Engineering Camelina sativa Seeds as a Green Bioreactor for the Production of Affordable Human Pro-insulin that Demonstrates Anti-diabetic Efficacy in Rats.

The current production of recombinant insulin via fermenter-based platforms (Escherichia coli and yeast) could not fulfill its fast-growing commercial demands, thus leading to a great interest in its sustainable large-scale production at low cost using a plant-based system. In the present study, Agrobacterium tumefaciens-mediated nuclear stable genetic transformation of an industrial oilseed crop, Camelina sativa, to express pro-insulin (with three furin endoprotease cleavage sites) fused with cholera toxin B subunit (CTB) in their seeds was successfully achieved for the first time. The bar gene was used as a selectable marker for selecting transformants and producing herbicide-resistant camelina plants. The transformation process involved the infiltration of camelina inflorescences (at flower buds with partially opened flowers) with A. tumefaciens and harvesting the seeds (T0) at maturity. The T0 seeds were raised into the putative T1 plants sprayed with Basta herbicide (0.03%, v/v), and the survived green transformed plants tested positive for pro-insulin and bar genes. A transformation frequency of 6.96% was obtained. The integration and copy number of the pro-insulin transgene and its expression at RNA and protein levels were confirmed in T1 plants using Southern hybridization, semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqPCR), and quantitative real-time Time PCR (qPCR) and western blot analysis, respectively. Enzyme-linked immunosorbent Assay (ELISA) quantified the amount of expressed pro-insulin protein, and its anti-diabetic efficacy was validated in diabetic rats on oral feeding. Transgenic plants integrated the pro-insulin gene into their genomes and produced a maximum of 197 µg/100 mg of pro-insulin (0.804% of TSP) that had anti-diabetic efficacy in rats.

MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

Histological and molecular analysis of cellular leiomyoma with sclerosis: linked to HMGA2 overexpression.

HMGA2 overexpression is found in 10-15% of leiomyomas (LM). HMGA2 overexpression is common in variants of hydropic, intravenous and lipo-LM. Cellular or highly cellular LM (CLM) is a LM variant with a less well-defined molecular nature. In this study, we identified and examined 52 hypercellular LM with sclerotic collagen, herein defined as cellular leiomyoma with sclerosis (CLM-S). CLM-S shows large tumour size (average 12.2 cm) and characteristic histology of tumour cells, arranged in cellular fascicles, sheets and trabeculae with abundant dense, pink sclerotic extracellular matrix in bands and nodules and increased vascularity. Tumour cells are uniform with small, round-oval nuclei and scant, pale-eosinophilic to vacuolated cytoplasm reminiscent of pericytes. The differential diagnosis of CLM-S includes conventional CLM, endometrial stromal tumours and perivascular epithelioid cell tumour. Immunohistochemical profile [HMGA2, fumarate hydratase, smooth muscle markers, Melan A and HMB-45] and molecular alterations [by HMGA2 mRNA reverse transcription-polymerase chain reaction (RT-PCR), HMGA2 fluorescence in-situ hybridisation and MED12 sequencing] were analysed in comparison to matched myometrium and CLM controls. Remarkably, 96% (50 of 52) of CLM-S demonstrated diffuse positive immunoreactivity for HMGA2 and up to an 80-fold increase in HMGA2 mRNA, determined by RT-PCR. FISH analysis with break-part probes at intron 3 and the 5' UTR detected HMGA2 rearrangements in 47% (18 of 38) of CLM-S. All CLM-S retained expression of fumarate hydratase. No MED12 mutations were found in any CLM-S. Our findings show that CLM-S has unique and characteristic histomorphology probably driven by HMGA2 overexpression.

Epigenomic and enhancer dysregulation in uterine leiomyomas.

