The LCRs occur during diastolic depolarization (DD) and activate an inward Na+/Ca2+ exchange current that accelerates the DD rate prompting the next action potential (AP). Basal phosphodiesterases (PDEs) activation degrades cAMP, reduces basal cAMP/PKA-dependent phosphorylation, and suppresses normal spontaneous firing of SANCs. The cAMP-degrading PDE1, PDE3, and PDE4 represent major PDE activities in rabbit SANC, and PDE inhibition by 3-isobutyl-1-methylxanthine (IBMX) increases spontaneous firing of SANC by ∼50%. Though inhibition of single PDE1–PDE4 only moderately increases spontaneous SANC firing, dual PDE3 + PDE4 inhibition produces a synergistic effect hastening the spontaneous SANC beating rate by ∼50%. Here, we describe the expression and distribution of different PDE subtypes within rabbit SANCs, several specific targets (L-type Ca2+ channels and phospholamban) regulated by basal concurrent PDE3 + PDE4 activation, and critical importance of RyR Ca2+ releases for PDE-dependent regulation of spontaneous SANC firing. Colocalization of PDE3 and PDE4 beneath sarcolemma or in striated patterns inside SANCs strongly suggests that PDE-dependent regulation of cAMP/PKA signaling might be executed at the local level; this idea, however, requires further verification.Spontaneous firing of sinoatrial (SA) node cells (SANCs) is regulated by cyclic adenosine monophosphate (cAMP)-mediated, protein kinase A (PKA)-dependent (cAMP/PKA) local subsarcolemmal Ca The SA node automaticity is generated within the SA node pacemaker cells (SANCs), which fire spontaneous action potentials (APs) because of gradual depolarization of the membrane potential called diastolic depolarization (DD) linked to complex interactions of ‘coupled clock’ mechanisms. The ‘membrane clock’ refers to multiple voltage-gated ion channels and transporters in the cell membrane, including hyperpolarization-activated “funny” current I2+ via sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) and Ca2+ release channels, ryanodine receptors (RyRs). The SANCs can generate spontaneous local Ca2+ releases (LCRs) from RyR in the subsarcolemmal space during late DD before the AP upstroke  and RyRs in rabbit SANC . This clock entrainment provides an additional degree of flexibility and robustness to the generation of spontaneous APs by the cardiac pacemaker cells  and are equipped to cycle Ca next AP . Colocalbit SANC  permits bit SANC , manifeser cells .2+ clock” are regulated by cAMP and cAMP-mediated PKA-dependent phosphorylation. Funny current is directly activated by cAMP , RyR, and SERCA] are also regulated by PKA-dependent phosphorylation (The cyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that modulates multiple cell processes, e.g., cAMP-mediated protein kinase A (PKA)-dependent protein phosphorylation. Basal level of cAMP in rabbit SANCs is substantially higher than in ventricular myocytes (VM)  due to c by cAMP . SeveralIf, etc. . Proteinrylation . The genrylation .2+/calmodulin, PDE2 is stimulated by cGMP, PDE3 is inhibited by cGMP, and PDE4 is specific for cAMP. The PDE3 and PDE4 represent the major cAMP PDE activities in cardiac myocytes , the only known mechanism to degrade cAMP . The PDEmyocytes .Figure 1C). Combined activities of PDE3 and PDE4 represent the major basal PDE activities in the rabbit SA node, accounting for ∼50% in cytosolic and ∼90% in SR fractions , markedly increases the level of cAMP and concurrently increases the spontaneous SANC firing rate by ∼50%, which surpasses the positive chronotropic effect of the β-AR agonist isoproterenol . The PDE1 activity might have a greater impact at higher cAMP levels; indeed, stimulation of ACs with forskolin markedly increases both cAMP level and PDE1 activity in paced mouse VM , concurrent inhibition of PDE3 + PDE4 increases the spontaneous SANC beating rate by ∼48% . An acceleration of spontaneous SANC firing by concomitant PDE3 + PDE4 inhibition by ∼twofold exceeds the summed increases in the spontaneous firing produced by inhibition of PDE3 (∼20%) and PDE4 (∼5%) alone  are relatively small  alone , indicat%) alone .2+ cycling proteins phosphorylated in the basal state in rabbit SANC are PLB  increase in PLB phosphorylation at Ser16 site in SANC, but dual PDE3 + PDE4 inhibition increases PLB phosphorylation by ∼110%, an effect comparable to that of IBMX . Therefore, basal PLB phosphorylation at Ser16 site in SANC appeared to be regulated by synergism of concurrent PDE3 + PDE4 activation . Dual PDE3 + PDE4 inhibition increases ICa,L in rabbit SANC by ∼100%, markedly exceeding combined effects of separate PDE3 or PDE4 inhibition and creating effect comparable with that of IBMX . Therefore, dual PDE3 + PDE4 activation regulates basal ICa,L amplitude in rabbit SANC in a synergistic manner . An increase in cAMP-mediated PKA-dependent phosphorylation of Ca2+ cycling proteins concurrently elevates amount of Ca2+ (influx via ICaL) available for pumping into SR, accelerates the SR Ca2+ refilling (PLB), and likely alters the threshold for spontaneous Ca2+ releases (RyR), creating conditions required to boost spontaneous LCRs and speed up their appearance.The LCRs are also regulated by basal PDE activation both in intact and permeabilized rabbit SANCs . During P < 0.05) and decreases the LCR period by ∼15% (P < 0.05), while changes in these parameters by rolipram are relatively small. Dual PDE3 + PDE4 inhibition, however, produces a synergistic effect and augments both the LCR size and number by ∼45% (P < 0.01) each, as RyR activation becomes more synchronized via RyR recruitment and decreases the LCR period by ∼40% (P < 0.01). An amplification of local RyR Ca2+ release activates augmented INCX at earlier times leading to an increase in the DD rate and spontaneous SANC beating rate .In contrast, when RyR function is disabled by ryanodine, dual PDE3 + PDE4 inhibition failed to accelerate the DD rate or increase the spontaneous SANC beating rate , despiteplitudes , indicat2+ channels or PLB and likely others are regulated by concurrent PDE3 + PDE4 activation in a synergistic manner , changes in global intracellular cAMP in SANC did not follow this pattern. Specifically, dual PDE3 + PDE4 inhibition in SANC lysates increases cAMP level by only ∼90% less than the effect produced by IBMX (∼160%) or the sum of cAMP elevations created by inhibition of either PDE3 (∼45%) or PDE4 (∼56%) alone . Therefore, an increase in the spontaneous SANC beating rate by dual PDE3 + PDE4 inhibition  is not created by changes in global cAMP, but likely by changes in local cAMP levels produced in the vicinity of PLB or L-type Ca2+ channels  or RyR etc.Though L-type Ca2+ channels and HCN4 channels are also localized to caveolae , there is no information on how much of that activity might be inside or outside of caveolae.Rabbit SANC lack t-tubular system, but they have considerable number of caveolae, flask-like invaginated lipid rafts containing caveolin, which could provide abundant physical boundaries for localized cAMP signaling. In rabbit SANCs, caveolae increase the surface plasma membrane by ∼115 and ∼30% in VM . A variecaveolae . Caveolacaveolae . Though In VM, intracellular cAMP concentration in the basal conditions is close to 1 μmol/L, and this value is ∼10-fold higher during hormone or neurotransmitter activation . Multipl2/s) (Because affinity of endogenous PKA for cAMP is in the range 100–300 nmol/L  and free2/s) , cAMP wo2/s) . Therefo2/s) . The PDE2/s) .2+ channels or SR in VM. Indeed, PDE3A is colocalized with SERCA2-PLB-AKAP18 multiprotein complex or “signalosome” that regulates refilling of SR through modulation of PLB phosphorylation both in mouse and human VM . Compared with PDE3 or PDE4, PDE1 mRNA in rabbit SANC is relatively low, but PDE1A transcript abundance in SANC surpasses that in VM by fourfold .At the messenger RNA level, PDE3A, PDE4A, PDE4B, and PDE4D are the major cAMP-degrading PDE subtypes expressed in both rabbit SANC and VM . Expressfourfold . InteresFigure 2; Figure 2D) suggests that, like in the mouse heart, these PDE subtypes could work together limiting Ca2+ influx through L-type Ca2+ channels in a synergistic manner . The PDE3A is also detected in a striated pattern and colocalizes with the Z-line associated protein α-actinin in rabbit SANC. Similar to human or mouse VM . Colocalization of PDE3A and PDE4D with SERCA and PLB suggests that these PDE isoforms could likely regulate cAMP-mediated PKA-dependent phosphorylation of major SR proteins in SANC .Very little is known about the distribution of major PDE subtypes within SANC. Recent studies established that PDE1A, PDE3A, and PDE4B are localized beneath the sarcolemma of rabbit SANC . Since PDE3 affinity is ∼10–100 nmol/L, it is likely that PDE3 is active and degrades cAMP in the basal state, while PDE4 remains dormant. An increase in local cAMP level by inhibition of PDE3 alone may increase cAMP to a range required for PDE4 activation, concurrently elevating PKA-dependent phosphorylation of PDE4, which is associated with 2–6-fold increase in PDE4 activity  by synergistic dual PDE3 + PDE4 activation could be energetically beneficial, since slight variations in local cAMP levels at multiple locations could lead to substantial functional effects.Though dual PDE3 and PDE4 inhibition could interact synergistically to modulate functional effects mediated by cAMP in multiple cell types, the mechanisms underlying these synergistic effects remain unclear. This synergistic effect could be based on colocalization and interaction of different PDE3 and PDE4 subtypes (activity . ActivatRecent studies demonstrated that physiologically relevant cAMP signals operate within the nanometer range, creating local cAMP nanodomains, while ‘global cAMP’ is less involved in functional responses . Computai inhibition, leading to a diminished level of cAMP/PKA-dependent phosphorylation accompanied by a ∼50% decrease in spontaneous SANC firing rate (-1 μmol/L (Though it is helpful to directly measure PDE activity using classical biochemical assays, this experimental approach lacks spatial resolution. Local degradation of cAMP produced by PDEs creates cAMP gradients and forms nanodomain organization of cellular signaling . Real-tiing rate . Furthering rate . Changesing rate . Becauseing rate . Recentl1 μmol/L . In shorTV made substantial contributions to conception, design and writing of the manuscript, and approved the last version for publication. EK helped to draft the manuscript, revised its content, and approved the last version for publication. EL helped to draft the manuscript, critically reviewed its content, and approved the last version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium tuberculosis, the causative agent of tuberculosis, has surpassed HIV as the leading cause of death due to an infectious disease worldwide, being responsible for more than 1.5 million deaths in low-income countries. In response to a pandemic threat by drug resistant strains, the tuberculosis research community is searching for new chemical entities with novel mechanisms of action to avoid drug resistance and shorten treatment regimens using combinatorial chemotherapy. Herein, we have identified several novel chemical scaffolds, GSK97C (spiro-oxazolidin-2-one), GSK93A , which target M. tuberculosis aspartyl-tRNA synthetase (Mt-AspRS), an essential component of the protein synthesis machinery of tuberculosis, using a whole-cell target-based screening strategy against a genetically modified Mycobacterium bovis BCG strain. We also provide further evidence of protein inhibition and inhibitor profiling through a classical aminoacylation reaction and a tRNA-independent assay, respectively. Altogether, our results have identified a number of hit new molecules with novel mechanism of action for further development through medicinal chemistry as hits and leads. It represents a major threat to global health with one-third of the world’s population infected with latent tuberculosis, and