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Background Abnormal alternative splicing is frequently associated with carcinogenesis. In B-cell acute lymphoblastic leukemia (B-ALL), double homeobox 4 fused with immunoglobulin heavy chain (DUX4/IGH) can lead to the aberrant production of E-26 transformation-specific family related gene abnormal transcript (ERGalt ) and other splicing variants. However, the molecular mechanism underpinning this process remains elusive. Here, we aimed to know how DUX4/IGH triggers abnormal splicing in leukemia. Methods The differential intron retention analysis was conducted to identify novel DUX4/IGH-driven splicing in B-ALL patients. X-ray crystallography, small angle X-ray scattering (SAXS), and analytical ultracentrifugation were used to investigate how DUX4/IGH recognize double DUX4 responsive element (DRE)-DRE sites. The ERGalt biogenesis and B-cell differentiation assays were performed to characterize the DUX4/IGH crosslinking activity. To check whether recombination-activating gene 1/2 (RAG1/2) was required for DUX4/IGH-driven splicing, the proximity ligation assay, co-immunoprecipitation, mammalian two hybrid characterizations, in vitro RAG1/2 cleavage, and shRNA knock-down assays were performed. Results We reported previously unrecognized intron retention events in C-type lectin domain family 12, member A abnormal transcript (CLEC12Aalt ) and chromosome 6 open reading frame 89 abnormal transcript (C6orf89alt ), where also harbored repetitive DRE-DRE sites. Supportively, X-ray crystallography and SAXS characterization revealed that DUX4 homeobox domain (HD)1-HD2 might dimerize into a dumbbell-shape trans configuration to crosslink two adjacent DRE sites. Impaired DUX4/IGH-mediated crosslinking abolishes ERGalt , CLEC12Aalt , and C6orf89alt biogenesis, resulting in marked alleviation of its inhibitory effect on B-cell differentiation. Furthermore, we also observed a rare RAG1/2-mediated recombination signal sequence-like DNA edition in DUX4/IGH target genes. Supportively, shRNA knock-down of RAG1/2 in leukemic Reh cells consistently impaired the biogenesis of ERGalt , CLEC12Aalt , and C6orf89alt . Conclusions All these results suggest that DUX4/IGH-driven DNA crosslinking is required for RAG1/2 recruitment onto the double tandem DRE-DRE sites, catalyzing V(D)J-like recombination and oncogenic splicing in acute lymphoblastic leukemia.

MicroRNA-491-5p (miR-491-5p) plays an important role in regulating cell proliferation and migration; however, the effect of miR-491-5p on neovascularization after traumatic brain injury remains poorly understood. In this study, a controlled cortical injury model in C57BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro, respectively. In the in vivo model, quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5p increased or decreased following the intracerebroventricular injection of an miR-491-5p agomir or antagomir, respectively, and the expression of miR-491-5p decreased slightly after traumatic brain injury. To detect the neuroprotective effects of miR-491-p, neurological severity scores, Morris water maze test, laser speckle techniques, and immunofluorescence staining were assessed, and the results revealed that miR-491-5p downregulation alleviated neurological dysfunction, promoted the recovery of regional cerebral blood flow, increased the number of lectin-stained microvessels, and increased the survival of neurons after traumatic brain injury. During the in vitro experiments, the potential mechanism of miR-491-5p on neovascularization was explored through quantitative real-time-polymerase chain reaction, which showed that miR-491-5p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5p mimic or inhibitor, respectively. Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5p. Cell counting kit 8 (CCK-8) assay, flow cytometry, and 2?,7?-dichlorofluorescein diacetate (DCFH-DA) assay results confirmed that the downregulation of miR-491-5p increased brain microvascular endothelial cell viability, reduced cell apoptosis, and alleviated oxidative stress under oxygen-glucose deprivation conditions. Cell scratch assay, Transwell assay, tube formation assay, and western blot assay results demonstrated that miR-491-5p downregulation promoted the migration, proliferation, and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway. These findings confirmed that miR-491-5p downregulation promotes neovascularization, restores cerebral blood flow, and improves the recovery of neurological function after traumatic brain injury. The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress. All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China (approval No. 2020-304) on June 22, 2020.

Studies have shown that downregulation of nuclear-enriched autosomal transcript 1 (Neat1) may adversely affect the recovery of nerve function and the increased loss of hippocampal neurons in mice. Whether Neat1 has protective or inhibitory effects on neuronal cell apoptosis after secondary brain injury remains unclear. Therefore, the effects of Neat1 on neuronal apoptosis were observed. C57BL/6 primary neurons were obtained from the cortices of newborn mice and cultured in vitro, and an oxygen and glucose deprivation cell model was established to simulate the secondary brain injury that occurs after traumatic brain injury in vitro. The level of Neat1 expression in neuronal cells was regulated by constructing a recombinant adenovirus to infect neurons, and the effects of Neat1 expression on neuronal apoptosis after oxygen and glucose deprivation were observed. The experiment was divided into four groups: the control group, without any treatment, received normal culture; the oxygen and glucose deprivation group were subjected to the oxygen and glucose deprivation model protocol; the Neat1 overexpression and Neat1 downregulation groups were treated with Neat1 expression intervention techniques and were subjected to the in oxygen and glucose deprivation protocol. The protein expression levels of neurons p53-induced death domain protein 1 (PIDD1, a pro-apoptotic protein), caspase-2 (an apoptotic priming protein), cytochrome C (a pro-apoptotic protein), and cleaved caspase-3 (an apoptotic executive protein) were measured in each group using the western blot assay. To observe changes in the intracellular distribution of cytochrome C, the expression levels of cytochrome C in the cytoplasm and mitochondria of neurons from each group were detected by western blot assay. Differences in the cell viability and apoptosis rate between groups were detected by cell-counting kit 8 assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, respectively. The results showed that the apoptosis rate, PIDD1, caspase-2, and cleaved caspase-3 expression levels significantly decreased, and cell viability significantly improved in the Neat1 overexpression group compared with the oxygen and glucose deprivation group; however, Neat1 downregulation reversed these changes. Compared with the Neat1 downregulation group, the cytosolic cytochrome C level in the Neat1 overexpression group significantly decreased, and the mitochondrial cytochrome C level significantly increased. These data indicate that Neat1 upregulation can reduce the release of cytochrome C from the mitochondria to the cytoplasm by inhibiting the PIDD1-caspase-2 pathway, reducing the activation of caspase-3, and preventing neuronal apoptosis after oxygen and glucose deprivation, which might reduce secondary brain injury after traumatic brain injury. All experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China, on December 19, 2020 (approval No. 2020-895).

The black soldier fly larvae (BSFL) (Hermetia illucens) are a good candidate for poultry and livestock manure treatment. The harvested insect bodies and feces can be used as animal feed and organic fertilizer. However, heavy metals have a negative impact on the transformation process of the manure by BSFL. Here we evaluated the effects of Cd, Cr and As in pig manure on the growth of BSFL, as well as the mobility and changes in speciation of the metals during the transformation process. The results showed that As significantly reduced the weight of BSFL, but Cr and Cd had no significant effects on BSFL growth. The bioaccumulation factors (BAFs) at various concentrations of Cd (2.8–3.7) were significantly higher than those of Cr (0.44–0.62) and As (0.43–0.45). The heavy metals in pig manure were mainly transferred to BSFL feces, and the distribution percentages of Cd, Cr and As in the BSFL feces were 52.6–62.0%, 90.3–94.2% and 93.0–93.3%, respectively. Cd concentrations in BSFL feces were significantly lower than those in the pig manure, while there were no significant differences in the concentrations of Cr and As between BSFL feces and pig manure (except for the treatment with the addition of 150 mg Cr kg−1). The metal speciation (weak acid soluble, reducible, oxidizable and residual fractions) in BSFL feces obviously changed when compared with pig manure. In BSFL feces, the reducible proportion of Cr decreased while the oxidizable proportion increased. Cd mainly existed in the weak acid soluble and reducible states in pig manure and BSFL feces. For As, the proportions of weak acid soluble and reducible states decreased in BSFL feces. After the BSFL transformation process, the bioavailable fraction of Cr decreased by 17.3–23.1%, but those of Cd and As did not change significantly, except for As in the CK group. These findings contribute to our understanding of the roles of BSFL during the biotransformation process of livestock manure as well as the safe utilization of transformed products.

The conversion of CO2 into value-added chemicals has attracted a lot of attention, not only reducing the concentration of CO2 in the air, but also generating chemicals and fuels, which are of great importance to solve the growing environmental and energy problems. Ni catalysts with different Zr/Al ratios were prepared by co-precipitation and impregnation for CO2 methanation. The activity test results showed that almost 100% CH4 selectivity and 82.6% CO2 conversion were obtained at a reaction temperature of 350 °C when the Zr/Al ratio was 1. The physicochemical properties of the catalysts were investigated using XRD, N2 adsorption–desorption, H2-TPR, H2-TPD, CO2-TPD, XPS and TEM, and the surface-generating substances and methanation reaction mechanism of the catalysts were explored using in situ DRIFTS characterisation. The experimental results show that the addition of Al2O3 to Ni/ZrO2 weakens the Ni–ZrO2 interaction and promotes the reduction and dispersion of NiO through the solid solution bonding of ZrO2–Al2O3. The in situ DRIFTS results show that there are more formate adsorbed species on the surface of the Ni/ZrO2–Al2O3 catalyst compared to the Ni/ZrO2 catalyst, which is the main formation substance for CO2 methanation.

The hydrogenation of carbon dioxide to methanol is perceived as an effective way to mitigate global warming and drive fossil-fuel companies towards sustainable energy sources. In this study, a series of Cu/ZnO–CeO2 catalysts were prepared by a co-precipitation method, and the effect of the CeO2 content on the catalytic performance of the Cu/ZnO–CeO2 catalysts for the hydrogenation of CO2 to CH3OH was analysed. The physicochemical properties of the catalysts were investigated using XRD, H2-TPR, CO2-TPD, BET, XPS, TEM, and DRIFTS. The CZC-3 catalyst (CZC stands for Cu/ZnO–CeO2 catalyst and 3 indicates a 30% CeO2 content) had the best catalytic activity at 280 °C in a 3 MPa CO2/H2 (1/3) atmosphere, exhibiting a CO2 conversion rate of 15.6% and a methanol yield of 10.06%. More dispersed CuO species and oxygen vacancies were detected on the CZC-3 catalyst, leading to good activation of H2 and CO2, respectively. In situ DRIFTS results for the catalysts showed that the hydrogenation of CO2 to methanol over Cu/ZnO–CeO2 catalysts occurs via the formate pathway. The CeO2-rich Cu/ZnO–CeO2 catalyst facilitated the conversion of formate to CH3OH, and the presence of a large number of intermediate species on the surface of the CZC-3 catalyst greatly contributed to its catalytic performance for CO2 hydrogenation.

Spinel-structured lithium nickel manganese oxide (LiNi0.5Mn1.5O4, LNMO) is a low-cost battery cathode material with a high operating voltage (∼4.7 V vs Li+/Li), relatively high capacity (∼147 mA h ...

Tanshinone IIA (tan IIA), a key component of Salvia miltiorrhiza Bunge (Danshen), has been proven to play a significant role in suppressing inflammation. However, the molecular mechanisms underlying the anti-inflammatory properties of tan IIA against lipopolysaccharide (LPS)-induced neuroinflammation and neurotoxicity in human U87 astrocytoma cells have not been well justified. Therefore, in this study, U87 cells were pretreated with tan IIA (1, 5 and 10 μM) for 30 min, followed by stimulation with LPS for 24 h. Immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blotting were performed to investigate the effects of tan IIA on neuroinflammatory responses. The findings demonstrated that tan IIA prevented LPS-induced cell viability decrease, inhibited U87 cells activation, and suppressed the expression of glial fibrillary acidic protein (GFAP). Furthermore, tan IIA significantly reduced the mRNA expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in LPS-stimulated U87 cells. Meanwhile, the increased protein levels of IL-1β, TNF-α, and IL-6 in cell culture supernatants were also markedly inhibited by tan IIA. Moreover, tan IIA significantly alleviated the phosphorylation of IκBα, nuclear factor-kappa B (NF-κB), p38, and JNK induced by LPS. Additionally, tan IIA suppressed the upstream signaling adaptor molecules toll-like receptor 4 (TLR4), myeloid differentiation primary response protein 88 (MyD88), and tumor necrosis factor receptor-associated factor 6 (TRAF6). Blockade of NF-κB, p38, and JNK obviously attenuated IL-1β, TNF-α, and IL-6 in U87 cells. In conclusion, the present results suggested that tan IIA can attenuate LPS-induced neurotoxicity and neuroinflammation partly by inhibiting TLR4/NF-κB/MAPKs signaling pathways in U87 cells.

