Transgenic early japonica rice: Integration and expression characterization of stem borer resistance Bt gene.

BACKGROUND: Transgenic insect-resistant rice offers an environmentally friendly approach to mitigate yield losses caused by lepidopteran pests, such as stem borers. Bt (Bacillus thuringiensis) genes encode insecticidal proteins and are widely used to confer insect resistance to genetically modified crops. This study investigated the integration, inheritance, and expression characteristics of codon-optimised synthetic Bt genes, cry1C* and cry2A*, in transgenic early japonica rice lines. METHODS: The early japonica rice cultivar, Songgeng 9 (Oryza sativa), was transformed with cry1C* or cry2A*, which are driven by the ubi promoter via Agrobacterium tumefaciens-mediated transformation. Molecular analyses, including quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Southern blot analysis were performed to confirm transgene integration, inheritance, transcriptional levels, and protein expression patterns across different tissues and developmental stages. RESULTS: Stable transgenic early japonica lines exhibiting single-copy transgene integration were established. Transcriptional analysis revealed variations in Bt gene expression among lines, tissues, and growth stages, with higher expression levels observed in leaves than in other organs. Notably, cry2A* exhibited consistently higher mRNA and protein levels than cry1C* across all examined tissues and developmental time points. Bt protein accumulation followed the trend of leaves > stem sheaths > young panicles > brown rice, with peak expression during the filling stage in the vegetative tissues. CONCLUSIONS: Synthetic cry2A* displayed markedly elevated transcription and translation compared to cry1C* in the transgenic early japonica rice lines examined. Distinct spatiotemporal patterns of Bt gene expression were elucidated, providing insights into the potential insect resistance conferred by these genes in rice. These findings will contribute to the development of insect-resistant japonica rice varieties and facilitate the rational deployment of Bt crops.

Trifolium repens L. recruits root-associated Microbacterium species to adapt to heavy metal stress in an abandoned Pb-Zn mining area.

Root-associated microbiota provide great fitness to hosts under environmental stress. However, the underlying microecological mechanisms controlling the interaction between heavy metal-stressed plants and the microbiota are poorly understood. In this study, we screened and isolated representative amplicon sequence variants (strain M4) from rhizosphere soil samples of Trifolium repens L. growing in areas with high concentrations of heavy metals. To investigate the microecological mechanisms by which T. repens adapts to heavy metal stress in abandoned mining areas, we conducted potting experiments, bacterial growth promotion experiments, biofilm formation experiments, and chemotaxis experiments. The results showed that high concentrations of heavy metals significantly altered the rhizosphere bacterial community structure of T. repens and significantly enriched Microbacterium sp. Strain M4 was demonstrated to significantly increased the biomass and root length of T. repens under heavy metal stress. Additionally, L-proline and stigmasterol could promote bacterial growth and biofilm formation and induce chemotaxis for strain M4, suggesting that they are key rhizosphere secretions of T. repens for Microbacterium sp. recruitment. Our results suggested that T. repens adapted the heavy metal stress by reshaping rhizosphere secretions to modify the rhizosphere microbiota.

Immobilization of Bacillus thuringiensis Cry1Ac in metal-organic frameworks through biomimetic mineralization for sustainable pest management.

Traditional chemical pesticide dosage forms and crude application methods have resulted in low pesticide utilization, increased environmental pollution, and the development of resistance. Compared to traditional pesticides, nanopesticides enhance the efficiency of pesticide utilization and reduce the quantity required, thereby decreasing environmental pollution. Herein, Cry1Ac insecticidal crystal protein from Bacillus thuringiensis Subsp. Kurstaki HD-73 was encapsulated in a metal-organic framework (zeolite imidazolate framework-8, ZIF-8) through biomimetic mineralization to obtain Cry1Ac@ZIF-8 nanopesticides. The Cry1Ac@ZIF-8 nanopesticides exhibited a dodecahedral porous structure, and the introduction of Cry1Ac did not affect the intrinsic crystal structure of ZIF-8. The indoor toxicity analysis revealed that the toxicity of Cry1Ac towards Ostrinia furnacalis (Guenée), Helicoverpa armigera Hubner, and Spodoptera litura Fabricius was not affected by ZIF-8 encapsulation. Surprisingly, Cry1Ac@ZIF-8 still exhibited excellent pest management efficacy even after exposure to heat, UV irradiation, and long-term storage. More importantly, the encapsulation of ZIF-8 significantly enhanced the internal absorption performance of Cry1Ac in maize leaves and extended its persistence period. Thus, ZIF-8 could potentially serve as a promising carrier for the preparation of nanopesticides with enhanced applicability, stability, and persistence period, providing a powerful strategy to improve the application of Cry1Ac in future agricultural pest management.

Reshaping the root endophytic microbiota in plants to combat mercury-induced stress.

