Bidirectional associations between adiposity and physical activity: a longitudinal study from pre-puberty to early adulthood.

Objective: This study aimed to investigate directional influences in the association between adiposity and physical activity (PA) from pre-puberty to early adulthood. Methods: In the Calex-study, height, weight, body fat and leisure-time physical activity (LTPA) were measured at age11.2-years, 13.2-years and 18.3-years in 396 Finnish girls. Body fat was measured by dual-energy X-ray absorptiometry, calculating fat mass index (FMI) as total fat mass in kilograms divided by height in meters squared. LTPA level was evaluated using a physical activity questionnaire. In the European Youth Heart Study (EYHS), height, weight and habitual PA were measured at age 9.6-years, 15.7-years and 21.8-years in 399 Danish boys and girls. Habitual PA and sedentary behaviour were assessed with an accelerometer. Directional influences of adiposity and PA were examined using a bivariate cross-lagged path panel model. Results: The temporal stability of BMI from pre-puberty to early adulthood was higher than the temporal stability of PA or physical inactivity over the same time period both in girls and boys. In the Calex-study, BMI and FMI at age 11.2-years were both directly associated with LTPA at age 13.2-years (ß = 0.167, p = 0.005 and ß = 0.167, p = 0.005, respectively), whereas FMI at age 13.2-years showed an inverse association with LTPA at age 18.3-years (ß = - 0.187, p = 0.048). However, earlier LTPA level was not associated with subsequent BMI or FMI. In the EYHS, no directional association was found for physical inactivity, light-, moderate-, and vigorous-PA with BMI during the follow-up in girls. In boys, BMI at age 15.7-years was directly associated with moderate PA (ß = 0.301, p = 0.017) at age 21.8-years, while vigorous PA at age 15.7-years showed inverse associations with BMI at age 21.8-years (ß = - 0.185, p = 0.023). Conclusion: Our study indicates that previous fatness level is a much stronger predictor of future fatness than level of leisure-time or habitual physical activity during adolescence. The directional associations between adiposity and physical activity are not clear during adolescence, and may differ between boys and girls depending on pubertal status.

Impact of Functional Training on Functional Movement and Athletic Performance in College Dragon Boat Athletes.

Functional training has become a popular training method in different sports, yet limited studies have focused on paddle sports. The purpose of this study was to evaluate the effects of functional training on functional movement and athletic performance in college dragon boat athletes. A total of 42 male athletes were divided into 2 groups: a functional training (FT) group (n = 21, 21 ± 1.47 years) and a regular training (RT) group (n = 21, 22 ± 1.50 years). The FT group participated in an 8-week (16-session) functional-training program, while the RT group trained with strength-training sessions. Functional movement screen (FMS), Y-balance test (YBT) and athletic performance evaluations were conducted before and after the intervention. Repeated measure ANOVA and t-test evaluations were employed to examine differences for both groups. The FT group was significantly improved in FMS scores (F = 0.191, p < 0.001) and YBT scores (F = 2.59, p = 0.027), and it also showed significantly improved muscular fitness (pull-ups: F = 0.127, p < 0.001; push-ups: F = 1.43, p < 0.001) and rowing speed (F = 4.37, p = 0.004). It is recommended to include functional training as a part of training and routine exercise, as it appears to be an effective way of improving FMS and athletic performance in paddle sports.

Changes in health behaviors and conditions during COVID-19 pandemic strict campus lockdown among Chinese university students.

