An End-to-End Platform for Digital Pathology Using Hyperspectral Autofluorescence Microscopy and Deep Learning-Based Virtual Histology.

Conventional histopathology involves expensive and labor-intensive processes that often consume tissue samples, rendering them unavailable for other analyses. We present a novel end-to-end workflow for pathology powered by hyperspectral microscopy and deep learning. First, we developed a custom hyperspectral microscope to nondestructively image the autofluorescence of unstained tissue sections. We then trained a deep learning model to use autofluorescence to generate virtual histologic stains, which avoids the cost and variability of chemical staining procedures and conserves tissue samples. We showed that the virtual images reproduce the histologic features present in the real-stained images using a randomized nonalcoholic steatohepatitis (NASH) scoring comparison study, where both real and virtual stains are scored by pathologists (D.T., A.D.B., R.K.P.). The test showed moderate-to-good concordance between pathologists' scoring on corresponding real and virtual stains. Finally, we developed deep learning-based models for automated NASH Clinical Research Network score prediction. We showed that the end-to-end automated pathology platform is comparable with an independent panel of pathologists for NASH Clinical Research Network scoring when evaluated against the expert pathologist consensus scores. This study provides proof of concept for this virtual staining strategy, which could improve cost, efficiency, and reliability in pathology and enable novel approaches to spatial biology research.

Kilohertz two-photon brain imaging in awake mice.

Two-photon microscopy is a mainstay technique for imaging in scattering media and normally provides frame-acquisition rates of ~10-30 Hz. To track high-speed phenomena, we created a two-photon microscope with 400 illumination beams that collectively sample 95,000-211,000 µm2 areas at rates up to 1 kHz. Using this microscope, we visualized microcirculatory flow, fast venous constrictions and neuronal Ca2+ spiking with millisecond-scale timing resolution in the brains of awake mice.

Propofol Causes Sustained Ca2+ Elevation in Endothelial Cells by Stimulating Ryanodine Receptor and Suppressing Plasmalemmal Ca2+ Pump.

ABSTRACT: Propofol, a general anesthetic administered intravenously, may cause pain at the injection site. The pain is in part due to irritation of vascular endothelial cells. We here investigated the effects of propofol on Ca2+ transport and pain mediator release in human umbilical vein endothelial cells (EA.hy926). Propofol mobilized Ca2+ from cyclopiazonic acid (CPA)-dischargeable pool but did not cause Ca2+ release from the lysosomal Ca2+ stores. Propofol-elicited Ca2+ release was suppressed by 100 µM ryanodine, suggesting the participation of ryanodine receptor channels. Propofol did not affect ATP-triggered Ca2+ release but abolished the Ca2+ influx triggered by ATP; in addition, propofol also suppressed store-operated Ca2+ entry elicited by CPA. Ca2+ clearance during CPA-induced Ca2+ discharge was unaffected by a low Na+ (50 mM) extracellular solution, but strongly suppressed by 5 mM La3+ (an inhibitor of plasmalemmal Ca2+ pump), suggesting Ca2+ extrusion was predominantly through the plasmalemmal Ca2+ pump. Propofol mimicked the effect of La3+ in suppressing Ca2+ clearance. Propofol also stimulated release of pain mediators, namely, reactive oxygen species and bradykinin. Our data suggest propofol elicited Ca2+ release and repressed Ca2+ clearance, causing a sustained cytosolic [Ca2+]i elevation. The latter may cause reactive oxygen species and bradykinin release, resulting in pain.

Three-photon imaging of mouse brain structure and function through the intact skull.

Optical imaging through the intact mouse skull is challenging because of skull-induced aberrations and scattering. We found that three-photon excitation provided improved optical sectioning compared with that obtained with two-photon excitation, even when we used the same excitation wavelength and imaging system. Here we demonstrate three-photon imaging of vasculature through the adult mouse skull at >500-µm depth, as well as GCaMP6s calcium imaging over weeks in cortical layers 2/3 and 4 in awake mice, with 8.5 frames per second and a field of view spanning hundreds of micrometers.

A basal level of γ-linolenic acid depletes Ca2+ stores and induces endoplasmic reticulum and oxidative stresses to cause death of breast cancer BT-474 cells.

