Adjuvant Therapy-Free Strategy for Stage IB to IIIA Non-Small-Cell Lung Cancer Patients After Radical Resection Based on Longitudinal Undetectable Molecular Residual Disease: Prospective, Multicenter, Single-Arm Study (CTONG 2201).

BACKGROUND: The utility of circulating tumor DNA to monitor molecular residual disease (MRD) has been clinically confirmed to predict disease recurrence in non-small cell lung cancer (NSCLC) patients after radical resection. Patients with longitudinal undetectable MRD show a favorable prognosis and might not benefit from adjuvant therapy. PATIENTS AND METHODS: The CTONG 2201 trial is a prospective, multicenter, single-arm study (ClinicalTrials.gov identifier, NCT05457049), designed to evaluate the hypothesis that no adjuvant therapy is needed for patients with longitudinal undetectable MRD. Pathologically confirmed stage IB-IIIA NSCLC patients who have undergone radical resection will be screened. Only patients with 2 consecutive rounds of undetectable MRD will be enrolled (first at days 3-10, second at days 30 ± 7 after surgery), and admitted for imaging and MRD monitoring every 3 months without adjuvant therapy. The primary endpoint is the 2-year disease-free survival rate for those with longitudinal undetectable MRD. The recruitment phase began in August 2022 and 180 patients will be enrolled. CONCLUSIONS: This prospective trial will contribute data to confirm the negative predictive value of MRD on adjuvant therapy for NSCLC patients. CLINICAL TRIAL REGISTRATION: NCT05457049 (CTONG 2201).

A Mushroom P450-Monooxygenase Enables Regio- and Stereoselective Biocatalytic Synthesis of Epoxycyclohexenones.

An epoxycyclohexenone (ECH) moiety occurs in natural products of both bacteria and ascomycete and basidiomycete fungi. While the enzymes for ECH formation in bacteria and ascomycetes have been identified and characterized, it remained obscure how this structure is biosynthesized in basidiomycetes. In this study, we i) identified a genetic locus responsible for panepoxydone biosynthesis in the basidiomycete mushroom Panus rudis and ii) biochemically characterized PanH, the cytochrome P450 enzyme catalyzing epoxide formation in this pathway. Using a PanH-producing yeast as a biocatalyst, we synthesized a small library of bioactive ECH compounds as a proof of concept. Furthermore, homology modeling, molecular dynamics simulation, and site directed mutation revealed the substrate specificity of PanH. Remarkably, PanH is unrelated to ECH-forming enzymes in bacteria and ascomycetes, suggesting that mushrooms evolved this biosynthetic capacity convergently and independently of other organisms.

Biochemical and Genetic Basis of Guanacastane Diterpene Biosynthesis in Basidiomycete Fungi.

Guanacastane diterpenoids with an unusual 5/7/6 tricyclic skeleton mainly produced by basidiomycete fungi represent a structurally intriguing class of natural products. While the chemical synthesis of several members has been achieved, the biochemical and genetic basis of their biosynthesis remain unknown. Herein, we present the identification and characterization of two crucial enzymes in the biosynthesis of guanacastane diterpenoids in Psathyrella candolleana. Heterologous expression reveals that PsaD, a typical class I diterpene synthase, catalyzes the cyclization of geranylgeranyl diphosphate to form a new guanacastane-type diterpene, guanacasta-1,3-diene (7). Moreover, we demonstrate that PsaA, a cytochrome P450 monooxygenase, can catalyze multiple oxidations of 7 to yield guanacastepene U (8). These results provide new opportunities for genome mining and metabolic engineering of guanacastane diterpenoids.

A flavin-monooxygenase catalyzing oxepinone formation and the complete biosynthesis of vibralactone.

Oxepinone rings represent one of structurally unusual motifs of natural products and the biosynthesis of oxepinones is not fully understood. 1,5-Seco-vibralactone (3) features an oxepinone motif and is a stable metabolite isolated from mycelial cultures of the mushroom Boreostereum vibrans. Cyclization of 3 forms vibralactone (1) whose ß-lactone-fused bicyclic core originates from 4-hydroxybenzoate, yet it remains elusive how 4-hydroxybenzoate is converted to 3 especially for the oxepinone ring construction in the biosynthesis of 1. In this work, using activity-guided fractionation together with proteomic analyses, we identify an NADPH/FAD-dependent monooxygenase VibO as the key enzyme performing a crucial ring-expansive oxygenation on the phenol ring to generate the oxepin-2-one structure of 3. The crystal structure of VibO reveals that it forms a dimeric phenol hydroxylase-like architecture featured with a unique substrate-binding pocket adjacent to the bound FAD. Computational modeling and solution studies provide insight into the likely VibO active site geometry, and suggest possible involvement of a flavin-C4a-OO(H) intermediate.

