Mechanical metamaterials as broadband electromagnetic wave absorbers: investigating relationships between geometrical parameters and electromagnetic response.

The utilization of low-density and robust mechanical metamaterials rises as a promising solution for multifunctional electromagnetic wave absorbers due to their structured porous structures, which facilitates impedance matching and structural absorption. However, the various geometrical parameters involved in constructing these metamaterials affect their electromagnetic response, necessitating a comprehensive understanding of underlying absorbing mechanisms. Through experimentally validated numerical analysis, this study delves into the influence of geometrical factors on the electromagnetic response of representative low-density, high strength mechanical metamaterials, namely octet-truss and octet-foam. By juxtaposing electromagnetic response under varying volume fractions, cell lengths, and multilayer configurations of octet-truss and octet-foam, distinct absorption mechanisms emerge as geometrical parameters evolve. These mechanisms encompass diminished reflection owing to porous structures, effective medium approximations within subwavelength limits, and transmission-driven or reflection-driven phenomena originating from the interplay of open- and closed-cell structures. Through analyses on these mechanical metamaterials, we demonstrate the viability of employing them as tunable yet scalable structures that are lightweight, robust, and broadband electromagnetic wave absorption.

Analysis of Polymeric Immunoglobulin Receptor Expression in Olive Flounder (Paralichthys olivaceus) against Viral Hemorrhagic Septicemia Virus.

Polymeric immunoglobulin receptor (pIgR) mediates the transfer of polymeric immunoglobulin to protect organisms and is one of the most important mucosal effectors. In this study, the developmental stage- and tissue-specific expression of pIgR were observed before virus inoculation in olive flounder. pIgR was gradually expressed until the formation of immune tissue, exhibiting high expression in the late juvenile period; thereafter, pIgR expression gradually decreased and exhibited high expression in the spleen and skin. Moreover, pIgR expression after viral hemorrhagic septicemia virus infection was high in the kidney and spleen tissues at high density and low at low density. The results of this study can provide a basis for future studies on breeding density, virus expression, and immune system studies in fish.

Effect of fermentation on antioxidant, antimicrobial, anti-inflammatory, and anti-Helicobacter pylori adhesion activity of Ulmus davidiana var. japonica root bark.

The limited yield of Ulmus davidiana var. japonica root bark (URB) extract is considered an economic loss to the food industry. Improving extraction yield and bioactivity through fermentation increase the industrial usage of URB. The study aims to optimize the fermentation with cellulolytic and pectinolytic bacteria and evaluate the bioactivity and anti-Helicobacter pylori activity of the fermented URB extract. URB fermentation with the Bacillus licheniformis FLa3, isolated from salted seafood (Sardinella zunasi), under optimal conditions (37 °C, pH 6, 10% inoculum dose, and 36 h) improved the extraction yield by 36% compared to the control. The antioxidant and antimicrobial activity of the fermented extract were significantly higher than non-fermented extract. High-performance liquid chromatography results confirmed that the fermentation increased the proportion of bioactive components such as catechin (171.7%), epicatechin (144.3%), quercetin (27.3%), and kaempferol (16.7%). The results confirmed that the fermentation increased both the extraction yield and bioactivity.

A supervised machine learning approach to discriminate the effect of carcass leachate on shallow groundwater quality around on-farm livestock mortality burial sites.

The evaluation of leachate leakage at livestock mortality burial sites is challenging, particularly when groundwater is previously contaminated by agro-livestock farming. Supervised machine learning was applied to discriminate the impacts of carcass leachate from pervasive groundwater contamination in the following order: data labeling, feature selection, synthetic data generation, and classification. Physicochemical data of 359 water samples were collected from burial pits (LC), monitoring wells near pits (MW), pre-existing shallow household wells (HW), and background wells with pervasive contamination (BG). A linear classification model was built using two representative groups (LC and BG) affected by different pollution sources as labeled data. A classifier was then applied to assess the impact of leachate leakage in MW and HW. As a result, leachate impacts were observed in 40% of MW samples, which indicates improper construction and management of some burial pits. Leachate impacts were also detected in six HW samples, up to 120 m downgradient, within one year. The quantitative decision-making tool to diagnose groundwater contamination with leachate leakage can contribute to ensuring timely responses to leakage. The proposed machine learning approach can also be used to improve the environmental impact assessment of water pollution by improper disposal of organic waste.

