Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling.

Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.

DNA G-Quadruplex Recognition In Vitro and in Live Cells by a Structure-Specific Nanobody.

G-quadruplexes (G4s) are four-stranded DNA secondary structures that occur in the human genome and play key roles in transcription, replication, and genome stability. G4-specific molecular probes are of vital importance to elucidate the structure and function of G4s. The scFv antibody BG4 has been a widely used G4 probe but has various limitations, including relatively poor in vitro expression and the inability to be expressed intracellularly to interrogate G4s in live cells. To address these considerations, we describe herein the development of SG4, a camelid heavy-chain-only derived nanobody that was selected against the human Myc DNA G4 structure. SG4 exhibits low nanomolar affinity for a wide range of folded G4 structures in vitro. We employed AlphaFold combined with molecular dynamics simulations to construct a molecular model for the G4-nanobody interaction. The structural model accurately explains the role of key amino acids and Kd measurements of SG4 mutants, including arginine-to-alanine point mutations that dramatically diminish G4 binding affinity. Importantly, predicted amino acid-G4 interactions were subsequently confirmed experimentally by biophysical measurements. We demonstrate that the nanobody can be expressed intracellularly and used to image endogenous G4 structures in live cells. We also use the SG4 protein to positionally map G4s in situ and also on fixed chromatin. SG4 is a valuable, new tool for G4 detection and mapping in cells.

Glomerular filtration rate early after liver transplantation independently predicts atherosclerotic events.

Cardiovascular disease (CVD) is an important cause of mortality among liver transplantation (LT) recipients; however, the data on CVD risk stratification following LT are limited. Thus, the primary aim of this study was to evaluate the association between decline in renal function early after LT and atherosclerotic events. This retrospective study included all patients receiving LT between 2007 and 2019. Early renal function was quantified as estimated glomerular filtration rate (GFR) 6 months after LT. The primary endpoint for the study was a composite atherosclerotic cardiovascular event of three-point major adverse cardiovascular events (MACEs), which includes nonfatal myocardial infarction (MI), nonfatal stroke, or death from CVD. A total of 553 LT recipients met entry criteria. After a median follow-up of 74 months (interquartile range 46-111), 94 (17%) LT recipients died and CVD-associated death occurred in 20 patients. MACE-3 occurred in 66 (12%) patients, with nonfatal MI being the most common event (n = 30). A strong inverse relationship between early GFR and MACE-3 was noted in unadjusted analysis with hazard ratio (HR) 0.96 (95% confidence interval [CI] 0.95-0.98; p = 0.0001) and remained significant even after accounting for age, sex, coronary artery disease, diabetes mellitus, hypertension, calcineurin inhibitor use, and Framingham Risk Score (FRS; HR 0.96, 95% CI 0.95-0.97; p = 0.0001 per unit increase in GFR). Furthermore, an independent interaction between GFR, FRS, and likelihood of developing an MACE-3 was noted. GFR 6 months following LT is a strong predictor of developing atherosclerotic events. This relationship is independent of traditional CVD risk stratification models (e.g. FRS) and thus has the potential to be incorporated into CVD risk assessment after LT but requires further validation.

The addition of oral Tranexamic acid to knee arthroplasty patients does not further improve blood loss: a double blinded randomized control trial.

Perioperative intravenous (IV) TA has become routine in knee and hip arthroplasty. Less evidence exists on the administration of oral TA in the post- operative period. Our study aims to identify the efficacy and safety of combined perioperative IV and post-operative oral TA on blood loss and Hemoglobin (Hb) drop compared to perioperative IV TA alone. Patients undergoing primary elective knee arthro- plasty at our institution were invited to participate in the study (n=50). A computer-generated randomisation sequence was created online (www.randomization. org), with an allocation ratio of 1:1 and a block size of 50. Group A received perioperative IV TA alone and post-operative oral TA (n= 26) and Group B received perioperative IV TA plus 48 hours additional oral placebo (n= 24). Day 3 total blood loss and Hb drop was calculated. Continuous, normally distributed data (total blood loss) was compared utilising using one-way analysis of variance with post hoc Tukey test. Continuous skewed data (Hb drop) was compared using the Kruskal-Wallis test. P <0.05 was considered statistically significant. Group A demonstrated a trend in decreased total blood loss that was close to statistical significance ( p = 0.072). No difference in Hb drop was identified between the 2 groups. Increased nausea was also observed in Group A. The administration of oral TA to post-operative knee arthroplasty patients does not improve further blood loss compared to patients receiving perioperative IV TA pre-operatively and at wound closure.