p70S6K is involved in cellular response, such as tumor metastases, the immune response and tissue repair in vertebrates. The role of p70S6K in these physiological processes in crustaceans remains, however, unknown. In this study, the Lvp70S6K was identified, containing a 5' UTR of 294 bp, an ORF of 1494 bp ad a 3' UTR of 211 bp, encoding 497 amino acids with a theoretical molecular weight of 70 kDa and an estimated isoelectric point of (pI) of 5.16. The multiple alignment found that Lvp70S6K was highly homologous with other invertebrates. Lvp70S6K mRNA was detected in all the tested tissues and the Lvp70S6K expression levels was significantly down-regulated and reached the lowest level (0.44-fold, p 0.01) at 1.5 h after low temperature stress. The subcellular localization of Lvp70S6K could be detected in cytoplasm. ROS production was significantly up-regulation (1.19-fold, p 0.01), total hemocyte count (THC) was significantly down-regulation (0.22-fold, p 0.01), apoptosis rate was markedly increased (1.09-fold, p 0.01), apoptosis-related genes of LvPDCD4 (1.61-fold, p 0.01) and LvCyt.C (1.23-fold, p 0.01) were up-regulated, and anti-apoptotic gene of LvBcl-2 (0.69-fold, p 0.01), LvIAP1 (0.68-fold, p 0.01) and LvIAP2 (0.45-fold, p 0.01) were decreased after low temperature stress in hemolymph of Lvp70S6K-silenced shrimp at 1.5 h. Silencing of LvPTEN significantly increased Lvp70S6K, LvPI3K, LvAKT and LvmTOR expression. In summary, these results indicated that Lvp70S6K play a crucial role in oxidative and apoptosis, which was able to negatively regulate by PTEN.

Background: The role of glucose transporters in the transport of flavonoids remains ambiguous. Objective: In this study, we examined whether quercitrin would be absorbed intactly in modeled Caco-2 cells, as well as determined the involvement of sodium-dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) in its transmembrane transport. Design: The first experiment was conducted to examine the uptake of quercitrin into Caco-2 cells 24 h after they were seeded and the second experiment was conducted to determine the transport across the apical and basolateral membrane of Caco-2 cells after they were cultured for 21 days in a Millicell system. Quercitrin was administered at 3, 9, or 18 μg/mL; and the time points of sampling were 30, 60, 90, 120, and 150 min. Results: In the uptake experiment, the highest intracellular quercitrin concentration was observed in the cells treated with 18 μg/mL quercitrin at 60 min, with a bell-shaped kinetic curve. Quercetin, isorhamnetin, and tamarixetin were detected inside the cells, particularly when treated with a high dose. In the transport experiment, quercitrin was transported from the apical to basolateral side and vice versa; its concentrations depended on dose, time, and transport direction (P < 0.0001). Only trace amounts of isorhamnetin and tamarixetin were detected in the apical chamber when quercitrin was added to the basolateral chamber. Phloridzin and phloretin, potent inhibitors of SGLT1 and GLUT2, respectively, significantly diminished quercitrin transport from the apical to basolateral side; and phloretin had a greater inhibitory effect compared to phloridzin. Conclusion: Our results demonstrate that quercitrin is absorbed intactly and then effluxed out of Caco-2 cells through both apical and basolateral membranes probably via SGLT1 and GLUT2.

In this paper, the structure and magnetic behaviour of antiperovskite InxFe4-xN have been investigated systematically. The crystal lattice becomes larger and the Curie temperature decreases with increasing x. The magnetic state changes from a ferromagnetic to a glassy state. In addition, an obvious spin glass (SG) behaviour has been revealed in In0.6Fe3.4N (x = 0.6) with a freezing temperature of T0 = 73 K, dynamical exponent of zν = 5.51, and flipping time of τ0 = 4.26 × 10−11 s. The origin of the SG behaviour in In0.6Fe3.4N may be attributed to atomic disorders at Wyckoff position 1a or ferromagnetic frustrations, or the both.

The development of a stringent sensor to detect low levels of acetone, yielding the potential for the point-of-care clinical diagnosis of diabetes, is still a great challenge but is urgently required. Most studies have focused on Ti3C2Tx, yet other types of MXenes with good performance are rare. Herein, an emerging kind of MXene, V4C3Tx, has been prepared from V4AlC3 via the selective etching of the Al layer using aqueous HF at room temperature (RT), and its performance as an acetone sensor is presented. A V4C3Tx based acetone sensor delivers good performance, as demonstrated by its low working temperature of 25 °C, low detection limit of 1 ppm (lower than the 1.8 ppm diabetes diagnosis threshold), and high selectivity towards acetone in a mixed gas of acetone and water vapor, hopefully showing promise for application in the much faster and earlier diagnosis of diabetes. V4C3Tx MXene is used for the first time in the field of acetone detection in this work, hopefully opening up a path for the investigation of applications of MXene in gas sensors, and such exciting findings distinguish V4C3Tx as a comparable material to the well-known Ti3C2Tx. In addition, we used DFT calculations to explore the mechanisms that result in the superior selectivity for acetone with respect to water vapor. Hopefully, the proposed mechanisms combining experimental results and theoretical study will shed light on the design and production of new high-performance acetone sensors.

