Synergistic activity of agents targeting growth factor receptors, CDKs and downstream signaling molecules in a panel of pancreatic cancer cell lines and the identification of antagonistic combinations : implications for future clinical trials in pancreatic cancer

Pancreatic cancer is one of the most aggressive, heterogeneous and fatal type of human cancers for which more effective therapeutic agents are urgently needed. Here, we investigated the sensitivity of a panel of seven human pancreatic cancer cell lines (HPCCLs) to treatment with various tyrosine kinase inhibitors (TKIs), cyclin‑dependent kinase (CDK) inhibitors, an inhibitor of STAT3 stattic, and a cytotoxic agent gemcitabine both as single agents and in combination. The membranous expression of various receptors and the effect of selected agents on cell cycle distribution, cell signaling pathways and migration was determined using flow cytometry, western blot analysis and scratch wound healing assays, respectively. While the expression of both HER‑3 and HER‑4 was low or negative, the expression of EGFR and HER2 was high or intermediate in all HPCCLs. Of all the agents examined, the CDK1/2/5/9 inhibitor, dinacicilib, was the most potent agent which inhibited the proliferation of all seven HPCCLs with IC50 values of ≤10 nM, followed by SRC targeting TKI dasatinib (IC50 of ≤258 nM), gemcitabine (IC50 of ≤330 nM), stattic (IC50 of ≤2 µM) and the irreversible pan‑HER TKI afatinib (IC50 of ≤2.95 µM). Treatment with afatinib and dasatinib inhibited the ligand‑induced phosphorylation of EGFR and SRC respectively. Statistically significant associations were found between HER2 expression and response to treatment with the ALK/IGF‑IR/InsR inhibitor ceritinib and fibroblast growth factor receptor (FGFR)1/2/3 inhibitor AZD4547, HER3 and IGF‑IR expression and their response to treatment with TKIs targeting HER family members (erlotinib and afatinib), and c‑MET and ALK7 expression and their response to treatment with stattic. Interestingly, treatment with a combination of afatinib with dasatinib and gemcitabine with dasatinib resulted in synergistic tumor growth inhibition in all HPCCLs examined. In contrast, the combination of afatinib with dinaciclib was found to be antagonistic. Finally, the treatment with afatinib, dasatinib and dinaciclib strongly inhibited the migration of all HPCCLs examined. In conclusion, the CDK1/2/5/9 inhibitor dinaciclib, irreversible pan‑HER TKI afatinib and SRC targeting TKI dasatinib were most effective at inhibiting the proliferation and migration of HPCCLs and the combination of afatinib with dasatinib and gemcitabine with dasatinib led to synergistic tumor growth inhibition in all HPCCLs examined. Our results support further investigation on the therapeutic potential of these combinations in future clinical trials in pancreatic cancer

YPTB3816 of 'Yersinia pseudotuberculosis' strain IP32953 is a virulence-related metallo-oligopeptidase

Background: although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. Results: in this study we found that oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. Conclusions: the results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity

Co-expression and prognostic significance of putative CSC markers CD44, CD133, wild-type EGFR and EGFRvIII in metastatic colorectal cancer

The presence of colorectal cancer stem cells (CSCs) have been associated with tumour initiation and resistance to therapy. This study investigated the co-expression and prognostic significance of the CSCs biomarkers CD44 and CD133 with wild-type EGFR (wtEGFR) and EGFRvIII in colorectal cancer (CRC). The expression of these biomarkers were determined in tumours from 70 patients with metastatic CRC by immunohistochemistry, and in a panel of human CRC cell lines, and their variants with acquired-resistance to EGFR inhibitors, by flow cytometry. The expression of CD44, CD133, wtEGFR and EGFRvIII were present in 17%, 23%, 26% and 13% of cases and the co-expression of CD44/CD133 with wtEGFR and EGFRvIII were present in 9% and 3% of the cases respectively. Only co-expression of CSCs/EGFRvIII (P = 0.037), and amphiregulin (P = 0.017) were associated with worse overall survival. Interestingly, disease-free survival was improved in BTC expressing patients (P = 0.025). In vitro CD133 expression and its co-expression with CD44 were associated with primary-resistance to irinotecan and acquired-resistance to anti-EGFR inhibitors respectively. Our results suggest co-expression of CSCs and EGFRvIII could be potential biomarkers of worse overall survival and resistance to therapy in patients with mCRC and warrants further validation in a larger cohort

