Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase.

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+-binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+-dependent bioluminescence responses. Through extensive validation in cell lines, primary neurons, and live mice, we demonstrated the efficacy of this new tool for detecting K+. Our research demonstrates an innovative concept of incorporating sensory moieties into luciferins to modulate luciferase activity. This approach has great potential for developing a wide range of bioluminescent indicators, advancing bioluminescence imaging (BLI), and enabling the study of various analytes in biological systems.

A fast, high-affinity fluorescent serotonin biosensor engineered from a tick lipocalin.

Serotonin (5-HT) is an important signaling monoamine and neurotransmitter. We report structure-guided engineering of a green fluorescent, genetically encoded serotonin sensor (G-GESS) from a 5-HT-binding lipocalin in the soft tick Argas monolakensis. G-GESS shows fast response kinetics and high affinity, specificity, brightness and photostability. We used G-GESS to image 5-HT dynamics in cultured cells, brain slices and behaving mice.

Prognostic Utility of Left Atrial Strain From MRI Feature Tracking in Ischemic and Nonischemic Dilated Cardiomyopathy: A Multicenter Study.

BACKGROUND. MRI-based prognostic evaluation in patients with dilated cardiomyopathy (DCM) has historically used markers of late gadolinium enhancement (LGE) and feature tracking (FT)-derived left ventricular global longitudinal strain (LVGLS). Early data indicate that FT-derived left atrial strain (LAS) parameters, including reservoir, conduit, and booster, may also have prognostic roles in such patients. OBJECTIVE. The purpose of our study was to evaluate the prognostic utility of LAS parameters, derived from MRI FT, in patients with ischemic or nonischemic DCM, including in comparison with the traditional parameters of LGE and LVGLS. METHODS. This retrospective study included 811 patients with ischemic or nonischemic DCM (median age, 60 years; 640 men, 171 women) who underwent cardiac MRI at any of five centers. FT-derived LAS parameters and LVGLS were measured using two- and four-chamber cine images. LGE percentage was quantified. Patients were assessed for a composite outcome of all-cause mortality or heart failure hospitalization. Multivariable Cox regression analyses including demographic characteristics, cardiovascular risk factors, medications used, and a wide range of cardiac MRI parameters were performed. Kaplan-Meier analyses with log-rank tests were also performed. RESULTS. A total of 419 patients experienced the composite outcome. Patients who did, versus those who did not, experience the composite outcome had larger LVGLS (-6.7% vs -8.3%, respectively; p < .001) as well as a smaller LAS reservoir (13.3% vs 19.3%, p < .001), LAS conduit (4.7% vs 8.0%, p < .001), and LAS booster (8.1% vs 10.3%, p < .001) but no significant difference in LGE (10.1% vs 11.3%, p = .51). In multivariable Cox regression analyses, significant independent predictors of the composite outcome included LAS reservoir (HR = 0.96, p < .001) and LAS conduit (HR = 0.91, p < .001). LAS booster and LGE were not significant independent predictors in the models. LVGLS was a significant independent predictor only in a model that initially included LAS booster but not the other LAS parameters. In Kaplan-Meier analysis, all three LAS parameters were significantly associated with the composite outcome (p < .001). CONCLUSION. In this multicenter study, LAS reservoir and LAS conduit were significant independent prognostic markers in patients with ischemic or nonischemic DCM, showing greater prognostic utility than the currently applied markers of LVGLS and LGE. CLINICAL IMPACT. FT-derived LAS analysis provides incremental prognostic information in patients with DCM.

How to study a highly toxic protein to bacteria: A case of voltage sensor domain of mouse sperm-specific sodium/proton exchanger.

