Single Ligation Research Articles

Placement of interlocking fenestrated clips for a large broad-based middle cerebral artery aneurysm with atherosclerosis: Technical case report

IntroductionClip ligation of a large or giant middle cerebral artery (MCA) aneurysm is sometimes challenging, since these aneurysms commonly involve the efferent arteries and often show atherosclerotic changes in the aneurysm wall. We present herein a surgical case of large MCA aneurysm with atherosclerosis, and demonstrate a technique that enables adjustment of the patency of efferent arteries and the dog-eared remnant of the complex bifurcation aneurysm by placement of interlocking fenestrated clips. Case illustrationA 63-year-old woman was referred to our hospital for treatment of a relatively large unruptured left MCA saccular aneurysm (dome height × neck width: 9.5 mm × 7.6 mm). She underwent microsurgical clip ligation of the aneurysm. Due to the atherosclerotic changes in the aneurysm neck and efferent arteries, a large amount of dog-eared remnant of the aneurysm neck or clip slippage was likely to occur following single clip ligation. The interlocking clip application technique was thus considered using fenestrated clips with high closure force at the tips, resulting in successful aneurysm obliteration without complicated replacement of the clips. ConclusionAlthough complex clip application techniques for large MCA aneurysms with atherosclerosis have been reported, the present technique could provide an alternative.

Double ligation is useful to prevent recurrence for paediatric inguinal hernia in the laparoscopic percutaneous extraperitoneal closure procedure

AbstractAimAlthough laparoscopic percutaneous extraperitoneal closure (LPEC) is the standard procedure for paediatric inguinal hernia repair in our country, recurrence is an important problem. In this study, we examined the usefulness of double ligation in preventing recurrence in the LPEC procedure.MethodsWe retrospectively examined children with inguinal hernias who underwent the LPEC procedure. Cases from October 2012 to June 2016 who underwent surgery for inguinal hernias with single ligation were defined as Group A. Children operated on from July 2016 to April 2019, when double ligation was the standard procedure in our institution, were defined as Group B. We reviewed the operative time and postoperative complications including recurrence rate. Double ligation was performed by holding two threads on the LPEC needle and then binding them individually.ResultsGroup A consisted of 401 patients and Group B consisted of 360 patients. No significant differences were noted between the groups with respect to operative time, infection, metachronous contralateral hernia or testicular atrophy. The recurrence rate was significantly higher in Group A with recurrence occurring in seven cases compared to none in Group B.ConclusionsDouble ligation in the LPEC procedure is useful for preventing recurrence.

Rubber band ligation of symptomatic hemorrhoids: an old solution to an everyday problem.

The aim of this prospective study is to present the results of rubber band ligation (RBL) in 2635 consecutive patients with 2nd, 3rd, and 4th degree symptomatic hemorrhoids, the pain risk factors, and the applicability of the method in patients with liver cirrhosis and portal hypertension. A total of 1256 patients with 2nd, 1159 with 3rd, and 220 with 4th degree hemorrhoids were included in the study. Fifty-seven patients with hemorrhoids had liver cirrhosis and portal hypertension. RBL was performed using St Marks', McGinvey, and suction ligators. Single ligation was done in 178 patients, while 2457 patients had synchronous multiple ligations, in one (272), two (1289), and three (896 patients) sessions. After the end of treatment, 86.8% of our patients were asymptomatic and 84.5% remained asymptomatic 2 years later. A total of 593 patients had complications. Thirty required hospitalization, while pain was the most frequent complication (16.16 %). Multiple banding, young age, male sex, and external hemorrhoids were pain risk factors. RBL proved to be safe in 57 patients with coagulation disorders due to cirrhosis. Symptomatic recurrence was detected in 327 out of 2110 patients (15.49%), with repeat RBL in 219 cases and surgery in 108 cases. RBL is a safe, effective method for treating symptomatic 2nd and 3rd degree hemorrhoids. It can also be applied in selected cases of 4th degree hemorrhoids and patients with cirrhosis and portal hypertension.

V.S. Gruzdev. On the question of a single cord ligation. (Doctor, 1895, No. 7, 8 and 9)

From the extensive literary essay given by the author, it is clear that at the present time, there are various views among obstetricians regarding the merits of this or that way of maintaining the last period. In general, the methods of conducting the last period are reduced to expectant processes (Ahlfeld) and to active intervention (Cred). The author, noting the shortcomings of both methods, asks the question, will it not be possible to have a single ligation of the umbilical cord (fruit end), smooth out the bad sides of the Cred method and Ahlfeld's method, while preserving their advantages.

