Nuclei Isolation from Ocular Tissues of the Embryonic Chicken for Single-Nucleus Profiling.

The generation of quality data from a single-nucleus profiling experiment requires nuclei to be isolated from tissues in a gentle and efficient manner. Nuclei isolation must be carefully optimized across tissue types to preserve nuclear architecture, prevent nucleic acid degradation, and remove unwanted contaminants. Here, we present an optimized workflow for generating a single-nucleus suspension from ocular tissues of the embryonic chicken that is compatible with various downstream workflows. The described protocol enables the rapid isolation of a high yield of aggregate-free nuclei from the embryonic chicken eye without compromising nucleic acid integrity, and the nuclei suspension is compatible with single-nucleus RNA and ATAC sequencing. We detail several stopping points, either via cryopreservation or fixation, to enhance workflow adaptability. Further, we provide a guide through multiple QC points and demonstrate proof-of-principle using two commercially available kits. Finally, we demonstrate that existing in silico genotyping methods can be adopted to computationally derive biological replicates from a single pool of chicken nuclei, greatly reducing the cost of biological replication and allowing researchers to consider sex as a variable during analysis. Together, this tutorial represents a cost-effective, simple, and effective approach to single-nucleus profiling of embryonic chicken eye tissues and is likely to be easily modified to be compatible with similar tissue types.

Density and distribution patterns of seafloor macrolitter in the eastern Red Sea.

The constant production of plastic and incessant growth of waste pollution continues to alter the marine environment from the coasts and surface waters to the deep sea. The quantification and investigation of macrolitter on the vast seabed of the ocean are challenging tasks that must be undertaken to elucidate the impact of anthropogenic activity on the marine environment and facilitate subsequent implementation of legally binding waste management regulations. In this study, we analyzed >60,000 images collected during 84 dives surveying 62.1 km of seabed in the eastern Red Sea to quantify the abundance and density of seafloor macrolitter. The surveyed depth of the seabed varied between 35 and 2415 m, and litter was observed at depths ranging from 93 to 2415 m. The litter density varied between 0 and 73,798 items km-2, with the mean (± SE) and median densities of 4069 ±â€¯1188 and 1371 items km-2, respectively. Plastic was the main litter category, comprising 46 % of all litter. The density of litter was higher at deeper depths (>1400 m) and increased significantly at distances farther from the shore. The results of this study suggest that maritime traffic and the possible direct litter discharge from vessels are the main anthropogenic sources of seafloor litter in the eastern Red Sea. Thus, we emphasize the urgency of conservation efforts and strict waste regulations to preserve the marine ecosystem of the Red Sea.

Lisdexamfetamine maintenance treatment for binge-eating disorder following successful treatments: randomized double-blind placebo-controlled trial.

BACKGROUND: Controlled research examining maintenance treatments for responders to acute interventions for binge-eating disorder (BED) is limited. This study tested efficacy of lisdexamfetamine (LDX) maintenance treatment amongst acute responders. METHODS: This prospective randomized double-blind placebo-controlled single-site trial, conducted March 2019 to September 2023, tested LDX as maintenance treatment for responders to acute treatments with LDX-alone or with cognitive-behavioral therapy (CBT + LDX) for BED with obesity. Sixty-one (83.6% women, mean age 44.3, mean BMI 36.1 kg/m2) acute responders were randomized to LDX (N = 32) or placebo (N = 29) for 12 weeks; 95.1% completed posttreatment assessments. Mixed-models and generalized-estimating equations comparing maintenance LDX v. placebo included main/interactive effects of acute (LDX or CBT + LDX) treatments to examine their predictive/moderating effects. RESULTS: Relapse rates (to diagnosis-level binge-eating frequency) following maintenance treatments were 10.0% (N = 3/30) for LDX and 17.9% (N = 5/28) for placebo; intention-to-treat binge-eating remission rates were 59.4% (N = 19/32) and 65.5% (N = 19/29), respectively. Maintenance LDX and placebo did not differ significantly in binge-eating but differed in weight-loss and eating-disorder psychopathology. Maintenance LDX was associated with significant weight-loss (-2.3%) whereas placebo had significant weight-gain (+2.2%); LDX and placebo differed significantly in weight-change throughout treatment and at posttreatment. Eating-disorder psychopathology remained unchanged with LDX but increased significantly with placebo. Acute treatments did not significantly predict/moderate maintenance-treatment outcomes. CONCLUSIONS: Adults with BED/obesity who respond to acute lisdexamfetamine treatment (regardless of additionally receiving CBT) had good maintenance during subsequent 12-weeks. Maintenance lisdexamfetamine, relative to placebo, did not provide further benefit for binge-eating but was associated with significantly better eating-disorder psychopathology outcomes and greater weight-loss.