Resurrection of Chronically Occluded Prosthetic Bypass Grafts in a Single Stage with Suction Thrombectomy and Intervention.

BACKGROUND: Peripheral arterial occlusive disease (PAOD) continues to be a vexing problem despite the advent of endovascular techniques augmenting traditional open repair. At our institution, we have found there is a growing number of patients with PAOD who are vein-challenged and have undergone prosthetic bypass previously for infrainguinal arterial reconstruction. When occluded, these grafts have been abandoned for a new bypass strategy or amputation. We present a novel technique of reestablishing flow through chronically occluded prosthetic bypass grafts. METHODS: A retrospective review of a prospectively maintained database compiled at 2 institutions between 2016 and 2019 was performed. Six patients had previous prosthetic bypass grafts with 4 patients having femoral to popliteal grafts, 1 patient with a femoral to femoral graft, and 1 with a femoral to posterior tibial bypass graft. All patients had an attempted single-stage intervention to clear chronically occluded grafts. RESULTS: A total of 6 patients were included in the study. Indications for intervention were chronic, critical limb ischemia with tissue loss (3), severe claudication (2), and acute on chronic limb ischemia (1). Average time from bypass to suction thrombectomy was 29 months (6-60 months). Mean patency duration is 13 months (1-28 months). Adjunctive procedures include overnight lysis to improve outflow in 1 patient (16.6%), drug-coated balloon angioplasty (83.3%), or stents (83.3%). There were no embolic complications during these procedures. All (2) wounds healed and all are maintained on full-dose anticoagulation and/or antiplatelet therapy. CONCLUSIONS: Often, the timing of bypass graft occlusion is unknown, and the risk of embolism with lysis for chronically occluded bypass grafts is concerning with traditional peripheral intervention techniques. We report a new and unique minimally invasive approach to resurrect chronically occluded prosthetic bypass grafts often successful in just one stage. This tool offers an alternative technique for limb salvage in complex patients and as use increases, requires further interrogation.

Regorafenib is effective against neuroblastoma in vitro and in vivo and inhibits the RAS/MAPK, PI3K/Akt/mTOR and Fos/Jun pathways.

BACKGROUND: Regorafenib is an inhibitor of multiple kinases with aberrant expression and activity in neuroblastoma tumours that have potential roles in neuroblastoma pathogenesis. METHODS: We evaluated neuroblastoma cells treated with regorafenib for cell viability and confluence, and analysed treated cells for apoptosis and cell cycle progression. We evaluated the efficacy of regorafenib in vivo using an orthotopic xenograft model. We evaluated regorafenib-mediated inhibition of kinase targets and performed reverse-phase protein array (RPPA) analysis of neuroblastoma cells treated with regorafenib. Lastly, we evaluated the efficacy and effects of the combination of regorafenib and 13-cis-retinoic acid on intracellular signalling. RESULTS: Regorafenib treatment resulted in reduced neuroblastoma cell viability and confluence, with both induction of apoptosis and of cell cycle arrest. Regorafenib treatment inhibits known receptor tyrosine kinase targets RET and PDGFRß and intracellular signalling through the RAS/MAPK, PI3K/Akt/mTOR and Fos/Jun pathways. Regorafenib is effective against neuroblastoma tumours in vivo, and the combination of regorafenib and 13-cis-retinoic acid demonstrates enhanced efficacy compared with regorafenib alone. CONCLUSIONS: The effects of regorafenib on multiple intracellular signalling pathways and the potential additional efficacy when combined with 13-cis-retinoic acid represent opportunities to develop treatment regimens incorporating regorafenib for children with neuroblastoma.

Initial Results of Selected Use of Covered Stents in Transcarotid Artery Revascularization.