Gamma-linolenic acid (GLA), a natural fatty acid obtained from oils of various vegetables and seeds, has been demonstrated as an anticancer agent. In this work, we investigated the anticancer effects of GLA on breast cancer BT-474 cells. GLA at 30 µM, a concentration reportedly within the range of circulating concentrations in clinical studies, caused apoptotic cell death. GLA caused an elevation in mitochondrial Ca2+ level and a decrease in mitochondrial membrane potential. GLA treatment depleted cyclopiazonic acid (CPA)-sensitive Ca2+ store and triggered substantial Ca2+ influx. Intracellular Ca2+ release triggered by GLA was suppressed by 3 µM xestospongin C (XeC, IP3 receptor-channel blocker) and 100 µM ryanodine (ryanodine receptor-channel blocker), suggesting that the Ca2+ release was via IP3 receptor-channel and ryanodine receptor-channel. Increased expressions of p-eIF2α and CHOP were observed in GLA-treated cells, suggesting GLA-treated cells had increased expressions of p-eIF2α and CHOP, which suggest endoplasmic reticulum (ER) stress. In addition, GLA elicited increased production of reactive oxygen species. Taken together, our results suggest a basal level of GLA induced apoptotic cell death by causing Ca2+ overload, mitochondrial dysfunction, Ca2+ store depletion, ER stress, and oxidative stress. This is the first report to show that GLA caused Ca2+ store depletion and ER stress. GLA-induced Ca2+ store depletion resulted from opening of IP3 receptor-channel and ryanodine receptor-channel.

Attenuating HIV Tat/TAR-mediated protein expression by exploring the side chain length of positively charged residues.

RNA is a drug target involved in diverse cellular functions and viral processes. Molecules that inhibit the HIV TAR RNA-Tat protein interaction may attenuate Tat/TAR-dependent protein expression and potentially serve as anti-HIV therapeutics. By incorporating positively charged residues with mixed side chain lengths, we designed peptides that bind TAR RNA with enhanced intracellular activity. Tat-derived peptides that were individually substituted with positively charged residues with varying side chain lengths were evaluated for TAR RNA binding. Positively charged residues with different side chain lengths were incorporated at each Arg and Lys position in the Tat-derived peptide to enhance TAR RNA binding. The resulting peptides showed enhanced TAR RNA binding affinity, cellular uptake, nuclear localization, proteolytic resistance, and inhibition of intracellular Tat/TAR-dependent protein expression compared to the parent Tat-derived peptide with no cytotoxicity. Apparently, the enhanced inhibition of protein expression by these peptides was not determined by RNA binding affinity, but by proteolytic resistance. Despite the high TAR binding affinity, a higher binding specificity would be necessary for practical purposes. Importantly, altering the positively charged residue side chain length should be a viable strategy to generate potentially useful RNA-targeting bioactive molecules.

Effect of side chain length on intrahelical interactions between carboxylate- and guanidinium-containing amino acids.

The charge-containing hydrophilic functionalities of encoded charged amino acids are linked to the backbone via different numbers of hydrophobic methylenes, despite the apparent electrostatic nature of protein ion pairing interactions. To investigate the effect of side chain length of guanidinium- and carboxylate-containing residues on ion pairing interactions, α-helical peptides containing Zbb-Xaa (i, i + 3), (i, i + 4) and (i, i + 5) (Zbb = carboxylate-containing residues Aad, Glu, Asp in decreasing length; Xaa = guanidinium residues Agh, Arg, Agb, Agp in decreasing length) sequence patterns were studied by circular dichroism spectroscopy (CD). The helicity of Aad- and Glu-containing peptides was similar and mostly pH independent, whereas the helicity of Asp-containing peptides was mostly pH dependent. Furthermore, the Arg-containing peptides consistently exhibited higher helicity compared to the corresponding Agp-, Agb-, and Agh-containing peptides. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb-Xaa (i, i + 3) and (i, i + 4) interactions mainly involved the χ 1 dihedral combinations (g+, g+) and (g-, g+), respectively. These low energy conformations were also observed in intrahelical Asp-Arg and Glu-Arg salt bridges of natural proteins. Accordingly, Asp and Glu provides variation in helix characteristics associated with Arg, but Aad does not provide features beyond those already delivered by Glu. Importantly, nature may have chosen the side chain length of Arg to support helical conformations through inherent high helix propensity coupled with stabilizing intrahelical ion pairing interactions with the carboxylate-containing residues.

