Cell Extracts Research Articles

P-791 Supplementation of mitochondria from endometrial mesenchymal stem cells (EnMSCs) improves oocyte quality in aged mice

Abstract Study question To improve the quality of aging oocytes by supplementing the mitochondria from endometrial mesenchymal stem cells (EnMSCs) via microinjection. Summary answer Supplementation with EnMSC-derived mitochondria can improve the quality of oocytes and promote embryo development in aging mice. What is known already Maternal aging is one of the major causes of reduced ovarian reserve and low oocyte quality in elderly women. Decreased oocyte quality is the main cause of age-related infertility. Mitochondria are multifunctional energy stations that determine the oocyte quality. The mitochondria in aging oocytes display functional impairments with mtDNA damage, which leads to reduced competence and developmental potential of oocytes. To improve oocyte quality, mitochondrial supplementation is carried out as a potential therapeutic approach. However, the selection of suitable cells as the source of mitochondria remains controversial. Study design, size, duration Study design: Eight-month-old ICR mice were purchased and then were further raised to ten months of age for use in the experiments. EnMSCs were stably cultivated and oocytes were extracted from ten-month-old ICR mice. Study size: About 300 GV oocytes and 134 MII oocytes were counted . 80 fertilized oocytes were transplanted into the fallopian tubes of surrogate mothers. Study duraton: About one half a year. Participants/materials, setting, methods Participants:ten-month-old ICR mice. Setting and methods: We cultivated EnMSCs from aged mice and extracted mitochondria from EnMSCs. And then GV oocytes were supplemented with mitochondria via microinjection. To further assess the effects of mitochondrial supplementation on embryo development, MII oocytes from aging mice were fertilized by ICSI combining EnMSCs mitochondria microinjection.To test whether mitochondrial supplementation could improve in vivo embryo development in aging mice, embryo transplantation was performed after mitochondrial microinjection combined with ICSI. Main results and the role of chance In this study, we found that the mitochondria derived from EnMSCs could significantly improve the quality of aging oocytes.Supplementation with EnMSC mitochondria significantly increased the blastocyst ratio of MII oocytes from aging mice after intracytoplasmic sperm injection (ICSI). We also found that the birth rate of mitochondria-injected aging oocytes was significantly increased after embryo transplantation. Limitations, reasons for caution Microinjection into oocytes was invasive and some oocytes died after microinjection. The ability of this method to improve oocyte quality was limited because the MII rate of oocytes after mitochondria supplementation was 35.65±1.75% while the rate of oocytes from 6–8 weeks of mice in a previous study was nearly 80%. Wider implications of the findings For clinical treatment, the EnMSCs can be obtained from the patient's own menstrual blood as the sources of mitochondrial supplementation. A noninvasive procedure for cell extraction ensures safety, and the findings of this work might provide a valuable strategy for patients with declining oocyte quality due to aging. Trial registration number Not applicable

IRES-mediated Pichia pastoris cell-free protein synthesis

Cell-free protein synthesis (CFPS) system is an ideal platform for fast and convenient protein research and has been used for macromolecular assembly, unnatural amino acid embedding, glycoprotein production, and more. To realize the construction of an efficient eukaryotic CFPS platform with the advantages of low cost and short time, a CFPS system based on the yeast Pichia pastoris was built in this study. The internal ribosomal entry site (IRES) can independently initiate translation and thus promote protein synthesis. The Kozak sequences can facilitate translation initiation. Therefore, the screening of IRES and its combination with Kozak was performed, in which cricket paralysis virus (CRPV) exhibited as the best translation initiation element from 14 different IRESs. Furthermore, the system components and reaction environment were explored. The protein yield was nearly doubled by the addition of RNase inhibitor. The cell extract amount, energy regeneration system (phosphocreatine and phosphocreatine kinase), and metal ions (K+ and Mg2+) were optimized to achieve the best protein synthesis yield. This P. pastoris CFPS system can extend the eukaryotic CFPS platform, providing an enabling technology for fast prototyping design and functional protein synthesis.Graphical