Exploring Dietary Restraint as a Mediator of Behavioral and Cognitive-Behavioral Treatments on Outcomes for Patients With Binge-Eating Disorder With Obesity.

OBJECTIVE: To explore dietary-restraint as a mediator of binge eating and weight-loss outcomes within a randomized controlled trial comparing cognitive-behavioral therapy (CBT) and behavioral weight loss (BWL) for binge-eating disorder (BED) with obesity. METHODS: Ninety participants were randomly assigned to CBT or BWL and assessed by evaluators blinded to conditions at pretreatment, throughout-, and post-treatment (6 months). Three dietary-restraint measures (Eating Disorder Examination-Questionnaire [EDE-Q]-Restraint, Three-Factor Flexible-Restraint and Rigid-Restraint) were administered at pretreatment and after 2 months of treatment. Regression models examined whether changes at 2-months in the restraint scales mediated the effects of treatment (CBT versus BWL) on binge eating and weight-loss outcomes at post-treatment. RESULTS: CBT and BWL had similar binge-eating outcomes and similar changes in EDE-Q-restraint and flexible-restraint. BWL had greater 2-month increases in rigid-restraint and greater weight-loss at posttreatment than CBT, with results suggesting 2-month changes in rigid-restraint mediated the greater difference (>7 pounds) in weight-loss. The observed mediation effect of 2.92 suggests 39% of total treatment-effect on weight-loss was mediated through 2-month increases in rigid-restraint. DISCUSSION: This secondary analysis within a trial comparing CBT and BWL for BED suggests early-change in rigid-restraint has a mediating effect of BWL on weight-loss. Findings indicate that BWL improves binge eating and challenge views that dietary-restraint might exacerbate binge eating in BED with obesity. Findings require confirmation using hypothesis-testing in future trials. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00537758 ("Treatment for Obesity and Binge Eating Disorder").

Revisiting the Role of Balloon Atrial Septostomy Prior to the Arterial Switch Operation.

OBJECTIVES: Patients with dextro-transposition of the great arteries (d-TGA) frequently undergo balloon atrial septostomy (BAS) prior to the arterial switch operation (ASO) to promote atrial-level mixing. Balloon atrial septostomy has inherent risks as an invasive procedure and may not always be necessary. This study revisits the routine utilization of BAS prior to ASO. METHODS: Single-center, retrospective review of d-TGA patients undergoing the ASO from July 2018 to March 2023. Preoperative patient characteristics, pulse oximetry oxygen saturations (SpO2), cerebral/renal near-infrared spectroscopy (NIRS) readings along with prostaglandin status at the time of the ASO were analyzed with descriptive and univariate statistics. RESULTS: Thirty patients underwent the ASO. Of these, 7 (23%) were female, 25 (83%) were white, and median weight at ASO was 3.2 kg (range 0.8-4.2). Twenty-two (73%) patients underwent BAS. There were no demographic differences between BAS and no-BAS patients. Of those who underwent BAS, there was a significant increase in SpO2 (median 83% [range 54-92] to median 87% [range 72-95], P = .007); however, there was no change in NIRS from pre-to-post BAS. Six (27%) patients in the BAS group were prostaglandin-free at ASO. Balloon atrial septostomy patients underwent the ASO later compared with no-BAS patients (median 8 [range 3-32] vs 4 [range 2-10] days old, P = .016) and had a longer hospital length of stay (median 13 [range 7-43] vs 10 [range 8-131] days, P = .108). CONCLUSIONS: While BAS is an accepted preoperative procedure in d-TGA patients to improve oxygen saturations, it is also an additional invasive procedure, does not guarantee prostaglandin-free status at the time of ASO, and may increase the interval to ASO. Birth to direct early ASO, with prostaglandin support, should be revisited as an alternative, potentially more expeditious strategy.

Lower Extremity Arterial Calcification Predicts Referral to a Closed Unit After Primary Total Hip Arthroplasty.

