Visualization and Identification of Silicone Oil Emulsification Using Dynamic Infrared Confocal Scanning Laser Ophthalmoscopy.

Introduction: Silicone oil (SO) is a crucial agent used as an intraocular tamponade in the treatment of complex vitreoretinal diseases. Despite its effectiveness, SO is prone to emulsification, which can lead to significant and sometimes irreversible complications in both the anterior and posterior segments of the eye. The detection and monitoring of SO emulsification are therefore of paramount importance. Traditional imaging modalities have limitations in visualizing SO, leading to the exploration of more advanced imaging techniques. This study introduces the application of dynamic infrared confocal scanning laser ophthalmoscopy (IRcSLO) for this purpose and evaluates its effectiveness. Case Presentation: We report on 2 patients who underwent pars plana vitrectomy with subsequent SO injection for the management of retinal detachment. Postsurgery, both patients were imaged using the Heidelberg Retina Tomography Spectralis IRcSLO. The focus was on the visualization of the SO status, including the presence and distribution of emulsified SO droplets. The IRcSLO imaging technique demonstrated its capability to effectively visualize emulsified SO droplets. Interestingly, this was also true for cases where the SO had been removed. The emulsified droplets were observed as micron-sized, spherical entities with a nonuniform distribution throughout the vitreous cavity. Conclusion: Dynamic IRcSLO has proven to be an effective imaging modality for visualizing the emulsification of SO, offering a novel perspective into the characterization of SO droplets. It facilitates the analysis of droplet count, motility, and precise localization within the vitreous cavity. The findings from the case presentations underscore the variability of SO emulsification patterns and the sensitivity of IRcSLO in detecting even minuscule emulsified droplets. This imaging technique has significant potential for future research, particularly in understanding the timing of emulsification, the factors contributing to it, and the development of possible preventive strategies. Additionally, it allows for a more in-depth analysis of the behavior of emulsified SO droplets across different SO viscosities, which could be instrumental in optimizing patient outcomes.

Simulating A Subcondylar Mandibular Fracture With Intraoral Open Reduction and Internal Fixation: A Novel Education Tool for Residents.

Study Design: Face and content validation of a surgical simulation model. Objective: Open reduction and internal fixation in displaced subcondylar mandibular fractures is standard care. This requires an extraoral (eg: retromandibular, transparotideal) or intraoral approach. An intraoral approach requires further training since specialized instrumentation such as the 90° screwdriver system and endoscopes might be needed. Currently, no simulation models are available for training residents in intraoral reduction and fixation of subcondylar mandibular fractures. Therefore, we present a validated simulation model for intraoral treatment of subcondylar mandibular fractures. Methods: Based on a computer tomography data set, we designed and printed a 3D model of a mandible with a unilateral subcondylar fracture. To simulate intraoral work depth, it was positioned inside a dental phantom. We tested the model by a group of experts (n = 8), simulating intraoral reduction and fixation of a unilateral subcondylar fracture, using a 90° screwdriver system, a 1.0 subcondylar plate (lambda), and 5-6 mm screws.We assessed Face and Content validity by survey. Results: We provided an open-source printable fracture model. Printing costs were approximately US $10. Experts "Agreed" the model resembling the real scenario and its use for training intraoral reduction and fixation of subcondylar mandibular fractures. Conclusions: We developed a low cost, reproducible, open-source simulator for subcondylar mandibular fractures. Face and Content validity was achieved through evaluation by a group of experts.

A novel aminotransferase gene and its regulator acquired in Saccharomyces by a horizontal gene transfer event.

BACKGROUND: Horizontal gene transfer (HGT) is an evolutionary mechanism of adaptive importance, which has been deeply studied in wine S. cerevisiae strains, where those acquired genes conferred improved traits related to both transport and metabolism of the nutrients present in the grape must. However, little is known about HGT events that occurred in wild Saccharomyces yeasts and how they determine their phenotypes. RESULTS: Through a comparative genomic approach among Saccharomyces species, we detected a subtelomeric segment present in the S. uvarum, S. kudriavzevii, and S. eubayanus species, belonging to the first species to diverge in the Saccharomyces genus, but absent in the other Saccharomyces species. The segment contains three genes, two of which were characterized, named DGD1 and DGD2. DGD1 encodes dialkylglicine decarboxylase, whose specific substrate is the non-proteinogenic amino acid 2-aminoisobutyric acid (AIB), a rare amino acid present in some antimicrobial peptides of fungal origin. DGD2 encodes putative zinc finger transcription factor, which is essential to induce the AIB-dependent expression of DGD1. Phylogenetic analysis showed that DGD1 and DGD2 are closely related to two adjacent genes present in Zygosaccharomyces. CONCLUSIONS: The presented results show evidence of an early HGT event conferring new traits to the ancestor of the Saccharomyces genus that could be lost in the evolutionary more recent Saccharomyces species, perhaps due to loss of function during the colonization of new habitats.

Adaptive evolution in the Saccharomyces kudriavzevii Aro4p promoted a reduced production of higher alcohols.

The use of unconventional yeast species in human-driven fermentations has attracted a lot of attention in the last few years. This tool allows the alcoholic beverage industries to solve problems related to climate change or the consumer demand for newer high-quality products. In this sense, one of the most attractive species is Saccharomyces kudriavzevii, which shows interesting fermentative traits such as the increased and diverse aroma compound production in wines. Specifically, it has been observed that different isolates of this species can produce higher amounts of higher alcohols such as phenylethanol compared with Saccharomyces cerevisiae. In this work, we have shed light on this feature relating it to the S. kudriavzevii aromatic amino acid anabolic pathway in which the enzyme Aro4p plays an essential role. Unexpectedly, we observed that the presence of the S. kudriavzevii ARO4 variant reduces phenylethanol production compared with the S. cerevisiae ARO4 allele. Our experiments suggest that this can be explained by increased feedback inhibition, which might be a consequence of the changes detected in the Aro4p amino end such as L26 Q24 that have been under positive selection in the S. kudriavzevii specie.

Functional divergence in the proteins encoded by ARO80 from S. uvarum, S. kudriavzevii and S. cerevisiae explain differences in the aroma production during wine fermentation.

Phenylethanol (PE) and phenylethyl acetate (PEA) are commonly desired compounds in wine because of their rose-like aroma. The yeast S. cerevisiae produces the PE either through de novo biosynthesis by shikimate pathway followed by the Ehrlich pathway or the direct phenylalanine catabolism via Ehrlich pathway, and then converted into PEA. Previous work demonstrated that, compared to S. cerevisiae, other Saccharomyces species, such as S. kudriavzevii and S. uvarum, produce higher concentrations of PE and PEA from the precursor phenylalanine, which indicates differential activities of the biosynthetic-involved enzymes. A previous in-silico analysis suggested that the transcriptional activator Aro80p is one of the best candidates to explain these differences. An improved functional analysis identified significant radical amino acid changes in the S. uvarum and S. kudriavzevii Aro80p that could impact the expression of the catabolic genes ARO9 and ARO10, and hence, the production of PE from phenylalanine. Indeed, wine S. cerevisiae strains carrying the S. uvarum and S. kudriavzevii ARO80 alleles increased the production of both compounds in the presence of phenylalanine by increasing the expression of ARO9 and ARO10. This study provides novel insights of the unidentified Aro80p regulatory region and the potential usage of alternatives ARO80 alleles to enhance the PE and PEA concentration in wine.

Analysis by real-time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID-19 diagnosis in Santiago of Chile.

Due to the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N, and DNA/RNA Shield™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis.

Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis.

Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.

An Out-of-Patagonia migration explains the worldwide diversity and distribution of Saccharomyces eubayanus lineages.

