Simultaneous Measurement and Distribution Analysis of Urinary Nicotine, Cotinine, Trans-3'-Hydroxycotinine, Nornicotine, Anabasine, and Total Nicotine Equivalents in a Large Korean Population.

Measurement of multiple nicotine metabolites and total nicotine equivalents (TNE) might be a more reliable strategy for tobacco exposure verification than measuring single urinary cotinine alone. We simultaneously measured nicotine, cotinine, 3-OH cotinine, nornicotine, and anabasine using 19,874 urine samples collected from the Korean National Health and Nutrition Examination Survey. Of all samples, 18.6% were positive for cotinine, 17.4% for nicotine, 17.3% for nornicotine, 17.6% for 3-OH cotinine, and 13.2% for anabasine. Of the cotinine negative samples, less than 0.3% were positive for all nicotine metabolites, but not for anabasine (5.7%). The agreement of the classification of smoking status by cotinine combined with nicotine metabolites was 0.982-0.994 (Cohen's kappa). TNE3 (the molar sum of urinary nicotine, cotinine, and 3-OH cotinine) was most strongly correlated with cotinine compared to the other nicotine metabolites; however, anabasine was less strongly correlated with other biomarkers. Among anabasine-positive samples, 30% were negative for nicotine or its metabolites, and 25% were undetectable. Our study shows that the single measurement of urinary cotinine is simple and has a comparable classification of smoking status to differentiate between current smokers and non-smokers relative to the measurement of multiple nicotine metabolites. However, measurement of multiple nicotine metabolites and TNE3 could be useful for monitoring exposure to low-level or secondhand smoke exposure and for determining individual differences in nicotine metabolism. Geometric or cultural factors should be considered for the differentiation of tobacco use from patients with nicotine replacement therapy by anabasine.

Hypoxia-Directed and Self-Immolative Theranostic Agent: Imaging and Treatment of Cancer and Bacterial Infections.

The impact of bacteria on cancer progression and treatment is becoming increasingly recognized. Cancer-associated bacteria are linked to metastases, reduced efficacy, and survival challenges. In this study, we present a sensitive hypoxia-activated prodrug, NR-NO2, which comprises an antibiotic combined with a chemotherapeutic. This prodrug demonstrates rapid and robust fluorescence enhancement and exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria as well as tumor cells. Upon activation, NR-NO2 produces a distinct "fluorescence-on" signal, enabling real-time drug release monitoring. By leveraging elevated nitroreductase in cancer cells, NR-NO2 gives rise to heightened bacterial cytotoxicity while sparing normal cells. In A549 solid tumor-bearing mice, NR-NO2 selectively accumulated at tumor sites, displaying fluorescence signals under hypoxia superior to those of a corresponding prodrug-like control. These findings highlight the potential of NR-NO2 as a promising cancer therapy prodrug that benefits from targeted release, antibacterial impact, and imaging-based guidance.

Effect of Two Cystatin C Reagents and Four Equations on Glomerular Filtration Rate Estimations After Standardization.

Background: Serum cystatin C (cysC), which is less affected by sex, race, and muscle mass than creatinine, is a useful biomarker of the estimated glomerular filtration rate (eGFR). The standardization of cysC measurements remains controversial, although a certified reference material (ERM-DA471/IFCC) is available. Moreover, the effect of combinations of cysC reagents and equations for eGFR is unclear. Methods: We conducted a simulation analysis of cysC measured using two reagents standardized against ERM-DA471/IFCC-Gentian cystatin C immunoassay (Gentiancys; GentianAS, Moss, Norway) and Roche Tina-quant Cystatin C Gen.2 (Rochecys; Roche, Mannheim, Germany)-on a Cobas c702 system (Roche) and eGFR generated by eight combinations of four equations: 2012 cystatin C-based Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPIcys); the Caucasian, Asian, pediatric, and adult equation (CAPAeq); full age spectrum equation (FASeq); and 2023 cystatin C-based European Kidney Function Consortium equation (EKFCcys). Results: A total of 148 participants (mean age, 60.5±14.5 years; 43% female) were enrolled. The mean cysC was 1.72±1.44 mg/L for Gentiancys and 1.71±1.35 mg/L for Rochecys. Regression analysis showed concordance between the reagents within 0.85-4.40 mg/L when using ±7.61% total allowable error. Lin's concordance correlation coefficient of eGFR, by combining the measuring system and equation, varied from 0.73 to 1.00. Conclusions: The equivalence of cysC values at low concentrations (<0.85 mg/L) between the two reagents was unsatisfactory. Results obtained with different measurement systems could lead to larger differences in eGFR varying with the combination.

