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CRM197EK is a derivative of diphtheria toxoid cross-reactive material-197 (CRM197) with two-point mutations (K51E and E148K) to improve its properties for a vaccine conjugate and drug delivery. A previous study has shown that intracellularly expressing CRM197EK in Escherichia coli (E. coli) host formed inclusion bodies that need a complicated purification and refolding step. Protein purification from inclusion bodies can be overcome by solubilization of inclusion bodies by using N-lauroyl sarcosine (sarkosyl). In this work, recombinant CRM197EK (rCRM197EK) was expressed in E. coli BL21 (DE3) as inclusion bodies, then solubilized using sarkosyl to form a soluble rCRM197EK without the need for a renaturation process. Furthermore, rCRM197EK was purified using the Ni-NTA column, characterized by SDS-PAGE and Western Blot, and its biological activity was assayed through its DNase activity. Moreover, its binding affinity with anti-diphtheria toxin (DT) antibody was measured using the surface plasmon resonance (SPR). The result showed that solubilization with sarkosyl form soluble rCRM197EK (61.61 kDa) was confirmed by SDS-PAGE and Western Blot with a yield of 2.8 mg/mL. rCRM197EK shows DNase activity, and the SPR assay shows that it can interact with an anti-DT antibody with a binding energy of - 9.2 kcal/mol.
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Introduction: This investigation assessed the clinical characteristics of patients who received care through telemedicine and the clinical impact telemedicine service had on breast cancer patients in a low-income country. Materials and Methods: This natural experimental study assessed the impact of telemedicine service on cancer outcomes among breast cancer patients at Shaukat Khanum Memorial Trust (SKMT), Pakistan, between January 1st, 2018, to December 31st, 2022. The study group (hybrid group) consisted of patients that had both face-to-face and telemedicine appointments, and the control group (physical group) included patients with only face-to-face encounters. Results: A total of 3,205 patients were included in the analysis. Among those included in the analysis, 3,188 (99.5 %) were females, and the mean age of the cohort was 48.10 ± 11.94 years. Statistically significant differences were observed between the two groups in age, demographic distribution, disease stage, average number of emergency room visits, mean length of stay in the Intensive care unit, and the final patient status (alive at the end of observation period). However, the binary logistic regression model (forward-LR) suggested that the final patient outcome was related to disease relapse, COVID-19 infection, and age. Conclusion: Telemedicine clinics, when conducted in parallel with physical clinics (hybrid setup), are safe and have a clinical impact similar to having just physical encounters among breast cancer patients in a low-income country.
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BACKGROUND: There are still many patients newly diagnosed with HIV at an advanced stage in Indonesia. We aimed to identify factors associated with 1-year mortality among cytomegalovirus (CMV)-infected people living with HIV (PLHIV). METHODS: This retrospective cohort study was carried out at a tertiary-care hospital in Jakarta, Indonesia (January 2017 to December 2022). We included PLHIV with CMV end-organ disease (EOD) and CMV syndrome. The presence of CMV infection was confirmed by fulfilling one of the following criteria: (1) positive PCR from plasma, urine, cerebrospinal fluid, or other body fluids, or associated tissue for CMV EOD; (2) positive immunoglobulin M (IgM); or (3) consistent symptoms and signs of CMV retinitis. RESULTS: Out of 1737 PLHIV, 147 (8.5%, 95% CI: 7.2 to 9.9%) were diagnosed with CMV infection. Forty (27.2%, 95% CI: 20.6 to 35.1%) patients died within 1 year of being diagnosed. Only anti-retroviral therapy (ART) defaulting (aHR 3.31, 95% CI: 1.12 to 9.73) was found to be significantly associated with 1-year mortality in multivariate analysis. CONCLUSION: Defaulted ART status is significantly associated with reduced 1-year survival after CMV infection diagnosis. Patients with low CD4 counts, especially those with <50 cells/µL, should be assessed for CMV infection, monitored, and treated accordingly.