Electromagnetic navigation bronchoscopic localization versus percutaneous CT-guided localization for thoracoscopic resection of small pulmonary nodules.

BACKGROUND: Identification of small pulmonary nodules is challenging in a limited intrathoracic field during minimally invasive video-assisted thoracoscopic surgery (VATS), and preoperative localization is required. Various techniques have been reported with some failure and complications. Here, we compare the feasibility and safety between electromagnetic navigation bronchoscopic marking and computed tomography (CT)-guided percutaneous marking using indocyanine green (ICG) and iopamidol. METHODS: A total of 47 patients with small-sized pulmonary nodules, scheduled to undergo video-assisted thoracoscopic limited resection, were enrolled in this study. A mixture of diluted ICG and iopamidol was injected into the lung parenchyma as a marker, using CT-guided percutaneous or electromagnetic navigation bronchoscopic injection techniques and the results were examined and compared. RESULTS: A total of 35 and 12 patients underwent preoperative marking by percutaneous injection and electromagnetic navigation bronchoscopic injection, respectively, in which a marker was detected in 33/35 (94.3%) and 12/12 (100%) patients. No combination of these procedures was performed in any patient. All markers were successfully detected in three patients who underwent injection marking at two different lesion sites. Pneumothorax occurred in five patients (14%) in the percutaneous marking group, which was relieved in all patients without the necessity for chest tube drainage. No other complication was observed in this study. CONCLUSIONS: Electromagnetic navigation bronchoscopic injection techniques using indocyanine green fluorescence plus iopamidol are safe and effective, and comparable with CT-guided localization. Furthermore, a bronchoscopic approach enables marking of multiple lesion areas without increasing patient risk, especially for puncture-related pneumothorax. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: Either computed tomography (CT)-guided percutaneous or electromagnetic navigation bronchoscopic injection techniques can be used for preoperative marking of pulmonary nodules with indocyanine green (ICG) fluorescence. WHAT THIS STUDY ADDS: Indocyanine green (ICG) is a safe and easily detectable fluorescent marker for video-assisted thoracoscopic surgery (VATS). A bronchoscopic injection approach enables marking of multiple lesion areas without increasing the risk of pneumothorax.

Construction of a meroterpenoid-like compound collection by precursor-assisted biosynthesis.

Natural products (NPs) and their derivatives play a pivotal role in drug discovery due to their complexity and diversity. The strategies to rapidly generate NP-like compounds offer unique opportunities to access bioactive compounds. Here we present a new approach, precursor-assisted biosynthesis (PAB), for the creation of NP-like compounds by combination of artificial supplementation of common precursors and divergent post-modifications of precursor-deficient fungi. This method was applied to construct a meroterpenoid-like compound collection containing 43 compounds with diverse molecular scaffolds. Extensive bioactive screening of the collection revealed novel STING (stimulator of interferon genes) inhibitors, cytotoxic and antifungal compounds. This result indicates that PAB is an effective methodology for producing compound collections for the purpose of drug discovery.

Cerebral venous sinus thrombosis following transsphenoidal surgery for craniopharyngioma: A case report.

BACKGROUND: Cerebral venous sinus thrombosis (CVST) is a rare condition in patients with craniopharyngioma following transsphenoidal surgery. CASE SUMMARY: A 56-year-old man who underwent transsphenoidal surgery for craniopharyngioma 26 d ago presented gradual headache and cerebrospinal fluid leakage while vomiting 5 d post-discharge and required readmission to our department of neurosurgery. After admission, head imaging examination showed a hyperdense shadow in the superior sagittal sinus and right transverse sinus, edema at the bilateral parietal lobe, and hemorrhage at the left parietal lobe and right occipital lobe; the venous phase of cerebral angiography revealed CVST. The patient was treated immediately by intravenous thrombolysis, endovascular thrombolysis, and mechanical thrombectomy after the definite diagnosis. However, the neurological status of the patient continued to deteriorate and he died on the fourth day after readmission. CONCLUSION: For craniopharyngioma undergoing transsphenoidal surgery, it is vital to take an effective strategy to manage the postoperative complications, such as diabetes insipidus, severe electrolyte imbalance, and cerebrospinal fluid leakage. Additionally, the early differential diagnosis of CVST is essential when it develops clinical symptoms, especially in patients following transsphenoidal surgery with a high risk of CVST. Subsequently, the timely and effective treatment of the CVST is critical for preventing neurological deterioration.

