Single Ligation Research Articles

Synthetic Antiangiogenic Vascular Endothelial Growth Factor-A Splice Variant Revascularizes Ischemic Muscle in Peripheral Artery Disease.

Alternative splicing in the eighth exon C-terminus of VEGF-A (vascular endothelial growth factor-A) results in the formation of proangiogenic VEGF165a and antiangiogenic VEGF165b isoforms. The only known difference between these 2 isoform families is a 6-amino acid switch from CDKPRR (in VEGF165a) to SLTRKD (in VEGF165b). We have recently shown that VEGF165b can induce VEGFR2-activation but fails to induce VEGFR1 (VEGF receptor 1)-activation. The molecular mechanisms that regulate VEGF165b's ability toward differential VEGFR2 versus VEGFR1 activation/inhibition are not yet clear. Hypoxia serum starvation was used as an invitro peripheral artery disease model. Unilateral single ligation of the femoral artery was used as a preclinical peripheral artery disease model. VEGFR1 activating ligands have 2 arginine (RR) residues in their eighth exon C-terminus, that were replaced by lysine-aspartic acid (KD) in VEGF165b. A synthetic anti-angiogenic VEGF165b splice variant in which the KD residues were switched to RR (VEGF165bKD→RR) activated both VEGFR1- and VEGFR2-signaling pathways to induce ischemic-endothelial cell angiogenic capacity invitro and enhance perfusion recovery in a severe experimental-peripheral artery disease model significantly higher than VEGF165a. Phosphoproteome arrays showed that the therapeutic efficacy of VEGF165bKD→RR over VEGF165a is due to its ability to induce P38-activation in ischemic endothelial cells. Our data shows that the KD residues regulate VEGF165b's VEGFR1 inhibitory property but not VEGFR2. Switching these KD residues to RR resulted in the formation of a synthetic/recombinant VEGF165bKD→RR isoform that has the ability to activate both VEGFR1- and VEGFR2-signaling and induce ischemic-endothelial cell angiogenic and proliferative capacity that matched the angiogenic requirement necessary to achieve perfusion recovery in a severe experimental-peripheral artery disease model.

Contrast-Enhanced Ultrasound of Mouse Models of Hindlimb Ischemia Reveals Persistent Perfusion Deficits and Distinctive Muscle Perfusion Patterns

ObjectiveMouse models of hindlimb ischemia (HLI) are used to study peripheral arterial disease and evaluate novel therapies. Contrast-enhanced ultrasound (CEUS) is a noninvasive perfusion measurement technique that is increasingly being employed in these models. The objective of this study was to evaluate two models of severe HLI by CEUS to characterize perfusion recovery and muscle perfusion patterns. MethodsMice undergoing double femoral artery ligation were measured by CEUS and laser Doppler perfusion imaging (LDPI) at baseline and 1–150 d postsurgery. A second group undergoing femoral artery ligation and excision was measured 1–28 d postsurgery. ResultsBy LDPI, both surgeries showed robust perfusion recovery by 14 d postsurgery. However, by CEUS only a ∼40% perfusion recovery plateau was reached in either group. These results are consistent with our previous work, employing a less severe single femoral artery ligation, that showed perfusion in the ischemic limb does not return to normal by 150 d postsurgery. Cluster analysis of muscle perfusion patterns indicated 3–5 different patterns at day 1 postsurgery. The double ligation model yielded significantly less variable perfusion patterns, suggesting that it can provide more reproducible results. ConclusionContrary to LDPI, perfusion as measured by CEUS never fully recovers after hindlimb surgery, even when followed 28–150 d postsurgery. Individual mice can manifest different patterns of muscle perfusion to the same surgery, but these patterns are conserved within and between different surgical techniques. These results may have significant implications for the evaluation of novel therapeutics to treat PAD in mice.

