Fluorescent Indicator Research Articles

Mitochondrial temperature homeostasis resists external metabolic stresses

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

Mitochondrial temperature homeostasis resists external metabolic stresses.

Based on studies with a fluorescent reporter dye, Mito Thermo Yellow (MTY), and the genetically encoded gTEMP ratiometric fluorescent temperature indicator targeted to mitochondria, the temperature of active mitochondria in four mammalian and one insect cell line was estimated to be up to 15°C above that of the external environment to which the cells were exposed. High mitochondrial temperature was maintained in the face of a variety of metabolic stresses, including substrate starvation or modification, decreased ATP demand due to inhibition of cytosolic protein synthesis, inhibition of the mitochondrial adenine nucleotide transporter and, if an auxiliary pathway for electron transfer was available via the alternative oxidase, even respiratory poisons acting downstream of oxidative phosphorylation (OXPHOS) complex I. We propose that the high temperature of active mitochondria is an inescapable consequence of the biochemistry of OXPHOS and is homeostatically maintained as a primary feature of mitochondrial metabolism.

Quantitative source tracking for organic foulants in ultrafiltration membrane using stable isotope probing approach

Quantitatively identifying the primary sources of organic membrane fouling is essential for the effective implementation of membrane technology and optimal water resource management prior to the treatment. This study leveraged carbon stable isotope tracers to estimate the quantitative contributions of various organic sources to membrane fouling in an ultrafiltration system. Effluent organic matter (EfOM) and aquatic natural organic matter (NOM), two common sources, were combined in five different proportions to evaluate their mixed effects on flux decline and the consequent fouling behaviors. Generally, biopolymer (BP) and low molecular weight neutral (LMWN) size fractions - abundantly present in EfOM - were identified as significant contributors to reversible and irreversible fouling, respectively. Fluorescence spectroscopy disclosed that a protein-like component notably influenced overall membrane fouling, whereas humic-like components were predominantly responsible for irreversible fouling rather than reversible fouling. Fluorescence index (FI) and biological index (BIX), common fluorescence source tracers, showed promise in determining the source contribution for reversible foulants. However, these optical indices were insufficient in accurately determining individual source contributions to irreversible fouling, resulting in inconsistencies with the observed hydraulic analysis. Conversely, applying a carbon stable isotope-based mixing model yielded reasonable estimates for all membrane fouling. The contribution of EfOM surpassed 60 % for reversible fouling and increased with its content in DOM source mixtures. In contrast, aquatic NOM dominated irreversible fouling, contributing over 85 %, regardless of the source mixing ratios. This study emphasizes the potential of stable isotope techniques in accurately estimating the contributions of different organic matter sources to both reversible and irreversible membrane fouling.

High-throughput ligand profile characterization in novel cell lines expressing seven heterologous insect olfactory receptors for the detection of volatile plant biomarkers

Agriculturally important crop plants emit a multitude of volatile organic compounds (VOCs), which are excellent indicators of their health status and their interactions with pathogens and pests. In this study, we have developed a novel cellular olfactory panel for detecting fungal pathogen-related VOCs we had identified in the field, as well as during controlled inoculations of several crop plants. The olfactory panel consists of seven stable HEK293 cell lines each expressing a functional Drosophila olfactory receptor as a biosensing element along with GCaMP6, a fluorescent calcium indicator protein. An automated 384-well microplate reader was used to characterize the olfactory receptor cell lines for their sensitivity to reference VOCs. Subsequently, we profiled a set of 66 VOCs on all cell lines, covering a concentration range from 1 to 100 μM. Results showed that 49 VOCs (74.2%) elicited a response in at least one olfactory receptor cell line. Some VOCs activated the cell lines even at nanomolar (ppb) concentrations. The interaction profiles obtained here will support the development of biosensors for agricultural applications. Additionally, the olfactory receptor proteins can be purified from these cell lines with sufficient yields for further processing, such as structure determination or integration with sensor devices.

