Isolation, Characterization and Cytotoxicity of Phytochemicals from Sekobang Kechil (Anaxagorea Javanica)

Thirty-one plant extracts were obtained from the extract bank of Natural Product Laboratory, Institute of Bioscience, Universiti Putra Malaysia. The whole plant extracts were tested for cytotoxic effect on human breast cancer (MCF-7), prostate cancer (DU-145) and lung cancer (H-460) cell lines using MTT assay. The results of the preliminary cytotoxicity tests showed that eight extracts exhibited very strong activity against one or more of the cell lines at 100 μg ml-1, with cell viability of 10% or less. The methanolic extract of Anaxagorea javanica leaves exhibited the strongest activity against all three cell lines with cell viability of less than 2% and further dose-response tests against the MCF-7 cell line showed that it had an IC50 value of 2.4 μg ml-1. This sample was thus selected and recollected in larger quantities for further phytochemical investigation. The dichloromethane (DCM) fraction of the first collection was subjected to chromatographic purification from which a known flavonoid, 3',4',5-trihydroxy-3,7-dimethoxyflavone (41), was obtained. From the chromatographic separation of the DCM extract of the second collection batch, a mixture of long chain alkanes, predominated by nonacosane (42) and a mixture of stigmasterol (43) and β-sitosterol (40) were isolated, in addition to three pure phytochemicals, namely an aliphatic acid, hexadecanoic acid (44), and two alkaloids, 11-methoxyeupolauridine (45) and 4,11-dimethoxyeupolauridine (46). The latter two compounds were found to be new naphthyridine alkaloids with eupolauridine nuclei and reported for the first time for this species. Compounds that had been obtained in sufficient quantities were tested for cytotoxic activity against the MCF-7 cell line. The samples assayed were 3',4',5-trihydroxy-3,7-dimethoxyflavone (41), nonacosane (42), stigmasterol (43) and β-sitosterol (40) mixture, and 11-methoxyeupolauridine (45). Only 3',4',5-trihydroxy-3,7-dimethoxyflavone (41) showed cytotoxic effect with an IC50 value of 3.4 μM, and this was its first report for this activity. Plausible biogenetic pathways of the new compounds were also discussed

Cooperativity between MYC and other oncogenic factors : implications for tumorigenesis and targeting of MYC

