Implementation rate and related factors of confirmatory tests following an abnormal Pap smear: a nationwide study from the National Health Insurance.

OBJECTIVE: This study aims to investigate the implementation rate and influencing factors of confirmatory tests for women with abnormal cervical cytology results in the Korean nationwide cervical cancer screening program. METHODS: The National Health Insurance Service (NHIS) database was utilized to identify all Korean women who have participated in the Korean nationwide cervical cancer screening program from January 2011 and December 2021 using the NHIS database. Multiple logistic regression analysis was performed to estimate the multivariate odds ratio and evaluate the patients' characteristics. RESULTS: The rate of abnormal Papanicolaou (Pap) smears showed an initial increase from 2011 to 2015 and subsequently reached a plateau after 2016. When examining specific subcategories, cases of atypical squamous cells of undetermined significance (ASC-US) increased from 28,546 cases (1.1%) in 2011 to 62,850 cases (1.7%) in 2021. In contrast, cases of HSIL and SCC declined from 3,535 cases (0.14%) to 2,763 cases (0.07%) and from 383 cases (0.01%) to 179 cases (0.005%), respectively. Furthermore, the implementation rate of confirmatory tests for women with abnormal cytology increased from 8,865 cases (21.0%) in 2011 to 39,045 cases (51.2%) in 2021. Regarding the specific subcategory of ASC-US, the number of confirmatory tests exhibited a substantial increase from 4,101 cases (14.4%) in 2011 to 30,482 cases (48.5%) in 2021. For SCC, there was no significant change, with 216 cases (56.4%) in 2011 and 102 cases (57.0%) in 2021. The implementation rate of confirmatory tests was found to be significantly associated with results of abnormal Pap smear, age, and residence. Notably, economic status did not emerge as a significant factor affecting the likelihood of undergoing confirmatory tests. CONCLUSIONS: The severity of abnormal Pap smear results is a reliable indicator of the probability of undergoing a confirmatory test. Additional endeavors are required to improve the implementation rate among women who have received abnormal Pap smear results.

Nutlin-3a induces KRAS mutant/p53 wild type lung cancer specific methuosis-like cell death that is dependent on GFPT2.

BACKGROUND: Oncogenic KRAS mutation, the most frequent mutation in non-small cell lung cancer (NSCLC), is an aggressiveness risk factor and leads to the metabolic reprogramming of cancer cells by promoting glucose, glutamine, and fatty acid absorption and glycolysis. Lately, sotorasib was approved by the FDA as a first-in-class KRAS-G12C inhibitor. However, sotorasib still has a derivative barrier, which is not effective for other KRAS mutation types, except for G12C. Additionally, resistance to sotorasib is likely to develop, demanding the need for alternative therapeutic strategies. METHODS: KRAS mutant, and wildtype NSCLC cells were used in vitro cell analyses. Cell viability, proliferation, and death were measured by MTT, cell counting, colony analyses, and annexin V staining for FACS. Cell tracker dyes were used to investigate cell morphology, which was examined by holotomograpy, and confocal microscopes. RNA sequencing was performed to identify key target molecule or pathway, which was confirmed by qRT-PCR, western blotting, and metabolite analyses by UHPLC-MS/MS. Zebrafish and mouse xenograft model were used for in vivo analysis. RESULTS: In this study, we found that nutlin-3a, an MDM2 antagonist, inhibited the KRAS-PI3K/Akt-mTOR pathway and disrupted the fusion of both autophagosomes and macropinosomes with lysosomes. This further elucidated non-apoptotic and catastrophic macropinocytosis associated methuosis-like cell death, which was found to be dependent on GFPT2 of the hexosamine biosynthetic pathway, specifically in KRAS mutant /p53 wild type NSCLC cells. CONCLUSION: These results indicate the potential of nutlin-3a as an alternative agent for treating KRAS mutant/p53 wild type NSCLC cells.