The NiO/ZnxZr1−x (x represents the molar mass of Zn) catalyst was prepared by the impregnation method and tested in CO2 methanation. The activity results show that NiO/Zn0.3Zr0.7 has a higher CO2 conversion rate and methane selectivity than NiO/ZnO and NiO/ZnO–ZrO2. Combined with N2 adsorption–desorption, H2-TPR, CO2-TPD, H2-TPD, XRD, TEM, XPS and FTIR and other characterization methods, the physical and chemical properties of NiO/ZnO–ZrO2 were studied. The incorporation of ZnO into NiO/ZrO2 forms a ZnO–ZrO2 solid solution, and the combination of the solid solution weakens the interaction between NiO and the oxide support, thereby promoting the reduction and dispersion of NiO. The H2-TPR experiment results show that, because ZnO–ZrO2 forms a solid solution, NiO is better dispersed on the surface, resulting in a significant reduction in the reduction temperature of NiO. Using FTIR to conduct CO2 adsorption and methanation experiments on NiO/ZnxZr1−x to determine the adsorbed species and intermediates, the results show that CO2 methanation follows the formate pathway.

Background Primary malignant melanoma of the esophagus accounts for 0.1%-0.2% of all esophageal malignancies, including melanotic and amelanotic melanomas. Primary amelanotic malignant melanoma of the esophagus is extremely rare, and only about 20 cases have been published in the literature to date. Most primary malignant melanomas of the esophagus are diagnosed following development of metastatic lesions and thus have a very poor prognosis. The median survival duration of patients with metastatic melanoma has been reported to be 6.2 mo. Case summary A 49-year-old woman was referred to our hospital with a diagnosis of esophageal cancer. Endoscopy, biopsy, imaging evaluation, and physical examination at our hospital indicated a diagnosis of advanced primary amelanotic malignant melanoma of the esophagus. Immunohistochemical staining confirmed melanoma. Nuclear medicine examination revealed a left iliac bone metastatic lesion. After discharge, the patient self-administered apatinib for 3 mo, followed by oral treatment with Chinese medicines (also self-administered) for 2 mo. No treatments had been taken since then. The patient has survived with no growth out to the most recent follow-up (24 mo post diagnosis), and she always presented with a positive attitude about her condition during this period. Conclusion Survival following metastatic melanoma might be related to the pharmaceutical and Chinese medicine treatment and the patient's positive attitude.

A fluorescence-enhanced and lysosome-targeted Al3+ probe (1) based on a pyrazole-containing hydrazone was developed in this work. The fluorescent probe 1 displays an OFF–ON fluorescence response to Al3+ in the lysosome pH environment. In addition, the resultant 1–Al3+ complex could act as an ON–OFF fluorescence sensor for F−. Fluorescence imaging shows that 1 is suitable for visualization of Al3+ in lysosomes of HeLa cells.

Three kinds of porous catalysts CuO-ZnO/SBA-15 (CZ/SBA-15), CuO-ZnO-MnO2/SBA-15 (CZM/SBA-15) and CuO-ZnO-ZrO2/SBA-15 (CZZ/SBA-15) were synthesized by impregnation method with a siliceous framework mesoporous molecular sieve SBA-15. The performance of all catalysts for catalytic hydrogenation of CO2 to methanol was evaluated on a fixed bed reactor, combined with N2 adsorption-desorption (BET), X-ray diffraction (XRD), H2 temperature-programmed reduction (H2-TPR), temperature programmed desorption (H2-TPD, CO2-TPD), N2O titration, X-ray photoelectron spectroscopy (XPS) and transmission electron microscope (TEM) . The results show that the introduction of metal oxide in the catalyst changes the pore size and specific surface area of the SBA-15 molecular sieve support. The CuO-ZnO-MnO2/SBA-15 and CuO-ZnO-ZrO2/SBA-15 have high copper dispersion (DCu), large specific surface area (ACu), small surface CuO particle size, and easy to be reduced. Compared with the Mn-O cluster, the Zr-O cluster enhances the basic site and improves the methanol selectivity. In addition, CuO-ZnO-ZrO2/SBA-15 has the highest oxygen vacancy concentration and better catalytic activity among three catalysts. The methanol selectivity of CuO-ZnO-ZrO2/SBA-15 is 25.02%, which is 28% and 136.9% higher than those of CuO-ZnO/SBA-15 and CuO-ZnO-MnO2/SBA-15, respectively.

C35H32N6O2, triclinic, P 1 ‾ $‾{1}$ (no. 2), a = 10.902(2) Å, b = 12.434(2) Å, c = 13.481(4) Å, α = 102.881(5)°, β = 104.935(5)°, γ = 114.542(3)°, V = 1488.6(6) Å3, Z = 2, R gt (F) = 0.0511, wR ref (F 2) = 0.1429, T = 296 K.

C31H29N3O7, monoclinic, P21/c (no. 14), a = 13.6280(14) Å, b = 14.4801(15) Å, c = 14.4962(15) Å, β = 97.586(2)°, V = 2835.6(5) Å3, Z = 4 R gt (F) = 0.0532, wR ref (F 2) = 0.1569, T = 296 K.

Liquid chromatography (LC) is a common and straight forward approach used in the evaluation of the quality of Traditional Chinese Medicines (TCMs). Quality control is a critical step when systematically assessing the efficacy of TCMs. In the present study, the spectrum-effect correlation method was used to identify pharmacologically active substances. The aim of the present study was to investigate the underlying correlations between common chemical compounds with antipyretic effects and the anti-endotoxin activity of Lonicera japonica. The common chemical constituents of Lonicera japonica were analyzed using LC, and the antipyretic effects and anti-endotoxin activity were determined using ELISAs. Combining the results of bivariate and principal component analysis methods, eight active constituents were qualitatively and quantitatively analyzed. The results of these analyses indicated that neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and isochlorogenic acids A, B and C served a synergistic role with respect to antipyretic effects and anti-endotoxin activity. The present study lays a foundation for the future clinical use of Lonicera japonica.

Trifluoromethylthiolative difunctionalization of alkenes, a cheap and abundant feedstock, which installs a trifluoromethylthiol (SCF3) group and another unique functional group across the carbon–carbon double bonds, provides an ideal strategy for the preparation of β-functionalized alkyl trifluoromethyl sulfides and has become a hot topic recently. This review aims to summarize the major progress in this exciting research area, with particular emphasis on the mechanistic aspects of the reaction pathways.

C30H34N8O4S2Cu, triclinic, P1̄ (no. 2), a = 12.9537(9) Å, b = 15.2802(10) Å, c = 17.1041(11) Å, α = 78.748(1)°, β = 79.684(1)°, γ = 79.270(1)°, V = 3226.4(4) Å3, Z = 4, R gt(F) = 0.0488, wR ref(F 2) = 0.1178, T = 296 K.

C41H41N9O5Co, monoclinic, P21/n (no. 14), a = 10.666(4) Å, b = 18.538(6) Å, c = 19.585(7) Å, β = 104.036(7)°, V = 3757(2) Å3, Z = 4, R gt(F) = 0.0420, wR ref(F 2) = 0.1062, T = 296 K.

C30H28N8O2Br2Cd, triclinic, P1̄ (no. 2), a = 8.359(7) Å, b = 12.394(10) Å, c = 15.189(12) Å, α = 95.757(14)°, β = 90.593(16)°, γ = 106.961(15)°, V = 1496(2) Å3, Z = 2, R gt(F) = 0.0467, wR ref(F 2) = 0.0998, T = 296(2) K.

A novel pyrazine hydrazone 1 has been designed and synthesized, which acts as a multiprobe for selective detection of Al3+, Mg2+, and Zn2+ in DMSO solution based on fluorescent enhancement with three different emission colors. The higher affinity of 1 toward Al3+ and Zn2+ compared with Mg2+ facilitates the establishment of a simple ratiometric fluorescent method for the detection of Al3+ and Zn2+ based on the 1 + Mg2+ system. Conversely, probe 1 selectively recognized Al3+ in DMSO-H2O cosolvent with a turn-on response, and the resultant 1 + Al3+ complex could determine F− via a fluorescence quenching signal. In addition, the probe is applied for imaging of Al3+ and F− in living cells.

A rhodamine hydrazone 1 bearing coumarin moiety was designed and prepared. Compound 1 exhibited high selectivity toward Co2+ and trivalent metal ions with fluorescence enhancement in CH3OH solution. However, 1 selectively responded to Al3+ in nearly pure H2O media and was further applied to monitor Al3+ in live cells. Moreover, 1 could also act as a colorimetric probe toward Cu2+ in either CH3OH or H2O solution. In addition, sensor 1 displayed aggregation-induced ratiometric emission (AIRE) activities in mixed H2O/CH3OH solution.

A simple asymmetric hydrazone 1 based on salicylaldehyde was designed and prepared, and exhibited an evident aggregation-induced emission (AIE) at a long wavelength of 570 nm in aqueous medium. Probe 1 can selectively sense Al3+ in CH3OH solution through the chelation-enhanced fluorescence mechanism and also recognize Cu2+ via fluorescence quenching in H2O solution through the Cu2+-induced assembly of aggregates. In addition, the probe can be applied for detecting Cu2+ in biological samples.

A novel rhodamine-based Schiff base derivative was obtained via the simple condensation of substituted formyl-1H-pyrrole and rhodamine 6G hydrazone. Fluorescence resonance energy transfer enabled the subsequent use of the derivative as a naked-eye colorimetric and ratiometric fluorescent sensor for Cu2+ in semi-aqueous solution, and the existence of the morpholine group enabled the further application of the sensor in imaging Cu2+ in the lysosomes of HeLa cells.

Proline (Pro) metabolism is intimately associated with stress adaptation. The catabolism of Pro includes two dehydrogenation reactions catalyzed by proline dehydrogenase (ProDH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDh). P5CDh is a mitochondrial matrix NAD+-dependent dehydrogenase that is critical in preventing P5C-Pro intensive cycling and avoiding ROS production from electron run-off. Little is known about the roles of P5CDh in invertebrates, however. We cloned the P5CDh sequence in the Pacific white shrimp, Litopenaeus vannamei, and found that LvP5CDh is expressed predominantly in pleopod, hepatopancreas and gill. Subcellular localization analysis revealed that LvP5CDh protein was mainly found in the cytoplasm. In addition, overexpressing LvP5CDh in cells reduced ROS formation and inhibited apoptosis induced by LC50 Cd2+. Shrimp were exposed to various stress factors including infection with Vibrio alginolyticus, (½ LC50 and LC50) Cd2+, acid (pH 5.6) and alkali stress (pH 9.3). Both biotic and abiotic stress resulted in increased LvP5CDh expression and Pro accumulation; V. alginolyticus infection, pH 9.3 and LC50 Cd2+ stress apparently stimulated the Glu pathway of Pro synthesis, while pH 5.6 and ½ LC50 Cd2+ stress promoted the Orn pathway of Pro synthesis. Silencing of Lvp53 in shrimp attenuated LvP5CDh expression during Cd2+ stress, but had no effect on LvP5CDh mRNA levels if no Cd2+ stress was imposed. Our study contributes to the functional characterization of LvP5CDh in biotic and abiotic stress and reveals it to protect against ROS generation, damage to the cell, including the mitochondria, and apoptosis. Thus, LvP5CDh plays a critical role in immune defense and antioxidant responses.

Endoplasmic reticulum (ER) stress caused by β-amyloid protein (Aβ) may play an important role in the pathogenesis of Alzheimer disease (AD). Our previous data have indicated that tanshinone IIA (tan IIA) protected primary neurons from Aβ induced neurotoxicity. To further explore the neuroprotection of tan IIA, here we study the effects of tan IIA on the ER stress response in oligomeric Aβ1-42 (oAβ1-42)-induced SH-SY5Y cell injury. Our data showed that tan IIA pretreatment could increase cell viability and inhibit apoptosis caused by oAβ1-42. Furthermore, tan IIA markedly suppressed ER dilation and prevented oAβ1-42-induced abnormal expression of glucose regulated protein 78 (GRP78), initiation factor 2α (eIF2α), activating transcription factor 6 (ATF6), as well as inhibited the activation of C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) pathways. Moreover, tan IIA ameliorated oAβ1-42-induced Bcl-2/Bax ratio reduction, prevented cytochrome c translocation into cytosol from mitochondria, reduced oAβ1-42-induced cleavage of caspase-9 and caspase-3, suppressed caspase-3/7 activity, and increased mitochondrial membrane potential (MMP) and ATP content. Meanwhile, oAβ1-42-induced cell apoptosis and activation of ER stress can also be attenuated by the inhibitor of ER stress 4-phenylbutyric acid (4-PBA). Taken together, these data indicated that tan IIA protects SH-SY5Y cells against oAβ1-42-induced apoptosis through attenuating ER stress, modulating CHOP and JNK pathways, decreasing the expression of cytochrome c, cleaved caspase-9 and cleaved caspase-3, as well as increasing the ratio of Bcl-2/Bax, MMP and ATP content. Our results strongly suggested that tan IIA may be effective in treating AD associated with ER stress.

