NanoCore: core-genome-based bacterial genomic surveillance and outbreak detection in healthcare facilities from Nanopore and Illumina data.

Genomic surveillance enables the early detection of pathogen transmission in healthcare facilities and contributes to the reduction of substantial patient harm. Fast turnaround times, flexible multiplexing, and low capital requirements make Nanopore sequencing well suited for genomic surveillance purposes; the analysis of Nanopore data, however, can be challenging. We present NanoCore, a user-friendly method for Nanopore-based genomic surveillance in healthcare facilities, enabling the calculation and visualization of cgMLST-like (core-genome multilocus sequence typing) sample distances directly from unassembled Nanopore reads. NanoCore implements a mapping, variant calling, and multilevel filtering strategy and also supports the analysis of Illumina data. We validated NanoCore on two 24-isolate data sets of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). In the Nanopore-only mode, NanoCore-based pairwise distances between closely related isolates were near-identical to Illumina-based SeqSphere+ distances, a gold standard commercial method (average differences of 0.75 and 0.81 alleles for MRSA and VRE; sd = 0.98 and 1.00), and gave an identical clustering into closely related and non-closely related isolates. In the "hybrid" mode, in which only Nanopore data are used for some isolates and only Illumina data for others, increased average pairwise isolate distance differences were observed (average differences of 3.44 and 1.95 for MRSA and VRE, respectively; sd = 2.76 and 1.34), while clustering results remained identical. NanoCore is computationally efficient (<15 hours of wall time for the analysis of a 24-isolate data set on a workstation), available as free software, and supports installation via conda. In conclusion, NanoCore enables the effective use of the Nanopore technology for bacterial pathogen surveillance in healthcare facilities. IMPORTANCE: Genomic surveillance involves sequencing the genomes and measuring the relatedness of bacteria from different patients or locations in the same healthcare facility, enabling an improved understanding of pathogen transmission pathways and the detection of "silent" outbreaks that would otherwise go undetected. It has become an indispensable tool for the detection and prevention of healthcare-associated infections and is routinely applied by many healthcare institutions. The earlier an outbreak or transmission chain is detected, the better; in this context, the Oxford Nanopore sequencing technology has important potential advantages over traditionally used short-read sequencing technologies, because it supports "real-time" data generation and the cost-effective "on demand" sequencing of small numbers of bacterial isolates. The analysis of Nanopore sequencing data, however, can be challenging. We present NanoCore, a user-friendly software for genomic surveillance that works directly based on Nanopore sequencing reads in FASTQ format, and demonstrate that its accuracy is equivalent to traditional gold standard short read-based analyses.

Antibacterial Activity of Zinc-Doped Hydroxyapatite and Vancomycin-Loaded Gelatin Nanoparticles against Intracellular Staphylococcus aureus in Human THP-1 Derived Macrophages.

Treating bone infections with common antibiotics is challenging, since pathogens like Staphylococcus aureus can reside inside macrophages. To target these intracellular bacteria, we have proposed nanoparticles (NPs) as drug carriers. This study aims to investigate the efficacy of hydroxyapatite and gelatin NPs, selected in view of their bone mimicry and potential for targeted delivery, as carriers for the antibacterial agents zinc and vancomycin. Therefore, two distinct NPs are fabricated: zinc-doped hydroxyapatite (ZnHA) and vancomycin-loaded gelatin (VGel) NPs. The NPs are characterized based on morphology, size, chemical composition, cellular internalization, and intracellular bactericidal efficacy. Specifically, the intracellular bactericidal efficacy is tested using a validated coculture model of human THP-1 derived macrophages and phagocytosed S. aureus bacteria. Scanning electron microscopy (SEM) and Fourier transform-infrared spectroscopy (FTIR) results show that the spherical NPs are synthesized successfully. These NPs are internalized by THP-1 cells and show >75% colocalization with lysosomes without compromising the viability of the THP-1 cells. Both ZnHA and VGel NPs substantially reduce the intracellular survival of S. aureus compared to the direct addition of dissolved zinc and vancomycin. Concluding, our NPs are highly effective drug delivery vehicles to kill intracellular S. aureus, which stress the potential of these NPs for future clinical translation.

Differential Efficacy of Weight Loss Interventions in Patients with Versus Without Diabetes.

Obesity is both a major risk factor for diabetes and a serious comorbidity of the condition. The twin epidemics of obesity and diabetes have spread globally over the past few decades. Treatment of obesity in patients with diabetes provides a host of clinical benefits that encompass virtually all body systems. Despite this, multiple lines of evidence suggest that the efficacy of most therapies for weight loss is significantly reduced among patients with diabetes. With this background, we summarize the evidence of a differential effect of lifestyle, pharmacological, and surgical treatments for obesity in patients with existing diabetes, and explore the potential mechanisms involved in this phenomenon. This information is then used to formulate strategies to improve weight loss outcomes for patients with diabetes.

Transplant Outcomes in Myelofibrosis: Impact of Donor Type (Cord Blood Grafts Supported by CD34+ selected Cells [Haplo-Cord] Versus Matched Donors).

Despite the established potentially curative role of allogeneic hematopoietic cell transplantation (allo-HCT) in managing myelofibrosis (MF), the choice of alternative donors for patients lacking matched donors remains a challenge, and the optimal graft source in this disease entity continues to be an ongoing debate. We aimed to evaluate the impact of donor type: umbilical cord blood transplant supported with CD34+ selected haploidentical donor (haplo-cord) versus adult matched related donor (MRD) and matched unrelated donor (MUD) in 40 adult patients with primary or secondary MF, including those progressing to accelerated phase (AP) or blast phase (BP), who underwent their first allo-HCT. The primary objective of this study was to analyze the impact of stem cell source on primary endpoints of overall survival (OS), graft-versus-host disease, and non-relapse mortality (NRM). Median follow-up for all alive patients was 53 months (range 0.3-63 months). Nine patients (22.5%) underwent an MRD allo-HCT, 15 patients (37.5%) underwent a MUD allo-HCT, and 16 patients (40%) underwent a haplo-cord allo-HCT. Four patients died without neutrophil engraftment: 3 (19%) in haplo-cord group and one (4%) in MRD/MUD group. The cumulative incidence of absolute neutrophil engraftment by day 60 was 80% (95% CI 45-94) in the haplo-cord group and 92% (95% CI 65-98) in the MRD/MUD group (P = .09). The cumulative incidence of platelet engraftment by day 60 was 59% (95% CI 27-81) in haplo-cord group and 75% (95% CI 51-88) in MRD/MUD group (P = .4). OS was 62% at 1 year (95% CI 49-79) and 34% at 3 years (95% CI 21-55). The 3-year OS was similar between the haplo-cord group and the MRD/MUD (37% versus 32%, P = .9). The 1-year OS for AP/BP patients was 50% (95% CI 27-93) in the haplo-cord group, compared to 40% (95% CI 19-86) in the MRD/MUD. The 1-year OS for chronic phase CP patients was 83% (95% CI 58-100) in the haplo-cord group, compared to 79% (95% CI 60-100) in the MRD/MUD group. The cumulative incidence of relapse at 3 years in the haplo-cord group was 13% (95% CI 1.8-34), and in the MRD/MUD group was 28% (95% CI 10-49) (P = .36). One-year NRM was 38% (95% CI 15-61) in the haplo-cord group and 33% (95% CI 15-52) in the MRD/MUD group. Three-year NRM was 48% (95% CI 19-72) in the haplo-cord group and 54% (95% CI 29-73) in MRD/MUD group (P = .95). We showed no significant difference in OS, relapse, and NRM outcomes after haplo-cord transplant compared to adult matched donors' grafts (MRD or MUD) in MF patients. However, there were more graft failures in patients transplanted with a haplo-cord transplants and delayed engraftments with inadequate haplo myeloid bridges. Despite the small sample size in our study, considering our findings and the availability of other alternative donors, using haplo-cord platforms may no longer be justified for MF unless the UCB engraftment dynamics can be optimized.

Spatial multiplexed immunofluorescence analysis reveals coordinated cellular networks associated with overall survival in metastatic osteosarcoma.