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This paper aims to study the natural convective magneto-hydrodynamic flow of fluid through vertical concentric annuli with iso-flux heating under the conditions of constant internal heat absorption and an induced magnetic field. By solving the set of dimensionless coupled governing equations, we were able to obtain exact expressions for the temperature field, velocity field, and induced magnetic field. We also managed to derive the formulas for skin friction, mass flux, and induced current density. We also examined the effects of non-dimensional parameters on skin friction and mass flux. For easy comprehension and interpretation, the results are provided graphically and in tabular form. The heat absorption parameter, the induced current density, the induced magnetic field, and velocity exhibit a negative trend as the Hartmann number (Ha) value increases. The induced magnetic field has the effect of raising both the induced current density and velocity profile. It is found that, when a fluid absorbs heat, the heat absorption parameter experiences reverse flow. For the heat-absorbing fluids, the radii ratio has the effect of increasing velocity, induced magnetic field, and induced current density. The numerical values of skin friction and mass flux at cylindrical walls increase (decrease) with increasing heat absorption parameter and generally it has decreasing tendency with increasing Hartmann number.
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Diabetes mellitus (DM) is a chronic endocrine disorder that affects more than 20 million people in the United States. DM-related complications affect multiple organ systems and are a significant cause of morbidity and mortality among people with DM. Of the numerous acute and chronic complications, atherosclerosis due to diabetic dyslipidemia is a condition that can lead to many life-threatening diseases, such as stroke, coronary artery disease, and myocardial infarction. The nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway is an emerging antioxidative pathway and a promising target for the treatment of DM and its complications. This review aims to explore the Nrf2 pathway's role in combating diabetic dyslipidemia. We will explore risk factors for diabetic dyslipidemia at a cellular level and aim to elucidate how the Nrf2 pathway becomes a potential therapeutic target for DM-related atherosclerosis.
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Aterosclerosis , Dislipidemias , Factor 2 Relacionado con NF-E2 , Transducción de Señal , Humanos , Factor 2 Relacionado con NF-E2/metabolismo , Aterosclerosis/metabolismo , Aterosclerosis/etiología , Dislipidemias/metabolismo , Dislipidemias/complicaciones , Animales , Complicaciones de la Diabetes/metabolismo , Diabetes Mellitus/metabolismoRESUMEN
Single Chain Variable Fragment (scFv), a small fragment of antibody can be used to substitute the monoclonal antibody for diagnostic purposes. Production of scFv in Escherichia coli host has been a challenge due to the potential miss-folding and formation of inclusion bodies. This study aimed to express anti-CHIKV E2 scFv which previously designed specifically for Asian strains by co-expression of three chaperones that play a role in increasing protein solubility; GroEL, GroES, and Trigger Factor. The scFv and chaperones were expressed in Origami B E. coli host under the control of the T7 promoter, and purified using a Ni-NTA column. Functional assay of anti-CHIKV-E2 scFv was examined by electrochemical immunosensor using gold modified Screen Printed Carbon Electrode (SPCE), and characterized by differential pulses voltammetry (DPV) using K3[Fe(CN)6] redox system and scanning microscope electron (SEM). The experimental condition was optimized using the Box-Behnken design. The results showed that co-expression of chaperone increased the soluble scFv yield from 54.405 µg/mL to 220.097 µg/mL (~5×). Furthermore, scFv can be used to detect CHIKV-E2 in immunosensor electrochemistry with a detection limit of 0.74048 ng/mL and a quantification limit of 2,24388 ng/mL. Thus, the scFv-anti-CHIKV-E2 can be applied as a bioreceptor in another immunoassay method.
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Técnicas Biosensibles , Técnicas Electroquímicas , Escherichia coli , Chaperonas Moleculares , Anticuerpos de Cadena Única , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Chaperonas Moleculares/inmunología , Inmunoensayo/métodosRESUMEN
Enzymatic reactions can be modulated by the incorporation of organic solvents, leading to alterations in enzyme stability, activity, and reaction rates. These solvents create a favorable microenvironment that enables hydrophobic reactions, facilities enzyme-substrate complex formation, and reduces undesirable water-dependent side reactions. However, it is crucial to understand the impact of organic solvents on enzymatic activity, as they can also induce enzyme inactivation. In this study, the enzymatic performance of Aspergillus oryzae α-amylase (Taka-amylase) in various organic solvents both experimentally and computationally was investigated. The results demonstrated that ethanol and ether sustain Taka-amylase activity up to 20% to 25% of the organic solvents, with ether providing twice the stability of ethanol. Molecular dynamics simulations further revealed that Taka-amylase has a more stable structure in ether and ethanol relative to other organic solvents. In addition, the analysis showed that the loop located near the active site in the AB-domain is a vulnerable site for enzyme destabilization when exposed to organic solvents. The ability of Taka-amylase to preserve the secondary loop structure in ether and ethanol contributed to the enzyme's activity. In addition, the solvent accessibility surface area of Taka-amylase is distributed throughout all enzyme structures, thereby contributing to the instability of Taka-amylase in the presence of most organic solvents.
