Chromosomal instability and its effect on cell lines.

BACKGROUND: Cancer cell lines are invaluable model systems for biomedical research because they provide an almost unlimited supply of biological materials. However, there is considerable skepticism regarding the reproducibility of data derived from these in vitro models. RECENT FINDINGS: Chromosomal instability (CIN) is one of the primary issues associated with cell lines, which can cause genetic heterogeneity and unstable cell properties within a cell population. Many of these problems can be avoided with some precautions. Here we review the underlying causes of CIN, including merotelic attachment, telomere dysfunction, DNA damage response defects, mitotic checkpoint defects and cell cycle disturbances. CONCLUSION: In this review we summarize studies highlighting the consequences of CIN in various cell lines and provide suggestions on monitoring and controlling CIN during cell culture.

Discovery and Mechanism of Action of a Novel Antimicrobial Peptide from an Earthworm.

The robust innate immune system of the earthworm provides a potential source of natural antimicrobial peptides (AMPs). However, the cost and high rediscovery rate of direct separation and purification limits their discovery. Genome sequencing of numerous earthworm species facilitates the discovery of new antimicrobial peptides. Through predicting potential antimicrobial peptides in the open reading frames of the Eisenia andrei genome and sequence optimization, a novel antimicrobial peptide, named EWAMP-R (RIWWSGGWRRWRW), was identified. EWAMP-R demonstrated good activity against various bacteria, including drug-resistant strains. The antibacterial mechanisms of EWAMP-R were explored through molecular simulation and wet-laboratory experiments. These experiments demonstrated that the bacterial membrane may be one of the targets of EWAMP-R but that there may be different interactions with Gram-negative and Gram-positive bacterial membranes. EWAMP-R can disrupt bacterial membrane integrity; however, at low concentrations, it appears that EWAMP-R may get through the membrane of Escherichia coli instead of damaging it directly, implying the existence of a secondary response. Gene expression studies identified that in E. coli, only the apoptosis-like cell death (ALD) pathway was activated, while in Staphylococcus aureus, the MazEF pathway was also upregulated, limiting the influence of the ALD pathway. The different antimicrobial actions against Gram-positive and -negative bacteria can provide important information on the structure-activity relationship of AMPs and facilitate AMP design with higher specificity. This study identified a new source of antibacterial agents that has the potential to address the increasingly serious issue of antibiotic resistance. IMPORTANCE Drug-resistant bacteria are a great threat to public health and drive the search for new antibacterial agents. The living environment of earthworms necessitates a strong immune system, and therefore, they are potentially a rich resource of novel antibiotics. A novel AMP, EWAMP-R, with high antibacterial activity was found through in silico analysis of the Eisenia andrei genome. Molecular analysis investigating the interactions between EWAMP-R and the cell membrane demonstrated the importance of tryptophan and arginine residues to EWAMP-R activity. Additionally, the different secondary responses found between E. coli and S. aureus were in accordance with a common phenomenon where some antibacterial agents only target specific species of bacteria. These results provided useful molecular information to support further AMP research and design. Our study expands the sources of antimicrobial peptides and also helps to explain the adaptability of earthworms to their environment.

LOT and HOT or not. The proliferation of clinically insignificant and poorly characterised types of renal neoplasia.

The classification of malignant tumours is influenced by both immunohistochemical and molecular genetic findings. This is highlighted in the latest World Health Organization classification of renal neoplasia, which has a tumour category of 'tumours that are molecularly defined'. This implies that the defining molecular features are integral to tumourigenesis, which may not necessarily be the case. Renal oncocytoma is recognised as a benign tumour with variable morphology and immunoexpression. A variant of these tumours is hybrid oncocytic chromophobe tumour, which has features of both oncocytoma and chromophobe renal cell carcinoma and may, on rare occasions, show malignant behaviour. Recent reports have proposed two further entities with eosinophilic cytoplasm and varying nuclear pleomorphism, designated low grade oncocytic tumour (LOT) and eosinophilic vacuolated tumour (EVT), formally known as high grade oncocytic tumour (HOT). The diagnosis of these apparently benign tumours was made on the basis of morphological and immunohistochemical features. More recently it has been claimed that the mutations in the mTOR pathway are also a diagnostic feature and it is further suggested that these mutations are key to the pathogenesis of these tumours. As is seen in oncocytoma, immunohistochemical expression of tumours included in series of LOT and EVT is variable. The mutations in the mTOR pathway, where detected, were not constant, with any combination of mTOR, TSC1 and/or TSC2 being involved. A major issue is that in many of the studies full comparative genomic hybridisation results are not presented. In addition it is well recognised that mTOR mutations are seen in a variety of renal tumours. In view of these conflicting results, the rarity of these tumours and their apparent benign nature, raises questions as to why these tumours should be considered specific entities.