multi-drug resistant (MDR) and extensively drug resistant (XDR) strains posing a significant potential threat to health care systems if left unaddressed. To effectively treat patients with MDR-TB, a 24-month-treatment regimen with second-line drugs, such as aminoglycosides and fluoroquinolones is needed, which unavoidably increases pill burden and potential side effects due to nephrotoxicity and damage to the central nervous system2. In order to achieve effective eradication of MDR and XDR-TB safer and more effective drugs are urgently needed with entirely novel mechanism of action3.Tuberculosis (TB) ranks first as the leading cause of death due to an infectious disease worldwide, overcoming HIVMycobacterium tuberculosis have provided many promising new hits, representing a shifting strategy from classical target-based approaches4. Whole genome sequencing (WGS) of spontaneous resistant isolates generated against HTS hits in vitro has proven to be a valid initial starting point for target identification5. The discovery of TMC2077, now licensed as the FDA-approved drug bedaquiline8, was one of the first hits to be characterised using this approach of WGS of resistant isolates, highlighting the success of phenotypic screening campaigns9. However, further detailed biochemical and genetic evidence is required to elucidate the precise mode of action of small molecule hits as exemplified by the recent studies of inhibitors targeting MmpL312.Phenotypic high-throughput screening (HTS) strategies against 14, their specificity towards their cognate amino acid and tRNA15 and their evolution16. Their utility as the target of anti-infective agents is demonstrated by the use of the clinically approved isoleucyl-tRNA inhibitor, pseudomonic acid A17, although drug discovery efforts against these targets has remained challenging due to: (I) the lack of translational whole-cell inhibitory activities, (II) off-target effects due to ATP competitiveness and (III) poor pharmacokinetic profiles18. A rhodanine compound was previously identified to target the aspartyl-tRNA synthetase of TB by WGS approaches19, which was then biochemically validated in a tRNA-independent assay20, encouraging further screening campaigns to find more potent and chemically tractable hits against this target. Herein, we have identified Mt-AspRS inhibitors by a whole-cell target-based screening of the so-called TB box21, a GSK library of 11,000 compounds (previously assessed against M. tuberculosis H37Rv) against the surrogate Mycobacterium bovis BCG strain genetically engineered to constitutively express the TB AspRS open-reading frame in a replicative pMV261 plasmid. Combining whole-cell and target-based screening methods allows the discovery of new chemical entities with potential to shorten early drug discovery programmes.Aminoacyl-tRNA synthetases have extensively been studied by many academic research groups to elucidate the kinetics of their two-step reaction mechanismMycobacterium bovis BCG genetically modified to constitutively express the Mt-AspRS open-reading frame. The GSK TB box compound collection of 11,000 compounds21 was used at three independent concentrations  and initial hits were confirmed based on inhibition shift between the two strains  minus % inhibition of M. bovis BCG pMV261::Mt-AspRS [based upon duplicate data]) on ActivityBase (IDBS). Assay quality was monitored in an inter-plate manner with the statistical Z′, the gold standard to assess assay quality and reproducibility in HTS assays22. Plates with Z′ values below 0.4 were discarded for further analysis due to poor assay robustness.In this study we report the identification of a number of biochemically validated Mt-AspRS inhibitors identified using a target-based whole-cell screening assay in Initial hits (250) were cherry-picked for further validation in a dose-response assay at a concentration range of 0.1 up to 100 μM to assess whole-cell potency and confirmation of whole-cell target-engagement (MIC50 shift) using the previously reported rhodanine entity as a tool control compound . Reagent stability was previously assessed over the overall period of the assay and robustness was monitored in an inter-plate manner (Z′ > 0.4). Interestingly, the parent compound GSK87A had failed to show potency in the biochemical validation assays against Mt-AspRS. However, two analogues of GSK87A  we show here how these inhibitors display non-competitive binding mechanism against Mt-AspRS with respect to a β-γ-methyladenosine triphosphate substrate as an ATP surrogate in a tRNA-independent reaction  lack of penetration through bacterial membranes, the latter explaining the shifting strategy from target-based to whole-cell phenotypic approaches. We have expanded the chemical space available against Mt-AspRS that had traditionally been limited by these unspecific, ATP-competitive binding compounds or insufficient whole-cell inhibitory activities, further proving the versatility of Mt-AspRS as potential target for tuberculosis drug discovery. Diversifying the pipeline with inhibitors with novel modes of action is essential to ensure the overall survival of TB drug discovery projects with the potential to reach in vivo stages. Moreover, the success of integrating whole-cell and target-based screening strategies against other targets has previously been demonstrated, allowing the straightforward identification of small molecules against virtually any essential target with acceptable druggability features26. This has the potential to shorten the preclinical turnaround times of drug development projects. In this context, we have shown that expanding the chemical space available for Mt-AspRS inhibitors can expand existing possibilities for drug development by exploiting new hits with novel modes of action and mechanistic features, expanding the pipeline of available preclinical antitubercular agents. The identification of inhibitors targeting conserved binding sites that retain selectivity has been shown to be achievable27 although as we show here, mechanistic studies are needed to derisk potential ATP-competitive inhibitors. However, despite the availability of increasing numbers of preclinical candidates for the treatment of TB, it is important to consider that small molecule drug discovery is not enough to tackle the epidemic. Combinatorial chemotherapy needs to be complemented with rapid diagnostic tools and effective prophylaxis to contain the spread of drug resistant strains. In addition, improved health care is needed in low-income countries so that tuberculosis patients can effectively access new medicines. Altogether, our results represent another step toward the final eradication of TB by expanding the chemical space available against tRNA synthetases of this organism.The availability of new chemical scaffolds with sufficient whole-cell and on-target inhibitory activities displaying novel modes of action represents a promising starting point in early TB drug discovery efforts. Traditionally, drug discovery efforts with aminoacyl tRNA inhibitors had been limited due (I) poor pharmacokinetic properties, (II) off-target effects caused by competitive binding mechanismsAll experiments were performed in accordance with the relevant ethical guidelines and regulations approved by the University of Birmingham and Diseases of the Developing World (DDW-GSK) Ethical Committees where required and there are no other ethical issues to report.M.bovis BCG that had been previously transformed with a pMV261 containing the Mt-AspRS20. Selected transformants were grown using 7H9 media supplemented with 0.025% Tween, 10% ADC  and kanamycin (25 μg/ml).Whole-cell screening experiments were conducted using 4 cells in 25 μL was added into each assay well of a 384-well-plate with white opaque walls using a MultidropTMCombi dispenser (Thermo Scientific). To control evaporation, plates were individually sealed with parafilm, covered with aluminium foil and stored in plastic boxes inside a 37 °C and 5% CO2 incubator with humidity control. At day 7, reconstituted Bac-titer GLO reagent (25 μL) were added into each assay well, which causes cell lysis and allows the luminescent detection of ATP consumption, briefly shaken for 8 minutes and read for luminescence using a Spectra Max M5 reader. Raw luminescence values were standardized to cell survival percentages using positive and negative controls for cell growth. The effect of a given inhibitor was calculated as: % inhibition = 100* ((data- control 1)/(control 2- control 1)) where control 1 = maximum activity , and control 2 = bacterial growth completely inhibited (by treatment with 10 μM rifampicin). Assay performance statistics  were calculated using templates in ActivityBase XE . Dose-response confirmation studies were performed at a concentration range 0.1 up to 100 μM in a 1:3 dilution fashion and raw luminescence values were standardized to the maximum and minimum percentage of inhibition as stated for single-shot experiments. Dose-response curves were obtained on Tibco Spotfire and whole-cell target engagement confirmed by the MIC shift, reported here as the ratio between the mean of the MIC values for each strain.The GSK TB box collection was screened for Mt-AspRS inhibitors based on resistance upon Mt-AspRS overexpression. We selected three different concentrations  based on compound potency in order not to miss any potential hit. A standard inoculum size of 1020. To establish accurate inhibitor potency, prior biochemical characterization is essential to obtain optimised substrate concentrations for aspartic acid (L-Asp), adenosine triphosphate (ATP) and tRNA. For speed and convenience, a pool of commercially available tRNA was used since a high degree of sequence identity is shared between E. coli and M. tuberculosis tRNA molecules. The Mt-AspRS functional characterisation was developed using a classical TCA precipitation method in standard 96-well-polystyrene plates. The aminoacylation reaction was performed in a final assay mixture of 80 μL consisting of 20 mM HEPES buffer pH 7.7, 4 mM MgCl2, 50 mM KCl and a range of substrate and enzyme concentrations in order to determine the initial rate and the kinetic constants of the aminoacylation reaction. The values obtained in the experiments described below were therefore obtained under steady-state conditions following classical Michaelis-Menten guidelines for enzyme kinetic studies. For the determination of the initial rate of the aminoacylation reaction, several enzyme concentrations  were incubated at 37 °C in the presence of 1 mg/mL tRNA from E. coli 600 MRE, 15 μM L-Asp  and 4 mM ATP. The reaction was quenched at several time points over a period of fifteen minutes with 50 μL of 10% TCA. When the time-course was complete, the plates were incubated at 4 °C during 30 minutes to allow for proper tRNA precipitation. The aminoacylated product was then transferred onto GF/C 96-well filter plates (Packard), extensively washed in an excess volume of TCA (10%), once with 95% ethanol and then dried under a heat lamp prior to scintillation counting in a Microbeta Trilux 1450 model, basically in accordance with the method previously described for classical studies of aminoacyl-tRNA synthetases28. For KM determination, varying substrate concentrations were assayed keeping the others at saturating concentrations. For L-Asp kinetic studies, a starter concentration of radiolabelled L-Asp of 15 μM was serially diluted with non-radioactive L-Asp to a final concentration of 900 μM where saturation was observed. The obtained data points were fitted to a Sigmoidal dose-response curve on Graph Pad Prism 7.0, and its IC50 value correSponds to the KM plus the concentration of radiolabelled L-Asp used in the assay.Pure, recombinant Mt-AspRS was obtained as previously describedM values of L-Asp and tRNA, while keeping ATP at a saturating concentration in order to filter out ATP-competitive inhibitors. Compound stocks were made as 10 mM solutions