Identification of exosomal microRNA panel as diagnostic and prognostic biomarker for small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) has an exceptionally poor prognosis; as most of the cases are initially diagnosed as extensive disease with hematogenous metastasis. Therefore, the early diagnosis of SCLC is very important and may improve its prognosis. METHODS: To investigate the feasibility of early diagnosis of SCLC, we examined exosomal microRNAs (miRNAs) present in serum obtained from patients with SCLC. First, exosomes were isolated in serum from patients with SCLC and healthy individuals and were characterized using particle size and protein markers. Additionally, miRNA array was performed to define SCLC-specific exosomal miRNAs. Second, the obtained miRNAs were further validated employing a large cohort. Finally, the ability to diagnose SCLC was estimated by area under the curve (AUC), and intracellular mRNA change patterns were verified through validated miRNAs. RESULTS: From the miRNA array results, we selected 51-miRNAs based on p-values and top 10 differentially expressed genes, and 25-miRNAs were validated using quantitative reverse transcription-polymerase chain reaction. The 25-miRNAs were further validated employing a large cohort. Among them, 7-miRNAs showed significant differences. Furthermore, 6-miRNAs (miR-3565, miR-3124-5p, miR-200b-3p, miR-6515, miR-3126-3p and miR-9-5p) were up-regulated and 1-miRNA (miR-92b-5p) was down-regulated. The AUC value of each miRNA sets between 0.64 and 0.76, however the combined application of 3-miRNAs (miR-200b-3p, miR-3124-5p and miR-92b-5p) remarkably improved the diagnostic value (AUC = 0.93). Gene ontology analysis revealed that the 3-miRNA panel is linked to various oncogene pathways and nervous system development. When the 3-miRNAs were introduced to cells, the resulting changes in total mRNA expression strongly indicated the presence of lung diseases, including lung cancer. In addition, the 3-miRNA panel was significantly associated with a poorer prognosis, although individual miRNAs have not been validated as prognostic markers. CONCLUSION: Our study identified SCLC-specific exosomal miRNAs, and the 3-miRNAs panel (miR-200b-3p, miR-3124-5p and miR-92b-5p) may serve as a diagnostic and prognostic marker for SCLC.

AXL receptor tyrosine kinase inhibition improves the anti-tumor effects of CD8+ T cells by inducing CD103+ dendritic cell-mediated T cell priming.

AXL is a member of TAM receptor family and has been highlighted as a potential target for cancer treatment. Accumulating evidence has uncovered the critical role of the AXL signaling pathway in tumor growth, metastasis, and resistance against anti-cancer drugs, as well as its association with cancer immune escape. However, the function of AXL as a manipulator of the immune system in the tumor microenvironment (TME) remains unclear. Therefore, in this study, we investigated the impact of AXL on immune cells in the TME of a syngeneic tumor model using AXL knockout (AXL-/-) mice. Compared to AXL wild-type (AXL+/+) mice, tumor growth was significantly suppressed in AXL-/- mice, and an induced population of tumor-infiltrated CD8+ T cells and CD103+ dendritic cells (DCs) was observed. The change of CD8+ T cells and CD103+ DCs was also confirmed in tumor-draining lymph nodes (TdLN). In addition, the clonal expansion of OVA-specific CD8+ T cells was dominant in AXL-/- mice. Finally, anti-PD-1 treatment evidenced synergistic anti-cancer effects in AXL-/- mice. Overall, our data indicate that AXL signaling may inhibit the clonal expansion of tumor-specific CD8+ T cells through the regulation of the migration of CD8+ T cells and DCs in TME. Thus, AXL may be a powerful molecular target to improve anti-cancer effects through single or combined therapy with immune checkpoint inhibitors (ICI).

Prolapsed Uterine Smooth Muscle Tumor of Uncertain Malignant Potential: A Case Report and Review of Radiologic Findings.

INTRODUCTION: Uterine smooth muscle tumor of uncertain malignant potential (STUMP) is a rare tumor that arises in the myometrium of the uterus. It is regarded as an intermediate malignant tumor according to the recent World Health Organization classification. Few studies have reported the radiologic findings of STUMP, and the differentiation of STUMP from leiomyoma remains controversial. CASE DESCRIPTION: A 42-year-old nulliparous female presented at our institution with massive vaginal bleeding. Radiological studies, including ultrasonography, computed tomography (CT), and magnetic resonance imaging, revealed an oval-shaped mass with well-defined margins in the uterus protruding into the vagina. The patient underwent a total abdominal hysterectomy, and the final pathology was confirmed as STUMP. CONCLUSION: Distinguishing STUMP from leiomyomas based solely on radiological findings can be challenging. However, if the uterine mass appears as a single mass lacking acoustic shadowing on ultrasound and demonstrates diffusion restriction with high T2 signal intensity on magnetic resonance imaging, consideration of STUMP may be necessary for proper patient management, given the poor prognosis associated with this tumor.