Effect of experimental warming on dissolved organic matter and bacterial diversity in a forest swamp soil

Forest swamp ecosystems play an important role in the global carbon cycle, yet they are often overlooked. With global climate warming, it is inevitable that it will impact the relationship between soil microorganisms and organic matter. Dissolved organic matter (DOM) in the soil environment is extremely sensitive to environmental changes. Understanding the effects of climate change on DOM and microorganisms is crucial for assessing the stability of carbon (C) in forest swamp soils. Therefore, we conducted a 142-day laboratory warming incubation experiment (control: 10 °C, and warming: 20 °C) to investigate the response of forest swamp soil microbial and DOM properties to warming. Excitation-emission matrix (EEM) fluorescence spectroscopy was used to explore the changes in different material compositions of DOM over time at different incubation temperatures, and high-throughput sequencing was used to investigate the changes in soil bacteria. The DOM content (dissolved organic C, DOC) and the degree of humification index (HIX) increased with an increase in incubation time under warming conditions (p < 0.05). In contrast, microbial humic substances (C3) decreased with increasing time. Long-term warming has separated bacterial communities and gradually tightened the degree of connectivity between bacterial networks. The degree of soil humification (Mantel test r = 0.58, p < 0.01) and DOC (r = 0.21, p < 0.05) were the most critical indicators that changed the diversity of the bacterial community. Our findings suggest a degree of interaction between changes in forest swamp soil microbial communities and DOM under warming conditions. The results of this study contribute to our understanding of changes in DOM fluorescence indices in forest swamp soils under the influence of climate change and their associated microbial mechanisms.

Fjords Are Potential Hotspots of Refractory Dissolved Organic Matter Production: Insights From 1 Year of Weekly Time‐Series Observations of Fluorescent Dissolved Organic Matter in the Bedford Basin, a Northwestern Atlantic Fjord

AbstractFjords are representative marine ecosystems that play an important role in regulating coastal carbon cycling. As fingerprints of the dissolved organic matter (DOM) pool, fluorescent DOM (FDOM) is widely used to study the ocean carbon cycle. Here, we report on 1‐year of weekly observations of FDOM (from May 2018 to May 2019) in the Bedford Basin, a well‐studied northwestern Atlantic fjord. Results showed that differences in FDOM indices (biological index, fluorescence index, and humification indices) between the euphotic and aphotic layers changed during the investigation period, suggesting that classical definitions of these indices may not apply consistently. Spearman rank correlations showed that the surface FDOM was affected by physical and biological factors, while water mass renewal was the major control for the deeper layer. Humic‐like FDOM (FDOMH) changed following the Atlantic deep‐water intrusion, indicating that FDOMH may be used to trace water mass movements in this region. FDOMH shows a significantly positive linear correlation with apparent oxygen utilization (AOU). Their slopes were higher than those from the open ocean, suggesting that the Bedford Basin is a hotspot of RDOM production. Hence, we proposed a possible carbon sequestration mechanism that fjords play as “RDOM producing hot spots” and that the produced RDOM is then transported to the open ocean via water mass movement and finally stored in the ocean for extended periods of time. The proposed biogeochemical processes may also occur, more generally, in the global coastal ocean.

Quantification of sedimentary organic carbon sources in a land–river–lake continuum combined with multi-fingerprint and un-mixing models

Identifying organic carbon (OC) sources in lake sediment is essential for elucidating biogeochemical cycling processes and effectively supporting watershed management. However, the complexity of sources as well as environments in the land–river–lake continuum makes it challenging to accurately identify OC sources. Accordingly, the current study utilized a systematic approach to identify and validate OC sources in a typical land–river–lake continuum. Two tracer groups (group 1: δ13C and δ15N; group 2: fluorescence index and biotic index, respectively (where C is carbon and N is nitrogen)) and one model (MixSIAR) were eventually selected from five tracer groups and two models to identify the OC sources in a land–river–lake continuum according to a consistency evaluation and virtual mixing test. The results showed that the distribution of OC sources in lake sediment was spatially heterogeneous. Closer to the lake center (from sampling site S1 to S3), the autochthonous contributions increased while the allochthonous contributions decreased. Downstream of the inlet river (site S1) was dominated by allochthonous contributions (78.6%), especially cropland (28.7% ± 0.5%, where ± indicates a standard deviation range) and urban land (30.5% ± 2.5%). From site S1 to S2, the allochthonous contribution decreased 11.4%. Autochthonous OC gradually became the major source closer to the lake center (site S3: phragmites: 48% ± 4.5%). This distribution of OC sources in the land–river–lake system was attributed to the mixing effect of the autochthonous sources, selective transport of sediment, and human activities. The current findings may aid in validating the ability of different tracers and models to identify OC sources in complex ecosystems and also provide a theoretical basis for watershed management.

Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy.