Effect of each guanidinium group on the RNA recognition and cellular uptake of Tat-derived peptides.

The six arginine (Arg) residues in the human immunodeficiency virus transactivator of transcription protein (HIV Tat protein) basic region (residues 47-57) are crucial for two bioactivities: RNA recognition and cellular uptake. Herein, we report a systematic study to investigate the role of the guanidinium group on Arg at each position in Tat-derived peptides for the two bioactivities. Tat-derived peptides, in which each guanidinium-bearing arginine was replaced with a urea-bearing citrulline (Cit) or an ammonium-bearing Lys, were synthesized by solid phase peptide synthesis. RNA recognition of the peptides was studied by electrophoretic mobility shift assays, and cellular uptake into Jurkat cells was determined by flow cytometry. Our results showed that removing the positive charge and altering the hydrogen bonding capacity of Arg affect the two biological functions differently. Furthermore, the effects are position dependent. These findings should be useful for the development of functional molecules containing guanidinium, urea, and ammonium groups for RNA recognition to affect biological processes and for cellular uptake for drug delivery.

Overcoming the Blood-Brain Tumor Barrier with Docetaxel-Loaded Mesoporous Silica Nanoparticles for Treatment of Temozolomide-Resistant Glioblastoma.

While temozolomide (TMZ) has been a cornerstone in the treatment of newly diagnosed glioblastoma (GBM), a significant challenge has been the emergence of resistance to TMZ, which compromises its clinical benefits. Additionally, the nonspecificity of TMZ can lead to detrimental side effects. Although TMZ is capable of penetrating the blood-brain barrier (BBB), our research addresses the need for targeted therapy to circumvent resistance mechanisms and reduce off-target effects. This study introduces the use of PEGylated mesoporous silica nanoparticles (MSN) with octyl group modifications (C8-MSN) as a nanocarrier system for the delivery of docetaxel (DTX), providing a novel approach for treating TMZ-resistant GBM. Our findings reveal that C8-MSN is biocompatible in vitro, and DTX@C8-MSN shows no hemolytic activity at therapeutic concentrations, maintaining efficacy against GBM cells. Crucially, in vivo imaging demonstrates preferential accumulation of C8-MSN within the tumor region, suggesting enhanced permeability across the blood-brain tumor barrier (BBTB). When administered to orthotopic glioma mouse models, DTX@C8-MSN notably prolongs survival by over 50%, significantly reduces tumor volume, and decreases side effects compared to free DTX, indicating a targeted and effective approach to treatment. The apoptotic pathways activated by DTX@C8-MSN, evidenced by the increased levels of cleaved caspase-3 and PARP, point to a potent therapeutic mechanism. Collectively, the results advocate DTX@C8-MSN as a promising candidate for targeted therapy in TMZ-resistant GBM, optimizing drug delivery and bioavailability to overcome current therapeutic limitations.

Receptor Ligand-Free Mesoporous Silica Nanoparticles: A Streamlined Strategy for Targeted Drug Delivery across the Blood-Brain Barrier.

Mesoporous silica nanoparticles (MSNs) represent a promising avenue for targeted brain tumor therapy. However, the blood-brain barrier (BBB) often presents a formidable obstacle to efficient drug delivery. This study introduces a ligand-free PEGylated MSN variant (RMSN25-PEG-TA) with a 25 nm size and a slight positive charge, which exhibits superior BBB penetration. Utilizing two-photon imaging, RMSN25-PEG-TA particles remained in circulation for over 24 h, indicating significant traversal beyond the cerebrovascular realm. Importantly, DOX@RMSN25-PEG-TA, our MSN loaded with doxorubicin (DOX), harnessed the enhanced permeability and retention (EPR) effect to achieve a 6-fold increase in brain accumulation compared to free DOX. In vivo evaluations confirmed the potent inhibition of orthotopic glioma growth by DOX@RMSN25-PEG-TA, extending survival rates in spontaneous brain tumor models by over 28% and offering an improved biosafety profile. Advanced LC-MS/MS investigations unveiled a distinctive protein corona surrounding RMSN25-PEG-TA, suggesting proteins such as apolipoprotein E and albumin could play pivotal roles in enabling its BBB penetration. Our results underscore the potential of ligand-free MSNs in treating brain tumors, which supports the development of future drug-nanoparticle design paradigms.

