Chemical Variation in Essential Oils from the Oleo‐gum Resin of Boswellia carteri: A Preliminary Investigation

Frankincense, the oleo‐gum resin of Boswellia species, has been an important element of traditional medicine for thousands of years. Frankincense is still used for oral hygiene, to treat wounds, and for its calming effects. Different Boswellia species show different chemical profiles, and B. carteri, in particular, has shown wide variation in essential oil composition. In order to provide insight into the chemical variability in authentic B. carteri oleoresin samples, a hierarchical cluster analysis of 42 chemical compositions of B. carteri oleo‐gum resin essential oils has revealed at least three different chemotypes, i) an α‐pinene‐rich chemotype, ii) an α‐thujene‐rich chemotype, and iii) a methoxydecane‐rich chemotype.     

Pherophorins: a family of extracellular matrix glycoproteins fromVolvox structurally related to the sex-inducing pheromone

Pherophorins are extracellular matrix (ECM) glycoproteins from Volvox that share homology with the sex-inducing pheromone. A novel pherophorin (pherophorin III) was characterized both with respect to expression pattern and proteolytic processing in vivo. Furthermore, it was shown that the pherophorins represent a protein family of ECM glycoproteins exhibiting a modular composition: their N-terminally located domain is a homolog of a domain found in the ECM glycoprotein SSG 185. Together with SSG 185, pherophorin I is a main component of the cellular zone within the ECM. The Volvox genome contains a tandem arrangement of genes encoding pherophorin II-related polypeptides. Inhibition of proteolytic processing of pherophorin II and III in vivo appears to result in the suppression of sexual induction.Abbreviations ECM extracellular matrix - PCR polymerase chain reaction - RACE rapid amplification of cDNA ends We wish to thank Dr. R. Deutzmann (Universität Regensburg, Germany) for sequencing peptides. This work was supported by the Deutsche Forschungsgemeinschaft (SFB43).     

Germline stem cell arrest inhibits the collapse of somatic proteostasis early in Caenorhabditis elegans adulthood

All cells rely on highly conserved protein folding and clearance pathways to detect and resolve protein damage and to maintain protein homeostasis (proteostasis). Because age is associated with an imbalance in proteostasis, there is a need to understand how protein folding is regulated in a multicellular organism that undergoes aging. We have observed that the ability of Caenorhabditis elegans to maintain proteostasis declines sharply following the onset of oocyte biomass production, suggesting that a restricted protein folding capacity may be linked to the onset of reproduction. To test this hypothesis, we monitored the effects of different sterile mutations on the maintenance of proteostasis in the soma of C. elegans. We found that germline stem cell (GSC) arrest rescued protein quality control, resulting in maintenance of robust proteostasis in different somatic tissues of adult animals. We further demonstrated that GSC‐dependent modulation of proteostasis requires several different signaling pathways, including hsf‐1 and daf‐16/kri‐1/tcer‐1, daf‐12, daf‐9, daf‐36, nhr‐80, and pha‐4 that differentially modulate somatic quality control functions, such that each signaling pathway affects different aspects of proteostasis and cannot functionally complement the other pathways. We propose that the effect of GSCs on the collapse of proteostasis at the transition to adulthood is due to a switch mechanism that links GSC status with maintenance of somatic proteostasis via regulation of the expression and function of different quality control machineries and cellular stress responses that progressively lead to a decline in the maintenance of proteostasis in adulthood, thereby linking reproduction to the maintenance of the soma.     