Huge Primary Clear Cell Hepatocellular Carcinoma: a Case Report and Review of Radiologic Findings.

INTRODUCTION: Clear cell hepatocellular carcinoma (HCC) is a rare subtype of HCC. Histologically, clear cell HCC is characterized by the cytoplasmic accumulation of glycogen with a clear cell appearance, constituting > 80% of tumor cells. Radiologically, clear cell HCC demonstrates early enhancement and washout similar to conventional HCC. Occasionally, enhancing capsule and intratumoral fat are accompanied by clear cell HCC. CASE DESCRIPTION: A 57-year-old male presented to our hospital with right upper quadrant abdominal pain. Ultrasonography, computed tomography, and magnetic resonance imaging revealed a large mass with a well-defined margin in the right hemiliver. The patient underwent a right hemihepatectomy, and the final histopathology revealed clear cell-type HCC. CONCLUSION: Distinguishing clear cell types from other types of HCC solely based on radiological findings is challenging. If hepatic tumors exhibit encapsulated margins, enhancing rims, intratumoral fat, and arterial phase hyperenhancement/washout pattern despite their large size, consideration of clear cell subtypes in the differential diagnosis list will aid patient management, implying better prognosis than not-otherwise-specified HCC.

GPX4 overexpressed non-small cell lung cancer cells are sensitive to RSL3-induced ferroptosis.

Ferroptosis can be induced by inhibiting antioxidant enzymes GPX4 or system Xc-, increased intracellular iron concentrations, and lipid peroxidation. Recently, it has been suggested that ferroptosis can be an effective way to induce cancer cell death, although the specific relevance and mechanism of ferroptosis have not been fully elucidated. Here, we investigated the anticancer effects of ferroptosis inducers erastin and RSL3 on non-small cell lung cancer (NSCLC) cells. RSL3 induced cell death more effectively in NSCLC cells than erastin, with limited cytotoxicity in BEAS-2B normal bronchial epithelial cells. The sensitivity of NSCLC cells to RSL3 induced death was dependent on GPX4 expression levels; the effect of RSL3 was reversed by ferrostatin-1 (a ferroptosis inhibitor) but not by Z-VAD-FMK, chloroquine, bafilomycin A1, or necrostatin-1. RSL3 induced ferroptosis by promoting lipid peroxidation, elevating intracellular LIP concentration and ROS level, and blocking GSH-to-GSSH conversion through the inhibition of GPX4 and induction of Nrf2/HO1. Furthermore, RSL3 induced autophagosomes but disrupted the formation of autolysosomes with lysosomal membrane destabilization. GPX4 knockdown had a similar effect on ferroptosis phenotypes as RSL3. Taken together, RSL3-induced ferroptosis depends on the regulation of GPX4-Nrf2/HO1 in NSCLC cells. These results may be useful in predicting the ferroptosis response in NSCLC as well as drug resistant cancer cells.

Integrative modeling of tumor genomes and epigenomes for enhanced cancer diagnosis by cell-free DNA.

Multi-cancer early detection remains a key challenge in cell-free DNA (cfDNA)-based liquid biopsy. Here, we perform cfDNA whole-genome sequencing to generate two test datasets covering 2125 patient samples of 9 cancer types and 1241 normal control samples, and also a reference dataset for background variant filtering based on 20,529 low-depth healthy samples. An external cfDNA dataset consisting of 208 cancer and 214 normal control samples is used for additional evaluation. Accuracy for cancer detection and tissue-of-origin localization is achieved using our algorithm, which incorporates cancer type-specific profiles of mutation distribution and chromatin organization in tumor tissues as model references. Our integrative model detects early-stage cancers, including those of pancreatic origin, with high sensitivity that is comparable to that of late-stage detection. Model interpretation reveals the contribution of cancer type-specific genomic and epigenomic features. Our methodologies may lay the groundwork for accurate cfDNA-based cancer diagnosis, especially at early stages.

