FEBS Open Bio

The accumulation of amyloid-beta (A{beta}) plaques is a hallmark of Alzheimers disease (AD), with A{beta}42 representing the predominant and most aggregation-prone isoform. Reliable preparation of monomeric A{beta}42 is essential for investigating the kinetics and mechanisms of its aggregation into oligomers and fibrils. This study provides a direct comparison of two monomerization protocols for recombinantly expressed A{beta}42: one incorporating size-exclusion chromatography (SEC) and the other relying solely on chemical denaturation, using agents such as NaOH and NH4OH. A{beta}42 was produced in E. coli, purified through urea solubilization followed by HPLC, and subjected to monomerization via the respective methods. Monomeric preparations were evaluated using Thioflavin T (ThT) fluorescence to assess aggregation kinetics, TEM to detect fibrils and preformed aggregates, and NMR spectroscopy. SEC-isolated monomers displayed sigmoidal aggregation profiles in ThT assays, featuring distinct lag, growth, and plateau phases consistent with secondary nucleation-dominated models as determined by AmyloFit analysis. Increasing the initial peptide concentration resulted in higher fibril yields, which was further supported by TEM images showing extensive fibrillization following incubation. In contrast, non-SEC preparations containing pre-existing aggregates detectable by TEM and showed attenuated NMR signals, leading to impaired aggregation behavior. NaOH-denatured samples predominantly exhibited flat ThT curves, whereas NH4OH-denatured samples displayed extended lag phases. NH4OH performance better than NaOH, likely because its gradual pH neutralization reduced peptide structural perturbation. Overall, these findings demonstrate that SEC is critical for obtaining highly pure monomeric A{beta}42 and improving the reproducibility of aggregation assays, highlighting the importance of standardized monomer preparation protocols in AD research. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=49 SRC="FIGDIR/small/724608v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1a3b9caorg.highwire.dtl.DTLVardef@1fa85d2org.highwire.dtl.DTLVardef@67a83dorg.highwire.dtl.DTLVardef@1564f77_HPS_FORMAT_FIGEXP M_FIG C_FIG

Seismic shifts within academia over the last several decades have seen the growth of biomedical PhD recipients alongside the relative stagnation of tenure-track research-intensive faculty careers (RIFCs). This hypercompetitive academic job market has prompted interest in the paths of those who attain RIFCs. Understanding what drives recent biomedical PhDs to make their career decisions and persist toward them requires a clear picture of how career perceptions, motivations, and intentions develop and crystallize over time. Using annual in-depth interviews across nearly two decades, this report explores the evolution of career thinking and differentiation among 40 who attained a RIFC from diverse starting points to their attainment of a RIFC. Participants strategies for navigating early scientific experiences were patterned by their varied educational and socioeconomic backgrounds. Nearly half of participants did not start with or maintain stable interest in RIFCs, exhibiting changes in both PhD and postdoctoral phases. Participants highlighted six drivers toward RIFCs including desire for independence/autonomy and contributing to knowledge/health. Our results are instructive for trainees and mentors guiding career exploration and differentiation.

Understanding what is requisite for attaining a biomedical faculty career is crucial for guiding trainees preparing for these roles. For nearly two decades, we have collected accounts of biomedical training and career transitions from a large cohort through annual in-depth interviews and tracking of competencies and achievements. This paper elucidates the common and varied credentials of 40 who entered research-intensive faculty careers (RIFCs). Participants completed PhDs and postdocs in a range of research-intensive institutional settings. Developing research independence and a niche were essential to RIFC attainment, and mentors played a crucial role in this development. Counter to common assumptions, high-prestige publications and grants were not in and of themselves necessary for RIFC attainment. Our findings can aid RIFC aspirants and mentors who guide them.

