Polarization-independent microchannel in a high-speed-scan femtosecond laser-assisted etching of fused silica.

Microchannels fabricated by femtosecond laser-assisted chemical etching are of great use in biochemical analysis. In this paper, we study the morphology change of etched microchannels in fused silica by controlling the laser scan speed, and we find a significant difference between the chemical etched length and volume. The fabricated microchannels would gradually become tapered along the scan direction, which influences the flow of the hydrofluoric (HF) reagent and the etching rate. As a result, the difference ratios of the etched length and volume, respectively, reach -5.56% and -41.83% followed by the scan speed increasing from 5 to 200 µm/s. Microchannels with polarization independence and better aspect ratio could be obtained in a high-speed-scan mode. We suggest that laser-induced structural transformation from interconnected microcracks to nanogratings could be responsible for this change. Aforementioned results offer a feasible approach to achieve polarization-independent microchannels, which is in favor of accelerating the fabrication of three-dimensional microfluidic devices.

Research Note: Expression of T cell-related cytokines in chicken cecal and spleen tissues following Eimeria tenella infection in vivo.

The T cell-mediated immune response plays an important role in coccidiosis. To reveal the host T cell immune response following Eimeria tenella (E. tenella) infection in chickens, we performed quantitative real-time PCR to analyze the dynamic expression of the Th1-related cytokines IFN-γ, IL-2, and IL-12; the Th17-related cytokines IL-17A, IL-17F, and IL-22; and the Treg-related cytokines IL-10, TGF-ß, and CTLA-4 in the cecum and spleen at 0, 2, 4, 6, 8, and 10 d postinfection (dpi). In the cecal tissue, the expression of the Th1-related cytokine IFN-γ was significantly higher at 6 and 8 dpi than at other time points (11.97-fold and 39.78-fold, respectively, compared with 0 dpi, P < 0.05). IL-2 and IL-12 expression was significantly higher at 6 and 8 dpi than at 0, 2 and 10 dpi (P < 0.05). The expression of the Th17-related cytokines IL-17A and IL-17F at 2 and 4 dpi and IL-22 expression at 4 dpi were significantly higher than those at 0, 6, 8 and 10 dpi (P < 0.05). The expression of the Treg-related cytokines IL-10, TGF-ß and CTLA-4 was significantly higher at 6 and 8 dpi than at 0, 2 and 4 dpi (P < 0.05). In the spleen, IFN-γ and IL-12 expression peaked at 4 dpi, while IL-2 expression peaked at 10 dpi. IL-17A, IL-17F and IL-22 expression was significantly higher at 2 and 4 dpi than at 0, 6, 8 and 10 dpi (P < 0.05). Treg-related cytokine TGF-ß expression was almost unchanged and significantly decreased at only 4 dpi (P < 0.05), while CTLA-4 expression showed an overall decreasing trend from 0 to 8 dpi but increased significantly at 10 dpi (P < 0.05). The expression patterns of three T cell subset-related cytokines were different in the cecum and spleen. Furthermore, Th1 and Treg cells participate in the immune response mainly in the latter stage of coccidia infection (6 and 8 dpi), while Th17 cells play a role mainly in the early stages of infection (2 and 4 dpi). Our data will help to deepen the understanding of the complex T cell immune response after coccidia infection.

Comprehensive Analyses of circRNA Expression Profiles and Function Prediction in Chicken Cecums After Eimeria tenella Infection.