Anti-inflammatory effects of Allium cepa L. peel extracts via inhibition of JAK-STAT pathway in LPS-stimulated RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Allium cepa L. (A. cepa) is one of the oldest cultivated plants in the world. A. cepa has been used in traditional folk medicine to treat inflammatory disease in several regions, such as Palestine and Serbia. A. cepa peel has a higher content of flavonoids, such as quercetin, than the edible parts. These flavonoids alleviate inflammatory diseases. However, the anti-inflammatory effects of A. cepa peel extract-obtained using various extraction methods-and their underlying mechanisms require further investigation. AIM OF THE STUDY: Although research to find safe anti-inflammatory substances in various natural products has been actively conducted for many years, it is important to continue identifying potential anti-inflammatory effects in natural materials. The purpose of this study was to investigate the ethnopharmacological properties of the A. cepa peel extract, whose efficacy when obtained through different extraction methods and underlying action mechanisms is not well known. The present study specifically aimed to observe the anti-inflammatory effects of the A. cepa peel extracts obtained using various extraction methods and the related detailed mechanisms of A. cepa peel extracts in lipopolysaccharide (LPS)-induced RAW264.7 cells. MATERIALS AND METHODS: The total flavonoid content of the A. cepa peel extracts was determined the diethylene glycol colorimetric method and measured using a calibration curve prepared using quercetin as a standard solution. The antioxidant activity was evaluated using the ABTS assay, and cytotoxicity was measured using the MTT assay. NO production was measured using Griess reagent. Protein levels were measured by western blotting, and mRNA expression was measured by RT-qPCR. Secreted cytokines were analyzed using ELISA or cytokine arrays. In the GSE160086 dataset, we calculated Z-scores for individual genes of interest and displayed using a heat map. RESULTS: Of the three A. cepa peel extracts obtained using different extraction methods, the A. cepa peel 50% EtOH extract (AP50E) was the most effective at inhibiting LPS-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Furthermore, AP50E significantly reduced the levels of pro-inflammation cytokines interleukin (IL)-1α, IL-1ß, IL-6, and IL-27. Additionally, AP50E directly inhibited the Janus kinase-signaling transducer and activator of transcription (JAK-STAT) pathway. CONCLUSIONS: These results showed that AP50E exhibited an anti-inflammatory effect in LPS-induced RAW264.7 mouse macrophages by directly inhibiting JAK-STAT signaling. Based on these findings, we propose AP50E as a potential candidate for the development of preventive or therapeutic agents against inflammatory diseases.

