Supplemental Digital Content is available in the text Thoracic-surgery-related unilateral vocal fold paralysis (UVFP) may cause severe morbidity and can cause profound functional impairment and psychosocial stress in patients with pre-existing thoracic diseases. In-office intracordal hyaluronate (HA) injections have recently been applied to improve voice and quality of life in patients with vocal incompetence, but their effect on thoracic-surgery-related UVFP remains inconclusive. We therefore conducted a prospective study to clarify the effect of early HA injection on voice and quality of life in patients with thoracic-surgery-related UVFP.Patients with UVFP within 3 months after thoracic surgery who received office-based HA injection were recruited. Quantitative laryngeal electromyography, videolaryngostroboscopy, voice-related life quality (voice outcome survey), laboratory voice analysis, and health-related quality of life (SF-36) were evaluated at baseline, and at 1 month postinjection.A total of 104 consecutive patients accepted office-based HA intracordal injection during the study period, 34 of whom were treated in relation to thoracic surgery and were eligible for inclusion. Voice-related life quality, voice laboratory analysis, and most generic quality of life domains were significantly improved at 1 month after in-office HA intracordal injection. No HA-related complications were reported.Single office-based HA intracordal injection is a safe and effective treatment for thoracic-surgery-related UVFP, resulting in immediate improvements in patient quality of life, voice quality, and swallowing ability. The above findings indicate the importance of early intervention to improve laryngeal function in these patients, and to alleviate the impact on their quality of life.Unilateral vocal fold paralysis (UVFP) is a common complication of some surgeries, such as thyroidectomy, cervical discectomy, and esophagectomy.6 Although this was well tolerated in most patients, it failed in 1 patient as a result of a side effect of general anesthesia induction. Furthermore, patients may already be debilitated after major thoracic surgery, preventing them from undergoing conventional laryngoplasty to restore the laryngeal function.7 The development of digital imaging systems and distal-chip laryngoscopy has allowed the introduction of office-based injection laryngoplasty, which represents a candidate standard therapy for early phase thoracic-surgery-related UVFP. Grant et al reported that 14 of 15 patients with thoracic-surgery-induced UVFP successfully accepted awake injection laryngoplasty.8 They suggested that it was a helpful technique for the immediate correction of swallowing and vocal impairment. However, its use remains inconclusive because of the use of a variety of materials for injection, and a lack of objective pre- and postoperative measurements.Acute injection laryngoplasty under general anesthesia has been used for thoracic-surgery-related UVFP.9 Voice quality and related quality of life improved immediately, with some long-term effects. However, the general health status of patients with UVFP and its causes differ, and thus its impacts may also differ. Patients with UVFP as a result of thoracic surgery are likely to have poorer health and stay longer in hospital for intensive care10 than those with UVFP from other causes. Some patients may be at increased risk of other complications, especially in relation to general anesthesia. The need for early treatment of UVFP and its effects should thus be evaluated for each specific cause.9We recently demonstrated the benefit of office-based hyaluronate (HA) injection in acute UVFP.11 and office-based injections have thus increased in popularity. This case-series study aimed to evaluate the feasibility of early intracordal HA injection in patients with thoracic-surgery-related UVFP and to determine the short-term postoperative outcomes. We hypothesized that HA injection for these patients can improve their voice and quality of life.The effect of injection laryngoplasty may differ according to the injectable material used. The safety and effectiveness of injection laryngoplasty using HA has been reported for the management of UVFP. HA injections immediately improved the voice in patients with acute UVFP,®, Q-Med AB, Uppsala, Sweden) and received the follow-up assessment. The inclusion of eligibility for all patients within a period of time was used to avoid selection bias. Written informed consent was obtained from each participant before recruitment. We recruited patients with thoracic-surgery-related UVFP and received office-based HA injection. The date of thoracic surgery should be within 3 months before our first outpatient assessment. The patient's voice should be adequate before the thoracic surgery and UVFP-associated symptoms occurred within 1 month following the thoracic surgery. The diagnosis of UVFP was confirmed by both laryngoscopy and laryngeal electromyography (LEMG) , videolaryngostroboscopy, Mandarin Chinese-version Voice Outcome Survey (VOS) Table , laborat14 Specifically, videolaryngoscopy captured images whereas the patient vocalized /eee/ at modal pitch and regular loudness. The glottal gap was calculated (in square pixels) and membranous vocal-fold length was measured from the point of the anterior commissure to the point of the tip of the vocal process (in linear pixels). The NGGA was computed by equation (1): The normalized glottal gap area (NGGA) was analyzed using Image J software , following the method defined by Omori et alGlottal gaps were measured individually during the open and closed phases in the phonatory cycle.15 consists 5 items that evaluate the UVFP-relative physical and social problems. Each survey item and the total scores were normalized on a 0 (worst) to 100 (best) scale. The Mandarin Chinese version of VOS questionnaire has been validated by the present research team.16The VOS questionnaire, developed by Gliklich et al,Each patient was asked to read a standard passage and a sustained vowel at a conversational pitch and loudness in a sound-insulated room. Their voice was captured using a unidirectional dynamic microphone  with a distance of 10 cm between mouth and the microphone and an off-axis angle of 45°. Voice was then recorded and sampled using voice-analysis software  with a sampling rate of 25.6 kHz and a 16-bit quantization. Modal fundamental frequency, perturbation of frequency (jitter) and amplitude (shimmer), and harmonic-to-noise ratio (HNR) were tabulated from the recorded voice. Each parameter reflects a specific voice dimension. The values of jitter and shimmer reflect the deviation from voice periodicity and tend to increase in patients with voice problems. HNR quantifies the amount of additive noise produced by turbulent glottal airflow and was suggested to be more analogous to the perception evaluation. The maximal phonation time (MPT) represented the longest duration of sustaining a vowel /a/. The SZ ratio was the ratio of the voicing duration of /s/ to /z/, which represents the patient's vocal control, with the ideal reference value being close to 1.0. Patients with UVFP tend to have shorter MPT and longer SZ ratio as compared to healthy subjects.Intracordal HA injection is getting popular in recent years, but this procedure is an off-label use. Patients received intracordal HA injection in an office-based setting Figure . Before t tests for parametric data and χ2 tests for categorical data. The α-value was defined as 0.05.Data were analyzed using SPSS . One out of 34 patients had missing data in laboratory voice analysis and the patient was not analyzed specifically for the laboratory voice parameters. Parameter changes over time were compared using Student's During the study period, 123 patients with UVFP accepted in-office HA injection postinjection assessment, among whom 20 were excluded . Among the remaining 103 patients, 34 patients were related to thoracic surgery. Patient demographics, type of surgery, and peak turn frequencies of LEMG of thyroarytenoid–lateral cricoarytenoid muscles are listed in Table In 9 of the 34 cases, UVFP derived from surgery for benign diseases. Twenty-five patients developed UVFP after cancer surgery; 8 for lung cancer, 16 for esophageal cancer, and 1 for mediastinal malignancy. The detailed characteristics of patients in terms of cancer-related surgeries are shown in Table P < 0.001). The acoustic and aerodynamic parameters measured in the voice laboratory showed significant improvements in all but the fundamental frequencies , role limitations owing to physical health (P = 0.026), role limitations owing to emotional problems (P = 0.017), general health bodily pain (P = 0.031), and social functioning (P = 0.001)  and vitality (P = 0.202) were also improved, but the differences were not significant. Voice-related quality of life measured by VOS showed significant improvements in all items  and in the composite score (P < 0.001) .Recurrent laryngeal nerve injury that manifests with UVFP is a common complication of thoracic surgery. Patients with thoracic-surgery-related UVFP tend to suffer from breathy voice and swallowing disturbances, and are at increased risk of aspiration pneumonia and death. Most patients require intensive care immediately following thoracic surgery, and compromised cardiopulmonary function may make them ineligible for injection laryngoplasty under general anesthesia. Given that oral feeding may subsequently be delayed in these patients solely as a result of a correctable UVFP, office-based injection laryngoplasty represents a unique opportunity for providing a safe and effective early intervention.6 Office-based injection laryngoplasty, in which the procedure is performed in awake patients, might thus offer a safe alternative method for early intervention. Indeed, we previously reported the use of office-based HA injection for acute UVFP.13 The injected HA would remain in the vocal cord temporarily17 and may thus not interfere with the spontaneous recovery of vocal-fold motion. Given that general anesthesia may not be tolerated by most patients immediately after thoracic surgery, office-based HA injection may help to restore their voice function, with minimal risk. Subjects demonstrated fast recovery after office-based HA injection and were able to talk and eat on the same day. The shortened recovery period also enabled patients to undergo appropriate early adjuvant therapy (eg chemoradiation therapy) for the superimposed thoracic disease.Injection laryngoplasty has been an established treatment for UVFP for decades and has recently been suggested for early-phase UVFP. However, although early injection laryngoplasty with a temporary agent under general anesthesia showed promising results in patients with thoracic surgery-related UVFP, 1 of 20 cases was complicated by pneumonitis caused by induction anesthesia.Five patients in the current series had died by the end of follow-up, at a mean of 13 months post-HA injection, all as a result of their thoracic malignancies. Although the survival of patients with thoracic malignancies may be relatively short, the procedure nevertheless improved their life quality without the need for a prolonged hospital stay.The present