Coccidiosis is an important intestinal parasitic disease that causes great economic losses to the global poultry production industry. Circular RNAs (circRNAs) are long non-coding RNAs that play important roles in various infectious diseases and inflammatory responses. However, the expression profiles and functions of circRNAs during Eimeria tenella (E. tenella) infection remain unclear. In this study, high-throughput sequencing was carried out to detect circRNAs in chicken cecal tissues from the control (JC), resistant (JR), and susceptible (JS) groups on day 4.5 postinfection (pi), respectively. A total of 104 circRNAs were differentially expressed, including 47 circRNAs between the JS and JC groups, 38 between the JR and JS groups, and 19 between the JR and JC groups. Functional analyses indicated that these differentially expressed circRNAs were involved in pathways related to E. tenella infection; the adaptive immune response was enriched in the JS vs JC group, the NF-kappa B signaling and natural killer cell-mediated cytotoxicity pathways were enriched in the JS vs JC and JR vs JC groups, while the B cell receptor signaling pathway was enriched in only the JR vs JC group. Moreover, the coexpression network of differentially expressed circRNAs and mRNAs suggested that circRNA2202 and circRNA0759 associated with DTX1 in the JS vs JC group, circRNA4338 associated with VPREB3 and CXCL13L3 in the JR vs JC group, and circRNA2612 associated with IL8L1 and F2RL2 in the JR vs JS group were involved in the immune response upon E. tenella infection. In conclusion, our results provide valuable information on the circRNAs involved in the progression of chicken E. tenella infection and advance our understanding of the circRNA regulatory mechanisms of host resistance and susceptibility to E. tenella infection in chickens.

Transcriptome Analysis of Long Noncoding RNAs and mRNAs in Granulosa Cells of Jinghai Yellow Chickens Illuminated With Red Light.

Jinghai Yellow chickens are a new indigenous breed with a dual purpose in China, but their egg laying performance is limited. Compared with white light (WL), exposure to red light (RL) can improve the egg laying performance of hens. Herein, to elucidate the molecular mechanism by which RL affects the egg laying performance, RNA sequencing was used to analyze long noncoding RNAs (lncRNAs) and mRNAs from granulosa cells of small yellow follicles from Jinghai Yellow chickens in RL and WL groups. A total of 12,466 lncRNAs were identified among the assembled transcripts, of which 168 lncRNAs were significantly different between the RL and WL groups (101 downregulated and 67 upregulated). Additionally, 1182 differentially expressed mRNAs were identified (958 downregulated and 224 upregulated). Integrated network analysis demonstrated that numerous differential mRNAs were involved in follicular development through steroid hormone synthesis, oocyte meiosis, and the PI3K-Akt signaling pathway. The impact of lncRNAs on cis and trans target mRNAs indicates that some lncRNAs play important roles in follicular development of small yellow follicles. The results provide a starting point for studies aimed at understanding the molecular mechanisms by which monochromatic light affects follicular development and egg production in hens.

MicroRNA-7 Targets the KLF4 Gene to Regulate the Proliferation and Differentiation of Chicken Primary Myoblasts.

The proliferation and differentiation of chicken primary myoblasts (CPMs) play an important role in the development of skeletal muscle. In our previous research, RNA-seq analysis showed that microRNA-7 (miR-7) was relatively highly expressed in the proliferation phase of CPMs, but its expression level decreased significantly after CPMS-induced differentiation. Meanwhile, the mechanism by which the miR-7 regulates the proliferation and differentiation of CPMs is still unknown. In this study, we found that the expression levels of miR-7 and the Krüppel-like factor 4 (KLF4) gene were negatively correlated during the embryonic phase, and in vitro induced differentiation. A dual-luciferase assay and a rescue experiment show that there is a target relationship between miR-7 and the KLF4 gene. Meanwhile, the results show that overexpression of miR-7 inhibited the proliferation and differentiation of CPMs, while inhibition of miR-7 had the opposite effects. Furthermore, overexpression of the KLF4 gene was found to significantly promote the proliferation and differentiation of CPMs. Conversely, inhibition of the KLF4 gene was able to significantly decrease the proliferation and differentiation of CPMs. Our results demonstrate, for the first time, that miR-7 inhibits the proliferation and differentiation of myoblasts by targeting the KLF4 gene in chicken primary myoblasts.

Research Note: Correlation analysis of interleukin-6, interleukin-8, and C-C motif chemokine ligand 2 gene expression in chicken spleen and cecal tissues after Eimeria tenella infection in vivo.

