Label-free metabolic optical biomarkers track stem cell fate transition in real time.

Tracking stem cell fate transition is crucial for understanding their development and optimizing biomanufacturing. Destructive single-cell methods provide a pseudotemporal landscape of stem cell differentiation but cannot monitor stem cell fate in real time. We established a metabolic optical metric using label-free fluorescence lifetime imaging microscopy (FLIM), feature extraction and machine learning-assisted analysis, for real-time cell fate tracking. From a library of 205 metabolic optical biomarker (MOB) features, we identified 56 associated with hematopoietic stem cell (HSC) differentiation. These features collectively describe HSC fate transition and detect its bifurcate lineage choice. We further derived a MOB score measuring the "metabolic stemness" of single cells and distinguishing their division patterns. This score reveals a distinct role of asymmetric division in rescuing stem cells with compromised metabolic stemness and a unique mechanism of PI3K inhibition in promoting ex vivo HSC maintenance. MOB profiling is a powerful tool for tracking stem cell fate transition and improving their biomanufacturing from a single-cell perspective.

Timing of Biomarker Changes in Sporadic Alzheimer's Disease in Estimated Years from Symptom Onset.

OBJECTIVE: A clock relating amyloid positron emission tomography (PET) to time was used to estimate the timing of biomarker changes in sporadic Alzheimer disease (AD). METHODS: Research participants were included who underwent cerebrospinal fluid (CSF) collection within 2 years of amyloid PET. The ages at amyloid onset and AD symptom onset were estimated for each individual. The timing of change for plasma, CSF, imaging, and cognitive measures was calculated by comparing restricted cubic splines of cross-sectional data from the amyloid PET positive and negative groups. RESULTS: The amyloid PET positive sub-cohort (n = 118) had an average age of 70.4 ± 7.4 years (mean ± standard deviation) and 16% were cognitively impaired. The amyloid PET negative sub-cohort (n = 277) included individuals with low levels of amyloid plaque burden at all scans who were cognitively unimpaired at the time of the scans. Biomarker changes were detected 15-19 years before estimated symptom onset for CSF Aß42/Aß40, plasma Aß42/Aß40, CSF pT217/T217, and amyloid PET; 12-14 years before estimated symptom onset for plasma pT217/T217, CSF neurogranin, CSF SNAP-25, CSF sTREM2, plasma GFAP, and plasma NfL; and 7-9 years before estimated symptom onset for CSF pT205/T205, CSF YKL-40, hippocampal volumes, and cognitive measures. INTERPRETATION: The use of an amyloid clock enabled visualization and analysis of biomarker changes as a function of estimated years from symptom onset in sporadic AD. This study demonstrates that estimated years from symptom onset based on an amyloid clock can be used as a continuous staging measure for sporadic AD and aligns with findings in autosomal dominant AD. ANN NEUROL 2024;95:951-965.

Evaluation of the Electronic Clinical Dementia Rating for Dementia Screening.

Importance: The Clinical Dementia Rating (CDR) is a well-validated instrument widely used to detect and stage dementia due to Alzheimer disease. The digital Electronic Clinical Dementia Rating (eCDR) can be remotely self-administered and automatically scored, with potential to facilitate efficient dementia screening and staging. Objective: To evaluate the association of the eCDR with the CDR and other in-clinic assessments for screening older adults for cognitive impairment. Design, Setting, and Participants: This multisite, cross-sectional study used baseline data from a longitudinal, observational study from 2020 to 2023, including up to 3 years of follow-up. Participants were enrolled from 3 Alzheimer Disease Research Centers and the Brain Health Registry. Participants (aged ≥55 years, with a study partner, and no acute or unstable major medical conditions) were recruited during in-clinic visits or by automated emails. Exposures: Participants completed the Uniform Data Set Version 3 (UDS; including the CDR) in supervised clinical research settings, and then completed the eCDR remotely, online and unsupervised, using their own device. Main Outcomes and Measures: The primary outcomes were eCDR scores (item; categorical box and global; continuous box and global), CDR scores (item; categorical box and global), and UDS assessment scores. Associations were evaluated using linear and logistic regressions. Results: A total of 3565 participants were contacted, and 288 were enrolled. Among 173 participants with item-level data (mean [SD] age, 70.84 [7.65] years; 76 women [43.9%]), eCDR to CDR concordance was 90% or higher for 33 items (63%) and 70% to 89% for 13 items (25%). Box (domain) level concordance ranged from 80% (memory) to 99% (personal care). The global score concordance rate was 81%. κ statistics were fair to moderate. Among 206 participants with box and global scores (mean [SD] age, 71.34 [7.68] years; 95 women [46.1%]), eCDR continuous global score was associated with CDR global (categorical) score with an area under the receiver operating characteristic curve of 0.79 (95% CI, 0.70-0.87). Correlations between eCDR and in-clinic UDS assessments were similar to those between CDR sum of box scores and the same in-clinic assessments. Conclusions and Relevance: These findings suggest that the eCDR is valid and has potential use for screening and assessment of older adults for cognitive and functional decline related to Alzheimer disease. Instrument optimization and validation in diverse cohorts in remote settings are crucial for evaluating scalability and eCDR utility in clinical research, trials, and health care settings.