BACKGROUND: Transcarotid artery revascularization (TCAR) is a novel, hybrid approach to treating carotid disease in the treatment of stroke and stroke prevention. Early results of this hybrid approach to carotid stenting using flow reversal have been promising, with reported stroke rates around 1-2.8%.1,2 Currently, carotid stenting, regardless of approach, is performed with uncovered stents, which incurs the risk of plaque protrusion through the stent and in-stent restenosis. Overall, plaque protrusion is a rare event, with a reported incidence of 2.8% on angiography, but it is associated a high rate of ischemic complications (up to 66.7%).3 The use of covered stents could eliminate the risk of plaque protrusion and therefore short to midterm embolic phenomenon during the remodeling process. It also may improve rates of in-stent restenosis as it is a fully covered stent. Adoption of this technique has the potential to further improve the safety, efficacy, and durability of TCAR. METHODS: We performed a retrospective review of a prospectively maintained database of patients undergoing TCAR with covered stents between September 2018 and December 2019. Procedures were performed by the same operator at 2 separate institutions. Indications included severe asymptomatic or symptomatic carotid stenosis with high-risk lesions defined as lesions 2 cm lesions or longer and/or >50% of the lesion containing soft plaque or bleeding carotid pseudoaneurysm. Our primary outcomes included periprocedural and 30-day stroke rates. Secondary outcomes included stent patency and other procedure-related complications. All patients were maintained on clopidogrel postprocedure for 3 months and then transitioned to aspirin, unless otherwise indicated. RESULTS: A total of 6 patients underwent TCAR with covered stent angioplasty during this time period. Patient demographics included 5 males and 1 female, with an average age of 70.8 ± 4.6 years. Indications for stenting included 4 patients with asymptomatic >70% carotid stenosis and 1 patient with transient ischemic attack-like symptoms and >70% stenosis, and 1 patient with bleeding carotid pseudoaneurysm. Gore Viabahn covered stents were used in all patients. There were no periprocedural or postprocedural ischemic events at 30 days. All 6 stents remained patent at follow-up on duplex ultrasound, and all patients remained asymptomatic on clinical follow-up (average 3.4 [1.4-6.9] months). CONCLUSIONS: The use of covered stents for TCAR appears to be a safe and effective in select patients requiring carotid intervention. It holds the potential to decrease ischemic events from plaque protrusion and in-stent restenosis in the long-term. Further investigation in device design or clinical evaluation is warranted.

Novel Approach to Treatment of Chronic Iliocaval Occlusion Utilizing Covered Stents.

BACKGROUND: Chronic iliocaval obstruction is a challenging clinical entity to treat. Endovenous iliocaval stenting is becoming the treatment of choice for central vein stenosis and occlusion. However, outcomes in thrombotic disease have not been as robust as nonthrombotic disease. In this article, we describe our experience utilizing covered stents as a novel tool for chronic total occlusions of the iliocaval veins. METHODS: We performed a retrospective review of a prospectively maintained database of all patients undergoing endovenous stenting with a covered stent for chronic occlusive iliocaval disease over a 3-year period at our institution. Patients were followed clinically and with venous duplexes to assess the feasibility, safety, and outcomes of iliocaval endovenous stenting with covered stents. RESULTS: A total of 10 patients (8 men and 2 women) underwent iliocaval stenting with covered stents from July 2015 to May 2018. A total of 20 self-expanding covered stents (SECS) and 13 balloon expandable covered stents (BECS) were deployed in the central veins of the 10 patients. Six SECS and 5 BECS were deployed in the inferior vena cava, 10 SECS and 6 BECS were deployed in the common iliac veins (CIVs) (5 patients had bilateral CIV BECS and 2 patients had bilateral CIV SECS), and 4 SECS and 2 BECS were deployed in the external iliac veins (2 patients had bilateral SECS placed). Median follow-up time was 12.1 (range 0.5-35.0) months. There were no perioperative or postoperative complications. Nine (90%) patients maintained primary stent patency during our follow-up time. One patient (10%) had rethrombosis of his stent due to undertreated common femoral vein disease in the setting of a new myeloproliferative neoplasm and an inappropriate cessation of therapeutic anticoagulation. All patients who were symptomatic preoperatively had improvement in their pain, venous ulceration, and venous claudication. Eight of nine (89%) patients had improvement in their lower extremity edema. CONCLUSIONS: Covered endovenous stenting of chronically occluded central veins is a safe and promising procedure. Their use may improve the short- and long-term outcomes in this challenging patient population. Further studies are required to evaluate the long-term efficacy and cost of their use.

Carnoy's solution fixation with compression significantly increases the number of lymph nodes yielded from colorectal cancer specimens.