A Hydrolase-Catalyzed Cyclization Forms the Fused Bicyclic ß-Lactone in Vibralactone.

Vibralactone is isolated from the basidiomycete fungus Boreostereum vibrans as one of the strongest lipase inhibitors. Its unusual ß-lactone-fused bicycle is derived from an aryl ring moiety by an oxidative ring-expansion prior to an intramolecular cyclization. Herein, we report the discovery of the cyclase VibC which belongs to the α/ß-hydrolase superfamily and is involved in the vibralactone biosynthesis. Biochemical and crystal studies suggest that VibC may catalyze an aldol or an electrocyclic reaction initiated by the Ser-His-Asp catalytic triad. For the aldol and pericyclic chemistry in living cells, VibC is a unique hydrolase performing the carbocycle formation of an oxepinone to a fused bicyclic ß-lactone. This presents a naturally occurring, new enzymatic reaction in both aldol and hydrolase (bio)chemistry that will guide future exploitation of these enzymes in synthetic biology for chemical-diversity expansion of natural products.

Rapid discovery and functional characterization of diterpene synthases from basidiomycete fungi by genome mining.

Basidiomycete fungi are a rich source of bioactive diterpenoid secondary metabolites. However, compared with the large number of diterpene synthases (di-TPSs) identified in plants and ascomycete fungi, only three di-TPSs have been described from basidiomycete fungi. Large scale genome sequencing projects combined with the development of synthetic biology techniques now has enabled the rapidly discovery and characterization of di-TPSs from basidiomycete fungi. In this study, we discovered and functionally characterized four di-TPSs from 220 genome sequenced basidiomycete fungi by a combined strategy of genomic data mining, phylogenetic analysis and fast products characterization with synthetic biology techniques. Among them, SteTC1 of Stereum histurum was characterized as the first fungal cembrane diterpene synthase; PunTC of Punctularia strigosozonata and SerTC of Serpula lacrymans were characterized as ent-kauran-16α-ol synthase and DenTC3 of Dentipellis sp was characterized as a cyathane synthase. Our results provide opportunities for the discovery of new diterpenoids from basidiomycete fungi by genome mining.

Diterpene synthases facilitating production of the kaurane skeleton of eriocalyxin B in the medicinal plant Isodon eriocalyx.

The Isodon plants (Lamiaceae) have been used in traditional Chinese medicine to alleviate sufferings from inflammations and cancers. This feature has been attributed to the presence of pharmacologically active ent-kaurane diterpenoids such as eriocalyxin B and oridonin. The Isodon eriocalyx (Dunn) Kudô species native to southwest China can accumulate a particularly high content of ent-kaurane diterpenoids (∼1.5% w/w of dried leaves). We previously identified diterpene synthases IeCPS1 and IeCPS2 as ent-copalyl diphosphate synthases (ent-CPS) potentially involved in Isodon ent-kaurane diterpenoids biosynthesis. In this study, analysis of RNA-seq transcriptome of the I. eriocalyx plant revealed three other diterpene synthase genes (IeCPS3, IeKS1, and IeKSL1). Their functional characterization through coupled in vitro enzyme assays has confirmed that IeCPS3 is an ent-CPS specifically producing ent-copalyl diphosphate (ent-CPP). IeKS1 accepted ent-CPP to produce exclusively ent-kaurene and may thus be defined as an ent-kaurene synthase (ent-KS). When IeKSL1 was combined with IeCPS2 or IeCPS3, no product was detected. Based on tissue-specific expression and metabolic localization studies, the IeCPS3 and IeKS1 transcripts were significantly accumulated in leaves where the ent-kaurane diterpenoid eriocalyxin B dominates, whereas weak expression of both were observed in germinating seeds in which gibberellin biosynthetic pathway is normally active. Our findings suggest that both IeCPS3 and IeKS1 possess dual roles in general (gibberellins) and specialized diterpenoid metabolism, such as that of the Isodon ent-kaurane diterpenoids.