Identification of an in-frame homozygous KIF1A variant causing a mild SPG30 phenotype in a Korean family.

SPG30 is a newly categorized type of HSP caused by variants in the kinesin family member 1A gene (KIF1A). Advances in next-generation sequencing have resulted in a limited number of studies describing the clinical, electrophysiological, and radiological features of HSP, with variable manifestations. Most known pathogenic KIF1A variants affect the motor domain, although some rare pathogenic variants have been identified that affect the non-motor domain. Here, we report a Korean family with a rare homozygous autosomal-recessive form of SPG30. A 59-year-old man and his father presented with an uncomplicated, mild SPG30 phenotype, characterized by a progressive, spastic gait. Familial co-segregation analysis revealed a pathogenic c.2751_2753delGGA KIF1A variant that affects the non-motor domain. Our case broadens the genetic and clinical variability of SPG30, warranting similar studies to consolidate the pathogenicity of SPG30.

Single Cell Analysis of Human Thyroid Reveals the Transcriptional Signatures of Aging.

The thyroid gland plays a critical role in the maintenance of whole-body metabolism. However, aging frequently impairs homeostatic maintenance by thyroid hormones due to increased prevalence of subclinical hypothyroidism associated with mitochondrial dysfunction, inflammation, and fibrosis. To understand the specific aging-related changes of endocrine function in thyroid epithelial cells, we performed single-cell RNA sequencing (RNA-seq) of 54 726 cells derived from pathologically normal thyroid tissues from 7 patients who underwent thyroidectomy. Thyroid endocrine epithelial cells were clustered into 5 distinct subpopulations, and a subset of cells was found to be particularly vulnerable with aging, showing functional deterioration associated with the expression of metallothionein (MT) and major histocompatibility complex class II genes. We further validated that increased expression of MT family genes are highly correlated with thyroid gland aging in bulk RNAseq datasets. This study provides evidence that aging induces specific transcriptomic changes across multiple cell populations in the human thyroid gland.

An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone.

Tumor progression is intimately associated with the vasculature, as tumor proliferation induces angiogenesis and tumor cells metastasize to distant organs via blood vessels. However, whether tumor invasion is associated with blood vessels remains unknown. As glioblastoma (GBM) is featured by aggressive invasion and vascular abnormalities, we characterized the onset of vascular remodeling in the diffuse tumor infiltrating zone by establishing new spontaneous GBM models with robust invasion capacity. Normal brain vessels underwent a gradual transition to severely impaired tumor vessels at the GBM periphery over several days. Increasing vasodilation from the tumor periphery to the tumor core was also found in human GBM. The levels of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) showed a spatial correlation with the extent of vascular abnormalities spanning the tumor-invading zone. Blockade of VEGFR2 suppressed vascular remodeling at the tumor periphery, confirming the role of VEGF-VEGFR2 signaling in the invasion-associated vascular transition. As angiopoietin-2 (ANGPT2) was expressed in only a portion of the central tumor vessels, we developed a ligand-independent tunica interna endothelial cell kinase 2 (Tie2)-activating antibody that can result in Tie2 phosphorylation in vivo. This agonistic anti-Tie2 antibody effectively normalized the vasculature in both the tumor periphery and tumor center, similar to the effects of VEGFR2 blockade. Mechanistically, this antibody-based Tie2 activation induced VE-PTP-mediated VEGFR2 dephosphorylation in vivo. Thus, our study reveals that the normal-to-tumor vascular transition is spatiotemporally associated with GBM invasion and may be controlled by Tie2 activation via a novel mechanism of action.

Ultra-Low Level Somatic Mutations and Structural Variations in Focal Cortical Dysplasia Type II.

