Chitosan and nematophagous fungi for sustainable management of nematode pests

Plants are exposed to large number of threats caused by herbivores and pathogens which cause important losses on crops. Plant pathogens such as nematodes can cause severe damage and losses in food security crops worldwide. Chemical pesticides were extendedly used for nematode management. However, due to their adverse effects on human health and the environment, they are now facing strong limitations by regulatory organisations such as EFSA (European Food Safety Authority). Therefore, there is an urgent need for alternative and efficient control measures, such as biological control agents or bio-based plant protection compounds. In this scenario, chitosan, a non-toxic polymer obtained from seafood waste mainly, is becoming increasingly important. Chitosan is the N-deacetylated form of chitin. Chitosan is effective in the control of plant pests and diseases. It also induces plants defence mechanisms. Chitosan is also compatible with some biocontrol microorganisms mainly entomopathogenic and nematophagous fungi. Some of them are antagonists of nematode pests of plants and animals. The nematophagous biocontrol fungus Pochonia chlamydosporia has been widely studied for sustainable management of nematodes affecting economically important crops and for its capability to grow with chitosan as only nutrient source. This fungus infects nematode eggs using hyphal tips and appressoria. Pochonia chlamydosporia also colonizes plant roots endophytically, stimulating plant defences by induction of salicylic and jasmonic acid biosynthesis and favours plant growth and development. Therefore, the combined use of chitosan and nematophagous fungi could be a novel strategy for the biological control of nematodes and other root pathogens of food security crops.This research was funded by PID2020-119734RB-I00 Project from the Spanish Ministry of Science and Innovation and by European Project H2020 MUSA no. 727624

Putative LysM Effectors Contribute to Fungal Lifestyle

Fungal LysM effector proteins can dampen plant host–defence responses, protecting hyphae from plant chitinases, but little is known on these effectors from nonpathogenic fungal endophytes. We found four putative LysM effectors in the genome of the endophytic nematophagous fungus Pochonia chlamydosporia (Pc123). All four genes encoding putative LysM effectors are expressed constitutively by the fungus. Additionally, the gene encoding Lys1—the smallest one—is the most expressed in banana roots colonised by the fungus. Pc123 Lys1, 2 and 4 display high homology with those of other strains of the fungus and phylogenetically close entomopathogenic fungi. However, Pc123 Lys3 displays low homology with other fungi, but some similarities are found in saprophytes. This suggests evolutionary divergence in Pc123 LysM effectors. Additionally, molecular docking shows that the NAcGl binding sites of Pc123 Lys 2, 3 and 4 are adjacent to an alpha helix. Putative LysM effectors from fungal endophytes, such as Pc123, differ from those of plant pathogenic fungi. LysM motifs from endophytic fungi show clear conservation of cysteines in Positions 13, 51 and 63, unlike those of plant pathogens. LysM effectors could therefore be associated with the lifestyle of a fungus and give us a clue of how organisms could behave in different environments.This research was funded by European Project H2020 MUSA, Grant Number 727624

Molecular Mechanisms of Chitosan Interactions with Fungi and Plants

Chitosan is a versatile compound with multiple biotechnological applications. This polymer inhibits clinically important human fungal pathogens under the same carbon and nitrogen status as in blood. Chitosan permeabilises their high-fluidity plasma membrane and increases production of intracellular oxygen species (ROS). Conversely, chitosan is compatible with mammalian cell lines as well as with biocontrol fungi (BCF). BCF resistant to chitosan have low-fluidity membranes and high glucan/chitin ratios in their cell walls. Recent studies illustrate molecular and physiological basis of chitosan-root interactions. Chitosan induces auxin accumulation in Arabidopsis roots. This polymer causes overexpression of tryptophan-dependent auxin biosynthesis pathway. It also blocks auxin translocation in roots. Chitosan is a plant defense modulator. Endophytes and fungal pathogens evade plant immunity converting chitin into chitosan. LysM effectors shield chitin and protect fungal cell walls from plant chitinases. These enzymes together with fungal chitin deacetylases, chitosanases and effectors play determinant roles during fungal colonization of plants. This review describes chitosan mode of action (cell and gene targets) in fungi and plants. This knowledge will help to develop chitosan for agrobiotechnological and medical applications.This work was supported by European Project H2020 MUSA 727624

CPEB4 Increases Expression of PFKFB3 to Induce Glycolysis and Activate Mouse and Human Hepatic Stellate Cells, Promoting Liver Fibrosis

BACKGROUND & AIMS: We investigated mechanisms of hepatic stellate cell (HSC) activation, which contributes to liver fibrogenesis. We aimed to determine whether activated HSCs increase glycolysis, which is regulated by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and whether this pathway might serve as a therapeutic target. METHODS: We performed studies with primary mouse HSCs, human LX2 HSCs, human cirrhotic liver tissues, rats and mice with liver fibrosis (due to bile duct ligation [BDL] or administration of carbon tetrachlo- ride), and CPEB4-knockout mice. Glycolysis was inhibited in cells and mice by administration of a small molecule antagonist of PFKFB3 (3-[3-pyridinyl]-1-[4-pyridinyl]-2- propen-1-one [3PO]). Cells were transfected with small interfering RNAs that knock down PFKFB3 or CPEB4. RESULTS: Up-regulation of PFKFB3 protein and increased glycolysis were early and sustained events during HSC activation and accompanied by increased expression of markers of fibrogenesis; incubation of HSCs with 3PO or knockdown of PFKFB3 reduced their activation and prolif- eration. Mice with liver fibrosis after BDL had increased hepatic PFKFB3; injection of 3PO immediately after the surgery prevented HSC activation and reduced the severity of liver fibrosis compared with mice given vehicle. Levels of PFKFB3 protein were increased in fibrotic liver tissues from patients compared with non-fibrotic liver. Up-regulation of PFKFB3 in activated HSCs did not occur via increased transcription, but instead via binding of CPEB4 to cyto- plasmic polyadenylation elements within the 3'-untranslated regions of PFKFB3 messenger RNA. Knockdown of CPEB4 in LX2 HSCs prevented PFKFB3 overexpression and cell acti- vation. Livers from CPEB4-knockout had decreased PFKFB3 and fibrosis after BDL or administration of carbon tetra- chloride compared with wild-type mice. CONCLUSIONS: Fibrotic liver tissues from patients and rodents (mice and rats) have increased levels of PFKFB3 and glycolysis, which are essential for activation of HSCs. Increased expression of PFKFB3 is mediated by binding of CPEB4 to its untranslated messenger RNA. Inhibition or knockdown of CPEB4 or PFKFB3 prevents HSC activation and fibrogenesis in livers of mice

Chitosan Induces Plant Hormones and Defenses in Tomato Root Exudates

In this work, we use electrophysiological and metabolomic tools to determine the role of chitosan as plant defense elicitor in soil for preventing or manage root pests and diseases sustainably. Root exudates include a wide variety of molecules that plants and root microbiota use to communicate in the rhizosphere. Tomato plants were treated with chitosan. Root exudates from tomato plants were analyzed at 3, 10, 20, and 30 days after planting (dap). We found, using high performance liquid chromatography (HPLC) and excitation emission matrix (EEM) fluorescence, that chitosan induces plant hormones, lipid signaling and defense compounds in tomato root exudates, including phenolics. High doses of chitosan induce membrane depolarization and affect membrane integrity. 1H-NMR showed the dynamic of exudation, detecting the largest number of signals in 20 dap root exudates. Root exudates from plants irrigated with chitosan inhibit ca. twofold growth kinetics of the tomato root parasitic fungus Fusarium oxysporum f. sp. radicis-lycopersici. and reduced ca. 1.5-fold egg hatching of the root-knot nematode Meloidogyne javanica.This work was supported by AGL 2015 66833-R Grant from the Spanish Ministry of Economy and Competitiveness and H2020 MUSA 727624 European Project

Impact of intrapartum antimicrobial prophylaxis upon the intestinal microbiota and the prevalence of antibiotic resistance genes in vaginally delivered full-term neonates

Background: Disturbances in the early establishment of the intestinal microbiota may produce important implications for the infant's health and for the risk of disease later on. Different perinatal conditions may be affecting the development of the gut microbiota. Some of them, such as delivery mode or feeding habits, have been extensively assessed whereas others remain to be studied, being critical to identify their impact on the microbiota and, if any, to minimize it. Antibiotics are among the drugs most frequently used in early life, the use of intrapartum antimicrobial prophylaxis (IAP), present in over 30% of deliveries, being the most frequent source of exposure. However, our knowledge on the effects of IAP on the microbiota establishment is still limited. The aim of the present work was to evaluate the impact of IAP investigating a cohort of 40 full-term vaginally delivered infants born after an uncomplicated pregnancy, 18 of which were born from mothers receiving IAP. Results: Fecal samples were collected at 2, 10, 30, and 90 days of age. We analyzed the composition of the fecal microbiota during the first 3 months of life by 16S rRNA gene sequencing and quantified fecal short chain fatty acids by gas chromatography. The presence of genes for resistance to antibiotics was determined by PCR in the samples from 1-month-old infants. Our results showed an altered pattern of intestinal microbiota establishment in IAP infants during the first weeks of life, with lower relative proportions of Actinobacteria and Bacteroidetes and increased of Preoteobacteria and Firmicutes. A delay in the increase on the levels of acetate was observed in IAP infants. The analyses of specific antibiotic resistance genes showed a higher occurrence of some beta-lactamase coding genes in infants whose mothers received IAP. Conclusions: Our results indicate an effect of IAP on the establishing early microbiota during the first months of life, which represent a key moment for the development of the microbiota-induced host homeostasis. Understanding the impact of IAP in the gut microbiota development is essential for developing treatments to minimize it, favoring a proper gut microbiota development in IAP-exposed neonates

Epigenetic Modulation of Gremlin-1/NOTCH Pathway in Experimental Crescentic Immune-Mediated Glomerulonephritis

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treatment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provides limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory disorders and experimental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs could regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model resembling human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, restoring podocyte numbers. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by modulation of NOTCH pathway. JQ1 inhibited the gene expression of the NOTCH effectors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis

How much does HIV self-testing cost in low and middle income countries? A systematic review of evidence from economic studies

ObjectivesHIV self-testing (HIVST) has been proposed as an innovative strategy to diagnose human immunodeficiency virus (HIV). While HIVST offers the potential to broaden accessibility of early HIV diagnosis and treatment initiation, this testing strategy incurs additional cost and requires confirmatory testing and treatment. We have conducted the first systematic review to summarize the current economic literature for HIVST in low- and middle-income countries (LMICs).DesignA search strategy was developed including key terms for HIV, self-testing and cost-effectiveness and was conducted in Medline and Embase databases. Studies were included that reported costs per outcome and included both cost-effectiveness and cost-utility outcome measures. The search strategy identified publications up until August 15, 2023 were included. Abstract and full text screening was conducted and a standardized data abstraction form was used for included studies. Costs are reported in USD, 2020.ResultsOur search strategy identified 536 total titles from the search strategy, which were screened down to 25 relevant studies that provided both cost and outcome data on HIVST. There was significant heterogeneity in the HIVST intervention, study population, costs and outcomes reported among included studies. Cost per person tested ranged from $1.09–155. Cost per case diagnosed ranged from$20–1,277. Cost-utility estimates ranged from cost-saving to $1846 per DALY averted. Higher cost-effectiveness estimates were associated with more expensive testing algorithms with increased support for linkage to care and post-test counseling.ConclusionAll studies considered HIVST cost-effective although major drivers were identified included underlying HIV prevalence, testing cost and linkage to care. HIVST is likely to be cost-effective in a LMIC context, however policy makers should be aware of the drivers of cost-effectiveness when implementing HIVST programs as these underlying factors can impact the overall cost-effectiveness of HIVST

Lateral flow test engineering and lessons learned from COVID-19

The acceptability and feasibility of large-scale testing with lateral flow tests (LFTs) for clinical and public health purposes has been demonstrated during the COVID-19 pandemic. LFTs can detect analytes in a variety of samples, providing a rapid read-out, which allows self-testing and decentralized diagnosis. In this Review, we examine the changing LFT landscape with a focus on lessons learned from COVID-19. We discuss the implications of LFTs for decentralized testing of infectious diseases, including diseases of epidemic potential, the ‘silent pandemic’ of antimicrobial resistance, and other acute and chronic infections. Bioengineering approaches will play a key part in increasing the sensitivity and specificity of LFTs, improving sample preparation, incorporating nucleic acid amplification and detection, and enabling multiplexing, digital connection and green manufacturing, with the aim of creating the next generation of high-accuracy, easy-to-use, affordable and digitally connected LFTs. We conclude with recommendations, including the building of a global network of LFT research and development hubs to facilitate and strengthen future diagnostic resilience