study showed that office-based intracordal HA injection was safe and effective during the early phase of UVFP and improved voice and quality of life in patients after thoracic surgery. The procedure can be accomplished within 30 min. In our experience, the main difficulty associated with awake injection is handling acid reflux and gag reflex, especially in patients after esophagectomy. Some patients are required to clear their throat several times to expose the larynx, to avoid its being obscured by secretions. We suggest performing the injection in a sitting, rather than a supine position. The patient should be instructed not to take food for at least 2 h before the procedure and should be prepared by adequate mucosal numbing.Voice may be restored immediately following office-based HA injection. Although HA may not alter motor unit recruitment, it can correct the glottal gap, voice, and the associated quality of life immediately after injection and at 1-month follow-up. The complication of fluid aspiration may also be reduced, because the protective effect of vocal closure is re-established. Quality of life analysis also showed an immediate positive effect of early HA injection not only on physical function, but also on psychosocial well-being.18 Friedman et al showed that 63% of patients who received HA injection within 6 months of symptom onset retained adequate voice at long-term follow-up.19 In addition, early HA injection reduced the need for transcervical medialization laryngoplasty in patients with UVFP.20 However, the present study focused on the short-term effects of office-based HA injection for thoracic-surgery-related UVFP, and further studies are needed to investigate its long-term outcome in these patients.Although the injected HA is absorbed over time, it may still have a long-lasting effect if given during the early phase of UVFP, especially in patients with a large glottal gap.The left recurrent laryngeal nerve has a longer passage in the thoracic cage than the right recurrent laryngeal nerve, which explains why most cases of thoracic-surgery-related UVFP occurred on the left side. However, the 4 right UVFP cases in the present study resulted from thoracoscopic surgical procedures involving the upper mediastinum or right upper lobe. Popularization of the technique of minimally invasive endoscopy might be associated with an increase in the incidence of right-side vocal paralysis. It is therefore important to note the surgical anatomy of the right recurrent laryngeal nerve in cases requiring upper thoracic procedures.Office-based intracordal HA injection can provide immediate improvements in voice and swallowing in patients with UVFP, allowing an earlier return to normal social functioning. The procedure is safe and effective, and is thus highly recommended as an early intervention in these patients, including those with thoracic-surgery-related UVFP.


Desulfovibrio vulgaris under planktonic and biofilm growth modes, a single-cell based RT-qPCR approach was applied to determine gene expression levels of 8 selected target genes in four sets of the 31 individual cells isolated from each growth condition  and planktonic cultures, exponential and stationary phases). The results showed obvious gene-expression heterogeneity for the target genes among D. vulgaris single cells of both biofilm and planktonic cultures. In addition, an increased gene-expression heterogeneity in the D. vulgaris biofilm when compared with the planktonic culture was also observed for seven out of eight selected genes at exponential phase, and six out of eight selected genes at stationary phase, respectively, which may be contributing to the increased complexity in terms of structures and morphology in the biofilm. Moreover, the results showed up-regulation of DVU0281 gene encoding exopolysaccharide biosynthesis protein, and down-regulation of genes involved in energy metabolism , stress responses  and response regulator  in the D. vulgaris biofilm cells. Finally, the gene (DVU2571) involved in iron transportation was found down-regulated, and two genes (DVU1340 and DVU1397) involved in ferric uptake repressor and iron storage were up-regulated in D. vulgaris biofilm, suggesting their possible roles in maintaining normal metabolism of the D. vulgaris biofilm under environments of high concentration of iron. This study showed that the single-cell based analysis could be a useful approach in deciphering metabolism of microbial biofilms.Sulfate-reducing bacteria (SRB) biofilm formed on metal surfaces can change the physicochemical properties of metals and cause metal corrosion. To enhance understanding of differential gene expression in  Desulfovibrio vulgaris on a steel surface  analysis , and to further confirm the relationship between the selected genes and biofilm metabolism at a single-cell level, we applied a real-time reverse-transcription quantitative PCR (RT-qPCR) approach (D. vulgaris biofilm was cultivated on mild steel (SS) slides to mimic microenvironments of metal corrosion. As an attempt to quantify heterogeneity levels of gene-expression between single cells in the D. vulgaris biofilm, the study could contribute to the further understanding of biofilm metabolism related to metal corrosion in natural environments.In this study, with major aims to determine gene-expression heterogeneity between approach . To do tDesulfovibrio vulgaris subsp. vulgaris strain Hildenborough DSM 644 used in this study was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen  and cultured in mineral medium as described by a previous publication  were purchased from BioSurface Technologies . The SS slides were cleaned according to a previous study (595 nm = 0.4), and all cultivation experiments were carried out at 35°C under anaerobic conditions, by using an anaerobic chamber  for this purpose. These anaerobic conditions were achieved according to our previous publication .Cells collected from the biofilm and planktonic cultures were re-suspended in 1 mL of 50 mM PBS (pH 6.5). For 1 mL cell suspension, 10 μL of phenylmethylsulfonyl fluoride (PMSF) was added. After cell suspensions were repeatedly frozen in liquid nitrogen and thawed at 25°C room temperature for three times, the suspended cells were broken by ultrasonication with an ultrasonic disrupter  with ultrasonic amplitude transformer probe. The conditions for ultrasonic disruption was 5 s working time, 5 s interval time, 10 min total time under 60% ultrasonic power. Protein and carbohydrate levels were measured for the samples to confirm the growth status (biofilm vs. planktonic). The carbohydrate concentrations were measured using the anthrone-sulfuric acid colorimetry with sucrose as standard , 1955. PD. vulgaris biofilm, 8 target genes  were selected for the following single-cell analysis  data obtained from RT-qPCR analyses against an internal control gene  (gap-1), and recombinase A gene  (D. vulgaris single cells (each containing 12 single cells) from D. vulgaris biofilm and planktonic culture, respectively, conducted RT-qPCR analysis of three internal control genes for all single cells, and then calculated the mean and standard deviations (SDs) of CT values from single-cell RT-qPCR using the OriginPro 8.0 software.To minimize differences between single cells due to sample preparation , it was supposed to normalize the resulting threshold cycle (Crol gene . So far oculture . To obta(Dv16SA) , glycera0, recA) . Toward g). To keep their gene expression profiles intact, the planktonic cells were put into a RNALater solution immediately   .D. vulgaris biofilms, superficial cells of the mature biofilm formed on the surface of SS slides were slightly washed off with 50 mM oxygen-free phosphate-buffered saline  using a dropper under anaerobic conditions in the anaerobic chamber, then inner mature biofilm cells were scraped using a sterile razor blade from the surface of SS slides. To keep their gene expression profiles intact, the biofilm cells were also put into a RNALater solution immediately   . The sussolation .Single cells were isolated using an inverted IX71 microscope connected with a single cell manipulator  from the cell suspension according to previous publications . Total RD. vulgaris cells were randomly isolated from three culture bottles under the identical growth and subjected to the single-cell RT-qPCR analysis from each biological sample of the biofilm and the planktonic cultures. To ensure no significant change of gene expression profiles, the sample cells were put into a RNALater solution and the single-cell isolation process for 31 cells was completed within 30 min  as the internal reference to normalize the resulting threshold cycle (CT) data. Non-parametric statistic tests were used to analyze the distribution variation of gene expression levels of single cells  as electron donor and sulfate (50 mM) as electron acceptor. Typically, when D. vulgaris cells was cultured at 35°C for 6 days, the SS slides were completely covered by D. vulgaris biofilm with average 1-2 mm in thickness . To monitor growth status of D. vulgaris in both biofilm and planktonic cultures and determine sampling points for further single-cell based analysis, both protein and carbohydrate levels in the biofilm and planktonic cells were measured as previously described . The results showed that protein and carbohydrate levels approached steady-state stages at 36 h for planktonic culture and at 144 h for biofilm, respectively . At the steady-state stages, the planktonic cultures had protein concentration of 167 ± 5.2 μg/mL, carbohydrate concentration of 15.9 ± 0.6 μg/mL and a carbohydrate: protein ratio (C:P) of approximately 0.10 (μg/μg), while the biofilm had a protein level of 88 ± 7.0 μg/cm2 and a carbohydrate level of 9.7 ± 0.2 μg/cm2 with an approximately C:P ratio of 0.11 (μg/μg), consistent with a previous conclusion that D. vulgaris did not produce a carbohydrate-rich biofilm matrix  . To minimize variation during the sampling process, the total sampling time was completed within 2 h from the collection of the samples to the synthesis of the cDNA to keep their gene expression profiles intact  potentially related to formation and metabolism of the D. vulgaris biofilm and three candidate genes of internal reference  were selected . Compared with the other two potential internal control genes, the SD of DVU1090 (recA) were lower among individual cells from both biofilm and planktonic cultures  were selected for the following single-cell analysis  and eight selected target genes  were analyzed in each of the 31 individual cells isolated from each growth condition . A quality control was manually conducted for a total 3,348 reactions . As a quality control for reliable data, poor RT-qPCR reactions, i.e., wrong amplification peaks in the melting curves; large variations  among analytical triplicates, were removed, resulting in a total of 29, 30, 28, and 28 cells out of 31 individual cells with all nine genes successfully analyzed at exponential phase in biofilm and planktonic cultures, and at stationary phase in biofilm and planktonic cultures, respectively. The amplification efficiency of all RT-qPCR data was greater than 93%. The results indicated that variations among technical replicates of