Status of clipless laparoscopic cholecystectomy in a tertiary care hospital of Nepal: A descriptive cross-sectional study

Background & Objectives: In this modern age, the preferred course of treatment for gallstone disease symptoms is laparoscopic cholecystectomy. For securing both the cystic duct and artery with a single ligation (SLAD), is a quicker and safe procedure. The study was aimed to determine the status of clipless laparoscopic cholecystectomy in a tertiary care hospital of Nepal. Materials and Methods: A total of 205 patients with gallstone, who underwent laparoscopic cholecystectomy at tertiary center of Nepal of age group 16 - 70 years were included. During the procedure the cystic duct and artery was tied with single ligation. Data were subjected to Microsoft excel 16 for analysis. Results: Laparoscopic cholecystectomy was performed in 205 patients with gallstone. Average age of patients was 31.54 yrs. There was female predominance in the study. The cystic duct and artery were tied in single ligature using nonabsorbable Silk 2-0. The mean time taken for ligation was 120.78 ± 26.92seconds. Bile leakage and intra-op bleeding were found in two patients without any mortality. Conclusion: In laparoscopic cholecystectomy, a viable and secure alternate technique for securing the cystic artery and duct is single ligation with the use of non-absorbable thread. Even though it is a common procedure, it has an additional benefit over clips when dealing with thick, edematous cystic artery and duct complexes. It lowers the threat of surgical morbidity and does not increase the intra operative time.

The Temporal Relationship between Blood-Brain Barrier Integrity and Microglial Response following Neonatal Hypoxia Ischemia.

Blood-brain barrier (BBB) dysfunction and neuroinflammation are key mechanisms of brain injury. We performed a time-course study following neonatal hypoxia-ischemia (HI) to characterize these events. HI brain injury was induced in postnatal day 10 rats by single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). At 6, 12, 24, and 72 h (h) post-HI, brains were collected to assess neuropathology and BBB dysfunction. A significant breakdown of the BBB was observed in the HI injury group compared to the sham group from 6 h in the cortex and hippocampus (p < 0.001), including a significant increase in albumin extravasation (p < 0.0033) and decrease in basal lamina integrity and tight-junction proteins. There was a decrease in resting microglia (p < 0.0001) transitioning to an intermediate state from as early as 6 h post-HI, with the intermediate microglia peaking at 12 h (p < 0.0001), which significantly correlated to the peak of microbleeds. Neonatal HI insult leads to significant brain injury over the first 72 h that is mediated by BBB disruption within 6 h and a transitioning state of the resident microglia. Key BBB events coincide with the appearance of the intermediate microglial state and this relationship warrants further research and may be a key target for therapeutic intervention.

Is the double-ligation method safe and effective in laparoscopic paediatric inguinal hernia repair? A retrospective cohort study.

Laparoscopic paediatric hernia repair has since been performed more frequently. Recently, a double-ligation method was introduced to prevent recurrence in laparoscopic surgery. The objective of our study was to compare the postoperative outcomes of patients who underwent double and single ligations. We performed a retrospective review of patients with inguinal hernia who underwent laparoscopic paediatric inguinal hernia repair from 2014 to 2018 at our institution. Patients were divided into two groups and underwent single ligation and double ligation. Three hundred and six patients (single-ligation group: 142 and double-ligation group: 164) were included. Four patients experienced recurrence, and the recurrence rate was 1.29%. The recurrence rate was significantly higher in the single-ligation group. There were no statistically significant differences in the postoperative complications between the two groups. Double-ligation method in laparoscopic paediatric hernia repair was relatively safe and helpful.

An Optimized High-Resolution Mapping Method for Glucocorticoid Receptor-DNA Binding in Mouse Primary Macrophages.

