Polyphyllin VII as a potential medication for targeting epithelial mesenchymal transitionin in thyroid cancer.

The need for novel anti-thyroid cancer (TC) medications is urgent due to the rising incidence and metastatic rates of malignant TC. In this study, we investigated the effect of Polyphyllin VII (PPVII) to TC cells, and explored their potential mechanism. B-CPAP and TPC-1 cells, were used to analyze the antitumor activity of PPVII by quantifying cell growth and metastasis as well as to study the effect on epithelial mesenchymal transition (EMT). The results showed that PPVII dramatically reduced the capacity of B-CPAP and TPC-1 cells to proliferate and migrate in a dose-response manner. Following PPVII treatment of TC cells, the expression levels of E-cadherin progressively increased and were higher than the control group, while the expression levels of EMT-related genes Vimentin, N-cadherin, Slug, Zeb-1, and Foxe1 gradually declined and were lower than the control group. It was proposed that PPVII might prevent TC from undergoing EMT. The Foxe1 gene was shown to be significantly expressed in TC, and a statistically significant variation in Foxe1 expression was observed across clinical stages of the disease, according to a bioinformatics database study. There was a strong link between the expression of the Foxe1 gene and the EMT-related gene. In the meantime, TC cells' expression of Foxe1 can be inhibited by PPVII. In conclusion, our results showed that PPVII may as a potential medication for targeting EMT in thyroid cancer.

Preliminary Investigation Into the Antidepressant Effects of a Novel Curcumin Analogue (CACN136) In Vitro and In Vivo.

The aim of this study was to develop a novel antidepressant with high activity. Based on the findings of molecular docking, eight novel curcumin analogues were evaluated in vitro to check for antidepressant efficacy. Among them, CACN136 had the strongest antidepressant effect. Firstly, CACN136 had a stronger 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical ion scavenging ability (IC50: 17.500 ± 0.267 µg/mL) compared to ascorbic acid (IC50: 38.858 ± 0.263 µg/mL) and curcumin (27.189 ± 0.192 µg/mL). Secondly, only CACN136 demonstrated clear protective effects on cells damaged by glutamate and oxidative stress at all concentrations. Finally, only CACN136 showed ASP + inhibition and was more effective than fluoxetine hydrochloride (FLU) at low concentrations. To further confirm the antidepressant effect of CACN136 in vivo, the CUMS model was established. Following 28 days of oral administration of CUMS mice, CACN136 increased the central area residence time in the open-field test, significantly increased the sucrose preference rate in the sucrose preference test (P < 0.001) and significantly reduced the immobility period in the tail suspension test (P < 0.0001), all of which were more effective than those of FLU. Subsequent research indicated that the antidepressant properties of CACN136 were linked to a decrease in the metabolism of 5-HT and the modulation of oxidative stress levels in vivo. In particular, the activation of the Keap1-Nrf2/BDNF-TrkB signaling pathway by CACN136 resulted in elevated levels of antioxidant enzymes, enhancing the antioxidant capability in mice subjected to CUMS. In conclusion, CACN136 has the potential to treat depression and could be an effective antidepressant.

Synthesis, antimicrobial activity and application of polymers of praseodymium complexes based on pyridine nitrogen oxide.

The traditional pyridine nitrogen oxide-based antimicrobial agents are often associated with health risks due to heavy metal enrichment. To mitigate this concern, we synthesized two novel complexes, Pr2(mpo)6(H2O)2 and Pr(hpo)(mpo)2(H2O)2, and integrated rare-earth salts, Hhpo (2-hydroxypyridine-N-oxide) and Nampo (2-mercapto-pyridine-N-oxide sodium salt). These complexes were characterized through infrared analysis, elemental analysis, thermogravimetric analysis, and X-ray crystallographic analysis. Our comparative analyses demonstrate that the synthesized rare-earth complexes exhibit stronger antimicrobial activity against Staphylococcus aureus (S. aureus ATCC6538) and Escherichia coli (E. coli ATCC25922) compared to the ligands and rare-earth salts alone. Quantitative results revealed the lowest inhibitory concentrations of the two complexes against S. aureus ATCC6538 and E. coli ATCC25922 at 3.125 µg mL-1, 6.25 µg mL-1, 3.125 µg mL-1 and 6.25 µg mL-1, respectively. Preliminary investigations indicated that the antibacterial mechanism of these complexes involved promoting intracellular substance exudation to achieve antibacterial effects. Incorporation of these complexes into polymeric antimicrobial films resulted in a potent antimicrobial effect, achieving a 100% inhibition rate against S. aureus ATCC6538 and E. coli ATCC25922 at a low addition level of 0.6 wt%. Our results suggest that nitrogen oxide-based praseodymium complexes have potential for various antimicrobial applications.