OBJECTIVE: Brain somatic mutations in mTOR pathway genes are a major genetic etiology of focal cortical dysplasia type II (FCDII). Despite a greater ability to detect low-level somatic mutations in the brain by deep sequencing and analytics, about 40% of cases remain genetically unexplained. METHODS: We included 2 independent cohorts consisting of 21 patients with mutation-negative FCDII without apparent mutations on conventional deep sequencing of bulk brain. To find ultra-low level somatic variants or structural variants, we isolated cells exhibiting phosphorylation of the S6 ribosomal protein (p-S6) in frozen brain tissues using fluorescence-activated cell sorting (FACS). We then performed deep whole-genome sequencing (WGS; >90×) in p-S6+ cells in a cohort of 11 patients with mutation-negative. Then, we simplified the method to whole-genome amplification and target gene sequencing of p-S6+ cells in independent cohort of 10 patients with mutation-negative followed by low-read depth WGS (10×). RESULTS: We found that 28.6% (6 of 21) of mutation-negative FCDII carries ultra-low level somatic mutations (less than 0.2% of variant allele frequency [VAF]) in mTOR pathway genes. Our method showed ~34 times increase of the average mutational burden in FACS mediated enrichment of p-S6+ cells (average VAF = 5.84%) than in bulky brain tissues (average VAF = 0.17%). We found that 19% (4 of 21) carried germline structural variations in GATOR1 complex undetectable in whole exome or targeted gene sequencing. CONCLUSIONS: Our method facilitates the detection of ultra-low level somatic mutations, in specifically p-S6+ cells, and germline structural variations and increases the genetic diagnostic rate up to ~80% for the entire FCDII cohort. ANN NEUROL 2023;93:1082-1093.

Clinical Features, Neuropathology, and Surgical Outcome in Patients With Refractory Epilepsy and Brain Somatic Variants in the SLC35A2 Gene.

BACKGROUND AND OBJECTIVES: The SLC35A2 gene, located at chromosome Xp11.23, encodes for a uridine diphosphate-galactose transporter. We describe clinical, genetic, neuroimaging, EEG, and histopathologic findings and assess possible predictors of postoperative seizure and cognitive outcome in 47 patients with refractory epilepsy and brain somatic SLC35A2 gene variants. METHODS: This is a retrospective multicenter study where we performed a descriptive analysis and classical hypothesis testing. We included the variables of interest significantly associated with the outcomes in the generalized linear models. RESULTS: Two main phenotypes were associated with brain somatic SLC35A2 variants: (1) early epileptic encephalopathy (EE, 39 patients) with epileptic spasms as the predominant seizure type and moderate to severe intellectual disability and (2) drug-resistant focal epilepsy (DR-FE, 8 patients) associated with normal/borderline cognitive function and specific neuropsychological deficits. Brain MRI was abnormal in all patients with EE and in 50% of those with DR-FE. Histopathology review identified mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy in 44/47 patients and was inconclusive in 3. The 47 patients harbored 42 distinct mosaic SLC35A2 variants, including 14 (33.3%) missense, 13 (30.9%) frameshift, 10 (23.8%) nonsense, 4 (9.5%) in-frame deletions/duplications, and 1 (2.4%) splicing variant. Variant allele frequencies (VAFs) ranged from 1.4% to 52.6% (mean VAF: 17.3 ± 13.5). At last follow-up (35.5 ± 21.5 months), 30 patients (63.8%) were in Engel Class I, of which 26 (55.3%) were in Class IA. Cognitive performances remained unchanged in most patients after surgery. Regression analyses showed that the probability of achieving both Engel Class IA and Class I outcomes, adjusted by age at seizure onset, was lower when the duration of epilepsy increased and higher when postoperative EEG was normal or improved. Lower brain VAF was associated with improved postoperative cognitive outcome in the analysis of associations, but this finding was not confirmed in regression analyses. DISCUSSION: Brain somatic SLC35A2 gene variants are associated with 2 main clinical phenotypes, EE and DR-FE, and a histopathologic diagnosis of MOGHE. Additional studies will be needed to delineate any possible correlation between specific genetic variants, mutational load in the epileptogenic tissue, and surgical outcomes.

Estradiol-17 ß levels as a tool for sex determination in Farmed Anguilla japonica.

In this study, the levels of plasma estradiol-17ß (E2) in farmed Anguilla japonica were measured to determine their sex. The analyses were performed for two different size groups (large group, Total length (TL): 61-69 cm; small group, TL: 53-60 cm). The anatomical and histological observations showed that the large group consisted of 29% males and 71% females; the small group, 54% males and 45% females. The gonad histology showed that in the large group, 88% of the eels had immature gonads with ongoing sexual differentiation, 12% were mature with completed sexual differentiation. In the small group, 87% of the eels had immature gonads. The plasma E2 hormone levels were higher in the females of both sizes. In the large group, the average plasma E2 in females was 415 pg/ml, which was significantly higher than the average of 109 pg/ml in males (P < 0.05). In the small group, the average plasma E2 hormone level was 618 pg/ml, which was much higher than the average of 108 pg/ml in males. Quantitative real-time PCR showed that zygote arrest 1 (zar 1) and zona pellucida glycoprotein 3 (zp3) were more highly expressed in females than male. In the H-E staining, an eel in the oil droplet containing ovary stage had a high level of plasma E2 (1500 pg/ml), while an eel with testis in the spermatocyte stage had a low (60 pg/ml). E2 is a potentially useful tool and could play an important role in sex determination in broodstocks.

Identification of a pleiotropic effect of ADIPOQ on cardiac dysfunction and Alzheimer's disease based on genetic evidence and health care records.

Observations of comorbidity in heart diseases, including cardiac dysfunction (CD) are increasing, including and cognitive impairment, such as Alzheimer's disease and dementia (AD/D). This comorbidity might be due to a pleiotropic effect of genetic variants shared between CD and AD/D. Here, we validated comorbidity of CD and AD/D based on diagnostic records from millions of patients in Korea and the University of California, San Francisco Medical Center (odds ratio 11.5 [8.5-15.5, 95% Confidence Interval (CI)]). By integrating a comprehensive human disease-SNP association database (VARIMED, VARiants Informing MEDicine) and whole-exome sequencing of 50 brains from individuals with and without Alzheimer's disease (AD), we identified missense variants in coding regions including APOB, a known risk factor for CD and AD/D, which potentially have a pleiotropic role in both diseases. Of the identified variants, site-directed mutation of ADIPOQ (268 G > A; Gly90Ser) in neurons produced abnormal aggregation of tau proteins (p = 0.02), suggesting a functional impact for AD/D. The association of CD and ADIPOQ variants was confirmed based on domain deletion in cardiac cells. Using the UK Biobank including data from over 500000 individuals, we examined a pleiotropic effect of the ADIPOQ variant by comparing CD- and AD/D-associated phenotypic evidence, including cardiac hypertrophy and cognitive degeneration. These results indicate that convergence of health care records and genetic evidences may help to dissect the molecular underpinnings of heart disease and associated cognitive impairment, and could potentially serve a prognostic function. Validation of disease-disease associations through health care records and genomic evidence can determine whether health conditions share risk factors based on pleiotropy.

Adrenomedullin2 stimulates progression of thyroid cancer in mice and humans under nutrient excess conditions.

Thyroid cancer is associated with genetic alterations, e.g. BRAFV600E , which may cause carcinomatous changes in hormone-secreting epithelial cells. Epidemiological studies have shown that overnutrition is related to the development and progression of cancer. In this study, we attempted to identify the cell nonautonomous factor responsible for the progression of BRAFV600E thyroid cancer under overnutrition conditions. We developed a mouse model for inducible thyrocyte-specific activation of BRAFV600E , which showed features similar to those of human papillary thyroid cancer. LSL-BrafV600E ;TgCreERT2 showed thyroid tumour development in the entire thyroid, and the tumour showed more abnormal cellular features with mitochondrial abnormalities in mice fed a high-fat diet (HFD). Transcriptomics revealed that adrenomedullin2 (Adm2) was increased in LSL-BrafV600E ;TgCreERT2 mice fed HFD. ADM2 was upregulated on the addition of a mitochondrial complex I inhibitor or palmitic acid with integrated stress response (ISR) in cancer cells. ADM2 stimulated protein kinase A and extracellular signal-regulated kinase in vitro. The knockdown of ADM2 suppressed the proliferation and migration of thyroid cancer cells. We searched The Cancer Genome Atlas and Genotype-Tissue Expression databases and found that increased ADM2 expression was associated with ISR and poor overall survival. Consistently, upregulated ADM2 expression in tumour cells and circulating ADM2 molecules were associated with aggressive clinicopathological parameters, including body mass index, in thyroid cancer patients. Collectively, we identified that ADM2 is released from cancer cells under mitochondrial stress resulting from overnutrition and acts as a secretory factor determining the progressive properties of thyroid cancer. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Analysis of low-level somatic mosaicism reveals stage and tissue-specific mutational features in human development.