ChIP-exo is a powerful tool for achieving enhanced sensitivity and single-base-pair resolution of transcription factor (TF) binding, which utilizes a combination of chromatin immunoprecipitation (ChIP) and lambda exonuclease digestion (exo) followed by high-throughput sequencing. ChIP-nexus (chromatin immunoprecipitation experiments with nucleotide resolution through exonuclease, unique barcode, and single ligation) is an updated and simplified version of the original ChIP-exo method, which has reported an efficient adapter ligation through the DNA circularization step. Building upon an established method, we present a protocol for generating NGS (next-generation sequencing) ready and high-quality ChIP-nexus library for glucocorticoid receptor (GR). This method is specifically optimized for bone marrow-derived macrophage (BMDM) cells. The protocol is initiated by the formation of DNA-protein cross-links in intact cells. This is followed by chromatin shearing, chromatin immunoprecipitation, ligation of sequencing adapters, digestion of adapter-ligated DNA using lambda exonuclease, and purification of single-stranded DNA for circularization and library amplification.

Inguinal Arteriovenous Malformation

Arteriovenous malformations (AVMs) are rare disorders, with inguinal region involvement being particularly uncommon, as they typically manifest in the neck, limbs, trunk, and intracranial or extracranial sites. Despite their varied presentation across organs and tissues, AVMs can lead to cardiac failures due to high output, necessitating management even in asymptomatic cases. We present the case of a 37-year-old male with a known history of hypertension and severe depressive disorder, referred to the urology department for bilateral renal calculi evaluation. The patient complained of sudden-onset, moderate-intensity left-sided flank pain, with no signs of infection or constitutional symptoms. A non-contrast CT scan revealed bilateral renal calculi and an intriguing mass in the scroto-inguinal region. Further investigation through contrast-enhanced computed tomography identified an AVM originating from the right internal iliac artery, located a few millimeters next to the scrotum. A multidisciplinary team, including interventional radiology, urology, and vascular surgery, collaborated to develop a curative strategy. Angioembolization was performed successfully in a single session, involving catheterization and ligation of the feeder vessel using a lipiodol-glue mixture. Postoperatively, the patient remained stable with mild pyrexia (99°F), a heart rate of 76 bpm, and blood pressure fluctuating between 110-125 mmHg systolic and 72-80 mmHg diastolic. The absence of glue leakage upon saline injection confirmed the effectiveness of embolization. The patient experienced relief from pain and was mobilized on the second postoperative day. This case underscores the importance of a comprehensive approach in managing rare presentations of AVMs, emphasizing successful angioembolization as a viable therapeutic option

Neuroprotective Action of Tacrolimus before and after Onset of Neonatal Hypoxic-Ischaemic Brain Injury in Rats.

(1) Background: Neonatal brain injury can lead to permanent neurodevelopmental impairments. Notably, suppressing inflammatory pathways may reduce damage. To determine the role of neuroinflammation in the progression of neonatal brain injury, we investigated the effect of treating neonatal rat pups with the immunosuppressant tacrolimus at two time points: before and after hypoxic-ischaemic (HI)-induced injury. (2) Methods: To induce HI injury, postnatal day (PND) 10 rat pups underwent single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). Pups received daily tacrolimus (or a vehicle) starting either 3 days before HI on PND 7 (pre-HI), or 12 h after HI (post-HI). Four doses were tested: 0.025, 0.05, 0.1 or 0.25 mg/kg/day. Pups were euthanised at PND 17 or PND 50. (3) Results: All tacrolimus doses administered pre-HI significantly reduced brain infarct size and neuronal loss, increased the number of resting microglia and reduced cellular apoptosis (p < 0.05 compared to control). In contrast, only the highest dose of tacrolimus administered post-HI (0.25 mg/kg/day) reduced brain infarct size (p < 0.05). All doses of tacrolimus reduced pup weight compared to the controls. (4) Conclusions: Tacrolimus administration 3 days pre-HI was neuroprotective, likely mediated through neuroinflammatory and cell death pathways. Tacrolimus post-HI may have limited capacity to reduce brain injury, with higher doses increasing rat pup mortality. This work highlights the benefits of targeting neuroinflammation during the acute injurious period. More specific targeting of neuroinflammation, e.g., via T-cells, warrants further investigation.