RT-qPCR analysis were small for all eight genes, suggestive of good reproducibility of the data  . Similarity between gene-expression distributions was evaluated using p-values determined by non-parametric two-sample Kolmogorov–Smirnov tests. In addition, in the box plots, similarity of gene expression between conditions was evaluated using p-values calculated by two-tailed non-parametric Mann–Whitney statistical significance tests. To more accurately describe the heterogeneity levels, we first defined degree of gene-expression heterogeneity as the fold between the highest and the lowest gene expression levels for each target gene in all single cells under each growth condition, and data was presented in Table 2 accordingly. The results showed that the distribution span of relative gene-expression levels along the horizontal axis reached up to 15, e.g., DVU1397 at stationary phase in the biofilm , suggesting that all target genes carried significant gene-expression heterogeneity in all four conditions. For instance, the differences in terms of relative gene-expression levels could be greater than approximately 80 folds between single cells for DVU0281 in the biofilm at exponential phase, and approximately 50 folds between single cells for DVU3062 at stationary phase in the planktonic culture (Table 2). These results demonstrated that the gene-expression heterogeneity might be a common phenomenon in both biofilm and planktonic cultures. In addition, among all eight genes, the gene-expression heterogeneity for DVU0281 encoding exopolysaccharide biosynthesis protein tended to be the largest among single cells under exponential phase in D. vulgaris biofilm, while DVU2571 involved in ferrous iron transport tended to be the largest under stationary phase in D. vulgaris biofilm (Table 2). Although it is still unknown the specific relationship between the heterogeneity and functionality and physiology of biofilms, high level of heterogeneity for various cellular components is commonly found in biofilm. For example, studies have demonstrated that the distribution of exopolysaccharides in biofilms is highly uneven, which may be related to the formation of the three-dimensional biofilm structures . The lower heterogeneity levels of regulatory genes in biofilm might be important in maintaining delicate regulation of the genes involved in biofilm formation and maintenance, as proper regulation and assembly of the matrix components are key determinant to the biofilm formation . Although chemical heterogeneity and physiological adaptation to the local environment explain much of the biological heterogeneity in biofilms, it is likely that stochastic gene expression also contributes to the phenotypic heterogeneity . By comparing gene-expression heterogeneity of all eight genes under the four growth conditions, the results indicated that gene-expression heterogeneity of six genes  were increased at both exponential and stationary phases for the biofilm when compared with the planktonic culture, and gene-expression heterogeneity of DVU3062 was increased slightly at exponential phase while decreased significantly at stationary phase for the biofilm (Table 2). The only exception was DVU1340 whose gene-expression heterogeneity was decreased at both exponential and stationary phases in the D. vulgaris biofilm (Table 2). In addition, the results showed decreased heterogeneity levels for two regulatory genes  at stationary phase in biofilm, which may worth further investigation for possible mechanism. Overall, compared with the planktonic culture, gene-expression heterogeneity of target genes was increased at both exponential and stationary phases for the biofilm. To demonstrate that the gene-expression heterogeneity resulted from the growth states of biofilm instead of the variances among biological replications, we compared the gene-express histograms and heterogeneity boxplots  and the heterogeneity frequency distribution histograms  for target gene DVU0281 that encodes a exopolysaccharide biosynthesis protein, relevant to biofilm formation) from biological replicates in both planktonic and biofilm cultures. The results showed that the mean gene-expression heterogeneity levels of three biological replicates for the biofilm were about the same ; similarly, the mean gene-expression heterogeneity levels of three biological replicates for planktonic culture were also about the same . Meanwhile, the mean heterogeneity levels between biofilm and planktonic cultures were more than five times different for DVU0281 under the exponential phase, suggesting that different heterogeneity levels were resulted from different growth state (biofilm vs. planktonic) rather than from variation between biological replicates. In addition, based on the studies on aerobic and facultative bacteria, the phenotypic heterogeneity and complexity within biofilm were greater when compared with the planktonic culture , with an emphasis to the first two pairs of comparisons that may reveal genes functionally closely related to the biofilm metabolism. The comparison of planktonic culture vs. biofilm is presented in Figure 2 for the exponential-phase cells, while Figure 3 presents the same comparison for the stationary-phase cells. The results showed that: (i) the distributions of three genes  were clearly shifted toward right side in the plots at both growth phases in the biofilm, representing possible up-regulation of gene expression in single cells of the D. vulgaris biofilm, suggesting that these genes may be related to formation and metabolism of biofilm ; (ii) both p-values of the distribution and box plots analysis of DVU1340 were greater than 0.05 at stationary phase , suggesting that DVU1340 may be functional primarily at fast-growing exponential phase; (iii) the distributions of three target genes, DVU0434, DVU0588, and DVU2571 were shifted toward left side at both growth phases in the biofilm with compared to the planktonic culture, suggesting that the genes were down-regulated and they may not be directly related to the formation and metabolism of biofilm ; (iv) DVU2410 and DVU3062 were down-regulated at exponential phase in the biofilm but up-regulated at stationary phase in the biofilm , suggesting the two genes may be related to late-stage function of mature biofilm, such as maintenance, although further proof is still needed.To determine the differentially regulated genes between the different growth conditions using single-cell datasets, we established four pairs of comparisons for eight target genes  family transcriptional regulators and DVU1397 encoding ion storage protein bacterioferritin were up-regulated, while DVU2571 encoding a ferrous iron transport protein was down-regulated in the D. vulgaris biofilm (Table 3), suggesting that these genes may be important in maintaining the normal metabolism of the D. vulgaris biofilm under a high concentration of iron, although further investigation is still needed to reveal the related mechanism.It has been found that excess iron could be detrimental to biofilms . In thisFigures 4 and 5). The results showed that the distribution patterns of 4 out of the 8 genes  were shifted toward right at the exponential phase in the biofilm , with statistical significances of both non-parametric two-sample Mann–Whitney and Kolmogorov–Smirnov tests less than 0.05, suggesting that they might be functional primarily in exponential phase in the D. vulgaris biofilm. Meanwhile, the distribution patterns of three genes  shifted toward left at exponential phase in the biofilm with the p-values less than 0.05 .To determine the functioning periods of the genes in biofilm and planktonic cultures, the gene-expression distribution was also compared between exponential and stationary phases , and another was based on the relationship between target genes and four growth conditions where the conditions were represented by the CT means of all 31 single cells . The CA analysis showed that a cumulative inertia (that represents the explanation levels of each dimension to the differences between the various categories of the variables) of 91.2 and 95.6% could be explained by the first two dimensions in Figures 6A,B, respectively, suggesting that the plots explained most of the variance quite well. Several key features can be observed from the plots: (i) all target genes were well separated in the plots , confirmed by the Kruskal–Wallis ANOVA tests with p-values significantly less than 0.05 (Table 4), suggesting that the RT-qPCR and CA analysis was able to differentiate the independent gene-expression distribution patterns exhibited by each of the target genes , single cells at exponential phase all distributed in the left, while single cells at stationary phase all distributed in the right of the CA plots. In addition, single cells in the biofilm located in the upper of the CA plot, while single cells in the planktonic culture located in the bottom of the CA plot, indicating significant differences between single cells from four growth conditions and the distributions of four growth conditions in the CA plot were independent as also confirmed by the statistical analyses; (iii) in the CA plot based on the relationship between target genes and four growth conditions , DVU0281 and DVU1340 genes were more close to the exponential phase in the biofilm, while DVU1397 was more close to the stationary phase in the biofilm, suggesting that they could be functionally related to the corresponding condition according to the CA theory  inA theory . SimilarD. vulgaris grown in biofilm and planktonic cultures. While significant gene-expression heterogeneity was found for the selected genes in both the D. vulgaris biofilm and planktonic culture, the analysis showed that gene-expression heterogeneities of 7 out of 8 selected genes were clearly increased in single cells isolated from the D. vulgaris biofilm when compared with those from the planktonic culture, implying a possible linkage between gene-expression heterogeneity and structural and phenotypic complexities of the D. vulgaris biofilms. Interestingly, the analysis showed that compared to planktonic culture, expression levels of DVU1340 encoding ferric uptake repressor family transcriptional regulators and DVU1397 encoding the iron storage protein bacterioferritin were up-regulated for the D. vulgaris biofilms, while the expression level of DVU2571 encoding ferrous iron transport protein were down-regulated, suggesting their roles in maintaining the normal metabolism of the D. vulgaris biofilm under high concentration of iron. Finally, the results demonstrated that single-cell RT-qPCR analysis could be a valuable tool to reveal cell-level and micro-scale differences in the D. vulgaris biofilm, which could be applied in the future to understand molecular mechanism related to growth, maintenance and functions of various microbial biofilms.In this study, we applied a single-cell RT-qPCR based approach to compare gene-expression levels of selected target genes in ZQ performed experiments and data analysis, and wrote the manuscript. LC conceived, designed experiments and wrote the manuscript. WZ conceived and designed experiments, analyzed the data and wrote the 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.