Heterologous expression systems have been used as a powerful experimental strategy to study the function of many proteins, particularly ion transporters. For this experiment, it is fundamental to prepare an expression vector encoding a protein of interest. However, we encountered problems in vector preparation of the voltage sensor domain (VSD) of murine sperm-specific Na+/H+ exchanger (sNHE) due to its severe toxicity to bacteria. We overcame the problems by insertion of an amber stop codon or a synthetic intron into the coding sequence of the VSD in the expression vectors. Both methods allowed us to express the protein of interest in HEK293 cells (combined with a stop codon suppression system for amber codon). The VSD of mouse sNHE generates voltage-dependent outward ionic currents, which is a probable cause of toxicity to bacteria. We propose these two strategies as practical solutions to study the function of any protein toxic to bacteria.

Structural origin and rational development of bright red noncanonical variants of green fluorescent protein.

The incorporation of noncanonical amino acids (ncAAs) into fluorescent proteins is promising for red-shifting their fluorescence and benefiting tissue imaging with deep penetration and low phototoxicity. However, ncAA-based red fluorescent proteins (RFPs) have been rare. The 3-aminotyrosine modified superfolder green fluorescent protein (aY-sfGFP) represents a recent advance, yet the molecular mechanism for its red-shifted fluorescence remains elusive while its dim fluorescence hinders applications. Herein, we implement femtosecond stimulated Raman spectroscopy to obtain structural fingerprints in the electronic ground state and reveal that aY-sfGFP possesses a GFP-like instead of RFP-like chromophore. Red color of aY-sfGFP intrinsically arises from a unique "double-donor" chromophore structure that raises ground-state energy and enhances charge transfer, notably differing from the conventional conjugation mechanism. We further developed two aY-sfGFP mutants (E222H and T203H) with significantly improved (∼12-fold higher) brightness by rationally restraining the chromophore's nonradiative decay through electronic and steric effects, aided by solvatochromic and fluorogenic studies of the model chromophore in solution. This study thus provides functional mechanisms and generalizable insights into ncAA-RFPs with an efficient route for engineering redder and brighter fluorescent proteins.

Modeling the dynamic changes in Plasmopara viticola sporangia concentration based on LSTM and understanding the impact of relative factor variability.

Reliable disease management can guarantee healthy plant production and relies on the knowledge of pathogen prevalence. Modeling the dynamic changes in spore concentration is available for realizing this purpose. We present a novel model based on a time-series modeling machine learning method, i.e., a long short-term memory (LSTM) network, to analyze oomycete Plasmopara viticola sporangia concentration dynamics using data from a 4-year field experiment trial in North China. Principal component analysis (PCA)-based high-quality input screening and simulation result calibration were performed to ensure model performance, obtaining a high determination coefficient (0.99), a low root mean square error (0.87), and a low mean bias error (0.55), high sensitivity (91.5%), and high specificity (96.5%). The impact of the variability of relative factors on daily P. viticola sporangia concentrations was analyzed, confirming that a low daily mean air temperature restricts pathogen development even during a long period of high humidity in the field.

Improved Red Fluorescent Redox Indicators for Monitoring Cytosolic and Mitochondrial Thioredoxin Redox Dynamics.

Thioredoxin (Trx) is one of the major thiol-dependent antioxidants in living systems. The study of Trx functions in redox biology was impeded by the lack of practical tools to track Trx redox dynamics in live cells. Our previous work developed TrxRFP1, the first genetically encoded fluorescent indicator for Trx redox. In this work, we report an improved fluorescent indicator, TrxRFP2, for tracking the redox of Trx1, which is primarily cytosolic and nuclear. Furthermore, because mitochondria specifically express Trx2, we have created a new genetically encoded fluorescent indicator, MtrxRFP2, for the redox of mitochondrial Trx. We characterized MtrxRFP2 as a purified protein and used subcellularly localized MtrxRFP2 to image mitochondrial redox changes in live cells.

Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo.