A Combination of Bio-Orthogonal Supramolecular Clicking and Proximity Chemical Tagging as a Supramolecular Tool for Discovery of Putative Proteins Associated with Laminopathic Disease.

Protein mutations alter protein-protein interactions that can lead to a number of illnesses. Mutations in lamin A (LMNA) have been reported to cause laminopathies. However, the proteins associated with the LMNA mutation have mostly remained unexplored. Herein, a new chemical tool for proximal proteomics is reported, developed by a combination of proximity chemical tagging and a bio-orthogonal supramolecular latching based on cucurbit[7]uril (CB[7])-based host-guest interactions. As this host-guest interaction acts as a noncovalent clickable motif that can be unclicked on-demand, this new chemical tool is exploited for reliable detection of the proximal proteins of LMNA and its mutant that causes laminopathic dilated cardiomyopathy (DCM). Most importantly, a comparison study reveals, for the first time, mutant-dependent alteration in LMNA proteomic environments, which allows to identify putative laminopathic DCM-linked proteins including FOXJ3 and CELF2. This study demonstrates the feasibility of this chemical tool for reliable proximal proteomics, and its immense potential as a new research platform for discovering biomarkers associated with protein mutation-linked diseases.

Osteoarthritis improvement effect of Chrysanthemum zawadskii var. latilobum extract in relation to genotype.

Objectives: To determine whether SNPs of osteoarthritis (OA)-related genes predict the effect of Chrysanthemum zawadskii var. latilobum (CZ) extract in OA patients with OA. Subjects/methods: To analyze correlations between CZ extract effects in humans and their genotypes, 121 Korean patients with OA were recruited. Patients ingested 600 mg/day of the CZ extract GCWB106 (one tablet daily), including 250-mg CZ, or placebo (one tablet daily) for 12 weeks. Twenty SNPs were genotyped in 11 genes associated with OA pathogenesis, including tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinases (MMPs), and 9 genes involved in OA-related dietary intervention. The Visual Analogue Scale (VAS) and Korean Western Ontario and McMaster Universities (K-WOMAC) were measured as indicators of GCWB106 effect. Statistical comparisons were performed using Kruskal-Wallis tests to identify associations between these scales and genotyped loci in patients with OA. Results: Three SNPs (PPARG rs3856806, MMP13 rs2252070, and ZIP2 rs2234632) were significantly associated with the degree of change in VAS pain score. Homozygous CC genotype carriers of rs3856806, G allele carriers (GA or GG) of rs2252070, and T allele carriers (GT or TT) of rs2234632 showed lower VAS score (i.e., less severe symptoms) in the GCWB106 group (n=53) than the placebo group (n=57) (p=0.026, p=0.009, and p=0.025, respectively). Gene-gene interaction effects on GCWB106-mediated pain relief were then examined, and it was found that the addition of each genotype resulted in a greater decrease in VAS pain score in the GCWB106 group (p=0.0024) but not the placebo group (p=0.7734). Conclusions: These novel predictive markers for the pain-relieving effects of GCWB106 may be used in the personalized treatment of patients with OA.

Effect of the respiratory motion of pulmonary nodules on CT-guided percutaneous transthoracic needle biopsy.

BACKGROUND: Computed tomography (CT)-guided percutaneous transthoracic needle biopsy (PTNB) is highly affected by respiratory motion; however, respiratory motion of target nodule during the PTNB and its effect on CT-guided lung biopsy have not been studied. PURPOSE: To investigate the effect of the respiratory motion of pulmonary nodules on CT-guided PTNB. MATERIAL AND METHODS: We retrospectively reviewed the procedural CT scans of 426 pulmonary nodules that underwent PTNB during quiet breathing. Maximal and average respiratory motions were measured using the difference of table position of the targeted nodule between multiple procedural scans. Diagnostic performance, complications, and technical factors of PTNB in nodules with large motion (maximal motion >1 cm) were compared with those in nodules with small motion (≤1 cm). RESULTS: The mean maximal and average respiratory motions between tidal volume breathing were 5.4 ± 4.4 and 2.7 ± 2.6 mm, respectively. Sensitivity and accuracy were 93.1% and 96.1% in nodules with large motion, compared with 94.7% and 95.9% in nodules with small motion, respectively. Respiratory targeting (P < 0.001), needle modulation (P < 0.001), motion artifact of target (P < 0.001), target disappearance from scans (P < 0.001), and number of performed CT scans (P < 0.001) were significantly higher in the large motion group, with no significant difference in radiation dose and complications between the groups. CONCLUSION: The respiratory motion of pulmonary nodules during CT-guided PTNB may cause technical difficulties but does not affect diagnostic performance nor complications associated with PTNB.

Generation of genetically engineered mice for lung cancer with mutant EGFR.

Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown dramatic response and improvement in treating lung cancer with mutant EGFR, the emergence of drug resistance remains a major problem. In particular, some mutations including T790 M and C797S have been recognized as mechanisms of acquired resistance because they weaken binding affinity to drugs. To date, many attempts have been made to develop a new drug for overcoming acquired resistance to EGFR-TKIs, including secondary mutations. However, an appropriate animal model to evaluate in vivo efficacy during novel drug development remains lacking. In this study, we generated a novel transgenic mouse model that conditionally expresses human EGFRL858R/T790M/C797S and firefly luciferase using Cas9-mediated homology-independent targeted integration. Using a lung-specific Sftpc-CreERT2 mouse line, we induced expression of both the human EGFRL858R/T790M/C797S transgene and firefly luciferase in the lungs of adult mice. The expression of these genes and lung cancer occurrence was monitored using an in vivo imaging system and magnetic resonance imaging, respectively. Overall, our mouse model can be utilized to develop new drugs for overcoming C797S-mediated resistance to osimertinib; further, such knock-in systems for expressing oncogenes may be applied to study tumorigenesis and the development of other targeted agents.

Microbiota manipulation to increase macrophage IL-10 improves colitis and limits colitis-associated colorectal cancer.

Inflammatory bowel disease (IBD) is a chronic life-long inflammatory disease affecting almost 2 million Americans. Although new biologic therapies have been developed, the standard medical treatment fails to selectively control the dysregulated immune pathways involved in chronic colonic inflammation. Further, IBD patients with uncontrolled colonic inflammation are at a higher risk for developing colorectal cancer (CRC). Intestinal microbes can impact many immune functions, and here we asked if they could be used to improve intestinal inflammation. By utilizing an intestinal adherent E. coli that we find increases IL-10 producing macrophages, we were able to limit intestinal inflammation and restrict tumor formation. Macrophage IL-10 along with IL-10 signaling to the intestinal epithelium were required for protection in both inflammation and tumor development. Our work highlights that administration of immune modulating microbes can improve intestinal outcomes by altering tissue inflammation.

The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis.

Ferroptosis is a unique form of cell death caused by excessive iron-dependent lipid peroxidation. The level of the anabolic reductant NADPH is a biomarker of ferroptosis sensitivity. However, specific regulators that detect cellular NADPH levels, thereby modulating downstream ferroptosis cascades, are largely unknown. We show here that the transmembrane endoplasmic reticulum MARCHF6 E3 ubiquitin ligase recognizes NADPH through its C-terminal regulatory region. This interaction upregulates the E3 ligase activity of MARCHF6, thus downregulating ferroptosis. We also found that MARCHF6 mediates the degradation of the key ferroptosis effectors ACSL4 and p53. Furthermore, inhibiting ferroptosis rescued the growth of MARCHF6-deficient tumours and peri-natal lethality of Marchf6-/- mice. Together, these findings identify MARCHF6 as a previously unknown NADPH sensor in the ubiquitin system and a crucial regulator of ferroptosis.

Enhanced half-life and antitumor activity of interleukin-15 through genetic fusion of a serum albumin-specific protein binder.