BACKGROUND: Uterine leiomyomas, also known as uterine fibroids or myomas, are the most common benign gynecological tumors and are found in women of reproductive and postmenopausal age. There is an exceptionally high prevalence of this tumor in women by the age of 50 years. Black women are particularly affected, with an increased incidence, earlier age of onset, larger and faster growing fibroids and greater severity of symptoms as compared to White women. Although advances in identifying genetic and environmental factors to delineate these fibroids have already been made, only recently has the role of epigenomics in the pathogenesis of this disease been considered. OBJECTIVE AND RATIONALE: Over recent years, studies have identified multiple epigenomic aberrations that may contribute to leiomyoma development and growth. This review will focus on the most recent discoveries in three categories of epigenomic changes found in uterine fibroids, namely aberrant DNA methylation, histone tail modifications and histone variant exchange, and their translation into altered target gene architecture and transcriptional outcome. The findings demonstrating how the altered 3D shape of the enhancer can regulate gene expression from millions of base pairs away will be discussed. Additionally, translational implications of these discoveries and potential roadblocks in leiomyoma treatment will be addressed. SEARCH METHODS: A comprehensive PubMed search was performed to identify published articles containing keywords relevant to the focus of the review, such as: uterine leiomyoma, uterine fibroids, epigenetic alterations, epigenomics, stem cells, chromatin modifications, extracellular matrix [ECM] organization, DNA methylation, enhancer, histone post-translational modifications and dysregulated gene expression. Articles until September 2021 were explored and evaluated to identify relevant updates in the field. Most of the articles focused on in the discussion were published between 2015 and 2021, although some key discoveries made before 2015 were included for background information and foundational purposes. We apologize to the authors whose work was not included because of space restrictions or inadvertent omission. OUTCOMES: Chemical alterations to the DNA structure and of nucleosomal histones, without changing the underlying DNA sequence, have now been implicated in the phenotypic manifestation of uterine leiomyomas. Genome-wide DNA methylation analysis has revealed subsets of either suppressed or overexpressed genes accompanied by aberrant promoter methylation. Furthermore, differential promoter access resulting from altered 3D chromatin structure and histone modifications plays a role in regulating transcription of key genes thought to be involved in leiomyoma etiology. The dysregulated genes function in tumor suppression, apoptosis, angiogenesis, ECM formation, a variety of cancer-related signaling pathways and stem cell differentiation. Aberrant DNA methylation or histone modification is also observed in altering enhancer architecture, which leads to changes in enhancer-promoter contact strength, producing novel explanations for the overexpression of high mobility group AT-hook 2 and gene dysregulation found in mediator complex subunit 12 mutant fibroids. While many molecular mechanisms and epigenomic features have been investigated, the basis for the racial disparity observed among those in the Black population remains unclear. WIDER IMPLICATIONS: A comprehensive understanding of the exact pathogenesis of uterine leiomyoma is lacking and requires attention as it can provide clues for prevention and viable non-surgical treatment. These findings will widen our knowledge of the role epigenomics plays in the mechanisms related to uterine leiomyoma development and highlight novel approaches for the prevention and identification of epigenome targets for long-term non-invasive treatment options of this significantly common disease.

Epigenomic tensor predicts disease subtypes and reveals constrained tumor evolution.

Understanding the epigenomic evolution and specificity of disease subtypes from complex patient data remains a major biomedical problem. We here present DeCET (decomposition and classification of epigenomic tensors), an integrative computational approach for simultaneously analyzing hierarchical heterogeneous data, to identify robust epigenomic differences among tissue types, differentiation states, and disease subtypes. Applying DeCET to our own data from 21 uterine benign tumor (leiomyoma) patients identifies distinct epigenomic features discriminating normal myometrium and leiomyoma subtypes. Leiomyomas possess preponderant alterations in distal enhancers and long-range histone modifications confined to chromatin contact domains that constrain the evolution of pathological epigenomes. Moreover, we demonstrate the power and advantage of DeCET on multiple publicly available epigenomic datasets representing different cancers and cellular states. Epigenomic features extracted by DeCET can thus help improve our understanding of disease states, cellular development, and differentiation, thereby facilitating future therapeutic, diagnostic, and prognostic strategies.

Tks adaptor proteins at a glance.

Tyrosine kinase substrate (Tks) adaptor proteins are considered important regulators of various physiological and/or pathological processes, particularly cell migration and invasion, and cancer progression. These proteins contain PX and SH3 domains, and act as scaffolds, bringing membrane and cellular components in close proximity in structures known as invadopodia or podosomes. Tks proteins, analogous to the related proteins p47phox, p40phox and NoxO1, also facilitate local generation of reactive oxygen species (ROS), which aid in signaling at invadopodia and/or podosomes to promote their activity. As their name suggests, Tks adaptor proteins are substrates for tyrosine kinases, especially Src. In this Cell Science at a Glance article and accompanying poster, we discuss the known structural and functional aspects of Tks adaptor proteins. As the science of Tks proteins is evolving, this article will point out where we stand and what still needs to be explored. We also underscore pathological conditions involving these proteins, providing a basis for future research to develop therapies for treatment of these diseases.

Physiological growth and galactose utilization by dairy yeast Kluyveromyces marxianus in mixed sugars and whey during fermentation.

The dairy yeast Kluyveromyces marxianus represents a promising industrial strain useful for the production of bioethanol from cheese whey. Physiology of the five K. marxianus strains on galactose was examined during batch cultivation under controlled aerobic conditions on minimal media and one of the strains designated K. marxianus strain 6C17 which presented the highest specific galactose consumption rate. A maximum specific growth rate of 0.34 and 0.37 h-1, respectively, was achieved using batch cultivation in a minimal medium and a complex medium amended with galactose (50 g/L) at 37 °C. The sugar was metabolized for the production of ethanol as the chief metabolite with a maximum ethanol yield of 0.39 g/g of galactose. Different growth behaviors were observed when galactose was used with other sugar such as glucose, lactose and fructose. The growth rates on hydrolyzed cheese whey were also measured, and a maximum specific growth rate of 0.39 and 0.32 h-1 was observed with glucose and galactose, respectively, with the maximum flux diverted toward ethanol production. This approach of studying the physiology of thermotolerant K. marxianus on hydrolyzed whey during fermentation would be helpful in achieving higher yields of ethanol.

