Ethanol extract of Kalopanax septemlobus leaf inhibits HepG2 human hepatocellular carcinoma cell proliferation via inducing cell cycle arrest at G_1 phase

Objective:To investigate the effects of an ethanol extract of Kalopanax septemlobus(Thunb.)Koidz.leaf(EEKS) on cell proliferation in human hepatocellular carcinoma cells and its mechanisms of action.Methods:Cells were treated with EEKS and subsequently analyzed for cell proliferation and flow cytometry analysis.Expressions of cell cycle regulators were determined by reverse transcriptase polymerase chain reaction analysis and Western blotting,and activation of eyclin-associaled kinases studied using kinase assays.Results:The EEKS suppressed cell proliferation in both HepG2 and Hep3 B cells,but showed a more sensitive anli-proliferative activity in HepG2 cells.Flow cytometry analysis revealed an association between the growth inhibitory effect of EEKS and with G_1 phase cell cycle arrest in HepG2 cells,along with the dephosphorylation of retinoblastoma protein(pRB) and enhanced binding of pRB with the E2 F transcription factor family proteins.Treatment with EEKS also increased the expression of cyclin-dependent kinase(CDK) inhibitors,such as p21WAF1/CIP1 and p27KIP1.without any noticeable changes in G_1 cyclins and CDKs(except for a slight decrease in CDK4).Treatment of HepG2 cells with EEKS also increased the binding of p21 and p27 with CDK4 and CDK6.which was paralleled by a marked decrease in the cyclin D- and cyclin E-associated kinase activities.Conclusions:Overall,our findings suggest that EEKS may be an effective treatment for liver cancer through suppression of cancer cell proliferation via G_1,cell cycle arrest Further studies arc required to identify the active compounds in EEKS.

Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

AIM: To establish a novel, sensitive and high-throughput gelatinolytic assay to define new inhibitors and compare domain deletion mutants of gelatinase B/matrix metalloproteinase (MMP)-9. METHODS: Fluorogenic Dye-quenched (DQ)TM-gelatin was used as a substrate and biochemical parameters (substrate and enzyme concentrations, DMSO solvent concentrations) were optimized to establish a highthroughput assay system. Various small-sized libraries (ChemDiv, InterBioScreen and ChemBridge) of hetero-cyclic, drug-like substances were tested and compared with prototypic inhibitors. RESULTS: First, we designed a test system with gelatin as a natural substrate. Second, the assay was validated by selecting a novel pyrimidine-2,4,6-trione (barbitu- rate) inhibitor. Third, and in line with present structural data on collagenolysis, it was found that deletion of the O-glycosylated region significantly decreased gelatinolytic activity (kcat/kM ± 40% less than full-length MMP-9). CONCLUSION: The DQTM-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.

Ethanol extracts of Hizikia fusiforme induce apoptosis in human prostate cancer PC3 cells via modulating a ROS-dependent pathway

Objective:To investigate whether ethanol extracts of Hizikia fusiforme could induce apoptosis in human prostate cancer PC3 cells.Methods:Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.Apoptosis and mitochondrial membrane potential(MMP)were measured using flow cytometry in PC3 cells.DNA damage was assessed by nuclear staining and DNA fragmentation assay.Expressions of apoptosis-associated proteins were determined by Western blotting assays.Activities of caspase-3,-8,and-9 were determined by colorimetric assay.Moreover,intracellular reactive oxygen species(ROS)generation was detected using a flow cytometer and fluorescence microscope.Results:Treatment of PC3 cells with ethanol extracts of Hizikia fusiforme inhibited proliferation,which was associated with induction of apoptosis,and accompanied by increased expression of Fas,Fas-ligand(Fas L),Bax and t Bid,and decreased expression of Bcl-2.In addition,ethanol extracts of Hizikia fusiforme reduced c-Flip expression and activated caspase-8,-9 and-3,resulting in an increase in poly(ADP-ribose)polymerase(PARP)cleavage.However,in the presence of a pan-caspase inhibitor,ethanol extracts of Hizikia fusiforme-mediated growth inhibition and apoptosis were significantly attenuated.Ethanol extracts of Hizikia fusiforme also destroyed the integrity of mitochondria due to the loss of MMP,leading to cytosolic release of cytochrome c.Moreover,the levels of ROS were markedly increased by treatment with ethanol extracts of Hizikia fusiforme,which was significantly suppressed by the ROS scavenger N-acetyl-L-cysteine.Further investigation of whether ethanol extracts of Hizikia fusiforme-induced apoptosis was related to the generation of ROS was conducted and the results showed that N-acetyl-L-cysteine fully blocked ethanol extracts of Hizikia fusiforme-induced apoptotic events including loss of MMP,activation of caspase-3,the cytosolic release of cytochrome c and cytotoxicity.Conclusions:Ethanol extracts of Hizikia fusiforme have chemopreventive potential via induction of ROS-dependent apoptosis.Therefore,ethanol extracts of Hizikia fusiforme may be useful for developing effective and selective natural sources to inhibit cancer cell proliferation.

