Intracellular Amount Research Articles

Accumulation of chlorinated paraffins in adipocytes is determined by cellular lipid content and chlorination level.

Chlorinated paraffins (CPs) are environmental pollutants extensively used in industries. While the use of short-chain chlorinated paraffins (SCCPs) has been restricted since 2017, the use of medium-chain chlorinated paraffins (MCCPs) has risen as their replacement. Due to lipophilic character, it can be expected that CPs enter the cells; however, the in vitro accumulation potential of CPs remains poorly understood. In this study, we aimed to explore the ability of SCCPs and MCCPs to accumulate in fat cells. We utilized an in vitro model of mouse 3T3-L1 preadipocytes and adipocytes. Using gas chromatography coupled with high-resolution mass spectrometry operated in negative chemical ionization mode, we determined the intracellular amounts of CPs. These compounds accumulated at rates of 8.5 ± 0.1µg/gcells/h for SCCPs and 7.8 ± 0.3µg/gcells/h for MCCPs when an initial concentration of 120ng/ml was present in the medium. This rate increased approximately tenfold when the concentration of CPs was raised to 1200ng/ml. CPs content in adipocytes steadily increased over 5days, whereas preadipocytes accumulated 15-20 times less CPs. This highlights the importance of cellular lipid content, which was about 12 times higher in adipocytes. Furthermore, we found that the level of chlorine content in the CPs molecules significantly influenced their accumulation. Our results demonstrate that MCCPs exhibit a similar accumulation potential to SCCPs, with lipid content playing a crucial role. As with SCCPs, restrictions on the use of MCCPs in industry should be considered to mitigate their environmental and health impacts.

Efficacy of glutathione inhibitor eprenetapopt against the vulnerability of glutathione metabolism in SMARCA4-, SMARCB1- and PBRM1-deficient cancer cells

Mutation of genes related to the SWI/SNF chromatin remodeling complex is detected in 20% of all cancers. The SWI/SNF chromatin remodeling complex comprises about 15 subunits and is classified into three subcomplexes: cBAF, PBAF, and ncBAF. Previously, we showed that ovarian clear cell carcinoma cells deficient in ARID1A, a subunit of the cBAF complex, are synthetic lethal with several genes required for glutathione (GSH) synthesis and are therefore sensitive to the GSH inhibitor eprenetapopt (APR-246). However, we do not know whether cancer cells deficient in SWI/SNF components other than ARID1A are selectively sensitive to treatment with eprenetapopt. Here, we show that SMARCA4-, SMARCB1-, and PBRM1-deficient cells are more sensitive to eprenetapopt than SWI/SNF-proficient cells. We found that deficiency of SMARCA4, SMARCB1, or PBRM1 attenuates transcription of the SLC7A11 gene (which supplies cysteine as a raw metabolic material for GSH synthesis) by the failure of recruitment of cBAF and PBAF to the promotor and enhancer regions of the SLC7A11 locus, thereby reducing basal levels of GSH. In addition, eprenetapopt decreased the amount of intracellular GSH and increased the intracellular amount of reactive oxygen species (ROS), followed by induction of apoptosis. Taken together, eprenetapopt could be a promising selective agent for SWI/SNF-deficient cancer cells derived from SMARCA4-deficient lung cancers, SMARCB1-deficient rhabdoid tumors, and PBRM1-deficient kidney cancers.

Analysis of cellular effects by continuous exposure AT low concentration of tritium.

This study investigated the induction of DNA double-strand breaks (DSBs) in the hTERT-immortalized normal human diploid epithelial cells (RPE1-hTERT) continuously exposed to 6000Bq/ml of tritiated water (HTO) and organically bound tritium (OBT). The relationship of the DSBs induction with the intracellular amount as well as the localization of tritium was also examined. Tritium-labeled thymidine (3H-Thy) and palmitic acid (3H-PA) were used as OBT. The average number of DSBs, which were indicated as co-localized foci of 53BP1 with phosphorylated H2AX, per cell was higher in the order of treatments with 3H-Thy, 3H-PA, and HTO. This order was consistent with that of tritium localization in the insoluble nuclear fraction but not with the intracellular amount of tritium. In addition to the intracellular amount of tritium, we showed that the subcellular localization of tritium is an important factor in cellular effects stimulated by DSBs.

De Novo Production of the Bioactive Phenylpropanoid Artepillin C Using Membrane-Bound Prenyltransferase in Komagataella phaffii.

