Delfi-metoda istraživanja i verifikacija procjene videozapisa o kemijskim eksperimentima

The purpose of this study was to use the Delphi method to combine consensus opinion to develop indicators for pre-service chemistry teachers to assess videos of chemical experiments. This study had two stages. The first was to construct the Video Assessment Questionnaire using a modified Delphi consensus process delivering three rounds of face-to-face or online surveys. The 50 participants included professors, associate professors, undergraduate students, and middle school teachers. The Video Assessment Questionnaire has a high degree of consensus as shown by the coefficients of variation and Kendall’s W of the second and third rounds. The final version included 20 indicators in three major aspects: content organization, experiment operation, and video quality. In the second stage, a quasi-experiment was designed to test the effect of using the questionnaire in Chemistry Teaching Methodology Course. The experimental group (n = 22) used the questionnaire to evaluate their own videos and reflect on their performance, whereas the control group (n = 11) carried out the same tasks without the tool. We found that the pre-service teachers who used the Video Assessment Questionnaire had better laboratory instruction (t(31) = 4.25, p<.001), especially the operation in experiments (t(31) = 4.95, p<.001) than those who only made videos. This study verified the effectiveness of the Video Assessment Questionnaire in terms of helping pre-service chemistry teachers to improve experiment operations skills.Cilj ovoga istraživanja bio je primijeniti Delfi-metodu za dobivanje i kombiniranje podudarnih mišljenja, kako bi se izradili pokazatelji pomoću kojih budući nastavnici kemije mogu procijeniti videozapise o kemijskim eksperimentima. Ovo istraživanje provedeno je u dvije faze. U prvoj je izrađen Upitnik za procjenu videozapisa o kemijskim eksperimentima pomoću modificirane Delfi metode. Na taj su način utvrđena podudarna mišljenja i provedena tri kruga anketiranja uživo ili u online obliku. Uzorak se sastojao od 50 ispitanika – profesora, docenata, studenata dodiplomskih studija te srednjoškolskih nastavnika. Upitnik za procjenu videozapisa pokazao je visoku razinu konsenzusa, što potvrđuju koeficijent varijacije i Kendallov W koeficijent podudarnosti u drugom i trećem krugu anketiranja. Finalna verzija obuhvaća 20 pokazatelja za tri glavna aspekta: organizaciju sadržaja, izvođenje eksperimenta i kvalitetu videozapisa. U drugoj fazi provedenen je kvazieksperiment kako bi se testirao učinak primjene Upitnika u kolegiju Metodika nastave kemije. Eksperimentalna skupina (n = 22) koristila je upitnik kako bi procijenila vlastite videozapise i provela refleksiju o izvedbi eksperimenta, dok je kontrolna skupina (n = 11) odradila iste zadatke, no bez upitnika. Utvrđeno je da su budući nastavnici koji su koristili Upitnik za procjenu videozapisa o kemijskim eksperimentima izvodili bolje nastavu u laboratoriju (t(31) = 4,25, p < .001) i kemijske eksperimente (t(31) = 4,95, p < .001) od onih koji su samo izrađivali videozapise. Istraživanje je potvrdilo učinkovitost Upitnika za procjenu videozapisa o kemijskim eksperimentima kao pomoć budućim nastavnicima Kemije u poboljšanju vještina izvođenja kemijskih eksperimenata

Characterization of hypoxia-inducible factor α and its response to hypoxia in Callosobruchus chinensis (Coleoptera, Bruchidae)

