KCNK5 Regulating Potassium Efflux and Inducing Pyroptosis in Corneal Epithelial Cells Through TNFSF10-Mediated Autophagy in Dry Eye.

Purpose: The purpose of this study was to elucidate the involvement of potassium two pore domain channel subfamily K member 5 (KCNK5)-mediated potassium efflux in the pathogenesis of dry eye and to unravel the underlying molecular mechanisms. Methods: To induce experimental dry eye in adult wild-type C57BL/6 mice, scopolamine was administered via subcutaneous injection, and the mice were subjected to desiccating stress. To create an in vitro model of dry eye, desiccation stress was applied to the human corneal epithelial cell line (HCE-T). Intracellular potassium concentration was quantified using inductively coupled plasma mass spectrometry. Cellular death was assessed through lactate dehydrogenase assays. Gene expression profiling was conducted through both RNA sequencing and quantitative real-time PCR. Protein analysis was carried out through Western blotting and immunofluorescence staining. Assessment of the corneal epithelial defect area was conducted through fluorescein sodium staining. Tear secretion was quantified using the phenol red cotton thread method. Results: Potassium efflux was observed to further facilitate corneal epithelial pyroptosis. KCNK5 exhibited upregulation in both in vivo and in vitro models of dry eye. The overexpression of KCNK5 was observed to induce potassium efflux and activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome-mediated pyroptosis in vitro. Silencing KCNK5 effectively mitigated pyroptosis in dry eye. Additionally, the overexpression of KCNK5 results in the downregulation of TNF superfamily member 10 (TNFSF10) and subsequent impairment of autophagy. TNFSF10 supplementation could promote autophagy and mitigate pyroptosis in dry eye. Conclusions: The upregulation of KCNK5 mediates TNFSF10 to impair autophagy and induce pyroptosis in dry eye. Consequently, targeting KCNK5 may represent a novel and promising approach to therapeutic intervention in the management of dry eye.

Drug screening at single-organoid resolution via bioprinting and interferometry.

High throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms use two-dimensional cultures which do not accurately reflect the biology of human tumors. More clinically relevant model systems such as three-dimensional tumor organoids can be difficult to scale and screen. Manually seeded organoids coupled to destructive endpoint assays allow for the characterization of treatment response, but do not capture transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We present a pipeline to generate bioprinted tumor organoids linked to label-free, time-resolved imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D structures with unaltered tumor histology and gene expression profiles. HSLCI imaging in tandem with machine learning-based segmentation and classification tools enables accurate, label-free parallel mass measurements for thousands of organoids. We demonstrate that this strategy identifies organoids transiently or persistently sensitive or resistant to specific therapies, information that could be used to guide rapid therapy selection.

Study on the Changes in Immobilized Petroleum-Degrading Bacteria Beads in a Continuous Bioreactor Related to Physicochemical Performance, Degradation Ability, and Microbial Community.

Continuous bioreactors for petroleum degradation and the effect factors of these bioreactors have rarely been mentioned in studies. In addition, indigenous bacteria living in seawater could influence the performance of continuous bioreactors with respect to petroleum degradation in practice. In this paper, a bioreactor fitted with immobilized petroleum-degrading bacteria beads was designed for further research. The results indicated that the diesel degradation rate of the bioreactor could remain above 50% over 27 days, while degradation performance decreased with bioremediation time. Intriguingly, the diameters of immobilized petroleum-degrading bacteria beads were reduced by 32.49% after 45 days remediation compared with the initial size of the immobilized petroleum-degrading bacteria beads. Change in immobilized petroleum-degrading bacteria beads was considered to correlate remarkably with reduced degradation efficiency. Therefore, this paper will be helpful for further study and improvement of bioreactors in the practical context of oil-spill accident recovery.

Complementary Photo-Synapses Based on Light-Stimulated Porphyrin-Coated Silicon Nanowires Field-Effect Transistors (LPSNFET).

