Profiling Walnut Fungal Pathobiome Associated with Walnut Dieback Using Community-Targeted DNA Metabarcoding.

Walnut dieback can be caused by several fungal pathogenic species, which are associated with symptoms ranging from branch dieback to fruit necrosis and blight, challenging the one pathogen-one disease concept. Therefore, an accurate and extensive description of the walnut fungal pathobiome is crucial. To this end, DNA metabarcoding represents a powerful approach provided that bioinformatic pipelines are evaluated to avoid misinterpretation. In this context, this study aimed to determine (i) the performance of five primer pairs targeting the ITS region in amplifying genera of interest and estimating their relative abundance based on mock communities and (ii) the degree of taxonomic resolution using phylogenetic trees. Furthermore, our pipelines were also applied to DNA sequences from symptomatic walnut husks and twigs. Overall, our results showed that the ITS2 region was a better barcode than ITS1 and ITS, resulting in significantly higher sensitivity and/or similarity of composition values. The ITS3/ITS4_KYO1 primer set allowed to cover a wider range of fungal diversity, compared to the other primer sets also targeting the ITS2 region, namely, GTAA and GTAAm. Adding an extraction step to the ITS2 sequence influenced both positively and negatively the taxonomic resolution at the genus and species level, depending on the primer pair considered. Taken together, these results suggested that Kyo set without ITS2 extraction was the best pipeline to assess the broadest fungal diversity, with a more accurate taxonomic assignment, in walnut organs with dieback symptoms.

Differential carbohydrate-active enzymes and secondary metabolite production by the grapevine trunk pathogen Neofusicoccum parvum Bt-67 grown on host and non-host biomass.

Neofusicoccum parvum is one of the most aggressive Botryosphaeriaceae species associated with grapevine trunk diseases. This species may secrete enzymes capable of overcoming the plant barriers, leading to wood colonization. In addition to their roles in pathogenicity, there is an interest in taking advantage of N. parvum carbohydrate-active enzymes (CAZymes), related to plant cell wall degradation, for lignocellulose biorefining. Furthermore, N. parvum produces toxic secondary metabolites that may contribute to its virulence. In order to increase knowledge on the mechanisms underlying pathogenicity and virulence, as well as the exploration of its metabolism and CAZymes for lignocellulose biorefining, we evaluated the N. parvum strain Bt-67 capacity in producing lignocellulolytic enzymes and secondary metabolites when grown in vitro with two lignocellulosic biomasses: grapevine canes (GP) and wheat straw (WS). For this purpose, a multiphasic study combining enzymology, transcriptomic, and metabolomic analyses was performed. Enzyme assays showed higher xylanase, xylosidase, arabinofuranosidase, and glucosidase activities when the fungus was grown with WS. Fourier transform infrared (FTIR) spectroscopy confirmed the lignocellulosic biomass degradation caused by the secreted enzymes. Transcriptomics indicated that the N. parvum Bt-67 gene expression profiles in the presence of both biomasses were similar. In total, 134 genes coding CAZymes were up-regulated, where 94 of them were expressed in both biomass growth conditions. Lytic polysaccharide monooxygenases (LPMOs), glucosidases, and endoglucanases were the most represented CAZymes and correlated with the enzymatic activities obtained. The secondary metabolite production, analyzed by high-performance liquid chromatography-ultraviolet/visible spectophotometry-mass spectrometry (HPLC-UV/Vis-MS), was variable depending on the carbon source. The diversity of differentially produced metabolites was higher when N. parvum Bt-67 was grown with GP. Overall, these results provide insight into the influence of lignocellulosic biomass on virulence factor expressions. Moreover, this study opens the possibility of optimizing the enzyme production from N. parvum with potential use for lignocellulose biorefining.

Botryosphaeriaceae gene machinery: Correlation between diversity and virulence.

The Botryosphaeriaceae family comprises numerous fungal pathogens capable of causing economically meaningful diseases in a wide range of crops. Many of its members can live as endophytes and turn into aggressive pathogens following the onset of environmental stress events. Their ability to cause disease may rely on the production of a broad set of effectors, such as cell wall-degrading enzymes, secondary metabolites, and peptidases. Here, we conducted comparative analyses of 41 genomes representing six Botryosphaeriaceae genera to provide insights into the genetic features linked to pathogenicity and virulence. We show that these Botryosphaeriaceae genomes possess a large diversity of carbohydrate-active enzymes (CAZymes; 128 families) and peptidases (45 families). Botryosphaeria, Neofusicoccum, and Lasiodiplodia presented the highest number of genes encoding CAZymes involved in the degradation of the plant cell wall components. The genus Botryosphaeria also exhibited the highest abundance of secreted CAZymes and peptidases. Generally, the secondary metabolites gene cluster profile was consistent in the Botryosphaeriaceae family, except for Diplodia and Neoscytalidium. At the strain level, Neofusicoccum parvum NpBt67 stood out among all the Botryosphaeriaceae genomes, presenting a higher number of secretome constituents. In contrast, the Diplodia strains showed the lowest richness of the pathogenicity- and virulence-related genes, which may correlate with their low virulence reported in previous studies. Overall, these results contribute to a better understanding of the mechanisms underlying pathogenicity and virulence in remarkable Botryosphaeriaceae species. Our results also support that Botryosphaeriaceae species could be used as an interesting biotechnological tool for lignocellulose fractionation and bioeconomy.

Pathogenicity Factors of Botryosphaeriaceae Associated with Grapevine Trunk Diseases: New Developments on Their Action on Grapevine Defense Responses.

Botryosphaeriaceae are a family of fungi associated with the decay of a large number of woody plants with economic importance and causing particularly great losses in viticulture due to grapevine trunk diseases. In recent years, major advances in the knowledge of the pathogenicity factors of these pathogens have been made possible by the development of next-generation sequencing. This review highlights the knowledge gained on genes encoding small secreted proteins such as effectors, carbohydrate-associated enzymes, transporters and genes associated with secondary metabolism, their representativeness within the Botryosphaeriaceae family and their expression during grapevine infection. These pathogenicity factors are particularly expressed during host-pathogen interactions, facilitating fungal development and nutrition, wood colonization, as well as manipulating defense pathways and inducing impacts at the cellular level and phytotoxicity. This work highlights the need for further research to continue the effort to elucidate the pathogenicity mechanisms of this family of fungi infecting grapevine in order to improve the development of control methods and varietal resistance and to reduce the development and the effects of the disease on grapevine harvest quality and yield.

Long-term outcomes in a series of idiopathic retinal vasculitis, aneurysms, and neuroretinitis (IRVAN) syndrome.

OBJECTIVE: Idiopathic retinal vasculitis, aneurysms, and neuroretinitis (IRVAN) syndrome is a rare entity with a potentially poor visual prognosis. Our objective is to review the clinical presentation and long-term outcomes of patients with IRVAN syndrome. DESIGN: This is a retrospective case series. METHODS: We reviewed the charts of all the patients diagnosed with IRVAN syndrome at our tertiary care centre from 2002 to 2015. RESULTS: We included the long-term clinical outcomes of 7 eyes (5 patients) diagnosed with IRVAN syndrome. After a mean follow-up of 84.9 months, best-corrected visual acuity was 20/40 or better in the majority of eyes (70%). Four (57.1%) patients had systemic conditions, namely, multiple sclerosis, ischemic stroke, and positive antiphospholipid titres. All eyes were treated with laser photocoagulation. Four (40%) eyes received adjunctive intravitreal bevacizumab injections. CONCLUSION: IRVAN is an important diagnosis for clinicians to recognize. When treated in a timely manner, long-term visual outcomes can be favourable.

ENDOPHTHALMITIS IN A PATIENT TREATED WITH BACILLUS CALMETTE-GUÉRIN IMMUNOTHERAPY.