Soil-transmitted helminths detected from environmental samples in a campus of southern Brazil

Soil harbours enormous biodiversity,essential for maintaining environmental and human health.However,soil can also be a reservoir of various parasitic pathogens,such as soil-transmitted helminths(STH).We evaluated the presence of STH(e.g.,hookworms,roundworms and whipworms)in soil samples collected at twenty points within the perimeter of Campus do Vale(a university campus belonging to the Federal University of Rio Grande do Sul-UFRGS),during 2022 winter season.Considering the One Health perspective,human,animal and environmentrelated data from each sampling point were collected.All soil samples showed nematode larvae,representing natural components of soil biodiversity.Considering STH eggs,35%(n=7)of soil samples showed hookworm eggs(e.g.,from Necator americanus or Ancylostoma duodenale),10%(n=2)showed roundworm(Ascaris lumbricoides)eggs,and 5%(n=1)showed whipworm(Trichuris trichiura-like)eggs.Of note,10%of the sampling points showed the presence of rhabditiform hookworm larvae,5%showed Strongyloides stercoralis rhabditiform larvae and 5%had the presence of filariform hookworm larvae,indicating a risk of human percutaneous infection.The significant people circulation in Campus do Vale,in association with other environment-related factors,help to explain the prevalence of STH observed in this study.

JAK/STAT signaling regulates tissue outgrowth and male germline stem cell fate in Drosophila

In multicellular organisms, biological activities are regulated by cell signaling. The various signal transduction path- ways regulate cell fate, proliferation, migration, and polarity. Miscoordination of the communicative signals will lead to disasters like cancer and other fatal diseases. The JAK/STAT signal transduction pathway is one of the pathways, which was first identified in vertebrates and is highly conserved throughout evolution. Studying the JAK/STAT signal transduc- tion pathway in Drosophila provides an excellent opportunity to understand the molecular mechanism of the cell regu- lation during development and tumor formation. In this review, we discuss the general overview of JAK/STAT signaling in Drosophila with respect to its functions in the eye development and stem cell fate determination.

Brazil's heavy metal pollution harms humans and ecosystems

This letter draws attention to the worrying situation of heavy metal pollution in Brazil, especially concerning the Amazon's Indigenous peoples affected by mercury contamination from illegal gold mining activities. Heavy metal pollution is also an emerging problem in other Brazilian biomes besides the Amazon Forest (e.g., Pampa biome in southern Brazil), as well as in coastal ecosystems/regions and large cities. Despite being a neglected problem, Brazil's heavy metal pollution causes significant detrimental impacts on human health and ecosystems. Finally, some alternatives to overcome this problem are suggested.

