Advancing characterisation with statistics from correlative electron diffraction and X-ray spectroscopy, in the scanning electron microscope.

The routine and unique determination of minor phases in microstructures is critical to materials science. In metallurgy alone, applications include alloy and process development and the understanding of degradation in service. We develop a correlative method, exploring superalloy microstructures, which are examined in the scanning electron microscope (SEM) using simultaneous energy dispersive X-ray spectroscopy (EDS) and electron backscatter diffraction (EBSD). This is performed at an appropriate length scale for characterisation of carbide phases' shape, size, location, and distribution. EDS and EBSD data are generated using two different physical processes, but each provide a signature of the material interacting with the incoming electron beam. Recent advances in post-processing, driven by 'big data' approaches, include use of principal component analysis (PCA). Components are subsequently characterised to assign labels to a mapped region. To provide physically meaningful signals, the principal components may be rotated to control the distribution of variance. In this work, we develop this method further through a weighted PCA approach. We use the EDS and EBSD signals concurrently, thereby labelling each region using both EDS (chemistry) and EBSD (crystal structure) information. This provides a new method of amplifying signal-to-noise for very small phases in mapped regions, especially where the EDS or EBSD signal is not unique enough alone for classification

Correlative statistical microstructural assessment of precipitates and their distribution, with simultaneous electron backscatter diffraction and energy dispersive X-ray spectroscopy

Modern engineering alloys have bespoke microstructures, where features such as precipitates are used to control properties. In many Ni-based alloys, carbo-nitride precipitates are introduced to strengthen and improve performance. These precipitates can be distributed throughout the microstructure and niobium rich carbides are often found at grain boundaries. In this work, we used combined energy dispersive X-ray spectroscopy (EDS) and electron backscatter diffraction (EBSD) to characterise the population of these precipitates. Processing of the EDS signal is used to label the Mo/Nb-rich precipitates, and their size and location are measured from maps using a circular Hough transform. This label map is combined with the grain boundary network (from EBSD analysis). Statistical analysis, using ANOVA testing, reveals differences in chemistry between carbides found in Ni-rich matrix grain interiors, on random high angle boundaries and on special boundaries (Σ3 and Σ9). These results are compared between wrought and power metallurgy product forms. These distributions are discussed in the context of their performance within demanding environments, such as reactor core internals

Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms

Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3′-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability

Antiplasmodial and trypanocidal activity of violacein and deoxyviolacein produced from synthetic operons.

BACKGROUND: Violacein is a deep violet compound that is produced by a number of bacterial species. It is synthesized from tryptophan by a pathway that involves the sequential action of 5 different enzymes (encoded by genes vioA to vioE). Violacein has antibacterial, antiparasitic, and antiviral activities, and also has the potential of inducing apoptosis in certain cancer cells. RESULTS: Here, we describe the construction of a series of plasmids harboring the complete or partial violacein biosynthesis operon and their use to enable production of violacein and deoxyviolacein in E.coli. We performed in vitro assays to determine the biological activity of these compounds against Plasmodium, Trypanosoma, and mammalian cells. We found that, while deoxyviolacein has a lower activity against parasites than violacein, its toxicity to mammalian cells is insignificant compared to that of violacein. CONCLUSIONS: We constructed E. coli strains capable of producing biologically active violacein and related compounds, and propose that deoxyviolacein might be a useful starting compound for the development of antiparasite drugs

A 'synthetic-sickness' screen for senescence re-engagement targets in mutant cancer backgrounds.

Senescence is a universal barrier to immortalisation and tumorigenesis. As such, interest in the use of senescence-induction in a therapeutic context has been gaining momentum in the past few years; however, senescence and immortalisation remain underserved areas for drug discovery owing to a lack of robust senescence inducing agents and an incomplete understanding of the signalling events underlying this complex process. In order to address this issue we undertook a large-scale morphological siRNA screen for inducers of senescence phenotypes in the human melanoma cell line A375P. Following rescreen and validation in a second cancer cell line, HCT116 colorectal carcinoma, a panel of 16 of the most robust hits were selected for further validation based on significance and the potential to be targeted by drug-like molecules. Using secondary assays for detection of senescence biomarkers p21, 53BP1 and senescence associated beta-galactosidase (SAβGal) in a panel of HCT116 cell lines carrying cancer-relevant mutations, we show that partial senescence phenotypes can be induced to varying degrees in a context dependent manner, even in the absence of p21 or p53 expression. However, proliferation arrest varied among genetic backgrounds with predominantly toxic effects in p21 null cells, while cells lacking PI3K mutation failed to arrest. Furthermore, we show that the oncogene ECT2 induces partial senescence phenotypes in all mutant backgrounds tested, demonstrating a dependence on activating KRASG13D for growth suppression and a complete senescence response. These results suggest a potential mechanism to target mutant KRAS signalling through ECT2 in cancers that are reliant on activating KRAS mutations and remain refractory to current treatments

Genomic instability in human cancer: molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology

Hrk1 Plays Both Hog1-Dependent and -Independent Roles in Controlling Stress Response and Antifungal Drug Resistance in Cryptococcus neoformans

The HOG (High Osmolarity Glycerol response) pathway plays a central role in controlling stress response, ergosterol biosynthesis, virulence factor production, and differentiation of Cryptococcus neoformans, which causes fatal fungal meningoencephalitis. Recent transcriptome analysis of the HOG pathway discovered a Hog1-regulated gene (CNAG_00130.2), encoding a putative protein kinase orthologous to Rck1/2 in Saccharomyces cerevisiae and Srk1 in Schizosaccharomyces pombe. Its function is not known in C. neoformans. The present study functionally characterized the role of Hrk1 in C. neoformans. Northern blot analysis confirmed that HRK1 expression depends on the Hog1 MAPK. Similar to the hog1Δ mutant, the hrk1Δ mutant exhibited almost complete resistance to fludioxonil, which triggers glycerol biosynthesis via the HOG pathway. Supporting this, the hrk1Δ mutant showed reduced intracellular glycerol accumulation and swollen cell morphology in response to fludioxonil, further suggesting that Hrk1 works downstream of the HOG pathway. However, Hrk1 also appeared to have Hog1-independent functions. Mutation of HRK1 not only further increased osmosensitivity of the hog1Δ mutant, but also suppressed increased azole-resistance of the hog1Δ mutant in an Erg11-independent manner. Furthermore, unlike the hog1Δ mutant, Hrk1 was not involved in capsule biosynthesis. Hrk1 was slightly involved in melanin production but dispensable for virulence of C. neoformans. These findings suggest that Hrk1 plays both Hog1-dependent and –independent roles in stress and antifungal drug susceptibility and virulence factor production in C. neoformans. Particularly, the finding that inhibition of Hrk1 substantially increases azole drug susceptibility provides a novel strategy for combination antifungal therapy

Identification of potential inhibitors of casein kinase 2 alpha of Plasmodium falciparum with potent in vitro activity