BACKGROUND: Intravesical Bacillus Calmette-Guérin (BCG) instillation has become one of the mainstays of adjunctive therapy in the treatment of superficial bladder cancer. Ophthalmologic complications are rare, but few cases are reported in the literature. METHODS: Retrospective observational case report. RESULTS: The authors report a case of unilateral Mycobacterium bovis BCG endophthalmitis after intravesical BCG instillations. Despite appropriate systemic antituberculous and corticosteroid therapy, the patient almost completely lost sight in the affected eye. This is the fourth case in the literature of proven M. bovis endophthalmitis suggesting a direct choroidal mycobacterial infection and not only a hypersensitivity immunologic reaction as previously suggested. CONCLUSION: This case highlights the direct choroidal mycobacterial infection of the disease after BCG instillations for bladder cancer and failure of treatment despite culture-proven drug sensitivity, thus suggesting the need to revaluate adequate treatment to avoid loss of vision.

Choroidal extranodal marginal zone lymphoma diagnosed by full-thickness retinochoroidal biopsy: case report and review of the literature.

The case of an 89-year-old man who was referred for a painless decrease of vision in his right eye (RE) is reported. Fundus examination of the RE showed an elevated amelanotic lesion located in the posterior pole with an adjacent focal round pigmented lesion. There was also a more peripheral amelanotic lesion extending from 6 to 9 o'clock clockwise inferotemporally. Uveitis workup and imaging studies of brain and orbits were normal. A retinochoroidal biopsy was done and showed the presence of choroidal extranodal marginal zone lymphoma. The patient was treated with external beam radiotherapy. This report presents a review of the literature of all reported cases of choroidal extranodal marginal zone lymphoma.

Transgenic mice expressing nitroreductase gene under the control of the podocin promoter: a new murine model of inductible glomerular injury.

The present work identifies a new mouse model of inductible acute glomerular injury leading to focal segmental glomerulonephritis. We take advantage of the suicide gene/prodrug nitroreductase/CB1954 combination, in which nitroreductase converts CB1954, a monofunctional alkylating agent, into its toxic form. We generate two lines of transgenic mice in which the nitroreductase gene was placed under the control of the podocyte-specific gene podocin. The functional analysis of transgenic mice lines showed that CB1954 treatment induced a severe but transitory proteinuria. Sequential histopathological analysis was performed on serial kidney biopsies. Injured glomeruli showed acute lesions with early podocyte vacuolization and detachment, podocyte apoptosis, and cellular proliferation leading to a marked hypercellularity of the urinary space that was associated with collapsing of the glomerular tuft. After 1 month, progressive scarring lead to focal segmental glomerulosclerosis with fibrous capsular adhesion, hyalinosis, and podocytosis associated with interstitial fibrosis. The phenotype of podocytes was changed exhibiting dedifferentiation characterized by the loss of podocyte specific proteins/transcription factor and the expression of injury markers. Bowman's capsule cells were also involved in the cellular changes in a manner suggesting epithelial to mesenchymal transition. This model of podocyte injury in transgenic mice provides new insights into the cellular mechanisms of podocytopathies and their progression to scarring.

Incidence of cystoid macular edema after cataract surgery in patients with and without uveitis using optical coherence tomography.

PURPOSE: To determine the incidence of cystoid macular edema (CME) after cataract surgery among eyes with and without uveitis using optical coherence tomography (OCT) and to determine risk factors for postoperative CME among eyes with uveitis. DESIGN: Prospective, comparative cohort study. METHODS: Single-center, academic practice. Forty-one eyes with uveitis and 52 eyes without uveitis underwent clinical examination and OCT testing within 4 weeks before cataract surgery and at 1-month and 3-month postoperative visits. The main outcome measure was incidence of CME at 1 and 3 months after surgery. RESULTS: Both uveitic and control eyes gained approximately 3 lines of vision (P = .6). Incidence of CME at 1 month was 12% (5 eyes) for uveitis and 4% (2 eyes) for controls (P = .2). Incidence of CME at 3 months was 8% (3 eyes) for uveitis and 0% for eyes without uveitis (P = .08). Eyes with uveitis treated with perioperative oral corticosteroids had a 7-fold reduction in postoperative CME (relative risk [RR], 0.14; P = .05). In uveitic eyes, active inflammation within 3 months before surgery increased the risk of CME when compared with eyes without inflammation (RR, 6.19; P = .04). CME was significantly associated with poorer vision (P = .01). CONCLUSIONS: Eyes with well-controlled uveitis may obtain similar outcomes to control eyes after cataract surgery (up to 3 months). Use of perioperative oral corticosteroids and control of uveitis for more than 3 months before surgery seemed to decrease the risk of postoperative CME among uveitic eyes in this study.

