Abstract Background Hypomethylating agents (HMA), such as azacytidine (AZA) and decitabine (DAC), are epigenetic therapies used to treat some patients with acute myeloid leukaemia (AML) and myelodysplastic syndrome. HMAs act in a replication-dependent manner to remove DNA methylation from the genome. However, AML cells targeted by HMA therapy are often quiescent within the bone marrow, where oxygen levels are low. In this study, we investigate the effects of hypoxia on HMA responses in AML cells. Results AML cell lines (MOLM-13, MV-4-11, HL-60) were treated with DAC (100 nM) or AZA (500–2000 nM) in normoxic (21% O2) and hypoxic (1% O2) conditions. Hypoxia significantly reduced AML cell growth across all cell lines, with no additional effects observed upon HMA treatment. Hypoxia had no impact on the extent of DNA hypomethylation induced by DAC treatment, but limited AZA-induced loss of methylation from the genome. Transcriptional responses to HMA treatment were also altered, with HMAs failing to up-regulate antigen presentation pathways in hypoxia. In particular, cell surface expression of the MHC class II receptor, HLA-DR, was increased by DAC treatment in normoxia, but not hypoxia. Conclusion Our results suggest that HMA-induced antigen presentation may be impaired by hypoxia. This study highlights the need to consider microenvironmental factors when designing co-treatment strategies to improve HMA therapeutic efficacy.

Cancer therapy-related cardiovascular toxicity (CTR-CVT) is now recognised as one of the leading causes of long-term morbidity and mortality in cancer patients. To date, potential overlapping cardiotoxicity mechanism(s) across different chemotherapeutic classes have not been elucidated. Doxorubicin, an anthracycline, and Carfilzomib, a proteasome inhibitor, are both known to cause heart failure in some patients. Given this common cardiotoxic effect of these chemotherapies, we aimed to investigate differential and common mechanism(s) associated with Doxorubicin and Carfilzomib-induced cardiac dysfunction. Primary human cardiomyocyte-like cells (HCM-ls) were treated with 1 µM of either Doxorubicin or Carfilzomib for 72 h. Both Doxorubicin and Carfilzomib induced a significant reduction in HCM cell viability and cell damage. DNA methylation analysis performed using MethylationEPIC array showed distinct and common changes induced by Doxorubicin and Carfilzomib (10,270 or approximately 12.9% of the DMPs for either treatment overlapped). RNA-seq analyses identified 5,643 differentially expressed genes (DEGs) that were commonly dysregulated for both treatments. Pathway analysis revealed that the PI3K-Akt signalling pathway was the most significantly enriched pathway with common DEGs, shared between Doxorubicin and Carfilzomib. We identified that there are shared cardiotoxicity mechanisms for Doxorubicin and Carfilzomib pathways that can be potential therapeutic targets for treatments across 2 classes of anti-cancer agents.

Bacterial cellulose presents itself as an alternative to traditional textiles due to its versatility, aesthetics, mechanical similarity to leather and, most importantly, sustainability in production processes. However, its use depends on surface modifications that promote suitable performance in fashion products, due to its high hydrophilicity. A less polluting alternative is the plasma treatment, which does not involve effluents or solid or liquid waste. In this context, this study aims to use the cathodic cage plasma process, an environmentally friendly technique, to reduce the hydrophilic nature of bacterial cellulose for its application in the fashion industry. Samples were produced from kombucha and treated through a modified low-pressure cold plasma system, named cathodic cage plasma. The influence of the gas mixture (Ar/acetylene and Ar/acetylene/H2), treatment time and duty cycle was evaluated. The samples were characterized through wetting behavior (contact angle), FTIR, TG and dTG, XPS and FEG-SEM. The cultivated bacterial cellulose exhibited apparent flexibility, roughness, and some transparency. The plasma treatment was proven effective, forming a thin hydrocarbon film on the surface of the BC, sufficient to decrease the hydrophilicity and purify it without degradation. The bacterial cellulose could be made hydrophobic by using parameters such as a longer treatment time, a combination of gases without the addition of hydrogen, and a lower duty cycle (from the most to the least influential factor, respectively). Plasma treatment mitigated the common issues of hydrophilic bacterial cellulose without the need to introduce processes that generate effluents or waste while maintaining the material’s biodegradability.

