Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice.

Articular cartilage (AC) is most susceptible to degeneration in knee osteoarthritis (OA); however, the existing treatments for OA do not target the core link of the pathogenesis-"decreased tissue cell function activity and extracellular matrix (ECM) metabolic disorders" for effective intervention. iMSC hold lower heterogeneity and great promise in biological research and clinical applications. Rps6ka2 may play an important role in the iMSC to treat OA. In this study, the CRISPR/Cas9 gene editing Rps6ka2-/- iMSC were obtained. Effect of Rps6ka2 on iMSC proliferation and chondrogenic differentiation was evaluated in vitro. An OA model was constructed in mice by surgical destabilization of medial meniscus (DMM). The Rps6ka2-/- iMSC and iMSC were injected into the articular cavity twice-weekly for 8 weeks. In vitro experiments showed that Rps6ka2 could promote iMSC proliferation and chondrogenic differentiation. In vivo results further confirmed that Rps6ka2 could improve iMSC viability to promote ECM production to attenuate OA in mice.

Cleaved SPP1-rich extracellular vesicles from osteoclasts promote bone regeneration via TGFß1/SMAD3 signaling.

Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) to maintain bone architecture and systemic mineral homeostasis throughout life. However, the mechanisms responsible for the coupling between OCs and OBs have not been fully elucidated. Herein, we first validate that secreted extracellular vesicles by osteoclasts (OC-EVs) promote osteogenic differentiation of mesenchymal stem cells (MSCs) and further demonstrate the efficacy of osteoclasts and their secreted EVs in treating tibial bone defects. Furthermore, we show that OC-EVs contain several osteogenesis-promoting proteins as cargo. By employing proteomic and functional analysis, we reveal that mature osteoclasts secrete thrombin cleaved phosphoprotein 1 (SPP1) through extracellular vesicles which triggers MSCs osteogenic differentiation into OBs by activating Transforming Growth Factor ß1 (TGFß1) and Smad family member 3 (SMAD3) signaling. In conclusion, our findings prove an important role of SPP1, present as cargo in OC-derived EVs, in signaling to MSCs and driving their differentiation into OBs. This biological mechanism implies a paradigm shift regarding the role of osteoclasts and their signaling toward the treatment of skeletal disorders which require bone formation.

Stem Cell-Based Therapeutic Strategies for Premature Ovarian Insufficiency and Infertility: A Focus on Aging.

Reproductive aging is on the rise globally and inseparable from the entire aging process. An extreme form of reproductive aging is premature ovarian insufficiency (POI), which to date has mostly been of idiopathic etiology, thus hampering further clinical applications and associated with enormous socioeconomic and personal costs. In the field of reproduction, the important functional role of inflammation-induced ovarian deterioration and therapeutic strategies to prevent ovarian aging and increase its function are current research hotspots. This review discusses the general pathophysiology and relative causes of POI and comprehensively describes the association between the aging features of POI and infertility. Next, various preclinical studies of stem cell therapies with potential for POI treatment and their molecular mechanisms are described, with particular emphasis on the use of human induced pluripotent stem cell (hiPSC) technology in the current scenario. Finally, the progress made in the development of hiPSC technology as a POI research tool for engineering more mature and functional organoids suitable as an alternative therapy to restore infertility provides new insights into therapeutic vulnerability, and perspectives on this exciting research on stem cells and the derived exosomes towards more effective POI diagnosis and treatment are also discussed.

Systematic molecular analysis of the human secretome and membrane proteome in gastrointestinal adenocarcinomas.

