items dependent on a lot of exploration and clinical preliminaries and clarify restorative plants with hostile to diabetic advantages in an extensive way, zeroing in on hormonal guidelines and metabolic guidelines."}
+{"text": "Exaggerated local field potential bursts of activity at frequencies in the low beta band are a well-established phenomenon in the subthalamic nucleus of patients with Parkinson\u2019s disease. However, such activity is only moderately correlated with motor impairment. Here we test the hypothesis that beta bursts are just one of several dynamic states in the subthalamic nucleus local field potential in Parkinson\u2019s disease, and that together these different states predict motor impairment with high fidelity.Local field potentials were recorded in 32 patients (64 hemispheres) undergoing deep brain stimulation surgery targeting the subthalamic nucleus. Recordings were performed following overnight withdrawal of anti-parkinsonian medication, and after administration of levodopa. Local field potentials were analysed using hidden Markov modelling to identify transient spectral states with frequencies under 40 Hz.Findings in the low beta frequency band were similar to those previously reported; levodopa reduced occurrence rate and duration of low beta states, and the greater the reductions, the greater the improvement in motor impairment. However, additional local field potential states were distinguished in the theta, alpha and high beta bands, and these behaved in an opposite manner. They were increased in occurrence rate and duration by levodopa, and the greater the increases, the greater the improvement in motor impairment. In addition, levodopa favoured the transition of low beta states to other spectral states. When all local field potential states and corresponding features were considered in a multivariate model it was possible to predict 50% of the variance in patients\u2019 hemibody impairment OFF medication, and in the change in hemibody impairment following levodopa. This only improved slightly if signal amplitude or gamma band features were also included in the multivariate model. In addition, it compares with a prediction of only 16% of the variance when using beta bursts alone.We conclude that multiple spectral states in the subthalamic nucleus local field potential have a bearing on motor impairment, and that levodopa-induced shifts in the balance between these states can predict clinical change with high fidelity. This is important in suggesting that some states might be upregulated to improve parkinsonism and in suggesting how local field potential feedback can be made more informative in closed-loop deep brain stimulation systems. et al. report that multiple spectral states in the STN LFP have a bearing on motor impairment, and that levodopa-induced shifts in the balance between these states can predict clinical change with high fidelity. This is important in suggesting how LFP feedback can be made more informative in closed-loop DBS systems.Khawaldeh  Parkinson\u2019s disease is a neurodegenerative condition involving degeneration of dopaminergic neurons in the substantia nigra of the midbrain and characterized by the cardinal symptoms of bradykinesia, rigidity and tremor. Symptomatology is partially reversed by the dopamine prodrug levodopa, but more advanced disease may necessitate deep brain stimulation (DBS). This therapy involves the chronic electrical stimulation of key targets in the basal ganglia, like the subthalamic nucleus (STN), that have been functionally compromised by the effects of dopaminergic denervation.More recently, it has been stressed that beta activity consists of transient bursts in Parkinson's disease and that burst characteristics such as rate and duration may correlate more strongly with motor impairment than mean beta power.,,et al.,,However, in the correlative studies cited above, beta power, or even beta bursting, is only moderately indicative of the severity of motor impairment. So what of other oscillatory activities in the STN LFP? Broadly, the STN LFP can be divided into lower-frequency activities in the theta, alpha, low beta and high beta bands and higher frequency activities such as finely tuned gamma,Here, we use hidden Markov modelling (HMM) to objectively detect different types of LFP states in the theta (4\u20137 Hz), alpha (8\u201312 Hz), low beta (13\u201321 Hz) and high beta (22\u201335 Hz) frequencies.We investigated spectral states in the STN LFP before and after administration of levodopa in 32 patients (64 hemispheres) with advanced Parkinson's disease undergoing DBS surgery targeting the STN. Patients had an average age of 58.5 years (range 34\u201379 years) and an average disease duration of 11.7 years (range 5\u201318 years). Twelve were female . All patDBS electrodes were temporarily externalized prior to connection to the implantable pulse generator. LFP recordings were performed with the patient quietly seated with their eyes open following overnight withdrawal of anti-parkinsonian medication and were repeated before and after administration of levodopa (see below) 3\u20137 days after lead implantation. In 18 patients LFPs were recorded online from adjacent bipolar contact pairs  to limit volume conduction.Patients were evaluated using the UPDRS part III after omitting all dopaminergic medication overnight and after administration of at least 200 mg of levodopa, with the exception of case 11 who received 100 mg of levodopa. Clinical evaluation was performed at the same time as LFP recordings, except for 14 patients in whom it was performed up to 3 months preoperatively. Changes in motor impairment with levodopa was calculated using hemibody scores for bradykinesia\u2013rigidity  and tremor (sum of UPDRS motor score sub-items 20\u201321 for arm and leg), either in combination or separately, contralateral to the recording site. The clinical improvement was defined as the percentage of the reduced UPDRS hemibody score in ON condition compared with OFF condition.https://ohba-analysis.github.io/osl-docs/], data were imported into MATLAB , where all further signal processing steps took place. Signal durations ranged from 127.9 to 639.9 s with a mean \u00b1 standard deviation (SD) signal duration of 392.4 \u00b1 111.9 s for both the OFF and the ON conditions (data duration OFF and ON medication was matched in each subject).,,z-normalized on a per-session basis to facilitate comparison across different recording systems and to minimize the effects of targeting variance. All processed LFPs were then concatenated into one vector representing the selected contact pair from all medication conditions and hemispheres. The concatenated signal was then fed to the time-delay embedded hidden Markov model (TDE-HMM), which was compared against a percentile thresholding model.The bipolar contact pair with the highest beta power in the average of the OFF and ON drug conditions was selected for further analysis based on the presumption that this contact pair was most likely to pick up from the dorsolateral \u2018motor\u2019 STN.A principled way to detect bursts is to use HMMs. This can overcome many limitations of standard threshold-based approaches to burst detection, such as an overdependence on the choice of arbitrary thresholds.,,In general, HMMs assume that a time series can be described using a hidden sequence of a finite number of states.z-normalized and concatenated) LFP data; i.e. copies of the LFP data, each shifted by different time lags which define the time window around the point of interest, are fed in concurrently as multiple data channels to the HMM inference. Each HMM state then captures periods of time that have distinct autocovariance, which translates into distinct spectral content. The net result is that the TDE-HMM models the data as a sequence of state visits, where each state can be interpreted as a transient spectral event of a certain type.The variant of HMM used here is the TDE-HMM. This uses a multivariate Gaussian distribution as the observation model, but, crucially, the HMM is inferred on a TDE version of the (K (5\u201318 states) and time lags L (3\u201315 lags). The time lag L determines the window duration so that a lag of 10 means that the window includes 10 data-points either side of that representing the time under analysis. Longer windows allow better frequency resolution at the expense of temporal resolution . The state number K denotes the number of states to be detected. For every combination, TDE-HMM states were categorized by their predominant frequency content by cross-correlating the states\u2019 time courses with the Hilbert envelopes of the theta, alpha, low beta and high beta frequency bands. States were estimated using the TDE-HMM model for 56 different combinations of state number This assessment was repeated for the OFF and ON medication conditions. Various extracted measures of interest were extracted from the TDE-HMM models, and changes in them between the two medication conditions were correlated with the contralateral UPDRS hemibody scores to investigate any link to parkinsonian motor impairment. The extracted measures were: probability of transitioning between different states and within states , fractional occupancy (referring to how much time the HMM spends in each state on average), life time (defined as the average duration per visit of each state), interval time (defined as the average time between each state visit) and switching rate between states. Occurrence rate (how often a state gets visited over time) and relative number of visits were deduced from the life time measure as further features describing bursting dynamics. The occurrence rate and relative number of visits are related to the burst rate and relative burst rate reported with the thresholding technique and described below. Inherently, some of these different summary measures of the bursting dynamics are interrelated, e.g. a state with an increase in occurrence or burst rate will see a corresponding decrease in interval time if the probability of transitioning out of that state remains the same. As such, some of these measures if associated with motor impairment might overall tend to correlate with motor state with opposite polarity, such as the occurrence or burst rate in a given frequency band and the interval time.,,,HMM method results were contrasted with those from the standard thresholding method of defining beta bursts, to allow comparison with relevant previous work.X is the features matrix, y is the predicted score, k is the ridge parameter and I is the identity matrix. Small, positive values of k improve the conditioning of the problem and reduce the variance of the estimates. While biased, the reduced variance of ridge estimates often results in a smaller mean squared error when compared to least-squares estimates. The k parameter was set by varying the k-value in the model between \u201330 and 30, and the final k-value chosen when model prediction power converged and stopped changing. To avoid overfitting, we performed leave-one-out cross-validation. Finally, to evaluate the results we used the previously described method to ascribe states defined in each HMM model to our chosen frequency bands so that we could then attribute the corresponding ridge coefficients to these frequency bands.Ridge regression was used to create parsimonious regression models given that the number of extracted features (i.e. regressors) could exceed the number of STN recordings.Note that the TDE-HMM approach is also able to identify the frequency of signal segments that are shorter than the period of the signal frequency.Statistical analyses were performed using MATLAB  and SPSS . The HMM and thresholding extracted features were tested with MATLAB Lilliefors test to check whether they were normally distributed. State feature and other distributions were tested against the null hypothesis using permutation testing.The original data are available upon reasonable request to the corresponding authors.TDE-HMM was run on the STN LFP data to identify a temporal sequence of states in each STN LFP channel, where each state can be interpreted as a transient spectral event of a certain type, including, but not limited to, beta bursts.Initially, we focused on activities with frequencies under 40 Hz, as these are more consistently found and more readily registered with the ultra-low-power amplifiers that are necessary in chronic recording systems that might, in the future, deliver closed-loop DBS. Identified LFP states with clear spectral peaks were then ascribed to the theta, alpha, low beta and high beta bands, or to a background state if no dominant peak existed . OverallThe occurrence rate of LFP states  significantly increased when ON compared to OFF levodopa for the state visits of the shortest duration in all frequency bands, except low and high beta . In cont,To compare the present findings in the beta band with previously reported results of the standard thresholding technique, we also plotted the change in the relative rate of different durations of states  within the beta frequency band with levodopa . There wIn addition, there were significant decreases in fractional occupancy and life time and corresponding increases in state-interval time in the low beta band ON levodopa, and significant converse changes in the theta and, to a lesser extent, alpha and high beta bands . ChangesThe levodopa-induced changes in low beta and theta-alpha states were also associated with opposing effects on motor impairment . The chaTo compare the present findings with previously reported results of standard thresholding in the beta band, we also estimated the correlation with the relative number of states in each frequency band . As withChanges in the absolute and relative occurrence rate of states in the different frequency bands were linked to frequency-specific and often opposing changes in motor impairment. However, the bivariate correlations reported above and illustrated in We also considered the contributions made to the regression model by states in the different frequency bands and their features . The norThe change in contralateral UPDRS Part III hemibody scores predicted above in r2) values were about half of those of the combined model , rate of occurrence and duration of low beta states, with this reduction correlating with the improvement in motor impairment. These observations are consistent with earlier reports using a standard thresholding approach to state or burst identification in patients with Parkinson's disease, although these did not distinguish between the low and high beta band and only determined the relative numbers of states of a given duration, and hence could not unequivocally detect whether short or long duration beta bursts were independently detrimental as measured by motor UPDRS score.,,In addition, bivariate correlations and the normalized regression coefficients from ridge regression models suggested that STN LFP states at other frequencies (up to 40 Hz) had opposing effects to the low beta state. Thus the preponderance, occurrence rate and duration of LFP states in the theta, alpha and high beta frequency ranges negatively correlated with motor impairment in patients withdrawn from their anti-parkinsonian medication, and levodopa increased the preponderance, occurrence rate and duration of these states, with the increase correlating with the improvement in motor impairment. Changes in LFP state behaviour were biggest in the theta frequency band. Although averaged power spectra of LFP need not faithfully mirror LFP state behaviour in the STN, there are past reports of theta and, to a lesser extent, alpha activity being increased by levodopa in Parkinson's disease.The opposing effects of the low beta state and theta, alpha and high beta states were together able to account for just over 50% of the variance in patients\u2019 hemibody impairment OFF medication, and in the change in hemibody impairment following ingestion of the dopamine prodrug, levodopa. The features of the different states contributing to the regression model for hemibody UPDRS scores OFF medication were similar to those contributing to the regression model predicting the change in contralateral hemibody scores with medication. This suggests that the LFP features that predict motor impairment OFF medication are those that are modulated by treatment with levodopa to achieve improvement in motor state. Hence these features, like the elements of the motor impairment which they predict, are modulated by the current status of dopaminergic activity.,Our core analysis sought features of HMM states with a frequency of under 40 Hz that predicted motor impairment. However, we did not include amplitude information and omitted the effects of gamma band activities. In a secondary analysis we therefore included these features in our ridge regression. Information on signal amplitude and inclusion of HMM states in the gamma band led to modest further increases in the prediction of motor impairment off medication and in the change in this with levodopa treatment. Perhaps the most parsimonious explanation for the modest effect of incorporating amplitude information is that the pattern of spectral states under 40 Hz determines signal amplitude or vice versa. Thus adding features related to absolute amplitude did not greatly improve predictions. Gamma activity may have had modest impact because it is not a consistent feature in LFP recordings across patients, and because the information contained within may be shared by reciprocal changes at lower frequencies.The HMM approach serves to define states on objective statistical grounds and does not necessitate the application of arbitrary filtering within a canonical frequency band or of arbitrary thresholds. Indeed, the derived features were consistently more predictive of motor impairment than features drawn from simple, standard thresholding used for burst detection. However, the HMM will identify as many states as are defined in the model, and if one or more related states react in a particular way then the converse may be reflected in the remaining state features, where these are considered relative to other states. Yet this is unlikely to explain the reciprocal nature of low beta and non-low beta features, both in terms of their response to levodopa and their prediction of motor impairment. This is because multiple (5\u201318) states were prescribed and systematic differences between frequency bands other than low beta were identified. Thus the major reciprocal effects occurred in the theta band and the least were seen in the background activity. Moreover, had changes in the non-low beta band been secondary to those in the low beta band, then we should not have seen additional predictive value from non-low beta band features; the ridge regression model with all features was able to predict much more of the variance in motor UPDRS than the model with just low beta band features. Finally, not all of the state features considered in this study were relative to other states. Life time, interval time and occurrence rate take absolute values, and yet were also reciprocal in nature in the low beta and non-low beta bands.A clear limitation of the HMM is that only one state is visited at any given time. Although this state will have the most evidence and can be considered dominant at that moment in time, our approach will miss independent subsidiary states that might overlap in time, as might come from other neural generators.post hoc analysis step performed after HMM state identification and feature prediction by ridge regression), we facilitated comparison with the existing literature, but this approach meant that multiple HMM states could get combined together into a given frequency band, and we could therefore be overlooking detail available in the underlying states. For example, it is possible that different HMM states could be differentially affected by medication status  and this detail gets lost when states are combined into a single frequency band. In addition, the number of states to be classified by HMM has to be predefined, as does the number of lags utilized, although by collapsing the results across many realizations of the HMM, as here, we can identify robust features in the data. We should also note that the attribution of different HMM states to canonical frequency bands was not perfect, partly because the frequency ranges of these bands are somewhat arbitrary when applied to the basal ganglia and predominantly defined on the basis of cortical physiology.By focusing on frequency bands (as a ,,,,It is tempting to assume that theta, alpha and high beta states promote normal movement, but we did not assess the presence of levodopa-induced dyskinesias in our cohort and so it remains to be seen whether any of these LFP states are linked to dyskinesias ON medication. Indeed, there is some evidence that excessive theta activity in dorsolateral motor STN might be related to dyskinesias.,,,,With respect to our clinical data it should be noted that we had a mix of motor UPDRS values determined at the time of the recording in some subjects and values determined up to 3 months preoperatively in others. This may increases the variance in our clinical measure, as clinical impairment may have been greater in those assessed preoperatively given that the postoperative stun effect would have been absent. This may have potentially led us to underestimate the regression coefficients linking burst features to motor impairment. Finally, it should be noted that bursts can be defined by means other than the original thresholding method.,,,The most immediate therapeutic significance of the present findings lies in the potential identification of a more informative feedback signal for closed-loop DBS than beta activity alone.,,However, several points need addressing before the tracking of the pattern of multiple LFP states can be used as feedback. First, it should be noted that LFP state correlations with motor impairment were shown across subjects rather than within subjects. However, recent studies relating beta bursts to trial-to-trial variations in motor performance make it possible that our findings will extrapolate to the individual.Our findings are also important in suggesting that while some LFP states might be usefully downregulated for therapeutic benefit, others could potentially be upregulated to better mimic the effect of dopaminergic therapy and improve parkinsonism. Current conventional DBS serves to suppress low beta activity in the STN, but also potentially beneficial activities in other frequency bands.awab264_Supplementary_DataClick here for additional data file."}
