Likelihood-based deconvolution of bulk gene expression data using single-cell references.

Direct comparison of bulk gene expression profiles is complicated by distinct cell type mixtures in each sample that obscure whether observed differences are actually caused by changes in the expression levels themselves or are simply a result of differing cell type compositions. Single-cell technology has made it possible to measure gene expression in individual cells, achieving higher resolution at the expense of increased noise. If carefully incorporated, such single-cell data can be used to deconvolve bulk samples to yield accurate estimates of the true cell type proportions, thus enabling one to disentangle the effects of differential expression and cell type mixtures. Here, we propose a generative model and a likelihood-based inference method that uses asymptotic statistical theory and a novel optimization procedure to perform deconvolution of bulk RNA-seq data to produce accurate cell type proportion estimates. We show the effectiveness of our method, called RNA-Sieve, across a diverse array of scenarios involving real data and discuss extensions made uniquely possible by our probabilistic framework, including a demonstration of well-calibrated confidence intervals.

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm2 that is well below damage thresholds (25 J/cm2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent versus activated (CD3/CD28/CD2) primary human T cells, and quiescent versus activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

In vitro and in vivo anti-tumor effect of Trichobakin fused with urokinase-type plasminogen activator ATF-TBK.

BACKGROUND: Trichobakin (TBK), a member of type I ribosome-inactivating proteins (RIPs), was first successfully cloned from Trichosanthes sp Bac Kan 8-98 in Vietnam. Previous study has shown that TBK acts as a potential protein synthesis inhibitor; however, the inhibition efficiency and specificity of TBK on cancer cells remain to be fully elucidated. METHODS AND RESULTS: In this work, we employed TBK and TBK conjugated with a part of the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), which contains the Ω-loop that primarily interacts with urokinase-type plasminogen activator receptor, and can be a powerful carrier in the drug delivery to cancer cells. Four different human tumor cell lines and BALB/c mice bearing Lewis lung carcinoma cells (LLC) were used to evaluate the role of TBK and ATF-TBK in the inhibition of tumor growth. Here we showed that the obtained ligand fused RIP (ATF-TBK) reduced the growth of four human cancer cell lines in vitro in the uPA receptor level-dependent manner, including the breast adenocarcinoma MDA-MB 231 cells and MCF7 cells, the prostate carcinoma LNCaP cells and the hepatocellular carcinoma HepG2 cells. Furthermore, the conjugate showed anti-tumor activity and prolonged the survival time of tumor-bearing mice. The ATF-TBK also did not cause the death of mice with doses up to 48 mg/kg, and they were not significantly distinct on parameters of hematology and serum biochemistry between the control and experiment groups. CONCLUSIONS: In conclusion, ATF-TBK reduced the growth of four different human tumor cell lines and inhibited lung tumor growth in a mouse model with little side effects. Hence, the ATF-TBK may be a target to consider as an anti-cancer agent for clinical trials.

Human visceral adipose tissue microvascular endothelial cell isolation and establishment of co-culture with white adipocytes to analyze cell-cell communication.

Communication between adipocytes and endothelial cells (EC) is suggested to play an important role in the metabolic function of white adipose tissue. In order to generate tools to investigate in detail the physiology and communication of EC and adipocytes, a method for isolation of adipose microvascular EC from visceral adipose tissue (VAT) biopsies of subjects with obesity was developed. Moreover, mature white adipocytes were isolated from the VAT biopsies by a method adapted from a previously published Membrane aggregate adipocytes culture (MAAC) protocol. The identity and functionality of the cultivated and isolated adipose microvascular EC (AMvEC) was validated by imaging their morphology, analyses of mRNA expression, fluorescence activated cell sorting (FACS), immunostaining, low-density lipoprotein (LDL) uptake, and in vitro angiogenesis assays. Finally, we established a new trans filter co-culture system (membrane aggregate adipocyte and endothelial co-culture, MAAECC) for the analysis of communication between the two cell types. EC-adipocyte communication in this system was validated by omics analyses, revealing several altered proteins belonging to pathways such as metabolism, intracellular transport and signal transduction in adipocytes co-cultured with AMvEC. In reverse experiments, induction of several pathways including endothelial development and functions was found in AMvEC co-cultured with adipocytes. In conclusion, we developed a robust method to isolate EC from small quantities of human VAT. Furthermore, the MAAECC system established during the study enables one to study the communication between primary white adipocytes and EC or vice-versa and could also be employed for drug screening.