Objective: To explore how a stringent campus lockdown affects the physical activity (PA), sleep and mental health of Chinese university students living in student dormitories during the COVID-19 pandemic. Methods: Data on PA, sleep and mental health were collected between 24 March and 4 April 2022 from 2084 university students (mean age = 22.4 years, 61.1% male students) via an online questionnaire distributed by the students' advisers of each dormitory. The Chinese short version of the International Physical Activity Questionnaire (IPAQ-C), Athens Insomnia Scale (CAIS) and General Health Questionnaire 12-item (GHQ-12) were applied. The Mann-Whitney test and Kruskal-Wallis tests were used to evaluate the PA profile differences between genders, before and during the lockdown period and between students' living environments. Chi-squared (χ2) or Fisher's exact test was used to assess changes in health behaviors by gender and students' living environment compared to before the lockdown. A mediation model was used to examine whether sleep disorder mediated the relationship between PA and mental health in different students' living environments. Results: Participants reported a significant decrease in weekly total PA levels (63.9%). Mean daily sedentary time increased by 21.4% and daily lying time increased by 10.7% compared to before lockdown. Among the participants, 21.2% had experienced insomnia, and 39.0% reported having high mental distress. Female students reported 10% higher rates of sleep disorders than male students (p < 0.001), and also experienced a higher incidence of mental disorders (p < 0.001). Students living with three roommates had a larger decrease in frequencies and durations of participation in light PA than other students (p < 0.001). PA was negatively associated with sleep and mental health, and sleep disorder was a mediating factor between PA and mental health in the students living with two and three roommates. Conclusion: This study showed that strict lockdowns within university dormitories during the COVID-19 pandemic had a negative effect on the health of university students by changing their health behaviors, physical activity and sleep. Our findings indicate a need for strategies to promote an active lifestyle for students in space-limited dormitories in order to maintain health during a prolonged lockdown.

Validity of three smartwatches in estimating energy expenditure during outdoor walking and running.

Commercially wrist-worn devices often present inaccurate estimations of energy expenditure (EE), with large between-device differences. We aimed to assess the validity of the Apple Watch Series 6 (AW), Garmin FENIX 6 (GF) and Huawei Watch GT 2e (HW) in estimating EE during outdoor walking and running. Twenty young normal-weight Chinese adults concurrently wore three index devices randomly positioned at both wrists during walking at 6 km/h and running at 10 km/h for 2 km on a 400- meter track. As a criterion, EE was assessed by indirect calorimetry (COSMED K5). For walking, EE from AW and GF was significantly higher than that obtained by the K5 (p < 0.001 and 0.002, respectively), but not for HW (p = 0.491). The mean absolute percentage error (MAPE) was 19.8% for AW, 32.0% for GF, and 9.9% for HW, respectively. The limits of agreement (LoA) were 44.1, 150.1 and 48.6 kcal for AW, GF, and HW respectively. The intraclass correlation coefficient (ICC) was 0.821, 0.216 and 0.760 for AW, GF, and HW, respectively. For running, EE from AW and GF were significantly higher than the K5 (p < 0.001 and 0.001, respectively), but not for HW (p = 0.946). The MAPE was 24.4%, 21.8% and 11.9% for AW, GF and HW, respectively. LoA were 62.8, 89.4 and 65.6 kcal for AW, GF and HW, respectively. The ICC was 0.741, 0.594, and 0.698 for AW, GF and HW, respectively. The results indicate that the tested smartwatches show a moderate validity in EE estimations for outdoor walking and running.

Assessment of sleep disturbances with the athlete sleep screening questionnaire in Chinese athletes.

This study investigated the factors that are associated with sleep disturbances among Chinese athletes. Sleep quality and associated factors were assessed by the Athlete Sleep Screening Questionnaire (ASSQ, n â€‹= â€‹394, aged 18-32 years, 47.6% female). Sleep difficulty score (SDS) and level of sleep problem (none, mild, moderate, or severe) were used to classify participants' sleep quality. Categorical variables were analyzed by Chi-square or fisher's exact tests. An ordinal logistic regression analysis was used to explore factors with poor sleep (SDS ≥8). Approximately 14.2% of participants had moderate to severe sleep problem (SDS ≥8). Fifty-nine percent of the athletes reported sleep disturbance during travel, while 43.3% experienced daytime dysfunction when travelling for competition. No significant difference was found in the SDS category between gender, sports level and events. Athletes with evening chronotype were more likely to report worse sleep than athletes with morning and intermediate chronotype (OR, 2.25; 95%CI, 1.44-3.52; p â€‹< â€‹0.001). For each additional year of age, there was an increase of odds ratio for poor sleep quality (OR, 1.15; 95%CI, 1.04-1.26; p â€‹= â€‹0.004), while each additional year of training reduced the odds ratio (OR, 0.95; 95%CI, 0.91-0.99; p â€‹= â€‹0.044). To improve sleep health in athletes, chronotype, travel-related issues, age and years of training should be taken into consideration.

Changes in Fat Oxidation and Body Composition after Combined Exercise Intervention in Sedentary Obese Chinese Adults.

(1) Background: Evidence suggests that aerobic exercise and high-intensity interval training (HIIT) might increase fat oxidation and reduce fat. However, limited research has examined the effects of combining progressive aerobic exercise and HIIT interventions in sedentary adults with overweight and obesity, and differences in its effects between men and women remain unclear. The purpose of this study was to investigate the effects of combined progressive aerobic exercise and HIIT (CAEH) on fat oxidation and fat reduction in sedentary Chinese adults and compare sex differences in sedentary adults after seven weeks. (2) Methods: Eighty-four sedentary obese adults were enrolled and allocated to two groups in baseline (experimental (EXP) group:42; control (CON) group:42), and fifty-six subjects (EXP:31; CON:25) completed the experiments and were included in the final analysis. Subjects in the EXP group performed CAEH three times per week for seven weeks. Subjects in the CON group were advised to continue with their normal daily activities. Anthropometric, lipid profile, cardiorespiratory fitness, and fat oxidation outcomes were assessed before and after the intervention. (3) Results: After seven weeks of the CAEH intervention, compared with the CON group, the EXP group showed significant increases in fat oxidation at rest (FO_rest) (+0.03 g/min, p < 0.01), maximal fat oxidation (MFO) (+0.05 g/min, p < 0.01), and maximal oxygen intake (VO2max) (+3.2 mL/kg/min, p < 0.01). The changes in the percentages of the FO_rest (+57%) and the VO2max (+16%) were significantly greater (+20%, +6%) in males than in females (p < 0.05, p < 0.05). The body mass index (BMI) (-1.2 kg/m2, p < 0.01), body fat percentage (-3.2%, p < 0.001), visceral fat area (-12.8 cm2, p < 0.001), and total cholesterol (TC) levels (-0.4 mmol/L, p < 0.05) were significantly decreased in the EXP group. (4) Conclusions: Seven weeks of the CAEH intervention effectively improved FO_rest, MFO, and VO2max in sedentary obese adults, and the improvements in FO_rest and VO2max were more pronounced in males than in females. CAEH also improved body composition and TC levels in sedentary obese adults.

Maximal isometric strength indices are associated with the oxygen cost of walking and running in recreationally active men and women.

This study assessed the associations of maximal isometric strength and movement economy in 126 recreationally active men and women. Oxygen consumption was assessed through a graded treadmill test with 4-minute increments (4-12 km∙h-1). Maximal isometric leg extensor, leg flexor and handgrip strength were assessed by isometric dynamometry. Models of best fit for gross oxygen cost and gross caloric unit cost were observed across the majority of velocities when the leg extensor/flexor strength ratio and handgrip strength were combined (R2 = 0.207-0.525 and R2 = 0.152-0.475, respectively). Additionally, the oxygen cost differed statistically for the majority of velocities when participants were split by the median of leg extensor strength (12.3-26.3 ml∙kg-1∙km-1, p < 0.05) and the average of all strength variables (13.9-30.3 ml∙kg-1∙km-1, p < 0.05). Our data underline the importance of maintaining maximal strength in order to perform activities with low to moderate oxygen demands.

The Effect of a Ketogenic Low-Carbohydrate, High-Fat Diet on Aerobic Capacity and Exercise Performance in Endurance Athletes: A Systematic Review and Meta-Analysis.

A low-carbohydrate, high-fat (LCHF) diet has been proposed to enhance the fat utilization of muscle and the aerobic capacity of endurance athletes, thereby improving their exercise performance. However, it remains uncertain how the macronutrient intake shift from carbohydrate to fat affects endurance exercise training and performance. This study performed a systematic review and meta-analysis to explore the effects of a ketogenic low-carbohydrate, high-fat (K-LCHF) diet on aerobic capacity and exercise performance among endurance athletes. Searches were carried out in five electronic databases, and we followed the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines. The search included studies using an LCHF diet as an intervention protocol and compared data on factors such as maximum oxygen uptake (VO2max) and rating of perceived exertion (RPE) from the graded exercise test. In this case, 10 studies met the criteria and were included in the meta-analysis. We did not find a significant effect of K-LCHF diet interventions on VO2max, time to exhaustion, HRmax or RPE. However, a significant overall effect in the substrate oxidation response to respiratory exchange rate was observed. The meta-analysis showed that K-LCHF diets did not affect aerobic capacity and exercise performance. Therefore, high-quality interventions of a K-LCHF diet are needed to illustrate its effect on various endurance training programs.

