Iron-sulfur protein odyssey: exploring their cluster functional versatility and challenging identification.

Iron-sulfur (Fe-S) clusters are an essential and ubiquitous class of protein-bound prosthetic centers that are involved in a broad range of biological processes (e.g. respiration, photosynthesis, DNA replication and repair and gene regulation) performing a wide range of functions including electron transfer, enzyme catalysis, and sensing. In a general manner, Fe-S clusters can gain or lose electrons through redox reactions, and are highly sensitive to oxidation, notably by small molecules such as oxygen and nitric oxide. The [2Fe-2S] and [4Fe-4S] clusters, the most common Fe-S cofactors, are typically coordinated by four amino acid side chains from the protein, usually cysteine thiolates, but other residues (e.g. histidine, aspartic acid) can be found. While diversity in cluster coordination ensures the functional variety of the Fe-S clusters, the lack of conserved motifs makes new Fe-S protein identification challenging especially when the Fe-S cluster is also shared between two proteins as observed in several dimeric transcriptional regulators and in the mitoribosome. Thanks to the recent development of in cellulo, in vitro and in silico approaches, new Fe-S proteins are still regularly identified, highlighting the functional diversity of this class of proteins. In this review, we will present three main functions of the Fe-S clusters and explain the difficulties encountered to identify Fe-S proteins and methods that have been employed to overcome these issues.

Regulations of mitoNEET by the key redox homeostasis molecule glutathione.

Human mitoNEET (mNT) and CISD2 are two NEET proteins characterized by an atypical [2Fe-2S] cluster coordination involving three cysteines and one histidine. They act as redox switches with an active state linked to the oxidation of their cluster. In the present study, we show that reduced glutathione but also free thiol-containing molecules such as ß-mercaptoethanol can induce a loss of the mNT cluster under aerobic conditions, while CISD2 cluster appears more resistant. This disassembly occurs through a radical-based mechanism as previously observed with the bacterial SoxR. Interestingly, adding cysteine prevents glutathione-induced cluster loss. At low pH, glutathione can bind mNT in the vicinity of the cluster. These results suggest a potential new regulation mechanism of mNT activity by glutathione, an essential actor of the intracellular redox state.

A novel electrospun polylactic acid silkworm fibroin mesh for abdominal wall hernia repair.

Objective: Abdominal wall hernias are common abdominal diseases, and effective hernia repair is challenging. In clinical practice, synthetic meshes are widely applied for repairing abdominal wall hernias. However, postoperative complications, such as inflammation and adhesion, are prevalent. Although biological meshes can solve this problem to a certain extent, they face the problems of heterogeneity, rapid degradation rate, ordinary mechanical properties, and high-cost. Here, a novel electrospinning mesh composed of polylactic acid and silk fibroin (PLA-SF) for repairing abdominal wall hernias was manufactured with good physical properties, biocompatibility and low production cost. Materials and methods: FTIR and EDS were used to demonstrate that the PLA-SF mesh was successfully synthesized. The physicochemical properties of PLA-SF were detected by swelling experiments and in vitro degradation experiments. The water contact angle reflected the hydrophilicity, and the stress‒strain curve reflected the mechanical properties. A rat abdominal wall hernia model was established to observe degradation, adhesion, and inflammation in vivo. In vitro cell mesh culture experiments were used to detect cytocompatibility and search for affected biochemical pathways. Results: The PLA-SF mesh was successfully synthesized and did not swell or degrade over time in vitro. It had a high hydrophilicity and strength. The PLA-SF mesh significantly reduced abdominal inflammation and inhibited adhesion formation in rat models. The in vitro degradation rate of the PLA-SF mesh was slower than that of tissue remodeling. Coculture experiments suggested that the PLA-SF mesh reduced the expression of inflammatory factors secreted by fibroblasts and promoted fibroblast proliferation through the TGF-ß1/Smad pathway. Conclusion: The PLA-SF mesh had excellent physicochemical properties and biocompatibility, promoted hernia repair of the rat abdominal wall, and reduced postoperative inflammation and adhesion. It is a promising mesh and has potential for clinical application.

Isolation and structural identification of a potassium ion channel Kv4.1 inhibitor SsTx-P2 from centipede venom. / 蜈蚣毒液中钾离子通道Kv4.1抑制剂SsTx-P2的分离和结构鉴定.

