Development of a non-chemical RNAi-based strategy for Amaranthus hybridus L. weed management

Weeds are one of the major issues in cropping systems, responsible for significant yield losses. Herbicide applications are the most effective strategy to control weeds, but stricter legislation has resulted in a significant reduction in the number of herbicides available on the market. Furthermore, the recent European legislation on the sustainable use of pesticides will require farmers to drastically reduce chemical use over the next ten years while promoting integrated weed management strategies that improve environmental sustainability and lower the risks to animal and human health. In addition, the over-reliance on chemical control has resulted in the evolution of resistant biotypes. As a result, new technologies to effectively manage weeds and weed resistance should be developed. In this regard, the development of a non-chemical weed control strategy based on RNA interference (RNAi) technology could: i) represent a potential non-chemical weed control strategy, ii) provide an emerging GMO-free strategy for managing invasive and resistant weeds, and iii) provide a valid opportunity to go inside the molecular mechanisms of weed biology. In this study, the acetolactate synthase (ALS) gene of Amaranthus hybridus L. has been used as the target to assess the effectiveness and applicability of in-vitro synthesized double-stranded RNAs (dsRNAs) direct application for endogenous gene silencing and weed control. A. hybridus is a monoecious and self-pollinated weed that has evolved multiple resistance to herbicides with different sites of action, including ALS inhibitors, which are the most used herbicides in soybean. ALS represents an ideal target for the development and future application of dsRNA-mediated gene silencing because it is an intronless, nucleotide-stable, and single-copy gene. We have produced dsRNAs of various lengths (ranging from 218 to 460bp) targeting three distinct ALS regions: the 5’- and 3’-ends, and a central region. dsRNAs molecules were transcribed in-vitro by T7 RNA polymerase and externally applied to the abaxial leaf surface of A. hybridus plants at 4-6 true leaves developmental stage by: i) mechanical inoculation, or ii) high-pressure spraying. Despite the expression of ALS gene transcripts was found to be lightly downregulated when synthetic 2 ALS-dsRNAs were applied, no phenotypic effects were observed. Our current research focuses on the determination of the effectiveness of ALS-dsRNAs silencing using agroinfiltration techniques, and on dsRNAs delivery techniques through the use of nanomaterials to maximize the effectiveness of gene silencing by exogenous dsRNAs application. This second approach was preliminary studied by RNA electrophoretic mobility of functionalized nanomaterial and by means of confocal microscopy on A. hybridus leaves. In parallel, we are examining the expression patterns of genes thought to be involved in the RNAi pathway in A. hybridus to verify if their expression is triggered by dsRNA applications

The Suicide Gene Therapy Challenge: How to Improve a Successful Gene Therapy Approach

The transfer of a suicide gene into donor lymphocytes to control alloreactivity in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT) represents the widest clinical application of T-cell based gene transfer, as shown by more than 100 patients treated worldwide to date, several phase I–II studies completed, and a registrative phase III study, sponsored by a biotech firm, about to begin. In this mini-review, we will summarize the clinical results obtained to date, and attempt to identify the steps envisaged to optimize the suicide gene therapy approach

Anastomosis configuration and technique following ileocaecal resection for Crohn's disease: a multicentre study

A limited ileocaecal resection is the most frequently performed procedure for ileocaecal CD and different anastomotic configurations and techniques have been described. This manuscript audited the different anastomotic techniques used in a national study and evaluated their influence on postoperative outcomes following ileocaecal resection for primary CD. This is a retrospective, multicentre, observational study promoted by the Italian Society of Colorectal Surgery (SICCR), including all adults undergoing elective ileocaecal resection for primary CD from June 2018 May 2019. Postoperative morbidity within 30 days of surgery was the primary endpoint. Postoperative length of hospital stay (LOS) and anastomotic leak rate were the secondary outcomes. 427 patients were included. The side to side anastomosis was the chosen configuration in 380 patients (89%). The stapled anastomotic (n = 286; 67%), techniques were preferred to hand-sewn (n = 141; 33%). Postoperative morbidity was 20.3% and anastomotic leak 3.7%. Anastomotic leak was independent of the type of anastomosis performed, while was associated with an ASA grade ≥ 3, presence of perianal disease and ileocolonic localization of disease. Four predictors of LOS were identified after multivariate analysis. The laparoscopic approach was the only associated with a reduced LOS (p = 0.017), while age, ASA grade ≥ 3 or administration of preoperative TPN were associated with increased LOS. The side to side was the most commonly used anastomotic configuration for ileocolic reconstruction following primary CD resection. There was no difference in postoperative morbidity according to anastomotic technique and configuration. Anastomotic leak was associated with ASA grade ≥ 3, a penetrating phenotype of disease and ileo-colonic distribution of CD