Effect of charged amino acid side chain length at non-hydrogen bonded strand positions on ß-hairpin stability.

ß-Sheets have been implicated in various neurological disorders, and ∼20% of protein residues adopt a sheet conformation. Therefore, studies on the structural origin of sheet stability can provide fundamental knowledge with potential biomedical applications. Oppositely charged amino acids are frequently observed across one another in antiparallel ß-sheets. Interestingly, the side chains of natural charged amino acids Asp, Glu, Arg, Lys have different numbers of hydrophobic methylenes linking the backbone to the hydrophilic charged functionalities. To explore the inherent effect of charged amino acid side chain length on antiparallel sheets, the stability of a designed hairpin motif containing charged amino acids with varying side chain lengths at non-hydrogen bonded positions was studied. Peptides with the guest position on the N-terminal strand and the C-terminal strand were investigated by NMR methods. The charged amino acids (Xaa) included negatively charged residues with a carboxylate group (Asp, Glu, Aad in increasing length), positively charged residues with an ammonium group (Dap, Dab, Orn, Lys in increasing length), and positively charged residues with a guanidinium group (Agp, Agb, Arg, Agh in increasing length). The fraction folded and folding free energy for each peptide were derived from the chemical shift deviation data. The stability of the peptides with the charged residues at the N-terminal guest position followed the trends: Asp > Glu > Aad, Dap < Dab < Orn ∼ Lys, and Agb < Arg < Agh < Agp. The stability of the peptides with the charged residues at the C-terminal guest position followed the trends: Asp < Glu < Aad, Dap ∼ Dab < Orn ∼ Lys, and Agb < Arg ∼ Agp < Agh. These trends were rationalized by thermodynamic sheet propensity and cross-strand interactions.

Altering the Tat-derived peptide bioactivity landscape by changing the arginine side chain length.

Mutations of proteins with dual activities that lead to enhancement of one activity are frequently accompanied by attenuation of the other activity. However, this mutational negative trade-off phenomenon typically only involves the canonical 20 amino acids. To test the effect of non-canonical amino acids on the negative trade-off phenomenon, two bioactivities of HIV-1 Tat-derived peptides were monitored upon changing the Arg side chain length. In contrast to the expected mutational negative trade-off, shortening Arg by one methylene resulted in both higher TAR RNA binding specificity and higher cellular uptake. These results suggest that introducing previously unexploited building blocks, even if the difference is only one methylene, can alter the peptide bioactivity landscape leading to the enhancement of multiple bioactivities.

Hybrid photoacoustic and fast super-resolution ultrasound imaging.

The combination of photoacoustic (PA) imaging and ultrasound localization microscopy (ULM) with microbubbles has great potential in various fields such as oncology, neuroscience, nephrology, and immunology. Here we developed an interleaved PA/fast ULM imaging technique that enables super-resolution vascular and physiological imaging in less than 2 seconds per frame in vivo. By using sparsity-constrained (SC) optimization, we accelerated the frame rate of ULM up to 37 times with synthetic data and 28 times with in vivo data. This allows for the development of a 3D dual imaging sequence with a commonly used linear array imaging system, without the need for complicated motion correction. Using the dual imaging scheme, we demonstrated two in vivo scenarios challenging to image with either technique alone: the visualization of a dye-labeled mouse lymph node showing nearby microvasculature, and a mouse kidney microangiography with tissue oxygenation. This technique offers a powerful tool for mapping tissue physiological conditions and tracking the contrast agent biodistribution non-invasively.

Afatinib triggers a Ni2+ -resistant Ca2+ influx pathway in A549 non-small cell lung cancer cells.