in 100% DMSO and dispensed into 96-well-polystetyrene plates in a 1:3 dilution manner up to a final concentration of 100 μM. In the maximum (100% activity) and minimum (0% activity) controls similar DMSO concentrations were added to check the absence of solvent-related inhibition. The highest concentration of DMSO and CHAPS used in the assay were 1% and 0.5%, respectively. Precipitable radioactivity was transferred onto GF/C filter plates for scintillation counting.The IC50 dose response assays for each were performed at 0.2 μM Mt-AspRS and matching apparent K2, 50 mM KCl, 1 mM DTT, 2 mM ADPCP, 10 mM D-glucose, 0.5 mM NADP+, 10 mM L- Asp, 3 μg of yeast hexokinase and L. mesenteroides glucose-6- phosphate dehydrogenase mixture and 0.5 μM of Mt-AspRS in a final assay volume of 20 μL. The amount of coupled enzymes used was high enough to minimise assay intereference. Stock compounds were dispensed with the Echo 555 (Labcyte) into black bottom 384 plates (Greiner 781101) and were stored at −80 °C until needed. Enzymes and substrates were kept separately in two working solutions prepared at 2X the final assay concentrations. Reactions were triggered with the addition of substrate solution into plates pre-plated with enzyme and no-enzyme control wells. The assay was performed at room temperature and fluorescence values were continuously monitored in EnVision (Perkin Elmer) during the period of the assay (4 minutes). Similar amounts of DMSO (1%) were added into control wells to check for absence of solvent-related inhibition. The raw data was normalised to the positive and negative controls for enzyme activity in Excel and was further processed in Excel and GraphPad Prism 6.0 for IC50 determination.A number of structurally related analogues to GSK97C, GSK93A and GSK87A from the GSK TB box collection were evaluated in a dose-response manner (0.1–100 μM) in a 384-well black-bottom microplate-adapted ATP release assay optimised for HTS purposes. The reaction mixture consisted of 20 mM HEPES pH 7.6, 4 mM MgClM). The reaction was triggered with the addition of 10 μL of a 2X substrate mix solution to a 384 black-bottom polystyrene plate (Corning) containing 10 μl of a 2X buffer solution with 1 μM Mt-AspRS in 40 mM HEPES pH 7.6, 8 mM MgCl2 and 100 mM KCl. Plates were then briefly centrifuged and read on EnVision (Perkin Elmer) using NADPH fluorescence in the kinetic mode as the method of detection. Slopes corresponding to initial reaction rates were plotted versus substrate concentration for each inhibitor concentration and data points were fitted to standard hyperbolic Michaelis Menten curves on GraphPad Prism 6.0 for KM and VMAX determination.The inhibitory activities of GSK97C and GSK93A against Mt-AspRS was assessed at several β-γ-methyladenosine triphosphate (0–8000 μM) and inhibitor concentrations  while keeping fixed amounts of L-Asp and PPi at saturating values (10*KSupplementary Information


The medial geniculate body (MGB) is the target of excitatory and inhibitory inputs from several neural sources. Among these, the inferior colliculus (IC) is an important nucleus in the midbrain that acts as a nexus for auditory projections, ascending and descending, throughout the rest of the central auditory system and provides both excitatory and inhibitory inputs to the MGB. In our study, we assessed the relative contribution from presumed excitatory and inhibitory IC neurons to the MGB in mice. Using retrograde tract tracing with cholera toxin beta subunit (CTβ)-Alexa Fluor 594 injected into the MGB of transgenic, vesicular GABA transporter (VGAT)-Venus mice, we quantitatively analyzed the projections from both the ipsilateral and contralateral IC to the MGB. Our results demonstrate inhibitory projections from both ICs to the MGB that likely play a significant role in shaping auditory processing. These results complement prior studies in other species, which suggest that the inhibitory tectothalamic pathway is important in the regulation of neuronal activity in the auditory forebrain. The medial geniculate body (MGB) is a critical part of the central auditory system, acting as a neural hub for ascending and descending axonal projections ,2,3,4,5.The major source of ascending input to the MGB arises from the inferior colliculus (IC), which integrates input from lower brainstem nuclei . This teThe goal of this project was to examine the contribution of the excitatory and inhibitory tectothalamic projections to the MGB in the mouse. Here, we used a vesicular GABA transporter (VGAT)-Venus, transgenic mouse model which enabled the rapid identification of inhibitory neurons in these structures ,32. We eThe following procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Louisiana State University School of Veterinary Medicine. Transgenic mice  (colony breeder mice kindly provided by Dr. Janice Naegele at Wesleyan University) expressing the Venus fluorescent protein  in inhibitory, vesicular GABA transporter (VGAT)–positive neurons were used. These transgenic mice were raised on a C57BL/6J background. ® 594 Conjugate , a fluorescent tracer dye, according to the manufacturer’s recommendations for retrograde tracing volume , typically spreading from ~25–50% of the volume of the MGB  ,32. The  the MGB C. After Each animal was then recovered for three to five days to allow for the tracer to transport optimally and were monitored for health during this period. After this transport period, the animal was deeply anesthetized with isoflurane and sacrificed via transcardiac perfusion, using 1ml of 10 mM phosphate-buffered saline (PBS) solution, followed by 6 mL of 4% paraformaldehyde (PFA) in 10 mM PBS fixative. The brain was then removed and postfixed in 4% PFA/in 10 mM PBS at 4 °C for 24 h, after which it was stored in a 4% PFA with 30% sucrose solution in 10 mM PBS for 24 h for cryoprotection at 4 °C. Subsequently, the brain was prepared for cryosectioning; the brain was blocked with a 35-degree cut on the dorsal surface and sliced to preserve the IC, MGB and AC, that is, the tectothalamic sectioning plane that we have previously described . Using aA 1:2 series of sections was mounted and coverslipped on slides using Vectashield anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) . Once the slides had dried, they were scanned using a Nanozoomer digital slide scanner A HamamaB Olympu. NeuronsRetrograde tract tracing of convergent inputs to the MGB with CTβ-AlexaFluor 594 in VGAT-Venus mice showed a significant number of single- and double-labeled cells . The VGAThe CTβ injection sites displayed limited diffusion (<500 μm) of the tracer dye, similar to that seen in our prior studies ,34,35, aFollowing these retrograde tracer injections in the MGB, we observed in the IC both single-labeled cells, identified as Venus-negative, retrograde-positive cells A. MorphoThe distributions of single- and double-labeled retrograde cells were counted, from the confocal and Nanozoomer digital microscopy images (see Methods). The percentages of single- and double-labeled retrograde cells in both ICs were generally similar, but with approximately an order of magnitude more retrogradely-labeled cells counted in the ipsilateral IC . TopograThe ipsilateral inferior colliculus (IC) contained single- and double-labeled retrograde cells projecting to the medial geniculate body (MGB); 30 ± 2% of the ipsilateral IC’s retrograde cells were double-labeled, presumed GABAergic inhibitory, while the rest were VGAT-negative, presumed glutamatergic excitatory, as suggested based on previous studies ,37,38,39In this study, we used retrograde tract tracing with the CTβ-AlexaFluor 594 tracer dye to quantitatively characterize the neuronal pathways arising from the IC that project to the MGB. Our data, using the VGAT-Venus mouse, reveal excitatory and inhibitory projections from the IC to the MGB. These data strengthen past findings of homologous regions in various species, such as the cat, rat, and guinea pig ,26,27,28One drawback with the VGAT-Venus mice in neuroanatomical studies of the auditory pathway are that they are raised on the C57BL/6J background, which exhibits a well-characterized, hearing loss at high frequencies beginning at 6 months of age . In our Our results correlate with previous studies of the tectothalamic projection in cats , rats 11, and thThe inhibitory tectothalamic projection is one of several potential sources of inhibition to MGB relay neurons, which also receive local inhibition from interneurons (in non-rodents) and feedback inhibition from the thalamic reticular nucleus (TRN) that is driven by the thalamus and corticothalamic neurons in layer 6  13,42,4,44,45. TIn addition, our study shows that the contralateral IC provides a less intense, but apparent inhibitory projection to the contralateral MGB, which may enable further ascending inhibitory control of auditory processing. The overall proportion was similar to that from the ipsilateral IC, but an order of magnitude fewer in number. Such a contralateral IC projection has been noted in other studies, and our results are similar in also exhibiting a crossed ascending inhibitory tectothalamic projection ,26,27,28In conclusion, these results demonstrate ascending excitatory and inhibitory inputs to the medial geniculate body from both the ipsilateral and contralateral inferior colliculi in the mouse, confirming similar findings in other species ,26,27,28


SLC12A3 gene, encoding NCC, gives rise to three isoforms, of which only the third isoform (NCC3) has been extensively investigated so far. However, recent studies unraveled the importance of the isoforms 1 and 2, collectively referred to as NCC splice variant (NCCSV), in several (patho)physiological conditions. In the human kidney, NCCSV localizes to the apical membrane of the DCT and could constitute a functional route for renal sodium-chloride reabsorption. Analysis of urinary extracellular vesicles (uEVs), a non-invasive method for measuring renal responses, demonstrated that NCCSV abundance changes in response to acute water loading and correlates with patients’ thiazide responsiveness. Furthermore, a novel phosphorylation site at serine 811 (S811), exclusively present in NCCSV, was shown to play an instrumental role in NCCSV as well as NCC3 function. This review aims to summarize these new insights of NCCSV function in humans that broadens the understanding on NCC regulation in blood pressure control.The renal thiazide-sensitive sodium-chloride cotransporter (NCC), located in the distal convoluted tubule (DCT) of the kidney, plays an important role in blood pressure regulation by fine-tuning sodium excretion. The human  Hypertension (high blood pressure) is a major risk factor for stroke, myocardial infarction, heart failure, chronic kidney disease and overall increased risk of mortality –4. It isSLC12A3 gene, encoding NCC, in relation to Gitelman syndrome  [It is well known that salt intake plays a significant role in the development of hypertension. The body’s salt homeostasis is maintained by the kidneys through regulated processes of sodium (and chloride) reabsorption . Specifi263,800) –16. This263,800) ,18.SLC12A3 gene yields three separate isoforms as a result of an alternative splicing. NCC isoforms 1 and 2 (NCC1 and NCC2), collectively termed NCCSV, are nine amino acids longer than the third isoform (NCC3) [SV and its role in renal salt handling in various (patho)physiological conditions. A better understanding of all three isoforms is crucial to unravel the molecular events underlying the pathogenesis of essential hypertension, and to develop effective therapeutic approaches to combat the dysregulation of blood pressure.The human m (NCC3) –20. In tSLC12A3 gene [et al. reported an mRNA sequence encoding a 1,030 amino acid protein, which was NCC1 [et al. reported later the mRNA sequence corresponding to the NCC3, which encoded for a protein of 1,021 amino acids [3 is nine amino acids shorter in comparison to NCC1. A possible cryptic