Objective: To identify Dickkopf-1 (DKK1) as a prognostically relevant candidate in head and neck squamous cell carcinoma and to evaluate whether DKK1 and cytoskeleton-associated protein 4 (CKAP4) expression is associated with cervical lymph node metastasis in tongue squamous cell carcinoma (TSCC). Methods: DKK1 was screened using the Human Protein Atlas Pathology Atlas. Immunohistochemical expression of DKK1 and CKAP4 was examined in 54 patients with primary TSCC (cT1-4N0) treated surgically between 2015 and 2020. Nine cases were excluded because of insufficient tissue blocks or inadequate staining quality, leaving 45 evaluable cases. Associations with delayed cervical lymph node metastasis were assessed together with conventional clinicopathological factors, including infiltrative growth pattern (INF) and pathological depth of invasion (pDOI). Results: In public database analysis, high DKK1 expression was associated with poorer overall survival in head and neck squamous cell carcinoma. In the TSCC cohort, pDOI [≥]5 mm and INF pattern c were significantly associated with cervical lymph node metastasis. Positive DKK1 and CKAP4 expression were also significantly associated with cervical lymph node metastasis. Furthermore, combined DKK1/CKAP4 positivity, when incorporated with INF and pDOI, provided additional risk stratification, and cases with all 3 factors showed a markedly increased likelihood of cervical lymph node metastasis. Conclusions: Expression of DKK1 and CKAP4 was associated with cervical lymph node metastasis in TSCC. Combined assessment of DKK1/CKAP4 expression with INF and pDOI may improve pathological risk stratification and may help identify patients who require closer neck evaluation and postoperative management.

Graduate-level genomics courses require students to integrate dense material across subfields, concepts and methods. In modular, multi-instructor courses, students may struggle because the coherence between lectures can be difficult to navigate, while the course structure may be visible to instructors. We evaluated a 2025 navigation redesign of BIO322, a graduate genomics course at the Norwegian University of Life Sciences, while preserving course content, multi-instructor teaching, modular organization and assessment framework. The redesign includes introducing a standardized self-learning guide, expanded syllabus, enriched online quiz feedback, and added support for a final group research proposal. Using anonymized course evaluation scores from 2021-2025 and aggregated learning management system access data from 2023-2025, we examined student experience and resource use. In 2025, five of six course evaluation items reached their highest observed BIO322 scores, while one, lecture-specific score remained within the previous range. The consolidated self-learning guide was accessed by nearly all students, whereas access to optional readings declined across the course sequence, despite comparatively stable page views per accessing student. These course-level findings are consistent with improved perceived navigability following the introduction of standardized learning support. However, some students continued to report difficulty identifying priorities and connections among course components, indicating that challenges in perceived course coherence remained for part of the cohort despite the redesign. Practitioner PointsO_LIMaking course structure explicit may improve students perceived navigability in multi-instructor graduate genomics courses. C_LIO_LIA centralized self-learning guide can broaden access to preparatory guidance without changing core course content or assessment. C_LIO_LIOptional learning supports may be used unevenly, so resource availability should not be assumed to translate into uniform resource access. C_LI

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

Abstract Introduction: MET tyrosine kinase (TKI) therapy has improved outcomes in patients with non-small cell lung cancer (NSCLC) harboring MET alterations. However, primary and acquired resistance ultimately limits durability of response. This study evaluated the safety and efficacy of the MET inhibitor capmatinib with the MEK inhibitor trametinib in patients with metastatic MET-driven NSCLC who had progressed on prior treatment with at least one MET inhibitor. Methods: A multicenter phase I study evaluated capmatinib in combination with trametinib in patients with advanced stage NSCLC harboring activating MET alterations and prior exposure to at least one MET TKI. A 3+3 dose-escalation design was employed to assess safety and tolerability of the combination. Results: Three patients (n = 3) were enrolled in the study and completed a median of 3 cycles of therapy. Dose-limiting toxicities, including rash, edema, and nausea, necessitated dose reductions in the first two patients and initiation of the third patient at a lower dose level. Ultimately, all patients discontinued therapy due to treatment-related adverse events. The study was terminated early due to poor accrual and TRAEs. No radiographic objective responses were observed. Conclusions: In this phase I trial, capmatinib plus trametinib was associated with significant treatment-related adverse events and treatment was discontinued in all participants. Based on these findings, further investigation of this combination of MET and MEK inhibitors is not recommended.