IL-6, IL-8, and C-C motif chemokine ligand 2 (CCLi2) are important factors in inflammatory and immune responses. To investigate their relationships in the spleen and cecum and between coccidiosis-infected and uninfected states, we performed quantitative real-time PCR to compare the relative expression difference of IL-6, IL-8, and CCLi2 in the same tissues between the infection and control groups. In addition, the correlations of the relative expression levels of these 3 genes were determined in the same and different tissues within the same group. The results showed that the expression levels of IL-6, IL-8, and CCLi2 in the spleen and cecum of the infected group were all higher than those of the uninfected group (P < 0.05). The correlation coefficients among the IL-6, IL-8, and CCLi2 expression levels in the spleen or cecum were all positive in both the infection and control groups. In the spleen tissues, CCLi2 expression was strongly correlated with IL-6 and IL-8 in the uninfected group (P < 0.01), and the correlation coefficients reached 0.853 (R2 = 0.728) and 0.996 (R2 = 0.992), respectively. The expression of CCLi2 was also strongly correlated with IL-8 (R reached 0.890, R2 = 0.792) in the infected group. In the cecal tissues, the expression levels of the 3 genes were all extremely significantly correlated in the uninfected group (P < 0.01), and the correlation coefficients ranged from 0.498 to 0.765, indicating moderate correlations. The expression of IL-6 was extremely significantly positively correlated with IL-8 and CCLi2 in the infected group (P < 0.01), with moderate correlations (R ranged from 0.469-0.639). In addition, the expression levels of the 3 genes were not significantly correlated (P > 0.05) between the spleen and cecum tissues in either the infection group or the control group. These results indicate that IL-6, IL-8, and CCLi2 were correlated and play an important role in coccidiosis infection of Jinghai yellow chicken. Our data also provide a basis for further exploring the role of these 3 genes in genetic breeding for coccidiosis resistance.

Establishing a Model for Evaluating Chicken Coccidiosis Resistance Based on Principal Component Analysis.

To establish a coccidiosis resistance evaluation model for chicken selection, the different parameters were compared between infected and control Jinghai yellow chickens. Validation parameters were selected for principal component analysis (PCA), and an optimal comprehensive evaluation model was selected based on the significance of a correlation coefficient between coccidiosis resistance parameters and principal component functions. The following six different parameters were identified: body weight gain 3-5 days post infection and catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and γ-interferon (IFN-γ) concentrations on the eight day post inoculation. Six principal components and one accumulated contribution of up to 80% of the evaluation models were established by PCA. The results showed that the first model was significantly or highly significantly related to nine resistance parameters (p < 0.01 or p < 0.05), especially to cecal lesions (p < 0.01). The remaining models were related to only 2-3 parameters (p < 0.01 or p < 0.05) and not to cecal lesions (p > 0.05). The values calculated by the optimal model (first model) were significantly negatively correlated with cecal lesion performance; the larger the value, the more resistant to coccidiosis. The model fi1 = -0.636 zxi1 + 0.311 zxi2 + 0.801 zxi3 - 0.046 zxi4 - 0.076 zxi5 + 0.588 zxi6 might be the best comprehensive selection index model for chicken coccidiosis resistance selection.

Transcriptome Analysis of Differentially Expressed Genes Related to the Growth and Development of the Jinghai Yellow Chicken.

The growth traits are important traits in chickens. Compared to white feather broiler breeds, Chinese local broiler breeds have a slow growth rate. The main genes affecting the growth traits of local chickens in China are still unclear and need to be further explored. This experiment used fast-growth and slow-growth groups of the Jinghai Yellow chicken as the research objects. Three males and three females with similar body weights were selected from the two groups at four weeks old and eight weeks old, respectively, with a total of 24 individuals selected. After slaughter, their chest muscles were taken for transcriptome sequencing. In the differentially expressed genes screening, all of the genes obtained were screened by fold change ≥ 2 and false discovery rate (FDR) < 0.05. For four-week-old chickens, a total of 172 differentially expressed genes were screened in males, where there were 68 upregulated genes and 104 downregulated genes in the fast-growth group when compared with the slow-growth group. A total of 31 differentially expressed genes were screened in females, where there were 11 upregulated genes and 20 downregulated genes in the fast-growth group when compared with the slow-growth group. For eight-week-old chickens, a total of 37 differentially expressed genes were screened in males. The fast-growth group had 28 upregulated genes and 9 downregulated genes when compared with the slow-growth group. A total of 44 differentially expressed genes were screened in females. The fast-growth group had 13 upregulated genes and 31 downregulated genes when compared with the slow-growth group. Through gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, many genes were found to be related to cell proliferation and differentiation, muscle growth, and cell division such as SNCG, MCL1, ARNTL, PLPPR4, VAMP1, etc. Real-time PCR results were consistent with the RNA-Seq data and validated the findings. The results of this study will help to understand the regulation mechanism of the growth and development of Jinghai Yellow chicken and provide a theoretical basis for improving the growth rate of Chinese local chicken breeds.