A simple monocarbazone 1 derived from 2H-benzo[h]chromene-3-carbaldehyde and carbazide exhibited aggregation-induced ratiometric emission upon the addition of water to CH3OH solution (Δλem= 30 nm). Upon Cu2+ treatment, the fluorescence emission of 1 displayed a significant red shift in both CH3OH and H2O solutions. In addition, 1 can detect Hg2+ through the fluorescence quenching in H2O solution. The response mechanism to Cu2+ involved an intermolecular charge transfer coupled with a metal-induced assembly, whereas that to Hg2+ was probably due to a photo-induced electron transfer coupled with metal-induced assembly, which were thoroughly investigated by spectral methods and theoretical calculation. In addition, the sensor could successfully detect the corresponding ions in HeLa cells.

Amyloid β peptide 1-42 (Aβ1-42) could induce cognitive deficits through oxidative stress, inflammation, and neuron death in Alzheimer's disease (AD). MAPK pathways have been thought to mediate Aβ1-42-induced neuroinflammation responses, neuron death and cognitive decline in AD. The α7 nicotinic acetylcholine receptor (α7nAChR) exerts a neuroprotective effect. However, whether α7nAChR alleviates Aβ1-42-induced neurotoxicity through MAPKs (p38, ERK, JNK) in vivo remains unclear. In our study, memory was assessed in C57BL/6 mice using a Y-maze test. Cell death was assessed by Nissl and Hoechst staining and Bax, Bcl-2, Caspase 3, and Cytochrome C levels using Western blotting. Oxidative stress was assayed by superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) levels. Inflammation was examined with GFAP and Iba1 using immunohistochemistry. The Aβ degrading enzymes insulin degrading enzyme (IDE) and neprilysin (NEP) were tested using Western blotting. We found that activating α7nAChR or inhibiting p38 or JNK pathway alleviated Aβ1-42-induced cognitive deficits and neuron loss and death by reducing oxidative stress. In addition, activating α7nAChR or inhibiting p38 or JNK pathway also reduced inflammation, which was observed as reduced GFAP and Iba1 levels with different effects on Aβ degrading enzymes. Finally, we found that the activation of α7nAChR led to the downregulation of pp38 and pJNK levels. Conversely, the inhibition of p38 or JNK resulted in the upregulation of α7nAChR levels in the hippocampus and cortex. Our data indicate that the activation of α7nAChR alleviates Aβ1-42-induced neurotoxicity, and this protective effect might act through the downregulation of p38 and JNK MAPKs.

Clofibric acid (CFA), a drug and personal care product, has been identified as ubiquitous in the aquatic system and surface water, causing pollution to the environment. In this study, after environmental (4 µg/L) levels of CFA challenge, the LvFABP, LvACS gene expressions, total haemocyte count (THC), relative enzymes (SOD1 and GST) activities in Litopenaeus vannamei were observed to decrease. In the meantime LvFATP, LvRXR expression and the level of NEFA were upregulated in L. vannamei body. LvFABP expression in vivo was knocked down by dsRNA-mediated RNA interference (RNAi), which led to significantly decreased levels of PPARα (including LvFATP, LvRXR and LvACS). When exposed to environmental CFA after 4 days, LvFABP knocked down group had a sharp upregulation of LvFATP, LvRXR, LvACS expression, GST activity and NEFA amount, following decreased THC and SOD1 activity. These results suggested that environmental concentration CFA may have some toxicological effect on L. vannamei, following fatty acids metabolism and oxidative stress responses by LvFABP via the PPARα/RXR signaling pathway, including LvFATP, LvRXR and LvACS.

Hypochlorite plays a significant role in physiological processes, particularly regulation of lysosomal functions, and is involved in various diseases. Thus, it is crucial to develop highly sensitive and selective molecule tools to detect HClO in lysosomes. Herein, a novel 2H-benzo[h]chromene-pyridine derivative (1) was synthesized through condensation reaction, which exhibited a notable deep-red emission at 640 nm in pure water. This deep-red emission was specifically quenched by adding ClO−. The response of probe 1 toward ClO− was rapid (within 10 s), sensitive (detection limit of 0.012 μM), and effective over a wide range of pH (1.0–12.0). Due to the existence of morpholine as the lysosome-targeting unit, the probe was successfully utilized to monitor lysosomal ClO−. Moreover, the probe 1 was also applied to detecting ClO− in zebrafish.

Heart aging is characterized by structural and diastolic dysfunction of the heart. However, there is still no effective drug to prevent and treat the abnormal changes in cardiac function caused by aging. Here, we present the preventive effects of emodin and its derivative Kanglexin (KLX) against heart aging. We found that the diastolic dysfunction and cardiac remodeling in mice with D-galactose (D-gal)-induced aging were markedly mitigated by KLX and emodin. In addition, the senescence of neonatal mouse cardiomyocytes induced by D-gal was also reversed by KLX and emodin treatment. However, KLX exhibited better anti-heart aging effects than emodin at the same dose. Dysregulated mitophagy was observed in aging hearts and in senescent neonatal mouse cardiomyocytes, and KLX produced a greater increase in mitophagy than emodin. The mitophagy-promoting effects of KLX and emodin were ascribed to their abilities to enhance the protein stability of Parkin, a key modulator in mitophagy, with different potencies. Molecular docking and SPR analysis demonstrated that KLX has a higher affinity for the ubiquitin-like (UBL) domain of Parkin than emodin. The UBL domain might contribute to the stabilizing effects of KLX on Parkin. In conclusion, this study identifies KLX and emodin as effective anti-heart aging drugs that activate Parkin-mediated mitophagy and outlines their putative therapeutic importance.