Genomic and transcriptomic analyses reveal differential regulation of diverse terpenoid and polyketides secondary metabolites in Hericium erinaceus.

The lion's mane mushroom Hericium erinaceus is a famous traditional medicinal fungus credited with anti-dementia activity and a producer of cyathane diterpenoid natural products (erinacines) useful against nervous system diseases. To date, few studies have explored the biosynthesis of these compounds, although their chemical synthesis is known. Here, we report the first genome and tanscriptome sequence of the medicinal fungus H. erinaceus. The size of the genome is 39.35 Mb, containing 9895 gene models. The genome of H. erinaceus reveals diverse enzymes and a large family of cytochrome P450 (CYP) proteins involved in the biosynthesis of terpenoid backbones, diterpenoids, sesquiterpenes and polyketides. Three gene clusters related to terpene biosynthesis and one gene cluster for polyketides biosynthesis (PKS) were predicted. Genes involved in terpenoid biosynthesis were generally upregulated in mycelia, while the PKS gene was upregulated in the fruiting body. Comparative genome analysis of 42 fungal species of Basidiomycota revealed that most edible and medicinal mushroom show many more gene clusters involved in terpenoid and polyketide biosynthesis compared to the pathogenic fungi. None of the gene clusters for terpenoid or polyketide biosynthesis were predicted in the poisonous mushroom Amanita muscaria. Our findings may facilitate future discovery and biosynthesis of bioactive secondary metabolites from H. erinaceus and provide fundamental information for exploring the secondary metabolites in other Basidiomycetes.

Discovery and Characterization of a New Family of Diterpene Cyclases in Bacteria and Fungi.

Diterpene cyclases from bacteria and basidiomycete fungi are seldom studied. Here, we presented the identification and verification of EriG, a member of the UbiA superfamily, as the enzyme responsible for the cyclization of the cyathane skeleton in the mushroom Hericium erinaceum. Genome mining using the EriG protein sequence as a probe led to the discovery of a new family of ubiquitous UbiA-related diterpene cyclases in bacteria and fungi. We successfully characterized seven new diterpene cyclases from bacteria or basidiomycete fungi with the help of an engineered Escherichia coli strain and determined the structures of their corresponding products. A new diterpene with an unusual skeleton was generated during this process. The discovery of this new family of diterpene cyclases provides new insight into the UbiA superfamily.

Antioxidants and α-glucosidase inhibitors from "Liucha" (young leaves and shoots of Sibiraea laevigata).

The young leaves and shoots of Sibiraea laevigata, known as "Liucha", are used as tea by Tibetans to improve digestion after meals. Long-term consumption of "Liucha" will cause weight loss. In present work, we reported on the isolation and NMR and chemical analysis-based elucidation of seven new sorbitol O-caffeic acid ester derivatives named sorbitol esters A-G (1-7) and eighteen known phenolic compounds from S. laevigata. All of the isolates were evaluated for their antioxidant and α-glucosidase inhibitory activities. Among them sorbitol ester A (1), sorbitol ester D (4), sorbitol ester F (6), sorbitol ester G (7), isoferulic acid (15), methyl caffeate (18), trans-p-hydroxycinnamic acid (19), and kaempferol 3-O-ß-d-(6″-E-p-coumaroyl)-glucopyranoside (25) showed more potent α-glucosidase inhibitory activity than the clinical drug acarbose.

Isolation and identification of antioxidant and α-glucosidase inhibitory compounds from fruit juice of Nitraria tangutorum.

