lncRNA- RP11-156p1.3, novel diagnostic and therapeutic targeting via CRISPR/Cas9 editing in hepatocellular carcinoma.

We aim to characterize the expression of RNA panel in HCC. We assessed the expression of HCC-associated mRNA, miRNA and lncRNA network by real time PCR in sera and tissue samples. In a proof-of-principle approach, CRISPR cas9 mediated knock out for lncRNA- RP11-156p1.3 was performed in HEPG2 cell line to validate the role of the chosen RNA in HCC pathogenesis. The differential expression of RFTN1 mRNA, lncRNA- RP11-156p1.3 and miRNA-4764-5p was statistically different among the studied groups. After CRISPR cas9 mediated knockout of lncRNA- RP11-156p1.3 in HEPG2 cells, there was significant decrease in cell count and viability with reversal of the expression of the chosen RNAs. The chosen RNAs play a significant role in HCC pathogenesis and may be potential diagnostic and therapeutic targets.

Dysregulation in the expression of (lncRNA-TSIX, TP53INP2 mRNA, miRNA-1283) in spinal cord injury.

AIM: The objective of this study is to examine the alterations in the levels of expression of serum lncRNA-TSIX, TP53INP2 mRNA, miRNA-1283 in spinal cord injured (SCI) patients versus healthy control. METHOD: The expression of the selected RNAs in the sera was determined in 23 patients suffering from acute spinal cord injury, 41 individuals with chronic spinal cord injury, and 36 healthy control using real-time reverse-transcription polymerase chain reaction method. RESULTS: The results showed that lncRNA-TSIX and the TP53INP2 mRNA expression levels in SCI patients was overexpressed in comparison to the control group alongside with a significant downregulation of miR-1283. Statistically,there was a highly significant positive correlation between lnc-RNA-TRIX and TP53INP2 mRNA with inverse correlation between miRNA-1283 and lnc-RNA-TRIX based on fold changes. CONCLUSION: Up-regulation of lncRNA-TSIX, TP53INP2 mRNA with downregulation of miRNA-1283 might be closely associated with progression of SCI.

Clinical significance of serum DRAM1 mRNA, ARSA mRNA, hsa-miR-2053 and lncRNA-RP1-86D1.3 axis expression in malignant pleural mesothelioma.

AIM AND BACKGROUND: Malignant pleural mesothelioma (MPM) is a lethal cancer mainly caused by chronic exposure of asbestos. In this pilot study, we aimed to assess the expression of serum RNA-based biomarker panel exploring their clinical utility as diagnostic and prognostic biomarkers for MPM. METHODS: We have selected an MPM-specific RNA-based biomarker panel through bioinformatics analysis based on the integration of DNA damage regulated autophagy modulator 1 (DRAM1) and arylsulfatase A ( ARSA) gene expression with their epigenetic regulators microRNA ( miR-2053) and long noncoding RNA ( lncRNA-RP1-86D1.3). Then, quantitative real-time polymerase chain reaction (qPCR) validation in sera of 60 MPM patients, 20 chronic asbestos exposure patients, and 20 healthy volunteers was done. Lastly, the prognostic power of the selected panel was assessed. RESULTS: The expression of serum DRAM1 messenger RNA (mRNA), ARSA mRNA, hsa-miR-2053 and lncRNA-RP1-86D1.3 were positive in 78.3%, 90%, 85%, and 83.3% of MPM patients, respectively. The RNA-based biomarker panel was able to discriminate between MPM patients and controls with high accuracy and their combined sensitivity reached 100% for the diagnosis of MPM. Kaplan-Meier analysis showed that hsa-miR-2053 is an independent prognostic factor of MPM. CONCLUSION: Our preliminary data revealed that the chosen RNAs play an important role in driving MPM development and progression.

circRNAs (hsa_circ_00156, hsa_circ _000224, and hsa_circ _000520) are novel potential biomarkers in hepatocellular carcinoma.

