Molecular Mechanisms of Natural Compounds : Compound Kushen Injection (CKI) in Cancer

Chemotherapy is a treatment that uses cytotoxic drugs to kill rapidly dividing cancer cells. There are many anti-cancer chemotherapeutic drugs used alone or in combination with others to kill cancerous cells, and some of these, are of plant origin. Naturally occurring compounds, such as Taxol, are used in chemotherapy and have very specific, unique, molecular targets. However, according to the World Health Organization (Ekor, 2014), approximately eighty percent of the world’s population depends on natural compounds from traditional medicine and these compounds are widely used in complementary medicine as anti-cancer drugs (Foster et al., 2000). Traditional Chinese medicine (TCM) uses treatments that contain multiple natural compounds, a number of which have been claimed to be of therapeutic benefit to cancer sufferers (Chung et al., 2015). Some TCM preparations have shown anti-cancer, anti-migratory and anti-metastatic properties in laboratory settings (Wang et al., 2009;Pan et al., 2011;Qu et al., 2016). Research suggests that TCM natural compound mixtures might synergistically trigger therapeutic benefits through the action of multiple components affecting multiple regulatory signaling targets (Wang et al., 2008). Compound Kushen injection (CKI) is a TCM anticancer agent which has been approved by the Chinese State Food and Drug Administration to treat solid tumors in combination with chemotherapy drugs in clinics for pain relief, cancer metastasis and enhancement of the immune system since 1995, and is used to treat approximately 30,000 patients daily. Although a large body of evidence has suggested CKI has anti-cancer properties (Xu et al., 2011;Gao et al., 2018) the anti-cancer mechanisms attributable to specific compounds within the mixture remain unknown. CKI contains multiple alkaloid and flavonoid compounds and the main bioactive compounds such as matrine and oxymatrine have shown to affect cancer cells in the lab. However, other medicinal herbs containing these two compounds as main components have demonstrated patient toxicity. It is therefore important to better understand the effects of CKI, particularly with respect the contributions of individual compounds within the mixture. In this thesis, I describe a multi-disciplinary approach including analytical chemistry, cellular assays and transcriptome analysis to explore the effects of several major compounds present in CKI. Through the application of a subtractive fractionation method that removed individual compounds one, two or three at a time, I have been able to map these compounds and their interactions to specific pathways based on altered gene expression profiles. This has illuminated the roles of several major compounds of CKI, that on their own, have no, or minimal, activity in our bioassay. This approach has enabled us to identify the interactions between compounds in a mixture as shown by the response of cancer cell cultures. Using a systems biology approach along with cellular migration and invasion assays, I have mapped the activity of related proteins and pathways which may contribute to the migrastatic activity of CKI. Altogether, this thesis presents an initial characterization of the underlying mechanistic changes induced by CKI. First, by comparing differentially expressed genes across treatment combinations generated using our subtractive fractionation approach, I identified specific candidate pathways that were altered by the removal of compounds from the mixture. Second, by using transcriptome data of a breast cancer cell line, the effects of CKI on candidate anti-migratory pathways for six different cancer cell lines were assessed. These experiments identified specific candidate target pathways through which CKI might act. These approaches can be used to understand the roles and interactions of individual compounds from any complex natural compound mixture whose biological activity cannot be associated with purified compounds.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 201

Participatory Visioning and Future Planning. Backcasting with Myanmar Farmers for a more Sustainable Future. Methodological Report

This report describes the methodology of participatory visioning and future planning, including a technique called backcasting. Based on literature and experience, we developed and applied this methodology to support smallholder farmers in southern Myanmar. In a highly participatory bottom-up approach, we co-created and documented the vision of these farmers – in direct collaboration with them – including their desirable futures, and we jointly explored possible pathways and action plans to reach these futures. We also co-implemented needs-based actions including concrete trainings, study trips, and a community-led micro-loan system to strengthen their agriculture – their main source of livelihood. Unfortunately, beginning in 2020, Myanmar experienced two overlapping waves of crisis: the health crisis as a result of the COVID-19 pandemic; and the political crisis following the coup d’etat in 2021. The final two years of the project were implemented under these extremely challenging conditions. We frequently had to adapt our operationalization to the very dynamic, changing circum-stances. Fortunately, our methodology allowed for this kind of adaptive management. Despite the challenging circumstances, the farmers were positive in their assessment of the project outcomes. This suggests that the methodology of participatory visioning and future planning can be effective even under difficult circumstances. We conclude the report by presenting lessons learnt and recommendations. Firstly, we reflect on how useful this methodology was and how it can be applied in similar or different projects (research, development, etc.). Secondly, we share our practical insights and recommendations regarding the application of the methodology in the given or similar contexts. Finally, we provide practical recommendations for project planning for those who want to include participatory visioning and future planning in their projects

Objective assessment of tumor infiltrating lymphocytes as a prognostic marker in melanoma using machine learning algorithms