Nitraria tangutorum Bor., having edible berries, is valued for reputed health benefits in Qinghai-Tibet plateau. The phytochemical research on the fruit juice of N. tangutorum led to the isolation of twenty-six compounds including five new compounds, tangutorids A-D (1, 2, 3a, and 3b), and (3E,5E)-7-O-ß-glucosyl-4-(2-methoxy-2-oxoethyl)hepta-3,5-dienoic acid (15). The structures of these compounds were elucidated through comprehensive spectroscopic analyses. Tangutorids A-F were the first examples of glucose-derived ß-carbolines from natural products. The biogenetic pathways of 1-8 were proposed to involve Pictet-Spengler reactions and described starting from the co-isolated tryptophan (10) and corresponding aldehydes. All isolates were evaluated for their antioxidant and α-glucosidase inhibitory activities. Compounds 21, 22, and 24 showed antioxidant activity with SC50 values ranging from 12.2±1.9 to 30.4±2.7µg/mL, and compound 1 showed strong α-glucosidase inhibitory effect with IC50 value of 63.3±4.6µg/mL.

Use of etanercept to treat rheumatoid arthritis in an HIV-positive patient: a case-based review.

Rheumatoid arthritis (RA) is a relatively common autoimmune disease that is associated with progressive disability and systemic complications, with a relatively high socioeconomic burden. The treatment of RA has been revolutionized by the use of biological drugs, such as anti-tumor necrosis factor (TNF) agents. A wide spectrum of RA disease severity has been reported among patients with human immunodeficiency virus (HIV) infection. Yet, only a few cases using anti-TNF therapy have been described in this clinical population. Therefore, the aim of our case-based review was to describe the successful use of etanercept in a 38-year-old female patient with RA concomitant with HIV infection, who had been resistant to the first-line anti-rheumatic therapies. As per routine care guidelines, the patient was screened for hepatitis virus infection, latent tuberculosis, and other infectious conditions, prior to the initiation of etanercept treatment. CD4 cell count, HIV viral load, and adverse effects were closely monitored during the treatment. The HIV infection remained stable with etanercept treatment, without the need for anti-retrovirus agents. No adverse effects and serious infections were identified during the treatment. Therefore, anti-TNF therapy is a viable alternative for the treatment of RA in patients with HIV, who do not respond to conventional anti-rheumatic therapies. The relationship between TNF-α and HIV infection, as well as cautionary guidelines regarding the utilization of anti-TNF therapy in this clinical population, is discussed.

Low Serum Levels of Brain-Derived Neurotrophic Factor Were Associated with Poor Short-Term Functional Outcome and Mortality in Acute Ischemic Stroke.

Circulating brain-derived neurotrophic factor (BDNF) has been highlighted as being a key regulator of rehabilitation-induced recovery after stroke. The aim of this study was to evaluate the association between serum levels of BDNF and functional outcome and mortality events in a 3-month follow-up study in a cohort of patients with an acute ischemic stroke (AIS). From January 2015 to December 2015, consecutive first-ever AIS patients admitted to the Department of Emergency of our hospital were identified. Serum BDNF levels were measured at admission. Functional outcome was evaluated at 3 months using the modified Rankin scale (m-Rankin). We used logistic regression models to assess the relationship between BDNF levels and functional outcome or mortality. In this study, 204 patients were included. Patients with poor outcomes and non-survivors had significantly lower BDNF levels on admission (P < 0.0001 all). Multivariate logistic regression analysis adjusted for common risk factors showed that BDNF levels in the lowest interquartile (≤1st 9.2 ng/ml) was an independent predictor of functional outcome (odds ratios [OR] = 3.75; 95 % confidence interval [CI], 2.43-8.12) and mortality (OR = 4.04; 95 % CI, 2.07-9.14). The area under the receiver operating characteristic curve of BDNF was 0.77 (95 % CI, 0.70-0.84) for functional outcome and 0.79 (95 % CI, 0.71-0.86) for mortality. The findings indicated that low serum levels of BDNF at admission were significantly associated with poor short-term functional outcome and mortality, suggesting that BDNF may serve as a biomarker of poor function outcome after stroke.

Identification and Characterization of a δ-Cadinol Synthase Potentially Involved in the Formation of Boreovibrins in Boreostereum vibrans of Basidiomycota.

