Expanding the horizon of transient CAR T therapeutics using virus-free technology.

The extraordinary success that chimeric antigen receptor (CAR) T cell therapies have shown over the years on fighting hematological malignancies is evidenced by the six FDA-approved products present on the market. CAR T treatments have forever changed the way we understand cellular immunotherapies, as current research in the topic is expanding even outside the field of cancer with very promising results. Until now, virus-based strategies have been used for CAR T cell manufacturing. However, this methodology presents relevant limitations that need to be addressed prior to wide spreading this technology to other pathologies and in order to optimize current cancer treatments. Several approaches are being explored to overcome these challenges such as virus-free alternatives that additionally offer the possibility of developing transient CAR expression or in vivo T cell modification. In this review, we aim to spotlight a pivotal juncture in the history of medicine where a significant change in perspective is occurring. We review the current progress made on viral-based CAR T therapies as well as their limitations and we discuss the future outlook of virus-free CAR T strategies to overcome current challenges and achieve affordable immunotherapies for a wide variety of pathologies, including cancer.

Association between urinary fluoride concentrations and the prevalence of metabolic syndrome in adult individuals from the Central Region of Mexico.

Emerging scientific studies have supported the hypothesis that exposure to environmental chemicals increases the incidence of diverse human metabolic disorders. Therefore, this study aimed to evaluate the association between fluoride exposure and metabolic syndrome (MetS) prevalence in people from the Central Region of Mexico. This research included 575 adult individuals. Urinary fluoride concentrations were determined using a potentiometric method. Anthropometric measurements and blood pressure were also acquired. Serum fasting glucose and lipid levels were quantified. For the MetS screening, we used the International Diabetes Federation (IDF) and National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria. The mean urinary fluoride levels were 3.50 ± 2.50 mg/L. The prevalence of MetS was approximately 27 and 25% according to NCEP ATP III and IDF criteria, respectively. A multivariate logistic regression analysis showed significant associations (p < 0.05) between urinary fluoride concentrations and MetS occurrence using NCEP ATP III criteria (OR = 1.60; 95% CI 1.05-2.10) and IDF criteria (OR = 1.35; 95% CI 0.95-1.45). These findings emphasize the need to discover the underlying mechanisms that fluoride exposure has on MetS pathogenesis.

The aryl hydrocarbon receptor (AhR) activation mediates benzo(a)pyrene-induced overexpression of AQP3 and Notch1 in HaCaT cells.

The aim of this study was twofold: (1) evaluate the effect of benzo[a]pyrene (BaP) on expression levels of AQP3 and Notch1 genes in HaCaT cells exposed "in vitro" and (2) investigate the possible biological role of assessed genes by bioinformatics methods. Cells were exposed to increasing concentrations of BaP (0.0-4.0 µM) for 1-4 days. After treatments, cell viability and expression levels of AhR, CYP1A1, AQP3, and Notch1 genes were evaluated. The possible biological role of assessed genes was evaluated using bioinformatics tools. Low cytotoxicity in HaCaT cells dosed with BaP was detected. A significant overexpression (p < .05) of CYP1A1, AQP3, and Notch1 was found in exposed HaCaT cells. The gene expression upregulation was dependent on AhR activation. The bioinformatics analysis showed that these genes were enriched in related cancer signaling pathways. The findings suggest that AQP3 and Notch1 are upregulated by AhR activation in HaCaT cells exposed to BaP.

Assessment of Different Niosome Formulations for Optogenetic Applications: Morphological and Electrophysiological Effects.

Gene therapy and optogenetics are becoming promising tools for treating several nervous system pathologies. Currently, most of these approaches use viral vectors to transport the genetic material inside the cells, but viruses present some potential risks, such as marked immunogenicity, insertional mutagenesis, and limited insert gene size. In this framework, non-viral nanoparticles, such as niosomes, are emerging as possible alternative tools to deliver genetic material, avoiding the aforementioned problems. To determine their suitability as vectors for optogenetic therapies in this work, we tested three different niosome formulations combined with three optogenetic plasmids in rat cortical neurons in vitro. All niosomes tested successfully expressed optogenetic channels, which were dependent on the ratio of niosome to plasmid, with higher concentrations yielding higher expression rates. However, we found changes in the dendritic morphology and electrophysiological properties of transfected cells, especially when we used higher concentrations of niosomes. Our results highlight the potential use of niosomes for optogenetic applications and suggest that special care must be taken to achieve an optimal balance of niosomes and nucleic acids to achieve the therapeutic effects envisioned by these technologies.