Construction of Multiple DNAzymes Driven by Single Base Elongation and Ligation for Single-Molecule Monitoring of FTO in Cancer Tissues.

Fat mass and obesity-associated proteins (FTO) play an essential role in the reversible regulation of N6-methyladenosine (m6A) epigenetic modification, and the overexpression of FTO is closely associated with the occurrence of diverse human diseases (e.g., obesity and cancers). Herein, we demonstrate the construction of multiple DNAzymes driven by single base elongation and ligation for the single-molecule monitoring of FTO in cancer tissues. When target FTO is present, the m6A-RNA is specifically demethylated and subsequently acts as a primer to combine with the padlock probe, initiating single-base elongation and ligation reaction to generate a closed template probe. Upon the addition of phi29 DNA polymerase, a rolling circle amplification (RCA) reaction is initiated to produce large numbers of Mg2+-dependent DNAzyme repeats. Subsequently, the DNAzymes cyclically digest the signal probes, liberating numerous Cy5 molecules that can be precisely counted by single-molecule imaging. Taking advantage of the sequence specificity of the polymerase/ligase-mediated gap-filling and ligation as well as the high amplification efficiency of RCA, this biosensor shows excellent specificity and high sensitivity with a detection limit of 5.96 × 10-16 M. It can be applied to screen FTO inhibitors and quantify FTO activity at the single-cell level. Moreover, the proposed strategy can accurately distinguish the FTO expression level in tissues of healthy individuals and breast cancer patients, providing a new platform for drug discovery, m6A modification-related research, and clinical diagnostics.

Optimized infrared photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (IR-PAR-CLIP) protocol identifies novel IGF2BP3-interacting RNAs in colon cancer cells.

The conserved family of RNA-binding proteins (RBPs), IGF2BPs, plays an essential role in posttranscriptional regulation controlling mRNA stability, localization, and translation. Mammalian cells express three isoforms of IGF2BPs: IGF2BP1-3. IGF2BP3 is highly overexpressed in cancer cells, and its expression correlates with a poor prognosis in various tumors. Therefore, revealing its target RNAs with high specificity in healthy tissues and in cancer cells is of crucial importance. Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies the binding sites of RBPs on their target RNAs at nucleotide resolution in a transcriptome-wide manner. Here, we optimized the PAR-CLIP protocol to study RNA targets of endogenous IGF2BP3 in a human colorectal carcinoma cell line. To this end, we first established an immunoprecipitation protocol to obtain highly pure endogenous IGF2BP3-RNA complexes. Second, we modified the protocol to use highly sensitive infrared (IR) fluorescent dyes instead of radioactive probes to visualize IGF2BP3-crosslinked RNAs. We named the modified method "IR-PAR-CLIP." Third, we compared RNase cleavage conditions and found that sequence preferences of the RNases impact the number of the identified IGF2BP3 targets and introduce a systematic bias in the identified RNA motifs. Fourth, we adapted the single adapter circular ligation approach to increase the efficiency in library preparation. The optimized IR-PAR-CLIP protocol revealed novel RNA targets of IGF2BP3 in a human colorectal carcinoma cell line. We anticipate that our IR-PAR-CLIP approach provides a framework for studies of other RBPs.

Production of Sitobion avenae-resistant Triticum aestivum cvs using laccase as RNAi target and its systemic movement in wheat post dsRNA spray.