splicing at the donor site leads to both long and short forms of exon 20 [1, the isoform 2 (NCC2) lacks one amino acid at glutamine residue 95 within amino (N)-terminal domain, making these two isoforms practically indistinguishable (SV), which is only present in humans and higher primates.The molecular cloning of human NCC took place in 1996, showing that there exist three different isoforms of the protein as a result of alternative splicing of the 2A3 gene ,21. Simowas NCC1 . Mastroiuishable . Hence, 3 and NCCSV was shown in human urinary extracellular vesicles (uEVs) using mass spectrometry and immunoblot analysis [SV and NCC3, which revealed that SCL12A3 leads to the expression of a mixture of NCC3 and at least one but possibly both NCC1 and NCC2 [SV and ~ 50% of NCC3 [SV to NCC3 mRNA expression in human kidney varied between 20 and 60%, which is likely the result of a highly regulated splicing at exon 20 [Recently, the presence of both NCCanalysis . This stand NCC2 . Total N of NCC3 . However exon 20 .SV led to confirmation of NCCSV abundance at the apical membrane of the DCT in human kidney, as well in kidney membrane fractions and uEVs [SV in renal salt handling.Generation of antibodies specific against NCCand uEVs . This deNCC is a member of the solute carrier 12 (SLC12) family of electroneutral cation-coupled chloride cotransporters. It is a membrane glycoprotein and contains 12 transmembrane segments (S) with large intracellular N- and carboxy (C)-terminal regions, and a large hydrophilic extracellular loop between S7 and S8 . This loThese phosphorylation sites (apart from S124) are targets of the serine-threonine kinases SPAK  and OSR1 (oxidative stress responsive protein type 1), which are activated by with no lysine (WNK) kinases ,41. MutaSV was identified [et al. further investigated the role of NCCSV S811 phosphorylation on activity of both NCCSV and NCC3 [SV, but not the phospho-mimetic S811D, prevents the phosphorylation at T55 and T60. Importantly, NCCSV S811A was able to inhibit NCC3 phosphorylation at T55 and T60 upon co-expression of NCCSV and NCC3 [SV activity, but also affects the function of NCC3 in a dominant-negative fashion  consensus site. Interestingly, several hormonal systems have been shown to activate the cAMP-PKA pathway [Although it is acknowledged that T60 and T55 are phosphorylated via activation of the WNK-SPAK signaling pathway ,49, the  pathway ,52. For  pathway ,54. Addi pathway   system and AVP has been studied extensively and is well documented . It willOver the past years, uEVs have been established as a non-invasive tool to study NCC regulation in several physiological and pathophysiological conditions. The uEVs are composed of nucleic acids and proteins that reflect the (patho)physiological state of cells lining the nephron and the urinary tract. They contain membrane and cytosolic proteins, DNA and RNA that are conserved and protected from degradation ,66. The SV and NCC3 are present as glycosylated oligomeric structures in uEVs [SVin vivo. In response to water loading, a strong decrease in expression of NCC3 as well as NCCSV was shown [SV abundance in water-loaded subjects is likely the result of the decline in RAAS activity and AVP release [SV, like NCC3, is highly regulated under physiological conditions and suggests a key role in renal salt handling. As abovementioned, future studies should examine whether AVP or the RAAS hormones are directly involved in phosphorylation of NCCSV.Both NCC in uEVs ,75. Hencas shown . Water las shown . AVP is as shown . Two recas shown ,78. In las shown ,78. Sincas shown ), the de release . So far,SV in pathophysiological conditions, the abundance and phosphorylation of all three NCC isoforms were compared in uEVs of essential hypertensive patients before and after hydrochlorothiazide or valsartan treatment [To understand the role of NCCreatment . The thireatment , whereasreatment .SV and NCC3 was increased upon chronic thiazide treatment, but not by valsartan [SV and NCC3 abundance was significantly higher in responders than in patients who did not respond to thiazide treatment. There was no significant difference of the phosphorylated form of NCC (T55/T60 abundance) [SV abundance in uEVs and with plasma potassium levels. Together, these observations demonstrate the importance of NCCSV in pathophysiological processes and its involvement in blood pressure control.While plasma sodium levels remained the same in both treatments, plasma renin levels increased considerably after valsartan treatment. Plasma potassium concentrations were reduced in patients treated with hydrochlorothiazide but not upon valsartan treatment . Interesalsartan . Howeverundance) . These dSV is significantly expressed in the human kidney and is not a redundant transcription product, but a fully functional thiazide-sensitive sodium-chloride transporting protein. Besides, the phosphorylation site S811 in NCCSV acts as a dominant-negative regulatory site for phosphorylation of T55 and T60 in all isoforms. While the WNK-SPAK kinases are a well-studied signaling mechanism for NCC regulation, it is yet unclear whether which molecular pathways and kinases are involved in phosphorylation of S811. Future experiments should focus on delineating the regulation of NCCSV. These discoveries reveal a new regulatory mechanism of NCC function, which might be important in renal salt handling, consequently playing a significant role in the pathogenesis of essential hypertension. Future experiments developing S811 phospho-specific antibodies and samples from different human pathological states could provide further understanding of the precise role of NCCSV. Here, uEVs can serve as a non-invasive biomarker source as recent data revealed its potential in guiding anti-hypertensive therapy in patients with essential hypertension. This could ultimately lead to personalized anti-hypertensive treatment. The development and standardization of high-throughput assays for uEV analysis will be necessary for future clinical applications.NCC


The four-screw extruder (FSE) is a novel equipment for polymer processing. In this paper, from a new viewpoint of Lagrangian coherent structures (LCS), two-dimensional fluid transport and chaotic mixing characteristics within three kinds of novel industrial FSEs are explored based on LCS to better understand the flow and mixing natures in the FSEs. Firstly, based on the finite-time invariant manifold theory, the finite-time Lyapunov exponent (FTLE) and LCS of FSEs are calculated by considering the different initial time. Hyperbolic LCSs from the FTLE maps are adopted to identify chaotic mixing manifolds in FSEs. Moreover, particle tracking and Poincaré sections are used to illustrate the different fluid motions in the above three isolated regions. Finally, the effects of relative rotating directions and layout of four screws on the chaotic manifolds in FESs are discussed in order to enhance local mixing performance. Furthermore, quantitative mixing measures, such as the segregation scale, logarithmic of stretching, and mean-time mixing efficiency are employed to compare the mixing efficiencies in three kinds of FSEs. The results show that the relative rotating directions and positions of four screws can change the chaotic manifolds and increase mixing performance in local poor mixing regions. FTLE and LCS analysis are helpful to better understand the chaotic mixing nature in the novel screw extruders. As a classic piece of mixing equipment, single-screw and twin-screw extruders are widely used in the polymer-processing industry . With thThe key mixing mechanisms of laminar flow in screw extruders include dispersive and distributive mixing from a Eulerian viewpoint. In general, most of the studies have focused on the dispersive and distributive mixing to understand the mixing mechanisms in single- and twin-screw extruders ,10,11,12Many studies have proved that chaotic mixing is an effective method to enhance mixing efficiency in the laminar flow of polymer processing in order to conquer the nature of laminar flow ,17,18,19Recently, as a new measurement of chaotic mixing, Lagrangian coherent structures (LCS) were proposed to identify the chaotic manifold ,24,25 inAs a novel extrusion device, four-screw extruders share distinct fluid-transport and mixing mechanisms in comparison to traditional twin-screw extruders. This paper explores the chaotic mixing and fluid-transport characteristics within three kinds of novel industrial four-screw extruders by employing FTLE, LCSs, and Poincaré sections. We focus on the two-dimensional chaotic advection and mixing in four-screw extruders using computational fluid dynamics. The chaotic manifold structures of the four-screw extruders were visualized by extracting the hyperbolic LCSs from the FTLE maps. Moreover, the Poincaré sections are used to assist in explore the mixing process in extruders and how material transport characteristics respond to changes in LCSs. Furthermore, quantitative mixing measures by means of the segregation scale, logarithmic of stretching. and mean-time mixing efficiency are employed to provide a rigorous method for comparison three kinds of four-screw extruders. The primary aim of this article is to better understand the nature and inherent of flow and mixing natures in the four-screw extruder.p is the pressure, τ is the extrastress tensor, and v is the velocity vector.In this study, 2D non-Newtonian and transient flow conditions are employed by using the finite-element method (FEM). The forms of continuity and momentum equations can be expressed as follows :(1)∇⋅v=0The stress tensor is given as:η is the viscosity, In which η is the dynamic viscosity, λ is a model-specific relaxation time, and D is the second invariant of the rate of deformation tensor and n is the power-law index. In this study, the material parameters of the HDPE melts at 200 °C are as follows: η0 = 113088 Pa·s, λ = 3.11 s, n = 0.36, a = 0.26.In this study, HDPE is processed in the four-screw extruders. It is appropriate to use the Carreau–Yasuda model to describe its rheological behavior as follows:The Lagrangian description is considered as a better method to understand the idiosyncrasies of steady fluid flow. The LCS was used to describe the coherent structures of two-dimensional turbulent flow that defined as manifolds upon the dynamics. These manifolds are useful to understand the results of material transport from experimental and numerical flow data , and espx is a fluid point trajectory on an arbitrary interval of time . The solution of the dynamical system given in Equation (5) in a certain time as a flow map can be viewed as a flow map. It is denoted by The two-dimension flow dynamical system of four-screw extruders, the fluid point trajectory satisfied as: follows :(6)φt0t:x(t) of the dynamical system is obtained by C is the Cauchy–Green deformation tensor. The maximum and minimum eigenvectors of the C in Equation (7) imply that there are compression and expansion along the trajectory, respectively; and the maximum stretching occurs in direction aligned with the eigenvector associated with the maximum eigenvalue of C.Then, a finite-time version of Cauchy–Green deformation tensor by displacement grads tensor that form the trajectory So, the FTLE with a finite integral time T can be defined as:T is associated to point x∈D at time t0; λmax(C) is the maximum eigenvalue of C. The LCSs are approximately obtained by the ridge of the FTLE field at time t for the initial position [In which, position  and reprFor a periodic flow described in this paper, the LCSs corresponds to the hyperbolic invariant manifolds. The geometry of the hyperbolic manifolds can be found by calculating the spatial distribution of the finite-time Lyapunov exponent (FTLE) ,24,25,26Based on the typical FSE, we developed two other types of new configurations. Therefore, four-screw extruders mainly involve three types geometries according to the arranged forms