CT-based intratumoral and peritumoral deep transfer learning features prediction of lymph node metastasis in non-small cell lung cancer.

BACKGROUND: The main metastatic route for lung cancer is lymph node metastasis, and studies have shown that non-small cell lung cancer (NSCLC) has a high risk of lymph node infiltration. OBJECTIVE: This study aimed to compare the performance of handcrafted radiomics (HR) features and deep transfer learning (DTL) features in Computed Tomography (CT) of intratumoral and peritumoral regions in predicting the metastatic status of NSCLC lymph nodes in different machine learning classifier models. METHODS: We retrospectively collected data of 199 patients with pathologically confirmed NSCLC. All patients were divided into training (n = 159) and validation (n = 40) cohorts, respectively. The best HR and DTL features in the intratumoral and peritumoral regions were extracted and selected, respectively. Support Vector Machine (SVM), k-Nearest Neighbors (KNN), Light Gradient Boosting Machine (Light GBM), Multilayer Perceptron (MLP), and Logistic Regression (LR) models were constructed, and the performance of the models was evaluated. RESULTS: Among the five models in the training and validation cohorts, the LR classifier model performed best in terms of HR and DTL features. The AUCs of the training cohort were 0.841 (95% CI: 0.776-0.907) and 0.955 (95% CI: 0.926-0.983), and the AUCs of the validation cohort were 0.812 (95% CI: 0.677-0.948) and 0.893 (95% CI: 0.795-0.991), respectively. The DTL signature was superior to the handcrafted radiomics signature. CONCLUSIONS: Compared with the radiomics signature, the DTL signature constructed based on intratumoral and peritumoral areas in CT can better predict NSCLC lymph node metastasis.

Triggering High Capacity and Superior Reversibility of Manganese Oxides Cathode via Magnesium Modulation for Zn//MnO2 Batteries.

Aqueous zinc-ion batteries (ZIBs) have attracted extensive attention in recent years because of its high volumetric energy density, the abundance of zinc resources, and safety. However, ZIBs still suffer from poor reversibility and sluggish kinetics derived from the unstable cathodic structure and the strong electrostatic interactions between bivalent Zn2+ and cathodes. Herein, magnesium doping into layered manganese dioxide (Mg-MnO2 ) via a simple hydrothermal method as cathode materials for ZIBs is proposed. The interconnected nanoflakes of Mg-MnO2 possess a larger specific surface area compared to pristine δ-MnO2 , providing more electroactive sites and boosting the capacity of batteries. The ion diffusion coefficients of Mg-MnO2 can be enhanced due to the improved electrical conductivity by doped cations and oxygen vacancies in MnO2 lattices. The assembled Zn//Mg-MnO2 battery delivers a high specific capacity of 370 mAh g-1 at a current density of 0.6 A g-1 . Furthermore, the reaction mechanism confirms that Zn2+ insertion occurred after a few cycles of activation reactions. Most important, the reversible redox reaction between Zn2+ and MnOOH is found after several charge-discharge processes, promoting capacity and stability. It believes that this systematic research enlightens the design of high-performance of ZIBs and facilitates the practical application of Zn//MnO2 batteries.

SARS-CoV-2 Omicron XBB subvariants exhibit enhanced fusogenicity and substantial immune evasion in elderly population, but high sensitivity to pan-coronavirus fusion inhibitors.

Numerous emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have shown significant immune evasion capacity and caused a large number of infections, as well as vaccine-breakthrough infections, especially in elderly populations. Recently emerged Omicron XBB was derived from the BA.2 lineage, but bears a distinct mutant profile in its spike (S) protein. In this study, we found that Omicron XBB S protein drove more efficient membrane-fusion kinetics on human lung-derived cells (Calu-3). Considering the high susceptibility of the elderly to the current Omicron pandemic, we performed a comprehensive neutralization assessment of elderly convalescent or vaccine sera against XBB infection. We found that the sera from elderly convalescent patients who experienced with BA.2 infection or breakthrough infection potently inhibited BA.2 infection, but showed significantly reduced efficacy against XBB. Moreover, recently emerged XBB.1.5 subvariant also showed more significant resistance to the convalescent sera of BA.2- or BA.5-infected elderly. On the other hand, we found that the pan-CoV fusion inhibitors EK1 and EK1C4 can potently block either XBB-S- or XBB.1.5-S-mediated fusion process and viral entry. Moreover, EK1 fusion inhibitor showed potent synergism when combined with convalescent sera of BA.2- or BA.5-infected patients against XBB and XBB.1.5 infection, further indicating that EK1-based pan-CoV fusion inhibitors are promising candidates for development as clinical antiviral agents to combat the Omicron XBB subvariants.

N-Fluorobenzamide-Directed Formal [4+2] Cycloaddition Reaction with Maleic Anhydride: Access to Fluorescent Aminonaphthalic Anhydrides.

We have developed a formal [4+2] cycloaddition reaction of N-fluorobenzamides and maleic anhydride in the presence of CuI and LiOH, and a series of fluorescent 1-amino-2,3-naphthalic anhydrides were produced in good yields. This reaction proceeded via a multistep process involving nitrogen-centered radical generation, 1,5-hydrogen atom transfer, and benzylic radical addition to the amide carbonyl oxygen to generate an N-(tert-butyl) isobenzofuran-1(3H)-imine intermediate, which isomerized to an N-(tert-butyl) isobenzofuran-1-amine via deprotonation and protonation with the aid of LiOH; finally, the amine underwent a [4+2] cycloaddition reaction with maleic anhydride to give the 1-amino-2,3-naphthalic anhydride product upon dehydrating aromatization. Notably, the corresponding naphthalic anhydride products could be transformed into a diverse array of naphthalimides. Both the naphthalic anhydrides and the naphthalimides exhibited similar fluorescent features.

Microbiome and function alterations in the gastric mucosa of asymptomatic patients with Helicobacter pylori infection.

BACKGROUND: Most patients with Helicobacter pylori (H. pylori) infection have no clinical symptoms, numerous studies reported the gastric microbiome in H. pylori-infected patients, but asymptomatic patients have not been distinguished. How the microbiome and function changes in asymptomatic patients with H. pylori infection remains poorly understood. METHODS: A total of 29 patients were divided into H. pylori-infected asymptomatic group (10 patients), H. pylori-infected symptomatic group (11 patients) and H. pylori-uninfected group (8 patients). Gastric mucosa specimens were taken for histopathological examination, special staining, and 16 S rDNA sequencing. High-throughput results were evaluated by community composition analysis, indicator species analysis, alpha diversity analysis, beta diversity analysis, and function prediction. RESULTS: The gastric microbiota composition at phylum and genus level of H. pylori-infected asymptomatic patients were similar with H. pylori-infected symptomatic group, but different from H. pylori-uninfected patients. The diversity and richness of gastric microbial community declined significantly in H. pylori-infected asymptomatic group comparing with H. pylori-uninfected group. Sphingomonas may be an indicator between symptomatic and asymptomatic patients with H. pylori infection, the AUC value of Sphingomonas is 0.79. Interactions between species increased and altered notably after H. pylori infection. More genera were affected by Helicobacter in H. pylori-infected asymptomatic patients. The function condition changed significantly in asymptomatic patients with H. pylori infection, there was no difference comparing with symptomatic ones. Amino acid metabolism and lipid metabolism strengthened but carbohydrate metabolism remained constant after H. pylori infection. The metabolism of fatty acid and bile acid was disturbed after infection with H. pylori. CONCLUSION: The gastric microbiota composition and function mode changed significantly after H. pylori infection regardless of the presence of clinical symptoms, there was no difference between H. pylori-infected asymptomatic and symptomatic patients. The difference in gastric microbiota composition and interactions between species might be responsible for presence of digestive symptoms.

Dietary Bacillus cereus LS2 protects juvenile sea cucumber Apostichopus japonicus against Vibrio splendidus infection.