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Developing therapeutics such as neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is essential to halt the Covid-19 infection. However, antibody production is expensive and relatively inaccessible to many low-income countries. Therefore, a more efficient and smaller antibody fragment, such as a single-chain variable fragment (scFv), derived from a known neutralizing antibody structure, is of interest due to the lower cost of recombinant protein production and the ability to tailor scFvs against circulating viruses. In this study, we used computational design to create an scFv based on the structure of a known neutralizing antibody, S230, for SARS-CoV-1. By analyzing the interaction of S230 with the RBD of both SARS-CoV-1 and SARS-CoV-2, five mutations were introduced to improve the binding of the scFv to the RBD of SARS-CoV-2. These mutations were Ser32Thr, Trp99Val, Asn57Val, Lys65Glu, and Tyr106Ile. Molecular dynamics simulations were used to evaluate the stability and affinity of the designed scFv. Our results showed that the designed scFv improved binding to the RBD of SARS-CoV-2 compared to the original S230, as indicated by principal component analysis, distance analysis, and MM/GBSA interaction energy. Furthermore, a positive result in a spot test lateral flow assay of the expressed scFv against the RBD indicated that the mutations did not alter the protein's structure. The designed scFv showed a negative result when tested against human serum albumin as a negative control, indicating reasonable specificity. We hope that this study will be useful in designing a specific and low-cost therapeutic agent, particularly during early outbreaks when information on neutralizing antibodies is limited.Communicated by Ramaswamy H. Sarma.
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COVID-19 , Anticuerpos de Cadena Única , Humanos , Glicoproteína de la Espiga del Coronavirus/metabolismo , SARS-CoV-2/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Anticuerpos Antivirales/metabolismo , Anticuerpos Neutralizantes/química , Unión ProteicaRESUMEN
Platelets have a pivotal role in maintaining cardiovascular homeostasis. They are kept docile by endothelial-derived mediators. Aberration in haemostatic balance predisposes an individual to an elevated risk of a prothrombotic environment. Anti-platelet therapy has been a key component to reduce this risk. However, understanding how these medications affect the balance between the activation and inhibition of platelets is critical. There is no evidence that a key anti-platelet therapy - aspirin, may not be the most efficacious medicine of choice, as it can compromise both platelet inhibition and activation pathways. In this review, the rationale of aspirin as an anti-thrombotic drug has been critically discussed. This review looks at how recently published trials are raising key questions about the efficacy and safety of aspirin in countering cardiovascular diseases. There is an increasing portfolio of evidence that identifies that although aspirin is a very cheap and accessible drug, it may be used in a manner that is not always beneficial to a patient, and a more nuanced and targeted use of aspirin may increase its clinical benefit and maximize patient response. The questions about the use of aspirin raise the potential for changes in its clinical use for dual anti-platelet therapy. This highlights the need to ensure that treatment is targeted in the most effective manner and that other anti-platelet therapies may well be more efficacious and beneficial for CVD patients in their standard and personalized approaches.
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The thermostability of enzymes plays a significant role in the starch hydrolysis process in the industry. The structural difference between thermostable Bacillus licheniformis α-amylase (BLA) and thermolabile Aspergillus niger α-amylase (ANA) is interesting to be explored. This work aimed to study the thermostability-determining factor of BLA as compared to a non-thermostable enzyme, ANA, using molecular dynamics (MD) simulation at a high temperature. A 100 ns of classical MD, which was followed by 200 ns accelerated MD was conducted to explore the conformational changes of the enzyme. It is revealed that the intramolecular interactions through salt bridge interactions and the presence of calcium ions dominates the stability effect of BLA, despite the absence of a disulfide bond in the structure. These results should be useful in designing a thermostable enzyme that can be used in industrial processes.Communicated by Ramaswamy H. Sarma.