Sesquiterpenoids are very common among natural products. A large number of sesquiterpene synthase genes have been cloned and functionally characterized. However, until now there is no report about the δ-cadinol synthase predominantly forming δ-cadinol (syn. torreyol) from farnesyl diphosphate. Sesquiterpenoids boreovibrins structurally similar to δ-cadinol were previously isolated from culture broths of the basidiomycete fungus Boreostereum vibrans. This led us to expect a corresponding gene coding for a δ-cadinol synthase that may be involved in the biosynthesis of boreovibrins in B. vibrans. Here we report the cloning and heterologous expression of a new sesquiterpene synthase gene from B. vibrans. The crude and purified recombinant enzymes, when incubating with farnesyl diphosphate as substrate, gave δ-cadinol as its principal product and thereby identified as a δ-cadinol synthase. A new sesquiterpene synthase gene was cloned from the basidiomycete fungus Boreostereum vibrans and heterologously expressed in E. coli. The purified recombinant enzyme gave δ-cadinol as its principal product from farnesyl diphosphate and thereby identified as a δ-cadinol synthase (BvCS).

A Monooxygenase from Boreostereum vibrans Catalyzes Oxidative Decarboxylation in a Divergent Vibralactone Biosynthesis Pathway.

The oxidative decarboxylation of prenyl 4-hydroxybenzoate to prenylhydroquinone has been frequently proposed for the biosynthesis of prenylated (hydro)quinone derivates (sometimes meroterpenoids), yet no corresponding genes or enzymes have so far been reported. A FAD-binding monooxygenase (VibMO1) was identified that converts prenyl 4-hydroxybenzoate into prenylhydroquinone and is likely involved in the biosynthesis of vibralactones and other meroterpenoids in the basidiomycete Boreostereum vibrans. Feeding of 3-allyl-4-hydroxybenzylalcohol, an analogue of the vibralactone pathway intermediate 3-prenyl-4-hydroxybenzylalcohol, generated 20 analogues with different scaffolds. This demonstrated divergent pathways to skeletally distinct compounds initiating from a single precursor, thus providing the first insight into a novel biosynthetic pathway for 3-substituted γ-butyrolactones from a shikimate origin.

Procalcitonin Is a Stronger Predictor of Long-Term Functional Outcome and Mortality than High-Sensitivity C-Reactive Protein in Patients with Ischemic Stroke.

Inflammatory markers have been associated with functional outcome and mortality of stroke. We investigated the changes in procalcitonin (PCT) and high-sensitivity C-reactive protein (Hs-CRP) levels during the acute period of ischemic stroke and evaluated the relationship between these levels and the long-term functional outcome and mortality. We prospectively studied 376 patients with acute ischemic stroke (AIS) who were admitted within 24 h after the onset of symptoms. PCT, Hs-CRP, and NIH Stroke Scale (NIHSS) were measured at the time of admission. Long-term functional outcome were measured by modified Rankin scale (mRS) at 1 year after admission. The correlations between the levels of PCT, Hs-CRP, and mortality at 1 year after stroke onset were analyzed. Patients with poor with functional outcome and non-survivors had significantly increased PCT and Hs-CRP levels on admission. Multivariate logistic regression analysis showed that PCT was an independent prognostic marker of 1-year functional outcome and death [odds ratio (OR) 2.33 (95% CI, 1.33-3.44) and 3.11 (2.02-4.43), respectively, P < 0.0001 for both, adjusted for age, NIHSS, other predictors, and vascular risk factors] in patients with AIS. The area under the receiver operating characteristic curve of PCT was 0.77 (95% CI, 0.72-0.83) for functional outcome and 0.88 (95% CI, 0.84-0.93) for mortality. PCT improved the area under the receiver operating characteristic curve of the NIHSS score for functional outcome from 0.74 (95% CI, 0.66-0.81) to 0.85 (95% CI, 0.76-0.92; P < 0.0001) and for mortality from 0.77 (95% CI, 0.70-0.83) to 0.94 (95% CI, 0.89-0.97; P < 0.0001). Serum level of PCT at admission was an independent predictor of long-term functional outcome and mortality after ischemic stroke in Chinese sample.

Two new phenol derivatives from Stereum hirsutum FP-91666.

Two new phenol derivatives, 2-(3-methyl-2-buten-1-yl)-4-methoxyethyl-phenol (1) and 5-hydroxy-4-(hydroxymethyl)-2-(3-methylbut-2-en-1-yl)cyclohex-4-en-1-one (2), together with eight known compounds consisting of phenol derivatives (3 and 4), niacinamide (5), and five ergosta type compounds (6-10), were isolated from solid fermentation products of Stereum hirsutum FP-91666. Two new structures were elucidated by extensive spectroscopic methods, including 1D NMR and 2D NMR, and HR-EI-MS experiments.