OBJECTIVES: To isolate a potassium ion channel Kv4.1 inhibitor from centipede venom, and to determine its sequence and structure. METHODS: Ion-exchange chromatography and reversed-phase high-performance liquid chromatography were performed to separate and purify peptide components of centipede venom, and their inhibiting effect on Kv4.1 channel was determined by whole-cell patch clamp recording. The molecular weight of isolated peptide Kv4.1 channel inhibitor was identified with matrix assisted laser desorption ionization-time-of-flight mass spectrometry; its primary sequence was determined by Edman degradation sequencing and two-dimensional mass spectrometry; its structure was established based on iterative thread assembly refinement online analysis. RESULTS: A peptide SsTx-P2 was separated from centipede venom with the molecular weight of 6122.8, and its primary sequence consists of 53 amino acid residues NH2-ELTWDFVRTCCKLFPDKSECTKACATEFTGGDESRLKDVWPRKLRSGDSRLKD-OH. Peptide SsTx-P2 potently inhibited the current of Kv4.1 channel transiently transfected in HEK293 cell, with 1.0 µmol/L SsTx-P2 suppressing 95% current of Kv4.1 channel. Its structure showed that SsTx-P2 shared a conserved helical structure. CONCLUSIONS: The study has isolated a novel peptide SsTx-P2 from centipede venom, which can potently inhibit the potassium ion channel Kv4.1 and displays structural conservation.

[Sulfonated magnetic graphite carbon nitride solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry for screening malachite green and leucomalachite green in freshwater fish].

Malachite green (MG) and its metabolite, leucomalachite green (LMG), exert toxic effects on the human body. The use of these dyes is illegal, but they are still detected in aquatic products. Freshwater fish are aquatic products with the high non-qualified rates. Therefore, the sensitive screening of MG and LMG in freshwater fish is of great importance to ensure the safety of aquatic products. Owing to the low contents of MG and LMG in fish and the complex matrix of actual samples, sample preparation is required before detection to purify impurities and enrich the target compounds. Graphite carbon nitride (GCN), a polymer material composed of C, N, and H, has good chemical and thermal stability, a large specific surface area, and a large number of active sites. It has a wide range of application prospects in adsorption and can be used in food safety testing when compounded with Fe3O4 to form magnetic graphite carbon nitride (MGCN). In this study, sulfonated magnetic graphite carbon nitride (S-MGCN) was prepared by further functionalizing MGCN with sulfonic acid. After characterization by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and vibrating sample magnetometry (VSM), a magnetic solid-phase extraction (MSPE) method based on S-MGCN was established to extract MG and LMG from freshwater fish. The targets were screened using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Following sulfonic acid functionalization, S-MGCN showed increased electrostatic interactions based on the MGCN adsorption mechanism, which includes hydrogen bonds and π-π interactions; thus, its adsorption efficiency was significantly improved. The matrix effects were -42.21% and -33.77% before functionalization, -11.40% and -7.84% after functionalization, thus confirming that S-MGCN has significant matrix removal ability. Given that S-MGCN demonstrated excellent efficiency as an MSPE adsorbent, the adsorption conditions for S-MGCN were optimized. The optimal conditions were as follows: adsorbent dosage, 15 mg; adsorption time, 2 min; solution pH, 5; and ionic strength, not adjusted. Under these conditions, the adsorption efficiency of S-MGCN could reach 94.2%. Different organic solvents were used to elute adsorbed MG and LMG, and the desorption efficiency peaked when 1%(v/v) ammonia acetonitrile was used as the elution solvent. The elution volume was also optimized, and a maximum desorption efficiency of 93.2% was obtained when 1 mL of 1%(v/v) ammonia acetonitrile was added to S-MGCN. The limits of detection (LODs) and quantification (LOQs) of the two targets were determined at signal-to-noise ratios (S/N) of 3 and 10, respectively. The LODs and LOQs were 0.075 µg/kg and 0.25 µg/kg, respectively. The linear ranges of the two target compounds were 0.25-20.0 µg/kg with correlation coefficients (r) greater than 0.998. To assess accuracy and precision, we prepared spiked samples at three levels (low, medium, and high) with six parallel samples per level (n=6). The recoveries ranged from 88.8% to 105.9%. The intra- and inter-day relative standard deviations were 5.4%-13.7% (n=6) and 3.3%-11.1% (n=3), respectively. Compared with the national standard method, the proposed method features simpler sample pretreatment procedures, less use of organic reagents (5 mL), and a shorter extraction time (2 min); moreover, the method does not require complicated elution steps, and the eluent can be directly analyzed by UPLC-MS/MS. The test results of actual samples were consistent with those obtained via the national standard method, thus confirming the practical feasibility of the developed method. The proposed MSPE method based on S-MGCN is an efficient and environmentally friendly method that could provide a new methodological reference for the sensitive screening of MG and LMG in actual samples.