PPP2R1A regulates migration persistence through the NHSL1-containing WAVE Shell Complex

The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. PPP2R1A is found to associate at the lamellipodial edge with an alternative form of WAVE complex, the WAVE Shell Complex, that contains NHSL1 instead of the Arp2/3 activating subunit WAVE, as in the canonical WAVE Regulatory Complex. PPP2R1A is required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement is abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impair WAVE Shell Complex binding and migration regulation, suggesting that the coupling of PPP2R1A to the WAVE Shell Complex is essential to its function.

Deciphering the biosynthesis of a novel lipid in Mycobacterium tuberculosis expands the known roles of the nitroreductase superfamily

Mycobacterium tuberculosis's (Mtb) success as a pathogen is due in part to its sophisticated lipid metabolic programs, both catabolic and biosynthetic. Several of Mtb lipids have specific roles in pathogenesis, but the identity and roles of many are unknown. Here, we demonstrated that the tyz gene cluster in Mtb, previously implicated in resistance to oxidative stress and survival in macrophages, encodes the biosynthesis of acyl-oxazolones. Heterologous expression of tyzA (Rv2336), tyzB (Rv2338c) and tyzC (Rv2337c) resulted in the biosynthesis of C12:0-tyrazolone as the predominant compound, and the C12:0-tyrazolone was identified in Mtb lipid extracts. TyzA catalyzed the N-acylation of l-amino acids, with highest specificity for l-Tyr and l-Phe and lauroyl-CoA (kcat/KM= 5.9± 0.8×103M-1s-1). In cell extracts, TyzC, a flavin-dependent oxidase (FDO) of the nitroreductase (NTR) superfamily, catalyzed the O2-dependent desaturation of the N-acyl-L-Tyr produced by TyzA, while TyzB, a ThiF homolog, catalyzed its ATP-dependent cyclization. The substrate preference of TyzB and TyzC appear to determine the identity of the acyl-oxazolone. Phylogenetic analyses revealed that the NTR superfamily includes a large number of broadly distributed FDOs, including five in Mtb that likely catalyze the desaturation of lipid species. Finally, TCA1, a molecule with activity against drug-resistant and persistent tuberculosis, failed to inhibit the cyclization activity of TyzB, the proposed secondary target of TCA1. Overall, this study identifies a novel class of Mtb lipids, clarifies the role of a potential drug target, and expands our understanding of the NTR superfamily.

High-Throughput SARS-CoV-2 Antiviral Testing Method Using the Celigo Image Cytometer

The COVID-19 pandemic has created a worldwide public health crisis that has since resulted in 6.8 million reported deaths. The pandemic prompted the immediate response of researchers around the world to engage in rapid vaccine development, surveillance programs, and antiviral testing, which resulted in the delivery of multiple vaccines and repurposed antiviral drug candidates. However, the emergence of new highly transmissible SARS-CoV-2 variants has renewed the desire for discovering new antiviral drug candidates with high efficacy against the emerging variants of concern. Traditional antiviral testing methods employ the plaque-reduction neutralization tests (PRNTs), plaque assays, or RT-PCR analysis, but each assay can be tedious and time-consuming, requiring 2–3 days to complete the initial antiviral assay in biologically relevant cells, and then 3–4 days to visualize and count plaques in Vero cells, or to complete cell extractions and PCR analysis. In recent years, plate-based image cytometers have demonstrated high-throughput vaccine screening methods, which can be adopted for screening potential antiviral drug candidates. In this work, we developed a high-throughput antiviral testing method employing the Celigo Image Cytometer to investigate the efficacy of antiviral drug candidates on SARS-CoV-2 infectivity using a fluorescent reporter virus and their safety by measuring the cytotoxicity effects on the healthy host cell line using fluorescent viability stains. Compared to traditional methods, the assays defined here eliminated on average 3–4 days from our standard processing time for antiviral testing. Moreover, we were able to utilize human cell lines directly that are not typically amenable to PRNT or plaque assays. The Celigo Image Cytometer can provide an efficient and robust method to rapidly identify potential antiviral drugs to effectively combat the rapidly spreading SARS-CoV-2 virus and its variants during the pandemic.

