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1.
Prev Med ; 174: 107619, 2023 09.
Article in English | MEDLINE | ID: mdl-37451552

ABSTRACT

Diabetes seems to be a severe protracted disease or combination of biochemical disorders. A person's blood glucose (BG) levels remain elevated for an extended period because tissues lack and non-reaction to hormones. Such conditions are also causing longer-term obstacles or serious health issues. The medical field handles a large amount of very delicate data that must be handled properly. K-Nearest Neighbourhood (KNN) seems to be a common and straightforward ML method for creating illness threat prognosis models based on pertinent clinical information. We provide an adaptable neuro-fuzzy inference K-Nearest Neighbourhood (AF-KNN) learning-dependent forecasting system relying on patients' behavioural traits in several aspects to obtain our aim. That method identifies the best proportion of neighborhoods having a reduced inaccuracy risk to improve the predicting performance of the final system.


Subject(s)
Algorithms , Diabetes Mellitus , Humans , Diabetes Mellitus/diagnosis , Forecasting , Multivariate Analysis
2.
PLoS One ; 18(6): e0286550, 2023.
Article in English | MEDLINE | ID: mdl-37319242

ABSTRACT

Classic homocystinuria is an inborn error of metabolism caused mainly by missense mutations leading to misfolded and/or unstable human cystathionine ß-synthase (CBS) protein, causing the accumulation of excess total homocysteine (tHcy) in tissues. Previously, it has been shown that certain missense containing human CBS proteins can be functionally rescued in mouse models of CBS deficiency by treatment with proteasome inhibitors. The rescue by proteasome inhibitors is thought to work both by inhibiting the degradation of misfolded CBS protein and by inducing the levels of heat-shock chaperone proteins in the liver. Here we examine the effectiveness of two FDA approved protease inhibitors, carfilzomib and bortezomib, on various transgenic mouse models of human CBS deficiency. Our results show that although both drugs are effective in inducing the liver chaperone proteins Hsp70 and Hsp27, and are effective in inhibiting proteasome function, bortezomib was somewhat more robust in restoring the mutant CBS function. Moreover, there was no significant correlation between proteasome inhibition and CBS activity, suggesting that some of bortezomib's effects are via other mechanisms. We also test the use of low-doses of bortezomib and carfilzomib on various mouse models for extended periods of time and find that while low-doses are less toxic, they are also less effective at restoring CBS function. Overall, these results show that while restoration of mutant CBS function is possible with proteasome inhibitors, the exact mechanism is complicated and it will likely be too toxic for long-term patient treatment.


Subject(s)
Cystathionine beta-Synthase , Homocystinuria , Humans , Mice , Animals , Cystathionine beta-Synthase/genetics , Cystathionine beta-Synthase/metabolism , Proteasome Inhibitors/pharmacology , Proteasome Inhibitors/therapeutic use , Homocystinuria/drug therapy , Homocystinuria/genetics , Homocystinuria/metabolism , Bortezomib/pharmacology , Bortezomib/therapeutic use , Proteasome Endopeptidase Complex , Mice, Transgenic
3.
Ann Med Surg (Lond) ; 85(2): 111-121, 2023 Feb.
Article in English | MEDLINE | ID: mdl-36845768

ABSTRACT

The Alvarado score (AS) has not been widely used for diagnosing acute appendicitis although it has shown to be a good predictor for diagnosing appendicitis. The aim was to perform a systematic review of the available literature and synthesize the evidence. Methods: A systematic review was performed as per the PRISMA guidelines using search engines like Ovid, PubMed, and Google Scholar with predefined, strict inclusion and exclusion criteria. The quality assessment of included studies was performed using the QUADAS 2 tool. Summary statistics were performed for all variables. A linear regression model was performed between dependent and independent variables using STATA software. Heterogeneity testing showed significant heterogeneity within the included studies; hence, a forest plot with pooled estimates could not be constructed, and therefore a meta-regression was performed. Results: Seventeen full-text articles met inclusion and exclusion criteria. Ten of which were identified as low-risk studies. Five studies were included in final data pooling with total patients being 2239 and mean age of 31.9 years. (1) Linear regression demonstrated an association between 'histological appendicitis' and 'AS 7-0' with patients receiving intervention, with a significant P value of less than 0.005. (2) Meta-regression demonstrated a positive coefficient (0.298), a positive Z score of 2.20 with a significant P value of 0.028 for patients with 'high AS' who received interventions that were significantly proven to be 'histologically appendicitis', indicating a cause-and-effect relationship. Conclusion: High AS (7 and above) is a significant predictor of acute appendicitis. The authors recommend further prospective randomized clinical trials to establish a cause-and-effect relationship.

