Effective Identification of Low-Gliadin Wheat Lines by Near Infrared Spectroscopy (NIRS): Implications for the Development and Analysis of Foodstuffs Suitable for Celiac Patients

Scope

The aim of this work was to assess the ability of Near Infrared Spectroscopy (NIRS) to distinguish wheat lines with low gliadin content, obtained by RNA interference (RNAi), from non-transgenic wheat lines. The discriminant analysis was performed using both whole grain and flour. The transgenic sample set included 409 samples for whole grain sorting and 414 samples for flour experiments, while the non-transgenic set consisted of 126 and 156 samples for whole grain and flour, respectively.

Methods and Results

Samples were scanned using a Foss-NIR Systems 6500 System II instrument. Discrimination models were developed using the entire spectral range (400–2500 nm) and ranges of 400–780 nm, 800–1098 nm and 1100–2500 nm, followed by analysis of means of partial least square (PLS). Two external validations were made, using samples from the years 2013 and 2014 and a minimum of 99% of the flour samples and 96% of the whole grain samples were classified correctly.

Conclusions

The results demonstrate the ability of NIRS to successfully discriminate between wheat samples with low-gliadin content and wild types. These findings are important for the development and analysis of foodstuff for celiac disease (CD) patients to achieve better dietary composition and a reduction in disease incidence.     

Loss of a reporter gene for green fluorescent protein during tumor progression suggests the recruitment of host cells in rats with experimentally induced colon cancer

The interactions between a host's normal cells and tumor cells appear to be of significant importance during the development of tumors. In the present study, we examined this issue using a cancer model in vivo in which tumor cells were tagged with a reporter gene for green fluorescent protein (GFP). We used a model of colon cancer in immunocompetent rats, which were given a subcutaneous injection of tumor cells that had been transfected with a gene for GFP. We found that the number of fluorescent cells decreased with the progression of the primary tumors and that lymph node and lung metastases were never macroscopically fluorescent. No GFP-encoding sequences were detected by PCR in many of the long-term primary tumors, in most lymph node metastases (86%) and in all lung metastases, whereas the detection of mutated k-ras, which identified such cells as tumor cells, was always positive. To explain these findings, we present a brief review of the literature and postulate that tumor growth did not occur exclusively as a result of the division of the injected cells, but also involved recruitment of host cells.     

Effects of wire-bottom caging on heart rate, activity and body temperature in telemetry-implanted rats

Some experimental procedures are associated with placement of animals in wire-bottom cages. The goal of this study was to evaluate stress-related physiological parameters (heart rate [HR], body temperature [BT], locomotor activity [LA], body weight [BW] and food consumption) in rats under two housing conditions, namely in wire-bottom cages and in bedding-bottom cages. Telemetry devices were surgically implanted in male Sprague-Dawley rats. HR, BT and LA were recorded at 5 min intervals. Analysis under each housing condition was performed from 16:00 to 08:00 h of the following day (4 h light, 12 h dark). During almost all of the light phase, the HR of rats housed in wire-bottom cages remained high (371 ± 35 bpm; mean ± SD; n = 6) and was significantly different from that of rats housed in bedding-bottom cages (340 ± 29 bpm; n = 6; P < 0.001; Student's t-test). In general, BT was similar under the two housing conditions. However, when rats were in wire-bottom cages, BT tended to fluctuate more widely during the dark phase. LA decreased when animals were housed in wire-bottom cages, in particular during the dark phase. Moreover, there was a significant difference with respect to the gain in BW: BW of rats housed in bedding-bottom cages increased 12 ± 2 g, whereas that of rats in wire-bottom cages decreased by 2 ± 3 g (P < 0.001). Our results demonstrate that housing rats in wire-bottom cages overnight leads to immediate alterations of HR, BW and LA, which might be related to a stress response.     

Circulating nucleic acids in plasma and serum (CNAPS) and its relation to stem cells and cancer metastasis: state of the issue

The presence of circulating cell-free nucleic acids has been demonstrated both in disease and health. In the last decade, a burst of papers about Circulating Nucleic Acids in Plasma and Serum (CNAPS) have been found in the literature, showing the scientific interest raised by this phenomenon and their putative clinical interest, especially in the field of cancer. Today, the detection of extracellular tumor-derived DNA and/or RNA is considered by many authors as a new molecular marker for situations such as cancer diagnosis, monitoring the outcome of a disease and, even, as a treatment response indicator. Furthermore, in some studies it has been suggested a possible role of tumor CNAPS in the development of metastasis. Specifically, the hypothesis known as the "genometastasis hypothesis" proposes that stem cells might be naturally transfected with dominant oncogenes as a result of dissemination of such genes in the plasma. On the other hand, current studies concerned with the biology of metastatic cells are increasingly being focused on the striking similarities found between these cells and stem cells. In this review we intend to expound and integrate two theories about metastatization: the "genometastasis hypothesis" and the idea of stem cells as cancer stem cells.     

Rat model of anal sphincter injury and two approaches for stem cell administration

AIM To establish a rat model of anal sphincter injury and test different systems to provide stem cells to injured area.METHODS Adipose-derived stem cells(ASCs) were isolated from BDIX rats and were transfected with green fluorescent protein(GFP) for cell tracking. Biosutures(sutures covered with ASCs) were prepared with 1.5 × 10~6 GFPASCs, and solutions of 10~6 GFP-ASCs in normal saline were prepared for injection. Anorectal normal anatomy was studied on Wistar and BDIX female rats. Then, we designed an anal sphincter injury model consisting of a 1-cm extra-mucosal miotomy beginning at the anal verge in the anterior middle line. The sphincter lesion was confirmed with conventional histology(hematoxylin and eosin) and immunofluorescence with 4', 6-diamidino-2-phenylindole(commonly known as DAPI), GFP and α-actin. Functional effect was assessed with basal anal manometry, prior to and after injury. After sphincter damage, 36 BDIX rats were randomized to three groups for:(1) Cell injection without repair;(2) biosuture repair; and(3) conventional suture repair and cell injection. Functional and safety studies were conducted on all the animals. Rats were sacrificed after 1, 4 or 7 d. Then, histological and immunofluorescence studies were performed on the surgical area.RESULTS With the described protocol, biosutures had been covered with at least 820000-860000 ASCs, with 100% viability. Our studies demonstrated that some ASCs remained adhered after suture passage through the muscle. Morphological assessment showed that the rat anal anatomy is comparable with human anatomy; two sphincters are present, but the external sphincter is poorly developed. Anal sphincter pressure data showed spontaneous, consistent, rhythmic anal contractions, taking the form of "plateaus" with multiple twitches(peaks) in each pressure wave. These basal contractions were very heterogeneous; their frequency was 0.91-4.17 per min(mean 1.6980, SD 0.57698), their mean duration was 26.67 s and mean number of peaks was 12.53. Our morphological assessment revealed that with the aforementioned surgical procedure, both sphincters were completely sectioned. In manometry, the described activity disappeared and was replaced by a gentle oscillation of basal line, without a recognizable pattern. Surprisingly, these findings appeared irrespective of injury repair or not. ASCs survived in this potentially septic area for 7 d, at least. We were able to identify them in 84% of animals, mainly in the muscular section area or in the tissue between the muscular endings. ASCs formed a kind of "conglomerate" in rats treated with injections, while in the biosuture group, they wrapped the suture. ASCs were also able to migrate to the damaged zone. No relevant adverse events or mortality could be related to the stem cells in our study. We also did not find unexpected tissue growths. CONCLUSION The proposed procedure produces a consistent sphincter lesion. Biosutures and injections are suitable for cell delivery. ASCs survive and are completely safe in this clinical setting.     

Stem cell therapy applied for digestive anastomosis: Current state and future perspectives

BACKGROUND Digestive tract resections are usually followed by an anastomosis.Anastomotic leakage,normally due to failed healing,is the most feared complication in digestive surgery because it is associated with high morbidity and mortality.Despite technical and technological advances and focused research,its rates have remained almost unchanged the last decades.In the last two decades,stem cells(SCs)have been shown to enhance healing in animal and human studies;hence,SCs have emerged since 2008 as an alternative to improve anastomoses outcomes.AIM To summarise the published knowledge of SC utilisation as a preventative tool for hollow digestive viscera anastomotic or suture leaks.METHODS PubMed,Science Direct,Scopus and Cochrane searches were performed using the key words“anastomosis”,“colorectal/colonic anastomoses”,“anastomotic leak”,“stem cells”,“progenitor cells”,“cellular therapy”and“cell therapy”in order to identify relevant articles published in English and Spanish during the years of 2000 to 2021.Studies employing SCs,performing digestive anastomoses in hollow viscera or digestive perforation sutures and monitoring healing were finally included.Reference lists from the selected articles were reviewed to identify additional pertinent articles.METHODS Given the great variability in the study designs,anastomotic models,interventions(SCs,doses and vehicles)and outcome measures,performing a reliable meta-analysis was considered impossible,so we present the studies,their results and limitations.RESULTS Eighteen preclinical studies and three review papers were identified;no clinical studies have been published and there are no registered clinical trials.Experimental studies,mainly in rat and porcine models and occasionally in very adverse conditions such as ischaemia or colitis,have been demonstrated SCs as safe and have shown some encouraging morphological,functional and even clinical results.Mesenchymal SCs are mostly employed,and delivery routes are mainly local injections and cell sheets followed by biosutures(sutures coated by SCs)or purely topical.As potential weaknesses,animal models need to be improved to make them more comparable and equivalent to clinical practice,and the SC isolation processes need to be standardised.There is notable heterogeneity in the studies,making them difficult to compare.Further investigations are needed to establish the indications,the administration system,potential adjuvants,the final efficacy and to confirm safety and exclude definitively oncological concerns.CONCLUSION The future role of SC therapy to induce healing processes in digestive anastomoses/sutures still needs to be determined and seems to be currently far from clinical use.     

Development of a simple and sensitive technique for detection of point mutations in the K-ras oncogene

We sought to develop a simple and sensitive method based on mutant allele-specific amplification (MASA) for the detection of point mutations in the k-ras oncogene in blood samples. We used MASA and three nested MASA methods to detect a point mutation (GGT→GAT) in rat DHD cells at codon 12 of exon 1 of the k-ras gene. MASA allowed us to detect one k-ras mutated cell on a background of 10⁷ normal cells. The third nested-MASA (nested-MASA.c) method that we developed allowed us to detect one mutated cell among 10¹⁰ normal cells. Our methods should allow the detection of small amounts of mutant k-ras DNA in tissue, serum, and plasma, combining speed with efficiency and specificity.     

Tumor DNA circulating in the plasma might play a role in metastasis. The hypothesis of the genometastasis.

BACKGROUND: Clinical and experimental observations suggest that more than one pathway might be involved in the development of metastases. In the present study, we examined the presence of tumor DNA in plasma using an experimental model in which tumor cells were modified with a genome-associated tag. We also investigated whether plasma of tumor-bearing rats had any effect on cultured cells and healthy animals. METHODS: Transfected cancer cells (DHD/K12-PROb stably transfected with pCDNA3.1CAT.) were injected subcutaneously into the chest of BD-IX rats. Animals were divided into ten groups according to the time between injection of tumor cells and euthanasia. Prior to euthanasia (2-14 week), blood samples were collected by cardiac puncture. To detect circulating tumor cells and CAT-encoding DNA in plasma, we performed PCR with nested primers. Fifty samples of plasma were chosen at random to supplement the medium of fifty cultures of DHD cells for 10-12 days. PCR for the detection of CAT DNA in cells was performed approximately one to two months later. Four healthy rats received an intraperitoneal injection of plasma from a tumor-bearing rat five times at week for 4 to 6 weeks. Animals were sacrificed and samples of liver, kidney, spleen, omentum, blood and lung were processed by PCR for the detection of CAT DNA. RESULTS: Detection of CAT DNA in plasma was slightly more frequent than in the buffy-coat fraction. All surviving cultures that had been supplemented with plasma were positive at some point for CAT DNA. In all four healthy animals injected with plasma of tumor-bearing rats, the marker gene for CAT was found in extracts of lungs. CONCLUSION: Our present observation lead us to propose the following hypothesis. Metastases might develop as a result of transfection of susceptible cells in distant target organs with dominant oncogenes that are present in the circulating plasma and are derived from the primary tumor.