Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity.

Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism

Generation and characterization of human induced pluripotent stem cell line METUi001-A from a 25-year-old male patient with relapsing-remitting multiple sclerosis

© 2021Multiple sclerosis is a chronic disease characterized by inflammation, demyelination, and axonal damage in the central nervous system. Here, we established an induced pluripotent stem cell (iPSC) line METUi001-A from the peripheral blood mononuclear cells of a 25-year-old male individual with clinically diagnosed Relapsing-Remitting Multiple Sclerosis (RRMS) using the integration-free Sendai reprogramming method. We demonstrated that the iPSCs are free of exogenous Sendai reprogramming vectors, have a normal male karyotype, express pluripotency markers, and differentiate into the three germ layers. The iPSC line can serve as a valuable resource to generate cellular model systems to investigate molecular mechanisms underlying RRMS

Central venous catheterization in open heart surgery: Internal jugular vein or supraclavicular subclavian vein approach?

Amaç: Bu çalışmada santral venöz kateter (SVK) takılan hastalarda komplikasyon gelişimine etki eden faktörler araştırıldı. Çalışma planı: Kasım 2007 - Ocak 2009 tarihleri arasında kliniğimizde SVK uygulanan 94 hasta (69 erkek, 25 kadın; ort. yaş 60.3±13.2 yıl; dağılım 4-84 yıl) ileriye yönelik olarak incelendi. Hastalar rastgele iki gruba ayrıldı. Santral venöz kateter grup 1’de (n=49), sağ internal juguler ven (İJV) yoluyla, grup 2’de (n=45) ise sağ supraklaviküler subklaviyen ven (SpSV) yoluyla uygulandı. Tüm SVK’ler aynı hekim tarafından Seldinger tekniği ile takıldı. Bulgular: Demografik özellikler açısından iki grup arasında anlamlı fark yoktu. Ameliyat sırası ve sonrası dönemde, iki grup arasında pnömotoraks, hemotoraks, malpozisyon, hematom gelişimi, nörolojik hasar, infeksiyon ve kateter kalış süresi açısından anlamlı fark bulunmadı. Dokuz hastada arter ponksiyonu [grup 1 (n=8) ve grup 2 (n=1); p=0.020], yedi hastada pinch-off fenomeni [grup 1 (n=0) ve grup 2 (n=7); p=0.004] ve 19 hastada iki veya daha fazla girişim [grup 1 (n=16) ve grup 2 (n=3); p=0.002] gerçekleşti. Çokdeğişkenli analizde; arter ponksiyonu için vücut kütle indeksi (VKİ) (p=0.028), pinch-off fenomeni için VKİ (p=0.040) ve SpSV yaklaşım (p=0.022); iki veya daha fazla girişim için ise İJV yaklaşım (p=0.007) güçlü öngörücü faktörler olarak saptandı. Sonuç: Başarılı bir girişim için hasta anatomisi, VKİ ve hekimin deneyimi göz önünde bulundurulmalı ve bu seçenekler arasında SpSV kateterizasyonunun da olabileceği düşünülmelidir.Background: In this study we investigated the factors that affect the development of complications in patients who had central venous catheter (CVC) insertion. Methods: Ninety-four patients (69 males, 25 females; mean age 60.3±13.2 years; range 4 to 84 years) who had CVC between November 2007 - January 2009 in our clinic were investigated prospectively. Patients were randomized to two groups; in group 1 (n=49), CVC was performed through right internal jugular vein (IJV), and in group 2 (n=45), CVC was performed through right supraclavicular subclavian vein (SpSV). All of the CVC's were performed by means of Seldinger's technique by the same operator. Results: There were no significant differences in the demographic features between the two groups. There were no significant differences between the groups with regard to pneumothorax, hemothorax, malposition, hematoma development, neurological damage, infection and catheter stay in the intra- and postoperative periods. Nine cases had arterial puncture [group 1 (n=8) and group 2 (n=1); p=0.020], seven cases had pinch-off phenomenon [group 1 (n=0) and group 2 (n=7); p=0.004], and 19 cases had two or more interventions [group 1 (n=16) and group 2 (n=3); p=0.002]. In the multivariate analysis, the powerful predictive factor for arterial puncture was the body mass index (BMI) (p=0.028), for the pinch-off phenomenon BMI (p=0.040) and the SpSV approach (p=0.022); the predictive factor for two or more insertional attempts was IJV approach (p=0.007). Conclusion: The patient's anatomy, BMI and the physician's experience should be considered for a successful attempt. We think that SpSV catheterization may be among the preferences