+{"text": "Frailty is conceptually defined as a state of increased vulnerability in older persons, resulting from age-associated declines in physiological reserve and function as they cope with everyday life stressors. To date, the prevalence of frailty has been assessed in various ways. The objective of this study was to identify the prevalence of the condition and the assessment tools used to determine its occurrence among Malaysian older individuals. A systematic literature search was performed on electronic bibliographic databases, namely, Web of Science, Scopus, EBSCOHost: MEDLINE Complete and Google Scholar. Ten eligible articles were reviewed and evaluated using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses and the Newcastle-Ottawa Scale. Frailty was diagnosed using Fried\u2019s frailty phenotype and the Groningen Frailty Indicator (GFI). Assessment tools that included physical performance tests detected a lower prevalence of frailty than that determined using questionnaire-based tools. The prevalence of frailty ranged from as low as 6% to as high as 76%, and there was a higher prevalence among older persons living in aged care homes. This review suggests increasing prospective and interventional studies on frailty to establish a cause\u2013effect relationship between standardised tools of assessing frailty and its prevalence among Malaysian older persons and provide guidelines for health professionals on promoting active lifestyles among older populations. Malaysia is expected to reach an ageing nation status by 2035, during which more than 15% of its population will have reached the age of 60 years old and older , 5. RegaAccording to a systematic review, the global mean prevalence of frailty is 10.7% . Pooled As can be seen, a variety of methods are used to assess the prevalence of frailty among older people. To date, no study has pooled information regarding frailty studies in Malaysia. The objective of the current work was to identify the prevalence of frailty among the Malaysian older population and the tools used to assess the occurrence of the condition. The identification of such prevalence and corresponding assessment tools may provide evidence that can be used by the government to advance public health strategies.In December 2020, a systematic literature search was performed on Web of Science, Scopus, EBSCOHost: MEDLINE Complete and Google Scholar to identify prospective papers for review. The search was managed using the primary search terms (frail OR frailty) and the following secondary search terms: (older person OR older adult OR elderly OR senior OR geriatric OR aged OR ageing) AND .Studies involving participants aged 60 years old and older and full-text articles written in English and published between January 2011 and April 2021 were included in the analysis. Studies that were conducted only in Malaysia, that were intended to inform the prevalence of frailty and that used validated frailty measurements that include physical performance tools were also deemed eligible for review. Finally, studies with participants who lived in either community dwellings or residential areas were covered in the analysis. The entries in the bibliographic databases were manually screened for duplication.Two reviewers (MIS and NFNAR) independently chose the articles for analysis on the basis of the inclusion criteria. The selection was conducted by screening article titles and abstracts, after which full texts were examined to determine their eligibility for inclusion. The initial sample of articles was compared in a consensus meeting and disagreement was resolved through discussion. Articles that both reviewers deemed suitable were included in the final sample. The selected articles were reviewed following PRISMA guidelines , and theData from the selected articles were retrieved using structured sheets containing demographic information, including ages, the total number of participants and the location of the studies. Information regarding standardised assessment and physical performance tools and the studies\u2019 outcomes, limitations and strengths were also included.The reviewers used the Newcastle-Ottawa Scale to assess the methodological quality of the selected articles. Eight quality assessment tools with three main components  were used to evaluate the cohort and case control studies. The maximum score that could be derived was 9 points and studies were considered high quality if they scored at least 5 points .The database search yielded 1,565 articles, of which 1,254 were duplicate studies and therefore excluded. The titles and abstracts of the remaining 311 articles were screened, leaving 21 for potential inclusion. Following full-text assessment and screening on the basis of the inclusion criteria, 10 studies were deemed eligible for analysis. The PRISMA flowchart in The characteristics of the 10 studies are summarised in The studies derived broadly varying results on the prevalence of frailty, with percentages ranging from as low as 6%  to as hiIn terms of tools for identifying frailty, the studies can be divided into those that adopted and excluded performance tests in their assessments. Seven studies determined frailty using either the modified or standardised version of Fried\u2019s frailty phenotype \u201319, 22, Standardised Fried\u2019s criteria were used in three studies , 18, 22 Other standardised assessment tools used in the study were cognitive assessments: the GDS, Mini-Mental State Examination, Montreal Cognitive Assessment, Digit Span, Rey Auditory Verbal Learning Test, Digit symbol forward and backward and Addenbrooke\u2019s Cognitive Examination , 22. A sFrailty was positively associated with advanced age , 18, 22 Frailty was also significantly associated with poor self-rated health, being single, low body mass index, abdominal obesity, physical disability, a history of falls, lack of balance, low peak respiratory rates, slow gait speeds, low skeletal muscle mass, high serum CRP levels and institutionalisation , 15\u201319. As previously stated, this study was aimed at determining the prevalence of frailty and the standardised tools used to assess it among Malaysian older persons. This condition is widely prevalent in the country, and this prevalence is even higher among older individuals living in aged care homes\u2014a situation similar to that described in a nationally representative study in the United States, where people in such institutions are twice as likely to be frail than those living in residential areas . A prospVariations in prevalence depend on the types of assessment tools used to identify frailty. This study uncovered that the use of self-report tools detected the prevalence of frailty to a higher degree than that achieved with physical performance tests. The findings are in line with a recent study that reported a higher prevalence when the condition is assessed using self-report questionnaires , 29. HowThe advantage of self-report tools for determining frailty is their quick and easy implementation. Such tools do not require training and a unique instrument for administration. Even so, the validity of a self-report questionnaire is influenced by the cognitive level of a participant. Severe cognitive impairment may lead to inaccurate assessments of frailty . ConversThe reviewed studies used two primary standardised assessments to identify frailty: Fried\u2019s frailty phenotype and the GFI. Both include multiple components in identifying frailty, but the former involves physical performance testing, whereas the latter depends entirely on a self-report questionnaire. Nevertheless, the GFI features broader domains for identifying frailty, including physical, cognitive, social and psychological aspects.Frailty is a multidimensional factor that is not limited to physical performance capabilities and components, including psychological, cognitive, clinical and social aspects , 33. A oIn summary, there are variations in the prevalence of frailty among afflicted individuals mainly because of the use of different assessment tools. Longitudinal and prospective studies may help to establish a cause-effect relationship with respect to frailty. Intervention studies on physical activity can also be conducted to provide guidelines for health professionals, especially physiotherapists, in implementing evidence-based practice as they promote the pursuit of active lifestyles among older persons. Frailty is reversible , 34 and"}
+{"text": "The management of large malignant tracheo-esophageal fistulas (TEF) is not standardized. Herein, we report a case with a malignant TEF associated with esophageal post-transplant lymphoproliferative disorder (PTLD) for whom we successfully performed a surgical repair. This contributes to the knowledge on how to treat large acquired malignant TEFs.A 69-year old male presented with a one-week history of fever, productive cough and bilateral coarse crackles. In addition, he described a weight loss of 10\u00a0kg during the past three months. The patient's history included a kidney transplantation twenty years ago. Esophagogastroduodenoscopy with a biopsy of the esophagus was performed nine days before. Histopathology showed a PTLD of diffuse large B-cell lymphoma subtype. Subsequent diagnostics revealed a progressive TEF (approx. 2.0\u00a0\u00d7\u00a01.5\u00a0cm) 3.0\u00a0cm above the carina. PET-CT scan showed an esophagus with slight tracer uptake in the middle third . After decision against stenting, transthoracic subtotal esophagectomy with closure of the tracheal mouth of the fistula by a pedicled flap was performed. PTLD was treated with prednisone and rituximab. Tumor progression (brain metastasis) led to death 95\u00a0days after surgery.The treatment of a malignant TEF is complex and personalized while both the consequences of the esophago-tracheal connection and those of the underlying responsible diagnosis have to be considered concurrently. In this case, we considered surgery as the best treatment option due to a relatively good prognosis of the underlying diagnosis (PTLD) and a large fistula. Esophageal or dual stenting, the treatment of choice for small malignant TEF, would have been associated with a high risk of failure due to the wide trachea, extensively dilated esophagus, proximal location and large diameter of the fistula.Surgery can be considered for patients with a large acquired malignant TEF and positive long-term prognosis of the underlying diagnosis. Due to the complexity of TEF management, immediate pre-operative multidisciplinary discussion is advised. \u2022Surgical repair of a large malignant tracheo-esophageal fistula\u2022Immediate pre-operative multidisciplinary discussion is needed for large malignant trachea-esophageal fistulas.\u2022Esophageal post-transplant lymphoproliferative disorder (PTLD) of diffuse large B-cell lymphoma (DLBCL) subtype\u2022Esophagectomy and closure of the trachea by a pedicled deepithelized muscle flap Acquired TEF is divided into benign and malignant categories, whereas the latter accounts for more than 50\u00a0% of the acquired cases In this case, we successfully managed a large malignant TEF by performing a transthoracic subtotal esophagectomy and a closure of the defect in the membranous part of the trachea with a pedicled deepithelized latissimus dorsi flap.This work has been reported in line with the SCARE 2020 criteria 2Candida albicans, cytomegalovirus (CMV) infection, and others.A 56-year old male patient presented with a one-week history of fever, chills, productive cough, and bilateral coarse crackles. Furthermore, he described a weight loss of 10\u00a0kg during the past three months. The patient's history included a kidney transplantation twenty years ago due to a diabetes mellitus type 1-induced nephropathy. Since then, immunosuppression lead to at least three periods of polymicrobial esophageal thrush, including Serratia marcescens, Lacticaseibacillus rhamnosus, Streptococcus mittis, Streptococcus anginosus, Candida albicans, CMV) and, additionally, a monomorphic post-transplant lymphoproliferative disorder (PTLD) of diffuse large B-cell lymphoma (DLBCL) subtype. CMV viremia was confirmed by polymerase chain reaction, which in the large majority of cases corresponds to a reactivation of latent CMV infection.Nine days before presentation, elective esophagogastroduodenoscopy with a biopsy of the esophagus was performed. Histopathology showed polymicrobial esophageal thrush  scan revealed an extensively dilated and wall-thickened esophagus (42\u00a0\u00d7\u00a023\u00a0mm), a TEF (6\u00a0\u00d7\u00a013\u00a0mm) on the level of the T3 vertebral body, and signs of aspiration pneumonia a. PET-CTSix days later, after transfer to our institution, gastro-/bronchoscopy confirmed a large communicating TEF in the membranous part of the trachea approx. 2.0\u00a0cm in craniocaudal direction and 3.0\u00a0cm above the carina a. After Pathological examination of the esophagectomy specimen revealed transmural infiltrates of a monomorphic, mass-forming lymphoid infiltrate with circumscribed perforation of the esophageal wall . ImmunohTwo and a half months after surgery, head MRI \u2013 performed due to persistent headache \u2013 revealed multiple large necrotic lesions in the cerebrum. Due to polymorbidity, a decision against combination chemotherapy with curative intent was made. Due to missing consequences, brain biopsy was not performed and progress of PTLD was assumed; there was no clinical or radiographic evidence of a secondary neoplasia. After whole-brain radiotherapy and treatment with dexamethasone and rituximab, tumor progression led to death 95\u00a0days after surgery.During a follow-up six weeks after surgery, the patient reported an improved lung function and mobility; however, he still suffered from a quick fatigue and lost an additional four kilograms of weight. Since the gastric pull up could not be performed due to tumor progression, a final detailed assessment of the patient and his perspective on the procedure was not done.Length of hospital stay was 56\u00a0days , followed by 19\u00a0days in a rehabilitation center. Before the patient was discharged to a palliative care home, the patient stayed another 22\u00a0days in a regional tertiary hospital due to hyperglycemia and re-staging (head MRI).3In this case, we considered surgery as the best treatment option due to a relatively good prognosis of the underlying diagnosis and a large fistula with high risk of stent failure.The patient suffered from an extended esophageal monomorphic PTLD of diffuse large B-cell lymphoma (DLBCL) subtype which is an exceedingly rare diagnosis. In a multicenter, international analysis approximately 40\u00a0% of patients with primary central nervous system PTLD showed a three-year overall survival after therapy with rituximab and/or combination chemotherapy In addition, esophageal or dual stenting, the treatment of choice for small malignant TEF, would have been associated with a high risk of failure due to the wide trachea, extensively dilated esophagus, proximal location and large diameter of the fistula without great potential of spontaneous closure The tracheal mouth of the fistula was indirectly closed by a pedicled deepithelized myocutaneous flap of latissimus dorsi muscle. A pedicled flap is connected to its original blood supply, thereby avoiding the insufficient healing and early recurrence associated with former ways of patching, using fascia, skin and other foreign materials. Moreover, to limit further spread of the malignancy, therapy with prednisone and rituximab was initiated. Other approaches would have been to resect or directly suture the tracheal part of the fistula. Direct suture is not an option in large malignant fistulas due to airway stenosis. Nowadays, planned resections of up to 4\u00a0cm of the trachea will mostly be tolerated, depending on the surgical approach  and individual conditions  Overall survival after surgery in this patient was 95\u00a0days. The patient spent all of the time in a hospital (9\u00a0weeks) or in other institutions .In general, since both the consequences of the esophago-tracheal connection and those of the underlying responsible diagnosis have to be considered concurrently, treatment of an acquired malignant TEF is complex and personalized. Therefore, the individual approach should ideally be discussed within a multidisciplinary team including thoracic and upper gastrointestinal surgeons, interventional pulmonologists, gastroenterologists, oncologists, and anesthesiologists.4Surgery can be considered for patients with a large acquired malignant TEF, stable physical condition, and relatively positive long-term prognosis regarding tumor histology and stage. Nonetheless, immediate pre-operative multidisciplinary discussion is needed.Written informed consent was obtained from the daughter of the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Not commissioned, externally peer reviewed.In Switzerland, case reports are not required to be voted by the Swiss Ethics. If required, this specific confirmation can be given later.None declared.Isabelle Opitz.N/a.1.Conceptualization: JS, CAG, II, IO.2.Data curation: JS, CAG, II, VHK, IO.3.Formal analysis: JS, CAG, II, VHK, IO.4.Funding acquisition: n/a.5.Investigation & Methodology: JS, CAG, II, VHK, IO.6.Project administration: IO.7.Resources: JS, CAG, II, IO.8.Software: n/a.9.Supervision: IO.10.Validation: JS, II, IO.11.Visualization: JS, II, VHK, IO.12.Writing \u2013 original draft: JS.13.Writing \u2013 review & editing: JS, II, VHK, IO.IO reports the following disclosures: Roche , AstraZeneca (Advisory Board and Speakers Bureau), MSD (Advisory Board), BMS (Advisory Board), Medtronic , Intuitive (Proctorship). All other authors state that they have no conflict of interest."}
+{"text": "This new artificial metalloenzyme has been purified and characterized by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), circular dichroism, and UltraViolet\u2013Visible spectroscopy. Using theoretical experiments, the structure of this biohybrid and the positioning of the residues near the metal complex were examined, which made it possible to complete the coordination of the cobalt ion by an axial glutamine Gln283 ligand. While the Co(III)\u2013porphyrin catalyst alone showed weak catalytic activity for both reactions, 10 times more H2 and four times more CO2 were produced when the Co(III)\u2013porphyrin complex was buried in the hydrophobic cavity of the protein. This study thus provides a solid basis for further improvement of these biohybrids using well-designed modifications of the second and outer coordination sphere by site-directed mutagenesis of the host protein.The covalent insertion of a cobalt heme into the cavity of an artificial protein named alpha Rep (\u03b1Rep) leads to an artificial cobalt hemoprotein that is active as a catalyst not only for the photo-induced production of H H2 production processes are numerous  and the resources used in these processes are equally as abundant . To meet the fast-growing global demand for H2, the development of catalysts for the production of hydrogen is highly sought after. Specifically, new original molecular complexes, most often bioinspired, based on non-noble metals such as nickel, cobalt, or molybdenum have been synthesized and studied to replace platinum, a catalyst that is certainly effective but scarce and expensive 2+  was chosen as the photosensitizer and ascorbic acid as the sacrificial electron donor agent, since it has previously been shown that under irradiation at 405 nm, the excited state of [Ru(bpy)3]2+ is reduced by ascorbic acid to [Ru(bpy)3]+, which in turn transfers electrons to the catalyst, generating its active form, which is capable of reducing substrates such as protons 2+ as a photosensitizer and either 7 \u03bcM (A3A3\u2032)Y26C-4 or 7 \u03bcM Co(III)PPIX as a catalyst in 100 mM potassium phosphate buffer at pH 7.0 under irradiation with a white LED light at \u03bb > 405 nm. 2 production over time for 25 h. It appears that in the presence of the A3A3\u2032Y26C-4 biohybrid, the production of H2 increases over time to reach a value of 163 TON after 25 h. In contrast, when the metalloporphyrin CoPPIX was used as a catalyst, only a low production of H2 not exceeding 13 TON was observed after 25 h whereas a negligible H2 production was observed in the presence of the non-modified (A3A3\u2032)Y26C protein or in the absence of a catalyst. These results clearly show that the production of H2 catalyzed by the cobalt(III)\u2013porphyrin was greatly enhanced by a factor of at least 10, when the latter was buried in the hydrophobic cavity of the (A3A3\u2032)Y26C protein. In addition, it is noteworthy that the TON value obtained with the biohybrid (A3A3\u2032)Y26C-4 as a catalyst was similar to that described by Ghirlanda\u2019s group using Co-cytochrome b 562 as the catalyst [4 did not show any signs of degradation and continued to show a steady production of H2. To assay the production of Hcatalyst . It is a2 catalyzed by (A3A3\u2032)Y26C-4 (7 \u00b5M) was also studied at pH 7.0 and 20 \u00b0C in a CO2 saturated potassium phosphate buffer containing 100 mM ascorbic acid as a sacrificial electron donor and 1 mM [Ru(bpy)3]2+ as a photosensitizer. 4 is replaced by the CoPPIX metalloporphyrin, a plateau at TON = 20 is reached after 7 h. In the control experiments, no CO production was observed in the presence of the non-modified (A3A3\u2032)Y26C protein as a catalyst or in the absence of a catalyst. Here also, our results clearly showed that the production of CO catalyzed by the cobalt porphyrin was greatly enhanced by a factor of 4 when the latter was inserted in the hydrophobic cavity of the (A3A3\u2032)Y26C protein. Interestingly, the TON value obtained with the biohybrid (A3A3\u2032)Y26C-4 as a catalyst was twice higher than that described by Ghirlanda\u2019s group using Co-cytochrome b 562 as the catalyst (42 TON) under similar reaction conditions : found. 577.278, calc. 577.280.MS-ESI  = found. 699.327, calc. 699.326.MS-ESI  = found: 755.235, calc. 755.239.MS-ESI Cl2 and 100 mM NaAsc, the biohybrid protein was added to lead to a final concentration of 7 \u00b5M in a total volume of 3 mL of 0.1 M phosphate buffer, pH 7. The solution was further degassed with Ar and then irradiated with a SugarCube white LED  for 25 h. During irradiation, the solution was stirred and thermostated at 20 \u00b0C with the RM6 Lauda cooling system. Gas samples from the overhead space were automatically injected into a MicroGC Fusion Gas Analyzer  every hour. For CO2 reduction, the cell was saturated with CO2 gas. All experiments were made at least in duplicate.To a degassed solution of 1 mM [Ru4 biohybrid were recorded and compared to the spectra of other Co-PPIX\u2013protein complexes previously reported in the literature. The 1 mL samples of a 7.5 \u03bcM solution of purified (A3A3\u2032)Y26C-4 were prepared in argon-sparged 10 mM HEPES, NaCl 150 mM buffer, pH 8.0 and placed in a septum-sealed cuvette to ensure anaerobic conditions. The reduction was initiated via the addition of 1.5 mM sodium dithionite. Spectra were recorded every five minutes at RT until the absorbance changes were no longer observed.Electronic absorption spectra of the oxidized and reduced (A3A3\u2032)Y26C-\u22121, a response time of 0.5 s, and a bandwidth of 2 nm using quartz cells with a 1 mm path length, on a Jasco J810 dichrograph. Each spectrum was recorded five times and averaged. The CD signal was corrected with buffer subtraction, resulting in the ellipticity (\u03b8) in mdeg. Data were acquired from a 2.2 \u00b5M sample of the biohybrid previously desalted on a Zeba spin desalting column  equilibrated with 20 mM sodium phosphate buffer, pH 7.5.Circular dichroism (CD) spectra were recorded from 185 nm to 260 nm with a data pitch of 0.2 nm, a scan speed of 50 nm min4 structure Y26C of an artificial bidomain protein named alpha Rep (\u03b1Rep). This new artificial metalloenzyme (A3A3\u2032)Y26C-4 was characterized by Maldi-TOF MS, circular dichroism, and UV\u2013Visible spectrophotometry. A comparison of the maxima of absorption of (A3A3\u2032)Y26C-4 with those of various Co(II)\u2013 and Co(III)\u2013PPIX-substituted hemoproteins Mal-PPIXMME proteins  suggeste4 biohybrid was then shown to be able to catalyze, not only the photoinduced production of H2, but also the reduction of CO2 into CO. While the Co(III)porphyrin catalyst alone showed a low activity with respect to these two reactions, more than 10 times more H2 and 4 times more CO2 were produced in the reaction, whereby the Co(III)porphyrin was buried within the hydrophobic cavity of the protein. Based on these results, we will thus be able in the future to optimize our biohybrid by carrying out different mutants for axial coordination and also to introduce positive charges on the proximal face of the biohybrid, which should increase the local concentration of CO2 in the enzymatic cavity to enhance its conversion into CO.The (A3A3\u2032)Y26C-"}
