Andrea Romano

Particle bombardment and the genetic enhancement of crops: myths and realities

DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation method, not limited by cell type, species or genotype. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene cotransformation is straightforward. The perceived disadvantages of particle bombardment compared to Agrobacterium-mediated transformation, i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. There is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals and legumes, and other non-model systems. Indeed, a major advantage of particle bombardment is that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This has been demonstrated in recently reported strategies that favor the recovery of transgenic plants containing intact, single-copy integration events, and demonstrating high-level transgene expression. At the current time, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation. In this review, we discuss recent data highlighting the positive impact of particle bombardment on the genetic transformation of plants, focusing on the fate of exogenous DNA, its organization and its expression in the plant cell. We also discuss some of the most important applications of this technology including the deployment of transgenic plants under field conditions.

Increased Production of 17β-Estradiol in Endometriosis Lesions Is the Result of Impaired Metabolism

CONTEXT substantial evidence suggests that the expression of steroid metabolizing enzymes in endometriosis is altered, turning the ectopic endometrium into a source of 17beta-estradiol. However, whether these differences result in a net increase in local 17beta-estradiol production/activity has not been shown. SUBJECTS AND METHODS The activities of the most important steroidogenic enzymes synthesizing and inactivating 17beta-estradiol were determined by HPLC in matched eutopic and ectopic tissue from patients with endometriosis (n = 14) and in endometrium from controls (n = 20). RESULTS Aromatase activity is negligible in the ectopic endometrium, whereas the activity of estrogen sulfatase is high though not different between ectopic, eutopic and control endometrium. The activity of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) converting estrone into 17beta-estradiol is higher in the ectopic compared to the eutopic endometrium in patients. The activity of 17beta-HSDs converting 17beta-estradiol back to estrone is significantly lower in the ectopic compared to the eutopic endometrium of both patients and controls. To evaluate the net metabolic capacity of tissues to synthesize 17beta-estradiol, we calculated the activity ratio between 17beta-HSDs synthesizing versus 17beta-HSDs inactivating 17beta-estradiol. This ratio is significantly higher in the ectopic compared to the eutopic endometrium of patients and controls, indicating a high synthesis of 17beta-estradiol in the ectopic locations. This is further supported by the elevated mRNA levels of the estrogen-responsive gene TFF1 in all ectopic compared to eutopic endometria. CONCLUSION Endometriotic lesions have higher production of 17beta-estradiol than the eutopic endometrium of patients and controls. This is mostly the result of impaired metabolism.

Transgene organisation in potato after particle bombardment-mediated (co-)transformation using plasmids and gene cassettes.

Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transformed with a plasmid containing the selectable marker contained one and two additional non-selected genes, respectively. When gene cassettes were used in transformation, six out of eight plants were co-transformed. Expression analysis showed that 75–80% of the plants transformed with two transgenes expressed both of them, irrespective of the use of plasmids or gene cassettes. Thirty-eight plants containing the gusA reporter-gene and the nptII selectable-marker have been characterised with respect to the molecular organisation of the donor DNAs. Seventeen out of 49 (35%) gusA sites of integration contained one copy of the gene. Only 11 gusA sites (22%) were linked to the site of integration of the selectable marker. When one site of integration contained several copies of the transgene, a predominance of 3′–3′ inverted re-arrangement repeats was observed.

Nonnutritive Sucking during Heelstick Procedures Decreases Behavioral Distress in the Newborn Infant

We investigated if nonnutritive sucking (NNS) during heelstick procedures alleviates behavioral distress in neonates. In our NICU, 26 neonates without severe complications (mean Minde score 0.8, range 0–3), undergoing heelstick procedures at least twice a day, in the first 2 weeks of life, were enrolled in the trial (mean gestational age 33.9 weeks, range 26–39 weeks, mean birth weight 1,988.5 g, range 1,200–4,010 g, mean Apgar score at the first minute 6.7, range 4–10, at the fifth minute 8.5, range 6–10). Two heelpricks were performed in each neonate with NNS randomly assigned. Behavioral states, transcutaneous oxygen tension (TcPO2), heart rate, and respiratory rate were monitored before, during and after the heelstick procedures. Heelstick procedures lasted for a mean of 109 s (range 50–230 s) with NNS, and a mean of 128.8 s (range 20–420 s) without NNS. Compared with baseline, heart rate and behavioral distress increased and respiratory rate decreased during heelstick and after heelstick. Oxygen tension did not change. Nonnutritive sucking had no effect on respiratory rate or transcutaneous oxygen tension, but reduced the time of crying and the heart rate increase during the procedure. In conclusion, NNS can be recommended to reduce distress in newborns undergoing invasive routine procedures. Further studies are needed to evaluate the effects of NNS on respiratory rate and blood gas levels.

Next-Generation Sequencing in Oncology: Genetic Diagnosis, Risk Prediction and Cancer Classification

Next-generation sequencing (NGS) technology has expanded in the last decades with significant improvements in the reliability, sequencing chemistry, pipeline analyses, data interpretation and costs. Such advances make the use of NGS feasible in clinical practice today. This review describes the recent technological developments in NGS applied to the field of oncology. A number of clinical applications are reviewed, i.e., mutation detection in inherited cancer syndromes based on DNA-sequencing, detection of spliceogenic variants based on RNA-sequencing, DNA-sequencing to identify risk modifiers and application for pre-implantation genetic diagnosis, cancer somatic mutation analysis, pharmacogenetics and liquid biopsy. Conclusive remarks, clinical limitations, implications and ethical considerations that relate to the different applications are provided.