Circular RNAs (circRNAs) are a newly validated type of noncoding RNAs recently found to be deregulated in several human cancers. More accurate and specific noninvasive biomarkers are strongly needed for better diagnosis and prognosis of hepatocellular carcinoma (HCC). We performed a bioinformatics analysis to retrieve a novel panel of circRNAs potentially relevant to HCC. We examined their expression in the sera of 68 patients with HCC, 60 patients with chronic hepatitis C, and 36 healthy controls using quantitative polymerase chain reaction. We examined the performance characteristics of the selected circRNA biomarker panel in comparison with alpha-fetoprotein (AFP). In addition, we performed a survival analysis to correlate between their expression levels and patient survival. The circRNAs hsa_circ _00224 and hsa_circ _00520 showed a strong biomarker potential with relatively high sensitivities and specificities compared with AFP. The combined panel including the three circRNAs showed superior performance characteristics relative to those of AFP. The median follow-up period was 26 months. hsa_circ_00520 expression has been shown to be associated with relapse-free survival (P < 0.005). circRNAs hsa_circ_00156, hsa_circ_000224, and hsa_circ_000520 are novel potential biomarkers of high sensitivity and specificity, which could potentially be used in the diagnosis of HCC.

Identification of Novel Molecular Network Expression in Acute Myocardial Infarction.

BACKGROUND: In the current study, we aimed to analyze the hypothesis that human myocardial-specific extracellular RNAs expression could be used for acute myocardial injury(AMI) diagnosis. METHODOLOGY: We used bioinformatics' analysis to identify RNAs linked to ubiquitin system and specific to AMI, named, (lncRNA-RP11-175K6.1), (LOC101927740), microRNA-106b-5p (miR-106b-5p) and Anaphase, promoting complex 11 (ANapc11mRNA). We measured the serum expression of the chosen RNAs in 69 individuals with acute coronary syndromes, 31 individuals with angina pectoris without MI and non-cardiac chest pain and 31 healthy control individuals by real-time reverse-transcription PCR. RESULTS: Our study revealed a significant decrease in both lncRNA-RP11-175K6.1 and ANapc11mRNA expression of in the sera samples of AMI patients compared to that of the two control groups alongside with significant upregulation of miR-106b-5p. CONCLUSION: Of note, the investigated serum RNAs decrease the false discovery rate of AMI to 3.2%.

Mechanistic effects of mesenchymal and hematopoietic stem cells: New therapeutic targets in myocardial infarction.

Myocardial infarction (MI) results in dysfunction and irreversible loss of cardiomyocytes and is of the most serious health threats today. Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have been explored as promising cell therapy in MI and regenerative therapy. Recently, reports investigated the potential therapeutic effects of MSCs or HSCs transplantation after MI in numerous experimental and clinical studies; however, their results are controversy and needs more explorations. The current review is an attempt to clarify the therapeutic potentials of MSCs and HSCs in MI therapy, as well as their possible effects; especially the paracrine one and the exosome-derived stem cell among animal models as well as clinical trials conducted within the last 10 years. In this context, various sources of MSCs and HSCs have been addressed in helping cardiac regeneration by either revitalizing the cardiac stem cells niche or revascularizing the arteries and veins of the heart. In addition, both MSCs and HSCs could produce paracrine mediators and growth factors which led to cardiomyocytes protection, angiogenesis, immunemodulation, antioxidants, anti-apoptotic, anti-inflammatory, antifibrotic, as well as increasing cardiac contractility. Recently, microRNAs (miRNAs), post-transcriptional regulators of gene expression, and long non-coding RNA (lncRNA), a miRNA sponge, are recent stem cell-derived mediators can be promising targets of MSCs and HSCs through their paracrine effects. Although MSCs and HSCs have achieved considerable achievements, however, some challenges still remain that need to be overcome in order to establish it as a successful technique. The present review clarified the mechanistic potentials of MSCs and HSCs especially paracrine effects involved in MI including human and animal studies and the challenges challenges regarding type, differentiation, route, and number of injections.

Mesenchymal stem cell therapy: A promising cell-based therapy for treatment of myocardial infarction.