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of the mitochondrial network allows healthy mitochondria to meet cellular demands; however, the loss of this capacity has been related to the development or progression of different pathologies. In skeletal muscle, mitochondrial density and distribution changes are observed in physiological and pathological conditions such as exercise, aging, and obesity, among others. Therefore, the study of the mitochondrial network may provide a better understanding of mechanisms related to those conditions. Here, a protocol for mitochondria imaging of live-skeletal muscle fibers from rats is described. Fibers are manually dissected in a relaxing solution and incubated with a fluorescent live-cell imaging indicator of mitochondria (tetramethylrhodamine ethyl ester, TMRE). The mitochondria signal is recorded by confocal microscopy using the XYZ scan mode to obtain confocal images of the intermyofibrillar mitochondrial (IMF) network. After that, the confocal images are processed by thresholding and binarization. The binarized confocal image accounts for the positive pixels for mitochondria, which are then counted to obtain the mitochondrial density. The mitochondrial network in skeletal muscle is characterized by a high density of IMF population, which has a periodic longitudinal distribution similar to that of T-tubules (TT).The Fast Fourier Transform (FFT) is a standard analysis technique performed to evaluate the distribution of TT that allows finding the distribution frequency and the level of their organization. In this protocol, the implementation of the FFT algorithm is described for the analysis of the longitudinal mitochondrial distribution in skeletal muscle.

Fast, portable, selective, and ratiometric determination of ochratoxin A (OTA) by a fluorescent supramolecular sensor

Ochratoxin A (OTA), a mycotoxin found in various food items, possesses significant health risks due to its carcinogenic and toxic properties. Thus, detecting OTA is crucial to ensure food safety. Among the reported analytical methods, there has yet to be one that achieves fast, selective, and portable detection of OTA. In this study, we explore a novel supramolecular sensor, DOCE@ALB, utilizing human serum albumin as the host and a flavonoid fluorescent indicator as the guest. On the basis of indicator displacement assay, this sensor boasts an ultra-fast response time of just 5 s, high sensitivity with a limit of detection at 0.39 ppb, exceptional selectivity, and a noticeable ratiometric fluorescence response to OTA. This discernible color change and portability of the sensor make it suitable for on-site OTA detection in real food samples, including flour, beer, and wine, simply using a smartphone. In comparison to previously reported methods, our approach has showcased notable advantages in both response time and portability, addressing a critical need for food safety and regulatory compliance.

CD22 CAR T Cell-Related IEC-HS Is Associated with an IFN-γ Cytokine Signature

Introduction Chimeric antigen receptor T-cell therapy targeting CD22 (CAR22) shows promise for patients with relapsed / refractory B-cell acute lymphoblastic leukemia (ALL) and large B cell lymphoma (LBCL) (Fry et al. Nat Med 2018; Baird et al. Blood 2020). The overall response rate for CAR22 in adults who received prior CD19 CAR therapy is 100% and 68% in ALL and LBCL respectively (Tandem 2023) (NCT04088890). However, 33% of pediatric ALL patients who received CAR22 developed immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) (Shah et al. JCO 2020). IEC-HS is characterized by hyperferritinemia, cytopenias, coagulopathy and end organ damage. Pediatric ALL data suggest many cytokines including IFN-γ, IL-1β, IL-18 and TNF-α are upregulated in IEC-HS (Ishii et al. JCI 2020, Lichtenstein et al. Blood 2021). Here, we report the clinical features and cytokines associated with IEC-HS after CAR22 infusion in predominantly adult subjects with ALL and LBCL. Methods Plasma was collected weekly from Day 0 to 28 for 54 patients (ALL: n = 16, LBCL: n = 38; adult: n = 51, pediatrics: n = 3) who were treated with CAR22 manufactured using an automated, closed-system Miltenyi CliniMACS Prodigy device. Three dose levels were tested (ALL: 3 x 10 5 cells/kg, LBCL dose level 1 (DL1): 1 x 10 6 cells/kg, LBCL dose level 2 (DL2): 3 x 10 6 cells/kg). Clinical characteristics associated with IEC-HS were retrospectively collected and graded according to consensus criteria (Hines et al. TCT 2023). Eighty cytokines were quantified in batch via multiplexed fluorescence immunoassay (Luminex) to generate mean fluorescent indices (MFIs). Unsupervised hierarchical clustering using Euclidean distance and ward.D2 linkage were employed to analyze cytokine dynamics. Statistical analyses were performed using R (v 4.2.2). Results 8 of 54 (15%) patients developed IEC-HS, all were ≥18 years old. 5 patients had LBCL, 3 had ALL. 5 cases were grade 2, 3 were grade 4. Median onset was D+12 (range 8 - 21). All patients had prior CRS with median onset at D+1 (range 0 - 9). The 3 ALL patients had ongoing CRS and spectral overlap with IEC-HS at time of diagnosis whereas LBCL patients' CRS resolved prior to IEC-HS onset. IEC-HS patients had significant increases in inflammatory markers (ferritin, CRP, LDH, triglycerides), coagulation markers (INR, PTT), cytopenias (hemoglobin, platelets) and transaminases (Table 1). Patients most commonly received high-dose glucocorticoids, tocilizumab and anakinra. Additional agents for grade 4 IEC-HS included anti-thymoglobulin, dasatanib and emapalumab. 5 of 8 patients developed serious infections requiring IV antimicrobials including bacteremia, invasive fungal infections and viral reactivation. 5 patients with IEC-HS died after achieving complete response: 3 due to IEC-HS and concomitant infection, 1 from t-AML and 1 lost to follow up. IEC-HS occurred more frequently at higher doses in LBCL where multiple doses were tested (DL2: 33% vs DL1: 7%, p = 0.08). Peak serum CAR22 concentration was markedly elevated in patients who developed IEC-HS compared to those who did not (median 648 vs 42 cells/µL, p&amp;lt;0.001). Unsupervised hierarchical clustering performed on serial cytokine measurements identified a signature of IFN-γ-related cytokines including CXCL9, CXCL10, TNFα, FasL, IL6 and IL18 as being closely related in IEC-HS (Figure 1). Most cytokines in the signature peaked at D+14, consistent with onset of clinical IEC-HS. Peak levels of nearly all cytokines in the interferon-γ cluster were significantly elevated in IEC-HS patients compared to patients who did not develop IEC-HS, and this cluster contained most cytokines with significant increases in peak levels (Figure 1). Conclusions IEC-HS is a potentially life-threatening toxicity associated with CAR22 across age and underlying malignancy. The high rates of non-relapse mortality and severe infections in this study highlight the importance of understanding the immune dysregulation underlying IEC-HS. Higher CAR22 dose and expansion were associated with IEC-HS, indicating optimal dosing as a potential preventive strategy. The observed IFN-γ cytokine signature is consistent with myeloid cell activation and Th1 T-cell skewing that likely contributes to IEC-HS pathogenesis. These cytokines may be amenable to therapeutic intervention and further study of approaches targeting JAK/STAT and IFN-γ are warranted.