Redox-Based Strategies against Infections by Eukaryotic Pathogens.

Redox homeostasis is an equilibrium between reducing and oxidizing reactions within cells. It is an essential, dynamic process, which allows proper cellular reactions and regulates biological responses. Unbalanced redox homeostasis is the hallmark of many diseases, including cancer or inflammatory responses, and can eventually lead to cell death. Specifically, disrupting redox balance, essentially by increasing pro-oxidative molecules and favouring hyperoxidation, is a smart strategy to eliminate cells and has been used for cancer treatment, for example. Selectivity between cancer and normal cells thus appears crucial to avoid toxicity as much as possible. Redox-based approaches are also employed in the case of infectious diseases to tackle the pathogens specifically, with limited impacts on host cells. In this review, we focus on recent advances in redox-based strategies to fight eukaryotic pathogens, especially fungi and eukaryotic parasites. We report molecules recently described for causing or being associated with compromising redox homeostasis in pathogens and discuss therapeutic possibilities.

Adenylate Kinase Fused to Spidroin as a Catalyst for Decreasing Leakage out of 3D-Bioprinted Hydrogels and for ATP Regeneration.

Numerous metabolic reactions and pathways use adenosine 5'-triphosphate (ATP) as an energy source and as a phosphorous or pyrophosphorous donor. Based on three-dimensional (3D)-printing, enzyme immobilization can be used to improve ATP regeneration and operability and reduce cost. However, due to the relatively large mesh size of 3D-bioprinted hydrogels soaked in a reaction solution, the lower-molecular-weight enzymes cannot avoid leaking out of the hydrogels readily. Here, a chimeric adenylate-kinase-spidroin (ADK-RC) is created, with ADK serving as the N-terminal domain. The chimera is capable of self-assembling to form micellar nanoparticles at a higher molecular scale. Although fused to spidroin (RC), ADK-RC remains relatively consistent and exhibits high activity, thermostability, pH stability, and organic solvent tolerance. Considering different surface-to-volume ratios, three shapes of enzyme hydrogels are designed, 3D bioprinted, and measured. In addition, a continuous enzymatic reaction demonstrates that ADK-RC hydrogels have higher specific activity and substrate affinity but a lower reaction rate and catalytic power compared to free enzymes in solution. With ATP regeneration, the ADK and ADK-RC hydrogels significantly increase the production of d-glucose-6-phosphate and obtain an efficient usage frequency. In conclusion, enzymes fused to spidroin might be an efficient strategy for maintaining activity and reducing leakage in 3D-bioprinted hydrogels under mild conditions.

A secretory system for extracellular production of spider neurotoxin huwentoxin-I in Escherichia coli.

Due to their advantages in structural stability and versatility, cysteine-rich peptides, which are secreted from the venom glands of venomous animals, constitute a naturally occurring pharmaceutical arsenal. However, the correct folding of disulfide bonds is a challenging task in the prokaryotic expression system like Escherichia coli due to the reducing environment. Here, a secretory expression plasmid pSE-G1M5-SUMO-HWTX-I for the spider neurotoxin huwentoxin-I (HWTX-I) with three disulfides as a model of cysteine-rich peptides was constructed. By utilizing the signal peptide G1M5, the fusion protein 6 × His-SUMO-HWTX-I was successfully secreted into extracellular medium of BL21(DE3). After enrichment using cation-exchange chromatography and purification utilizing the Ni-NTA column, 6 × His-SUMO-HWTX-I was digested via Ulp1 kinase to release recombinant HWTX-I (rHWTX-I), which was further purified utilizing RP-HPLC. Finally, both impurities with low and high molecular weights were completely removed. The molecular mass of rHWTX-I was identified as being 3750.8 Da, which was identical to natural HWTX-I with three disulfide bridges. Furthermore, by utilizing whole-cell patch clamp, the sodium currents of hNav1.7 could be inhibited by rHWTX-I and the IC50 value was 419 nmol/L.