1803-P: Antifibrotic Effect of a Novel Long-Acting GLP-1/GIP/Glucagon Triple Agonist (HM15211) in BDL-Induced Liver Fibrosis Mice

Fibrosis due to nonalcoholic steatohepatitis (NASH) remains a major cause of liver-related mortality. However, no medication has been approved despite accelerated drug development. Since multiple biological pathways are involved, strategy simultaneously targeting multiple pathways might be required to effectively treat fibrosis. To address this, we developed a novel long-acting GLP-1/GIP/Glucagon triple agonist, HM15211. Previously, HM15211 significantly improved fibrosis as well as steatosis and inflammation in MCD-diet mice. In addition to the diet-induced models, bile duct ligation (BDL) has also been extensively used as an animal model of fibrosis. Here, we further investigated the anti-fibrotic effects of HM15211 in BDL-induced fibrosis mice. Two days post BDL (single ligation), the mice were administered either with HM152111 or obeticholic acid (OCA) for 2 weeks, followed by histological analysis. HM15211 treatment showed greater reduction in hepatic hydroxyproline (HP) (450.5, 351.7 and 253.6 nmol/g liver for vehicle, OCA and HM15211) and fibrosis score (1.7, 1.8 and 1.0 for vehicle, OCA and HM15211) compared to OCA. Consistently, HM15211, but not OCA, treatment was associated with the reduction of serum surrogate markers of fibrosis including TGF-β (-61.4, 9.1% vs. vehicle for HM15211, OCA), TIMP-1 (-71.6, -13.9% vs. vehicle), and hyaluronic acid (-68.9, 25.0% vs. vehicle). To further clarify its benefits in fibrosis, BDL (double ligation) mice with an extended induction period (up to 2 weeks) was used. Notably, despite the progression to advanced liver fibrosis, HM15211 treatment still attenuated HP (571.5 and 334.3 nmol/g liver for vehicle and HM15211) and fibrosis score markedly. Considering the pathologic features of BDL mice (liver fibrosis without liver fat accumulation), these results demonstrate the direct anti-fibrotic effect of HM15211. Further studies are needed to assess the clinical relevance of these findings. Disclosure J. Kim: Employee; Self; Hanmi Pharmaceutical. J. Lee: Employee; Self; Hanmi Pharmaceutical. H. Kwon: Employee; Self; Hanmi Pharmaceutical. J. Lee: Employee; Self; Hanmi Pharmaceutical. D. Kim: Board Member; Self; Hanmi Pharmaceutical. S. Bae: Employee; Self; Hanmi Pharmaceutical. S. Lee: Employee; Self; Hanmi Pharmaceutical. I. Choi: Board Member; Self; Hanmi Pharmaceutical.

Comparison of single versus double versus triple site band ligation for haemorrhoids in a single visit: a case-controlled double blind randomized study

Background: Rubber band ligation is considered one of the most popular non-surgical procedures available, representing the most reasonable balance between efficacy, pain and potential of complication. The present study was a controlled double blinded randomized study to compare Single versus double versus triple site band ligation for haemorrhoids in single treatment visit with special regards to its safety and efficacy in our patient population.Methods: There were 78 patients equally randomized in three groups by closed enveloped method. The number of patients in each group was 26, In Group A, a single, in Group B, two, and in Group C three major haemorrhoidal groups were rubber band ligated, in a single treatment visit on outpatient basis under topical anaesthesia with lignocaine 2% jelly.Results: Rubber band ligation was effective with significant symptom improvement seen in all patients with 88% patient satisfaction at end of 30 days period. Post ligation pain score and number of analgesic requirement in immediate post band ligation period, at 12 hours, at 1,7,14, 21 and 30 days post band ligation were similar in all three groups with p value >0.05. Multiple site haemorrhoidal bandings was done safely at single session without severe discomfort, pain or bleeding or severe complications requiring hospitalization.Conclusions: The study finding shows that triple site band ligation in a single session is a safe way of treating symptomatic haemorrhoids with similar post-procedure pain and patient satisfaction as conventional single or double site band ligation.

Loss of Endothelial Glycocalyx Hyaluronan Impairs Endothelial Stability and Adaptive Vascular Remodeling After Arterial Ischemia.

We recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such derangements in the glycocalyx may also impair the adaptive response to vascular ischemia. Endothelial specific conditional hyaluronan synthase 2-KO (Has2-cKO) mice revealed reduced endothelial HA expression and lower hindlimb perfusion at baseline compared to control mice. After a single ligation of the common femoral artery in these mice, we observed dysregulated angiogenesis in the gastrocnemius muscle which did not restore capillary perfusion. Mechanistically, decreased endothelial binding of the pericyte-derived molecule angiopoietin1 (Ang1) could be observed in the Has2-cKO mouse. In vitro angiogenesis assays with an endothelial cell-pericyte coculture confirmed such disturbed Ang1-TIE2 signaling resulting in excessive angiogenesis upon loss of HA. These data could be of relevance to diabetes patients, where we confirm loss of endothelial HA in the microcirculation of muscle tissue, indicating that this may contribute to the known disturbed adaptation to ischemia in these patients. In summary, loss of endothelial HA results in impaired microvascular perfusion and endothelial stability in ischemic gastrocnemius muscle. Endothelial HA is a potential target to improve angiogenic therapy in diabetic patients with critical limb ischemia.

Reoperation After Laparoscopic Inguinal Hernia Repair in Children: A Retrospective Review.

Background: Laparoscopic inguinal hernia repair (LIHR) is increasingly widely accepted in pediatric surgery. However, reoperation risks remain unknown. We summarized our single-center experience with reoperations after laparoscopic percutaneous extraperitoneal closure (LPEC) and single-incision LPEC (SILPEC). Materials and Methods: We retrospectively reviewed reoperation cases of hernia repair greater than or equal to two times between 2000 and 2018, wherein the first hernia repair was performed laparoscopically. Primary outcomes were recurrence type and screening sufficiency for contralateral patent processus vaginalis (cPPV). Secondary outcomes were associated with details of recurrences. Results: Of the 2112 patients who underwent LPEC/SILPEC, 14 (recurrence rate = 0.7%) showed recurrences after treatment and 8 (incidence rate = 0.4%) showed contralateral metachronous inguinal hernia (CMIH). Concerning the primary outcome of recurrence type, the orifice was inside the previous ligation (Inside group), suggesting loosened first ligation, in 6 (42.9%) patients and outside the previous ligation (Outside group) in 7 (50.0%); and 1 (7.1%) patient showed no orifice. Regarding CMIH, 3 (37.5%) patients were suspected of insufficient screening for cPPV, 1 (12.5%) underwent sufficient screening, and 1 (12.5%) had cPPV but treatment was deemed unnecessary. Concerning secondary outcomes, 4 (66.7%) and 6 (85.7%) patients from the Inside and Outside groups were treated with single ligation, respectively. One patient from the Outside group (14.3%) had a massive peritoneum injury during the first operation. Conclusions: Some preventable factors, such as loosened ligation, torn peritoneum, and use of single ligation in recurrences and insufficient screening for cPPV in CMIH, were observed. These should be taken care to prevent reoperations in LIHR.

Experimental Characterization of the Chronic Constriction Injury-Induced Neuropathic Pain Model in Mice.

Number of ligations made in the chronic constriction injury (CCI) neuropathic pain model has raised serious concerns. We compared behavioural responses, nerve morphology and expression of pain marker, c-fos among CCI models developed with one, two, three and four ligations. The numbers of ligation(s) on sciatic nerve shows no significant difference in displaying mechanical and cold allodynia, and mechanical and thermal hyperalgesia throughout 84days. All groups underwent similar levels of nerve degeneration post-surgery. Similar c-fos level in brain cingulate cortex, parafascicular nuclei and amygdala were observed in all CCI models compared to sham-operated group. Therefore, number of ligations does not impact intensity of pain symptoms, pathogenesis and neuronal activation. A single ligation is sufficient to develop neuropathic pain, in contrast to the established model of four ligations. This study dissects and characterises the CCI model, ascertaining a more uniform animal model to surrogate actual neuropathic pain condition.

Glucocorticoid Receptor-Tethered Mineralocorticoid Receptors Increase Glucocorticoid-Induced Transcriptional Responses.