Soluble and Membrane P-Glycoprotein Expression in Lymphocytes from Diffuse Large B Cell Non-Hodgkin's Lymphoma Patients Treated with R-CHOP

Introduction Diffuse large B cell lymphoma (DLBC) accounts for 25% of all Non-Hodgkin's Lymphoma (NHL) in the United States. Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) has been the first-line therapy for patients with DLBC-NHL since the early 2000s. Nearly 40% relapse in the following 2-years after complete response and 15% are refractory to first-line therapy. Despite many efforts, other chemotherapy combinations have failed to improve the response rate of NHL patients. Mechanisms of resistance to cytotoxic drugs are vast and include augmented drug efflux mediated through ATP-dependent pumpsas P-glycoprotein (P-gp), which has been related to lesser efficacy. This is due to an over-expression of the glycoprotein with the consequent increase of the drug's efflux from the cytosol. Almost all components of the R-CHOP therapy are P-gp substratesand thereby are affected by P-gp activity. Objectives Analyze serum, plasmatic levels, and membrane expression of P-gp in lymphocytes from patients with DLBC-NHL. Methods A case-control study was conducted to measure serum and plasmatic levels of P-gp, and membrane P-gp expression at the end of treatment on DLBC-NHL patients at “Hospital Civil de Guadalajara Fray Antonio Alcalde,” diagnosed from April to September 2022. Inclusion criteria were age ≥ 18 years, without cancer history and without P-gp inductors or inhibitors. Exclusion criteria were pregnancy, hepatitis, autoimmune disease, Richter syndrome and infection 15 days before blood draw. Using whole blood samples, we identified T and NK cells expressing membrane P-gp by flow cytometry on Attune® NxT (Life Technologies) using the following antibodies and fluorochrome conjugates: CD3-APC/Cy7, CD4-AlexaFlour488, CD8-APC, CD56-BrilliantViolet, CD243-PE. Data was analyzed with FlowJo v10 software. Serum and plasmatic P-gp measurement were done using the Human Permeability Glycoprotein (P-gp) ELISA kit (MyBioSource). Statistical Analysis Proportion comparison between groups was made using Chi-Square or Fisher exact tests. A quantitative variable comparison was done using the U Mann-Witney test. Correlation analysis was made using the Spearman correlation coefficient. Statistical significance was calculated using the p&amp;lt;0.05 two-tailed test. All statistical analysis was made using the software R version 4.1.2 and ggpurb/ggplot2 package. Results Only 15 patients and 30 control individuals were included. Group characteristics and comparisons are summarized in table 1. Regarding the DLBC-NHL group, 27% patients were classified as Lugano I, II and IV stages respectively and 20% as Lugano III stage. Nearly 27% of the patients received radiotherapy as consolidation therapy. Also, 40% of patients showed lymphopenia with a median of 1.23 x10 3/μL (IQR 0.94 - 1.34). Serum and plasmatic P-gp concentrations were similar between groups (p&amp;gt;0.05). The CD8+, CD4+, and CD56+ lymphocyte percentage with membrane P-gp expression comparison between the DLBC-NHL group and the control group presented no significant differences (p&amp;gt;0.05). However, the P-gp median fluorescence index (MFI) in CD8+, CD4+, and CD56+ lymphocytes was higher in the control group than DLBC-NHL group (p&amp;lt;0.05), as displayed in figure 1. Discussion When analyzing only the DLBC-NHL group, we identified a negative correlation between serum P-gp concentration and its expression in NK cell membrane and a positive correlation with membrane expression in CD4+ and CD8+ lymphocytes. Kato et al. reported that P-gp can be secreted through exosomes in patients with drug resistance. Lettau et al. found that CD4+ and CD8+ T lymphocytes have higher exosome expulsion compared to NK cells. These could be related to our finding of negative correlation between serum P-gp and membrane P-gp expression in NK lymphocytes. Ghetie et al. found that Rituximab-an anti-CD20 monoclonal antibody-inhibits P-gp efflux function and therefore makes multi-drug-resistant cell cultures chemosensitive again. B-cells affected by rituximab may influence T-cell P-gp expression and this may be the reason why MFI was lower on DLBC-NHL group than control group. This study helps to expand the understanding of P-gp's role in DLBC-NHL patients as well as R-CHOP therapy effects on the expression of the P-gp in circulating T-cells.