BACKGROUND AND OBJECTIVES: Carnoy's fixation and compression represents a novel technique to enhance lymph node evaluation and accuracy of staging after colorectal cancer resection. METHODS: This study was performed in all adults undergoing colorectal cancer operations by Kaiser Permanente surgeons at two separate facilities. Patients were assigned to either location based upon surgeon and patient availability. One group of patients had their lymph nodes examined with current standard manual technique (MT). The other group had their specimens fixed with Carnoy's solution and then compressed (CT) to assess for lymph nodes. RESULTS: A total of 157 patients were enrolled. Seventy-eight patient specimens underwent MT and 79 patient specimens underwent the new compression technique (CT). CT resulted in a significant increase in total lymph node yield per specimen (37.6 ± 18.5 nodes with CT vs 18.9 ± 8.8 nodes with MT; P < 0.0001). CT also resulted in sufficient lymph node sampling (>12 nodes) in all 79 patients in the group compared with 13 of 78 patients (17%) with an insufficient lymph node evaluation in the MT group ( P = 0.0002). CONCLUSION: This study demonstrated that Carnoy's fixation with compression can significantly increase lymph node yields in colorectal cancer specimens and allow for a higher rate of adequate lymph node sampling.

Alterations in sociability and functional brain connectivity caused by early-life seizures are prevented by bumetanide.

There is a well-described association between infantile epilepsy and pervasive cognitive and behavioral deficits, including a high incidence of autism spectrum disorders. Despite the robustness of the relationship between early-life seizures and the development of autism, the pathophysiological mechanism by which this occurs has not been explored. As a result of increasing evidence that autism is a disorder of brain connectivity we hypothesized that early-life seizures would interrupt normal brain connectivity during brain maturation and result in an autistic phenotype. Normal rat pups underwent recurrent flurothyl-induced seizures from postnatal (P)days 5-14 and then tested, along with controls, for developmental alterations of development brain oscillatory activity from P18-P25. Specifically we wished to understand how normal changes in rhythmicity in and between brain regions change as a function of age and if this rhythmicity is altered or interrupted by early life seizures. In rat pups with early-life seizures, field recordings from dorsal and ventral hippocampus and prefrontal cortex demonstrated marked increase in coherence as well as a decrease in voltage correlation at all bandwidths compared to controls while there were minimal differences in total power and relative power spectral densities. Rats with early-life seizures had resulting impairment in the sociability and social novelty tests but demonstrated no evidence of increased activity or generalized anxiety as measured in the open field. In addition, rats with early-life seizures had lower seizure thresholds than controls, indicating long-standing alterations in the excitatory/inhibition balance. Bumetanide, a pharmacological agent that blocks the activity of NKCC1 and induces a significant shift of ECl toward more hyperpolarized values, administration at the time of the seizures precluded the subsequent abnormalities in coherence and voltage correlation and resulted in normal sociability and seizure threshold. Taken together these findings indicate that early-life seizures alter the development of oscillations and result in autistic-like behaviors. The altered communication between these brain regions could reflect the physiological underpinnings underlying social cognitive deficits seen in autism spectrum disorders.

Symptom changes in five dimensions of the Positive and Negative Syndrome Scale in refractory psychosis.

Refractory psychosis units currently have little information regarding which symptoms profiles should be expected to respond to treatment. In the current study, we provide this information using structural equation modeling of Positive and Negative Syndrome Scale (PANSS) ratings at admission and discharge on a sample of 610 patients admitted to a treatment refractory psychosis program at a Canadian tertiary care unit between 1990 and 2011. The hypothesized five-dimensional structure of the PANSS fit the data well at both admission and discharge, and the latent variable scores are reported as a function of symptom dimension and diagnostic category. The results suggest that, overall, positive symptoms (POS) responded to treatment better than all other symptoms dimensions, but for the schizoaffective and bipolar groups, greater response on POS was observed relative to the schizophrenia and major depression groups. The major depression group showed the most improvement on negative symptoms and emotional distress, and the bipolar group showed the most improvement on disorganization. Schizophrenia was distinct from schizoaffective disorder in showing reduced treatment response on all symptom dimensions. These results can assist refractory psychosis units by providing information on how PANSS symptom dimensions respond to treatment and how this depends on diagnostic category.

Impact of Liver Transplantation on Adipose Tissue Compartments and Its Association With Metabolic Sequela.