The human secretome and membrane proteome are a large source of cancer biomarkers. Membrane-bound and secreted proteins are promising targets for many clinically approved drugs, including for the treatment of tumours. Here, we report a deep systematic analysis of 957 adenocarcinomas of the oesophagus, stomach, colon and rectum to examine the cancer-associated human secretome and membrane proteome of gastrointestinal tract adenocarcinomas (GIACs). Transcriptomic data from these GIACs were applied to an innovative majority decision-based algorithm. We quantified significantly expressed protein-coding genes. Interestingly, we found a consistent pattern in a small group of genes found to be overexpressed in GIACs, which were associated with a cytokine-cytokine interaction pathway (CCRI) in all four cancer subtypes. These CCRI associated genes, which spanned both one secretory and one membrane isoform were further analysed, revealing a putative biomarker, interleukin-1 receptor accessory protein (IL1RAP), which indicated a poor overall survival, a positive correlation with cancer stemness and a negative correlation with several kinds of T cells. These results were further validated in vitro through the knockdown of IL1RAP in two human gastric carcinoma cell lines, which resulted in a reduced indication of cellular proliferation, migration and markers of invasiveness. Following IL1RAP silencing, RNA seq results showed a consistent pattern of inhibition related to CCRI, proliferation pathways and low infiltration of regulatory T cells (Tregs) and CD8 naive cells. The significance of the human secretome and membrane proteome is elucidated by these findings, which indicate IL1RAP as a potential candidate biomarker for cytokine-mediated cancer immunotherapy in gastric carcinoma.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Hold Lower Heterogeneity and Great Promise in Biological Research and Clinical Applications.

Mesenchymal stem cells (MSC) isolated from different tissue sources exhibit multiple biological effects and have shown promising therapeutic effects in a broad range of diseases. In order to fulfill their clinical applications in context of precision medicine, however, more detailed molecular characterization of diverse subgroups and standardized scalable production of certain functional subgroups would be highly desired. Thus far, the generation of induced pluripotent stem cell (iPSC)-derived MSC (iMSC) seems to provide the unique opportunity to solve most obstacles that currently exist to prevent the broad application of MSC as an advanced medicinal product. The features of iMSC include their single cell clone origins, and defined and controllable cultural conditions for their derivation and proliferation. Still, comprehensive research of the molecular and functional heterogeneity of iMSC, just like MSC from any other tissue types, would be required. Furthered on previous efforts on iMSC differentiation and expansion platform and transcriptomic studies, advantages of single cell multi-omics analysis and other up-to-dated technologies would be taken in order to elucidate the molecular origin and regulation of heterogeneity and to obtain iMSC subgroups homogeneous enough for particular clinical conditions. In this perspective, the current obstacles in MSC applications, the advantages of iMSC over MSC and their implications for biological research and clinical applications will be discussed.

LP1 from Lentinula edodes C91-3 Induces Autophagy, Apoptosis and Reduces Metastasis in Human Gastric Cancer Cell Line SGC-7901.

Present study aimed to elucidate the anticancer effect and the possible molecular mechanism underlying the action of Latcripin 1 (LP1), from the mushroom Lentinula edodes strain C91-3 against gastric cancer cell lines SGC-7901 and BGC-823. Cell viability was measured by Cell Counting Kit-8 (CCK-8); morphological changes were observed by phase contrast microscope; autophagy was determined by transmission electron microscope and fluorescence microscope. Apoptosis and cell cycle were assessed by flow cytometer; wound-healing, transwell migration and invasion assays were performed to investigate the effect of LP1 on gastric cancer cell's migration and invasion. Herein, we found that LP1 resulted in the induction of autophagy by the formation of autophagosomes and conversion of light chain 3 (LC3I into LC3II. LP1 up-regulated the expression level of autophagy-related gene (Atg7, Atg5, Atg12, Atg14) and Beclin1; increased and decreased the expression level of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) proteins respectively, along with the activation of Caspase-3. At lower-doses, LP1 have shown to arrest cells in the S phase of the cell cycle and decreased the expression level of matrix metalloproteinase MMP-2 and MMP-9. In addition, it has also been shown to regulate the phosphorylation of one of the most hampered gastric cancer pathway, that is, protein kinase B/mammalian target of rapamycin (Akt/mTOR) channel and resulted in cell death. These findings suggested LP1 as a potential natural anti-cancer agent, for exploring the gastric cancer therapies and as a contender for further in vitro and in vivo investigations.

Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies.

The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (- 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein's activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.

Recombinant latcripin 11 of Lentinula edodes C91-3 suppresses the proliferation of various cancer cells.

Lentinula edodes C91-3 is an edible mushroom that has demonstrated a remarkable anti-tumor effect in various cancer cells both in vitro and in vivo. In the present study, we report the ability of recombinant thioredoxin-like latcripin 11 (LP-11) of Lentinula edodes C91-3 to suppress the proliferation of various cancer cells. The LP-11 gene of Lentinula edodes C91-3 was cloned in the pET-32a(+) expression vector and expressed in a prokaryotic system. The expressed protein was refolded by gradual dialysis and purified by affinity gel filtration chromatography. The antioxidant activity of LP-11 was tested by 1,1-dipheny l-2-picrylhydrazyl (DPPH) assay. The anti-tumor activity of recombinant LP-11 was tested in eight kinds of tumor cell lines by CCK-8 assay. Recombinant LP-11 significantly suppressed the proliferation of various cancer cells, but not normal human umbilical vein endothelial cells. Human lymphoma U937 cells exhibited the most sensitivity to LP-11 protein. U937 cell apoptosis was assessed by Annexin V staining coupled with flow cytometry, and mitochondrial morphology was analyzed by light and electron microscopy. It was revealed that recombinant LP-11 induced apoptosis in human leukemic monocyte lymphoma U937 cells. Our findings suggest that recombinant LP-11 is a promising agent for the treatment of lymphoma.

A Preclinical Evaluation of the Antitumor Activities of Edible and Medicinal Mushrooms: A Molecular Insight.

Cancer is the leading cause of morbidity and mortality around the globe. For certain types of cancer, chemotherapy drugs have been extensively used for treatment. However, severe side effects and the development of resistance are the drawbacks of these agents. Therefore, development of new agents with no or minimal side effects is of utmost importance. In this regard, natural compounds are well recognized as drugs in several human ailments, including cancer. One class of fungi, "mushrooms," contains numerous compounds that exhibit interesting biological activities, including antitumor activity. Many researchers, including our own group, are focusing on the anticancer potential of different mushrooms and the underlying molecular mechanism behind their action. The aim of this review is to discuss PI3K/AKT, Wnt-CTNNB1, and NF-κB signaling pathways, the occurrence of genetic alterations in them, the association of these aberrations with different human cancers and how different nodes of these pathways are targeted by various substances of mushroom origin. We have given evidence to propose the therapeutic attributes and possible mode of molecular actions of various mushroom-originated compounds. However, anticancer effects were typically demonstrated in in vitro and in vivo models and very limited number of studies have been conducted in the human population. It is our belief that this review will help the research community in designing concrete preclinical and clinical studies to test the anticancer potential of mushroom-originated compounds on different cancers harboring particular genetic alteration(s).

Combined effect of linolenic acid and tobramycin on Pseudomonas aeruginosa biofilm formation and quorum sensing.

Pseudomonas aeruginosa is a ubiquitous Gram negative opportunistic pathogen capable of causing severe nosocomial infections in humans, and tobramycin is currently used to treat P. aeruginosa associated lung infections. Quorum sensing regulates biofilm formation which allows the bacterium to result in fatal infections forcing clinicians to extensively use antibiotics to manage its infections leading to emerging multiple drug resistant strains. As a result, tobramycin is also becoming resistant. Despite extensive studies on drug discovery to alleviate microbial drug resistance, the continued microbial evolution has forced researchers to focus on screening various phytochemicals and dietary compounds for antimicrobial potential. Linolenic acid (LNA) is an essential fatty acid that possesses antimicrobial actions on various microorganisms. It was hypothesized that LNA may affect the formation of biofilm on P. aeruginosa and improve the potency of tobramycin. The present study demonstrated that LNA interfered with cell-to-cell communication and reduced virulence factor production. It further enhanced the potency of tobramycin and synergistically inhibited biofilm formation through P. aeruginosa quorum sensing systems. Therefore, LNA may be considered as a potential agent for adjunctive therapy and its utilization may decrease tobramycin concentration in combined treatment thereby reducing aminoglycoside adverse effects.