Rapid cooling preserves the ischaemic myocardium against mitochondrial damage and left ventricular dysfunction.

AIMS: We investigated whether rapid cooling instituted by total liquid ventilation (TLV) improves cardiac and mitochondrial function in rabbits submitted to ischaemia-reperfusion. METHODS AND RESULTS: Rabbits were chronically instrumented with a coronary artery occluder and myocardial ultrasonic crystals for assessment of segment length-shortening. Two weeks later they were re-anaesthetized and underwent either a normothermic 30-min coronary artery occlusion (CAO) (Control group, n = 7) or a comparable CAO with cooling initiated by a 10-min hypothermic TLV and maintained by a cold blanket placed on the skin. Cooling was initiated after 5 or 15 min of CAO (Hypo-TLV and Hypo-TLV(15') groups, n = 6 and 5, respectively). A last group underwent normothermic TLV during CAO (Normo-TLV group, n = 6). Wall motion was measured in the conscious state over three days of reperfusion before infarct size evaluation and histology. Additional experiments were done for myocardial sampling in anaesthetized rabbits for mitochondrial studies. The Hypo-TLV procedure induced a rapid decrease in myocardial temperature to 32-34 degrees C. Throughout reperfusion, segment length-shortening was significantly increased in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (15.1 +/- 3.3%, 16.4 +/- 2.3%, 1.8 +/- 0.6%, and 1.1 +/- 0.8% at 72 h, respectively). Infarct sizes were also considerably attenuated in Hypo-TLV and Hypo-TLV(15') vs. Control and Normo-TLV (4 +/- 1%, 11 +/- 5%, 39 +/- 2%, and 42 +/- 5% infarction of risk zones, respectively). Mitochondrial function in myocardial samples obtained at the end of ischaemia or after 10 min of reperfusion was improved by Hypo-TLV with respect to ADP-stimulated respiration and calcium-induced opening of mitochondrial permeability transition pores (mPTP). Calcium concentration opening mPTP was, e.g., increased at the end of ischaemia in the risk zone in Hypo-TLV vs. Control (157 +/- 12 vs. 86 +/- 12 microM). Histology and electron microscopy also revealed better preservation of lungs and of cardiomyocyte ultrastructure in Hypo-TLV when compared with Control. CONCLUSION: Institution of rapid cooling by TLV during ischaemia reduces infarct size as well as other sequelae of ischaemia, such as post-ischaemic contractile and mitochondrial dysfunction.

Sirolimus interacts with pathways essential for podocyte integrity.

BACKGROUND: The specific mTor inhibitor sirolimus has been implicated in the pathogenesis of renal glomerular lesions and nephrotic syndrome appearance after transplantation. Podocyte injury and focal segmental glomerulosclerosis have been related to sirolimus therapy in some patients but the pathways underlying these lesions remain hypothetical. METHODS: To go further in the comprehension of these mechanisms, primary cultures of human podocytes were exposed to therapeutic-range concentrations of sirolimus. RESULTS: Cell viability was not affected after 2 days' exposure to the drug but changes in cell phenotype and cytoskeleton reorganization were observed. We also evidenced that vascular endothelial growth factor (VEGF) synthesis and Akt phosphorylation were decreased by sirolimus addition. We did not observe any loss of podocyte differentiation markers with the notable exception of WT1, a transcription factor essential for maintaining podocyte integrity. WT1 gene and protein expression in podocytes were decreased in a dose-dependent manner after incubation with sirolimus. CONCLUSION: Taken together, these data suggest that sirolimus could impair pathways essential for podocyte integrity and therefore predisposes to glomerular injury.

GDF15 triggers homeostatic proliferation of acid-secreting collecting duct cells.

