Pulmonary inflammation and viral replication define distinct clinical outcomes in fatal cases of COVID-19.

COVID-19 has affected more than half a billion people worldwide, with more than 6.3 million deaths, but the pathophysiological mechanisms involved in lethal cases and the host determinants that determine the different clinical outcomes are still unclear. In this study, we assessed lung autopsies of 47 COVID-19 patients and examined the inflammatory profiles, viral loads, and inflammasome activation. Additionally, we correlated these factors with the patient's clinical and histopathological conditions. Robust inflammasome activation was detected in the lungs of lethal cases of SARS-CoV-2. Experiments conducted on transgenic mice expressing hACE2 and infected with SARS-CoV-2 showed that Nlrp3-/- mice were protected from disease development and lethality compared to Nlrp3+/+ littermate mice, supporting the involvement of this inflammasome in disease exacerbation. An analysis of gene expression allowed for the classification of COVID-19 patients into two different clusters. Cluster 1 died with higher viral loads and exhibited a reduced inflammatory profile than Cluster 2. Illness time, mechanical ventilation time, pulmonary fibrosis, respiratory functions, histopathological status, thrombosis, viral loads, and inflammasome activation significantly differed between the two clusters. Our data demonstrated two distinct profiles in lethal cases of COVID-19, thus indicating that the balance of viral replication and inflammasome-mediated pulmonary inflammation led to different clinical outcomes. We provide important information to understand clinical variations in severe COVID-19, a process that is critical for decisions between immune-mediated or antiviral-mediated therapies for the treatment of critical cases of COVID-19.

Organoselenium Has a Potent Fungicidal Effect on Cryptococcus neoformans and Inhibits the Virulence Factors.

Cryptococcosis therapy is often limited by toxicity problems, antifungal tolerance, and high costs. Studies approaching chalcogen compounds, especially those containing selenium, have shown promising antifungal activity against pathogenic species. This work aimed to evaluate the in vitro and in vivo antifungal potential of organoselenium compounds against Cryptococcus neoformans. The lead compound LQA_78 had an inhibitory effect on C. neoformans planktonic cells and dispersed cells from mature biofilms at similar concentrations. The fungal growth inhibition led to an increase in budding cells arrested in the G2/M phase, but the compound did not significantly affect structural cell wall components or chitinase activity, an enzyme that regulates the dynamics of the cell wall. The compound also inhibited titan cell (Tc) and enlarged capsule yeast (NcC) growth and reduced the body diameter and capsule thickness associated with increased capsular permeability of both virulent morphotypes. LQA_78 also reduced fungal melanization through laccase activity inhibition. The fungicidal activity was observed at higher concentrations (16 to 64 µg/mL) and may be associated with augmented plasma membrane permeability, ROS production, and loss of mitochondrial membrane potential. While LQA_78 is a nonhemolytic compound, its cytotoxic effects were cell type dependent, exhibiting no toxicity on Galleria mellonella larvae at a dose ≤46.5 mg/kg. LQA_78 treatment of larvae infected with C. neoformans effectively reduced the fungal burden and inhibited virulent morphotype formation. To conclude, LQA_78 displays fungicidal action and inhibits virulence factors of C. neoformans. Our results highlight the potential use of LQA_78 as a lead molecule for developing novel pharmaceuticals for treating cryptococcosis.

Snakebite envenoming in Brazilian children: clinical aspects, management and outcomes.

Snakebite envenoming is currently considered a neglected tropical disease, which affects over 5 million people worldwide, and causes almost 150 000 deaths every year, as well as severe injuries, amputations and other sequelae. Snakebite envenoming in children, although proportionally less frequent, is generally more severe, and represents an important challenge for pediatric medicine, since they often result in worse outcomes. In Brazil, given its ecological, geographic and socioeconomic characteristics, snakebites are considered an important health problem, presenting approximately 30 000 victims per year, approximately 15% of them in children. Even with low snakebite incidence, children tend to have higher snakebite severity and complications due to the small body mass and same venom volume inoculated in comparison to adults, even though, due to the lack of epidemiological information about pediatric snakebites and induced injuries, it is difficult to measure the treatment effectiveness, outcomes and quality of emergency medical services for snakebites in children. In this review, we report how Brazilian children are affected by snakebites, describing the characteristics of this affected population, clinical aspects, management, outcomes and main challenges.