Population-level sampling and whole-genome sequences of different individuals allow one to identify signatures of hybridization, gene flow and potential molecular mechanisms of environmental responses. Here, we report the isolation of 160 Saccharomyces eubayanus strains, the cryotolerant ancestor of lager yeast, from ten sampling sites in Patagonia along 2,000 km of Nothofagus forests. Frequency of S. eubayanus isolates was higher towards southern and colder regions, demonstrating the cryotolerant nature of the species. We sequenced the genome of 82 strains and, together with 23 available genomes, performed a comprehensive phylogenetic analysis. Our results revealed the presence of five different lineages together with dozens of admixed strains. Various analytical methods reveal evidence of gene flow and historical admixture between lineages from Patagonia and Holarctic regions, suggesting the co-occurrence of these ancestral populations. Analysis of the genetic contribution to the admixed genomes revealed a Patagonian genetic origin of the admixed strains, even for those located in the North Hemisphere. Overall, the Patagonian lineages, particularly the southern populations, showed a greater global genetic diversity compared to Holarctic and Chinese lineages, in agreement with a higher abundance in Patagonia. Thus, our results are consistent with a likely colonization of the species from peripheral glacial refugia from South Patagonia. Furthermore, fermentative capacity and maltose consumption resulted negatively correlated with latitude, indicating better fermentative performance in northern populations. Our genome analysis, together with previous reports in the sister species S. uvarum suggests that a S. eubayanus ancestor was adapted to the harsh environmental conditions of Patagonia, a region that provides the ecological conditions for the diversification of these ancestral lineages.

Molecular profiling of beer wort fermentation diversity across natural Saccharomyces eubayanus isolates.

The utilization of S. eubayanus has recently become a topic of interest due to the novel organoleptic properties imparted to beer. However, the utilization of S. eubayanus in brewing requires the comprehension of the mechanisms that underlie fermentative differences generated from its natural genetic variability. Here, we evaluated fermentation performance and volatile compound production in ten genetically distinct S. eubayanus strains in a brewing fermentative context. The evaluated strains showed a broad phenotypic spectrum, some of them exhibiting a high fermentation capacity and high levels of volatile esters and/or higher alcohols. Subsequently, we obtained molecular profiles by generating 'end-to-end' genome assemblies, as well as metabolome and transcriptome profiling of two Patagonian isolates exhibiting significant differences in beer aroma profiles. These strains showed clear differences in concentrations of intracellular metabolites, including amino acids, such as valine, leucine and isoleucine, likely impacting the production of 2-methylpropanol and 3-methylbutanol. These differences in the production of volatile compounds are attributed to gene expression variation, where the most profound differentiation is attributed to genes involved in assimilatory sulfate reduction, which in turn validates phenotypic differences in H2 S production. This study lays a solid foundation for future research to improve fermentation performance and select strains for new lager styles based on aroma and metabolic profiles.

Distinct Transcriptional Changes in Response to Patulin Underlie Toxin Biosorption Differences in Saccharomyces Cerevisiae.

Patulin (4-hydroxy-4H-furo[3,2c]pyran-2[6H]-one) is a mycotoxin produced by a suite of fungi species. Patulin is toxic to humans and is a sporadic contaminant in products that were made from fungi-infected fruits. The baker yeast Saccharomyces cerevisiae (S. cerevisiae) has been shown to decrease patulin levels likely by converting it to the less harmful E-ascladiol, yet this capacity is dependent on the strain utilized. In this study we show that four representative strains of different S. cerevisiae lineages differ in their ability to tolerate and decrease patulin levels in solution, demonstrating that some strains are better suitable for patulin biocontrol. Indeed, we tested the biocontrol capacities of the best patulin-reducer strain (WE) in contaminated apple juice and demonstrated their potential role as an efficient natural biocontrol solution. To investigate the mechanisms behind the differences between strains, we explored transcriptomic changes of the top (WE strain) and worst (WA strain) patulin-biocontroller strains after being exposed to this toxin. Large and significant gene expression differences were found between these two strains, the majority of which represented genes associated with protein biosynthesis, cell wall composition and redox homeostasis. Interestingly, the WE isolate exhibited an overrepresentation of up-regulated genes involved in membrane components, suggesting an active role of the membrane towards patulin detoxification. In contrast, WA upregulated genes were associated with RNA metabolism and ribosome biogenesis, suggesting a patulin impact upon transcription and translation activity. These results suggest that different genotypes of S. cerevisiae encounter different stresses from patulin toxicity and that different rates of detoxification of this toxin might be related with the plasma membrane composition. Altogether, our data demonstrates the different molecular mechanisms in S. cerevisiae strains withstanding patulin exposure and opens new avenues for the selection of new patulin biocontroller strains.