Faculty perceptions regarding an individually tailored, flexible length, outcomes-based curriculum for undergraduate medical students / 한국의학교육

Purpose@#The perception of faculty members about an individually tailored, flexible-length, outcomes-based curriculum for undergraduate medical students was studied. Their opinion about the advantages, disadvantages, and challenges was also noted. This study was done to help educational institutions identify academic and social support and resources required to ensure that graduate competencies are not compromised by a flexible education pathway. @*Methods@#The study was done at the International Medical University, Malaysia, and the University of Lahore, Pakistan. Semi-structured interviews were conducted from 1st August 2021 to 17th March 2022. Demographic information was noted. Themes were identified, and a summary of the information under each theme was created. @*Results@#A total of 24 (14 from Malaysia and 10 from Pakistan) faculty participated. Most agreed that undergraduate medical students can progress (at a differential rate) if they attain the required competencies. Among the major advantages mentioned were that students may graduate faster, learn at a pace comfortable to them, and develop an individualized learning pathway. Several logistical challenges must be overcome. Providing assessments on demand will be difficult. Significant regulatory hurdles were anticipated. Artificial intelligence (AI) can play an important role in creating an individualized learning pathway and supporting time-independent progression. The course may be (slightly) cheaper than a traditional one. @*Conclusion@#This study provides a foundation to further develop and strengthen flexible-length competency-based medical education modules. Further studies are required among educators at other medical schools and in other countries. Online learning and AI will play an important role.

Novel Insights into Redox-Based Mechanisms for Auranofin-Induced Rapid Cancer Cell Death.

Auranofin (Ridaura®, AUF) is a gold complex originally approved as an antirheumatic agent that has emerged as a potential candidate for multiple repurposed therapies. The best-studied anticancer mechanism of AUF is the inhibition of thioredoxin reductase (TrxR). However, a number of reports indicate a more complex and multifaceted mode of action for AUF that could be cancer cell type- and dose-dependent. In this study, we observed that AUF displayed variable cytotoxicity in five triple-negative breast cancer cell lines. Using representative MDA-MB-231 cells treated with moderate and cytotoxic doses of AUF, we evidenced that an AUF-mediated TrxR inhibition alone may not be sufficient to induce cell death. Cytotoxic doses of AUF elicited rapid and drastic intracellular oxidative stress affecting the mitochondria, cytoplasm and nucleus. A "redoxome" proteomics investigation revealed that a short treatment with a cytotoxic dose AUF altered the redox state of a number of cysteines-containing proteins, pointing out that the cell proliferation/cell division/cell cycle and cell-cell adhesion/cytoskeleton structure were the mostly affected pathways. Experimentally, AUF treatment triggered a dose-dependent S-phase arrest and a rapid disintegration of the actin cytoskeleton structure. Our study shows a new spectrum of AUF-induced early effects and should provide novel insights into the complex redox-based mechanisms of this promising anticancer molecule.

Incidence of extrauterine growth retardation and its risk factors in very preterm infants during hospitalization: a multicenter prospective study. / ä½é™¢æžæ—©äº§å„¿å®«å¤–ç”Ÿé•¿è¿Ÿç¼“åŠç›¸å ³å› ç´ çš„å¤šä¸­å¿ƒå‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To investigate the incidence of extrauterine growth retardation (EUGR) and its risk factors in very preterm infants (VPIs) during hospitalization in China. METHODS: A prospective multicenter study was performed on the medical data of 2 514 VPIs who were hospitalized in the department of neonatology in 28 hospitals from 7 areas of China between September 2019 and December 2020. According to the presence or absence of EUGR based on the evaluation of body weight at the corrected gestational age of 36 weeks or at discharge, the VPIs were classified to two groups: EUGR group (n=1 189) and non-EUGR (n=1 325). The clinical features were compared between the two groups, and the incidence of EUGR and risk factors for EUGR were examined. RESULTS: The incidence of EUGR was 47.30% (1 189/2 514) evaluated by weight. The multivariate logistic regression analysis showed that higher weight growth velocity after regaining birth weight and higher cumulative calorie intake during the first week of hospitalization were protective factors against EUGR (P<0.05), while small-for-gestational-age birth, prolonged time to the initiation of total enteral feeding, prolonged cumulative fasting time, lower breast milk intake before starting human milk fortifiers, prolonged time to the initiation of full fortified feeding, and moderate-to-severe bronchopulmonary dysplasia were risk factors for EUGR (P<0.05). CONCLUSIONS: It is crucial to reduce the incidence of EUGR by achieving total enteral feeding as early as possible, strengthening breastfeeding, increasing calorie intake in the first week after birth, improving the velocity of weight gain, and preventing moderate-severe bronchopulmonary dysplasia in VPIs.