Among the wheat biotic stresses, Sitobion avenae is one of the main factors devastating the wheat yield per hectare. The study's objective was to find out the laccase (lac) efficacy; as a potential RNAi target against grain aphids. The Sitobion avenae lac (Salac) was confirmed by Reverse Transcriptase-PCR. Gene was sequenced and accession number "ON703252" was allotted by GenBank. ERNAi tool was used to design 143 siRNA and one dsRNA target. 69% mortality and 61% reduction in lac expression were observed 8D-post lac DsRNA feeding. Phylogenetic analysis displayed the homology of grain aphid lac gene with peach potato, pea, and Russian wheat aphids. While Salac protein was found similar to the Russian grain, soybean, pea, and cedar bark aphid lac protein multi-copper oxidase. The dsRNAlac spray-induced silencing shows systematic translocation from leaf to root; with maximum lac expression found in the root, followed by stem and leaf 9-13D post-spray; comparison to control. RNAi-GG provides the Golden Gate cloning strategy with a single restriction ligation reaction used to achieve lac silencing. Agrobacterium tumefaciens mediated in planta and in-vitro transformation was used in the study. In vitro transformation, Galaxy 2012 yielded a maximum transformation efficiency (1.5%), followed by Anaj 2017 (0.8%), and Punjab (0.2%). In planta transformation provides better transformation efficiencies with a maximum in Galaxy 2012 (16%), and a minimum for Punjab (5%). Maximum transformation efficiency was achieved for all cultivars with 250 μM acetosyringone and 3h co-cultivation. Galaxy 2012 exhibited maximum transformation efficiency, and aphid mortality post-feeding transgenic wheat.

Peptide-Based Fibrin-Targeting Fluorescent Probes for Murine Stasis Venous Thrombus Resolution

Background- The lack of a stasis venous thrombosis model (VT) in female mice has limited investigation of sex differences of anti-VT therapies, such as acetylsalicylic acid (ASA), which appears efficacious in preventing recurrent VT. Here we (i) develop a new extended stasis VT model; and (ii) examine the effects of ASA on stasis VT resolution in both female and male mice using serial intravital microscopy (IVM). Method- To extend stasis thrombus persistence beyond a single ligation (SL) model, the femoral vein was ligated, followed by a second ligation of saphenous vein near popliteal vein (Fig. A). Following FITC-dextran injection (10 mg/kg), FITC light irradiation (475/35nm, 20x) for three minutes across three illumination fields generated stasis VT. Subsequently, Dylight-649-GPIbβ agent was injected (100ug/kg, ex 630nm) to observe platelets. At T30’, fibrin targeting agent-FTP11-CyAm7 (150nmol/kg, ex 750 nm) was injected. ASA pre-treatment was done for 7 days (3mg/kg, n=5-6). Serial epifluorescence IVM images were acquired up to 24 hours (90i, NIS Elements software, Nikon). Thrombus area was quantified (NIH ImageJ). Results- DL thrombus was significantly larger and visualized through 24 hours (Fig B-G) and 48 hours (data not shown) versus SL thrombus (Fig. E). Corresponding sections of thrombus at T60’, T6h and T24h showed formation of a persistent thrombus core consisting platelets and fibrin (Fig. H-J). In the DL model, females developed reproducible stasis VT burden and resolution profiles as in males (Fig. K). ASA pre-treatment resulted in significant reduction in thrombus induction (T0’), and accelerated resolution in both sexes (Fig. L-P). ASA inhibited platelet recruitment to thrombi within T30’, consistent with its known anti-platelet effect via irreversible inhibition of Thromboxane A2. Furthermore, ASA inhibited fibrinogenesis, suggestive of an impaired platelet-fibrin interface. Conclusion- Here we establish a new extended stasis VT model applicable to both female and male mice that develops thrombus for up to 48 hours. Moreover, this model is amenable to study vein wall fibrosis-key aspect of post-thrombotic syndrome. Furthermore, ASA inhibits thrombus formation and accelerates thrombus resolution in a sex-independent manner, supporting its use in prevention of recurrent VT. AHA 953662; NIH 1R21AR081535-01; NIH R01GM126062 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Rapid assembly of SARS-CoV-2 genomes reveals attenuation of the Omicron BA.1 variant through NSP6

Although the SARS-CoV-2 Omicron variant (BA.1) spread rapidly across the world and effectively evaded immune responses, its viral fitness in cell and animal models was reduced. The precise nature of this attenuation remains unknown as generating replication-competent viral genomes is challenging because of the length of the viral genome (~30 kb). Here, we present a plasmid-based viral genome assembly and rescue strategy (pGLUE) that constructs complete infectious viruses or noninfectious subgenomic replicons in a single ligation reaction with >80% efficiency. Fully sequenced replicons and infectious viral stocks can be generated in 1 and 3 weeks, respectively. By testing a series of naturally occurring viruses as well as Delta-Omicron chimeric replicons, we show that Omicron nonstructural protein 6 harbors critical attenuating mutations, which dampen viral RNA replication and reduce lipid droplet consumption. Thus, pGLUE overcomes remaining barriers to broadly study SARS-CoV-2 replication and reveals deficits in nonstructural protein function underlying Omicron attenuation.

Comparison of the long-term efficacy and safety of multiple endoscopic rubber band ligations in a single session for varying grades of internal hemorrhoids.

There is no consensus onthe number of ligation bandsforvarying grades of internal hemorrhoids (IH) in a single sessionof endoscopic rubber band ligation (ERBL). The aims of this study were to investigate this issue. Patients withIHwere treated with ERBL. The primary endpointwas no hemorrhoid symptoms 6weeks after ERBL.The secondary endpointswerepostoperative complications and recurrence. One hundred twenty-three eligible patients met the inclusion criteria, and 3 patients were excluded. The average bands were 3.9 ± 1.1, and grade II hemorrhoids required significantly fewer bands than grade III hemorrhoids (3.45 ± 0.85 vs 4.35 ± 1.06, P = 0.000). 78.8%of patients achieved the primary endpoint. The efficacy of grade II hemorrhoids was higher than that of grade III hemorrhoids (87.8% vs 69.2%). The incidence of postoperative complications was not significantly correlated with the grade of hemorrhoids (P = 0.201) and the number of bands (P = 0.886). The median follow-up time was 14.3 ± 7.9 (1.3-30.9) months. The overall recurrence rate was 25.0%, with grade III significantly higher than grade I-II (32.8% vs 14.6%, P = 0.027). For grade III hemorrhoids, the recurrence rate was lower with more than 4 bands of ligation than with less than 4 bands (26.5% vs 53.3%). The average ERBL treatment of IH requires nearly four bands, and grade II hemorrhoids require less banding than grade III hemorrhoids, while the efficacy was better. Increasing the number of bands improves the treatment efficacy for grade III IH, which could be a strategy to reduce the recurrence rate without increasing postoperative complications.

Mouse Model of Spinal Cord Hypoperfusion with Immediate Paralysis Caused by Endovascular Repair of Thoracic Aortic Aneurysm.

A clinically relevant mouse model of thoracic endovascular aortic repair-induced ischemic spinal cord injury has been lacking since the procedure was first employed in 1991. The hypothesis was that ligation of mouse intercostal arteries would simulate thoracic endovascular aortic repair-induced ischemic spinal cord injury and behavioral deficit. The aim was to create a mouse model of thoracic endovascular aortic repair-induced spinal cord hypoperfusion by ligating five pairs of mouse intercostal vessels. Mice were divided into sham (n = 53) and ligation (n = 60) groups. The procedures called for double ligation of three pairs and single ligation of two pairs of thoracic intercostal arteries in adult C57BL/6 mice. A laser Doppler probe was used in vivo on the spinal cords and intercostal arteries to document the extent of arterial ligation and spinal cord hypoperfusion. The Basso Mouse Scale for Locomotion, histological studies, and electron microscopy demonstrated postligation locomotive and histopathological changes. Ligation induced a significant and instantaneous drop in blood flow in the intercostal arteries (% change; mean = -63.81; 95% CI, -72.28 to -55.34) and the thoracic spinal cord (% change; mean = -68.55; 95% CI, -80.23 to -56.87). Paralysis onset was immediate and of varying degree, with behavioral deficit stratified into three groups: 9.4% exhibited severe paralysis, 37.5% moderate paralysis, and 53.1% mild paralysis at day 1 (n = 32; P < 0.001). Mild and moderate paralysis was transient, gradually improving over time. Severe paralysis showed no improvement and exhibited a higher mortality rate (83%; n = 15 of 18) compared to moderately (33%; n = 6 of 18) and mildly (24%; n = 6 of 25) paralyzed mice (P < 0.001). The overall ligation group survival rate (84%; n = 46 of 55) was significantly lower than the sham group (100%; n = 48 of 48) with P = 0.003. The mouse model generates reproducible spinal cord hypoperfusion and accompanying histopathological ischemic spinal cord damage. The resulting anatomical changes and variable behavioral deficits mimic the variability in radiological and clinical findings in human patients.