Emerging evidence suggests that plants experiencing abiotic stress actively seek help from soil microbes. However, the empirical evidence supporting this strategy is limited, especially in response to heavy metal stress. We used integrated microbial community profiling and culture-based methods to investigate the interaction between mercury (Hg) stress, the entophytic root microbiome, and maize seedlings. The results of the pot experiment showed that soil Hg (20 mg/kg) strongly inhibited maize growth, indicating its strong phytotoxicity. Furthermore, Hg stress significantly altered the structure of the bacterial and fungal communities and enriched the potentially pathogenic Fusarium sp., suggesting that soil Hg stress may enhance the bio-stress induced by Fusarium species in maize. Additionally, soil Hg also led to the enrichment of beneficial bacterial members of Streptomyces, Lysobacter, and Sphingomonas (defined as differential species), which were also identified as keystone species in the Hg treatment by the analysis of co-occurrence networks. Therefore, it can be postulated that the members of Streptomyces, Lysobacter, and Sphingomonas function as stress-alleviating microbes. We successfully isolated the representatives of these stress-alleviating microbes. As expected, these strains mitigated the detrimental effects of Hg stess for the maize seedlings, suggesting that plants recruit the stress-alleviated microbiota to combat Hg stress. This study provides insights into the potential of manipulating the root microbiome to enhance plant growth in polluted environments.

Rational design and application of broad-spectrum antibodies for Bt Cry toxins determination.

Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.

Maternal Perceptions and Responsiveness to Cry in Armed Conflict Zones: Links to Child Behavior Problems.

Crying is a typical infant behavior that activates parental caregiving behaviors, acting as "human alarms" important for the infant's survival. When living under war-related threat, the auditory system may be sensitized given its importance for survival, potentially impacting maternal cry processing. Children living in armed-conflict zones are at increased risk for behavior problems, which may relate to both direct exposure and indirect effects through their parents' perceptions and behaviors. This hypothesis was examined in a sample of mothers and their first-born children (aged 10-45 months) living in the Gaza vicinity area in Israel, chronically exposed to missile alarms (high-exposure; n = 45), and a comparison group (low-exposure; n = 86). Group differences in child behavior problems and maternal perceptions of and responsiveness to cry were investigated. A moderated indirect-effect of maternal cry perceptions on child behavior problems via maternal responsiveness to cry was examined. In the high-exposure group, children had more externalizing problems and mothers rated cries as more aversive. Maternal cry perception was indirectly related to child behavior problems via responsiveness to cry only in the high-exposure group: higher perceptions of cry as aversive or the child as distressed were related to faster responding to crying, and faster cry responsiveness was linked with fewer behavior problems. Results suggest that in armed-conflict zones with auditory warning signals, the parental caring system may be easily activated by cries due to the strong association between alarms and threat. Furthermore, children may need their mothers to react faster when feeling distressed, possibly because of the surrounding threat.

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Circadian rhythms in the Drosophila eye may regulate adaptation of vision to light intensity.

The sensitivity of the eye at night would lead to complete saturation of the eye during the day. Therefore, the sensitivity of the eye must be down-regulated during the day to maintain visual acuity. In the Drosophila eye, the opening of TRP and TRPL channels leads to an influx of Ca++ that triggers down-regulation of further responses to light, including the movement of the TRPL channel and Gα proteins out of signaling complexes found in actin-mediated microvillar extensions of the photoreceptor cells (the rhabdomere). The eye also exhibits a light entrained-circadian rhythm, and we have recently observed that one component of this rhythm (BDBT) becomes undetectable by antibodies after exposure to light even though immunoblot analyses still detect it in the eye. BDBT is necessary for normal circadian rhythms, and in several circadian and visual mutants this eye-specific oscillation of detection is lost. Many phototransduction signaling proteins (e.g., Rhodopsin, TRP channels and Gα) also become undetectable shortly after light exposure, most likely due to a light-induced compaction of the rhabdomeric microvilli. The circadian protein BDBT might be involved in light-induced changes in the rhabdomere, and if so this could indicate that circadian clocks contribute to the daily adaptations of the eye to light. Likewise, circadian oscillations of clock proteins are observed in photoreceptors of the mammalian eye and produce a circadian oscillation in the ERG. Disruption of circadian rhythms in the eyes of mammals causes neurodegeneration in the eye, demonstrating the importance of the rhythms for normal eye function.

Draft genome sequence of Bacillus thuringiensis strain V-AB8.18, a novel isolate with potential nematicidal activity.

We report the draft genome of Bacillus thuringiensis strain V-AB8.18, comprising 308 contigs totaling 6,182,614 bp, with 35% G + C content. It contains 6,151 putative protein-coding genes, including App6 and Cry5-like crystal proteins, exhibiting 99% pairwise identity to nematicidal proteins App6Aa2 and Cry5Ba2, active against Meloidogyne incognita and Meloidogyne hapla.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

A Narrative Review on Different Novel Machine Learning Techniques for Detecting Pathologies in Infants From Born Baby Cries.