Prognostic significance of percentage Gleason grade 5 prostatic adenocarcinoma in needle biopsies from patients treated by radical prostatectomy.

Previous studies have shown that the percentage of high grade prostatic adenocarcinoma (Gleason patterns 4 and 5) in a biopsy correlates with outcome parameters. It has also been shown that the percentage Gleason pattern 4/5 tumour correlates with biochemical failure and overall survival. There are little data relating to the prognostic significance of quantifying the percentage of Gleason pattern 5 in isolation. We investigated the prognostic predictive value of quantifying the percentage of Gleason pattern 5 tumour in needle biopsies from a series of 196 cases of Gleason score 4+5=9 prostate adenocarcinoma from patients who had also undergone radical prostatectomy. Division of cases according to the percentage of Gleason pattern 5 present (based upon the core with the highest grade) and analysing these with tumour grouped as Gleason score 4+5 with <5% pattern 5 (GS 4+5 <5%), Gleason score 4+5 with 5-20% pattern 5 (GS 4+5 5-20%) and Gleason score 4+5 with 21-49% pattern 5 (GS 4+5 21-49%) showed no difference in outcome determined as time interval to prostate specific antigen biochemical failure. The results showed that each of the subgroups of GS 4+5 tumours had a significantly shorter biochemical recurrence-free survival than for a control group of 179 patients with Gleason score 4+3=7 (GS 4+3) cancer. Similar results were obtained when grading was based upon percentage of Gleason pattern 5 present in all the cores taken from the same patient (case-based grade). Adverse findings at radical prostatectomy showed each of the subgroups of GS 4+5 tumours to have a higher incidence of extraprostatic extension and seminal vesicle invasion than the GS 4+3 group of controls. Further, the differences in incidence between each of the subgroups were not significant for either extraprostatic extension or seminal vesicle invasion. These observations applied to both the highest core-based grade and the case-based grade. Our study has shown that any proportion of Gleason pattern 5 tumour in a needle biopsy is associated with a worse prognosis when compared to GS4+3 tumours and that these results are similar for grading that is core- or case-based.

Eco-risk management of tylosin fermentation residues using vermicomposting.

Tylosin fermentation residues (TFR) pose an ecotoxicological risk through antibiotic resistant bacteria (ARBs) and their corresponding genes (ARGs). This study evaluated the ecotoxicity of TFR to soil biological activity, and further explored the mechanisms of vermicomposting to reduce the toxicological risk. The results showed that tylosin (TYL) was moderately degradable with a half-life (t1/2) of 37.5 d, inducing 28-44% inhibition rate of nitrogen transformation in soil, and the EC50 of earthworm avoidance was 880 mg/kg. The 30-d vermicomposting reduced the pH and OM content, while increased the EC and TN content, accelerated compost maturation (C/N ratio up to 20), and enriched the microbial community. ARGs were reduced by earthworm through removal of TYL (>70% degradation, t1/2 of <20 d), inhibiting abundance of intI1 and ARBs. We conclude that vermicomposting is an efficient method for TFR treatment and its eco-risk management.

Empowering Women in the Face of Body Ideals: A Scoping Review of Health Promotion Programs.