Optimisation of ultrasonication pretreatment on microalgae Chlorella Vulgaris &Nannochloropsis Oculata for lipid extraction in biodiesel production

A wide variety of nutritious products and biofuels can be produced from microalgae. Their applications span across various sectors such as food, medical, and energy industries. Microalgal cell disruption is a crucial aspect in the production processes as it facilitates the extraction of bio-products stored within them. One of the commonly extracted compounds from microalgae is lipids, which constitute a high percentage of their composition. In this study, we conducted ultrasonic pre-treatment on two different microalgae species, namely Chlorella vulgaris and Nannochloropsis oculata. The pre-treatment involved ultrasonic frequencies of 37 and 80 kHz, exposure times of 10, 15, and 20 min, and extraction times of 2, 3, and 4 h. By utilising ultrasonic frequencies, we induced acoustic cavitation to break the microalgae cell walls and extract lipids. The experimental design followed a Response Surface Methodology (RSM) to evaluate the effect of ultrasonication pretreatment parameters on lipid extraction. In comparison to previously published studies, our findings indicate that higher frequencies of ultrasonication lead to increased lipid extraction from microalgae. The highest lipid yield achieved in our study was 11.54% for Chlorella vulgaris and 17.91% for Nannochloropsis oculata, using the input factors of 80 kHz frequency, 20 min of running time, and 4 h of extraction time. Our results contribute to the existing body of knowledge by demonstrating the efficacy of ultrasonic pre-treatment in lipid extraction from microalgae. By providing a quantitative comparison with previous studies, this study highlight the novel aspects of work and its potential implications for the production of biofuels and other valuable products from microalgae.

Metformin-Induced Receptor Turnover Alters Antibody Accumulation in HER-Expressing Tumors.

Metformin has effects beyond its antihyperglycemic properties, including altering the localization of membrane receptors in cancer cells. Metformin decreases human epidermal growth factor receptor (HER) membrane density. Depletion of cell-surface HER decreases antibody-tumor binding for imaging and therapeutic approaches. Here, we used HER-targeted PET to annotate antibody-tumor binding in mice treated with metformin. Methods: Small-animal PET annotated antibody binding in HER-expressing xenografts on administration of an acute versus a daily dose schedule of metformin. Analyses at the protein level in the total, membrane, and internalized cell extracts were performed to determine receptor endocytosis, HER surface and internalized protein levels, and HER phosphorylation. Results: At 24 h after injection of radiolabeled anti-HER antibodies, control tumors had higher antibody accumulation than tumors treated with an acute dose of metformin. These differences were temporal, and by 72 h, tumor uptake in acute cohorts was similar to uptake in control. Additional PET imaging revealed a sustained decrease in tumor uptake on daily metformin treatment compared with control and acute metformin cohorts. The effects of metformin on membrane HER were reversible, and after its removal, antibody-tumor binding was restored. The time- and dose-dependent effects of metformin-induced HER depletion observed preclinically were validated with immunofluorescence, fractionation, and protein analysis cell assays. Conclusion: The findings that metformin decreases cell-surface HER receptors and reduces antibody-tumor binding may have significant implications for the use of antibodies targeting these receptors in cancer treatment and molecular imaging.