Importance of PNO1 for growth and survival of urinary bladder carcinoma: Role in core‐regulatory circuitry

PNO1 (partner of Nob1) was known as a RNA‐binding protein in humans, and its ortholog PNO1 was reported to participate ribosome and proteasome biogenesis in yeasts. Yet there have been few studies about its functions in mammalian cells, and so far its role in human cells has never been reported, especially in urinary bladder cancer (UBC).We interrogated the cellular functions and clinical significance of PNO1 in, and its molecular mechanism through microarrays and bioinformatics analysis. Our findings support that PNO1 participates in promoting proliferation and colonogenesis, while reducing apoptosis of UBC cells, and is also predicted to be associated with the migration and metastasis of UBC PNO1 knockdown (KD) attenuated the tumorigenesis ability of UBC in mouse. PNO1 KD led to the altered expression of 1543 genes that are involved in a number of signalling pathways, biological functions and regulation networks. CD44, PTGS2, cyclin D1, CDK1, IL‐8, FRA1, as well as mTOR, p70 S6 kinase, p38 and Caspase‐3 proteins were all down‐regulated in PNO1 KD cells, suggesting the involvement of PNO1 in inflammatory responses, cell cycle regulation, chemotaxis, cell growth and proliferation, apoptosis, cell migration and invasiveness. This study will enhance our understanding of the molecular mechanism of UBC and may eventually provide novel targets for individualized cancer therapy.     

SHARPIN regulates collagen architecture and ductal outgrowth in the developing mouse mammary gland

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells (Sharpin^cpdm), and mice with a stromal (S100a4‐Cre) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin^cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin^cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin^cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro. Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.     

Local oestrogen therapy modulates extracellular matrix and immune response in the vaginal tissue of post‐menopausal women with severe pelvic organ prolapse

This study investigates the effect of local oestrogen therapy (LET) on the expression of proteins participating in collagen/elastin biogenesis and immune markers in vaginal tissues of post‐menopausal women with severe pelvic organ prolapse (POP). Vaginal biopsies were collected from the anterior vaginal wall of informed and consented 52 post‐menopausal women with severe POP undergoing total hysterectomy. Twenty‐nine of the 52 women were treated with LET (in the form of vaginal oestrogen cream or tablet), while the remaining 23 untreated patients served as the controls. This study was approved by Sinai Health System REB. Vaginal tissue specimens were analysed for gene and protein expression using real‐time RT‐PCR and Luminex assays, protein localization and immune cell infiltration were assessed by immunohistochemistry. Forty‐four cytokines were detected. We found that LET application: (a) significantly increased (P < 0.05) gene and protein expression levels of extracellular matrix (ECM) structural proteins, collagen and elastin, as well as the expression of ECM maturation enzyme BMP1; (b) decreased protein expression level of ECM degradation enzymes MMP1, MMP2 and MMP3 accompanied by an increase in their tissue inhibitors, TIMP1 and TIMP4; (c) significantly increased (P < 0.05) the gene and protein expression levels of 14 vaginal cytokines involved in leucocyte infiltration, which was confirmed by immunohistochemistry. Our results indicate that LET plays an important role in the activation of immune system within the local vaginal environment, limiting the undesirable ECM degradation, which supports the strengthening of vaginal ECM in post‐menopausal women, therefore resisting menopause/age‐related changes and inducing urogenital tract tissue regeneration.     

Urokinase receptor and CXCR4 are regulated by common microRNAs in leukaemia cells

The urokinase‐type plasminogen activator (uPA) receptor (uPAR) focuses uPA proteolytic activity on the cell membrane, promoting localized degradation of extracellular matrix (ECM), and binds vitronectin (VN), mediating cell adhesion to the ECM. uPAR‐bound uPA and VN induce proteolysis‐independent intracellular signalling, regulating cell adhesion, migration, survival and proliferation. uPAR cross‐talks with CXCR4, the receptor for the stroma‐derived factor 1 chemokine. CXCR4 is crucial in the trafficking of hematopoietic stem cells from/to the bone marrow, which involves also uPAR. Both uPAR and CXCR4 are expressed in acute myeloid leukaemia (AML), with a lower expression in undifferentiated and myeloid subsets, and higher expression in myelomonocytic and promyelocytic subsets. We hypothesized a microRNA (miR)‐mediated co‐regulation of uPAR and CXCR4 expression, which could allow their cross‐talk at the cell surface. We identified three miRs, miR‐146a, miR‐335 and miR‐622, regulating the expression of both uPAR and CXCR4 in AML cell lines. Indeed, these miRs directly target the 3′untranslated region of both uPAR‐ and CXCR4‐mRNAs; accordingly, uPAR/CXCR4 expression is reduced by their overexpression in AML cells and increased by their specific inhibitors. Overexpression of all three miRs impairs migration, invasion and proliferation of myelomonocytic cells. Interestingly, we observed an inverse relationship between uPAR/CXCR4 expression and miR‐146a and miR‐335 levels in AML blasts, suggesting their possible role in the regulation of uPAR/CXCR4 expression also in vivo.     