The poor prognosis of brain tumors, including IDH-wild-type glioblastoma (GB), as well as brain and leptomeningeal metastases, is partly related to the blood-brain barrier (BBB), which limits the delivery of hydrophilic anticancer drugs to the tumor site and surrounding brain parenchyma. Early studies using vital dyes demonstrated that intracranial injection could bypass the BBB in cats. We confirmed that, in guinea pigs, the vital dye Bleu Patente V diffused efficiently into the brain after a bolus intracranial injection, whereas the brain remained unstained after intravenous administration. Similarly, brain concentrations of the hydrophilic anticancer drug gemcitabine were significantly higher following intracranial injection than after intravenous administration. Consistent with these findings, Bleu Patente penetrated deeply into the cerebral cortex of sheep after a 24-hour intraventricular infusion. At the end of a 24-hour intraventricular infusion of 20 mg gemcitabine in sheep, mean gemcitabine concentrations reached 1,415 {micro}g/L in cerebrospinal fluid and 850 {micro}g/kg in brain tissue. These concentrations exceeded the IC90 values of gemcitabine for A172, U87-MG, and U118-MG human glioblastoma cell lines, as determined in vitro after 24 hours of incubation. We hypothesize that Bleu Patente dye and gemcitabine circumvent the blood-brain barrier (BBB) by utilizing the glymphatic system. Tolerance of a single 24-hour intraventricular infusion of gemcitabine at doses of 5, 10, and 20 mg was good. Taken together, these encouraging preclinical results support the resumption of Phase I clinical trials evaluating intraventricular infusion of gemcitabine in patients with refractory primary or secondary brain tumors.

De novo vessel formation (vasculogenesis) in vitro is a key step in tissue engineering to preserve tissue viability for long-term assays and testing therapeutic agents. However, in vitro vasculogenesis is often unreliable due to differences in vascular-supporting cells, including endothelial cells and stromal cells such as smooth muscle cells (SMCs) and fibroblasts. Here, we developed a robust co-culture system of HUVECs and SMCs to generate stable vascular networks capable of maintaining tissue viability over extended periods. Given that SMC plasticity is a major limitation in supporting endothelial network formation, we systematically evaluated the effects of passage number, confluency, and freezing on primary SMC function. To overcome this limitation, we generated immortalized supportive SMCs, which preserved their vasculogenic gene program and functional capacity even at high passage. In addition, we identified and validated key genes associated with endothelial support, including CD248, C3, and FBLN1, all essential for vasculogenesis. Immortalized SMCs consistently maintained expression of these genes and supported robust vessel formation under variable culture conditions. Collectively, this study demonstrates that immortalized SMCs provide a stable, reproducible platform for endothelial-SMC co-cultures, enabling long-term vascularized tumor models suitable for functional studies and therapeutic screening.

The neural crest (NC) is a transient stem cell population which migrates throughout the developing embryo to contribute to diverse tissues dependent on axial origin. For example, cranial NC can give rise to bone and cartilage, while more posterior NC populations give rise to peripheral nervous system and neuroendocrine tissues. Perturbations in neural crest development can lead to severe congenital anomalies and cancers, with over 700 neurocristopathies reported. In humans, early NC development remains poorly understood due to the inaccessibility of tissue samples, thus necessitating the development of in vitro models. Currently, a limited number of NC organoid protocols are available, but these mainly focus on cranial NC and lack relevant tissue architecture. Here, we describe a novel bioengineered pipeline to derive human pluripotent stem cell (hPSC)-derived neuroepithelial organoids, "neurocrestoids" featuring physiologically-relevant tissue architecture. We show that neurocrestoids recapitulate the dynamics of induction, delamination, and migration of human neural crest cells (NCCs), and can be directly compared to murine NC explants for cross-species validation. Organoids express an array of HOX genes indicating the successful generation of cranial, vagal and trunk NCCs. Moreover, we have integrated our neurocrestoids with a customised micropatterned substrate suitable for live visualisation and guided separation of SOX10-positive migratory human NCCs. Our "NCC migration on-chip" are reproducible across multiple hPSC lines and should be scalable for future diagnostic and therapeutic applications, significantly improving our ability to study human NC pathologies.

BackgroundThe current biomarker framework for the diagnosis and staging of Alzheimers disease (AD) relies mainly on neuropathological features; thus, its performance for diagnosis is limited prior to the initiation of neurodegeneration. Here, we leveraged transcriptomic data to develop a new framework for omic-informed blood-based diagnostic biomarkers for AD from early-stage. MethodsMicroglial gene expression from single-nucleus (sn)RNA-seq data was analyzed via 6 statistical methods to identify candidate panels of genes predictive of AD. A total of 78 gene panels, 30-2000 genes in size, were selected and evaluated for their ability to distinguish AD patients from controls. Three top-ranked panels of 300, 50 and 30 genes were transferred to blood (monocyte) transcriptomic data obtained from living subjects via a graph-based mapping approach based on optimal transport statistics. ResultsThe 300-panel method resulted in an AUC of 0.7 and moderate accuracy (75%) in classifying AD; however, the accuracy in predicting cognitively normal patients was lower (53%). While the 300 genes provided high accuracy, inspection of the distribution of p values for the gene set revealed that the panel could be greatly reduced in size to capture the most significant differences between AD patients and cognitively normal individuals. The accuracy and specificity of the 50 and 30 panels demonstrated similar AUC values but improved the balance between the prediction of AD patients and normal controls. Specifically, the 50-gene panel resulted in an AUC of 0.7, with 65% AD accuracy and 71% normal accuracy. ConclusionsIntegrating multiomics datasets into the AD biomarker discovery pipeline offers a powerful modality to increase precision and comprehensiveness in AD research and clinical applications.

