Anticancer efficacy of biosynthesized silver nanoparticles loaded with recombinant truncated parasporin-2 protein.

Bacterial toxins have received a great deal of attention in the development of cancer treatments. Parasporin-2 (PS2Aa1 or Mpp46Aa1) is a Bacillus thuringiensis parasporal protein that preferentially destroys human cancer cells while not harming normal cells, making it a promising anticancer treatment. With the efficient development and sustainable silver nanoparticles (AgNPs) synthesis technology, the biomedical use of AgNPs has expanded. This study presents the development of a novel nanotoxin composed of biosynthesized silver nanoparticles loaded with the N-terminal truncated PS2Aa1 toxin. MOEAgNPs were synthesized using a biological method, with Moringa oleifera leaf extract and maltose serving as reducing and capping agents. The phytochemicals present in M. oleifera leaf extract were identified by GC-MS analysis. MOEAgNPs were loaded with N-terminal truncated PS2Aa1 fused with maltose-binding protein (MBP-tPS2) to formulate PS2-MOEAgNPs. The PS2-MOEAgNPs were evaluated for size, stability, toxin loading efficacy, and cytotoxicity. PS2-MOEAgNPs demonstrated dose-dependent cytotoxicity against the T-cell leukemia MOLT-4 and Jurkat cell lines but had little effect on the Hs68 fibroblast or normal cell line. Altogether, the current study provides robust evidence that PS2-MOEAgNPs can efficiently inhibit the proliferation of T-cell leukemia cells, thereby suggesting their potential as an alternative to traditional anticancer treatments.

Mosquitocidal toxin-like islands in Bacillus thuringiensis S2160-1 revealed by complete-genome sequence and MS proteomic analysis.

Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.

Silence of Aminopeptidase N 2 gene reveals the trade-offs for acquiring Cry1Ac resistance in Plutella xylostella.

Bacillus thuringiensis (Bt) is widely used as a biopesticide worldwide. To date, at least eight pest species have been found to be resistant to Bt in the field. As the first pest that was reported having resistance to Bt in the field, considerable research has been done on the mechanisms of Bt resistance in Plutella xylostella. However, whether the acquisition of Bt resistance by P. xylostella comes at a fitness cost is also a valuable question. In this study, Aminopeptidase-N 2 (APN2), a Cry toxin receptor gene of P. xylostella, was knocked down by RNA interference, resulting in improved resistance to Cry1Ac. It was also found that larval mortality of APN2 knockdown P. xylostella was significantly higher than that of the control, while the pupation rate, pupal weight, eclosion rate, fecundity (egg/female), hatchability, and female adult longevity were significantly lower in APN2 knockdown P. xylostella than in the control. These results illustrate that if Cry1Ac resistance was obtained only through the reduction of APN2 expression, P. xylostella would need to incur some fitness costs for it.

[Application of ultrasonic irrigation combined with chlorhexidine in root canal treatment of pulpitis].

PURPOSE: To explore the clinical effect of ultrasonic irrigation combined with chlorhexidine in root canal treatment of pulpitis. METHODS: A total of 120 patients with pulpitis treated with root canal therapy were randomly divided into a study group (n=60, 72 affected teeth) and a control group (n=60, 70 affected teeth). During root canal preparation, the study group was treated with chlorhexidine combined with ultrasonic irrigation, while the control group was treated with chlorhexidine conventional irrigation. The bacterial count and endotoxin content in the root canal before and after root canal preparation were compared between the two groups, as well as the endodontic inter-appointment pain (EIAP), lateral branch root canal filling rate, and degree of tooth pain after root canal treatment. The success rate of treatment was statistically analyzed after one-year follow-up. Statistical analysis was performed with SPSS 19.0 software package. RESULTS: After root canal preparation, the number of colonies in experimental group and control group was significantly decreased compared with that before root canal preparation(P＜0.05), and the number of colonies in experimental group was significantly lower than that in control group(P＜0.05). After root canal preparation, endotoxin levels in experimental group and control group were significantly lower than those before root canal preparation(P＜0.05), and the level in experimental group was significantly lower than that in control group(P＜0.05). The lateral branch root canal filling rate in the study group and the control group was 29.17% and 11.43%, respectively, with significant difference between the groups(P＜0.05). The incidence of EIAP was 4.17% and 14.29%, respectively, with significant difference between the two groups(P＜0.05). At 48 hours after surgery, the visual analogue score (VAS) of the study group and the control group was (2.74±0.61) and (3.29±0.68), respectively, which were significantly lower than at before surgery(P＜0.05). There was a significant difference in VAS score between the two groups 48 hours after surgery(P＜0.05). One week after surgery, the VAS score in the study group and the control group was (1.52±0.34) and (1.81±0.42), respectively, significantly lower than that before and 48 hours after surgery(P＜0.05). There was a significant difference in VAS score between the two groups at one week after surgery (P＜0.05). The successful rate of treatment in the control group was 84.62%, and 95.71% in the study group, with a significant difference between the two groups(P＜0.05). CONCLUSIONS: The application of ultrasonic irrigation combined with chlorhexidine in the treatment of pulpitis root canals can help reduce the level of bacteria and endotoxin after root canal preparation, alleviate the degree of postoperative tooth pain, and improve the filling rate of lateral branch root canals, with superior curative effects.