Achieving women's health equity and empowerment is a global priority. In a Western context, women are often disempowered by the value society places on body size, shape or weight, which can create a barrier to health. Health promotion programs can exacerbate women's preoccupations with their bodies by focusing outcomes toward achieving an "ideal" body size. Women's health promotion activities should be empowering if the desired outcomes are to improve their health and well-being long-term. This review sought to identify key elements from health promotion programs that aimed to empower women. A search was conducted in PubMed, MEDLINE, Web of Science, Scopus, CINAHL complete, and Academic Search Premiere databases. The search yielded 27 articles that collectively reported on 10 different programs. Through thematic synthesis, each article was analyzed for (1) key program features employed to empower women and (2) how such programs evaluated women's health. Seven themes resulted, of which five describe key empowering features (active participation, social support, sustainable change, holistic health perspective, strength-based approach) and two evaluation characteristics (assessment across multiple health domains and a mixed-method design). The findings from this review can assist health promoters to design and improve initiatives that aim to empower women.

Molecular Profiling of Endometrial Cancer: An Exploratory Study in Aotearoa, New Zealand.

BACKGROUND: Aotearoa, New Zealand, has one of the fastest-rising rates of endometrial cancer (EC) worldwide, increasing particularly in younger Maori and Pasifika women. There is a move towards using molecular profiling to direct treatment for each EC subtype. AIM: This study aimed to explore the molecular profiling of primary EC tissue in Aotearoa. METHODS: We used the PORTEC guidelines for the molecular subtyping of 90 patients' samples into four categories: POLE-mutated, p53 abnormal, mismatch repair deficient (MMRd) and no specific molecular profile (NSMP). The CTNNB1 mutation and L1CAM expression were also included in the analysis. POLE and CTNNB1 mutations were analysed using targeted next-generation sequencing (NGS). Novel mutations were assessed using VarSome. MMRd, L1CAM and p53 abnormalities were analysed using immunohistochemistry. RESULTS: In total, 15 samples were MMRd, 9 were p53 abnormal, 8 were POLE-mutated and the rest (56) were NSMP. Eleven samples had exon 3 CTNNB1 mutations and eleven novel POLE mutations were described. CONCLUSION: Surrogate markers for POLE mutations should be investigated. The validation of POLE variants and CTNNB1 mutations as part of an Aotearoa-based molecular panel is warranted.

The epigenome: key to understanding and predicting gout flares.

Gout is a form of arthritis, resulting from an inflammatory reaction to the deposition of monosodium urate (MSU) crystals in the synovial fluid of the joint space. It is characterised by periods of acute inflammation in the affected joint, or joints (known as gout flares), separated by asymptomatic periods. There seems to be substantial overlap between environmental triggers of gout flares and common environmental modifiers (diet, pharmaceuticals, and stress) of epigenetic markers (DNA methylation, histone modifications, and ncRNA). Very few studies have looked at whether environment is influencing gout through epigenetic mechanisms. The pathogenesis of gouty inflammation is well understood but understanding the variation of response to hyperuricaemia in terms of gout flare initiation is less well known. In this review, we will examine the potential of epigenomics in understanding how gout flares may occur, both in terms of development of hyperuricaemia and the inflammatory response. Looking at the epigenome and its intersection with lifestyle could help identify new targets and strategies for effective management of gout flares.

Prognostic significance of morphological patterns of Gleason grade 5 prostatic adenocarcinoma diagnosed on needle biopsy.

Grading is one of the best prognostic indicators of prostate cancer with Gleason grade 5 having the worst outcome. The prognostic influence of grade 5 patterns remains uncertain. A total of 646 prostate needle biopsy sets with Gleason score (GS) 9-10 prostatic adenocarcinoma were prospectively analysed. Patterns of grade 5 were correlated with radical prostatectomy (RP) adverse findings of high tumour volume (TV), extra-prostatic extension (EPE), seminal vesicle invasion (SVI) and lymph node involvement (LNI) in 472 and biochemical recurrence (BCR) in 338 patients after RP. Mean age and serum PSA were 69 years (range 37-91) and 26.1 ng/mL (range 1.4-1800), respectively. Gleason scores were 4+5=9 in 539 (83%), 5+4=9 in 94 (15%) and 5+5=10 in 13 cases (2%). Clusters/cords, single cells, sheets and comedocarcinoma were found in 86%, 69%, 26% and 18% with a pure pattern in 25% of cases. Comparing cases with and without sheets, there were no significant differences with RP high TV (p=0.8577), EPE (p=0.5372), SVI (p=0.5183) and LNI (p=0.4323). However, the presence of sheets predicted a significantly higher BCR rate (p=0.0033), while for tumours with single cells, the interval to BCR was significantly shorter (p<0.0001). Comparing cases with and without the other patterns, two other significant differences were found. Comedocarcinoma predicted high TV (p=0.0230) and single cells predicted EPE (p=0.0101). This study shows that all patterns currently used to assign a Gleason grade 5, including sheets, comedocarcinoma, single cells and clusters/cords, are associated with aggressive outcomes validating their inclusion in grade 5.

Staging of renal cell carcinoma: current progress and potential advances.

Formal staging classifications for renal cell carcinoma (RCC) were first proposed in 1978 and were incorporated into the Tumour, Nodes, Metastases (TNM) system initially published by the Union Internationale Contre le Cancer (UICC) in 1978. There has been a gradual evolution of grading criteria through six separate editions of the UICC TNM Classification, with the latest edition being published in 2016. Somewhat surprisingly there were no changes to the T category criteria from the 2009 to the 2016 editions of the classification, although an erratum has subsequently been published that incorporated the minor changes included in the eighth edition of the TNM Classification published by the American Joint Committee on Cancer. Localised tumours are staged according to the size of the primary tumour, with the TNM classification recognising that these tumours may exceed 10 cm in diameter. This is unfortunate as there is good evidence to demonstrate that, for clear cell RCC, virtually all tumours >7 cm in diameter and a substantial proportion of tumours <7 cm in diameter, show extra-renal spread. Infiltration of tumour beyond the renal capsule into the peri-renal fat is also categorised as T3a, however the clinical importance of this remains unclear. The classification of microvascular invasion within the renal sinus requires clarification, as does the prognostic significance of tumour in small vessels within the kidney.

Gene of the month: DICER1: ruler and controller.

DICER1 is a highly conserved RNaseIII endoribonuclease that has a critical role in the biogenesis of microRNAs (miRNAs). miRNAs are small regulatory RNAs responsible for post-transcriptional gene silencing, controlling more than half of human protein-coding genes. This is achieved through the targeting and regulation of complementary RNA transcripts and has a well-documented role in post-transcriptional gene regulation and transposon repression. DICER1 deficiency results in dysregulation of miRNAs, changing the expression of many genes. DICER1 syndrome represents a collection of benign and malignant tumours arising from an autosomally inherited germline mutation leading to an inherited predisposition to cancer. The syndrome represents an unusual form of Knudson's two-hit hypothesis, where individuals with a pathogenic germline DICER1 variant acquire a second trans-somatic missense DICER1 mutation. This somatic mutation appears to have to occur in one of five hotspots codons and may contribute towards the incomplete penetrance observed within DICER1 syndrome families. In this case, DICER1 is haploinsuffcient with only one deletion required and partial loss of function being advantageous to tumours over complete loss of function. As increasing data emerge reaffirming the pivotal role of DICER1 in the maintenance of human physiology, DICER1 is likely to become an increasingly attractive target for novel therapeutic strategies.

Gene of the month: TMPRSS2 (transmembrane serine protease 2).

Transmembrane serine protease 2 is encoded by the TMPRSS2 gene. The gene is widely conserved and has two isoforms, both being autocatalytically activated from the inactive zymogen form. A fusion gene between the TMPRSS2 gene and ERG (erythroblast-specific-related gene), an oncogenic transcription factor, is the most common chromosomal aberration detected in prostate cancer, responsible for driving carcinogenesis. The other key role of TMPRSS2 is in priming the viral spike protein which facilitates viral entry essential for viral infectivity. The protease activates a diverse range of viruses. Both SARS-CoV and SARS-CoV-2 (COVID-19) use angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 to facilitate entry to cells, but with SARS-CoV-2 human-to-human transmission is much higher than SARS-CoV. As TMPRSS2 is expressed outside of the lung, and can therefore contribute to extrapulmonary spread of viruses, it warrants further exploration as a potential target for limiting viral spread and infectivity.

Granular necrosis: a distinctive form of cell death in malignant tumours.

Foci of necrosis are frequently seen in malignant tumours and may be due to a variety of causes. Different types of necrosis are given various names based upon their morphological features and presumed pathogenesis, such as coagulative, liquefactive and fibrinoid necrosis. Here, we propose the term 'granular necrosis' (GN) for a specific form of tumour necrosis characterised by the presence of well-defined necrotic foci being sharply demarcated from adjacent viable tumour. A constant feature is loss of architecture resulting in an amorphous necrotic mass containing granular nuclear and cytoplasmic debris, without an associated neutrophilic infiltrate. There is usually extensive karyorrhexis, which in larger tumours is more prominent at the periphery. These foci are often microscopic but may range up to several millimetres or larger in size. This distinctive form of necrosis has been erroneously given a variety of names in the literature including coagulative necrosis and microscopic necrosis, which on the basis of the aforementioned gross and microscopic findings is inappropriate. It is apparent that this is a specific form of necrosis, hence the descriptive term 'granular necrosis' that differentiates this form of necrosis from other types. The presence of GN is recognised as occurring in a variety of tumour types, being commonly seen in renal cell carcinoma, where it has been shown to have independent prognostic significance. In some epithelial and stromal tumours of the uterus, the presence of GN also has prognostic significance and is a defining feature for the differentiation of uterine leiomyoma and leiomyosarcoma. The pathogenesis of GN is unresolved. It does not show the features of apoptosis and in recent studies has been shown to have some of the molecular changes associated with necroptosis.

Uncovering social structures and informational prejudices to reduce inequity in delivery and uptake of new molecular technologies.

Advances in molecular technologies have the potential to help remedy health inequities through earlier detection and prevention; if, however, their delivery and uptake (and therefore any benefits associated with such testing) are not more carefully considered, there is a very real risk that existing inequities in access and use will be further exacerbated. We argue this risk relates to the way that information and knowledge about the technology is both acquired and shared, or not, between health practitioners and their patients.A healthcare system can be viewed as a complex social network comprising individuals with different worldviews, hierarchies, professional cultures and subcultures and personal beliefs, both for those giving and receiving care. When healthcare practitioners are not perceived as knowledge equals, they would experience informational prejudices, and the result is that knowledge dissemination across and between them would be impeded. The uptake and delivery of a new technology may be inequitable as a result. Patients would also experience informational prejudice when they are viewed as not being able to understand the information that is presented to them, and information may be withheld.Informational prejudices driven by social relations and structures have thus far been underexplored in considering (in)equitable implementation and uptake of new molecular technologies. Every healthcare interaction represents an opportunity for experiencing informational prejudice, and with it the risk of being inappropriately informed for undertaking (or offering) such screening or testing. Making knowledge acquisition and information dissemination, and experiences of informational prejudice, explicit through sociologically framed investigations would extend our understandings of (in)equity, and offer ways to affect network relationships and structures that support equity in delivery and uptake.

Intraductal carcinoma of the prostate is an aggressive form of invasive carcinoma and should be graded.

Infiltration of the prostatic ducts by prostatic adenocarcinoma occurs relatively frequently, being most commonly associated with high grade disease. It is now recognised that intraductal carcinoma of the prostate (IDCP) has an associated poor prognosis and this is reflected in its histological, molecular and immunohistochemical features. The current recommendation of the World Health Organization is that IDCP not be taken into consideration when grading prostate adenocarcinoma. It is apparent that Gleason did not differentiate between IDCP and stromal invasive carcinoma when developing and validating his grading system, and recent studies suggest that the incorporation of IDCP grading into the overall grading of the specimen provides additional prognostic information.

Reflection on the current status of endometrial cancer in New Zealand.

Incidence of endometrial cancer is increasing rapidly in the developed world and is the most common gynaecological cancer in Australia and New Zealand. In line with obesity rates, the landscape and average age of women diagnosed with endometrial cancer are changing. There is still unmet need in early diagnosis, directed treatment, management of comorbidities and prevention strategies. This opinion piece aims to reflect on the current status of endometrial cancer in New Zealand in parallel to Australia, drawing out areas for future research and discussion.

Using NGS-methylation profiling to understand the molecular pathogenesis of young MI patients who have subsequent cardiac events.