BACKGROUND The prognostic value of tumor-infiltrating lymphocytes (TILs) assessed by machine learning algorithms in melanoma patients has been previously demonstrated but has not been widely adopted in the clinic. We evaluated the prognostic value of objective automated electronic TILs (eTILs) quantification to define a subset of melanoma patients with a low risk of relapse after surgical treatment. METHODS We analyzed data for 785 patients from 5 independent cohorts from multiple institutions to validate our previous finding that automated TIL score is prognostic in clinically-localized primary melanoma patients. Using serial tissue sections of the Yale TMA-76 melanoma cohort, both immunofluorescence and Hematoxylin-and-Eosin (H&E) staining were performed to understand the molecular characteristics of each TIL phenotype and their associations with survival outcomes. FINDINGS Five previously-described TIL variables were each significantly associated with overall survival (p<0.0001). Assessing the receiver operating characteristic (ROC) curves by comparing the clinical impact of two models suggests that etTILs (electronic total TILs) (AUC: 0.793, specificity: 0.627, sensitivity: 0.938) outperformed eTILs (AUC: 0.77, specificity: 0.51, sensitivity: 0.938). We also found that the specific molecular subtype of cells representing TILs includes predominantly cells that are CD3+ and CD8+ or CD4+ T cells. INTERPRETATION eTIL% and etTILs scores are robust prognostic markers in patients with primary melanoma and may identify a subgroup of stage II patients at high risk of recurrence who may benefit from adjuvant therapy. We also show the molecular correlates behind these scores. Our data support the need for prospective testing of this algorithm in a clinical trial. FUNDING This work was also supported by a sponsored research agreements from Navigate Biopharma and NextCure and by grants from the NIH including the Yale SPORE in in Skin Cancer, P50 CA121974, the Yale SPORE in Lung Cancer, P50 CA196530, NYU SPORE in Skin Cancer P50CA225450 and the Yale Cancer Center Support Grant, P30CA016359

Preference in learning gross anatomy among IIUM students

Both medical (Year I, Year II) and dental (Year I) students in IIUM are exposed to three types of teaching aids in learning gross anatomy. They are cadavers, prosected wet specimens and plastic models. This study aimed at exploring the students’ preference on teaching aids in learning gross anatomy and reasons for their preference. A cross-sectional comparative study was carried out among 185 medical and dental students by using the pretested, semi structured, self administrated questionnaires including open-ended questions. Significance of preferences were analysed by X2 test. Year I (99%) and Year II (97%) medical students preferred the plastic models as the best approach to learn gross anatomy because of their handleability and portability. Year I dental students (96%) preferred the prosected wet specimens because they were real human structures and well preserved. The preferred and less preferred rates were 86% and 4% for plastic model, 84% and 10% for prosected wet specimen and 77% and 17% for cadaver. These differences were statistically significant (p <0.05). Students didn’t prefer the cadavers most but they agreed that the cadavers are more realistic, informative and easier to remember. This study indicates that students prefer all three types of teaching aids while the most preferred one is the plastic model. The quality of teaching aid is the reason for their preference. Students’ suggestion to use the advanced technologies such as three dimensional animations or simulated videos should be considered to get discernable learning outcomes

Sustainable Development Under Competing Claims on Land: Three Pathways Between Land-Use Changes, Ecosystem Services and Human Well-Being

Competition over land is at the core of many sustainable development challenges in Myanmar: villagers, companies, governments, ethnic minority groups, civil society organisations and non-governmental organisations from local to the international level claim access to and decision-making power over the use of land. Therefore, this article investigates the actor interactions influencing land-use changes and their impacts on the supply of ecosystem services and human well-being. We utilise a transdisciplinary mixed-methods approach and the analytical lens of the social-ecological systems framework. Results reveal that the links between land-use changes, ecosystem services and human well-being are multifaceted; For example ecosystem services can decline, while human well-being increases. We explain this finding through three different pathways to impact (changes in the resource systems, the governance systems or the broader social, economic and political context). We conclude with implications of these results for future sustainable land governance

Survival analysis of hospitalized COVID-19 patients in New Yangon General Hospital by prognostic indicators

Background: Coronavirus disease (COVID-19) is an infectious disease of the SARS-CoV-2 virus and it can infect anyone resulting in serious illness and death. Methods: A retrospective cohort study was conducted in New Yangon General Hospital (NYGH), Yangon between September and November, 2022. Previous records of COVID-19 in-patients admitted to NYGH from 1st June to 31st October, 2021, were reviewed. COVID-19 in-patients who tested positive by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) or rapid diagnostic test (RDT) were included in this study. Multivariate analysis by Cox proportional-hazards (CPH) model was used to identify the prognostic indicators associated with the survival of COVID-19 inpatients. Results: Of (460) COVID-19 positive patients, there were 133 (28.9%) deaths with mortality rate of 16.9 per 1000 person-days. Then, 97 (72.9%) deaths occurred within 21 days of symptom onset, with median survival time of 28 (95% CI: 25-36) days. The results of the CPH model showed that the abnormal chest X-ray (CXR) [aHR=3.8, 95% CI: (1.1, 12.6), p=0.032], SpO2 level below 92% [aHR=3.7, 95% CI: 2.3, 5.9, p&lt;0.001)], serum creatinine level more than 133 µmol/L [aHR=1.9, 95% CI: 1.1, 3.2, p=0.025] and C-reactive protein level (CRP) more than 10 mg/L [aHR=3.9, 95% CI: 1.2, 12.9, p=0.027] were the prognostic indicators of COVID-19 death among inpatients in NYGH. Conclusions: Patients with abnormal CXR result, low SpO2 level, high serum creatinine level, and high CRP level may have increased risks of death among COVID-19 inpatients in NYGH. Thus, close monitoring of the hospitalized COVID-19 inpatients by using these prognostic indicators should be emphasized

