Inheritance of seedlessness and the molecular characterization of the INO gene in Annonaceae

The inheritance of the seedless fruit characteristic of Annona squamosa has not yet been explained. Molecular techniques may aid breeding programs, mainly in the assisted selection of the target gene. The INO gene may be related to seed development in these fruits. The objective of the present paper was to investigate the inheritance of seedlessness in the 'Brazilian seedless' sugar apple and INO gene conservation in Annona squamosa and Annona cherimola x Annona squamosa genotypes by assessing their homology with the INO database genes. The F1 generation was obtained by crossing the mutant 'Brazilian seedless' (male genitor) (P1) with the wild-type A. squamosa with seeds (M1 and M2, female genitors). The INO gene was studied in mutant and wild-type A. squamosa (P1, M1, M2 and M3) and in the Gefner atemoya (A. cherimola x A. squamosa) (M4) cultivar. The DNA was extracted from young leaves, and four sets of specific primers flanking the INO gene were amplified. The seedless characteristic was identified as stenospermatic in the fruits of parental P1, suggesting monogenic inheritance with complete dominance. High sequence similarity of the INO gene amplifications in the sugar apple accessions (M1, M2, M3) and the atemoya cultivar Gefner (M4) reinforces the hypothesis of their conservation.

Activated Platelets Autocrine 5-Hydroxytryptophan Aggravates Sepsis-Induced Acute Lung Injury by Promoting Neutrophils Extracellular Traps Formation

Excessive neutrophil extracellular trap (NET) formation is an important contributor to sepsis-induced acute lung injury (ALI). Recent reports indicate that platelets can induce neutrophil extracellular trap formation. However, the specific mechanism remains unclear. Tph1 gene, which encodes the rate-limiting enzyme for peripheral 5-hydroxytryptophan (5-HT) synthesis, was knocked out in mice to simulate peripheral 5-HT deficiency. Cecal ligation and puncture (CLP) surgery was performed to induce sepsis. We found that peripheral 5-HT deficiency reduced NET formation in lung tissues, alleviated sepsis-induced lung inflammatory injury, and reduced the mortality rate of CLP mice. In addition, peripheral 5-HT deficiency was shown to reduce the accumulation of platelets and NETs in the lung of septic mice. We found that platelets from wild-type (WT), but not Tph1 knockout (Tph1−/−), mice promote lipopolysaccharide (LPS)-induced NET formation. Exogenous 5-HT intervention increased LPS-induced NET formation when Tph1−/− platelets were co-cultured with WT neutrophils. Therefore, our study uncovers a mechanism by which peripheral 5-HT aggravated sepsis-induced ALI by promoting NET formation in the lung of septic mice.

Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T5015, and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

Disease Outcome and Brain Metabolomics of Cyclophilin-D Knockout Mice in Sepsis

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction resulting from a systemic inflammatory response to infection, but the mechanism remains unclear. The mitochondrial permeability transition pore (MPTP) could play a central role in the neuronal dysfunction, induction of apoptosis, and cell death in SAE. The mitochondrial isomerase cyclophilin D (CypD) is known to control the sensitivity of MPTP induction. We, therefore, established a cecal ligation and puncture (CLP) model, which is the gold standard in sepsis research, using CypD knockout (CypD KO) mice, and analyzed the disease phenotype and the possible molecular mechanism of SAE through metabolomic analyses of brain tissue. A comparison of adult, male wild-type, and CypD KO mice demonstrated statistically significant differences in body temperature, mortality, and histological changes. In the metabolomic analysis, the main finding was the maintenance of reduced glutathione (GSH) levels and the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio in the KO animals following CLP. In conclusion, we demonstrate that CypD is implicated in the pathogenesis of SAE, possibly related to the inhibition of MPTP induction and, as a consequence, the decreased production of ROS and other free radicals, thereby protecting mitochondrial and cellular function.

Naive arthritogenic SKG T cells have a defect in anergy and a repertoire pruned by superantigen

How autoreactive CD4 T cells develop to cause rheumatoid arthritis remains unknown. We used a reporter for antigen-receptor signaling in the SKG autoimmune arthritis model to profile a T cell subpopulation enriched for arthritogenic naive CD4 T cells before arthritis onset by bulk and single cell RNA and T cell antigen-receptor (TCR) sequencing. Our analyses reveal that despite their impaired proximal TCR signaling, a subset of SKG naive CD4 T cells that have recently encountered endogenous antigen upregulate gene programs associated with positive regulation of T cell activation and cytokine signaling at higher levels than wild type cells in the pre-disease state. These arthritogenic cells also induce genes associated with negative regulation of T cell activation but do so less efficiently than wild type cells. Furthermore, their TCR sequences exhibit a previously unrecognized biased peripheral TCR Vβ repertoire likely driven by endogenous viral superantigens. These particular Vβs, known to recognize endogenous mouse mammary tumor virus (MMTV) superantigen, are further expanded in arthritic joints. Our results demonstrate that autoreactive naive CD4 T cells which recognize endogenous viral superantigens are poised to cause disease by their altered transcriptome.

Outer membrane protein A (OmpA) deficient Salmonella Typhimurium displays enhanced susceptibility towards β-lactam antibiotics: third-generation cephalosporins (ceftazidime) and carbapenems (meropenem)

The invasive non-typhoidal serovar of Salmonella enterica, namely Salmonella Typhimurium ST313, causes bloodstream infection in sub-Saharan Africa. Like other bacterial pathogens, the development of antimicrobial resistance is a severe problem in curing non-typhoidal Salmonella infection. In this work, we have investigated the role of four prominent outer membrane porins of S. Typhimurium, namely OmpA, OmpC, OmpD, and OmpF, in resistance against broad-spectrum β-lactam antibiotics- ceftazidime and meropenem. We found that deleting OmpA from Salmonella makes the bacteria susceptible to β-lactam drugs. The MIC for both the antibiotics reduced significantly for STM ΔompA compared to the wild-type and the ompA complemented strains. Despite the presence of antibiotics, the uninterrupted growth of STM ΔompC, ΔompD, and ΔompF endorsed the dispensability of these three porins in antimicrobial resistance. The β-lactam antibiotics caused massive depolarization in the outer membrane of the bacteria in the absence of OmpA. We have proved that none of the extracellular loops but the complete structure of perfectly folded OmpA is required by the bacteria for developing antimicrobial resistance. Our data revealed that STM ΔompA consumed more antibiotics than the wild-type and the complemented strain, resulting in severe damage of the bacterial outer membrane and subsequent killing of the pathogen by antibiotic-mediated oxidative stress. Upon deleting ompA, the steady decrease in the relative proportion of antibiotic-resistant persisters and the clearance of the STM ΔompA from the liver and spleen of C57BL/6 mice upon treatment with ceftazidime proved the role of OmpA in rendering protection against β-lactam antibiotics.

Real-time monitoring of Ralstonia solanacearum infection progress in tomato and Arabidopsis using bioluminescence imaging technology

Background Ralstonia solanacearum, one of the most devastating bacterial plant pathogens, is the causal agent of bacterial wilt. Recently, several studies on resistance to bacterial wilt have been conducted using the Arabidopsis-R. solanacearum system. However, the progress of R. solanacearum infection in Arabidopsis is still unclear. Results We generated a bioluminescent R. solanacearum by expressing plasmid-based luxCDABE. Expression of luxCDABE did not alter the bacterial growth and pathogenicity. The light intensity of bioluminescent R. solanacearum was linearly related to bacterial concentrations from 104 to 108 CFU·mL−1. After root inoculation with bioluminescent R. solanacearum strain, light signals in tomato and Arabidopsis were found to be transported from roots to stems via the vasculature. Quantification of light intensity from the bioluminescent strain accurately reported the difference in disease resistance between Arabidopsis wild type and resistant mutants. Conclusions Bioluminescent R. solanacearum strain spatially and quantitatively measured bacterial growth in tomato and Arabidopsis, and offered a tool for the high-throughput study of R. solanacearum-Arabidopsis interaction in the future.

Phenotypical Characterization of a Mutant dea1 With Developmental Defect in Female Inflorescence and Mutantion Impact on Yield in Maize

The establishment of female inflorescence morphology is of great significance to the formation of final maize yield. defective ear1 (dea1) is a novel maize mutant with developmental defect of female inflorescence caused by natural variation. Morphological analysis revealed that the mutant dea1 was characterized as a “scar-like” crack on the adaxial side of the top of the ear, accounting for 28.6-100.0% of the ear length, with an average of 32.4%. The results of scanning electron microscope showed that there was collapse in the formation of paired spikelet primordium at the base of the axillary meristem. Most of investigated botanical and agronomical traits of dea1 were lower than those of wild type, except for ear length and hundred grain weight. The grain yield per ear of mutant dea1 was 35.93% lower than that of wild type, and the width of mutation crack contributed the most to the yield loss per ear. The identification of the mutant dea1 and the characteristically phenotypic analysis provide a theoretical basis for the study of the molecular regulation mechanism of ear development and the application of high-yield breeding in maize.The establishment of female inflorescence morphology is of great significance to the formation of final maize yield. defective ear1 (dea1) is a novel maize mutant with developmental defect of female inflorescence caused by natural variation. Morphological analysis revealed that the mutant dea1 was characterized as a “scar-like” crack on the adaxial side of the top of the ear, accounting for 28.6-100.0% of the ear length, with an average of 32.4%. The results of scanning electron microscope showed that there was collapse in the formation of paired spikelet primordium at the base of the axillary meristem. Most of investigated botanical and agronomical traits of dea1 were lower than those of wild type, except for ear length and hundred grain weight. The grain yield per ear of mutant dea1 was 35.93% lower than that of wild type, and the width of mutation crack contributed the most to the yield loss per ear. The identification of the mutant dea1 and the characteristically phenotypic analysis provide a theoretical basis for the study of the molecular regulation mechanism of ear development and the application of high-yield breeding in maize.