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.

The colocatome as a spatial -omic reveals shared microenvironment features between tumour-stroma assembloids and human lung cancer.

Computational frameworks to quantify and compare microenvironment spatial features of in-vitro patient-derived models and clinical specimens are needed. Here, we acquired and analysed multiplexed immunofluorescence images of human lung adenocarcinoma (LUAD) alongside tumour-stroma assembloids constructed with organoids and fibroblasts harvested from the leading edge (Tumour-Adjacent Fibroblasts;TAFs) or core (Tumour Core Fibroblasts;TCFs) of human LUAD. We introduce the concept of the "colocatome" as a spatial -omic dimension to catalogue all proximate and distant colocalisations between malignant and fibroblast subpopulations in both the assembloids and clinical specimens. The colocatome expands upon the colocalisation quotient (CLQ) through a nomalisation strategy that involves permutation analysis and thereby allows comparisons of CLQs under different conditions. Using colocatome analysis, we report that both TAFs and TCFs protected cancer cells from targeted oncogene treatment by uniquely reorganising the tumour-stroma cytoarchitecture, rather than by promoting cellular heterogeneity or selection. Moreover, we show that the assembloids' colocatome recapitulates the tumour-stroma cytoarchitecture defining the tumour microenvironment of LUAD clinical samples and thereby can serve as a functional spatial readout to guide translational discoveries.

Medical student attitudes and perceptions of psychedelic-assisted therapies.

Introduction: Although certain psychedelic agents may soon gain federal approval for use in treating specific psychiatric conditions, the utilization of such therapies in clinical practice will depend largely on the attitudes of healthcare providers. Therefore, this study assesses the current attitudes, knowledge, exposure, and acceptance of psychedelics and psychedelic-assisted therapies amongst medical students. Methods: In fall semester of 2022, surveys were emailed to 580 medical students attending medical institutions in the state of Nevada in the United States. Utilizing knowledge and attitude items from previously published studies, the survey collected demographic data and assessed student attitudes with five-point Likert-scale variables. Data was analyzed using summary statistics and Kruskal-Wallis tests for differences in mean survey scores (i.e., attitudes towards psychedelics) based on demographic factors. Results: 132 medical students participated in the survey (22.7% response rate). Medical students demonstrated overall positive attitudes towards psychedelics, lack of knowledge regarding psychedelics, and uncertainty towards neurocognitive risks of psychedelics. Overall, 78.6% of students agreed that psychedelics have therapeutic potential, while 95.2% agreed that psychedelics deserves further research in assessing this potential. Additionally, there was no statistically significant effect of demographic variables, including age, sex, and level of training, on attitudes. Discussion: Although students are overall curious and optimistic about psychedelics, they demonstrate a lack of knowledge regarding recent research efforts. As the field of psychiatry prepares to implement psychedelics and psychedelic-assisted therapies, education and awareness of such agents should be initiated early on in medical clinical training.

p53 governs an AT1 differentiation programme in lung cancer suppression.

Lung cancer is the leading cause of cancer deaths worldwide1. Mutations in the tumour suppressor gene TP53 occur in 50% of lung adenocarcinomas (LUADs) and are linked to poor prognosis1-4, but how p53 suppresses LUAD development remains enigmatic. We show here that p53 suppresses LUAD by governing cell state, specifically by promoting alveolar type 1 (AT1) differentiation. Using mice that express oncogenic Kras and null, wild-type or hypermorphic Trp53 alleles in alveolar type 2 (AT2) cells, we observed graded effects of p53 on LUAD initiation and progression. RNA sequencing and ATAC sequencing of LUAD cells uncovered a p53-induced AT1 differentiation programme during tumour suppression in vivo through direct DNA binding, chromatin remodelling and induction of genes characteristic of AT1 cells. Single-cell transcriptomics analyses revealed that during LUAD evolution, p53 promotes AT1 differentiation through action in a transitional cell state analogous to a transient intermediary seen during AT2-to-AT1 cell differentiation in alveolar injury repair. Notably, p53 inactivation results in the inappropriate persistence of these transitional cancer cells accompanied by upregulated growth signalling and divergence from lung lineage identity, characteristics associated with LUAD progression. Analysis of Trp53 wild-type and Trp53-null mice showed that p53 also directs alveolar regeneration after injury by regulating AT2 cell self-renewal and promoting transitional cell differentiation into AT1 cells. Collectively, these findings illuminate mechanisms of p53-mediated LUAD suppression, in which p53 governs alveolar differentiation, and suggest that tumour suppression reflects a fundamental role of p53 in orchestrating tissue repair after injury.