Pharmacological anti-tumor effects of natural Chamaecyparis obtusa (siebold & zucc.) endl. Leaf extracts on breast cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (C. obtusa, cypress species) is a plant that grows mainly in the temperate Northern Hemisphere and has long been used as a traditional anti-inflammatory treatment in East Asia. C. obtusa contains phytoncides, flavonoids, and terpenes, which have excellent anti-cancer effects and have been reported to prevent the progression of various cancers. However, the detailed mechanisms underlying the anti-cancer effects of C. obtusa extracts are unknown. AIM OF THE STUDY: We sought to confirm the anti-cancer effects of C. obtusa leaf extracts and to reveal the mechanism of action, with the possibility of its application in the treatment or prevention of cancer. MATERIAL &METHODS: The cytotoxicity of C. obtusa leaf extracts was confirmed using an MTT assay. Intracellular changes in protein levels were measured by immunoblotting, and mRNA levels were measured with qRT-PCR. Wound healing assay and transwell migration assay were used to evaluate the metastatic potential of breast cancer cells. The extract-induced apoptosis was observed using IncuCyte Annexin V Red staining analysis. A syngeneic breast cancer mouse model was established by injecting 4T1-Luc mouse breast cancer cells into the fat pad of female BALB/c mice, and the extract was administered orally. Luciferin solution was injected intraperitoneally to assess primary tumor development and metastasis by bioluminescence. RESULTS: C. obtusa leaf extracts were extracted with boiling water, 70% EtOH, and 99% EtOH. Among the extracts, the 99% EtOH extract of C. obtusa leaf (CO99EL) most clearly inhibited the tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (pY-STAT3) in MDA-MB-231 breast cancer cells at a concentration of 25 and 50 µg/mL. In addition, CO99EL strongly inhibited not only endogenous pY-STAT3 levels but also IL-6-induced STAT3 activation in various types of cancer cells, including breast cancer. CO99EL inhibited metastatic potential by downregulating the expression of N-cadherin, fibronectin, TWIST, MMP2, and MMP9 in MDA-MB-231 breast cancer cells. CO99EL also induced apoptotic cell death by increasing cleaved caspase-3 and decreasing anti-apoptotic proteins Bcl-2 and Bcl-xL. In an in vivo syngeneic breast cancer mouse model, 100 mg/kg CO99EL suppressed tumor growth and induced apoptosis of cancer cells. Moreover, CO99EL significantly inhibited lung metastasis from primary breast cancer. CONCLUSIONS: Our study demonstrated that 100 mg/kg CO99EL has potent anti-tumor effects against breast cancer, thus suggesting that 100 mg/kg CO99EL has potential applications in the treatment and prevention of breast cancer.

Comprehensive Evaluation of the NeoBase 2 Non-derivatized MSMS Assay and Exploration of Analytes With Significantly Different Concentrations Between Term and Preterm Neonates.

Background: Despite the popularity of the NeoBase 2 Non-derivatized MSMS assay (PerkinElmer, Turku, Finland), there are no reports of its comprehensive evaluation, including the ability to distinguish transient tyrosinemia of the newborn (TTN) from tyrosinemia type 1 (TYR 1) using succinylacetone (SUAC). No newborn screening (NBS) cutoffs for preterm neonates in the Korean population have been suggested. We evaluated the NeoBase 2 assay and identified analytes requiring different cutoffs in preterm neonates. Methods: Residual NBS dried blood spot samples and proficiency testing (PT) materials of the Newborn Screening Quality Assurance Program and the Korean Association of External Quality Assessment Service were used. Precision, accuracy, limit of detection (LOD), lower limit of quantification (LLOQ), linearity, recovery, carryover, and performance of SUAC were evaluated. Cutoffs were determined, and analytes requiring different cutoffs in preterm neonates were investigated. Results: Mean CVs for within-run and between-day precision were within 15%. Accuracy analysis indicated high agreement with in-house derivatized assay results and results of other PT participants. All analytes demonstrated acceptable LOD, LLOQ, and linearity. Recoveries were acceptable, except for SUAC. Carryover was negligible. Cutoffs were established for all analytes; Tyr, adenosine, and C20:0-lysophosphatidylcholine required different cutoffs in preterm neonates. Differential diagnosis of TYR 1 and TTN was successful with simultaneous Tyr and SUAC measurement. Conclusions: The NeoBase 2 assay demonstrated satisfactory performance. The additional analytes provide a wider diagnostic coverage, and the simultaneous measurement of Tyr and SUAC is efficient in excluding TYR 1. The new cutoffs for preterm neonates may decrease false-positive rates, without compromising diagnostic sensitivity.

Diagnostic Performance Evaluation of the Novel Index Combining Urinary Cotinine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol in Smoking Status Verification and Usefulness for Trend Monitoring of Tobacco Smoking Exposure.

During the last decade in Korea, urinary cotinine concentrations in non-current smokers have decreased, making it difficult to distinguish secondhand smoke (SHS) exposure from nonsmokers because of overlapping values between non-current smokers with and without SHS exposure. Additionally, the importance of smoking status verification to avoid misclassification is increasing with the increased use of e-cigarettes. We developed a novel index combining urinary cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and evaluated its diagnostic performance for the classification of smoking status using the KNHANES VII dataset. A total of 10,116 and 5575 Korean participants aged >19 years with measured urinary cotinine concentrations were enrolled in a training set and validation set, respectively. When using 4.0 as the cutoff value for distinguishing current smokers from non-current smokers, urinary cotinine∙NNAL showed a better diagnostic performance than urinary cotinine or urinary NNAL. Among e-cigarette users, urinary cotinine∙NNAL showed more accurate classification rates than urinary NNAL. Furthermore, urinary cotinine∙NNAL had measurable values in non-current smokers, whereas urinary cotinine had unmeasurable values in one-fourth of all participants. This study shows that urinary cotinine∙NNAL might be a useful biomarker for smoking status verification and trend monitoring of tobacco smoking exposure with increased use of e-cigarettes.

Effect of Second-Hand Smoke Exposure on Establishing Urinary Cotinine-Based Optimal Cut-Off Values for Smoking Status Classification in Korean Adults.

Regulations for banning smoking in indoor public places and workplaces have increased worldwide in recent years. A consecutive Korean National Health and Nutrition Examination Survey (KNHANES) between 2008 and 2018 showed a trend toward significant decreases in self-reported tobacco smoke exposure and measured urinary cotinine concentrations. We established and compared each optimal cut-off value for assessing the effect of second-hand smoke (SHS) exposure on establishing urinary cotinine-based cut-off values for smoking status classification in a population setting controlled for racial and cultural diversity, using four KNHANES datasets consisting of the 2008, 2011, 2014, and 2018 surveys. A total of 18,229 Korean participants aged >19 years with measured urinary cotinine concentrations were enrolled. Self-reports of current smoking status showed that the prevalence of current smokers decreased from 22.9% to 18.2% between 2008 and 2018. During this period, the median value of urinary cotinine in nonsmokers decreased from 5.86 µg/L to 0.48 µg/L, whereas the median value showed no remarkable decrease in current smokers. The AUC-based optimal cut-off values of urinary cotinine concentration for distinguishing current smokers from nonsmokers decreased from 86.5 µg/L to 11.5 µg/L. Our study showed that decreased SHS exposure would result in decreased optimal cut-off values for distinguishing current smokers from nonsmokers. In addition, the study suggests that the range of urinary cotinine concentration to define SHS exposure for the trend monitoring of populationof SHS exposure is appropriate between 0.30 µg/L and 100 µg/L. In addition, our study showed the importance of determination of cotinine concentration, which would have allowed us to avoid mistakes in qualification to the study group in an increased use of e-cigarette setting.

Hypoxia-Responsive Luminescent CEST MRI Agent for In Vitro and In Vivo Tumor Detection and Imaging.

Hypoxia is a feature of most solid tumors and a key determinant of cancer growth and propagation. Sensing hypoxia effectively could lead to more favorable clinical outcomes. Here, we report a molecular antenna-based bimodal probe designed to exploit the complementary advantages of magnetic resonance (MR)- and optical-based imaging. Specifically, we describe the synthesis and evaluation of a dual-action probe (NO2-Eu) that permits hypoxia-activated chemical exchange saturation transfer (CEST) MR and optical imaging. In CT26 cells, this NO2-Eu probe not only provides an enhanced CEST MRI signal but also turns "on" the optical signal under hypoxic conditions. Time-dependent in vivo CEST imaging in a hypoxic CT26 tumor xenograft mouse model revealed probe-dependent tumor detection by CEST MRI contrast in the tumor area. We thus suggest that dual-action hypoxia probes, like that reported here, could have a role to play in solid tumor diagnosis and monitoring.

A modifiable universal cotinine-chimeric antigen system of NK cells with multiple targets.