Neuromorphic computing has emerged as the high-energy-efficiency and intelligent solution for processing sensory data. As a potential alternative to neuromorphic computing, photo-excited synaptic systems can integrate the functions of optoelectronic sensing and synaptic computing to realize the low-power and high-performance visual perception. However, one major challenge in high-efficient photo-excited synaptic system is to realize the complementarily enhanced and inhibited synaptic behaviors with small hardware cost as possible. Another challenge is to fabricate the photo-synapse devices with complementary metal oxide semiconductor (CMOS)-compatible process to achieve high enough integration density for practical application. Here, a CMOS-compatible Light-stimulated Porphyrin-coated Silicon Nanowire Field Effect Transistor (LPSNFET) technology is proposed and developed to form the complementary photo-synapses with only two CMOS-like transistors. LPSNFET exhibits fivefold improvement in photo-sensitivity compared to the bare silicon nanowire (SiNW) devices, and can still show obvious responses when incident illumination power is as low as 0.1 mW cm-2 . Moreover, it enables tunable dynamic synaptic plasticity and versatile synaptic functions. Especially, the complementarily enhanced and inhibited behaviors can be realized by modulating SiNW/porphyrin interface via simply changing the MOS type of LPSNFET, which acts like the photonic counterpart of CMOS technology to provide the basic brick for building complex neuromorphic circuits efficiently and economically. Finally, the CMOS process compatibility of LPSNFET provides potential application in future large scale in-sensor computing.

UPLC-MS/MS for the Herb-Drug Interactions of Xiao-Ai-Ping Injection on Enasidenib in Rats Based on Pharmacokinetics.

OBJECTIVE: To develop and validate a sensitive and rapid ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of enasidenib in rat plasma and to investigate the effect of Xiao-ai-ping injection (XAPI) on the pharmacokinetics of enasidenib in rats. METHODS: The rat plasma was precipitated with acetonitrile, enasidenib and internal standard (IS) were separated on an Acquity UPLC BEH C18 column, and acetonitrile and 0.1% formic acid were used as the mobile phase in gradient mode. Enasidenib and IS were monitored and detected by multiple reaction monitoring (MRM) using tandem mass spectrometry in positive ion mode. 12 Sprague-Dawley (SD) rats were randomly divided into control group (group A) and experimental group (group B), 6 rats in each group. Group B was intramuscularly injected with XAPI (0.3 mL/kg) every morning, 7 days in a row. Group A was intramuscularly injected with normal saline, 7 days in a row. On the seventh day, enasidenib (10 mg/kg) was given to both groups 30 min after injection of normal saline (group A) or XAPI (group B), and the blood was collected at different time points such as 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 9, 12, 24, and 48 h. The concentration of enasidenib was detected by UPLC-MS/MS, and the main parameters of pharmacokinetic of enasidenib were calculated using the DAS 2.0 software. RESULTS: Under the current experimental conditions, this UPLC method showed good linearity in the detection of enasidenib. Interday and intraday precision did not exceed 10%, the range of accuracy values were from -1.43% to 2.76%. The results of matrix effect, extraction recovery, and stability met the requirements of FDA approval guidelines of bioanalytical method validation. The C max of enasidenib in the group A and the group B was (458.87 ± 136.02) ng/mL and (661.47 ± 107.32) ng/mL, t 1/2 was (7.74 ± 0.91) h and (8.64 ± 0.42) h, AUC(0 - t) was (4067.24 ± 1214.36) ng·h/mL and (5645.40 ± 1046.30) ng·h/mL, AUC(0 - ∞) was (4125.79 ± 1235.91) ng·h/mL and (5759.61 ± 1078.59) ng·h/mL, respectively. The C max of enasidenib in group B was 44.15% higher than that in group A, and the AUC(0 - t) and AUC(0 - ∞) of enasidenib in group B were 38.80% and 39.60% higher than that in group A, respectively, and the t 1/2 was prolonged from 7.74 h to 8.64 h. CONCLUSION: An UPLC-MS/MS method for the determination of enasidenib in rat plasma was established. XAPI can inhibit the metabolism of enasidenib and increase the concentration of enasidenib in rats. It is suggested that when XAPI was combined with enasidenib, the herb-drug interaction and adverse reactions should be paid attention to, and the dosage should be adjusted if necessary.

Ma Xing Shi Gan Decoction Protects against PM2.5-Induced Lung Injury through Suppression of Epithelial-to-Mesenchymal Transition (EMT) and Epithelial Barrier Disruption.