The NanoLuc luciferase (NLuc) and its furimazine (FRZ) substrate have revolutionized bioluminescence (BL) assays and imaging. However, the use of the NLuc-FRZ luciferase-luciferin pair for mammalian tissue imaging is hindered by the low tissue penetration of the emitting blue photons. Here, we present the development of an NLuc mutant, QLuc, which catalyzes the oxidation of a synthetic QTZ luciferin for bright and red-shifted emission peaking at ∼585 nm. Compared to other small single-domain NLuc mutants, this amber-light-emitting luciferase exhibited improved performance for imaging deep-tissue targets in live mice. Leveraging this novel bioluminescent reporter, we further pursued in vivo immunobioluminescence imaging (immunoBLI), which used a fusion protein of a single-chain variable antibody fragment (scFv) and QLuc for molecular imaging of tumor-associated antigens in a xenograft mouse model. As one of the most red-shifted NLuc variants, we expect QLuc to find broad applications in noninvasive mammalian imaging. Moreover, the immunoBLI method complements immunofluorescence imaging and immuno-positron emission tomography (immunoPET), serving as a convenient and nonradioactive molecular imaging tool for animal models in basic and preclinical research.

Measuring response in metastatic castration-resistant prostate cancer using PSMA PET/CT: comparison of RECIST 1.1, aPCWG3, aPERCIST, PPP, and RECIP 1.0 criteria.

PURPOSE: To compare the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, the adapted Prostate Cancer Working Group Criteria 3 (aPCWG3), the adapted Positron Emission Tomography Response Criteria in Solid Tumors (aPERCIST), the PSMA PET Progression (PPP), and the Response Evaluation Criteria In PSMA-Imaging (RECIP) 1.0 for response evaluation using prostate-specific membrane antigen (PSMA)-PET/CT in men with metastatic castration-resistant prostate cancer (mCRPC) treated with 177Lu-PSMA radioligand therapy. METHODS: A total of 124 patients were included in this multicenter retrospective study. All patients received 177Lu-PSMA and underwent PSMA-PET/CT scans at baseline (bPET) and at 12 weeks (iPET). Imaging responses according to RECIST 1.1, aPCWG3, aPERCIST, PPP, and RECIP 1.0 were interpreted by consensus among three blinded readers. Changes in total tumor burden were obtained using the semi-automatic qPSMA software. The response according to each criterion was classified to progressive disease (PD) vs no-PD. Primary outcome measure was the prognostic value (by Cox regression analysis) for overall survival (OS). Secondary outcome measure was the inter-reader reliability (by Cohen's κ coefficient). RESULTS: A total of 43 (35%) of patients had non-measurable disease according to RECIST 1.1. Sixteen (13%), 66 (52%), 72 (58%), 69 (56%), and 39 (32%) of 124 patients had PD according to RECIST 1.1, aPCWG3, aPERCIST, PPP, and RECIP, respectively. PD vs no-PD had significantly higher risk of death according to aPCWG3 (HR = 2.37; 95%CI, 1.62-3.48; p < 0.001), aPERCIST (HR = 2.48; 95%CI, 1.68-3.66; p < 0.001), PPP (HR = 2.72; 95%CI, 1.85-4.01; p < 0.001), RECIP 1.0 (HR = 4.33; 95%CI, 2.80-6.70; p < 0.001), but not according to RECIST 1.1 (HR = 1.29; 95%CI, 0.73-2.27; p = 0.38). The κ index of RECIST 1.1, aPCWG3, aPERCIST 1.0, PPP, and RECIP 1.0 for identifying PD vs no-PD were 0.50 (95%CI, 0.32-0.76), 0.72 (95%CI, 0.63-0.82), 0.68 (95%CI, 0.63-0.73), 0.73 (95%CI, 0.63-0.83), and 0.83 (95%CI, 0.77-0.88), respectively. CONCLUSION: PSMA-PET-specific criteria for early response evaluation in men with mCRPC treated with 177Lu-PSMA achieved higher prognostic values and inter-reader reliabilities in comparison to conventional CT assessment or to criteria adapted to PSMA-PET from other imaging modalities. RECIP 1.0 identified the fewest patients with PD and achieved the highest risk of death for PD vs. no-PD, suggesting that other classification methods tend to overcall progression. Prospective validation of our findings on an independent patient cohort is warranted.