National variations in perioperative assessment and surgical management of Crohn's disease: a multicentre study

Aim: Crohn's disease (CD) requires a multidisciplinary approach and surgery should be undertaken by dedicated colorectal surgeons with audited outcomes. We present a national, multicentre study, with the aim to collect benchmark data on key performance indicators in CD surgery, to highlight areas where standards of CD surgery excel and to facilitate targeted quality improvement where indicated. Methods: All patients undergoing ileocaecal or redo ileocolic resection in the participating centres for primary and recurrent CD from June 2018 to May 2019 were included. The main objective was to collect national data on hospital volume and practice variations. Postoperative morbidity was the primary outcome. Laparoscopic surgery and stoma rate were the secondary outcomes. Results: In all, 715 patients were included: 457 primary CD and 258 recurrent CD with a postoperative morbidity of 21.6% and 34.7%, respectively. Laparoscopy was used in 83.8% of primary CD compared to 31% of recurrent CD. Twenty-five hospitals participated and the total number of patients per hospital ranged from 2 to 169. Hospitals performing more than 10 primary CD procedures per year showed a higher adoption of laparoscopy and bowel sparing surgery. Conclusions: There is significant heterogeneity in the number of CD surgeries performed per year nationally in Italy. Our data suggest that high-volume hospitals perform more complex procedures, with a higher adoption of bowel sparing surgery. The rate of laparoscopy in high-volume hospitals is higher for primary CD but not for recurrent CD compared with low-volume hospitals

Development and application of an exogenous RNAi-based approach for acetolactate synthase (ALS) gene silencing in Amaranthus hybridus L.

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Therapeutic and diagnostic applications of minor histocompatibility antigen HA-1 and HA-2 disparities in allogeneic hematopoietic stem cell transplantation: a survey of different populations.

Minor histocompatibility antigens (mHags) HA-1 and HA-2 are encoded by biallelic loci, with immunogenic variants, HA-1(H) and HA-2(V), which induce strong HLA-A2-restricted alloreactive T-cell responses, and nonimmunogenic counterparts, HA-1(R) and HA-2(M), which represent functional null alleles that are poorly presented by HLA class I molecules. HA-1 and HA-2 are potential targets of selective graft-versus-leukemia,e and graft-versus-tumor reactivity after allogeneic hematopoietic stem cell transplantation (HSCT); however, these applications are restricted to a limited number of patients. Here, we show that a far more frequent application of HA-1 and HA-2 disparity relies on their use as markers for the state of host chimerism after allogeneic HSCT. We have determined allelic frequencies of 29.3% and 70.7% for HA-1(H) and HA-1(R), respectively, and of 83.7% and 16.3% for HA-2(V) and HA-2(M), respectively, in > 200 healthy individuals from northern Italy. Similar frequencies were observed in nearly 100 patients affected by hematologic malignancies or solid tumors, thus showing that HA-1 and HA-2 variabilitv are not associated with the presence of cancer. On the basis of these data, we predict that HA-1 and HA-2 can be used in 32.8% and 23.5% of Italian transplant patients, respectively, as markers for the state of host chimerism, whereas exploitation of disparity for these mHags for targeted immunotherapy will be possible in 10.7% and 1.1% of Italian patients, respectively. Retrospective HA-2 typing of bone marrow., aspirates obtained from a patient during complete remission or recurrence of acute myeloid leukemia after haploidentical HSCT showed the feasibility of using HA-2 as a surrogate marker for disease monitoring. Because of an apparent north-south gradient for HA-1 allelic frequencies, with higher frequencies for the HA-1(H) variant reported in white populations from Southern Europe as compared with Northern Europe and North America, the diagnostic applicability of HA-1 disparity will be slightly more frequent in transplant patients from the north. Taken together, our data show that determination of HA-1 and HA-2 variability can be an important parameter for the selection of allogeneic stem cell donors, in particular for patients affected by hematologic malignancies without a tumor-specific molecular marker. (c) 2006 American Society for Blood and Marrow Transplantation

In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells

A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow-transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCMcontribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCMare able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings