An improved method for intracellular DNA (iDNA) recovery from terrestrial environments.

The simultaneous extraction of intracellular DNA (iDNA) and extracellular DNA (eDNA) can help to separate the living in situ community (represented by iDNA) from background DNA that originated both from past communities and from allochthonous sources. As iDNA and eDNA extraction protocols require separating cells from the sample matrix, their DNA yields are generally lower than direct methods that lyse the cells within the sample matrix. We, therefore, tested different buffers with and without adding a detergent mix (DM) in the extraction protocol to improve the recovery of iDNA from surface and subsurface samples that covered a variety of terrestrial environments. The combination of a highly concentrated sodium phosphate buffer plus DM significantly improved iDNA recovery for almost all tested samples. Additionally, the combination of sodium phosphate and EDTA improved iDNA recovery in most of the samples and even allowed the successful extraction of iDNA from extremely low-biomass iron-bearing rock samples taken from the deep biosphere. Based on our results, we recommend using a protocol with sodium phosphate in combination with either a DM (NaP 300 mM + DM) or EDTA (NaP + EDTA 300 mM). Furthermore, for studies that rely on the eDNA pool, we recommend using buffers solely based on sodium phosphate because the addition of EDTA or a DM resulted in a decrease in eDNA for most of the tested samples. These improvements can help reduce community bias in environmental studies and contribute to better characterizations of both modern and past ecosystems.

Evolution and Current Status of Influenza A Virus in Chile: A Review.

The influenza A virus (IAV) poses a significant global threat to public health and food security. Particularly concerning is the avian influenza virus (AIV) subtype H5N1, which has spread from Europe to North and Central/South America. This review presents recent developments in IAV evolution in birds, mammals, and humans in Chile. Chile's encounter with IAV began in 2002, with the highly pathogenic avian influenza (HPAI) H7N3 virus, derived from a unique South American low pathogenic avian influenza (LPAI) virus. In 2016-2017, LPAI H7N6 caused outbreaks in turkey, linked to wild birds in Chile and Bolivia. The pandemic influenza A (H1N1) 2009 (H1N1pdm09) virus in 2009 decreased egg production in turkeys. Since 2012, diverse IAV subtypes have emerged in backyard poultry and pigs. Reassortant AIVs, incorporating genes from both North and South American isolates, have been found in wild birds since 2007. Notably, from December 2022, HPAI H5N1 was detected in wild birds, sea lions, and a human, along Chile's north coast. It was introduced through Atlantic migratory flyways from North America. These findings emphasize the need for enhanced biosecurity on poultry farms and ongoing genomic surveillance to understand and manage AIVs in both wild and domestic bird populations in Chile.

Exoscopic Microsurgery: A Change of Paradigm in Brain Tumor Surgery? Comparison with Standard Operative Microscope.

BACKGROUND: The exoscope is a high-definition telescope recently introduced in neurosurgery. In the past few years, several reports have described the advantages and disadvantages of such technology. No studies have compared results of surgery with standard microscope and exoscope in patients with glioblastoma multiforme (GBM). METHODS: Our retrospective study encompassed 177 patients operated on for GBM (WHO 2021) between February 2017 and August 2022. A total of 144 patients were operated on with a microscope only and the others with a 3D4K exoscope only. All clinical and radiological data were collected. Progression-free survival (PFS) and overall survival (OS) have been estimated in the two groups and compared by the Cox model adjusting for potential confounders (e.g., sex, age, Karnofsky performance status, gross total resection, MGMT methylated promoter, and operator's experience). RESULTS: IDH was mutated in 9 (5.2%) patients and MGMT was methylated in 76 (44.4%). Overall, 122 patients received a gross total resection, 14 patients received a subtotal resection, and 41 patients received a partial resection. During follow-up, 139 (73.5%) patients experienced tumor recurrence and 18.7% of them received a second surgery. After truncation to 12 months, the median PFS for patients operated on with the microscope was 8.82 months, while for patients operated on with the exoscope it was >12 months. Instead, the OS was comparable in the two groups. The multivariable Cox model showed that the use of microscope compared to the exoscope was associated with lower progression-free survival (hazard ratio = 3.55, 95%CI = 1.66-7.56, p = 0.001). CONCLUSIONS: The exoscope has proven efficacy in terms of surgical resection, which was not different to that of the microscope. Furthermore, patients operated on with the exoscope had a longer PFS. A comparable OS was observed between microscope and exoscope, but further prospective studies with longer follow-up are needed.

Bacterial networks in Atlantic salmon with Piscirickettsiosis.

An unbalanced composition of gut microbiota in fish is hypothesized to play a role in promoting bacterial infections, but the synergistic or antagonistic interactions between bacterial groups in relation to fish health are not well understood. We report that pathogenic species in the Piscirickettsia, Aeromonas, Renibacterium and Tenacibaculum genera were all detected in the digesta and gut mucosa of healthy Atlantic salmon without clinical signs of disease. Although Piscirickettsia salmonis (and other pathogens) occurred in greater frequencies of fish with clinical Salmonid Rickettsial Septicemia (SRS), the relative abundance was about the same as that observed in healthy fish. Remarkably, the SRS-positive fish presented with a generalized mid-gut dysbiosis and positive growth associations between Piscirickettsiaceae and members of other taxonomic families containing known pathogens. The reconstruction of metabolic phenotypes based on the bacterial networks detected in the gut and mucosa indicated the synthesis of Gram-negative virulence factors such as colanic acid and O-antigen were over-represented in SRS positive fish. This evidence indicates that cooperative interactions between organisms of different taxonomic families within localized bacterial networks might promote an opportunity for P. salmonis to cause clinical SRS in the farm environment.

Salmo salar Skin and Gill Microbiome during Piscirickettsia salmonis Infection.

Maintaining the high overall health of farmed animals is a central tenant of their well-being and care. Intense animal crowding in aquaculture promotes animal morbidity especially in the absence of straightforward methods for monitoring their health. Here, we used bacterial 16S ribosomal RNA gene sequencing to measure bacterial population dynamics during P. salmonis infection. We observed a complex bacterial community consisting of a previously undescribed core pathobiome. Notably, we detected Aliivibrio wodanis and Tenacibaculum dicentrarchi on the skin ulcers of salmon infected with P. salmonis, while Vibrio spp. were enriched on infected gills. The prevalence of these co-occurring networks indicated that coinfection with other pathogens may enhance P. salmonis pathogenicity.

Genomics of Re-Emergent Aeromonas salmonicida in Atlantic Salmon Outbreaks.

Furunculosis, caused by Aeromonas salmonicida, poses a significant threat to both salmonid and non-salmonid fish in diverse aquatic environments. This study explores the genomic intricacies of re-emergent A. salmonicida outbreaks in Atlantic salmon (Salmo salar). Previous clinical cases have exhibited pathological characteristics, such as periorbital hemorrhages and gastrointestinal abnormalities. Genomic sequencing of three Chilean isolates (ASA04, ASA05, and CIBA_5017) and 25 previously described genomes determined the pan-genome, phylogenomics, insertion sequences, and restriction-modification systems. Unique gene families have contributed to an improved understanding of the psychrophilic and mesophilic clades, while phylogenomic analysis has been used to identify mesophilic and psychrophilic strains, thereby further differentiating between typical and atypical psychrophilic isolates. Diverse insertion sequences and restriction-modification patterns have highlighted genomic structural differences, and virulence factor predictions can emphasize exotoxin disparities, especially between psychrophilic and mesophilic strains. Thus, a novel plasmid was characterized which emphasized the role of plasmids in virulence and antibiotic resistance. The analysis of antibiotic resistance factors revealed resistance against various drug classes in Chilean strains. Overall, this study elucidates the genomic dynamics of re-emergent A. salmonicida and provides novel insights into their virulence, antibiotic resistance, and population structure.

Isolation of a New Infectious Pancreatic Necrosis Virus (IPNV) Variant from Genetically Resistant Farmed Atlantic Salmon (Salmo salar) during 2021-2022.

Infectious pancreatic necrosis (IPN), caused by IPNV, affects several species of farmed fish, particularly Atlantic salmon, and is responsible for significant economic losses in salmon aquaculture globally. Despite the introduction of genetically resistant farmed Atlantic salmon and vaccination strategies in the Chilean salmon industry since 2019, the number of IPN outbreaks has been increasing in farmed Atlantic salmon in the freshwater phase. This study examined gross and histopathological lesions of IPNV-affected fish, as well as the IPNV nucleotide sequence encoding the VP2 protein in clinical cases. The mortality reached 0.4% per day, and the cumulative mortality was from 0.4 to 3.5%. IPNV was isolated in the CHSE-214 cell line and was confirmed by RT-PCR, and VP2 sequence analysis. The analyzed viruses belong to IPNV genotype 5 and have 11 mutations in their VP2 protein. This is the first report of IPN outbreaks in farmed Atlantic salmon genetically resistant to IPNV in Chile. Similar outbreaks were previously reported in Scotland and Norway during 2018 and 2019, respectively. This study highlights the importance of maintaining a comprehensive surveillance program in conjunction with the use of farmed Atlantic salmon genetically resistant to IPNV.

Repeated implantation of skeletal myoblast in a swine model of chronic myocardial infarction.

AIMS: Although transplantation of skeletal myoblast (SkM) in models of chronic myocardial infarction (MI) induces an improvement in cardiac function, the limited engraftment remains a major limitation. We analyse in a pre-clinical model whether the sequential transplantation of autologous SkM by percutaneous delivery was associated with increased cell engraftment and functional benefit. METHODS AND RESULTS: Chronically infarcted Goettingen minipigs (n = 20) were divided in four groups that received either media control or one, two, or three doses of SkM (mean of 329.6 x 10(6) cells per dose) at intervals of 6 weeks and were followed for a total of 7 months. At the time of sacrifice, cardiac function was significantly better in animals treated with SkM in comparison with the control group. A significantly greater increase in the DeltaLVEF was detected in animals that received three doses vs. a single dose of SkM. A correlation between the total number of transplanted cells and the improvement in LVEF and DeltaLVEF was found (P < 0.05). Skeletal myoblast transplant was associated with an increase in tissue vasculogenesis and decreased fibrosis (collagen vascular fraction) and these effects were greater in animals receiving three doses of cells. CONCLUSION: Repeated injection of SkM in a model of chronic MI is feasible and safe and induces a significant improvement in cardiac function.

A comparison between percutaneous and surgical transplantation of autologous skeletal myoblasts in a swine model of chronic myocardial infarction.

OBJECTIVE: Our aim was to compare the efficacy of surgical versus percutaneous administration of skeletal myoblasts (SkM) in a swine model of chronic myocardial infarction and to determine the mechanism(s) involved in their beneficial effect. METHODS: Two months after induction of myocardial infarction (MI), Goettingen miniature pigs underwent autologous SkM transplant either by direct surgical injection (n=6) or percutaneous access and intramyocardial delivery under fluoroscopic and echocardiographic guidance (n=6). Control animals received media alone (n=4). Functional analysis was performed by 2D echocardiography. Myoblast engraftment, in vivo cell differentiation, vessel formation, fibrosis, and the ratio between collagen type I/III deposition were analyzed in the infarct (IA) and non-infarct area (NIA) by immunohistochemistry. RESULTS: Animals received a median of 407.55+/-115x10(6) BrdU-labeled autologous SkM. Myoblast transplant was associated with a statistically significant increase in left ventricular ejection fraction (p<0.01), increased vasculogenesis and decreased fibrosis (p<0.05), and reduced collagen type I/III ratio in the IA and NIA areas as compared with control animals. No differences were found between groups receiving SkM by percutaneous or surgical access. CONCLUSIONS: Our results indicate that increased vasculogenesis and changes in matrix remodeling with decreased fibrosis are associated with the beneficial effect of SkM transplant in chronic MI. The equivalent benefit observed from surgical and percutaneous delivery has important clinical implications.

Dietary supplementation with vitamins C and E prevents downregulation of endothelial NOS expression in hypercholesterolemia in vivo and in vitro.

Impaired endothelium-dependent vasodilation has been associated with decreased NO bioavailability in hypercholesterolemia. This study aimed to determine whether antioxidant vitamins C and E could improve hypercholesterolemia-derived endothelial dysfunction in the porcine model, and whether observed in vivo results could be reproduced in vitro by incubation of coronary endothelial cells (EC) in the presence of native low-density lipoproteins (LDL). Adult mini-pigs were fed standard (C), cholesterol rich (HC) or cholesterol rich diet supplemented with vitamins C and E (HCV). Endothelium-dependent blood flow increase in response to acetylcholine was determined. Endothelial nitric oxide synthase (eNOS) expression was measured in arterial samples and in EC incubated with LDL isolated from porcine plasma. Vasomotor response to acetylcholine in HC was significantly lower (P<0.05) than control and HCV. There was a significant (P<0.05) decrease in eNOS immunoreactivity in HC, compared with HCV and control. Native LDL from HC, but not from HCV, induced a significant decrease in eNOS expression. Vitamins C and E treatment improved the endothelium-dependent vasomotor capacity and prevented decreased expression of eNOS in hypercholesterolemic pigs. A similar effect could be demonstrated in vitro, by incubation of EC with native LDL, suggesting that the effect of physiologically-modified LDL on eNOS could have a role in recovering vascular function.

Treatment of reperfused ischemia with adipose-derived stem cells in a preclinical Swine model of myocardial infarction.

The aim of the study was to determine the long-term effect of transplantation of adipose-derived stromal cells (ADSCs) in a preclinical model of ischemia/reperfusion (I/R). I/R was induced in 28 Goettingen minipigs by 120 min of coronary artery occlusion followed by reperfusion. Nine days later, surviving animals were allocated to receive transendocardial injection of a mean of 213.6 ± 41.78 million green fluorescent protein (GFP)-expressing ADSCs (n = 7) or culture medium as control (n = 9). Heart function, cell engraftment, and histological analysis were performed 3 months after transplantation. Transplantation of ADSCs induced a statistically significant long-lasting (3 months) improvement in cardiac function and geometry in comparison with control animals. Functional improvement was associated with an increase in angiogenesis and vasculogenesis and a positive effect on heart remodeling with a decrease in fibrosis and cardiac hypertrophy in animals treated with ADSCs. Despite the lack of cell engraftment after 3 months, ADSC transplantation induced changes in the ratio between MMP/TIMP. Our results indicate that transplantation of ADSCs, despite the lack of long-term significant cell engraftment, increases vessel density and prevents adverse remodeling in a clinically relevant model of myocardial infarction, strongly suggesting a paracrine-mediated effect. ADSCs thus constitute an attractive candidate for the treatment of myocardial infarction.