Human interleukin-15 (hIL-15) has attracted a considerable attention as a promising cancer immunotherapeutic due to its function to directly stimulate the proliferation and cytotoxic activity of NK and T cells. Nevertheless, a relatively short half-life of hIL-15 requires repeated administration and higher doses, causing serious side effects. Here, we demonstrate an enhanced blood half-life and biological activity of hIL-15 through genetic fusion of a human serum albumin-specific protein binder (rHSA). The fusion construct (rHSA-IL15) was observed to maintain respective binding activities for both hIL-15 receptor α and human serum albumin. The rHSA-IL15 led to a significant increase in the secretion of Granzyme B and INF-γ by immune cells compare to free hIL-15, expanding the population of activated T cell subset such as CD4 + T and CD8+ T cells. The terminal half-life of the rHSA-IL15 was prolonged by around a 40-fold in transgenic mice expressing human serum albumin, compared to free hIL-15. The rHSA-IL15 resulted in distinct anti-tumor activities in xenograft SCC (squamous cell carcinoma) mouse and allograft melanoma mouse models through activation of NK and CD8+ T cells. The rHSA-IL15 is expected to be used in cancer immunotherapy, assisting in the development of other cytokines as immunotherapeutic agents with greater efficacy.

The reversible fourth-generation EGFR tyrosine kinase inhibitor OBX02-011 overcomes C797S-mediated resistance in lung cancer.

Osimertinib is an irreversible third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that was initially developed to overcome the EGFR T790M mutation and is used as a standard therapy in patients with advanced non-small cell lung cancer (NSCLC) with EGFR-activating mutations. Despite the remarkable initial efficacy, osimertinib, like other EGFR-TKIs, is limited by the emergence of acquired resistance. As the EGFR mutation C797S has been identified as a key driver of acquired resistance to osimertinib, development of a drug that targets this clinically relevant mutation could help improve patient outcomes. Here, we report the discovery and preclinical efficacy of OBX02-011, a reversible fourth-generation EGFR TKI that overcomes the EGFR C797S mutation. Compared to approved EGFR TKIs, OBX02-011 showed potent anticancer effects and inhibited EGFR-related signaling in various models, including those harboring the EGFR C797S mutation. Additionally, in transgenic mouse models (EGFRL858R/T790M/C797S), OBX02-011 treatment effectively inhibited tumor growth and EGFR activity, leading to enhanced survival. Collectively, these results suggest that OBX02-011 may be a promising new EGFR TKI to overcome C797S-mediated resistance in NSCLC.

Male breast cancer presenting in accessory axillary breast.

We present a rare case of male axillary accessory breast cancer, which is extremely rare and is indistinguishable from lymphadenopathy and other malignancies, such as lymphoma and skin-derived tumors. Clinicians should consider accessory breast cancer in the differential diagnosis even in men, particularly in those who present with superficially located tumors with adjacent accessory breast tissue.

Olfactory Stimulation with Volatile Aroma Compounds of Basil (Ocimum basilicum L.) Essential Oil and Linalool Ameliorates White Fat Accumulation and Dyslipidemia in Chronically Stressed Rats.

We explored the physiological effects of inhaling basil essential oil (BEO) and/or linalool and identified odor-active aroma compounds in BEO using gas chromatography/mass spectrometry (GC-MS) and GC-olfactometry (GC-O). Linalool was identified as the major volatile compound in BEO. Three groups of rats were administered BEO and linalool via inhalation, while rats in the control group were not. Inhalation of BEO for 20 min only reduced the total weight gain (190.67 ± 2.52 g) and increased the forced swimming time (47.33 ± 14.84 s) compared with the control group (219.67 ± 2.08 g, 8.33 ± 5.13 s). Inhalation of BEO for 5 min (392 ± 21 beats/min) only reduced the pulse compared with the control group (420 ± 19 beats/min). Inhalation of linalool only reduced the weight of white adipose tissue (5.75 ± 0.61 g). The levels of stress-related hormones were not significantly different among the groups. The total cholesterol and triglyceride levels decreased after inhalation of BEO for 20 min (by more than -10% and -15%, respectively). Low-density lipoprotein cholesterol levels were lowered (by more than -10%) by the inhalation of BEO and linalool, regardless of the inhalation time. In particular, BEO inhalation for 20 min was associated with the lowest level of low-density lipoprotein cholesterol (53.94 ± 2.72 mg/dL). High-density lipoprotein cholesterol levels increased after inhalation of BEO (by more than +15%). The atherogenic index and cardiac risk factors were suppressed by BEO inhalation. Animals exposed to BEO and linalool had no significant differences in hepatotoxicity. These data suggest that the inhalation of BEO and linalool may ameliorate cardiovascular and lipid dysfunctions. These effects should be explored further for clinical applications.