Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content

The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100–250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies.

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Management of urolithiasis associated with tuberous sclerosis complex

10.21203/rs.2.12454/v1 ◽

2019 ◽

Author(s):

Takashi Hatano ◽

Yu Imai ◽

Kei-ichiro Mori ◽

Jun Moritake ◽

Katsuhisa Endo ◽

...

Keyword(s):

Parathyroid Hormone ◽

Specific Gravity ◽

Tuberous Sclerosis ◽

Tuberous Sclerosis Complex ◽

Urine Specific Gravity ◽

Intact Parathyroid Hormone ◽

Urine Ph ◽

Extracorporeal Shock Wave ◽

Increased Risk ◽

Significant Difference

Abstract The most common renal symptoms of tuberous sclerosis complex (TSC) are angiomyolipomas (AMLs) and renal cysts; however, some patients with TSC also develop urolithiasis. In this study, we evaluated the incidence, risk factors and clinical courses of TSC-associated urolithiasis. We analyzed a total of 102 patients who met the diagnostic criteria for TSC, of whom 15 (14.7%) had urolithiasis. We compared urinary specific gravity, urine pH, serum calcium, intact parathyroid hormone and the use of antiepileptic agents of the urolithiasis and non-urolithiasis groups. The urine specific gravity and urine pH were higher in the urolithiasis group than in the non-urolithiasis group (P = 0.005, P = 0.042, respectively). No significant difference was found between the two groups with regard to the serum corrected calcium and intact parathyroid hormone levels. The antiepileptic drugs topiramate and zonisamide were more frequently administered to the urolithiasis group than the non-urolithiasis group (P <0.001, P = 0.039, respectively). Four patients in the urolithiasis group underwent surgery. Three patients could not receive extracorporeal shock wave lithotripsy due to the risk of bleeding from the AML, and therefore underwent transurethral lithotripsy. If urolithiasis is comorbid with TSC-associated AML, the treatment options are more limited in cases with multiple AMLs around the stone due to an increased risk of hemorrhage. Prevention and early detection of urolithiasis are beneficial to patients with TSC.

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Analysis of renal lesions in Chinese tuberous sclerosis complex patients with different conditions of TSC1/2 genetic mutations

10.21203/rs.3.rs-15210/v1 ◽

2020 ◽

Author(s):

Wenda Wang ◽

Yang Zhao ◽

Xu Wang ◽

Zhan Wang ◽

Guoyang Zheng ◽

...

Keyword(s):

Tuberous Sclerosis ◽

Tuberous Sclerosis Complex ◽

Renal Cysts ◽

Genetic Mutations ◽

Imaging Evaluation ◽

Female Ratio ◽

Renal Lesions ◽

Significant Difference ◽

Male Female Ratio ◽

And Function

Abstract Objective The renal manifestations of tuberous sclerosis complex (TSC) are complicated and various. We’d like to report the information of genetic mutations in TSC patients with renal lesions, and to discuss the relationship between features of renal lesions and genetic mutations, including mutant genes and mutant types. Methods TSC patients with renal lesions who came to Urology Department of our hospital from January 1st, 2015 to January 1st, 2020 were retrospectively analyzed. TSC patients who received next generation sequencing (NGS) of TSC1/2 and imaging examinations were screened out. When familial TSC patients were confirmed, only the probands were included. The patients who had imaging evaluation in our hospital before any treatment for TSC renal angiomyolipomas (AMLs) were also selected for further analysis of relationship between genetic mutations and AML sizes. Results 70 clinically or genetically diagnosed TSC patients with renal lesions were included. The average age was 29.3±8.3 years old. Male-female ratio was 1:1.5. 15 patients (21.4%) were probands of TSC families (3 TSC1 , 10 TSC2 , and 2 NMI). 67 patients (95.8%) had bilateral renal AMLs with one patient had a pathological diagnosis of epithelioid AML ( TSC2 mutation). One patient had multiple renal cysts ( TSC2 mutation), one had renal cell carcinomas (RCCs) ( TSC1 mutation) and one had Wilms tumors ( TSC1 mutation). Among the 70 included patients, 4 patients had TSC1 mutations, 51 had TSC2 mutations, and 15 had no mutation identified (NMI). There was no statistically significant difference between TSC2 mutations and NMI groups (11.4±5.7 vs. 8.0±5.6cm, P = 0.077) when considering AML sizes. There was also no statistically significant difference among AML sizes of patients with TSC2 mutation types of nonsense, missense, frameshift, slipping, and fragment deletion ( P = 0.712). And no statistically significant difference was found between maximus diameters in familial and sporadic patients, either (11.4±5.8 vs. 10.5±5.8, P = 0.663). Conclusions The conditions of TSC genetic mutations will affect type and severity of renal lesions. Other focuses such as protein structure and function need to be studied for renal manifestations. Except for patients with TSC1 and TSC2 genetic mutations, patients with NMI and familial patients are also needed more attention for the pathogenesis of them is still unknown.