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Epitope-based peptide vaccine can elicit T-cell immunity against SARS-CoV-2 to clear the infection. However, finding the best epitope from the whole antigen is challenging. A peptide screening using immunoinformatics usually starts from MHC-binding peptide, immunogenicity, cross-reactivity with the human proteome, to toxicity analysis. This pipeline classified the peptides into three categories, i.e., strong-, weak-, and non-binder, without incorporating the structural aspect. For this reason, the molecular detail that discriminates the binders from non-binder is interesting to be investigated. In this study, five CTL epitopes against HLA-A*02:01 were identified from the coarse-grained molecular dynamics-guided immunoinformatics screening. The strong binder showed distinctive activities from the non-binder in terms of structural and energetic properties. Furthermore, the second residue from the nonameric peptide was most important in the interaction with HLA-A*02:01. By understanding the nature of MHC-peptide interaction, we hoped to improve the chance of finding the best epitope for a peptide vaccine candidate.
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Antineoplásicos , COVID-19 , Humanos , Vacunas contra la COVID-19 , Epítopos de Linfocito T , SARS-CoV-2 , COVID-19/prevención & control , Simulación de Dinámica Molecular , Simulación del Acoplamiento Molecular , Péptidos , Vacunas de Subunidad , Antígenos HLA-A , Epítopos de Linfocito BRESUMEN
Marennine, a blue pigment produced by the blue diatom Haslea ostrearia, is known to have some biological activities. This pigment is responsible for the greening of oysters on the West Coast of France. Other new species of blue diatom, H. karadagensis, H. silbo sp. inedit., H. provincialis sp. inedit, and H. nusantara, also produce marennine-like pigments with similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms present a commercial potential for the aquaculture, food, cosmetics, and health industries. Unfortunately, for a hundred years, the exact molecular structure of this bioactive compound has remained a mystery. A lot of hypotheses regarding the chemical structure of marennine have been proposed. The recent discovery of this structure revealed that it is a macromolecule, mainly carbohydrates, with a complex composition. In this study, some glycoside hydrolases were used to digest marennine, and the products were further analyzed using nuclear magnetic resonance (NMR) and mass spectroscopy (MS). The reducing sugar assay showed that marennine was hydrolyzed only by endo-1,3-ß-glucanase. Further insight into the structure of marennine was provided by the spectrum of 1H NMR, MS, a colorimetric assay, and a computational study, which suggest that the chemical structure of marennine contains 1,3-ß-glucan.
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Studies on the interaction between gold nanoparticles (AuNPs) and functional proteins have been useful in developing diagnostic and therapeutic agents. Such studies require a realistic computational model of AuNPs for successful molecular design works. This study offers a new multilayer model of AuNPs to address the inconsistency between its molecular mechanics' interpretation and AuNP's plasmonic nature. We performed partial charge quantum calculation of AuNPs using Au13 and Au55 models. The result showed that it has partial negative charges on the surface and partial positive charges on the inner part, indicating that the AuNP model should be composed of multiatom types. We tested the partial charge parameters of these gold (Au) atoms in classical molecular dynamics simulation (CMD) of AuNPs. The result showed that our parameters performed better in simulating the adsorption of Na+ and dicarboxy acetone in terms of consistency with surface charge density than the zero charges Au in the interface force field (IFF). We proposed that the multiple-charged AuNP model can be developed further into a simpler four-atom type of Au in a larger AuNP size.
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Lipid nanoparticles (LNPs) have emerged as a promising delivery system, particularly for genetic therapies and vaccines. LNP formation requires a specific mixture of nucleic acid in a buffered solution and lipid components in ethanol. Ethanol acts as a lipid solvent, aiding the formation of the nanoparticle's core, but its presence can also affect LNP stability. In this study, we used molecular dynamics (MD) simulations to investigate the physicochemical effect of ethanol on LNPs and gain a dynamic understanding of its impact on the overall structure and stability of LNPs. Our results demonstrate that ethanol destabilizes LNP structure over time, indicated by increased root mean square deviation (RMSD) values. Changes in the solvent-accessible surface area (SASA), electron density, and radial distribution function (RDF) also suggest that ethanol affects LNP stability. Furthermore, our H-bond profile analysis shows that ethanol penetrates the LNP earlier than water. These findings emphasize the importance of immediate ethanol removal in lipid-based systems during LNP production to ensure stability.