The added value of PSMA PET/MR radiomics for prostate cancer staging.

PURPOSE: To evaluate the performance of combined PET and multiparametric MRI (mpMRI) radiomics for the group-wise prediction of postsurgical Gleason scores (psGSs) in primary prostate cancer (PCa) patients. METHODS: Patients with PCa, who underwent [68 Ga]Ga-PSMA-11 PET/MRI followed by radical prostatectomy, were included in this retrospective analysis (n = 101). Patients were grouped by psGS in three categories: ISUP grades 1-3, ISUP grade 4, and ISUP grade 5. mpMRI images included T1-weighted, T2-weighted, and apparent diffusion coefficient (ADC) map. Whole-prostate segmentations were performed on each modality, and image biomarker standardization initiative (IBSI)-compliant radiomic features were extracted. Nine support vector machine (SVM) models were trained: four single-modality radiomic models (PET, T1w, T2w, ADC); three PET + MRI double-modality models (PET + T1w, PET + T2w, PET + ADC), and two baseline models (one with patient data, one image-based) for comparison. A sixfold stratified cross-validation was performed, and balanced accuracies (bAcc) of the predictions of the best-performing models were reported and compared through Student's t-tests. The predictions of the best-performing model were compared against biopsy GS (bGS). RESULTS: All radiomic models outperformed the baseline models. The best-performing (mean ± stdv [%]) single-modality model was the ADC model (76 ± 6%), although not significantly better (p > 0.05) than other single-modality models (T1w: 72 ± 3%, T2w: 73 ± 2%; PET: 75 ± 5%). The overall best-performing model combined PET + ADC radiomics (82 ± 5%). It significantly outperformed most other double-modality (PET + T1w: 74 ± 5%, p = 0.026; PET + T2w: 71 ± 4%, p = 0.003) and single-modality models (PET: p = 0.042; T1w: p = 0.002; T2w: p = 0.003), except the ADC-only model (p = 0.138). In this initial cohort, the PET + ADC model outperformed bGS overall (82.5% vs 72.4%) in the prediction of psGS. CONCLUSION: All single- and double-modality models outperformed the baseline models, showing their potential in the prediction of GS, even with an unbalanced cohort. The best-performing model included PET + ADC radiomics, suggesting a complementary value of PSMA-PET and ADC radiomics.

A general strategy to red-shift green fluorescent protein-based biosensors.

Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic ß-cells.

Illegal drugs and periodontal conditions.

In recent years, the practice of dentistry and periodontology has become complicated by several risk factors, including the treatment of an increasing number of patients with substance use disorder. This review presents an update in the current literature of the impact of illegal drug use on periodontal conditions and their possible effect as risk factors or indicators. The main illegal drugs that may have an impact on periodontal health and conditions are described, including their effect, medical manifestations, risks, and the overall effect on oral health and on the periodontium. Where available, data from epidemiologic studies are analyzed and summarized. The clinical management of periodontal patients using illegal drugs is reported in a comprehensive approach inclusive of the detection of illicit drug users, screening, interviewing and counseling, the referral to treatment, and the dental and periodontal management. With regard to the impact of illegal substance use on periodontal conditions, there is moderate evidence that regular long-term use of cannabis is a risk factor for periodontal disease, manifesting as a loss of periodontal attachment, deep pockets, recessions, and gingival enlargements. Limited evidence also shows that the use of cocaine can cause a series of gingival conditions that mostly presents as chemical induced-traumatic lesions (application of cocaine on the gingiva) or necrotizing ulcerative lesions. There is a scarcity of data regarding the impact of other drug use on periodontal health. There is evidence to suggest that regular long-term use of cannabis is a risk factor for periodontal disease and that the use of cocaine can cause a series of periodontal conditions. The dental treatment of subjects that use illegal substances is becoming more common in the daily clinical practice of periodontists and other dental clinicians. When the clinicians encounter such patients, it is essential to manage their addiction properly taking into consideration the impact of it on comprehensive dental treatment. Further studies and clinical observations are required to obtain sound and definitive information.