Funding Information: The authors are incredibly grateful to Brazilian funding agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), CNPq, CAPES, Fundação de Apoio à Pesquisa do Estado de Goiás (FAPEG), and to the Swedish Research Council (grants 2016–05627 and 2021–03667). K.C.P.T. thanks FAPESP (grants 2018/05926–2, and 2019/17062–5). FAPESP funded T.A.T. (2019/27626-3), J.V.B.B. (2019/21854-4), K.B.M. and R.M.C. (2014/50897-0), V.M.A. (2022/00743-2), G.C.C. (2015/20774-6), and F.T.M.C. (grants 2017/18611–7 and 2018/07007–4). L.C.S.A. was funded by CNPq (162117/2018– 3). E.B. was supported by FAPESP (2015/03553-6 and 2018/07007–4) and CAPES (88887.304810/2018–00). J.V.B.B. and J.T.M.F. were supported by CAPES (Finance Code 001). C.H.A. and M.M. thanks FAPEG (grants 20171026700006 and 202010267000272). C.H.A. thanks the “L'Oréal-UNESCO-ABC Para Mulheres na Ciência” and “L’Oréal-UNESCO International Rising Talents” for the awards and fellowships received, which partially funded this work. C.H.A. and F.T.M.C. are CNPq research fellows. We are thankful to Liam B. King for his critical review of the report. Publisher Copyright: © 2023 American Society for Microbiology. All Rights Reserved.Drug resistance to commercially available antimalarials is a major obstacle in malaria control and elimination, creating the need to find new antiparasitic compounds with novel mechanisms of action. The success of kinase inhibitors for oncological treatments has paved the way for the exploitation of protein kinases as drug targets in various diseases, including malaria. Casein kinases are ubiquitous serine/threonine kinases involved in a wide range of cellular processes such as mitotic checkpoint signaling, DNA damage response, and circadian rhythm. In Plasmodium, it is suggested that these protein kinases are essential for both asexual and sexual blood-stage parasites, reinforcing their potential as targets for multi-stage antimalarials. To identify new putative PfCK2α inhibitors, we utilized an in silico chemogenomic strategy involving virtual screening with docking simulations and quantitative structure-activity relationship predictions. Our investigation resulted in the discovery of a new quinazoline molecule (542), which exhibited potent activity against asexual blood stages and a high selectivity index (>100). Subsequently, we conducted chemical-genetic interaction analysis on yeasts with mutations in casein kinases. Our chemical-genetic interaction results are consistent with the hypothesis that 542 inhibits yeast Cka1, which has a hinge region with high similarity to PfCK2α. This finding is in agreement with our in silico results suggesting that 542 inhibits PfCK2α via hinge region interaction.publishersversionpublishe

The HOG Pathway Dictates the Short-Term Translational Response after Hyperosmotic Shock

In the global osmoshock translational response in yeast, some gene products were translationally mobilized without transcriptional up-regulation. Conversely, other transcriptionally up-regulated mRNAs were translationally inhibited. Analogous changes occurred on the protein level. These translational responses were strongly dependent on Hog1 and Rck2

Tissue invasion and metastasis: Molecular, biological and clinical perspectives

Cancer is a key health issue across the world, causing substantial patient morbidity and mortality. Patient prognosis is tightly linked with metastatic dissemination of the disease to distant sites, with metastatic diseases accounting for a vast percentage of cancer patient mortality. While advances in this area have been made, the process of cancer metastasis and the factors governing cancer spread and establishment at secondary locations is still poorly understood. The current article summarizes recent progress in this area of research, both in the understanding of the underlying biological processes and in the therapeutic strategies for the management of metastasis. This review lists the disruption of E-cadherin and tight junctions, key signaling pathways, including urokinase type plasminogen activator (uPA), phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene (PI3K/AKT), focal adhesion kinase (FAK), β-catenin/zinc finger E-box binding homeobox 1 (ZEB-1) and transforming growth factor beta (TGF-β), together with inactivation of activator protein-1 (AP-1) and suppression of matrix metalloproteinase-9 (MMP-9) activity as key targets and the use of phytochemicals, or natural products, such as those from Agaricus blazei, Albatrellus confluens, Cordyceps militaris, Ganoderma lucidum, Poria cocos and Silybum marianum, together with diet derived fatty acids gamma linolenic acid (GLA) and eicosapentanoic acid (EPA) and inhibitory compounds as useful approaches to target tissue invasion and metastasis as well as other hallmark areas of cancer. Together, these strategies could represent new, inexpensive, low toxicity strategies to aid in the management of cancer metastasis as well as having holistic effects against other cancer hallmarks