Minimal residual disease detection by next-generation sequencing in multiple myeloma: Promise and challenges for response-adapted therapy.

Assessment of minimal residual disease (MRD) is becoming a standard diagnostic tool for curable hematological malignancies such as chronic and acute myeloid leukemia. Multiple myeloma (MM) remains an incurable disease, as a major portion of patients even in complete response eventually relapse, suggesting that residual disease remains. Over the past decade, the treatment landscape of MM has radically changed with the introduction of new effective drugs and the availability of immunotherapy, including targeted antibodies and adoptive cell therapy. Therefore, conventional serological and morphological techniques have become suboptimal for the evaluation of depth of response. Recently, the International Myeloma Working Group (IMWG) introduced the definition of MRD negativity as the absence of clonal Plasma cells (PC) with a minimum sensitivity of <10-5 either by next-generation sequencing (NGS) using the LymphoSIGHT platform (Sequenta/Adaptative) or by next-generation flow cytometry (NGF) using EuroFlow approaches as the reference methods. While the definition of the LymphoSIGHT platform (Sequenta/Adaptive) as the standard method derives from its large use and validation in clinical studies on the prognostic value of NGS-based MRD, other commercially available options exist. Recently, the LymphoTrack assay has been evaluated in MM, demonstrating a sensitivity level of 10-5, hence qualifying as an alternative effective tool for MRD monitoring in MM. Here, we will review state-of-the-art methods for MRD assessment by NGS. We will summarize how MRD testing supports clinical trials as a useful tool in dynamic risk-adapted therapy. Finally, we will also discuss future promise and challenges of NGS-based MRD determination for clinical decision-making. In addition, we will present our real-life single-center experience with the commercially available NGS strategy LymphoTrack-MiSeq. Even with the limitation of a limited number of patients, our results confirm the LymphoTrack-MiSeq platform as a cost-effective, readily available, and standardized workflow with a sensitivity of 10-5. Our real-life data also confirm that achieving MRD negativity is an important prognostic factor in MM.

Live birth and clinical outcome of vitrification-warming donor oocyte programme: an experience of a single IVF unit.

Medically assisted reproductive (MAR) treatments using donated oocytes are commonly applied in several countries to treat women who cannot conceive with their own gametes. Historically, in Italy, gamete donation has been prohibited but, in 2014, the law changed and gamete donation became allowed for couples undergoing MAR treatments. Consequently, in the last decade, there has been an increase in application of the oocyte donation programme. This study reports an egg-donation programme's clinical efficacy, based on importing donated vitrified oocytes from cryo-banks located in a foreign country. For this, we conducted a retrospective analysis of data from a single reproductive unit located in Italy (Donna Salus Women's Health and Fertility, Bozen). The study group consisted of 681 vitrified oocytes, which were warmed and culture to be replaced in 100 recipients. The survival rate after warming was 79.1% (n = 539/681), whereas the fertilization and blastulation rates were 90.2% (n = 486/539) and 47.9% (n = 233/486), respectively. Positive pregnancy test, clinical pregnancy rates, and live-birth rates per embryo transfer were 37.8%, 31.1% and 28.4%, respectively. The multiple pregnancy rate was 0.7%. This study is one of the first to report on the efficacy of a donor oocyte programme in Italy using imported vitrified oocytes. The above data may reassure women who are undertaking donation programmes using vitrified oocytes imported from commercial egg banks.

Live birth following a single oocyte fertilized with ICSI and embryo biopsy at blastocyst stage: a case report.

In this report, we present a case of a couple who obtained a live birth with a single oocyte fertilized by ICSI. Two oocytes were collected at 35.5 hours (h) post trigger and both were at metaphase II when sperm injection was performed (38 h). At 18 h post injection, one oocyte was fertilized, developed to the blastocyst stage and underwent to trophectoderm biopsy for preimplantation genetic testing on day 5. Following biopsy, the blastocyst was vitrified and 4 h after warming procedure, the euploid blastocyst was replaced to the uterine cavity. Healthy live birth was obtained after 39 weeks of gestation.

Nutritional, antioxidant and sensory properties of functional beef burgers formulated with chia seeds and goji puree, before and after in vitro digestion.