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.

Exploratory comparative transcriptomic analysis reveals potential gene targets associated with Cry1A.105 and Cry2Ab2 resistance in fall armyworm (Spodoptera frugiperda).

Genetically modified (GM) crops, expressing Bacillus thuringiensis (Bt) insecticidal toxins, have substantially transformed agriculture. Despite rapid adoption, their environmental and economic benefits face scrutiny due to unsustainable agricultural practices and the emergence of resistant pests like Spodoptera frugiperda, known as the fall armyworm (FAW). FAW's adaptation to Bt technology in corn and cotton compromises the long-term efficacy of Bt crops. To advance the understanding of the genetic foundations of resistance mechanisms, we conducted an exploratory comparative transcriptomic analysis of two divergent FAW populations. One population exhibited practical resistance to the Bt insecticidal proteins Cry1A.105 and Cry2Ab2, expressed in the genetically engineered MON-89Ø34 - 3 maize, while the other population remained susceptible to these proteins. Differential expression analysis supported that Cry1A.105 and Cry2Ab2 significantly affect the FAW physiology. A total of 247 and 254 differentially expressed genes were identified in the Cry-resistant and susceptible populations, respectively. By integrating our findings with established literature and databases, we underscored 53 gene targets potentially involved in FAW's resistance to Cry1A.105 and Cry2Ab2. In particular, we considered and discussed the potential roles of the differentially expressed genes encoding ABC transporters, G protein-coupled receptors, the P450 enzymatic system, and other Bt-related detoxification genes. Based on these findings, we emphasize the importance of exploratory transcriptomic analyses to uncover potential gene targets involved with Bt insecticidal proteins resistance, and to support the advantages of GM crops in the face of emerging challenges.

Metabolic Endotoxemia: From the Gut to Neurodegeneration.

Metabolic endotoxemia is a severe health problem for residents in developed countries who follow a Western diet, disrupting intestinal microbiota and the whole organism's homeostasis. Although the effect of endotoxin on the human immune system is well known, its long-term impact on the human body, lasting many months or even years, is unknown. This is due to the difficulty of conducting in vitro and in vivo studies on the prolonged effect of endotoxin on the central nervous system. In this article, based on the available literature, we traced the path of endotoxin from the intestines to the blood through the intestinal epithelium and factors promoting the development of metabolic endotoxemia. The presence of endotoxin in the bloodstream and the inflammation it induces may contribute to lowering the blood-brain barrier, potentially allowing its penetration into the central nervous system; although, the theory is still controversial. Microglia, guarding the central nervous system, are the first line of defense and respond to endotoxin with activation, which may contribute to the development of neurodegenerative diseases. We traced the pro-inflammatory role of endotoxin in neurodegenerative diseases and its impact on the epigenetic regulation of microglial phenotypes.

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.

CRISPR-Cas9 knockout of membrane-bound alkaline phosphatase or cadherin does not confer resistance to Cry toxins in Aedes aegypti.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

BMAL2 promotes eCIRP-induced macrophage endotoxin tolerance.