+{"text": "A rapid curing method for the preparation of colloidal photonic crystal films is presented. Firstly, a colloidal crystal array template was prepared by self-assembly of nanospheres, and then a dilute polymer solution was poured into the gap of the template. Then the composite photonic film was obtained as the polymer solution was cured. Such films have good properties in mechanical strength, anti pH interference, rapid solvent response and are easy to preserve. The films show good linear response to ethanol aqueous solutions of different concentrations, and the response equilibrium takes less than 20\u00a0s. The films also show long-term stability and reusability, and further functionalization can make the films multi-sensitive. The preparation of high-efficiency, low-cost chemical sensors is facing increasing demand in many different fields . In partStimuli-responsive polymers have interesting characteristics such as shape memory, rehydratablity and volume phase transition, as the external environment changes slightly, the polymers have noticeable and reversible changes . PrecisePoly  (PVA) is a non-toxic, biodegradable polymer with good mechanical properties, which has certain resistance to organic solvents and film-forming ability . PVA-basIn the current study, we facilely prepared an easy-to-use PVA/PC material based on the characteristics of PVA bulk film. Specifically, a CPC template was prepared by self-assembly of monodisperse nanospheres, and the template was then infiltrated with the preferred PVA solution. The composite films were formed by curing the system via simple thermal treatment. Compared with traditional hydrogel-based PC materials, the current test-paper-like films have good mechanical properties . Such fi2O2, 30%) were purchased from Shanghai Adamas Reagent Ltd. Methacrylic acid , ammonium persulfate , sodium hydroxide , PVA , ethanol (99.7%) were purchased from Sinopharm Chemical Reagent Co., Ltd. Milli-Q water (18.2\u00a0M\u03a9\u00a0cm) was used throughout the experiment.Styrene  and hydrogen peroxide solution  were vertically placed in the diluted PS suspension. The suspension was dried in an oven at 60\u00b0C for 48\u00a0h, the PS nanospheres were self-assembled onto the glass slides with the evaporation of water due to the capillary force, and the resulted CPC showed bright structural colors.One millilitre of dialyzed monodisperse PS nanospheres suspension was diluted into 100\u00a0ml with water, and then the glass slides  and stored without further treatment. The construction of the whole PVA/CPC film is shown in Particle sizes and size distribution of PS nanospheres were analyzed by Zetasizer . The morphology of the CPC template was characterized by a scanning electron microscope . The optical photos of the samples were taken by a digital camera  with a macro lens . The diffraction spectrum of the sample was captured by a fiber-optical spectrometer  with a light source . The diffraction spectrum was collected with wavelength ranges between 400 and 900\u00a0nm.S) can be calculated as:w0 is the weight of the dried PVA/CPC film, and w1 is the weight of the swollen PVA/CPC film. The film was immersed in 20\u00a0ml of ethanol aqueous solution with different concentrations for 30\u00a0min to reach swelling equilibrium, and the excess water was wiped from the surface of the PVA/CPC film with tissue paper.In order to determine the ethanol sensitivity of PVA/CPC film, the film was immersed in ethanol aqueous solutions of different concentrations, and whose diffraction spectrum was measured as the swelling equilibrium of the film was reached. The equilibrium swelling degree (E) can be calculated as:To investigate the mechanical properties of PVA/CPC film, the elongation was tested by fixing the fully swollen PVA/CPC film on the ruler with a dovetail clamp, and the film was slowly and uniformly stretched till the film broke. During the stretching process, the diffraction wavelength of the PVA/CPC film was recorded and the elongation  and low hydrolyzed (<88%); on the other hand, according to the polymerization degree, usually from 300 to 6,000, the molecular weight difference in PVA molecule is rather huge. PVA with low alcoholysis degree can be dissolved at room temperature with low viscosity, which cannot form hydrogel via physical method due to the low hydroxyl content. PVA with high alcoholysis degree and medium molecular weight cannot be dissolved in water at room temperature, but only swells. Highly hydrolyzed PVA must be dissolved in water at high temperature and the related hydrogels can be formed through the freezing method, relying on enough hydrogen bonds. Hence, in the current study, a highly hydrolyzed, medium molecular weight PVA was chosen to fabricate bulk films combined with CPC. The as-prepared PVA/CPC films are insoluble but swellable in water at room temperature, which have higher mechanical strength and is easier to preserve than PVA hydrogel-based photonic crystal materials, and also have swelling sensing properties and funtionalization potentials.As the natural light is propagated along the CPC lattice, the refractive index remains constant, and the observed diffracted wavelength is then related to the lattice parameter of the cubic unit cell, in the case of CPC, that is the nearest-neighbor distance of PS nanospheres. As can be seen in The ethanol sensing properties of PVA/CPC films were investigated. Samples prepared from PS nanospheres of 186 , 209  and 252  nm are noted PVA/CPC-186, PVA/CPC-209 and PVA/CPC-252, respectively, which were first fully swollen in water and then immersed in ethanol aqueous solution with different concentrations to reach swelling equilibrium. The refractive index of ethanol is 1.362, which is slightly higher than that of water (1.333). Therefore, with the increase of ethanol concentration, the average refractive index of the solution increased accordingly. On the other hand, the equilibrium swelling degree of the PVA polymer film changed due to the change of free energy of mixing. According to Flory-Rehner theory , during In addition, the accuracy, response time and sensitivity of PVA/CPC film to ethanol, which are essential parameters, were also tested. Firstly, the diffraction peak shifts at the concentration of 0\u2013100% ethanol aqueous solution were tested repeatedly. It can be seen in As shown in Then, the reusability of sensors is investigated to evaluate the performance of the PVA/CPC sensor. The sensor film was alternately immersed in pure water and ethanol aqueous solutions with different concentrations  to detect whether the sensor could be reused. Although the PVA/CPC film sensor was constructed with biocompatible PVA material, the biocompatibility was also examined by cell proliferation activity and fluorescence micrographs observation. As presented in A simple preparation method of non-hydrogel polymer-based photonic crystal film was presented. The film was prepared by combining biocompatible PVA and structural color reflecting colloidal photonic crystal structure. The difference in swelling properties of PVA in water and ethanol leads to the difference in equilibrium swelling degree. Thus the film can be utilized as an ethanol sensor, and the color change of which can directly be distinguished by the naked eye. The sensor showed a good linear relationship with diffraction wavelength and ethanol concentrations with rapid response within 20\u00a0s. It also proved with good accuracy , repeatability (for at least six cycles), and stability (anti-interference in various pH)."}
+{"text": "Conclusion: Cognitive decline was detected in almost one third of adults with JIA after 24 months of follow-up. Systemic inflammatory activity in JIA patients was related to cognitive decline. Patients treated with biologics had a lower risk of decline in cognitive functions.Objective: To prospectively evaluate possible decline of cognitive functions in adult patients with juvenile idiopathic arthritis (JIA) and identify associated factors. Patients and methods: We performed a 24-month prospective observational study of adults (\u226516 years) with JIA. The primary outcome measure was decline in cognitive function defined as a worsening of \u22652 points on the scales of the subsets administered to evaluate the different cognitive areas using the Wechsler Adult Intelligence Scale (WAIS) after 24 months: attention/concentration (digit span); verbal function (vocabulary); visual-spatial organization (block design); working memory (letter-number sequencing); and problem solving (similarities). Other variables included average inflammatory activity using C-reactive protein and composite activity indexes, comorbidity, and treatment. Logistic regression was performed to identify factors associated with cognitive decline. Results: The study population comprised 52 patients with JIA. Of these, 15 (28.8%) had cognitive decline at V24. The most affected functions were working memory (17.3%), attention/concentration (9.6%), verbal function (7.7%), visual-spatial organization (7.7%), and problem solving (3.8%). There were no significant differences in the median direct or scale scores for the cognitive functions evaluated between V0 and V24 for the whole sample. The factors associated with cognitive decline in patients with JIA were average C-reactive protein (OR [95% CI], 1.377 [1.060\u20131.921];  Juvenile idiopathic arthritis (JIA) comprises the most frequent group of inflammatory rheumatic disorders in children and can lead to major morbidity and disability in the short and long terms . JIA incCognitive decline can be defined as the objective worsening of memory and other cognitive functions compared with a previous state. Cognitive decline is a physiological process that occurs with aging but also is pathologic when the worsening is greater than expected for normal aging with different functional repercussions in daily activities (mild cognitive impairment and dementia). Cognitive decline and cognitive impairment are comorbid conditions that can affect functioning and quality of life in patients with chronic joint diseases . SeveralWe performed a 24-month prospective, observational study of a cohort of patients with JIA. Recruitment ran from September 2019 to February 2022. The study was approved by the Research Ethics Committee of Hospital Regional Universitario de M\u00e1laga (HRUM) (Code 2627-N-21). All the subjects gave their written informed consent to participate.We recruited patients aged \u226516 years with JIA classified according to the criteria of ILAR 2001  who wereBaseline in 2019 (V0) and at 24 months from the initial evaluation (V24) , all of Two rheumatologists collected the clinical data and physical examination. One of the rheumatologists performed the physical examination of the patients and the other rheumatologist administrated WAIS subtests. The neuropsychological evaluation was corrected jointly by a neurologist and a neuropsychologist. The neuropsychological evaluation was performed using a series of tests following the appropriate manual. These included the Wechsler Adult Intelligence Scale  and the Beck Depression Inventory-II (BDI-II). All the tests were administered in the same clinic based on the same timetable as the first evaluation. Before administration of the neuropsychological battery, all patients underwent the Nine-Hole Peg Test to assess their manual dexterity.WAIS-III is the fifth version of the Wechsler Adult Intelligence Scale. It comprises 13 subsets divided between 2 scales: the verbal scale, with the subtests vocabulary, similarities, arithmetic, digit span, information, comprehension, and letternumber sequencing; and the performance scale, including picture completion, digit symbol coding, block design, matrix reasoning, picture arrangement, and symbol search . Some ofThe Beck Depression Inventory (BDI) 21-item self-assessment instrument is used for evaluating depressive symptoms. According to the score, depression can be classified as normal (0\u201313), mild (14\u201319), moderate 20\u201328), and severe (29\u201363) ), and smoking . Traditional cardiovascular risk factors were also recorded. Arterial hypertension was defined as blood pressure \u2265140/90 mmHg or treatment with antihypertensive medication [2) [We recorded demographic, laboratory, and treatment-related data. V24 was considered the cut-off for including patients in the study and V0 the baseline date for evaluation of cognitive function in 2019. The demographic and clinical variables included age (years), sex , educational level , body mass index : height [mdication . Diabetedication . Dyslipition [2) .The characteristics of patients with a JIA included date of onset of the disease , diagnostic delay (months from onset of symptoms to diagnosis of JIA), uveitis, and the following laboratory variables: rheumatoid factor (RF), positive if >20 IU/mL; anticitrullinated peptide antibody (ACPA), positive if >10 IU/mL; HLA27 (positive/negative); antinuclear antibody (ANA), positive if >1/80 at any point during the disease. We recorded all previous drugs, current medication, and changes in medication during the prospective follow-up, including disease-modifying antirheumatic drugs (DMARDs). These included the following: conventional synthetic DMARDs (csDMARDs), such as methotrexate, leflunomide, and sulfasalazine; and biologic DMARDS (bDMARDs), such as tumor necrosis factor inhibitors (anti-TNF), tocilizumab, abatacept, rituximab, ustekinumab, and Janus kinase inhibitors . We also recorded corticosteroids.2 test; the t test was used for quantitative variables. Finally, we ran a multivariate logistic regression analysis to identify those factors associated with cognitive decline in JIA. We used the same sample as in the evaluation in 2019, whose size was calculated based on data for patients with RA, owing to the lack of data in adults with JIA [First, we performed a descriptive analysis of the main patient-related variables. Qualitative variables were expressed as number and percentage. Quantitative variables were expressed as mean \u00b1 standard deviation (SD) for normally distributed data and as median \u00b1 interquartile range (IQR) for non-normally distributed data. The normality of the distribution of continuous variables was confirmed using the Kolmogorov\u2013Smirnov test. The main variables of cognitive function were compared after 24 months of follow-up (V0 vs. V24). We studied the characteristics of patients with JIA according to the degree of cognitive decline. Hypotheses for qualitative variables were tested using the Pearson \u03c7with JIA . Data weBetween September 2019 and February 2022, we prospectively followed 52 patients with JIA. There were no losses during recruitment or follow-up or exclusions . During p = 0.001), and although no differences were observed for most cardiovascular risk factors, more patients were smokers (p = 0.010) and were overweight or obese (p = 0.015). As for clinical-laboratory characteristics, more than half of the patients had longstanding oligoarticular JIA, and 40% had positive ANA titers. There were no significant differences at V24 compared with V0 in inflammatory activity assessed using JADAS27 (p = 0.751). The patients had a median JADAS27 of around 4.0 and an average CRP of 3.4 mg/dL (The main characteristics at V0 and at V24 are shown in .4 mg/dL .p = 0.785), bDMARDs (p = 0.182), or corticosteroids (p = 0.103). During follow-up, two patients suspended leflunomide owing to intolerance, and another patient switched leflunomide for sulfasalazine because she wished to become pregnant, although she did not become pregnant during this period. As for bDMARDs, three patients started a new treatment: one patient initiated adalimumab, one certolizumab, and one secukinumab. One patient discontinued tofacitinib owing to inefficacy and switched to golimumab.At V24, 20/52 patients (38.4%) were receiving monotherapy with bDMARDs, 13/52 (25.0%) were receiving monotherapy with csDMARDs, 11/52 patients (21.1%) were receiving combination therapy, and 8/52 patients (15.3%) were not receiving DMARDs because their symptoms were controlled . There wp = 0.035), and more patients fulfilled the criteria for depression (p = 0.046).A total of 15 patients (28.8%) had cognitive decline with worsening \u22652 scale points in the subtests administered to evaluate different cognitive areas. Cognitive decline was recorded for 10/15 patients (66.6%) in 1 subtest, for 4/15 patients (26.6%) in 2 subtests, and 1/15 patients (6.6%) in 3 subtests. The cognitive tests showing decline with respect to V0 in a larger percentage of patients were digit span (11.5%) and letter-number sequencing (9.6%), followed by vocabulary (7.7%), block design (7.7%), and, finally, similarities (3.8%) . Considep = 0.045), had a higher CRP value at baseline , and were less frequently taking biologics .p = 0.030), with more patients educated to secondary and university level among those who did not experience cognitive decline than among those who did. Furthermore, compared to the other patients, those with cognitive decline had a higher median (IQR) BMI (p = 0.032), a higher average CRP (p = 0.002), and a higher average JADAS27 (p = 0.048); fewer of these patients were receiving biologics (p = 0.016). Depression was more frequent in patients with cognitive decline (p = 0.001) and BDI scores were higher (p = 0.027).At the end follow-up, (V24) were found to have significant differences in educational level (p = 0.980), or at the end of follow-up .In patients with cognitive decline compared with the other patients, the median (IQR) of corticosteroids (grams) did not show significant differences with the baseline values  scores for the evaluation of cognitive decline. However, we have been able to describe our main objective of observing cognitive changes in the group of patients with JIA and associated factors. Albeit there were no significant changes in overall cognitive function of the sample, we found an individual cognitive decline in several patients that was significantly associated with several variables. To avoid variance bias in psychometric testing , we used a scaled score (adjusted by age) and considered decline when the variation is of 2 or more points and not in a single scaled score. Also, the clinical and treatment between V0 and V24 clearly can affect the neurological response; for this, we calculated the average inflammatory activity throughout the follow-up period using the mean JADAS27 and C-reactive protein values (mg/dL), and only seven patients (13%) changed treatment during follow-up. As we were unable to compare our results with those of similar studies, we made the comparison using data for other types of chronic arthritis, such as RA. Nevertheless, our study is the first to evaluate prospectively cognitive function in adults with JIA and these data are interesting to consider future clinical trials of biological drugs evaluating cognitive function in these patients. A very long-term follow-up would be recommended to determine if these patients present a higher risk for mild cognitive impairment or dementia. Additionally, it would be interesting if patients with cognitive decline improve in the follow-up and are related to improvement in RPC values or the use of anti-TNF. We must also remember that the SARS-CoV-2 pandemic may have led to greater impairment of patients\u2019 mood, although we identified other variables that were independently associated with cognitive decline in affected patients.In conclusion, almost one third of adult patients with JIA had cognitive decline after 24 months of follow-up. Executive functions seem to be more affected in this population. In adults with JIA, cognitive decline was directly associated with average inflammatory activity measured based on C-reactive protein and with depression. Decline was less frequent in patients receiving biologics. Our findings highlight that management of joint involvement should be accompanied by management of inflammatory activity and mental health. Given that inflammation may initiate and maintain cognitive decline in patients with JIA, cognitive functioning should be also assessed in these patients. Future studies should be performed to determine the mechanism of how inflammation affects cognition in young patients with JIA."}