Patients diagnosed with advanced osteosarcoma, often in the form of lung metastases, have abysmal five-year overall survival rates. The complexity of the osteosarcoma immune tumor microenvironment has been implicated in clinical trial failures of various immunotherapies. The purpose of this exploratory study was to spatially characterize the immune tumor microenvironment of metastatic osteosarcoma lung specimens. Knowledge of the coordinating cellular networks within these tissues could then lead to improved outcomes when utilizing immunotherapy for treatment of this disease. Importantly, various cell types, interactions, and cellular neighborhoods were associated with five-year survival status. Of note, increases in cellular interactions between T lymphocytes, positive for programmed cell death protein 1, and myeloid-derived suppressor cells were observed in the 5-year deceased cohort. Additionally, cellular neighborhood analysis identified an Immune-Cold Parenchyma cellular neighborhood, also associated with worse 5-year survival. Finally, the Osteosarcoma Spatial Score, which approximates effector immune activity in the immune tumor microenvironment through the spatial proximity of immune and tumor cells, was increased within 5-year survivors, suggesting improved effector signaling in this patient cohort. Ultimately, these data represent a robust spatial multiplexed immunofluorescence analysis of the metastatic osteosarcoma immune tumor microenvironment. Various communication networks, and their association with survival, were described. In the future, identification of these networks may suggest the use of specific, combinatory immunotherapeutic strategies for improved anti-tumor immune responses and outcomes in osteosarcoma.

Towards tunable exciton delocalization in DNA Holliday junction-templated indodicarbocyanine 5 (Cy5) dye derivative heterodimers.

We studied the exciton delocalization of indodicarbocyanine 5 dye derivative (Cy5-R) heterodimers templated by a DNA Holliday junction (HJ), which was quantified by the exciton hopping parameter Jm,n. These dyes were modified at the 5 and 5' positions of indole rings with substituent (R) H, Cl, tBu, Peg, and hexyloxy (Hex) groups that exhibit different bulkiness and electron-withdrawing/donating capacities. The substituents tune the physical properties of the dyes, such as hydrophobicity (log P) and solvent-accessible surface area (SASA). We tuned the Jm,n of heterodimers by attaching two Cy5-Rs in adjacent and transverse positions along the DNA-HJ. Adjacent heterodimers exhibited smaller Jm,n compared to transverse heterodimers, and some adjacent heterodimers displayed a mixture of H- and J-like aggregates. Most heterodimers exhibited Jm,n values within the ranges of the corresponding homodimers, but some heterodimers displayed synergistic exciton delocalization that resulted in larger Jm,n compared to their homodimers. We then investigated how chemically distinct Cy5-R conjugated to DNA can interact to create delocalized excitons. We determined that heterodimers involving Cy5-H and Cy5-Cl and a dye with larger substituents (bulky substituents and large SASA) such as Cy5-Peg, Cy5-Hex, and Cy5-tBu resulted in larger Jm,n. The combination provides steric hindrance that optimizes co-facial packing (bulky Cy5-R) with a smaller footprint (small SASA) that maximizes proximity. The results of this study lay a groundwork for rationally optimizing the exciton delocalization in dye aggregates for developing next-generation technologies based on optimized exciton transfer efficiency such as quantum information systems and biomedicine.

S100A9 and HMGB1 orchestrate MDSC-mediated immunosuppression in melanoma through TLR4 signaling.

BACKGROUND: Immunotherapies for malignant melanoma are challenged by the resistance developed in a significant proportion of patients. Myeloid-derived suppressor cells (MDSC), with their ability to inhibit antitumor T-cell responses, are a major contributor to immunosuppression and resistance to immune checkpoint therapies in melanoma. Damage-associated molecular patterns S100A8, S100A9, and HMGB1, acting as toll like receptor 4 (TLR4) and receptor for advanced glycation endproducts (RAGE) ligands, are highly expressed in the tumor microenvironment and drive MDSC activation. However, the role of TLR4 and RAGE signaling in the acquisition of MDSC immunosuppressive properties remains to be better defined. Our study investigates how the signaling via TLR4 and RAGE as well as their ligands S100A9 and HMGB1, shape MDSC-mediated immunosuppression in melanoma. METHODS: MDSC were isolated from the peripheral blood of patients with advanced melanoma or generated in vitro from healthy donor-derived monocytes. Monocytes were treated with S100A9 or HMGB1 for 72 hours. The immunosuppressive capacity of treated monocytes was assessed in the inhibition of T-cell proliferation assay in the presence or absence of TLR4 and RAGE inhibitors. Plasma levels of S100A8/9 and HMGB1 were quantified by ELISA. Single-cell RNA sequencing (scRNA-seq) was performed on monocytes from patients with melanoma and healthy donors. RESULTS: We showed that exposure to S100A9 and HMGB1 converted healthy donor-derived monocytes into MDSC through TLR4 signaling. Our scRNA-seq data revealed in patient monocytes enriched inflammatory genes, including S100 and those involved in NF-κB and TLR4 signaling, and a reduced major histocompatibility complex II gene expression. Furthermore, elevated plasma S100A8/9 levels correlated with shorter progression-free survival in patients with melanoma. CONCLUSIONS: These findings highlight the critical role of TLR4 and, to a lesser extent, RAGE signaling in the conversion of monocytes into MDSC-like cells, underscore the potential of targeting S100A9 to prevent this conversion, and highlight the prognostic value of S100A8/9 as a plasma biomarker in melanoma.

