Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

Two well-characterized carbohydrate epitopes are absent in humans but present in other mammals. These are galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc) which are introduced by the activities of two enzymes including α(1,3) galactosyltransferase (encoded by the GGTA1 gene) and CMP-Neu5Gc hydroxylase (encoded by the CMAH gene) that are inactive in humans but present in cattle. Hence, bovine-derived products are antigenic in humans who receive bioprosthetic heart valves (BHVs) or those that suffer from red meat syndrome. Using programmable nucleases, we disrupted (knockout, KO) GGTA1 and CMAH genes encoding for the enzymes that catalyse the synthesis of αGal and Neu5Gc, respectively, in both male and female bovine fibroblasts. The KO in clonally selected fibroblasts was detected by polymerase chain reaction (PCR) and confirmed by Sanger sequencing. Selected fibroblasts colonies were used for somatic cell nuclear transfer (SCNT) to produce cloned embryos that were implanted in surrogate recipient heifers. Fifty-three embryos were implanted in 33 recipients heifers; 3 pregnancies were carried to term and delivered 3 live calves. Primary cell cultures were established from the 3 calves and following molecular analyses confirmed the genetic deletions. FACS analysis showed the double-KO phenotype for both antigens confirming the mutated genotypes. Availability of such cattle double-KO model lacking both αGal and Neu5Gc offers a unique opportunity to study the functionality of BHV manufactured with tissues of potentially lower immunogenicity, as well as a possible new clinical approaches to help patients with red meat allergy syndrome due to the presence of these xenoantigens in the diet

Unexpected impairment of INa underpins reentrant arrhythmias in a knock-in swine model of Timothy syndrome

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Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell-derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and identified conditions for the robust and unlimited expansion of hPS cell-derived PP cells under GMP-compliant conditions through a hypothesis-driven iterative approach. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling, and inhibition of selected branches of the TGFβ and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over 10 passages and 40–45 d, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cell lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant β-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell-derived PP cells. They will enable the establishment of large banks of GMP-produced PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine, and cell therapies

Introduction of the TLA sequencing analyses for a better identification of the transgene integrations, to select porcine founder animals for breeding

Xenotransplantation of modified pig islets could be a possible medical treatment for T1/T2 diabetic patients. Tailored genetic engineering of a genomic safe harbor locus assisted by programmable nucleases (ZFNs, TALENs and CRISPR/Cas9) is making easier and reproducible also the generation of new transgenic large animal models. To validate in vivo our preliminary in vitro data (Mourad et al., 2017), we generated cloned transgenic porcine lines targeting the Insulin-GLP1Ser8M3R transgene (I-GS8M3R) into the genomic safe harbor V2G locus (Chromosome 9-EP2921048), using TALENs. Resulting transgenic pancreatic islets presented an increased glucose-stimulated insulin secretion. The aim of our work was to detect all the possible transgene integrations occurred in our targeted V2G/I-GS8M3R founder lines, introducing the Targeted Locus Amplification (TLA) technique as a new support to our genotyping procedure. TALENs-mediated integrations of transgene I-GS8M3R in V2G locus were achieved nucleofecting two different PERV-C negative and low PERV-A/B lines (female and male). Resulting Puromycin-resistant colonies were genotyped with 4 different PCR assays to select ones, having an intact transgene copy and a targeted V2G-allele, as nuclear donors for the Somatic Cell Nuclear Transfer (SCNT) experiments. At birth tissue biopsies were taken from alive piglets for genotyping analyses, identifying the donor colony, and to establish primary fibroblast cell lines for cryopreservation. TLA sequencing analyses of selected founders were outsourced to Cergentis (Netherlands). We selected by PCR, 6 V2G-targeted colonies (1 female = 1D5; 5 males = 1C2, 1F2, 2C2, 2E3 and 2E7) for SCNT. V2G-targeting of InsGLP1Ser8-M3R transgene resulted monoallelic in colonies 1D5, 2C2, 2E3 and 2E7 and bi-allelic in colonies 1C2 and 1F2. Characterization of alive born piglets revealed that colonies 1D5, 1C2, 1F2 and 2E3 produced healthy animals. TLA sequencing analyses confirmed the V2G-targeting of the I-GSer8M3R expression cassette for the male clones (1C2, 1F2 and 2E3), identifying also different transgenes tandems in lines 1C2 and 1F2. Instead, the TLA results on the 1D5 female line, revealed that an unexpected rearrangement occurred eliminating the I-GS8M3R transgene from the targeted V2G locus and that an intact copy of the I-GSer8M3R was randomly integrated only into the Chromosome 17. Conclusion: mono- or bi-allelic V2G-gene targeting of the I-GSer8M3R construct using TALENs was suitable for the generation of healthy pig clones. At the same time the access to the Next Generation Sequencing technologies benefits, integrating the TLA analyses in our classical molecular PCR screenings, permitted us to define better the characterization of the V2G-targeting events and to detect new random transgene integrations. Following this strategy, we will be able to select the founder animals suited for establishing a breeding colony

Rib-oriented Thoracoscopic Sympathetic Surgery for Hyperhidrosis: Prospective Long-term Results and Quality of Life

BACKGROUND: The optimal thoracoscopic sympathetic surgery for primary palmar and/or axillary hyperhidrosis (PPAH) is still unclear because of lack of uniform technique and qualitative/quantitative scales for definition of results. The aims of this study were to compare long-term outcomes based on the surgical technique and the level of sympathetic trunk interruption by clipping and to assess postoperative compensatory sweating (CS), patients' satisfaction, and quality of life (QoL).MATERIALS AND METHODS: Between September 2009 and April 2016, 94 patients who underwent 2-stage bilateral thoracoscopic rib-oriented (R) sympathetic clipping were prospectively followed up through the administration of standardized preoperative and postoperative questionnaires.Thirty-four (36.2%) patients underwent single-port transaxillary access instead of the standard two 5-mm incisions. The level of sympathetic clipping for PPAH was R3+4(top and bottom); in patients who complained associated facial or plantar hyperhidrosis R2-bottom and R5-top were clipped, respectively. Seventy-five patients completed bilateral surgery.RESULTS: There were no significant differences between single-port and biportal video-assisted thoracoscopic surgery in terms of operative times and postoperative results. At a mean follow-up of 72 (SD: 26) months, CS was reported in 42 (56%) patients, severe only in 6 (8%). It was higher in the case of R2-bottom clipping (P=0.03). Thirty-one of 60 (51.6%) patients who had a plantar hyperhidrosis declared an improvement of feet sweating after surgery. Postoperative satisfaction was excellent (86.11% on a 0 to 100 scale) and 95.4% of patients declared an improvement in QoL, which was statistically significant in all evaluated parameters. These results were not related to the level of clipping.CONCLUSION: Thoracoscopic R3 to R4 clipping appears to be a safe and effective treatment for PPAH. Although postoperative CS was common and higher after R2-bottom clipping, this did not seem to affect patients' satisfaction and improvement in QoL

Initial experience with uniportal video-assisted thoracic surgery esophagectomy

Background: Multiportal thoracoscopic approach is already a well standardized procedure for minimally invasive esophagectomy (MIE); conversely very few reports have been published about uniportal video-assisted thoracic surgery (VATS) technique till now. We present our preliminary experience with uniportal VATS esophagectomy, evaluating short-term outcomes as perioperative mortality, complications, oncological radicality, postoperative pain and cosmetic results. Methods: From December 2016 to November 2017, the prospectively collected clinical data of 12 patients, who underwent uniportal VATS esophagectomy and reconstruction with a stomach conduit, according to McKeown technique, were reviewed and outcomes evaluated. Results: The mean age of population was 60.67\ub18.61 years. Ten (83.3%) patients were males. The main histological type was a squamous cell carcinoma in six patients (50%). No patient had a local recurrence. After 4.33\ub13.31 months 10 patients (83.3%) were alive with no evidence of disease; 2 (16.7%) patients died of other causes. Two (16.7%) patients developed an anastomotic leak (treated conservatively) and one (8.3%) patient a chylothorax (which required a surgical treatment). The mean operative time of uniportal VATS esophagectomy was 104.67\ub120.66 min. Mean number of thoracic nodes removed was 10.44\ub13.94. Postoperative hospitalization was 15.73\ub114.29 days (median of 9 days). The mean level of pain was 1.92\ub10.90 in first postoperative day with a duration of 2.25\ub11.54 days. Cosmetic result was 2.42\ub10.79 on a 3-point scale. Conclusions: Uniportal VATS esophagectomy seems to be a safe, feasible and effective alternative to multiportal VATS in terms of operative time, postoperative mortality, hospital stay and oncological outcomes. Less postoperative pain and better cosmetic results seem to be some advantages in favor of Uniportal VATS, however further studies with longer follow-up are claimed

Uniportal vs. triportal video-assisted thoracic surgery in the treatment of primary pneumothorax - A propensity matched bicentric study

Background: The role of triportal video-assisted thoracoscopic surgery (VATS) is widely recognized for the treatment of primary spontaneous pneumothorax (PSP). The aim of this study was to assess the effectiveness and the potential advantages of uniportal VATS (U-VATS) for the treatment of PSP compared with triportal VATS. Methods: A total of 104 triportal (n=39) and uniportal (n=65) VATS procedures where performed for the treatment of PSP in two University hospitals. The prospectively collected data of postoperative outcomes were retrospectively reviewed and a 1:1 propensity score matching analysis was performed to compare the two VATS approaches. Results: No major adverse events occurred after operation. Compared with triportal-VATS, Uniportal-VATS showed the same effectiveness in terms of risk of recurrence (null in both groups), post-operative complications (P=1.000) and operating time (66.04±16.92 vs. 74.57±21.38 min, P=0.141). However, there was a statistically significant difference in favor of uniportal-VATS in terms of necessity of further access [0 vs. 7 (30.4%), P=0.004], chest tube duration (4.39±1.41 vs. 6.32±0.94 days, P<<0.001), postoperative hospital stay (4.78±1.31 vs. 6.61±1.67 days, P<<0.001), visual analogue pain score (VAS) at 24 hours (3.45±1.41 vs. 6.44±2.45, P<<0.001), number of patients who had pain after chest drain removal [1 (4.3%) vs. 16 (69.6%), P<<0.001], VAS after drainage removal (0.11±0.47 vs. 2.74±2.25, P<<0.001), postoperative pain duration (2.50±1.20 vs. 14.82±37.41 days, P<<0.001), pain killers intake (0.75±1.06 vs. 7.53±3.96 days, P=0.001), chronic paresthesia (level scale: 0 to 2; 0 vs. 0.52±0.66, P<<0.001), chronic neuralgia (0 vs. 0.43±0.59, P<<0.001) and cosmetic results (level scale: 0 to 3; 2.91±0.28 vs. 2.00±0.77, P<<0.001). Conclusions: U-VATS is feasible and safe and may be a less invasive alternative to triportal VATS for the treatment of PSP because of its effectiveness in reducing postoperative pain, paresthesia, hospital stay and in improving cosmetic results