The correlation between miR -34a-3p, miR -31, PLEK2 and the occurrence, development and prognosis of colorectal cancer.

The current study aimed to explore the correlation between Mir-34A-3p, Mir-31, PLEK2 and the occurrence, development and prognosis of colorectal cancer. For this paper, 120 patients with colorectal cancer were selected as the study group, and their adjacent normal tissues were selected as the control group. The quantitative real-time PCR (QRT-PCR) method was used to detect miR-34a-3p and miR-31 in tissues, and the immunohistochemistry EnVision two-step method was used to detect PLEK2 positive expression. The expressions of miR -34a-3p, miR -31, and PLEK2 in colon cancer tissues and normal cancer tissues were compared, and the correlation between miR -34a-3p, miR -31, and PLEK2 and clinic-pathological characteristics of colorectal cancer patients were analyzed. The results showed that expression of miR -34a-3p, miR -31 and positive expression rate of PLEK2 in colorectal cancer tissues were higher than those in normal adjacent tissues (P<0.05). The expression of miR -34a-3p was related to tumor size, degree of tissue differentiation, lymph node metastasis and TNM stage (P < 0.05). The 3-year survival rate of miR -34a-3p with low expression was lower than miR -34a-3p with high expression, which was a protective factor affecting the poor prognosis of colorectal cancer (P < 0.05). The expression of miR -31 was related to tumor size and TNM stage. The 3-year survival rate of the group with high expression of miR -31 was lower than the group with low expression of miR -31, which was a risk factor affecting the poor prognosis of colorectal cancer (P < 0.05). PLEK2 positive expression was associated with lymph node metastasis, and the 3-year survival rate of the PLEK2 positive group was lower than the PLEK2 low expression group, which was a risk factor for poor prognosis of colorectal cancer (P < 0.05). In general, miR -34a-3p, miR -31, and PLEK2 are closely associated with the occurrence and development of colorectal cancer, and they are all influential factors affecting the prognosis of patients with colorectal cancer, which can provide a basis for the evaluation and treatment of patients, and are worthy of widespread clinical application.

Contact network analysis of COVID-19 Delta variant outbreak in urban China -based on 2,050 confirmed cases in Xi'an, China.

BACKGROUND: The purpose of this paper is to study how the Delta variant spread in a China city, and to what extent the non-pharmaceutical prevention measures of local government be effective by reviewing the contact network of COVID-19 cases in Xi'an, China. METHODS: We organize the case reports of the Shaanxi Health Commission into a database by text coding and convert them into a network matrix. Then we construct a dynamic contact network for the corresponding analysis and calculate network indicators. we analyze the cases' dynamic contact network structure and intervals between diagnosis time and isolation time by using data visualization, network analysis method, and Ordinary Least Square (OLS) regression. RESULTS: The contact network for this outbreak in Xi'an is very sparse, with a density of less than 0.0001. The contact network is a scale-free network. The average degree centrality is 0.741 and the average PageRank score is 0.0005. The network generated from a single source of infection contains 1371 components. We construct three variables of intervals and analyze the trend of intervals during the outbreak. The mean interval (interval 1) between case diagnosis time and isolation time is - 3.9 days. The mean of the interval (interval 2) between the infector's diagnosis time and the infectee's diagnosis time is 4.2 days. The mean of the interval (interval 3) between infector isolation time and infectee isolation time is 2.9 days. Among the three intervals, only interval 1 has a significant positive correlation with degree centrality. CONCLUSIONS: By integrating COVID-19 case reports of a Chinese city, we construct a contact network to analyze the dispersion of the outbreak. The network is a scale-free network with multiple hidden pathways that are not detected. The intervals of patients in this outbreak decreased compared to the beginning of the outbreak in 2020. City lockdown has a significant effect on the intervals that can affect patients' network centrality. Our study highlights the value of case report text. By linking different reports, we can quickly analyze the spread of the epidemic in an urban area.

