ABSTRACT
PURPOSE: To introduce a novel technique of the aseptic manufacture of autologous serum eye drops (ASEDs) with a prefiltered closed system and to analyze the sterility of the produced ophtioles between 2018 and 2022. METHODS: This is a prospective single-center study conducted at the Department of Ophthalmology at a Swiss University Hospital between 2018 and 2022. For regulatory reasons, closed systems for manufacturing ASEDs are strongly recommended. We attached an upstream sterile filter (Sterivex PES0.22 µm Burlington, USA) to a commercially available closed system (COL System Modena, Italy) for manufacturing ASEDs. The goal of this novel approach was to reduce the microbiological contamination of the donated autologous blood. Using the presented manufacturing method, we are able to produce, on average, 56 ophtioles per batch, containing either 1.45 mL or 2.5 mL of autologous serum per ophtiole. For each batch of ASEDs, we performed a microbiological analysis by automated blood culture testing (BACTEC). This system examines the presence of bacteria and fungi. RESULTS: We analyzed all manufactured batches between 2018 and 2022. None of the 2297 batches and the resulting 129â060 ophtioles showed bacterial or mycotic contamination. During the analyzed period, two batches were discarded: one due to fibrin-lipid aggregations, further microbiological and histological work-up excluded any contamination; another due to false-positive HIV in serological testing. Overall, the contamination rate was 0%, and the batch discharge rate was 0.09%. CONCLUSIONS: The combination of upstream sterile filtration with a commercial closed system for manufacturing ASEDs proved to be effective in ensuring sterility without any contamination over the past 4 years. This is becoming crucial, as the demand for autologous blood products for treating ocular surface disorders, such as refractory dry eyes or nonhealing defects of the corneal epithelium, is on the rise.
Subject(s)
Drug Contamination , Ophthalmic Solutions , Serum , Humans , Drug Contamination/prevention & control , Prospective Studies , Sterilization/methods , Asepsis/methodsABSTRACT
PURPOSE: To establish a national consensus on contraindications for corneal donation for transplantation in Switzerland. METHODS: Swisstransplant (SWT), the Swiss national foundation coordinating tissue and organ donations, convened a working group consisting of six national corneal surgeons and eye bankers and donation experts to create a contraindication list for corneal donation. The group reviewed available national and international guidelines and recommendations, while adhering to Swiss law and transplant regulations. In cases of opposing opinions, the group held follow-up meetings until a consensus was reached. A consensus was defined as agreement among all parties present. RESULTS: From March 2021 to November 2021, the study group held six meetings and created a standardized minimal contraindication list for corneal donation in Switzerland. Thanks to this list, SWT has created a mandatory working and documentation file for donor coordinators to use when evaluating multiorgan donors for corneal harvesting. The authors agreed that while the national consensus list provides standardized minimal contraindication criteria, local eye banks may choose to introduce additional, more rigorous criteria. CONCLUSION: Given that corneal transplantation is the most commonly performed transplantation, establishing a consensus on contraindications is crucial for recipient safety. The creation of a consensus on contraindications for corneal donation in Switzerland is an essential contribution to fulfil the legal requirements concerning quality assurance and provides sufficient high-quality donor tissue within the country. Therefore, periodic review and revision of the consensus is considered critical.