P30 | Beneficial effect of fermented wheat germ extract in intestinal epithelial cells in case of lipopolysaccharide‐evoked inflammation

Journal of Veterinary Pharmacology and TherapeuticsVolume 46, Issue S1 p. 100-100 POSTER PRESENTATIONS P30 | Beneficial effect of fermented wheat germ extract in intestinal epithelial cells in case of lipopolysaccharide-evoked inflammation First published: 11 June 2023 https://doi.org/10.1111/jvp.13309Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume46, IssueS1Special Issue: 15th International Congress of the European Association for Veterinary Pharmacology and Toxicology held Bruges, Belgium, July 2–5, 2023June 2023Pages 100-100 RelatedInformation

Toxic copper level increases erythrocyte glycolytic rate, glutathione production and alters electrolyte balance in male Wistar rats

BackgroundCopper is a micronutrient vital to several cellular energy metabolic processes and drives erythropoiesis. However, it disrupts cellular biological activities and causes oxidative damage when in excess of cellular needs. This study investigated the effects of copper toxicity on erythrocyte energy metabolism in male Wistar rats. MethodsTen Wistar rats (150–170 g) were randomly divided into 2 groups: control (given 0.1 ml distilled water) and copper toxic (given 100 mg/kg copper sulphate). Rats were orally treated for 30 days. Blood, collected retro-orbitally after sodium thiopentone anaesthesia (50 mg/kg i.p.) into fluoride oxalate and EDTA bottles, was subjected to blood lactate assay and extraction of red blood cell respectively. Red blood cell nitric oxide (RBC NO), glutathione (RBC GSH), adenosine triphosphate (RBC ATP) levels, RBC hexokinase, glucose-6-phosphate (RBC G6P), glucose-6-phosphate dehydrogenase (RBC G6PDH), and lactate dehydrogenase (RBC LDH) activity was estimated spectrophotometrically. Values (Mean±SEM, n = 5) were compared by Student’s unpaired T-test at p < 0.05. Results and conclusionCopper toxicity significantly increased RBC hexokinase (23.41 ± 2.80 µM), G6P (0.48 ± 0.03 µM), G6PDH (71.03 ± 4.76nmol/min/ml) activities, ATP (624.70 ± 57.36 µmol/gHb) and GSH (3.08 ± 0.37 µM) level compared to control (15.28 ± 1.37 µM, 0.35 ± 0.02 µM, 330.30 ± 49.58 µmol/gHb, 54.41 ± 3.01nmol/min/ml and 2.05 ± 0.14 µM respectively, p < 0.05). Also, RBC LDH activity (145.00 ± 19.88mU/ml), NO (3.45 ± 0.25 µM) and blood lactate (31.64 ± 0.91 mg/dl) level were lowered significantly compared to control (467.90 ± 94.23mU/ml, 4.48 ± 0.18 µM and 36.12 ± 1.06 mg/dl respectively). This study shows that copper toxicity increases erythrocyte glycolytic rate and glutathione production. This increase could be connected to a compensatory mechanism for cellular hypoxia and increased free radical generation.

Study of hydrosalpinx on endometrial growth and expression of HOXA10mRNA and related factors

ObjectiveThe objective of the study is to extract the patient's endometrium at the time of proliferative stage using hydrosalpinx in order to culture the cells and decidualization induction in vitro. Further, the study is also intended at identifying the expression of HOXA10mRNA and related factors and understand the hydrosalpinx's impact upon the working mechanism of endometrial cells. MethodsOnce the extraction of the primary cells is over, the cells are cultured and other activities are performed such as the cell identification, CCK8 assay, cell decidua induction and HE staining. The researchers assessed the expression levels of HOXA10, IGFBP1 and avβ3 in either proliferation or secretion of the endometrium. This was accomplished using Western blot assay and real-time fluorescence quantitative PCR. ResultsThe results confirmed that at the time of endometrial proliferation, there was a decline in the expression of HOXA10 as a result of HS influence. This affected its expression in the secretory stage i.e., corresponding function. Further, a significant decline was observed in the levels of HOXA10mRNA of endometrial cells that were subjected to continuous HS, post decidualization. It was found that during decidualization, if the HS is removed, it is possible to restore the expression of HOXA10mRNA to a certain extent, though it is not possible to reach the general endometrial level. So, in terms of clinical aspects, the expression of HOXA10mRNA by the endometrial cells decreases significantly when blocking the hydrosalpinx. ConclusionsAmong hydrosalpinx patients, one of the major mechanisms that damage the endometrium was found to be the abnormal expression of HOXA10 followed by IGFBP1 and avβ3, its downstream genes. This further results in the implantation of the embryo as well. Though it is possible to gradually repair the damage after the removal of hydrosalpinx, the recovery is a time-consuming process.