Most somatic mutations that arise during normal development are present at low levels in single or multiple tissues depending on the developmental stage and affected organs. However, the effect of human developmental stages or mutations of different organs on the features of somatic mutations is still unclear. Here, we performed a systemic and comprehensive analysis of low-level somatic mutations using deep whole-exome sequencing (average read depth ~500×) of 498 multiple organ tissues with matched controls from 190 individuals. Our results showed that early clone-forming mutations shared between multiple organs were lower in number but showed higher allele frequencies than late clone-forming mutations [0.54 vs. 5.83 variants per individual; 6.17% vs. 1.5% variant allele frequency (VAF)] along with less nonsynonymous mutations and lower functional impacts. Additionally, early and late clone-forming mutations had unique mutational signatures that were distinct from mutations that originated from tumors. Compared with early clone-forming mutations that showed a clock-like signature across all organs or tissues studied, late clone-forming mutations showed organ, tissue, and cell-type specificity in the mutation counts, VAFs, and mutational signatures. In particular, analysis of brain somatic mutations showed a bimodal occurrence and temporal-lobe-specific signature. These findings provide new insights into the features of somatic mosaicism that are dependent on developmental stage and brain regions.

Protocol to analyze antitumor immunity of orthotopic injection and spontaneous murine high-grade glioma models using flow cytometry and single-cell RNA sequencing.

Despite the recognized importance of antitumor immunity, our understanding of brain tumor immunity is poor. Orthotopic injection models have been widely used for immunological analyses. However, these models have limitations in analysis of antitumor immunity because the approach involves drilling skulls and injecting tumor cells, which can induce adverse effects. We describe a protocol for the induction of spontaneous brain tumor model, isolation of single cells from brain tumor microenvironment, and analysis of the immune responses using scRNA-seq and flow cytometry. For complete details on the use and execution of this protocol, please refer to Park et al. (2021).

Characterization of TRIM16, a member of the fish-specific finTRIM family, in olive flounder Paralichthys olivaceus.

Tripartite motif-containing (TRIM) proteins are conserved throughout the metazoan kingdom, and the TRIM subset finTRIM is highly diversified in fish. We isolated TRIM16 cDNA, a member of the finTRIM family, from the olive flounder Paralichthys olivaceus (PoTRIM16). PoTRIM16 contained a 1,725-bp coding sequence encoding a 574-amino acid polypeptide, which in turn contained a really interesting new gene (RING) finger domain, B-box-type zinc finger (B-BOX), nuclease SbcCD subunit C (SbcC), structural maintenance of chromosome (SMC prok B), and stonustoxin (SNTX) subunit alpha (SPRY-PRY-SNTX). Multiple alignment of related sequences revealed that PoTRIM16 showed 86.63-97.40% identity with fish orthologues, and a phylogenetic tree was constructed of vertebrates. PoTRIM16 mRNA was detected in all tissues examined; levels were highest in the eye and ovary. PoTRIM16 mRNA expression was investigated during early development. Under VHSV infection, PoTRIM16 mRNA was downregulated in the liver of P. olivaceus. This is the first study to characterize fish-specific finTRIM in P. olivaceus, which may play a role in the immune response against virus infection.

Non-coding de novo mutations in chromatin interactions are implicated in autism spectrum disorder.

Three-dimensional chromatin interactions regulate gene expressions. The significance of de novo mutations (DNMs) in chromatin interactions remains poorly understood for autism spectrum disorder (ASD). We generated 813 whole-genome sequences from 242 Korean simplex families to detect DNMs, and identified target genes which were putatively affected by non-coding DNMs in chromatin interactions. Non-coding DNMs in chromatin interactions were significantly involved in transcriptional dysregulations related to ASD risk. Correspondingly, target genes showed spatiotemporal expressions relevant to ASD in developing brains and enrichment in biological pathways implicated in ASD, such as histone modification. Regarding clinical features of ASD, non-coding DNMs in chromatin interactions particularly contributed to low intelligence quotient levels in ASD probands. We further validated our findings using two replication cohorts, Simons Simplex Collection (SSC) and MSSNG, and showed the consistent enrichment of non-coding DNM-disrupted chromatin interactions in ASD probands. Generating human induced pluripotent stem cells in two ASD families, we were able to demonstrate that non-coding DNMs in chromatin interactions alter the expression of target genes at the stage of early neural development. Taken together, our findings indicate that non-coding DNMs in ASD probands lead to early neurodevelopmental disruption implicated in ASD risk via chromatin interactions.