The addition of chia seeds and goji puree (2.5 and/or 5%) was evaluated in terms of their effects on the fatty acid profile, lipid peroxidation, total phenols and antioxidant capacity of cooked beef burgers. In comparison to control burgers, polyunsaturated fatty acids doubled or tripled in samples containing chia seeds; polyphenols and antioxidant capacities (ORAC, ABTS, DPPH) increased up to 70% and malondialdehyde values were reduced up to 50% in burgers formulated with both ingredients. Polyphenols, antioxidant capacity and lipid peroxidation were also assessed after in vitro digestion. A marked increase of polyphenol bioaccessibility and antioxidant capacity was observed for all samples, but also malondialdehyde values were increased after digestion, especially in samples containing 5% chia seeds. Finally, hedonistic tests were conducted on young (18-30 years), adult (31-60 years) and elderly (>60 years) subjects and the burgers resulted acceptable by all groups, appointing to their potential application as functional burgers.

Synthesis and biological evaluation of novel heteroring-annulated pyrrolino-tetrahydroberberine analogues as antioxidant agents.

Tetrahydroberberine (THB), otherwise known as canadine, is a natural alkaloid showing significant pharmacological properties and antioxidant protection against oxidative damage. Herein, we synthetized structurally complex THB analogues, namely pyrrolino-tetrahydroberberines (PTHBs) 4a-g, containing the pyrrolino[2,3-b]pyridine system, by means of the reactions of 1,2-diaza-1,3-dienes and 7,8-dihydroberberine. Aim of the study was to explore the in vitro antioxidant properties of PTHBs in comparison to THB thus to identify the most effective against free radical-induced oxidative injury, by using three different antioxidant tests: the ORAC method, the DNA nicking assay, and the DCFH-DA cellular assay. As a result, PTHB 4d emerged among the other THB analogues by exhibiting the best antioxidant properties. First, it was the only compound having an ORAC value completely comparable to that of THB, indicating the same ability to neutralize peroxyl radicals. Secondly, 4d showed an even better antioxidant capacity than THB in protecting DNA against ferrous ion-induced strand breaks. These observations were also confirmed in NCTC-2544 human keratinocytes exposed to hydrogen peroxide. Indeed, 4d protected cells against oxidation more efficiently than THB both in the short (1 and 3 h) and long (24 h) period of incubation, possibly suggesting increased cell membrane permeability and/or intracellular stability of 4d as compared to THB.

A Combination of Moringin and Avenanthramide 2f Inhibits the Proliferation of Hep3B Liver Cancer Cells Inducing Intrinsic and Extrinsic Apoptosis.

Moringin (MOR), a glycosyl-isothiocyanate obtained by myrosinase-catalyzed hydrolysis of the precursor 4-(α-l-rhamnosyloxy)-benzyl glucosinolate (glucomoringin), found predominantly in the seeds of Moringa oleifera, shows anticancer effects against several cancer cell lines. Avenanthramide (AVN) 2f is a phytochemical purified from oats with antioxidant and anticancer properties. The aim of this study was to investigate the antiproliferative and proapoptotic effects of MOR and AVN 2f used alone and in combination on Hep3B cancer cells, which are highly resistant to conventional anticancer drugs. We found that a cocktail of MOR and AVN 2f significantly inhibited the Hep3B proliferation rate by markedly increasing the activity of caspases 2, 8, 9, and 3. Extrinsic apoptosis was induced by the AVN 2f-mediated activation of caspase 8, while the intrinsic apoptotic pathway was triggered by MOR-induced increase in the levels of intracellular reactive oxygen species, MOR-mediated activation of caspases 2 and 9 and the MOR-mediated downregulation of the prosurvival gene BIRC5. Our results suggest that the combination MOR + AVN 2f could be an effective chemopreventive cocktail against the development of hepatocarcinoma.

Novel insights into pericarp, protein body globoids of aleurone layer, starchy granules of three cereals gained using atomic force microscopy and environmental scanning electronic microscopy.