Effect of Chronic Exercise Training on Blood Lactate Metabolism Among Patients With Type 2 Diabetes Mellitus: A Systematic Review and Meta-Analysis.

Purpose: To assess the effect of chronic exercise training on blood lactate metabolism at rest (i.e., basal lactate concentrations) and during exercise (i.e., blood lactate concentration at a fixed load, load at a fixed blood lactate concentration, and load at the individual blood lactate threshold) among patients with type 2 diabetes mellitus (T2DM). Methods: PubMed (MedLine), Embase, Web of Science, and Scopus were searched. Randomized controlled trials, non-randomized controlled trials, and case-control studies using chronic exercise training (i.e., 4 weeks) and that assessed blood lactate concentrations at rest and during exercise in T2DM patients were included. Results: Thirteen studies were eligible for the systematic review, while 12 studies with 312 participants were included into the meta-analysis. In the pre-to-post intervention meta-analysis, chronic exercise training had no significant effect on changes in basal blood lactate concentrations (standardized mean difference (SMD) = -0.20; 95% CI, -0.55 to 0.16; p = 0.28), and the results were similar when comparing the effect of intervention and control groups. Furthermore, blood lactate concentration at a fixed load significantly decreased (SMD = -0.73; 95% CI, -1.17 to -0.29; p = 0.001), while load at a fixed blood lactate concentration increased (SMD = 0.40; 95% CI, 0.07 to 0.72; p = 0.02) after chronic exercise training. No change was observed in load at the individual blood lactate threshold (SMD = 0.28; 95% CI, -0.14 to 0.71; p = 0.20). Conclusion: Chronic exercise training does not statistically affect basal blood lactate concentrations; however, it may decrease the blood lactate concentrations during exercise, indicating improvements of physical performance capacity which is beneficial for T2DM patients' health in general. Why chronic exercise training did not affect basal blood lactate concentrations needs further investigation.

Bidirectional Influence of the COVID-19 Pandemic Lockdowns on Health Behaviors and Quality of Life among Chinese Adults.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has created challenges that have caused profound changes in health behaviors. This study aimed to explore how COVID-19 is affecting the health-related quality of life (QoL) among Chinese adults. METHODS: The data of health-related behaviors and QoL were collected via online surveys from 2289 adults (mean age = 27.8 ± 12 years) who had been isolated at home for an average of 77 days. RESULTS: More than 50% of the respondents reported that their time engaged in daily physical activity (PA) decreased, while sedentary behavior (SB) time increased compared with that before the lockdown. Only 20% of the respondents reported engaging in moderate-to-vigorous PA, 23% of adults reported changed their diets to be healthier, and 30% reported consuming more vegetables, fruits, and milk products than before home-isolation. During home-isolation, 75.2% of the adults rated their sleep quality as very good, and 65% reported that they were satisfied with their QoL. Sleep quality mediated the relationship between PA and QoL. CONCLUSION: The two-to-three-month home-isolation has had mixed effects on adult health behaviors in China. The participants were found to have focused more on their eating quality and patterns, which had a positive influence on their QoL. However, people should be encouraged to exercise at home with limited space to maintain a generally healthy lifestyle during a prolonged quarantine.

Effects of exercise and dietary interventions on serum metabolites in men with insomnia symptoms: A 6-month randomized controlled trial.

Accumulating evidence show that exercise and diet interventions are associated with improved sleep quality. Studies investigating the effects of exercise and dieting on circulating metabolomics in people with sleep disorders, particularly insomnia, are scarce. This 6-month randomized study aimed to assess the effects of exercise and dietary interventions on serum metabolites in men with insomnia symptoms. Seventy-two Finnish men (age: 51.6 ±â€¯10.1 years) with chronic insomnia symptoms who were assigned to different intervention groups completed this study (exercise, n = 24; diet, n = 27; and control, n = 21). The Shapiro-Wilk W-test, Levene test, Spearman correlation analysis, and analysis of variance were used for data analysis. We found that exercise and diet intervention were associated with improved sleep quality and with a number of metabolites across different biochemical pathways. Although we could not show causality, our findings provide new insight into the biological mechanisms underlying the health effects of physical activity, diet, and sleep quality. Further investigation is needed to better understand the link among lifestyle, sleep quality, and metabolic health.

Phagocytic intracellular digestion in amphioxus (Branchiostoma).

The digestive methods employed by amphioxus (Branchiostoma)-both intracellular phagocytic digestion and extracellular digestion-have been discussed since 1937. Recent studies also show that epithelial cells lining the Branchiostoma digestive tract can express many immune genes. Here, in Branchiostoma belcheri, using a special tissue fixation method, we show that some epithelial cells, especially those lining the large diverticulum protruding from the gut tube, phagocytize food particles directly, and Branchiostoma can rely on this kind of phagocytic intracellular digestion to obtain energy throughout all stages of its life. Gene expression profiles suggest that diverticulum epithelial cells have functional features of both digestive cells and phagocytes. In starved Branchiostoma, these cells accumulate endogenous digestive and hydrolytic enzymes, whereas, when sated, they express many kinds of immune genes in response to stimulation by phagocytized food particles. We also found that the distal hindgut epithelium can phagocytize food particles, but not as many. These results illustrate phagocytic intercellular digestion in Branchiostoma, explain why Branchiostoma digestive tract epithelial cells express typical immune genes and suggest that the main physiological function of the Branchiostoma diverticulum is different from that of the vertebrate liver.

Genome-wide comparison of the protein-coding repertoire reveals fast evolution of immune-related genes in cephalochordates and Osteichthyes superclass.

Amphioxus is used to investigate the origin and evolution of vertebrates. To better understand the characteristics of genome evolution from cephalochordates to Osteichthyes, we conducted a genome-wide pairwise comparison of protein-coding genes within amphioxus (a comparable group) and parallel analyses within Osteichthyes (two comparable groups). A batch of fast-evolving genes in each comparable group was identified. Of these genes, the most fast-evolving genes (top 20) were scrutinized, most of which were involved in immune system. An analysis of the fast-evolving genes showed that they were enriched into gene ontology (GO) terms and pathways primarily involved in immune-related functions. Similarly, this phenomenon was detected within Osteichthyes, and more well-known and abundant GO terms and pathways involving innate immunity were found in Osteichthyes than in cephalochordates. Next, we measured the expression responses of four genes belonging to metabolism or energy production-related pathways to lipopolysaccharide challenge in the muscle, intestine or skin of B. belcheri; three of these genes (HMGCL, CYBS and MDH2) showed innate immune responses. Additionally, some genes involved in adaptive immunity showed fast evolution in Osteichthyes, such as those involving "intestinal immune network for IgA production" or "T-cell receptor signaling pathway". In this study, the fast evolution of immune-related genes in amphioxus and Osteichthyes was determined, providing insights into the evolution of immune-related genes in chordates.

Transcriptome-wide analysis of immune-responsive microRNAs against poly (I:C) challenge in Branchiostoma belcheri by deep sequencing and bioinformatics.

Amphioxus is a key experimental animal for studying the evolution of vertebrate immune system. However, we still do not know about the roles of microRNAs (miRNAs) under viral stress in amphioxus. In this study, we sequenced six small RNA libraries (three biological replicates were included in the treatments challenged by the viral mimic, poly (I:C) (pIC) and control groups, respectively) from Branchiostoma belcheri. A total of 151 known miRNAs, 197 new miRNAs (named novel_mir, including nine conserved miRNAs) were identified by deep sequencing from the six libraries. We primarily focused on differentially expressed miRNAs (DEMs) after pIC challenge. Next, we screened a total of 77 DEMs, including 27 down- and 50 up-regulated DEMs in response to pIC challenge. Furthermore, we used real-time quantitative PCR (qRT-PCR) to verify the expression levels of 10 randomly selected DEMs. Target genes likely regulated by DEMs were predicted, and functional enrichment analyses of these targets were performed using bioinformatics approach. MiRNA targets of DEMs are primarily involved in immune response, diseases, cancer and regulation process, and could be largely linked to 14 immune-related signaling pathways, including NF-kappa B, NOD-like receptor, RIG-I-like receptor and endocytosis. The present study for the first time explores key regulatory roles of miRNAs in the innate antiviral immune response in amphioxus, and will provide insight into the molecular basis of antiviral immunity and evolution of immune-related miRNAs.

Exploring gene expression changes in the amphioxus gill after poly(I:C) challenge using digital expression profiling.

Amphioxus, a cephalochordate, is a key model animal for studying the evolution of vertebrate immunity. Recently, studies have revealed that microRNA (miRNA) expression profiles change significantly in the amphioxus gill after immune stimulation, but it remains largely unknown how gene expression responds to immune stress. Elucidating gene expression changes in the amphioxus gill will provide a deeper understanding of the evolution of gill immunity in vertebrates. Here, we used high-throughput RNA sequencing technology (RNA-seq) to conduct tag-based digital gene expression profiling (DGE) analyses of the gills of control Branchiostoma belcheri and of those exposed to the viral mimic, poly(I:C) (pIC). Six libraries were created for the control and treatment groups including three biological replicates per group. A total of 1999 differently expressed genes (DEGs) were obtained, with 571 and 1428 DEGs showing up- or down-regulation, respectively, in the treatment group. Enrichment analysis of gene ontology (GO) terms and pathways revealed that the DEGs were primarily related to immune and defense response, apoptosis, human disease, cancer, protein metabolism, enzyme activity, and regulatory processes. In addition, eight DEGs were randomly selected to validate the RNA-seq data using real-time quantitative PCR (qRT-PCR), and the results confirmed the accuracy of the RNA-seq approach. Next, we screened eight key responding genes to examine the dynamic changes in expression levels at different time points in more detail. The results indicated that expressions of TRADD, MARCH, RNF31, NF-κb, CYP450, TNFRSF6B, IFI and LECT1 were induced to participate in the antiviral response against pIC. This study provides a valuable resource for understanding the role of the amphioxus gill in antiviral immunity and the evolution of gill immunity in vertebrates.

Genome-wide gene expression analysis of amphioxus (Branchiostoma belcheri) following lipopolysaccharide challenge using strand-specific RNA-seq.

Amphioxus is the closest living proxy for exploring the evolutionary origin of the immune system in vertebrates. To understand the immune responses of amphioxus to lipopolysaccharide (LPS), 5 ribosomal RNA (rRNA)-depleted libraries of amphioxus were constructed, including one control (0 h) library and 4 treatment libraries at 6, 12, 24, and 48 h post-injection (hpi) with LPS. The transcriptome of Branchiostoma belcheri was analyzed using strand-specific RNA sequencing technology (RNA-seq). A total of 6161, 6665, 7969, and 6447 differentially expressed genes (DEGs) were detected at 6, 12, 24, and 48 hpi, respectively, compared with expression levels at 0 h. We identified amphioxus genes active during the acute-phase response to LPS at different time points after stimulation. Moreover, to better visualize the resolution phase of the immune process during immune response, we identified 6057 and 5235 DEGs at 48 hpi by comparing with 6 and 24 hpi, respectively. Through real-time quantitative PCR (qRT-PCR) analysis of 12 selected DEGs, we demonstrated the accuracy of the RNA-seq data in this study. Functional enrichment analysis of DEGs demonstrated that most terms were related to defense and immune responses, disease and infection, cell apoptosis, and metabolism and catalysis. Subsequently, we identified 1330, 485, 670, 911, and 1624 time-specific genes (TSGs) at 0, 6, 12, 24, and 48 hpi. Time-specific terms at each of 5 time points were primarily involved in development, immune signaling, signal transduction, DNA repair and stability, and metabolism and catalysis, respectively. As this is the first study to report the transcriptome of an organism with primitive immunity following LPS challenge at multiple time points, it provides gene expression information for further research into the evolution of immunity in vertebrates.

Genome-wide gene expression analysis in the amphioxus, Branchiostoma belcheri after poly (I: C) challenge using strand-specific RNA-seq.

The gene expression associated with immune response to bacteria/bacterial mimic has been extensively analyzed in amphioxus, but remains largely unknown about how gene are involved in the immune response to viral invasion at expression level. Here, we analyze the rRNA-depleted transcriptomes of Branchiostoma belcheri using strand-specific RNA-seq in response to the viral mimic, poly (I:C) (pIC). A total of 5,317 differentially expressed genes were detected at treatment group by comparing with control. The gene with the most significant expression changes (top 15) after pIC challenge and 7 immune-related categories involving 58 differently expressed genes were scrutinized. By functional enrichment analysis of differently expressed genes, gene ontology terms involving response to stress and stimulus, apoptosis, catabolic and metabolic processes and enzyme activity were overrepresented, and several pathways related to immune signaling, immune response, cancer, apoptosis, viral disease, metabolism were activated after pIC injection. A positive correlation between the qRT-PCR and strand-specific RNA-seq data confirmed the accuracy of the RNA-seq results. Additionally, the expression of genes encoding NLRC5, CASP1, CASP6, CYP450, CAT, and MDA5 were induced in B. belcheri under pIC challenge. Our experiments provide insight into the immune response of amphioxus to pIC and valuable gene expression information for studying the evolution of antiviral immunity in vertebrates.

Selection of reliable reference genes for normalization of quantitative RT-PCR from different developmental stages and tissues in amphioxus.

Amphioxus is a closest living proxy to the ancestor of cephalochordates with vertebrates, and key animal for novel understanding in the evolutionary origin of vertebrate body plan, genome, tissues and immune system. Reliable analyses using quantitative real-time PCR (qRT-PCR) for answering these scientific questions is heavily dependent on reliable reference genes (RGs). In this study, we evaluated stability of thirteen candidate RGs in qRT-PCR for different developmental stages and tissues of amphioxus by four independent (geNorm, NormFinder, BestKeeper and deltaCt) and one comparative algorithms (RefFinder). The results showed that the top two stable RGs were the following: (1) S20 and 18 S in thirteen developmental stages, (2) EF1A and ACT in seven normal tissues, (3) S20 and L13 in both intestine and hepatic caecum challenged with lipopolysaccharide (LPS), and (4) S20 and EF1A in gill challenged with LPS. The expression profiles of two target genes (EYA and HHEX) in thirteen developmental stages were used to confirm the reliability of chosen RGs. This study identified optimal RGs that can be used to accurately measure gene expression under these conditions, which will benefit evolutionary and functional genomics studies in amphioxus.

Measurement of cetuximab and panitumumab-unbound serum EGFR extracellular domain using an assay based on slow off-rate modified aptamer (SOMAmer) reagents.

BACKGROUND: Response to cetuximab (Erbitux®) and panitumumab (Vectibix®) varies among individuals, and even those who show response ultimately gain drug resistance. One possible etiologic factor is differential interaction between the drug and target. We describe the development of an assay based on Slow Off-rate Modified Aptamer (SOMAmer(™)) reagents that can distinguish drug-bound from unbound epidermal growth factor receptor (EGFR). METHODS: This quantitative assay uses a SOMAmer reagent specific for EGFR extracellular domain (ECD) as a capturing reagent. Captured SOMAmer is quantitated using PCR. Linearity and accuracy (recovery) of the assay were assessed using normal sera and purified EGFR ECD. RESULTS: This EGFR ECD assay showed linearity between 2.5 and 600 ng/mL. Average recovery was 101%. The assay detected EGFR but showed little cross-reactivity to other ErbB proteins: 0.4% for ErbB2, 6.9% for ErbB3, and 1.3% for ErbB4. Preincubation of normal serum with either cetuximab or panitumumab resulted in a dose-dependent decrease in EGFR ECD levels measured using the SOMAmer assay; preincubation did not affect measurement with an ELISA. CONCLUSIONS: This SOMAmer-based serum EGFR ECD assay accurately and specifically measures EGFR in serum. Detection of significant amounts of drug-unbound EGFR in patients undergoing cetuximab or panitumumab treatment could be an indicator of poor drug response. Further studies are needed to evaluate the utility of the assay as an indicator of drug efficacy or as a guide to dosing.