and rotating directions of the four screws, as follows: (i) The basic system (BS) is the corotating square-arranged four-screw extruder , in whicIn comparison with typical twin-screw extruders, the three types of four-screw extruders have more intermeshing regions than twin-screw extruders, as shown in dt = 0.5 s, which corresponds to 1.5° of screw rotation.In this article, two-dimensional finite-element (FE) models without considering axial movement are employed to better understand the influences of geometric configurations on the flow-transportation and chaotic-mixing characteristics of four-screw extruders at cross sections. This is useful to achieve optimum shape design of cross section and improve mixing efficiency in practical applications. Three types of four-screw extruders were constructed using Solidworks software , as shown in −4 in all the numerical cases. Moreover, based on the velocity fields in the extruders, the fluid-particle positions at time t + T were located by using the fourth-order Runge–Kutta scheme. Then, spatial gradient txdφ(t)/dx was used to determine the Cauchy–Green deformation tensor for each initial point, and the FTLE map in the mixer was obtained at time t of each grid point from Equation (8).In this paper, two-dimensional flow fields in the mixer are calculated by using a commercial CFD code, ANSYS Polyflow, based on the generalized Newtonian approach. The convergence criterion was set to 1 × 10x-directional velocities in three kinds of FE models are almost same. Considering the computational cost, the FE model consisting of 72,800 cells was selected to accurately study the mixing mechanisms in the four-screw extruders.The effects of cell numbers in FE models on the numerical results is employed to validate the grid-independent . Three kt = 1.0 s, dt = 0.5 s, and dt = 0.25 s, to detect velocity changing of the detected line in y direction, as shown in t = 0.5 s was chosen in our simulations considering the cost of computing.To illustrate the effect of time steps on prediction, the red detected line was selected in the flow domain and was advected for 100 s using three different time steps, namely, dT. When the initial condition is stable, in principle, LCS positions are uniquely determined by their end positions. From a Lagrangian perspective quantifying a fluid-transport process, it is important to define the original condition and the integration time. After many calculations with different integration times, rotating revolution T is chosen as integration time for all simulations.The Lagrangian method has high sensitivity to initial conditions. The spatial map of FTLE describes the dynamical evolution of each particle over interval time T = +a revolution and different initial time are shown in The forward- and backward-time FTLE maps in the BS with integration time t0 = 0 s and t0 = 10 s were redrawn, as shown in the To locate the position of the hyperbolic fixed point in the flow system, the main repelling and attracting LCSs, namely, hyperbolic LCSs, in the BS with initial time In the central region of the BS, the repelling and attracting LCSs tangle each other and emerge closed diamond of LCS structures, whose vertex is near the hyperbolic points. When a pair of hyperbolic LCSs come closer to being coincident, the tangle becomes a material transport barrier. The regions surrounded by the tangle only allow a small flux of fluid across the boundary. Therefore, we consider the tangle as the boundary that separates the physical flow domain into three portions with different mixing characteristics, as shown in To further understand the relative good or poor mixing characteristics in the three portions identified by the LCSs, particle tracking was used to illustrate the different fluid motions in the BS. Initially, we set free 3000 particles in a rectangle region located on the axis of the flow domain, which would pass through two hyperbolic periodic points, as shown in From the above analysis, we know that there are four hyperbolic periodic points at the center of the domain. Nearby, in every hyperbolic periodic fixed point, the particles would move apart faster. With the four screw rotations, the particle groups near hyperbolic periodic points 1 and 3 move along the unstable manifold into the left and right chambers, respectively, as shown in It is noted that the movement of those particles in the central region is limited by the transport boundary of LCSs. The closed-diamond LCSs mean that the tracers are substantially isolated and could not efficiently mix with the rest of the domain. Therefore, the dead zone in the central region formed by the closed LCSs must be considered to optimize the geometric deign and enhance the mixing efficiency of the BS. In the next section, in order to decrease or remove the dead zone, we study the effect of screw rotational direction and the arrayed manner of the four screws on the chaotic manifolds in four-screw extruders.Based on the base model of FSE, changing the direction of screw rotation is a relatively simple method to change the chaotic manifolds of a four-screw dynamic system. For the CRS, the two upper screws rotate counterclockwise, while the two screws below rotate clockwise. Based on the base model of BS, the relative positions of the four screws are varied to construct the SEDS model in order to obtain different flow domain from BS. For the SEDS, we explored the relationship between the four screw positions and the hyperbolic LCSs of the flow system, which are responsible for determining mixing performance.T = 120 s. Ridges in these FTLE plots show the location of the repelling LCSs and wraps around the four screws. In particular, two small-area closed LCSs appear in the left and right central regions, respectively. With screw rotation, the area of the left central region decreases, corresponding to the decrease of the closed LCS areas, as shown in The Poincaré section is a simple method for analyzing chaotic mixing flows. It is a powerful way to reveal regular zones and chaotic motions. The Poincaré section allows for a systematic reduction in the complexity of problems by reducing the number of dimensions. Previously, many researchers have investigated the relationship between a Poincaré section and chaotic flow . Here, wAs shown in In order to further understand the influences of the screw rotational manner and geometric configurations of four-screw extruders on chaotic mixing, the logarithm of stretching, segregation scale, and time-averaged efficiency were calculated and compared with the three types of four-screw extruders. Initially, 10,000 massless particles were injected in the flow domain, and particle statistics were employed to obtain the above parameters over five periods, as shown in x = χ, where initially x = χ for an infinitesimal material line segment x = χ, located at position x at time t, the length of stretch of a material line is defined as:The logarithmic of stretching is often used to evaluate fluid stretching . Given mR is the correlation coefficient for the concentration, and it gives the probability of finding a pair of random points with relative distance r with the same concentration [The segregation scale is a measure of homogeneous concentration in flow regions  and can S(t)=∫0ζRTime-average efficiency is often used to describe the stretching mixing efficiency during mixing. It is defined as:This paper explores the chaotic-mixing and fluid-transport characteristics within three kinds of novel industrial four-screw extruders by employing FTLE, LCSs, and Poincaré sections. We focused on two-dimensional chaotic advection and mixing in four-screw extruders using computational fluid dynamics. The manifold structures of the four-screw flow systems were visualized by extracting the hyperbolic LCSs from the FTLE maps. Hyperbolic LCSs as fluid-transport boundaries were used to analyze the material transport in two-dimension flow field of FSEs. Moreover, Poincaré sections were employed to indicate the chaotic and regular regions, which are key in improving local mixing. Furthermore, quantitative mixing measures by means of the segregation scale, logarithmic of stretching, and mean-time mixing efficiency were employed to provide a rigorous method for comparison of the three FSE systems.In the BS, namely, the corotating square-arranged four-screw extruder, the hyperbolic LCSs have four hyperbolic fixed points in the four intermesh regions, which are important to enhance mixing efficiency due to the fluid stretching and folding actions, which can help us better understand the better mixing performances in the intermeshing regions. Hyperbolic LCSs, as an underlying transportation boundary, separate the fluid flow of the mixer into three isolated regions, namely, the inner mixing region, outer mixing region, and central region. In the outer mixing region, the fluid was stretched strongly, indicating better mixing efficiency. In the inner mixing region, however, mixing was relatively poor. However, the central region, surrounded by the closed diamond LCSs, was a dead region, in which there was a large-size KAM island in the Poincaré sections of the BS. This obviously decreases the global mixing efficiency in the BS. Correspondingly, the mixing-efficiency measurement of the segregation scale, logarithmic of stretching, and average-time mixing efficiency was lower than the other two FSE systems. So, the central region should be considered to optimize the manifold geometry controlled by the BS profiles to enhance mixing efficiency.In the CRS, namely, the counter-rotating square-arrayed four-screw extruder, the geometry of the flow field was the same as that of the BS. However, the corotating and the counter-rotating manners of the four screws was relatively different from that of the BS. This caused the dead region in the central region of the flow domain to disappear from the analysis of the repelling LCS and Poincaré sections. Local mixing efficiency in the central region was obviously enhanced. Furthermore, the CRS had a more complex manifold structure and great numbers of kinks in the hyperbolic LCSs, resulting in stronger fluid stretching and transport. Therefore, the CRS had a smaller segregation scale, and greater logarithmic of stretching and average-time mixing efficiency than the other four-screw systems. So, the screw rotating manner has a great influence on the manifold structure and mixing efficiency in a four-screw flow system.The SEDS, namely, the equidistance-arrayed four-screw extruder, has five intermeshing regions and two central regions, which are different from the BS and CRS. The manifold structure of the SEDS has five hyperbolic fixed points in five intermeshing regions, implying better local mixing performance. In addition, several kinks appeared in the closed LCSs in the SEDS, implying perfect folding action of the fluid, and enhancing fluid exchange for both inward and outward of the closed LCSs. Therefore, the SEDS had a smaller segregation scale, and greater logarithmic of stretching and average time mixing efficiency than the BS. However, two small-area closed LCSs appeared in the left and right central regions, which corresponded to several little-size KAM islands. This resulted in relatively poor mixing in the SEDS compared to the CRS, because the CRS had no dead region in the central region. So, varying the relative positions of screws could change the manifold structure and enhance the mixing efficiency of the four-screw flow system.This paper has shown that a fresh Lagrangian perspective is more feasible than the traditional Eulerian method in numerically investigating the evolution of two-dimensional mixing performance within a novel screw extruder. FTLE and LCS are useful tools for analyzing chaotic mixing flow. This method is robust and flexible, and can be applied to other types of polymer-processing equipment. It provides a better understanding of the mixing mechanism in screw extruders.