Anti-inflammatory effects of the Aralia elata and Cirsium japonicum in Raw264.7 cells and in vivo colitis model in mice and dogs.

Ulcerative colitis (UC) is a severe inflammatory disease that has spread throughout the world. Cirsium japonicum (CJ) and Aralia elata (AE) are natural herbs with potent antioxidative antidiabetics and anti-inflammatory effects. In this investigation, we studied the defensive role of the combination of CJ and AE against LPS-induced inflammation in RAW 264.7 cells, dextran sulfate sodium (DSS)-induced colitis in mice, and acetic acid-induced colitis in dogs. MTT assay was performed to identify the toxic effect of CJ and AE extracts. NO, and MDA level was also measured by NO and MDA assay. To measure the pro-inflammatory protein expression, a western blot was performed. To induce colitis, 3% DSS was used for mice and 6% acetic acid was used for dogs. Histopathology and colonoscopy were executed to detect the effect of extracts. CJ and AE pretreatment reduced the level of NO, MDA, and the expression of pro-inflammatory proteins cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) in RAW 264.7. Compared to the separate doses of CJ and AE, the combined dose of CJ and AE significantly reduced clinical symptoms induced by DSS in mice and acetic acid in dogs including weight loss, bloody stool, shortening of the colon, and the severity of colitis and degree of histological damage in the colon. Therefore, these results indicated that a combined dose of CJ and AE has a protective effect against LPS-induced RAW 264.7 cells, DSS-mediated colonic inflammation in mice, and acetic acid-induced colitis in dogs.

The ILAE consensus classification of focal cortical dysplasia: An update proposed by an ad hoc task force of the ILAE diagnostic methods commission.

Ongoing challenges in diagnosing focal cortical dysplasia (FCD) mandate continuous research and consensus agreement to improve disease definition and classification. An International League Against Epilepsy (ILAE) Task Force (TF) reviewed the FCD classification of 2011 to identify existing gaps and provide a timely update. The following methodology was applied to achieve this goal: a survey of published literature indexed with ((Focal Cortical Dysplasia) AND (epilepsy)) between 01/01/2012 and 06/30/2021 (n = 1349) in PubMed identified the knowledge gained since 2012 and new developments in the field. An online survey consulted the ILAE community about the current use of the FCD classification scheme with 367 people answering. The TF performed an iterative clinico-pathological and genetic agreement study to objectively measure the diagnostic gap in blood/brain samples from 22 patients suspicious for FCD and submitted to epilepsy surgery. The literature confirmed new molecular-genetic characterizations involving the mechanistic Target Of Rapamycin (mTOR) pathway in FCD type II (FCDII), and SLC35A2 in mild malformations of cortical development (mMCDs) with oligodendroglial hyperplasia (MOGHE). The electro-clinical-imaging phenotypes and surgical outcomes were better defined and validated for FCDII. Little new information was acquired on clinical, histopathological, or genetic characteristics of FCD type I (FCDI) and FCD type III (FCDIII). The survey identified mMCDs, FCDI, and genetic characterization as fields for improvement in an updated classification. Our iterative clinico-pathological and genetic agreement study confirmed the importance of immunohistochemical staining, neuroimaging, and genetic tests to improve the diagnostic yield. The TF proposes to include mMCDs, MOGHE, and "no definite FCD on histopathology" as new categories in the updated FCD classification. The histopathological classification can be further augmented by advanced neuroimaging and genetic studies to comprehensively diagnose FCD subtypes; these different levels should then be integrated into a multi-layered diagnostic scheme. This update may help to foster multidisciplinary efforts toward a better understanding of FCD and the development of novel targeted treatment options.