Modes of action and potential as a peptide-based biofungicide of a plant defensin MtDef4.

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation-tolerant MtDef4 variant was generated that bound to ß-glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation-tolerant variant as a peptide-based fungicide.

Small Cationic Cysteine-Rich Defensin-Derived Antifungal Peptide Controls White Mold in Soybean.

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action possess potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active γ-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. GMA4CG_V6 exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro with an MIC value of 24 µM. At this concentration, internalization of this peptide into fungal cells occurred prior to discernible membrane permeabilization. GMA4CG_V6 markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Its spray application on soybean plants provided robust control of this disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has potential for development as a bio-fungicide for white mold control in soybean.

Evolution of antimicrobial cysteine-rich peptides in plants.

KEY MESSAGE: We analyzed the evolutionary pattern of cysteine-rich peptides (CRPs) to infer the relationship between CRP copy number and plant ecotype, and the origin of bi-domains CRPs. Plants produce cysteine-rich peptides (CRPs) that have long-lasting broad-spectrum antimicrobial activity to protect themselves from various groups of pathogens. We analyzed 240 plant genomes, ranging from algae to eudicots, and discovered that CRPs are widely distributed in plants. Our comparative genomics results revealed that CRP genes have been amplified through both whole genome and local tandem duplication. The copy number of these genes varied significantly across lineages and was associated with the plant ecotype. This may be due to their resistance to changing pathogenic environments. The conserved and lineage-specific CRP families contribute to diverse antimicrobial activities. Furthermore, we investigated the unique bi-domain CRPs that result from unequal crossover events. Our findings provide a unique evolutionary perspective on CRPs and insights into their antimicrobial and symbiosis characteristics.

Medicago Sativa Defensin1 as a tumor sensitizer for improving chemotherapy: translation from anti-fungal agent to a potential anti-cancer agent.

Plant defensins including Medicago Sativa defensin 1 (MsDef1) are cysteine-rich antifungal peptides which are known for potent broad-spectrum antifungal activity against bacterial or fungal pathogens of plants. The antimicrobial activities of these cationic defensins are attributed to their capacity to bind to cell membranes to create potentially structural defects tin the cell membranes to interact with intracellular target (s) and mediates cytotoxic effects. Our earlier work identified Glucosylceramide (GlcCer) of fungus F. graminearum as a potential target for biological activity. Multi-drug resistant (MDR) cancer cells overexpress GlcCer on the surface of plasma membrane. Hence, MsDef1 may have a potential to bind to GlcCer of MDR cancer cells to induce cell death. We have characterized the three-dimensional structure of MsDef1 and the solution dynamics using of 15N-labeled MsDef1 nuclear magnetic resonance (NMR) spectroscopy which showed that GlcCer binds MsDef1 at two specific sites on the peptide molecule. The ability of MsDef1 to permeate MDR cancer cells was demonstrated by measuring the release of apoptotic ceramide in drug resistant MCF-7R cells. It was also shown that MsDef1 activated dual cell death pathways ceramide and Apoptosis Stimulating Kinase ASK1 by disintegrating GlcCer and oxidizing tumor specific biomarker thioredoxin (Trx) respectively. As a result, MsDef1 sensitizes MDR cancer cells to evoke a better response from Doxorubicin, a front-line chemotherapy for triple negative breast cancer (TNBC) treatment. The combination of MsDef1 and Doxorubicin induced 5 to10-fold greater apoptosis in vitro MDR cells MDA-MB-231R compared to either MsDef1 or Doxorubicin alone. Confocal microscopy revealed that MsDef1 facilitates a) influx of Doxorubicin in MDR cancer cells, b) preferential uptake by MDR cells but not by normal fibroblasts and breast epithelial cells (MCF-10A). These results suggest that MsDef1 targets MDR cancer cells and may find utility as a neoadjuvant chemotherapy. Hence, the extension of antifungal properties of MsDef1 to cancer my result in addressing the MDR problems in cancer.

Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bio-inspired fungicide.

Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino-acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed that it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventive and curative suppression of grey mould disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development of bio-inspired fungicides.

Osteoporosis in Indian Patients Undergoing Elective Arthroplasty and Spinal Procedures: An Observational Study.