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Proton MR spectroscopy of the foramen of Monro region in patients with tuberous sclerosis complex

Arquivos de Neuro-Psiquiatria ◽

10.1590/s0004-282x2008000300003 ◽

2008 ◽

Vol 66 (2b) ◽

pp. 303-307 ◽

Cited By ~ 4

Author(s):

Arnolfo de Carvalho-Neto ◽

Isac Bruck ◽

Sérgio A. Antoniuk ◽

Edson Marchiori ◽

Emerson L. Gasparetto

Keyword(s):

Basal Ganglia ◽

Tuberous Sclerosis ◽

Tuberous Sclerosis Complex ◽

Control Group ◽

Resonance Spectroscopy ◽

1H Mrs ◽

Foramen Of Monro ◽

1H Magnetic Resonance Spectroscopy ◽

Significant Difference ◽

And Gender

PURPOSE: To evaluate with 1H-magnetic resonance spectroscopy (MRS) the metabolites rations of the foramen of Monro's region in patients with tuberous sclerosis complex (TSC). METHOD: Twelve patients with TSC and an age and gender-matched control group underwent MR imaging at a 1.5T scanner, and 1H-MRS at the foramen of Monro level with a multivoxel acquisition. Similar volumes of interest were selected in each side of the foramen of Monro and in the basal ganglia (lentiform nuclei). The obtained N-acetylaspartate (NAA), creatine (Cr) and choline (Cho) peak amplitude values and ratios were studied. The statistical analysis was performed and p<0.05 was considered statically significant. RESULTS: There was no significant difference between the NAA/Cr and Cho/Cr ratios near to the foramen of Monro and basal ganglia of the TSC patients compared with the controls (p>0.05). CONCLUSION: The NAA/Cr and Cho/Cr ratios near to the foramen of Monro and basal ganglia of TSC patients are similar to the rations obtained in the control group.

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Everolimus Versus Sirolimus for Angiomyolipoma Associated with Tuberous Sclerosis Complex: A Multi-Institutional Retrospective Study in China

10.21203/rs.3.rs-305370/v1 ◽

2021 ◽

Author(s):

luo cong ◽

Yu-shi Zhang ◽

Ming-Xin Zhang ◽

Min-Feng Chen ◽

Yuan Li ◽

...

Keyword(s):

Retrospective Study ◽

Adverse Events ◽

Tuberous Sclerosis ◽

Oral Mucositis ◽

Tuberous Sclerosis Complex ◽

National Cancer Institute ◽

Therapeutic Efficacy ◽

Efficacy And Safety ◽

First Choice ◽

Significant Difference

Abstract Purpose To evaluate the efficacy and safety of everolimus and sirolimus in patients with tuberous sclerosis complex-associated angiomyolipomas (TSC-AML). Materials and Methods We performed a multi-institutional retrospective study of TSC-AML patients treated with oral everolimus or sirolimus. Angiomyolipoma volume was estimated using orthogonal measurements by MRI or CT. Adverse events (AEs) were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events. All analyses were performed using SPSS 19.0 software. Results There were high response rates in both two groups. With the prolonged medication time, the therapeutic efficacy of two agents became more significant. Everolimus had a more significant TSC-AML volume reduction after 6 and 12 months than sirolimus. More than 1/2 of everolimus patients had ≥ 50% reduction and approximately 80% of them had ≥ 30% reduction, higher than that of the sirolimus patients. Regarding safety, there was no significant difference in the incidence of AEs between the two groups. And the most common AE was oral mucositis. Conclusions Both everolimus and sirolimus are excellent therapeutic choices for TSC-AML. However, everolimus had a better therapeutic efficacy than sirolimus, especially in reducing TSC-AML volume. Therefore, everolimus is recommended as the first choice.

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Identification of amylase activity from vannamei shrimps’ (Litopenaeus vannamei) digestive tract using size exclusion chromatography method

Jurnal Natural ◽

10.24815/jn.v21i3.18874 ◽

2021 ◽

Vol 21 (3) ◽

pp. 123-127

Author(s):

HARIYANTI HARIYANTI ◽

HANIFAH RAHMI

Keyword(s):