Mineralocorticoid and glucocorticoid receptors (MRs and GRs) constitute a functionally important dual receptor system detecting and transmitting circulating corticosteroid signals. High expression of MRs and GRs occurs in the same cells in the limbic system, the primary site of glucocorticoid action on cognition, behavior, and mood; however, modes of interaction between the receptors are poorly characterized. We used chromatin immunoprecipitation with nucleotide resolution using exonuclease digestion, unique barcode, and single ligation (ChIP-nexus) for high-resolution genome-wide characterization of MR and GR DNA binding profiles in neuroblastoma cells and demonstrate recruitment to highly similar DNA binding sites. Expressed MR or GR showed differential regulation of endogenous gene targets, including Syt2 and Ddc, whereas coexpression produced augmented transcriptional responses even when MRs were unable to bind DNA (MR-XDBD). ChIP confirmed that MR-XDBD could be tethered to chromatin by GR. Our data demonstrate that MR can interact at individual genomic DNA sites in multiple modes and suggest a role for MR in increasing the transcriptional response to glucocorticoids.

Single site robotic-assisted laparoscopic transperitoneal ligation of ovarian veins for treatment of pelvic congestion syndrome, a case report and review of literature

Single site robotic-assisted laparoscopic transperitoneal ligation of ovarian veins for treatment of pelvic congestion syndrome, a case report and review of literature

The differences in histological changes among pulmonary vessels divided with an energy device.

Histological changes after division of the pulmonary artery (PA) and the pulmonary vein (PV) using a vessel-sealing device are not fully understood. The goal of the present study was to clarify histologically and immunohistochemically how division with the device affects the wall layers of the pulmonary vasculature. This prospective cohort study analysed outcomes of 20 patients who underwent anatomical lung resection. After a single proximal ligation, the PA and the PV (diameter 2-7 mm) were divided using a LigaSure Blunt Tip (LSB). Histological findings and thermal damage were evaluated in vascular specimens from resected lungs. The PA has a well-developed media with rich elastic fibres and a thin adventitia, whereas the PV has a thinner media and a thicker adventitia with abundant collagen fibres. Vascular division of the PAs and PVs appeared complete to the naked eye. However, in all divided PAs, the area adjacent to the sealed zone comprised only adventitia and thin disrupted media. Additionally, thermal energy generated by the LSB resulted in a wide area of thermal necrosis over the histologically fragile region in all cases. Conversely, the wall layers of all divided PVs were completely fused without disruption. Thermal spread and disruption did not significantly differ between small (2-4 mm) and large (5-7 mm) PAs [187 (150-253) vs 236 (190-275) μm, P = 0.22; 180 (138-200) vs 210 (161-305) μm, P = 0.22]. Histological changes differed significantly between the pulmonary vessels after division using the LSB. Surgeons should consider that dividing the pulmonary vessels with a vessel-sealing device might have more histological impact on the layers of the wall of the PA than on those of the PV, although it remains unclear whether these findings constitute a clinical risk.

Evaluation of a pinhole castration technique in ponies: Comparing single with double ligation (using silk or catgut) of the spermatic cord

SummaryThe objective of the study was to evaluate a pinhole castration technique in male ponies. Adult ponies (n = 12) were randomly allotted to one of the two equal groups. Both of the groups were anaesthetised with xylazine–ketamine–diazepam and had single (SLS, SLC) ligation of the spermatic cord on one side and double (DLS, DLC) ligation on the other side using silk (Group T1) and catgut (Group T2). Single ligation whether using silk or catgut (SLS and SLC) was completed in 3.50 ± 0.34 min. DLS took 6.66 ± 0.49 min and DLC 7.16 ± 0.47 min. Scrotal oedema was noticed in all of the ponies from Day 1 to Day 15. The scrotal circumference and testicular volume in animals of both of the groups showed a significant (P<0.05) increase from Day 1 to Day 4. Orchiectomy to recover testicular remnants was performed in all of the animals on Day 41. Straw‐coloured fluid accumulation was noticed in seven testicular remnants (4 T1 and 3 T2). One testicle from Group DLS showed suppuration. Multiple gross and histological abnormalities were detected in all double ligated testes. The changes were more severe in the DLS than the DLC group and their epididymis (n = 3) also showed necrosis, fibroplasia and an obstructed lumen. Severe adhesions had developed in three and one testicle only from the SLS and SLC groups respectively. The remaining testes and epididymis in both of these groups showed only mild‐to‐moderate adhesions. From this study, it was concluded that castration may not be achieved by percutaneous single ligation of the spermatic cord in ponies. Although double ligation induces marked gross and histopathological changes, assessments of the testosterone levels and sperm analysis are required before recommending this procedure. Use of silk for ligation of the spermatic cord is advantageous over catgut but maintenance of strict asepsis is mandatory.

Conformational Dynamics Modulate Activation of the Ubiquitin Conjugating Enzyme Ube2g2.