+{"text": "Cucumis sativus L. var. Krak) seedlings exposed to salinity. RBOH activity and gene expression, as well as H2O2 content, were determined in the roots of plants treated with 50 or 100 mM NaCl for 1 h, and 50 mM NaCl for 1 or 6 days. It was found that enzyme activity increased in parallel with an enhancement in the H2O2 level in roots exposed to 100 mM NaCl for 1 h, and to 50 mM NaCl for 1 day. The expression of some CsRboh genes was induced by salt. Moreover, an increase in the activity of G6PDH, providing the substrate for the NADPH oxidase, was observed. In seedlings subjected to salinity for a longer time, antioxidant enzymes\u2014including superoxide dismutase, catalase, and ascorbate peroxidase\u2014were activated, participating in maintaining a steady-state H2O2 content in the root cells. In conclusion, NADPH oxidase and endogenous H2O2 up-regulation seem to be early events in cucumber response to salinity.Plasma membrane NADPH oxidases  are known as the main ROS generators involved in plant adaptation to stress conditions. In the present work, regulation of NADPH oxidase was analyzed in cucumber ( On the other hand, when present in low concentrations, they are essential signaling molecules involved in the regulation of multiple physiological processes that affect plant growth and development [Arabidopsis and barley Rboh mutants display impaired growth [In plants, reactive oxygen species (ROS), including the superoxide radical  motifs. They catalyze an electron transfer from cytosolic donor NADPH to extracellular acceptor O to H2O2 . Studies binding . More recucumber ,6. Diffeisoforms .2O2 generation can also be mediated by oxidative degradation of polyamines (PAs) [2O2 [2O2 pool appears. It has been suggested that H2O2 generated as a product of PA oxidation may be directly involved in cell signaling processes, as well as in plant adaptation to abiotic stresses [In addition to the plasma membrane NADPH oxidases, apoplastic Hes (PAs) . The celAs) [2O2 . Consequstresses . It is wstresses .2+ levels [+/H+ antiporter, AtSOS1, mRNA [+/K+ homeostasis via activation of plasma membrane Ca2+-permeable channels as well as inwardly rectifying K+ channels [In their natural environment, plants are exposed to unfavorable factors, including drought, salt, and extreme temperatures, which are perceived by cells. Tolerance of plants to salinity is governed by numerous physiological and molecular adaptive mechanisms . Some of+ levels , the staS1, mRNA , the regchannels , and NaCchannels .2O2, and thereby activating tolerance mechanisms [2O2 generation. H2O2 produced by PA catabolism may activate antioxidative defense responses [2O2 and superoxides O2\u2212. Both enzymes are a part of the same ROS regulatory network, in which NADPH seems to act upstream of apoplastic PAO [Other studies have revealed that salinity induces the PA catabolism pathway into the apoplast, generating Hchanisms . Tanou, chanisms , observeesponses . More reesponses . RBOH anstic PAO .2O2 level, and its interrelation with antioxidant enzymes  in the roots of cucumber seedlings exposed to salinity. Roots are the first organ exposed to unfavorable factors that get into plants from the soil. It was expected that under conditions of different time-exposure to salt, the plasma membrane NADPH oxidase activity would be modulated in a manner dependent on its gene expression and substrate (NADPH) delivery, and that, consequently, the content of the H2O2 would be modified. Moreover, it was assumed that changes in NADPH oxidase activity are related to the action of antioxidant enzymes.The aim of the present study was to understand the role of the plasma membrane NADPH oxidase in the regulation of the HCucumis sativus L. var. Krak) seeds  were germinated in darkness at 25 \u00b0C for 2 days. Seedlings were grown in containers (one container for each treatment) filled with nutrient solution (pH 6.5) consisting of 1.7 mM KNO3, 1.7 mM Ca(NO3)2\u00b74H2O, 0.33 mM KH2PO4, 0.33 mM MgSO4\u00b77H2O, and micronutrients: 75 \u03bcM ferric citrate, 10 \u03bcM MnSO4\u00b75H2O, 5 \u03bcM H3BO4, 1 \u03bcM CuSO4\u00b75H2O, 0.01 \u03bcM ZnSO4\u00b77H2O, 0.05 \u03bcM Na2MoO4\u00b72H2O without (control) or with the addition of salt. The plants were treated with 50 or 100 mM NaCl for 1 h immediately before analysis , 50 mM NaCl for 1 day  or 50 mM NaCl for 6 days . Concentration of 5 mM H2O2 was added to the control nutrient solution for 24 h. The plants were grown hydroponically under a 16-h photoperiod at temperatures of 25 \u00b0C/22 \u00b0C (day/night), photon flux density 180 mol m\u22122s\u22121, and about 70% relative humidity. Whole roots were collected from all the cucumber plants in the container , and used for analysis.Cucumber , was measured at 390 nm.H, et al. , with so, et al. , based oThe plasma membrane fractions were isolated from the cucumber roots in accordance with the method of Larsson , as modi2\u2212 radical was determined at 470 nm in the presence and absence of 50 units of CuZn-SOD. NADPH oxidase in-gel assays were performed according to the method described by Sagi and Fluhr [w/v) polyacrylamide gel. Electrophoresis was conducted at 110 V and 20 mA for 60 min. The NADPH-dependent O2\u2212 production was assayed in gel by a modified NBT (nitroblue tetrazolium) reduction method. After the NADPH addition, the appearance of blue formazan bands was monitored.The catalytic activity of the NADPH oxidase EC 1.6.3.1) was determined in the plasma membrane fractions, according to the modified method of Sagi and Fluhr , as desc.3.1 was nd Fluhr . The pla3 mol\u22121 cm\u22121).Diamine oxidase  activity was assayed in seedling roots spectrophotometrically, using putrescine as a substrate, in accordance with the method of Holmsted, et al. , as modiThe activities of enzymes generating NADPH, 6PGDH , G6PDH , NADP-ICDH , and NADP-ME , were determined in the cucumber roots in accordance with the method of Li, et al. , as desc2O2 decomposition was monitored spectrophotometrically by measuring the reduction in absorbance at 240 nm. One unit of CAT is defined as the amount of enzyme that breaks down 1 \u00b5mol of H2O2 per min.Catalase  activity was determined according to the method of Aebi , as desc2O2-dependent oxidation of ascorbic acid was followed by monitoring the decrease of absorbance at 290 nm, assuming an absorption coefficient value of 2.8 mM\u22121 cm\u22121. One unit of APX is defined as the amount of enzyme that oxidizes 1 \u00b5mol of ascorbic acid per min.Ascorbate peroxidase  activity was determined in accordance with the method of Chen and Asada , as descSuperoxide dismutase  activity was measured in accordance with the method (xanthine/xanthine oxidase test) of Beauchamp and Fridovich . The phoProtein content was determined according to the method of Bradford , using bThe total RNA was isolated from the cucumber roots using Tri Reagent , and cDNA was synthetized with a High-Capacity cDNA Reverse Transcription Kit , in accordance with the manufacturer\u2019s instructions, as described by Jakubowska, et al. .CsTIP41, and the clathrin adaptor complex subunit, CsCACS, were used as the internal standards [The expression profile of the NADPH oxidase and SOD encoding genes in the cucumber roots was analyzed with the LightCycler 480 system . The cDNA was used as a template in a real-time PCR reaction with the Real-Time 2\u00d7 PCR Master Mix SYBR kit , performed in accordance with the manufacturer\u2019s instructions, as described by Jakubowska, et al. . The amptandards . Primer p < 0.05) were used for statistical analysis, performed with Statistica 13.3 .All data presented are expressed as the means of at least three biological replicates  \u00b1 standard deviation (SD). One-way ANOVA and Duncan\u2019s post hoc analysis  was observed  and polyamine oxidase (PAO) appeared to be the other enzymes responsible for Hd plants A,B. On tCsCSD1, 2, and 3 belonging to Cu/Zn-SODs, CsMSD representing Mn-SODs as well as CsFSD2 and 3 identified as Fe-SOD\u2014indicated that the expression of one of them, CsMSD, was upregulated about twice in seedlings exposed to 50 mM NaCl for both 1 h and 6 days. On the other hand, the transcript level of CsCSD2 significantly increased  only after plant exposure to salt for 6 days (The activity of the plasma membrane NADPH oxidase is related to the action of the superoxide dismutase (SOD). It was found in cucumber that SOD activity was enhanced by salt, similarly to NADPH oxidase. However, a significant increase, by about 60%, was observed in the roots of the plants treated with salt for 6 days A,B. Analr 6 days C,D.The activity of NADPH oxidase can be dependent on NADPH as its metabolic substrate. For this reason, the role of four NADPH-generating enzymes, i.e., glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-isocitrate dehydrogenase (NADP-ICDH), and NADP-malic enzyme (NADP-ME), was examined in cucumber plants exposed to salinity . Among t2O2-metabolizing enzymes, including catalase (CAT) and ascorbate peroxidase (APX). In the cucumber root tissues, CAT activity was reduced by about 30% after one day of plant treatment with 50 mM NaCl, as compared to the control plants. Longer plant exposure to this salt concentration (6 days) caused a significant increase in enzyme activity, which achieved above 150% of the control value. CAT activity did not change significantly in the roots treated with salt for 1 h  exhibit increased Na+ hypersensitivity, and lack of ROS accumulation in the xylem vessels [Although knowledge about salinity sensing by plant cells is still limited, several sensory mechanisms have been proposed . Moreovemulation ,35. Arab vessels ,36. Thus vessels .Brassica juncea , and that it remains unaffected in a halophyte Sesuvium portulacastrum  under salt stress conditions. Decrease in the ROS level related to inhibited RBOH activity has been shown in maize seedlings treated with NaCl [2O2 content was significantly lower in salt-treated tissues compared to the controls. This correlated with decreased NADPH oxidase activity and down-regulation of RBOH gene transcription [In contrast to the above results, NaCl can also negatively affect the NADPH oxidase. Srivastava, et al. , have deith NaCl . Similarcription .2O2 level occurred in cucumber roots stressed with 100 mM NaCl for 1 h and 50 mM NaCl for 1 day. At the same time, NADPH oxidase activity was significantly stimulated. The observed increase in enzyme activity was related to the enhanced expression of two Rboh genes, CsRbohD and CsRbohF1. Similarly, Niu, et al. [2O2 content is rapidly elevated in the roots of salt-treated pumpkin-grafted cucumber plants, reaching a peak at 3 h and decreasing during the 3\u201312 h period. This increase is related to the NaCl-induced NADPH oxidase activity, as well as enhanced expression of RbohD and RbohF transcription level.Our results indicate that an enhanced H, et al. , using 72O2 content required for cell wall loosening and leaf growth [2O2 generation in the apoplast in cucumber roots under salt stress. As we demonstrated earlier, the H2O2 content only slightly increased in 1-day stressed roots, whereas it was very high in 6-day treated roots of Cucumis sativus var. Wisconsin under salinity condition [2O2 production in the cell wall of stressed plants. In contrast, salt stress was found to both strongly promote DAO activity and to stimulate polyamine degradation in the root tissues of maize seedlings [2O2 production, depending on the plant species/cultivar and its sensitivity to salt as well as treatment conditions.The generation of ROS in the apoplastic space could be related not only to the activation of the cell membrane-bound RBOH but also the apoplastic polyamine or diamine oxidases . In saltf growth . In our ondition . It is iondition , using tondition . The auteedlings  and lupieedlings . Thus, iArabidopsis has revealed some regulatory mechanisms of RBOH proteins, which depend on different signaling effectors. Thus, it has been suggested that differences in regulatory mechanisms may be responsible for diverse RBOH functions in plant cells [+ accumulation followed by Ca2+ elevation can lead to the activation of RBOH and, consequently, to the accumulation of apoplastic H2O2 [2O2 clearly activated NADPH oxidase, indicating that this enzyme remains under feedback control. However, the observed effect did not include the gene expression level, suggesting some post-translational regulatory mechanisms or additional signaling events, such as nitrosylation [2S-dependent persulfidation [The activity of plasma membrane NADPH oxidase can be regulated in several ways . Researcnt cells . A few stic H2O2 . In our sylation , H2S-depfidation , and revfidation .Arabidopsis seedlings, the NADP-ICDH, which activity increased, appeared to act as a barrier in the defense mechanisms activated against salt stress [A. thaliana exposed to high salinity, the cytosolic G6PDH isozyme (G6PD6) was subjected to phosphorylation, and this modification was responsible for the observed enzyme activation [Arabidopsis mutants revealed that both the G6PD enzymatic activity and the expression of G6PD5 and G6PD6 decreased in atrbohD1, atrbohF1, and atrbohD1/F1 mutants in comparison to the WT plants, confirming the involvement of cytosolic G6PDH in RBOH-dependent ROS generation [Substrate availability appears to be another factor involved in controlling NADPH oxidase activity. Several enzymes are responsible for NADPH delivery function in plant cells . It has t stress . In contt stress . Among tt stress . As it wtivation . Studiesneration . Scharteneration , have pr2\u2212 radicals, which are transformed by superoxide dismutases to produce less reactive H2O2. Related to the metal cofactor at the active center, three distinct isoenzymes function in plant cells: Mn-SODs are present in the mitochondria and peroxisomes; Cu/Zn-SODs mainly in the cytosol, mitochondria, and plastids; while Fe-SODs are localized in the cytosol, mitochondria, chloroplasts, and peroxisomes [CsMSD and CsCSD2 expression increased significantly in the roots of seedlings exposed to 50 mM NaCl for 6 days, when NADPH oxidase activity and H2O2 level were lowered to nearly control values. This suggests that hydrogen peroxide generated by SOD may be effectively decomposed by H2O2-metabolizing systems. Hossain, et al. [2O2 content is always significantly lower in the salt-treated leaves in comparison to the control. This was correlated with the suppressed NADPH oxidase activity and gene expression (with the exception of RBOHB), as well as with the up-regulation of SOD activity and increased transcription of genes encoding this enzyme (with the exception of FeSOD1). Such a system, together with enhanced expression of genes for alternative oxidase and plastid terminal oxidase, is responsible for maintaining the low ROS level under severe salinity [Plasma membrane-localized NADPH oxidases are a major source of cellular Ooxisomes . Many stoxisomes . In cucu, et al. , have shsalinity .2O2 acts as a signal to induce antioxidant systems and prevent subsequent ROS generation and ROS-dependent cell damage [Arabidopsis under short-term salt treatments, RbohD and RbohF were suggested to be required in this process [2O2 are catalase, with the highest turnover rates, and ascorbate peroxidase, exhibiting higher affinity for H2O2 than CAT [2O2 level under prolonged salt exposure. In contrast, catalase activity was inhibited in roots treated with salt for 1 day. Stimulation of both APX and CAT in response to NaCl has been reported in many plant species. It has been shown to be associated with decreased oxidative damage and improved tolerance to salinity. Similar to SOD, a greater increase in CAT activity was observed in salt-tolerant plants/genotypes than in more sensitive ones [It is assumed that early production of Hl damage . In Arab process . The maithan CAT ,61. In aive ones . Furtherive ones .Cucumis sativus var. Krak) seedlings. This activation correlated with an observed enhancement in H2O2 content, suggesting the involvement of NADPH oxidase in the generation of H2O2, which may function as a signaling molecule participating in stress response. The observed stimulation of NADPH oxidase may have been at least partially related to the induction of some CsRboh genes, as well as the increased activity of G6PDH, providing NADPH. Another factor which may contribute to positive RBOH regulation at the post-translational level, could be H2O2. This proposal, however, requires additional studies concerning direct or indirect H2O2 action.At the early stages of salt stress (1 h and 1-day-treatment), the plasma membrane NADPH oxidase was activated in the cucumber (2O2 level, were diminished. This protected the cells against the toxic effect of high H2O2 concentrations. Additionally, antioxidant enzymes, including SOD, APX, and CAT, were significantly activated to maintain low H2O2 content in cells, and to avoid ROS-dependent damages. However, to explain the precise mechanism of NADH oxidase regulation, and its interrelation with the antioxidant system in cucumber seedlings subjected to salinity, further analysis is needed.After 6-day exposure to salt stress, the plasma membrane NADPH oxidase activity, and consequently the H"}
+{"text": "Here, we report quantitative reporters for Mpro function in living cells in which protease inhibition by genetic or chemical methods results in robust signal readouts by fluorescence (enhanced green fluorescent protein [eGFP]) or bioluminescence (firefly luciferase). These gain-of-signal systems are scalable to high-throughput platforms for quantitative discrimination between Mpro mutants and/or inhibitor potencies as evidenced by validation of several reported inhibitors. Additional utility is shown by single Mpro amino acid variants and structural information combining to demonstrate that both inhibitor conformational dynamics and amino acid differences are able to influence inhibitor potency. We further show that a recent variant of concern (Omicron) has an unchanged response to a clinically approved drug, nirmatrelvir, whereas proteases from divergent coronavirus species show differential susceptibility. Together, we demonstrate that these gain-of-signal systems serve as robust, facile, and scalable assays for live cell quantification of Mpro inhibition, which will help expedite the development of next-generation antivirals and enable the rapid testing of emerging variants.The main protease, M For activity , 13. Catectively . Inhibitpro activity. Cellular systems also add value by reflecting other key parameters of small-molecule development, including membrane permeability, metabolic liability, and off-target effects, including toxicity. A common live cell assay used currently is based on the FlipGFP concept  and reconstitution of the full GFP \u03b2-barrel and fluorescence emission. However, this assay has yielded variable signal/noise ratios in multiple different labs  fluorescence or luciferase luminescence as experimental readouts. Amino acid changes that disrupt Mpro function, including catalytic, allosteric, and dimerization mutants, elicit substantial increases in fluorescent or luminescent signal compared to the fully active wild-type enzyme. Similarly, treatment of cells expressing the wild-type Mpro reporter with known inhibitors of Mpro causes dose-responsive increases in signal, with the luciferase readout showing a 100-fold dynamic range, sensitivity into the low-nanomolar range, and adaptability to a 1,536-well plate format for HTS. This system was used to test the impact of amino acid changes surrounding the active site and identify a circulating variant with increased susceptibility to boceprevir but unchanged susceptibility to GC376, which could be explained by the flexibility of GC376 binding to Mpro in crystal structures. Testing of a broader panel of reported inhibitors underscores the high stringency of the assay for ruling out likely off-target molecules and the utility for rapid testing of variants of concern against clinical-grade antivirals. Together, these results illustrate the broad utility of our assay for aiding in the development of Mpro antiviral compounds and understanding the cis-acting determinants that impart Mpro activity.Here, we demonstrate highly sensitive and reproducible gain-of-signal assays for quantifying genetic or chemical inhibition of SARS2 Mtrans-activator of transcription (Tat) to bind to an integrated HIV-1 long terminal repeat sequence to activate transcriptional elongation of a downstream reporter such as eGFP or firefly luciferase. As the transcriptional function of Tat relies on nuclear localization, we hypothesized that if Tat could be sequestered in the cytoplasm by an Mpro-cleavable cytosolic membrane anchor such as the N-terminal myristoylation domain from the Src kinase, then Mpro-catalyzed cleavage during infection would cause relocalization of Tat to the nucleus to activate expression of a reporter construct. However, before testing this concept with infectious virus, we sought to determine whether cis-cleavage of an in-frame Mpro would result in the anticipated relocalization phenotype.These studies initiated with the goal of developing a cell line that would be able to report SARS2 infection analogous to established HIV-1 assays , 25. Reppro amino acid sequence with cognate N- and C-terminal self-cleavage sites, HIV-1 Tat, and eGFP  (pro (C145A or H41A) resulted in high levels of eGFP fluorescence, suggesting that proteolytic activity is responsible for the apparent lack of