Normal cell homeostasis in a tissue requires a delicate balance between cell growth, maintenance and death, tightly controlled by an intricate interplay between proto-oncogenes and tumor suppressor genes. When the balance is tipped due to genetic or epigenetic lesions in such genes, diseases such as cancer may arise. MYC transcription factors has been known to regulate up to 15% of mammalian genes involved in diverse intracellular programs, including cell cycle regulation, cell growth, differentiation, apoptosis, and senescence, and is deregulated in many human cancers. MYC recruits different co-factors for activation or repression of transcription, such as MAX or Miz- 1, respectively. MYC is tightly regulated at multiple levels, including transcription, posttranslational modification and turnover. Ubiquitylation is one such control, and although ubiquitylation is mainly associated with proteasomal degradation, it has also been shown to be involved in non-proteolytic functions such as DNA replication and repair. Tumorigenesis is a multistep process that involves activating or inactivating mutations or epigenetic changes in more than one gene to confer growth advantages to the cell. MYC is known to cooperate with another oncoprotein, RAS, to transform rodent cells. While RAS has been found to suppress MYC-induced apoptosis, MYC also inhibits RAS-induced senescence, thereby blocking two main anti-tumorigenic mechanisms in the cell and may, at least in part, explain the basis for the MYC/RAS cooperativity. Inactivation of MYC in mouse tumor models demonstrated tumor regression with welltolerated side effects, suggesting that MYC is a potential and suitable target for anti-cancer therapy. However, pharmacological targeting of transcription factors is considered difficult and no anti-MYC drugs are clinically available today. In this thesis, we deepen our understanding on MYC biology by studying different proteins that cooperate and interact with MYC (Paper I to III), and identify small molecules that would target specific interactions involving MYC (Paper IV). In Paper I, we found that oncogenic MYC and RAS do not cooperate to cancel out each other’s intrinsic anti-tumorigenic barrier, namely apoptosis and senescence, in normal human fibroblasts as they do in murine fibroblasts, even in the absence of the tumor suppressor p53. This is in contrast to previous results from human melanocytes, where MYC was reported to suppress BRAF- and partially NRAS-induced senescence, thus suggesting that these antitumor barriers are orchestrated differently in different species and in different cell types. In Papers II and III, we discovered new regulatory mechanisms for MYC. In Paper II, we found that the cyclin-dependent kinase (CDK) inhibitor p27KIP1 (p27) binds MYC and targets it for degradation. p27 is upregulated by interferon-ɣ (IFN-ɣ) and by other growth inhibitory signals. We also found that IFN-ɣ treatment leads to the degradation of MYC, mediated by upregulation of p27. There is significant clinical relevance between high activity of nuclear p27 levels and low MYC expression in tumor samples, and this correlates with a good prognosis and a positive clinical outcome. This may provide insights into strategies to target MYC-driven tumors, for example by finding ways to upregulate p27 expression and activity, utilizing IFN-ɣ in treatment modalities, stimulating immune cells to produce IFN-ɣ by immunotherapy and finding methods to combine these strategies to combat MYC-driven tumors. In Paper III, we uncovered a novel F-box protein, FBXO28, that ubiquitylates MYC in a nonproteolytic manner, and enhances MYC transcriptional activity and downstream pathways. Phosphorylation of SCFFBXO28 by CDK1/2 during the cell cycle is required for its efficient ubiquitylation of MYC. Depletion of FBXO28 or expression of its dominant negative F-box mutant, negates this function and results in reduction of MYC-driven transcription, transformation and tumorigenesis. High MYC expression coupled with high FBXO28 expression and phosphorylation are strong and independent predictors of poor prognosis in human breast cancer. Our data suggests that the CDK-FBXO28-MYC axis is a potential molecular drug target in MYC-driven cancers, including breast cancer. In Paper IV, we conducted a small molecule screen and found, MYCMI-6, that binds MYC, inhibits MYC/MYCN:MAX interactions, and impeded tumor cell growth in a MYCdependent manner in a variety of tumor cell cultures and in a mouse tumor model of MYCNamplified neuroblastoma. Importantly, this compound is highly specific and potent, has a good therapeutic window and does not have severe side effects. This discovery provides proof of principle of protein-protein targeting. MYCMI-6 can be used as a molecular tool to study MYC:MAX interactions and is a good candidate for drug development. Altogether, the projects involved in this thesis provide insights into molecular pathways involved in MYC oncogenic activity, regulation, and transcription functions, shed light in MYC-RAS cooperativity, identified new proteins interacting with MYC and small molecules interfering with MYC function. This is of importance not only to increase the basic knowledge on mechanisms through which MYC contributes to tumor development, but will hopefully also contribute to the development of new therapeutic strategies to combat MYCdriven cancer in the future

Comparison of the antioxidant activity of Malaysian ginger (Zingiber officinale Roscoe) extracts with that of selected natural products and its effect on the viability of myoblast cells in culture

Ginger has been proven to possess various therapeutic effects, including antibacterial, anticancer, anti-inflammatory, and antioxidant effects. However, data on the comparison of ginger antioxidant activity with that of other natural products are still lacking. This study aimed to analyse and compare the antioxidant properties of two types of Malaysian ginger extracts (GE1 and GE2) with that of selected natural products. The antioxidant activities were measured by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and ferric reducing antioxidant power (FRAP) assays, while cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) assay. The order of the DPPH scavenging activities was as follows: vitamin C > palm tocotrienol-rich fraction (TRF) > a-tocopherol > N-acetylcysteine (NAC) > Ficus deltoidea > butylated hydroxytoluene (BHT) > Centella asiatica > GE2 > GE1 > Moringa oleifera > Kelulut honey; the order of the mean FRAP value was as follows: NAC > a-tocopherol > BHT > TRF > Ficus deltoidea > Moringa oleifera > GE2 = GE1 > Centella asiatica > Kelulut honey. The viability assays showed that both ginger extracts significantly increased the percentage of viable cells (p < 0.05). In conclusion, neither of the ginger extracts was cytotoxic toward cells and both possessed comparable antioxidant properties, indicating their potential for ameliorating oxidative stress

Zingiber officinale Roscoe prevents DNA damage and improves muscle performance and bone integrity in old Sprague Dawley rats

Age-related loss of skeletal muscle mass and strength or sarcopenia is attributed to the high level of oxidative stress and inadequate nutritional intake. The imbalance in oxidative status with increased production of free radicals results in damage to the DNA which leads to cell dysfunction. This study aimed to determine the effect of Zingiber officinale Roscoe (ginger) on muscle performance and bone integrity in Sprague Dawley (SD) rats. SD rats aged three (young), nine (adult), and twenty-one (old) months old were treated with either distilled water or ginger extract at a concentration of 200 mg/kg body weight (BW) daily for 3 months via oral gavage. Muscle performance was assessed at 0, 1, 2, and 3 months of treatment by measuring muscle strength, muscle function, and bone integrity while DNA damage was determined by comet assay. Muscle cell histology was analyzed by hematoxylin and eosin (H&E) staining. Young and adult ginger-treated rats showed a significant improvement in muscle strength after 3 months of supplementation. Bone mineral density (BMD) and bone mineral content (BMC) were increased while fat free mass (FMM) was decreased after 3 months of ginger supplementation in young rats but not changed in adult and old ginger supplemented groups. Interestingly, supplementation of ginger for 3 months to the old rats decreased the level of damaged DNA. Histological findings showed reduction in the size of muscle fibre and fascicles with heterogenous morphology of the muscle fibres indicating sarcopenia was evident in old rats. Treatment with ginger extract improved the histological changes even though there was evidence of cellular infiltration (mild inflammation) and dilated blood vessels. In conclusion, Z. officinale Roscoe prevents DNA damage and improves muscle performance and bone integrity in SD rats indicating its potential in alleviating oxidative stress in ageing and thus delaying sarcopenia progression

CDK‐mediated activation of the SCFFBXO28 ubiquitin ligase promotes MYC‐driven transcription and tumourigenesis and predicts poor survival in breast cancer

SCF (Skp1/Cul1/F‐box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F‐box protein, FBXO28 that controls MYC‐dependent transcription by non‐proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2‐mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F‐box mutant unable to support MYC ubiquitylation results in an impairment of MYC‐driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK‐FBXO28‐MYC axis as a potential molecular drug target in MYC‐driven cancers, including breast cancer

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Identification of Y chromosomal material in turner syndrome by Fluorescence in Situ Hybridisation (FISH)

Turner syndrome is one of the most common chromosomal abnormalities affecting newborn females. More than half of patients with Turner syndrome have a 45X karyotype. The rest of the patients may have structurally abnormal sex chromosomes or are mosaics with normal or abnormal sex chromosomes. Mosaicism with a second X sex chromosome is not usually of clinical significance. However, Turner syndrome patients having a second Y chromosome or Y chromosomal material are at risk of developing gonadoblastoma later in life. The aim of this study is to compare the results of conventional (karyotyping) and molecular cytogenetics (FISH), and discuss the advantages and limitations in the diagnosis of Turner syndrome. We also aim to compare the degree of mosaicism identified using conventional cytogenetics and FISH techniques. Conventional cytogenetics and FISH analyses were performed on eight peripheral blood samples of patients with Turner syndrome collected between 2004 and 2006. From this study, two out of eight patients with Turner syndrome were found to have the sex determining region on the Y chromosome (SRY) gene by FISH analysis. Our results showed that the rate of detection of mosaic cases in Turner syndrome was also increased to 88% after using the FISH technique. We concluded that FISH is more superior to conventional cytogenetics in the detection of the Y chromosomal material. FISH is also a quick and cost effective method in diagnosing Turner syndrome and assessing the degree of mosaicis