Lentiviral overexpression of VEGFC in transplanted MSCs leads to resolution of swelling in a mouse tail lymphedema model.

Dysfunction of the lymphatic system following injury, disease, or cancer treatment can lead to lymphedema, a debilitating condition with no cure. Despite the various physical therapy and surgical options available, most treatments are palliative and fail to address the underlying lymphatic vascular insufficiency driving lymphedema progression. Stem cell therapy provides a promising alternative in the treatment of various chronic diseases with a wide range of therapeutic effects that reduce inflammation, fibrosis, and oxidative stress, while promoting lymphatic vessel (LV) regeneration. Specifically, stem cell transplantation is suggested to promote LV restoration, rebuild lymphatic circulation, and thus potentially be utilized towards an effective lymphedema treatment. In addition to stem cells, studies have proposed the administration of vascular endothelial growth factor C (VEGFC) to promote lymphangiogenesis and decrease swelling in lymphedema. Here, we seek to combine the benefits of stem cell therapy, which provides a cellular therapeutic approach that can respond to the tissue environment, and VEGFC administration to restore lymphatic drainage. Specifically, we engineered mesenchymal stem cells (MSCs) to overexpress VEGFC using a lentiviral vector (hVEGFC MSC) and investigated their therapeutic efficacy in improving LV function and tissue swelling using near infrared (NIR) imaging, and lymphatic regeneration in a single LV ligation mouse tail lymphedema model. First, we showed that overexpression of VEGFC using lentiviral transduction led to an increase in VEGFC protein synthesis in vitro. Then, we demonstrated hVEGFC MSC administration post-injury significantly increased the lymphatic contraction frequency 14-, 21-, and 28-days post-surgery compared to the control animals (MSC administration) in vivo, while also reducing tail swelling 28-days post-surgery compared to controls. Our results suggest a therapeutic potential of hVEGFC MSC in alleviating the lymphatic dysfunction observed during lymphedema progression after secondary injury and could provide a promising approach to enhancing autologous cell therapy for treating lymphedema.

Abstract 121: Acetylsalicylic Acid Accelerates Murine Stasis Venous Thrombus Resolution, Independent Of Sex