expression of the WT construct. In support of this interpretation, anti-GFP immunoblotting also indicated no reporter expression in cells expressing the WT construct; however, both catalytic mutants yielded a single band at the predicted molecular weight of the full-length Src-Mpro-Tat-eGFP polyprotein  . Surprisization)  and c. Hyprotein .pro is active as a homodimer, and mutations that disrupt dimerization also abrogate catalytic activity  values of 0.03 to 0.19\u2009\u03bcM and 1.6 to 8.0\u2009\u03bcM, respectively  of Tat (pro-Tat-eGFP protein at high drug concentrations (pro-Tat-eGFP band by immunoblotting extracts from cells treated with 10\u2009\u03bcM GC376 (At high concentrations of GC376 (100\u2009\u03bcM), the subcellular localization of the WT construct phenocopies the C145A catalytic mutant with cytoplasmic membrane targeting due to the N-terminal myristoyl anchor . However) of Tat   following incubation with DMSO or 10 \u03bcM GC376. A parallel anti-\u03b2-actin blot was done as a loading control. Download FIG\u00a0S1, PDF file, 0.4 MB.Accumulation of the shorter Tat-eGFP fragment requires a functional SARS2 MCopyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pro inhibitors, we next asked whether assay sensitivity might be increased by switching the readout from eGFP to firefly luciferase, which is capable of providing an enzyme-catalyzed signal amplification (pro inhibition (Z\u2032 score of 0.70 using 10\u2009\u03bcM GC376 as a positive control (To be able to detect lower-potency Mfication . As antification  and f. Mfication a and b.  control  to e.10.1128/mbio.00784-22.2FIG\u00a0S2pro inhibition. (a) GC376 dose responsiveness of the WT Luc-based reporter expressed in 293T cells. All values normalized to the luminescence of the DMSO-treated condition . (b) Mean signal to background (S/B) ratios for the experiment shown in panel a showing a 100-fold dynamic range. (c) Schematic of the 1,536-well plate experimental setup in which Luc-based SARS2 Mpro reporter-expressing cells are subjected to dose-response testing with GC376 and boceprevir. A representative luminescent image is shown below captured using the ViewLux microplate imager. (d and e) Histograms comparing the 1,536-well format dose responsiveness of the Luc-based reporter to GC376 and boceprevir, respectively . Download FIG\u00a0S2, PDF file, 2.5 MB.Sensitivity and miniaturization of Luc-based reporter system for MCopyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pro catalytic activity, we next asked whether it might be compatible with proteases from distantly related coronaviruses. The Mpro enzymes from two human-infecting alpha-coronaviruses, HCoV-229E and HCoV-NL63, were therefore analyzed with flanking cognate cleavage sites in the context of the luciferase-based reporter  Overview of the Mpro dimer, with GC376 bound in the active site shown in yellow sticks. (b) 2mFo-DFc electron density map contoured at 1.0 \u03c3 shown for the active site. (c) Superposition between our Mpro-GC376 structure  and complexes reported earlier. Structures and corresponding PDB ID numbers are color coded. (d) Multiple amino acid sequence alignment for the Mpro enzymes of the indicated coronaviruses . SARS2 Mpro M49, P168, and Q189 are boxed, and identical residues are highlighted with blue. (e) Histogram showing background levels of luminescence for the indicated Luc-based SARS2 Mpro mutant constructs. Significantly increased luminescence indicates weakened protease activity . Download FIG\u00a0S3, PDF file, 1.1 MB.Crystal structures of GC376-bound SARS2 MCopyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pro reporter system to ask how single amino acid changes might affect inhibitor efficacy. Guided by existing cocrystal structures of GC376 and boceprevir . In contrast, boceprevir inhibited wild-type Mpro with an IC50 of 14\u2009\u03bcM, and P168G and P168S showed approximately 10-fold lower IC50 values of 1.6\u2009\u03bcM and 2.1\u2009\u03bcM, respectively, consistent with the cell-based results above . P168G has yet to be observed in circulating isolates, likely because this substitution requires the simultaneous occurrence of two different nucleotide changes. Taken together, we propose that P168 single amino acid substitutions increase the flexibility of the Mpro active site and improve binding to boceprevir but not to GC376. Most importantly, these results highlight the usefulness of the luciferase-based system described here for rapidly and quantitatively comparing the drug susceptibilities of Mpro amino acid variants , and they also underscore the need to carefully compare results from assays in living cells and biochemical assays in vitro with purified enzymes.To further investigate the apparent heightened Mby GC376  and g (Its above . The selts above . It is t10.1128/mbio.00784-22.5FIG\u00a0S5pro. (a) Cell viability in response to GC376 treatment in cells transfected with reporter relative to a DMSO-treated control . (b) Schematic of a two-color reporter construct encoding Src-Mpro-Tat-eGFP driven from a CMV promoter and mCherry driven from an SV40 promoter  and signal fold change of eGFP and mCherry in response to GC376 as measured by flow cytometry with background signal normalized to 1 . (c) Schematic of the construct with an antisense U6 promoter (RNA PolIII) driving expression of a firefly luciferase fragment in addition to the sense-oriented CMV-driven Src-Mpro-Tat-eGFP (pT = RNA PolIII transcription termination sequence) with quantification of Src-Mpro-Tat-eGFP mRNA relative to the U6-driven transcript in the cytosolic fraction of 293T cells transfected with either WT plasmid (\u00b150 \u03bcM GC376) or the C145A mutant plasmid. The RNA ratio under WT conditions is normalized to 1 for comparison . Download FIG\u00a0S5, PDF file, 0.3 MB.Evaluating a general mechanism of expression inhibition caused by MCopyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pro inhibitors have gone through rigorous characterization and structure-activity relationships have been established, other candidates have been identified through high-throughput screens in drug repurposing efforts, and on-target efficacies remain controversial  of action of these putative Mpro inhibitors and suggest that the reported antiviral activities of these compounds are likely to be nonspecific.In contrast to the 5 different compounds that inhibited SARS2 Mtrations . Carmofue enzyme , 47, 48.tivation . Last, mtivation . To ensutivation . As our 10.1128/mbio.00784-22.8TABLE\u00a0S1Table\u00a0S1, PDF file, 0.08 MB.Inhibitor sources. Download Copyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pro inhibitor (https://www.fda.gov/media/155049/download), we assessed the broader-spectrum potential of this drug using distantly and closely related coronavirus variants. Interestingly, nirmatrelvir showed much stronger activity against the Mpro enzyme of 229E in comparison to that of NL63, despite the fact that these two alpha-coronavirus enzymes are closely related (71% identity) (pro (P132H), retains sensitivity to nirmatrelvir even at low-nanomolar concentrations, which bodes well for clinical application  . These rlication .pro by chemical inhibition or mutational inactivation. The original reporter, Src-Mpro-Tat-eGFP, shows very low protein expression, which can be rescued by mutating residues required for protease function . These genetic results suggested that reporter signal is inversely related to Mpro function and that chemical inhibitors may be similarly effective at recovering signal. Indeed, SARS2 Mpro inhibitors, GC376 and boceprevir, both cause a dose-dependent restoration of reporter signal, with the former compound being more effective, consistent with prior work  that does not significantly change the potency of nirmatrelvir  experiments requiring pathogenic viral isolates. Gain-of-signal assays have advantages over loss-of-signal assays because toxic and/or off-target compounds are less likely to score positive. For instance, compounds such as carmofur, ebselen, ethacridine, and masitinib, which were identified in loss-of-signal assays . (b) Immunoblot time course of 293T cells transfected with eGFP-based reporter using a monoclonal anti-Mpro antibody and an anti-\u03b1-tubulin antibody as a loading control. Download FIG\u00a0S4, PDF file, 0.6 MB.Kinetics of reporter expression levels. (a) Representative time course experiment showing luciferase signal accumulation of 293T cells transfected with the WT Luc-based SARS2 MCopyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the GTG-GGT-CAT-CTA-TCA-CCT-CAG-CTG-TTT-TGC-AGT-CTG-GTT-TTA-GGA-AAA-TGG-CGT-TCC-3\u2032 and 5\u2032-CCC-CCT-GAC-CCG-GTA-CCC-TTG-ATT-GTT-CTT-TTC-ACT-GCA-CTC-TGG-AAA-GTG-ACC-CCA-CTG-3\u2032. The sequence encoding HIV-1 Tat residues 1 to 72 was amplified from an HIV-1 BH10 full molecular clone  using 5\u2032-GCA-GCC-GGG-ATC-CAT-CGC-CAC-CGA-AGA-CGC-CAA-AAA-CAT-AAA-GAA-AGG-CC-3\u2032 and 5\u2032-TCG-AGC-GGC-CGC-TTT-ACA-ATT-TGG-ACT-TTC-CGC-CCT-TCT TG-3\u2032 and subcloned into the Src-Mpro-Tat-eGFP construct by restriction ligation using the BamHI and NotI cut sites flanking eGFP  Domain descriptions and corresponding amino acid sequences with every other domain shaded gray to facilitate comparison. (b) Complete nucleotide sequence of the Src-Mpro-Tat-eGFP construct from the HindIII 5\u2032 restriction site to the NotI 3\u2032 restriction site. The sequence is gray shaded to match the protein coding domains detailed above in panel a. The DNA sequences for Src and Mpro are codon optimized for expression in human cells. Download FIG\u00a0S6, PDF file, 0.2 MB.Amino acid and nucleotide sequences of the Src-MCopyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00784-22.7FIG\u00a0S7pro-Tat-Luc construct. (a) Domain descriptions and corresponding amino acid sequences with every other domain shaded gray to facilitate comparison. (b) Complete nucleotide sequence of the Src-Mpro-Tat-Luc construct from the HindIII 5\u2032 restriction site to the NotI 3\u2032 restriction site. The sequence is gray shaded to match the protein coding domains detailed above in panel a. The DNA sequences for Src and Mpro are codon optimized for expression in human cells. Download FIG\u00a0S7, PDF file, 0.2 MB.Amino acid and nucleotide sequences of the Src-MCopyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00784-22.9TABLE\u00a0S2Table\u00a0S2, PDF file, 0.01 MB.Site-directed mutagenesis and qPCR primers. Download Copyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pro-Tat-eGFP construct was generated by amplifying the mCherry coding sequence using 5\u2032-TTT-TTT-GGA-GGC-CTA-GGC-TTT-TGC-AAA-AAG-GCC-ACC-ATG-GTG-AGC-AAG-GGC-GAG-3\u2032 and 5\u2032-CAA-GCT-CCC-GGG-AGT-TAC-TTG-TAC-AGC-TCG-TCC-ATG-CC-3\u2032 and subcloning this fragment into the AvrII and SmaI sites downstream of the simian virus 40 (SV40) promoter in the parental Src-Mpro-Tat-eGFP construct. The U6-driven reporter RNA construct was generated by overlap extension PCR of DNA fragments, which included the U6 promoter amplified using 5\u2032-CAG-ATA-TAC-GCG-TCC-CCA-GTG-GAA-AGA-CGC-G-3\u2032 and 5\u2032-CCT-TTC-TTT-ATG-TTT-TTG-GCG-TCT-TCC-GGT-GTT-TCG-TCC-TTT-CCA-CAA-GAT-ATA-TAA-AGC-3\u2032 and a small fragment of luciferase amplified using 5\u2032- GAA-GAC-GCC-AAA-AAC-ATA-AAG-AAA-GGC-C-3\u2032 and 5\u2032-ACT-ATT-AAT-AAC-TAG-TCA-ATA-ATC-AAT-GTC-ATT-CAT-AGC-TTC-TGC-CAA-CCG-AAC-3\u2032. The combined fragment was subcloned into the Src-Mpro-Tat-eGFP construct by restriction and ligation into the MluI site upstream of the cytomegalovirus (CMV) promoter.The two-color Src-M2 in RPMI 1640  supplemented with 10% fetal bovine serum  and penicillin-streptomycin . 293T cells were seeded in a 24-well plate at 1.5\u2009\u00d7\u2009105 cells/well and transfected 24\u2009h later with 200\u2009ng of the wild-type or mutant chimeric reporter construct . Forty-eight hours post-transfection, cells were washed twice with phosphate-buffered saline (PBS) and resuspended in 500 \u03bcL PBS. One-fifth of the cell suspension was transferred to a 96-well plate, mixed with TO-PRO3 ReadyFlow reagent for live/dead staining per manufacturer\u2019s protocol , incubated at 37\u00b0C for 20\u2009min, and analyzed by flow cytometry (BD LSRFortessa). The remaining four-fifths of the cell suspension was pelleted, resuspended in 50 \u03bcL PBS, mixed with 2\u00d7 reducing sample buffer, and analyzed by immunoblotting (below).293T cells were maintained at 37\u00b0C and 5%CO6 cells were seeded in a 10-cm dish and transfected 24\u2009h later with 2\u2009\u03bcg of the Src-Mpro-Tat-fLuc construct. Four hours post-transfection, cells were washed once with PBS-EDTA, trypsinized, resuspended, and counted. Cells were diluted to yield a suspension containing 4\u2009\u00d7\u2009105 cells/mL, and 50 \u03bcL of the suspension was plated into a 96-well plate with 50 \u03bcL of media containing 2\u00d7 the desired drug concentration yielding a final 1\u00d7 drug concentration and 2\u2009\u00d7\u2009104 cells/well. Forty-four hours after plating into 96-well plates, medium was removed, and 50 \u03bcL of Bright-Glo reagent  was added, followed by a 5-min incubation before transferring the Bright-Glo and cell lysate into a white flat 96-well plate for measuring luminescence on a Tecan Spark plate reader. Cells were prepared in an identical manner as the luciferase assay for 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays, and cell viability was assessed using the manufacturer\u2019s protocol .For luciferase-based assays, 3\u2009\u00d7\u200910Compounds were purchased as powders from the commercial vendors listed in Fifty thousand 293T cells were plated in a 24-well plate and allowed to adhere overnight. The next day, cells were transfected with 150\u2009ng of each plasmid. Images were collected 48\u2009h post-transfection at \u00d710 magnification using a the Cytation 5 multimode imager (Biotek).Whole-cell lysates in 2\u00d7 reducing sample buffer  were denatured at 98\u00b0C for 15\u2009min, fractionated using SDS-PAGE , and transferred to a polyvinylidene difluoride (PVDF) membrane . Immunoblots were probed with mouse anti-GFP  or rabbit anti-SARS-CoV-2 3C-like protease  and rabbit anti-\u03b2-actin  or mouse anti-\u03b1-tubulin  followed by goat/sheep anti-mouse IgG IRDye 680  or goat anti-rabbit IgG-horseradish peroxidase . HRP secondary antibody was visualized using the SuperSignal West Femto maximum sensitivity substrate . Images were acquired using the LI-COR Odyssey Fc imaging system.E. coli codon-optimized synthetic gene for SARS2 Mpro (Twist Bioscience) was cloned into the BsaI restriction site of pE-SUMO vector (LifeSensors) via golden gate assembly. Expression plasmids for Mpro P168G and P168S were generated by site-directed mutagenesis. All plasmids were verified by Sanger DNA sequencing. A single colony of E. coli strain BL21(DE3) transformed with each expression plasmid was grown overnight to saturation in 25\u2009mL LB medium supplemented with 100\u2009\u03bcg mL\u22121 carbenicillin. The starter culture was then used to inoculate 3 L of ZYP-5052 auto-induction medium  on a Superdex 75\u2009pg column operating with 20\u2009mM Tris-HCl, pH 8.0, 150\u2009mM NaCl, and 1\u2009mM dithiothreitol (DTT). The peak fractions of SEC showing single band for Mpro in SDS-PAGE were pooled and concentrated to 12\u2009mg mL\u22121 as determined by UV absorbance measured on a NanoDrop 8000 spectrophotometer and flash frozen in liquid nitrogen for storage at \u221280\u00b0C.An n medium  supplemepro was analyzed using a quenched fluorescent peptide substrate DABCYL-KTSAVLQ\u2193SGFRKM-EDANS , which has been used in multiple recent studies . For inhibition studies, Mpro was incubated with various concentrations of GC376 or boceprevir  for 1\u2009h in the reaction buffer containing BSA prior to addition of the substrate to initiate the reaction. Fluorescence intensity was measured once per minute on a Tecan Spark 10M plate reader.Biochemical activity of M studies , 34, 60.pro at 12\u2009mg mL\u22121 was mixed with 2\u2009mM GC376 in 20\u2009mM Tris, pH 8.0, 150\u2009mM NaCl, 1\u2009mM DTT, and 2.5% DMSO and subjected to crystallization screening. The best diffracting crystals were obtained by sitting drop vapor diffusion method with a reservoir solution consisting of 0.2 M potassium thiocyanate, 20% polyethylene glycol 3350 (PEG 3350), and 0.1 M Bis-Tris propane buffer, pH 6.5. Crystals under this condition typically grew as a cluster of thick plates, from which single crystals could be isolated for data collection. X-ray diffraction data to ~1.7\u2009\u00c5 resolution were collected at the Northeastern Collaborative Access Team (NE-CAT) beamline 24-ID-C of the Advanced Photon Source  and processed using XDS (pro-GC376 complex was determined by molecular replacement with Phaser (pro structure as a search model (PDB ID 7C8U). Iterative model building and refinement were performed using Coot . Msing XDS . The strh Phaser , using aing Coot  and Phening Coot , respectructures , 42, 65,6U [7CBT , 7CB7 [6BT [7CB7 , 7JSU [6B7 [7JSU , and 6WTSU [6WTK ). Our hi10.1128/mbio.00784-22.10TABLE\u00a0S3Table\u00a0S3, PDF file, 0.07 MB.X-ray data collection and model refinement statistics. Download Copyright \u00a9 2022 Moghadasi et al.2022Moghadasi et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the pro-Tat-eGFP mRNA relative to an RNA PolIII-driven control transcript, the U6-Luc-Src-Mpro-Tat-eGFP plasmid (To quantify the relative levels of Src-M plasmid  was tranpro-GC376 complex structure have been deposited in the RCSB Protein Data Bank with accession code PDB 7TGR (The atomic coordinates and structure factors for our MPDB 7TGR ."}
+{"text": "Colorectal cancer (CRC) is one of the leading causes of mortality for cancer in industrialized countries. The link between diet and CRC is well-known, and presumably CRC is the type of cancer which is most influenced by dietary habits. In Western countries, an inadequate dietary intake of fibers is endemic, and this could be a driving factor in the increase of CRC incidence. Indeed, several epidemiologic studies have elucidated an inverse relationship between daily fiber intake and risk of CRC. Long-term prognosis in CRC survivors is also dependent on dietary fibers. Several pathogenetic mechanisms may be hypothesized. Fibers may interfere with the metabolism of bile acids, which may promote colon carcinogenesis. Further, fibers are often contained in vegetables which, in turn, contain large amounts of antioxidant agents like resveratrol, polyphenols, or phytoestrogens. Moreover, fibers can be digested by commensal flora, thus producing compounds such as butyrate, which exerts an antiproliferative effect. Finally, fibers may modulate gut microbiota, whose composition has shown to be associated with CRC onset. In this regard, dietary interventions based on high-fiber-containing diets are ongoing to prevent CRC development, especially in patients with high potential for this type of tumor. Despite the fact that outcomes are preliminary, encouraging results have been observed. Colorectal cancer (CRC) is one of the most common malignancies, being the third leading cause of cancer death worldwide, mostly in Europe and the United States, although there has been a rise in its incidence in Asia over recent decades. In 2018, the incidence of new cases was approximately 2 million, while it caused 1 million deaths worldwide [Colorectal carcinoma is classified into sporadic (70%), inherited (5%), and familial 25%). There are three main genetic pathways that can lead to this situation: chromosomal instability (CIN), microsatellite instability (MSI), and CpG island methylator phenotype (CIMP). The CIN pathway is one of the main causes of CRC (85%) and is characterized by chromosomal imbalance that leads to loss of heterozygosity. The microsatellite instability pathway is caused by a hypermutable phenotype due to loss of DNA repair mechanisms. Epigenetic instability, which is responsible for the CpG island methylator phenotype, is another common feature in CRC. The main characteristic of CIMP tumors is the hypermethylation of oncogene promoters, which leads to genetic silencing and a loss of protein expression [5%. ThereIn the CIN scenario, some genetic alterations involved in hereditary forms of CRC are also responsible for sporadic forms. In particular, Wnt/\u03b2-catenin, TGF-\u03b2 receptor, Notch, and Hedgehog pathways are involved.The Wnt/\u03b2-catenin pathway is influenced by mutations of the APC gene, which generates an incorrect translocation of the protein \u03b2-catenin to the nucleus. The APC gene encodes for a specific tumor suppressor that creates the destruction complex, in association with axin, a cyclin-dependent kinase inhibitor (CKI), and glycogen synthase kinase 3 (GSK-3), a serine/threonine protein kinase [This complex generates a \u03b2-TrCP (protein involved in the regulation of the cell cycle)  recognitThe interaction between Frizzled (Fz), a transmembrane receptor, and its ligand Wnt, regulates \u03b2-catenin synthesis. Accumulation of \u03b2-catenin in the nucleus leads to an interaction with many Transcription Factors, as a way to modulate mitosis and cell proliferation. Mutations of the APC gene generate an abnormal accumulation of \u03b2-catenin in the nucleus. The consequence is an incorrect activation of transcription of Wnt target genes, which leads to tumoral cell proliferation.TGF-\u03b2/SMAD is a signaling pathway involved in cell proliferation, differentiation, and apoptosis. Ligand TGF-\u03b2 interacts with type II TGF-\u03b2 receptors. Consequently, the receptor phosphorylates type SMAD Transcription Factors, SMAD2 and SMAD3. These Transcription Factors create a complex with SMAD4 and migrate in the nucleus, in order to promote cyclin and apoptosis-associated proteins. TGF-\u03b2 has a tumor suppressor role. Anyway, alterations in the expression of the gene encoding for TGF-\u03b2 receptor can promote tumoral cell proliferation.Notch and Hedgehog pathways are linked to gut epithelium proliferation. The Notch pathway is based on the link between Notch ligands and their transmembrane receptors. These receptors migrate in the nucleus in order to regulate the determination of cell fate, cell differentiation, and oncogenesis. Anyway, in the case of cancer, point mutations and genetic alteration can constitutively activate this pathway, leading to indiscriminate cell proliferation.Finally, the Hedgehog signaling pathway is associated with protein ligands Indian, Sonic, and Desert. Their receptor is the Patched protein, which suppresses the activity of Smoothened, a G protein-coupled receptor. This signaling pathway regulates the expression of many target genes. Abnormal activation of Hedgehog signaling is linked to the development of CRC .Dietary fibers are an environmental factor associated with risk for colorectal cancer. The effects of their ingestion might impact the complex events that characterize colorectal oncogenesis . The defMoreover, functional fibers are defined as isolated, non-digestible carbohydrates  with positive physiological effects for the human organism. Another classification of dietary fibers is based on their water solubility . SolubleThe correct fiber intake for adults is 30\u201335 g per day for men and 25\u201332 g per day for women, bringing benefits to the gut microbiome and metabolic health, as well as reducing cardiovascular diseases and the risk of colonic cancer .Major food sources of dietary fiber are cereals/grains, vegetables, fruits, and legumes. Dietary fibers from different food sources are heterogeneous with respect to chemical composition, physicochemical properties, and solubility and thus are hypothesized to present varying degrees of anticarcinogenic properties. However, the evidence on the relationships of dietary fiber intake from different food sources with colorectal cancer and adenoma risks is mixed. Further, little is known about the shape of the relationships as well as the relative importance of fiber source .The mechanisms relating diet with CRC are complex and still poorly understood. A diet with a high intake of whole grains, fruits, and vegetables is accompanied by the intake of lower-energy-density foods and lower intake of foods with high glycemic index, glycemic load, and fat, which has been shown to be related to decreased risk of obesity and diabetes, both identified risk factors for CRC. Nonetheless, studies that had adjusted the risk for obesity and diabetes reported that associations persisted in adjusted analyses. Further possible explanations for the reduced risk of CRC and intake of these food groups might be driven by the high content of fiber, also including resistant starch, oligosaccharides, and lignins, which are related to increased stool mass, decreasing colonic transit time, prebiotic effects such as action of bacterial enzymes, and fecal bile acid concentration, and are suggested to play a role in colorectal carcinogenesis. Moreover, whole grains, fruits, and vegetables are a source of minerals , and, in particular, fruit contains a wide range of antioxidant vitamins, flavonoids, and carotenoids, and studies suggested a potential protective role against CRC .13 microorganisms of different species, with significant abundance of Firmicutes and Bacteroidetes phyla [Moreover, dietary fiber may influence the composition of gut microflora. The human gut contains a complex microbial community, composing the microbiota. By means of metagenomic, meta-transcriptomic, and bioinformatics tools, it is possible to define the microbial population, to monitor its compositional changes, and to understand how microbial communities interact with the gut environment. The healthy adult human gut microbiota consists of about 10es phyla . Table 1Dietary formulation can impact gut microbiota composition, diversity, and richness. In particular, dietary fiber provides various substrates for fermentation reactions carried out by species of microbes that have specific enzymes to degrade these complex carbohydrates . In factThis biochemical process, operated by the gut flora, gives Short-Chain Fatty Acids (SCFAs) as products, including butyrate, acetate, and propionate ,14. ThesRuminococcaceae _UCG-014, Rikenellaceae _RC9_gut_group, and Bifidobacterium) increased in the fiber inclusion diets, and pathogenic genera  were increased in the basal diet. Moreover, the intestinal flora of growing pigs adapted more easily and quickly to the SBP diet compared to the DFRB diet, as reflected by the concentration of propionate, butyrate, isovalerate, and total SCFA [For example, three different fiber-rich diets were given to growing pigs, one replacing corn, soybean meal, and soybean oil in the diet with 20% sugar beet pulp (SBP), defatted rice bran (DFRB), or soybean hull (SBH). The analysis of fresh feces demonstrated that principal coordinate analysis (PCoA) revealed distinctly different microbial communities on the DFRB diet and SBH diet across different times; additionally, the stool microbiota of the four diet groups displayed remarkably dissimilar clusters at each time point. With adaptation time increased from 7 to 21 d, cellulose-degrading bacteria and SCFA-producing bacteria  inhibitor, stopping cell proliferation or inducing apoptosis. In a physiological situation, low doses of butyrate are not sufficient for HDAC inhibition, and consequently, this promotes histone acetylation, leading to colonic cell proliferation. In particular, butyrate is metabolized in the mitochondria, where it is transformed into acetyl-CoA by beta-oxidation. Then, acetyl-CoA condenses with oxalacetate, forming citrate as a result. Citrate goes out of the mitochondria and interacts with the enzyme ATP citrate lyase (ACL), being reconverted into cytosolic and nuclear acetyl-CoA, useful for lipid biosynthesis and as a cofactor for histone acetyltransferases, respectively. On the contrary, high doses of butyrate promote HDAC inhibition, with the final consequence of promoting cell apoptosis. In this case, this compound is not metabolized in mitochondria, but migrates into the nucleus, acting as an HDAC inhibitor; in this way, histones cannot alter the structure of nucleosomes, stopping colonic cell proliferation and increasing apoptosis .In the colorectal cancer scenario, cells undergo the Warburg effect, the mechanism by which tumoral cells prefer glycolysis instead of oxidative phosphorylation, as a source of energy. As a consequence, even lower doses of butyrate do not enter mitochondria, but accumulate in the nucleus of cancerous colonocytes, acting as HDAC inhibitors and apoptosis promoters.Additionally, another important function of butyrate related to colorectal cancer is its anti-inflammatory action. SCFAs can be ligands for G-coupled receptors called GPR, which regulate different pathways. In particular, butyrate can bind the receptor GPR43 located on the surface of T cells, promoting an anti-inflammatory effect, positive for colorectal cancer prevention .p = 0.02). Additionally, CRC incidence was higher in individuals with lower levels of SCFAs , compared to healthy individuals. Qualitative analyses identified 70.4% of studies reporting significantly lower concentrations of fecal acetic, propionic, and butyric acid or total SCFAs in those at higher risk of CRC, while 66.7% reported significantly lower concentrations of fecal acetic and butyric acid in CRC patients compared to healthy controls. In conclusion, lower fecal concentrations of the three major SCFAs were associated with higher risk of CRC and incidence of CRC [Alvandi et al. designed a study to better understand the link between SCFA concentration in CRC risk and incidence. The analyses have been divided on the available evidence into two outcomes: (1) CRC-risk and (2) incidence. Combined analysis of acetic, propionic, and butyric acid revealed significantly lower concentrations of these SCFAs in individuals with a high risk of CRC  and oxaliplatinum (OXA) at the animal level, created a murine model in which subcutaneous implantation of CRC cells was performed and 16S sequencing technology was used to detect intestinal bacteria. GC-MS was used to detect metabolites in mouse stools. NAB was a differential metabolite that affected the efficacy of OXA. It was found that NAB and oxaliplatin could synergically inhibit cell proliferation, migration, and invasion and induce cell apoptosis in cell lines, while animal experiments confirmed the inhibitory effect of oxaliplatin and sodium butyrate on tumors in mice. Furthermore, the intestinal microbe detection and microbial metabolite detection in fecal samples from mice showed a significant increase in species producing butyrate such as Bacteroides, especially when NAB and OXA were administered in combination .In a study performed by Kang et al., an azoxymethane/dextran sodium sulfate-induced mouse CRC model was used to explore the role and mechanism of butyrate in regulating colon cancer and its intestinal microecological balance. Butyrate alleviated weight loss, disease activity index, and survival in CRC mice and inhibited tumor number and progression. Further research revealed that butyrate restrained the aggregation of harmful flora while promoting the colonization of beneficial flora, such as Actinobacteriota, Bifidobacteriales, and Muribaculacea, through 16S rDNA sequence analysis. This study confirmed that butyrate can ameliorate CRC by repairing intestinal microecology, providing ideas and evidence for chemical prophylactic agents, such as butyrate, to counteract tumor growth and regulate tumor microbiota .Garavaglia et al. studied how mutations in APC (Adenomatous Polyposis Coli) are reflected in \u03b2-Catenin hyperactivation and loss of proliferation control. Certain intestinal bacteria metabolites have shown the ability to limit CRC cell proliferation and CRC pathogenesis. This study investigated the molecular mechanism underlying the anti-proliferative activity of butyrate, a microbiota-derived short-chain fatty acid, in two CRC cell lines, namely HCT116 and SW620, which bear a mutation in \u03b2-Catenin and APC, respectively. Butyrate reduced CRC cell proliferation, as witnessed by the downregulation of proliferation markers. In CRC cells, regardless of the mutational state of APC or \u03b2-Catenin genes, butyrate caused the autophagy-mediated degradation of \u03b2-Catenin, thus preventing its transcriptional activity .Dietary fibers also have an important role in colorectal cancer prevention, due to their interactions with bile acids. These compounds are amphiphilic molecules synthesized from cholesterol, in the liver. They are involved in the absorption of lipids in the small bowel and also create micelles to solubilize cholesterol. Bile acids are metabolized in the large bowel; bacterial flora helps to deconjugate and to dehydroxylate primary bile acids, generating secondary bile acids, which have been invoked in colonic carcinogenesis. Experimental evidence in humans and animals showed that bile acids are cytotoxic to colonocytes, stimulating cell proliferation .Anyway, dietary fiber intake can counteract this phenomenon. In fact, fiber is involved in the increase in bowel movement, thus acting on an important etiological factor in colon cancer. Also, fibers bind bile acids, modifying the gut\u2013liver axis. This reduces cholesterol levels, also decreasing the risk of colorectal cancer growth ,27.From a molecular point of view, fibers entrap polyphenols, promoting their bioavailability for gut bacteria metabolism. Indeed, the gut microbiota can modulate oxidative stress positively or negatively, due to the degradation of fibers in phenolic compounds. These compounds act as antioxidants after being absorbed by the intestine and sent to the bloodstream . Figure The last beneficial feature of dietary fibers concerns specifically soluble ones. It is shown, in fact, that these molecules, at low concentrations, have great water-holding capacity by forming polysaccharidic gel. In opposition to fruit and vegetable cells, wheat bran cells have lignified walls; this confers to wheat bran gel walls a lower water-holding capacity. The hydrophobic characteristics of these cell walls are important for cancer prevention. Indeed, carcinogens can easily bind to hydrophobic walls, so that they can pass out of the alimentary tract in the stools, reducing their interaction with colonic mucosal cells. Because of their indigestibility, dietary fibers can also increase the bulk of the feces, with the consequence of their shorter transit time in the gut. In this way, there will be a lower concentration of carcinogens interacting with colonic mucosal cells .The main mechanisms by which fibers may counteract CRC development are summarized in World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) suggest that decreased consumption of grains and dairy products and the Western diet, characterized by a high consumption of red, processed meat and fats, increase the risk of CRC .The hypothesis of the protective role of fiber against CRC started in 1971 from Burkitt, who reported that colorectal cancer was rare among rural Africans. In fact, the incidence of colorectal cancer in men 35\u201364 years of age was 3.5/100,000 in Kampala (Uganda), as opposed to 51.8/100,000 in Connecticut (USA). These data were explained by the fact that Africans had small amounts of meat and a lot of fiber from fruits, grains, and vegetables in their diet .In recent decades, many clinical trials have investigated the topic and showed that the prevalence of CRC increases inversely to the intake of dietary fiber, with few exceptions. A 2018 meta-analysis conducted by Yu Ma et al. investigated the relationship between dietary fiber intake and subsite-specific colorectal cancer. In fact, CRC can be classified into two different types, in relation to the site: proximal colon cancer (PCC) and distal colon cancer (DCC), with different genetic and environmental risk factors. The result of the analysis was that fiber intake was inversely associated with the risk of CRC in both subgroups, with no significant heterogeneity. The risk ratio (RR) of PCC for individuals with the highest fiber intake compared to those with the lowest intake was 0.86; for DCC the RR was 0.79, i.e., 21% lower than individuals with the lowest fiber intake .A 2017 update of the Cochrane review has analyzed the effect of dietary fiber on the recurrence of adenomatous polyps and on the incidence of CRC in people with a known history of adenomatous polyps (but no previous CRC) which had been removed to achieve a polyp-free colon at baseline. No statistically significant difference was found between the intervention and control groups for the number of participants with at least one adenoma, more than one adenoma, or at least one adenoma 1 cm or greater at three to four years. After 8 years of comprehensive dietary intervention, no statistically significant difference was found in the number of participants with at least one recurrent adenoma or with more than one adenoma. However, authors themselves claimed that the results should be interpreted cautiously because of the high rate of loss to follow-up and also because adenomatous polyp is a surrogate outcome for the true endpoint, which is CRC .The Polyp Prevention Trial (PPT), a multicenter randomized clinical trial, evaluated the effects of a high-fiber, high-fruit and vegetable, and low-fat diet on the recurrence of adenomatous polyps in the large bowel over a period of 4 years. Similarly, the risk of recurrent adenomas was not significantly different from that of the controls. The PPT-Continued Follow-up Study (PPT-CFS) was a post-intervention observation of PPT participants for an additional 4 years from the end of the trial; the results showed no significant intervention\u2013control group differences in the relative risk for recurrence of an advanced adenoma or multiple adenomas .On the bases of these results, we can hypothesize that dietary fiber is able to prevent the evolution of polyps into CRC more than their onset.Since CRC is one of the types of cancer most affected by diet, a healthy diet can be essential to set up its natural and permanent chemoprevention. Fiber is an essential component of a healthy diet and its beneficial effects in preventing the development of CRC have been clearly elucidated in several studies. These benefits can be obtained through various mechanisms, which contribute to their achievement. On these bases, it appears evident that a correct nutritional education could be fundamental for reducing the incidence of CRC in populations at risk. This education should start from childhood in order to promote the prevention not only of metabolic diseases, but also of neoplastic diseases more influenced by dietary factors . In thisFinally, it should be mentioned that the waste of fibers is a topic that deserves further discussion, since it may underpower the beneficial effect on CRC. Indeed, it is known that different types and sources of dietary fibers can derive from agri-food by-products. Food processing industrial wastes include bio-actives such as dietary fibers, pigments, essential minerals, fatty acids, and antioxidant polyphenolic compounds. Therefore, ample amounts of fibers that derive from non-edible and edible parts of fruits and vegetables are often wasted along the entire agri-food supply chain ,48,49."}
+{"text": "While physicians tend to emphasize on physical medical problems, behavioral and cognitive disorders of geriatric patients are usually missed, especially in emergency settings. The aim of the study was to determine the prevalence of delirium, dementia, and depression (3D) among older patients (\u226565 years old) in the Emergency Department (ED) and to evaluate the effect of geriatric 3D on the 6-month and 5-year mortality.This was a prospective, observational cohort study, including 415 patients from eligible 512 consecutive older patients, who are 65 years of age or older, presenting to the ED of a tertiary care university hospital. Geriatric delirium, dementia, and depression were prospectively evaluated using Confusion Assessment Method, Quick Confusion Scale, and Geriatric Depression Scale-15, respectively. Premorbid functional status was determined by Barthel Index. The Charlson Comorbidity Index was used to measure the comorbid burden. After enrollment, patients were screened for 6-month and 5-year survival rates via the Government Death Reporting System records. The Kaplan\u2013Meier method and Cox proportional hazards analysis was used for survival analysis.Among the study population, the prevalence of geriatric 3D was found as 10.6% (n = 44/415) for delirium, 45.6% (n = 160/351) for dementia, and 35.1% (n = 123/350) for depression. Delirium, dementia, and depression all had higher mortality rates among older ED patients covering the 5-year period. However, only delirium was predictive of both 6-month and 5-year mortality rates.Aside from the medical and surgical issues of geriatric patients, the prevalences of dementia and depression are much higher than expected in the emergency department. Delirium was a predictor for 6-month and 5-year mortality. We suggest that EDs should have screening tools for geriatric 3D mental health disorders to increase the quality of life for the geriatric population. The geriatric population in the world is growing fast, and the proportion of emergency department (ED) visits by older patients is expected to increase over the next decades . ED is oGeriatric 3D represents a common and challenging set of diagnoses for older patients. These disorders are interconnected, increasing the risk of each other, and are all associated with an increased risk of mortality and morbidity. Despite this, emergency physicians and other ED healthcare professionals do not routinely evaluate older patients for the disorders and may miss the critical diagnoses  by the research coordinators. Subsequently, a pilot practical training session on mental health disorder tests was conducted with ten older patients for two consecutive days. Following training, all interviewers were supervised by an emergency medicine attending physician (I.K.) and a geriatric attending physician (Z.U.) for initial screens to ensure that the test was administered in a standardized manner. Any unclear queries were adjudicated by the attending physicians I. K. and Z.U. jointly.The screening of delirium, dementia, and depression of the patients were prospectively evaluated using the Confusion Assessment Method (CAM), Quick Confusion Scale (QCS), and short-form of Geriatric Depression Scale-15 (GDS), respectively. ED personnel were blinded to the findings of the assessment to avoid influencing treatment decisions.Every patient was first assessed for delirium in the ED and Glasgow Coma Scale (GCS) was obtained. It was not possible to further assess dementia or depression for patients with acute delirium. If the patient does not have delirium and is engaging with the investigator, the evaluation was continued with dementia and depression screening tests. We should note that these screening tests have varying degree of sensitivities and specificities for exact diagnosis.Delirium was assessed with the CAM developed by Inouye and colleagues. CAM is the most widely used delirium assessment tool in the ED with high sensitivity (94%\u2013100%) and specificity 90%\u201395%) [% [5]. MoThe Quick Confusion Scale Assessment (QCS) was used to assess the level of cognitive functioning in the study. QCS is a six-item questionnaire to evaluate the orientation, memory, and concentration weighted to give a best total score of 15 . PatientThe presence of dementia was also confirmed by screening the past medical history, dementia medications and questioning the family and relatives. As previously described, QCS was inapplicable in delirious patients. If the patient did not have delirium, all study participants underwent the QCS test.Depression was assessed with the Short Form of Geriatric Depression Scale (GDS), specifically known as GDS-15. The GDS was originally 30-item questionnaires (GDS-30) created by Brink . The GDSPremorbid functional status was assessed by using the Barthel Index (BI). This widely used assessment is an ordinal scale measuring the ability of an individual to perform ten basic activities of daily living related to self-care, continence, and mobility, including bathing, grooming (0 and 5 points), feeding, dressing, fecal control, urinary control, climbing stairs , transfers , and walking  in an independent manner. The final score ranges from 0 (completely dependent) to 100  points with the 5-point intervals. BI scores were grouped further by functional categories using points; 0\u201320 total disability, 21\u201361 severe disability, 62\u201390 moderate disability, 91\u201399 mild disability, 100 points no disability.The Charlson Comorbidity Index (CCI) was used to measure the comorbid burden. CCI, designed by Charlson and et al. in 1987, and it is a morbidity score that reflects mortality risk . It is bhttps://obs.saglik.gov.tr) developed by the Ministry of Health Information Technology, Turkey. Notification of deaths is obligatory in Turkey, and no burial can be carried out without this report approved by the State Physicians. The death report includes the information related to the identification numbers, identity information, date, and time of death of the individual.The data for 6-month and 5-year mortality rates were collected from electronic medical records. Information on mortality regarding the date of death for patients was obtained from the Death Reporting System records  and MedCalc version 15.8 . Demographic and clinical variables were presented as descriptive statistics. The continuous variables were presented as median values, and interquartile ranges (IQRs), and the categorical variables were summarized as frequencies and percentages. The normality of the continuous variables was evaluated using the Kolmogorov\u2013Smirnov test. The statistical differences between the two groups of continuous variables were determined using the Mann\u2013Whitney U test. The categorical variables were compared using Pearson\u2019s \u03c72 or Fisher\u2019s exact test. The odds ratios (ORs) were presented with 95% confidence intervals (95% CIs). A critical \u03b1 value of 0.05 was accepted as statistically significant.Kaplan\u2013Meier estimation was performed to generate the observed survival curves for geriatric 3D mental health disorders, and hazard ratios (HR) were calculated. Univariate and multivariate Cox regression analysis was applied to evaluate the prognostic relationship between mortality and geriatric 3D mental health disorders and other parameters. Only one of the parameters with a high correlation factor was included in this analysis.During the study, a total of 4463 patients presented to our ED. Of whom, 512 consecutive patients were at the age of 65 or older. 415 patients, meeting our inclusion criteria, were enrolled in the study . A totalAmong 415 patients included in the study, delirium evaluations were performed using CAM, and 44 (10.6%) delirium positivity was detected. Of these 415 patients, dementia assessment (QCS) could not be performed in 64 patients because 44 had acute delirium, 9 patients were illiterate, 7 patients had aphasia or deafness, 3 patients did not want to respond to dementia tests, and 1 patient had a language barrier. In the remaining 351 patients, dementia positivity was detected in 160 (45.6%) patients. In the same way, the depression test (GDS-15) could not be done in 65 patients because 44 patients had acute delirium, 10 patients had advanced dementia, 7 patients had aphasia or deafness, 3 patients did not want to respond to depression tests, and 1 patient had a language barrier. In the remaining 350 patients, depression positivity was detected in 123 (35.1%) patients.The prevalence of delirium was 10.6% (n = 44/415). Hazard ratio of delirium was 4.5 (95% CI 1.96\u201310.34) for 6-month mortality and was 3.4 (95% CI 1.90\u20136.22) for 5-year mortality . In the The prevalence for dementia was 45.6% (n = 160/351). The presence of dementia does not affect 6-month mortality in Kaplan\u2013Meier survival analysis (OR: 1.6 with a 95% CI 0.89\u20132.84), (p = 0.1168). However, dementia positivity was relevant for the 5-year mortality (OR: 1.98 with a 95% CI 1.42\u20132.77) (p < 0.001) . In the The prevalence of depression was 35.1% (n = 123/350). The hazard ratio of depression was 2.5 (95% CI 1.34\u20134.64) for 6-month mortality and 2.14 (95% CI 1.49\u20133.09) for 5-year mortality . In the In this study, we investigated the mental health disorders in the older patients presented to the ED, and we observed a silent epidemic with a prevalence of 10.6% for delirium, 45.6% for dementia, and 35.1% for depression, respectively. Considering that 3D mental health disorders are seen together with a high prevalence and have worse outcomes in terms of mortality, EDs have a vital role regarding screening of older adults after their presentation to the ED. EDs may be considered as the first point of contact for the screening of Geriatric 3D in many patients.Older patients are the population at highest risk for decompensation if not diagnosed and managed early, due to reduced physiological reserve, atypical presentation of symptoms, more accompanying comorbidities, underlying fragility associated with aging, as well as hearing and visual impairments. Given the underlying medical fragility and complex presentation of older patients, the causes of mental and behavioral changes are likely to be misdiagnosed. In addition, they are likely to benefit from early intervention. For these reasons, our research questions have a significant potential impact on vulnerable older patients in the ED.Delirium and agitation are among the most common problems in the geriatric population, occurring in approximately 25% of hospitalized geriatric patients , 17. PatDelirium in the ED is widely studied in the literature. According to reported studies, the delirium prevalence in patients older than 65 years of age presenting to ED is reported as 8.3%  and 12% In our study, the prevalence of dementia was found to be relatively high. In the literature, the prevalence of dementia in EDs was reported to be between 20% and 38% . There mDepression in older patients, another important topic of our study, is common and untreated in this population. It may coexist with dementia and increases the risk of delirium. It is also associated with higher morbidity and mortality rates, together with increased healthcare utilization. Depression may be present in up to one-third of older ED patients , 31. In Our finding of depression prevalence is 35.1%. It is higher when compared to the studies done in the ED of developed countries [16.5% (USA) , 17% (USAnother aim of this study was to investigate whether geriatric 3D has an effect on short-term (6-month) and long-term (5-year) mortality. In our study, we found that geriatric patients diagnosed with one of the mental health disorders had higher mortality rates than those without, and delirium alone predicted mortality in both 6-month and 5-year periods. In literature, Kakuma and et al. reported that inability to detect delirium in the ED might be associated with increased mortality within 6 months after discharge . Also, HOlder adults with dementia have a higher rate of ED admission, hospitalization, and ED revisits . A largeThe relationship between the diagnosis of depression and mortality in the ED was also examined, and mortality was found to be high in patients diagnosed with depression . In our Our findings have to be considered in conjunction with the study limitations. First, we used three validated instruments to identify the geriatric 3D, the CAM for delirium diagnosis, QCS for dementia, GDS for depression as a reference standard in our study. While they are widely used for research and clinical purposes, they are not \u201cgold standard\u201d diagnostic instruments. Thus, our results should be interpreted with caution by the possibility of an imperfect gold standard bias . SecondlApart from medical and surgical problems of the geriatric patient population, incidences of dementia and depression are much higher than expected in the emergency department. We suggest that emergency departments should have screening tools for dementia, delirium, and depression (3D) to increase the quality of life for the geriatric patient population."}
+{"text": "The effect of the decreasing maternal DHA and MeHg intake on infant IQ was estimated by using the equation developed by the FAO/WHO. Net IQ change (the difference in IQ gain by DHA and IQ loss by MeHg) was constant or even increasing, depending on the assumption, in this period while seafood consumption was significantly decreasing. This was due to the decreasing adverse effect of MeHg, along with saturated DHA-derived benefits on infant IQ, even at the decreased seafood consumption of Japanese women of childbearing age. It was indicated that the recent decreasing trend in seafood consumption in Japan did not have an unfavorable effect on infant IQ.National statistics show that seafood consumption in Japan is decreasing since the mid-1990s. The risks and benefits of this decreasing seafood consumption was assessed in this study. Intake of docosahexaenoic acid (DHA) and methylmercury (MeHg) of women of childbearing age were estimated by using seafood consumption data of women of age 20\u201339 in the period 2011\u20132019 and seafood DHA and MeHg content data to find significantly ( The National Health and Nutrition Survey (NHNS) of the Ministry of Health, Labor and Welfare of Japan (MHLW)  revealedThe decreasing seafood consumption was paralleled with increasing meat consumption, so the daily protein intake level in the last two decades has been relatively constant at around 70 g/person/day. From the nutritional viewpoint, one of the most important issues potentially related to this decrease in seafood consumption is the associated decrease in the intake of n-3 polyunsaturated fatty acid (PUFA). In fact, there has been a decreasing trend in n-3 PUFA intake from 2005 to the present in Japan. Seafood is a rich source of PUFA, though not the sole source. PUFA is known to be related with decreased incidence of cardiovascular diseases in adults and the enhanced cognitive development of infants ,5. On thThus, the decreasing seafood consumption of the Japanese can have both adverse and beneficial effects on the cognitive development of infants due to decreased intake of PUFA, particularly that of docosahexaenoic acid (DHA), and the decreased intake of MeHg, respectively, of their mother during pregnancy. The aim of this study was to quantitatively estimate adverse and beneficial effects, and the net effect, on the IQ of infants due to decreased seafood consumption of pregnant Japanese women. Average DHA and MeHg intakes from seafood and their temporal trend were estimated by using the national statistics on the intake of the categories of seafood and published DHA and MeHg concentrations of fish and shellfish species and products. The effect of the DHA and MeHg intake of Japanese women of childbearing age on infant IQ was calculated with an equation developed by the FAO/WHO , and itstsukudani\u201d (seafood boiled in sweetened soy sauce), \u201cfish paste\u201d and \u201cfish ham and sausage\u201d) categories. Daily consumption of 13 categories of seafood by the Japanese population is available in the results of the NHNS, MHLW, which has been conducted yearly by involving randomly selected persons in Japan . The categories of seafood include 8 seafood categories  and 5 seafood products  . The metThe average DHA and MeHg contents of seafood of the 13 categories were obtained by averaging the DHA and MeHg contents of fish and shellfish species belonging to each of the categories. Daily intakes of DHA (mg/day) and MeHg (\u03bcg Hg/day) of women of childbearing age in 2011\u20132019 were calculated by multiplying the average contents of DHA (mg/g) and MeHg (\u03bcg Hg/g) by daily consumption for each of the 13 seafood categories (g/day) (see DHA is the daily intake of DHA (mg/day); 0.04 is the slope of regression between DHA intake and IQ; MeHg is the daily intake of MeHg (\u03bcg Hg/day); 9.3 is the factor converting from daily intake to maternal hair Hg level; \u22120.18 or \u22120.7 are the center value and upper bound estimate of regression slope between hair Hg level and IQ; and 53.2 is the average body weight of Japanese women of age 20\u201339.An increase and decrease in IQ of infant is expected due to maternal DHA and MeHg intake during pregnancy, respectively, and net IQ change was estimated by the following Equation (1):Equation (1) is a slightly modified version of the equation proposed in an FAO/WHO document  in whichp < 0.001). Seafood consumption of this age grade (20\u201339 years old) was approximately 75% of that of general Japanese .The intake of DHA and MeHg of Japanese women of childbearing age was calculated by multiplying the average contents in p < 0.05). The slope of the regression indicated that intake of DHA and MeHg decreased by 2.8 mg/day and 0.19 \u03bcg/day per year, respectively, in the 2011\u20132019 period.The mean intake of DHA during the 2011\u20132019 period was calculated to be 277 \u00b1 11 (min\u2013max: 262\u2013292) mg/day and that of MeHg was 6.77 \u00b1 0.77 (5.60\u20137.62) \u03bcg Hg/day. Both of the intakes are linearly decreasing toward the present  in propo2 under the moderate condition and +5.0 \u00b1 0.1 under the upper-bound condition. The net IQ was constantly on the positive side for both conditions, which meant that the seafood intake of the Japanese women of childbearing age resulted in the IQ gain of offspring, and the between-year variation of the net IQ change was small. Under the upper bound condition, net IQ change, i.e., IQ gain, seems to increase as seafood consumption decreases. This trend was not apparent by visual inspection of Based on the estimated maternal daily intakes of DHA and MeHg, the net IQ change of offspring for each year was calculated by Equation (1). The net IQ change denotes gain/loss of IQ from the infant IQ attained when no seafood was consumed by the mother. In It is generally believed that seafood is a healthy food. Therefore, the recent steadily decreasing trend of seafood consumption in Japan  has raisAs shown in As mentioned, the estimated IQ gain of infants in 2019 was greater than that in 1997, when the decrease in seafood consumption started, by 0.49 points at the maximum estimation. It is well recognized that IQ in childhood is positively associated with economic productivity in adulthood. Grosse and Zhou  estimateIt is supposed that the decreasing trend of seafood consumption by the Japanese will continue in the future. Benefits from seafood consumption would eventually disappear when seafood consumption further decreases. Therefore, it is of interest to predict how much reduction in seafood consumption would result in an apparent unfavorable IQ effect. n = 32 and 135 in 2011 and 2012, respectively) into consideration, suggesting that reduced seafood consumption of women after diagnosis of pregnancy was not apparent in Japan. This suggestion justifies the use of seafood consumption data of women of childbearing age for that of pregnant women in this study. In fact, the reported daily DHA intake of Japanese pregnant women was consistent with the estimated intakes of the present study: Shiraishi et al. [In this study, we assumed that the seafood consumption of pregnant Japanese women is not different from that of women of age 20\u201339. However, it is possible that a woman may reduce seafood consumption after she becomes pregnant, as the MHLW announced in 2003 that women who are pregnant or who are expected to be pregnant should restrict the frequency of the consumption of specific species of seafood  because maternal consumption of these species with elevated MeHg content could bring about adverse effects on infant development . This anThe decreasing seafood consumption in Japan was not found to have a negative effect on infant IQ gain, but even had a positive effect (more IQ gain). This was because, even though the seafood consumption is decreasing, the consumption is still abundant enough to supply adequate DHA to keep upper-limit IQ gain (+5.8 points), and the decreasing consumption resulted in decreasing adverse effects from MeHg intake. The benefit we have at present can be maintained until seafood consumption is further decreased by 45% of the present level on the assumption that the present seafood composition is constant. It is warranted to examine if the decreasing seafood consumption in Japan has adverse effects on other outcomes, e.g., cardiovascular and cerebrovascular disease/mortality, to further evaluate health effects of the dietary habit change."}
+{"text": "Larotrectinib and entrectinib are FDA-approved therapies for patients with non-small cell lung cancer (NSCLC) with neurotrophic receptor tyrosine kinase gene fusion (TRK fusion-positive) whose cancer has metastasized and progressed. Early evidence indicates that these targeted therapies may offer dramatic survival benefits versus traditional cytotoxic regimens, but it remains uncertain how larotrectinib and entrectinib compare with each other.To simulate and compare expected life-years and quality-adjusted life-years (QALYs) for both TRK inhibitors.We developed a partitioned survival model to project the long-term comparative effectiveness of larotrectinib versus entrectinib in second-line treatment of metastatic NSCLC. Larotrectinib survival data were derived from a 13-month follow-up of 12 patients with TRK fusion-positive NSCLC in the NCT02122913 (phase 1) and NCT02576431 (NAVIGATE) trials. Entrectinib survival data were derived from a 13-month follow-up of 10 patients with TRK fusion-positive NSCLC in the ALKA-372-001, STARTRK-1, and STARTRK-2 trials. For larotrectinib and entrectinib progression-free survival and overall survival (OS), in-trial survival was extrapolated using parametric curve fits. Exponential fits were selected for all survival models based on minimal Bayesian information criteria and clinical plausibility. Lifetime survival curves were used to estimate expected mean/median survival. QALYs were estimated by applying preprogression and postprogression health state utilities derived from the literature.In the base case, treatment with larotrectinib and entrectinib resulted in 5.4 and 1.2 median preprogression life-years and 7.0 and 1.8 median total life-years, respectively. Mean preprogression life-years (QALYs) were 7.5 (5.0) and 1.9 (1.2), and mean total life-years (QALYs) were 9.2 (5.8) and 4.4 (2.4), respectively.Among TRK inhibitors for metastatic NSCLC, larotrectinib is estimated to provide improved life-year and QALY outcomes versus entrectinib based on parametric extrapolations of in-trial survival data. Our analysis is limited by lack of NSCLC-specific data on entrectinib OS, the small samples of patients with NSCLC in the trials, and a cross-trial comparison. Future studies should re-evaluate the comparative effectiveness of larotrectinib versus entrectinib as more patients are treated and as long-term survival data mature. What is already known about this subjectEarly evidence indicates that the receptor tyrosine kinase (TRK) inhibitors larotrectinib and entrectinib may offer dramatic survival benefits versus traditional cytotoxic regimens for treatment in TRK fusion-positive metastatic non-small cell lung cancer (NSCLC).No study has evaluated the potential comparative effectiveness of larotrectinib versus entrectinib for second-line treatment of TRK fusion-positive metastatic NSCLC.What this study addsWe used larotrectinib and entrectinib clinical trial data and simulation modeling to project the potential long-term comparative effectiveness of the 2 FDA-approved TRK inhibitors for treatment in TRK fusion-positive metastatic NSCLC.We found that larotrectinib is expected to provide improved life-year and quality-adjusted life-year outcomes compared with entrectinib based on parametric extrapolations of in-trial survival data.Future studies should reevaluate the comparative effectiveness of larotrectinib and entrectinib as greater numbers of patients are treated and as long-term survival data mature.2Larotrectinib and entrectinib are therapies approved by the U.S. Food and Drug Administration (FDA) for adult patients with neurotrophic receptor tyrosine kinase gene fusion (TRK fusion-positive) found in multiple solid tumor types whose cancer has metastasized and progressed. Entrectinib also was approved for treatment in adults with non-small cell lung cancer (NSCLC) with ROS1 fusion-positive tumors. Emerging evidence indicates that these targeted therapies may offer potentially dramatic survival benefits for patients compared with traditional cytotoxic regimens.3 However, data on larotrectinib and entrectinib comparative effectiveness and safety remain uncertain, in part because of the design of the clinical research programs used to support FDA approval. Both clinical research programs employed single-arm, basket trial designs without concurrent or historical control groups in relatively small populations of patients.2 The trial populations were relatively similar across key characteristics, and the available data to date indicate longer median progression-free (PFS) and overall survival (OS) in samples of all patients treated across tumor types with larotrectinib  as compared with entrectinib .2 In small samples of patients with NSCLC, larotrectinib median PFS and OS were not reached (n = 12), while entrectinib median PFS was 14.9 months, and OS results have not been reported (n = 10).Guidelines recommend that patients who initiate on either larotrectinib or entrectinib do not use the alternate TRK inhibitor as a subsequent line of treatment, that is, clinicians and patients need to decide between these 2 products for their only chance at using a TRK inhibitor.4 The objective of this study was to use available evidence to extrapolate and compare expected life-years and QALYs for both TRK inhibitors in metastatic NSCLC to inform initial stakeholder considerations about potential comparative effectiveness.Of importance to clinicians and decision makers is that extrapolation of within-trial survival outcomes using standard parametric curve fit methods can provide more comprehensive estimates of the potential comparative effectiveness of larotrectinib and entrectinib in treatment for NSCLC and can allow for estimation on mean life expectancy and quality-adjusted life-years (QALYs).5 The simulation evaluated preprogression and OS over a lifetime horizon . The model was populated with larotrectinib and entrectinib survival, response, and health-related quality of life data drawn from several clinical trials reported at recent major oncology conferences. It should be noted that all source studies had limited sample size (n < 60) and median follow-up duration (13 months).6 We modeled parametric curve fits to in-trial PFS and OS data