BACKGROUND: Loss of skeletal muscle can be accompanied by an increase in adipose tissue leading to sarcopenic obesity. There are limited data on how liver transplantation (LT) might impact adipose tissue compartments, particularly among patients with metabolically active disease, such as nonalcoholic steatohepatitis (NASH) and subsequent metabolic sequela. METHODS: Skeletal muscle, visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) were measured using cross-sectional imaging performed in 190 patients pre-LT, 6 mo post-LT and 12 mo post-LT. Changes in adipose tissue and their impact on metabolic diseases were determined in patients transplanted for NASH versus non-NASH. RESULTS: Skeletal muscle, VAT, and SAT were similar in patients with NASH and non-NASH pre-LT despite a higher burden of metabolic diseases in patients with NASH. Following LT, no significant differences between skeletal muscle and SAT were observed in the entire cohort and among patients with NASH (versus non-NASH). LT recipients with the highest muscle mass pre-LT were at the greatest risk for muscle loss post-LT. A time-dependent increase in VAT was noted post-LT, which was more robust among patients with a history of NASH cirrhosis. In adjusted multivariate analysis, NASH versus non-NASH was a strong predictor of post-LT increase in VAT (ß-coefficient 3.00, P = 0.04). Pre-LT VAT was an independent predictor of post-LT serum triglycerides (ß-coefficient 5.49 ± 2.78, P = 0.05) and low-density lipoprotein cholesterol (ß-coefficient 1.80 ± 0.75, P = 0.02). A trend between pre-LT VAT and diabetes was noted but did not reach statistical significance. CONCLUSIONS: VAT but not SAT increases rapidly after LT, especially among patients transplanted for NASH cirrhosis and predicts future metabolic burden.

Subsidence of monoblock and modular titanium fluted tapered stems in revision hip arthroplasty: A retrospective multicentre comparison study.

Background: Tapered, fluted, titanium (TFT) stems have shown good clinical outcomes in revision total hip arthroplasty (rTHA), however concerns exist regarding early subsidence. This study compares subsidence between a modern monoblock 3-degree and a modular 2-degree TFT stem in rTHA. Methods: A retrospective, international multicentre comparative study was conducted including 64 rTHA in 63 patients. A monoblock TFT stem was used in 37 cases and a modular TFT stem was used in 27 cases. Patient demographics, Paprosky femoral bone loss classification, bicortical contact and stem subsidence were recorded at minimum four week follow up. Results: There was no statistically significant difference in overall subsidence (p = 0.318) or the rate of subsidence >10 mm between stems. Mean subsidence was 2.13 mm in the monoblock group and 3.15 mm in the modular group. Two stems subsided >10 mm: one in each group. There was no difference in bicortical contact between groups (p = 0.98). No re-revisions were performed. Conclusions: We found no difference in subsidence between the two stems. Surgeons may consider the use of monoblock stems in rTHA as they have comparably low rates of subsidence and eliminate the small but potentially catastrophic risk of implant fracture at modular junctions associated with modular stems.

G-quadruplex DNA structures in human stem cells and differentiation.

The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.

Using temperature to modify the reaction conditions and outcomes of polymers formed using transfer-dominated branching radical telomerisation (TBRT).

Transfer-dominated Branching Radical Telomerisation (TBRT) enables the production of branched polymers with step-growth backbones using radical telomerisation chemistry. By conducting identical TBRTs over a broad temperature range, the role of temperature in telomer formation and branching has been evaluated. Elevated temperature limits telomer length, thereby allowing a >10% reduction in the amount of telogen required to produce near identical high molecular weight branched polymers.

Legal reform to enhance global text and data mining research.

Outdated copyright laws around the world hinder research.

Anticonvulsant neuropeptides as drug leads for neurological diseases.

Anticonvulsant neuropeptides are best known for their ability to suppress seizures and modulate pain pathways. Galanin, neuropeptide Y, somatostatin, neurotensin, dynorphin, among others, have been validated as potential first-in-class anti-epileptic or/and analgesic compounds in animal models of epilepsy and pain, but their therapeutic potential extends to other neurological indications, including neurodegenerative and psychatric disorders. Disease-modifying properties of neuropeptides make them even more attractive templates for developing new-generation neurotherapeutics. Arguably, efforts to transform this class of neuropeptides into drugs have been limited compared to those for other bioactive peptides. Key challenges in developing neuropeptide-based anticonvulsants are: to engineer optimal receptor-subtype selectivity, to improve metabolic stability and to enhance their bioavailability, including penetration across the blood­brain barrier (BBB). Here, we summarize advances toward developing systemically active and CNS-penetrant neuropeptide analogs. Two main objectives of this review are: (1) to provide an overview of structural and pharmacological properties for selected anticonvulsant neuropeptides and their analogs and (2) to encourage broader efforts to convert these endogenous natural products into drug leads for pain, epilepsy and other neurological diseases.