Globally, ischaemic heart disease is a major contributor to premature morbidity and mortality. A significant number of young Myocardial Infarction (MI) patients (aged <55 y) have subsequent cardiac events within a year of their index event. This study used Next Generation Sequencing (NGS) methylation to understand the pathogenesis in this subset of young MI patients, comparing them to a cohort of patients without recurrent events. Cases and controls were matched for age, gender, ethnicity, and comorbidities. Differential methylation analyses were performed on Reduced Representation Bisulphite Sequencing (RRBS) data. Across the group and within case-control pairs' variation were analysed. Pairwise comparisons across each matched case-control pair resulted in a list of genes that were consistently significantly differentially methylated between all 16 matched pairs. This gene list was input into pathway analysis databases. Of particular relevance to cardiac pathology the following pathways were identified as over-represented in the patients with recurrent events; cell adhesion, transcription regulation and cardiac electrical conduction, specifically relating to calcium channel activity. This study looked at methylation differences between two populations of young MI patients. There were significantly different methylation profiles between the two groups studied; key pathways were identified as specifically affected in the patients with recurrent cardiac events. Matched pairwise comparisons and detailed interpretations of DNA methylation data may help to elucidate complex pathogeneses within and between clinical subtypes. Further analysis will determine whether these epigenomic differences can be useful as predictive biomarkers of clinical progression.

Identification of potential 'lifestyle-responsive' epigenomic biomarkers in healthy women aged 18-40.

CONTEXT: Human health is complex and multifaceted; there is a need for biomarkers that reflect the multidimensional nature of health. OBJECTIVE: To identify potential epigenomic biomarkers of health in women aged 18-40 participating in a six-month lifestyle intervention, next level health. MATERIALS AND METHODS: Methylation data were obtained by reduced representation bisulphite sequencing of 21 female intervention participants as well as three non-participants. The Differential Methylation Analysis Package (DMAP) was used to investigate inter- and intra-individual variability and to identify potential targets of transient epigenetic control in the population studied. RESULTS: Eleven genes were identified as significantly differentially methylated post- intervention in all 21 participants. 1884 genomic locations were found to be differentially methylated amongst the total female population studied representing potential epigenomic biomarkers. CONCLUSIONS: The ability to demonstrate epigenetic changes arising from a lifestyle intervention can provide key information on the relationship between gene regulation, human behaviour and health.

Effect of enrofloxacin and roxarsone on CYP450s in pig.

Enrofloxacin (ENR) and roxarsone (ROX) have been widely used in animal breeding. In this study, the daily clinical dosage of ENR and daily additive amount of ROX were administrated to Bama pigs. After 5days, the activity and protein expression of three important enzymes in the cytochrome P450 family were measured in the porcine liver. CYP1A2 was induced by both ENR and ROX independently. CYP2E1 and CYP3A4 were inhibited by ENR, but not affected by ROX. The combined administration of ENR and ROX were antagonistic to CYP1A2 and CYP2E1, but not to CYP3A4. Drug-drug interactions should be considered during the administration of ENR, ROX and for their co-administration with other drugs to minimize adverse reactions.

Effect of enrofloxacin on the proteome of earthworms.

The environmental and human health risks of veterinary drugs are becoming public health issues. Enrofloxacin (EF) is an extensively used animal-specific antibacterial agent that leaves drug residues in the environment. This study investigated the proteomic response of the earthworm Eisenia fetida to EF exposure. Earthworms were exposed to EF in soil at 1-500mg·kg-1, and samples were collected at intervals during a 28 day period. The extracted proteins were separated by two dimensional electrophoresis to detect differentially expressed proteins (DEPs) in EF-exposed earthworms. In total, 35 unique DEPs were found. These proteins were subjected to MALDI-TOF/TOF-MS analysis and identified through comparison of their mass spectra with those in protein databases. The DEPs were grouped on the basis of their function, into metabolism, stress-related, transport, transcription, and predicted/hypothetical protein categories. Knowledge of proteins that are induced or repressed by EF in earthworms could provide insight into mechanisms of sub-clinical physiological effects of xenobiotic residues in the environment, and may also help understand synergy between pollutants. As several DEPs in E. fetida showed similarity to human protein sequences, E. fetida has potential as an indicator species to assess the environmental and biological risks of drug residues.