Background This is an observational study conducted to determine the prevalence of osteoporosis and osteopenia in patients undergoing elective arthroplasty and spinal procedures in India. Methods This observational, multicentre study included both male and female patients. Their bone mineral density and fracture risk were measured using dual-energy x-ray absorptiometry (DEXA) and Fracture Risk Assessment Tool (FRAX®: Centre for Metabolic Bone Diseases, University of Sheffield, UK), respectively, in compliance with the guidelines for Good Epidemiological Practice (ISRCTN: 14543098). Results The study revealed that majority (76.4%; 97/127) of the patients had low BMD; over one-third had osteoporosis (39.4%; 50/127) or osteopenia (37%; 47/127). Among those undergoing total knee replacement (TKR)/total hip replacement (THR), majority (75.6%; 59/78) had low BMD (osteoporosis: 38.5% {30/78}; osteopenia: 37.2% {29/78}). Among the patients undergoing spinal procedures, all except two (93.10%; 27/29) had low BMD, two-thirds had osteoporosis (65.5%; 19/29), and around one-fourth had osteopenia (27.6%; 8/29). Radial BMD measurements showed higher prevalence of osteoporosis and osteopenia. Based on FRAX score, nearly 30% of patients were at a high risk of hip fracture in the next 10 years. As per National Osteoporosis Foundation (NOF) guidelines, most (59.79%; 58/97) patients with osteoporosis/osteopenia met criteria for pharmacological treatment. Conclusions Regular preoperative bone health evaluation should be adopted and osteoporosis/osteopenia patients should be adequately managed pharmacologically in India.

Redox-dependent structure and dynamics of macrophage migration inhibitory factor reveal sites of latent allostery.

Macrophage migration inhibitory factor (MIF) is a multifunctional immunoregulatory protein that is a key player in the innate immune response. Given its overexpression at sites of inflammation and in diseases marked by increasingly oxidative environments, a comprehensive understanding of how cellular redox conditions impact the structure and function of MIF is necessary. We used NMR spectroscopy and mass spectrometry to investigate biophysical signatures of MIF under varied solution redox conditions. Our results indicate that the MIF structure is modified and becomes increasingly dynamic in an oxidative environment, which may be a means to alter the MIF conformation and functional response in a redox-dependent manner. We identified latent allosteric sites within MIF through mutational analysis of redox-sensitive residues, revealing that a loss of redox-responsive residues attenuates CD74 receptor activation. Leveraging sites of redox sensitivity as targets for structure-based drug design therefore reveals an avenue to modulate MIF function in its "disease state."

A Cysteine Variant at an Allosteric Site Alters MIF Dynamics and Biological Function in Homo- and Heterotrimeric Assemblies.

Macrophage migration inhibitory factor (MIF) is an inflammatory protein with various non-overlapping functions. It is not only conserved in mammals, but it is found in parasites, fish, and plants. Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro1 as a catalytic base, activates the receptors CD74, CXCR2, and CXCR4, has functional interactions in the cytosol, and is reported to be a nuclease. There is a solvent channel down its 3-fold axis with a recently identified gating residue as an allosteric site important for regulating, to different extents, the enzymatic activity and CD74 binding and signaling. In this study we explore the consequence of converting the allosteric residue Tyr99 to cysteine (Y99C) and characterize its crystallographic structure, NMR dynamics, stability, CD74 function, and enzymatic activity. In addition to the homotrimeric variant, we develop strategies for expressing and purifying a heterotrimeric variant consisting of mixed wild type and Y99C for characterization of the allosteric site to provide more insight.

Adiponectin ameliorates hyperoxia-induced lung endothelial dysfunction and promotes angiogenesis in neonatal mice.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a common respiratory disease of preterm infants. Lower circulatory/intrapulmonary levels of the adipokine, adiponectin (APN), occur in premature and small-for-gestational-age infants and at saccular/alveolar stages of lung development in the newborn rat. However, the role of low intrapulmonary APN during hyperoxia exposure in developing lungs is unknown. METHODS: We test the hypothesis that treatment of hyperoxia-exposed newborn mice with recombinant APN protein attenuates the BPD phenotype characterized by inflammation, impaired alveolarization, and dysregulated vascularization. We used developmentally appropriate in vitro and in vivo BPD modeling systems as well as human lung tissue. RESULTS: We observed reduced levels of intrapulmonary APN in experimental BPD mice and human BPD lungs. APN-deficient (APN-/-) newborn mice exposed to moderate (60% O2) hyperoxia showed a worse BPD pulmonary phenotype (inflammation, enhanced endothelial dysfunction, impaired pulmonary vasculature, and alveolar simplification) as compared to wild-type (WT) mice. Treatment of hyperoxia-exposed newborn WT mice with recombinant APN protein attenuated the BPD phenotype (diminished inflammation, decreased pulmonary vascular injury, and improved pulmonary alveolarization) and improved pulmonary function tests. CONCLUSIONS: Low intrapulmonary APN is associated with disruption of lung development during hyperoxia exposure, while recombinant APN protein attenuates the BPD pulmonary phenotype. IMPACT: Intrapulmonary APN levels were significantly decreased in lungs of experimental BPD mice and human BPD lung tissue at various stages of BPD development. Correlative data from human lung samples with decreased APN levels were associated with increased lung adhesion markers (intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin). Decreased APN levels were associated with endothelial dysfunction and moderate BPD phenotype in APN-deficient, as compared to WT, experimental BPD mice. WT experimental BPD mice treated with recombinant APN protein had an improved pulmonary structural and functional phenotype. Exogenous APN may be considered as a potential therapeutic agent to prevent BPD.

A structurally preserved allosteric site in the MIF superfamily affects enzymatic activity and CD74 activation in D-dopachrome tautomerase.

The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.

Small Immunomodulatory Molecules as Potential Therapeutics in Experimental Murine Models of Acute Lung Injury (ALI)/Acute Respiratory Distress Syndrome (ARDS).

BACKGROUND: Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS) is a severe inflammatory pulmonary process triggered by a variety of insults including sepsis, viral or bacterial pneumonia, and mechanical ventilator-induced trauma. Currently, there are no effective therapies available for ARDS. We have recently reported that a novel small molecule AVR-25 derived from chitin molecule (a long-chain polymer of N-acetylglucosamine) showed anti-inflammatory effects in the lungs. The goal of this study was to determine the efficacy of two chitin-derived compounds, AVR-25 and AVR-48, in multiple mouse models of ALI/ARDS. We further determined the safety and pharmacokinetic (PK) profile of the lead compound AVR-48 in rats. METHODS: ALI in mice was induced by intratracheal instillation of a single dose of lipopolysaccharide (LPS; 100 µg) for 24 h or exposed to hyperoxia (100% oxygen) for 48 h or undergoing cecal ligation and puncture (CLP) procedure and observation for 10 days. RESULTS: Both chitin derivatives, AVR-25 and AVR-48, showed decreased neutrophil recruitment and reduced inflammation in the lungs of ALI mice. Further, AVR-25 and AVR-48 mediated diminished lung inflammation was associated with reduced expression of lung adhesion molecules with improvement in pulmonary endothelial barrier function, pulmonary edema, and lung injury. Consistent with these results, CLP-induced sepsis mice treated with AVR-48 showed a significant increase in survival of the mice (80%) and improved lung histopathology in the treated CLP group. AVR-48, the lead chitin derivative compound, demonstrated a good safety profile. CONCLUSION: Both AVR-25 and AVR-48 demonstrate the potential to be developed as therapeutic agents to treat ALI/ARDS.

miR34a: a novel small molecule regulator with a big role in bronchopulmonary dysplasia.

Preterm infants with bronchopulmonary dysplasia (BPD), characterized by pulmonary inflammation leading to impaired alveolarization and vascular dysregulation, have an increased risk of abnormal lung function in infancy, childhood, and adulthood. These include a heightened risk of pulmonary hypertension, and respiratory illnesses. MicroRNAs (miRNAs) are known to disrupt normal lung development and function by interrupting alveolarization and vascularization resulting in the development of BPD. Among the various miRs involved in BPD, miR34a has been shown to have a significant role in BPD pathogenesis. Targeting miR34a or its downstream targets may be a promising therapeutic intervention for BPD. In this review, we summarize the data on cellular arrest, proliferation, differentiation, epithelial-mesenchymal transition, mitochondrial dysfunction, and apoptosis impacted by miR34a in the development of BPD pulmonary phenotypes while predicting the future perspective of miR34a in BPD.

α1,3-Fucosyltransferase-IX, an enzyme of pulmonary endogenous lung stem cell marker SSEA-1, alleviates experimental bronchopulmonary dysplasia.

BACKGROUND: Endogenous pulmonary stem cells (PSCs) play an important role in lung development and repair; however, little is known about their role in bronchopulmonary dysplasia (BPD). We hypothesize that an endogenous PSC marker stage-specific embryonic antigen-1 (SSEA-1) and its enzyme, α1,3-fucosyltransferase IX (FUT9) play an important role in decreasing inflammation and restoring lung structure in experimental BPD. METHODS: We studied the expression of SSEA-1, and its enzyme FUT9, in wild-type (WT) C57BL/6 mice, in room air and hyperoxia. Effects of intraperitoneal administration of recombinant human FUT9 (rhFUT9) on lung airway and parenchymal inflammation, alveolarization, and apoptosis were evaluated. RESULTS: On hyperoxia exposure, SSEA-1 significantly decreased at postnatal day 14 in hyperoxia-exposed BPD mice, accompanied by a decrease in FUT9. BPD and respiratory distress syndrome (RDS) in human lungs showed decreased expression of SSEA-1 as compared to their term controls. Importantly, intraperitoneal administration of FUT9 in the neonatal BPD mouse model resulted in significant decrease in pulmonary airway (but not lung parenchymal) inflammation, alveolar-capillary leakage, alveolar simplification, and cell death in the hyperoxia-exposed BPD mice. CONCLUSIONS: An important role of endogenous PSC marker SSEA-1 and its enzyme FUT9 is demonstrated, indicating early systemic intervention with FUT9 as a potential therapeutic option for BPD. IMPACT: Administration of rhFUT9, an enzyme of endogenous stem cell marker SSEA-1, reduces pulmonary airway (but not lung parenchymal) inflammation, alveolar-capillary leak and cell death in the BPD mouse model. SSEA-1 is reported for the first time in experimental BPD models, and in human RDS and BPD. rhFUT9 treatment ameliorates hyperoxia-induced lung injury in a developmentally appropriate BPD mouse model. Our results have translational potential as a therapeutic modality for BPD in the developing lung.

Novel biomarkers of bronchopulmonary dysplasia and bronchopulmonary dysplasia-associated pulmonary hypertension.

OBJECTIVE: To quantify and compare levels of potential biomarkers in neonates with (i) Bronchopulmonary dysplasia (BPD); (ii) BPD-associated pulmonary hypertension (BPD-PH); (iii) PH without BPD; and (iv) neonates without lung disease at ~36 weeks postmenstrual age. STUDY DESIGN: Multiple potential biomarkers were measured in plasma samples of 90 patients using a multi-spot enzyme-linked immunosorbent assay. Statistical tests done included one-way ANOVA to compare levels of biomarkers between different groups. RESULTS: Higher levels of ICAM-1 were present in infants with BPD and correlated with its severity. Infants with BPD have significantly higher levels of ANG-2 and lower levels of ANG-1. Infants with PH have higher levels of: IL-6, IL-8, IL-10, and TNF-α. Infants with BPD-PH have significantly lower levels of MCP-1 and higher levels of IL-1ß than infants with PH without BPD. CONCLUSION: ICAM-1 may be used as a specific biomarker for diagnosis of BPD and its severity.

Chitin Analog AVR-25 Prevents Experimental Bronchopulmonary Dysplasia.

Infants born extremely preterm are at a high risk of developing bronchopulmonary dysplasia (BPD) which is characterized by large, simplified alveoli, increased inflammation, disrupted and dysregulated vasculogenesis, decreased cell proliferation, and increased cell death in the lungs. Due to lack of specific drug treatments to combat this condition, BPD and its long-term complications have taken a significant toll of healthcare resources. AVR-25, a novel immune modulator experimental compound, was able to partially recover the pulmonary phenotype in the hyperoxia-induced experimental mouse model of BPD. We anticipate that AVR-25 will have therapeutic potential for managing human BPD.

Innovative recruitment and clinical orientation programme to manage NHS junior doctor shortfall: A district hospital experience.

BACKGROUND: International medical graduates (IMGs) contribute significantly towards the NHS care provision. No standardised clinical orientation programme (COP) for IMGs new to the NHS exists. OBJECTIVE: Our objective was to describe recruitment and retention strategies for junior doctors (JDs) in general medicine and develop a framework to anticipate outcomes of these interventions using the realist evaluation methodology. METHODS: We performed quality improvement interventions of recruitment and COP for new entrant IMGs in our organisation employed between December 2017 and April 2019. RESULTS: Twenty-three IMGs were recruited, 96% successfully completed the COP with a mean contract duration of 13±5 months. From the academic year 2017/18 to 2018/19, mean JD post occupancy increased from 54±3 to 73±4 JDs (p<0.001) and JD locum spend fell by £1.9 million. CONCLUSION: Our structured COP provides a stable, trained and financially sustainable JD workforce. Application in broader NHS settings is recommended.

Antifungal symbiotic peptide NCR044 exhibits unique structure and multifaceted mechanisms of action that confer plant protection.

In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel ß-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.

Correction to: Inhibition of microRNA-451 is associated with increased expression of Macrophage Migration Inhibitory Factor and mitigation of the cardio-pulmonary phenotype in a murine model of Bronchopulmonary Dysplasia.

An amendment to this paper has been published and can be accessed via the original article.