Natural killer (NK) cells are immune effector cells with outstanding features for adoptive immunotherapy. Immune effector cells with chimeric antigen receptors (CARs) are promising targeted therapeutic agents for various diseases. Because tumor cells exhibit heterogeneous antigen expression and lose cell surface antigen expression during malignant progression, many CARs fixed against only one antigen have limited efficacy and are associated with tumor relapse. To expand the utility of CAR-NK cells, we designed a split and universal cotinine-CAR (Cot-CAR) system, comprising a Cot-conjugator and NK92 cells (α-Cot-NK92 cells) engineered with a CAR containing an anti-Cot-specific single-chain variable fragment and intracellular signaling domain. The efficacy of the Cot-CAR system was assessed in vitro using a cytolysis assay against various tumor cells, and its single- or multiple- utility potential was demonstrated using an in vivo lung metastasis model by injecting A549-Red-Fluc cells. The α-Cot-NK92 cells could switch targets, logically respond to multiple antigens, and tune cytolytic activation through the alteration of conjugators without re-engineering. Therefore the universal Cot-CAR system is useful for enhancing specificity and diversity of antigens, combating relapse, and controlling cytolytic activity. In conclusion, this universal Cot-CAR system reveals that multiple availability and controllability can be generated with a single, integrated system.

Diagnostic Value of Multiple Serum Biomarkers for Vancomycin-Induced Kidney Injury.

Acute kidney injury (AKI) is a major contributor to in-hospital morbidity and mortality. Vancomycin, one of the most commonly used antibiotics in a clinical setting, is associated with AKI, with its incidence ranging up to 43%. Despite the high demand, few studies have investigated serum biomarkers to detect vancomycin-induced kidney injury (VIKI). Here, we evaluated the diagnostic value of nine candidate serum biomarkers for VIKI. A total of 23,182 cases referred for vancomycin concentration measurement from January 2018 to December 2019 were screened and 28 subjects with confirmed VIKI were enrolled (VIKI group). Age- and sex- matched control group consisted of 21 subjects who underwent vancomycin therapy without developing VIKI (non-VIKI group), and 23 healthy controls (HC group). The serum concentrations of clusterin, retinol binding protein 4 (RBP4), interleukin-18 (IL-18), tumor necrosis factor receptor 1 (TNF-R1), C-X-C motif chemokine ligand 10 (CXCL10), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin, trefoil factor-3 (TFF3), and cystatin C were compared among the three groups, and their correlations with estimated glomerular filtration rate (eGFR) and diagnostic values for VIKI were assessed. All of the biomarkers except clusterin and RBP4 exhibited significant elevation in the VIKI group. Serum TFF3, cystatin C, TNF-R1, and osteopontin demonstrated an excellent diagnostic value for VIKI (TFF3, area under the curve (AUC) 0.932; cystatin C, AUC 0.917; TNF-R1, AUC 0.866; osteopontin, AUC 0.787); and except osteopontin, a strong negative correlation with eGFR (TFF3, r = -0.71; cystatin C, r = -0.70; TNF-R1, r = -0.60). IL-18, CXCL10, and NGAL showed weak correlation with eGFR and moderate diagnostic value for VIKI. This study tested multiple serum biomarkers for VIKI and showed that serum TFF3, cystatin C, TNF-R1, and osteopontin could efficiently discriminate VIKI patients. Further studies are warranted to clarify the diagnostic value of these biomarkers in VIKI.

Proteomic Analysis of the Meniscus Cartilage in Osteoarthritis.

The distribution of differential extracellular matrix (ECM) in the lateral and medial menisci can contribute to knee instability, and changes in the meniscus tissue can lead to joint disease. Thus, deep proteomic identification of the lateral and medial meniscus cartilage is expected to provide important information for treatment and diagnosis of various knee joint diseases. We investigated the proteomic profiles of 12 lateral/medial meniscus pairs obtained from excess tissue of osteoarthritis patients who underwent knee arthroscopy surgery using mass spectrometry-based techniques and measured 75 ECM protein levels in the lesions using a multiple reaction monitoring (MRM) assay we developed. A total of 906 meniscus proteins with a 1% false discovery rate (FDR) was identified through a tandem mass tag (TMT) analysis showing that the lateral and medial menisci had similar protein expression profiles. A total of 131 ECM-related proteins was included in meniscus tissues such as collagen, fibronectin, and laminin. Our data showed that 14 ECM protein levels were differentially expressed in lateral and medial lesions (p < 0.05). We present the proteomic characterization of meniscal tissue with mass spectrometry-based comparative proteomic analysis and developed an MRM-based assay of ECM proteins correlated with tissue regeneration. The mass spectrometry dataset has been deposited to the MassIVE repository with the dataset identifier MSV000087753.

