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3D printing results in anisotropy in the microstructure and mechanical properties. The focus of this study is to investigate the structure, texture and phase evolution of the as-printed and heat treated IN718 superalloy. Cylindrical specimens, printed by powder-bed additive manufacturing technique, were subjected to two post-treatments: homogenization (1100 °C, 1 h, furnace cooling) and hot isostatic pressing (HIP) (1160 °C, 100 MPa, 4 h, furnace cooling). The Selective laser melting (SLM) printed microstructure exhibited a columnar architecture, parallel to the building direction, due to the heat flow towards negative z-direction. Whereas, a unique structural morphology was observed in the x-y plane due to different cooling rates resulting from laser beam overlapping. Post-processing treatments reorganized the columnar structure of a strong 002 texture into fine columnar and/or equiaxed grains of random orientations. Equiaxed structure of about 150 µm average grain size, was achieved after homogenization and HIP treatments. Both δ-phase and MC-type brittle carbides, having rough morphologies, were formed at the grain boundaries. Delta-phase formed due to γ″-phase dissolution in the γ matrix, while MC-type carbides nucleates grew by diffusion of solute atoms. The presence of (Nb0.78Ti0.22)C carbide phase, with an fcc structure having a lattice parameter a = 4.43 Å, was revealed using Energy dispersive spectrometer (EDS) and X-ray diffractometer (XRD) analysis. The solidification behavior of IN718 alloy was described to elucidate the evolution of different phases during selective laser melting and post-processing heat treatments of IN718.

The integration of additive manufacturing into traditional manufacturing processes presents the future of engineered components with similar or superior performance levels to wrought or cast materials. In this work, the hot-deformation behavior and the microstructural changes of heat-treated SLM-printed IN718 specimens are investigated. Samples having the same shape and size were 3D-printed, homogenized (1100 °C, 1 h, and furnace-cooled), and hot-compressed, using a Gleeble® 3800 physical simulator at 1000 and 1050 °C and 0.1 and 0.01 s −1 strain rates. A 3D diagram showing the effect of temperature and strain rate on the flow stress behavior of IN718 during hot deformation is plotted. The dynamic recrystallization (DRX) mechanism was dominant in all specimens tested at 0.1 s −1 , while dynamic recovery (DRV) dominated in 0.01 s −1 tests. Changing the strain rate from 0.01 to 0.1 s −1 at 1000 °C increased the peak stress from 150 to 290 MPa (93%), while with a temperature decrease from 1050 to 1000 °C at 0.1 s −1 , the peak stress increased by 45%. Thus, the mechanical behavior was found to be more dependent on the strain rate than on the temperature. The DRX structure showed new grains developing at the boundaries of the original grains, whereas with the DRV structure, new grains grew within the original grains. A phenomenological model based on the Zener-Hollomon parameter was proposed in order to predict the size of recrystallized grains during hot deformation of the SLM-printed IN718 superalloy. The thermal softening due to recrystallization was compensated by precipitation hardening, as was revealed by phase analysis.

Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance. Kumari et al. show that host-derived extracellular vesicles capture systemic LPS and transfer it to the cytosol of immune cells via CD14-dependent endocytosis, triggering caspase-11-mediated gasdermin D activation and pyroptosis.

The development of multicellular organisms relies on interconnected genetic programs that control progression through their life cycle. MicroRNAs (miRNAs) and transcription factors (TFs) play key roles in such regulatory circuits. Here, we describe how three evolutionary conserved miRNA-TF pairs interact to form multiple checkpoints during reproductive development of Arabidopsis thaliana. Genetic, cellular, and physiological experiments show that miR159- and miR319-regulated MYB and TCP transcription factors pattern the expression of miR167 family members and their ARF6/8 targets. Coordinated action of these miRNA-TF pairs is crucial for the execution of consecutive hormone-dependent transitions during flower maturation. Cross-regulation includes both cis- and trans-regulatory interactions between these miRNAs and their targets. Our observations reveal how different miRNA-TF pairs can be organized into modules that coordinate successive steps in the plant life cycle.

Disease-modifying therapies (DMT) administered to patients with multiple sclerosis (MS) can influence immune responses to SARS-CoV-2 and vaccine efficacy. However, data on the detailed phenotypic, functional and metabolic characteristics of antigen (Ag)-specific cells following the third dose of mRNA vaccine remain scarce. Here, using flow cytometry and 45-parameter mass cytometry, we broadly investigate the phenotype, function and the single-cell metabolic profile of SARS-CoV-2-specific T and B cells up to 8 months after the third dose of mRNA vaccine in a cohort of 94 patients with MS treated with different DMT, including cladribine, dimethyl fumarate, fingolimod, interferon, natalizumab, teriflunomide, rituximab or ocrelizumab. Almost all patients display functional immune response to SARS-CoV-2. Different metabolic profiles characterize antigen-specific-T and -B cell response in fingolimod- and natalizumab-treated patients, whose immune response differs from all the other MS treatments. SARS-CoV-2 immune responses after vaccination in individuals with multiple sclerosis (MS) remain poorly understood. Here, using mass cytometry, the authors demonstrate that, following three doses of mRNA vaccine, patients with MS have distinct metabolic profiles in antigen-specific B and T cells.