Scientific supervision is central to the experience of early-career researchers (ECRs), yet its role in shaping wellbeing and retention remains underexamined from the ECR perspective. We analyzed 2,604 anonymous survey responses from predoctoral, postdoctoral and former researchers across 65 countries. Overall, 76% of respondents reported that their supervisors attitude had a moderate or severe impact on mental health. Although most entered academia for vocational reasons, negative experiences with supervisors were among the most frequently reported reasons for leaving among former researchers (48%), comparable to job insecurity and financial instability. Harm was most often associated with poor communication, disregard for wellbeing, micromanagement and competitiveness. In contrast, ECRs valued supportive rather than boss-like supervision, regular communication, realistic expectations and respect for personal time. These findings identify supervisory behavior as a major and modifiable determinant or ECRs wellbeing and retention, and highlight the need for stronger institutional accountability, mentor training and funding incentives that recognize mentorship as a core component of research culture.

PurposeLow-dose radiation-induced adaptive response (LDRIAR) is well documented, but its role in early DNA damage signalling remains unclear. This study aimed to investigate whether adaptive response influences initial DNA double-strand break (DSB) recognition, as reflected by {gamma}H2AX foci formation, and to evaluate its time-dependent expression in human lymphocytes. Materials and MethodsPeripheral blood lymphocytes from three healthy donors were exposed to a priming dose followed by a challenging dose at defined time intervals. DNA damage was assessed using {gamma}H2AX foci analysis, comparing acute and split-dose exposures in both PHA-stimulated (large) and non-stimulated (small) lymphocytes. ResultsA clear time-dependent adaptive response was observed. No significant reduction in {gamma}H2AX foci was detected at 1 h (p > 0.05). At 2 h, a significant decrease was observed ([~]7-8% in large and [~]13% in small lymphocytes; p < 0.01), which increased at 4 h ([~]12% and [~]22%, respectively; p < 0.001). The maximal response occurred at 15 h, with reductions of [~]40- 43% in large and [~]27% in small lymphocytes (p < 0.001). Small lymphocytes exhibited an earlier response, while large lymphocytes showed a greater magnitude at later time points. The temporal trend was consistent across donors, with minor variability at later intervals. ConclusionsThe findings demonstrate that LDRIAR is reflected at the level of DNA damage signalling and follows a defined temporal pattern with cell-type specificity. This suggests that adaptive response may influence early DSB-associated processes, contributing to a better understanding of radiation response mechanisms in radiobiology.