Artepillin C is a diprenylated phenylpropanoid with various pharmacological benefits for human health. Its natural occurrence is limited to a few Asteraceae plants, such as Baccharis species, necessitating a stable supply through synthetic biology. In Saccharomyces cerevisiae, the utilization of aromatic substrates within the cell was limited, resulting in very low production of artepillin C. In this study, we used AcPT1, a p-coumaric acid (p-CA)-specific diprenyltransferase, in Komagataella phaffii to produce artepillin C. Detailed studies revealed that the critical bottleneck in K. phaffii was the supply of prenyl diphosphates, not phenylpropanoid flux. By enhancing the prenyl substrate pathway through overexpression of isopentenyl diphosphate isomerase and a truncated HMG-CoA reductase, we achieved a strong increase in artepillin C production. A major part of artepillin C was accumulated in yeast cells. One of the advantages of K. phaffii is its superior growth and ability to achieve high cell density cultivation compared to that of S. cerevisiae. Therefore, fed-batch cultivation with glycerol was performed. As a result, the dry cell weight (DCW) reached 61.0 g/L, and the intracellular amount of de novo produced artepillin C reached 187.3 μg/DCW. Analysis of intermediates revealed that the supply of p-CA constituted a bottleneck in artepillin C production in the engineered strain. By enhancing the p-CA supply, the intracellular accumulation of artepillin C reached 1200 μg/DCW even in batch cultivation. Moreover, the total intra- and extracellular amounts of artepillin C reached 12.5 mg/L, marking the highest de novo synthesis amount of artepillin C reported thus far, even under batch cultivation conditions.

Trafficking of adhesion and aggregation-modulating proteins during the early stages of Dictyostelium development

The social amoeba Dictyostelium discoideum has been studied for close to a century to better understand conserved cellular and developmental processes. The life cycle of this model eukaryote is composed of a unicellular growth phase and a multicellular developmental phase that is induced by starvation. When starved, individual cells undergo chemotactic aggregation to form multicellular mounds that develop into slugs. Terminal differentiation of cells within slugs forms fruiting bodies, each composed of a stalk that supports a mass of viable spores that germinate and restart the life cycle when nutrients become available. Calcium-dependent cell adhesion protein A (CadA) and countin (CtnA) are two proteins that regulate adhesion and aggregation, respectively, during the early stages of D. discoideum development. While the functions of these proteins have been well-studied, the mechanisms regulating their trafficking are not fully understood. In this study, we reveal pathways and cellular components that regulate the intracellular and extracellular amounts of CadA and CtnA during aggregation. During growth and starvation, CtnA localizes to cytoplasmic vesicles and punctae. We show that CtnA is glycosylated and this post-translational modification is required for its secretion. Upon autophagy induction, a signal peptide for secretion facilitates the release of CtnA from cells via a pathway involving the μ subunit of the AP3 complex (Apm3) and the WASP and SCAR homolog, WshA. Additionally, CtnA secretion is negatively regulated by the D. discoideum orthologs of the human non-selective cation channel mucolipin-1 (Mcln) and sorting receptor sortilin (Sort1). As for CadA, it localizes to the cell periphery in growth-phase and starved cells. The intracellular and extracellular amounts of CadA are modulated by autophagy genes (atg1, atg9), Apm3, WshA, and Mcln. We integrate these data with previously published findings to generate a comprehensive model summarizing the trafficking of CadA and CtnA in D. discoideum. Overall, this study enhances our understanding of protein trafficking during D. discoideum aggregation, and more broadly, provides insight into the multiple pathways that regulate protein trafficking and secretion in all eukaryotes.

Antiproliferative Effect of 7-Ketositosterol in Breast and Liver Cancer Cells: Possible Impact on Ceramide, Extracellular Signal-Regulated Kinases, and Nuclear Factor Kappa B Signaling Pathways.

Background: This study aimed to examine the effect of 7-Ketositosterol (7-KSS), on sphingomyelin/ceramide metabolites and apoptosis in human breast MCF-7 and human liver HepG2 cancer cells. Methods: Anti-proliferative effects of 7-KSS treatment were assessed at different concentrations and periods. Cell viability was assessed through MTT analysis, whereas the levels of sphingosine-1-phosphate (S1P), sphingomyelins (SMs), and ceramides (CERs) were measured using LC-MS/MS. Phosphorylated 44/42 ERK1/2 and NF-κB p65 (Ser536) protein levels were measured by Western blot analysis and immunofluorescence staining. Apoptosis was evaluated by TUNEL staining and flow cytometric assessment of annexin-V and propidium iodide (PI) labeling. Results: Treatment with 7-KSS significantly decreased cell survival and S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels in cancer cells compared to controls. A substantial rise was detected in intracellular amounts of C16-C24 CERs and apoptosis in cancer cells incubated with 7-KSS. Conclusions: 7-KSS stimulated ceramide accumulation and apoptosis while decreasing cell proliferation via downregulating S1P, p-44/42 ERK1/2, and p-NF-κB p65 protein levels.

DNA Framework-Enabled 3D Organization of Antiarrhythmic Drugs for Radiofrequency Catheter Ablation.

Preorganizing molecular drugs within a microenvironment is crucial for the development of efficient and controllable therapeutic systems. Here, the use of tetrahedral DNA framework (TDF) is reported to preorganize antiarrhythmic drugs (herein doxorubicin, Dox) in 3D for catheter ablation, a minimally invasive treatment for fast heartbeats, aiming to address potential complications linked to collateral tissue damage and the post-ablation atrial fibrillation (AF) recurrence resulting from incomplete ablation. Dox preorganization within TDF transforms its random distribution into a confined, regular spatial arrangement governed by DNA. This, combined with the high affinity between Dox and DNA, significantly increases local Dox concentration. The exceptional capacity of TDF for cellular internalization leads to a 5.5-fold increase in intracellular Dox amount within cardiomyocytes, effectively promoting cellular apoptosis. In vivo investigations demonstrate that administering TDF-Dox reduces the recurrence rate of electrical conduction after radiofrequency catheter ablation (RFCA) to 37.5%, compared with the 77.8% recurrence rate in the free Dox-treated group. Notably, the employed Dox dosage exhibits negligible adverse effects in vivo. This study presents a promising treatment paradigm that strengthens the efficacy of catheter ablation and opens a new avenue for reconciling the paradox of ablation efficacy and collateral damage.

