Biological characterisation of a novel and naturally isolated indole alkaloid

Natural products play a pivotal role in the treatment of cancer; identification of compounds such as taxanes and the vinca alkaloids were seminal landmarks in natural product drug discovery. Jerantinine A (JA), a novel Aspidosperma alkaloid isolated from plant species Tabernaemontana corymbosa, was previously reported to possess cytotoxic activity against vincristine-resistant nasopharyngeal carcinoma cells and is therefore an ideal candidate for biological investigation. Furthermore, Tabernaemontana corymbosa has been placed in the endangered list of threatened species by the International Union for Conservation of Nature (IUCN) thus making it a priority to elucidate the biological activity of this alkaloid. Herein, we report detailed biological evaluation of JA on various human-derived carcinoma cell lines. Our preliminary screens showed that significant inhibition of cell growth and colony formation accompanied time- and dose-dependent induction of apoptosis in human cancer cell lines after treatment with JA. Dose-dependent accumulations of cleaved PARP and caspase 3 further confirmed apoptosis. Profound G2/M cell cycle arrest was observed 24 h after treatment in all cell lines. Characteristics of mitotic arrest including inhibition of tubulin polymerisation, microtubule disruption, and aneuploidy were clearly observed. DNA fragmentation was also evident in cells treated with JA. Indeed, significant increases in phosphorylated-γH2AX were indicative of DNA damage caused by double strand breaks and were relatively similar to levels caused by vincristine. Investigations into JA’s ability to overcome vincristine resistance demonstrated that it is not a substrate of Pgp. The role of reactive oxygen species (ROS) in acquired resistance and cell death have also been widely studied. JA induced significant levels of ROS in treated cells, possibly contributing to their apoptotic destiny. Proteomic analyses also corroborated the phenotype of JA-treated cells with increased expression of ROS-neutralising enzymes, aberrant expression of proteins involved in the spindle assembly checkpoint critical to mitosis, and decreased expression in all tubulin proteins detected by LC-MS/MS. A genome-wide RNAi screen revealed several candidate genes involved in mediating sensitivity to JA. The genes corresponding to c-Jun-N-terminal kinases, JNK1/2, were selected for subsequent investigation based on their involvement in multiple pathways that were identified using bioinformatic tools. JNK1/2 were knocked down in MCF-7 and MDA-468 cells and then treated with JA. MTT assays revealed some loss of sensitivity, suggesting that these proteins were indeed involved in mediating cell sensitivity to JA

Biological characterisation of a novel and naturally isolated indole alkaloid

Natural products play a pivotal role in the treatment of cancer; identification of compounds such as taxanes and the vinca alkaloids were seminal landmarks in natural product drug discovery. Jerantinine A (JA), a novel Aspidosperma alkaloid isolated from plant species Tabernaemontana corymbosa, was previously reported to possess cytotoxic activity against vincristine-resistant nasopharyngeal carcinoma cells and is therefore an ideal candidate for biological investigation. Furthermore, Tabernaemontana corymbosa has been placed in the endangered list of threatened species by the International Union for Conservation of Nature (IUCN) thus making it a priority to elucidate the biological activity of this alkaloid. Herein, we report detailed biological evaluation of JA on various human-derived carcinoma cell lines. Our preliminary screens showed that significant inhibition of cell growth and colony formation accompanied time- and dose-dependent induction of apoptosis in human cancer cell lines after treatment with JA. Dose-dependent accumulations of cleaved PARP and caspase 3 further confirmed apoptosis. Profound G2/M cell cycle arrest was observed 24 h after treatment in all cell lines. Characteristics of mitotic arrest including inhibition of tubulin polymerisation, microtubule disruption, and aneuploidy were clearly observed. DNA fragmentation was also evident in cells treated with JA. Indeed, significant increases in phosphorylated-γH2AX were indicative of DNA damage caused by double strand breaks and were relatively similar to levels caused by vincristine. Investigations into JA’s ability to overcome vincristine resistance demonstrated that it is not a substrate of Pgp. The role of reactive oxygen species (ROS) in acquired resistance and cell death have also been widely studied. JA induced significant levels of ROS in treated cells, possibly contributing to their apoptotic destiny. Proteomic analyses also corroborated the phenotype of JA-treated cells with increased expression of ROS-neutralising enzymes, aberrant expression of proteins involved in the spindle assembly checkpoint critical to mitosis, and decreased expression in all tubulin proteins detected by LC-MS/MS. A genome-wide RNAi screen revealed several candidate genes involved in mediating sensitivity to JA. The genes corresponding to c-Jun-N-terminal kinases, JNK1/2, were selected for subsequent investigation based on their involvement in multiple pathways that were identified using bioinformatic tools. JNK1/2 were knocked down in MCF-7 and MDA-468 cells and then treated with JA. MTT assays revealed some loss of sensitivity, suggesting that these proteins were indeed involved in mediating cell sensitivity to JA

Microstructure and mechanical characterization of aa6061/tic in situ aluminium matrix composites synthesized by in situ reaction of silicon carbide and potassium fluotitanate

Abstract: In situ method of synthesizing aluminum matrix composites (AMCs) has been widely recognized and followed by researchers due to numerous merits over conventional stir casting. Aluminum alloy AA6061 reinforced with various amounts (0, 2.5 and 5 wt. %) of TiC particles were synthesized by the in situ reaction of inorganic salt K2TiF6 and ceramic particle SiC with molten aluminum. The casting was carried out at an elevated temperature and held for a longer duration to decompose SiC to release carbon atoms. X-ray diffraction patterns of the prepared AMCs clearly revealed the formation of TiC particles without the occurrence of any other intermetallic compounds. The microstructure of the prepared AA6061/TiC AMCs was studied using field emission scanning electron microscope

In vitro anticancer properties and biological evaluation of novel natural alkaloid jerantinine B

Natural products play a pivotal role in medicine especially in the cancer arena. Many drugs that are currently used in cancer chemotherapy originated from or were inspired by nature. Jerantinine B (JB) is one of seven novel Aspidosperma indole alkaloids isolated from the leaf extract of Tabernaemontana corymbosa. Preliminary antiproliferative assays revealed that JB and JB acetate significantly inhibited growth and colony formation, accompanied by time- and dose-dependent apoptosis induction in human cancer cell lines. JB significantly arrested cells at the G2/M cell cycle phase, potently inhibiting tubulin polymerisation. Polo-like kinase 1 (PLK1; an early trigger for the G2/M transition) was also dose-dependently inhibited by JB (IC50 1.5 µM). Furthermore, JB provoked significant increases in reactive oxygen species (ROS). Annexin V+ cell populations, dose-dependent accumulation of cleaved-PARP and caspase 3/7 activation, and reduced Bcl-2 and Mcl-1 expression confirm apoptosis induction. Preclinical in silico biopharmaceutical assessment of JB calculated rapid absorption and bioavailability >70%. Doses of 8–16 mg/kg JB were predicted to maintain unbound plasma concentrations >GI50 values in mice during efficacy studies. These findings advocate continued development of JB as a potential chemotherapeutic agent

Sphingosine kinase 1 regulates the survival of breast cancer stem cells and non-stem breast cancer cells by suppression of STAT1

Cancer stem cells (CSCs) represent rare tumour cell populations capable of self-renewal, differentiation and tumour initiation, and are highly resistant to chemotherapy and radiotherapy. Thus, therapeutic approaches which can effectively target CSCs and tumour cells could be the key towards efficient tumour treatment. In this study, we explored the function of SPHK1 in breast CSCs and non-CSCs. We showed that RNAi-mediated knockdown of SPHK1 inhibits cell proliferation and induces apoptosis in both breast CSCs and non-CSCs, while ectopic expression of SPHK1 enhances breast CSC survival and mammosphere forming efficiency. We identified STAT1 and IFN signalling as key regulatory targets of SPHK1 and demonstrated that an important mechanism by which SPHK1 promotes cancer cell survival is through the suppression of STAT1. We further demonstrate that SPHK1 inhibitors, FTY720 and PF543 synergized with doxorubicin in targeting both breast CSCs and non-CSCs. In conclusion, we provide important evidence that SPHK1 is a key regulator of cell survival and proliferation in breast CSCs and non-CSCs and is an attractive target for the design of future therapies

The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU-NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces

Silencing Nociceptor Neurons Reduces Allergic Airway Inflammation

Lung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8(+) sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4(+) and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma

Functional implications of glycans and their curation:insights from the workshop held at the 16th Annual International Biocuration Conference in Padua, Italy

Dynamic changes in protein glycosylation impact human health and disease progression. However, current resources that capture disease and phenotype information focus primarily on the macromolecules within the central dogma of molecular biology (DNA, RNA, proteins). To gain a better understanding of organisms, there is a need to capture the functional impact of glycans and glycosylation on biological processes. A workshop titled "Functional impact of glycans and their curation" was held in conjunction with the 16th Annual International Biocuration Conference to discuss ongoing worldwide activities related to glycan function curation. This workshop brought together subject matter experts, tool developers, and biocurators from over 20 projects and bioinformatics resources. Participants discussed four key topics for each of their resources: (i) how they curate glycan function-related data from publications and other sources, (ii) what type of data they would like to acquire, (iii) what data they currently have, and (iv) what standards they use. Their answers contributed input that provided a comprehensive overview of state-of-the-art glycan function curation and annotations. This report summarizes the outcome of discussions, including potential solutions and areas where curators, data wranglers, and text mining experts can collaborate to address current gaps in glycan and glycosylation annotations, leveraging each other's work to improve their respective resources and encourage impactful data sharing among resources. Database URL: https://wiki.glygen.org/Glycan_Function_Workshop_2023

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care