Subject(s)
Corneal Transplantation , Tissue and Organ Procurement , Switzerland , Corneal Transplantation/legislation & jurisprudence , Humans , Tissue and Organ Procurement/legislation & jurisprudence , Tissue Donors/legislation & jurisprudence , Consensus , Eye Banks/legislation & jurisprudence , Contraindications, ProcedureABSTRACT
PURPOSE: Proof of concept of ex vivo retinal vessel diameter measurements in human postmortem eyes. METHODS: En face near-infrared (IR) images and optical coherence tomography (OCT) of the optic nerve head (ONH) were captured ex vivo with a Heidelberg Engineering Spectralis (Spectralis, version 7.0.4, Image Capture Module, version 1.2.4, Heidelberg Heidelberg, Germany) device, using a custom-made eye chamber holding and positioning the eyes during the image process. Thirty-two formaldehyde-fixated eyes of 16 patients were imaged. In the IR images, two independent graders measured retinal vessel diameters at the intersection of a drawn circle centered on the ONH with diameters of 2.0 mm and 3.4 mm, respectively. The anatomically corresponding measurements between both graders were statistically analyzed using a Wilcoxon signed-rank test. RESULTS: A total of 246 matched measurements of both graders were analyzed across all 32 imaged eyes. Statistically significant differences between the graders were found for arterioles at 2 mm from the ONH. The other measurements did not show statistically significant intergrader differences. The mean values for arteriole diameters were 72.2 µm at 2.0 mm and 61.5 µm at 3.4 mm for grader 1, and 66.4 µm at 2.0 mm and 63.2 µm at 3.4 mm for grader 2. The mean diameter for venules were 75.5 µm at 2.0 mm and 79.3 µm at 3.4 mm for grader 1, and 67.4 µm at 2 mm and 79.1 µm at 3.4 mm for grader 2. CONCLUSION: To the best of our knowledge, this is the first study to present IR image-based retinal vessel diameters in ex vivo postmortem eyes. Retinal IR/OCT imaging is possible, and measurements are reproducible in formaldehyde-fixated human eyes. Fixation artefacts result in lower image quality, and this can impose challenges in correctly detecting, classifying, and measuring retinal vessels.
Subject(s)
Optic Disk , Retinal Vessels , Tomography, Optical Coherence , Humans , Tomography, Optical Coherence/methods , Retinal Vessels/diagnostic imaging , Retinal Vessels/pathology , Female , Male , Optic Disk/diagnostic imaging , Optic Disk/blood supply , Optic Disk/pathology , Aged , Reproducibility of Results , Middle Aged , Sensitivity and Specificity , Aged, 80 and over , Autopsy/methods , CadaverABSTRACT
Aspergillus fumigatus can cause severe fatal invasive aspergillosis in immunocompromised patients but is also found in the environment. A. fumigatus infections can be treated with antifungals agents among which azole and echinocandins. Resistance to the class of azoles has been reported not only from patient samples but also from environmental samples. Azole resistance mechanisms involve for most isolates alterations at the site of the azole target (cyp51A); however, a substantial number of isolates can also exhibit non-cyp51A-mediated mechanisms.We aimed here to identify novel A. fumigatus genes involved in azole resistance. For this purpose, we designed a functional complementation system of A. fumigatus cDNAs expressed in a Saccharomyces cerevisiae isolate lacking the ATP Binding Cassette (ABC) transporter PDR5 and that was therefore more azole-susceptible than the parent wild type. Several genes were recovered including two distinct ABC transporters (atrF, atrI) and a Major Facilitator transporter (mdrA), from which atrI (Afu3g07300) and mdrA (Afu1g13800) were not yet described. atrI mediated resistance to itraconazole and voriconazole, while atrF only to voriconazole in S. cerevisiae Gene inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The expression of the transporters was addressed in clinical and environmental isolates with several azole resistance profiles. Our results show that atrI and mdrA tended to be expressed at higher levels than atrF in normal growth conditions. atrF was upregulated in 2/4 of azole-resistant environmental isolates and was the only gene with a significant association between transporter expression and azole resistance. In conclusion, this work showed the potential of complementation to identify functional transporters. The identified transporters were suggested to participate in azole resistance of A. fumigatus; however, this hypothesis will need further approaches to be verified.