This study aimed to investigate the effects of Bacillus cereus LS2 on the growth performance, innate immunity, intestinal microbiota, and disease resistance of sea cucumber Apostichopus japonicus. After feeding with LS2 for 30 days, results showed that dietary with LS2 had a significant improvement in the growth rate and immune parameters (including total coelomocytes counts, phagocytosis, respiratory burst, and immune-related enzymes) of juvenile sea cucumbers. Subsequently, transcriptome sequencing and qRT-PCR verification were performed to analyze the potential mechanism of LS2 diet and thus improve the immune response of A. japonicus. GO and KEGG pathway analysis indicated that LS2 can primarily activate the "Lectins" and "complement and coagulation cascades" pathways to modulate the innate immunity of the sea cucumbers. Furthermore, 16S rRNA sequencing was used to analyze the intestinal microbial composition of sea cucumbers after dietary with LS2. Results showed that Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes were the most prevalent phyla in A. japonicus intestinal microbiota. The abundance of Actinobacteria (46.20%) and Bacteroidetes (12.80%) were significantly higher in the LS2 group, whereas the relative abundance of Proteobacteria (49.98%) and Firmicutes (14.97%) were higher in the control group. The LDA scores of Nocardiaceae and Rhodococcus were also the highest taxa after the dietary administration of LS2, indicating that Actinobacteria phylum played a pivotal role in the intestinal microbial function of A. japonicus. Overall, these results suggested that feeding with Bacillus LS2 may be beneficial for A. japonicus farming.

Hepatitis E Virus Capsid as a Carrier of Exogenous Antigens for the Development of Chimeric Virus-Like Particles.

INTRODUCTION: Virus-like particles (VLPs), self-assembled multiprotein structures, can stimulate robust immune responses due to their structural similarity to native virions that allow the presentation of multiple copies of the target epitopes. Utilizing VLPs as vaccine platforms to present exogenous antigens is a promising and challenging approach in the vaccine development field. This study investigates the potential of the truncated hepatitis E virus (HEV) capsid as a VLP platform to present foreign antigens. METHODS: The S and M domains of the HEV capsid protein were selected as the optimal carrier (CaSM). The exogenous antigen Seq8 containing 3 neutralizing epitopes from 3 different foot-and-mouth disease virus (FMDV) strains was linked to the C-terminal of CaSM to construct a chimeric VLP (CaSM-Seq8). The chimeric particles were produced in Escherichia coli, and their morphology, physicochemical properties, antigenicity, and immunogenicity were analyzed. RESULTS: Morphological analysis showed that CaSM-Seq8 self-assembled into VLPs similar to CaSM VLPs (∼26 nm in diameter) but smaller than native HEV virions. Further, the thermal stability and the resistance to enzymatic proteolysis of Seq8 were enhanced when it was attached to the CaSM carrier. The antigenicity analysis revealed a more robust reactivity against anti-FMDV antibodies when Seq8 was presented on CaSM particles. Upon injection into mice, FMDV-specific IgGs induced by CaSM-Seq8 appeared earlier, increased faster, and maintained higher levels for a longer time than those induced by Seq8 alone or the inactivated FMDV vaccine. CONCLUSION: This study demonstrated the potential of utilizing the truncated HEV capsid as an antigen-presenting platform for the development of chimeric VLP immunogens.

ISG15 enhances glioma cell stemness by promoting Oct4 protein stability.

The effects of ISG15 or ISGylation on tumor progression have been widely revealed; however, its roles in glioma progression are largely unknown. This study aims to explore the roles and underlying mechanisms of ISG15 in glioma progression. Here, ISG15 level was found to be upregulated in glioma tissues compared to the paired/unpaired normal tissues, and positively correlated with the level of stemness markers in glioma tissues. Loss of functional experiments indicated that ISG15 positively regulated glioma cell stemness, as evident by the increase of sphere formation ability, ALDH activity, stemness marker expression, and tumor-initiating ability. Further mechanistic studies revealed that ISG15 directly interacted with Oct4 protein, a critical stemness promoter, induced the ISGylation of Oct4 protein, and thus enhanced Oct4 protein stability. Additionally, it was found that Oct4 was ISGylated at lysine 284 (K284), which has been confirmed to be the ubiquitination site of Oct4 protein, and ISG15 knockdown did not degrade K284R mutant Oct4. Furthermore, ISG15 knockdown-induced downregulation of glioma cell stemness was rescued by Oct4 overexpression, but not by K284R mutant Oct4. Altogether, we suggest that ISG15-induced ISGylation of Oct4 protein is essential for glioma cell stemness.

Vibrio parahaemolyticus CadC regulates acid tolerance response to enhance bacterial motility and cytotoxicity.