4.
J Cancer Res Clin Oncol ; 149(7): 2951-2961, 2023 Jul.
Article in English | MEDLINE | ID: mdl-35834010

ABSTRACT

PURPOSE: The COVID-19 pandemic changed diagnostic and treatment pathways in oncology. We compared the safety and efficacy of pembrolizumab amongst advanced nonsmall cell lung cancer (NSCLC) patients with a PD-L1 tumor proportion score (TPS) ≥ 50% before and during the pandemic. METHODS: Advanced NSCLC patients initiating pembrolizumab between June 2015 and December 2019 ("pre-pandemic cohort") and between March 2020 and March 2021 ("pandemic cohort") at BC Cancer were identified retrospectively. Multivariable logistic regression evaluated risk factors for immune-related adverse events (irAE) ≥ grade 3 at the 6 week, 3 month, and 6 month landmarks. Cox regression models of overall survival (OS) were constructed. RESULTS: The study population comprised 417 patients in the pre-pandemic cohort and 111 patients in the pandemic cohort. Between March and May 2020, 48% fewer advanced NSCLC cases with PD-L1 TPS ≥ 50% were diagnosed compared to similar intervals in 2018-2019. Telemedicine assessment [new patient consultations (p < 0.001) and follow-up (p < 0.001)] and extended interval pembrolizumab dosing (p < 0.001) were more common in the pandemic cohort. Patients initiating pembrolizumab after February 2020 (vs. before January 2020) experienced similar odds of developing severe irAE. 2/111 (1.8%) patients receiving pembrolizumab during the pandemic tested positive for COVID-19. On multivariable analysis, no association between pembrolizumab treatment period (before vs. during the COVID-19 pandemic) and OS was observed (p = 0.18). CONCLUSION: Significant changes in healthcare delivery in response to the pandemic did not result in increased high grade toxicity or lower survival outcomes in patients with advanced NSCLC treated with pembrolizumab.


Subject(s)
COVID-19 , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Lung Neoplasms/metabolism , Pandemics , Retrospective Studies , B7-H1 Antigen/metabolism
5.
Cureus ; 14(12): e32583, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36540322

ABSTRACT

Cowper's syringocele is a rare but underdiagnosed cystic dilatation of the main ducts of Cowper's gland. It is becoming more widely known in the adult population. Recent research proposes that syringoceles should be categorized according to the intraductal pressures causing ductal dilatation from mild to gross ultimately involving the gland itself. Although there may be some overlap in the clinical manifestations of different syringoceles, mildly dilated ducts are frequently asymptomatic. Moreover, moderate to gross duct dilatations can manifest as lower urinary tract symptoms (LUTS) or obstructive symptoms. A valid differential diagnosis is essential because these symptoms can be found in a wide range of severe illnesses. Syringocele can be diagnosed by ultrasonography in combination with voiding retrograde/antegrade cystourethrogram (VCUG), nevertheless, other procedures like cystourethroscopy, CT scan, and MRI scans can be helpful. Initially, conservative surveillance is advised, but if necessary, endoscopic marsupialization or surgical excision is the preferred treatment modality to address persistent problems.

6.
Ann Med Surg (Lond) ; 82: 104643, 2022 Oct.
Article in English | MEDLINE | ID: mdl-36268326

ABSTRACT

Perforated peptic ulcer (PPU) treatment guidelines are still up for discussion. Due to the morbidity and mortality linked to each, the use of both operative and non-operative management, including conservative and endoscopic treatment, is still debatable. A standardized protocol has been used to write a best evidence topic. The discussion focused on whether operative management for PPU is preferable to non-operational management or vice versa. MEDLINE, the Cochrane Library, Scopus, and the Web of Science were the databases used to conduct an electronic search of the pertinent literature. We found 56 articles, out of these only 5 studies were found to be appropriate to answer the question. The outcome assessed was failure of management. The best evidence showed that both operative and non-operative management can be used with similar outcomes depending on the patient selection for each category.