Strains of Faecalibacterium prausnitzii and its extracts reduce blood glucose levels, percent HbA1c, and improve glucose tolerance without causing hypoglycemic side effects in diabetic and prediabetic mice

IntroductionThe commensal bacterium Faecalibacterium prausnitzii is a prominent member of the microbiome of animals and humans, and it plays an important role in several physiological processes. Numerous studies have correlated...

Effect of 6-hydroxydopamine increase the glutathione level in SH-SY5Y human neuroblastoma cells.

Treatment of human neuroblastoma SH-SY5Y cells with a catecholaminergic neurotoxin, 6-hydroxydopamine (6-OHDA) is an acknowledged in vitro experimental model of Parkinson disease (PD). A decrease in the glutathione content occurs in PD. Higher concentrations of 6-OHDA lowered the glutathione level in SH-SY5Y cells, nonetheless, we and other authors found a considerable increase in these cells' glutathione content after 24 h treatment with 60 μM 6-OHDA. A synthetic antioxidant, 4-aminotetramethylpiperidine-1-oxyl (4-AT) exerted a similar effect. The aim of the present study was to explain this surprising effect by monitoring the time course of changes in the levels of reduced (GSH) and oxidized glutathione (GSSG), total antioxidant activity (TAC) of human neuroblastoma cell SH-SY5Y extracts as well as the level of reactive oxygen species and activities of enzymes of glutathione metabolism after treatment of the cells with 60 µM 6-OHDA and/or 4-AT for 30 min - 24 h. A transient decrease in the level of GSH and TAC of cell extracts, increase in the level of GSSG, and decrease in the activities of glutathione peroxidase, glutathione reductase, glutathione S-transferase and γ-glutamyl-cysteine ligase activities were found followed by normalization or overshoot of the GSH level, TAC and enzyme activities. Increased activity of γ-glutamyl-cysteine ligase activity starting after 4-6 h was responsible for the elevation of the level of GSH and TAC in cells treated with 6-OHDA, 4-AT, and both compounds. The 6-OHDA-induced increase in the GSH content is a result of an overcompensatory response. The antioxidant 4-AT may be useful for the induction of an increase in the level of GSH in neural cells, without the negative effect of 6-OHDA.

The Rock-Hosted Biosphere

Our understanding of Earth's rock-hosted subsurface biosphere has advanced over the past two decades through the collection and analysis of fluids and rocks from aquifers within the continental and oceanic crust. Improvements in cell extraction, cell sorting, DNA sequencing, and techniques for detecting cell distributions and activity have revealed how the combination of lithology, permeability, and fluid mixing processes controls the diversity and heterogeneous distribution of microbial communities in fractured rock systems. However, the functions of most organisms, and the rates of their activity and growth, remain largely unknown. To mechanistically understand what physiochemical and hydrological factors control the rock-hosted biosphere, future studies are needed to characterize the physiology of microorganisms adapted to mineral-associated growth under energy- and nutrient-limited conditions. Experiments should be designed to detect synergistic interactions between microorganisms, and between microorganisms and minerals, at highly variable turnover rates. ▪ The heterogeneous distribution of the rock-hosted biosphere is controlled by variations in porosity, permeability, and chemical disequilibrium. ▪ Several imaging and chemical techniques can sensitively detect microbial activity within the rock-hosted biosphere. ▪ The physiology and turnover rates of the subsurface rock-hosted biosphere remain poorly known.