Afatinib is used to treat non-small cell lung cancer cells (NSCLC), and its mechanism involves irreversible inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase. In this study, we examined if afatinib had cytotoxic action against NSCLC other than inhibition of tyrosine kinase. Afatinib (1-30 µM) caused apoptotic death in A549 NSCLC in a concentration-dependent manner. Afatinib triggered Ca2+ influx without causing Ca2+ release, and the Ca2+ influx was unaffected by sodium orthovanadate (SOV, an inhibitor of tyrosine phosphatase), suggesting that afatinib-triggered Ca2+ response was unrelated to its inhibition of tyrosine kinase. Addition of afatinib also promoted Mn2+ influx. Ca2+ influx triggered by afatinib was resistant to SKF96365 and ruthenium red (two general blockers of TRP channels) and, unexpectedly, Ni2+ (a non-specific Ca2+ channel blocker). Afatinib caused an increase in mitochondrial Ca2+ level, an initial mitochondrial hyperpolarization (4 h) and followed by mitochondrial potential collapse (24-48 h). Afatinib-induced cell death was slightly but significantly alleviated in low extracellular Ca2+ condition or under pharmacological block of mitochondrial permeability transition pore (MPTP) opening by cyclosporin A. Therefore, in addition to tyrosine kinase inhibition as a major anti-cancer mechanism of afatinib, stimulation of an atypical Ca2+ influx pathway, mitochondrial Ca2+ overload, and potential collapse in part contribute to afatinib-induced cell death.

Ultracold fermionic Feshbach molecules of 23Na40K.

We report on the formation of ultracold weakly bound Feshbach molecules of 23Na40K, the first fermionic molecule that is chemically stable in its absolute ground state. The lifetime of the nearly degenerate molecular gas exceeds 100 ms in the vicinity of the Feshbach resonance. The measured dependence of the molecular binding energy on the magnetic field demonstrates the open-channel character of the molecules over a wide field range and implies significant singlet admixture. This will enable efficient transfer into the singlet vibrational ground state, resulting in a stable molecular Fermi gas with strong dipolar interactions.

Helix formation and capping energetics of arginine analogs with varying side chain length.

Arginine (Arg) has been used for recognizing negatively charged biological molecules, cell penetration, and oligosaccharide mass signal enhancement. The versatility of Arg has inspired the need to develop Arg analogs and to research the structural effects of incorporating Arg analogs. Accordingly, we investigated the effect of Arg side chain length on helix formation by studying 12 Ala-based peptides containing the Arg analogs (S)-2-amino-6-guanidino-hexanoic acid (Agh), (S)-2-amino-4-guanidinobutyric acid (Agb), and (S)-2-amino-3-guanidinopropionic acid (Agp). Solid phase guanidinylation with orthogonal protection strategies was necessary to synthesize Agb- and Agp-containing peptides using Fmoc-based chemistry. The fraction helix for the peptides was determined by circular dichroism spectroscopy, and used to derive the statistical mechanical parameters and energetics for N-capping, C-capping, and helix propagation (propensity). All four Arg analogs were unfavorable for N-capping. The C-cap parameter followed the trend AgpAgh, highlighting the uniqueness of the Arg side chain length in helix formation. Molecular mechanics calculations and a survey on protein structures were consistent with the experimental results. Furthermore, calculations and survey both showed that the g- conformation for the χ1 dihedral was present for the first two residues at the N-terminus of helices, but not favored in the center or C-terminus of helices due to sterics. These results should serve as the foundation for developing Arg-related bioactive compounds and technologies.

Clinical implication of genetic composition and molecular mechanism on treatment strategies of HER2-positive breast cancers.

The current clinical management model of HER2-positive breast cancers is commonly based on guidelines, which in turn are based on the design and outcome of clinical trials. While this model is useful to most practicing clinicians, the treatment outcome of individual patient is not certain at the start of treatment. As the understanding of the translational research of carcinogenesis and the related changes in cancer genetics and tumor microenvironment during treatment is critical in the selection of right choice of treatment to maximize the successful clinical outcome for the patient, this review article intends to discuss the latest developments in the genetic and molecular mechanisms of cancer progression and treatment resistance, and how they influence the planning of the treatment strategies of HER2-positive breast cancers.

Nobiletin, a citrus flavonoid, suppresses invasion and migration involving FAK/PI3K/Akt and small GTPase signals in human gastric adenocarcinoma AGS cells.