Towards enhanced treatment of piggery biogas slurry in tidal flow constructed wetlands by regulation of hydraulic retention time

Livestock farming wastewater is complex in composition, containing a large number of pathogenic microorganisms and ammonia nitrogen (NH4+-N) with high concentration, which makes biological treatment difficult. In this study, tidal flow constructed wetlands were applied to treat piggery biogas slurry. Compared with a continuous flow constructed wetland, two tidal influent modes (hydraulic retention time: idling time for 12 h:12 h and 20 h:4 h, namely TFCW1 and TFCW2) were set up to explore treatment performances on chromaticity, turbidity, chemical oxygen demand (COD), total nitrogen (TN), NH4+-N and total phosphorus (TP), and determine the relationship between nitrogen transfer and dissolved oxygen (DO) contents. The fluorescence spectra of dissolved organic matter (DOM) in constructed wetlands were analyzed by excitation-emission matrix spectroscopy-parallel factor analysis to reveal its composition and content of humus. The results showed that tidal flow constructed wetlands obviously improved the removal of chromaticity and turbidity by 44.6% and 10.1%, in comparison to continuous flow constructed wetland. TFCW2 had enhanced reduction on pollutants, with removal efficiencies of COD, NH4+-N and TP at 78.6%, 82.1% and 84.0%, respectively. Furthermore, three fluorescent components were detected in TFCW2, and the value of Fmax was increased in the effluent, among which the Fmax of C3 (Humic-like substances) increased obviously from 0.320 to 9.39. The ratio of C2 (Aquatic fulvic acids) to C3 showed a slow increasing trend in the daily outflow of TFCW2, while C1 (dark-incubation substances) remained relatively constant. Fluorescence index (FI), humification index (HIX) and biological index (BIX) increased from 1.35 to 2.09, 0.210–1.51, and 0.240–1.05, respectively, showing that the effluent from the tidal flow constructed wetland had a high microbial activity. This study provided a new insight for the advanced treatment of livestock biogas slurry with a high NH4+-N content.

