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Non-alcoholic fatty liver disease (NAFLD), encompassing fatty liver and its progression into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), is one of the rapidly rising health concerns worldwide. SIRT6 is an essential nuclear sirtuin that regulates numerous pathological processes including insulin resistance and inflammation, and recently it has been implicated in the amelioration of NAFLD progression. SIRT6 overexpression protects from formation of fibrotic lesions. However, the underlying molecular mechanisms are not fully delineated. Moreover, new allelic variants of SIRT6 (N308K/A313S) were recently associated with the longevity in Ashkenazi Jews by improving genome maintenance and DNA repair, suppressing transposons and killing cancer cells. Whether these new SIRT6 variants play different or enhanced roles in liver diseases is currently unknown. In this study, we aimed to clarify how these new centenarian-associated SIRT6 genetic variants affect liver metabolism and associated diseases. We present evidence that overexpression of centenarian-associated SIRT6 variants dramatically altered the metabolomic and secretomic profiles of unchallenged immortalized human hepatocytes (IHH). Most amino acids were increased in the SIRT6 N308K/A313S overexpressing IHH when compared to IHH transfected with the SIRT6 wild-type sequence. Several unsaturated fatty acids and glycerophospholipids were increased, and ceramide tended to be decreased upon SIRT6 N308K/A313S overexpression. Furthermore, we found that overexpression of SIRT6 N308K/A313S in a 3D hepatic spheroid model formed by the co-culture of human immortalized hepatocytes (IHH) and hepatic stellate cells (LX2) inhibited collagen deposition and fibrotic gene expression in absence of metabolic or dietary challenges. Hence, our findings suggest that novel longevity associated SIRT6 N308K/A313S variants could favor the prevention of NASH by altering hepatocyte proteome and lipidome. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11357-022-00713-1.

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

Water-in-oil droplets allow researchers to perform massive experimental parallelization and high-throughput studies, such as single-cell experiments. However, the analysis of such vast arrays of droplets usually requires advanced expertise and sophisticated workflow tools, which limits the accessibility for wider user base in chemistry and biology. Thus, there is a need for more user-friendly tools for droplet analysis. In this article, we deliver a set of analytical pipelines for user-friendly analysis of typical scenarios in droplet-based experiments. We build the pipelines combining different open-source image-analysis software with the custom-developed data visualization tool “EasyFlow”. Our pipelines are designed to be applicable for the typical experimental scenarios users encounter with droplets: i) mono- and polydisperse droplets, ii) brightfield and fluorescent images, iii) droplet and object detection, iv) signal profile of droplets and objects (e.g., fluorescence).

Droplet microfluidic assays are rapidly gaining popularity because they enable analysis of biochemical reactions, individual cells or small cell populations with high sensitivity, precision and accuracy in a high-throughput manner. Nonetheless, there is a demand for user-friendly and low-cost droplet analysis technology. In this article, we meet this demand by developing two droplet analysis pipelines via free open-source software CellProfiler (CP) and its companion (CellProfiler Analyst, CPA). To illustrate the competence of the pipelines as an independent, and freely accessible droplet analysis tool for any researcher without the need of programming skills, we show (i) droplet digital quantification of viable fluorescent bacteria using single-color images and (ii) analysis of a multi-color fluorescence image with droplets containing different chemical compositions.

Droplet microfluidics has revealed innovative strategies in biology and chemistry. This advancement has delivered novel quantification methods, such as droplet digital polymerase chain reaction (ddPCR) and an antibiotic heteroresistance analysis tool. For droplet analysis, researchers often use image-based detection techniques. Unfortunately, the analysis of images may require specific tools or programming skills to produce the expected results. In order to address the issue, we explore the potential use of standalone freely available software to perform image-based droplet detection. We select the four most popular software and classify them into rule-based and machine learning-based types after assessing the software's modules. We test and evaluate the software's (i) ability to detect droplets, (ii) accuracy and precision, and (iii) overall components and supporting material. In our experimental setting, we find that the rule-based type of software is better suited for image-based droplet detection. The rule-based type of software also has a simpler workflow or pipeline, especially aimed for non-experienced users. In our case, CellProfiler (CP) offers the most user-friendly experience for both single image and batch processing analyses.

Background: Sarcoma is a heterogeneous group of malignancies comprising almost 80 subtypes of bone and soft tissue cancers. Previously, all subtypes were managed identically. Advancements in biological and genetic studies have revealed that sarcoma subtypes display varying characteristics and therefore require tailored treatments. Locally advanced soft tissue malignancies of both the trunk and the extremities can present significant challenges for treatment. At present, a negative surgical resection margin is the only definitive treatment despite attempts to use neoadjuvant and adjuvant therapies. In patients with locally advanced non-resectable soft tissue sarcoma (STS), the current practice would advocate amputation. However, studies suggest that limb salvage may be possible with radiotherapy or regional chemotherapy using isolated limb perfusion or isolated limb infusion (ILI). An ideal treatment modality for non-resectable STS would strive for preservation of anatomy and functionality as well as improve quality of life. The aim of the study was to investigate the efficacy of isolated limb infusion as an alternative treatment modality for non-resectable locally advanced STS. Methods: The efficacy of ILI was retrospectively investigated in 10 patients with STS. All patients received ILI with melphalan and actinomycin at the North Estonia Medical Centre Foundation, Tallinn, Estonia from September 1, 2014 to May 31, 2018. The procedures were performed in a lower extremity in 8 patients and in an upper extremity in 2 patients. The 6-month overall response rate was 78% and the overall limb salvage rate was 100%. The distant metastatis-free survival was longer for responders than for non-responders. Results and Conclusions: ILI is an alternative treatment modality for regional disease control and limb preservation in patients with cutaneous and soft tissue malignant neoplasms of the extremities. The short-term response rates are encouraging and the median overall survival shows good results in this highly complex patient population.

Droplet microfluidic assays are rapidly gaining popularity as the result of the ability to manipulate and monitor single biological molecules, individual cells or small populations of bacteria in pico- and nanoliter droplets, with high sensitivity, precision and accuracy in a high-throughput manner. Nonetheless, there is a demand for user-friendly and low-cost droplet analysis technology. In this article we meet this demand with free open-source software CellProfiler (CP). To illustrate the competence of CP as a droplet analysis tool, we show droplet digital quantification of viable fluorescent bacteria.

Background Isolated limb infusion (ILI) with cytotoxic agents is a simple and effective treatment option for patients with melanoma in-transit metastases (ITMs) confined to an extremity. Data for ILIs performed in Europe are sparse and to date no Eastern European ILI experience has been reported. The aim of the current study was to evaluate the efficacy of ILI in Estonia. Patients and methods Data for twenty-one patients were collected and analysed. All patients had melanoma ITMs and underwent an ILI between January 2012 and May 2018. The cytotoxic drug combination of melphalan and actinomycin-D was used. Drug circulation times were 20–30 min under mildly hyperthermic conditions (38–39 °C). Primary outcome measures were treatment response and overall survival. Results Nineteen lower limb and two upper limb ILIs were performed. The female to male ratio was 18:3. The overall response rate (complete + partial response) was 76% (n = 16), with a complete response in 38% (n = 8). The overall long-term limb salvage rate was 90% (n = 19). During follow-up, eight patients (38%) died, two due to metastatic melanoma. Five-year overall survival was 57%. Conclusion This first Eastern European report of ILI for melanoma ITMs shows results comparable to those from other parts of the world. In this era of effective targeted and immune therapies, ILI remains a useful treatment option, with a high overall response rate and durable responses in patients with melanoma ITMs confined to a limb.

ABCE1 is a highly conserved protein universally present in eukaryotes and archaea, which is crucial for the viability of different organisms. First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several stages of eukaryotic translation and ribosome biogenesis. The nature of vital functions of ABCE1, however, remains unexplained. Here, we study the role of ABCE1 in human cell proliferation and its possible connection to translation. We show that ABCE1 depletion by siRNA results in a decreased rate of cell growth due to accumulation of cells in S phase, which is accompanied by inefficient DNA synthesis and reduced histone mRNA and protein levels. We infer that in addition to the role in general translation, ABCE1 is involved in histone biosynthesis and DNA replication and therefore is essential for normal S phase progression. In addition, we analyze whether ABCE1 is implicated in transcript-specific translation via its association with the eIF3 complex subunits known to control the synthesis of cell proliferation-related proteins. The expression levels of a few such targets regulated by eIF3A, however, were not consistently affected by ABCE1 depletion.

Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals.

Background The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport. Results Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils. Conclusions We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes.

Hepatocellular carcinoma (HCC) is a deadly malignancy characterized at the epigenetic level by global DNA hypomethylation and focal hypermethylation on the promoter of tumor suppressor genes. In most cases it develops on a background of liver steatohepatitis, fibrosis, and cirrhosis. Guadecitabine (SGI-110) is a second-generation hypomethylating agent, which inhibits DNA methyltransferases. Guadecitabine is formulated as a dinucleotide of decitabine and deoxyguanosine that is resistant to cytidine deaminase (CDA) degradation and results in prolonged in vivo exposure to decitabine following small volume subcutaneous administration of guadecitabine. Here we found that guadecitabine is an effective demethylating agent and is able to prevent HCC progression in pre-clinical models. In a xenograft HCC HepG2 model, guadecitabine impeded tumor growth and inhibited angiogenesis, while it could not prevent liver fibrosis and inflammation in a mouse model of steatohepatitis. Demethylating efficacy of guadecitabine on LINE-1 elements was found to be the highest 8 d post-infusion in blood samples of mice. Analysis of a panel of human HCC vs. normal tissue revealed a signature of hypermethylated tumor suppressor genes (CDKN1A, CDKN2A, DLEC1, E2F1, GSTP1, OPCML, E2F1, RASSF1, RUNX3, and SOCS1) as detected by methylation-specific PCR. A pronounced demethylating effect of guadecitabine was obtained also in the promoters of a subset of tumor suppressors genes (CDKN2A, DLEC1, and RUNX3) in HepG2 and Huh-7 HCC cells. Finally, we analyzed the role of macroH2A1, a variant of histone H2A, an oncogene upregulated in human cirrhosis/HCC that synergizes with DNA methylation in suppressing tumor suppressor genes, and it prevents the inhibition of cell growth triggered by decitabine in HCC cells. Guadecitabine, in contrast to decitabine, blocked growth in HCC cells overexpressing macroH2A1 histones and with high CDA levels, despite being unable to fully demethylate CDKN2A, RUNX3, and DLEC1 promoters altered by macroH2A1. Collectively, our findings in human and mice models reveal novel epigenetic anti-HCC effects of guadecitabine, which might be effective specifically in advanced states of the disease.