This paper reviews the research work on the analysis and classification of pathological infant cries in the last 50 years. The literature review mainly covers the need and role of early clinical diagnosis, pathologies detected from cry samples, challenges in pathological cry signal data acquisition, signal processing techniques, and signal classifiers. The signal processing techniques include preprocessing, feature extraction from domains, such as time, spectral, time-frequency, prosodic, wavelet, etc, and feature selection for selecting dominant features. Literature covers traditional machine learning classifiers, such as Bayesian networks, decision trees, K-nearest neighbor, support vector machine, Gaussian mixture model, etc, and recently added neural network models, such as convolutional neural networks, regression neural networks, probabilistic neural networks, graph neural networks, etc. Significant experimental results of pathological cry identification and classification are listed for comparison. Finally, it suggests future research in the direction of database preparation, feature analysis and extraction, neural network classifiers to provide a non-invasive and robust automatic infant cry analysis model.

Chronotoxici-Plate Containing Droplet-Engineered Rhythmic Liver Organoids for Drug Toxicity Evaluation.

The circadian clock coordinates the daily rhythmicity of biological processes, and its dysregulation is associated with various human diseases. Despite the direct targeting of rhythmic genes by many prevalent and World Health Organization (WHO) essential drugs, traditional approaches can't satisfy the need of explore multi-timepoint drug administration strategies across a wide range of drugs. Here, droplet-engineered primary liver organoids (DPLOs) are generated with rhythmic characteristics in 4 days, and developed Chronotoxici-plate as an in vitro high-throughput automated rhythmic tool for chronotherapy assessment within 7 days. Cryptochrome 1 (Cry1) is identified as a rhythmic marker in DPLOs, providing insights for rapid assessment of organoid rhythmicity. Using oxaliplatin as a representative drug, time-dependent variations are demonstrated in toxicity on the Chronotoxici-plate, highlighting the importance of considering time-dependent effects. Additionally, the role of chronobiology is underscored in primary organoid modeling. This study may provide tools for both precision chronotherapy and chronotoxicity in drug development by optimizing administration timing.

A Comparative Evaluation of Pain Experience and Time of Onset of 2% Lignocaine and 4% Articaine in Inferior Alveolar Nerve Block among Pediatric Population: A Clinical Study.

Aim: The objective of this research was to conduct a comparison and evaluate the pain perception and time of onset of 2% lignocaine 1:80,000 epinephrine with 4% articaine 1:100,000 epinephrine in the pediatric population. Materials and methods: A split-mouth randomized control trial was conducted on 50 children aged 9-14 years who required inferior alveolar nerve block (IANB) anesthesia for bilateral dental treatment in the mandibular arch. The time of onset was recorded when no sensation was reported even when maximum electrical stimulus was applied in an electric pulp testing (EPT). The pain perception was assessed using a visual analog scale (VAS) rated by the patient for subjective symptoms and face, legs, activity, cry, and consolability (FLACC) scale for objective pain rated by the operator. Results: The mean onset of time, pain-VAS, and FLACC score decreased by 1.31, 12.07, and 18.39%, respectively in 4% articaine as compared to 2% lignocaine but the difference did not reach statistical significance (p > 0.05), that is, found to be statistically the same.In conclusion, it can be inferred that the utilization of 4% articaine is as potent as 2% lignocaine solution but showed slightly better onset of anesthesia and pain experience among the children although the findings were not statistically significant. Clinical significance: Local anesthesia (LA) is one of the main methods of pain management in pediatric practice which makes it essential to choose an LA agent with a shorter time of onset and less pain on administration. How to cite this article: Singh SS, Koul M. A Comparative Evaluation of Pain Experience and Time of Onset of 2% Lignocaine and 4% Articaine in Inferior Alveolar Nerve Block among Pediatric Population: A Clinical Study. Int J Clin Pediatr Dent 2024;17(1):67-71.

Genetically engineered eucalyptus expressing pesticidal proteins from Bacillus thuringiensis for insect resistance: a risk assessment evaluation perspective.