Peptides synthesized in endocrine cells in the gastrointestinal tract and neurons are traditionally considered regulators of metabolism, energy intake, and appetite. However, recent work has demonstrated that many of these peptides act on corticostriatal-limbic circuitry and, in turn, regulate addictive behaviors. Given that alcohol is a source of energy and an addictive substance, it is not surprising that increasing evidence supports a role for gut-brain peptides specifically in alcohol use disorders (AUD). In this review, we discuss the effects of several gut-brain peptides on alcohol-related behaviors and the potential mechanisms by which these gut-brain peptides may interfere with alcohol-induced changes in corticostriatal-limbic circuitry. This review provides a summary of current knowledge on gut-brain peptides focusing on five peptides: neurotensin, glucagon-like peptide 1, ghrelin, substance P, and neuropeptide Y. Our review will be helpful to develop novel therapeutic targets for AUD. Peptides secreted by endocrine cells and enteric neurons in the gastrointestinal (GI) tract and neurons in the central nervous system (CNS), have been implicated in the pathophysiology of alcohol use disorders (AUD). Gut-brain peptides have different functions when administered into the brain relative to the periphery since the blood-brain barrier (BBB) restricts access of peripheral peptides to the CNS. In addition, gut-brain peptides typically have a short half-life due to rapid inactivation by peptidases. Although gut-brain peptides have numerous site-specific functions, they are generally considered to act as regulators of energy intake and expenditure. Alcohol is both an addictive substance and a source of energy. Therefore, it is not surprising that there are peptidergic signaling links between appetitive- and alcohol-related behaviors.There are numerous lines of evidence indicating a mechanistic link between alcohol drinking and food intake. For example, there is a high comorbidity between AUD and eating disorders, especially bulimia  binge/intoxication, 2) preoccupation/anticipation, 3) withdrawal/negative affect , operant self-administration or binge-like drinking-in-the-dark  is a 13-amino-acid (a.a.) peptide originally isolated from the bovine hypothalamus  and AUD in humans  were found to be significantly associated with consumption factor scores in 827 young adult twin women from the Missouri Adolescent Female Twin Registry  and alcohol dependence in Han Chinese males  by healthy men transiently increased plasma NT levels  induces hypothermia , but more time in non-REM sleep  projecting to the VTA and NAc are the most likely source of endogenous GLP-1. However, the mechanism by which GLP-1 in the mesolimbic pathway reduces food or drug intake is not clear  in the pituitary to stimulate growth hormone release  revealed ghrelin increased the activity of the VTA and the regions it projects to  when viewing pictures of food  dose of oral alcohol in humans decreased total and acyl-ghrelin levels  in exon 1 of the Preclinical studies have shown both normal baseline and decreased alcohol consumption by ghrelin KO mice relative to wildtype mice Table . GHS-R1ai.c.v to C57BL/6J mice  is an 11-a.a. peptide and a member of the tachykinin family. SP was originally isolated from the horse intestine and brain , neurokinin 2 receptor (NK2R), and neurokinin 3 receptor (NK3R)  and alcohol dependence  were also associated with blood oxygen level dependent (BOLD) responses to the taste of alcohol in the medial PFC, putamen and insula, as measured by fMRI in heavy drinkers  and symptoms of alcohol dependence.The importance of SP/NK1R system in AUD is supported by the observation of significant associations between the NK1R gene  of alcohol-naïve P rats relative to NP rats  in Wistar rats that were previously exposed to alcohol vapor  seem promising for the treatment of heavy drinking that is driven by anxiety or negative reinforcement. However, very little is known about the mechanism behind the anti-drinking effects of Y1R agonists and Y2R antagonists. Microinjection of Y1R or Y2R-selective pharmacological agents into the amygdala has yielded conflicting results in regard to alcohol consumption. Considering that Y1R and Y2R are expressed in other brain regions within corticostriatal-limbic circuitry, more work is needed to determine the mechanism behind the therapeutic effect of these drugs.Accumulating preclinical and early clinical work suggests that drugs targeting gut-brain peptide signaling are promising therapeutics for the treatment of AUD. Before moving drugs into clinical trials, however, it is essential to validate the therapeutic potential in humans through in-depth behavioral tests and translational research, which includes pharmacodynamic and pharmacokinetic studies in larger animal models. The gut-brain peptides highlighted in this review so far appear to effectively reduce particular addictive behaviors. AUD is a complex heterogeneous disorder. There are numerous reasons why people begin drinking and why they cannot control their use. Comorbid psychiatric and neurological disorders, such as anxiety or chronic pain, may increase propensity to drink and promote relapse. Here, we discussed evidence for why drugs targeting SP and NPY may be useful for the treatment of alcohol dependence with comorbid anxiety. NTS2 or NTS1 agonists may be useful for treating alcohol dependence with comorbid pain-related problems or psychosis. GHS-R1a antagonists have been implicated in alcohol craving, while GLP-1R agonists may be particularly effective in heavy drinkers. For future studies, it will be important to identify subgroups of individuals with AUD that would receive the most benefit and least side effects from potential peptide therapeutics for AUD. Although a daunting task, individualized treatment for AUD seems to be the next frontier for the field. The identification of biomarkers and longitudinal studies will help to demonstrate whether these peptides play a major role in AUD.Chelsea A. Vadnie and David J. Hinton constructed the figures. Chelsea A. Vadnie, Jun Hyun Park, Noha Abdel Gawad, Ada Man Choi Ho, David J. Hinton, and Doo-Sup Choi wrote the 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.


Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth. Glucose, a primary substrate for fetal growth, is transported across the human placenta via the GLUT1 glucose transporter that is found in both the microvillous and basal membranes of the syncytiotrophoblast barrier layer. GLUT1 glucose transporters are asymmetrically distributed, being several fold higher on the microvillous  than basal  membrane Although these changes in GLUT1 expression are important for fetal growth, the regulatory mechanisms controlling these changes have yet to be elucidated. Our prior work has focused on factors associated with the aberrant fetal growth observed in conditions such as fetal growth restriction or macrosomia Despite the evidence demonstrating a role for IGF-I in fetoplacental growth, there is an absence of information on the specific mechanisms by which this interaction might be mediated. One possible mechanism is the regulation by maternal and/or fetal circulating IGF-I of the trans-syncytial nutrient fluxes mediated via glucose, amino acid and other transporters. GLUT1 expression is regulated by IGF-I in a variety of tissues Human placental tissue was obtained from normal, term singleton pregnancies following non-laboring, elective Cesarean section. Written, informed consent was obtained using a protocol approved by the New Jersey Medical School Institutional Review Board (explant and cell studies) or a protocol approved by the ethical committee of the Canton of Berne, Switzerland (perfusion studies). Subjects were excluded if there was evidence of fetal anomalies, intrauterine growth restriction, diabetes, hypertension, anemia, tobacco or drug use or other medical or obstetric complications.2 incubator. Following this incubation period, IGF-I (200 ng/mL) was added to the experimental samples. The incubation for control (no addition) and experimental samples was then continued for another 18 hr. After this incubation, the fragments were pelleted by centrifugation at 500×g for 2 minutes and used for the preparation of syncytial microvillous and basal membranes (see below).Villous tissue was washed in cold PBS and dissected into 3–5 mm fragments immediately following delivery. Dissected fragments were maintained in ice-cold PBS during the dissection period. Fragments  were incubated in T75 tissue culture flasks in 40 mL of DMEM containing 5 mM glucose, 0.5% BSA and 1% penicillin/streptomycin/gentamycin for 3 hr. on an oscillating shaker in a humidified CO2/5% CO2 and contained 3 mM D-glucose. The intervillous space  was perfused with medium containing [3H] 3-O-methyl-D-glucose and [14C] L-glucose  for periods of 30 minutes  to establish rates of transfer from the maternal to fetal circulation. For all placentas used in the perfusion studies, tissue samples were taken from placental lobules that were not used for perfusion. These are identified as “immediate control” samples. Villous tissue from immediate controls was prepared by removal of a decidual layer and the chorionic plate. This tissue was then washed in ice cold 0.9% saline and utilized for the preparation of syncytial microvillous and basal membranes. Following termination of the perfusion, the perfused lobule was excised and villous tissue was prepared in the same manner to yield control or experimental (IGF-I treated) samples.Perfusion was performed as described previously Washed villous tissue or previously incubated villous tissue fragments were used to prepare syncytial microvillous and basal membranes using a previously described method BeWo cells  containing 10% FBS under a humidified air/5% CO2 atmosphere. Purity and syncytialization were tested as described previously Cytotrophoblast cells were prepared as described previously To determine GLUT1 protein expression, slot-blotting was performed on membrane samples as described previously Protein concentration was measured by the Bradford assay All data is presented as mean ± SEM. BeWo GLUT1 expression and glucose transport, syncytial and explant GLUT1 expression data were analyzed by paired t test (two-tailed) or ANOVA (Dunnett’s test). Perfusion GLUT1 expression was assessed by a one-sample t test. Glucose transport in the perfusion model was analyzed by repeated measures ANOVA with a linear trend post hoc test.3H] 2-deoxy-D-glucose was obtained from American Radiochemicals . [3H] 3-O-methyl-D-glucose and [14C] L-glucose were obtained from New England Nuclear . Nitrocellulose membranes (Hybond ECL) were obtained from GE Healthcare and chemiluminescence kits  were obtained from Pierce Biotechnology  for growing BeWo cells were obtained from Corning . All other reagents were obtained from Sigma Chemical Co.  or Bio-Rad .Insulin-like growth factor I (IGF-I) was obtained from Life Technologies  and the anti-GLUT1 antibody was obtained from Thermo Scientific . Protease inhibitor and the HRP-labeled anti-rabbit IgG antibodies were obtained from Sigma Chemical Company .  2-deoxy-D-glucose). Uptake was measured in serum-starved control and IGF-I treated cells. Using BeWo cells plated on permeable Transwell inserts, glucose uptake into BeWo cells was measured from the upper reservoir, across the apical membrane or from the lower reservoir, across the basolateral membrane using a radiolabeled tracer . Following treatment, cells were washed×3 with cold PBS and extracted with RIPA. GLUT1 expression in syncytial samples was measured by slot blotting and normalized to ß-actin expression. The results  show thaWe compared GLUT1 expression in syncytial microvillous and basal membranes prepared from explants which had been incubated in the presence or absence of IGF-I (200 ng/ml) for 18 hr. 3H] 3-O-methyl-D-glucose, 3-OMG). The percent transfer from maternal to fetal circulation was calculated from the radiolabeled tracer levels and fetal flow rate. The results in 14C] L-glucose, remained constant over the perfusion period in both groups.We performed separate control and experimental perfusions. Both employed an open maternal perfusion circuit and the protocol shown in Syncytial microvillous and basal membranes were prepared from both the control and experimental (IGF-I treated) perfused tissue. In order to control for differences in function over time following removal from the uterus, membranes were also generated from the un-perfused villous tissue of both groups, obtained at the time of the initiation of the perfusion (“immediate controls”). GLUT1 protein expression was measured by slot-blotting both the immediate control and perfused samples. The results of these measurements are given in These studies examined the role of IGF-I in the regulation of GLUT1 protein expression in trophoblast cells. We hypothesized that IGF-I would up-regulate GLUT1 expression on the basal surface, the rate limiting step in maternal to fetal glucose transport. Several different trophoblast models were used to examine the effects of IGF-I. We found that IGF-I increased GLUT1 expression in BeWo choriocarcinoma cells, associated with an increase in glucose uptake across the basolateral but not the apical surface. IGF-I treatment also increased BeWo transepithelial glucose transport. Treatment of primary syncytial cells with IGF-I increased GLUT1 expression. Administration of IGF-I to human placental explants increased syncytial basal membrane GLUT1 content compared to untreated explants, whereas microvillous membrane content was not altered. In placental perfusion studies, control perfusions demonstrated a significant decrease in the maternal-to-fetal transfer of glucose over the course of the perfusion compared to experiments in which the fetal perfusate contained IGF-I. In the control perfusions there was a decrease in basal membrane GLUT1 protein expression over the course of the experiment. In comparison, placental tissue perfused with IGF-I via the fetal circulation demonstrated an increase in basal membrane GLUT1. These results support the hypothesis that GLUT1 protein expression on the basal membrane of human trophoblast cells is regulated by IGF-I. This has significant consequences for conditions in which there are changes in circulating IGF-I concentrations such fetal growth restriction, preeclampsia or macrosomia.in vitro perfusion to examine the role of IGF-I in the regulation of GLUT1. BeWo choriocarcinoma cells model the syncytial layer in vivo, enabling measurement of transport properties that cannot be performed with primary syncytial cells due to the absence of a transporting monolayer. Limitations of the BeWo model are that while they are trophoblastic in nature, they are transformed cells that may respond differently compared to syncytial cells in vivo. Primary cytotrophoblast cells aggregate, fuse and differentiate to form syncytial cells, however current techniques do not allow for generation of a confluent monolayer, precluding the measurement of transepithelial glucose transfer or transport of glucose across the microvillous and basal membranes. Placental explants cannot be used for the measurement of transport but are comprised of the original cellular components of the tissue in their in vivo configuration and are therefore appropriate for examining the response of syncytial cells to external agents such as IGF-I. However previous studies have noted that syncytial degeneration begins to occur after six hours in culture in vivo situation. It allows for measurements of both transporter expression and maternal-fetal transport of glucose, albeit under conditions which are, in part, non-physiological (e.g perfusate with a high PO2 but low O2 content). Used singly, each of the models cited above has flaws that preclude definitive conclusions. Used in combination, the commonality of results between the models gives us confidence in the results reported here.Investigations of processes in the human placenta face a number of challenges as a result of the nature of tissue/cellular structures, requiring utilization of a variety of experimental models. We have used choriocarcinoma cells, primary trophoblast, placental explants and As we noted in an earlier publication, there is very little GLUT1 present in syncytiotrophoblast which is not localized to the microvillous or basal membrane in vivo maternal environment, maintain tissue viability and extend the period of experimentation.Not addressed in these studies is the mode of action of IGF-I. It is quite possible that IGF-I has more than one mode of action in stimulating GLUT1 expression and activity. For example the causes of the degenerative changes in the syncytium are unknown but given the anti-apoptotic nature of IGF-I, it is possible that the differences in GLUT1 expression in the IGF-I-treated explants or in the perfusion may be related to IGF-I inhibition of  apoptosis or other IGF-I effects promoting or supporting syncytial viability. IGF-I action to sustain tissue viability is supported by the nature of IGF-I effects on glucose transport in the perfusion experiments, where the progressive loss of glucose transfer capacity is