Background: The disruption of the circadian clock is associated with inflammatory and immunological disorders. BMAL2, a critical circadian protein, forms a dimer with CLOCK, activating transcription. Extracellular cold-inducible RNA-binding protein (eCIRP), released during sepsis, can induce macrophage endotoxin tolerance. We hypothesized that eCIRP induces BMAL2 expression and promotes macrophage endotoxin tolerance through triggering receptor expressed on myeloid cells-1 (TREM-1). Methods: C57BL/6 wild-type (WT) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Serum levels of eCIRP 20 h post-CLP were assessed by ELISA. Peritoneal macrophages (PerM) were treated with recombinant mouse (rm) CIRP (eCIRP) at various doses for 24 h. The cells were then stimulated with LPS for 5 h. The levels of TNF-α and IL-6 in the culture supernatants were assessed by ELISA. PerM were treated with eCIRP for 24 h, and the expression of PD-L1, IL-10, STAT3, TREM-1 and circadian genes such as BMAL2, CRY1, and PER2 was assessed by qPCR. Effect of TREM-1 on eCIRP-induced PerM endotoxin tolerance and PD-L1, IL-10, and STAT3 expression was determined by qPCR using PerM from TREM-1-/- mice. Circadian gene expression profiles in eCIRP-treated macrophages were determined by PCR array and confirmed by qPCR. Induction of BMAL2 activation in bone marrow-derived macrophages was performed by transfection of BMAL2 CRISPR activation plasmid. The interaction of BMAL2 in the PD-L1 promoter was determined by computational modeling and confirmed by the BIAcore assay. Results: Serum levels of eCIRP were increased in septic mice compared to sham mice. Macrophages pre-treated with eCIRP exhibited reduced TNFα and IL-6 release upon LPS challenge, indicating macrophage endotoxin tolerance. Additionally, eCIRP increased the expression of PD-L1, IL-10, and STAT3, markers of immune tolerance. Interestingly, TREM-1 deficiency reversed eCIRP-induced macrophage endotoxin tolerance and significantly decreased PD-L1, IL-10, and STAT3 expression. PCR array screening of circadian clock genes in peritoneal macrophages treated with eCIRP revealed the elevated expression of BMAL2, CRY1, and PER2. In eCIRP-treated macrophages, TREM-1 deficiency prevented the upregulation of these circadian genes. In macrophages, inducible BMAL2 expression correlated with increased PD-L1 expression. In septic human patients, blood monocytes exhibited increased expression of BMAL2 and PD-L1 in comparison to healthy subjects. Computational modeling and BIAcore assay identified a putative binding region of BMAL2 in the PD-L1 promoter, suggesting BMAL2 positively regulates PD-L1 expression in macrophages. Conclusion: eCIRP upregulates BMAL2 expression via TREM-1, leading to macrophage endotoxin tolerance in sepsis. Targeting eCIRP to maintain circadian rhythm may correct endotoxin tolerance and enhance host resistance to bacterial infection.

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.

Characterization of airborne endotoxin in personal exposure to fine particulate matter (PM2.5) and bioreactivity for elderly residents in Hong Kong.

The heavy metals and bioreactivity properties of endotoxin in personal exposure to fine particulate matter (PM2.5) were characterized in the analysis. The average personal exposure concentrations to PM2.5 were ranged from 6.8 to 96.6 µg/m3. The mean personal PM2.5 concentrations in spring, summer, autumn, and winter were 32.1±15.8, 22.4±11.8, 35.3±11.9, and 50.2±19.9 µg/m3, respectively. There were 85 % of study targets exceeded the World Health Organization (WHO) PM2.5 threshold (24 hours). The mean endotoxin concentrations ranged from 1.086 ± 0.384-1.912 ± 0.419 EU/m3, with a geometric mean (GM) varied from 1.034 to 1.869. The concentration of iron (Fe) (0.008-1.16 µg/m3) was one of the most abundant transition metals in the samples that could affect endotoxin toxicity under Toll-Like Receptor 4 (TLR4) stimulation. In summer, the interleukin 6 (IL-6) levels showed statistically significant differences compared to other seasons. Spearman correlation analysis showed endotoxin concentrations were positively correlated with chromium (Cr) and nickel (Ni), implying possible roles as nutrients and further transport via adhering to the surface of fine inorganic particles. Mixed-effects model analysis demonstrated that Tumor necrosis factor-α (TNF-α) production was positively associated with endotoxin concentration and Cr as a combined exposure factor. The Cr contained the highest combined effect (0.205-0.262), suggesting that Cr can potentially exacerbate the effect of endotoxin on inflammation and oxidative stress. The findings will be useful for practical policies for mitigating air pollution to protect the public health of the citizens.