BACKGROUND: We aimed to determine the association between lower extremity arterial calcification (LEAC) and referral to a closed unit (CU), length of stay, 90-day readmissions, and 1-year mortality in primary total hip arthroplasty (THA) patients. METHODS: We retrospectively analyzed 705 patients who underwent primary THA, identifying 64 patients (9.13%) who had LEAC and 641 who did not have LEAC. Patients who had LEAC were older (77 ± 10.0 versus 67 ± 11.5 years; P < 0.001) and had more comorbidities, except for a history of thromboembolic and oncologic diseases (P > 0.05). A preoperative antero-posterior pelvic radiograph was used to assess the presence of LEAC. Admission to CU, length of stay, 90-day readmissions, and 1-year mortality were recorded. A logistic regression model was used to identify risk factors for referral to CU. RESULTS: Patients who had LEAC had a higher incidence of admission to the ICU (8 of 64 [12.5%] versus 8 of 641 [1.09%]; P < 0.001), a longer hospital stay (4.7 ± 1.8 versus 4.2 ± 1.3 days; P = 0.006), more readmissions (16 of 64 [25%] vs. 33 of 641 [5.15%]; P < 0.001), and a higher 1-year mortality rate (6 of 64 [9.3%] versus 0 of 641 [0%]; P < 0.001) than patients who did not have LEAC. Of the patients who had LEAC admitted to CU, only 3 of 8 had a previous indication to do so in the preoperative assessment performed by the Department of Anesthesiology, while all non-LEAC ones referred to CU did so. Logistic regression analysis showed that LEAC was a risk factor for admission to CU (odds ratio [OR] = 4.77; 95% confidence interval [CI]: 1.12 to 20.25, P = 0.034). CONCLUSIONS: The presence of LEAC was a risk factor for transfer to CU, longer in-hospital stays, more readmissions, and a higher 1-year mortality rate. Identifying patients who have LEAC can aid in the preoperative assessment and risk stratification of patients planned for primary THA.

Diversity of bacteria of the genus Sphingomonas associated with sugarcane (Saccharum spp.) culm apoplast fluid and their agrotechnological potential.

In sugarcane, sequences related to the genus Sphingomonas have been widely detected by microbiome studies. In this work, the presence of bacteria of this genus was confirmed using culture-dependent and independent techniques. A collection of thirty isolates was obtained using semispecific cultivation conditions, and a specific PCR assay was applied to help confirm the isolates as belonging to the genus. A series of laboratory evaluations were carried out to identify potential properties among the isolates in the collection, which consequently allowed the identification of some most promising isolates for the development of new agricultural bioinputs. In a separate analysis, the culture-independent fluorescence in situ hybridization (FISH) methodology was applied to demonstrate the natural occurrence of Sphingomonas in different organs and tissues of sugarcane. The results showed the presence of bacteria of the genus in the spaces between cells (apoplast) of the culm parenchyma, in vessels in the region of the leaf vein, on the adaxial surface of the leaf blade, and on the root surface, sometimes close to the base of root hairs, which suggests extensive colonization on the host plant. In summary, the present study corroborates previous metagenomic amplicon sequencing results that indicated a high occurrence of Sphingomonas associated with sugarcane. This is the first study that uses approaches other than amplicon sequencing to confirm the occurrence of the genus in sugarcane and, at the same time, demonstrates potentially beneficial activities to be explored by sugarcane cultivation.

Antarctic wastewater: A local source of microplastic pollution.

Microplastic (MP) particles can be found all around the planet, even in Antarctica where they can be locally originated or transported by marine currents and winds. In this communication, we identify and report for the first time the contribution of a wastewater treatment plant (WWTP) as a local source of MP particles in the region. The analysis of the entire sample using micro-Raman spectroscopy revealed an MP concentration that ranged from 64 to 159 particles per liter of wastewater. >90 % of the identified particles were smaller than 50 µm. Among those analyzed, microplastics were identified as polyethylene, polypropylene, polyvinyl chloride, polytetrafluoroethylene, polyethylene terephthalate, and polystyrene. These findings demonstrate the need for urgent policies and technologies to mitigate this MP contamination source.

Wriggly woes: Helminths stirring up T cell trouble.

Understanding determinants of immune response variation is central to developing treatment options. Even et al. show that naive CD4+ T cell transcriptional heterogeneity is altered by helminth infection leading to impaired immune responses independent of commensals.

Enhanced Assessment of Cross-Reactive Antigenic Determinants within the Spike Protein.

Despite successful vaccination efforts, the emergence of new SARS-CoV-2 variants poses ongoing challenges to control COVID-19. Understanding humoral responses regarding SARS-CoV-2 infections and their impact is crucial for developing future vaccines that are effective worldwide. Here, we identified 41 immunodominant linear B-cell epitopes in its spike glycoprotein with an SPOT synthesis peptide array probed with a pool of serum from hospitalized COVID-19 patients. The bioinformatics showed a restricted set of epitopes unique to SARS-CoV-2 compared to other coronavirus family members. Potential crosstalk was also detected with Dengue virus (DENV), which was confirmed by screening individuals infected with DENV before the COVID-19 pandemic in a commercial ELISA for anti-SARS-CoV-2 antibodies. A high-resolution evaluation of antibody reactivity against peptides representing epitopes in the spike protein identified ten sequences in the NTD, RBD, and S2 domains. Functionally, antibody-dependent enhancement (ADE) in SARS-CoV-2 infections of monocytes was observed in vitro with pre-pandemic Dengue-positive sera. A significant increase in viral load was measured compared to that of the controls, with no detectable neutralization or considerable cell death, suggesting its role in viral entry. Cross-reactivity against peptides from spike proteins was observed for the pre-pandemic sera. This study highlights the importance of identifying specific epitopes generated during the humoral response to a pathogenic infection to understand the potential interplay of previous and future infections on diseases and their impact on vaccinations and immunodiagnostics.

LncRNA PTENP1/miR-21/PTEN Axis Modulates EMT and Drug Resistance in Cancer: Dynamic Boolean Modeling for Cell Fates in DNA Damage Response.

It is well established that microRNA-21 (miR-21) targets phosphatase and tensin homolog (PTEN), facilitating epithelial-to-mesenchymal transition (EMT) and drug resistance in cancer. Recent evidence indicates that PTEN activates its pseudogene-derived long non-coding RNA, PTENP1, which in turn inhibits miR-21. However, the dynamics of PTEN, miR-21, and PTENP1 in the DNA damage response (DDR) remain unclear. Thus, we propose a dynamic Boolean network model by integrating the published literature from various cancers. Our model shows good agreement with the experimental findings from breast cancer, hepatocellular carcinoma (HCC), and oral squamous cell carcinoma (OSCC), elucidating how DDR activation transitions from the intra-S phase to the G2 checkpoint, leading to a cascade of cellular responses such as cell cycle arrest, senescence, autophagy, apoptosis, drug resistance, and EMT. Model validation underscores the roles of PTENP1, miR-21, and PTEN in modulating EMT and drug resistance. Furthermore, our analysis reveals nine novel feedback loops, eight positive and one negative, mediated by PTEN and implicated in DDR cell fate determination, including pathways related to drug resistance and EMT. Our work presents a comprehensive framework for investigating cellular responses following DDR, underscoring the therapeutic potential of targeting PTEN, miR-21, and PTENP1 in cancer treatment.

SNHG1: Redefining the Landscape of Hepatocellular Carcinoma through Long Noncoding RNAs.

Hepatocellular carcinoma (HCC) represents a global health concern, ranking as the sixth most common malignancy worldwide and the third leading cause of cancer-related mortality. Despite advances in research, the diagnosis and prognosis of such malignancy remain challenging. Alpha-fetoprotein, the current serum biomarker used in the management of HCC, has limited sensitivity and specificity, making early detection and effective management more difficult. Thus, new management approaches in diagnosis and prognosis are needed to improve the outcome and survival of HCC patients. SNHG1 is a long noncoding RNA mainly expressed in the cell and cytoplasm of cells and is consistently upregulated in tissues and cell lines of HCC, where it acts as an important regulator of various processes: modulation of p53 activity, sponging of microRNAs with consequent upregulation of their target mRNAs, regulation of fatty acid, iron and glucose metabolism, and interaction with immune cells. The deregulation of these processes results in abnormal cell division, angiogenesis, and apoptosis, thus promoting various aspects of tumorigenesis, including proliferation, invasion, and migration of cells. Clinically, a higher expression of SNHG1 predicts poorer clinical outcomes by significantly correlating with bigger, less differentiated, and more aggressive tumors, more advanced disease stages, and lower overall survival in HCC patients. This article comprehensively summarizes the current understanding of the multifaceted roles of SNHG1 in the pathogenesis of HCC, while also highlighting its clinicopathological correlations, therefore concluding that it has potential as a biomarker in HCC diagnosis and prognosis.

Image Quality, Radiation Dose, and Patient Comfort Associated with Wireless Sensors in Digital Radiography: A Systematic Review.