prevented by IGF-I. While the time course of IGF-I action in the perfusion is shorter than that employed in the cell or explant experiments, we have previously shown the effects of hypoxia mimetics on GLUT1 expression over a similar time scale Other verified trophoblast glucose transporters are GLUT3, GLUT8 and GLUT9 Another aspect worthy of consideration is the location of IGF-I action. It is likely that IGF-I is able to act on type 1 IGF receptors on both the microvillous and basal/basolateral membranes in the cell models used here. It is less likely however that external, hydrophilic agents such as IGF-I are able to reach the basal  face of the syncytial cells in the cultured explants and so we must assume that the IGF-I added to explants only interacts with the microvillous  surface of the syncytial cells. In the perfusion, by contrast, IGF-I was added only to the fetal circulation, thus its action was limited to receptors on the basal face of the syncytium. Despite this, our results show that IGF-I has the same effect whether it binds to receptors on the maternal- or fetal-facing membrane, increasing GLUT1 on the syncytial basal membrane. As the type 1 IGF receptor is found on both microvillous and basal membranes The role played by IGF-I as a growth factor is widely recognized. Equally, its clear association with fetoplacental growth has long been known. The results presented here connect these processes and provide a mechanism by which IGF-I is able to regulate fetal growth, through its up-regulation of the GLUT1 glucose transporter protein on the basal membrane of the syncytiotrophoblast. Agents such as IGF-I which alter basal GLUT1 expression will have significant effects on placental glucose transfer capacity and consequently on the levels of fetal circulating glucose. There is also strong evidence for IGF-I involvement in the regulation of placental amino acid transporters


This open-label pilot study aimed to investigate the efficacy of canakinumab in colchicine-resistant familial Mediterranean fever (FMF) patients.Patients with one or more attacks in a month in the preceding 3 months despite colchicine were eligible to enter a 30-day run-in period. Patients who had an attack during the first run-in period advanced to a second 30-day period. At the first attack, patients started to receive three canakinumab 150 mg subcutaneous injections at 4-week intervals, and were then followed for an additional 2 months. Primary efficacy outcome measure was the proportion of patients with 50 % or more reduction in attack frequency. Secondary outcome measures included time to next attack following last canakinumab dose and changes in quality of life assessed by SF-36.Thirteen patients were enrolled in the run-in period and 9 advanced to the treatment period. All 9 patients achieved a 50 % or more reduction in attack frequency, and only one patient had an attack during the treatment period. C-reactive protein and serum amyloid A protein levels remained low throughout the treatment period. Significant improvement was observed in both physical and mental component scores of the Short Form-36 at Day 8. Five patients had an attack during the 2-month follow-up, occurring median 71  days after the last dose. Adverse events were similar to those observed in the previous canakinumab trials.Canakinumab was effective at controlling the attack recurrence in patients with FMF resistant to colchicine. Further investigations are warranted to explore canakinumab’s potential in the treatment of patients with colchicine resistant FMF.NCT01088880. Registered 16 March 2010.ClinicalTrials.gov The online version of this article (doi:10.1186/s13075-015-0765-4) contains supplementary material, which is available to authorized users. Familial Mediterranean fever (FMF), the most common form of hereditary autoinflammatory disorder, is characterized by recurrent attacks of fever with serosal or synovial inflammation, generally lasting 12 to 72 hours [Colchicine, the standard of care for patients with FMF, has been considered as safe and effective in the majority of the patients for reducing both the frequency of inflammatory episodes and the risk of developing amyloidosis –4. HowevMEFV gene, which encodes pyrin protein [MEFV mutations, which affect pyrin-mediated regulation of caspase 1 activity in the inflammasomes, are associated with increased IL-1β production in mice and humans [The majority of FMF patients have autosomal recessive inheritance associated with mutations in the  protein . FMF-reld humans , 6.The objective of this study was to evaluate the efficacy and safety of canakinumab, a fully human anti-IL-1β monoclonal antibody with a half-life of approximately 4 weeks, that binds to human IL-1β and neutralizes its proinflammatory effects, in adolescent and adult patients with FMF, who are resistant or intolerant to higher doses of colchicine.MEFV gene. Patients with end-organ dysfunction due to secondary amyloidosis, active tuberculosis or any other infectious diseases, or a history of malignancy within the last 5 years were excluded from the study.The present study was an investigator-initiated, open-label exploratory trial that included adolescent and adult FMF patients with active disease despite receiving the highest tolerable doses of colchicine (1.5 to 2.0 mg/day). All patients had a typical type I phenotype, fulfilling the criteria for FMF diagnosis , along wColchicine-compliant patients with a history of one or more attacks per month within 3 months before the screening were eligible to enter the first 30-day run-in period. Patients who had at least one attack during that period advanced to a second 30-day period, and they received their first dose of canakinumab upon the first attack they experienced during this second 30-day run-in period.The treatment period started with the first injection, and patients received a total of three subcutaneous injections of canakinumab 150 mg at 4-week intervals. The canakinumab dose could be increased to 300 mg, if an attack occurred between the first and second doses. Stable doses of colchicine (1.5 to 2.0 mg/day) were allowed throughout the study without any dose modification, and compliance was followed tightly throughout the study. After the 12-week treatment period, the patients were subsequently followed for up to 2 months or until the next attack.FMF attacks were confirmed by presence of fever, clinical findings of serositis/arthritis, and elevated C-reactive protein (CRP) levels. Details of each attack  were recorded in diaries. Diaries were dispensed to the colchicine-resistant FMF (crFMF) patients at each visit to record the occurrence of any attacks between the scheduled visits.The primary outcome measure was the proportion of patients with 50 % or more reduction in time-adjusted frequency of attacks. Due to the unequal pre-treatment and treatment periods, attack rates were adjusted to the 84-day treatment period compared with the pre-treatment periods. Secondary outcome measures included the percentage of patients with no attacks in the treatment period, time to next attack after the last canakinumab administration, changes in quality of life assessed by the 36-item short-form health survey (SF-36), and serum levels of CRP and serum amyloid A (SAA) proteins. Physicians’ and patients’ global assessments of control of FMF since the last visit and response to treatment at the end of the treatment period were also measured using a modified 5-point scale [Istanbul Faculty of Medicine Ethics Committee and Ministry of Health approved the study protocol, and all patients provided written informed consent before the screening. Exploratory analyses were performed using descriptive statistics.MEFV genotypes are summarized in Additional file A total of 13 patients with crFMF were screened and included in the first 30-day run-in period. Nine patients  had ≥1 attack during that period, advancing to the second 30-day period, and began their treatment period by starting canakinumab treatment at the first observed attack within this period. Patient baseline demographic characteristics and All nine patients in the treatment period achieved the primary endpoint of ≥50 % reduction in frequency of attacks compared with the time-adjusted pre-treatment frequency of attacks. During the treatment period, only one patient, who was p.Met694Val homozygous and receiving 2 mg/day colchicine, had an attack of peritonitis on day 54. No patient qualified for a canakinumab dose increase between the first and second injections. The time-adjusted frequency of attacks over 84 days observed in the screening and run-in periods, including the baseline attack , decreased dramatically during the treatment period .Five patients, all p.Met694Val homozygous and receiving 2 mg/day colchicine, subsequently experienced an attack within the 2-month follow up, which occurred at a median 71 days (range 31 to 78 days) after the last canakinumab injection  and upper respiratory tract infection (n = 2) were the only adverse events reported by more than one patient Table . All advOne of the patients became pregnant during the treatment period after the third canakinumab dose and gave birth to a healthy son.This open-label pilot trial showed that monthly canakinumab 150 mg subcutaneous injections prevented attacks in patients with crFMF, and only one of nine patients experienced an attack during canakinumab treatment. The safety profile of canakinumab in this small group of patients was similar to that of larger controlled trials in other hereditary autoinflammatory conditions –10.Colchicine has long been used to prevent inflammatory attacks and reduce the risk of secondary amyloidosis in patients with FMF. Despite the efficacy of colchicine, some manifestations, such as arthritis, are less responsive to colchicine . FurtherMEFV variations with increased production of IL-1β, several case reports of patients treated with either anakinra or canakinumab have suggested the potential efficacy of IL-1 blockade in colchicine-resistant patients and those with amyloidosis [Given the association of type and number of loidosis , 6. Addiloidosis . All thrloidosis , 9. TherThe limitations of this small exploratory study were its open-label design, relatively short treatment period, and lack of formal definitions for disease severity and colchicine resistance. However, inclusion of mainly patients with FMF with two penetrant mutations and ≥1 attack per month despite receiving the highest tolerable doses of colchicine, together with confirmation of attacks and colchicine-compliance during the run-in period, represent a real-world patient cohort with few treatment options.In conclusion, the results of the present pilot trial suggest that canakinumab may be an effective and safe treatment option for colchicine-resistant and colchicine-intolerant patients with FMF, and warrant further investigations to explore its efficacy, safety and optimum dosage and administration intervals in this subset of FMF patients.