Field migration of veterinary antibiotics via surface runoff from chicken-raising orchard in responding to natural rainfalls

Knowledge on runoff transport of manure-sourced antibiotics from farmland soil to aquatic environment is limited due to complexity of hydrological regime and pathways. This study monitored natural rainfalls in sloping orchard plots with free-range chickens, with an attempt to investigate the migration characteristics of typical antibiotics via surface runoff as well as the impact of manure presence. Results showed that rainstorms continuously carried away antibiotics in surface runoff and all target antibiotics including those with high affinities to soil were detected at the beginning of runoff production. Concentration of antibiotics was found to respond strongly to the instantaneous rainfall intensity, showing consistent fluctuations during rainfalls. Concentrations of sulfonamides and florfenicol were two orders of magnitude higher than that of tetracyclines and fluoroquinolones. Compared to the control without raising chickens, antibiotics migration was considerably increased with the increased runoff production due to soil surface changes caused by chicken activities. Additionally, dynamics of antibiotic concentration significantly correlated with variations of fluorescent DOM components. Chicken manure-derived DOM mainly contained tryptophan moiety, and laboratory fluorescence quenching test with 2D-COS analysis indicated that all antibiotics interacted more strongly and preferentially with tryptophan than humic-like species. Antibiotics bonded to manure DOM with an affinity corresponding to the significance level of their correlations. In this light, potential use of fluorescence indices based on the established correlations may provide a convenient tool for tracing runoff migration of antibiotics during rainfalls.

Optogenetic Microwell Array Screening System: A High-Throughput Engineering Platform for Genetically Encoded Fluorescent Indicators.

Genetically encoded fluorescent indicators (GEFIs) are protein-based optogenetic tools that change their fluorescence intensity when binding specific ligands in cells and tissues. GEFI encoding DNA can be expressed in cell subtypes while monitoring cellular physiological responses. However, engineering GEFIs with physiological sensitivity and pharmacological specificity often requires iterative optimization through trial-and-error mutagenesis while assessing their biophysical function in vitro one by one. Here, the vast mutational landscape of proteins constitutes a significant obstacle that slows GEFI development, particularly for sensors that rely on mammalian host systems for testing. To overcome these obstacles, we developed a multiplexed high-throughput engineering platform called the optogenetic microwell array screening system (Opto-MASS) that functionally tests thousands of GEFI variants in parallel in mammalian cells. Opto-MASS represents the next step for engineering optogenetic tools as it can screen large variant libraries orders of magnitude faster than current methods. We showcase this system by testing over 13,000 dopamine and 21,000 opioid sensor variants. We generated a new dopamine sensor, dMASS1, with a >6-fold signal increase to 100 nM dopamine exposure compared to its parent construct. Our new opioid sensor, μMASS1, has a ∼4.6-fold signal increase over its parent scaffold's response to 500 nM DAMGO. Thus, Opto-MASS can rapidly engineer new sensors while significantly shortening the optimization time for new sensors with distinct biophysical properties.

Bioactive peptides PDBSN improve mitochondrial function and suppression the oxidative stress in human adiposity cells

ABSTRACT Introduction: Mitochondria are essential for generating cellular energy and are significant in the pathogenesis of obesity. Peptide PDBSN has been demonstrated to inhibit the adipogenic differentiation of adipocytes in vitro and improves metabolic homoeostasis in vivo. Therefore, in this study, we further investigated the effects of PDBSN on the morphology, synthesis, and function of adipocyte mitochondria. Methods: Human visceral and subcutaneous primary preadipocytes (HPA-v and HPA-s) were cultured into mature adipocytes. Intracellular triglyceride content was assessed using oil-red O staining and tissue triglyceride determination. Gene and protein levels associated with mitochondrial synthesis were detected using real-time quantitative polymerase chain reaction and western blotting. Mitochondrial membrane potentials and ROS were detected using fluorescent indicators. Morphological changes were observed by electron microscopy. Results: PDBSN significantly increased mitochondrial membrane potential (MMP), while decreasing intracellular triglyceride (TG) and intracellular reactive oxygen species (ROS) levels. On the other hand, the transcription and protein levels of genetic marker genes PGC1-α and MTFA were significantly up-regulated after PDBSN administration. Further studies showed that transcriptional and protein levels of mitochondrial fusion and fission genetic markers MFN1, MFN2, NRF1, and DRP1 increased. Conclusion: PDBSN significantly reduces intracellular TG and ROS levels and increases MMP. The maximum respiratory capacity in adults significantly increases after PDBSN administration, and ROS levels are significantly reduced. This suggests that PDBSN improves mitochondrial function to some extent, which not only provides an essential basis for the pathophysiology of obesity but also provides insights for the development of new drugs to treat obesity and metabolic diseases.