Radiography facilities face challenges with the positioning of digital radiography detectors. This study evaluates the image quality, radiation dose, and patient comfort associated with wireless sensors in digital radiography. A systematic exploration was performed across PubMed/MEDLINE, EMBASE, SCOPUS, Web of Science, and SCIELO. Nine papers met the eligibility criteria, including three observational studies with 111 patients, four in vitro experiments with 258 extracted human teeth, and two ex vivo investigations with 16 cadaver mandibles. All studies consistently reported high-quality images produced by wireless sensors. Two studies demonstrated the superiority of wireless sensors, one found comparable accuracy with conventional radiography, and another indicated similar image quality among the sensors. Both wireless and wired sensors significantly reduced radiation doses compared to conventional X-rays. The Visual Analog Scale (VAS) did not reveal a clear superiority of wireless over wired sensors, though both were generally less comfortable than traditional film. The wireless sensors consistently produce high-quality images, comparable to or superior to other digital devices. Both wireless and wired sensors significantly reduce radiation doses compared to conventional X-rays, emphasizing their safety and efficacy. Patient comfort levels vary, with neither sensor type showing clear superiority over the other, and both being less comfortable than traditional film.

Discarded substrates from soilless hydroponic horticulture as potential amendments for metal-contaminated soils.

Soil contamination with metals is a major threat for the environment and public health since most metals are toxic to humans and to non-human biota, even at low concentrations. Thus, new sustainable remediation approaches are currently needed to immobilize metals in soils to decrease their mobility and bioavailability. In this work, we explore the application of discarded substrates from hydroponic cultivation, namely coconut shell and a mixture of coconut shell and pine bark, for immobilization of metals (Cd, Cr, Ni, Cu, Pb, Hg, Sb and As) in a naturally contaminated soil from a mining region in Portugal. The immobilization capacity of substrates (added to the soil at 5% mass ratio) was assessed both individually and also combined with other traditional agriculture soil additives (limestone and gypsum, at 2% mass ratio) and nanoparticles of zero-valent iron (nZVI) at 1-3% mass ratio. The overall results obtained after a 30-d incubation showed that the discarded substrates are a viable, economic, and environmental-friendly solution for metal remediation in soils, with the capacity of immobilization ranging from 20 to 91% for the metals and metalloids studied. Furthermore, they showed the capacity to reduce the soil toxicity (EC50 ∼ 6000 mg/L) to non-toxic levels (EC50 > 10000 mg/L) to the bacteria Aliivrio fischeri.

Self-sensing magnetic actuator based on sustainable collagen hybrid nanocomposites.

Taking into account that natural polymers are renewable and biodegradable, hybrid materials based on natural polymers are required for advanced technological applications with reduced environmental footprint. In this work, sustainable composites have been developed based on collagen as a polymeric matrix and different magnetic fillers, in order to tailor magnetic response. The composites were prepared by solution casting with 30 wt% of magnetite nanoparticles (Fe3O4 NPs), magnetite nanorods (Fe3O4 NRs) or cobalt ferrite nanoparticles (CoFe2O4 NPs). It is shown that the magnetic filler type has no bearing on the morphology, physical-chemical, or thermal characteristics of the composites, whereas the mechanical properties are determined by the magnetic filler, leading to a reduction in tensile strength, with values of 4.95 MPa for Fe3O4 NPs, 9.20 MPa for Fe3O4 NRs and 5.21 MPa for CoFe2O4 NPs containing samples. However, the highest magnetization saturation is obtained for Fe3O4 NPs (44 emu.g-1) and the higher coercive field for CoFe2O4 NPs (2062 Oe). In order to prove functionality of the developed composites, a self-sensing magnetic actuator device has been developed with the composite film with CoFe2O4 NPs, showing high stability over cycling.

Rapid Autopsy to Define Dendritic Cell Spatial Distribution and T Cell Association in Lung Adenocarcinoma.

Immunotherapy response is associated with the presence of conventional dendritic cells (cDCs). cDC type 1 (cDC1) is critically important for CD8+ T cell activation, cDC type 2 (cDC2) regulates CD4+ T cell responses, and mature regulatory cDCs may dampen T cell responses in the tumor microenvironment (TME). However, we lack a clear understanding of cDC distribution in the human TME, cDC prevalence in metastatic sites, and cDC differences in early- versus late-stage disease. Rapid autopsy specimens of 10 patients with lung adenocarcinoma were evaluated to detect cDCs and immune cells via multiplex immunofluorescence using 18 markers and 42 tumors. First, we found that T cells, cDC1, and cDC2 were confined to stroma, whereas mature regulatory DCs were enriched in tumor, suggesting unique localization-specific functions. Second, lung and lymph node tumors were more enriched in T cells and cDCs than liver tumors, underscoring differences in the TME of metastatic sites. Third, although the proportion of T cells and cDC1 did not differ in different stages, an increase in the proportion of cDC2 and macrophages in late stage suggests potential differences in regulation of T cell responses in different stages. Collectively, these findings provide new, to our knowledge, insights into cDC biology in human cancer that may have important therapeutic implications.