Gastrointestinal symptoms as the first sign of chronic granulomatous disease in a neonate: A case report.

BACKGROUND: Chronic granulomatous disease (CGD) characterized by recurrent and severe bacterial and fungal infections is most common in childhood. CASE SUMMARY: We reported a 24-d-old male infant who developed gastrointestinal symptoms as the first sign of CGD. CONCLUSION: Gastrointestinal symptoms representing the first sign of CGD are very rare, and prompt diagnosis and treatment with broad-spectrum antibiotics were of crucial importance.

Characteristics of electrospun membranes in different spidroin/PCL ratios.

Spider silk is a protein fiber with the highest strength and elasticity known in nature, even higher than that of silkworm silk. It was a biological and technical reserve material with great potential. However, the low yield of natural spider silk limits the application of spider silk, and the development of genetic engineering provides opportunities for the mass production of spider silk. We constructed a mini-recombinant spidroin NRC based on spider silk gene fromAraneus ventricosusand successfully expressed it through Prokaryotic expression that provide a high production for application using electrospinning, which is a mature technique to produce micro-nano scale fibers as thin as natural spider silks. By blending the purified and lyophilized NRC with polycaprolactone (PCL) in different mass ratio for electrospinning, different electrospun membranes were obtained, and then characterized in terms of morphology, chemical structure, mechanical and Schwann cell proliferation. Compared the difference between polycaprolactone (PCL) and NRC, the fiber diameter decreased from 1.0779 µm to 0.5785 µm, water contact angel decreased from 104.1 ± 2° to 56.9 ± 5°, and elongation decreased from 240.97 ± 89% to 37.76 ± 13%, while tensile strength increased from 1.74 ± 1.2 MPa to 3.18 ± 0.9 MPa and Young's Module increased from 3.05 ± 1.6 MPa to 16.54 ± 6.7 MPa. In this study, we obtained a thinner fiber, hydrophilicity and high strengthen electrospinning spidroin contained membrane, which can also promote Schwann cell proliferation and adhesion.

A dynamic and multilocus metabolic regulation strategy using quorum-sensing-controlled bacterial small RNA.

Metabolic regulation strategies have been developed to redirect metabolic fluxes to production pathways. However, it is difficult to screen out target genes that, when repressed, improve yield without affecting cell growth. Here, we report a strategy using a quorum-sensing system to control small RNA transcription, allowing cell-density-dependent repression of target genes. This strategy is shown with convenient operation, dynamic repression, and availability for simultaneous regulation of multiple genes. The parameters Ai, Am, and RA (3-oxohexanoyl-homoserine lactone [AHL] concentrations at which half of the maximum repression and the maximum repression were reached and value of the maximum repression when AHL was added manually, respectively) are defined and introduced to characterize repression curves, and the variant LuxRI58N is identified as the most suitable tuning factor for shake flask culture. Moreover, it is shown that dynamic overexpression of the Hfq chaperone is the key to combinatorial repression without disruptions on cell growth. To show a broad applicability, the production titers of pinene, pentalenene, and psilocybin are improved by 365.3%, 79.5%, and 302.9%, respectively, by applying combinatorial dynamic repression.

Fe-S coordination defects in the replicative DNA polymerase delta cause deleterious DNA replication in vivo and subsequent DNA damage in the yeast Saccharomyces cerevisiae.

B-type eukaryotic polymerases contain a [4Fe-4S] cluster in their C-terminus domain, whose role is not fully understood yet. Among them, DNA polymerase delta (Polδ) plays an essential role in chromosomal DNA replication, mostly during lagging strand synthesis. Previous in vitro work suggested that the Fe-S cluster in Polδ is required for efficient binding of the Pol31 subunit, ensuring stability of the Polδ complex. Here, we analyzed the in vivo consequences resulting from an impaired coordination of the Fe-S cluster in Polδ. We show that a single substitution of the very last cysteine coordinating the cluster by a serine is responsible for the generation of massive DNA damage during S phase, leading to checkpoint activation, requirement of homologous recombination for repair, and ultimately to cell death when the repair capacities of the cells are overwhelmed. These data indicate that impaired Fe-S cluster coordination in Polδ is responsible for aberrant replication. More generally, Fe-S in Polδ may be compromised by various stress including anti-cancer drugs. Possible in vivo Polδ Fe-S cluster oxidation and collapse may thus occur, and we speculate this could contribute to induced genomic instability and cell death, comparable to that observed in pol3-13 cells.

