Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.

Use of Polyamide (Nylon) Cable Ties for Vascular Ligation of Healthy Equine Jejunal Mesentery

Jejunal vascular ligation is an essential step in performing jejunojejunostomy. Hand sewn ligation is typically used and can increase operative time with long sections of bowel to be removed. Nylon cable ties (NCT) have been used for vascular ligation in horses but are yet to be investigated for application on the mesenteric vasculature of the gastrointestinal tract. Our objective was to evaluate the efficacy and short-term safety of NCT jejunal mesenteric vessel ligation in healthy horses. Eight healthy adult horses underwent midline celiotomy. A segment of jejunal mesentery was identified (≥4 arcades). Briefly, three fenestrations (proximal, middle, distal) were made 5–10 mm apart adjacent to the first and last vascular arcade to be ligated. Two sterilized NCT were passed to encircle the mesentery through the proximal and middle fenestrations, separated by intact mesentery. NCT were closed tightly and the vascular pedicle transected with Mayo scissors through the distal fenestration. Jejunojejunostomy was then performed and the mesentery sutured closed. The number of vascular arcades and time to ligate using NCT were recorded. At 2 weeks, horses underwent repeat celiotomy to assess the healing of the NCT ligation site and an equal number of vascular arcades were hand sewn double ligated using 2-0 Polyglactin 910 as a timed comparison. NCT mesenteric ligation was significantly faster than hand sewn methods (P < 0.01). Effective hemostasis was achieved in all cases. There was no evidence of local infection or adhesions at 14 days post-operatively. Further investigation in the long-term effects in horses as well as horses with strangulating jejunal lesions are needed for clinical application.

Short- and long-term outcomes of rectal cancer patients with high or improved low ligation of the inferior mesenteric artery

The ligation site of the inferior mesenteric artery (IMA) during laparoscopic radical resection for rectal cancer has been controversial. Consecutive patients (n = 205) with rectal cancer who underwent laparoscopic-assisted low anterior resection from January 2009 to December 2015 were retrospectively analyzed. The patients were divided into high ligation (n = 126) and improved low ligation groups (n = 79). A total of 205 rectal cancer patients underwent laparoscopic assisted anterior resection: 126 patients in the high ligation group and 79 patients in the improved low ligation group. The improved low ligation group was better than the high ligation group in terms of postoperative flatus time and postoperative defecation time. There were no differences between the groups in terms of blood loss, operation time, total number of lymph nodes, anastomotic leakage, postoperative time to first liquid diet and postoperative hospital stay. There were also no differences in 5-year overall survival (OS). Compared to high ligation, the improved low ligation ensures the extent of lymph node dissection, and promotes the early recovery of postoperative gastrointestinal function, but does not increase the operation time, bleeding risk, or anastomotic leakage. A ligation site of the IMA in laparoscopic rectal cancer surgery may not influence oncological outcomes.

Pulsed Radiofrequency Reduced Neuropathic Pain Behavior in Rats Associated with Upregulation of GDNF Expression

Background: Pulsed radiofrequency (PRF) is a novel nondestructive interventional technique for the treatment of neuropathic pain (NP). However, this intervention is still lack of relevant regulation and the mechanism of action is insofar not clear. Historically, most studies have reported that PRF can relieve reduce hyperalgesia in multiple NP animal models by acting on the dorsal root ganglion. However, a few recent studies have shown that PRF can effectively treat hyperalgesia in pain models by a direct application on injured peripheral nerves. Objectives: To observe changes in pain behavior and the pathology of the sciatic nerve (SN) after applying PRF at the ligation site in a chronic constriction injury (CCI) rat model and to investigate the effect of PRF on the expression of glia cell line-derived neurotrophic factor (GDNF) in nervous tissue. Study Design: A randomized, experimental trial. Setting: Experimental Animal Center, Beijing Tiantan Hospital, Capital Medical University. Methods: Thirty-six adult Sprague-Dawley rats were randomly divided into 3 groups: Sham-Sham (SS), CCI-Sham (CS), and CCI-PRF (CP). The right SNs of the rats in the CS and CP groups were ligated to create a CCI model. For the SS group, the right SN was separated without ligation. On the 14th fourteenth day after surgery, PRF treatment was applied at the ligation site of the SN for the rats in the CP group using a 45 V output voltage at 42°C for 3 minutes. The electrode was placed in rats in the SS and CS groups without electricity applied. The hindpaw withdrawal threshold (HWT) and thermal withdrawal latency (TWL) were measured at various time points before and after the treatments in each group. Optical microscopic scores and electron microscopic observation were given to the right SN ligation sites of the rats in each group 14 days after the treatment . Meanwhile, the GDNF expression levels in the ligation site of the SN and in the L4-L6 spinal cord segments were determined for each group by enzyme-linked immunosorbent assay (ELISA). Results: Fourteen days after PRF treatment, the HWT and TWL values in the CP group were significantly increased compared to those of the CS group (P < 0.01). Under the optical microscope, the axonal number, axonal diameter, and myelin sheath thickness in the CP group were significantly increased compared to those of the CS group 14 days after PRF treatment (P < 0.01). Under the electron microscope, the degeneration at the SN ligation site was significantly improved in the CP group compared to the CS group. The GDNF expression levels at the ligation site of the SN and the L4-L6 spinal segments in the CP and CS groups were increased compared to those of the SS group (P < 0.01). In addition, the GDNF expression in the CP group was significantly higher than that in the CS group (P < 0.01). Limitations: GDNF expression was only measured at day 14 after the treatment rather than at various time points during the experiment. Conclusions: The findings suggest that the application of PRF at the impaired SN relieved reduced the CCI-induced NP by through regulating the upregulation of the GDNF expression in the nervous tissues. Key words: Pulsed radiofrequency, chronic constriction injury, sciatic nerve, spinal cord, hind paw withdrawal threshold, thermal withdrawal latency, optical microscopic, electron microscope, glia cell line-derived neurotrophic factor, enzyme-linked immunosorbent assay

The Role of Hemoclips Reinforcement in the Ligation-Assisted Endoscopic Enucleation for Small GISTs in Gastric Fundus

Background.Endoscopic ultrasonography- (EUS-) assisted band ligation has been proven to be a safe and effective procedure for the treatment of small gastrointestinal stromal tumors (GISTs) apart from the relatively high risk of the postligation perforation of the gastric fundus. The aim of this study is to investigate the efficacy of hemoclip reinforcement in treating small GISTs in the gastric fundus.Method.During a standard endoscopy, a transparent cap attached to the endoscopic tip was placed over the lesion to exert sustained maximal aspiration before a rubber band was released. Once a definite ligation was confirmed by EUS, the tumor was enucleated. Four to 6 hemoclips were placed on the folds around the ligation band to reduce the tension of the ligation site.Results.The small GISTs were resected completely in 192 patients. Two cases of delayed perforation were found 72 hours after the procedure and successfully treated with an ordinary conservative method.Conclusion.Hemoclip-reinforced endoscopic band ligation with systematic follow-up using EUS appears to be a simple and effective technique for the resection of small GISTs in the gastric fundus.

Assessment of the Impact of Enforced Expression of Platelet Derived Growth Factor Beta in Human Mesenchymal Stem Cells and Contribution to Blood Flow Restoration.

Abstract 3568 Poster Board III-505 Both mesenchymal stem cells (MSC) and platelet derived growth factor beta (PDGFB) have been shown to promote angiogenesis in vivo and are therefore interesting therapeutic agents for the restoration of blood flow to ischemic tissues encountered in peripheral artery disease and critical limb ischemia. Careful evaluation of the mechanisms responsible for neovessel formation in stem cell-based therapeutic angiogenesis is critical for developing efficient treatment strategies. Previous studies have shown that MSCs correspond to pericytes, which are lost in PDGFB-null mice, suggesting that MSCs strongly rely on PDGFB signaling in vivo. Our major aim was to establish a highly sensitive method to assess whether overexpression of PDGFB in MSCs could modulate expression of pro-angiogenic cytokines and improve the restoration of bloodflow in an established murine xenograft model of hind limb ischemia. Human bone marrow derived MSCs (passage 2-5) were transduced with either a lentiviral vector containing the coding sequence of PDGFB or the same vector without PDGFB (control). As expected, overexpression of PDGFB strongly enhanced the proliferation of MSCs, reducing the doubling time from 51 to 20 hours (n=4, p<0.05). This effect correlated with a shift of 15% of cells from the G0/G1 to the S/G2/M phase, as compared to controls, when measured by flow cytometry of propidium iodide stained cells. Next, we induced a unilateral hind limb ischemia in NODSCIDbeta2null mice by excision of the femoral artery and performed intravenous transplantation of 2.5×10e5 MSCs (control MSC vs. PDGFB-MSC) the day following surgery. Recovery of blood flow to the affected limb was assessed at days 0 ((before transplantation), n=11), 4 (n=11), 7 (n=9), 10 (n=6), and 14 (n=6) by laser doppler imaging and values were normalized to the non-ischemic contralateral leg. Treated animals of both groups (control and PDGFB overexpressing MSC) exhibited a steadily progressive recovery in blood flow in the region proximal to the primary ligation site, reaching near normal levels by day 10. Surprisingly, no significant difference was seen in the blood flow recovery in animals receiving the PDGF over expressing MSCs. Donor MSCs (control and PDGFB) homed to the site of injury, as assessed by immunofluorescent staining of the ischemic muscle in the regions proximal and distal to the primary ligation site at days 4 and 7. However, the rapid recovery in blood flow did not appear to be attributed to a direct differentiation or integration of MSC into the vasculature, but rather through the expression of paracrine angiogenic factors. Interestingly, overexpression of PDGF reduced FGF2 mRNA expression levels in MSC (n=4, p=0.056), suggesting that high levels of PDGFB may indeed lead to a reduction of other key angiogenic factors, explaining the mild effect of PDGFB in vivo. Our results show that, although overexpression of PDGFB in bone marrow-derived MSCs enhanced the proliferation of MSC in vitro, it did not impact the speed or magnitude of blood flow recovery to the ischemic hind limb as compared to native MSCs, likely due to down modulation of other key angiogenic signals including FGF2. Disclosures: No relevant conflicts of interest to declare.