Evaluating the cumulative and time-lag effects of vegetation response to drought in Central Asia under changing environments

Central Asia (CA) is an arid region sensitive to climate change, and with the increase in drought events due to global warming, droughts are posing more severe threats to the vegetation in CA. However, the response of vegetation in CA to the cumulative and time-lag effects of prolonged drought remains unclear. This has limited our understanding of the mechanisms by which vegetation responds to meteorological drought in arid regions. To clarify the response of vegetation to drought in CA, this study analyzed the effects of drought and meteorological factors on vegetation growth dynamics. The extent of vegetation response to the cumulative and time-lag effects from 2001 to 2020 was evaluated using three satellite-derived vegetation indices (NDVI, EVI, NIRv) and the solar-induced chlorophyll fluorescence (SIF) and standardized precipitation evapotranspiration index (SPEI). We used the Copula-Bayes conditional probability method to assess the drought thresholds for vegetation under two scenarios: mild and severe losses. The effects of soil moisture (SM) and vapor pressure deficit (VPD) on vegetation dynamics were analyzed based on an improved partial wavelet coherence (PWC). The results show that the average response time of vegetation to the cumulative and lagged effects of drought was mostly concentrated within 4–6 months, and the lagged effect was stronger than the cumulative effect. The vegetation response to drought is nonlinear along the precipitation gradient. The SPEI values for triggering mild vegetation loss (below the 40th percentile) drought threshold range from −0.7 to −1.0, while the SPEI values for triggering severe vegetation loss (below the 10th percentile) drought threshold range from −2.0 to −2.5. The SM was the main driver affecting vegetation dynamics. This study provides deeper insight into the response of vegetation to drought in the arid zone of CA, which will further provide theoretical support for addressing global climate change and extreme drought events.

TMIC-26. TAMS AND HYPOXIA ENHANCE GLIOBLASTOMA STEM-LIKE CELL ENRICHMENT AND MAY IMPACT RADIORESISTANCE

Abstract The dismal survival rates of glioblastoma (GBM) patients are due, at least in part, to therapeutic resistance of GBM stem-like cells (GSCs) and their ability to repopulate recurrent tumors. Our goal was to determine if tumor-associated macrophages (TAMs) or hypoxia enhance the enrichment of GSCs in a manner that could impact radioresistance. First, we established GBM-brain cortical organoids (GBM-BCOs) to model human GBM. We co-cultured GFP-labeled GBM neurospheres with BCOs for 14 days and observed a tumor take rate of 100%. Next, we evaluated whether hypoxia enhanced TAM migration into GBM-BCOs by incorporating THP-1 cells into GFP-GBM-BCOs and incubating them for 24 h under normoxic or hypoxic conditions. We found that hypoxia significantly increased THP-1 cell influx into GBM-BCOs. Further, we investigated whether TAMs influenced the enrichment of GSCs in GBM neurospheres and in GBM-BCOs. We found that co-culture of THP-1 cells with either GBM neurospheres or with GBM-BCOs enhanced the CD133+ population after 72 h hypoxia, correlating with our IF studies of human GBM samples that show increased CD133+ and IBA1+ cells in hypoxic peri-necrotic zones compared to non-necrotic regions. Next, we established Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), an advanced methodology for monitoring cell cycle phases for investigating the role of hypoxia and radiation on GBM. Flow cytometry analysis of patient-derived GBM cells revealed that the fraction of G1 cells decreased and the fraction of G2/M cells increased 24 h after 3 Gy radiation compared to non-irradiated cells in normoxia. In contrast, GBM cells exposed to hypoxia do not show such cell cycle shifts after radiation and have an enriched population of cells in G0 in both radiated and non-irradiated cells. Our results indicate that hypoxia and TAMs play critical roles in GSC enrichment and may impact radioresistance.

Development and validation of a colorimetric antifungal susceptibility testing method for the dimorphic fungus Talaromyces marneffei.

Antifungal drug resistance is an emerging cause of treatment failure in invasive fungal infections, and antifungal susceptibility testing (AFST) may inform treatment decisions. Currently, there are no established AFST guidelines for Talaromyces marneffei (Tm) or other dimorphic fungi. We developed a colorimetric AFST method using a fluorescent redox indicator alamarBlue, which changes from blue to pink in proportion to cellular metabolic activity. We determined the optimal time for alamarBlue addition to be 24h post-inoculation and for MIC reading to be 72h post-inoculation. Our method allows three ways to determine minimum inhibitory concentration (MIC): visual inspection of color change, optical density, and fluorescence intensity. We validated the assay by determining the MICs for seven antifungals against 32 Tm clinical isolates and assessed the essential agreement (EA) and inter-rater reliability between our alamarBlue and the Clinical Laboratory Standard Institute (CLSI) broth microdilution methods. The MIC ranges (from low to high) were: 0.008-0.025 μg/ml for itraconazole, 0.004-0.13 μg/ml for voriconazole, 0.03-0.13μg/ml for posaconazole, 0.06-0.5µg/ml for flucytosine, 0.5-1µg/ml for amphotericin B, 0.5-4µg/ml for caspofungin, and 0.5-16µg/ml for fluconazole. The EAs were 100% between all three MIC readouts of the alamarBlue method, and 94%-100% between the alamarBlue and CLSI methods. Our alamarBlue method had substantially higher inter-rater agreement and offers a more reliable method that can be standardized across laboratories in both high- and low-resource settings compared to the established CLSI methodology.

Pregabalin inhibits purinergic P2Y2 receptor and TRPV4 to suppress astrocyte activation and to relieve neuropathic pain

BackgroundsTransient receptor potential vanilloid 4 (TRPV4)-mediated astrocyte activation is critical to neuropathic pain. Pregabalin, a widely used drug to treat chronic pain, is reported to lower the intracellular calcium level. However, the molecular mechanism by which pregabalin decreases the intracellular calcium level remains unknown. Purinergic P2Y2 receptor—a member of the G protein-coupled receptor (GPCR) family—regulates calcium-related signal transduction in astrocyte activation. We investigated whether P2Y2 receptor is involved in the pharmacological effects of pregabalin on neuropathic pain. MethodsNeuropathic pain was induced by chronic compression of the dorsal root ganglion (CCD) in rats. Paw withdrawal mechanical threshold (PWMT) was used for behavioral testing. Intracellular calcium concentration was measured using a fluorescent calcium indicator (Fluo-4 AM). ResultsWe found that P2Y2 receptor protein was upregulated and astrocytes were activated in the experimental rats after CCD surgery. Lipopolysaccharide (LPS) increased the intracellular calcium concentration and induced astrocyte activation in cultured astrocytes but was prevented via P2Y2 receptor inhibitor AR-C118925 or pregabalin. Furthermore, plasmid-mediated P2Y2 receptor overexpression induced an elevation of the intracellular calcium levels and inflammation in astrocytes, which was abolished by the TRPV4 inhibitor HC-067047. AR-C118925, HC-067047, and pregabalin relieved neuropathic pain and inflammation in rats after CCD surgery. Finally, plasmid-mediated P2Y2 receptor overexpression induced neuropathic pain in rats, which was abolished by pregabalin administration. ConclusionsPathophysiological variables that upregulated the P2Y2 receptor/TRPV4/calcium axis contribute to astrocyte activation in neuropathic pain. Pregabalin exerts an analgesic effect by inhibiting this pathway.

Comparing Phenology of a Temperate Deciduous Forest Captured by Solar-Induced Fluorescence and Vegetation Indices

A shifting phenology in deciduous broadleaf forests (DBFs) can indicate forest health, resilience, and changes in the face of a rapidly changing climate. The availability of satellite-based solar-induced fluorescence (SIF) from the Orbiting Carbon Observatory-2 (OCO-2) promises to add to the understanding of the regional-level DBF phenology that has been developed, for instance, using proxies of gross primary productivity (GPP) from the Moderate Imaging Spectroradiometer (MODIS). It is unclear how OCO-2 and MODIS metrics compare in terms of capturing intra-annual variations and benchmarking DBF seasonality, thus necessitating a comparison. In this study, spatiotemporally matched OCO-2 SIF metrics (at footprint level) and corresponding MODIS GPP, normalized difference vegetation index (NDVI), and enhanced vegetation index (EVI) products within a temperate DBF were used to compare the phenology captured by the productivity metrics. Additionally, an estimate of the SIF yield (SIFy), derived from OCO-2 SIF measurements, and a MODIS fraction of photosynthetically active radiation (fPAR) were tested. An examination of the trends and correlations showed relatively few qualitative differences among productivity metrics and environmental variables, but it highlighted a lack of seasonal signal in the calculation of SIFy. However, a seasonality analysis quantitatively showed similar seasonal timings and levels of seasonal production in and out of the growing season between SIF and GPP. In contrast, NDVI seasonality was least comparable to that of SIF and GPP, with senescence occurring approximately one month apart. Taken together, we conclude that satellite-based SIF and GPP (and EVI to a smaller degree) provide the most similar measurements of forest function, while NDVI is not sensitive to the same changes. In this regard, phenological metrics calculated with satellite-based SIF, along with those calculated with GPP and EVI from MODIS, can enhance our current understanding of deciduous forest structures and functions and provide additional information over NDVI. We recommend that future studies consider metrics other than NDVI for phenology analyses.