Subject(s)
Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Aspergillus fumigatus/drug effects , Aspergillus fumigatus/enzymology , Drug Resistance, Fungal , Genes, Fungal , Membrane Transport Proteins/metabolism , Aspergillus fumigatus/genetics , Aspergillus fumigatus/isolation & purification , Azoles/metabolism , Azoles/pharmacology , Environmental Microbiology , Gene Expression , Genetic Complementation Test , Humans , Membrane Transport Proteins/genetics , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/geneticsABSTRACT
BACKGROUND: Photoreceptor degeneration is a main hallmark of many blinding diseases making protection of photoreceptors crucial to prevent vision loss. Thus, regulation of endogenous neuroprotective factors may be key for cell survival and attenuation of disease progression. Important neuroprotective factors in the retina include H2O2 generated by injured photoreceptors, and leukemia inhibitory factor (LIF) expressed in Müller glia cells in response to photoreceptor damage. RESULTS: We present evidence that H2O2 connects to the LIF response by inducing stabilization of Lif transcripts in Müller cells. This process was independent of active gene transcription and p38 MAPK, but relied on AU-rich elements (AREs), which we identified within the highly conserved Lif 3'UTR. Affinity purification combined with quantitative mass spectrometry identified several proteins that bound to these AREs. Among those, interleukin enhancer binding factor 3 (ILF3) was confirmed to participate in the redox-dependent Lif mRNA stabilization. Additionally we show that KH-type splicing regulatory protein (KHSRP) was crucial for maintaining basal Lif expression levels in non-stressed Müller cells. CONCLUSIONS: Our results suggest that H2O2-induced redox signaling increases Lif transcript levels through ILF3 mediated mRNA stabilization. Generation of H2O2 by injured photoreceptors may thus enhance stability of Lif mRNA and therefore augment neuroprotective LIF signaling during degenerative conditions in vivo.
Subject(s)
Ependymoglial Cells/metabolism , Leukemia Inhibitory Factor/metabolism , RNA Stability/genetics , Retina/metabolism , Animals , Cell Line , Gene Expression Regulation , Hydrogen Peroxide/metabolism , Mice , Oxidation-Reduction , Rats , Retinal Degeneration/metabolism , Signal Transduction/geneticsABSTRACT
Background: Burn wounds pose significant challenges in medical treatment due to their devastating nature and resource-intensive requirements. Temporary coverage of burn wounds using synthetic or biological dressings allows for reepithelization before definitive skin grafting. Allogenic skin grafts have been widely used but come with drawbacks such as rejection and disease transmission. The use of amniotic membranes (AMs) offers a promising alternative for temporary coverage, as they possess biological properties that promote faster healing and improved scar quality. The various components of the amniotic membrane, including pluripotent stem cells, extracellular matrix proteins, and regenerative factors, contribute to cell growth, migration, and differentiation, as well as preservation of the original epithelial phenotype. Objective: Reliable information on the treatment of burn wounds with AM is needed. The knowledge gained in this project may help to include this advantageous modern concept of biological dressings in clinical practice. The purpose of this study is to use human amniotic membranes from our in hospital laboratory, as an allogenic biological dressing after enzymatic debridement in superficial partial thickness, deep partial thickness or full thickness burn wounds. Methods: We will include 30 patients in a randomized-controlled trial with each patient receiving the study intervention and the control intervention. Two 7 × 7 cm burn wound areas will be compared regarding percentage of skin graft take, healing time, healing percentage value and total healing time. Human amniotic membranes will be compared to allogenic skin grafts.
ABSTRACT
Age-related macular degeneration (AMD) is a progressive disease of the retinal pigment epithelium (RPE) and the retina leading to loss of central vision. Polymorphisms in genes involved in lipid metabolism, including the ATP-binding cassette transporter A1 (ABCA1), have been associated with AMD risk. However, the significance of retinal lipid handling for AMD pathogenesis remains elusive. Here, we study the contribution of lipid efflux in the RPE by generating a mouse model lacking ABCA1 and its partner ABCG1 specifically in this layer. Mutant mice show lipid accumulation in the RPE, reduced RPE and retinal function, retinal inflammation and RPE/photoreceptor degeneration. Data from human cell lines indicate that the ABCA1 AMD risk-conferring allele decreases ABCA1 expression, identifying the potential molecular cause that underlies the genetic risk for AMD. Our results highlight the essential homeostatic role for lipid efflux in the RPE and suggest a pathogenic contribution of reduced ABCA1 function to AMD.