In this study, we applied Environmental Scanning Electron Microscopy-Energy Dispersive Spectroscopy (ESEM-EDS) and Atomic Force Microscopy (AFM) analysis to three different cereal caryopses: barley, oat and einkorn wheat. The morphological structures, chemical elemental composition and surface characteristics of the three cereals were described. Regarding the morphology, barley showed the thickest pericarp, providing a strong barrier digestion and absorption of nutrients. The aleurone layer of each cereal type contained protein body globoids within its cells. Large type-A and small type-B starchy granules were revealed in the endosperm of barley and einkorn wheat, whereas irregular starchy granules were found in oats. The starchy granule elemental composition, detected by ESEM-EDS, was rather homogenous in the three cereals, whereas the pericarp and protein body globoids showed heterogeneity. In the protein body globoids, oats showed higher P and K concentrations than barley and einkorn wheat. Regarding the topographic profiles, detected by AFM, einkorn wheat starchy granules showed a surface profile that differed significantly from that of oats and barley, which were quite similar to one another. The present work provides insights into the morphological and chemical makeup of the three grains shedding light on the higher bio-accessibility of einkorn wheat nutrients compared to barley and oats, providing important suggestions for human nutrition and technological standpoints.

Antiproliferative activity of vitexin-2-O-xyloside and avenanthramides on CaCo-2 and HepG2 cancer cells occurs through apoptosis induction and reduction of pro-survival mechanisms.

PURPOSE: CaCo-2 colon cancer cells and HepG2 liver cancer cells represent two malignant cell lines, which show a high resistance to apoptosis induced by the conventional anticancer drugs. Vitexin-2-O-xyloside (XVX) and avenanthramides (AVNs) are naturally occurring dietary agents from Beta vulgaris var. cicla L. and Avena sativa L., respectively. The aim of this work was to evaluate the antiproliferative effects and the reduction of the pro-survival mechanisms exerted by XVX and AVNs, used individually and in combination, in CaCo-2 and HepG2 cancer cells. METHODS: XVX and AVNs were isolated by liquid chromatography and characterized by HPLC-PDA-MS. The XVX and AVN antiproliferative effects were evaluated through sulforhodamine B method, while their pro-apoptotic effects through caspase activity assays. RTqPCR was used to investigate the modulation of the pro-survival factors baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), hypoxia inducible factor 1 A (HIF1A), and vascular endothelial growth factor A (VEGFA). Cellular antioxidant activity (CAA) was investigated by means of DCFH-DA assay, whereas chemical antioxidant capacity was evaluated by the ORAC method. RESULTS: XVX and AVNs, both individually and in combination, inhibited the proliferation of CaCo-2 and HepG2 cancer cells, through activation of caspases 9, 8, and 3. XVX and AVNs downregulated the pro-survival genes BIRC5, HIF1A, and VEGFA. The CAA assay showed that AVNs exhibited strong antioxidant activity inside both CaCo-2 and HepG2 cells. CONCLUSIONS: The antiproliferative activity of the XVX + AVNs mixture represents an innovative treatment, which is effective against two types of cancer cells characterized by high resistance to the conventional anticancer drugs.

The effect of mechanical processing on avenanthramide and phenol levels in two organically grown Italian oat cultivars.

Avenanthramides (AVNs), free and bound phenols and their antioxidant capacities (ORAC) were evaluated in two Avena sativa L. cultivars, Donata and Flavia. The cultivars (cvs.) were grown in loamy and medium texture soils and assessed after industrial dehulling and milling. Total dietary fiber, ß-glucan, starch and proteins were also evaluated. Cv. Donata showed 2.8 fold higher AVN storage as compared to cv. Flavia, which was linked with genotype. The accumulation of AVN content was also influenced by the texture of the soil. Dehulling resulted in a 75 and 37% AVN decrease in cv. Donata and Flavia, respectively. The dehulled grains of cv. Donata showed 40% reduction in free phenolic content, whereas the dehulled grains of both cvs. showed 67% reduction in bound phenols. Milling affected the bound phenolics and their antioxidant capacity. Cv. Flavia showed 1.3 fold higher ß-glucan than that of cv. Donata. Total dietary fiber was reduced by 50 and 12% after dehulling and milling, respectively, while marginal changes in proteins were observed after milling. The results suggest that the choice of genotype and the kind of dehulling processes that are employed are essential considerations in the production of oat-based products with high AVN content and extra health benefits.

C-Glycosyl Flavonoids from Beta vulgaris Cicla and Betalains from Beta vulgaris rubra: Antioxidant, Anticancer and Antiinflammatory Activities-A Review.

The green beet (Beta vulgaris var. cicla L.) and red beetroot (B. vulgaris var. rubra L.) contain phytochemicals that have beneficial effects on human health. Specifically, the green beet contains apigenin, vitexin, vitexin-2-O-xyloside and vitexin-2-O-rhamnoside, while the red beetroot is a source of betaxanthins and betacyanins. These phytochemicals show considerable antioxidant activity, as well as antiinflammatory and antiproliferative activities. Vitexin-2-O-xyloside, in combination with betaxanthins and betacyanins, exerts antiproliferative activity in breast, liver, colon and bladder cancer cell lines, through the induction of both intrinsic and extrinsic apoptotic pathways. A significant body of evidence also points to the role of these phytochemicals in the downregulation of the pro-survival genes, baculoviral inhibitor of apoptosis repeat-containing 5 and catenin beta-1, as well as the genes controlling angiogenesis, hypoxia inducible factor 1A and vascular endothelial growth factor A. The multi-target action of these phytochemicals enhances their anticancer activity. Vitexin-2-O-xyloside, betaxanthins and betacyanins can be used in combination with conventional anticancer drugs to reduce their toxicity and overcome the multidrug resistance of cancer cells. In this review, we describe the molecular mechanisms that enable these dietary phytochemicals to block the proliferation of tumor cells and inhibit their pro-survival pathways. Copyright © 2017 John Wiley & Sons, Ltd.

Quantity and quality of secoiridoids and lignans in extra virgin olive oils: the effect of two- and three-way decanters on Leccino and Raggiola olive cultivars.

In this investigation, 14 extra virgin olive oils (EVOOs), produced with Leccino and Raggiola olive cultivars, by a new two-way (2W) decanter were compared with 14 EVOOs produced by means of a conventional three-way (3W) decanter. The 2W EVOOs had higher phenol concentrations, as shown by high-performance liquid chromatography/diode array detection (HPLC-DAD) analysis and yielded a higher extraction of the 3,4-DHPEA-EDA (oleacein), 3,4-DHPEA-EA (oleuropein aglycone) and p-HPEA-EDA (oleocanthal). The concentrations of lignans, (+)-pinoresinol and (+)-1-acetoxypinoresinol, detected by HPLC-FLD equipment, were higher in the 2W EVOOs than they were in EVOOs produced using the 3W system. Total phenols, detected by the Folin-Ciocalteu assay, were lower than those obtained by HPLC, but they significantly correlated (p < 0.05). The antioxidant capacity (ORAC) values of 2W EVOOs were higher than those of 3W EVOOs. In conclusion, the 2W system provided high-quality phenol EVOOs and became an indispensable tool when adverse climatic conditions reduced the olive secoiridoid concentration.

Histone demethylase JARID1C inactivation triggers genomic instability in sporadic renal cancer.

Mutations in genes encoding chromatin-remodeling proteins are often identified in a variety of cancers. For example, the histone demethylase JARID1C is frequently inactivated in patients with clear cell renal cell carcinoma (ccRCC); however, it is largely unknown how JARID1C dysfunction promotes cancer. Here, we determined that JARID1C binds broadly to chromatin domains characterized by the trimethylation of lysine 9 (H3K9me3), which is a histone mark enriched in heterochromatin. Moreover, we found that JARID1C localizes on heterochromatin, is required for heterochromatin replication, and forms a complex with established players of heterochromatin assembly, including SUV39H1 and HP1α, as well as with proteins not previously associated with heterochromatin assembly, such as the cullin 4 (CUL4) complex adaptor protein DDB1. Transcription on heterochromatin is tightly suppressed to safeguard the genome, and in ccRCC cells, JARID1C inactivation led to the unrestrained expression of heterochromatic noncoding RNAs (ncRNAs) that in turn triggered genomic instability. Moreover, ccRCC patients harboring JARID1C mutations exhibited aberrant ncRNA expression and increased genomic rearrangements compared with ccRCC patients with tumors endowed with other genetic lesions. Together, these data suggest that inactivation of JARID1C in renal cancer leads to heterochromatin disruption, genomic rearrangement, and aggressive ccRCCs. Moreover, our results shed light on a mechanism that underlies genomic instability in sporadic cancers.