Sinaptozomların Fare Kök Hücrelerinden Başkalaştırılmış Motor Nöronlardan Geniş Ölçekte İzole Edilmesi ve Karakterizasyonu

Sinaptik fonksiyonların bozulması Alzheimer ve Parkinson hastalıkları gibi çeşitli nörodejeneratif hastalıklar, ve benzer şekilde epilepsi dahil olmak üzere çeşitli nörodejeneratif ve nöropsikiyatrik hastalıklar ile ilişkilendirilmiştir. Bu nedenle sinapsların oluşmasında ve normal aktivitelerini devamlarında önemli rol oynayan moleküler mekanizmaların anlaşılması hem temel araştırmalar açısından hem de sinaptik iletimi etkileyen hastalıkların tedavisinde yeni terapötiklerin geliştirilmesi bakımından kritik öneme sahiptir. Bu çalışmaları kolaylaştırmak ve hızlandırmak için, masraf seviyesi düşük ve iyi karakterize edilmiş in vitro modellere ihtiyaç vardır. Bu amaçla, sinir hücrelerinin terminal uçları, yani sinaptozomların, hücre parçalarının ayrıştırılması yoluyla izole edilebileceği gösterilmiştir. Amiyotropik Lateral Skleroz (ALS) gibi motor nöron hastalıklarında sinapslar hastalık sürecinde birinci derecede etkilenen yapılardır. Benzer şekilde bazı nörotoksinler de (örneğin Botulinum nörotoksin) motor nöronları hedef alıp nöromusküler kavşaklardaki sinyal iletimini durdurur. Bildiğimiz ve literatürde araştırdığımız kadarıyla, sinaptozomların embriyonik kök hücre kaynaklı motor nöronlardan izolasyonu bildirilmemiştir. Kök hücrelerden yüksek miktarlarda motor nöron oluşturulması mümkün olduğu için, bu projede sinaptozomların fare embriyonik kök hücrelerinden türetilmiş motor nöronlardan geniş ölçekte izole edilip karakterizasyonu yapılması amaçlanmaktadır

Quantitative Analysis of MAP-Mediated Regulation of Microtubule Dynamic Instability In Vitro—Focus on Tau

The regulation of microtubule growing and shortening dynamics is essential for proper cell function and viability, and microtubule-associated proteins (MAPs) such as the neural protein tau are critical regulators of these dynamic processes. Further, we and our colleagues have proposed that misregulation of microtubule dynamics may contribute to tau-mediated neuronal cell death and dementia in Alzheimer's and related diseases. In the first part of this chapter, we present a general background on microtubule dynamics and then focus in on tau. We review the literature on the roles of tau in normal neuronal cell biology, the tau structure-function relationship, regulatory mechanisms influencing tau action, and pathological tau action, including normal and aberrant regulation of microtubule dynamics. In the second part of this chapter, we present detailed protocols for various in vitro procedures often used in studying tau-mediated regulation of microtubule dynamics, including purification and characterization of necessary reagents, microtubule assembly assays, and microtubule dynamics assays. Importantly, these assays are readily adaptable to examine other regulators of microtubule dynamics besides tau. In the final analysis, in vitro analyses of MAP-mediated regulation of microtubule dynamics will provide extremely valuable insights into our understanding of normal and pathological cell biology

Recent advances in botulinum neurotoxin inhibitor development.

Botulinum neurotoxins (BoNTs) are endopeptidases that target motor neurons and block acetylcholine neurotransmitter release. This action results in the muscle paralysis that defines the disease botulism. To date, there are no FDA-approved therapeutics to treat BoNT-mediated paralysis after intoxication of the motor neuron. Importantly, the rationale for pursuing treatments to counter these toxins is driven by their potential misuse. Current drug discovery efforts have mainly focused on small molecules, peptides, and peptidomimetics that can directly and competitively inhibit BoNT light chain proteolytic activity. Although this is a rational approach, direct inhibition of the Zn2+ metalloprotease activity has been elusive as demonstrated by the dearth of candidates undergoing clinical evaluation. Therefore, broadening the scope of viable targets beyond that of active site protease inhibitors represents an additional strategy that could move the field closer to the clinic. Here we review the rationale, and discuss the outcomes of earlier approaches and highlight potential new targets for BoNT inhibition. These include BoNT uptake and processing inhibitors, enzymatic inhibitors, and modulators of neuronal processes associated with toxin clearance, neurotransmitter potentiation, and other pathways geared towards neuronal recovery and repair