Mesenchymal stromal cell delivery as a potential therapeutic strategy against COVID-19:Promising evidence from in vitro results

BACKGROUND Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is responsible for the coronavirus disease 2019(COVID-19)pandemic,which was initiated in December 2019.COVID-19 is characterized by a low mortality rate(<6%);however,this percentage is higher in elderly people and patients with underlying disorders.COVID-19 is characterized by mild to severe outcomes.Currently,several therapeutic strategies are evaluated,such as the use of anti-viral drugs,prophylactic treatment,monoclonal antibodies,and vaccination.Advanced cellular therapies are also investigated,thus representing an additional therapeutic tool for clinicians.Mesenchymal stromal cells(MSCs),which are known for their immunoregulatory properties,may halt the induced cytokine release syndrome mediated by SARS-CoV-2,and can be considered as a potential stem cell therapy.AIM To evaluate the immunoregulatory properties of MSCs,upon stimulation with COVID-19 patient serum.METHODS MSCs derived from the human Wharton’s Jelly(WJ)tissue and bone marrow(BM)were isolated,cryopreserved,expanded,and defined according to the criteria outlined by the International Society for Cellular Therapies.Then,WJ and BM-MSCs were stimulated with a culture medium containing 15%COVID-19 patient serum,1%penicillin-streptomycin,and 1%L-glutamine for 48 h.The quantification of interleukin(IL)-1 receptor a(Ra),IL-6,IL-10,IL-13,transforming growth factor(TGF)-β1,vascular endothelial growth factor(VEGF)-a,fibroblast growth factor(FGF),platelet-derived growth factor(PDGF),and indoleamine-2,3-dioxygenase(IDO)was performed using commercial ELISA kits.The expression of HLA-G1,G5,and G7 was evaluated in unstimulated and stimulated WJ and BMMSCs.Finally,the interactions between MSCs and patients’macrophages were established using co-culture experiments.RESULTS Thawed WJ and BM-MSCs exhibited a spindle-shaped morphology,successfully differentiated to“osteocytes”,“adipocytes”,and“chondrocytes”,and in flow cytometric analysis were characterized by positivity for CD73,CD90,and CD105(>95%)and negativity for CD34,CD45,and HLA-DR(<2%).Moreover,stimulated WJ and BM-MSCs were characterized by increased cytoplasmic granulation,in comparison to unstimulated cells.The HLA-G isoforms(G1,G5,and G7)were successfully expressed by the unstimulated and stimulated WJ-MSCs.On the other hand,only weak expression of HLA-G1 was identified in BM-MSCs.Stimulated MSCs secreted high levels of IL-1Ra,IL-6,IL-10,IL-13,TGF-β1,FGF,VEGF,PDGF,and IDO in comparison to unstimulated cells(P<0.05)after 12 and 24 h.Finally,macrophages derived from COVID-19 patients successfully adapted the M2 phenotype after co-culturing with stimulated WJ and BM-MSCs.CONCLUSION WJ and BM-MSCs successfully produced high levels of immunoregulatory agents,which may efficiently modulate the over-activated immune responses of critically ill COVID-19 patients.

Disrupted Ca^(2+) h homeostasis and immunodeficiency in patients with functional IP_(3) receptor subtype 3 defects

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca^(2+)) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP_(3)R), a homo- or heterotetramer of the IP_(3)R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca^(2+) from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP_(3)R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP_(3)R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca^(2+) signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca^(2+) signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP_(3)R3 in IP_(3)R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype–phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca^(2+) channels and immunodeficiency and identify IP_(3)Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca^(2+)-associated immunodeficiency from store-operated entry to impaired Ca^(2+) mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca^(2+) signaling.

Mannan-binding lectin-associated serine protease-2 （MASP-2） deficiency in two patients with pulmonary tuberculosis and one healthy control

Hereditary complement deficiencies are usually associated with increased susceptibility to infections and/ or autoimmune diseases. Generally, they are rare： the majority have been detected in no more than a few dozen individuals. However, due to lack of population-based studies, such cases are usually detected by chance, e.g., in severely ill persons and their family members. An exception is mannan-binding lectin （MBL） deficiency, affecting perhaps 5%-10% of the population. MBL, like collectin-10, -11 and the ficolins （M-, L-, H-）, is a pattern-recognition molecule, cooperating with MBL- associated serine proteases （MASPs） in the initiation of complement activation via the lectin pathway. In contrast, deficiency