This research was designed to explore the effect of Ma Xing Shi Gan decoction (MXD) in alleviating particulate matter less than 2.5 µm in diameter (PM2.5) induced lung injury from the perspective of epithelial barrier protection and inhibition of epithelial-to-mesenchymal transition (EMT). Rats were exposed to PM2.5 to establish a lung injury model in vivo, and a PM2.5-stimulated primary cultured type II alveolar epithelial cell model was introduced in vitro. Our results indicated that MXD alleviated the weight loss and pathologic changes and improved the epithelial barrier dysfunction. MXD also significantly inhibited the TGF-ß/Smad3 pathway, increased the level of ZO-1 and claudin-5, and reversed the EMT process. Notably, the protection of MXD was abolished by TGF-ß in vitro. Our results indicated that MXD has a protection against PM2.5-induced lung injury. The proposed mechanism is reversing PM2.5-induced EMT through inhibiting TGF-ß/Smad3 pathway and then upregulating the expression of tight-junction proteins.

Ma xing shi gan decoction eliminates PM2.5-induced lung injury by reducing pulmonary cell apoptosis through Akt/mTOR/p70S6K pathway in rats.

The present study was designed to investigate the anti-apoptosis effect of Ma xing shi gan decoction (MXD) on PM2.5-induced lung injury via protein kinase B (Akt)/mTOR/p70S6K pathway. A UPLC-MS/MS system was introduced for component analysis of MXD. Rats were instilled with PM2.5 solution suspension intratracheally to induce acute lung injury. The rats were then orally administered with MXD (16, 8, and 4 g/kg) once a day for 7 consecutive days. The therapeutic effects of MXD were evaluated by Hematoxylin and Eosin (HE) staining. The apoptotic cell death was analyzed by terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay. The alterations in cytochrome c (Cytc) and cleaved-caspase-3 (C-caspase-3) were measured by immunohistochemistry (IHC). The expressions of Bax, B-cell lymphoma 2 (Bcl-2), p-Akt, p-mTOR and p-p70S6K were detected by Western blot. In vitro, PM2.5 exposure model was introduced in A549 cell, followed by incubation with MXD-medicated serum. Hoechst staining was used to determine apoptotic rate. The levels of Bax, Bcl-2, p-Akt, p-mTOR and p-p70S6K were detected by Western blot. Our results in vivo indicated that treatment with MXD decreased histopathological changes score, TUNEL-positive cells rate, expressions of Cytc and C-caspase-3. The in vitro results revealed that incubation with MXD-mediated serum decreased apoptotic rate. Both results in vivo and in vitro demonstrated that MXD inhibited pro-apoptotic protein Bax and promoted anti-apoptotic protein Bcl-2 expression. Likewise, MXD activated Akt/mTOR/p70S6K signal pathway, which was also confirmed by Western immunoblotting. In conclusion, MXD attenuates lung injury and the underlying mechanisms may relate to regulating the apoptosis via Akt/mTOR/p70S6K signaling pathway activation.

Ma Xing Shi Gan Decoction Attenuates PM2.5 Induced Lung Injury via Inhibiting HMGB1/TLR4/NFκB Signal Pathway in Rat.

Ma Xing Shi Gan Decoction (MXD), a classical traditional Chinese medicine prescription, is widely used for the treatment of upper respiratory tract infection. However, the effect of MXD against particulate matters with diameter of less than 2.5 µm (PM2.5) induced lung injury remains to be elucidated. In this study, rats were stimulated with PM2.5 to induce lung injury. MXD was given orally once daily for five days. Lung tissues were harvested to assess pathological changes and edema. Myeloperoxidase (MPO) activity and malonaldehyde (MDA) content in lung were determined to evaluate the degree of injury. To assess the barrier disruption, the bronchoalveolar lavage fluid (BALF) was collected to determine the total protein content and count the number of neutrophils and macrophages. For evaluating the activation of macrophage in lung tissue, CD68 was detected using immunohistochemistry (IHC). The levels of inflammatory factors including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), and interleukin-6 (IL-6) in BALF and serum were measured. In vitro, a PM2.5-activated RAW 264.7 macrophages inflammatory model was introduced. To evaluate the protective effect of MXD-medicated serum, the cell viability and the release of inflammatory factors were measured. The effects of MXD on the High mobility group box-1/Toll-like receptor 4/Nuclear factor-kappa B (HMGB1/TLR4/NFκB) pathway in lung tissue and RAW 264.7 cells were assessed by Western blot. For further confirming the protective effect of MXD was mediated by inhibiting the HMGB1/TLR4/NFκB pathway, RAW 264.7 cells were incubated with MXD-medicated serum alone or MXD-medicated serum plus recombinant HMGB1 (rHMGB1). MXD significantly ameliorated the lung injury in rats, as evidenced by decreases in the pathological score, lung edema, MPO activity, MDA content, CD68 positive macrophages number, disruption of alveolar capillary barrier and the levels of inflammatory factors. In vitro, MXD-medicated serum increased cell viability and inhibited the release of inflammatory cytokines. Furthermore, MXD treatment was found to inhibit HMGB1/TLR4/NFκB signal pathway both in vivo and in vitro. Moreover, the protection of MXD could be reversed by rHMGB1 in RAW 264.7. Taken together, these results suggest MXD protects rats from PM2.5 induced acute lung injury, possibly through the modulation of HMGB1/TLR4/NFκB pathway and inflammatory responses.