Pathogens adapted to sub-lethal acidic conditions could increase the virulence and survival ability under lethal conditions. In the aquaculture industry, feed acidifiers have been used to increase the growth of aquatic animals. However, there is limited study on the effects of acidic condition on the virulence and survival of pathogens in aquaculture. In this study, we investigated the survival ability of Vibrio parahaemolyticus at lethal acidic pH (4.0) after adapted the bacteria to sub-lethal acidic pH (5.5) for 1 hr. Our results indicated that the adapted strain increased the survival ability at lethal acidic pH invoked by an inorganic (HCl) or organic (citric) acid. RNA-sequencing (RNA-seq) results revealed that 321 genes were differentially expressed at the sub-lethal acidic pH including cadC, cadBA and groES/groEL relating to acid tolerance response (ATR), as well as genes relating to outer membrane, heat-shock proteins, phosphotransferase system and flagella system. Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that cadC and cadBA were upregulated under sub-lethal acidic conditions. The CadC protein could directly regulate the expression of cadBA to modulate the ATR in V. parahaemolyticus. RNA-seq data also indicated that 113 genes in the CadC-dependent way and 208 genes in the CadC-independent way were differentially expressed, which were related to the regulation of ATR. Finally, the motility and cytotoxicity of the sub-lethal acidic adapted wild type (WT) were significantly increased compared with the unadapted strain. Our results demonstrated that the dietary acidifiers may increase the virulence and survival of V. parahaemolyticus in aquaculture.

Synthesis of methylene cyclopropane-fused chromenes and dihydroquinolines by sequential [4 + 2]- and [1 + 2]-annulation.

A base promoted sequential [4 + 2]- and [1 + 2]-annulation of 2-hydroxychalcones or 2-tosylaminochalcones with prop-2-ynylsulfonium salts has been developed to give the corresponding methylene cyclopropane fused dihydroquinolines or chromenes in moderate to good yields. This transformation has advantage of wide substrate scope and functional group tolerance as well as excellent regioselectivity. Prop-2-ynylsulfonium salts act as both C2 and C1 synthons in the tandem processes.

The Depletion of ABI3BP by MicroRNA-183 Promotes the Development of Esophageal Carcinoma.

BACKGROUND: Esophageal cancer (EC), as a serious threat to human life and health, is one of the most common cancers around the world. Many studies have suggested that many microRNAs are involved in tumorigenesis and progression. METHODS: To search for a novel and promising predictive therapeutic target or biomarker to achieve the goal of the early diagnosis and treatment of EC, we used the EC cell lines Eca-109 and KYSE-150 and normal human esophageal epithelial cells (HEECs) to investigate the effect of ABI3BP on EC. RESULTS: We found that ABI family member 3 binding protein (ABI3BP) was downregulated in EC and suppressed the proliferation, activity, migration, and invasion of EC cells. ABI3BP was downregulated by miR-183, which plays the role of an oncogene. CONCLUSION: ABI3BP and miR-183 can be considered potential biomarkers for the diagnosis of patients with EC and can be effective targets for antitumor therapy.

Risk factors related to intracranial infections after transsphenoidal pituitary adenomectomy under endoscope.

BACKGROUND AND PURPOSE: Background - Up to now, the risk factors related to intracranial infections after transsphenoidal pituitary adenomectomy remain controversial. Purpose - To analyze the risk factors related to intracranial infections after transsphenoidal pituitary adenomectomy under an endoscope, and to provide evidence for preventing and controlling the occurrence and development of infections. METHODS: A total of 370 patients receiving endoscopic transsphenoidal pituitary adenomectomy in our hospital from January 2014 to October 2017 were selected. The risk factors related to postoperative intracranial infections were analyzed. The hospitalization lengths and expenditures of patients with and without intracranial infections were compared. RESULTS: Of the 370 patients, 18 underwent postoperative intracranial infections, with the infection rate of 4.86%. Intraoperative blood loss >120 mL, cerebrospinal leakage, diabetes, preoperative use of hormones, macroadenoma as well as surgical time >4 h all significantly increased the infection rate (P<0.05). Preoperative use of antibacterial agents prevented intracranial infection. Compared with patients without intracranial infections, the infected ones had significantly prolonged hospitalization length and increased expenditure (P<0.05). Discussion - It is of great clinical significance to analyze the risk factors related to intracranial infection after endoscopic transsphenoidal pituitary adenomectomy, aiming to prevent and to control the onset and progression of infection. CONCLUSION: Intracranial infections after endoscopic transsphenoidal pituitary adenomectomy were affected by many risk factors, also influencing the prognosis of patients and the economic burden.