Enzymatic and structural properties of human glutamine:fructose-6-phosphate amidotransferase 2 (hGFAT2).

Glycoconjugates play a central role in several cellular processes, and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in Escherichia coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics, and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.

Color heterogeneity and individual color changes in dentin and enamel bleached in the presence of a metallic orthodontic bracket.

OBJECTIVE: This in-vitro study evaluated the color changes in enamel and dentin bleached in the presence of an orthodontic bracket, as well as the heterogeneity of the color and contribution of each tissue to the color change (CTCC). MATERIALS AND METHODS: Enamel-dentin specimens from extracted bovine teeth were bleached before or after bracket removal or maintained without any treatment (control). The colors of the dentin and enamel were measured individually and after recombining these substrates. The changes in the color (ΔE00 ) and whitening index (ΔWID) were estimated by the color difference between the bleached specimens and those of the control. The CTCC was also calculated by recombining the tissues from different treatments. Images of specimens obtained from a stereomicroscope were used to analyze the color homogeneity within each tissue. RESULTS: The highest values of ΔE00 and ΔWID were observed in the specimens bleached in the absence of a bracket. Bleaching in the presence of a bracket resulted in enamel with a center that was whiter than the periphery. Dentin and enamel presented similar CTCC values. CONCLUSIONS: Tooth bleaching performed in the presence of a metallic bracket resulted in a reduced bleaching effect and increased color heterogeneity within both tissues. CLINICAL SIGNIFICANCE: Tooth bleaching before the orthodontic bracket removal reduces the bleaching effect in both dentin and enamel. Moreover, a heterogeneous color of enamel surface can be observed after tooth bleaching in the presence of a nickel-free metallic orthodontic bracket.

Technical benefit on apple fruit of controlled atmosphere influenced by 1-MCP at molecular levels.

The apple is a highly perishable fruit after harvesting and, therefore, several storage technologies have been studied to provide the consumer market with a quality product with a longer shelf life. However, little is known about the apple genome that is submitted to the storage, and even less with the application of ripening inhibitors. Due to these factors, this study sought to elucidate the transcriptional profile of apple cultivate Gala stored in a controlled atmosphere (AC) treated and not treated with 1-methyl cyclopropene (1-MCP). Through the genetic mapping of the apple, applying the microarray technique, it was possible to verify the action of treatments on transcripts related to photosynthesis, carbohydrate metabolism, response to hormonal stimuli, nucleic acid metabolism, reduction of oxidation, regulation of transcription and metabolism of cell wall and lipids. The results showed that the transcriptional profile in the entire genome of the fruit showed significant differences in the relative expression of the gene, this in response to CA in the presence and absence of 1-MCP. It should be noted that the transcription genes involved in the anabolic pathway were only maintained after six months in fruits treated with 1-MCP. The data in this work suggests that the apple in the absence of 1-MCP begins to prepare its metabolism to mature, even during the storage period in AC. Meanwhile, in the presence of the inhibitor, the transcriptional profile of the fruit is similar to that at the time of harvest. It was also found that a set of genes that code for ethylene receptors, auxin homeostasis, MADS Box, and NAC transcription factors may be involved in the regulation of post-harvest ripening after storage and in the absence of 1-MCP.

Presence of dynapenia and association with anthropometric variables in cancer patients.