For decades, mesenchymal stem (MSCs) cells have been used for cardiovascular diseases as regenerative therapy. This review is an attempt to summarize the types of MSCs involved in myocardial infarction (MI) therapy, as well as its possible mechanisms effects, especially the paracrine one in MI focusing on the studies (human and animal) conducted within the last 10 years. Recently, reports showed that MSC therapy could have infarct-limiting effects after MI in both experimental and clinical trials. In this context, various types of MSCs can help cardiac regeneration by either revitalizing the cardiac stem cells or revascularizing the arteries and veins of the heart. Furthermore, MSCs could produce paracrine growth factors that increase the survival of nearby cardiomyocytes, as well as increase angiogenesis through recruitment of stem cell from bone marrow or inducing vessel growth from existing capillaries. Recent research suggests that the paracrine effects of MSCs could be mediated by extracellular vesicles including exosomes. Exosomal microRNAs (miRNAs) released by MSCs are promising therapeutic hotspot target for MI. This could be attributed to the role of miRNA in cardiac biology, including cardiac regeneration, stem cell differentiation, apoptosis, neovascularization, cardiac contractility and cardiac remodeling. Furthermore, gene-modified MSCs could be a recent promising therapy for MI to enhance the paracrine effects of MSCs, including better homing and effective cell targeted tissue regeneration. Although MSC therapy has achieved considerable attention and progress, there are critical challenges that remains to be overcome to achieve the most effective successful cell-based therapy in MI.

Phytochemicals in cervical cancer: an epigenetic overview.

Cervical cancer is the fourth most common female malignancy worldwide and a complex disease that typically starts with HPV infection. Various genetic and epigenetic alterations are implicated in its development. The current cervical cancer therapies have unsatisfactory outcomes due to their serious adverse effects, necessitating the need for safe, effective preventive and therapeutic modalities. Phytochemicals have been addressed in cervical cancer prevention and treatment, and further understanding the epigenetics of cervical cancer pathogenesis is critical to investigate new preventive and therapeutic modalities. Addressing the epigenetic mechanisms of potential phytochemicals will provide an overview of their use individually or in combination. The primary aim of this review is to highlight the epigenetic effects of the phytochemicals addressed in cervical cancer therapy.

Influence of pentoxifylline on gene expression of PAG1/ miR-1206/ SNHG14 in ischemic heart disease.

The regulation by immune checkpoint is able to prevent excessive tissue damage caused by ischemia reperfusion (I/R); therefore, the study aims to investigate the behavior of phosphoprotein associated with glycosphingolipid-enriched microdomains 1 (PAG1) mRNA, miR-1206 and small nucleolar RNA host gene 14 (SNHG14) during I/R and intake of pentoxifylline (PTX) as a protective drug. The relative expression level of PAG1/miR-1206/SNHG14 was determined by qRT-PCR. Cardiac tissue levels of cytotoxic T-lymphocyte associated antigen 4 (CTLA4) and PAG1 protein expression were determined by ELISA technique. The regulatory T cells achieved by the flow cytometry. The results found that the relative expression of SNHG14 was significantly upregulated in I/R, but suppressed in PTX treated groups with enhancement of the relative expression level of miR-1206. The gene and protein expression of PAG1 were downregulated with effective doses of PTX. The results showed that (30 and 40 mg/kg bwt) PTX dose suppressed the CTLA4 development significantly. The mean of the regulatory T cell in PTX protective groups is significantly reduced at (p < 0.001) in a comparison with I/R group. Spearman's correlation analysis revealed a significant negative correlation between SNHG14 and miR-1206, but a significant positive correlation between SNHG14 and PAG1 in I/R heart tissue. The results indicated that miR-1206 and SNHG14 can be used as biomarkers with perfect sensitivity and specificity. Using PTX reduced cardiac tissue damage. SNHG14 and miR-1206 can be used as a diagnostic tool in I/R.

Role of extracellular LncRNA-SNHG14/miRNA-3940-5p/NAP12 mRNA in colorectal cancer.

BACKGROUND: We aim to identify and analyze the expression of dyregulated RNAs in colorectal cancer (CRC). METHODS: We selected a panel of RNAs specific to CRC composed of Nucleosome Assembly Protein 1 Like 2 (NAP1L2) mRNA, LNCRNA SNHG14 small nucleolar RNA host gene 14 (LNCRNA SNHG14) and homo sapiens microRNA-3940-5p(hsa-miRNA-3940-5p) from genetic and epigenetic databases. Validation of the chosen RNAs was achieved by real time quantitative PCR in sera of patients with CRC, versus controls groups (benign lesions and healthy individual). RESULTS: We found that LLNCRNA SNHG14, hsa-miRNA-3940-5p and NAP1L2 mRNA had an excellent performance characteristics and more superior than CEA, and CA19.9 for differentiating CRC from controls. Combined expression of lncRNA SNHG14- hsa-miR-3940-5p and NAP1L2 mRNA had reached 100% sensitivity with accuracy 93%. Interestingly, serum hsa-miRNA-3940-5p could be an independent prognostic factor in CRC. CONCLUSION: The extracellular lncRNA SNHG14- hsa-miR-3940-5p - NAP1L2 mRNA may aid in CRC management.KEY MESSAGESThe extracellular RNAs provide a potential class of noninvasive biomarkers with high specificity, accuracy and stability for detection of CRC.We used insilico data analysis followed by qPCR for detection of differential NAP1L2 gene expression with the selected epigenetic regulators.Our data presented interesting biomarker panel (NAP1L2 gene, lncRNA-SNHG14 and hsa-miR-3940-5p) that may be potential for CRC diagnosis and prognosis.