RNA Sequencing Analysis of Chicken Cecum Tissues Following Eimeria tenella Infection in Vivo.

Eimeria tenella (E. tenella) is one of the most frequent and pathogenic species of protozoan parasites of the genus Eimeria that exclusively occupies the cecum, exerting a high economic impact on the poultry industry. To investigate differentially expressed genes (DEGs) in the cecal tissue of Jinghai yellow chickens infected with E. tenella, the molecular response process, and the immune response mechanism during coccidial infection, RNA-seq was used to analyze the cecal tissues of an E. tenella infection group (JS) and an uninfected group (JC) on the seventh day post-infection. The DEGs were screened by functional and pathway enrichment analyses. The results indicated that there were 5477 DEGs (p-value < 0.05) between the JS and the JC groups, of which 2942 were upregulated, and 2535 were downregulated. GO analysis indicated that the top 30 significantly enriched GO terms mainly involved signal transduction, angiogenesis, inflammatory response, and blood vessel development. KEGG analysis revealed that the top significantly enriched signaling pathways included focal adhesion, extracellular matrix-receptor interaction, and peroxisome proliferator-activated receptor. The key DEGs in these pathways included ANGPTL4, ACSL5, VEGFC, MAPK10, and CD44. These genes play an important role in the infection of E. tenella. This study further enhances our understanding of the molecular mechanism of E. tenella infection in chickens.

Quantitative analysis of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in eggs via liquid chromatography-electrospray ionization tandem mass spectrometry.

A widely applicable method involving liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (LC-ESI/MS/MS) was developed for the simultaneous determination of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v) and defatted with hexane saturated with acetonitrile. The analysis was carried out on a mass spectrometer via an electrospray interface operated in the positive and negative ionization modes using deuterated chloramphenicol-d5 as the internal standard. The limits of detection and limits of quantification were 0.04-0.5 µg/kg and 0.1-1.5 µg/kg in eggs, respectively. The average recoveries of the four analytes from egg samples were 90.84-108.23%, with relative standard deviations of less than 9.61%. The corresponding intra-day and inter-day variations were found to be less than 8.11% and 11.30%, respectively. Finally, the new approach was successfully applied to the quantitative determination of these analytes in 50 commercial eggs from local supermarkets.

Quantification of piperazine in chicken and pig tissues by gas chromatography-electron ionization tandem mass spectrometry employing pre-column derivatization with acetic anhydride.

This paper describes a novel method that combines acetic anhydride derivatization with gas chromatography-electron ionization tandem mass spectrometry (GC-EI/MS/MS) for the sensitive and selective determination of piperazine in chicken and pig tissues. Samples were extracted using an accelerated solvent extraction (ASE) apparatus, purified by solid-phase extraction (SPE) and derivatized with acetic anhydride. This optimized method was validated according to the requirements defined by the European Union and the Food and Drug Administration. At the limit of quantification (LOQ) spiked levels of 50.0, 100.0, 500.0, 1000.0 and 2000.0µg/kg, the average recoveries of piperazine in chicken and pig tissues were 77.46-96.26%, with relative standard deviations (RSDs) of 1.55-6.64%. The intra-day RSDs were 1.39-5.92%, and the inter-day RSDs were 2.24-8.39%. The limits of detection (LODs) and the LOQs were 1.4-1.6µg/kg and 4.8-5.2µg/kg, respectively. The decision limits (CCα) were 102.02-105.17µg/kg, and the detection capabilities (CCß) were 104.03-109.09µg/kg. Finally, the new approach was verified for the quantitative determination of piperazine in 30 commercial chicken and pig tissues from local supermarkets.