Eucalyptus covers approximately 7.5 million hectares in Brazil and serves as the primary woody species cultivated for commercial purposes. However, native insects and invasive pests pose a significant threat to eucalyptus trees, resulting in substantial economic losses and reduced forest productivity. One of the primary lepidopteran pests affecting eucalyptus is Thyrinteina arnobia (Stoll, 1782) (Lepidoptera: Geometridae), commonly referred to as the brown looper caterpillar. To address this issue, FuturaGene, the biotech division of Suzano S.A., has developed an insect-resistant (IR) eucalyptus variety, which expresses Cry pesticidal proteins (Cry1Ab, Cry1Bb, and Cry2Aa), derived from Bacillus thuringiensis (Bt). Following extensive safety assessments, including field trials across various biomes in Brazil, the Brazilian National Technical Commission of Biosafety (CTNBio) recently approved the commercialization of IR eucalyptus. The biosafety assessments involved the analysis of molecular genomics, digestibility, thermostability, non-target organism exposure, degradability in the field, and effects on soil microbial communities and arthropod communities. In addition, in silico studies were conducted to evaluate allergenicity and toxicity. Results from both laboratory and field studies indicated that Bt eucalyptus is as safe as the conventional eucalyptus clone for humans, animals, and the environment, ensuring the secure use of this insect-resistant trait in wood production.

CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

[Developmental toxicity of Cry1Ab protein in the embryonic stem-cell model].

OBJECTIVE: To evaluate the developmental toxicity of Cry1Ab protein by studying its effects on cell proliferation and differentiation ability using a developmental toxicity assessment model based on embryonic stem-cell. METHODS: Cry1Ab protein was tested in seven dose groups (31.25, 62.50, 125.00, 250.00, 320.00, 1 000.00, and 2 000.00 µg/L) on mouse embryonic stem cells D3 (ES-D3) and 3T3 mouse fibroblast cells, with 5-fluorouracil (5-FU) used as the positive control and phosphate buffer saline (PBS) as the solvent control. Cell viability was detected by CCK-8 assay to calculate the 50% inhibitory concentration (IC50) of the test substance for different cells. Additionally, Cry1Ab protein was tested in five dose groups (125.00, 250.00, 320.00, 1 000.00, and 2 000.00 µg/L) on ES-D3 cells, with PBS as the solvent control and 5-FU used for model validation. After cell treatment, cardiac differentiation was induced using the embryonic bodies (EBs) culture method. The growth of EBs was observed under a microscope, and their diameters on the third and fifth days were measured. The proportion of EBs differentiating into beating cardiomyocytes was recorded, and the 50% inhibition concentration of differentiation (ID50) was calculated. Based on a developmental toxicity discrimination function, the developmental toxicity of the test substances was classified. Furthermore, at the end of the culture period, mRNA expression levels of cardiac differentiation-related markers (Oct3/4, GATA-4, Nkx2.5, and ß-MHC) were quantitatively detected using real-time quantitative polymerase chain reaction (qPCR) in the collected EBs samples. RESULTS: The IC50 of 5-FU was determined as 46.37 µg/L in 3T3 cells and 32.67 µg/L in ES-D3 cells, while the ID50 in ES-D3 cells was 21.28 µg/L. According to the discrimination function results, 5-FU was classified as a strong embryotoxic substance. There were no statistically significant differences in cell viability between different concentrations of Cry1Ab protein treatment groups and the control group in both 3T3 cells and ES-D3 cells (P>0.05). Moreover, there were no statistically significant differences in the diameter of EBs on the third and fifth days, as well as their morphology, between the Cry1Ab protein treatment groups and the control group (P>0.05). The cardiac differentiation rate showed no statistically significant differences between different concentrations of Cry1Ab protein treatment groups and the control group (P>0.05). 5-FU significantly reduced the mRNA expression levels of ß-MHC, Nkx2.5, and GATA-4 (P < 0.05), showing a dose-dependent trend (P < 0.05), while the mRNA expression levels of the pluripotency-associated marker Oct3/4 exhibited an increasing trend (P < 0.05). However, there were no statistically significant differences in the mRNA expression levels of mature cardiac marker ß-MHC, early cardiac differentiation marker Nkx2.5 and GATA-4, and pluripotency-associated marker Oct3/4 between the Cry1Ab protein treatment groups and the control group (P>0.05). CONCLUSION: No developmental toxicity of Cry1Ab protein at concentrations ranging from 31.25 to 2 000.00 µg/L was observed in this experimental model.

Noise phenomena in distress cries of term and very preterm infants at term-equivalent age.

BACKGROUND AND AIM: To examine whether the noise components in distress cries of term infants differed from very preterm infants whose cries were collected at a comparable "corrected" gestational age. METHODS: Distress cries were collected from 20 term and 20 preterm infants. The cries were acoustically examined for the occurrence of aperiodic phonatory behavior within and across moments of crying. RESULTS: The findings indicated no significant differences between term and preterm infants at term age in the occurrence of noise. CONCLUSIONS: Distress cries of both term and term-equivalent preterm infants appear to contain high instances of phonatory noise. The high arousal associated with distress crying and associated increase in subglottal pressure appeared to influence both term and term-equivalent preterm infants similarly.

The emerging tools for precisely manipulating microtubules.

Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions.

Utilization of Diverse Molecules as Receptors by Cry Toxin and the Promiscuous Nature of Receptor-Binding Sites Which Accounts for the Diversity.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori. Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.