Prospects for repurposing CNS drugs for cancer treatment.

Drug repurposing is the idea of using an already approved drug for another disease or disorder away from its initial use. This new approach ensures the reduction in high cost required for developing a new drug in addition to the time consumed, especially in the tumor disorders that show an unceasing rising rate with an unmet success rate of new anticancer drugs. In our review, we will review the anti-cancer effect of some CNS drugs, including both therapeutic and preventive, by searching the literature for preclinical or clinical evidence for anticancer potential of central nervous system drugs over the last 8 years period (2010-2018) and including only evidence from Q1 journals as indicated by Scimago website (www.scimagojr.com). We concluded that Some Central Nervous system drugs show a great potential as anti-cancer in vitro, in vivo and clinical trials through different mechanisms and pathways in different types of cancer that reveal a promising evidence for the repurposing of CNS drugs for new indications.

Computational prediction of vaccine potential epitopes and 3-dimensional structure of XAGE-1b for non-small cell lung cancer immunotherapy.

BACKGROUND: XAGE-1b is shown to be overexpressed in lung adenocarcinoma and to be a strong immunogenic antigen among non-small cell lung cancer (NSCLC) patients. However, 3D structure of XAGE-1b is not available and its confirmation has not been solved yet. METHODS: Multiple sequence alignment was run to select the most reliable templates. Homology modeling technique was performed using computer-based tool to generate 3-dimensional structure models, eight models were generated and assessed on basis of local and global quality. Immune Epitope Database (IEDB) tools were then used to determine potential B-Cell epitopes while NetMHCpan algorithms were used to enhance the determination for potential epitopes of both Cytotoxic T-lymphocytes and T-helper cells. RESULTS: Computational prediction was performed for B-Cell epitopes, prediction results generated; 3 linear epitopes where XAGE-1b (13-21) possessed the best score of 0.67, 5 discontinuous epitopes where XAGE-1b (40-52) possessed the best score of 0.67 based on the predicted model of the finest quality. For a potential vaccine design, computational prediction yielded potential Human Leukocyte Antigen (HLA) class I epitopes including HLA-B*08:01-restricted XAGE-1b (3-11) epitope which was the best with 0.2 percentile rank. Regarding HLA Class II epitopes, HLA-DRB1*12:01-restricted XAGE-1b (25-33) was the most antigenic epitope with 5.91 IC50 value. IC50 values were compared with experimental values and population coverage percentages of epitopes were computed. CONCLUSIONS: This study predicted a model of XAGE-1b tertiary structure which could explain its antigenic function and facilitate usage of predicted peptides for experimental validation towards designing immunotherapies against NSCLC.

Emerging role of nutrition and the non-coding landscape in type 2 diabetes mellitus: A review of literature.

With the advent of recent advances in molecular techniques and whole genome sequencing, we have come to know that the non-coding landscape (including non-coding RNAs, tRNAs and even telomeres) plays a major role in the regulation of cellular processes. Furthermore, the deregulation of this landscape has been found to contribute to and even bring about the pathogenesis of a large number of diseases. One of such diseases is diabetes mellitus (type 2 specifically) whose incidence rate and global burden is constantly increasing. Nutrition has been proven to be a key player in the development, onset and control of type 2 diabetes mellitus. Additionally, non-coding DNA based molecular markers are emerging as biomarkers of T2D, susceptibility, and perhaps dietary supplements can modulate non-coding DNA based markers expression and function in T2D management. In this review, we provide a brief overview of the developmental origins and genetics of type 2 diabetes mellitus, how each component of the non-coding landscape contributes to the development and progression of the disease and finally we discuss how dietary interventions modulate the non-coding landscape in T2D.