RUNX1 is required in granulocyte-monocyte progenitors to attenuate inflammatory cytokine production by neutrophils.

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancy (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 was shown to regulate inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to epigenetically repress two inflammatory signaling pathways in neutrophils: Toll-like receptor 4 (TLR4) and type I interferon (IFN) signaling. RUNX1 loss in GMPs augments neutrophils' inflammatory response to the TLR4 ligand lipopolysaccharide through increased expression of the TLR4 coreceptor CD14. RUNX1 binds Cd14 and other genes encoding proteins in the TLR4 and type I IFN signaling pathways whose chromatin accessibility increases when RUNX1 is deleted. Transcription factor footprints for the effectors of type I IFN signaling-the signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRFs)-were enriched in chromatin that gained accessibility in both GMPs and neutrophils when RUNX1 was lost. STAT1::STAT2 and IRF motifs were also enriched in the chromatin of retrotransposons that were derepressed in RUNX1-deficient GMPs and neutrophils. We conclude that a major direct effect of RUNX1 loss in GMPs is the derepression of type I IFN and TLR4 signaling, resulting in a state of fixed maladaptive innate immunity.

Design and feasibility of an Alzheimer's disease blood test study in a diverse community-based population.

INTRODUCTION: Alzheimer's disease (AD) blood tests are likely to become increasingly important in clinical practice, but they need to be evaluated in diverse groups before use in the general population. METHODS: This study enrolled a community-based sample of older adults in the St. Louis, Missouri, USA area. Participants completed a blood draw, Eight-Item Informant Interview to Differentiate Aging and Dementia (AD8® ), Montreal Cognitive Assessment (MoCA), and survey about their perceptions of the blood test. A subset of participants completed additional blood collection, amyloid positron emission tomography (PET), magnetic resonance imaging (MRI), and Clinical Dementia Rating (CDR® ). RESULTS: Of the 859 participants enrolled in this ongoing study, 20.6% self-identified as Black or African American. The AD8 and MoCA correlated moderately with the CDR. The blood test was well accepted by the cohort, but it was perceived more positively by White and highly educated individuals. DISCUSSION: Studying an AD blood test in a diverse population is feasible and may accelerate accurate diagnosis and implementation of effective treatments. HIGHLIGHTS: A diverse group of older adults was recruited to evaluate a blood amyloid test. The enrollment rate was high and the blood test was well accepted by participants. Cognitive impairment screens have moderate performance in a diverse population. Alzheimer's disease blood tests are likely to be feasible for use in real-world settings.

RUNX1 is required in granulocyte-monocyte progenitors to attenuate inflammatory cytokine production by neutrophils.

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancies (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 regulates inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to restrict the inflammatory response of neutrophils to toll-like receptor 4 (TLR4) signaling. Loss of RUNX1 in GMPs increased the TLR4 coreceptor CD14 on neutrophils, which contributed to neutrophils’ increased inflammatory cytokine production in response to the TLR4 ligand lipopolysaccharide. RUNX1 loss increased the chromatin accessibility of retrotransposons in GMPs and neutrophils and induced a type I interferon signature characterized by enriched footprints for signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRF) in opened chromatin, and increased expression of interferon-stimulated genes. The overproduction of inflammatory cytokines by neutrophils was reversed by inhibitors of type I IFN signaling. We conclude that RUNX1 restrains the chromatin accessibility of retrotransposons in GMPs and neutrophils, and that loss of RUNX1 increases proinflammatory cytokine production by elevating tonic type I interferon signaling.

Membrane-bound SCF and VCAM-1 synergistically regulate the morphology of hematopoietic stem cells.

Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized cues and regulate the long-term functions of adult stem cells, yet little is known about the underlying mechanisms. Here, we used a supported lipid bilayer (SLB) to recapitulate the membrane-bound interactions between hematopoietic stem cells (HSCs) and niche stromal cells. HSCs cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a polarized morphology with aggregated mSCF under a large protrusion through a synergy with VCAM-1 on the bilayer, which drastically enhances HSC adhesion. These features are unique to mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF-VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC maintenance, and minimizes its loss induced by soluble SCF. Our work thus reveals a unique role and signaling mechanism of membrane-bound factors in regulating stem cell morphology and function.

A Microdevice Platform Recapitulating Hypoxic Tumor Microenvironments.

Hypoxia plays a central role in cancer progression and resistance to therapy. We have engineered a microdevice platform to recapitulate the intratumor oxygen gradients that drive the heterogeneous hypoxic landscapes in solid tumors. Our design features a "tumor section"-like culture by incorporating a cell layer between two diffusion barriers, where an oxygen gradient is established by cellular metabolism and physical constraints. We confirmed the oxygen gradient by numerical simulation and imaging-based oxygen sensor measurement. We also demonstrated spatially-resolved hypoxic signaling in cancer cells through immunostaining, gene expression assay, and hypoxia-targeted drug treatment. Our platform can accurately generate and control oxygen gradients, eliminates complex microfluidic handling, allows for incorporation of additional tumor components, and is compatible with high-content imaging and high-throughput applications. It is well suited for understanding hypoxia-mediated mechanisms in cancer disease and other biological processes, and discovery of new therapeutics.

Three-Dimensional Cone Beam Computed Tomography Volumetric Outcomes of rhBMP-2/Demineralized Bone Matrix versus Iliac Crest Bone Graft for Alveolar Cleft Reconstruction.

BACKGROUND: Recent studies indicate that recombinant human bone morphogenetic protein-2 (rhBMP-2) in a demineralized bone matrix scaffold is a comparable alternative to iliac bone autograft in the setting of secondary alveolar cleft repair. Postreconstruction occlusal radiographs demonstrate improved bone stock when rhBMP-2/demineralized bone matrix (DBM) scaffold is used but lack the capacity to evaluate bone growth in three dimensions. This study uses cone beam computed tomography to provide the first clinical evaluation of volumetric and density comparisons between these two treatment modalities. METHODS: A prospective study was conducted with 31 patients and 36 repairs of the alveolar cleft over a 2-year period. Twenty-one repairs used rhBMP-2/DBM scaffold and 14 repairs used iliac bone grafting. Postoperatively, occlusal radiographs were obtained at 3 months to evaluate bone fill; cone beam computed tomographic images were obtained at 6 to 9 months to compare volumetric and density data. RESULTS: At 3 months, postoperative occlusal radiographs demonstrated that 67 percent of patients receiving rhBMP-2/DBM scaffold had complete bone fill of the alveolus, versus 56 percent of patients in the autologous group. In contrast, cone beam computed tomographic data showed 31.6 percent (95 percent CI, 24.2 to 38.5 percent) fill in the rhBMP-2 group compared with 32.5 percent (95 percent CI, 22.1 to 42.9 percent) in the autologous population. Density analysis demonstrated identical average values between the groups (1.38 g/cc). CONCLUSIONS: These data demonstrate comparable bone regrowth and density values following secondary alveolar cleft repair using rhBMP-2/DBM scaffold versus autologous iliac bone graft. Cone beam computed tomography provides a more nuanced understanding of true bone regeneration within the alveolar cleft that may contribute to the information provided by occlusal radiographs alone. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

A cost-effective micromilling platform for rapid prototyping of microdevices.

Micromilling has great potential in producing microdevices for lab-on-a-chip and organ-on-a-chip applications, but has remained under-utilized due to the high machinery costs and limited accessibility. In this paper, we assessed the machining capabilities of a low-cost 3-D mill in polycarbonate material, which were showcased by the production of microfluidic devices. The study demonstrates that this particular mill is well suited for the fabrication of multi-scale microdevices with feature sizes from micrometers to centimeters.

A Point-of-Care Prothrombin Time Test on a Microfluidic Disk Analyzer Using Alternate Spinning.

In this study, we conducted a fully integrated point-of-care prothrombin time test on a microfluidic disk analyzer. The microfluidic functions integrated on the disk were capable of separating whole blood, decanting plasma, and mixing it with reagents in sequence under alternate spinning. The assay protocol was completed by alternate spinning without using microvalves or surface modification. Clinical sample tests on prothrombin time measurement were conducted by both the microfluidic disk analyzer and the reference instrument used in medical centers. The test results showed a good correlation and agreement between the two instruments.