Nobiletin, a compound isolated from citrus fruits, is a polymethoxylated flavone derivative shown to have anti-inflammatory, antitumor, and neuroprotective properties. This study has investigated that nobiletin exerted inhibitory effects on the cell adhesion, invasion, and migration abilities of a highly metastatic AGS cells under non-cytotoxic concentrations. Data also showed nobiletin could inhibit the activation of focal adhesion kinase (FAK) and phosphoinositide-3-kinase/Akt (PI3K/Akt) involved in the downregulation of the enzyme activities, protein expressions, messenger RNA levels of matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-2 (MMP-9). Also, our data revealed that nobiletin inhibited FAK/PI3K/Akt with concurrent reduction in the protein expressions of Ras, c-Raf, Rac-1, Cdc42, and RhoA by western blotting, whereas the protein level of RhoB increased progressively. Otherwise, nobiletin-treated AGS cells showed tremendously decreased in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, nobiletin significantly decreased the levels of phospho-Akt and MMP-2/9 in Akt1-cDNA-transfected cells concomitantly with a marked reduction in cell invasion and migration. These results suggest that nobiletin can reduce invasion and migration of AGS cells, and such a characteristic may be of great value in the development of a potential cancer therapy.

Aqueous Extract of Shi-Liu-Wei-Liu-Qi-Yin Induces G2/M Phase Arrest and Apoptosis in Human Bladder Carcinoma Cells via Fas and Mitochondrial Pathway.

Shi-Liu-Wei-Liu-Qi-Yin (SLWLQY) was traditionally used to treat cancers. However, scientific evidence of the anticancer effects still remains undefined. In this study, we aimed to clarify the possible mechanisms of SLWLQY in treating cancer. We evaluated the effects of SLWLQY on apoptosis-related experiments inducing in TSGH-8301 cells by (i) 3-(4,5-dimethylthiazol-zyl)-2,5-diphenylterazolium bromide (MTT) for cytotoxicity; (ii) cell-cycle analysis and (iii) western blot analysis of the G2/M-phase and apoptosis regulatory proteins. Human bladder carcinoma TSGH-8301 cells were transplanted into BALB/c nude mice as a tumor model for evaluating the antitumor effect of SLWLQY. Treatment of SLWLQY resulted in the G2/M phase arrest and apoptotic death in a dose-dependent manner, accompanied by a decrease in cyclin-dependent kinases (cdc2) and cyclins (cyclin B1). SLWLQY stimulated increases in the protein expression of Fas and FasL, and induced the cleavage of caspase-3, caspase-9 and caspase-8. The ratio of Bax/Bcl(2) was increased by SLWLQY treatment. SLWLQY markedly reduced tumor size in TSGH-8301 cells-xenografted tumor tissues. In the tissue specimen, SLWLQY up-regulated the expression of Fas, FasL and Bax proteins, and down-regulated Bcl(2) as well as in in vitro assay. Our results showed that SLWLQY reduced tumor growth, caused cell-cycle arrest and apoptosis in TSGH-8301 cells via the Fas and mitochondrial pathway.

In Vitro Activity of 2-methoxy-1,4-naphthoquinone and Stigmasta-7,22-diene-3ß-ol from Impatiens balsamina L. against Multiple Antibiotic-Resistant Helicobacter pylori.

Infection with Helicobacter pylori is strongly associated with gastric cancer and gastric adenocarcinoma. WHO classified H. pylori as a group 1 carcinogen in 1994. Impatiens balsamina L. has been used as indigenous medicine in Asia for the treatment of rheumatism, fractures and fingernail inflammation. In this study, we isolated anti-H. pylori compounds from this plant and investigated their anti- and bactericidal activity. Compounds of 2-methoxy-1,4-naphthoquinone (MeONQ) and stigmasta-7,22-diene-3ß-ol (spinasterol) were isolated from the pods and roots/stems/leaves of I. balsamina L., respectively. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for MeONQ were in the ranges of 0.156-0.625 and 0.313-0.625 µg mL(-1), respectively, and in the ranges of 20-80 µg mL(-1) both of MICs and MBCs for spinasterol against antibiotic (clarithromycin, metronidazole and levofloxacin) resistant H. pylori. Notably, the activity of MeONQ was equivalent to that of amoxicillin (AMX). The bactericidal H. pylori action of MeONQ was dose-dependent. Furthermore, the activity of MeONQ was not influenced by the environmental pH values (4-8) and demonstrated good thermal (121°C for 15 min) stability. MeONQ abounds in the I. balsamina L. pod at the level of 4.39% (w/w db). In conclusion, MeONQ exhibits strong potential to be developed as a candidate agent for the eradication of H. pylori infection.