Investigating miRNA-661 and ATG4-B mRNA expression as potential biomarkers for hepatocellular carcinoma.

AIM: We aimed to examine the statistical association between serum expression of miRNA 661 (miR-661) and ATG-4B mRNA and hepatocellular carcinoma (HCC) based on in silico data analysis followed by clinical validation. PATIENTS & METHODS: Quantitative reverse-transcriptase real-time PCR was used to examine the expression of miR-661 and ATG-4B mRNA in the sera of HCC patients versus control. RESULTS: The expression of miR-661 and ATG-4B mRNA was positive in 97.14 and 77.14%, respectively, in HCC patients. The survival analysis showed that ATG-4B mRNA was an independent prognostic factor. CONCLUSION: Our data are the first report of its kind regarding the considerable clinical significance of miR-661 and ATG-4B mRNA in HCC patients.

The molecular mechanisms of action of the endocrine disrupting chemical bisphenol A in the development of cancer.

The endocrine disrupting chemical (EDC) is an exogenous substance or mixture that alters the function of the endocrine system and consequently causes adverse effects in intact organisms. Bisphenol A (BPA), one of the most common endocrine disrupting chemicals is a carbon-based synthetic compound used in the production of water bottles, cans, and teeth suture materials. It is known to be a xenoestrogen as it interacts with estrogen receptors and acts as agonist or antagonist via estrogen receptor-dependent signaling pathways. BPA has been associated with serious health effects in humans and wildlife. It elicits several endocrine disorders and plays a role in the pathogenesis of several hormone-dependent tumors such as breast, ovarian, prostate cancer and others. More complicate to this picture, its effects rely on several and diverse molecular and epigenetic mechanisms that converge upon endocrine and reproductive systems. The present review gives an overview of general hazards of BPA, its epigenetic modifications and the molecular mechanisms of BPA action in different types of cancers as the increase in information about responses and action mechanisms of BPA may bring a better understanding of the risks of BPA exposure in humans and provide an important platform on which human health can be improved.

Clinical evaluation of circulating miR-548a-3p and -20a expression in malignant pleural mesothelioma patients.

AIM: miRNAs may act as promising diagnostic and prognostic biomarkers of mesothelioma. This study integrates serum  miR-548a-3p and miR-20a expression based on in silico data analysis followed by clinical validation in malignant mesothelioma patients (malignant pleural mesothelioma [MPM]). PATIENTS & METHODS: Serum miR-548a-3p and  miR-20a level was assessed in the serum of patients with MPM, chronic asbestos exposure and healthy volunteers by quantitative real-time PCR. RESULTS: The expression of serum miR-548a-3p and  miR-20a was positive in 91.6 and 96.7% MPM patients, respectively. Both miRNAs were able to segregate between cases and controls. The sensitivity of the combined chosen serum miRNAs reached 100% in the diagnosis of MPM. CONCLUSION: The current work revealed that sera miR-548a-3p and miR-20a may serve as promising novel diagnostic tools for MPM.

Stop eating plastic, molecular signaling of bisphenol A in breast cancer.

Breast cancer is the second most common fatal cancer in women. Developing a breast cancer is a multi-factorial and hormonal-dependent process, which may be triggered by many risk factors. An endocrine disrupting substance known as bisphenol A (BPA), that is used greatly in the manufacture of plastic products, was suggested as a possible risk factor for developing breast cancer. BPA has a strong binding affinity to non-classical membrane estrogen receptors like estrogen-related and G protein-coupled (GPER) receptors. Based on animal and in vitro studies, results showed a link between BPA exposure and increased incidence of breast cancer. BPA has the ability to alter multiple molecular pathways in cells namely, G protein-coupled receptor (GPER) pathway, estrogen-related receptor gamma (ERRγ) pathway, HOXB9 (homeobox-containing gene) pathway, bone morphogenetic protein 2 (BMP2) and (BMP4), immunoregulatory cytokine disturbance in the mammary gland, EGFR-STAT3 pathway, FOXA1 in ER-breast cancer cells, enhancer of zeste homolog 2 (EZH2), and epigenetic changes. Thus, the aforementioned alterations cause undesired gene stimulation or repression that increase risk of developing breast cancer. So, restricting exposure to BPA should be considered to aid in lowering the risk of developing breast cancer.