The interplay of density functional selection and crystal structure for accurate NMR chemical shift predictions.

Ab initio chemical shift prediction plays a central role in nuclear magnetic resonance (NMR) crystallography, and the accuracy with which chemical shifts can be predicted relative to experiment impacts the confidence with which structures can be assigned. For organic crystals, periodic density functional theory calculations with the gauge-including projector augmented wave (GIPAW) approximation and the PBE functional are widely used at present. Many previous studies have examined how using more advanced density functionals can increase the accuracy of predicted chemical shifts relative to experiment, but nearly all of those studies employed crystal structures that were optimized with generalized-gradient approximation (GGA) functionals. Here, we investigate how the accuracy of the predicted chemical shifts in organic crystals is affected by replacing GGA-level PBE-D3(BJ) crystal geometries with more accurate hybrid functional PBE0-D3(BJ) ones. Based on benchmark data sets containing 132 13C and 35 15N chemical shifts, plus case studies on testosterone, acetaminophen, and phenobarbital, we find that switching from GGA-level geometries and chemical shifts to hybrid-functional ones reduces 13C and 15N chemical shift errors by ∼40-60% versus experiment. However, most of the improvement stems from the use of the hybrid functional for the chemical shift calculations, rather than from the refined geometries. In addition, even with the improved geometries, we find that double-hybrid functionals still do not systematically increase chemical shift agreement with experiment beyond what hybrid functionals provide. In the end, these results suggest that the combination of GGA-level crystal structures and hybrid-functional chemical shifts represents a particularly cost-effective combination for NMR crystallography in organic systems.

Enhancing medical anatomy education with the integration of virtual reality into traditional lab settings.

In medical education, traditional anatomy labs have relied heavily on the hands-on dissection of cadavers to teach the complex spatial relationships within the human body. However, the advent of virtual reality (VR) technology offers the potential for significantly enhancing this traditional approach by providing immersive, interactive 3D visualizations that can overcome some of the limitations of physical specimens. This study explores the integration of VR into a traditional gross anatomy lab to enrich the learning experience for medical students. Methods included the deployment of a VR application developed to complement the dissection process, featuring detailed 3D models of human anatomy that students could manipulate and explore digitally. Approximately 60 s-year medical students participated in the lab, where they engaged with both traditional dissection and the VR application. Results indicated that the VR integration not only increased engagement and satisfaction but also improved the students' ability to understand anatomical structures and their spatial relationships. Moreover, feedback from students suggested more efficient learning and retention than with traditional methods alone. We conclude that VR technology can significantly enhance medical anatomy education by providing an adjunct to traditional dissection, potentially replacing certain aspects of physical specimens with digital simulations that offer repeatable, detailed exploration without the associated logistical and ethical constraints.

Molecular Signatures of CB-6644 Inhibition of the RUVBL1/2 Complex in Multiple Myeloma.

Multiple myeloma is the second most hematological cancer. RUVBL1 and RUVBL2 form a subcomplex of many chromatin remodeling complexes implicated in cancer progression. As an inhibitor specific to the RUVBL1/2 complex, CB-6644 exhibits remarkable anti-tumor activity in xenograft models of Burkitt's lymphoma and multiple myeloma (MM). In this work, we defined transcriptional signatures corresponding to CB-6644 treatment in MM cells and determined underlying epigenetic changes in terms of chromatin accessibility. CB-6644 upregulated biological processes related to interferon response and downregulated those linked to cell proliferation in MM cells. Transcriptional regulator inference identified E2Fs as regulators for downregulated genes and MED1 and MYC as regulators for upregulated genes. CB-6644-induced changes in chromatin accessibility occurred mostly in non-promoter regions. Footprinting analysis identified transcription factors implied in modulating chromatin accessibility in response to CB-6644 treatment, including ATF4/CEBP and IRF4. Lastly, integrative analysis of transcription responses to various chemical compounds of the molecular signature genes from public gene expression data identified CB-5083, a p97 inhibitor, as a synergistic candidate with CB-6644 in MM cells, but experimental validation refuted this hypothesis.

Unlocking the potential of water-soluble gold(i)-NHC complexes: unveiling the role of carboxylic acid in cycloisomerization of alkynyl amino acid derivatives.

Hydrocarboxylation of alkyne-containing amino acid derivatives using water-soluble gold(i)-NHC complexes in an aqueous biphasic system at room temperature is described. Addition of silver salts is not required as the carboxylic acid group of the substrate is responsible for the activation of the gold catalyst at room temperature. Our results confirm that the steric bulk of the N-heterocyclic carbene ligands is an important factor in both the stability and the catalytic activity of gold(i) complexes in aqueous medium, and consequently in the recycling (at least 15 times without any loss of activity). The catalytic activity of our most active water-soluble gold(i)-NHC complex has been demonstrated in the cycloisomerization of amino acids derivatives with terminal and internal alkynes in aqueous media at room temperature.

Outcomes of Mitral Valve Regurgitation Management after Expert Multidisciplinary Valve Team Evaluation.

Background/Objectives: Mitral regurgitation (MR) affects millions worldwide, necessitating timely intervention. There are significant clinical challenges in the conservative management of MR, leaving a knowledge gap regarding the impact of multidisciplinary decision-making on treatment outcomes. This study aimed to provide insights into the impact of multidisciplinary decision-making on the survival outcomes of MR patients, focusing on conservative approaches. Methods: This study retrospectively analyzes 1365 patients evaluated by an expert multidisciplinary heart team (MDT) in a single center from 2015 to 2022. Treatments included surgery, catheter-based interventions, and conservative management. Propensity matching was utilized to compare surgery and conservative approaches. Results: Surgical intervention was associated with superior long-term survival outcomes compared to conservative and catheter-based treatments, particularly for degenerative MR (DMR). Survival rates of patients deemed by the MDT to have non-severe DMR were comparable to surgical patients (HR 1.07, 95% CI: 0.37-3.12, p = 0.90). However, non-severe functional MR (FMR) patients trended towards elevated mortality risk (HR 1.77, 95% CI: 0.94-3.31, p = 0.07). Pharmacological treatment for DMR was associated with significantly higher mortality compared to surgery (HR 8.0, 95% CI: 1.78-36.03, p = 0.001). Functional MR patients treated pharmacologically exhibited a non-significantly higher mortality risk compared to surgical intervention (HR 1.93, 95% CI: 0.77-4.77, p = 0.20). Conclusions: Survival analysis revealed significant benefits for surgical intervention, contrasting with elevated mortality risks associated with conservative management. "Watchful waiting" may be appropriate for non-severe DMR, while FMR may require closer monitoring. Further research is needed to assess the impact of regular follow-up or delayed surgery on survival rates, as pharmacological therapy has limited long-term efficacy for DMR.

Noninferior Immunogenicity and Consistent Safety of Respiratory Syncytial Virus Prefusion F Protein Vaccine in Adults 50-59 Years Compared to ≥60 Years of Age.

BACKGROUND: The adjuvanted respiratory syncytial virus (RSV) prefusion F protein-based vaccine (RSVPreF3 OA) is approved in adults aged ≥60 years. We evaluated RSVPreF3 OA immunogenicity and safety in adults aged 50-59 years without or with increased risk for RSV disease due to specific chronic medical conditions. METHODS: This observer-blind, phase 3, noninferiority trial included adults aged 50-59 years, stratified into 2 subcohorts: those with and those without predefined, stable, chronic medical conditions leading to an increased risk for RSV disease. Participants in both subcohorts were randomized 2:1 to receive RSVPreF3 OA or placebo. A control group of adults aged ≥60 years received RSVPreF3 OA. Primary outcomes were RSV-A and RSV-B neutralization titers (geometric mean titer ratios and sero-response rate differences) 1 month post-vaccination in 50-59-year-olds versus ≥60-year-olds. Cell-mediated immunity and safety were also assessed. RESULTS: The exposed population included 1152 participants aged 50-59 years and 381 participants aged ≥60 years. RSVPreF3 OA was immunologically noninferior in 50-59-year-olds versus ≥60-year-olds; noninferiority criteria were met for RSV-A and RSV-B neutralization titers in those with and those without increased risk for RSV disease. Frequencies of RSVPreF3-specific polyfunctional CD4+ T cells increased substantially from pre- to 1 month post-vaccination. Most solicited adverse events had mild-to-moderate intensity and were transient. Unsolicited and serious adverse event rates were similar in all groups. CONCLUSIONS: RSVPreF3 OA was immunologically noninferior in 50-59-year-olds compared to ≥60-year-olds, in whom efficacy was previously demonstrated. The safety profile in 50-59-year-olds was consistent with that in ≥60-year-olds. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: NCT05590403.

Transcriptional regulator MarT negatively regulates MarT-regulated motility gene I, a new gene involved in invasion and virulence of Salmonella enterica.

The speciation of Salmonella occurred by acquisition of genomic islands from other bacterial species and continued to diverge into subspecies and serovars with diferent range of host. S. enterica serovar Typhimurium (STM) is a generalist pathogen infecting hosts that include birds, mice, and humans, whilst S. enterica serovar Typhi (STY) is a restricted-host pathogen, infecting only humans. Despite their ranges of hosts, STM and STY possess 97-98% identity. Gain of genes by horizontal transference and loss of genes by mutations, are believed essential for differentiation of Salmonella. Salmonella pathogenicity island 3 (SPI-3) is an example combining these two processes. SPI-3 encodes misL and marT, among other genes. In STM, misL is required for gut colonization. Furthermore, protein MarT, positively regulates expression of misL by binding to misL-promoter. On the other hand, in SPI-3 of STY, marT and misL are pseudogenes. Interestingly, the gene t3766 (gene involved in resistance to H2O2) is present only in STY and is negatively regulated when marT STM is heterologously expressed in STY. Based on the view that MarT might regulate genes implicated in virulence, this work searched for new genes regulated by MarT. In silico searches for possible MarT target genes were performed, and 4 genes were selected for further analysis as they contained at least 2 copies of the consensus MarT-binding sequence in their promoters. Mutating marT in STM or heterologously expressing marT STM in STY confirmed that MarT negatively regulates ORF STY1408 or STM14_2003, its homologue in STM. STY1408 encodes for a putative protein with homology to methyl accepting chemotaxis proteins, which participate in chemotaxis and motility. Therefore, STY1408 was named mrmI (MarT-regulated motility gene I). Motility assays confirmed that the product of mrmI modulates motility. In addition, in vitro infection of cells with STM and STY mutants in mrmI reduces association with cells at 1, 3 and 24 h post-infection. Oral infection of mice showed that a mrmI null mutant was defective in producing systemic disease. Therefore, we conclude that MarT regulated mrmI, is involved in virulence of Salmonella. While pseudogenization of marT might modulate the fitness of narrow host range STY.

Acquisition of non-olfactory encoding improves odour discrimination in olfactory cortex.

Olfaction is influenced by contextual factors, past experiences, and the animal's internal state. Whether this information is integrated at the initial stages of cortical odour processing is not known, nor how these signals may influence odour encoding. Here we revealed multiple and diverse non-olfactory responses in the primary olfactory (piriform) cortex (PCx), which dynamically enhance PCx odour discrimination according to behavioural demands. We performed recordings of PCx neurons from mice trained in a virtual reality task to associate odours with visual contexts to obtain a reward. We found that learning shifts PCx activity from encoding solely odours to a regime in which positional, contextual, and associative responses emerge on odour-responsive neurons that become mixed-selective. The modulation of PCx activity by these non-olfactory signals was dynamic, improving odour decoding during task engagement and in rewarded contexts. This improvement relied on the acquired mixed-selectivity, demonstrating how integrating extra-sensory inputs in sensory cortices can enhance sensory processing while encoding the behavioural relevance of stimuli.

Influence of adjuvant therapies on organ-specific recurrence of cutaneous melanoma: A multicenter study on 1383 patients of the prospective DeCOG registry ADOReg.