The ubiquitin conjugating enzyme Ube2g2 together with its cognate E3 ligase gp78 catalyzes the synthesis of lysine-48 polyubiquitin chains constituting signals for the proteasomal degradation of misfolded proteins in the endoplasmic reticulum. Here, we employ NMR spectroscopy in combination with single-turnover diubiquitin formation assays to examine the role of the RING domain from gp78 in the catalytic activation of Ube2g2∼Ub conjugates. We find that approximately 60% of the Ube2g2∼Ub conjugates occupy a closed conformation in the absence of gp78-RING, with the population increasing to 82% upon gp78-RING binding. As expected, strong mutations in the hydrophobic patch residues of the ∼Ub moiety result in Ube2g2∼Ub populating only open states with corresponding loss of the ubiquitin conjugation activity. Less disruptive mutations introduced into the hydrophobic patch of the ∼Ub moiety also destabilize the closed conformational state, yet the corresponding effect on the ubiquitin conjugation activity ranges from complete loss to an enhancement of the catalytic activity. These results present a picture in which Ube2g2’s active site is in a state of continual dynamic flux with the organization of the active site into a catalytically viable conformation constituting the rate-limiting step for a single ubiquitin ligation event. Ube2g2’s function as a highly specific K48-polyubiquitin chain elongator leads us to speculate that this may be a strategy by which Ube2g2 reduces the probability of nonproductive catalytic outcomes in the absence of available substrate.

Targeted chromatin ligation, a robust epigenetic profiling technique for small cell numbers.

The complexity and inefficiency of chromatin immunoprecipitation strategies restrict their sensitivity and application when examining rare cell populations. We developed a new technique that replaces immunoprecipitation with a simplified chromatin fragmentation and proximity ligation step that eliminates bead purification and washing steps. We present a simple single tube proximity ligation technique, targeted chromatin ligation, that captures histone modification patterns with only 200 cells. Our technique eliminates loss of material and sensitivity due to multiple inefficient steps, while simplifying the workflow to enhance sensitivity and create the potential for novel applications.

A universal phage display system for the seamless construction of Fab libraries

The construction of Fab phage libraries requires the cloning of domains from both the light and the heavy chain of antibodies. Despite the advent of powerful strategies such as splicing-by-overlap extension PCR, obtaining high quality libraries with excellent coverage remains challenging. Here, we explored the use of type IIS restriction enzymes for the seamless cloning of Fab libraries. We analyzed human, murine and rabbit germline antibody repertoires and identified combinations of restriction enzymes that exhibit very few or no recognition sites in the antibody sequences. We describe three phagemid vectors, pUP-22Hb, pUP-22Mc and pUP-22Rc, which were employed for cloning the Fab repertoire of these hosts using BsmBI and SapI (human) or SapI alone (mouse and rabbit). Using human serum albumin as a model immunization, we built a mouse/human chimeric Fab library and a mouse Fab library in a single step ligation and successfully panned multiple cognate antibodies. The overall process is highly scalable and faster than PCR-based techniques, with a Fab insertion success rate of around 80%. By using carefully chosen overhangs on each end of the antibody domains, this approach paves the way to the universal, sequence- and vector-independent cloning and reformatting of antibody libraries.

New Hairpin RNAi Vector with Brassica rapa ssp. pekinensis Intron for Gene Silencing in Plants

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpinbased double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR ), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops

Intraoperative Computed Tomography (CT) for Treating Giant Carotid Intracavernous Aneurysms.

BackgroundGiant carotid intracavernous aneurysm refers to those lesions larger than 2.5 cm and derived from a cavernous segment, accounting for about 30% of all intracranial tumors. Dynamic CT perfusion imaging (PCT) is a common method recently employed to evaluate cerebral perfusion. This study investigated the efficacy and clinical application of intraoperative CT in the surgery for giant symptomatic carotid intracavernous aneurysm.Material/MethodsA retrospective analysis was performed on 23 cases with giant symptomatic carotid intracavernous aneurysm. BTO testing was performed before surgery. Differential treatments were performed based on the condition of aneurysm, and some patients received intraoperative PCT. Postoperative anti-coagulation was given with DSA or CTA follow-up examinations at 3–6 months, 1 year, and 2 years after surgery.ResultsA total of 17 patients received aneurysm isolation coupled with high-flow bypass surgery. Among those, 9 developed early-onset neurological function after surgery, with gradual recover within 6 months. One coma patient died 25 months after discharge. One patient had aneurysm isolation with clapping of anterior communicating artery, and the other 5 cases received artery clapping only. In those patients, 4 had improvement at early phase, while 1 patient had numbness of the oculomotor nerve. Six patients received surgery in the CT room, including 5 cases with single proximal ligation of the internal carotid artery plus 1 aneurysm isolation combined with high-flow bypass surgery.ConclusionsIntraoperative PCT can provide objective evidence and effective evaluation of cerebral perfusion.