Improved Analysis of Prostate Cancer: VIM3, ATG7 and P53 Form a Complex and Activate miRNA 371a-3p.

It is not possible to differentiate prostate carcinomas sufficiently to ensure that every patient receives the right therapy. New molecular markers are needed. Our objective was to identify a complex consisting of vimentin variant 3 (VIM3), autophagy-related protein 7 (ATG7) and tumor protein p53 (TP53) in prostate cancer cells and its effect on microRNA (miR)-371a-3p. Prostate cancer cell lines (PC3, DU145, LNCaP) and the benign prostatic hyperplasia cell line BPH-1 were cultured in growth medium for 24 h, then stimulated with endothelin 1 (EDN1) (50 nM) and withaferin A (2 nM) for 24 h. Cell extracts were then analyzed by western blot. The localization of VIM3, ATG7 and TP53 in the nucleus was demonstrated with immunofluorescence staining and complex formation was demonstrated by immunoprecipitation. Cancer cell migration was analyzed with a scratch assay and agarose drop analysis. The binding of the complex to the promoter of pri-miR-371a-3p was analyzed with a non-radioactive electrophoretic mobility shift assay. VIM3 knockdown using small interfering RNA and quantitative real-time polymerase chain reaction for miR-371a-3p were performed. The complex was present in the nucleus of prostate cancer cells and in the BPH-1 cell line. EDN1 increased the levels of the complex partners and cell migration, whereas withaferin A reduced the levels of the complex partners and migration. The complex bound to the promoter of pri-miR-371a-3p and might be involved in its transcription. Transfection with miR-371a-3p increased migration of prostate cancer cells. VIM3 knockdown reduced miR-371a-3p expression. The VIM3-ATG7-P53 complex, with its stimulatory effect on miR-371a-3p, may have the potential to be a marker for improved differentiation between prostate carcinomas, allowing tailored therapy.

The cytotoxic effects of Graptopetalum paraguayense (N.E.Br.) E. Walther extract on human melanoma cells

IntroductionThis study was conducted to investigate the mechanisms of apoptosis inducing by 50% ethanolic G. paraguayense (N.E.Br.) E. Walther extract (GE50) in human melanoma cells. MethodsThe polyphenols in GE50 were analyzed by HPLC. After GE50 treatment, cell viability, cell cycle distribution, DNA damage, Ca2+, mitochondrial membrane potential (MMP), caspase-3 activity, and cellular redox status were determined. Additionally, the expression of cell cycle-regulated and apoptosis-related proteins in GE50-treated cells was determined by Western blotting. The translocation of GADD153, Endo G, AIF, and cytochrome c proteins was examined by immunostaining. ResultsHPLC analysis showed that flavone, quercetin, and daidzein were the major constituents in GE50. GE50 significantly prohibits cell proliferation and arrests the cell cycle in the G2/M phase. Results of DAPI staining and DNA gel electrophoresis showed that GE50-treatment can cause chromatin condensation and DNA fragmentation in the cells. GE50 stimulated Ca2+ release, decreased MMP, but inhibited ROS production. GE50 promoted caspase-3 activity in cells. The protein levels of cyclin A, cyclin B, CDC2, CDC25c, and Bcl-2 were decreased; and that of CHK1, CHK2, Weel, p21, p53, GRP78, GADD153, caspase-7, caspase-9, Bax, and AIF were increased in GE50-treated cells. The proteins of GADD153, Endo G, AIF, and cytochrome c were released in the nucleus or cytosol. The GE50-treatment can significantly decrease the glutathione content and antioxidant enzyme activity but significantly increase the MDA levels in the cells. ConclusionsGE50 may have potential cytotoxic effect in human melanoma through G2/M phase arrest, ER stress, and intrinsic apoptotic pathways.