This study investigated whether adjuvant treatments in stage III cutaneous melanoma (CM) influenced patterns of recurrence. Patients with primary (n = 1033) or relapsed CM (n = 350) who received adjuvant therapies with Nivolumab (N), Pembrolizumab (P), or Dabrafenib and Trametinib (D + T) were extracted from the prospective multicenter real-world skin cancer registry ADOReg. Endpoints were progression-free survival (PFS), distant metastasis-free survival (DMFS), organ-specific DMFS, and overall survival (OS). For primary cases, D + T indicated an improved PFS (1- and 2-year PFS: 90.9%; 82.7%) as compared to P (81.0%, 73.9%; p = .0208), or N (83.8%, 75.2%; p = .0539). BRAF-mutated(mut) CM demonstrated significantly lower PFS (p = .0022) and decreased DMFS (p = .0580) when treated with immune checkpoint inhibitor (ICI) instead of D + T. Besides, NRAS-mut CM tended to perform worse than wt CM upon ICI (PFS: p = .1349; DMFS: p = .0540). OS was similar between the groups. Relapsed cases showed decreased PFS, DMFS, and OS in comparison to primary (all: p < .001), without significant differences between the subgroups. Organ-specific DMFS was significantly altered for primary cases with bone (p = .0367) or brain metastases (p = .0202). In relapsed CM, the frequency of liver (D + T: 1.5%; P: 12%; N: 9%) and LN metastases (D + T: 1.5%; P: 12%; N: 10.2%) was significantly lower with adjuvant D + T than ICI. NRAS-mut CM showed increased recurrence in primary and relapsed cases. These data show that adjuvant D + T is superior to ICI in primary BRAF-mut CM.

Cerebrospinal Fluid Cytokine and Chemokine Profiles in Central Nervous System Sarcoidosis: Diagnostic and Immunopathologic Insights.

OBJECTIVE: To evaluate the cerebrospinal fluid (CSF) cytokine/chemokine profile of central nervous system (CNS) neurosarcoidosis (NS), and its utility in differential diagnosis, treatment, and prognostication. METHODS: In this case-control study, we validated 17 cytokines/chemokines (interleukin [IL]-1-beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, BAFF, IL-8/CXCL8, CXCL9, CXCL10, CXCL13, GM-CSF, interferon-gamma, and tumor necrosis factor [TNF]-alpha) in a multiplexed automated immunoassay system (ELLA; Bio-Techne, Minneapolis, MN, USA), and assessed them in CSF and serum of symptomatic patients with probable or definite CNS NS (01/2011-02/2023) with gadolinium enhancement and/or CSF pleocytosis. Patients with multiple sclerosis, primary CNS lymphoma, aquaporin-4 immunoglobulin G positivity, non-inflammatory disorders, and healthy individuals were used as controls. RESULTS: A total of 32 NS patients (59% women; median age, 59 years [19-81]) were included; concurrent sera were available in 12. CSF controls consisted of 26 multiple sclerosis, 8 primary CNS lymphoma, 84 aquaporin-4 immunoglobulin G positive, and 34 patients with non-inflammatory disorders. Gadolinium enhancement was present in 31 of 32 NS patients, and CSF pleocytosis in 27 of 32 (84%). CSF IL-2, IL-6, IL-10, IL-13, BAFF, IL-8/CXCL8, CXCL9, CXCL10, CXCL13, GM-CSF, interferon-gamma, and TNF-alpha levels were significantly higher in NS patients compared with non-inflammatory controls (p ≤ 0.02); elevations were more common in CSF than serum. Concurrent elevation of IL-6, CXCL9, CXCL10, GM-CSF, interferon-gamma, and TNF-alpha was present in 18 of 32 NS patients, but only in 1 control. Elevated IL-6, IL-10, IL-13, CXCL9, CXL10, GM-CSF, and TNF-alpha associated with measures of disease activity. INTERPRETATION: NS CSF cytokine/chemokine profiles suggest T cell (mainly T helper cell type 1), macrophage, and B-cell involvement. These signatures aid in NS diagnosis, indicate disease activity, and suggest therapeutic avenues. ANN NEUROL 2024.

Oriented Antibody Coupling to an Antifouling Polymer Using Glycan Remodeling for Biosensing by Particle Motion.

Biosensors based on immobilized antibodies require molecular strategies that (i) couple the antibodies in a stable fashion while maintaining the conformation and functionality, (ii) give outward orientation of the paratope regions of the antibodies for good accessibility to analyte molecules in the biofluid, and (iii) surround the antibodies by antibiofouling molecules. Here, we demonstrate a method to achieve oriented coupling of antibodies to an antifouling poly(l-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) substrate, using glycan remodeling to create antibody-DNA conjugates. The coupling, orientation, and functionality of the antibodies were studied using two analysis methods with single-molecule resolution, namely single-molecule localization microscopy and continuous biosensing by particle motion. The biosensing functionality of the glycan-remodeled antibodies was demonstrated in a sandwich immunosensor for procalcitonin. The results show that glycan-remodeled antibodies enable oriented immobilization and biosensing functionality with low nonspecific binding on antifouling polymer substrates.

Interobserver Agreement and Performance of Concurrent AI Assistance for Radiographic Evaluation of Knee Osteoarthritis.