Inhalation of ferrate-disinfected Escherichia coli caused lung injury via endotoxin-induced oxidative stress and inflammation response.

Ferrate (Fe(VI)) is an environmentally friendly disinfectant that is widely used to eradicate microbes in reclaimed water. However, the potential health risks associated with inhalation of Fe(VI)-treated bacteria-laden reclaimed water remains uncertain. We aimed to explore the inhalation hazards and potential mechanisms of K2FeO4-treated Escherichia coli (E. coli, ATCC 25922). Our findings indicated that Fe(VI) disinfection induced a dose- and time-dependent E. coli inactivation, accompanied by a rapid release of the bacterial endotoxin, lipopolysaccharide (LPS). Scanning electron microscopy (SEM) observations indicate that Fe(VI)-induced endotoxin production consists of at least two stages: initial binding of endotoxin to bacteria and subsequent dissociation to release free endotoxin. Furthermore, Fe(VI) disinfection was not able to effectively eliminate pure or E. coli-derived endotoxins. The E. coli strain used in this study lacks lung infection capability, thus the inhalation of bacteria alone failed to induce severe lung injury. However, mice inhaled exposure to Fe(VI)-treated E. coli showed severe impairment of lung structure and function. Moreover, we observed an accumulation of neutrophil/macrophage recruitment, cell apoptosis, and ROS generation in the lung tissue of mice subjected to Fe(VI)-treated E. coli. RNA sequencing (RNA-seq) and PCR results revealed that genes involved with endotoxin stimuli, cell apoptosis, antioxidant defence, inflammation response, chemokines and their receptors were upregulated in response to Fe(VI)-treated E. coli. In conclusion, Fe(VI) is ineffective in eliminating endotoxins and can trigger secondary hazards owing to endotoxin release from inactivated bacteria. Aerosol exposure to Fe(VI)-treated E. coli causes considerable damage to lung tissue by inducing oxidative stress and inflammatory responses.

Abnormalities of Coagulation and Fibrinolysis Assessed by Thromboelastometry in an Endotoxic Shock Model in Piglets Treated with Nitric Oxide and Hydrocortisone.

This is an animal model study to investigate changes in hemostasis during endotoxemic shock and to determine whether the combination of inhaled nitric oxide (iNO) + intravenous hydrocortisone had an effect on clot formation and fibrinolysis. iNO selectively decreases pulmonary artery pressure, without affecting cardiac index or systemic vascular resistance; however, the results of studies on the possible consequences of iNO administration on coagulation are inconsistent and require further research. Thirty-four piglets were included. Administering endotoxin caused severe hypodynamic shock. Half of the animals received iNO (30 ppm) + hydrocortisone, starting 3 h after endotoxin infusion and continuing to the end of the study. All animals developed coagulation disorders, manifested by a tendency to hypocoagulation; at the same time, fibrinolysis was impaired. Coagulation and fibrinolysis disorders persisted after endotoxin infusion was discontinued, with worse severity in the animals that died before the study was terminated. Administering iNO + hydrocortisone did not cause further changes in coagulation and fibrinolysis parameters, either during or after the endotoxin challenge, suggesting that potential therapeutic interventions with iNO to lower pulmonary arterial pressure will not affect hemostasis.

Identification of novel biomarkers of acute phase response in chickens challenged with Escherichia coli lipopolysaccharide endotoxin.

BACKGROUND: The chicken's inflammatory response is an essential part of the bird's response to infection. A single dose of Escherichia coli (E. coli) lipopolysaccharide (LPS) endotoxin can activate the acute phase response (APR) and lead to the production of acute phase proteins (APPs). In this study, the responses of established chicken APPs, Serum amyloid A (SAA) and Alpha-1-acid-glycoprotein (AGP), were compared to two novel APPs, Hemopexin (Hpx) and Extracellular fatty acid binding protein (Ex-FABP), in 15-day old broilers over a time course of 48 h post E.coli LPS challenge. We aimed to investigate and validate their role as biomarkers of an APR. Novel plant extracts, Citrus (CTS) and cucumber (CMB), were used as dietary supplements to investigate their ability to reduce the inflammatory response initiated by the endotoxin. RESULTS: A significant increase of established (SAA, AGP) and novel (Ex-FABP, Hpx) APPs was detected post E.coli LPS challenge. Extracellular fatty acid binding protein (Ex-FABP) showed a similar early response to SAA post LPS challenge by increasing ~ 20-fold at 12 h post challenge (P < 0.001). Hemopexin (Hpx) showed a later response by increasing ∼5-fold at 24 h post challenge (P < 0.001) with a similar trend to AGP. No differences in APP responses were identified between diets (CTS and CMB) using any of the established or novel biomarkers. CONCLUSIONS: Hpx and Ex-FABP were confirmed as potential biomarkers of APR in broilers when using an E. coli LPS model along with SAA and AGP. However, no clear advantage for using either of dietary supplements to modulate the APR was identified at the dosage used.

Endotoxin, a novel biomarker for the rapid risk assessment of faecal contamination of coastal and transitional waters.

Current methods for testing water for faecal contamination rely on the culture of faecal indicator bacteria (FIB; Escherichia coli and Enterococci) that take 24-48 h, which leads to delays in taking proactive measures and poses a risk to public health. More rapid methods are therefore required. Here, we have tested a rapid, portable assay (Bacterisk) that detects the bacterial biomarker endotoxin in 30 min to quantify the bacterial biomass present, to evaluate 159 coastal water samples and to compare the results with the traditional culture of FIB. There was a significant correlation between the Bacterisk data given in endotoxin risk (ER) units and FIB culture that could accurately distinguish between poor and sufficient or good quality bathing water using the EU bathing directive values. Receiver operating characteristic analysis was used to determine the optimal ER threshold for coastal water samples, and the area under the curve was 0.9176 with a p-value of <0.0001. The optimal threshold was 7,300 ER units with a sensitivity of 95.45% and a specificity of 83.48%. In conclusion, we have shown that the Bacterisk assay provides a rapid and easy-to-use in situ method to assess bathing water quality.

Recombinant Beauveria bassiana expressing Bacillus thuringiensis toxin Cyt1Aa: a promising approach for enhancing Aedes mosquito control.

The entomopathogenic fungus Beauveria bassiana provides an eco-friendly substitute to chemical insecticides for mosquito control. Nevertheless, its widespread application has been hindered by its comparatively slow efficacy in eliminating mosquitoes. To augment the potency of B. bassiana against Aedes mosquitoes, a novel recombinant strain, Bb-Cyt1Aa, was developed by incorporating the Bacillus thuringiensis toxin gene Cyt1Aa into B. bassiana. The virulence of Bb-Cyt1Aa was evaluated against Aedes aegypti and Aedes albopictus using insect bioassays. Compared to the wild-type (WT) strain, the median lethal time (LT50) for A. aegypti larvae infected with Bb-Cyt1Aa decreased by 33.3% at a concentration of 1 × 108 conidia/mL and by 22.2% at 1 × 107 conidia/mL. The LT50 for A. aegypti adults infected with Bb-Cyt1Aa through conidia ingestion was reduced by 37.5% at 1 × 108 conidia/mL and by 33.3% at 1 × 107 conidia/mL. Likewise, the LT50 for A. aegypti adults infected with Bb-Cyt1Aa through cuticle contact decreased by 33.3% and 30.8% at the same concentrations, respectively. Furthermore, the Bb-Cyt1Aa strain also demonstrated increased toxicity against both larval and adult A. albopictus, when compared to the WT strain. In conclusion, our study demonstrated that the expression of B. thuringiensis toxin Cyt1Aa in B. bassiana enhanced its virulence against Aedes mosquitoes. This suggests that B. bassiana expressing Cyt1Aa has potential value for use in mosquito control. IMPORTANCE: Beauveria bassiana is a naturally occurring fungus that can be utilized as a bioinsecticide against mosquitoes. Cyt1Aa is a delta-endotoxin protein produced by Bacillus thuringiensis that exhibits specific and potent insecticidal activity against mosquitoes. In our study, the expression of this toxin Cyt1Aa in B. bassiana enhances the virulence of B. bassiana against Aedes aegypti and Aedes albopictus, thereby increasing their effectiveness in killing mosquitoes. This novel strain can be used alongside chemical insecticides to reduce dependence on harmful chemicals, thereby minimizing negative impacts on the environment and human health. Additionally, the potential resistance of B. bassiana against mosquitoes in the future could be overcome by acquiring novel combinations of exogenous toxin genes. The presence of B. bassiana that expresses Cyt1Aa is of significant importance in mosquito control as it enhances genetic diversity, creates novel virulent strains, and contributes to the development of safer and more sustainable methods of mosquito control.