Sepsis, a critical condition marked by systemic inflammation, profoundly impacts both innate and adaptive immunity, often resulting in lymphopenia. This immune alteration can spare regulatory T cells (Tregs) but significantly affects other lymphocyte subsets, leading to diminished effector functions, altered cytokine profiles, and metabolic changes. The complexity of sepsis stems not only from its pathophysiology but also from the heterogeneity of patient responses, posing significant challenges in developing universally effective therapies. This review emphasizes the importance of phenotyping in sepsis to enhance patient-specific diagnostic and therapeutic strategies. Phenotyping immune cells, which categorizes patients based on clinical and immunological characteristics, is pivotal for tailoring treatment approaches. Flow cytometry emerges as a crucial tool in this endeavor, offering rapid, low cost and detailed analysis of immune cell populations and their functional states. Indeed, this technology facilitates the understanding of immune dysfunctions in sepsis and contributes to the identification of novel biomarkers. Our review underscores the potential of integrating flow cytometry with omics data, machine learning and clinical observations to refine sepsis management, highlighting the shift towards personalized medicine in critical care. This approach could lead to more precise interventions, improving outcomes in this heterogeneously affected patient population.

Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for approximately 20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA-resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

The non-canonical inflammasome, which includes caspase-11 in mice and caspase-4 and caspase-5 in humans, is upregulated during inflammatory processes and activated in response to bacterial infections to carry out pyroptosis. Inadequate activity of the inflammasome has been associated with states of immunosuppression and immunopathological organ damage. However, the regulation of the receptors caspase-4 and caspase-5 during severe states of immunosuppression is largely not understood. We report that CASP4 and CASP5 are differentially regulated during acute-on-chronic liver failure and sepsis-associated immunosuppression, suggesting non-redundant functions in the inflammasome response to infection. While CASP5 remained upregulated and cleaved p20-GSDMD could be detected in sera from critically ill patients, CASP4 was downregulated in critically ill patients who exhibited features of immunosuppression and organ failure. Mechanistically, downregulation of CASP4 correlated with decreased gasdermin D levels and impaired interferon signaling, as reflected by decreased activity of the CASP4 transcriptional activators IRF1 and IRF2. Caspase-4 gene and protein expression inversely correlated with markers of organ dysfunction, including MELD and SOFA scores, and with GSDMD activity, illustrating the association of CASP4 levels with disease severity. Our results document the selective downregulation of the non-canonical inflammasome activator caspase-4 in the context of sepsis-associated immunosuppression and organ damage and provide new insights for the development of biomarkers or novel immunomodulatory therapies for the treatment of severe infections.

Background Sepsis-induced immunosuppression is a frequent cause of opportunistic infections and death in critically ill patients. A better understanding of the underlying mechanisms is needed to develop targeted therapies. Circulating bile acids with immunosuppressive effects were recently identified in critically ill patients. These bile acids activate the monocyte G-protein coupled receptor TGR5, thereby inducing profound innate immune dysfunction. Whether these mechanisms contribute to immunosuppression and disease severity in sepsis is unknown. The aim of this study was to determine if immunosuppressive bile acids are present in endotoxemia and septic shock and, if so, which patients are particularly at risk. Methods To induce experimental endotoxemia in humans, ten healthy volunteers received 2 ng/kg E. coli lipopolysaccharide (LPS). Circulating bile acids were profiled before and after LPS administration. Furthermore, 48 patients with early (shock onset within < 24 h) and severe septic shock (norepinephrine dose > 0.4 μg/kg/min) and 48 healthy age- and sex-matched controls were analyzed for circulating bile acids. To screen for immunosuppressive effects of circulating bile acids, the capability to induce TGR5 activation was computed for each individual bile acid profile by a recently published formula. Results Although experimental endotoxemia as well as septic shock led to significant increases in total bile acids compared to controls, this increase was mild in most cases. By contrast, there was a marked and significant increase in circulating bile acids in septic shock patients with severe liver failure compared to healthy controls (61.8 µmol/L vs. 2.8 µmol/L, p  = 0.0016). Circulating bile acids in these patients were capable to induce immunosuppression, as indicated by a significant increase in TGR5 activation by circulating bile acids (20.4% in severe liver failure vs. 2.8% in healthy controls, p  = 0.0139). Conclusions Circulating bile acids capable of inducing immunosuppression are present in septic shock patients with severe liver failure. Future studies should examine whether modulation of bile acid metabolism can improve the clinical course and outcome of sepsis in these patients. Graphical abstract

The palatine tonsils, localized in the oropharynx, are easily accessible secondary lymphoid tissue in humans. Inflammation of the palatine tonsils, local and chronic in case of chronic tonsillitis (CT) or acute in the presence of a peritonsillar abscess (PTA), ranks among the most common diseases in otolaryngology. However, the functionality of tonsillar immune cells, notably T-cells, in the context of these immune pathologies is poorly understood. We have examined the functional status of human tonsillar T-cells in CT and compared it to the acute inflammatory setting of a PTA. Patients presenting with CT (n = 10) or unilateral PTA (n = 7) underwent bilateral tonsillectomy and a subgroup of 8 patients underwent additional blood sampling. T-cells were purified via automated magnetic selection and subjected to flow cytometry-based immunophenotyping. In addition, the response to T-cell receptor (TCR) stimulation was assessed at the level of proximal signaling, activation marker expression and proliferation. We observed no difference between the percentage of T helper (CD4(+)) cells from tonsil tissue in CT and PTA, but observed a trend towards a higher percentage of T helper cells in the blood of patients with PTA versus CT, probably reflecting an acute, systemic bacterial infection in the former cohort. Tonsils from CT harbored more PD-1(+) CD4(+) T-cells, pointing to T-cell exhaustion due to chronic infection. This notion was supported by functional studies that showed a tendency to weaker TCR responses of tonsillar T-cells from CT. Intriguingly, tonsillar T-cells recurrently featured a dampened response to T-cell receptor stimulation at the level of receptor proximal signaling steps compared to peripheral T-cells. In sum, our study documents distinct differences in tonsillar T-cell class distribution and function between the various pathological conditions. Our observations are consistent with the concept that tonsillar T-cells react to infections by eliciting specific immunological responses in chronic versus acute settings of inflammation.