DNA hypomethylating agents (HMAs) are used to treat acute myeloid leukaemia (AML) and myelodysplasia patients who are unsuitable for intensive chemotherapy, but low response rates and therapy-resistant relapse remain significant challenges. To optimise HMA efficacy, we must understand how resistance and relapse arise from cells that survive treatment. Here we combine single-cell multi-omic analysis with parallel colony-forming assays to link HMA-induced molecular heterogeneity with functional consequences in AML cells. HMAs, azacytidine (AZA) and decitabine (DAC), induced global epigenetic heterogeneity associated with upregulation of inflammatory responses and cell death pathways in a subset of hypomethylated cells. Some AML cells maintained high DNA methylation during treatment, and these methylation-retaining cells had increased self-renewal capacity following DAC, but not AZA. Molecular profiling of individual colonies revealed upregulated cholesterol biosynthesis as an adaptation to HMA treatment, and inhibition by rosuvastatin enhanced DAC effects in vitro and in vivo . Thus, HMA-induced heterogeneity has important implications for AML cell growth and statins are a candidate co-treatment strategy to delay or prevent HMA-resistant relapse.### Competing Interest StatementA.K.E. declares the following competing interests: Advisory board/ Honoraria from AbbVie, Astellas, Gilead, Servier, Jazz, Otsuka/Astex and RACE Oncology; Speakers fees from AbbVie, Otsuka/Astex, Astellas and Jazz; Research Funding from RACE oncology and Otsuka/Astex.

The interaction of acute myeloid leukaemic (AML) blasts with the bone marrow (BM) microenvironment is a major determinant governing disease progression and resistance to treatment. The constitutive expression of E-selectin in the vascular compartment of BM, a key endothelial cell factor, directly mediates chemoresistance via E-selectin ligand/receptors. Despite the success of hypomethylating agent (HMA)-containing regimens to induce remissions in older AML patients, the development of primary or secondary resistance is common. We report that following treatment with 5-azacitidine, promoter regions regulating the biosynthesis of the E-selectin ligands, sialyl Lewis X, become further hypomethylated. The resultant upregulation of these gene products, in particular α(1,3)-fucosyltransferase VII (FUT7) and α(2,3)-sialyltransferase IV (ST3GAL4), likely causes functional E-selectin binding. When combined with the E-selectin antagonist uproleselan, the adhesion to E-selectin is reversed and the survival of mice transplanted with AML cells is prolonged. Finally, we present clinical evidence showing that BM myeloid cells from higher risk MDS and AML patients have the potential to bind E-selectin, and these cells are more abundant in 5-azacitidine-non-responsive patients. The collective data provide a strong rationale to evaluate 5-azacitidine in combination with the E-selectin antagonist, uproleselan, in this patient population.

Background Acute myeloid leukaemia (AML) is a deadly haematological malignancy that originates from mutated myeloid progenitor cells that lie quiescent in the hypoxic bone marrow. Elderly patients who cannot tolerate standard chemotherapies are administered low-dose hypomethylating agents (HMA) which act in a replication-dependent manner to reprogram the epigenome. Relapse is common following HMA treatment and may arise from quiescent leukaemia cells in the hypoxic bone marrow. Therefore, the effects of hypoxia on HMA efficacy may influence AML progression.Results AML cell lines (MOLM-13, MV-4-11, HL-60) were treated with decitabine (100nM) or azacitidine (500-2000nM) in normoxic (21% O2) and hypoxic (1% O2) conditions. Exposure to hypoxia significantly reduced AML cell growth across all cell lines, with no additional effects observed upon HMA treatment. This was associated with distinct effects on DNA methylation. The extent of hypomethylation induced by AZA treatment was reduced in hypoxia, whereas DAC-induced hypomethylation was maintained in low oxygen conditions. Transcriptional response to HMA treatment were also altered in hypoxia, with HMAs failing to up-regulate antigen presentation pathways in hypoxia. In particular, human leukocyte antigens (HLAs) such as HLA-DR were increased upon HMA treatment in normoxia, but not hypoxia.Conclusion Our results suggest that HMA-induced antigen presentation may be impaired in hypoxic tissues such as the bone marrow. This study highlights the need to consider microenvironmental factors when designing co-treatment strategies to improve HMA therapeutic efficacy.### Competing Interest StatementThe authors have declared no competing interest.

Hybrid laser arc welding (HLAW) has gained significant attention in recent years due to its ability to provide high-quality welds with improved productivity. One of the key challenges in HLAW is the efficient utilization of the welding arc energy. This work aims to analyze the HLAW process with gas metal buried arc welding (GMBAW), and compare the results obtained with typical results of laser welding and HLAW with non buried arc. Tests were conducted at two welding speeds, 1.0 m/min and 1.5 m/min, and the results were compared with autogenous laser bean welding (LBW) and HLAW without a buried arc, on ASTM A709 steel that was manufactured by Thermo-Mechanical Control Process (TMCP). The buried arc HLAW resulted in a more uniform weld bead, with a reduced heat-affected zone. Moreover, the laser power required to achieve full penetration of the weld joint was smaller for the buried arc HLAW variation.

BACKGROUND:Acute myeloid leukaemia (AML) is a deadly disease characterised by the uncontrolled proliferation of immature myeloid cells within the bone marrow. Altered regulation of DNA methylation is an important epigenetic driver of AML, where the hypoxic bone marrow microenvironment can help facilitate leukaemogenesis. Thus, interactions between epigenetic regulation and hypoxia signalling will have important implications for AML development and treatment.MAIN BODY:This review summarises the importance of DNA methylation and the hypoxic bone marrow microenvironment in the development, progression, and treatment of AML. Here, we focus on the role hypoxia plays on signalling and the subsequent regulation of DNA methylation. Hypoxia is likely to influence DNA methylation through altered metabolic pathways, transcriptional control of epigenetic regulators, and direct effects on the enzymatic activity of epigenetic modifiers. DNA methylation may also prevent activation of hypoxia-responsive genes, demonstrating bidirectional crosstalk between epigenetic regulation and the hypoxic microenvironment. Finally, we consider the clinical implications of these interactions, suggesting that reduced cell cycling within the hypoxic bone marrow may decrease the efficacy of hypomethylating agents.CONCLUSION:Hypoxia is likely to influence AML progression through complex interactions with DNA methylation, where the therapeutic efficacy of hypomethylating agents may be limited within the hypoxic bone marrow. To achieve optimal outcomes for AML patients, future studies should therefore consider co-treatments that can promote cycling of AML cells within the bone marrow or encourage their dissociation from the bone marrow.

Abstract Many powerful techniques are available for the analysis of single cell DNA methylation, but all have associated technical challenges. Throughput and coverage are common challenges, and expense is a limitation of practically all single cell DNA methylation and related multiomic methods. Single cell transposable element methylation sequencing (scTEM-seq) provides a simplified protocol for analysis of global DNA methylation that alleviates the costs associated with single cell sequencing. Paired with fluorescence activated cell sorting (FACS) and parallel transcriptome analysis for each cell, scTEM-seq allows analysis of the effects of global DNA methylation changes in various cell populations of interest. In this method, targeted bisulfite sequencing of high copy number transposable elements (TEs) is utilised to provide an accurate estimate for genome-wide methylation with very low sequencing demands

Global changes in DNA methylation are observed in development and disease, and single-cell analyses are highlighting the heterogeneous regulation of these processes. However, technical challenges associated with single-cell analysis of DNA methylation limit these studies. We present single-cell transposable element methylation sequencing (scTEM-seq) for cost-effective estimation of average DNA methylation levels. By targeting high-copy SINE Alu elements, we achieve amplicon bisulphite sequencing with thousands of loci covered in each scTEM-seq library. Parallel transcriptome analysis is also performed to link global DNA methylation estimates with gene expression. We apply scTEM-seq to KG1a acute myeloid leukaemia (AML) cells, and primary AML cells. Our method reveals global DNA methylation heterogeneity induced by decitabine treatment of KG1a cells associated with altered expression of immune process genes. We also compare global DNA methylation estimates to expression of transposable elements and find a predominance of negative correlations. Finally, we observe co-ordinated upregulation of many transposable elements in a sub-set of decitabine treated cells. By linking global DNA methylation heterogeneity with transcription, scTEM-seq will refine our understanding of epigenetic regulation in cancer and beyond.

ABSTRACTGlobal changes in DNA methylation are observed in developmental and disease contexts, and singlecell analyses are highlighting the heterogeneous regulation of these processes. However, technical challenges associated with single-cell analysis of DNA methylation limit these studies. We present single-cell transposable element methylation sequencing (scTEM-seq) for cost-effective estimation of global DNA methylation levels. By targeting high-copy LINE-1 and SINE Alu elements, we achieve amplicon bisulphite sequencing with thousands of loci covered in each library. Parallel transcriptome analysis is also performed to link global DNA methylation heterogeneity with gene expression. We apply scTEM-seq to KG1a acute myeloid leukaemia (AML) cells, and primary AML cells. Decitabine treatment of KG1a cells induces global DNA methylation heterogeneity associated with altered expression of immune process genes. We also compare global levels of DNA methylation to expression of transposable elements and find a predominance of negative correlations in both the KG1a and patient cells. Finally, we observe co-ordinated upregulation of many transposable elements in a sub-set of decitabine treated cells. By linking global DNA methylation heterogeneity with transcription, scTEM-seq will refine our understanding of epigenetic regulation in cancer and beyond.

Injection molds can fail after a certain period of use, which may compromise the final part's integrity and quality, but that can be solved using welding repair processes. This work aimed to investigate the influence of AISI P20 molds repaired by Gas Tungsten Arc Welding (GTAW) and Nd-YAG laser-welding processes on the properties of injection-molded parts with semi-crystalline (polypropylene) and amorphous (polycarbonate and acrylonitrile butadiene and styrene terpolymer) thermoplastics. Welds were prepared by machining 0.8 mm-deep 40 mm x 5 mm grooves to be filled by GTAW and Nd-YAG laser-welding deposits, in order to simulate the repair of AISI P20 molds. All polymers were injected into weld-repaired and unrepaired molds, and then evaluated in terms of microstructure, crystallinity degree, mechanical properties and gloss. The results suggest that the repaired region for both studied welding processes, despite the difference in hardness of the weld region, does not significantly affect the properties of semi-crystalline and amorphous injected polymers.

The GMAW torch orientation, whether pulling or pushing, influences both arc welding and hybrid processes. In hybrid laser-arc welding, for example, when the torch is pulled, a greater bead penetration is obtained. To promote greater penetration, the literature also indicates the use of a buried arc in GMAW, although it was initially developed to only operate with a vertically-positioned torch. Therefore, this work aims to investigate the influence of the push and pull techniques on the behavior of buried-arc GMAW at high welding speeds. Welds were performed with the push and pull techniques under the following conditions: buried and unburied (long) arc with welding speeds of 1.0, 1.5 and 2.0 m/min and current ranging from 450 to 470 A. The process tends to be more stable when pulling than when pushing (buried or long arc). Evidence of instability was only identified for the pushed buried arc, due to material accumulation at the front region of the molten pool, for the higher welding speeds. Only the 1.0 m/min buried-arc processes resulted in beads with an appropriate surface finish.

Nanotechnology is the domain of technological development and application of materials which go from 1 to 100 nm in at least one of their dimensions. The use of nanoparticles in engineering focuses on the development of new materials with special properties, which can be used as filler materials in the welding field. In this context, this study contributes to the stage preceding the deposition of the weld beads. This work aims to insert, using the colloidal processing technique, ceramic nanoparticles (ZrO2) into a Co-based superalloy (Stellite 6) and study the solidification of this "nanocomposite". 4 filler materials in the shape of compacted cylinders were melted by PTA: Stellite 6 with added Fe carriers with ceramic nanoparticles (ZrO2) (1%, 2.5% and 7.5% volume) and Stellite 6 without any additions. The molten materials were characterized through optical and scanning electron microscopy with field emission (SEM-FEG), XRD and Vickers microhardness. The results show that the colloidal processing technique was effective in adhering the ZrO2 nanoparticles to the carrier Fe particles, which were mixed with Stellite 6, as well as the relationship between microstructure refinement, phases and microhardness after PTA melting.

In response to the COVID-19 pandemic, a gradual and measured lockdown occurred in Australia from March 2020 and tightened in April 2020 This included both social restrictions as well as restrictions to elective surgical procedures in an effort to both mitigate and prepare for spread of the virus An unintended consequence has been significant reductions in cancer diagnosis and treatment Serum Prostate-Specific Antigen (PSA), prostate biopsy (trans-rectal or trans-perineal) and radical prostatectomy are key investigations and interventions recommended by clinical guidelines for prostate cancer (PCA) The non-emergent nature of PCA likely renders it more susceptible to COVID-19 related restrictions We hypothesize that the number of diagnostic and interventional procedures for PCA being performed in Australia have substantially reduced as a result of COVID-19-related social and clinical restrictions Method: Medicare Item Reports were obtained from publicly listed sources for all cystoscopies in Australia from December 2019 to May 2020 Comparisons were made between April and May 2020 to the preceding 3-month average and to the same month the previous year Results: The monthly average for prostatectomies performed in Australia between December 2019 and March 2020 was 1,458 In April 2020 that number dropped to 1,112, a 29 29% decrease A similar decline was noted in PSA tests ordered in April 2020, with 12,259 tests ordered compared to the average 18,372 tests over the preceding four months, a 35 36% drop Prostate biopsy numbers followed a similar trend, falling from a national average of 1,891 to 1,496, a 27 24% decrease Conclusion: Medicare data indicates a significant decrease in PCA screening and treatment throughout the COVID-19 lockdown in Australia As the pandemic persists, we must bear in mind the significant cost delayed diagnosis and treatment has on oncological outcomes for PCA patients

Breast cancer is the most commonly diagnosed and the second leading cause of cancer-related mortality among women worldwide. miR-518f-5p has been shown to modulate the expression of the metastasis suppressor CD9 in prostate cancer. However, the role of miR-518f-5p and CD9 in breast cancer is unknown. Therefore, this study aimed to elucidate the role of miR-518f-5p and the mechanisms responsible for decreased CD9 expression in breast cancer, as well as the role of CD9 in de novo tumor formation and metastasis. miR-518f-5p function was assessed using migration, adhesion, and proliferation assays. miR-518f-5p was overexpressed in breast cancer cell lines that displayed significantly lower CD9 expression as well as less endogenous CD9 3′UTR activity, as assessed using qPCR and dual luciferase assays. Transfection of miR-518f-5p significantly decreased CD9 protein expression and increased breast cell migration in vitro. Cd9 deletion in the MMTV/PyMT mouse model impaired tumor growth, but had no effect on tumor initiation or metastasis. Therefore, inhibition of miR-518f-5p may restore CD9 expression and aid in the treatment of breast cancer metastasis.

Treatment options for pancreatic cancer (PC) are severely limited due to late diagnosis, early metastasis and the inadequacy of chemotherapy and radiotherapy to combat the aggressive biology of the disease. In recent years, plant-derived bioactive compounds have emerged as a source of novel, anti-cancer agents. Used in traditional medicine worldwide, Elaeocarpus species have reported anti-inflammatory, antioxidant and anti-cancer properties. This study aimed to isolate and identify potential anti-PC compounds in the fruit of Elaeocarpus reticulatus Sm. A 50% acetone crude extract significantly decreased the viability of four pancreatic cell lines (≥ 10 µg/mL for BxPC-3 cells) and induced apoptosis in BxPC-3 and HPDE cells. Analysis by HPLC identified the triterpenoid Cucurbitacin I as a likely component of the extract. Furthermore, treatment with Cucurbitacin I significantly reduced the viability of HPDE and BxPC-3 cells, with results comparable to the same concentration of gemcitabine. Interestingly, attempts to isolate bioactive compounds revealed that the crude extract was more effective at reducing PC-cell viability than the fractionated extracts. This study provides initial insight into the bioactive constituents of E. reticulatus fruits.

Myelodysplastic syndrome (MDS) is a malignancy that disrupts normal blood cell production and commonly affects our ageing population. MDS patients are diagnosed using an invasive bone marrow biopsy and high-risk MDS patients are treated with hypomethylating agents (HMAs) such as decitabine and azacytidine. However, these therapies are only effective in 50% of patients, and many develop resistance to therapy, often resulting in bone marrow failure or leukemic transformation. Therefore, there is a strong need for less invasive, diagnostic tests for MDS, novel markers that can predict response to therapy and/or patient prognosis to aid treatment stratification, as well as new and effective therapeutics to enhance patient quality of life and survival. Epigenetic modifiers such as DNA methylation, long non-coding RNAs (lncRNAs) and micro-RNAs (miRNAs) are perturbed in MDS blasts and the bone marrow micro-environment, influencing disease progression and response to therapy. This review focusses on the potential utility of epigenetic modifiers in aiding diagnosis, prognosis, and predicting treatment response in MDS, and touches on the need for extensive and collaborative research using single-cell technologies and multi-omics to test the clinical utility of epigenetic markers for MDS patients in the future.

Selecting the most suited manufacturing process for a specific product, as well as optimizing the design regarding manufacture and assembly, is actions that will directly impact on cost and quality, aiming at finding the best match between the product’s functional requirements and the attributes of the processes, and it should be carried out in the first stages of product development. In this context, the literature presents some sheet metal joining process selection methods, which can be classified as: mechanical (forming), metallurgical (welding) and chemical (adhesive) processes. However, such methods are complex and not specific for thin sheet metal. Therefore, our goal is to propose a joining process selector for overlapping sheet metal, which can correlate the product’s functional requirements with the technical characteristics of the processes (clinching, rivets and welding) in early stages of product development. Unlike what is found in the literature, this selector is subdivided into five different types of clinching processes. The selector design was based on the Quality Function Deployment (QFD) principle, which easily converts a product’s functional requirements into an ordered joining process list. The joining process data collection was carried out from two approaches: quantitative (joining sheet thickness, joint dimensions, production batch and joining strength) and qualitative (type of material, surface finish and accessibility to perform the joining). Three products were chosen to validate the selector. The results were compared against the literature and commonly commercially employed processes. The application of the selector in commercial products showed compatibility with the literature as well as the commercially used processes. However, depending on the product, other requirements might be considered, such as availability of equipment and production costs.

Cancer is a disease of global epigenetic dysregulation. Mutations in epigenetic regulators are common events in multiple cancer types and epigenetic therapies are emerging as a treatment option in several malignancies. A major challenge for the clinical management of cancer is the heterogeneous nature of this disease. Cancers are composed of numerous cell types and evolve over time. This heterogeneity confounds decisions regarding treatment and promotes disease relapse. The emergence of single-cell epigenomic technologies has introduced the exciting possibility of linking genetic and transcriptional heterogeneity in the context of cancer biology. The next challenge is to leverage these tools for improved patient outcomes. Here we consider how single-cell epigenomic technologies may address the current challenges faced by cancer clinicians.