Subject(s)
ATP Binding Cassette Transporter 1/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 1/metabolism , Lipid Metabolism , Retinal Degeneration/physiopathology , Retinal Pigment Epithelium/physiopathology , ATP Binding Cassette Transporter 1/deficiency , ATP Binding Cassette Transporter, Subfamily G, Member 1/deficiency , Animals , Cell Line , Disease Models, Animal , Humans , Inflammation/pathology , Mice , Photoreceptor Cells/pathologyABSTRACT
Reduced choroidal blood flow and tissue changes in the ageing human eye impair oxygen delivery to photoreceptors and the retinal pigment epithelium. As a consequence, mild but chronic hypoxia may develop and disturb cell metabolism, function and ultimately survival, potentially contributing to retinal pathologies such as age-related macular degeneration (AMD). Here, we show that several hypoxia-inducible genes were expressed at higher levels in the aged human retina suggesting increased activity of hypoxia-inducible transcription factors (HIFs) during the physiological ageing process. To model chronically elevated HIF activity and investigate ensuing consequences for photoreceptors, we generated mice lacking von Hippel Lindau (VHL) protein in rods. This activated HIF transcription factors and led to a slowly progressing retinal degeneration in the ageing mouse retina. Importantly, this process depended mainly on HIF1 with only a minor contribution of HIF2. A gene therapy approach using AAV-mediated RNA interference through an anti-Hif1a shRNA significantly mitigated the degeneration suggesting a potential intervention strategy that may be applicable to human patients.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Oxidative Stress , Photoreceptor Cells, Vertebrate/metabolism , Retinal Degeneration/metabolism , Animals , Humans , Mice , NIH 3T3 Cells , Retinal Pigment Epithelium/metabolismABSTRACT
PURPOSE: Due to the low abundance of cone photoreceptors in the mouse retina and the scarcity of alternative animal models, little is known about mechanisms of cone degeneration. Nrl knockout mice develop exclusively the cone-type of photoreceptors. However, the cone photoreceptor layer in Nrl(-/-) mice displays an irregular morphology with severe rosette formation. Retinas of Rpe65(-/-);Nrl(-/-) mice have no rosettes due to the lack of 11-cis-retinal, but also are not functional. To develop a model with a functional all-cone retina that is morphologically well structured, we generated R91W;Nrl(-/-) double-mutant mice, which express a hypomorphic Rpe65 allele (R91W). METHODS: The following analyses were used to characterize the R91W;Nrl(-/-)mice: morphology by light and electron microscopy, protein distribution by immunofluorescence, cone function by electroretinography and optomotor response, RNA levels by RT-PCR, and chromophore levels by HPLC. Cone degeneration was assessed in R91W;Nrl(-/-) mice treated with MNU, and in triple R91W;Nrl(-/-);Cpfl1 and quadruple R91W;Nrl(-/-);Cpfl1;rd10 mutant mice. RESULTS: The all-cone retina of R91W;Nrl(-/-) mice is functional and relatively stable with only very slow age-related degeneration. Using triple and quadruple mutant mice, or a chemical treatment, we demonstrated that cone degeneration could be induced and analyzed in these mice. CONCLUSIONS: The reduced levels of visual chromophore prevented rosette formation and sustained function in the R91W;Nrl(-/-) retina. Thus, the R91W;Nrl(-/-) mouse allows study of the etiology of diseases related to cone degeneration in a "morphologically intact" and functional all-cone photoreceptor retina.
Subject(s)
Retinal Cone Photoreceptor Cells/physiology , Retinal Degeneration/pathology , Animals , Chromatography, High Pressure Liquid , Disease Models, Animal , Electroretinography , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Retina/metabolism , Retinal Degeneration/physiopathology , Retinaldehyde/deficiency , cis-trans-Isomerases/metabolismABSTRACT
The small GTPase CDC42 has pleiotropic functions during development and in the adult. These functions include intra- as well as intercellular tasks such as organization of the cytoskeleton and, at least in epithelial cells, formation of adherens junctions. To investigate CDC42 in the neuronal retina, we generated retina-specific Cdc42-knockdown mice (Cdc42-KD) and analyzed the ensuing consequences for the developing and postnatal retina. Lack of CDC42 affected organization of the developing retina as early as E17.5, prevented correct tissue lamination, and resulted in progressive retinal degeneration and severely reduced retinal function of the postnatal retina. Despite the disorganization of the retina, formation of the primary vascular plexus was not strongly affected. However, both deeper vascular plexi developed abnormally with no clear layering of the vessels. Retinas of Cdc42-KD mice showed increased expression of pro-survival, but also of pro-apoptotic and pro-inflammatory genes and exhibited prolonged Müller glia hypertrophy. Thus, functional CDC42 is important for correct tissue organization already during retinal development. Its absence leads to severe destabilization of the postnatal retina with strong degeneration and loss of retinal function.