Evaluation of phenobarbital for prevention of alcohol withdrawal in trauma patients.

BACKGROUND: Up to 30% of trauma patients experience alcohol withdrawal syndrome (AWS) during their hospital admission, which is associated with worse outcomes. While benzodiazepines and phenobarbital are the mainstay of AWS management, there are limited data on the prevention of AWS. The objective was to evaluate the safety and efficacy of phenobarbital for the prevention of AWS. METHODS: Adult patients admitted to a level 1 trauma center who received at least one dose of phenobarbital for the prevention of AWS between January 2019 and August 2021 were included. Patients were case matched to a control group managed with symptom-triggered therapy based on risk of AWS. Risk factors included sex, age, history of AWS/delirium tremens or withdrawal seizures, selected laboratory values, and screening questionnaires. The primary endpoint was the need for rescue therapy. Secondary endpoints included the time to rescue therapy, intensive care unit (ICU) length of stay (LOS), and hospital LOS. RESULTS: Overall, 110 patients were included with 55 patients in each group. The phenobarbital group had higher baseline Injury Severity Scores ( p = 0.03) and were more likely to be admitted to the ICU (44% vs. 24%; p = 0.03). The phenobarbital group required less rescue therapy (16% vs. 62%; p < 0.001) with a longer time to rescue therapy administration (26 vs. 11 hours; p = 0.01). The phenobarbital group had a longer hospital LOS (216 vs. 87 hours; p = 0.0001) but no difference in ICU LOS ( p = 0.36). There was no incidence of delirium tremens or seizures and no difference in intubation rates ( p = 0.68). There was no incidence of hypotension associated with phenobarbital. CONCLUSION: Patients managed with phenobarbital had a lower need for rescue therapy for AWS with no increased adverse effects. Further studies should evaluate a protocol to prevent alcohol withdrawal in the trauma population. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level III.

Innate immune cell activation causes lung fibrosis in a humanized model of long COVID.

COVID-19 remains a global pandemic of an unprecedented magnitude with millions of people now developing "COVID lung fibrosis." Single-cell transcriptomics of lungs of patients with long COVID revealed a unique immune signature demonstrating the upregulation of key proinflammatory and innate immune effector genes CD47, IL-6, and JUN. We modeled the transition to lung fibrosis after COVID and profiled the immune response with single-cell mass cytometry in JUN mice. These studies revealed that COVID mediated chronic immune activation reminiscent to long COVID in humans. It was characterized by increased CD47, IL-6, and phospho-JUN (pJUN) expression which correlated with disease severity and pathogenic fibroblast populations. When we subsequently treated a humanized COVID lung fibrosis model by combined blockade of inflammation and fibrosis, we not only ameliorated fibrosis but also restored innate immune equilibrium indicating possible implications for clinical management of COVID lung fibrosis in patients.

Results of the phase I CCTG IND.231 trial of CX-5461 in patients with advanced solid tumors enriched for DNA-repair deficiencies.

CX-5461 is a G-quadruplex stabilizer that exhibits synthetic lethality in homologous recombination-deficient models. In this multicentre phase I trial in patients with solid tumors, 40 patients are treated across 10 dose levels (50-650 mg/m2) to determine the recommended phase II dose (primary outcome), and evaluate safety, tolerability, pharmacokinetics (secondary outcomes). Defective homologous recombination is explored as a predictive biomarker of response. CX-5461 is generally well tolerated, with a recommended phase II dose of 475 mg/m2 days 1, 8 and 15 every 4 weeks, and dose limiting phototoxicity. Responses are observed in 14% of patients, primarily in patients with defective homologous recombination. Reversion mutations in PALB2 and BRCA2 are detected on progression following initial response in germline carriers, confirming the underlying synthetic lethal mechanism. In vitro characterization of UV sensitization shows this toxicity is related to the CX-5461 chemotype, independent of G-quadruplex synthetic lethality. These results establish clinical proof-of-concept for this G-quadruplex stabilizer. Clinicaltrials.gov NCT02719977.

Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA.

Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We developed an unsupervised machine learning algorithm, CELESTA, which identifies the cell type of each cell, individually, using the cell's marker expression profile and, when needed, its spatial information. We demonstrate the performance of CELESTA on multiplexed immunofluorescence images of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, and validate our findings in an independent cohort. By coupling our spatial analysis with single-cell RNA-sequencing data on proximal sections of the same specimens, we identify cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of CELESTA to facilitate identification of clinically relevant interactions.

Reconstructing codependent cellular cross-talk in lung adenocarcinoma using REMI.

Cellular cross-talk in tissue microenvironments is fundamental to normal and pathological biological processes. Global assessment of cell-cell interactions (CCIs) is not yet technically feasible, but computational efforts to reconstruct these interactions have been proposed. Current computational approaches that identify CCI often make the simplifying assumption that pairwise interactions are independent of one another, which can lead to reduced accuracy. We present REMI (REgularized Microenvironment Interactome), a graph-based algorithm that predicts ligand-receptor (LR) interactions by accounting for LR dependencies on high-dimensional, small-sample size datasets. We apply REMI to reconstruct the human lung adenocarcinoma (LUAD) interactome from a bulk flow-sorted RNA sequencing dataset, then leverage single-cell transcriptomics data to increase the cell type resolution and identify LR prognostic signatures among tumor-stroma-immune subpopulations. We experimentally confirmed colocalization of CTGF:LRP6 among malignant cell subtypes as an interaction predicted to be associated with LUAD progression. Our work presents a computational approach to reconstruct interactomes and identify clinically relevant CCIs.

Graphene Nanoribbon Gap Waveguides for Dispersionless and Low-Loss Propagation with Deep-Subwavelength Confinement.

Surface plasmon polaritons (SPPs) have been attracting considerable attention owing to their unique capabilities of manipulating light. However, the intractable dispersion and high loss are two major obstacles for attaining high-performance plasmonic devices. Here, a graphene nanoribbon gap waveguide (GNRGW) is proposed for guiding dispersionless gap SPPs (GSPPs) with deep-subwavelength confinement and low loss. An analytical model is developed to analyze the GSPPs, in which a reflection phase shift is employed to successfully deal with the influence caused by the boundaries of the graphene nanoribbon (GNR). It is demonstrated that a pulse with a 4 µm bandwidth and a 10 nm mode width can propagate in the linear passive system without waveform distortion, which is very robust against the shape change of the GNR. The decrease in the pulse amplitude is only 10% for a propagation distance of 1 µm. Furthermore, an array consisting of several GNRGWs is employed as a multichannel optical switch. When the separation is larger than 40 nm, each channel can be controlled independently by tuning the chemical potential of the corresponding GNR. The proposed GNRGW may raise great interest in studying dispersionless and low-loss nanophotonic devices, with potential applications in the distortionless transmission of nanoscale signals, electro-optic nanocircuits, and high-density on-chip communications.

Using the Fluorescent Dye, Rhodamine B, to Study Mating Competitiveness in Male Aedes aegypti Mosquitoes.

The success of sterile or incompatible insect technique-based population suppression programs depends on the ability of released males to compete for wild-type females and induce sterility in the target population. Hence, laboratory assessment of male mating competitiveness is essential for evaluating the release strain's fitness before field release. Conventionally, such an assay is performed by determining the proportion of viable eggs produced by the females after being simultaneously exposed to two sets of males (wild-type and release strains) for copulation. However, this process is time-consuming and laborious due to the need to first blood-feed the females for egg production and then hatch and enumerate the hatched eggs to determine egg viability.  Moreover, this method cannot discern the degree of competitiveness between two sterile or Wolbachia-infected mosquito lines as wild-type female mosquitoes will only produce non-viable eggs upon mating with both. To circumvent these limitations, this paper describes a more direct method of measuring male mosquito mating competitiveness in laboratory settings using the fluorescent dye, rhodamine B (RhB), which can be used to mark males by feeding them in sucrose solution containing RhB. After the mating assay, the presence of fluorescing sperms in the spermathecae of a female can be used to determine her mating partner. This method is cost-effective, reduces the experimental time by 90% and allows comparison of mating fitness between two sterile or Wolbachia-infected lines.

Ventricular tachycardia converts to sinus rhythm after administration of propofol.

Ventricular tachycardia (VT) is a major contributor to sudden cardiac death, and pharmacologic treatment options beyond antiarrhythmics are limited. Emerging data suggest sympathetic blocking agents such as propofol are a potential management option for VT refractory to first line antiarrhythmics. Previous literature has described fixed-dose propofol boluses and continuous infusions to convert ventricular arrhythmias; however, to our knowledge, there are no reports of a weight-based dosing strategy for VT. We present the case of a patient with amiodarone-refractory VT who received a 1 mg/kg propofol bolus in preparation for cardioversion and subsequently converted to normal sinus rhythm. The patient stabilized following these interventions, transferred to a tertiary care facility, and was discharged home with an implantable cardioverter defibrillator.

Palmitate-Induced IRE1-XBP1-ZEB Signaling Represses Desmoplakin Expression and Promotes Cancer Cell Migration.

Elevated uptake of saturated fatty acid palmitate is associated with metastatic progression of cancer cells; however, the precise signaling mechanism behind the phenomenon is unclear. The loss of cell adhesion proteins, such as desmoplakin (DSP), is a key driving event in the transformation of cancer cells to more aggressive phenotypes. Here, we investigated the mechanism by which palmitate induces the loss of DSP in liver and breast cancer cells. We propose that palmitate activates the IRE1-XBP1 branch of the endoplasmic reticulum (ER) stress pathway to upregulate the ZEB transcription factor, leading to transcriptional repression of DSP. Using liver and breast cancer cells treated with palmitate, we found loss of DSP leads to increased cell migration independent of E-cadherin. We report that the ZEB family of transcription factors function as direct transcriptional repressors of DSP. CRISPR-mediated knockdown of IRE1 confirmed that the transcription of ZEB, loss of DSP, and enhanced migration in the presence of palmitate is dependent on the IRE1-XBP1 pathway. In addition, by analyzing the somatic expression and copy number variation profiles of over 11,000 tumor samples, we corroborate our hypothesis and establish the clinical relevance of DSP loss via ZEB in human cancers. IMPLICATIONS: Provides mechanistic link on palmitate-induced activation of IRE1α to cancer cell migration.

Epigenomic programming in early fetal brain development.

Aim: To provide a comprehensive understanding of gene regulatory networks in the developing human brain and a foundation for interpreting pathogenic deregulation. Materials & methods: We generated reference epigenomes and transcriptomes of dissected brain regions and primary neural progenitor cells (NPCs) derived from cortical and ganglionic eminence tissues of four normal human fetuses. Results: Integration of these data across developmental stages revealed a directional increase in active regulatory states, transcription factor activities and gene transcription with developmental stage. Consistent with differences in their biology, NPCs derived from cortical and ganglionic eminence regions contained common, region specific, and gestational week specific regulatory states. Conclusion: We provide a high-resolution regulatory network for NPCs from different brain regions as a comprehensive reference for future studies.

Graphene Plasmon Resonances for Electrically-Tunable Sub-Femtometer Dimensional Resolution.

A coupled graphene structure (CGS) is proposed to obtain an electrically tunable sub-femtometer (sub-fm) dimensional resolution. According to analytical and numerical investigations, the CGS can support two branches of localized surface plasmon resonances (LSPRs), which park at the dielectric spacer between two pieces of graphene. The coupled efficiencies of the odd-order modes are even four orders of magnitude higher than that of the even-order modes. In particular, a sub-fm resolution for detecting the change in the spacer thickness can be reached using the lowest order LSPR mode. The LSPR wavelength and the dimensional differential resolution can be electrically-tuned from 9.5 to 33 µm and from 4.3 to 15 nm/pm, respectively, by modifying the chemical potential of the graphene via the gate voltage. Furthermore, by replacing the graphene ribbon (GR) at the top of the CGS with multiple GRs of different widths, a resonant frequency comb in the absorption spectrum with a tunable frequency interval is generated, which can be used to detect the changes in spacer thicknesses at different locations with sub-fm resolution.

Breast cancer survival and the expression of genes related to alcohol drinking.

Breast cancer is the leading cause of cancer-related disease in women. Cumulative evidence supports a causal role of alcohol intake and breast cancer incidence. In this study, we explore the change on expression of genes involved in the biological pathways through which alcohol has been hypothesized to impact breast cancer risk, to shed new insights on possible mechanisms affecting the survival of breast cancer patients. Here, we performed differential expression analysis at individual genes and gene set levels, respectively, across survival and breast cancer subtype data. Information about postdiagnosis breast cancer survival was obtained from 1977 Caucasian female participants in the Molecular Taxonomy of Breast Cancer International Consortium. Expression of 16 genes that have been linked in the literature to the hypothesized alcohol-breast cancer pathways, were examined. We found that the expression of 9 out of 16 genes under study were associated with cancer survival within the first 4 years of diagnosis. Results from gene set analysis confirmed a significant differential expression of these genes as a whole too. Although alcohol consumption is not analyzed, nor available for this dataset, we believe that further study on these genes could provide important information for clinical recommendations about potential impact of alcohol drinking on breast cancer survival.

Sample Tracking Using Unique Sequence Controls.

Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (<30 bases) and contain no long homopolymer (>7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.

Disgust regulation between menstrual cycle phases: Differential effects of emotional suppression and reappraisal.

BACKGROUND AND OBJECTIVE: Immunosuppression is characteristic of the luteal phase of the menstrual cycle and is accompanied by an adaptive disgust response to reduce contact with pathogens. However, research has not examined the effects of emotion regulation on disgust during the menstrual cycle. Accordingly, the present study examines the effect of suppression and reappraisal on disgust during the luteal and follicular phases of the menstrual cycle. METHOD: Menstrual cycle phase was estimated in a sample of naturally cycling women (n = 73), and those in the follicular or luteal phase were assigned to suppress or reappraise disgust while watching a disgust-inducing video. Physiological arousal during the video and avoidance of disgust cues in a public restroom after the video were also assessed. RESULTS: No differences were observed in self-reported disgust to the video between those who suppressed and those who reappraised in the luteal phase. However, women in the follicular phase who suppressed reported less disgust than those who reappraised. The emotion regulation strategies did not influence physiology during the video or avoidance after the video as a function of menstrual cycle phase. LIMITATIONS: Hormone assay data was not collected to confirm menstrual cycle phase and a relatively small sample of naturally cycling women was used. CONCLUSIONS: Suppression may be a more effective strategy than reappraisal for reducing verbal disgust in the follicular phase but not in the luteal phase. The implications of these findings for the treatment of disgust-based disorders among women are discussed.

Tunable photonic-like modes in graphene-coated nanowires.

In this study, the dispersion equations of a graphene-coated nanowire (GN) are solved. It is found that in this waveguide, besides the surface plasmon polaritons (SPPs), there is another branch of guided modes, called photonic-like modes. The propagation distances of the photonic-like modes can be five orders of magnitude longer than those of the SPPs. Moreover, they can be modulated in the range of 10-4 to 1 m by changing the chemical potential of graphene. In particular, the mode field distributions remain nearly unchanged during the modulation. Based on the analysis performed using COMSOL Multiphysics, we further demonstrated that the propagation losses of the photonic-like modes are dependent on not only the chemical potential of graphene, but also the mode power proportion inside graphene. The photonic-like modes have tremendous potential to be used in optical switches, modulators, and switches in magnetic fields at the nanoscale.