Although adult kidney cells are quiescent, enlargement of specific populations of epithelial cells occurs during repair and adaptive processes. A prerequisite to the development of regenerative therapeutics is to identify the mechanisms and factors that control the size of specific populations of renal cells. Unfortunately, in most cases, it is unknown whether the growth of cell populations results from transdifferentiation or proliferation and whether proliferating cells derive from epithelial cells or from circulating or resident progenitors. In this study, the mechanisms underlying the enlargement of the acid-secreting cell population in the mouse kidney collecting duct in response to metabolic acidosis was investigated. Acidosis led to two phases of proliferation that preferentially affected the acid-secreting cells of the outer medullary collecting duct. All proliferating cells displayed polarized expression of functional markers. The first phase of proliferation, which started within 24 h and peaked at day 3, was dependent on the overexpression of growth differentiation factor 15 (GDF15) and cyclin D1 and was abolished when phosphatidylinositol-3 kinase and mammalian target of rapamycin were inhibited. During this phase, cells mostly divided along the tubular axis, contributing to tubule lengthening. The second phase of proliferation was independent of GDF15 but was associated with induction of cyclin D3. During this phase, cells divided transversely. In summary, acid-secreting cells proliferate as the collecting duct adapts to metabolic acidosis, and GDF15 seems to be an important determinant of collecting duct lengthening.

A method of reporting macular edema after cataract surgery using optical coherence tomography.

OBJECTIVE: To validate a method of reporting postcataract macular edema (ME) using optical coherence tomography (OCT). METHODS: : Data were analyzed for 130 eyes followed prospectively for ME after uncomplicated cataract surgery. Each eye underwent OCT within 4 weeks before surgery and at 1 month and 3 months after surgery. ME was defined by observation of cystoid changes by OCT. RESULTS: Incidence of ME was 14% (95% confidence interval, 8-20). Average increase in baseline center point thickness (CPT) +/- SD at 1 month for eyes with and without ME was 202 +/- 113 microm and 8 +/- 19 microm, respectively (P < 0.001), which resulted in a 1-letter loss (-0.02 logMAR [logarithm of the minimum angle of resolution]) and a 3-line gain (0.29 logMAR) in vision, respectively (P < 0.001). Percent change in baseline CPT +/- SD for eyes with and without ME was 115 +/- 67% and 6 +/- 11%, respectively (P < 0.001). A > or =40% increase in baseline CPT accurately determined 100% of eyes with ME and 99% of eyes without ME. CONCLUSIONS: A > or =40% increase in baseline CPT, determined by OCT, offers a valid and objective method of reporting clinically relevant postcataract ME. Standardized reporting of postcataract ME would allow objective assessment and comparison of treatment outcomes among clinical studies.

Safety of intravitreal ketorolac and diclofenac: an electroretinographic and histopathologic study.

OBJECTIVE: To determine the clinical, histologic, and electroretinographic effects in the rabbit retina of escalating doses of two intravitreally delivered nonsteroidal anti-inflammatory drugs (NSAIDs): ketorolac and diclofenac. METHODS: Right eyes received a single 0.1 mL injection of either ketorolac (500-6000 microg/0.1 mL) or diclofenac (300-1500 microg/0.1 mL) prepared in balanced salt solution (BSS). Left eyes served as controls and received BSS. Dark- and light-adapted electroretinograms (ERG) were obtained at baseline and 4 and 8 weeks postinjection. Enucleated eyes were examined histologically. RESULTS: Ophthalmic examinations demonstrated no signs of intraocular inflammation or retinal toxicity. Intraocular pressure measurements remained similar between NSAID injected and control eyes. Histologic and ERG studies of eyes injected with 6000 microg ketorolac and >or=500 microg diclofenac demonstrated toxicity. In contrast, doses up to 3000 microg ketorolac demonstrated enhanced b-wave amplitude responses. Delayed drug toxicity was observed for the highest doses of both NSAIDs. CONCLUSIONS: Intravitreal 3000 microg ketorolac and 300 microg diclofenac were nontoxic in this animal study, and may offer an effective and safer alternative to intravitreal corticosteroids.

Long-term preservation of vision in progressive outer retinal necrosis treated with combination antiviral drugs and highly active antiretroviral therapy.