Non-lysosomal Activation in Macrophages of Atlantic Salmon (Salmo salar) After Infection With Piscirickettsia salmonis.

Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported.

GPD1 and ADH3 Natural Variants Underlie Glycerol Yield Differences in Wine Fermentation.

Glycerol is one of the most important by-products of alcohol fermentation, and depending on its concentration it can contribute to wine flavor intensity and aroma volatility. Here, we evaluated the potential of utilizing the natural genetic variation of non-coding regions in budding yeast to identify allelic variants that could modulate glycerol phenotype during wine fermentation. For this we utilized four Saccharomyces cerevisiae strains (WE - Wine/European, SA - Sake, NA - North American, and WA - West African), which were previously profiled for genome-wide Allele Specific Expression (ASE) levels. The glycerol yields under Synthetic Wine Must (SWM) fermentations differed significantly between strains; WA produced the highest glycerol yields while SA produced the lowest yields. Subsequently, from our ASE database, we identified two candidate genes involved in alcoholic fermentation pathways, ADH3 and GPD1, exhibiting significant expression differences between strains. A reciprocal hemizygosity assay demonstrated that hemizygotes expressing GPD1WA , GPD1SA , ADH3WA and ADH3SA alleles had significantly greater glycerol yields compared to GPD1WE and ADH3WE . We further analyzed the gene expression profiles for each GPD1 variant under SWM, demonstrating that the expression of GPD1WE occurred earlier and was greater compared to the other alleles. This result indicates that the level, timing, and condition of expression differ between regulatory regions in the various genetic backgrounds. Furthermore, promoter allele swapping demonstrated that these allele expression patterns were transposable across genetic backgrounds; however, glycerol yields did not differ between wild type and modified strains, suggesting a strong trans effect on GPD1 gene expression. In this line, Gpd1 protein levels in parental strains, particularly Gpd1pWE, did not necessarily correlate with gene expression differences, but rather with glycerol yield where low Gpd1pWE levels were detected. This suggests that GPD1WE is influenced by recessive negative post-transcriptional regulation which is absent in the other genetic backgrounds. This dissection of regulatory mechanisms in GPD1 allelic variants demonstrates the potential to exploit natural alleles to improve glycerol production in wine fermentation and highlights the difficulties of trait improvement due to alternative trans-regulation and gene-gene interactions in the different genetic background.

Genetic basis of mycotoxin susceptibility differences between budding yeast isolates.

Micophenolic acid (MPA) is an immunosuppressant mycotoxin which impairs yeast cell growth to variable degrees depending on the genetic background. Such variation could have emerged from several phenomena, including MPA gene resistance mutations and variations in copy number and localisation of resistance genes. To test this, we evaluated MPA susceptibility in four S. cerevisiae isolates and genetically dissected variation through the identification of Quantitative Trait Loci. Via linkage analysis we identified six QTLs, majority of which were located within subtelomeres and co-localised with IMD2, an inosine monophosphate dehydrogenase previously identified underlying MPA drug resistance in yeast cells. From chromosome end disruption and bioinformatics analysis, it was found that the subtelomere localisation of IMD2 within chromosome ends is variable depending on the strain, demonstrating the influence of IMD2 on the natural variation in yeast MPA susceptibility. Furthermore, GxE gene expression analysis of strains exhibiting opposite phenotypes indicated that ribosome biogenesis, RNA transport, and purine biosynthesis were impaired in strains most susceptible to MPA toxicity. Our results demonstrate that natural variation can be exploited to better understand the molecular mechanisms underlying mycotoxin susceptibility in eukaryote cells and demonstrate the role of subtelomeric regions in mediating interactions with the environment.

Identification of Nitrogen Consumption Genetic Variants in Yeast Through QTL Mapping and Bulk Segregant RNA-Seq Analyses.