Palladium-Catalyzed Synthesis of Nitrones Via Redox Cross-Coupling of Nitro Compounds and Alcohols.

A novel methodology for the synthesis of nitrones via palladium-catalyzed redox cross-coupling of nitro compounds and alcohols is established. The protocol is a mild, convenient, ligand-free, and scalable synthesis method that can be compatible with various nitro compounds and alcohols. Nitrone is a significant multifunctional platform synthon which can be synthesized directly and efficiently via this tactic from commercially available and cheap raw materials.

Reversible single-crystal-to-single-crystal transition in Gd(III) metal-organic frameworks induced by heat and solvents with a significant magnetocaloric effect.

The design and synthesis of a Gd(III) metal-organic framework with the formula [Gd4(BTDI)3(DMF)4]n (JXUST-40, H4BTDI = 5,5'-(benzo[c][1,2,5]thiadiazole-4,7-diyl)diisophthalic acid) are reported hererin. Interestingly, a reversible single-crystal-to-single-crystal transition between JXUST-40 and {[Gd4(BTDI)3(H2O)4]·6H2O}n (JXUST-40a) was achieved under the stimulation of heat and solvents. Both JXUST-40 and JXUST-40a exhibited good stability when soaked in common solvents and aqueous solutions with pH values of 1-12. Magnetic studies showed that JXUST-40a has a larger magnetocaloric effect with -ΔSmaxm = 26.65 J kg-1 K-1 at 2 K and 7 T than JXUST-40 due to its larger magnetic density. Structural analyses indicated that the coordinated solvent molecules play a crucial role in the coordination environment around the Gd(III) ions and the change in the framework, ultimately leading to the changes in the pore size and magnetism between JXUST-40 and JXUST-40a. In addition, both isomorphic [Dy4(BTDI)3(DMF)4]n (JXUST-41) and {[Dy4(BTDI)3(H2O)4]·6H2O}n (JXUST-41a) displayed slow magnetic relaxation behaviour.

Multistimuli-responsive multicolor solid-state luminescence tuned by NH-dependent switchable hydrogen bonds.

Revealing the stimuli-responsive mechanism is the key to the accurate design of stimuli-responsive luminescent materials. We report herein the multistimuli-responsive multicolor solid-state luminescence of a new dicopper(I) complex [{Cu(bpmtzH)}2(µ-dppa)2](ClO4)2 (1), and the multistimuli-responsive mechanism is clarified by investigating its four different solvated compounds 1·2CH3COCH3·2H2O, 1·2DMSO·2H2O, 1·4CH3OH, and 1·4CH2Cl2. It is shown that luminescence mechanochromism is associated with the breakage of the hydrogen bonds of bmptzH-NH with counter-ions such as ClO4- induced by grinding, while luminescence vapochromism is attributable to the breaking and forming of hydrogen bonds of dppa-NH with solvents, such as acetone, dimethylsulfoxide, and methanol, caused by heating and vapor fuming. In addition, those results might provide new insights into the design and synthesis of multistimuli-responsive multicolor luminescent materials by using various structure-sensitive functional groups, such as distinct N-H ones, to construct switchable hydrogen bonds.

Highly Stable Rare Earth Metal-Organic Frameworks for Fluorescence Recognition of Folic Acid, Proton Conduction, and Magnetic Refrigeration.

Four new isostructural rare earth metal-organic frameworks (RE-MOFs) were synthesized and full characterized, namely, {[(CH)2NH2]3[RE2(BTDBA)2(HCOO)]·5H2O·2DMF}n (H4BTDBA = (4',4'''-(benzo[c][1,2,5]thiadiazole-4,7-diyl)bis([1,1'-biphenyl]-3,5-dicarboxylic acid); RE = Eu (JXUST-34), Gd (JXUST-35), Tb (JXUST-36), and Dy (JXUST-37)). The single-crystal structures analysis shows that JXUST-34-37 are chain-based three-dimensional structures. Importantly, JXUST-34 exhibits excellent water, organic solvents, and acid-base stability, which can be used as a fluorescence sensor for folic acid and Al3+ with detection limits of 0.02 mM and 0.05 µM, respectively. The presence of free [(CH)2NH2]+ cations in the channels can engage the proton carrier during proton conduction. JXUST-34-37 display good proton conductivity, and the conductivities vary with relative humidity and temperatures, among which JXUST-37 has the highest conductivity of 9.66 × 10-3 S·cm-1 at 60 °C and 98% RH. The magnetic studies show that the -ΔSm of JXUST-35 reaches 16.13 J kg-1 K-1 at 2 K and ΔH = 7 T. JXUST-34-37 show multifunctional properties of fluorescence sensing, high proton conductivity, and magnetic refrigeration, which provides a new clue for the development of fluorescent-responsive, magnetic-refrigerant, and proton-conductive RE-MOF materials.

The therapeutic value of bifidobacteria in cardiovascular disease.

There has been an increase in cardiovascular morbidity and mortality over the past few decades, making cardiovascular disease (CVD) the leading cause of death worldwide. However, the pathogenesis of CVD is multi-factorial, complex, and not fully understood. The gut microbiome has long been recognized to play a critical role in maintaining the physiological and metabolic health of the host. Recent scientific advances have provided evidence that alterations in the gut microbiome and its metabolites have a profound influence on the development and progression of CVD. Among the trillions of microorganisms in the gut, bifidobacteria, which, interestingly, were found through the literature to play a key role not only in regulating gut microbiota function and metabolism, but also in reducing classical risk factors for CVD (e.g., obesity, hyperlipidemia, diabetes) by suppressing oxidative stress, improving immunomodulation, and correcting lipid, glucose, and cholesterol metabolism. This review explores the direct and indirect effects of bifidobacteria on the development of CVD and highlights its potential therapeutic value in hypertension, atherosclerosis, myocardial infarction, and heart failure. By describing the key role of Bifidobacterium in the link between gut microbiology and CVD, we aim to provide a theoretical basis for improving the subsequent clinical applications of Bifidobacterium and for the development of Bifidobacterium nutritional products.

Preparation of capsaicin-loaded ultrafine fiber film and its application in the treatment of oral ulcers in rats.

A drug-loaded diaphragm is an easy-to-use and effective drug delivery system that is often used to treat mouth ulcers. In this study, an ultrafine fiber film loaded with capsaicin was successfully prepared using the electrospinning technology. poly-L-lactic acid and gelatin were selected as the matrix materials to form the composite fiber, and trifluoroethanol was used as a co-solvent for poly-L-lactic acid, gelatin and capsaicin to prepare the spinning solution, which was simple to fabricate. The prepared fiber films were characterized based on their microscopic morphology and tested to derive their mechanical properties. Thereafter, the capsaicin release behavior of the film was investigated. In vitro experiments revealed certain anti-inflammatory and antibacterial abilities while animal experiments revealed that the capsaicin-loaded ultrafine fiber film could promote the healing of oral ulcers in rats. Healing of the tongue tissue in rats administered 10% capsaicin-loaded fiber film was found to be better than that in rats administered the commercial dexamethasone patch. Overall, this development strategy may prove to be promising for the development of oral ulcer patch formulations.

Characteristics variations of size-fractionated anammox granules and identification of the potential effects on these evolutions.

Anaerobic ammonium oxidation (anammox) granulation which contributed to system stabilization and performance improvement has great potential in the field of wastewater nitrogen removal. The researchers fractionated anammox granules into small-size (0.5-0.9 mm), medium-size (1.8-2.2 mm), and large-size (2.8-3.5 mm) categories to examine their properties and mechanisms. Various analyses, including high-throughput sequencing, determination of inorganic elements and extracellular polymeric substances (EPS), and microbial function prediction, were conducted to characterize these granules and understand their impact. The results revealed distinct characteristics among the different-sized granules. Medium-size granules exhibited the highest sphericity, EPS content, and anammox abundance. In contrast, large-size granules had the highest specific surface area, heme c content, specific anammox activity, biodiversity, and abundance of filamentous bacteria. Furthermore, the precipitates within the granules were identified as CaCO3 and MgCO3, with the highest inorganic element content found in the large-size granules. Microbial community and function annotation also varied with granule size. Based on systematic analysis, the researchers concluded that cell growth, chemical precipitation, EPS secretion, and interspecies interaction all played a role in granulation. Small-size granules were primarily formed through cell growth and biofilm formation. As granule size increased, EPS secretion and chemical precipitation became more influential in the granulation process. In the large-size granules, chemical precipitation and interspecies interaction, including synergistic effects with nitrifying, denitrifying, and filamentous bacteria, as well as metabolic cross-feeding, played significant roles in aggregation. This interplay ultimately contributed to higher anammox activity in the large-size granules. By fully understanding the mechanisms involved in granulation, this study provides valuable insights for the acclimation of anammox granules with optimal sizes under different operational conditions.

A highly stable chain-based EuIII metal-organic framework as a turn-on and blue-shift luminescent sensor for dipicolinic acid.

The development of a rapid and selective method for the identification of dipicolinic acid (DPA), a specific biomarker in Bacillus anthracis spores, is of great importance for the avoidance of anthrax infection. Herein, a chain-based EuIII metal-organic framework with the formula {[Eu3(BTDB)3(µ3-OH)3(H2O)]·solvents}n (JXUST-38, H2BTDB = (benzo[c][1,2,5]thiadiazole-4,7-diyl)dibenzoic acid) was obtained using 2-fluorobenzoic acid as the pH regulator. JXUST-38 exhibits good chemical and thermal stability and can specifically recognize DPA in N,N-dimethylformamide solution through luminescence enhancement and blue-shift effects with a detection limit of 0.05 µM. Furthermore, the significant luminescence enhancement and blue shift under UV lamps are obviously observable by the naked eye. The luminescence sensing mechanism is attributed to absorbance-induced enhancement between JXUST-38 and DPA. Test paper and mixed-matrix membrane based on JXUST-38 are designed for DPA detection. In addition, the feasibility of using JXUST-38 in biosensing is discussed in detail.

Mechanochromic and Selective Vapochromic Solid-State Luminescence of a Dinuclear Cuprous Complex.

The unraveling of the stimuli-responsive mechanism is crucial to the design and precise synthesis of stimuli-responsive luminescent materials. We report herein the mechanochromic and selective vapochromic solid-state luminescence properties of a new bimetallic cuprous complex [{Cu(bpmtzH)}2(µ-dppm)2](ClO4)2 (1), and the corresponding response mechanisms are elucidated by investigating its two different solvated polymorphs 1·2CH2Cl2 (1-g) and 1·2CHCl3 (1-c). Green-emissive 1-g and cyan-emissive 1-c can be interconverted upon alternate exposure to CHCl3 and CH2Cl2 vapors, which is principally attributable to a combined alteration of both intermolecular NHbpmtzH···OClO3- hydrogen bonds and intramolecular "triazolyl/phenyl" π···π interactions induced by different solvents. Solid-state luminescence mechanochromism present in 1-g and 1-c is mainly ascribed to the grinding-induced breakage of the NHbpmtzH···OClO3- hydrogen bonds. It is suggested that intramolecular π···π-triazolyl/phenyl interactions are affected by different solvents but not by grinding. The results provide new insights into the design and precise synthesis of multi-stimuli-responsive luminescent materials by the comprehensive use of intermolecular hydrogen bonds and intramolecular π···π interactions.

Clinical and genetic analysis of a case of late onset carbamoyl phosphate synthase I deficiency caused by CPS1 mutation and literature review.

BACKGROUND: Carbamoyl phosphate synthetase I defect (CPS1D) is a rare disease with clinical case reports mainly in early neonates or adults, with few reports of first onset in late neonatal to childhood. We studied the clinical and genotypic characteristics of children with childhood onset CPS1D caused by two loci mutations (one of these is a rarely reported non-frame shift mutation) in the CPS1. CASE PRESENTATION: We present a rare case of adolescent-onset CPS1D that had been misdiagnosed due to atypical clinical features, and further investigations revealed severe hyperammonemia (287µmol/L; reference range 11.2 ~ 48.2umol/L). MRI of the brain showed diffuse white matter lesions. Blood genetic metabolic screening showed elevated blood alanine (757.06umol/L; reference range 148.8 ~ 739.74umol/L) and decreased blood citrulline (4.26umol/L; reference range 5.45 ~ 36.77umol/L). Urine metabolic screening showed normal whey acids and uracil. Whole-exome sequencing revealed compound heterozygous mutations in the CPS1, a missense mutation (c.1145 C > T) and an unreported de novo non-frame shift mutation (c.4080_c.4091delAGGCATCCTGAT), respectively, which provided a clinical diagnosis. CONCLUSION: A comprehensive description of the clinical and genetic features of this patient, who has a rare age of onset and a relatively atypical clinical presentation, will facilitate the early diagnosis and management of this type of late onset CPS1D and reduce misdiagnosis, thus helping to reduce mortality and improve prognosis. It also provides a preliminary understanding of the relationship between genotype and phenotype, based on a summary of previous studies, which reminds us that it may help to explore the pathogenesis of the disease and contribute to genetic counselling and prenatal diagnosis.

Molecular mechanisms of rapid-acting antidepressants: New perspectives for developing antidepressants.

Major depressive disorder (MDD) is a chronic relapsing psychiatric disorder. Conventional antidepressants usually require several weeks of continuous administration to exert clinically significant therapeutic effects, while about two-thirds of the patients are prone to relapse of symptoms or are completely ineffective in antidepressant treatment. The recent success of the N-methyl-D-aspartic acid (NMDA) receptor antagonist ketamine as a rapid-acting antidepressant has propelled extensive research on the action mechanism of antidepressants, especially in relation to its role in synaptic targets. Studies have revealed that the mechanism of antidepressant action of ketamine is not limited to antagonism of postsynaptic NMDA receptors or GABA interneurons. Ketamine produces powerful and rapid antidepressant effects by affecting α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors, adenosine A1 receptors, and the L-type calcium channels, among others in the synapse. More interestingly, the 5-HT2A receptor agonist psilocybin has demonstrated potential for rapid antidepressant effects in depressed mouse models and clinical studies. This article focuses on a review of new pharmacological target studies of emerging rapid-acting antidepressant drugs such as ketamine and hallucinogens (e.g., psilocybin) and briefly discusses the possible strategies for new targets of antidepressants, with a view to shed light on the direction of future antidepressant research.

Construction of Curcumin and Paclitaxel Co-Loaded Lipid Nano Platform and Evaluation of Its Anti-Hepatoma Activity in vitro and Pharmacokinetics in vivo.

Purpose: The present study aimed to construct a co-loading platform encapsulating curcumin and paclitaxel at ratios of 2:1-80:1 (w/w) designated "CU-PTX-LNP" and explored the synergistic effects of CU-PTX at different composite proportions on liver cancer cells using the combination index (CI) method. Methods: The CU lipid nanoplatform (CU-LNP) formulation was optimized via single-factor and orthogonal experiments. Various concentrations of PTX were added to the optimal formulation of CU-LNP to generate CU-PTX-LNP and the nanoplatform characterized via differential scanning calorimetry (DSC), transmission electron microscope (TEM), X-ray diffraction (XRD), zeta potential, polydispersity index (PDI), and size analyses. The cumulative release, stability, and cytotoxicity of CU-PTX-LNP in LO2, HepG2, and SMMC-7221 cells were assessed in vitro, followed by safety investigation and pharmacokinetic studies in vivo. The anti-tumor activity of CU-PTX-LNP was also evaluated using nude mice. Results: CU-PTX-LNP formulations containing CU:PTX at a range of proportions (2:1-80:1; w/w) appeared as uniformly dispersed nanosized spherical particles with high entrapment efficiency (EE> 90%), sustained release and long-lasting stability. Data from in vitro cytotoxicity assays showed a decrease in the IC50 value of PTX of CU-PTX-LNP (by 5.47-332.7 times in HepG2 and 4.29-143.21 times in SMMC-7221 cells) compared to free PTX. In vivo, CU-PTX-LNP displayed excellent biosafety, significant anti-tumor benefits and enhanced pharmacokinetic behavior with longer mean residence time (MRT(0-t); CU: 4.31-fold, PTX: 4.61-fold) and half-life (t1/2z; CU: 1.83-fold, PTX: 2.28-fold) relative to free drugs. Conclusion: The newly designed CU-PTX-LNP platform may serve as a viable technological support system for the successful production of CU-PTX composite preparations.