This study investigates the tracking facilitation effect during categorical distinction between targets and distractors in the Multiple Identity Tracking task. We asked observers to track four targets in a total of eight moving objects, and manipulated categorical distinctions of targets and distractors across four experiments, with different combinations of inter-category and intra-category differences. Results show that tracking performance was significantly better when the targets and distractors were inter-category different, compared to when the targets and distractors were identical or intra-category distinctive. As the inter-category distinction between targets and distractors narrowed, tracking performance improved, but the inter-category facilitation effect decreased. These results may indicate a category-based grouping effect: the observers organized the targets within the same semantic category into one group and made the targets more easily and accurately rediscovered when lost during tracking. Furthermore, the tracking facilitation of categorical distinction diminished when all the objects were inverted. This proved that besides their visual distinctiveness, objects’ semantic category information also played an important role during tracking. Multiple Object Tracking (MOT) tasks have been used to study the ability of humans to keep track of multiple moving objects and the cognitive processing of visual attention . In the Previous researches have examined the effects of visual distinctiveness or the uniqueness of objects’ features on tracking performance. Furthermore, Besides simple surface features , Some studies used cartoon animals as tracking objects to investigate the role of object identity during tracking. These studies found that unique objects make tracking easier compared to the homogenous condition and the paired condition  .About the reason why simple unique objects could promote tracking, previous study have suggested that because the unique identities of objects are stored in visual working memory, once one or more targets were lost during tracking, observers could utilize the stored identities to recover the targets . HoweverAs we know, visual object knowledge can be organized into different categories innately in reality . These sWe hypothesized that categorical distinction between targets and distractors can be utilized to organize the targets in one group and segregate them from the distractors. The semantic categories may be processed automatically during the target designation phase, and continue to exert an influence over tracking. In the current study, we designed four experiments to investigate the tracking facilitation effect of categorical distinction between targets and distractors. We used typical and nameable objects in this study. These objects are common in the real world, and thus have high ecological validity. In Experiment 1, we used land mammals and furniture as natural and artificial categories, respectively, to test the category-based tracking facilitation effect. We narrowed the categorical difference to land mammals and fruits which are animals and plants in Experiment 2, and further narrowed the categorical difference to land mammals and birds which are both animals in Experiment 3, to examine whether the category-based tracking facilitation effect persisted when there was a smaller categorical difference between targets and distractors. Since we used pictures of line drawings as tracking stimuli, the visual distinctiveness between targets and distractors belonging to different categories was much more evident than that of the same category. In Experiment 4, we inverted pictures and compared the tracking facilitation effects of upright and inverted objects, in order to provide further evidence that the tracking facilitation effect was not only caused by the visual or perceptual distinctiveness between targets and distractors, but also caused by their categorical distinction.In Experiment 1, we used land mammals and furniture as natural and artificial categories to test the category-based tracking facilitation effect and the influence of target uniqueness on tracking performance. The neuropsychological research proposes that semantic knowledge is represented categorically in the brain, and knowledge related to different semantic categories is anatomically segregated accordingly . From boSD = 1.56) with normal or corrected-to-normal vision completed the experiment. All observers provided informed written consent. The study was approved by the Institutional Review Board (Ethics Committee) of the School of Psychology at Beijing Normal University. All observers received payment.Eleven undergraduate students  aged 21–26 years . There was a central gray fixation cross that subtended 40 × 40 pixels (1.6° × 1.6°). Sixteen pictures were selected from The initial locations of the pictures, moving at a horizontal and vertical velocity between -5 and +5 pixels per frame at random, were randomly assigned . Each obTable 1). In the homogenous level, all of the targets and distractors were the same and were randomly chosen from either the set of land mammals or furniture. Table 1 presents the details of our experiment design.The experiment was a single-factor within-subject design. The independent variable was the semantic category distinction of targets and distractors with eight conditions. In the intra-category conditions, the targets and distractors were selected from the same category. In the inter-category conditions, the targets were selected from one category, while the distractors were selected from another category. The homogenous condition provided the performance baseline for comparison with the other levels  and the distractors were another two in the same category. In the intra-category all-unique condition, eight unique objects were chosen from the same category. In the intra-category paired-two condition, the targets and distractors shared the same identities. They were both two land mammals/furniture .As shown in The inter-category conditions included three sub-conditions. In the inter-category homogenous condition, the targets were the same land mammals/furniture and the distractors were another different furniture/land mammals, with the constraint that the targets and distractors were chosen from different categories. For example, the targets could be one land mammal’s image chosen randomly from the set of eight possible land mammals and the distractors could be one furniture image chosen randomly from the set of possible furniture images. The inter-category four-unique condition was similar to the inter-category homogenous condition except that the targets contained two land mammals/furniture and the distractors were two furniture/land mammals in a different category. For example, half the targets were rabbits and the remaining targets were lambs, while half the distractors were couches and the remaining distractors were desks. In the inter-category all-unique condition, the targets were four unique land mammals/furniture images and the distractors were four unique furniture/land mammals images.The dependent variable was tracking accuracy defined as the average proportion of correctly identified targets. As 4 of the 8 objects were targets, chance accuracy was 50%.Observers sat approximately 57 cm away from the monitors so that each pixel subtended 0.04° of the visual angle. The observers were shown instructions for the tracking task on the screen. At the start of each trial, a gray fixation cross and eight pictures were displayed. The observers were encouraged to maintain their fixation on the central cross during tracking. Four of eight pictures flashed five times in 1 s for observers to identify them as the targets and distinguish them from the distractors. Then, all of the pictures began to move randomly, and the movement stopped at a random point within 5–8 s to avoid observers simply remembering the last frames of the motion instead of tracking the targets. At the end of motion, the pictures were masked by gray squares subtended 60 × 60 pixels (2.4° × 2.4°). Observers were given 20 s to select all four targets with the mouse. Selected squares were highlighted by red frames. Observers pressed the space bar to continue to the next trial. Unlike the traditional MIT task, observers of the present study were not required to bind the targets’ identities and locations together. At the end of the tracking, observers only need to report the locations of the targets. Therefore, observers could achieve tracking entirely by updating the motion trajectories of the targets without remembering their identities.The experiment began with eight practice trials, one for each condition. The experiment consisted of 160 trials  displayed in random order. Observers rested for at least 1 min every 40 trials.Figure 1. A repeated measures analysis of variance (ANOVA) indicated a significant main effect . Post hoc tests with Bonferroni correction showed that there was generally no significant difference (ps > 0.05) among the paired intra-category homogenous, the inter-category homogenous, the inter-category four-unique and the inter-category all-unique conditions. One exception is that the tracking performance of the inter-category four-unique condition was significantly better than that of the intra-category homogenous condition, That is, the inter-category difference of the targets and distractors, rather than the identity uniqueness of the targets, played a relatively more important role in improving observers’ tracking performance. However, when the targets and distractors belonged to the same category, the tracking performance was enhanced only when greater identity difference between the targets and distractors existed, such as in the intra-category homogenous condition where the targets were four same objects and the distractors were another four same objects.The results are shown in ps > 0.05). This result was different from a previous finding that unique objects could aid tracking in MIT tasks (ps < 0.05 (see Table 2).In addition, no significant difference was found when we compared tracking performances of the intra-category four-unique, intra-category all-unique, intra-category paired-two, and homogenous conditions in pairs  and the homogenous condition. This provided evidence that categorical distinction between targets and distractors significantly improved tracking performance. By grouping the targets into one virtual representation, observers might find the targets more easily. These findings are further discussed in the general discussion section.In Experiment 2, to further test the category-based tracking facilitation effect in MIT tasks, we decreased the categorical distinction between the targets and distractors to land mammals and fruits. Though land mammals and fruits are all natural objects, they have many different features, such as shape, status of static or moving, and the relationship with humans. We predicted that the category-based tracking facilitation effect in Experiment 2 decreased from Experiment 1, but still could be detected.SD = 2.72) with normal or corrected-to-normal vision completed the experiment. All observers provided informed written consent. The study was approved by the Institutional Review Board (Ethics Committee) of the School of Psychology at Beijing Normal University. All observers received payment for their time.Eleven undergraduate students  who did not participate in Experiment 1 aged 18–26 years .The materials adopted in Experiment 2 were eight fruits and eight land mammals, which all belong to natural categories. The eight land mammals were the same as those used in Experiment 1. The eight fruit pictures were of an apple, banana, grapes, lemon, peach, pear, pineapple, and strawberry, selected from Figure 2 presents the results for Experiment 2. A repeated measures ANOVA indicated a significant main effect . Post hoc tests with Bonferroni correction (see Table 4) showed that most of the pairwise comparisons were significant except for the intra-category homogenous, inter-category homogenous, inter-category four-unique, and inter-category all-unique conditions, for which no significant difference was found between each pair, ps > 0.05. This might have the same reason as that in Experiment 1. The objects used in our experiment were simple line drawings with small visual angles and high motion speeds, resulted hard for observers to use targets’ distinct identities to promote tracking. Observers mainly used the categorical distinction between the targets and distractors to aid tracking. Similar to the results of Experiment 1, no significant differences (ps > 0.05) were found between each paired of the intra-category four-unique, intra-category all-unique, intra-category paired-two and homogenous conditions.The results also show that the tracking performances of observers in inter-category conditions were significantly better than