Expression levels and network analysis of inflammamiRs in peripheral blood mononuclear cells exposed to DDE "in vitro".

Recent studies have demonstrated that dichlorodiphenyldichloroethylene (DDE) induced a pro-inflammatory condition in peripheral blood mononuclear cells (PBMC). However, the molecular mechanisms implicated in this condition are poorly understood. Therefore, this study aimed to evaluate miR-155, miR-126, and miR-21 expression levels in PBMC exposed "in vitro" to DDE. PBMC were dosed with increasing concentrations of DDE (10-80 µg mL-1) at different treatment times (0-24 h). The results showed an up-regulation in the expression levels of assessed miRNAs (miR-155, miR-146, and miR-21) after PBMCs were exposed to DDE. Besides, bioinformatic analysis was performed to understand the biological roles of assessed miRNAs. The bioinformatic analysis shows that assessed miRNAs are associated with regulating signaling pathways involved in cancer, apoptosis, cell cycle, inflammation, metabolism, etc. These findings offer new insights into the molecular mechanisms related to the inflammatory processes and their regulation induced by DDE in PBMC exposed "in vitro".

Highlights of the 'I Congress ecancer Choosing Wisely' March 30 and 31, 2022 Santa Cruz, Bolivia.

The ecancer 'Choosing Wisely' conference was held for the first time in Latin America in Santa Cruz, Bolivia. The event had more than 150 registered attendees in addition to 22 speakers from different countries and different specialities in the field of oncology, who presented topics on prevention, oncological surgery, clinical oncology and palliative care, in order to demonstrate the current evidence of how to approach a patient in daily clinical practice based on the human resources, materials and drugs available, trying to offer the maximum benefit to the patient based on current scientific evidence. In addition to addressing issues of vital importance in breast cancer, during the 2 days of the event, updated information generated in recent years was presented, the results of which will change clinical practice. All the experts were in favour of developing strategies and methods that help us to properly select treatments to optimise resources and reduce the economic toxicity of the most modern and current treatments. This conference was an event of vital importance because it was the first face-to-face event for ecancer and the physicians after difficult years due to COVID-19.

The interplay between exposure to PAHs and MTHFR C677T polymorphism on cardiovascular risk biomarkers in Mexican women.

Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality worldwide. Environmental and genetic factors are recognized as risk determinants in the onset and development of CVDs. However, the interaction between both factors on CVDs risk is not still completely clarified. Therefore, the objective of this study was to evaluate the effect of the interaction between methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and polycyclic aromatic hydrocarbon (PAH) exposure (gene-environment interaction) on cardiovascular risk biomarkers in Mexican women. A cross-sectional study was completed with the participation of 390 healthy women. For all enrolled women, anthropometric measurements, serum biochemical analyses, atherogenic indexes, and serum concentrations of biomolecules used as CVD risk biomarkers were obtained. 1-Hydroxypyrene (1-OHP) was measured in urine, as an exposure biomarker of PAHs. The mean urinary level of 1-OHP in the assessed population was 1.23 ± 1.40 µmol/mol creatinine. The allelic frequency (MTHFR C677T polymorphism) identified in the registered individuals was 68.0% for the mutant allele (T-allele). Significant positive associations were detected between urinary 1-OHP levels and serum asymmetric dimethylarginine (ADMA) concentrations (p < 0.05) and atherogenic index of plasma (AIP) values (p < 0.05). Also, women with the TT genotype of the MTHFR C677T enzyme have the highest serum ADMA levels (p < 0.05) and AIP values (p < 0.05) compared to women grouped as CC genotype and CT genotype. Besides, the findings in this study suggest an interaction between environmental (PAHs exposure) and genetic (MTHFR C677T polymorphism) factors on cardiovascular risk markers (ADMA and AIP). According to the usefulness of AIP and ADMA, an increased cardiovascular risk is notable in highly exposed individuals to PAHs with the polymorphic genotype (TT) of the MTHFR enzyme. Therefore, intervention programs in the target communities are required to diminish the cardiovascular risk of the assessed individuals.

Giant Toads (Rhinella marina) From the Industrial Zones of Low Basin of the Coatzacoalcos River (Veracruz, MX) Presents Genotoxicity in Erythrocytes.

The lower basin of Coatzacoalcos River is one of the most polluted regions of the southern Gulf of Mexico. Organochlorine compounds, polybrominated diphenyl ethers, polycyclic aromatic hydrocarbons, and heavy metals have been registered in this region. In the present study, genotoxicity was evaluated in the blood of giant toads (Rhinella marina) from Coatzacoalcos' rural and industrial zones, and compared with laboratory toads. Determination of the frequency of micronucleus and erythrocyte nuclear abnormalities by the light microscope and cell cycle and apoptosis by flow cytometry were used as biomarkers of genotoxicity. We found more variability in micronucleus and more nuclear buds in toads from industrial zones. Also, cell cycle alterations and an increase of apoptosis in erythrocytes were found in toads from rural and industrial zones. Multivariate statistics show that the toads from the industrial zone were more affected than toads from laboratory and rural zones.

Differential Expression of AhR in Peripheral Mononuclear Cells in Response to Exposure to Polycyclic Aromatic Hydrocarbons in Mexican Women.

The exposure to air pollutants causes significant damage to health, and inefficient cooking and heating practices produce high levels of household air pollution, including a wide range of health-damaging pollutants such as fine particles, carbon monoxide and PAHs. The exposure to PAHs has been associated with the development of neoplastic processes, asthma, genotoxicity, altered neurodevelopment and inflammation. The effects on the induction of proinflammatory cytokines are attributed to the activation of AhR. However, the molecular mechanisms by which the PAHs produce proinflammatory effects are unknown. This study was performed on a group of 41 Mexican women from two rural communities who had stoves inside their houses, used wood as biomass fuel, and, thus, were vulnerable. According to the urinary 1-OHP concentration, the samples were stratified into two groups for determination of the levels of TNF-α, AhR, CYP1B1, miR-125b and miR-155 expression. Our results showed that the CYP1B1, TNF-α, miR-125b and miR-155 expression levels were not statistically different between women with the lowest and highest levels of 1-OHP. Interestingly, high levels of PAHs promoted augmented expression of AhR, which is a protein involved in the modulation of inflammatory pathways in vivo, suggesting that cell signaling of AhR may be implicated in several pathogenesis processes.

Trends in Mortality at a Level II Rural Trauma Center.

BACKGROUND: Most studies of deaths from traumatic injury are from urban trauma centers. In contrast, rural areas have higher incidence of traumatic fatal injuries than urban areas. The objective of this research was to describe trends of injuries and mortality from a trauma center serving a largely rural population and compare results with reports from the National Trauma Data Bank (NTDB). METHODS: We conducted a retrospective study of patients admitted to a rural Wisconsin level II trauma center from 2000 through 2018. Details on injuries and deaths prior to discharge were obtained from the trauma registry. Event counts and fatality ratios were described by year, sex, age, mechanism of injury, and injury severity score (ISS). Trends were analyzed across 2000-2005, 2006-2011, and 2012-2018 calendar year eras. RESULTS: During 2000-2018, there were 17,334 injury events among 16,495 patients included in the trauma registry. Across the 3 eras, the proportion of injuries related to falls increased (35.6%, 40.6%, and 51.5%, respectively), and the proportion from on-road motor vehicle events decreased (37.0 %, 32.8, and 22.5%, respectively), similar to the trends from 3 corresponding NTDB reports for 2004, 2010, and 2016. There was a statistically significant decreasing trend (P < 0.001) in overall fatality ratios across the 3 eras, 5.3% (95% CI, 4.7%-6.0%), 4.1% (95% CI, 3.7%-4.6%), and 3.9 (95% CI, 3.4%-4.4%), respectively. The fatality ratios point estimates were similar to overall fatality ratios from the NTDB reports (4.7%, 4.0%, 4.3%, respectively). The median patient age increased significantly from 42, 45, and 55 years across the 3 eras (test for trend P < 0.0001). CONCLUSION: Long-term trends of traumatic injuries and mortality were generally similar to national trends, particularly in the shift to older patients and in the increasing proportion of injury events due to falls. Further research on traumatic injuries and deaths in rural populations is needed, particularly regarding immediate deaths at the scene and longer-term deaths after discharge.

How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

Lead (Pb) exposure is associated with changes in the expression levels of circulating miRNAS (miR-155, miR-126) in Mexican women.

The environmental contamination with lead (Pb) is considered a critical issue worldwide. Therefore, this study aimed to evaluate the expression levels of circulating miRNAs (miR-155, miR-126, and miR-145) in Mexican women exposed to Pb. Blood lead levels (BLL) were assessed in enrolled women (n = 190) using an atomic absorption method. Also, serum miRNAs expression levels were quantified through a real-time PCR assay. A mean BLL of 10.5 ± 4.50 µg/dL was detected. Overexpression of miR-155 was detected in highly exposed women. Besides, a significant simple positive relationship (p < 0.05) was found between BLL and serum miR-155 expression levels. Additionally, a significant inverse correlation (p < 0.05) was determined between BLL and serum miR-126 expression levels, as downregulation of miR-126 expression levels was observed in highly exposed women. The findings in this study are the concern, as epigenetic changes detected may represent a connection between health illnesses and Pb exposure.

Nondestructive biomarkers in giant toad (Rhinella marina) to assess the effect of complex mixture of pollutants in Coatzacoalcos River, Mexico.

In this study, we evaluated the usefulness of nondestructive biomarkers approach in giant toads (Rhinella marina). We obtained blood samples and the residual condition index of toads from rural and industrial zones from Coatzacoalcos River, Mexico (COA). In the blood samples, we determined the activity of enzymes, lipid peroxidation, and the presence of cell death (apoptosis). We found that the activity of the enzyme delta-aminolevulinic dehydratase was lower. Still, the glutathione s-transferase activity and the percentage of apoptosis in erythrocytes were higher in the toads of COA than laboratory toads. Meanwhile, some biomarkers in toads showed differences when compared between Industrial and Rural zones. These results and correlations between biomarkers showed how the response changed in the toads living near the industrial zones. We demonstrate that a nondestructive biomarkers approach can be useful in environmental studies with anuran amphibians.

Effect of gene-environment interaction (arsenic exposure - PON1 Q192R polymorphism) on cardiovascular disease biomarkers in Mexican population.

Cardiovascular diseases (CVDs) are the primary cause of death worldwide. However, little is known about how the interaction between risk factors affects CVDs. Therefore, the aim of this study was to evaluate the effect of the gene-environment interaction (arsenic exposure x PON1 Q192R polymorphism) on serum levels of CVDs biomarkers in Mexican women. Urinary arsenic levels (UAs) ranged from 5.50-145 µg/g creatinine. The allele frequency was 0.38 and 0.62 for the Q and R alleles, respectively. Moreover, significant associations (p<0.05) were detected between UAs and CVDs biomarkers (ADMA, FABP4, and miR-155). Comparable data were found when CVDs biomarkers were evaluated through PON1 genotype, significant (p<0.05) higher serum concentrations of CVDs biomarkers were identified in R allele carriers compared to levels found in Q allele carriers. Besides, a gene-environment interaction was documented. The results of this study we believe should be of significant interest to regulatory authorities worldwide.

Probabilistic human health risk assessment associated with fluoride and arsenic co-occurrence in drinking water from the metropolitan area of San Luis Potosí, Mexico.

A major public health concern in Mexico is the natural contamination of groundwater with fluoride and arsenic. Therefore, this work aimed to evaluate the magnitude of human health risk after determining fluoride and arsenic concentrations in groundwater samples (n = 50) from the Metropolitan area of the city of San Luis Potosi, Mexico. Fluoride levels in water were determined via a potentiometric method using an ion-selective electrode. Arsenic concentrations in water samples were determined with an Atomic Absorption technique. Subsequently, a probabilistic health risk assessment was developed (Monte Carlo Analysis). Fluoride levels in water ranged from 0.20 to 3.50 mg/L. For arsenic, the mean level found in the assessed water samples was 15.5 ± 5.50 µg/L (range: 2.50-30.0 µg/L). In addition, when the probabilistic health risk assessment was completed, a mean HI (cumulative hazardous index) of higher than 1 was detected, indicating a high NCR (non-carcinogenic risk) for children and adults. According to the results found in this study, exposure protection campaigns are imperative in the Metropolitan area of the city of San Luis Potosí, Mexico, to successfully diminish exposure to arsenic and fluoride and, as a consequence, decrease the NCR in the population living in that region of Mexico.

Influence of Human Paraoxonase-1 Polymorphism (Q192R) on Serum Levels of Clinical Biomarkers Indicatives of Cardiovascular Diseases Risk in Mexican Women.

Human paraoxonase-1 (PON1) is a hydrolytic enzyme related in the onset, establishment, and progression of inflammatory illnesses, including cardiovascular diseases (CVD). Therefore, the primary goal of this investigation was to determinate the influence of the PON1 gene polymorphism (Q192R) on prognostic biomarkers of CVD in women (n = 350) from San Luis Potosi, Mexico. For everyone enrolled, anthropometric, clinical, and biochemical measurements were acquired. Q192R polymorphism of the PON1 gene was assessed by real-time PCR using TaqMan probes. Also, quantification and/or calculation of suggested predictive CVD biomarkers were completed. The association between PON1(Q192R) polymorphism with clinical predictive CVD biomarkers was assessed using univariate and multivariate logistic regression models. Genotype PON1 (Q192R) frequencies were: 18%, 47% and 35% for QQ, QR, and RR, respectively. Besides, the frequency of the variant R allele was 0.58. Furthermore, robust significant associations were found between PON1 (Q192R) polymorphism with serum ADMA [OR (95% CI); 3.50 (1.20-5.00), p < 0.01]; FABP4 [2.50 (2.15-3.95), p < 0.01]; and miR-126 [1.50 (1.15-2.00), p < 0.01] levels after adjusting for probable confounders. Data found in this research strongly support the notion that proposes a crucial role of PON1 enzyme (Q192R polymorphism) as a genetic determinant in the CVD risk in Mexican women.

Atherogenic Index of Plasma: Novel Predictive Biomarker for Cardiovascular Illnesses.

BACKGROUND/AIM: Cardiovascular diseases (CVD) are the most important cause of mortality globally. Nevertheless, the World Health Organization have declared that a precise and quick recognition of susceptible individuals to develop CVD is imperative to combat those illnesses. Additionally, developing countries need affordable alternatives to effectively prognosticate cardiovascular events. Therefore, the objective of this study was to assess well-established clinical prognostic markers in Mexican women to identify affordable, specific, and useful tools to predict cardiovascular events. MATERIAL AND METHODS: A cross-sectional study was performed including 340 healthy women. Anthropometric and clinical measurements were acquired from all enrolled individuals. Also, a blood sample of each participant women was obtained to complete biochemical analyses (triglycerides, glucose, total cholesterol, LDL cholesterol, and HDL cholesterol), and serum asymmetric dimethylarginine (ADMA), and adipocyte-fatty acid binding protein (FABP4) determinations. Finally, with anthropometric, clinical and biochemical determinations, atherogenic indices (Framingham risk score, Castelli's risk index, and atherogenic index of plasma) were estimated. RESULTS: A mean value of 6.5 ± 7.2 was detected for the Framingham risk score, 3.7 ± 1.3 for Castelli's risk index, and 0.12 ± 0.22 for the atherogenic index of plasma (AIP). Circulating mean ADMA and FABP4 levels found in assessed women were 0.68 ± 0.34 mmol/L and 20.3 ± 16.6 ng/mL, respectively. Furthermore, strong positive relationships (p <0.05) between AIP and serum FABP4 and ADMA concentrations were detected after adjustment by traditional CVD risk factors. CONCLUSION: In conclusion, AIP could be recommended as a potential biomarker in the early diagnosis of CVD events in developing countries.

Circulating miRNA-126, -145 and -155 levels in Mexican women exposed to inorganic arsenic via drinking water.

The aim of this research was to investigate circulating expression levels of three miRNAs (miR-126, miR-155, and miR-145) proposed as predictive CVD biomarkers in Mexican women exposed to inorganic arsenic via drinking water. Mean UAs concentration of 19.5 ± 14.0 µg/g creatinine was found after urine samples were analyzed (n = 105). Significant associations between UAs levels and serum expression levels of miR-155 (p < 0.05) and miR-126 (p < 0.05) were observed after adjustment for assessed co-variables. Alterations in the serum expression levels of miR-155 and miR-126 may be associated with the onset and development of cardiovascular diseases, hence miRNAs could be proposed as prognostic CVD biomarkers. Data found in this study are of concern and risk reduction plans are necessary for the assessed communities to prevent cardiovascular events in this population of women.

Expression levels of circulating microRNAs-126, -155, and -145 in Mexican women exposed to polycyclic aromatic hydrocarbons through biomass fuel use.

Human exposure to polycyclic aromatic hydrocarbons (PAHs) has been considered a risk determinant for the development of cardiovascular diseases (CVD). Therefore, the aim of this study was to assess expression levels of vascular-related miRNAs, miR-126, miR-155, and miR-145, in plasma from women (aged 19-81 years) exposed (n = 100) and non-exposed (n = 20) to PAHs via biomass combustion smoke.1-hydroxypyrene (1-OHP) was determined in urine as a biomarker of exposure to PAHs using high-resolution liquid chromatography. Plasma expression levels of proposed miRNAs were determined by quantitative real-time PCR. Additionally, traditional risk factors (age, blood pressure, serum lipid profile, blood glucose, and among others) associated with CVD were evaluated. Urinary 1-OHP concentrations and plasma expression levels of miR-126 and miR-155 were significantly higher (P < 0.05) in women using wood as a fuel source in their homes (indoor) compared to women from the reference group (non-exposed to biomass smoke). Besides, multivariate linear regression analyses revealed that miR-126[ß = 0.61; 95% confidence interval (0.32-0.90)] and miR-155 [ß = 0.45; 95% confidence interval (0.13-0.84)] expression levels were significantly associated with urinary 1-OHP concentrations after being adjusted by traditional risk factors (P < 0.05). In contrast, no significant relationship was found between miR-145 and urinary 1-OHP levels. Furthermore, miRNAs assessed in this investigation are associated with CVD events. Consequently, actions to reduce exposure to PAHs in the evaluated population are warranted. Environ. Mol. Mutagen. 60:546-558, 2019. © 2019 Wiley Periodicals, Inc.

Design of an Auscultation System for Phonoangiography and Monitoring of Carotid Artery Diseases.

Cerebrovascular diseases such as stenosis of the carotid artery are accountable for about 1 million death per year across Europe. Diagnostic tools like US, angiography or MRI require specific hardware and highly depend on the experience of the examining clinician. In contrast auscultation with a stethoscope can be used to screen for bruits - audible vascular sounds associated with turbulent blood flow. Dynamical changes in the flow due to pathological narrowing of the vessel can indicate the need for additional diagnostic investigations. A reliable auscultation setup is prerequisite to ensure high signal quality, adequate processing and the objective evaluation of a still subjectively assessed audible signal. We propose a computer assisted auscultation device for the characterisation of carotid bruits to facilitate the assessment of long-term changes in the vessel condition. Main goal of this work are design considerations regarding the mechanical interface of the proposed system to the skin. An experimental setup was used to compare the signal quality and morphology of different setups to a digital stethoscope. A combined system with two different interface configurations is proposed, current limitations of the system and potential improvements are discussed.