Effects of Antenatal Melatonin Treatment on the Cerebral Vasculature in an Ovine Model of Fetal Growth Restriction

Chronic moderate hypoxia, such as occurs in fetal growth restriction (FGR) during gestation, compromises the blood-brain barrier (BBB) and results in structural abnormalities of the cerebral vasculature. We have previously determined the neuroprotective and antioxidant effects of maternal administration of melatonin (MLT) on growth-restricted newborn lambs. The potential of maternal MLT therapy for the treatment of cerebrovascular dysfunction-associated developmental hypoxia has also been demonstrated in newborn lambs. We assessed whether MLT had an effect on the previously reported structural and cerebral vascular abnormalities in chronically hypoxic FGR lambs. Single umbilical-artery ligation surgery was performed in fetuses at approximately 105 days of gestation (term: 147 days) to induce placental insufficiency and FGR, and treatment with either saline or an MLT infusion (0.1 mg/kg) was started 4 h after surgery. Ewes delivered naturally at term and lambs were euthanased 24 h later. We found a significant reduction in the number of laminin-positive blood vessels within the subcortical and periventricular white matter (SCWM and PVWM) and the subventricular zone (SVZ) in FGR (p < 0.0005) and FGR + MLT brains (p < 0.0005 vs. controls), with no difference found between FGR and FGR + MLT animals. This was associated with a significant decrease in VEGF immunoreactivity in FGR and FGR + MLT brains versus controls (p < 0.0005; SCWM and PVWM) and in the SVZ in FGR brains versus controls (p < 0.005) and also with significantly lower levels of proliferating blood vessels versus controls (p < 0.0005). Glucose transporter-1 immunoreactivity (vascular endothelium) was decreased in FGR versus control lambs (p < 0.0005) in SCWM, PVWM, and the SVZ; it was significantly increased in FGR + MLT lambs compared with FGR lambs in SCWM and PVWM (p < 0.005) and even more markedly in the SVZ (p < 0.0005). FGR brains showed a 72% reduction in pericyte coverage versus control lambs and 68% versus FGR + MLT in PVWM. In SCWM, we found a 77 and 73% reduction compared with control and FGR + MLT lambs, respectively, while in the SVZ, we observed a 68% reduction versus controls and a 70% reduction in FGR versus FGR + MLT lambs. Astrocyte end-feet coverage in the SCWM showed a significant 24% reduction in FGR versus control levels, a 42% decrease within the PVWM, and a 35% decrease within the SVZ versus controls. MLT normalized astrocyte attachment to blood vessels, with no difference seen between controls and FGR + MLT animals in any of the brain regions examined. We also observed a decrease in albumin extravasation and microhemorrhage in controls and FGR + MLT brains versus FGR lambs. Our results demonstrate that umbilicoplacental insufficiency is associated with FGR-produced vascular changes in the white matter and SVZ of FGR newborn brains and that maternal MLT prevented disruption of the BBB by protecting perivascular cells essential for the maintenance of vascular homeostasis and stability.

Synthesis of Grb2 SH2 Domain Proteins for Mirror-Image Screening Systems.

Grb2 is an adaptor protein that mediates cellular signal transduction. Grb2 contains an SH2 domain that interacts with phosphotyrosine-containing sequences in EGFR and other signaling molecules, and it is a promising molecular target for anticancer agents. To identify novel inhibitors of the Grb2 SH2 domain from natural products and their mirror-image isomers, screening systems using both enantiomers of a synthetic Grb2 SH2 domain protein were established. A pair of synthetic procedures for the proteins were investigated: one employed a single native chemical ligation (NCL) of two segment peptides, and the other used the N-to-C-directed NCL of three segment peptides for easier preparation. Labeling at the N-terminus or the Ala115 residue of the Grb2 SH2 domain provided functional probes to detect binding to a phosphotyrosine-containing peptide. The resulting synthetic-protein-based probes were applied to bioassays, including chemical array analysis and enzyme-linked immunosorbent assays.