PURPOSE: To report the successful long-term treatment of varicella zoster virus-associated progressive outer retinal necrosis (VZV-PORN) with aggressive antiviral combination drugs along with highly active antiretroviral therapy (HAART). DESIGN: Interventional case report. METHODS: Combined treatment of progressive outer retinal necrosis in a university-based tertiary eye hospital with ganciclovir implant, intravenous acyclovir (10 mg/kg every 8 h), intravitreal foscarnet (2.4 mg), and HAART. RESULTS: Successful treatment of progressive outer retinal necrosis with disease remission and preservation of 20/20 visual acuity out to 1 year. CONCLUSIONS: Combination antiviral therapy and HAART may improve long-term visual outcomes for VZV-PORN.

Expression of matrix metalloproteinases MMP-2 and MMP-9 is altered during nephrogenesis in fetuses from diabetic rats.

Remodeling of extracellular matrix (ECM) is an important physiological feature of normal growth and development. Recent studies have emphasized the role of matrix metalloproteinases (MMP-2 and MMP-9) in normal mouse nephrogenesis. We have demonstrated previously in the rat that in utero exposure to maternal diabetes impairs renal development leading to a 30% reduction in the nephron number. Transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) are known to mediate high glucose effects on matrix degradation. The aim of the present study was to address the expression of type IV collagenase and TGF-beta1/CTGF systems in rat kidney during normal development and after in utero exposure to maternal diabetes. Both MMP-2 and MMP-9 mRNA metanephric expressions and activities were dramatically downregulated in kidneys issued from diabetic fetuses and in metanephros cultured in the presence of high glucose concentration. TGF-beta1 and CTGF expressions were significantly enhanced in diabetic fetal kidneys and in high glucose cultured metanephroi. Conditioned media obtained from metanephroi grown with high glucose concentration upregulated functional TGF-beta activity in transfected ATDC5 cells. In conclusion, in impaired nephrogenesis resulting from in utero exposure to maternal diabetes, alteration of both type IV collagenase and TGF-beta1/CTGF systems may lead to abnormal remodeling of ECM, which may, in turn, induce defects in ureteral bud branching leading to the observed reduction in the nephron number with consequences later in life: progression of chronic renal disease and hypertension.

Alteration of cytochrome oxidase subunit I labeling is associated with severe mitochondriopathy in NRTI-related hepatotoxicity in HIV patients.

Liver mitochondrial toxicity induced by nucleoside reverse transcriptase inhibitors (NRTI) in human immunodeficiency virus (HIV) patients has been associated with a wide range of liver involvement ranging from low-grade hepatotoxicity, asymptomatic lactacidemia to severe liver insufficiency, with massive steatosis and life-threatening lactic acidosis. Considerable efforts have been made in the last few years to establish clinical guidelines to avoid life-threatening NRTI-associated lactic acidosis. However, the important issue of low-grade NRTI-associated hepatotoxicity still needs to be unravelled since its natural history is largely unknown. We have recently reported a series of 13 monoinfected HIV patients with low-grade NRTI-associated toxicity. Our results outlined the heterogeneity of NRTI-induced hepatotoxicity and raised the question of its diagnosis. The present study evaluates the expression of cytochrome oxidase (COX) subunits I and IV, encoded by mitochondrial and nuclear DNA, respectively, in NRTI hepatotoxicity. The aim of our study was to compare the detection rate of mitochondrial abnormalities of immunohistochemistry for COX subunit I with electron microscopy. COX subunit I and IV labeling was performed together with light microscopy and ultrastructural analysis in a series of 55 liver biopsies from HIV monoinfected and HIV-hepatitis C virus coinfected patients. Clinical data were also recorded. Our major findings were: (i) decreased COX subunit I labeling is associated with severe ultrastructural mitochondrial alterations and may represent overt NRTI-induced mitochondrial cytopathy; (ii) mild ultrastructural damage associated with normal COX subunit I labeling is of unknown clinical significance. The results of the study suggest that COX subunit I labeling may be a valuable tool for the diagnosis of mitochondrial liver disease in HIV patients.