to project long-term survival outcomes.7 Outcomes included expected 3-, 5-, and 10-year survival proportions as well as mean expected life-years over a lifetime horizon.We developed a partitioned survival simulation model in Microsoft Excel  to project long-term comparative effectiveness of larotrectinib and entrectinib in second-line treatment of metastatic NSCLC.2 Among patients in this sample, median age was 52.0 years; 50% (n = 6) were female; 92% (n = 11) had \u2265 1 previous systemic therapy; 50% (n = 6) had 3 or more systemic therapies; and 8% (n = 1) had no previous systemic therapy.Larotrectinib OS and PFS data were derived from a 13-month follow-up of 12 patients with TRK fusion-positive NSCLC in the NCT02122913 (phase 1) and NCT02576431 (NAVIGATE) trials.6 Among patients in this sample, median age was 62.5 years, 50% (n = 5) were female, 30% (n = 3) had 1 previous systemic therapy, 40% (n = 4) had 2 or more systemic therapies, and 30% (n =3) had no previous systemic therapy.Entrectinib NSCLC PFS data were derived from a 13-month follow-up of 10 patients with TRK fusion-positive NSCLC in the ALKA-372-001, STARTRK-1, and STARTRK-2 trials.6 This approach resulted in a mean of 4.38 expected life years\u201474% greater than the mean life-years estimated from the model (2.52) when OS was derived from pooled entrectinib results across 54 patients with any TRK fusion-positive tumor type in the ALKA-372-001, STARTRK-1, and STARTRK-2 trials and extrapolated with an exponential curve fit.While there have been reports of entrectinib OS pooled across TRK fusion-positive tumor types, there have not been reports of entrectinib OS data in the subgroup of patients with TRK fusion-positive NSCLC. To model survival in the absence of such data, we imputed entrectinib OS by applying the same monthly ratio of OS to PFS observed in patients receiving larotrectinib to entrectinib PFS outcomes in ALKA-372-001, STARTRK-1, and STARTRK-2 trials.7 For entrectinib PFS, an exponential model was fit to the median PFS estimate for 10 patients with TRK fusion-positive NSCLC.6 This approach was taken in the absence of publicly reported Kaplan-Meier survival curves showing monthly survival proportions over the 13-month follow-up period.In-trial survival outcomes were extrapolated using parametric curve fits  for larotrectinib PFS and OS as well as entrectinib OS.8 If parametric curve fit annual mortality rates were less than that of U.S. life tables for a given mean age of the cohort, then mortality rates reverted to those of the life tables.Based on minimal Bayesian information criteria, clinical plausibility, and visual inspection, exponential fits were selected to simulate long-term survival in all PFS and OS survival models. Lifetime survival curves were used to estimate expected mean and median PFS and OS. Lifetime survival curves were designed so that survival age-based mortality rates could not be lower than those for the general public as reported in U.S. life tables.9 These values were applied to survival time in preprogression and postprogression states to estimate expected QALYs over a lifetime horizon.Health state utility values for preprogression and postprogression health states  were derived from a societal based valuation study of 100 people in the United Kingdom using standard gamble methods.Due to the small samples of patients with NSCLC in the larotrectinib (n = 12) and entrectinib (n = 10) trials and the limited median follow-up duration of 13 months, there was a high degree of uncertainty about long-term survival with both therapies. As such, we undertook sensitivity analyses with wide uncertainty ranges to explore the implications of alternative survival outcomes that might be demonstrated with larger samples tracked over longer follow-up periods. Specifically, we evaluated survival and QALY outcomes in scenarios where (a) larotrectinib OS hazard was increased by 30%  versus the base case larotrectinib OS function, and (b) entrectinib OS was decreased by 30%  versus the base case entrectinib OS function. The magnitude of variation in survival functions (\u00b1 30%) was arbitrarily selected as a large but statistically plausible deviation in survival outcomes as compared with the base case. We took this approach in absence of reported 95% confidence intervals for survival outcomes. Furthermore, it would be expected that survival function confidence intervals derived from samples of 10 or 12 patients that could have highly variable survival durations would result in such extreme values that they would not be informative in our sensitivity analyses.Using the sensitivity analyses survival functions previously described, we conducted 3 additional pairwise comparisons of larotrectinib and entrectinib: (1) sensitivity analysis of larotrectinib survival versus base case entrectinib survival, (2) base case larotrectinib survival versus sensitivity analysis of entrectinib survival, and (3) sensitivity analysis of larotrectinib survival versus sensitivity analysis of entrectinib survival. These comparisons would assess (a) comparative effectiveness under less favorable larotrectinib survival, (b) comparative effectiveness under more favorable entrectinib survival, and (c) comparative effectiveness under an extreme scenario with less favorable larotrectinib survival and more favorable entrectinib survival, respectively. We conducted these analyses recognizing the possibility that if all resulted in positive incremental survival outcomes for larotrectinib, it could reinforce the likely favorable comparative effectiveness of larotrectinib versus entrectinib in the treatment for TRK fusion-positive NSCLC that would likely be demonstrated in the base case.In the base case, treatment with larotrectinib and entrectinib resulted in a median of 5.4 and 1.3 preprogression life-years and a median of 7.0 and 1.8 total-life years, respectively . Mean prIn the base case, treatment with larotrectinib and entrectinib resulted in mean preprogression QALYs of 5.0 and 1.2 and mean total QALYs of 5.8 and 2.4, respectively .In scenario 1, treatment with larotrectinib and entrectinib resulted in 5-year survival proportions of 53% and 22%, respectively. Mean preprogression life-years were 6.0 and 1.9, and mean QALYs were 4.0 and 1.2, respectively . Mean toIn scenario 2, treatment with larotrectinib and entrectinib resulted in 5-year survival proportions of 61% and 35%, respectively. Mean preprogression life-years were 7.5 and 2.6, and mean QALYs were 5.0 and 1.7, respectively . Mean toIn scenario 3, treatment with larotrectinib and entrectinib resulted in 5-year survival proportions of 53% and 35%, respectively. Mean preprogression life-years were 6.0 and 2.6, and mean QALYs were 4.0 and 1.7, respectively . Mean toUsing emerging clinical data, we set out to develop comparative effectiveness estimates of larotrectinib and entrectinib in patients with TRK fusion-positive metastatic NSCLC. We undertook this analysis to better inform clinical and health care system reimbursement decisions, in part because both of these targeted, tumor-agnostic agents were FDA-approved with very limited clinical evidence.We found that among TRK fusion protein-targeted therapies for metastatic NSCLC, larotrectinib is estimated to provide improved life-year and QALY outcomes versus entrectinib using extrapolations of in-trial survival data. Larotrectinib survival gains remain highly uncertain, but scenario analyses suggested that gains are expected to persist under a range of clinically plausible survival effects. Future studies should re-evaluate the comparative outcomes of larotrectinib and entrectinib as more patients are treated, survival data mature, and real-world evidence studies are conducted in TRK fusion-positive NSCLC.3 Five years ago, patients with TRK fusion-positive tumors were limited to untargeted products. With the arrival of the first TRK inhibitors, larotrectinib and entrectinib, these patients can potentially achieve unprecedented response rates and survival outcomes.6 However, the choice between TRK inhibitors is critical as guidelines recommend that patients receive only 1 of the 2 available treatment options. Given the evidence available in the peer-reviewed literature and conference proceedings, coupled with the estimates provided here, larotrectinib may be the optimal treatment option for patients who are TRK fusion-positive who have no satisfactory alternative treatments or that have progressed following cytotoxic treatment.In the past 20 years, the treatment landscape in advanced NSCLC has evolved from a few traditional chemotherapeutic options with limited effectiveness to now include many innovative products ranging from immunotherapeutics to agents targeted to a variety of tumors molecular profiles.6 As such, there is a high degree of uncertainty about how well survival data from these samples will generalize to larger samples of patients with TRK fusion-positive NSCLC treated with larotrectinib or entrectinib. Recognizing this issue, we conducted a series of sensitivity analyses that widely varied the OS hazard functions for each agent and found that larotrectinib was still expected to result in a potentially clinically meaningful gain in survival versus entrectinib even if the larotrectinib hazard was increased by 30% and the entrectinib hazard was decreased by 30%.Our analysis has several important limitations that should be noted. First, in-trial survival data were derived from small samples tracked for a median of 13 months.6Second, there is limited publicly reported data about OS outcomes in patients with TRK fusion-positive NSCLC treated with entrectinib. To deal with this, we imputed entrectinib OS by applying the same monthly ratio of OS to PFS that was modeled for larotrectinib to the parametric extrapolation of in-trial PFS for entrectinib. This approach closely replicated reported OS in a pooled sample of TRK fusion-positive tumors treated with entrectinib and is also expected to minimize differences in comparative survival between larotrectinib versus entrectinib\u2014an important factor to mitigate bias in our incremental analyses.8 It is also possible that there are unmeasured prognostic factors that could vary between samples, which could impact comparative effectiveness estimates.Finally, this study involves a naive, direct  comparison of survival with larotrectinib and entrectinib. As samples were not derived from a single randomized trial, there is possible imbalance in prognostic factors between samples. For example, median age in the larotrectinib sample (52.0 years) was lower than median age in the entrectinib sample (62.5 years). This imbalance may account for some fraction of the more favorable survival we demonstrated for larotrectinib versus entrectinib. Still, given the relatively short survival duration of all patients diagnosed with advanced NSCLC, this imbalance likely only explains a limited amount of the mean survival differences we estimated. For example, U.S. life tables for the general public demonstrate that mortality rates for persons aged 52 years is only 1.09-fold greater than those aged 62 years, and our sensitivity analyses examined incremental outcomes under much larger uncertainty ranges.Our results should be interpreted with these notes in mind, and comparative effectiveness should be reassessed using rigorous observational research designs as each agent has increased real-world use in clinical practice.Among TRK inhibitors for treatment of metastatic NSCLC, larotrectinib is estimated to provide improved life-year and QALY outcomes compared with entrectinib based on parametric extrapolations of in-trial survival data. Our analysis is limited by a paucity of NSCLC-specific data on entrectinib OS; the small sample size of patients with NSCLC in source trials; and a naive, direct  comparison. Future studies should re-evaluate the comparative effectiveness of larotrectinib and entrectinib as greater numbers of patients are treated and as long-term survival data mature."}
+{"text": "The need for high-speed communication has created a way to design THz antennas that operate at high frequencies, speeds, and data rates. In this manuscript, a THz MIMO antenna is designed using a metamaterial. The two-port antenna design proposed uses a complementary split-ring resonator patch. The design results are also compared with a simple patch antenna to show the improvement. The design shows a better isolation of 50 dB. A broadband width of 8.3 THz is achieved using this complementary split-ring resonator design. The percentage bandwidth is 90%, showing an ultrabroadband response. The highest gain of 10.34 dB is achieved with this design. Structural parametric optimization is applied to the complementary split-ring resonator MIMO antenna design. The designed antenna is also optimized by applying parametric optimization to different geometrical parameters. The optimized design has a 20 \u00b5m ground plane, 14 \u00b5m outer ring width, 6 \u00b5m inner ring width, and 1.6 \u00b5m substrate thickness. The proposed antenna with its broadband width, high gain, and high isolation could be applied in high-speed communication devices. Antennas are a type of transducer used for communicating wirelessly between two devices. Antennas, which in the past were designed to have a huge aperture size, have now been reduced to small and compact nanoantennas. This reduction in the size of the antenna also extensively reduced its gain; therefore, there is now a need for high-gain compact antennas. There are various ways of improving the gain and bandwidth of an antenna, and one of these ways is to incorporate metamaterials. Today, high-speed communication needs THz antennas to be operated at high speeds. The THz antenna has in recent times been researched by many researchers to be used in high-speed wireless communication devices. The THz antenna offers a higher bandwidth, which could be used to transfer more data at high speeds. The need for high-gain and high-bandwidth THz antennas has increased. The gain and bandwidth of these THz antennas can be improved using metamaterials.Metamaterials are artificial materials that can be used to improve many parameters of THz antennas . Split-rToday, THz MIMO antennas are used in high-speed communication devices because of their good isolation, high gain, and high bandwidth. MIMO array antennas have been designed with graphene materials, and high isolation has been achieved using a serpentine resonator. The antennas have been applied in THz communication . The twoThe MIMO antenna design has been investigated for use in 5G communication, and has also been presented for integration with portable devices . The useThe optimization of the structural design is essential in achieving the optimized parameters. The different types of optimization that can be applied to these designs are nonlinear parametric optimization and linear parametric optimization . The selThe growing demand for high-speed wireless communication devices has led the way toward the design of an antenna that works at the THz frequency. As such, we propose a MIMO antenna design that shows a broadband and high isolation response at the THz frequency range. The proposed antenna is designed with a CSRR-loaded patch antenna. The antenna design comprising a simple microstrip patch is also designed with similar dimensions to show the improvement in the design. Nonlinear parametric optimization is applied to different parameters to achieve the optimal MIMO antenna design. The proposed antenna could be essential for use in high-speed wireless communication devices. We show the design, results, and analysis in the upcoming sections.2 in size. The two elements of this patch were used to prepare the two-port MIMO antenna. The MIMO antenna was fed through with a matched microstrip line, as shown in The THz MIMO antenna design was first prepared with a simple square patch, complemented by split-ring resonator etching, and, finally, through preparing the design of the complementary split-ring resonator patch antenna. The design is presented in The antenna parameter calculation mainly depended on the following equation, where the length and width are inversely proportional to the frequency, as shown in Equations (1)\u2013(4):The envelope correlation coefficient (ECC) and diversity gain (DG) could be calculated as per Equations (5) and (6) .(5)ECC=The improvement in the SNR of the multiple-element system over a one-element system is referred to as diversity gain (DG). The DG was calculated using the following Equation (6).The results of the ECC and DG are discussed further in the results and discussions section.2 in size was simulated first, and its response in terms of the S-parameters and gain was obtained. The square patch design was then etched with a split-ring resonator, and a complementary split-ring resonator metamaterial patch design was obtained, given in The design presented in The highest gain for the simple patch MIMO antenna design was 4.3 dB, as shown in The different MIMO antenna parameters, such as the diversity gain (DG) and envelope correlation coefficient (ECC), were calculated in this section to show their effect on the analyzed spectrum ranging from 5 to 15 THz. Both parameters were analyzed using Equations (5) and (6). The S-parameter results were applied to the equation, and the results achieved for the ECC and DG were given in The optimization algorithms could be applied to different structural parameters to obtain optimized parameters that give not only the best results, but also a compact design. The parametric optimization method can be applied to these structural parameters to obtain different optimized parameters. There are two main types of parametric optimization algorithms . The firx that were nonlinear [The functions did not behave linearly, which gave this optimization. It had function onlinear .The optimization of different structural parameters, such as the substrate height, ground plane width, and CSRR ring widths, was carried out to optimize antenna results, such as the bandwidth and S-parameters.The ST optimization was carried out to obtain the highest bandwidth and good results for the MIMO antenna design. The optimization was applied to the CSRR metamaterial-loaded MIMO antenna design. The substrate of the design varied from 0.5 \u00b5m to 1.5 \u00b5m to observe its effect on the absorption results. The variation was kept at 1.5 \u00b5m, because increasing the substrate more than this would increase the overall area of the structure, as well as the cost of the structure, so it was preferential to increase it to a certain limit, keeping it to a 1.5 \u00b5m thickness, so that the substrate was kept to a limit, suitable for fabrication and also reducing the cost of the substrate. The variation in the figure clearly showed that the reflectance shown in 21 results of the design are presented in The GW optimization was carried out to obtain the highest bandwidth and good results for the MIMO antenna design. The optimization was applied to the CSRR metamaterial-loaded MIMO antenna design. The ground layer width of the design varied from 20 \u00b5m to 35 \u00b5m to observe its effect on the absorption results. The variation was kept from 20 \u00b5m to 35 \u00b5m, because increasing the ground layer width further would have given abrupt results, and the defected ground concept was implemented by etching the part of the ground plane. The variation in the figure clearly showed that the reflectance shown in 2) optimization was carried out to obtain the highest bandwidth and good results for the MIMO antenna design. The optimization was applied to the CSRR metamaterial-loaded MIMO antenna design. The inner ring width of the design varied from 6 \u00b5m to 8 \u00b5m to observe its effect on the absorption results. The variation was kept at 6 \u00b5m to 8 \u00b5m, because increasing the width of the inner ring further would mix with the outer ring of the CSRR. The change in the ring width would change the capacitance of the metamaterial design. The increase in the width increased the capacitance and degraded the results, as shown in 21 results also showed that for the increased inner width, the results of the transmittance showed a peak of 20 dB, which was not valid and could not be considered. The optimized value of the inner ring width was 6 \u00b5m.The inner ring width (R1) optimization was carried out to obtain the highest bandwidth and good results for the MIMO antenna design. The optimization was applied to the CSRR metamaterial-loaded MIMO antenna design. The outer ring width of the design varied from 14 \u00b5m to 16 \u00b5m to observe its effect on the absorption results. The variation was kept at 14 \u00b5m to 16 \u00b5m, because increasing the width of the outer ring would increase the capacitance of the structure. The increase in the width increased the capacitance and degraded the results, as shown in 21, with the results presented in The outer ring width (R4 A/m. The 2D radiation pattern plot is also presented in The permittivity, which is very important for metamaterials, was also obtained and presented in The THz MIMO antenna design for the CSRR metamaterial patch element and simple patch element was compared with outer designs and presented in We compared two THz MIMO antenna designs in our research, namely, a CSRR metamaterial design and a square patch design. The gain and S-parameters were compared for these two designs. The CSRR design showed better performance compared to the other design at a 50 dB isolation, 10.34 dB gain, and 8.3 THz ultrabroadband width. The investigation was carried out for a 5 to 15 THz frequency range. Nonlinear parametric optimization was applied to the ground layer width, substrate thickness, CSRR inner ring width, and CSRR outer ring width. The optimized design parameters were achieved through this optimization. The current distribution and radiation patterns were also presented for the CSRR metamaterial design. The permittivity of the CSRR metamaterial design showed a negative behavior for its real and imaginary parts. The designed MIMO antennas were also compared with other published works. Overall, the proposed MIMO antenna with its high isolation, high gain, and broadband response could be applied in high-speed wireless communication devices."
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