BackgroundHepatic stellate cells (HSC) are Vitamin A storing non-parenchymal cells of the liver. During injury and inflammation, HSCs are the major contributors of excessive extracellular matrix (ECM) leading to Liver Fibrosis (LF). Emerging evidence suggests a fibrosis-independent role of these cells as key regulators of liver homeostasis and liver regeneration, emphasising on the dual role of HSCs in liver. HSCs are known to secrete several growth factors through which they largely execute their functions. However, the role of secretome (exosomes) from early activated or undifferentiated HSCs in a fibrotic milieu nor its composition are completely understood. MethodsLX-2 cells were cultured in low to no serum conditions and their isolated exosomes were transplanted into fibrotic severe combined immune deficient (SCID) mice livers, followed by post-transplantation analysis of the liver tissue and compared to the untreated controls. Total proteomic profiling of cell and exosomal cargo was performed using mass spectrometry and the data analysed and compared with the total HSC cell proteome. ResultsSignificant reduction in collagen in the transplanted mice livers compared to untreated fibrotic controls was observed with both the cells and exosomes transplantation. Comparative analysis revealed distinct enrichment of proteins and signaling pathways associated with extracellular matrix regulation, cellular communication, and metabolism in exosomes. Notably, these pathways are prominently represented in the exosomal fraction, suggesting a selective packaging of functional mediators. ConclusionThis study suggests the potential role of HSCs in regulating the complex liver homeostasis via exosomal network of proteins that contribute significantly to liver repair by ECM remodelling and growth factor-mediated signalling to regulate metabolism, fibrosis and liver regeneration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/721862v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@99bbf4org.highwire.dtl.DTLVardef@1029dd0org.highwire.dtl.DTLVardef@c6f578org.highwire.dtl.DTLVardef@1dba81_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundChimeric antigen receptor (CAR)-T cell therapies efficacy in solid tumors remains limited, largely due to the profoundly immunosuppressive tumor microenvironment (TME) which drives CAR-T cells to dysfunction and poor persistence. A comprehensive understanding of the dynamic interplay between CAR-T cells and the TME is therefore critical for the rational design of more effective CAR-T strategies for solid cancers. MethodsHere, we performed single-cell RNA sequencing of tumor samples from immunocompetent mice treated with stroma-targeting EDA-CAR-T cells, profiling CAR-T cell states and TME programs at the peak of antitumor response and during subsequent tumor progression. ResultsOur analysis revealed a marked temporal remodeling of EDA-CAR-T cells within the TME, where early antitumor efficacy is associated with concurrent expansion of cytotoxic effector CD8 CAR-T cells and activation of memory CD4 CAR-T subsets. Moreover, EDA-CAR-T cells effectively engaged the myeloid compartment, resulting in strengthened communication networks involving T cell activation. However, by tumor progression, EDA-CAR-T cells suffered a widespread transcriptional reprogramming towards dysfunction, characterized by loss of effector programs alongside induction of exhaustion and immunoregulatory pathways within the TME, including PD-L1/PD-L2 and TGF{beta} signaling, which impairs sustained immune responses. Notably, early CAR-T cell activation led to increased susceptibility to TME-mediated immunosuppression, revealing EDA-CAR-T-specific soluble galectin-mediated cell-to-cell interaction networks. ConclusionsTogether, this works offers a high-resolution view of CAR-T cell dynamics within the solid TME, uncovering cellular and molecular mechanisms of rapid functional decline and identifying regulatory pathways within the TME that can be exploited to improve CAR-T cell therapy efficacy in solid tumors. KEY MESSAGES OF THE ARTICLEO_ST_ABSWhat is already known on this topicC_ST_ABSThe determinants of CAR-T cell therapeutic efficacy in solid tumors remain poorly defined, largely due to the complexity of the immunosuppressive tumor microenvironment. In this effort, it is necessary to perform comprehensive and detailed mechanistic studies that capture CAR-T cell dynamics within the solid tumor microenvironment to understand treatment failure. What this study addsWe performed single-cell profiling of stroma-targeting EDA-CAR-T cells, revealing their dynamic reprogramming toward dysfunction within the solid tumor microenvironment. We dissected CAR-T cell states and their cell-to-cell interactions with the tumor microenvironment across response and tumor progression and identified mechanisms linking CAR-T cell functionality and therapeutic failure. How this study might affect research, practice or policyThis study provides comprehensive mechanistic insights from an immunocompetent model that can be leveraged to identify shared determinants of CAR-T cell functionality in solid tumors and potentially guide the rational development of improved CAR-T cell therapies.

Pluripotent stem cells derived from livestock species represent valuable systems for studying early mammalian development and for establishing renewable, well-defined cell sources; however, direct comparative characterization of distinct pluripotent stem cell platforms in sheep remains limited. In this study, we established and evaluated two ovine pluripotent stem cell types: reprogrammed induced pluripotent stem cells (siPSCs) and embryonic disc-derived stem cells (sEDSCs). Both siPSCs and sEDSCs exhibited core features of pluripotency, including compact colony morphology, alkaline phosphatase activity, expression of key pluripotency-associated markers, and maintenance of a normal ovine karyotype. Flow cytometry and quantitative RT-PCR analyses revealed broadly overlapping yet distinguishable pluripotency marker expression profiles between the two cell types. Functional pluripotency was confirmed by embryoid body formation and in vitro differentiation into derivatives of all three germ layers. To further assess lineage-specific differentiation competence and compare functional outputs relevant to mesodermal differentiation, both pluripotent stem cell types were directed towards the adipogenic lineage. While siPSCs and sEDSCs were each capable of adipogenic differentiation, differences in differentiation efficiency and marker expression were observed. Together, these findings demonstrate that ovine siPSCs and sEDSCs share core pluripotency characteristics while retaining distinct molecular and functional properties, providing a robust comparative framework for studies of ovine pluripotency, lineage specification, and stem cell biology.