BACKGROUND: Dynapenia is defined as an age-related loss of muscle strength. There is little information on dynapenia in cancer patients and on how it relates to anthropometric variables. The aim of this study was to analyze the presence of dynapenia and its association with anthropometric variables in hospitalized cancer patients. METHODS: Participants comprised adult and elderly cancer patients evaluated within the first 48 h of hospital admission to a tertiary public hospital, a referral center for gastrointestinal tract surgery. Anthropometric variables were measured according to standardized protocols. Dynapenia was identified based on handgrip strength (HGS), according to the cutoff points defined by the European Working Group on Sarcopenia in Older People (EWGSOP2), with values for women < 16 kg and for men < 27 kg. Statistical analysis was performed using SPSS software, version 22.0, with a significance level of 5%. RESULTS: This study included 158 patients aged in average 59.5 ± 14.0 years; of these, 53.6% were elderly, 58.9% non-white and 59.5% had some degree of malnutrition. The most prevalent type of cancer was that of the lower gastrointestinal tract (33.5%). The presence of dynapenia was observed in 23.4% of the patients and cachexia in 36.1%. There was an association between dynapenia with age (p < 0.001), life stage (p = 0.002) and race/color (p = 0.027), and also with body mass index (BMI) (p = 0.001) and adductor pollicis muscle thickness (APMT) of both hands (p < 0.05). After logistic regression analysis, adjusted for the sociodemographic variables, the APMT of the dominant hand and the low weight determined by body mass index remained associated with the occurrence of dynapenia (p < 0.05). CONCLUSIONS: In this study we confirmed that dynapenia was present in cancer patients, being associated with APMT of the dominant hand and low weight. HSG was proven to be a reliable and complementary measure to be added to the process of assessing nutritional status, contributing to the nutritional diagnosis of these patients and to the detection of early muscle depletion.

Cytotoxicity of 4-substituted quinoline derivatives: Anticancer and antileishmanial potential.

Chemical modifications of quinoline moiety have been recognized as a useful strategy to development of new drugs. Here, the cytotoxicity of a set of twenty-four 4-substituted quinolines (named HTI) was screened for their antitumor and antileishmanial potential in vitro, and the underlying mechanisms investigated. HTI 21 and HTI 22 exhibited the highest cytotoxicity, being selected to the subsequent studies. Both derivatives induced caspase-dependent apoptosis associated to the dissipation of the mitochondrial transmembrane potential (ΔΨ) and ROS generation. HTI-induced cell death was calcium dependent, associated to thiol oxidation and cysteine proteases activation. In isolated mitochondria, HTI derivatives promoted mitochondrial permeabilization by different mechanisms. The inhibition of BCL-2 by venetoclax enhanced the HTI-induced cytotoxicity. Regarding the inhibition of cysteine proteases type B of Leishmania mexicana, HTI 15 exhibited the most potent inhibitory activity through a linear non-competitive mechanism. These data highlight the therapeutic potential of 4-substituted quinolines as antitumor and antileishmanial drugs.

Genetic evidence for a species complex within the piranha Serrasalmus maculatus (Characiformes, Serrasalmidae) from three Neotropical river basins based on mitochondrial DNA sequences.

Mitochondrial molecular markers (DNA sequences of D-loop, cytochrome b and cytochrome c oxidase I) were employed to characterize populations of the piranha Serrasalmus maculatus from Upper Paraná, Upper Paraguay and Tocantins River basins. D-loop sequences of S. maculatus population from Paraná-Paraguay River basin exhibited tandem repeats of short motifs (12 base pairs) and variable numbers depending on specimens, accounting for length variation. Concatenated mitochondrial sequences suggested that S. maculatus encompasses different mitochondrial DNA lineages. Although sampling was restricted to three river basins, phylogenetic analysis clearly indicated that the species currently recognized as S. maculatus presents high genetic variability. Maximum likelihood and Bayesian analysis clustered S. maculatus populations according to their locations. However, the highest genetic differentiation was identified between populations from Paraná-Paraguay system and Tocantins River basin. Three species delimitation analyses (PTP, GMYC, and ABGD) suggested that there are at least two species among the analyzed populations. The analysis of the mitochondrial sequences evidenced genetic differentiation among populations corresponding to related, but different species, suggesting that at least S. maculatus from the Tocantins River and Paraná-Paraguay River basins are most likely different species. Therefore, S. maculatus should be considered a species complex with morphologically cryptic diversity. An integrative revision is suggested.

Activity of silver nanoparticles on prokaryotic cells and Bothrops jararacussu snake venom.

Nanoparticle-conjugated venom-toxins of venomous animals and its therapeutic efficacy against emerging or neglecting diseases is a promising strategy. In this study, silver nanoparticles (AgNPs ∼50 nm, 0.081 mg mL-1) were studied against the neuromuscular blockade, myotoxic effects induced by Bothrops jararacussu venom (60 µg mL-1) and also against prokaryotic cells. The neurotoxicity was evaluated on ex vivo mouse phrenic nerve-diaphragm using traditional myographic technique, able to obtain functional contractile responses and to check the neurotransmission. The myotoxicity on mammalian cells was evaluated in muscles resulting from pharmacological assays using routine histological techniques and light microscopy. The toxicity to prokaryotic cells was evaluated on Salmonella typhimurium TA100 without metabolic activation. The in vitro preincubation model between AgNPs and venom was enough to abolish toxic effects of B. jararacussu venom, but mammalian cells were highly sensitive to AgNPs more than prokaryotic cells, by acting as dose-independently and dose-dependently parameters, respectively. These results allowed us to conclude that AgNPs showed promising activity as antivenom agent but for its safer use, the toxicity should be evaluated on experimental animals.

Prevalence and reliability of treatment-based classification for subgrouping patients with low back pain.

OBJECTIVES: To observe the distribution of patients who presented with low back pain (LBP) and to determine the between therapists' interrater reliability of assessments in a private outpatient setting using treatment-based classification (TBC) subgroups. METHODS: An observational and methodological study was conducted. Four hundred and twenty-nine patients (231 male; 198 female) presenting LBP symptoms and referred to conservative treatment were assessed by 13 physical therapists who conducted a 60-min examination process utilizing TBC subgroups. Interrater reliability analyses from six raters were assessed using Fleiss' kappa and previously recorded data (n = 30). RESULTS: In this study, 65.74% of patients were classified in only one subgroup, the most prevalent being stabilization (21.91%), followed by extension (15.38%), traction (11.89%), flexion (10.96%), manipulation (5.13%), and lateral shift (0.47%). Approximately 20.98% of patients were classified in two subgroups, where the most frequent overlaps were flexion + stabilization (7.46%), extension + stabilization (6.06%), flexion + traction (4.20%), extension + manipulation (1.86%), and 13.29% of patients were not classified in any TBC subgroup. Analysis of interrater reliability showed a kappa value of 0.62 and an overall agreement of 66% between raters. DISCUSSION: LBP is a heterogeneous clinical condition and several classification methods are proposed in the attempt to observe better outcomes for patients. Eighty-five percent of patients assessed were able to be classified when using the TBC assessment and reliability analysis showed a substantial agreement between raters. LEVEL OF EVIDENCE: 2c.

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity.

Minor snake venom proteins: Structure, function and potential applications.

Snake venoms present a great diversity of pharmacologically active compounds that may be applied as research and biotechnological tools, as well as in drug development and diagnostic tests for certain diseases. The most abundant toxins have been extensively studied in the last decades and some of them have already been used for different purposes. Nevertheless, most of the minor snake venom protein classes remain poorly explored, even presenting potential application in diverse areas. The main difficulty in studying these proteins lies on the impossibility of obtaining sufficient amounts of them for a comprehensive investigation. The advent of more sensitive techniques in the last few years allowed the discovery of new venom components and the in-depth study of some already known minor proteins. This review summarizes information regarding some structural and functional aspects of low abundant snake venom proteins classes, such as growth factors, hyaluronidases, cysteine-rich secretory proteins, nucleases and nucleotidases, cobra venom factors, vespryns, protease inhibitors, antimicrobial peptides, among others. Some potential applications of these molecules are discussed herein in order to encourage researchers to explore the full venom repertoire and to discover new molecules or applications for the already known venom components.

Functionalization of protected tyrosine via Sonogashira reaction: synthesis of 3-(1,2,3-triazolyl)-tyrosine.