New Insights of the NEET Protein CISD2 Reveals Distinct Features Compared to Its Close Mitochondrial Homolog mitoNEET.

Human CISD2 and mitoNEET are two NEET proteins anchored in the endoplasmic reticulum and mitochondria membranes respectively, with an Fe-S containing domain stretching out in the cytosol. Their cytosolic domains are close in sequence and structure. In the present study, combining cellular and biochemical approaches, we compared both proteins in order to possibly identify specific roles and mechanisms of action in the cell. We show that both proteins exhibit a high intrinsic stability and a sensitivity of their cluster to oxygen. In contrast, they differ in according to expression profiles in tissues and intracellular half-life. The stability of their Fe-S cluster and its ability to be transferred in vitro are affected differently by pH variations in a physiological and pathological range for cytosolic pH. Finally, we question a possible role for CISD2 in cellular Fe-S cluster trafficking. In conclusion, our work highlights unexpected major differences in the cellular and biochemical features between these two structurally close NEET proteins.

Attenuation of Antiviral Immune Response Caused by Perturbation of TRIM25-Mediated RIG-I Activation under Simulated Microgravity.

Microgravity is a major environmental factor of space flight that triggers dysregulation of the immune system and increases clinical risks for deep-space-exploration crews. However, systematic studies and molecular mechanisms of the adverse effects of microgravity on the immune system in animal models are limited. Here, we establish a ground-based zebrafish disease model of microgravity for the research of space immunology. RNA sequencing analysis demonstrates that the retinoic-acid-inducible gene (RIG)-I-like receptor (RLR) and the Toll-like receptor (TLR) signaling pathways are significantly compromised by simulated microgravity (Sµg). TRIM25, an essential E3 for RLR signaling, is inhibited under Sµg, hampering the K63-linked ubiquitination of RIG-I and the following function-induction positive feedback loop of antiviral immune response. These mechanisms provide insights into better understanding of the effects and principles of microgravity on host antiviral immunity and present broad potential implications for developing strategies that can prevent and control viral diseases during space flight.

A clinical evaluation of noninvasive and contactless radiofrequency technique in the treatment of abdominal fat.

BACKGROUND: There is an increasing demand for fat reduction and body contouring procedures. Noninvasive radiofrequency devices have been used to tighten skin and treat cellulite, but there are few studies confirming their efficacy for abdominal fat reduction. OBJECTIVE: This study explored the effects of four noninvasive radiofrequency (RF) treatments on abdominal fat in Asian subjects, evaluating body weight, body mass index (BMI), and waist circumference. METHODS: In this study, 16 patients with abdominal obesity were treated four times with a noninvasive and contactless selective RF device (VANQUISH ME™, BTL Aesthetics). Treatments were 7 days apart and lasted 45 min each. The BMI and circumference of the upper, middle, and lower abdomen were measured at baseline and after each treatment. RESULTS: There were statistically significant reductions in BMI and abdominal circumference in all 16 patients (P < .05). Most patients only experienced a slight abdominal heat sensation and minimal body sweating during the treatment, and no adverse reactions were observed after the treatment. CONCLUSION: The noninvasive and contactless selective RF technique was effective and safe in reducing fat, BMI, and abdominal circumference.

PiQSARS: A pipeline for quantitative and statistical analyses of ratiometric fluorescent biosensors.

Genetically encoded ratiometric fluorescent probes are cutting-edge tools in biology. They allow precise and dynamic measurement of various physiological parameters within cell compartments. Because data extraction and analysis are time consuming and may lead to inconsistencies between results, we describe here a standardized pipeline for•Semi-automated treatment of time-lapse fluorescence microscopy images.•Quantification of individual cell signal.•Statistical analysis of the data.First, a dedicated macro was developed using the FIJI software to reproducibly quantify the fluorescence ratio as a function of time. Raw data are then exported and analyzed using R and MATLAB softwares. Calculation and statistical analysis of selected graphic parameters are performed. In addition, a functional principal component analysis allows summarizing the dataset. Finally, a principal component analysis is performed to check consistency and final analysis is presented as a visual diagram. The method is adapted to any ratiometric fluorescent probe. As an example, the analysis of the cytoplasmic HyPer probe in response to an acute cell treatment with increasing amounts of hydrogen peroxide is shown. In conclusion, the pipeline allows to save time and analyze a larger amount of samples while reducing manual interventions and consequently increasing the robustness of the analysis.