Attention to renal involvement: report of 17 Joubert syndrome cases in children of a single center in China.

BACKGROUND: Joubert Syndrome (JS) is a rare genetic developmental disorder. We are aiming for increasing awareness of this disease especially kidney involvement in children with JS. METHODS: Clinical and genetic data of 17 cases of JS in Beijing children's hospital in the past 21 years were collected retrospectively. RESULTS: Twelve males and 5 females, aged from 12d to 15y8m. The most common involvement was neurological system involvement. The second most common involvement was renal involvement: end stage kidney disease in 6 cases (35%), hematuria in 5 cases (29%), proteinuria in 5 cases (29%), renal diffuse lesions in 4 cases (24%), renal cystic lesions in 2 cases (12%), and echogenic enhancement of parenchyma in 2 cases (12%). 10 cases did genetic tests. 3 cases with renal deficiency all had RPGRIP1L gene mutation. CONCLUSIONS: The most common involvement of JS is neurological involvement, and the second is renal involvement. Pediatricians should improve awareness of JS and conduct systemic evaluation of children. More attention should be paid to renal involvement which may be onset hidden but fatal. Early recognition and diagnosis are the goals to delay the start to dialysis and improve quality of patients' life. The RPGRIP1L gene mutation maybe the most common gene mutation in JS and may have correlations with renal involvement.

Compassion satisfaction and compassion fatigue in frontline nurses during the COVID-19 pandemic in Wuhan, China.

AIM: The aim of this study is to investigate the compassion satisfaction and compassion fatigue among Chinese frontline nurses during the COVID-19 pandemic in Wuhan, China and to explore the related factors. BACKGROUND: Frontline nurses undertake a huge nursing workload with a risk of infection, causing great pressure on them and making them face a risk of compassion fatigue during the pandemic. METHODS: A cross-sectional online survey was conducted from 9 March to 15 March 2020. A total of 1582 nurses caring for critical patients with COVID-19 participated. Compassion satisfaction and compassion fatigue (comprising burnout and secondary traumatic stress) were assessed with the Professional Quality of Life Scale, and resilience was measured with the Chinese 10-item Connor-Davidson Resilience Scale. RESULTS: Moderate levels of compassion satisfaction (36.99 ± 6.71), burnout (24.14 ± 5.33) and secondary traumatic stress (24.53 ± 5.24) were experienced by frontline nurses. Resilience and perceived work pressure were the main predictors. CONCLUSIONS: Frontline nurses demonstrated a moderate level of compassion satisfaction and compassion fatigue. IMPLICATIONS FOR NURSING MANAGEMENT: The compassion fatigue of frontline nurses should be considered. Strategies aiming to reduce stress and enhance resilience, such as training about psychological adjustment, developing professional skills and creating a supportive workplace environment, are several options. The trial is not registered. This study is a cross-sectional study, and according to China's clinical trial registration standards, such studies are not required to be registered. So the trial is not registered. However, oral consent was obtained from the ethics committee of the hospital before this study was conducted.

Case Report of Neonatal Sotos Syndrome with a New Missense Mutation in the NSD1 Gene and Literature Analysis in the Chinese Han Population.

Currently, no consensus exists regarding Sotos syndrome in the Chinese population. Here, we present a case of neonatal Sotos syndrome, followed by a retrospective analysis of five cases of neonatal Sotos syndrome, reported in China. The study subject was a twin premature infant, heavier than gestational age, with characteristic facial features, limb shaking, and hypertonia. Transient hypoglycemia, abnormal cranial magnetic resonance imaging, multiple nodules in polycystic kidneys and liver, abnormal hearing, patent ductus arteriosus, and an atrial septal defect were also noted. The subject showed overgrowth and developmental retardation at 3 months of age. Sequencing revealed a novel missense mutation, c.5000C>A, in the nuclear receptor binding the SET domain protein 1 gene, resulting in an alanine-to-glutamate substitution. The bioinformatics analysis suggested high pathogenicity at this site. This study provides insights into diagnosis of neonatal Sotos syndrome based on specific phenotypes. Subsequent treatment and follow-up should focus on developmental retardation, epilepsy, and scoliosis.