Rice RAD51 paralogs play essential roles in somatic homologous recombination for DNA repair

Synthesis‐dependent strand annealing (SDSA) and single‐strand annealing (SSA) are the two main homologous recombination (HR) pathways in double‐strand break (DSB) repair. The involvement of rice RAD51 paralogs in HR is well known in meiosis, although the molecular mechanism in somatic HR remains obscure. Loss‐of‐function mutants of rad51 paralogs show increased sensitivity to the DSB‐inducer bleomycin, which results in greatly compromised somatic recombination efficiencies (xrcc3 in SDSA, rad51b and xrcc2 in SSA, rad51c and rad51d in both). Using immunostaining, we found that mutations in RAD51 paralogs (XRCC3, RAD51C, or RAD51D) lead to tremendous impairment in RAD51 focus formation at DSBs. Intriguingly, the RAD51C mutation has a strong effect on the protein loading of its partners (XRCC3 and RAD51B) at DSBs, which is similar to the phenomenon observed in the case of blocking PI3K‐like kinases in wild‐type plant. We conclude that the rice CDX3 complex acts in SDSA recombination while the BCDX2 complex acts in SSA recombination in somatic DSB repair. Importantly, RAD51C serves as a fulcrum for the local recruitment of its partners (XRCC3 for SDSA and RAD51B for SSA) and is positively modulated by PI3K‐like kinases to facilitate both the SDSA and SSA pathways in RAD51 paralog‐dependent somatic HR.     

Sphingosine 1‐phosphate and its carrier apolipoprotein M in human sepsis and in Escherichia coli sepsis in baboons

Sphingosine 1‐phosphate (S1P) is an important regulator of vascular integrity and immune cell migration, carried in plasma by high‐density lipoprotein (HDL)‐associated apolipoprotein M (apoM) and by albumin. In sepsis, the protein and lipid composition of HDL changes dramatically. The aim of this study was to evaluate changes in S1P and its carrier protein apoM during sepsis. For this purpose, plasma samples from both human sepsis patients and from an experimental Escherichia coli sepsis model in baboons were used. In the human sepsis cohort, previously studied for apoM, plasma demonstrated disease‐severity correlated decreased S1P levels, the profile mimicking that of plasma apoM. In the baboons, a similar disease‐severity dependent decrease in plasma levels of S1P and apoM was observed. In the lethal E. coli baboon sepsis, S1P decreased already within 6–8 hrs, whereas the apoM decrease was seen later at 12–24 hrs. Gel filtration chromatography of plasma from severe human or baboon sepsis on Superose 6 demonstrated an almost complete loss of S1P and apoM in the HDL fractions. S1P plasma concentrations correlated with the platelet count but not with erythrocytes or white blood cells. The liver mRNA levels of apoM and apoA1 decreased strongly upon sepsis induction and after 12 hr both were almost completely lost. In conclusion, during septic challenge, the plasma levels of S1P drop to very low levels. Moreover, the liver synthesis of apoM decreases severely and the plasma levels of apoM are reduced. Possibly, the decrease in S1P contributes to the decreased endothelial barrier function observed in sepsis.     