Therapeutic Effect of BDNF-Overexpressing Human Neural Stem Cells (F3.BDNF) in a Contusion Model of Spinal Cord Injury in Rats.

The most common type of spinal cord injury is the contusion of the spinal cord, which causes progressive secondary tissue degeneration. In this study, we applied genetically modified human neural stem cells overexpressing BDNF (brain-derived neurotrophic factor) (F3.BDNF) to determine whether they can promote functional recovery in the spinal cord injury (SCI) model in rats. We transplanted F3.BDNF cells via intrathecal catheter delivery after a contusion of the thoracic spinal cord and found that they were migrated toward the injured spinal cord area by MR imaging. Transplanted F3.BDNF cells expressed neural lineage markers, such as NeuN, MBP, and GFAP and were functionally connected to the host neurons. The F3.BDNF-transplanted rats exhibited significantly improved locomotor functions compared with the sham group. This functional recovery was accompanied by an increased volume of spared myelination and decreased area of cystic cavity in the F3.BDNF group. We also observed that the F3.BDNF-transplanted rats showed reduced numbers of Iba1- and iNOS-positive inflammatory cells as well as GFAP-positive astrocytes. These results strongly suggest the transplantation of F3.BDNF cells can modulate inflammatory cells and glia activation and also improve the hyperalgesia following SCI.

Recurrent neonatal sepsis and progressive white matter injury in a premature newborn culture-positive for group B Streptococcus: A case report.

RATIONALE: Group B Streptococcus (GBS) remains a principal pathogen causing neonatal sepsis and meningitis, particularly in premature infants with relatively insufficient immunity. Recurrence may occur uncommonly, largely associated with subclinical mucosal persistence or repetitive exposure to exogenous sources. White matter injury (WMI) including cystic periventricular leukomalacia (PVL) has been associated with intrauterine infection/inflammation, and neonatal infection as a more significant predictor including postnatal sepsis and recurrent infection, even without microbial neuroinvasion. Furthermore, clinical and experimental evidence of WMI by some bacteria other than GBS without central nervous system invasion has been reported. However, there is little evidence of WMI associated with neonatal GBS sepsis in the absence of meningitis in the literature. PATIENT CONCERNS: A newborn at 30+4 weeks' gestation with low birthweight presented with 2 episodes (with a 13-day interval with no antibiotic therapy) of neonatal sepsis culture-proven for GBS with early-onset presentation after clinical chorioamnionitis via vertical GBS transmission and the associated conditions including prematurity-related neonatal immunodeficiency and persistent mucosal GBS carriage after the first antibiotic treatment. The perinatal GBS infection was complicated by progressive WMI presenting with ventriculomegaly and cystic PVL without a definite evidence of meningitis, intraventricular hemorrhage, and documented cerebral hypoxia or hypoperfusion conditions including septic shock. DIAGNOSES: Recurrent group B streptococcal sepsis and cystic PVL with ventriculomegaly. INTERVENTIONS: Two episodes of GBS sepsis were treated with 15-day parenteral antibiotic therapy, respectively. OUTCOMES: Resolution of the recurrent GBS sepsis without further relapses, however, complicated by WMI and subsequent about 6 months delay in motor development at 12 months' corrected age. LESSONS: This case suggests WMI associated with GBS bacteremia without central nervous system entry by viable GBS and also shows that in premature infants, intrauterine GBS infection with no interventions may lead to extensive and persistent GBS colonization, early-onset and recurrent GBS disease, and WMI. Postnatal as well as intrauterine infection/inflammation controls with maternal prophylaxis may be pivotal for prevention and limiting the magnitude of neurologic injury.

An Activatable T1-Weighted MR Contrast Agent: A Noninvasive Tool for Tracking the Vicinal Thiol Motif of Thioredoxin in Live Cells.