Five New Cucurbitane-Type Triterpenoid Glycosides from the Rhizomes of Hemsleya penxianensis with Cytotoxic Activities.

Five new cucurbitane-typetriterpenoid glycosides, named Xuedanoside F-J (1-5), were obtained from the rhizomes of Hemsleya penxianensis (Xue dan), which belongs to the family of Cucurbitaceae. These new compounds were elucidated byspectroscopic analysis, including 1D, 2D NMR, and HR-ESI-MS spectra. Additionally, all the isolates were evaluated for cytotoxicity against three human cancer cell lines (Hela, MCF-7, and A-549) with the IC50 ranging from 2.25 to 49.44 µM in vitro with treatment 48 h and showed low cytotoxicity in human normal liver L-02 cells (IC50 > 50 µM). Compound 5 showed the most significant cytotoxic activity with the IC50 value of 2.25, 4.72, and 5.33 µM in 48 h, respectively.

Whole-genome analysis of the colonization-resistant bacterium Phytobacter sp. SCO41T isolated from Bacillus nematocida B16-fed adult Caenorhabditis elegans.

Colonization resistance is an important attribute for bacterial interactions with hosts, but the mechanism is still not completely clear. In this study, we found that Phytobacter sp. SCO41T can effectively inhibit the in vivo colonization of Bacillus nematocida B16 in Caenorhabditis elegans, and we revealed the colonization resistance mechanism. Three strains of colonization-resistant bacteria, SCO41T, BX15, and BC7, were isolated from the intestines of the free-living nematode C. elegans derived from rotten fruit and soil. The primary characteristics and genome map of one of the three isolates was investigated to explore the underlying mechanism of colonization resistance in C. elegans. In addition, we performed exogenous iron supplementation and gene cluster knockout experiments to validate the sequencing results. The results showed that relationship was close among the three strains, which was identified as belonging to the genus Phytobacter. The type strain is SCO41T (= CICC 24103T = KCTC 52362T). Whole genome analysis showed that csgA, csgB, csgC, csgE, csgF, and csgG were involved in the curli adhesive process and that fepA, fepB, fepC, fepD, and fepG played important roles in SCO41T against the colonization of B. nematocida B16 in C. elegans by competing for iron. Exogenous iron supplementation showed that exogenous iron can increase the colonization of B. nematocida B16, which was additionally confirmed by a deletion mutant strain. The csg gene family contributes to the colonization of SCO41T in C. elegans. Curli potentially contribute to the colonization of SCO41T in C. elegans, and enterobactin has a key role in SCO41T to resist the colonization of B. nematocida B16 by competing for iron.

Sodium Tanshinone IIA Sulfonate Prevents Radiation-Induced Toxicity in H9c2 Cardiomyocytes.

The present study was designed to elucidate the key parameters associated with X-ray radiation induced oxidative stress and the effects of STS on X-ray-induced toxicity in H9c2 cardiomyocytes. Cytotoxicity of STS and radiation was assessed by MTT. Antioxidant activity was evaluated by SOD and MDA. Apoptosis was measured by the flow cytometry, Hoechst 33258, clonogenic survival assay, and western blot. It was found that the cell viability of H9c2 cells exposed to X-ray radiation was significantly decreased in a dose-dependent manner and was associated with cell cycle arrest at the G0/G1 phase as well as apoptosis. STS treatment significantly reversed the morphological changes, attenuated radiation-induced apoptosis, and improved the antioxidant activity in the H9c2 cells. STS significantly increased the Bcl-2 and Bcl-2/Bax levels and decreased the Bax and caspase-3 levels, compared with the cells treated with radiation alone. STS treatment also resulted in a significant increase in p38-MAPK activation. STS could protect the cells from X-ray-induced cell cycle arrest, oxidative stress, and apoptosis. Therefore, we suggest the STS could be useful for the treatment of radiation-induced cardiovascular injury.