Immunoglobulin E (IgE), though constitutively present at low levels, is most commonly studied in atopic disease where it plays a vital role in mast cell degranulation and in initiating a T helper 2 (Th2) response. With the advent of better detection assays, however, researchers are discovering the importance of IgE in actively contributing to many disease states and pathologies. This review will discuss the latest findings in IgE beyond its role in allergies and recently discovered roles for IgE in its cell-bound form on FcεRI-expressing effector cells like monocytes and dendritic cells. In terms of parasites, we will discuss helminth-induced IgE that appears to protect the worms from immune recognition and a tick-borne illness that elicits an IgE response against red meat. Next, we describe recent findings of how auto-reactive IgE can contribute to the progression of lupus and induce organ damage. Finally, we summarize the emerging roles of IgE in tumor surveillance and antibody-dependent cytotoxicity. We additionally discuss recent or ongoing clinical trials that either target harmful IgE or use the unique characteristics of the isotype. There are many hypotheses regarding the origin of the immunoglobulin E (IgE) response and its conservation throughout evolution. The more widely accepted theories include host protection against metazoan parasites, environmental toxins, and venoms. Although IgE-mediated degranulation is an effective means of initiating a protective immune response, the process can also cause significant damage to healthy tissue. IgE has long been viewed as the notorious Ig behind type I hypersensitivities and related diseases such as allergic rhinitis, anaphylaxis, and asthma. Classic IgE-mediated activation occurs through FcεRI on mast cells and basophils. Multivalent antigens cross-link the receptor-bound IgE to trigger degranulation and the release of pre-formed mediators. This pro-inflammatory milieu recruits immune cells and initiates a T helper 2 (Th2) immune response.1, which has been widely reviewed5. Briefly, this theory states that lower incidences of infection in developed countries lead to inappropriate immune activation by IgE against common environmental antigens and self-antigens. This hypothesis can be expanded to include the recent upsurge in food allergies, especially among young children7. Recent research has suggested that a healthy and diverse gut microbiota can prevent the development of IgE-mediated food sensitivities9.Today, millions of individuals suffer from allergies, asthma, and auto-immunity. This rapid rise in atopic disease may be explained by the “hygiene hypothesis”Because asthma and allergies are a wide-scale problem across the globe, the majority of research into IgE revolves around these pathologies. Advances in analytical techniques as well as expanding scientific knowledge, however, have propelled a new wave of research into the role of IgE beyond the field of allergies. This review will span the latest findings on IgE in non-atopic pathological conditions. We will discuss a new take on “protective” IgE in helminth infection, revelations within a tick-borne disease, self-reactive IgE in auto-immunity, and a novel role for IgE in both preventing and treating cancer. Furthermore, we will discuss recent or ongoing clinical trials using IgE as a therapeutic tool in a variety of these scenarios.10. In the mouse, however, there is a lesser-studied subset of B cell, the innate-like B1 cell, whose importance has begun to be revealed. Reports have described B1 cells as participants in resistance against intestinal bacteria, defense from viral pathogens, generating auto-immune responses, and responding to parasites14. In each case, the role of the B1 cell is to immediately respond to infection with the production of low-affinity IgM or IgG antibodies15. Their B-cell receptor (BCR) repertoire is composed of vastly poly-specific receptors, meaning that B1 BCRs recognize a broad range of antigens15. The resulting antibodies, however, are a much lower affinity than those produced by B2 cells.It is traditionally understood that antibodies are produced by B2 cells. These cells undergo isotype switching and affinity maturation to produce antibodies with very high specificity toward an antigen. The resulting high-affinity, antigen-specific antibody response is important within the adaptive arm of the immune system16. Interestingly, this IgE is not derived from traditional B2 cells but instead is made by the innate-like B1 cells. This challenges the current understanding that B1 cells produce only IgM and IgG. Furthermore, Martinet al. revealed a competition between high-affinity B2 IgE and low-affinity B1 IgE to trigger or block mast cell degranulation, respectively16. Although mast cell degranulation does not clear the infection alone, it can enhance the anti-helminth response. We hypothesized that helminth may have evolved to induce this low-affinity B1 IgE within the host toprevent mast cell degranulation as a protective means of immune evasion.Gastrointestinal helminths are widely known to elicit a robust antigen-specific IgE response, although the reasons why are unclear because mast cells do not appear to play a major role in helminth clearance. Recently, our group showed that helminth infection also induces the production of low-affinity, poly-specific IgE17. Human studies are also limited to circulating cells for collection and examination. For these reasons, it remains to be seen whether B1-derived IgE plays a clinical role in human disease.The translational relevance of murine B1 cells in the human system, however, remains controversial because they are not well defined. The current markers used to identify human B1 cells are similarly found on activated cells and memory populations18. The underlying cause is an IgE response against galactose-α-1,3-galactose , a carbohydrate moiety commonly found on glycoproteins. In particular, α-Gal is widely expressed within mammals but not within humans19. The unique nature of the intense hypersensitivity reaction to α-Gal has remained a curiosity. Some hypothesize that certain ticks produce α-Gal in order to hide from the immune system of mammals, which are tolerized to see α-Gal as “self”19. Humans, which are considered accidental hosts for ticks, however, will recognize the tick-expressed α-Gal as foreign and mount an immune response.Scientists have recently discovered a bizarre occurrence in certain tick-bitten individuals within the southern US and beyond. These patients develop a delayed anaphylactic reaction against red meat, and the average onset is 3 to 6 hours after ingestionAdditional questions remain surrounding the sensitization process following the tick bite as well as the delay in symptomology. The IgE responsible for red meat allergy is unique because of its specificity. Instead of recognizing a specific epitope on a specific protein, this IgE targets the α-Gal moiety that can be present on a variety of proteins and even lipids. This provides potential for “cross-reactivity” among all mammalian meats, including beef and pork.20. The authors further suggest that α-Gal may protect proteins from degradation, thereby slowing the process of major histocompatibility complex (MHC) presentation and antigen recognition20. As mentioned before, α-Gal is also present on lipids. Fattier meats have been reported to elicit more severe reactions in patients with red meat allergy21. One final theory attributes the late symptoms to the inherently slow metabolism of fatty lipids, resulting in the delayed release of α-Gal antigens.The several-hour delay was originally thought to be caused by ineffective dendritic cell (DC) function. A recent study, however, showed that the presence of α-Gal on modified proteins actually increases the efficiency of antigen uptake22. With the advent of better detection assays, however, it is now apparent that auto-reactive IgE is also present within these patients. In addition, researchers have recognized some self-antigens that also show cross-reactivity with exogenous or environmental allergens through molecular mimicry. Numerous reports have highlighted the elevated IgE seen in patients with atopic dermatitis23, chronic urticaria24, rheumatoid arthritis25, or bullous pemphigoid26. Indeed, there have been many reviews covering the decades of IgE studies in these diseases30. The latest published research, however, has focused on systemic lupus erythematosus (SLE), the most common form of lupus.At a basic level, auto-immunity is characterized by an immune response directed against “self”. Auto-reactive B or T cells recognize proteins or DNA fragments normally found in healthy tissue, causing severe and chronic damage. We have come to understand that some of these disorders are characterized by high levels of circulating auto-reactive IgM and IgG34. Work by Juan Rivera’s group has shown an important role for basophils in both murine models and SLE patients. The authors showed that auto-reactive IgE ICs activate basophils to produce interleukin-4 (IL-4) and contribute to an overall Th2 skewing31. This inflammatory environment resulted in increased auto-antibody production by plasma cells and the development of severe lupus nephritis31. The group went on to show that IgE deficiency delayed disease onset and prolonged survival35. To authenticate the clinical relevance, they found increased basophil activation in SLE patients above healthy controls as well as the abnormal presence of basophils in the lymph node and spleen of two patients with SLE35. Furthermore, the authors showed a clear association of high levels of anti-dsDNA IgE in SLE patients with lupus nephritis and a positive association between auto-reactive IgE and clinical disease activity36. Overall, the authors hypothesize that IgE and basophils contribute to the disease pathology byamplifying the immune dysfunction.SLE is characterized by chronic inflammation due to the overproduction of auto-antibodies. These antibodies, often against nuclear factors like DNA, form immune complexes (ICs) that deposit in tissues and cause organ damage. Several studies have revealed a role for IgE in SLE, especially in terms of IgE-DNA ICs37. Henaultet al. designed a novel anti-dsDNA IgE molecule, which they used in conjunction with free DNA to form ICs (DNA-IgE IC)34. These DNA-IgE ICs bound to FcεRI on pDCs to trigger robust IFNα production through endosomal Toll-like receptor 9 (TLR9) activation. When co-cultured with B cells, pDCs activated by DNA-IgE IC caused a significant expansion of plasma cells. Furthermore, the authors showed close proximity between pDCs and B cells in kidney biopsies from patients with SLE. This suggests that the pDC-mediated B-cell proliferation seenin vitro may also be operant in SLE patientsin vivo.Significant strides toward understanding the role of IgE in SLE were made when another group revealed a role for plasmacytoid DCs (pDCs). Traditionally, pDCs are a specialized DC subset that produce type I interferons (IFNs) in response to bacterial or viral pathogen-associated molecular patterns (PAMPs)As a testament to the importance of these and many other findings, a small clinical trial  was conducted to test the effectiveness of treating SLE with the anti-IgE monoclonal antibody, omalizumab. The investigators hypothesized that neutralization of the circulating auto-reactive IgE would limit basophil and pDC activation, thereby dampening IFNα production. In a newly published report summarizing the results, the authors describe a maintained improvement in disease activity, as evaluated by using a patient scoring system . Upon the conclusion of the 32-week omalizumab treatment period, patient scores began to worsen. The authors additionally analyzed type I IFN gene signatures and reported a trend toward improvement in patients receiving omalizumab. Although it did not reach significance within their small cohort, this attenuation effect was enhanced in those participants with high baseline scores. Additional studies will need to extend these results to include a larger cohort of participants. The results, however, are promising and lay an important framework for verifying the pathological relevance of IgE in SLE and other auto-immune diseases.39. The high-affinity IgE receptor, FcεRI, exists mainly as a tetramer (αβγ2), although some human cells express a trimeric form lacking the amplifying beta-chain (αγ2)41. In 1993, one group created a recombinant, soluble version of the human FcεRIα ectodomain (rsFcεRIα)42. Using rsFcεRIα bothin vitro andin vivo, the group was able to completely block mast cell and basophil activation42. In 2011, another group reported their discovery of soluble FcεRI (sFcεRI) within human sera43. The roughly 40-kDa soluble form described by Dehlinket al. consists solely of the IgE-binding alpha-chain43. sFcεRI has been demonstrated to compete with membrane-bound FcεRI to bind IgE44. Indeed, the authors hypothesize that this competitive mechanism could attenuate IgE-mediated DC activation41.Another potential approach to reducing the effects of circulating IgE is through the use of soluble IgE receptors. The low-affinity IgE receptor, FcεRII or CD23, is well known to exist in both soluble and membrane-bound forms. CD23 is involved in a complex regulatory axis that controls the level of production of IgEet al. sensitized mice with IgE in the presence of sFcεRI and observed a reduction in the severity of experimental anaphylaxis44. Interestingly, sFcεRI was also effective in attenuating anaphylaxis when given post-sensitization44. Within 24 hours of administration, sFcεRI caused a reduction in the level of IgE bound to the surface of circulating basophils. Although it was found to be as effective as omalizumab in blocking IgE/FcεRI interaction44, much more research is needed to fully understand the physiological role of sFcεRI.Recently, Moñino-Romero+ cytotoxic lymphocytes (CTLs). Tumors, however, can induce massive immune suppression to limit the activity of these effector T cells.Cancer is a disease of excessive cell proliferation. The malignant cells become invasive, disrupting organ architecture and leaching nutrients from healthy tissue. Because uncontrolled growth can lead to organ failure and death, early detection is vital for patient survival. The body’s defense against malignantly transformed cells relies mainly on the activity of CD8To prevent cancers from growing unchecked, there is great need to find new avenues to reverse the tolerance and induce an immune response within the tumor microenvironment. A popular antibody-based therapy consists of checkpoint inhibitors, which target T cell–deactivating molecules such as CTL-associated protein-4 (CTLA-4) and programmed death ligand protein-1 (PDL-1) in order to recover cytotoxic activity. Although many antibody drugs are on the market, they are all IgG-based. With five different antibody classes, it is possible that another isotype will become the next breakthrough in treating neoplastic disease.47. The overall conclusion suggests that allergic disease may grant some protection against certain types of malignancies. This has led to the emergence of research into AllergoOncology, and there are several reviews of the subject50. Elevated levels of IgE have been reported in patients with various types of cancer53. Although there has not been any conclusive evidence for what purpose this IgE may serve or what antigens it may be specific to, several groups have investigated the unique role of IgE in tumor protection and divulged mechanisms through a variety of effector cells.Several epidemiological studies have compared IgE levels with incidences of cancer54. In un-immunized mice, the authors saw no difference in the tumor growth rate compared with that of wild-type (WT) mice. However, immunization with irradiated tumor cells 2 weeks prior to proper tumor challenge caused delayed tumor growth in the WT but not IgE-KO mice. Furthermore, mice with higher levels of IgE-secreting B cells (KN1) grew smaller tumors than WT mice and were completely protected in the immunization model. This IgE-protective effect was shown to rely on FcεRI and CD8+ cells54.In a direct investigation of the role of IgE in tumor development, one group explored adenocarcinoma growth in IgE-knockout (IgE-KO) mice55. These exogenous antigens then can be routed through a specialized pathway for presentation on MHC class I. This process, known as cross-presentation, permits antigen-specific activation of CD8+ CTLs. A 2015 report described a novel mechanism by which DCs can capture low levels offree antigen on FcεRI-bound IgE56. The authors were able to successfully show cross-presentation of soluble antigen to CD8+ cytotoxic T cells, resulting in proliferation and CTL activity. Furthermore, using antigen-specific IgE in a tumor vaccine model resulted in prolonged survival and generation of memory response upon re-challenge56.In addition to the classically IgE-activated mast cells and basophils, other cell types express FcεRI or FcεRII (CD23) or both. In particular, DCs are vital to both immune surveillance and priming immune responses. DCs take up free antigen through phagocytosis or capture IgG ICs carrying bound antigen, a process known as receptor-mediated endocytosiset al. showed a very different role for IgE in preventing epithelial carcinogenesis57. Following topical application of a carcinogen, the group discovered an unexpected accumulation of IgE within the skin. DNA damage within the epithelial cells triggered a unique mechanism whereby γδ lymphocytes induced draining lymph node (dLN) B cells to produce a robust repertoire of auto-reactive IgE. Using mice that lacked either IgE (−/−IgH-7) or FcεRI (FcεRIα−/−), they specifically showed that IgE signaling was playing a protective role, as the genetic-deficient mice developed more aggressive tumors57. This gives important insight into a previously unexplored immune mechanism that prevents the progression to epithelial malignancy.Crawfordet al. showed that treatment with tumor antigen–specific IgE (MOv18) caused monocyte recruitment into the tumor site58. The group also reported prolonged survival in treated mice, explained in part by an increase in antibody-dependent cell-mediated cytotoxicity (ADCC) that they observedin vitro. Interestingly, when cytotoxicity experiments were performed with non-specific IgE or PBMCs obtained from allergic patients, there was a significant decrease in activity58. The same group later found that IgE can induce both monocyte-driven ADCC and antibody-dependent cell-mediated phagocytosis of ovarian tumor cells through FcεRI and CD23, respectively52.In experiments using purified human peripheral blood mononuclear cells (PBMCs) transferred into nude mice, Karagiannis59. Another group is working to optimize the process of cloning and mass-producing recombinant IgE for use in pre-clinical and clinical studies60. Together, these experiments suggest that tumor antigen–specific IgE can play a powerful role in eliciting and maintaining an immune response at the tumor site.An ongoing phase I clinical trial is using this chimeric MOv18 IgE, specific for the cancer-associated folate receptor-alphaAlthough IgE is normally the least abundant antibody found in circulation because of its short half-life, there are certain conditions that exhibit high levels of IgE. Typically, we associate this increased IgE titer with atopic diseases such as allergies and asthma. Furthermore, there is the additional nuance of discerning between antigen-specific and total IgE. This was particularly important in understanding the competition between antigen-specific B2 IgE and poly-clonal B1 IgE in helminth infection. This lesson can be applied to the many epidemiological studies of helminth infection in allergic patients. Whereas one study may find a positive correlation in IgE, another may not. Most likely, the difference in findings is due to the examination of total IgE rather than helminth-specific IgE. The additional translation between animal studies with helminth infection and human studies has been difficult, as experimental outcomes in human studies are minimal or self-described observations, and longevity studies are hard to find.Extremely high levels of other self-specific isotypes, such as IgM and IgG, are characteristic of certain auto-immune diseases. It was only recently that advances in basic research have allowed us to unmask the presence of auto-reactive IgE in a variety of pathologies. The expanded use of omalizumab in these conditions is seeing positive results with few side effects and thus will likely result in a powerful adjuvant therapy for diseases like SLE.Multiple groups have uncovered unique roles for IgE in detecting malignant cells, slowing tumor progression, and even maintaining a memory response. An interesting feature of allergies is the lifelong sensitivity that can continue for years even after removing exposure to the antigen. Although it is unclear exactly how this long-term hypersensitivity is maintained, the high-affinity memory response could be a beneficial avenue for surveying for tumor recurrence. Additionally, because IgE binds so tightly to its high-affinity receptor, FcεRI, cell-bound IgE could be retained within the tumor site for longer than other antibody classes. This could be a novel means of extending the activity of therapeutic drugs as well as a targeted means to elicit an immune response within the suppressive tumor microenvironment.


Tetragonula sapiens Cockerell) propolis and their structure–activity relationships. The structure of the triterpenes was determined to include mangiferolic acid (1), Cycloartenol (2), ambonic acid (3), mangiferonic acid (4), and ambolic acid (5). The inhibitory test results of all isolated triterpenes against α-glucosidase showed a high potential for inhibitory activity with an IC50 range between 2.46 and 10.72 µM. Among the compounds tested, mangiferonic acid (4) was the strongest α-glucosidase inhibitor with IC50 2.46 µM compared to the standard (–)-epicatechin (1991.1 µM), and also had antioxidant activities with IC50 values of 37.74 ± 6.55 µM. The study on the structure–activity relationships among the compounds showed that the ketone group at C-3 and the double bonds at C-24 and C-25 are needed to increase the α-glucosidase inhibitory activity. The carboxylic group at C-26 is also more important for increasing the inhibitory activity compared with the methyl group. This study provides an approach to help consider the structural requirements of cycloartane-type triterpenes from propolis as α-glucosidase inhibitors. An understanding of these requirements is deemed necessary to find a new type of α-glucosidase inhibitor from the cycloartane-type triterpenes or to improve those inhibitors that are known to help in the treatment of diabetes.This study reports on the antioxidant activity and α-glucosidase inhibitory activity of five cycloartane-type triterpenes isolated from Indonesian stingless bee ( Diabetes mellitus is a chronic disease caused by inherited and/or acquired deficiency in insulin production by the pancreas or by the ineffectiveness of the insulin produced. There are three main types of diabetes pathogeneses: Type 1 diabetes, also known as insulin-dependent, in which the pancreas fails to produce insulin, which is essential for survival. In type I diabetes, β-cells in the islets of Langerhans of the pancreas are damaged, and insulin is not secreted. Type 2 diabetes (known as non-insulin-dependent) results from the body’s inability to respond properly to the actions of insulin produced by the pancreas. Another type of diabetes is gestational diabetes mellitus (GDM) or hyperglycaemia in pregnancy. In type II diabetes, “insulin resistance” hyperglycemia is the result of inadequate production of insulin and the inability of the body to respond fully to insulin. Consequently, sugars flow into the blood and diabetes develops  . Thus, both the Km and the Vmax values are decreased by the same amount; this is the exact opposite of the competitive case [Modes of inhibition of isolated triterpenes were determined by analysis of Lineweaver–Burk plots and calculated by Michaelis–Menten kinetics. Compounds us 1/[S] a,b, whilcosidase a–c. The ive case .3, 4, and 5 showed mixed-type inhibition against α-glucosidase. A compound will behave as a mixture of a competitive and a non-competitive inhibitor or will show mixed inhibition when it can bind both to the free sites of enzymes (competitive) and the enzyme–substrate sites (uncompetitive) [max values.As illustrated in etitive) , which a3, 4, and 5 showed that they have a higher affinity for free enzymes than for enzyme–substrate, as the value of Ki was smaller than Ki’ in compound 3 (Ki = 2.11 µM and Ki’ = 6.42 µM), compound 4 (Ki = 0.58 µM and Ki’ = 3.23 µM), and compound 5 (Ki = 1.71 µM and Ki’ = 4.58 µM).Furthermore, the values of inhibiting constants (Ki and Ki’) of compounds For mixed inhibition, the inhibitor would be still effective at lower concentrations compared to a competitive inhibitor like acarbose. It was reported that in competitive inhibitors such as acarbose, higher inhibitor concentrations are needed for higher carbohydrate food intake to show the same inhibitory effect ,46.50 of ambonic acid (3) is almost the same as that of 4. We conclude that the substituents at C-3 are important components of the cycloartane skeleton that can strengthen the inhibitory activity of α-glucosidase in the order C =O > β-OH and that the substituents at C-26 are also important components in the order of –COOH > –CH3. As a result of the isolated compounds, cycloartenol (2) has the lowest IC50, where the carboxyl group on C-26 is replaced by a methyl group (4) to 5.52 µM (compound 1), but if the double bond in C-24–C-25 shifts to C-31, the activity will decreases by 0.3-fold (from 2.46 (compound 4) to 3.01 µM (compound 3)  ).2) on C-31 can also increase the activity of 5 compared to that of 1, but it slightly reduces the activity of 3 when compared to 4. The structure of compound 3 is similar to that of 5; it is different only in the substituents in C-3. However, this difference in constituents also results in differences in the strength of the α-glucosidase inhibitory in both. At 3, the substituent at C-3 is a ketone (=O), and if replaced with β-OH, as it is in 5, the inhibiting activity is reduced to half-fold (from 3.01 µM to 4.31 µM).Interestingly, the presence of the terminal double bond with methylene (=CH4) isolated from bark of Mangifera mekongensis in Vietnam had the highest inhibitory activity against α-glucosidase from Saccharomyces cerevises compared with the other isolates, mangiferolic acid (1) and ambolic acid (5) (where C-3 is β-OH); however, the inhibitory activity and their structure–activity relationships were not reported. Another cycloartane-type triterpene isolated from Schisandra chinensis, 24-methylenecycloartenone, which also contains a ketone group at C-3, is reported to have high inhibitory activity against α-glucosidase with an IC50 value of 2.36 μM [4) in this study. Nguyen et al. (2016)  reportedlic acid  (where CT. sapiens propolis as the main components have high inhibitory activity against α-glucosidase. This study demonstrated that the five cycloartane-type triterpenes isolated from Samples of raw propolis P1 and P2 were extracted and isolated according to methods described in previous studies  to obtaiw/v) sodium carbonate solution. The resulting mixture were shaken vigorously and left at room temperature for 20 min. Color development at 760 nm was measured with an Epoch microplate reader . The calibration curve was obtained from various concentration of gallic acid. Total phenolic contents were expressed as µg/mg .Total phenol contents in propolis were determined by the Folin–Ciocaltecu method according to Suzuki et al. (2016)  with som50 value (50% inhibition concentration) was calculated by the following formula:A0 is the absorbance of the mixture without a sample, and A1 is the absorbance of the mixture with a sample after 20 min. The inhibitory concentration that results in 50% scavenging of DPPH radicals (IC50) was estimated based on the plot of inhibition versus the final concentration of the propolis samples. The results are expressed in µg Trolox Equivalents (TE) per mL.The antioxidative activities of the fractions were assayed for the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) . Free radicals were assayed using a previously reported method  with somSaccharomyces cerevisiae  of the isolation compounds was determined using a previously reported procedure with modifications [v/v) were added to a 96-well plate with 40 µL of α-glucosidase solution (0.1 U/mL) in phosphate buffer (pH 7.0) and were mixed and incubated at 37 °C for 5 min. Then, 40 µL of 3 mM pNPG solution was added, and the mixture was incubated at 37 °C for 20 min. The reaction was stopped by adding a solution of Na2CO3 , and the absorbance (at 405 nm) of the resulting solution was measured via an Epoch microplate reader . All experiments were performed in three independent replicates. The inhibition of isolated compounds on α-glucosidase was calculated as follows:A0 is the absorbance of the control, and A1 is the absorbance of the propolis samples. The IC50 values were determined as the half-maximal inhibitory concentration of the inhibitor (isolated compound) which provides 50% inhibition.The inhibitory activity of α-glucosidase from ications ,36. p-Nig, −21 °C for 20 min) with a high-speed refrigerated centrifuge . The resulting supernatant was used as an enzyme preparation, and stored at −30 °C until use [S. cerevisiae). The concentration of the isolated compounds used is 0.1–10 µg/mL (0.2–23 µM).In a centrifuge tube at 4 °C, 200 mg of rat intestine acetone granules  was placed in 8 mL of physiological saline (0.9 g of NaCl was dissolved in 100 mL pure water). The resulting suspension was homogenized with sonication equipment  for 60 s  in an ice bath. The resulting homogenate was centrifuged  of p-NPG as a substrate in the presence or absence of different concentrations  of the compounds. The mode of inhibition was then determined by Lineweaver–Burk plot analysis, which was calculated using Michaelis–Menten kinetics. Inhibition constants (Ki) were determined by analysis of Dixon plots and plots of the Y-intercept of the Lineweaver–Burk plot versus the inhibitor ,49. All Tetragonula sapiens propolis were shown to have high inhibition activity against α-glucosidase from Saccharomyces cerevisiae, with mangiferonic acid being the most active inhibitor. According to an enzyme kinetics study, the inhibition types of the isolated cycloartane-type triterpenes were uncompetitive inhibition and mixed inhibition. The inhibitory activity of cycloartane-type triterpenes against α-glucosidase depends on the substituents located on C3, the double bonds on C24–C25, and a carboxyl group on C26. This is the first report of inhibitory activity of cycloartane-type triterpenes isolated from propolis against α-glucosidase. This study provides an approach to consider the structural requirements of cycloartane-type triterpenes from propolis as α-glucosidase inhibitors. An understanding of these requirements is needed to be able to find, improve, or design new types of cycloartane-type α-glucosidase inhibitors from triterpene to help in the treatment of diabetes. Some compounds  also have moderate-high activity where antioxidant activity can help accelerate healing and prevent complications in diabetics, which makes this compound have more ideal potential as an anti-diabetes compound.Five cycloartane-type triterpenes, namely mangiferolic acid, cycloartenol, ambonic acid, mangiferonic acid, and ambolic acid, isolated from