We have synthesized new magnetic resonance imaging (MRI) T1 contrast agents (CA1 and CA2) that permit the activatable recognition of the cellular vicinal thiol motifs of the protein thioredoxin. The contrast agents showed MR relaxivities typical of gadolinium complexes with a single water molecule coordinated to a Gd3+ center (i.e., ~4.54 mM-1s-1) for both CA1 and CA2 at 60 MHz. The contrast agent CA1 showed a ~140% relaxivity enhancement in the presence of thioredoxin, a finding attributed to a reduction in the flexibility of the molecule after binding to thioredoxin. Support for this rationale, as opposed to one based on preferential binding, came from 1H-15N-HSQC NMR spectral studies; these revealed that the binding affinities toward thioredoxin were almost the same for both CA1 and CA2. In the case of CA1, T1-weighted phantom images of cancer cells (MCF-7, A549) could be generated based on the expression of thioredoxin. We further confirmed thioredoxin expression-dependent changes in the T1-weighted contrast via knockdown of the expression of the thioredoxin using siRNA-transfected MCF-7 cells. The nontoxic nature of CA1, coupled with its relaxivity features, leads us to suggest that it constitutes a first-in-class MRI T1 contrast agent that allows for the facile and noninvasive monitoring of vicinal thiol protein motif expression in live cells.

Near-Infrared Fluorescent Probe Activated by Nitroreductase for In Vitro and In Vivo Hypoxic Tumor Detection.

Tumor hypoxia is correlated with increased resistance to chemotherapy and poor overall prognoses across a number of cancer types. We present here a cancer cell-selective and hypoxia-responsive probe (fol-BODIPY) designed on the basis of density functional theory (DFT)-optimized quantum chemical calculations. The fol-BODIPY probe was found to provide a rapid fluorescence "off-on" response to hypoxia relative to controls, which lack the folate or nitro-benzyl moieties. In vitro confocal microscopy and flow cytometry analyses, as well as in vivo near-infrared optical imaging of CT26 solid tumor-bearing mice, provided support for the contention that fol-BODIPY is more readily accepted by folate receptor-positive CT26 cancer cells and provides a superior fluorescence "off-on" signal under hypoxic conditions than the controls. Based on the findings of this study, we propose that fol-BODIPY may serve as a tumor-targeting, hypoxia-activatable probe that allows for direct cancer monitoring both in vitro and in vivo.

Ninjurin1 deficiency aggravates colitis development by promoting M1 macrophage polarization and inducing microbial imbalance.

Disruption of colonic homeostasis caused by aberrant M1/M2 macrophage polarization and dysbiosis contributes to inflammatory bowel disease (IBD) pathogenesis. However, the molecular factors mediating colonic homeostasis are not well characterized. Here, we found that Ninjurin1 (Ninj1) limits colon inflammation by regulating macrophage polarization and microbiota composition under homeostatic conditions and during colitis development. Ninj1 deletion in mice induced hypersusceptibility to colitis, with increased prevalence of colitogenic Prevotellaceae strains and decreased immunoregulatory Lachnospiraceae strains. Upon co-housing (CoH) with WT mice, Ninj1-/- mice showed increased Lachnospiraceae and decreased Prevotellaceae abundance, with subsequent improvement of colitis. Under homeostatic conditions, M1 macrophage frequency was higher in the Ninj1-/- mouse colons than wild-type (WT) mouse colons, which may contribute to increased basal colonic inflammation and microbial imbalance. Following colitis induction, Ninj1 expression was increased in macrophages; meanwhile Ninj1-/- mice showed severe colitis development and impaired recovery, associated with decreased M2 macrophages and escalated microbial imbalance. In vitro, Ninj1 knockdown in mouse and human macrophages activated M1 polarization and restricted M2 polarization. Finally, the transfer of WT macrophages ameliorated severe colitis in Ninj1-/- mice. These findings suggest that Ninj1 mediates colonic homeostasis by modulating M1/M2 macrophage balance and preventing extensive dysbiosis, with implications for IBD prevention and therapy.

A three-dimensional hyaluronic acid-based niche enhances the therapeutic efficacy of human natural killer cell-based cancer immunotherapy.