A proteomics approach to identifying novel protein targets involved in erinacine A–mediated inhibition of colorectal cancer cells’ aggressiveness

Erinacine A, a major active component of a diterpenoid derivative isolated from Hericium erinaceus mycelium, has been demonstrated to exert anticancer effects. Herein, we present an investigation of the molecular mechanism of erinacine A induction associated with cancer cells’ aggressive status and death. A proteomic approach was used to purify and identify the differentially expressed proteins following erinacine A treatment and the mechanism of its action in apoptotic and the targets of erinacine A. Our results demonstrate that erinacine A treatment of HCT‐116 and DLD‐1 cells increased cell cytotoxicity and reactive oxygen species (ROS) production as well as decreased cell proliferation and invasiveness. Ten differentially displayed proteins were determined and validated in vitro and in vivo between the erinacine A‐treated and untreated groups. In addition, erinacine A time‐dependent induction of cell death and inhibitory invasiveness was associated with sustained phosphorylation of the PI3K/mTOR/p70S6K and ROCK1/LIMK2/Cofilin pathways. Furthermore, we demonstrated that erinacine A–induced HCT‐116 and DLD‐1 cells viability and anti‐invasion properties by up‐regulating the activation of PI3K/mTOR/p70S6K and production of ROS. Experiments involving specific inhibitors demonstrated that the differential expression of cofilin‐1 (COFL1) and profilin‐1 (PROF1) during erinacine A treatment could be involved in the mechanisms of HCT‐116 and DLD‐1 cells death and decreased aggressiveness, which occurred via ROCK1/LIMK2/Cofilin expression, with activation of the PI3K/mTOR/p70S6K signalling pathway. These findings elucidate the mechanism of erinacine A inhibiting the aggressive status of cells by activating PI3K/mTOR/p70S6K downstream signalling and the novel protein targets COF1 and PROF1; this could be a good molecular strategy to limit the aggressiveness of CRC cells.     

KITLG is a novel target of miR‐34c that is associated with the inhibition of growth and invasion in colorectal cancer cells

MiR‐34c is considered a potent tumour suppressor because of its negative regulation of multiple target mRNAs that are critically associated with tumorigenesis and metastasis. In the present study, we demonstrated a novel target of miR‐34c, KITLG, which has been implicated in colorectal cancer (CRC). First, we found a significant negative relationship between miR‐34c and KITLG mRNA expression levels in CRC cell lines, including HT‐29, HCT‐116, SW480 and SW620 CRC cell lines. In silico analysis predicted putative binding sites for miR‐34c in the 3′ untranslated region (3′UTR) of KITLG mRNA. A dual‐luciferase reporter assay further confirmed that KITLG is a direct target of miR‐34c. Then, the cell lines were infected with lentiviruses expressing miR‐34c or a miR‐34c specific inhibitor. Restoration of miR‐34c dramatically reduced the expression of KITLG mRNA and protein, while silencing of endogenous miR‐34c increased the expression of KITLG protein. The miR‐34c‐mediated down‐regulation of KITLG was associated with the suppression on proliferation, cellular transformation, migration and invasion of CRC cells, as well as the promotion on apoptosis. Knockdown of KITLG by its specific siRNA confirmed a critical role of KITLG down‐regulation for the tumour‐suppressive effects of miR‐34c in CRC cells. In conclusion, our results demonstrated that miR‐34c might interfere with KITLG‐related CRC and could be a novel molecular target for CRC patients.     

Mitochondria mediate septin cage assembly to promote autophagy of Shigella

Septins, cytoskeletal proteins with well‐characterised roles in cytokinesis, form cage‐like structures around cytosolic Shigella flexneri and promote their targeting to autophagosomes. However, the processes underlying septin cage assembly, and whether they influence S. flexneri proliferation, remain to be established. Using single‐cell analysis, we show that the septin cages inhibit S. flexneri proliferation. To study mechanisms of septin cage assembly, we used proteomics and found mitochondrial proteins associate with septins in S. flexneri‐infected cells. Strikingly, mitochondria associated with S. flexneri promote septin assembly into cages that entrap bacteria for autophagy. We demonstrate that the cytosolic GTPase dynamin‐related protein 1 (Drp1) interacts with septins to enhance mitochondrial fission. To avoid autophagy, actin‐polymerising Shigella fragment mitochondria to escape from septin caging. Our results demonstrate a role for mitochondria in anti‐Shigella autophagy and uncover a fundamental link between septin assembly and mitochondria.