Novel aptasensor based on polyaniline functionalized carboxylated dobby carbon nanotubes and molybdenum disulfide for endotoxin detection.

Endotoxins, also known as lipopolysaccharides (LPS), are present within the cell walls of Gram-negative bacteria and are released upon cellular death, which can pose a significant risk to human and animal health. Due to the minimal amount of endotoxin required to trigger an inflammatory response in human body, the demand for sensitive methods with low endotoxin detection limits is essential necessary. This paper presents a straightforward aptamer sensor which can enhance the conductivity and specific surface area of molybdenum disulfide (MoS2) by incorporating carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) and polyaniline (PANI). Doping with gold nanoparticles (AuNPs) improves biocompatibility and sensitivity while providing binding sites for thiolated endotoxin-binding aptamers (LBA). This biosensor achieved a remarkable detection limit as low as 0.5 fg mL-1, enabling trace-level identification of LPS. It also exhibits excellent repeatability, selectivity, and stability, facilitating rapid and accurate LPS detection. Moreover, this method demonstrates high recovery rates and specificity for LPS analysis in food samples, showcasing its promising application prospects in trace-level LPS detection within the food industry.

Fall armyworm (Lepidoptera: Noctuidae): practical resistance of 2 Brazilian populations to Cry1A.105†+†Cry2Ab and Cry1F Bt maize.

In the Americas, transgenic crops producing insecticidal proteins from Bacillus thuringiensis Berliner (Bt, Bacillales: Bacillaceae) have been used widely to manage fall armyworm (FAW, Spodoptera frugiperda [J.E. Smith]). As resistance to Cry1 single-gene Bt maize (Zea mays L.) rapidly evolved in some FAW populations, pyramided Bt maize hybrids producing Cry1, Cry2, or Vip3Aa proteins were introduced in the 2010s. We examined field-evolved resistance to single- and dual-protein Bt maize hybrids in 2 locations in southeastern Brazil, where plant damage by FAW larvae far exceeded the economic threshold in 2017. We collected late-instar larvae in Cry1A.105 + Cry2Ab and Cry1F maize fields and established 2 FAW populations in the laboratory. The F1 offspring reared on the foliage of Bt and non-Bt maize plants (Cry1A.105 + Cry2Ab and Cry1F) showed neonate-to-adult survival rates as high as 70% for both populations. There was no significant difference in the life-table parameters of armyworms reared on non-Bt and Bt maize foliage, indicating complete resistance to Cry1A.105 + Cry2Ab maize. Larval survival rates of reciprocal crosses of a susceptible laboratory strain and the field-collected populations indicated nonrecessive resistance to Cry1F and a recessive resistance to Cry1A.105 + Cry2Ab maize. When relaxing the selection pressure, the armyworm fitness varied on Cry1A.105 + Cry2Ab and non-Bt maize; the resistance was somewhat stable across 12 generations, without strong fitness costs, although one of the lines died confounded by a depleted-quality, artificial rearing diet. To our knowledge, this is the first report documenting the practical resistance of FAW to a pyramided Bt crop. We discuss the implications for resistance management.