1,2,3-Triazol tyrosines were synthesized from tyrosine alkynes that were in turn prepared via Sonogashira cross-coupling reaction. The tyrosine alkynes were subjected to click-chemistry reaction conditions leading to the corresponding 3-(1,2,3-triazolyl)-tyrosines in yields ranging from moderate to good.

Evidence of ternary complex formation in Trypanosoma cruzi trans-sialidase catalysis.

Trypanosoma cruzi trans-sialidase (TcTS) is a key target protein for Chagas disease chemotherapy. In this study, we investigated the implications of active site flexibility on the biochemical mechanism of TcTS. Molecular dynamics studies revealed remarkable plasticity in the TcTS catalytic site, demonstrating, for the first time, how donor substrate engagement with the enzyme induces an acceptor binding site in the catalytic pocket that was not previously captured in crystal structures. Furthermore, NMR data showed cooperative binding between donor and acceptor substrates, supporting theoretical results. In summary, our data put forward a coherent dynamic framework to understand how a glycosidase evolved its highly efficient trans-glycosidase activity.

Trypanosoma cruzi Trans-sialidase: structural features and biological implications.

Trypanosoma cruzi trans-sialidase (TcTS) has intrigued researchers all over the world since it was shown that T. cruzi incorporates sialic acid through a mechanism independent of sialyltransferases. The enzyme has being involved in a vast myriad of functions in the biology of the parasite and in the pathology of Chagas' disease. At the structural level experiments trapping the intermediate with fluorosugars followed by peptide mapping, X-ray crystallography, molecular modeling and magnetic nuclear resonance have opened up a three-dimensional understanding of the way this enzyme works. Herein we review the multiple biological roles of TcTS and the structural studies that are slowly revealing the secrets underlining an efficient sugar transfer activity rather than simple hydrolysis by TcTS.

Replacing HEMA with alternative dimethacrylates in dental adhesive systems: evaluation of polymerization kinetics and physicochemical properties.

PURPOSE: To evaluate the mechanical and physical properties of experimental HEMA-containing and HEMA-free resin adhesives. MATERIALS AND METHODS: Experimental HEMA-free adhesives containing alternative dimethacrylates (bis-EMA 10 [B10], bis-EMA 30 [B30], PEG 400 [P400], PEG 1000 [P1000], PEG 400 UDMA [UP400]) were formulated and compared with a HEMA-containing adhesive (control). The adhesives were characterized by rheological analysis, polymerization kinetics (PK), water sorption (WS), and solubility (SL) tests. Flexural strength (FS) and flexural modulus (E) tests were performed under dry or wet conditions (distilled water or 70% ethanol solution). One-way and two-way ANOVA as well as Tukey's test were used to evaluate differences between groups (p < 0.05). RESULTS: The control group showed the lowest viscosity and was the only one with a degree of conversion lower than 50%. The control and the P1000 adhesive showed the statistically significantly highest WS (p < 0.05). The control and the UP400 adhesive showed the highest FS and E, and the dry-stored specimens showed more improved mechanical strength than did the wet-stored specimens (p < 0.05). CONCLUSION: The physicomechanical properties of some of the HEMA-free adhesives were substantially improved when compared with those of the control, indicating that they could be potential monomers for the development of HEMA-free adhesive systems.

Hyperglycosylation impairs the inhibitory activity of rCdtPLI2, the first recombinant beta-phospholipase A2 inhibitor.