Background: The lack of a stasis venous thrombosis (VT) model in female mice has limited investigation on sex differences of anti-VT therapies, such as acetylsalicylic acid (ASA), which appears efficacious in preventing recurrent VT. Here we (i) develop a new extended stasis VT model; and (ii) examine the effects of ASA on stasis VT resolution in female and male mice using serial intravital microscopy. Method: To extend stasis thrombus persistence, femoral vein was ligated followed by second ligation of saphenous vein (Fig. A), after 7 days of either PBS/ASA (3mg/kg, n=5-6) pre-treatment. Following FITC-dextran injection (10 mg/kg), FITC light irradiation (475/35nm, 20x) for three minutes across three illumination fields generated stasis VT. Dylight649-GPIbβ agent was injected (100ug/kg, ex 630nm) to observe platelets. At T30’, fibrin targeting agent-FTP11-CyAm7 (150nmol/kg, ex 750 nm) was injected. Serial epifluorescence IVM images were acquired (90i, NIS Elements software, Nikon). Results: Double ligation (DL) thrombus was significantly larger than single ligation (SL) and remained so through 24h (Fig B-G) and 48h (data not shown). Corresponding sections of thrombus showed a persistent core consisting platelets and fibrin (Fig. H-J). In DL, females developed reproducible stasis VT burden and resolution profiles as in males (Fig. K). ASA pre-treatment resulted in reduced thrombus (T0’), and accelerated resolution in both sexes (Fig. L-P). ASA inhibited platelet recruitment to thrombi within T30’, consistent with its known anti-platelet effect. Also, ASA inhibited fibrinogenesis, suggestive of an impaired platelet-fibrin interface. Conclusion: Here we establish a new extended stasis VT model applicable to both female and male mice. This model is amenable to study vein wall fibrosis-critical aspect of post-thrombotic syndrome. Furthermore, ASA inhibits thrombus formation and accelerates thrombus resolution in a sex-independent manner.

ScSPLAT, a scalable plate-based protocol for single cell WGBS library preparation

DNA methylation is a central epigenetic mark that has diverse roles in gene regulation, development, and maintenance of genome integrity. 5 methyl cytosine (5mC) can be interrogated at base resolution in single cells by using bisulfite sequencing (scWGBS). Several different scWGBS strategies have been described in recent years to study DNA methylation in single cells. However, there remain limitations with respect to cost-efficiency and yield. Herein, we present a new development in the field of scWGBS library preparation; single cell Splinted Ligation Adapter Tagging (scSPLAT). scSPLAT employs a pooling strategy to facilitate sample preparation at a higher scale and throughput than previously possible. We demonstrate the accuracy and robustness of the method by generating data from 225 single K562 cells and from 309 single liver nuclei and compare scSPLAT against other scWGBS methods.

Construction of \xe0 la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins

BackgroundQconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process.ResultsWe report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro.ConclusionsWe refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

A Rodent Model of Mild Neonatal Hypoxic Ischemic Encephalopathy.

In the brain of full-term newborns, Hypoxic Ischemic Encephalopathy (HIE), a consequence of severe hypoxia and ischemia due to low cardiac output, is frequently observed and results in cerebral injuries with dramatic consequences for life. To investigate the physiopathology of HIE, several animal models have been developed, but none closely replicate human cases, mostly because they are based on a single carotid ligation protocol. In the present study we aimed to develop a novel and more accurate HIE model in juvenile (post-natal days (PND) 14–16) rats. For this, we induced a 9 min hypoxic cardiac arrest (CA) by stopping mechanical ventilation of intubated, ventilated and curarized rats followed by a cardiopulmonary resuscitation. To evaluate the consequences of the CA we performed radiological (cerebral MRI), behavioral (Open Field, Elevated Plus Maze, Fear Conditioning), and histological (Cresyl Violet and Fluoro-Jade B) testing on treated animals. We found that rats in the CA group developed an anxiolytic-like behavioral profile in adulthood without any locomotor impairment, nor memory deficits. However, MRI investigation performed early after CA failed to reveal any change in apparent diffusion coefficient (ADC) in brain tissue (including the hippocampus, striatum, and thalamus), suggesting no massive anatomical lesion had occurred. In contrast, signs of neurodegeneration were found in the Dentate Gyrus and the CA1 region of the hippocampus at day 1 post-CA, suggesting that the anxiolytic-like phenotype observed in adulthood could be related to an abnormal degeneration of this brain region beginning immediately after CA. Thus, our model, despite not representing a severe condition of HIE, nonetheless constitutes a potential model for studying mild, yet persistent and region-specific cerebral injury resulting from an acute oxygen deprivation.