Saccharomyces cerevisiae is responsible for wine must fermentation. In this process, nitrogen represents a limiting nutrient and its scarcity results in important economic losses for the wine industry. Yeast isolates use different strategies to grow in poor nitrogen environments and their genomic plasticity enables adaptation to multiple habitats through improvements in nitrogen consumption. Here, we used a highly recombinant S. cerevisiae multi-parent population (SGRP-4X) derived from the intercross of four parental strains of different origins to identify new genetic variants responsible for nitrogen consumption differences during wine fermentation. Analysis of 165 fully sequenced F12 segregants allowed us to map 26 QTL in narrow intervals for 14 amino acid sources and ammonium, the majority of which represent genomic regions previously unmapped for these traits. To complement this strategy, we performed Bulk segregant RNA-seq (BSR-seq) analysis in segregants exhibiting extremely high and low ammonium consumption levels. This identified several QTL overlapping differentially expressed genes and refined the gene candidate search. Based on these approaches, we were able to validate ARO1, PDC1, CPS1, ASI2, LYP1, and ALP1 allelic variants underlying nitrogen consumption differences between strains, providing evidence of many genes with small phenotypic effects. Altogether, these variants significantly shape yeast nitrogen consumption with important implications for evolution, ecological, and quantitative genomics.

DNA methylation and small interference RNAs participate in the regulation of MADS-box genes involved in dormancy in sweet cherry (Prunus avium L.).

Epigenetic modifications can yield information about connections between genotype, phenotype variation and environmental conditions. Bud dormancy release in temperate perennial fruit trees depends on internal and environmental signals such as cold accumulation and photoperiod. Previous investigations have noted the participation of epigenetic mechanisms in the control of this physiological process. We examined whether epigenetic modifications were modulated in MADS-box genes, potential candidates for the regulation of bud dormancy and flowering in sweet cherry (Prunus avium L.). We identified and cloned two MADS-box genes homologous to the already-characterized dormancy regulators DORMANCY-ASSOCIATED MADS-box (DAM3 and DAM5) from Prunus persica (L.) Batsch. Bisulfite sequencing of the identified genes (PavMADS1 and PavMADS2), Methylated DNA Immunoprecipitation and small RNA deep sequencing were performed to analyze the presence of DNA methylations that could be guided by non-coding RNAs in the floral buds exposed to differential chilling hours. The results obtained reveal an increase in the level of DNA methylation and abundance of matching small interference RNAs (siRNAs) in the promoter of PavMADS1 when the chilling requirement is complete. For the first intron and 5' UTR of PavMADS1, de novo DNA methylation could be associated with the increase in the abundance of 24-nt siRNA matching the promoter area. Also, in the second large intron of PavMADS1, maintenance DNA methylation in all cytosine contexts is associated with the presence of homologous siRNAs in that zone. For PavMADS2, only maintenance methylation was present in the CG context, and no matching siRNAs were detected. Silencing of PavMADS1 and PavMADS2 coincided with an increase in Flowering Locus T expression during dormancy. In conclusion, DNA methylations and siRNAs appear to be involved in the silencing of PavMADS1 during cold accumulation and dormancy release in sweet cherry.

Topographic arrangement of the rotundo-entopallial projection in the pigeon (Columba livia).

The tectofugal pathway (retina--optic tectum--nucleus rotundus--entopallium) is a prominent route mediating visual discrimination in diurnal birds. Several lines of evidence have shown that at the tecto-rotundal stage this pathway is composed of multiple parallel channels. Anatomical studies show that the nucleus rotundus is composed of at least four subdivisions, according to differences in cytoarchitectonic, histochemical, and hodological properties. Each of these subdivisions is in receipt of a highly convergent, nontopographic tectal projection, originating from a distinct subset of tecto-rotundal neurons. Physiological studies show that neurons of different subdivisions respond specifically to different visual dimensions, such as color, luminance, two-dimensional motion, and in-depth motion. At present it is less clear whether or to what extent this channel segregation is preserved at the telencephalic stage of the tectofugal pathway. The entopallium shows no obvious subdivisions or laminations. Nevertheless, tract-tracing experiments show that separate portions of the entopallium receive efferent projections from different rotundal subdivisions, in a way that maintains the rostrocaudal order of these subdivisions. In the present study we investigate in detail the topography of the rotundo-entopallial projection by means of anterograde and retrograde neuronal tracers. Our results confirm the zonal topography proposed by previous studies and indicate that each zone in the entopallium receives a direct and topographically organized projection from its corresponding rotundal subdivision. These results suggest that the spatial arrangement of the different rotundal functional modules is preserved at the entopallial level.