The proteins related to the biosynthesis of salvianolic acids and lignins were regulated by smoke–water and karrikinolide in Salvia miltiorrhiza hairy roots. The effects of smoke–water (SW) and karrikinolide (KAR1) on the biosynthesis of salvianolic acids and lignins in Salvia miltiorrhiza hairy roots have been studied using proteomic technology. The results showed that a total of 1290 and 1678 differentially expressed proteins were respectively obtained in SW and KAR1 comparing to the control. Bioinformatics analysis indicated the differentially expressed proteins responding to SW and KAR1 treatments mainly involved in macromolecule metabolic process, cell part, binding, etc., and most of the proteins were located at the cytoplasm and cell membrane, followed by nuclear. In addition, the proteins involved in salvianolic acids biosynthesis were up-regulated, including 4-coumarate-CoA ligase (EC 6.2.1.12) and shikimate O-hydroxycinnamoyl-transferase (EC 2.3.1.133). Enzymes involved in lignins biosynthesis were also identified, e.g. cinnamyl-alcohol dehydrogenase (EC 1.1.1.195) and peroxidase (EC 1.11.1.7). The results indicated that proteins related to the biosynthesis of salvianolic acids and lignins were regulated by SW and KAR1 in S. miltiorrhiza hairy roots. This study will enhance our understanding of the mechanism by which SW and KAR1 on the biosynthesis of salvianolic acids and lignins in S. miltiorrhiza hairy roots.

BackgroundGlial activation and neuroinflammation play a crucial role in the pathogenesis and development of Alzheimer’s disease (AD). The receptor for advanced glycation end products (RAGE)-mediated signaling pathway is related to amyloid beta (Aβ)-induced neuroinflammation. This study aimed to investigate the neuroprotective effects of tanshinone IIA (tan IIA), a natural product isolated from traditional Chinese herbalSalvia miltiorrhizaBunge, against Aβ-induced neuroinflammation, cognitive impairment, and neurotoxicity as well as the underlying mechanisms in vivo and in vitro.MethodsOpen-field test, Y-maze test, and Morris water maze test were conducted to assess the cognitive function in APP/PS1 mice. Immunohistochemistry, immunofluorescence, thioflavin S (Th-S) staining, enzyme-linked immunosorbent assay (ELISA), real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and western blotting were performed to explore Aβ deposition, synaptic and neuronal loss, microglial and astrocytic activation, RAGE-dependent signaling, and the production of pro-inflammatory cytokines in APP/PS1 mice and cultured BV2 and U87 cells.ResultsTan IIA treatment prevented spatial learning and memory deficits in APP/PS1 mice. Additionally, tan IIA attenuated Aβ accumulation, synapse-associated proteins (Syn and PSD-95) and neuronal loss, as well as peri-plaque microgliosis and astrocytosis in the cortex and hippocampus of APP/PS1 mice. Furthermore, tan IIA significantly suppressed RAGE/nuclear factor-κB (NF-κB) signaling pathway and the production of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in APP/PS1 mice and cultured BV2 and U87 cells.ConclusionsTaken together, the present results indicated that tan IIA improves cognitive decline and neuroinflammation partly via inhibiting RAGE/NF-κB signaling pathway in vivo and in vitro. Thus, tan IIA might be a promising therapeutic drug for halting and preventing AD progression.

Glycans possess unparalleled structural complexity arising from chemically similar monosaccharide building blocks, configurations of anomeric linkages and different branching patterns, potentially giving rise to many isomers. This level of complexity is one of the main reasons that identification of exact glycan structures in biological samples still lags behind that of other biomolecules. Here, we introduce a methodology to identify isomeric N‐glycans by determining gas phase conformer distributions (CDs) by measuring arrival time distributions (ATDs) using drift‐tube ion mobility spectrometry‐mass spectrometry. Key to the approach is the use of a range of well‐defined synthetic glycans that made it possible to investigate conformer distributions in the gas phase of isomeric glycans in a systematic manner. In addition, we have computed CD fingerprints by molecular dynamics (MD) simulation, which compared well with experimentally determined CDs. It supports that ATDs resemble conformational populations in the gas phase and offer the prospect that such an approach can contribute to generating a library of CCS distributions (CCSDs) for structure identification., Well‐defined synthetic glycans were used to show that ATDs in IMS‐MS resemble gas‐phase conformational populations, which was also confirmed by molecular dynamics simulation. By applying a database with intrinsic gas‐phase conformer distribution fingerprints, protein‐released N‐glycans, including isomeric structures, could be identified rapidly and unambiguously without the need for MS/MS experiments.

The hydrazone sensor (1) derived from pyrazine-2-carbohydrazide and 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) was applied to the fluorescent “turn-on” detection of Al3+ in semi-aqueous solutions. The strongly emissive 1+Al3+ complex could identify F− among other tested anions. The detection limits of sensor 1 for Al3+ and F− were 0.18 and 0.20 μM, respectively, which are both much lower than the corresponding values recommended by the World Health Organization, namely, 7.41 and 79 μM. Several spectral methods, including single crystal X-ray analysis for the crystal structures of the Cu2+ and Ni2+ complexes of sensor 1, as well as the density-functional theory method, were used to elucidate the binding mode between the sensor and Al3+. 1 can also be used for a selective sensor to continuously monitor Al3+ and F− in living cells.

A simple hydrazone probe (1) was designed and synthesized for the successive detection of Al3+ and pyrophosphate (PPi) in almost 100% buffer environment. The probe provided O2N donor set for chelation with Al3+, leading to a distinct fluorescence boost at 510 nm. The in-situ formed 1-Al3+ complex detected PPi with an “on-off” behavior. The detection limits for Al3+ and PPi were 35.7 nM and 76 nM, respectively. Benefiting from the existence of morpholine as lysosome-targeting group, probe 1 was successfully applied to the detection of Al3+ and PPi in lysosomes.