Protein Content ◽

Digestive Tract ◽

Size Exclusion Chromatography ◽

Litopenaeus Vannamei ◽

Amylase Activity ◽

Gel Filtration ◽

Size Exclusion ◽

Font Size ◽

Chromatography Method ◽

Exclusion Chromatography

Vannamei shrimps (Litopenaeus vannamei), also known as white leg shrimps are widely cultivated and consumed by many people. However, most consumers have removed the shrimp’s head which integrates with its digestive tract. The digestive tract of white leg shrimp contains digestive enzymes, including amylase. This study aimed to determine the protein content and amylase activity from Vannamei shrimps’ digestive tract using the size exclusion chromatography method. The protein isolation of size exclusion chromatography was prepared in three steps, namely; centrifugation, precipitation, and dialysis. The protein from the dialysis step was purified by using gel filtration chromatography. Each obtained fraction was determined the protein content and amylase activity by using spectrophotometer UV-Vis method. The results showed that the fraction of 108th had the highest protein content and amylase activity with a value of 0.85 mg/ml and 25.66U/ml respectively. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536869121 1107305727 33554432 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0cm; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman",serif; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:EN-US;}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:10.0pt; mso-ansi-font-size:10.0pt; mso-bidi-font-size:10.0pt; mso-ansi-language:IN; mso-fareast-language:IN;}div.WordSection1 {page:WordSection1;}

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Extracellular Vesicle Separation Techniques Impact Results from Human Blood Samples: Considerations for Diagnostic Applications

International Journal of Molecular Sciences ◽

10.3390/ijms22179211 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9211

Author(s):

Theophilos Tzaridis ◽

Daniel Bachurski ◽

Shu Liu ◽

Kristin Surmann ◽

Felix Babatz ◽

...

Keyword(s):

Human Serum ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Cell Of Origin ◽

Separation Techniques ◽

Significant Difference ◽

Exclusion Chromatography ◽

Diagnostic Applications ◽

Immunoaffinity Isolation ◽

Mirna Content

Extracellular vesicles (EVs) are reminiscent of their cell of origin and thus represent a valuable source of biomarkers. However, for EVs to be used as biomarkers in clinical practice, simple, comparable, and reproducible analytical methods must be applied. Although progress is being made in EV separation methods for human biofluids, the implementation of EV assays for clinical diagnosis and common guidelines are still lacking. We conducted a comprehensive analysis of established EV separation techniques from human serum and plasma, including ultracentrifugation and size exclusion chromatography (SEC), followed by concentration using (a) ultracentrifugation, (b) ultrafiltration, or (c) precipitation, and immunoaffinity isolation. We analyzed the size, number, protein, and miRNA content of the obtained EVs and assessed the functional delivery of EV cargo. Our results demonstrate that all methods led to an adequate yield of small EVs. While no significant difference in miRNA content was observed for the different separation methods, ultracentrifugation was best for subsequent flow cytometry analysis. Immunoaffinity isolation is not suitable for subsequent protein analyses. SEC + ultracentrifugation showed the best functional delivery of EV cargo. In summary, combining SEC with ultracentrifugation gives the highest yield of pure and functional EVs and allows reliable analysis of both protein and miRNA contents. We propose this combination as the preferred EV isolation method for biomarker studies from human serum or plasma.

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Everolimus versus sirolimus for angiomyolipoma associated with tuberous sclerosis complex: a multi-institutional retrospective study in China

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-021-01932-z ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Cong Luo ◽

Yu-Shi Zhang ◽

Ming-Xin Zhang ◽

Min-Feng Chen ◽

Yuan Li ◽

...

Keyword(s):

Retrospective Study ◽

Adverse Events ◽

Tuberous Sclerosis ◽

Tuberous Sclerosis Complex ◽

National Cancer Institute ◽

Therapeutic Efficacy ◽

Efficacy And Safety ◽

First Choice ◽

Significant Difference ◽

Choice Of Therapy

Abstract Purpose To evaluate the efficacy and safety of everolimus and sirolimus in patients with tuberous sclerosis complex-associated angiomyolipomas (TSC-AML). Materials and methods We performed a multi-institutional retrospective study of TSC-AML patients treated with oral everolimus 10 mg or sirolimus 2 mg per day for at least 3 months. Angiomyolipoma volume was estimated using orthogonal measurements by MRI or CT. Adverse events (AEs) were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events. All analyses were performed using SPSS 19.0 software. Results Response rates were high in both groups. With the prolonged medication durations, the therapeutic efficacy of both agents became more significant. The TSC-AML volume reduction after 6 and 12 months was more pronounced in patients with everolimus than those with sirolimus. More than half of the patients treated with everolimus had ≥ 50% reduction, and approximately 80% of them had ≥ 30% reduction, which was higher than that in patients treated with sirolimus. Regarding safety, there was no significant difference in the incidence of AEs between the two groups. Conclusions Both everolimus and sirolimus are excellent therapeutic options for TSC-AML. However, everolimus has a better therapeutic efficacy than sirolimus, particularly in reducing TSC-AML volume. Everolimus is therefore recommended as the first choice of therapy for TSC-AML.

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