Prenatal Exposure to Endocrine Disruptors and Reprogramming of Adipogenesis: An Early-Life Risk Factor for Childhood Obesity.

Obesity is a global health problem. It is characterized by excess adipose tissue that results from either increase in the number of adipocytes or increase in adipocytes size. Adipocyte differentiation is a highly regulated process that involves the activation of several transcription factors culminating in the removal of adipocytes from the cell cycle and induction of highly specific proteins. Several other factors, including hormones, genes, and epigenetics, are among the most important triggers of the differentiation process. Although the main contributing factors to obesity are high caloric intake, a sedentary lifestyle, and genetic predisposition, strong evidence supports a role for life exposure to environmental pollutants. Endocrine-disrupting chemicals are exogenous, both natural and man-made, chemicals that disrupt the body signaling processes, thus interfering with the endocrine system. Several studies have shown that prenatal exposure to endocrine disruptors modulates the mechanisms, by which multipotent mesenchymal stem cells differentiate into adipocytes. This review discusses adipocytes differentiation and highlights the possible mechanisms of prenatal exposure to endocrine disruptors in reprogramming of adipogenesis and induction of obesity later in life. Therefore, this review provides knowledge that reduction of early life exposure to these chemicals could open the door for new strategies in the prevention of obesity, especially during childhood.

Clinical significance of miRNA-autophagy transcript expression in patients with hepatocellular carcinoma.

AIM: This study integrates autophagy transcripts miRNAs expression based on bioinformatic analysis followed by clinical validation. METHODOLOGY: Cellular jun proto-oncogene mRNA, LAMP2 mRNA, miR-16 and miR-146a level were investigated in the serum and tissue of patients with hepatocellular carcinoma (HCC), chronic hepatitis C and healthy volunteers by quantitative real-time PCR. The prognostic power of this serum RNA panel was explored. RESULTS: The expression of serum cellular jun proto-oncogene mRNA, LAMP2 mRNA, miR-16 and miR-146a were positive in 85.1, 94, 97.1 and 84.2% HCC patients, respectively and they were correlated with tissue levels. Our results suggested that the chosen panel is an independent prognostic factor for survival in patients with HCC. CONCLUSION: The current work provides four RNA-based biomarker panel for HCC diagnosis and prognosis.

A review on the efficacy and toxicity of different doxorubicin nanoparticles for targeted therapy in metastatic breast cancer.

In metastatic breast cancer (MBC), the conventional doxorubicin (DOX) has various problems due to lack of selectivity with subsequent therapeutic failure and adverse effects. DOX- induced cardiotoxicity is a major problem that necessitates the presence of new forms to decrease the risk of associated morbidity. Nanoparticles (NPs) are considered an important approach to selectively increase drug accumulation inside tumor cells and thus decreasing the associated side effects. Tumor cells develop resistance to chemotherapeutic agents through multiple mechanisms, one of which is over expression of efflux transporters. Various NPs have been investigated to overcome efflux mediated resistance. To date, only liposomal doxorubicin (LD) and pegylated liposomal doxorubicin (PLD) have entered phase II and III clinical trials and FDA- approved for clinical use in MBC. This review addresses the effects of LD and PLD on the hematological and palmar-plantar erythrodysesthesia (PPE) in anthracycline naïve and pretreated MBC patients. For evidence, studies to be included in this review were identified through PubMed, Cochrane and Google scholar databases. The results derived from: four phase III clinical trials that compared LD with the conventional DOX in naïve MBC patients, and ten non-comparative clinical trials investigated LD and PLD as monotherapy or combination in pretreated MBC. This work confirmed the cardiac tolerability profile of LD and PLD versus DOX, while hematological and skin toxicities were more common. Other DOX-NPs in preclinical trials were discussed in a chronological order. Finally, the modern preclinical development framework for DOX includes exosomal DOX (exo-DOX). Exosomal NPs are non-toxic, non-immunogenic, and can be engineered to have high cargo loading capacity and targeting specificity. These NPs have not been investigated clinically. Our study shows that the full clinical potentiality of DOX-NPs remains to be addressed to move the field forward.