Subject(s)
Microvessels/embryology , Morphogenesis/physiology , Retina/embryology , cdc42 GTP-Binding Protein/genetics , Animals , Animals, Newborn , Cell Survival , Embryo, Mammalian , Gene Expression , Gene Expression Profiling , Mice , Mice, Knockout , Microvessels/metabolism , Microvessels/pathology , Neuroglia/metabolism , Neuroglia/pathology , Neurons/metabolism , Neurons/pathology , Retina/metabolism , Retina/pathology , cdc42 GTP-Binding Protein/metabolismABSTRACT
PURPOSE: Hypoxic preconditioning activates hypoxia-inducible transcription factors (HIFs) in the retina and protects photoreceptors from light-induced retinal degeneration. The authors tested whether photoreceptor-specific activation of HIFs in normoxia is sufficient for protection. METHODS: Rod-specific Vhl knockdown mice were generated using the Cre-lox system with the rod opsin promoter controlling expression of CRE recombinase to stabilize HIF transcription factors in normoxic rods. Cell death was induced by light exposure and quantified by ELISA. Rhodopsin was quantified by spectrophotometry. Gene expression was analyzed by real-time PCR, and levels of proteins were determined by Western blotting. Morphology was investigated by light microscopy and retinal function tested by ERG. RESULTS: The rod-specific Vhl knockdown stabilized HIF-α proteins and induced expression of HIF target genes in retinas of 10-week-old mice under normoxic conditions. Retinal morphology and function were normal. At 36 hours after exposure to excessive light, Vhl knockdowns showed significantly less photoreceptor cell death than did wild-type controls. Ten days after light exposure, however, photoreceptor degeneration in Vhl knockdowns was similar to that of control animals. Vhl knockdowns expressed Fgf2 at higher basal levels before light exposure. After light exposure, however, expression of Fgf2 was not significantly different from that of wild-type controls. CONCLUSIONS: Artificial activation of HIF transcription factors in normoxic photoreceptors results in an increased basal expression of Fgf2 that may contribute to a transient protection of rods against light damage. Full photoreceptor protection may require a hypoxia-like response in additional retinal cell types and/or the differential regulation of additional mechanisms.
Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Retinal Degeneration/physiopathology , Retinal Rod Photoreceptor Cells , Transcription Factors/metabolism , Aging/pathology , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Cell Death/physiology , Cell Death/radiation effects , Female , Fibroblast Growth Factor 2/genetics , Genotype , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Ischemic Preconditioning , Light/adverse effects , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Retinal Degeneration/pathology , Retinal Rod Photoreceptor Cells/pathology , Retinal Rod Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/radiation effects , Transcription Factors/genetics , Von Hippel-Lindau Tumor Suppressor Protein/genetics , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.