CopA Protects Streptococcus suis against Copper Toxicity.

Streptococcus suis is a zoonotic pathogen that causes great economic losses to the swine industry and severe threats to public health. A better understanding of its physiology would contribute to the control of its infections. Although copper is an essential micronutrient for life, it is toxic to cells when present in excessive amounts. Herein, we provide evidence that CopA is required for S. suis resistance to copper toxicity. Quantitative PCR analysis showed that copA expression was specifically induced by copper. Growth curve analyses and spot dilution assays showed that the ΔcopA mutant was defective in media supplemented with elevated concentrations of copper. Spot dilution assays also revealed that CopA protected S. suis against the copper-induced bactericidal effect. Using inductively coupled plasma-optical emission spectroscopy, we demonstrated that the role of CopA in copper resistance was mediated by copper efflux. Collectively, our data indicated that CopA protects S. suis against the copper-induced bactericidal effect via copper efflux.

A multivariate analysis for delayed healing of facial muscle spasm after microvascular decompression.

OBJECTIVES: To analyze the related factors for delayed healing of facial muscle spasm after microvascular decompression. METHODS: After microvascular decompression, 116 of 425 patients with delayed healing were followed up, and their clinical data were analyzed. RESULTS: The incidence rate of postoperative delayed healing was 27.3%, which was not correlated with gender, age or intraoperative vascular compression. However, it was correlated with disease course, severity of preoperative symptoms, arteriosclerosis and abnormal facial muscle response. The duration of delayed healing was positively correlated with preoperative disease course. CONCLUSIONS: Delayed healing is a common phenomenon after microvascular decompression for facial muscle spasm, with an elusive reason. Therefore, the treatment outcomes should be evaluated after one year of follow-up.

Shifting trends in bacteriology and antimicrobial resistance among gastrointestinal fistula patients in China: an eight-year review in a tertiary-care hospital.

BACKGROUND: The purpose of this study was to determine the shifting trends in bacteriology and antimicrobial resistance of infectious specimens isolated from gastrointestinal (GI) fistula patients over eight years in China. METHODS: We retrospectively reviewed the microbial records of intra-abdominal specimens at a teaching hospital from 2008 to 2015. Study period was divided into the first half (2008-2011) and the second half (2012-2015). All isolates underwent antibiotic susceptibility testing by the micro dilution method. RESULTS: A total of 874 intra-abdominal isolates were consecutively collected from 502 GI fistula patients (mean age, 46.5 years, 71.1% male) during the study period. Patients in the second study period (2012-2015) were older (>65 years) and more likely to have experienced cancer. Over the entire study period, most infections were caused by E. coli (24.2%) and K. pneumonia (14.1%). There was a significant decrease in the proportion E. coli isolates that were extended- spectrum beta-lactamase (ESBL)-positive (P = 0.026). The proportion of E. coli resistant to imipenem increased from 14.3% in 2008-2011 to 25.9% in 2012-2015 (P = 0.037). Imipenem resistance prevalence was higher in ESBL-negative bacteria than ESBL-positive bacteria for both E. coli and K. pneumonia (P < 0.001). In Enterococcus, significant increase in resistance to ampicillin (P = 0.01) and moxifloxacin (P = 0.02) over time were observed. In Staphylococcus and fungi, rates of antibiotic resistance did not significantly change over the study period. CONCLUSIONS: Gram-negative bacteria predominated as causative agents of intra-abdominal infections in GI fistula patients, and there was an increase in levels of resistance to certain antibiotics, particularly carbapenems. Infection control and source control are important tools available to surgeons to prevent the emergence of antibiotic-resistant pathogens.

Early permanent disappearance of abnormal muscle response during microvascular decompression for hemifacial spasm: a retrospective clinical study.