The Germin-like protein OsGER4 is involved in promoting crown root development under exogenous jasmonic acid treatment in rice.

In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.

The Germin-like protein gene OsGER4 is involved in heat stress response in rice root development.

Rice (Oryza sativa L.) is one of the most important dietary carbohydrate sources for half of the world's population. However, it is not well adapted to environmental stress conditions, necessitating to create new and improved varieties to help ensure sufficient rice production in the face of rising populations and shrinking arable land. Recently, the development of the CRISPR/Cas9 gene editing system has allowed researchers to study functional genomics and engineer new rice varieties with great efficiency compared to conventional methods. In this study, we investigate the involvement of OsGER4, a germin-like protein identified by a genome-wide association study that is associated with rice root development under a stress hormone jasmonic acids treatment. Analysis of the OsGER4 promoter region revealed a series of regulatory elements that connect this gene to ABA signaling and water stress response. Under heat stress, osger4 mutant lines produce a significantly lower crown root than wild-type Kitaake rice. The loss of OsGER4 also led to the reduction of lateral root development. Using the GUS promoter line, OsGER4 expression was detected in the epidermis of the crown root primordial, in the stele of the crown root, and subsequently in the primordial of the lateral root. Taken together, these results illustrated the involvement of OsGER4 in root development under heat stress by regulating auxin transport through plasmodesmata, under control by both ABA and auxin signaling.

EGGTART: A tool to visualize the dynamics of biophysical transport under the inhomogeneous l-TASEP.

The totally asymmetric simple exclusion process (TASEP), which describes the stochastic dynamics of interacting particles on a lattice, has been actively studied over the past several decades and applied to model important biological transport processes. Here, we present a software package, called EGGTART (Extensive GUI gives TASEP-realization in Real Time), which quantifies and visualizes the dynamics associated with a generalized version of the TASEP with an extended particle size and heterogeneous jump rates. This computational tool is based on analytic formulas obtained from deriving and solving the hydrodynamic limit of the process. It allows an immediate quantification of the particle density, flux, and phase diagram, as a function of a few key parameters associated with the system, which would be difficult to achieve via conventional stochastic simulations. Our software should therefore be of interest to biophysicists studying general transport processes and can in particular be used in the context of gene expression to model and quantify mRNA translation of different coding sequences.

Time-domain single photon-excited autofluorescence lifetime for label-free detection of T cell activation.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique, capable of label-free assessment of the metabolic state and function within single cells. The FLIM measurements of autofluorescence were recently shown to be sensitive to the functional state and subtype of T cells. Therefore, autofluorescence FLIM could improve cell manufacturing technologies for adoptive immunotherapy, which currently require a time-intensive process of cell labeling with fluorescent antibodies. However, current autofluorescence FLIM implementations are typically too slow, bulky, and prohibitively expensive for use in cell manufacturing pipelines. Here we report a single photon-excited confocal whole-cell autofluorescence system that uses fast field-programmable gate array-based time tagging electronics to achieve time-correlated single photon counting (TCSPC) of single-cell autofluorescence. The system includes simultaneous near-infrared bright-field imaging and is sensitive to variations in the fluorescence decay profile of the metabolic coenzyme NAD(P)H in human T cells due to the activation state. The classification of activated and quiescent T cells achieved high accuracy and precision (area under the receiver operating characteristic curve, AUC = 0.92). The lower-cost, higher acquisition speed, and resistance to pile-up effects at high photon flux compared to traditional multiphoton-excited FLIM and TCSPC implementations with similar SNR make this system attractive for integration into flow cytometry, sorting, and quality control in cell manufacturing.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF.

Nerve growth factor (NGF) and pro-NGF increase low-density lipoprotein (LDL) receptors in neuronal cells partly by different mechanisms: role of LDL in neurite outgrowth.

Low-density lipoprotein receptors (LDLRs) mediate the uptake of lipoprotein particles into cells, as studied mainly in peripheral tissues. Here, we show that nerve growth factor (NGF) increases LDLR levels in PC6.3 cells and in cultured septal neurons from embryonic rat brain. Study of the mechanisms showed that NGF enhanced transcription of the LDLR gene, acting mainly via Tropomyosin receptor kinase A receptors. Simvastatin, a cholesterol-lowering drug, also increased the LDLR expression in PC6.3 cells. In addition, pro-NGF and pro-brain-derived neurotrophic factor, acting via the p75 neurotrophin receptor (p75NTR) also increased LDLRs. We further observed that Myosin Regulatory Light Chain-Interacting Protein/Inducible Degrader of the LDLR (Mylip/Idol) was down-regulated by pro-NGF, whereas the other LDLR regulator, proprotein convertase subtilisin kexin 9 (PCSK9) was not significantly changed. On the functional side, NGF and pro-NGF increased lipoprotein uptake by neuronal cells as shown using diacetyl-labeled LDL. The addition of serum-derived lipoprotein particles in conjunction with NGF or simvastatin enhanced neurite outgrowth. Collectively, these results show that NGF and simvastatin are able to stimulate lipoprotein uptake by neurons with a positive effect on neurite outgrowth. Increases in LDLRs and lipoprotein particles in neurons could play a functional role during brain development, in neuroregeneration and after brain injuries. Nerve growth factor (NGF) and pro-NGF induce the expression of low-density lipoprotein receptors (LDLRs) in neuronal cells leading to increased LDLR levels. Pro-NGF also down-regulated myosin regulatory light chain-interacting protein/inducible degrader of the LDLR (Mylip/Idol) that is involved in the degradation of LDLRs. NGF acts mainly via Tropomyosin receptor kinase A (TrkA) receptors, whereas pro-NGF stimulates p75 neurotrophin receptor (p75NTR). Elevated LDLRs upon NGF and pro-NGF treatments enhanced lipoprotein uptake by neurons. Addition of LDL particles further led to the stimulation of neurite outgrowth in PC6.3 cells after NGF or simvastatin treatments, suggesting a stimulatory role of lipoproteins on neuronal differentiation. In contrast, pro-NGF had no effect on neurite outgrowth either in the absence or presence of LDL particles. The precise mechanisms by which increased lipoproteins uptake can affect neurite outgrowth warrant further studies.

Peroxisome proliferator-activated receptor-γ coactivator-1α mediates neuroprotection against excitotoxic brain injury in transgenic mice: role of mitochondria and X-linked inhibitor of apoptosis protein.

Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is a transcriptional coactivator involved in the regulation of mitochondrial biogenesis and cell defense. The functions of PGC-1α in physiology of brain mitochondria are, however, not fully understood. To address this we have studied wild-type and transgenic mice with a two-fold overexpression of PGC-1α in brain neurons. Data showed that the relative number and basal respiration of brain mitochondria were increased in PGC-1α transgenic mice compared with wild-type mitochondria. These changes occurred concomitantly with altered levels of proteins involved in oxidative phosphorylation (OXPHOS) as studied by proteomic analyses and immunoblottings. Cultured hippocampal neurons from PGC-1α transgenic mice were more resistant to cell degeneration induced by the glutamate receptor agonist kainic acid. In vivo kainic acid induced excitotoxic cell death in the hippocampus at 48 h in wild-type mice but significantly less so in PGC-1α transgenic mice. However, at later time points cell degeneration was also evident in the transgenic mouse hippocampus, indicating that PGC-1α overexpression can induce a delay in cell death. Immunoblotting showed that X-linked inhibitor of apoptosis protein (XIAP) was increased in PGC-1α transgenic hippocampus with no significant changes in Bcl-2 or Bcl-X. Collectively, these results show that PGC-1α overexpression contributes to enhanced neuronal viability by stimulating mitochondria number and respiration and increasing levels of OXPHOS proteins and the anti-apoptotic protein XIAP.

Rotation from methadone to buprenorphine using a micro-dosing regime in patients with opioid use disorder and serious mental illness: A case series.

INTRODUCTION: Inducting buprenorphine from methadone has traditionally involved initial opioid withdrawal, with risk of mental state deterioration in patients with serious mental illness (SMI). Micro-dosing of buprenorphine, with small incremental doses, is a novel off-label approach to transitioning from methadone and does not require a period of methadone abstinence. Given the limited literature about buprenorphine microdosing, we aimed to evaluate the feasibility and safety of inducting buprenorphine in a series of patients on methadone with SMI. METHODS: For this retrospective case series, we reviewed the records of 16 patients with SMI at a Melbourne addiction treatment centre, from January 2021 to July 2022, who transitioned via micro-dosing, from high-dose methadone (>30 mg) to buprenorphine and depot-buprenorphine. Psychiatric diagnoses, mental state, other substance withdrawal, transfer success, transition time, opioid withdrawal symptoms and overall patient experience were collected via objective and subjective reporting. RESULTS: Methadone to buprenorphine transfer was completed by 88% of patients. Mental health measures remained stable with the exception of mildly increased anxiety. Median transfer time was 6.5 days for inpatients, 9 days for mixed setting and 10 days for outpatients. Most patients (93%) rated their experience 'manageable' reporting mild withdrawal symptoms. One patient met study criteria for precipitated withdrawal. DISCUSSION AND CONCLUSIONS: This retrospective case series provides evidence that the use of a micro-dosing buprenorphine induction for methadone to buprenorphine transitions, including to depot-buprenorphine, has negligible risk, is tolerated by patients with SMI and is unlikely to precipitate an exacerbation of their mental illness.

Disparities in incidence and survival for patients with Ewing sarcoma in Florida.

BACKGROUND: Ewing sarcoma (ES) is a malignant bone tumor most commonly affecting non-Hispanic White (NHW) adolescent males, though recognition among Hispanic individuals is rising. Prior population-based studies in the United States (US), utilizing Surveillance, Epidemiology, and End Results (SEER) have shown higher all-cause mortality among White Hispanics, Blacks, and those of low socioeconomic status (SES). Florida is not part of SEER but is home to unique Hispanic populations including Cubans, Puerto Ricans, South Americans that contrasts with the Mexican Hispanic majority in other US states. This study aimed to assess racial/ethnic disparities on incidence and survival outcomes among this diverse Florida patient population. METHODOLOGY: Our study examined all patients diagnosed with osseous ES (2005-2018) in Florida (n = 411) based on the state's population-based cancer registry dataset. Florida Age-adjusted Incidence Rates (AAIRs) were computed by sex and race-ethnicity and compared to the equivalent populations in SEER. Cause-specific survival disparities among Florida patients were examined using Kaplan-Meier analysis. Univariable and multivariable analyses using Cox regression were performed for race/ethnicity, with adjustment for age, sex, year of diagnosis, site of disease, staging, SES, and insurance type. RESULTS: There was a significantly higher incidence of osseous ES in Florida Hispanic males (AAIR 2.6/1,000,000); (95% CI: 2.0-3.2 per 1,000,000; n = 84) compared to the SEER Hispanic males (AAIR 1.2/1,000,000;1.1-1.4 per 1,000,000; n = 382). Older age, distant metastasis, lack of chemotherapy or surgical resection were statistically significant determinants of poor survival while SES, insurance status and race-ethnicity were not. However, among nonmetastatic ES, Florida Hispanics had an increased risk of death compared to Florida NHW (adjusted Hazard Ratio 2.32; 95%CI: 1.20-4.46; p = 0.012). CONCLUSIONS: Florida Hispanic males have a higher-than-expected incidence of osseous ES compared to the US. Hispanics of both sexes show remarkably worse survival for nonmetastatic disease compared to NHW. This disparity is likely multifactorial and requires further in-depth studies.

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375nm picosecond-pulsed diode laser operating at 50MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification ( i.e ., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5ms integration time per cell, resulting in a light dose of 2.65 J/cm 2 that is well below damage thresholds (25 J/cm 2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent vs. activated (CD3/CD28/CD2) primary human T cells, and quiescent vs. activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant.

A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83 %. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 min, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20 % loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83 % to approximately 92-95 %.

Metabolic priming of GD2 TRAC -CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence.

Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact T-cell phenotypes. CRISPR/Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant ( TRAC ) gene resulting in TRAC -CAR T cells with an enriched stem cell memory T-cell population, a process that could be further optimized through modifications to the media composition. In this study we generated anti-GD2 TRAC -CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine low media and then expanded in glucose/glutamine high media. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro and potency against human GD2+ xenograft neuroblastoma models in vivo . Compared to standard TRAC -CAR T cells, MP TRAC -CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity and reduced IFN-γ, IL-2, IP-10, IL-1ß, IL-17, and TGFß production at the end of manufacturing ex vivo , with increased central memory CAR T cells and better persistence observed in vivo . Metabolic priming with media during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo , which could lead to better responses against solid tumors in vivo .

Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence.

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex, with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro, and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells, MP TRAC-CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity, and reduced IFN-γ, IL-2, IP-10, IL-1ß, IL-17, and TGF-ß production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo, which could lead to better responses against solid tumors in vivo.

Touch-free optical technologies to streamline the production of T cell therapies.

Currently approved adoptive T cell therapy relies on autologous (obtained from the same patient) T cells, which often suffer from poor quality that diminishes treatment efficacy. Due to the heterogeneous nature of T cell quality between and within patients, significant efforts are aimed at optimizing cell manipulation and growth conditions for potent T cell products. We believe that touch-free imaging and sensing technologies are critical to monitor single-cell features during T cell manufacturing to ensure consistent and optimally timed methods for cell manipulation and growth. Here, we discuss emerging label-free optical imaging and sensing methods, along with machine learning techniques that could enable in-line feedback to optimize T cell quality at multiple stages during manufacturing. These methods have the potential to streamline current workflow, accelerate the manufacture of safe high-quality T cell therapies, and improve our understanding of the dynamic, heterogeneous processes of T cell manufacturing.

Water lettuce (Pistia stratiotes L.) increases biogas effluent pollutant removal efficacy and proves a positive substrate for renewable energy production.

Background: Aquatic plants play a crucial role in nature-based wastewater treatment and provide a promising substrate for renewable energy production using anaerobic digestion (AD) technology. This study aimed to examine the contaminant removal from AD effluent by water lettuce (WL) and produce biogas from WL biomass co-digested with pig dung (PD) in a farm-scale biogas digester. Methods: The first experiment used styrofoam boxes containing husbandry AD effluent. WLs were initially arranged in 50%, 25%, 12.5%, and 0% surface coverage. Each treatment was conducted in five replicates under natural conditions. In the second experiment, WL biomass was co-digested with PD into an existing anaerobic digester to examine biogas production on a farm scale. Results: Over 30 days, the treatment efficiency of TSS, BOD5, COD, TKN, and TP in the effluent was 93.75-97.66%, 76.63-82.56%, 76.78-82.89%, 61.75-63.75%, and 89.00-89.57%, respectively. Higher WL coverage increased the pollutant elimination potential. The WL biomass doubled after 12 days for all treatments. In the farm-scale biogas production, the biogas yield varied between 190.6 and 292.9 L kg VSadded-1. The methane content reached over 54%. Conclusions: WL removed AD effluent nutrients effectively through a phytoremediation system and generated significant biomass for renewable energy production in a farm-scale model.

Qualitative feasibility study of the mobile app Destroke for clinical stroke monitoring based on the NIH stroke scale.

Background: Stroke is a leading cause of severe disability in the United States, but there is no effective method for patients to accurately detect the signs of stroke at home. We developed a mobile app, Destroke, that allows remote performance of a modified NIH stroke scale (NIHSS) by patients. Aims: To assess the feasibility of a mobile app for stroke monitoring and education by patients with a history of stroke. Materials and methods: We enrolled 25 patients with a history of stroke in a prospective open-label study to evaluate the feasibility of the Destroke app in patients with stroke. Nineteen patients completed all study assessments, with a median time from stroke onset to enrollment of 5.6 years (range 0.1-12 years). We designed a modified NIHSS that assessed 12 out of 16 tasks on the NIHSS. Patients completed this test eight times over a 28-day period. We conducted pre-study surveys that assessed demographic information, stroke and cardiovascular history, baseline NIHSS, and experience using mobile technologies, and mid- and post-study surveys that assessed patient satisfaction on app usage and confidence in stroke detection. Results: Ten men and nine women participated in this study (median age of 64 (33-76)), representing ten US states and Washington D.C. Median baseline NIHSS was 0 (0-4). 15 patients reported using health apps. On a 5-point Likert scale, patients rated the app as 4.2 on being able to understand and use the app and 4.3 on using the app when instructed by their doctor. For eight patients with poor confidence in detecting the signs of a stroke before the study, six showed higher confidence after the study. Conclusions: The use of an at-home stroke monitoring app is feasible by patients with a history of stroke and improves confidence in detecting the signs of stroke.