HER2 expression is predictive of survival in cetuximab treated patients with RAS wild type metastatic colorectal cancer

The overexpressed HER2 is an important target for treatment with monoclonal antibody (mAb) trastuzumab, only in patients with breast and gastric cancers, and is an emerging therapeutic biomarker in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) mAbs cetuximab and panitumumab. In this study, we investigated the relative expression and predictive value of all human epidermal growth factor receptor (HER) family members in 144 cetuximab-treated patients with wild type RAS mCRC. The relative expression of EGFR and HER2 have also been examined in 21-paired primary tumours and their metastatic sites by immunohistochemistry. Of the 144 cases examined, 25%, 97%, 79%, 48%, and 10% were positive for EGFR, HER2, HER3, and HER4 and all four HER family members, respectively. The expression of EGFR was an indicator of poorer overall survival and the membranous expression of HER2 and HER3 3+ intensity was associated with a shorter progression free survival (PFS). In contrast, the cytoplasmic expression of HER2 was associated with better PFS. In 48% and 71% of the cases, there were discordance in the expression of EGFR or one or more HER family members in paired primary and related metastatic tumours, respectively. Our results implicate the importance of a large prospective investigation of the expression level and predictive value of not only the therapeutic target (i.e., EGFR protein) but also HER2 and other HER family members as therapeutic targets, or for response to therapy with anti-EGFR mAbs and other forms of HER inhibitors, in both the primary tumours and metastatic sites in mCRC

Implementation of U.K. Earth system models for CMIP6

We describe the scientific and technical implementation of two models for a core set of experiments contributing to the sixth phase of the Coupled Model Intercomparison Project (CMIP6). The models used are the physical atmosphere-land-ocean-sea ice model HadGEM3-GC3.1 and the Earth system model UKESM1 which adds a carbon-nitrogen cycle and atmospheric chemistry to HadGEM3-GC3.1. The model results are constrained by the external boundary conditions (forcing data) and initial conditions.We outline the scientific rationale and assumptions made in specifying these. Notable details of the implementation include an ozone redistribution scheme for prescribed ozone simulations (HadGEM3-GC3.1) to avoid inconsistencies with the model's thermal tropopause, and land use change in dynamic vegetation simulations (UKESM1) whose influence will be subject to potential biases in the simulation of background natural vegetation.We discuss the implications of these decisions for interpretation of the simulation results. These simulations are expensive in terms of human and CPU resources and will underpin many further experiments; we describe some of the technical steps taken to ensure their scientific robustness and reproducibility

CmeABC multidrug efflux pump contributes to antibiotic resistance and promotes 'Campylobacter jejuni' survival and multiplication in 'Acanthamoeba polyphaga'

Campylobacter jejuni is a foodborne pathogen recognized as the leading cause of human bacterial gastroenteritis. The wide use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter In addition to a role in multidrug resistance, the Campylobacter CmeABC RND-type efflux pump, which is associated with multidrug resistance (MDR), may also be involved in virulence. As a vehicle of pathogenic microorganisms, the protozoan Acanthamoeba is a good model for the investigation of bacterial survival in the environment and molecular mechanisms of pathogenicity. The interaction between C. jejuni 81-176 and A. polyphaga was investigated in this study by using a modified gentamicin protection assay. In addition, a possible role for the CmeABC MDR pump in this interaction was explored. Here we report that this MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni in A. polyphaga, but is dispensable for biofilm formation and motility.Importance The endosymbiotic relationship between amoebae and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications to human health. In this study we found that Campylobacter jejuni was able to survive and multiply inside Acanthamoeba. polyphaga Since these microorganisms can co-exist in the same environment (e.g. in poultry farms), the latter may increase the risk of infection with Campylobacter Our data suggests that, in addition to its role in antibiotic resistance, the CmeABC MDR efflux pump also plays a role in bacterial survival within amoebae. Furthermore, we demonstrated a synergistic effect of the CmeABC MDR efflux pump and TetO on bacterial resistance to tetracycline. Due to its role both in antibiotic resistance and virulence of C. jejuni, the CmeABC MDR efflux pump could be considered as a good target for the development of antibacterial drugs against this pathogen

Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of the HER inhibitors and cytotoxic drugs

Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer stem cell (CSC) markers (CD24, CD44, CD117/c-Kit), P-glycoprotein (P-gp), and HER family members and response to treatment with these agents. The sensitivity of 10 ovarian tumour cell lines to the treatment with various forms of HER TKIs (gefitinib, erlotinib, lapatinib, sapitinib, afatinib, canertinib, neratinib), as well as other TKIs (dasatinib, imatinib, NVP-AEW541, crizotinib) and cytotoxic agents (paclitaxel, cisplatin and doxorubicin), as single agents or in combination, was determined by SRB assay. The effect on these agents on the cell cycle distribution, and downstream signaling molecules and tumour migration were determined using flow cytometry, western blotting, and the IncuCyte Clear View cell migration assay respectively. Of the HER inhibitors, the irreversible pan-TKIs (canertinib, neratinib and afatinib) were the most effective TKIs for inhibiting the growth of all ovarian cancer cells, and for blocking the phosphorylation of EGFR, HER-2, AKT and MAPK in SKOV3 cells. Interestingly, while the majority of cancer cells were highly sensitive to treatment with dasatinib, they were relatively resistant to treatment with imatinib (i.e., IC50 >10 µM). Of the cytotoxic agents, paclitaxel was the most effective for inhibiting the growth of OCCLs, and of various combinations of these drugs, only treatment with a combination of NVP-AEW541 and paclitaxel produced a synergistic or additive anti-proliferative effect in all three cell lines examined (i.e., SKOV3, Caov3, ES2). Finally, of the TKIs, only treatment with afatinib, neratinib and dasatinib were able to reduce the migration of HER-2 overexpressing SKOV3 cells. We did not find any significant association between the expression of putative ovarian CSC marker, HER family members, c-MET, ALK, and IGF-IR and the response to the irreversible HER TKIs. Our results support the need for further investigations of the therapeutic potential of these irreversible HER family blockers in ovarian cancer, and the therapeutic potential of dasatinib when used in combination with the inhibitors of the HER family members in ovarian cancer

Observed controls on resilience of groundwater to climate variability in sub-Saharan Africa

Groundwater in sub-Saharan Africa supports livelihoods and poverty alleviation1,2, maintains vital ecosystems, and strongly influences terrestrial water and energy budgets. Yet the hydrological processes that govern groundwater recharge and sustainability—and their sensitivity to climatic variability—are poorly constrained4. Given the absence of firm observational constraints, it remains to be seen whether model-based projections of decreased water resources in dry parts of the region4 are justified. Here we show, through analysis of multidecadal groundwater hydrographs across sub-Saharan Africa, that levels of aridity dictate the predominant recharge processes, whereas local hydrogeology influences the type and sensitivity of precipitation–recharge relationships. Recharge in some humid locations varies by as little as five per cent (by coefficient of variation) across a wide range of annual precipitation values. Other regions, by contrast, show roughly linear precipitation–recharge relationships, with precipitation thresholds (of roughly ten millimetres or less per day) governing the initiation of recharge. These thresholds tend to rise as aridity increases, and recharge in drylands is more episodic and increasingly dominated by focused recharge through losses from ephemeral overland flows. Extreme annual recharge is commonly associated with intense rainfall and flooding events, themselves often driven by large-scale climate controls. Intense precipitation, even during years of lower overall precipitation, produces some of the largest years of recharge in some dry subtropical locations. Our results therefore challenge the ‘high certainty’ consensus regarding decreasing water resources in such regions of sub-Saharan Africa. The potential resilience of groundwater to climate variability in many areas that is revealed by these precipitation–recharge relationships is essential for informing reliable predictions of climate-change impacts and adaptation strategies

Acquired resistance to anti-EGFR mAb ICR62 in cancer cells is accompanied by an increased EGFR expression, HER-2/HER-3 signalling and sensitivity to pan HER blockers

Our results provide a novel mechanistic insight into the development of acquired resistance to EGFR antibody-based therapy in colorectal cancer cells and justify further investigations on the therapeutic benefits of pan-HER family inhibitors in the treatment of colorectal cancer patients once acquired resistance to EGFR antibody-based therapy is developed