Structural analysis of an endogenous 4-megadalton succinyl-CoA-generating metabolon

The oxoglutarate dehydrogenase complex (OGDHc) participates in the tricarboxylic acid cycle and, in a multi-step reaction, decarboxylates α-ketoglutarate, transfers succinyl to CoA, and reduces NAD+. Due to its pivotal role in metabolism, OGDHc enzymatic components have been studied in isolation; however, their interactions within the endogenous OGDHc remain elusive. Here, we discern the organization of a thermophilic, eukaryotic, native OGDHc in its active state. By combining biochemical, biophysical, and bioinformatic methods, we resolve its composition, 3D architecture, and molecular function at 3.35 Å resolution. We further report the high-resolution cryo-EM structure of the OGDHc core (E2o), which displays various structural adaptations. These include hydrogen bonding patterns confining interactions of OGDHc participating enzymes (E1o-E2o-E3), electrostatic tunneling that drives inter-subunit communication, and the presence of a flexible subunit (E3BPo), connecting E2o and E3. This multi-scale analysis of a succinyl-CoA-producing native cell extract provides a blueprint for structure-function studies of complex mixtures of medical and biotechnological value.

Proteogenomic landscape of gastric adenocarcinoma peritoneal metastases

Advanced gastric adenocarcinoma (GAC) often leads to peritoneal carcinomatosis (PC) and is associated with very poor outcome. Here we report the comprehensive proteogenomic study of ascites derived cells from a prospective GAC cohort (n= 26 patients with peritoneal carcinomatosis, PC). A total of 16,449 proteins were detected from whole cell extracts (TCEs). Unsupervised hierarchical clustering resulted in three distinct groups that reflected extent of enrichment in tumor cells. Integrated analysis revealed enriched biological pathways and notably, some druggable targets (cancer-testis antigens, kinases, and receptors) that could be exploited to develop effective therapies and/or tumor stratifications. Systematic comparison of expression levels of proteins and mRNAs revealed special expression patterns of key therapeutics target notably high mRNA and low protein expression of HAVCR2 (TIM-3), and low mRNA but high protein expression of cancer-testis antigens CTAGE1 and CTNNA2. These results inform strategies to target GAC vulnerabilities.

Alkaloid from Geissospermum sericeum Benth. & Hook.f. ex Miers (Apocynaceae) Induce Apoptosis by Caspase Pathway in Human Gastric Cancer Cells.

Gastric cancer is among the major causes of death from neoplasia leading causes of death worldwide, with high incidence rates and problems related to its treatment. Here, we outline how Geissospermum sericeum exerts antitumor activity on the ACP02 cell line (human gastric adenocarcinoma) and the mechanism of cell death. The ethanol extract and fractions, neutral fraction and alkaloid fraction, were characterized by thin-layer chromatography and HPLC-DAD, yielding an alkaloid (geissoschizoline N4-methylchlorine) identified by NMR. The cytotoxicity activity of the samples (ethanol extract, neutral fraction, alkaloid fraction, and geissoschizoline N4-methylchlorine) in HepG2 and VERO cells was determined by MTT. The ACP02 cell line was used to assess the anticancer potential. Cell death was quantified with the fluorescent dyes Hoechst 33342, propidium iodide, and fluorescein diacetate. The geissoschizoline N4-methylchlorine was evaluated in silico against caspase 3 and 8. In the antitumor evaluation, there was observed a more significant inhibitory effect of the alkaloid fraction (IC50 18.29 µg/mL) and the geissoschizoline N4-methylchlorine (IC50 12.06 µg/mL). However, geissoschizoline N4-methylchlorine showed lower cytotoxicity in the VERO (CC50 476.0 µg/mL) and HepG2 (CC50 503.5 µg/mL) cell lines, with high selectivity against ACP02 cells (SI 39.47 and 41.75, respectively). The alkaloid fraction showed more significant apoptosis and necrosis in 24 h and 48 h, with increased necrosis in higher concentrations and increased exposure time. For the alkaloid, apoptosis and necrosis were concentration- and time-dependent, with a lower necrosis rate. Molecular modeling studies demonstrated that geissoschizoline N4-methylchlorine could occupy the active site of caspases 3 and 8 energetically favorably. The results showed that fractionation contributed to the activity with pronounced selectivity for ACP02 cells, and geissoschizoline N4-methylchlor is a promising candidate for caspase inhibitors of apoptosis in gastric cancer. Thus, this study provides a scientific basis for the biological functions of Geissospermum sericeum, as well as demonstrates the potential of the geissoschizoline N4-methylchlorine in the treatment of gastric cancer.