The objective of this study is to explore the cause of early abnormal muscle response (AMR) disappearance during microvascular decompression for hemifacial spasm and the clinical outcomes of these patients. Three hundred seventy-two patients received microvascular decompression (MVD) under intraoperative electrophysiological monitoring in Nanjing Drum Tower Hospital in 2014; the characteristic AMR of HFS was observed in 359 patients during the operation. And the 359 patients were divided into two groups based on whether AMR had remained before the beginning of the decompression procedure for offending vessels. Thirty-three patients who showed a permanent disappearance of AMR before the beginning of decompression were regarded as group I. Dural opening and the succeeding CSF drainage produced a permanent disappearance of AMR in 13. During the dissection of lateral cerebellomedullary cistern, a permanent disappearance of AMR was found in 20 patients. Thirty-two patients were cured immediately; delayed resolution (7 days after surgery) was found in one patient. No complications were observed and no recurrence was found during the follow-up period in the 33 patients. In the other 326 patients (group II), AMR disappeared temporarily before the beginning of the decompression procedure for offending vessels in 42 patients. After decompression, AMR disappeared completely in 305 patients. Two hundred sixty-seven patients were cured immediately and 57 patients got a delayed resolution (2 days to 45 weeks after surgery). The two left did not get a complete abolition of spasm. Three cases of hearing loss, one hoarseness, and nine delayed facial paralysis were observed. The reason of early abnormal muscle response disappearance may be that the degree of neurovascular compression was not serious; these patients were more likely to get an immediate cure. Continuous intraoperative electrophysiological monitoring of AMR is necessary.

Failed microvascular decompression surgery for hemifacial spasm: a retrospective clinical study of reoperations.

BACKGROUND: To investigate the repeat microvascular decompression on hemifacial spasm patients who failed the first MVD. METHODS: Twenty-six patients underwent late redo MVD in our institution from January 1, 2011 to December 31, 2015. The clinical features, surgical findings, outcomes, and complications of the repeat MVD were analyzed retrospectively. RESULTS: Twenty-four (92.3 %) patients were cured immediately after the redo MVD. Delayed relief was found in two (7.7 %) patients; it took 6 days and 2 weeks for them to obtain complete relief. No recurrence was found during follow-up. Surgical complications including three (11.5 %) facial paralysis and one (3.8 %) hearing loss. CONCLUSIONS: We suggested that repeat MVD can be performed 2 years after the first MVD if the spasm was not resolved. Repeat MVD for HFS is effective.

Sparks of function by de novo protein design.

Information in proteins flows from sequence to structure to function, with each step causally driven by the preceding one. Protein design is founded on inverting this process: specify a desired function, design a structure executing this function, and find a sequence that folds into this structure. This 'central dogma' underlies nearly all de novo protein-design efforts. Our ability to accomplish these tasks depends on our understanding of protein folding and function and our ability to capture this understanding in computational methods. In recent years, deep learning-derived approaches for efficient and accurate structure modeling and enrichment of successful designs have enabled progression beyond the design of protein structures and towards the design of functional proteins. We examine these advances in the broader context of classical de novo protein design and consider implications for future challenges to come, including fundamental capabilities such as sequence and structure co-design and conformational control considering flexibility, and functional objectives such as antibody and enzyme design.

Circulating cell-free DNA-based methylation pattern in plasma for early diagnosis of esophagus cancer.

With the increased awareness of early tumor detection, the importance of detecting and diagnosing esophageal cancer in its early stages has been underscored. Studies have consistently demonstrated the crucial role of methylation levels in circulating cell-free DNA (cfDNA) in identifying and diagnosing early-stage cancer. cfDNA methylation pertains to the methylation state within the genomic scope of cfDNA and is strongly associated with cancer development and progression. Several research teams have delved into the potential application of cfDNA methylation in identifying early-stage esophageal cancer and have achieved promising outcomes. Recent research supports the high sensitivity and specificity of cfDNA methylation in early esophageal cancer diagnosis, providing a more accurate and efficient approach for early detection and improved clinical management. Accordingly, this review aims to present an overview of methylation-based cfDNA research with a focus on the latest developments in the early detection of esophageal cancer. Additionally, this review summarizes advanced analytical technologies for cfDNA methylation that have significantly benefited from recent advancements in separation and detection techniques, such as methylated DNA immunoprecipitation sequencing (MeDIP-seq). Recent findings suggest that biomarkers based on cfDNA methylation may soon find successful applications in the early detection of esophageal cancer. However, large-scale prospective clinical trials are required to identify the potential of these biomarkers.