Subject(s)
Aspergillus fumigatus/physiology , Fungal Proteins/metabolism , Molecular Chaperones/metabolism , Spores, Fungal/genetics , Stress, Physiological , Amino Acid Sequence , Animals , Aspergillosis , Aspergillus fumigatus/genetics , Aspergillus fumigatus/pathogenicity , Catalase/metabolism , Dithiothreitol/pharmacology , Enzyme Assays , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Humans , Hydrogen-Ion Concentration , MAP Kinase Signaling System , Mice , Molecular Chaperones/genetics , Molecular Sequence Data , Osmotic Pressure , Oxidation-Reduction , Peroxisomes/enzymology , Phenotype , Sequence Alignment , Sequence Deletion , Transcription, Genetic , Unfolded Protein Response/drug effectsABSTRACT
To investigate the possible association between different prophylactic sulfa drugs and the genotype of the Pneumocystis jiroveci dihydropteroate synthase (DHPS) gene, we examined DHPS polymorphisms in clinical specimens from 158 immunosuppressed patients (38 HIV-negative and 120 HIV-positive), using polymerase chain reaction-single-strand conformation polymorphism. Fifty-seven (36.1%) of 158 patients were infected with a mutant DHPS genotype. All patients who developed P. jiroveci pneumonia (PcP) while receiving pyrimethamine/sulfadoxine (PM/SD) prophylaxis (n=14) had a strain harboring DHPS with an amino acid change at position 57 (Pro-->Ser). This mutation was only present in 20 (14%) of 144 patients not receiving prophylaxis (P<.001). Hospitalization in a specific hospital was an independent risk factor for having P. jiroveci harboring the same DHPS mutation, which indirectly supports that interhuman transmission may affect the dissemination of the mutant strains.
Subject(s)
Anti-Bacterial Agents , Antibiotic Prophylaxis/methods , Dihydropteroate Synthase/genetics , Drug Therapy, Combination/pharmacology , HIV Infections/microbiology , HIV-1 , Pneumocystis Infections/microbiology , Pneumocystis/enzymology , Pyrimethamine/pharmacology , Sulfadoxine/pharmacology , Adolescent , Adult , Aged , Alleles , Bronchoalveolar Lavage Fluid/microbiology , Child , Child, Preschool , Drug Combinations , Female , HIV Infections/immunology , Humans , Immunocompromised Host , Male , Middle Aged , Pneumocystis/genetics , Pneumocystis Infections/immunology , Pneumocystis Infections/prevention & control , Pneumocystis Infections/virology , Point Mutation , Polymerase Chain Reaction , Polymorphism, Single-Stranded ConformationalABSTRACT
Failure of sulfa or sulfone prophylaxis is associated with mutations in Pneumocystis carinii gene coding for dihydropteroate synthase (DHPS). The DHPS genotype was analyzed in AIDS patients who had two separate episodes of P. carinii pneumonia. The results suggest that DHPS mutations can be selected de novo within patients by the pressure of a sulfa or sulfone drug.
Subject(s)
Dihydropteroate Synthase/genetics , Drug Resistance, Fungal/genetics , Mutation/genetics , Pneumocystis/genetics , Pneumonia, Pneumocystis/microbiology , Sulfonamides/pharmacology , Sulfones/pharmacology , Adult , Anti-Infective Agents/pharmacology , DNA, Fungal/analysis , Dihydropteroate Synthase/metabolism , Female , Genotype , Humans , Male , Middle Aged , Pneumocystis/drug effects , Pneumonia, Pneumocystis/drug therapy , RecurrenceABSTRACT
Small nucleolar RNAs (snoRNAs) guiding modifications of ribosomal RNAs and other RNAs display diverse modes of gene organization and expression depending on the eukaryotic system: in animals most are intron encoded, in yeast many are monocistronic genes and in plants most are polycistronic (independent or intronic) genes. Here we report an unprecedented organization: plant dicistronic tRNA-snoRNA genes. In Arabidopsis thaliana we identified a gene family encoding 12 novel box C/D snoRNAs (snoR43) located just downstream from tRNA(Gly) genes. We confirmed that they are transcribed, probably from the tRNA gene promoter, producing dicistronic tRNA(Gly)-snoR43 precursors. Using transgenic lines expressing a tagged tRNA-snoR43.1 gene we show that the dicistronic precursor is accurately processed to both snoR43.1 and tRNA(Gly). In addition, we show that a recombinant RNase Z, the plant tRNA 3' processing enzyme, efficiently cleaves the dicistronic precursor in vitro releasing the snoR43.1 from the tRNA(Gly). Finally, we describe a similar case in rice implicating a tRNA(Met-e) expressed in fusion with a novel C/D snoRNA, showing that this mode of snoRNA expression is found in distant plant species.