Hypoxia-inducible factor (HIF) plays an essential role in hypoxic adaptation in various organisms. Callosobruchus chinensis, a common food storage pest, is highly adapted to hypoxic conditions, while the underlying mechanism of which is currently unclear. Here, we isolated, sequenced, and bioinformatically analyzed HIF1α from C. chinensis, then examined its mRNA and protein expression profiles under hypoxic conditions for various developmental stages and tissue types. The effects of environmental hypoxia on other genes in the HIF pathway response, aspartyl-hydroxylase (FIH) and prolyl hydroxylase (PHD), were also examined. We found that FIH and PHD were also highly conserved and that their expression varied with developmental stage, tissue type, and environmental hypoxia. Overall, hypoxia caused an increase in HIF1α and FIH mRNA expression, and a decrease in PHD mRNA expression, while expression of their proteins showed different in response to hypoxia. HIF1α protein levels increased, PHD and FIH protein levels decreased under hypoxic conditions, compared to normoxic conditions. This study has preliminarily investigated the bioinformation of C. chinensis HIF1α response relationship between HIF1α and its hydroxylases under normoxic and hypoxic conditions, which might provide important clues for future work on clarifying the mechanism of stored-product insect unique hypoxia adaption.,Quantitative real-time PCR (qRT-PCR) qRT-PCR was used to examine the expression levels of genes related to the HIF pathway in various tissues and developmental stages, as described previously. Briefly, 1 μg of total RNA was reverse-transcribed to cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio). Three genes were amplified using SYBR Premix Ex Taq™ II Kit (Takara Bio) in a CFX connect Real-Time system (Bio-Rad, Hercules, CA, USA). β-actin was identified via RNA-Seq analysis and used as a reference gene. Primer specificity was examined by dissociation curve analysis; fold-change values were calculated using the 2−ΔΔCt method (Schmittgen and Livak, 2008). Each experiment was performed in triplicate with two technical replicates. The primers used for qRT-PCR are presented in Table S4. Western blot analysis Approximately 100 mg C. chinensis larvae or tissues were suspended in 1 mL of RIPA lysis buffer in 2 mL microtubes. The lysed tissues were then homogenized using a Tissue Lyser (Qiagen, Hilden, Germany) and centrifuged at 4°C, 12,000 rpm for 5 min; the protein-containing supernatant was used for downstream applications. The protein content of the supernatant was quantified using Bradford reagent (Bio-Rad). Then, 20 μg of protein suspended in loading buffer was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 5% gels, followed by Western blotting onto a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane containing the protein samples was washed three times with tris-buffered saline Tween-20 (TBST) buffer, and then incubated at 25°C for 2 h with the primary antibody anti-HIF1α (Boster, Pleasanton, CA, USA) at 1:500 dilution, with anti-glyceraldehyde 3-phosphate dehydrogenase (Boster) at 1:2,000 dilution used as a loading control. The membrane was washed three times with TBST buffer, and then incubated with the secondary antibodies anti-rabbit horseradish peroxidase (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) and anti-mouse horseradish peroxidase (Jackson Immuno Research Laboratories, Inc.) at 1:2,000 dilution and 25°C for 2 h. The PVDF membrane was washed five times with TBST, and the protein bands were visualized using a hypersensitive chemiluminescence kit (Boster). The PVDF membrane with bound antibodies was transferred into a plastic bag in a darkroom for photographing, developing, and fixing.,See README file.

Inhibition of mitochondrial respiration under hypoxia and increased antioxidant activity after reoxygenation of <i>Tribolium castaneum</i>

<div><p>Regulating the air in low-oxygen environments protects hermetically stored grains from storage pests damage. However, pests that can tolerate hypoxic stress pose a huge challenge in terms of grain storage. We used various biological approaches to determine the fundamental mechanisms of <i>Tribolium castaneum</i> to cope with hypoxia. Our results indicated that limiting the available oxygen to <i>T</i>. <i>castaneum</i> increased glycolysis and inhibited the Krebs cycle, and that accumulated pyruvic acid was preferentially converted to lactic acid via anaerobic metabolism. Mitochondrial aerobic respiration was markedly suppressed for beetles under hypoxia, which also might have led to mitochondrial autophagy. The enzymatic activity of citrate synthase decreased in insects under hypoxia but recovered within 12 h, which suggested that the beetles recovered from the hypoxia. Moreover, hypoxia-reperfusion resulted in severe oxidative damage to insects, and antioxidant levels increased to defend against the high level of reactive oxygen species. In conclusion, our findings show that mitochondria were the main target in <i>T</i>. <i>castaneum</i> in response to low oxygen. The beetles under hypoxia inhibited mitochondrial respiration and increased antioxidant activity after reoxygenation. Our research advances the field of pest control and makes it possible to develop more efficient strategies for hermetic storage.</p></div