Study of cellular heterogeneity and differential dynamics of autophagy in human embryonic kidney development by single-cell RNA sequencing.

BACKGROUND: Autophagy is believed to participate in embryonic development, but whether the expression of autophagy-associated genes undergoes changes during the development of human embryonic kidneys remains unknown. METHODS: In this work, we identified 36,151 human renal cells from embryonic kidneys of 9-18 gestational weeks in 16 major clusters by single-cell RNA sequencing (scRNA-seq), and detected 1350 autophagy-related genes in all fetal renal cells. The abundance of each cell cluster in Wilms tumor samples from scRNA-seq and GDC TARGET WT datasets was detected by CIBERSORTx. R package Monocle 3 was used to determine differentiation trajectories. Cyclone tool of R package scran was applied to calculate the cell cycle scores. R package SCENIC was used to investigate the transcriptional regulons. The FindMarkers tool from Seurat was used to calculate DEGs. GSVA was used to perform gene set enrichment analyses. CellphoneDB was utilized to analyze intercellular communication. RESULTS: It was found that cells in the 13th gestational week showed the lowest transcriptional level in each cluster in all stages. Nephron progenitors could be divided into four subgroups with diverse levels of autophagy corresponding to different SIX2 expressions. SSBpod (podocyte precursors) could differentiate into four types of podocytes (Pod), and autophagy-related regulation was involved in this process. Pseudotime analysis showed that interstitial progenitor cells (IPCs) potentially possessed two primitive directions of differentiation to interstitial cells with different expressions of autophagy. It was found that NPCs, pretubular aggregates and interstitial cell clusters had high abundance in Wilms tumor as compared with para-tumor samples with active intercellular communication. CONCLUSIONS: All these findings suggest that autophagy may be involved in the development and cellular heterogeneity of early human fetal kidneys. In addition, part of Wilms tumor cancer cells possess the characteristics of some fetal renal cell clusters.

Genetically Encoded Fluorescent Redox Indicators for Unveiling Redox Signaling and Oxidative Toxicity.

Redox-active molecules play essential roles in cell homeostasis, signaling, and other biological processes. Dysregulation of redox signaling can lead to toxic effects and subsequently cause diseases. Therefore, real-time tracking of specific redox-signaling molecules in live cells would be critical for deciphering their functional roles in pathophysiology. Fluorescent protein (FP)-based genetically encoded redox indicators (GERIs) have emerged as valuable tools for monitoring the redox states of various redox-active molecules from subcellular compartments to live organisms. In the first section of this review, we overview the background, focusing on the sensing mechanisms of various GERIs. Next, we review a list of selected GERIs according to their analytical targets and discuss their key biophysical and biochemical properties. In the third section, we provide several examples which applied GERIs to understanding redox signaling and oxidative toxicology in pathophysiological processes. Lastly, a summary and outlook section is included.

KLF2 induces the senescence of pancreatic cancer cells by cooperating with FOXO4 to upregulate p21.

Pancreatic cancer is one of the most common malignancies in the world. Senescence is frequently observed in the progression of pancreatic cancer. In a previous study, we showed that KLF2 inhibited the growth and migration of pancreatic cancer. However, the mechanisms are not fully understood. In this study, we showed that overexpression of KLF2 induced the senescence of pancreatic cancer cells and inhibited tumorigenesis, and knockdown of KLF2 inhibited senescence and p21 expression. In the molecular mechanism study, KLF2 was found to interact with FOXO4 and cooperated with FOXO4 to induce the expression of p21. Downregulation of p21 and FOXO4 impaired the induction of senescence by KLF2. Overall, this study revealed the functions and mechanisms of KLF2 in senescence and provided a novel explanation for the suppressive roles of KLF2 in pancreatic cancer.