Adoptive transfer of natural killer (NK) cells is becoming one of the most important parts of cancer immunotherapy. However, recent accomplishments have focused on the improvement of the targeting effects based on the engineering of chimeric antigen receptors (CARs) on cell surfaces. Despite the large quantity of therapeutic cells required for clinical applications, the technology for ex vivo expansion is not well developed. Herein, a three-dimensional (3D) engineered hyaluronic acid-based niche for cell expansion (3D-ENHANCE) is introduced. Compared with the conventional two-dimensional (2D) method, NK-92 cell lines and human EGFR-specific (CAR)-NK cells cultured in 3D-ENHANCE yield favorable mRNA expressions, elevated cytokine release, upregulated proliferative and tumor-lytic abilities, and result in enhanced antitumor efficacy. Furthermore, controllable degradation rates can be realized by tuning the formulation of 3D-ENHANCE so that it can be applied as an implantable cell reservoir at surgical sites. In vivo results with the incompletely resected MDA-MB-231 model confirm that the peri-operative implantation of 3D-ENHANCE prevents the relapse and metastases after surgery. Overall, 3D-ENHANCE presents an effective cytokine-free niche for ex vivo expansion and postsurgical treatment that enhances the low-therapeutic efficacy of human NK cells.

Oxaliplatin Pt(IV) prodrugs conjugated to gadolinium-texaphyrin as potential antitumor agents.

Described here is the development of gadolinium(III) texaphyrin-platinum(IV) conjugates capable of overcoming platinum resistance by 1) localizing to solid tumors, 2) promoting enhanced cancer cell uptake, and 3) reactivating p53 in platinum-resistant models. Side by side comparative studies of these Pt(IV) conjugates to clinically approved platinum(II) agents and previously reported platinum(II)-texaphyrin conjugates demonstrate that the present Pt(IV) conjugates are more stable against hydrolysis and nucleophilic attack. Moreover, they display high potent antiproliferative activity in vitro against human and mouse cell cancer lines. Relative to the current platinum clinical standard of care (SOC), a lead Gd(III) texaphyrin-Pt(IV) prodrug conjugate emerging from this development effort was found to be more efficacious in subcutaneous (s.c.) mouse models involving both cell-derived xenografts and platinum-resistant patient-derived xenografts. Comparative pathology studies in mice treated with equimolar doses of the lead Gd texaphyrin-Pt(IV) conjugate or the US Food and Drug Administration (FDA)-approved agent oxaliplatin revealed that the conjugate was better tolerated. Specifically, the lead could be dosed at more than three times (i.e., 70 mg/kg per dose) the tolerable dose of oxaliplatin (i.e., 4 to 6 mg/kg per dose depending on the animal model) with little to no observable adverse effects. A combination of tumor localization, redox cycling, and reversible protein binding is invoked to explain the relatively increased tolerability and enhanced anticancer activity seen in vivo. On the basis of the present studies, we conclude that metallotexaphyrin-Pt conjugates may have substantial clinical potential as antitumor agents.

Self-assembled amphiphilic fluorescent probe: detecting pH-fluctuations within cancer cells and tumour tissues.

Abnormal anaerobic metabolism leads to a lowering of the pH of many tumours, both within specific intracellular organelles and in the surrounding extracellular regions. Information relating to pH-fluctuations in cells and tissues could aid in the identification of neoplastic lesions and in understanding the determinants of carcinogenesis. Here we report an amphiphilic fluorescent pH probe (CS-1) that, as a result of its temporal motion, provides pH-related information in cancer cell membranes and selected intracellular organelles without the need for specific tumour targeting. Time-dependent cell imaging studies reveal that CS-1 localizes within the cancer cell-membrane about 20 min post-incubation. This is followed by migration to the lysosomes at 30 min before being taken up in the mitochondria after about 60 min. Probe CS-1 can selectively label cancer cells and 3D cancer spheroids and be readily observed using the green fluorescence channel (λ em = 532 nm). In contrast, CS-1 only labels normal cells marginally, with relatively low Pearson's correlation coefficients being found when co-incubated with standard intracellular organelle probes. Both in vivo and ex vivo experiments provide support for the suggestion that CS-1 acts as a fluorescent label for the periphery of tumours, an effect ascribed to proton-induced aggregation. A much lower response is seen for muscle and liver. Based on the present results, we propose that sensors such as CS-1 may have a role to play in the clinical and pathological detection of tumour tissues or serve as guiding aids for surgery.