The activity of (A) SOD, (B) CAT, (C) GST and (D) MDA concentration of <i>T</i>. <i>castaneum</i> under normoxia and hypoxia-reperfusion.

<p>The bars represent means ± SE of three replicates. One-way ANOVA followed by Tukey’s range test was used for pairwise comparison of the difference between groups for mean separation (<i>P</i> < 0.05).</p

GO enrichment analysis of genes involves in the hypoxia resistance.

<p>The GO terms are sorted by —Log 10 of the enrichment <i>P</i>-value, which represent the enrichment significance of GO terms. The enrichment of GO terms are shown by comparing DEG with the whole genome.</p

Screening of active constituents in traditional Chinese medicines as potential Salmonella Typhimurium virulence inhibitors targeting Salmonella pathogenicity island III

Objective: To screen active constituents of traditional Chinese medicines (TCMs) as potential virulence inhibitors of Salmonella pathogenicity island III (SPI-3). Methods: The potential binding of TCM constituents to the MgtC protein of SPI-3 was clarified using molecular docking. The β-galactosidase assay was used to evaluate the effect of the TCM constituents on mgtC transcription. Finally, the effect of the drug on bacterial growth was investigated by assessing the growth curves and transcription levels of the key metabolic genes. Results: All 27 candidate TCM constituents showed that they could potentially bind to MgtC. The addition of ferulic acid, p-hydroxycinnamic acid, arctiin, and palmatine resulted in a more than 15% reduction in the transcriptional activity of mgtC. The minimum inhibitory concentrations of those four constituents on mgtC transcription were as follows: ferulic acid, 16 μM; p-hydroxycinnamic acid, 40 μM; arctiin, 80 μM; and palmatine, 160 μM. Additionally, we confirmed that none of these four constituents inhibited bacterial growth. Conclusion: In this study, we established a screening method for Salmonella virulence inhibitors based on the β-galactosidase assay, targeting SPI-3. Twenty-seven TCM constituents were screened, and four were found to have potentially potent inhibitory effects on Salmonella virulence. This provides lead compounds from herbal medicines for the development of novel antibiotics in the future. This method can also be used to screen for the virulence inhibitors of other pathogenic bacteria

Variation in citrate synthase activity of <i>T</i>. <i>castaneum</i> under normoxia and hypoxia-reperfusion.

<p>The citrate synthase activity of different groups was analyzed by one-way ANOVA. Results are shown as mean ± SE. Tukey’s multiple test was used for pairwise comparison of the difference between treatment for mean separation (<i>P</i> < 0.05).</p

Some hypoxia-response DEGs in the RNA-seq.

<p>Some hypoxia-response DEGs in the RNA-seq.</p

qRT-PCR analysis of selected transcripts to confirm expression profiles identified by RNA-seq.

<p><i>Tc</i>ELOVL, elongation of very long chain fatty acids protein 7; <i>Tc</i>MRP, ATP-binding cassette subfamily C (CFTR/MRP) member 4; <i>Tc</i>HSP70, heat shock 70kDa protein; <i>Tc</i>MAPK, MAP kinase; <i>Tc</i>DUSP, Dual specificity MAP kinase phosphatase; <i>Tc</i>SD, superoxide dismutase, Cu-Zn family; <i>Tc</i>AG, alpha-glucosidase; <i>Tc</i>DACHS, DACHS, Hippo signling pathway; <i>Tc</i>FJBP Four-jointed box protein 1 (FJBP). Value represents mean ± SE of three independent PCR amplifications and quantifications.</p