A simple water soluble hydrazone sensor (1·Cl) has been developed to selectively chelate with Zn2+ via an O2N donor set, leading to specific fluorescence enhancements. The highly emissive 1-Zn2+ complex can serve as a reversible on-off sensor for S2−. The detection limits of 1·Cl for Zn2+ and S2− are 66.0 and 32.6 nM, respectively. The recognition mechanism was carefully investigated through FL, UV-vis, 1H NMR, ESI-MS, time-resolved fluorescence, and density functional theory (DFT) calculations. Owing to the mitochondria-targeting ability of the terminal quaternary ammonium moiety, 1·Cl was successfully applied to image mitochondrial Zn2+ and S2−.

A water-soluble salicylaldehyde-based thiosemicarbazone has been designed and applied to the selective fluorescent turn-off detection of Hg2+ ions. The probe-Hg2+ system acted as a turn-on probe toward biothiols, including cysteine, homocysteine, and glutathione. The limits of detection of the probe toward Hg2+, cysteine, homocysteine, and glutathione are 0.52 μM, 0.74 μM, 0.31 μM, and 0.27 μM, respectively. The mechanisms are carefully confirmed by fluorescence spectrometry, UV-visible spectrometry, electrospray ionization mass spectra, hydrogen nuclear magnetic resonance, time-resolved fluorescence spectrum, and density functional theory calculation. Moreover, due to the presence of a morpholine group, the probe was successfully used to monitor Hg2+ and biothiols in the lysosomes of HeLa cells.

Objectives: There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here we used Bacillus stearothermophilus as an indicator strain in the format of the carbapenem inactivation method (CIM) procedure to develop a rapid carbapenemase phenotype detection method: CIMB.S. Methods: The CIMB.S test was derived from the mCIM, where B. stearothermophilus replaced Escherichia coli as the indicator strain. The test bacteria were incubated in the presence of imipenem for 30 min, and then, aliquots were placed on colorimetric plates, and incubation was continued for 3.5 h at 60°C. We examined 134 clinical strains to evaluate the CIMB.S performance. Results: The CIMB.S can be completed in 4 h, and we successfully identified 38/39 (97.4%) carbapenemase-producing Enterobacteriaceae, including 17/18 (94.4%) carbapenemase-producing Pseudomonas aeruginosa and 18/19 (94.7%) carbapenemase-producing Acinetobacter baumannii. All non-carbapenemase producers we tested were negative and included Enterobacteriaceae (n = 36), P. aeruginosa (n = 17), and A. baumannii (n = 5). Conclusions: The CIMB.S test is a rapid carbapenemase phenotype detection method requiring only 4 h of total work time and displays high sensitivity and specificity.

Preterm birth is one of the most common complications during human pregnancy and is associated with a dramatic switch within the uterus from quiescence to contractility. However, the mechanisms underlying uterine remodeling are largely unknown. Protein kinases and phosphatases play critical roles in regulating the phosphorylation of proteins involved in the smooth muscle cell functions. In the present study, we found that Src-homology phosphatase type-1 (SHP-1, PTPN6) was significantly decreased in human myometrium in labor compared with that not in labor. Timed-pregnant mice injected intraperitoneally with the specific SHP-1 inhibitor protein tyrosine phosphatase inhibitor I (PTPI-1) manifested significantly preterm labor, with enriched plasmalemmal dense plaques between myometrial cells and increased phosphorylation at Tyr397 and Tyr576/577 sites of focal adhesion kinase (FAK) in myometrial cells, which remained to the time of labor, whereas the phosphorylation levels of ERK1/2 and phosphatidylinositol 3 kinase (PI3K) showed a rapid increase upon PTPI-1 injection but fell back to normal at the time of labor. The Tyr576/577 in FAK played an important role in the interaction between FAK and SHP-1. Knockdown of SHP-1 dramatically increased the spontaneous contraction of human uterine smooth muscle cells (HUSMCs), which was reversed by coinfection of a FAK-knockdown lentivirus. PGF2α downregulated SHP-1 via PLCβ-PKC-NF-κB or PI3K-NF-κB pathways, suggesting the regenerative downregulation of SHP-1 enhances the uterine remodeling and plasticity by activating FAK and subsequent focal adhesion pathway, which eventually facilitates myometrium contraction and leads to labor. The study sheds new light on understanding of mechanisms that underlie the initiation of labor, and interventions for modulation of SHP-1 may provide a potential strategy for preventing preterm birth.

Precisely controlling the behaviours of stem cells has far-reaching application potential in clinical trials. In this study, we have developed a self-assembled monolayer (SAM) of a cyclic RGD peptide (cycRGD) and bone forming peptide-1 (BFP-1) on a quartz substrate to regulate the behaviours of mesenchymal stem cells (MSCs). The results demonstrated that cycRGD can accelerate the cell adhesion on the substrate, thereby enhancing the ability of BFP-1 in mediating the osteogenic activity. And the synergistic effect between these two functional peptides in osteogenic differentiation of MSCs was confirmed in terms of immunofluorescent staining, Alizarin Red S staining for mineralization and alkaline phosphatase (ALP) activity assay. This finding might give a new insight into designing functional substrates to regulate desired differentiation of stem cells.

Guillain-Barré syndrome is often caused by Campylobacter jejuni infection that has induced antibodies to the lipo-oligosaccharide (LOS) that cross-react with gangliosides at peripheral nerves causing polyneuropathy. To examine fine specificities of anti-ganglioside antibodies and develop a more robust platform for diagnosis and disease monitoring, we developed a chemoenzymatic approach that provided an unprecedented panel of oligosaccharides composed of the inner-core of the LOS of C. jejuni extended by various ganglioside mimics. The compounds and corresponding ganglio-oligosaccharides were printed as a microarray to examine binding specificities of lectins, anti-ganglioside antibodies, and serum antibodies of GBS patients. Although lectins and anti-ganglioside antibodies did not differentiate the ganglio-oligosaccharides and mimics, the patient serum samples bound much more strongly to the ganglioside mimics. The data indicate that antibodies have been elicited to a foreign epitope that includes a heptosyl residue unique of bacterial LOS and that these antibodies subsequently cross-react with lower affinity to gangliosides. The microarray detected anti-GM1a antibodies with high sensitivity and will be attractive for diagnosis, disease monitoring, and immunological research.