Aberrations in the progesterone receptor gene and the risk of recurrent endometrial carcinoma

A case–control study was performed in order to determine whether expression of the progesterone receptor (PR) and/or aberrations of the PR gene contribute to the development of recurrent endometrial carcinoma. Primary tumours from 44 patients with recurrence of stage I endometrial carcinoma (patients) within 3 years after initial treatment were compared with tumours from 44 matched patients who were free of recurrence for a minimum of 3 years (controls). Paraffin wax‐embedded primary tumours (n = 88) and recurrent tumours (n = 32) were analysed immunohistochemically for PR expression. A staining index (SI = 0–9) based on the staining intensity and the number of stained cells was calculated. DNA extracted from paraffin wax‐embedded tissues was subjected to PCR–restriction fragment length polymorphism analysis (PCR–RFLP) for determination of the PROGINS DNA sequence alterations and the +331G/A‐promoter polymorphism. Low PR expression (SI < 1.0) was observed in 7% of primary tumours derived from controls, 25% of primary tumours from patients with recurrence, and 38% of recurrent tumours. The expression of PR was significantly lower in primary tumours from patients with recurrence (SI = 4.0 ± 0.5) than in the tumours in the control group (SI = 5.6 ± 0.5) (T‐test for paired analysis, p < 0.05). The PROGINS and +331G/A‐promoter polymorphism were not related to age at diagnosis, tumour grade or myometrial invasion. The +331G/A‐promoter polymorphism was present in 14% of primary tumours from patients without recurrence, compared with 17% of patients with recurrence. The PROGINS polymorphism was observed in 16% of primary tumours from patients without, and in 34% of patients with, recurrence (OR 2.6; 95% CI: 0.9–7.6). Most interestingly, patients who carried the PROGINS variant and in whom a PR‐expressing tumour was diagnosed were at significantly enhanced risk of relapse (OR 4.7; 95% CI: 1.3–17.1). In conclusion, low PR expression tended to be associated with recurrent disease, and PR expression in tumours from patients carrying the PROGINS allele was predictive of the risk of recurrence. Copyright

Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer

BackgroundAbout 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients.MethodsCTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn’s post-test was used for comparisons between groups. Statistical significance was set at p < 0.05.ResultsEpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis.ConclusionsOur results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.

Overexpression of 17β-hydroxysteroid dehydrogenase type 1 increases the exposure of endometrial cancer to 17β-estradiol.

CONTEXT The local interconversions between estrone (low activity) and 17β-estradiol (potent compound) by 17β-hydroxysteroid dehydrogenases (17β-HSDs) can lead to high 17β-estradiol generation in endometrial cancer (EC). OBJECTIVE Examine the balance between the 17β-HSDs reducing estrone to 17β-estradiol (types 1, 5, 12, and 7) and those oxidizing 17β-estradiol to estrone (2, 4, and 8), in EC. PATIENTS AND METHODS Reducing and oxidizing 17β-HSD activities (HPLC) and mRNA level (RT-PCR) were assessed in normal post-menopausal (n = 16), peritumoral endometrium (normal tissue beside cancer, n = 13), and 58 EC (29 grade 1, 18 grade 2, 11 grade 3). RESULTS Grade 1 EC displayed a shifted estrone reduction/17β-estradiol oxidation balance in favor of 17β-estradiol compared with controls. This was more pronounced among estrogen receptor-α (ER-α)-positive biopsies. Type 1 17β-HSD mRNA (HSD17B1 gene expression, real time PCR) and protein levels (immunohistochemistry) were higher in ER-α-positive grade 1 EC than controls. The mRNA coding for types 4, 5, 7, 8, and 12 17β-HSD did not vary, whereas that coding for type 2 17β-HSD was increased in high-grade lesions compared with controls. Three-dimensional ex vivo EC explant cultures demonstrated that 17β-HSD type 1 generated 17β-estradiol from estrone and increased tumor cell proliferation. Additional in vitro studies using EC cells confirmed that in the presence of 17β-HSD type 1, estrone induced estrogen signaling activation similarly to 17β-estradiol. Therefore, estrone was reduced to 17β-estradiol. CONCLUSIONS Type 1 17β-HSD increases 17β-estradiol exposure in grade 1 EC, thus supporting tumor growth. This enzyme represents a potential therapeutic target.

Deoxyribonucleic acid methyltransferases and methyl-CpG-binding domain proteins in human endometrium and endometriosis.

OBJECTIVE To determine [1] expression levels of both DNA methyltransferases (DNMTs) and methyl-CpG-binding domain proteins (MBDs) in human endometrium throughout the menstrual cycle and in eutopic and ectopic endometrium of patients with endometriosis and [2] hormone responsiveness of DNMT and MBD expression in explant cultures of proliferative phase endometrium. DESIGN In vitro study. SETTING Academic medical center. PATIENT(S) Premenopausal women with and without endometriosis. INTERVENTION(S) Explant cultures of proliferative phase endometrium were treated with vehicle, 17β-E(2), or a combination of E(2) and P (E(2) + P) for 24 hours. MAIN OUTCOME MEASURE(S) Expression levels of DNMT1, DNMT2, and DNMT3B and MBD1, MBD2, and MeCP2 with use of real-time quantitative polymerase chain reaction. RESULT(S) Expression levels of DNMT1 and MBD2 were significantly higher in secretory-phase endometrium compared with proliferative endometrium and menstrual endometrium. In explant cultures, treatment with E(2) + P resulted in significant up-regulation of DNMT1 and MBD2. Expression levels of several DNMTs and MBDs were significantly lower in endometriotic lesions compared with eutopic endometrium of women with endometriosis and disease-free controls. CONCLUSION(S) These findings suggest a role for DNMTs and MBDs in the growth and differentiation of the human endometrium and support the notion that endometriosis may be an epigenetic disease.

Wound administration of M2-polarized macrophages does not improve murine cutaneous healing responses.

Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds.