Extracellular 5'-methylthioadenosine inhibits intracellular symmetric dimethylarginine protein methylation of FUSE-binding proteins.

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.

Predicting diabetes with multivariate analysis an innovative KNN-based classifier approach.

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.

Analysis of differential neonatal lethality in cystathionine ß-synthase deficient mouse models using metabolic profiling.

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.

Theory, methods, and operational results of the Young Women's Health History Study: a study of young-onset breast cancer incidence in Black and White women.

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.

Global Trigger Tool: Proficient Adverse Drug Reaction Autodetection Method in Critical Care Patient Units.

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.

In silico and in vivo models for Qatari-specific classical homocystinuria as basis for development of novel therapies.

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.

Mouse modeling and structural analysis of the p.G307S mutation in human cystathionine ß-synthase (CBS) reveal effects on CBS activity but not stability.

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.

Analysis of the Qatari R336C cystathionine ß-synthase protein in mice.

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.

The c.797 G>A (p.R266K) cystathionine ß-synthase mutation causes homocystinuria by affecting protein stability.

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.

Lack of global epigenetic methylation defects in CBS deficient mice.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of metabolism in which patients have extremely elevated plasma total homocysteine and have clinical manifestations in the vascular, visual, skeletal, and nervous systems. Homocysteine is an intermediary metabolite produced from the hydrolysis of S-adenosylhomocysteine (SAH), which is a by-product of methylation reactions involving the methyl-donor S-adenosylmethionine (SAM). Here, we have measured SAM, SAH, DNA and histone methylation status in an inducible mouse model of CBS deficiency to test the hypothesis that homocysteine-related phenotypes are caused by inhibition of methylation due to elevated SAH and reduced SAM/SAH ratio. We found that mice lacking CBS have elevated cellular SAH and reduced SAM/SAH ratios in both liver and kidney, but this was not associated with alterations in the level of 5-methylcytosine or various histone modifications. Using methylated DNA immunoprecipitation in combination with microarray, we found that of the 241 most differentially methylated promoter probes, 89 % were actually hypermethylated in CBS deficient mice. In addition, we did not find that changes in DNA methylation correlated well with changes in RNA expression in the livers of induced and uninduced CBS mice. Our data indicates that reduction in the SAM/SAH ratio, due to loss of CBS activity, does not result in overall hypomethylation of either DNA or histones.

Betaine supplementation is less effective than methionine restriction in correcting phenotypes of CBS deficient mice.

Cystathionine beta synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by elevated serum total homocysteine (tHcy). Betaine supplementation, which can lower tHcy by stimulating homocysteine remethylation to methionine, is often given to CBS deficient patients in combination with other treatments such as methionine restriction and supplemental B-vitamins. However, the effectiveness of betaine supplementation by itself in the treatment of CBS deficiency has not been well explored. Here, we have examined the effect of a betaine supplemented diet on the Tg-I278T Cbs (-/-) mouse model of CBS deficiency and compared its effectiveness to our previously published data using a methionine restricted diet. Tg-I278T Cbs (-/-) mice on betaine, from the time of weaning until for 240 days of age, had a 40 % decrease in mean tHcy level and a 137 % increase in serum methionine levels. Betaine-treated Tg-I278T Cbs (-/-) mice also exhibited increased levels of betaine-dependent homocysteine methyl transferase (BHMT), increased levels of the lipogenic enzyme stearoyl-coenzyme A desaturase (SCD-1), and increased lipid droplet accumulation in the liver. Betaine supplementation largely reversed the hair loss phenotype in Tg-I278T Cbs (-/-) animals, but was far less effective than methionine restriction in reversing the weight-loss, fat-loss, and osteoporosis phenotypes. Surprisingly, betaine supplementation had several negative effects in control Tg-I278T Cbs (+/-) mice including decreased weight gain, lean mass, and bone mineral density. Our findings indicate that while betaine supplementation does have some beneficial effects, it is not as effective as methionine restriction for reversing the phenotypes associated with severe CBS deficiency in mice.

Cystathionine ß-synthase-deficient mice thrive on a low-methionine diet.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by elevated serum total homocysteine (tHcy). Previously, our laboratory developed a mouse model of CBS deficiency, TgI278T Cbs(-)/(-) (abbreviated as Cbs(-/-)), characterized by low weight, low adiposity, decreased Scd-1 expression, facial alopecia, and osteoporosis. To determine the potential benefit of a methionine-restricted diet (MRD), we fed Cbs(-/-) and Cbs(+/-) control mice either an MRD or a regular diet (RD) from weaning till 240 d of age. Cbs(-/-) mice fed the MRD had a 77% decrease in tHcy, 28% increase in weight, 130% increase in fat mass, 82% increase in Scd-1 expression, and 10.6% increase in bone density and entirely lacked the alopecia phenotype observed in age-matched Cbs(-/-) mice fed the RD. At the end of the study, Cbs(-/-) mice fed the MRD were phenotypically indistinguishable from Cbs(+/-) mice fed the RD. Notably, whereas the MRD diet was highly beneficial to Cbs(-/-) mice, it had nearly opposite effect on Cbs(+/-) mice. These studies show that a low-methionine diet can correct the phenotypic consequences of loss of CBS and provide a striking example of how genotype and diet can interact to influence phenotype in mammals.

Protein arginine hypomethylation in a mouse model of cystathionine ß-synthase deficiency.

Accumulation of the homocysteine (Hcy) precursor S-adenosylhomocysteine (AdoHcy) may cause cellular hypomethylation in the setting of hyperhomocysteinemia because of cystathionine ß-synthase (CBS) deficiency, an inborn error of metabolism. To test this hypothesis, DNA and protein arginine methylation status were assessed in liver, brain, heart, and kidney obtained from a previously described mouse model of CBS deficiency. Metabolite levels in tissues and serum were determined by high-performance liquid chromatography or liquid chromatography-electrospray ionization-tandem mass spectrometry. Global DNA and protein arginine methylation status were evaluated as the contents of 5-methyldeoxycytidine in DNA and of methylarginines in proteins, respectively. In addition, histone arginine methylation was assessed by Western blotting. CBS-deficient mice exhibited increased (>6-fold) Hcy and AdoHcy levels in all tissues examined compared with control levels. In addition, global DNA methylation status was not affected, but global protein arginine methylation status was decreased (10-35%) in liver and brain. Moreover, asymmetric dimethylation of arginine 3 on histone H4 (H4R3me2a) content was markedly decreased in liver, and no differences were observed for the other histone arginine methylation marks examined. Our results show that CBS-deficient mice present severe accumulation of tissue Hcy and AdoHcy, protein arginine hypomethylation in liver and brain, and decreased H4R3me2a content in liver. Therefore, protein arginine hypomethylation arises as a potential player in the pathophysiology of CBS deficiency.

Correction of cystathionine ß-synthase deficiency in mice by treatment with proteasome inhibitors.

Cystathionine beta-synthase (CBS) deficiency is an inborn error of metabolism characterized by extremely elevated levels of plasma total homocysteine. The vast majority of CBS-deficient patients have missense mutations located in the CBS gene that result in the production of either misfolded or unstable protein. Here, we examine the effect of proteasome inhibitors on mutant CBS function using two different mouse models of CBS deficiency. These mice lack mouse CBS and express a missense mutant human CBS enzyme (either p.I278T or p.S466L) that has less than 5% of normal liver CBS activity, resulting in a 10-30-fold elevation in plasma homocysteine levels. We show that treatment of these mice with two different proteasome inhibitors can induce liver Hsp70, Hsp40, and Hsp27, increase levels of active CBS, and lower plasma homocysteine levels to within the normal range. However, response rates varied, with 100% (8/8) of the p.S466L animals showing correction, but only 38% (10/26) of the p.I278T animals. In total, our data show that treatment with proteostasis modulators can restore significant enzymatic activity to mutant misfolded CBS enzymes and suggests that they may be useful in treating certain types of genetic diseases caused by missense mutations.

Hyperhomocysteinemia impairs endothelium-derived hyperpolarizing factor-mediated vasorelaxation in transgenic cystathionine beta synthase-deficient mice.

Hyperhomocysteinemia (HHcy) is associated with endothelial dysfunction (ED), but the mechanism is largely unknown. In this study, we investigated the role and mechanism of HHcy-induced ED in microvasculature in our newly established mouse model of severe HHcy (plasma total homocysteine, 169.5 µM). We found that severe HHcy impaired nitric oxide (NO)- and endothelium-derived hyperpolarizing factor (EDHF)-mediated, endothelium-dependent relaxations of small mesenteric arteries (SMAs). Endothelium-independent and prostacyclin-mediated endothelium-dependent relaxations were not changed. A nonselective Ca(2+)-activated potassium channel (K(Ca)) inhibitor completely blocked EDHF-mediated relaxation. Selective blockers for small-conductance K(Ca) (SK) or intermediate-conductance K(Ca) (IK) failed to inhibit EDHF-mediated relaxation in HHcy mice. HHcy increased the levels of SK3 and IK1 protein, superoxide (O(2)(-)), and 3-nitrotyrosine in the endothelium of SMAs. Preincubation with antioxidants and peroxynitrite (ONOO(-)) inhibitors improved endothelium-dependent and EDHF-mediated relaxations and decreased O(2)(-) production in SMAs from HHcy mice. Further, EDHF-mediated relaxation was inhibited by ONOO(-) and prevented by catalase in the control mice. Finally, L-homocysteine stimulated O(2)(-) production, which was reversed by antioxidants, and increased SK/IK protein levels and tyrosine nitration in cultured human cardiac microvascular endothelial cells. Our results suggest that HHcy impairs EDHF relaxation in SMAs by inhibiting SK/IK activities via oxidation- and tyrosine nitration-related mechanisms.

Activation of mutant enzyme function in vivo by proteasome inhibitors and treatments that induce Hsp70.

Missense mutant proteins, such as those produced in individuals with genetic diseases, are often misfolded and subject to processing by intracellular quality control systems. Previously, we have shown using a yeast system that enzymatic function could be restored to I278T cystathionine beta-synthase (CBS), a cause of homocystinuria, by treatments that affect the intracellular chaperone environment. Here, we extend these studies and show that it is possible to restore significant levels of enzyme activity to 17 of 18 (94%) disease causing missense mutations in human cystathionine beta-synthase (CBS) expressed in Saccharomyces cerevisiae by exposure to ethanol, proteasome inhibitors, or deletion of the Hsp26 small heat shock protein. All three of these treatments induce Hsp70, which is necessary but not sufficient for rescue. In addition to CBS, these same treatments can rescue disease-causing mutations in human p53 and the methylene tetrahydrofolate reductase gene. These findings do not appear restricted to S. cerevisiae, as proteasome inhibitors can restore significant CBS enzymatic activity to CBS alleles expressed in fibroblasts derived from homocystinuric patients and in a mouse model for homocystinuria that expresses human I278T CBS. These findings suggest that proteasome inhibitors and other Hsp70 inducing agents may be useful in the treatment of a variety of genetic diseases caused by missense mutations.

Examination of two different proteasome inhibitors in reactivating mutant human cystathionine ß-synthase in mice.

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.

A systematic review and meta-regression for validation of the Alvarado score as a tool for predicting acute appendicitis.

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.

Safety and efficacy of pembrolizumab for advanced nonsmall cell lung cancer: before and during the COVID-19 pandemic.

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.

Operative versus non-operative management for perforated peptic ulcer disease.

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.