those in the intra-category conditions and the homogenous condition. One exception is that no significant difference was found between the intra-category homogenous condition and inter-category conditions. These results suggest that the categorical distinction of targets and distractors also improved tracking performance when the categorical difference was between land mammals and fruits, which are all natural objects. A category-based grouping effect might also exist during tracking in Experiment 2.Experiment 3 examined whether semantic category distinction would improve tracking performance when the categorical differences were further decreased to land mammals and birds. Both of them are animals. The exemplars of land mammals and birds have many similar surface features in perception. They have similar shapes. They both have heads, body and legs. But from the animal classification perspective, they belong to two different categories. So we predicted that there was also a tracking facilitation effect with this categorical difference.SD = 1.79) with normal or corrected-to-normal vision completed the experiment. All observers provided informed written consent. The study was approved by the Institutional Review Board (Ethics Committee) of the School of Psychology at Beijing Normal University. All observers received payment.Eleven undergraduate students  that did not participate in Experiments 1 and 2 aged 18–24 years .In Experiment 3, the objects were land mammals and birds. The eight land mammals were the same as those used in Experiments 1 and 2. The eight bird pictures were of a bird (a generic flying bird), chicken, duck, eagle, ostrich, owl, swan, and penguin, selected from Figure 3. Similar to the results of Experiments 1 and 2, a repeated measures ANOVA indicated a significant main effect . Post hoc tests with Bonferroni correction (see Table 6) also showed that tracking performances of the inter-category homogenous, inter-category four-unique, and inter-category all-unique conditions had no pairwise significant differences, ps > 0.05. The tracking performances of the intra-category four-unique, intra-category all-unique, intra-category paired-two, and homogenous conditions all had no pairwise significant differences, ps > 0.05. The tracking performances of the intra-category four-unique, the inter-category four-unique, and the inter-category all-unique also had no pairwise significant differences, ps > 0.05.The results of Experiment 3 are presented in Moreover, no significant differences were found between the intra-category homogenous vs. inter-category all-unique conditions and the intra-category all-unique vs. inter-category homogenous conditions.The tracking performances in the inter-category conditions were significantly better than that of the homogenous condition, suggesting that the categorical distinction between the targets and distractors enhanced tracking performance, even when the categorical difference was between two kinds of animals: land mammals and birds. However, there are some exceptions, such as no significant improvement was found between the intra-category four-unique vs. inter-category four-unique conditions and the intra-category four-unique vs. inter-category all-unique conditions; and accordingly, the category-based tracking facilitation effect decreased to some extent, compared to Experiments 1 and 2. One possible explanation is that the different animal exemplars had a similar overall shape and more detailed category which we seldom categorized them accurately in daily life according to observers’ general knowledge, then rendering identity differentiation difficulty.Since the typical and nameable cartoon pictures were used as tracking objects in the present study, both the visual distinctiveness and semantic categories between targets and distractors in the inter-category conditions were larger than that of intra-category and homogenous conditions, the tracking benefits of inter-category conditions might be caused by visual distinctiveness among the pictures. To distinguish between the effects of categorical distinction and visual distinctiveness, we inverted the objects (targets and distractors) in Experiment 4 and compared the tracking facilitation effects of upright and inverted objects. The inverted pictures weaken the category representation but leave all the low-level features and consequently the visual distinctiveness intact. If the objects’ perceptual or visual distinctiveness was the only source of the tracking benefit, the inter-category tracking facilitation effect of upright and inverted objects should have no difference. However, if the upright inter-category facilitation effect was significantly larger than the inverted one, further semantic processes might be involved in the task and affected observers’ tracking performances.SD = 1.83) with normal or corrected-to-normal vision completed the experiment. They all did not participate in Experiments 1, 2, and 3. All observers provided informed written consent. The study was approved by the Institutional Review Board (Ethics Committee) of the School of Psychology at Beijing Normal University.Twelve undergraduate students  aged 18–25 years .Figure 4. A two-way repeated-measures ANOVA revealed a significant main effect of the upright/inverted factor, F = 18.580, p = 0.001, η2 = 0.628, suggesting that observers’ tracking performances were better in the upright conditions than the inverted conditions. The main effect of the categorical distinction factor was also significant, F = 36.796, p < 0.001, η2 = 0.770, and so was the interaction between the two factors, F = 4.072, p = 0.031, η2 = 0.270.The results of Experiment 4 are presented in Post hoc multiple comparisons (Bonferroni correction) revealed that, in the inverted conditions, the tracking performance of inter-category all unique was significantly better than that of intra-category  and homogenous . There was no significant difference between intra-category and homogenous . The results of the upright conditions were consistent with Experiment 1. Tracking performance of inter-category was significantly better than that of intra-category  and homogenous , and there was also no significant difference between intra-category and homogenous .F = 0.22, p = 0.652]. However, for both intra-category and inter-category conditions, the tracking performances of observers with the upright objects were significantly better than that with the inverted objects  = 24.52, p < 0.001; upright and inverted inter-category: F = 23.76, p < 0.001). If only the perceptual process was involved in the tracking task, the tracking performances of upright and inverted conditions should have no difference. So these results suggested that the cognitive processing of objects with semantic meaning entered into the semantic level, and not just stayed on the perceptual level.Simple main effects analyses revealed that the tracking performances between the inverted and upright homogenous conditions had no significant difference . Since the visual distinctiveness and perceptual process were the same for the upright and inverted objects, If only the perceptual difference was involved in the tracking task, the upright and inverted inter-category facilitation effects should have no difference. So we conjecture that the stronger upright inter-category facilitation effect was caused by the categorical distinction between the targets and distractors. The above two results provided evidences that besides the visual distinctiveness, the semantic category processing was also involved in the task and contributed the tracking benefit in the inter-category conditions.To further compare the difference of inter-category facilitation effect between the upright and inverted conditions, we introduced the performance difference between inter-category and homogenous conditions (inter-category minus homogenous) as the index of inter-category facilitation effect. The goal of this study was to examine whether tracking performance would be enhanced when there were categorical or non-categorical distinctions between the targets and distractors. Experiment 1 established the principal finding that categorical distinction between targets and distractors significantly facilitated observers’ tracking performance and that a category-based grouping effect might exist during tracking. In Experiment 2, a tracking facilitation effect was observed in the inter-category conditions when the categorical difference between the targets and distractors narrowed from natural and artificial objects to land mammals and fruits. The results of Experiment 3 were consistent with those of Experiments 1 and 2, except that the tracking performance in the intra-category homogenous condition  was significantly better than that in the inter-category conditions, when the categorical difference was further narrowed to land mammals and birds, both of which are animals. That is, the identity difference of the targets and distractors played a more important role in facilitating tracking than categorical information. Thus, the categorical distinction advantage narrowed with the decreasing categorical difference.The results of Experiment 4 provided evidence that tracking facilitation of the targets and distractors chosen from different categories was not only caused by the visual distinctiveness. Although tracking facilitation effect existed in both upright and inverted conditions, observers displayed a significantly larger inter-category facilitation effect in the upright condition than in the inverted condition. This result suggested that, besides the perceptual grouping process, objects’ semantic category information also play an important role during tracking. It also indicated that the cognitive processing of objects in dynamic scenes is not just staying on the perceptual level, but enter to a higher level of object recognition and semantic classification.The present results supported our category-based grouping hypothesis. It was demonstrated that the human visual system improved tracking performance as semantic category difference between targets and distractors increased. This tracking facilitation effect was detected even when the categorical difference was relatively small. We propose that the tracking facilitation effect in the inter-category conditions compared to the intra-category conditions  was due to the processing of the objects’ semantic category. The reasoning will be illustrated in the following paragraphs. The grouping hypothesis proposed by In the current study, all of the objects moved randomly and independently, no spatiotemporal information could promote the perceptual grouping of targets. We manipulated the categorical differences systemically from animals vs. furniture, animals vs. fruits to land mammals vs. birds and found the consistent inter-category facilitation effect, even when the features of two categories were similar in perception (as shown in Experiment 3). Therefore, it is very likely that observers’ processing of the objects’ semantic category causes the facilitation effect detected in Experiments 1–3. Specifically, based on our results, we conjecture that there exists a category-based grouping mechanism during tracking when targets and distractors are categorical distinct. Observers used the categorical difference between the targets and distractors to organize them into two separate groups. They might treat the targets in the same category as one unit during tracking; therefore make the working memory load of targets lighter and the target recovery strategy easier and more effective . Once onThe results of Experiments 1, 2, and 3 also consistently showed that except the tracking performance of intra-category homogenous condition significantly better than the homogenous condition, there was no significant difference among other intra-category conditions and the homogenous condition. This result was inconsistent with a previous finding that unique objects could aid tracking in MIT tasks , and migMultiple Identity Tracking study in semantic and conceptual level is a new idea in the realm of cognitive processing of visual objects. This topic is worthy to investigate from both the theoretical and practical perspectives. The results of the present study suggested that observers could use the semantic categorical difference between the targets and distractors to improve their tracking performance in MIT tasks. This tracking facilitation effect decreased when the categorical distinction was narrowed. Observers might organize the targets of the same category into one group and treat them as a whole during tracking. They might find the lost objects more quickly and accurately based on their conceptual category.Conceived and designed the experiments: LW, XZ. Performed the experiments: LW, CL. Analyzed the data: LW. Contributed to the writing of the manuscript: LW, XZ, CL, ZL.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.