CD147 stimulates hepatoma cells escaping from immune surveillance of T cells by interaction with Cyclophilin A.

T cells play an important role in tumor immune surveillance. CD147 is a member of immunoglobulin superfamily present on the surface of many tumor cells and mediates malignant cell behaviors. Cyclophilin A (CypA) is an intracellular protein promoting inflammation when released from cells. CypA is a natural ligand for CD147. In this study, CD147 specific short hairpin RNAs (shRNA) were transfected into murine hepatocellular carcinoma Hepa1-6 cells to assess the effects of CD147 on hepatoma cells escaping from immune surveillance of T cells. We found extracellular CypA stimulated cell proliferation through CD147 by activating ERK1/2 signaling pathway. Downregulation of CD147 expression on Hepa1-6 cells significantly suppressed tumor progression in vivo, and decreased cell viability when co-cultured with T cells in vitro. Importantly, knockdown of CD147 on Hepa1-6 cells resulted in significantly increased T cells chemotaxis induced by CypA both in vivo and in vitro. These findings provide novel mechanisms how tumor cells escaping from immune surveillance of T cells. We provide a potential therapy for hepatocellular carcinoma by targeting CD147 or CD147-CypA interactions.

GalNAc-T4 putatively modulates the estrogen regulatory network through FOXA1 glycosylation in human breast cancer cells.

GALNT4 belongs to a family of N-acetylgalactosaminyltransferases, which catalyze the transfer of GalNAc to Serine or Threonine residues in the initial step of mucin-type O-linked protein glycosylation. This glycosylation type is the most complex post-translational modification of proteins, playing important roles during cellular differentiation and in pathological disorders. Most of the breast cancer subtypes are estrogen receptor positive, and hence, the estrogen pathway represents a key regulatory network. We investigated the expression of GalNAc-T4 in a panel of mammary epithelial cell lines and found its expression is associated with the estrogen status of the cells. FOXA1, a key transcription factor, functions to promote estrogen responsive gene expression by acting as a cofactor to estrogen receptor alpha (ERα), but all the aspects of this regulatory mechanism are not fully explored. This study found that knockdown of GALNT4 expression in human breast cancer cells attenuated the protein expression of ERα, FOXA1, and Cyclin D1. Further, our immunoprecipitation assays depicted the possibility of FOXA1 to undergo O-GalNAc modifications with a decrease of GalNAc residues in the GALNT4 knockdown cells and also impairment in the FOXA1-ERα association. Rescuing GALNT4 expression could restore the interaction as well as the glycosylation of FOXA1. Together, these findings suggest a key role for GalNAc-T4 in the estrogen pathway through FOXA1 glycosylation.

Phosphocreatine protects endothelial cells from oxidized low-density lipoprotein-induced apoptosis by modulating the PI3K/Akt/eNOS pathway.

Endothelial apoptosis triggered by oxidized low-density lipoprotein (oxLDL) can accelerate the progression of endothelial dysfunction atherosclerosis. Phosphocreatine (PCr) is a natural compound, which has been used in cardiac disease and cardiopulmonary resuscitation. However, its protective effects on atherosclerosis and its mechanism have not been clarified. In the present study, we investigated the anti-apoptotic effect of phosphocreatine in human umbilical vein endothelial cells (HUVECs) exposed to oxLDL and explored the possible mechanisms. HUVECs were pre-treated with 10-30 mM PCr and then stimulated with oxLDL. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, CCK assay, and flow cytometry respectively. Levels of Bax, Bcl-2, protein expression of protein kinase B (Akt), eNOS and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Lactate dehydrogenase (LDH), malondialdehyde (MDA), nitric oxide (NO) and superoxide dismutase (SOD) contents were determined by spectrophotometer. Our results showed that PCr dose-dependently prevented oxLDL associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, LDH and MDA leakage and loss of SOD, decrease of Bcl-2/Bax protein ratio, activation of caspase-3 and 9, and ROS generation. In addition, the antiapoptotic effect of PCr was partially inhibited by a PI3K inhibitor (LY294002) and also enhanced p-Akt/Akt protein ratio, eNOS activation and NO production. In conclusion, our data show that the inhibition of oxLDL-induced endothelial apoptosis by PCr is due, at least in part to its anti-oxidant activity and its ability to modulate the PI3K/Akt/eNOS signaling pathway.