7.
Life (Basel) ; 12(10)2022 Oct 20.
Article in English | MEDLINE | ID: mdl-36295099

ABSTRACT

The aim of this systematic review is to assess the impact of vitamin D on the outcomes of kidney transplantation and investigate whether its deficiency is associated with a negative impact. Methods: We conducted a systematic literature search in PubMed, Scopus and Cochrane databases, as well as gray literature. Ultimately, 16 articles with an average of 255.75 patients were included in this review. These articles compared the long-term outcomes of vitamin D deficiency and/or vitamin D supplementation therapy on kidney transplant recipients by assessing various parameters. Results: Most of the included studies showed a negative effect of vitamin D deficiency on kidney transplantation by being associated with a worse graft function, higher incidence of acute rejection episodes, higher incidence of proteinuria and lower overall graft and patient survival rate. Conclusions: We suggest that patients awaiting kidney transplantation have a careful evaluation in order to assess their vitamin D status and the optimal supplementation therapy. Regular follow-up of vitamin D levels post-transplant is also suggested. Prospective studies will be needed to establish the positive effects of vitamin D supplementation therapy on kidney transplant outcomes.

8.
J Biol Chem ; 298(9): 102367, 2022 09.
Article in English | MEDLINE | ID: mdl-35963436

ABSTRACT

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway that converts the polyamine synthesis byproduct 5'-deoxy-5'-methylthioadenosine (MTA) into methionine. Inactivation of MTAP, often by homozygous deletion, is found in both solid and hematologic malignancies and is one of the most frequently observed genetic alterations in human cancer. Previous work established that MTAP-deleted cells accumulate MTA and contain decreased amounts of proteins with symmetric dimethylarginine (sDMA). These findings led to the hypothesis that accumulation of intracellular MTA inhibits the protein arginine methylase (PRMT5) responsible for bulk protein sDMAylation. Here, we confirm that MTAP-deleted cells have increased MTA accumulation and reduced protein sDMAylation. However, we also show that addition of extracellular MTA can cause a dramatic reduction of the steady-state levels of sDMA-containing proteins in MTAP+ cells, even though no sustained increase in intracellular MTA is found because of catabolism of MTA by MTAP. We determined that inhibition of protein sDMAylation by MTA occurs within 48 h, is reversible, and is specific. In addition, we have identified two enhancer-binding proteins, FUBP1 and FUBP3, that are differentially sDMAylated in response to MTAP and MTA. These proteins work via the far upstream element site located upstream of Myc and other promoters. Using a transcription reporter construct containing the far upstream element site, we demonstrate that MTA addition can reduce transcription, suggesting that the reduction in FUBP1 and FUBP3 sDMAylation has functional consequences. Overall, our findings show that extracellular MTA can inhibit protein sDMAylation and that this inhibition can affect FUBP function.


Subject(s)
Arginine , Deoxyadenosines , Purine-Nucleoside Phosphorylase , Arginine/analogs & derivatives , DNA-Binding Proteins/metabolism , Humans , Methionine/metabolism , Methylation , Polyamines , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , Purine-Nucleoside Phosphorylase/genetics , Purine-Nucleoside Phosphorylase/metabolism , RNA-Binding Proteins/metabolism , Sequence Deletion , Thionucleosides
9.
JNCI Cancer Spectr ; 5(5)2021 10.
Article in English | MEDLINE | ID: mdl-34585063

ABSTRACT

Background: Childhood cancer survivors (CCS) face increased risk of morbidity and are recommended to receive lifelong cancer-related follow-up care. Identifying factors associated with follow-up care can inform efforts to support the long-term health of CCS. Methods: Eligible CCS (diagnosed between 1996 and 2010) identified through the Los Angeles County Cancer Surveillance Program responded to a self-report survey that assessed demographic, clinical, health-care engagement, and psychosocial risk and protective factors of recent (prior 2 years) cancer-related follow-up care. Weighted multivariable logistic regression was conducted to identify correlates of care. All statistical tests were 2-sided. Results: The overall response rate was 44.9%, with an analytical sample of n = 1106 (54.2% Hispanic; mean [SD] ages at survey, diagnosis, and years since diagnosis were 26.2 [4.9], 11.6 [5.4], and 14.5 [4.4] years, respectively). Fifty-seven percent reported a recent cancer-related visit, with lower rates reported among older survivors. Having insurance, more late effects, receipt of a written treatment summary, discussing long-term care needs with treating physician, knowledge of the need for long-term care, having a regular source of care, and higher health-care self-efficacy were statistically significantly associated with greater odds of recent follow-up care, whereas older age, Hispanic or Other ethnicity (vs non-Hispanic White), and years since diagnosis were associated with lower odds of recent care (all Ps < .05). Conclusions: Age and ethnic disparities are observed in receipt of follow-up care among young adult CCS. Potential intervention targets include comprehensive, ongoing patient education; provision of written treatment summaries; and culturally tailored support to ensure equitable access to and the utilization of care.


Subject(s)
Aftercare , Cancer Survivors , Healthcare Disparities/ethnology , Neoplasms/therapy , Adolescent , Age Factors , Cancer Survivors/statistics & numerical data , Child , Cohort Studies , Female , Hispanic or Latino , Humans , Logistic Models , Male , Neoplasms/ethnology , Self Report/statistics & numerical data , White People , Young Adult
10.
Cancer Causes Control ; 32(10): 1129-1148, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34292440

ABSTRACT

PURPOSE: The etiology of young-onset breast cancer (BC) is poorly understood, despite its greater likelihood of being hormone receptor-negative with a worse prognosis and persistent racial and socioeconomic inequities. We conducted a population-based case-control study of BC among young Black and White women and here discuss the theory that informed our study, exposures collected, study methods, and operational results. METHODS: Cases were non-Hispanic Black (NHB) and White (NHW) women age 20-49 years with invasive BC in metropolitan Detroit and Los Angeles County SEER registries 2010-2015. Controls were identified through area-based sampling from the U.S. census and frequency matched to cases on study site, race, and age. An eco-social theory of health informed life-course exposures collected from in-person interviews, including socioeconomic, reproductive, and energy balance factors. Measured anthropometry, blood (or saliva), and among cases SEER tumor characteristics and tumor tissue (from a subset of cases) were also collected. RESULTS: Of 5,309 identified potentially eligible cases, 2,720 sampled participants were screened and 1,812 completed interviews (682 NHB, 1140 NHW; response rate (RR): 60%). Of 24,612 sampled control households 18,612 were rostered, 2,716 participants were sampled and screened, and 1,381 completed interviews (665 NHB, 716 NHW; RR: 53%). Ninety-nine% of participants completed the main interview, 82% provided blood or saliva (75% blood only), and SEER tumor characteristics (including ER, PR and HER2 status) were obtained from 96% of cases. CONCLUSIONS: Results from the successfully established YWHHS should expand our understanding of young-onset BC etiology overall and by tumor type and identify sources of racial and socioeconomic inequities in BC.


Subject(s)
Breast Neoplasms , Adult , Black or African American , Breast Neoplasms/epidemiology , Case-Control Studies , Female , Humans , Incidence , Middle Aged , White People , Young Adult
11.
FASEB J ; 35(6): e21629, 2021 06.
Article in English | MEDLINE | ID: mdl-33949005

ABSTRACT

Cystathionine beta-synthase (CBS) is a key enzyme of the trans-sulfuration pathway that converts homocysteine to cystathionine. Loss of CBS activity due to mutation results in CBS deficiency, an inborn error of metabolism characterized by extreme elevation of plasma total homocysteine (tHcy). C57BL6 mice containing either a homozygous null mutation in the cystathionine ß-synthase (Cbs-/- ) gene or an inactive human CBS protein (Tg-G307S Cbs-/- ) are born in mendelian numbers, but the vast majority die between 18 and 21 days of age due to liver failure. However, adult Cbs null mice that express a hypomorphic allele of human CBS as a transgene (Tg-I278T Cbs-/- ) show almost no neonatal lethality despite having serum tHcy levels similar to mice with no CBS activity. Here, we characterize liver and serum metabolites in neonatal Cbs+/- , Tg-G307S Cbs-/- , and Tg-I278T Cbs-/- mice at 6, 10, and 17 days of age to understand this difference. In serum, we observe similar elevations in tHcy in both Tg-G307S Cbs-/- and Tg-I278T Cbs-/- compared to control animals, but methionine is much more severely elevated in Tg-G307S Cbs-/- mice. Large scale metabolomic analysis of liver tissue confirms that both methionine and methionine-sulfoxide are significantly more elevated in Tg-G307S Cbs-/- animals, along with significant differences in several other metabolites including hexoses, amino acids, other amines, lipids, and carboxylic acids. Our data are consistent with a model that the neonatal lethality observed in CBS-null mice is driven by excess methionine resulting in increased stress on a variety of related pathways including the urea cycle, TCA cycle, gluconeogenesis, and phosphatidylcholine biosynthesis.


Subject(s)
Cystathionine beta-Synthase/physiology , Disease Models, Animal , Liver Failure/pathology , Metabolome , Mutation , Animals , Animals, Newborn , Female , Liver Failure/etiology , Liver Failure/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Phenotype
12.
Cancer Discov ; 11(2): 446-479, 2021 02.
Article in English | MEDLINE | ID: mdl-33127842

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211.


Subject(s)
Adenocarcinoma/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Netrins/metabolism , Pancreatic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Humans , Immunosuppression Therapy , Nutritional Support , Tumor Microenvironment
13.
BMJ Case Rep ; 13(7)2020 Jul 08.
Article in English | MEDLINE | ID: mdl-32641313

ABSTRACT

A 78-year-old man with no surgical history or recent trauma presented to the emergency department with sudden onset right-sided chest pain and dyspnoea. He was admitted under the physicians for investigations and was subsequently diagnosed with empyema of the right thorax. After no improvement with intravenous antibiotics, a chest drain was inserted; no pus was drained. He worsened clinically; a repeated CT scan demonstrated an incarcerated loop of small bowel within the right thoracic cavity secondary to a diaphragmatic hernia (DH). The patient had emergency surgery to remove necrotic small bowel and to lavage the thorax. Strangulated DH should be considered as a differential diagnosis where presentation is unusual and empyema does not improve after initial management.


Subject(s)
Empyema, Pleural/diagnosis , Hernia, Diaphragmatic/diagnosis , Intestine, Small/pathology , Necrosis/diagnosis , Aged , Diagnosis, Differential , Hernia, Diaphragmatic/complications , Humans , Male , Necrosis/etiology , Thoracic Cavity/pathology
14.
Indian J Crit Care Med ; 24(3): 172-178, 2020 Mar.
Article in English | MEDLINE | ID: mdl-32435095

ABSTRACT

BACKGROUND: Emergency department (ED) being the most crucial part of hospital, where adverse drug reactions (ADRs) often go undetected. Trigger tools are proficient ADR detection methods, which have only been applied for retrospective surveillance. We did a prospective analysis to further refine the trigger tool application in healthcare settings. OBJECTIVE: To estimate the prevalence of ADRs and prospectively evaluate the importance of using trigger tools for their detection. MATERIALS AND METHODS: A prospective study was conducted in the ED for the presence of triggers in patient records to monitor and report ADRs by applying the Institute for Healthcare Improvement (IHI) trigger tool methodology. RESULTS: Four hundred sixty-three medical records were analyzed randomly using 51 trigger tools, where triggers were found in 181 (39.09%) and ADRs in 62 (13.39%) patients. The prevalence of ADR was 13.39%. According to the World Health Organization (WHO)-Uppsala Monitoring Centre (UMC) causality scale, 47 (75.8%) were classified as probable and 15 (24.2%) as possible, wherein 39 (62.9%) were predictable and 8 (12.9%) were definitely preventable. Most common triggers were abrupt medication stoppage (34.98%), antiemetic use (25.91%), and time in ED >6 hours (17.49%). The positive predictive values (PPVs) of triggers such as international normalized ratio (INR) > 4 (p = 0.0384), vitamin K administration (p = 0.002), steroid use (p = 0.0001), abrupt medication stoppage (p = 0.0077), transfusion of blood or blood products (p = 0.004), and rash (p = 0.0042) showed statistically significant results, which make the event detection process more structured when these triggers are positive. Presence of five or more triggers has statistically significant chances of developing an ADR (p < 0.05). CONCLUSION: Trigger tool could be a viable method to identify ADRs when compared to the traditional ADR identification methods, but there is insufficient data on IHI tool and its use to identify ADRs in the general outpatient setting. Healthcare providers may benefit from better trigger tools to help them detect ADRs. HOW TO CITE THIS ARTICLE: Pandya AD, Patel K, Rana D, Gupta SD, Malhotra SD, Patel P. Global Trigger Tool: Proficient Adverse Drug Reaction Autodetection Method in Critical Care Patient Units. Indian J Crit Care Med 2020;24(3):172-178.

15.
J Inherit Metab Dis ; 42(5): 831-838, 2019 09.
Article in English | MEDLINE | ID: mdl-31240737

ABSTRACT

Classical homocystinuria is a recessive inborn error of metabolism caused by mutations in the cystathionine beta-synthase (CBS) gene. The highest incidence of CBS deficiency in the world is found in the country of Qatar due to the combination of high rates of consanguinity and the presence of a founder mutation, c.1006C>T (p.R336C). This mutation does not respond to pyridoxine and is considered severe. Here we describe the creation of a mouse that is null for the mouse Cbs gene and expresses human p.R336C CBS from a zinc-inducible transgene (Tg-R336C Cbs -/- ). Zinc-treated Tg-R336C Cbs -/- mice have extreme elevation in both serum total homocysteine (tHcy) and liver tHcy compared with control transgenic mice. Both the steady-state protein levels and CBS enzyme activity levels in liver lysates from Tg-R336C Cbs -/- mice are significantly reduced compared to that found in Tg-hCBS Cbs -/- mice expressing wild-type human CBS. Treatment of Tg-R336C Cbs -/- mice with the proteasome inhibitor bortezomib results in stabilization of liver CBS protein and an increase in activity to levels found in corresponding Tg-hCBS Cbs -/- wild type mice. Surprisingly, serum tHcy did not fully correct even though liver enzyme activity was as high as control animals. This discrepancy is explained by in vitro enzymatic studies of mouse liver extracts showing that p.R336C causes reduced binding affinity for the substrate serine by almost 7-fold and significantly increased dependence on pyridoxal phosphate in the reaction buffer. These studies demonstrate that the p.R336C alteration effects both protein stability and substrate/cofactor binding.


Subject(s)
Cystathionine beta-Synthase/genetics , Homocystinuria/genetics , Alleles , Animals , Bortezomib/pharmacology , DNA Mutational Analysis , Female , Homocysteine/blood , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Mutation , Proteasome Inhibitors/chemistry , Pyridoxine/chemistry
16.
Hum Mutat ; 40(2): 230-240, 2019 02.
Article in English | MEDLINE | ID: mdl-30408270

ABSTRACT

Homocystinuria is a rare inborn error of methionine metabolism caused by cystathionine ß-synthase (CBS) deficiency. The prevalence of homocystinuria in Qatar is 1:1,800 births, mainly due to a founder Qatari missense mutation, c.1006C>T; p.R336C (p.Arg336Cys). We characterized the structure-function relationship of the p.R336C-mutant protein and investigated the effect of different chemical chaperones to restore p.R336C-CBS activity using three models: in silico, ΔCBS yeast, and CRISPR/Cas9 p.R336C knock-in HEK293T and HepG2 cell lines. Protein modeling suggested that the p.R336C induces severe conformational and structural changes, perhaps influencing CBS activity. Wild-type CBS, but not the p.R336C mutant, was able to restore the yeast growth in ΔCBS-deficient yeast in a complementation assay. The p.R336C knock-in HEK293T and HepG2 cells decreased the level of CBS expression and reduced its structural stability; however, treatment of the p.R336C knock-in HEK293T cells with betaine, a chemical chaperone, restored the stability and tetrameric conformation of CBS, but not its activity. Collectively, these results indicate that the p.R336C mutation has a deleterious effect on CBS structure, stability, and activity, and using the chemical chaperones approach for treatment could be ineffective in restoring p.R336C CBS activity.


Subject(s)
Cystathionine beta-Synthase/genetics , Homocystinuria/genetics , Molecular Chaperones/genetics , Mutant Proteins/genetics , Computer Simulation , Cystathionine beta-Synthase/chemistry , Enzyme Stability , Gene Expression Regulation, Enzymologic/genetics , HEK293 Cells , Hep G2 Cells , Homocystinuria/metabolism , Homocystinuria/pathology , Humans , Methionine/metabolism , Molecular Chaperones/chemistry , Mutant Proteins/chemistry , Mutation, Missense/genetics , Protein Folding , Protein Structure, Tertiary , Qatar , Structure-Activity Relationship
17.
Mol Genet Metab Rep ; 15: 15-21, 2018 Jun.
Article in English | MEDLINE | ID: mdl-30023284

ABSTRACT

Elevated plasma total homocysteine (tHcy) is associated with a number of human diseases including coronary artery disease, stroke, osteoporosis and dementia. It is highly correlated with intracellular S-adenosylhomocysteine (SAH). Since SAH is a strong inhibitor of methyl-transfer reactions involving the methyl-donor S-adenosylmethionine (SAM), elevation in SAH could be an explanation for the wide association of tHcy and human disease. Here, we have created a transgenic mouse (Tg-hAHCY) that expresses human S-adenosylhomocysteine hydrolase (AHCY) from a zinc-inducible promoter in the liver and kidney. Protein analysis shows that human AHCY is expressed well in both liver and kidney, but elevated AHCY enzyme activity (131% increase) is only detected in the kidney due to the high levels of endogenous mouse AHCY expression in liver. Tg-hAHCY mice were crossed with mice lacking cystathionine ß-synthase activity (Tg-I278T Cbs-/- ) to explore the effect to AHCY overexpression in the context of elevated serum tHcy and elevated tissue SAM and SAH. Overexpression of AHCY had no significant effect on the phenotypes of Tg-I278T Cbs-/- mice or any effect on the steady state concentrations of methionine, total homocysteine, SAM, SAH, and SAM/SAH ratio in the liver and kidney. Furthermore, enhanced AHCY activity did not lower serum and tissue tHcy or methionine levels. Our data suggests that enhancing AHCY activity does not alter the distribution of methionine recycling metabolites, even when they are greatly elevated by Cbs mutations.

18.
J Biol Chem ; 293(36): 13921-13931, 2018 09 07.
Article in English | MEDLINE | ID: mdl-30030379

ABSTRACT

Mutations in the cystathionine ß-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The p.G307S mutation is the most frequent cause of CBS deficiency in Ireland, which has the highest prevalence of CBS deficiency in Europe. Individuals homozygous for this mutation tend to be severely affected and are pyridoxine nonresponsive, but the molecular basis for the strong effects of this mutation is unclear. Here, we characterized a transgenic mouse model lacking endogenous Cbs and expressing human p.G307S CBS protein from a zinc-inducible metallothionein promoter (Tg-G307S Cbs-/-). Unlike mice expressing other mutant CBS alleles, the Tg-G307S transgene could not efficiently rescue neonatal lethality of Cbs-/- in a C57BL/6J background. In a C3H/HeJ background, zinc-induced Tg-G307S Cbs-/- mice expressed high levels of p.G307S CBS in the liver, and this protein variant forms multimers, similarly to mice expressing WT human CBS. However, the p.G307S enzyme had no detectable residual activity. Moreover, treating mice with proteasome inhibitors failed to significantly increase CBS-specific activity. These findings indicated that the G307S substitution likely affects catalytic function as opposed to causing a folding defect. Using molecular dynamics simulation techniques, we found that the G307S substitution likely impairs catalytic function by limiting the ability of the tyrosine at position 308 to assume the proper conformational state(s) required for the formation of the pyridoxal-cystathionine intermediate. These results indicate that the p.G307S CBS is stable but enzymatically inert and therefore unlikely to respond to chaperone-based therapy.