Crotoxin, a phospholipase A2 (PLA2) complex and the major Crotalus venom component, is responsible for the main symptoms described in crotalic snakebite envenomings and a key target for PLA2 inhibitors (PLIs). PLIs comprise the alpha, beta and gamma families, and, due to a lack of reports on beta-PLIs, this study aimed to heterologously express CdtPLI2 from Crotalus durissus terrificus venom gland to improve the knowledge of the neglected beta-PLI family. Thereby, recombinant CdtPLI2 (rCdtPLI2) was produced in the eukaryotic Pichia pastoris system to keep some native post-translational modifications. rCdtPLI2 (~41 kDa) presents both N- and O-linked glycans. Alpha-mannosidase digested-rCdtPLI2 (1 mol) strongly inhibited (73 %) CB-Cdc catalytic activity (5 mol), demonstrating that glycosylations performed by P. pastoris affect rCdtPLI2 action. Digested-rCdtPLI2 also inhibited PLA2s from diverse Brazilian snake venoms. Furthermore, rCdtPLI2 (1 mol) abolished the catalytic activity of Lmr-PLA2 (5 mol) and reduced the CTx-Cdc (5 mol) enzyme activity by 65 %, suppressing basic and acidic snake venom PLA2s. Additionally, crotalic antivenom did not recognize rCdtPLI2, suggesting a lack of neutralization by antivenom antibodies. These findings demonstrate that studying snake venom components may reveal interesting novel molecules to be studied in the snakebite treatment and help to understand these underexplored inhibitors.

The complex repertoire of Tityus spp. venoms: Advances on their composition and pharmacological potential of their toxins.

Animal venoms are a rich and complex source of components, including peptides (such as neurotoxins, anionic peptides and hypotensins), lipids, proteins (such as proteases, hyaluronidases and phospholipases) and inorganic compounds, which affect all biological systems of the envenoming victim. Their action may result in a wide range of clinical manifestations, including tachy/bradycardia, hyper/hypotension, disorders in blood coagulation, pain, edema, inflammation, fever, muscle paralysis, coma and even death. Scorpions are one of the most studied venomous animals in the world and interesting bioactive molecules have been isolated and identified from their venoms over the years. Tityus spp. are among the scorpions with high number of accidents reported in the Americas, especially in Brazil. Their venoms have demonstrated interesting results in the search for novel agents with antimicrobial, anti-viral, anti-parasitic, hypotensive, immunomodulation, anti-insect, antitumor and/or antinociceptive activities. Furthermore, other recent activities still under investigation include drug delivery action, design of anti-epileptic drugs, investigation of sodium channel function, treatment of erectile disfunction and priapism, improvement of scorpion antivenom and chelating molecules activity. In this scenario, this paper focuses on reviewing advances on Tityus venom components mainly through the modern omics technologies as well as addressing potential therapeutic agents from their venoms and highlighting this abundant source of pharmacologically active molecules with biotechnological application.

Pressurized liquid extraction assisted by high-intensity ultrasound to obtain hesperidin from lime waste: Integration of in-line purification and on-line chromatographic analysis.

Hesperidin is a phenolic compound usually found in citrus fruits, which is known for its anti-inflammatory and antioxidant properties. This bioactive compound has already been used to formulate medications to treat chronic venous insufficiency. In this work, through a system which allows the in-line coupling of the pressurized liquid extraction (PLE) and high-intensity ultrasound (HIUS) with solid phase extraction (SPE), and analysis by high-performance liquid chromatography with UV-Vis detector (HPLC-UV) in on-line mode, a method was developed to obtain, separate, and quantify hesperidin from the industrial waste of lime. An eco-friendly approach with water and ethanol as extraction solvents was used. Parameters such as temperature (80, 100, and 120 °C) and HIUS power (0, 200, and 400 W) were evaluated regarding hesperidin yield. In this context, the higher hesperidin yield (18.25 ± 1.52 mg/g) was achieved using water at a subcritical state (120 °C and 15 MPa). The adsorbent SepraTM C-18-E isolated hesperidin from the other extracted compounds employing 50% ethanol in the SPE elution. The possibility ofon-lineanalysis coupling a high-performance liquid chromatograph to an ultraviolet detector (HPLC-UV) system was studied and shown to be a feasible approach for developing integrated technologies. Conventional extractions and their antioxidant capacities were evaluated, highlighting the advantages of the HIUS-PLE-SPE extractive method. Furthermore, the on-linechromatographic analysis showed the potential of the HIUS-PLE-SPE- HPLC-UV system to quantify the extracted compounds in real time.