Background Due to conflicting findings in the literature, there are concerns about a lack of objectivity in grading knee osteoarthritis (KOA) on radiographs. Purpose To examine how artificial intelligence (AI) assistance affects the performance and interobserver agreement of radiologists and orthopedists of various experience levels when evaluating KOA on radiographs according to the established Kellgren-Lawrence (KL) grading system. Materials and Methods In this retrospective observer performance study, consecutive standing knee radiographs from patients with suspected KOA were collected from three participating European centers between April 2019 and May 2022. Each center recruited four readers across radiology and orthopedic surgery at in-training and board-certified experience levels. KL grading (KL-0 = no KOA, KL-4 = severe KOA) on the frontal view was assessed by readers with and without assistance from a commercial AI tool. The majority vote of three musculoskeletal radiology consultants established the reference standard. The ordinal receiver operating characteristic method was used to estimate grading performance. Light kappa was used to estimate interrater agreement, and bootstrapped t statistics were used to compare groups. Results Seventy-five studies were included from each center, totaling 225 studies (mean patient age, 55 years ± 15 [SD]; 113 female patients). The KL grades were KL-0, 24.0% (n = 54); KL-1, 28.0% (n = 63); KL-2, 21.8% (n = 49); KL-3, 18.7% (n = 42); and KL-4, 7.6% (n = 17). Eleven readers completed their readings. Three of the six junior readers showed higher KL grading performance with versus without AI assistance (area under the receiver operating characteristic curve, 0.81 ± 0.017 [SEM] vs 0.88 ± 0.011 [P < .001]; 0.76 ± 0.018 vs 0.86 ± 0.013 [P < .001]; and 0.89 ± 0.011 vs 0.91 ± 0.009 [P = .008]). Interobserver agreement for KL grading among all readers was higher with versus without AI assistance (κ = 0.77 ± 0.018 [SEM] vs 0.85 ± 0.013; P < .001). Board-certified radiologists achieved almost perfect agreement for KL grading when assisted by AI (κ = 0.90 ± 0.01), which was higher than that achieved by the reference readers independently (κ = 0.84 ± 0.017; P = .01). Conclusion AI assistance increased junior readers' radiographic KOA grading performance and increased interobserver agreement for osteoarthritis grading across all readers and experience levels. Published under a CC BY 4.0 license. Supplemental material is available for this article.

A customizable and defined medium supporting culturing of Candida albicans, Staphylococcus aureus, and human oral epithelial cells.

Candida albicans, an opportunistic oral pathogen, synergizes with Staphylococcus aureus, allowing bacteria to co-invade and systemically disseminate within the host. Studying human-microbe interactions creates the need for a universal culture medium that supports fungal, bacterial, and human cell culturing, while allowing sensitive analytical approaches such as OMICs and chromatography techniques. In this study, we established a fully defined, customizable adaptation of Dulbecco's modified Eagle medium (DMEM), allowing multi-kingdom culturing of S. aureus, C. albicans, and human oral cell lines, whereas minimal version of DMEM (mDMEM) did not support growth of S. aureus, and neither did supplementation with dextrose, MEM non-essential amino acids, pyruvate, and Glutamax. This new medium composition, designated as "mDMEM-DMP," promoted growth of all tested S. aureus strains. Addition of 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) further improved growth, while higher concentrations did not improve growth any further. Higher concentrations of HEPES did result in prolonged stabilization of medium pH. mDMEM-DMP promoted (hyphal) C. albicans monoculturing and co-culturing on both solid and semi-solid surfaces. In contrast to S. aureus, addition of HEPES reduced C. albicans maximum culture optical density (OD). Finally, only buffered mDMEM-DMP (100 mM HEPES) was successful in maintaining the metabolic activity of human oral Ca9-22 and HO1N1 cell lines for 24 hours. Altogether, our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions. IMPORTANCE: Interaction between microbes and the host are in the center of interest both in disease and in health. In order to study the interactions between microbes of different kingdoms and the host, alternative media are required. Synthetic media are useful as they allow addition of specific components. In addition, well-defined media are required if high-resolution analyses such as metabolomics and proteomics are desired. We describe the development of a synthetic medium to study the interactions between C. albicans, S. aureus, and human oral epithelial cells. Our findings show that mDMEM-DMP is a versatile and potent culture medium for both microbial and human cell culturing, providing a customizable platform to study human as well as microbial molecular physiology and putative interactions.