Upregulated ß1-6 branch N-glycan marks early gliomagenesis but exhibited biphasic expression in the progression of astrocytic glioma.

Glioma is the world's commonest primary brain malignancy with much of its biology relating to translational and post-translational events still unknown. In this study, we investigated the clinicopathological significance of N-linked ß1-6-GlcNAc branches and GnT-V enzyme in the development and progression of astrocytic glioma. Expression of GnT-V and its GlcNAc-ß1-6 oligosaccharides by-product together with Con-A binding sugars were assessed immunohistochemically on tissue microarrays of 16 normal brain and 159 tissue samples of astrocytomas of variable grades and histology. Although tissues of both grade I astrocytomas and normal brain showed considerably higher GnT-V expression, GlcNAc-ß1-6 expression was significantly high only in tissues of grade I astrocytomas (p < 0.001), which is attributable to elevated levels of the precursor Con-A binding sugar moieties (p < 0.001). The activity of GnT-V enzyme was found to be dependent on the degree of glioma pathogenesis, as the GlcNAc-ß1-6 branched expression diminished with every progressive grade of glioma, reaching minimum in glioblastoma (p < 0.001). Having biphasic activity in gliomagenesis, the role of GnT-V in glioma was deciphered by generating different ectopic GnT-V expressions in U-87 cells, which showed the highest GnT-V expression among selected glioma cell lines. Transient GnT-V rescue was achieved in knockdown clones by transfection with GnT-V expression vector. Suppression of GnT-V in U-87 cells slowed cell proliferation with G0/G1 cell cycle phase arrest. Reduced tumorigenicity, invasiveness and cell-ECM interactions were also associated with suppressed in vitro GnT-V activity suggesting GnT-V may act as an oncoprotein. We report for the first time that GnT-V products are involved in early gliomagenesis but their reduced expression, correlating with low Con-A binding sugars level found in high tumor grades predicts the loss of total N-glycosylation in glioma development and may be of potential diagnostic and/or prognostic value in astrocytoma.

Downregulated protein O-fucosyl transferase 1 (Pofut1) expression exerts antiproliferative and antiadhesive effects on hepatocytes by inhibiting Notch signalling.

The Notch signalling pathway is essential for proper cell growth and development. Many factors interact in the cellular context to bring about its effects. Critical to this process is the influence of Pofut1 whose function is to fucosylate Notch receptors and ligands on the cell surface for proper interaction. Of the three liver cell lines, HepG2, SMMC-7721 (hepatoma cell lines) and L-02 (normal fetal liver cell line) were investigated. In the current study, Pofut1 was silenced in L-02 due to its significantly high level of expression, (P<0.05) using transient SiRNA. Notch1, Cyclin D1 and p53 were significantly suppressed consequently. The effect on cell cycle, proliferation, adhesion and migration were investigated. Evidence adduced, indicate a general modification of cellular function. While proliferation and adhesion were significantly inhibited, the cell cycle arrest was obvious (P<0.05), migration was not affected. The effects seen are akin to those reported in previous studies in hepatoma cells mimicking some of the effects of notch1 silencing. Results of this study indicate a possible role of Pofut1 conferring a tumor suppressor role through the Notch signalling pathway.