Comparative Analysis of Oxidative Stress During Aging of Kluyveromyces marxianus in Synthetic and Whey Media.

During the aging of yeast culture, Kluyveromyces marxianus undergoes a number of changes in physiology and these changes play a significant role during fermentation. Aged stationary phase cells were found to contain more reactive oxygen species. Additionally, the level of oxidant is counteracted by the antioxidant defense system of the cells. Comparison of 3-day-old culture of K. marxianus with 45-day stationary phase culture represents an increased level of ROS inside the cells. Moreover, a decrease in glutathione content was observed over the set of the incubation period. The increased level of superoxide dismutase (SOD) and catalase also revealed that there is oxidative stress during the long period incubation of the stationary phase cells of K. marxianus. The actual phenomenon of aging in dairy yeast K. marxianus is a complex process, but the present study signifies that role of antioxidant defense system during aging in stationary phase cells of K. marxianus.

Utilization of Cheese Whey Using Synergistic Immobilization of ß-Galactosidase and Saccharomyces cerevisiae Cells in Dual Matrices.

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the ß-galactosidase using whey. The maximum ß-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, ß-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.

Mdm2 inhibition confers protection of p53-proficient cells from the cytotoxic effects of Wee1 inhibitors.

Pharmacological inhibition of the cell cycle regulatory kinase Wee1 represents a promising strategy to eliminate cancer cells. Wee1 inhibitors cooperate with chemotherapeutics, e. g. nucleoside analogues, pushing malignant cells from S phase towards premature mitosis and death. However, considerable toxicities are observed in preclinical and clinical trials. A high proportion of tumor cells can be distinguished from all other cells of a patient's body by inactivating mutations in the tumor suppressor p53. Here we set out to develop an approach for the selective protection of p53-proficient cells against the cytotoxic effects of Wee1 inhibitors. We pretreated such cells with Nutlin-3a, a prototype inhibitor of the p53-antagonist Mdm2. The resulting transient cell cycle arrest effectively increased the survival of cells that were subsequently treated with combinations of the Wee1 inhibitor MK-1775 and/or the nucleoside analogue gemcitabine. In this constellation, Nutlin-3a reduced caspase activation and diminished the phosphorylation of Histone 2AX, an indicator of the DNA damage response. Both effects were strictly dependent on the presence of p53. Moreover, Nutlin pre-treatment reduced the fraction of cells that were undergoing premature mitosis in response to Wee1 inhibition. We conclude that the pre-activation of p53 through Mdm2 antagonists serves as a viable option to selectively protect p53-proficient cells against the cytotoxic effects of Wee1 inhibitors, especially when combined with a nucleoside analogue. Thus, Mdm2 antagonists might prove useful to avoid unwanted side effects of Wee1 inhibitors. On the other hand, when a tumor contains wild type p53, care should be taken not to induce its activity before applying Wee1 inhibitors.

Wee1 is required to sustain ATR/Chk1 signaling upon replicative stress.

The therapeutic efficacy of nucleoside analogues, e.g. gemcitabine, against cancer cells can be augmented by inhibitors of checkpoint kinases, including Wee1, ATR, and Chk1. We have compared the chemosensitizing effect of these inhibitors in cells derived from pancreatic cancer, a tumor entity where gemcitabine is part of the first-line therapeutic regimens, and in osteosarcoma-derived cells. As expected, all three inhibitors rendered cancer cells more sensitive to gemcitabine, but Wee1 inhibition proved to be particularly efficient in this context. Investigating the reasons for this potent sensitizing effect, we found that Wee1 inhibition or knockdown not only blocked Wee1 activity, but also reduced the activation of ATR/Chk1 in gemcitabine-treated cells. Combination of several inhibitors revealed that Wee1 inhibition requires Cyclin-dependent kinases 1 and 2 (Cdk1/2) and Polo-like kinase 1 (Plk1) to reduce ATR/Chk1 activity. Through activation of Cdks and Plk1, Wee1 inhibition reduces Claspin and CtIP levels, explaining the impairment in ATR/Chk1 activity. Taken together, these results confer a consistent signaling pathway reaching from Wee1 inhibition to impaired Chk1 activity, mechanistically dissecting how Wee1 inhibitors not only dysregulate cell cycle progression, but also enhance replicative stress and chemosensitivity towards nucleoside analogues.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity.

DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.