H3K4me3 demethylation by the histone demethylase KDM5C/JARID1C promotes DNA replication origin firing.

DNA replication is a tightly regulated process that initiates from multiple replication origins and leads to the faithful transmission of the genetic material. For proper DNA replication, the chromatin surrounding origins needs to be remodeled. However, remarkably little is known on which epigenetic changes are required to allow the firing of replication origins. Here, we show that the histone demethylase KDM5C/JARID1C is required for proper DNA replication at early origins. JARID1C dictates the assembly of the pre-initiation complex, driving the binding to chromatin of the pre-initiation proteins CDC45 and PCNA, through the demethylation of the histone mark H3K4me3. Fork activation and histone H4 acetylation, additional early events involved in DNA replication, are not affected by JARID1C downregulation. All together, these data point to a prominent role for JARID1C in a specific phase of DNA replication in mammalian cells, through its demethylase activity on H3K4me3.

Rescue of Hippo coactivator YAP1 triggers DNA damage-induced apoptosis in hematological cancers.

Oncogene-induced DNA damage elicits genomic instability in epithelial cancer cells, but apoptosis is blocked through inactivation of the tumor suppressor p53. In hematological cancers, the relevance of ongoing DNA damage and the mechanisms by which apoptosis is suppressed are largely unknown. We found pervasive DNA damage in hematologic malignancies, including multiple myeloma, lymphoma and leukemia, which leads to activation of a p53-independent, proapoptotic network centered on nuclear relocalization of ABL1 kinase. Although nuclear ABL1 triggers cell death through its interaction with the Hippo pathway coactivator YAP1 in normal cells, we show that low YAP1 levels prevent nuclear ABL1-induced apoptosis in these hematologic malignancies. YAP1 is under the control of a serine-threonine kinase, STK4. Notably, genetic inactivation of STK4 restores YAP1 levels, triggering cell death in vitro and in vivo. Our data therefore identify a new synthetic-lethal strategy to selectively target cancer cells presenting with endogenous DNA damage and low YAP1 levels.

Targeting Mcl-1 for multiple myeloma (MM) therapy: drug-induced generation of Mcl-1 fragment Mcl-1(128-350) triggers MM cell death via c-Jun upregulation.

Myeloid cell leukemia-1 (Mcl-1, HGNC: 6943), a pro-survival member of the Bcl-2 family, plays a crucial role in Multiple Myeloma (MM) pathogenesis and drug resistance, thus representing a promising therapeutic target in MM. A novel strategy to inhibit Mcl-1 activity is the induction of ubiquitin-independent Mcl-1 degradation. Our own and other previous studies have demonstrated caspase-dependent generation of a 28kDa Mcl-1 fragment, Mcl-1(128-350), which inhibits MM cell proliferation and survival. Here, we show that similar to bortezomib, the novel proteasome inhibitors carfilzomib and ixazomib, as well as staurosporine and adaphostin, induce the generation of Mcl-1(128-350) in MM cells. Next, the molecular sequelae downstream of Mcl-1(128-350), which mediate its pro-apoptotic activity, were delineated. Surprisingly, we observed nuclear accumulation of drug-induced or exogenously overexpressed Mcl-1(128-350), followed by elevated mRNA and protein levels of c-Jun, as well as enhanced AP-1 reporter activity. Moreover, drug-induced AP-1 activity was blocked after introducing a point mutation into the highly conserved Mcl-1 caspase-cleavage site Asp127, but not Asp157. Consequently, drug-triggered cell death was significantly decreased in MM cells transfected with Mcl-1 D127A, but not with Mcl-1 D157A. Consistent with these data, treatment with bortezomib triggered c-Jun upregulation followed by apoptosis in Mcl-1(wt/wt), but not Mcl-1(Δ/null) murine embryonic fibroblasts (MEFs). Transfection of a plasmid carrying Mcl-1(wt) into Mcl-1(Δ/null) MEFs restored bortezomib-induced Mcl-1 fragmentation, c-Jun upregulation and AP-1 reporter activity. Finally, our data indicate that drug-induced generation of a pro-apoptotic Mcl-1 fragment followed by c-Jun upregulation may also be a novel therapeutic approach in other tumor entities.