O_LIPlants and fungi are major sources of natural products beneficial to society, making the study of distinct species essential for discovering new drugs and bioactive compounds. The medicinal mushroom "Lingzhi" or "Reishi" (Ganoderma lingzhi) is widely used in traditional medicine and extensively studied for its bioactive triterpenoids, yet it is commonly identified as Ganoderma lucidum, the type species of the genus, which lacks a type specimen. C_LIO_LIWe sequenced a G. lucidum specimen preserved in the Kew fungarium, which agreed with the original description and was collected from wood of Corylus avellana in southern England. Using this reference specimen, we compiled genomic and ITS barcoding datasets to explore the genetic and geographic variation within this species. C_LIO_LIWe showed that G. lingzhi and G. lucidum diverged more than 12 million years ago and that all seven "G. lucidum" genomes deposited in public databases belong to other species. More than 1000 barcoding sequences showed that the widely used homology-based ITS barcoding is not working in this group, which can be mitigated by a phylogenetic placement approach. The 149 sequences assigned to G. lucidum with high confidence showed a Eurasian distribution and introductions to North and South America and Africa. C_LIO_LIOur study underscores the importance of accurate species identification and provides guidance for a group of pharmaceutical and socially significant species. To further support future studies and the wider public in differentiating between G. lingzhi and G. lucidum, we propose using "False Lingzhi" as the English name for G. lucidum. C_LI Societal Impact StatementTraditional Chinese Medicine has expanded far beyond Asia, with growing markets in North America and Europe for supplements and functional foods. Lingzhi or Reishi (Ganoderma lingzhi), a well-known medicinal mushroom, is valued for its anti-inflammatory and anticancer properties. However, it is often misidentified with species that may not provide the same health benefits. This confusion poses risks to consumer safety, product regulation, and research. Here, we establish a reference using morphological and molecular tools for the most commonly misidentified species (Ganoderma lucidum) and propose the name "False Lingzhi" to distinguish it, supporting accurate identification, safer product development, and reliable research.

Aims/hypothesisQuantitative nanoscale analysis of insulin secretory granules (ISGs) in human pancreatic tissue has been limited by the lack of imaging methods that combine high resolution with large-scale sampling. We aimed to establish expansion microscopy (ExM) as a platform for in situ, quantitative analysis of ISG organisation in human {beta}-cells and to assess whether type 2 diabetes (T2D) is associated with alterations in granule size, abundance or spatial organisation. MethodsWe applied Magnify ExM to PFA-fixed, paraffin-embedded pancreatic tissue sections from 6 human donors, 3 non-diabetic (ND) and 3 T2D, enabling super-resolution optical imaging of insulin-labelled granules. Insulin-positive structures were segmented and analysed using a morphometric pipeline to quantitatively assess size, shape and spatial features. Granule clustering was quantified based on combined area and roundness criteria. ResultsThe diameter distribution of highly circular granules was similar between ND and T2D samples and estimates of granule number per cell indicated only a modest reduction in T2D ([~]25%). In contrast, mapping insulin-positive structures in a roundness-area space revealed a marked enrichment of large, irregular objects consistent with granule clustering in T2D. The fraction of clustered granules was significantly increased in T2D and strongly inversely correlated with insulin stimulation index (r = -0.85). Conclusions/interpretationThese results establish expansion microscopy as a powerful platform for quantitative nanoscale analysis of human pancreatic tissue and identify altered spatial organisation of insulin granules, rather than marked granule depletion, as a prominent feature associated with {beta}-cell dysfunction in T2D. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LI{beta}-cell dysfunction in type 2 diabetes is often attributed to reduced insulin content or {beta}-cell loss. C_LIO_LIInsulin secretory granules (ISGs) have been characterised ultrastructurally, but quantitative analysis in human tissue remains limited. C_LIO_LISuper-resolution approaches, including expansion microscopy, are emerging tools for nanoscale imaging in biological tissues. C_LI What is the key question?O_LIIs {beta}-cell dysfunction in type 2 diabetes associated with depletion of insulin granules or with altered spatial organisation? C_LI What are the new findings?O_LIInsulin granule size distribution is largely preserved in type 2 diabetes, with only a modest reduction in granule number per cell. C_LIO_LIA significant increase in insulin granule clustering is observed in diabetic {beta}-cells. C_LIO_LIGranule clustering is strongly inversely correlated with insulin secretion in the same donor tissues. C_LI How might this impact on clinical practice in the foreseeable future?O_LIIdentifying altered granule organisation as a feature of {beta}-cell dysfunction may help refine the understanding of disease mechanisms and guide future strategies targeting {beta}-cell function. C_LI

Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection and is increasingly explored for other dysbiosis-related disorders. However, its implementation as a regulated therapeutic strategy still requires robust donor screening, biosafety frameworks, and standardized processing workflows. Here, we describe the establishment of the first fecal microbiota biobank in the south of Brazil and evaluate the incorporation of metagenomic sequencing as a complementary layer of donor safety assessment. A structured donor selection pipeline based on international guidelines was implemented, integrating clinical screening, biochemical and serological testing, and microbiological analyses. Of 100 screened candidates, only four donors met all eligibility criteria and were included in the biobank, highlighting the stringency of the selection process. Shotgun metagenomic sequencing revealed a diverse resistome across all donors, including a shared core set of resistance-related genes alongside marked interindividual variability. Dominant antibiotic resistance genes included tetracycline-associated determinants, as well as ermF, CfxA-type {beta}-lactamases, and aminoglycoside-modifying enzymes, each linked to specific gut taxa. Notably, the relatively high abundance of tetW and ermF in Bacteroides fragilis suggests that this dominant commensal species may act as a reservoir for tetracycline and multidrug resistance determinants within the intestinal microbiota. Rather than serving as exclusion criteria, such determinants highlight the importance of integrating functional genomic profiling into donor characterization. Overall, this study provides a framework for microbiota biobank implementation and supports the use of metagenomics as a complementary strategy to improve biosafety and functional assessment in FMT.

1.Abiotic stresses like nitrogen deficiency and soil salinity are major factors contributing to low crop yields. The use of selective biofertilizers alleviates both types of stress. In this study, we investigated the biofertilizer activity and plant growth-promoting properties (PGP) of Rhodococcus jialingiae RS1 through cytosolic proteome remodelling. We cultured RS1 under two conditions, i) without and ii) with 6% NaCl, in nitrogen-deficient defined Burks medium. Under dual stress of nitrogen limitation and salt stress, Orbitrap LC-MS/MS proteomics revealed one-quarter of the proteome remodelling, particularly the upregulation of ribosomal synthesis and protein repair systems. As expected, we found high expression of EctC, an ectoine synthase, a key enzyme in osmolyte biosynthesis. Additionally, ribosomal and translational-associated factors, including RpsL, RpsS, RpsT, RpsR1, RplV, RplL, RplA, and elongation factor Tuf, were highly expressed, suggesting enhanced translational fidelity under dual stress. High levels of DNA protection protein, Dps suggest dual stress may lead to DNA damage. Upregulation of chaperones, environmental sensors (KinE), and redox transcriptional factors like WhiB3, Hsp18, AhpC, and MetE suggests protein misfolding and oxidative stress. Metabolic modulations were evident through high expression of IlvA, NAD-dependent glutamate dehydrogenase, lipid/envelope-remodelling enzymes, cutinase/esterases, lipases, endopeptidases like NlpC/P60 and transport systems. In contrast, proteins involved in urease structural components (urea-G), nitrogen regulators and ammonium transporters (GlnK and Amt) were downregulated. Dual stress may lead to an energy crisis, prompting strategic shifts away from high-ATP-dependent ureolytic nitrogen-scavenging pathways towards lower-energy nitrogen-assimilating routes, such as IlvA-mediated deamination and NAD-dependent glutamate dehydrogenation. Genetic manipulations of the above-mentioned genes or their homologues across the genera of microbes, plants, and crops may enhance resilience to abiotic stresses. Our studies reveal stress-responsive genes and biochemical pathways that could be used to improve transgenic efficacy in nitrogen-limited, saline soil and other (a)biotic stresses. Global Proteome Profiling of Rhodococcus jialingiae RS1 to Develop Transgenics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/724437v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1719d80org.highwire.dtl.DTLVardef@1b6b59org.highwire.dtl.DTLVardef@24d367org.highwire.dtl.DTLVardef@1b33224_HPS_FORMAT_FIGEXP M_FIG C_FIG