The application of extracellular vesicles in colorectal cancer metastasis and drug resistance: recent advances and trends.

Colorectal cancer (CRC) has high incidence and mortality rates and is one of the most common cancers of the digestive tract worldwide. Metastasis and drug resistance are the main causes of cancer treatment failure. Studies have recently suggested extracellular vesicles (EVs) as a novel mechanism for intercellular communication. They are vesicular particles, which are secreted and released into biological fluids, such as blood, urine, milk, etc., by a variety of cells and carry numerous biologically active molecules, including proteins, nucleic acids, lipids, metabolites, etc. EVs play a crucial part in the metastasis and drug resistance of CRC by delivering cargo to recipient cells and modulating their behavior. An in-depth exploration of EVs might facilitate a comprehensive understanding of the biological behavior of CRC metastasis and drug resistance, which might provide a basis for developing therapeutic strategies. Therefore, considering the specific biological properties of EVs, researchers have attempted to explore their potential as next-generation delivery systems. On the other hand, EVs have also been demonstrated as biomarkers for the prediction, diagnosis, and presumed prognosis of CRC. This review focuses on the role of EVs in regulating the metastasis and chemoresistance of CRC. Moreover, the clinical applications of EVs are also discussed.

Solvothermal synthesis and device fabrication of a Eu3+-based metal-organic framework as a turn-on and blue-shift fluorescence sensor toward Cr3+, Al3+ and Ga3.

A novel three-dimensional Eu3+-based metal-organic framework with the formula {[(CH3)2NH2][Eu(BTDI)]·H2O·DMF}n (JXUST-25) was prepared by solvothermal method based on Eu3+ and 5,5'-(benzothiadiazole-4,7-diyl)diisophthalic acid (H4BTDI) with benzothiadiazole (BTD) luminescent groups. Due to the presence of Eu3+ and organic fluorescence ligand, JXUST-25 displays turn-on and blue-shift fluorescence toward Cr3+, Al3+ and Ga3+ with limits of detection (LOD) of 0.073, 0.006 and 0.030 ppm, respectively. Interestingly, the alkaline environment can change the fluorescence of JXUST-25 toward Cr3+/Al3+/Ga3+ and the addition of HCl solution realizes the reversible change of the fluorescence of JXUST-25 toward Cr3+/Al3+/Ga3+. It is noteworthy that the fluorescent test paper and light-emitting diode lamp based on JXUST-25 can effectively detect Cr3+, Al3+ and Ga3+ by the visual changes. In addition, the turn-on and blue-shift fluorescence between JXUST-25 and M3+ ions may be caused by the host-guest interaction and the absorbance caused enhancement mechanism.

Highly pH-Responsive Sensor Based on a EuIII Metal-Organic Framework with Efficient Recognition of Arginine and Lysine in Living Cells.

A lanthanide-based three-dimensional metal-organic framework with excellent water, acid/base, and solvent stability, namely {[(CH3)2NH2]0.7[Eu2(BTDBA)1.5(lac)0.7(H2O)2]·2H2O·2DMF·2CH3CN}n (JXUST-29, H4BTDBA = 4',4‴-(benzo[c][1,2,5]thiadiazole-4,7-diyl)bis([1,1'-biphenyl]-3,5-dicarboxylic acid), Hlac = lactic acid), has been synthesized and characterized. Since the N atoms of the thiadiazole group will not coordinate with lanthanide ions, JXUST-29 has a free basic N-site accessible to small H+ ions, which allows it to be used as a promising pH fluorescence sensor. Interestingly, the luminescence signal was significantly enhanced, with an approximately 54-fold enhancement in the emission intensity when the pH value was increased from 2 to 5, which is the typical behavior of pH probes. In addition, JXUST-29 can also be used as a luminescence sensor to detect l-arginine (Arg) and l-lysine (Lys) in an aqueous solution through fluorescence enhancement and the blue-shift effect. The detection limits were 0.023 and 0.077 µM, respectively. In addition, JXUST-29-based devices were designed and developed to facilitate detection. Importantly, JXUST-29 is also capable of detecting and sensing Arg and Lys in living cells.