Subject(s)
Cystathionine beta-Synthase/genetics , Mutation , Amino Acid Substitution , Animals , Catalysis , Cystathionine beta-Synthase/chemistry , Cystathionine beta-Synthase/metabolism , Homocystinuria/genetics , Humans , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Proteasome Inhibitors/pharmacology , Protein Conformation , Protein Stability , Pyridoxine/pharmacology
19.
Hum Mutat ; 38(7): 863-869, 2017 07.
Article in English | MEDLINE | ID: mdl-28488385

ABSTRACT

Mutations in the cystathionine beta-synthase (CBS) gene are the cause of classical homocystinuria, the most common inborn error in sulfur metabolism. The c.797 G>A (p.R266K) mutation in CBS was originally described in several Norwegian pyridoxine responsive CBS deficient patients, and heterologous gene expression studies have shown that the protein has near wild-type levels of enzyme activity. Here, we characterize a transgenic mouse lacking endogenous Cbs and expressing p.R266K human CBS protein from a zinc inducible metallothionein promoter (Tg-R266K Cbs-/- ). Unlike mice expressing other mutant CBS alleles, the Tg-R266K transgene is unable to efficiently rescue neonatal lethality of Cbs-/- on a C57BL/6J background. On a C3H/HeJ background, zinc-induced Tg-R266K Cbs-/- mice express CBS mRNA, but have very low levels of CBS protein and enzyme activity, resulting in extreme elevations in serum total homocysteine (tHcy). Treatment with pyridoxine did not have any appreciable effect on tHcy, indicating this allele is not pyridoxine responsive in mice. However, treatment with the proteasome inhibitor bortezomib resulted in an 97% reduction in tHcy and a 2381% increase in liver CBS activity. These studies show that the p.R266K mutation causes increased proteasomal degradation in vivo, and that treatments that stabilize the protein can be used to reverse its effect.


Subject(s)
Cystathionine beta-Synthase/genetics , Homocystinuria/genetics , Alleles , Animals , Bortezomib/pharmacology , DNA Mutational Analysis , Female , Homocysteine/blood , Humans , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Proteasome Inhibitors/chemistry , Pyridoxine/chemistry
20.
Aging Cell ; 16(2): 273-280, 2017 04.
Article in English | MEDLINE | ID: mdl-27896923

ABSTRACT

Elevated levels of homocysteinemia (Hcy), a risk factor for late-onset Alzheimer's disease (AD), have been associated with changes in cell methylation. Alzheimer's disease is characterized by an upregulation of the 5-lipoxygenase (5LO), whose promoter is regulated by methylation. However, whether Hcy activates 5LO enzymatic pathway by influencing the methylation status of its promoter remains unknown. Brains from mice with high Hcy were assessed for the 5LO pathway and neuronal cells exposed to Hcy implemented to study the mechanism(s) regulating 5LO expression levels and the effect on amyloid ß formation. Diet- and genetically induced high Hcy resulted in 5LO protein and mRNA upregulation, which was associated with a significant increase of the S-adenosylhomocysteine (SAH)/S-adenosylmethionine ratio, and reduced DNA methyltrasferases and hypomethylation of 5-lipoxygenase DNA. In vitro studies confirmed these results and demonstrated that the mechanism involved in the Hcy-dependent 5LO activation and amyloid ß formation is DNA hypomethylation secondary to the elevated levels of SAH. Taken together these findings represent the first demonstration that Hcy directly influences 5LO expression levels and establish a previously unknown cross talk between these two pathways, which is highly relevant for AD pathogenesis. The discovery of such a novel link not only provides new mechanistic insights in the neurobiology of Hcy, but most importantly new therapeutic opportunities for the individuals bearing this risk factor for the disease.


Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , DNA Methylation/genetics , Homocysteine/pharmacology , Amyloid beta-Peptides/metabolism , Animals , Brain/drug effects , Brain/enzymology , DNA Methylation/drug effects , Diet , Humans , Hyperhomocysteinemia/enzymology , Hyperhomocysteinemia/genetics , Male , Mice , Neurons/drug effects , Neurons/metabolism , Protein Biosynthesis/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic/drug effects
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