Dipeptidyl peptidase-1 inhibition with brensocatib reduces the activity of all major neutrophil serine proteases in patients with bronchiectasis: results from the WILLOW trial

BackgroundBrensocatib is an oral, selective, reversible inhibitor of dipeptidyl peptidase-1 (DPP-1), responsible for activating neutrophil serine proteases (NSPs) including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG). In chronic inflammatory lung diseases such as non-cystic fibrosis bronchiectasis (NCFBE), neutrophils accumulate in the airways resulting in excess active NSPs that cause damaging inflammation and lung destruction.MethodsThe 24-week WILLOW trial (NCT03218917) was a randomized, double-blind, placebo-controlled, parallel-group trial in patients with NCFBE conducted at 116 sites across 14 countries. In this trial, treatment with brensocatib was associated with improvements in clinical outcomes including time to first exacerbation, reduction in exacerbation frequency and a reduction in NE activity in sputum. An exploratory analysis of NE activity in white blood cell (WBC) extracts and NE, PR3 and CatG activity in sputum was conducted to further characterize brensocatib’s effect and identify potential correlated effects.ResultsNE, PR3 and CatG activities were reduced in sputum and NE activity was reduced in WBC extracts in a dose-dependent manner after four weeks of brensocatib treatment, with a return to baseline four weeks after the end of treatment. Brensocatib produced the greatest reduction in the sputum activity of CatG, followed by NE and then PR3. Positive correlations among the sputum NSPs were observed both at baseline and in response to treatment, with the strongest correlation among the sputum NSPs for NE and CatG.ConclusionsThese results suggest a broad anti-inflammatory effect of brensocatib underlying its clinical efficacy observed in NCFBE patients.Trial registration: The study was approved by the corresponding ethical review boards of all participating centers. The trial was approved by the Food and Drug Administration and registered at clinicaltrials.gov (NCT03218917) on July 17, 2017 and approved by the European Medicines Agency and registered at the European Union Clinical trials Register (EudraCT No. 2017-002533-32). An independent, external data and safety monitoring committee (comprising physicians with pulmonary expertise, a statistician experienced in the evaluation of clinical safety, and experts in periodontal disease and dermatology) reviewed all adverse events.

A Stiff‐Soft Composite Fabrication Strategy for Fiber Optic Tethered Microtools

AbstractThe ability to capture, manipulate, and release microscale objects using autonomous systems can enable widespread applications—from microsurgery and selective cell extraction to the assembly of complex microdevices. With continuing development of smart and environmentally responsive materials compatible with 3D printing, microgrippers with environmental adaptability can give rise to biocompatible devices. In this paper, the design, fabrication, and testing of hybrid stiff‐soft microgrippers using compliant synthetic polymers and hydrogels to achieve autonomous actuation while avoiding an overly complex mechanical gripping system are described. Building microgrippers using 2‐photon polymerization additive manufacturing based on nonplanar, bioinspired architectures on the end of an optical fiber is investigated. To control actuation, current regulated growth of hydrogels within the printed architecture is demonstrated. Forces are generated by expansive gels actuated by changes in pH, temperature, and application of light via optical fiber. The resulting multimaterial actuators incorporate inert skeletal components with active hydrogels on the tip of a 200 µm diameter core fiber. The response time, accuracy, and cyclic durability of hybrid grippers are evaluated. This work provides a foundation to integrate stimuli‐responsive mechanical functions with microscale optical devices to expand the suite of tools available for minimally invasive surgical procedures.