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Etanol , Nanopartículas , Simulación de Dinámica Molecular , Lípidos/química , Solventes , Nanopartículas/química , ARN Interferente Pequeño/genéticaRESUMEN
Heart failure (HF) is a global pandemic with increasing prevalence and mortality rates annually. Its main cause is myocardial infarction (MI), followed by rapid cardiac remodeling. Several clinical studies have shown that probiotics can improve the quality of life and reduce cardiovascular risk factors. This systematic review and meta-analysis aimed to investigate the effectiveness of probiotics in preventing HF caused by a MI according to a prospectively registered protocol (PROSPERO: CRD42023388870). Four independent evaluators independently extracted the data using predefined extraction forms and evaluated the eligibility and accuracy of the studies. A total of six studies consisting of 366 participants were included in the systematic review. Probiotics are not significant in intervening left ventricular ejection fraction (LVEF) and high-sensitivity C-reactive protein (hs-CRP) when compared between the intervention group and the control group due to inadequate studies supporting its efficacy. Among sarcopenia indexes, hand grip strength (HGS) showed robust correlations with the Wnt biomarkers (p < 0.05), improved short physical performance battery (SPPB) scores were also strongly correlated with Dickkopf-related protein (Dkk)-3, followed by Dkk-1, and sterol regulatory element-binding protein 1 (SREBP-1) (p < 0.05). The probiotic group showed improvement in total cholesterol (p = 0.01) and uric acid (p = 0.014) compared to the baseline. Finally, probiotic supplements may be an anti-inflammatory, antioxidant, metabolic, and intestinal microbiota modulator in cardiac remodeling conditions. Probiotics have great potential to attenuate cardiac remodeling in HF or post-MI patients while also enhancing the Wnt signaling pathway which can improve sarcopenia under such conditions.
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Microbioma Gastrointestinal , Probióticos , Sarcopenia , Humanos , Antioxidantes , Calidad de Vida , Volumen Sistólico , Fuerza de la Mano , Remodelación Ventricular , Función Ventricular Izquierda , Ensayos Clínicos Controlados Aleatorios como Asunto , AntiinflamatoriosRESUMEN
Lansium domesticum Corr. is a member of the Meliaceae family that is widely spread in tropical and subtropical region of Asia and America. Traditionally, the fruit of this plant has been consumed because of its sweet taste. However, the fruit peels and the seeds of this plant have been rarely utilized. The previous chemical investigation of this plant showed the presence of secondary metabolites with many biological activities, including cytotoxic triterpenoid. Triterpenoids is a class of secondary metabolites which contain thirty carbon atoms in the main skeleton. The high modification of this type of compound, including the ring opening, highly oxygenated carbons, and the degradation of its carbon chain to give the nor-triterpenoid structure, is responsible for its cytotoxic activity. In this paper, we isolated and elucidated the chemical structure of two new onoceranoid triterpenes, kokosanolides E (1) and F (2), from the fruit peels of L. domesticum Corr., along with a new tetranortriterpenoid, kokosanolide G (3), from the seeds of L. domesticum Corr. The structural determination of compounds 1-3 was undertaken through FTIR spectroscopic analysis, 1D and 2D NMR, mass spectrometry, as well as through a comparison of the chemical shifts of the partial structures of compounds 1-3 with the literature data. The cytotoxic properties of compounds 1-3 were tested against MCF-7 breast cancer cells using the MTT assay. Moderate activity was shown by compounds 1 and 3, with IC50 values of 45.90 and 18.41 µg/mL, respectively, while compound 2 showed no activity (IC50 168.20 µg/mL). For the onoceranoid-type triterpene, the high symmetrical structure of compound 1 is presumably the reason for its better cytotoxic activity compared with that of compound 2. Compound 3 showed moderate activity, mainly because of the presence of the furan ring, which, based on the literature, gives better cytotoxic activity in a tetranortriterpenoid-type structure. The findings of three new triterpenoid compounds from L. domesticum indicate the significant value of this plant as a source of new compounds.