Virulence factors and genetic characteristics of methicillin-resistant and - susceptible staphylococcus aureus isolates in Myanmar

Staphylococcus aureus produces virulence factors, including various exotoxins and adhesins, which are associated with a variety of symptoms caused by its infections. In the present study, the prevalence of these virulence factors was analyzed for 23 S. aureus strains isolated from wound infections in hospitals, nasal swabs, or vomit from patients and cooks in a food poisoning case and from healthy adults in Yangon, Myanmar. Among these strains, five were methicillin-resistant S. aureus (MRSA) derived from pus (four strains, SCCmec III, ST239) and a healthy adult (one strain, SCCmec-IVa, ST5). The Panton-Valentine leukocidine (PVL) gene was detected in five methicillin-susceptible S. aureus (MSSA) clinical strains belonging to ST121 (CC121). The MRSA clinical strains had only a few or no staphylococcal enterotoxin (SE) genes, whereas PVL-positive MSSA and an MRSA strain from a healthy adult possessed an enterotoxin gene cluster (seg, sei, sem, sen, seo, and selu). Strains from the food poisoning case had either SE genes or only etd and edin-B. Adhesin genes, which are associated with binding to fibronectin, fibrinogen, and elastin, were detected in all the MRSA and most of the MSSA strains examined. However, the bone sialoprotein-binding protein gene (bbp) and the variant form of the elastin-binding protein gene (ebpS-v) with an internal 180 bp deletion were identified only in the MSSA strains harboring the PVL gene. These findings suggest that those genetic traits are characteristic of PVL-positive ST121 S. aureus strains in Myanmar

Sustainable Development Under Competing Claims on Land: Three Pathways Between Land-Use Changes, Ecosystem Services and Human Well-Being

Competition over land is at the core of many sustainable development challenges in Myanmar: villagers, companies, governments, ethnic minority groups, civil soci-ety organisations and non-governmental organisations from local to the international level claim access to and decision-making power over the use of land. Therefore, this article investigates the actor interactions influencing land-use changes and their impacts on the supply of ecosystem services and human well-being. We utilise a transdisciplinary mixed-methods approach and the analytical lens of the social-eco-logical systems framework. Results reveal that the links between land-use changes, ecosystem services and human well-being are multifaceted; For example ecosys-tem services can decline, while human well-being increases. We explain this find-ing through three different pathways to impact (changes in the resource systems, the governance systems or the broader social, economic and political context). We con-clude with implications of these results for future sustainable land governance

Identification of candidate anti-cancer molecular mechanisms of compound kushen injection using functional genomics

Compound Kushen Injection (CKI) has been clinically used in China for over 15 years to treat various types of solid tumours. However, because such Traditional Chinese Medicine (TCM) preparations are complex mixtures of plant secondary metabolites, it is essential to explore their underlying molecular mechanisms in a systematic fashion. We have used the MCF-7 human breast cancer cell line as an initial in vitro model to identify CKI induced changes in gene expression. Cells were treated with CKI for 24 and 48 hours at two concentrations (1 and 2 mg/mL total alkaloids), and the effect of CKI on cell proliferation and apoptosis were measured using XTT and Annexin V/Propidium Iodide staining assays respectively. Transcriptome data of cells treated with CKI or 5-Fluorouracil (5-FU) for 24 and 48 hours were subsequently acquired using high-throughput Illumina RNA-seq technology. In this report we show that CKI inhibited MCF-7 cell proliferation and induced apoptosis in a dose-dependent fashion. We integrated and applied a series of transcriptome analysis methods, including gene differential expression analysis, pathway over-representation analysis, de novo identification of long non-coding RNAs (lncRNA) as well as co-expression network reconstruction, to identify candidate anti-cancer molecular mechanisms of CKI. Multiple pathways were perturbed and the cell cycle was identified as the potential primary target pathway of CKI in MCF-7 cells. CKI may also induce apoptosis in MCF-7 cells via a p53 independent mechanism. In addition, we identified novel lncRNAs and showed that many of them might be expressed as a response to CKI treatment.Zhipeng Qu, Jian Cui, Yuka Harata-Lee, Thazin Nwe Aung, Qianjin Feng, Joy M. Raison, Robert Daniel Kortschak, David L. Adelso