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Antineoplásicos , Limoninas , Meliaceae , Triterpenos , Triterpenos/química , Limoninas/análisis , Semillas/química , Frutas/química , Antineoplásicos/análisis , Meliaceae/química , Estructura MolecularRESUMEN
Purpose: Considering the significance of rice as a staple food in Indonesia, this study aims to analyze the impact of various factors, i.e., domestic price production level, exchange rate, international rice price, and Gross Domestic Product (GDP) per capita, on domestic rice prices and the resultant disparity in income distribution and inequality in the country. Methods: A simulation analysis has been performed to assess the impact of the above-mentioned factors on rice prices and the resultant income disparity in Indonesia. For that, input data from 2006 to 2020 was used to depict the change in domestic rice prices from 2021 to 2026 due to independent variables changes. Findings: Results revealed that a regular increase in rice production decreases rice prices in the longer term. Besides, a rise in the exchange rate decreases rice prices, and a fall in the exchange rate results in higher rice prices. Results also showed the insignificant impact of international rice prices on domestic rice prices in Indonesia. In contrast, an increase in per capita income reflected an increase in rice prices. Moreover, the result of the study exemplifies that the expenditure for rice has a very low elasticity (0.0975) compared to the expenditure on non-rice food (0.4096). More than half of the total household expenditure (50.71%) is spent on food, while the rest (49.29%) is used for non-food. The increase in rice prices affects the rise of income amongst farmers and declines the income of non-farmers. Hence it affects the decline of disparity in households' income distribution. Originality/value: This study adds value to the existing literature with several implications for practitioners, policymakers, and government organizations to take necessary measures to stabilize rice prices and income distribution among the various income groups.
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A detection method based on an electrochemical aptasensor has been developed as an alternative fast, portable, simple, inexpensive, and high-accuracy detection method for detecting the SARS-CoV-2 Spike Receptor Binding Domain (spike RBD). The CeO2@NH2 functionalized Screen Printed Carbon Electrode (SPCE) was used to immobilize an aminated aptamer of spike RBD protein via glutaraldehyde as a linker. The aptamer's interaction with the SARS-CoV-2 Spike RBD was measured via the [Fe(CN)6]4-/3- redox system signal. Experimental conditions were optimized using a Box-Behnken experimental design and showed that the optimal conditions of the SARS-CoV-2 aptasensor were 1.5 ng mL-1 of aptamer, immobilization of aptamer for 60 minutes, and Spike RBD incubation for 10 minutes. The developed aptasensor was able to detect the standard SARS-CoV-2 Spike RBD with a detection limit of 0.017 ng mL-1 in the range of 0.001-100 ng mL-1. This aptasensor was used to detect salivary and oropharyngeal swab samples of normal individuals with the addition of Spike RBD, and the recoveries were 92.96% and 96.52%, respectively. The testing on nasopharyngeal swab samples of COVID-19 patients showed that the aptasensor results were comparable with the qRT-PCR results. Thus, the developed aptasensor has the potential to be applied as a SARS-CoV-2 rapid test method for clinical samples.
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The fast-economic development and population growth in Nigeria have resulted in huge quantities of air pollutants emission which have implications on the environment. Detailed sectoral emission inventory to serve as the basis for policy formation to mitigate the condition is still lacking. This study builds detailed sectoral emission inventory using the emission factor approach to estimates various pollutant emissions from different sources. Five major sources of pollutant emissions were identified which include transportation, energy, municipal solid waste, wood fuel, and agricultural sectors. An increasing trend in emissions from 1980 to 2020 was observed for total emission of CO, NOx, PM2.5, PM10, SO2, NH3 and NMVOC in Nigeria that increased from 1 736-6 210; 143-338; 126-551; 171-717; 19-60; 4-28; and 471-1 587 Gg, respectively. Wood fuel, transportation, and municipal waste sectors are the major sources that contributed to 63%, 16%, and 15% of the total CO emission. Three mitigation scenarios for emission reduction for the future were analyzed. CO emission reductions of 38%, 24%, and 38% will be obtained from the liquefied petroleum gas (LPG) intervention, waste to energy (WTE) technology, and vehicle inspection and maintenance (VIM) policy scenarios, respectively, through to the year 2050.