Multifunctional liposomal complex for detection of the acquired resistance to EGFR-TKIs and synergistic anticancer phototherapy

The emergence of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) poses formidable obstacles to non-small cell lung cancer (NSCLC) therapy. Real-time monitoring of the efficacy of EGFR-TKIs to avoid invalid treatment remains a great challenge. Herein, we rationally designed a EGFR-TKI-modified liposomal complex co-loaded with a iodine-containing contrast agent ioversol (IOV) and a photosensitizer indocyanine green (ICG), namely ELIG, for simultaneous EGFR-TKI-mediated computed tomography/fluorescence dual-modal imaging to monitor drug resistance during the treatment, and combined photodynamic therapy (PDT) and photothermal therapy (PTT) to reverse drug resistance. ELIG exhibited good physicochemical properties, favorable photothermal effects, and high singlet oxygen production. ELIG can protect ICG and IOV from rapid degradation and prolong the circulation time. In addition, ELIG had a great ability to recognize drug-sensitive EGFR-mutated NSCLC cells, and could monitor the changes in the intracellular amounts of the imaging agents to detect the drug resistance and predict the efficacy during the treatments. Additionally, the heavy atom effect of IOV can increase the PDT effect of ICG obviously. With 808 nm laser irradiation, ELIG showed satisfactory anticancer effects to reverse drug resistance by the synergism of PDT and PTT in vitro and in vivo. Hence, this work holds great promise for providing a new idea and an effective strategy for predicting and managing the efficacy of EGFR-TKIs.

Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Blocking lipid synthesis induces DNA damage in prostate cancer and increases cell death caused by PARP inhibition.

Androgen deprivation therapy (ADT) is the primary treatment for prostate cancer; however, resistance to ADT invariably develops, leading to castration-resistant prostate cancer (CRPC). Prostate cancer progression is marked by increased de novo synthesis of fatty acids due to overexpression of fatty acid synthase (FASN), making this enzyme a therapeutic target for prostate cancer. Inhibition of FASN results in increased intracellular amounts of ceramides and sphingomyelin, leading to DNA damage through the formation of DNA double-strand breaks and cell death. We found that combining a FASNi with the poly-ADP ribose polymerase (PARP) inhibitor olaparib, which induces cell death by blocking DNA damage repair, resulted in a more pronounced reduction in cell growth than that caused by either drug alone. Human CRPC organoids treated with a combination of PARP and FASNi were smaller, had decreased cell proliferation, and showed increased apoptosis and necrosis. Together, these data indicate that targeting FASN increases the therapeutic efficacy of PARP inhibitors by impairing DNA damage repair, suggesting that combination therapies should be explored for CRPC.

Oxaloacetate as new inducer for osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells in vitro.

Mitochondrial organelles play a crucial role in cellular metabolism so different cell types exhibit diverse metabolic and energy demands. Therefore, alternations in the intracellular distribution, quantity, function, and structure of mitochondria are required for stem cell differentiation. Finding an effective inducer capable of modulating mitochondrial activity is critical for the differentiation of specific stem cells into osteo-like cells for addressing issues related to osteogenic disorders. This study aimed to investigate the effect of oxaloacetate (OAA) on the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADSCs) in vitro. First, the most favorable OAA concentration was measured through MTT assay and subsequently confirmed using acridine orange staining. Human ADSCs were cultured in osteogenic medium supplemented with OAA and analyzed on days 7 and 14 of differentiation. Various assays including alkaline phosphatase assay (ALP), cellular calcium content assay, mineralized matrix staining with alizarin red, catalase (CAT) and superoxide dismutase (SOD) activity, and real-time RT-PCR analysis of three bone-specific markers (ALP, osteocalcin, and collagen type I) were conducted to characterize the differentiated cells. Following viability assessment, OAA at a concentration of 1 µM was considered the optimal dosage for further studies. The results of osteogenic differentiation assays showed that OAA at a concentration of 1 × 10- 6 M significantly increased ALP enzyme activity, mineralization, CAT and SOD activity and the expression of bone-specific genes in differentiated cells compared to control groups in vitro. In conclusion, the fundings from this study suggest that OAA possesses favorable properties that make it a potential candidate for application in medical bone regeneration.

Global transcriptome analysis reveals Salmonella Typhimurium employs nitrate metabolism to combat bilestress.

Salmonella Typhimurium is an enteric pathogen that is highly tolerant to bile. Next-generation mRNA sequencing was performed to analyze the adaptive responses to bile in two S. Typhimurium strains: wild type (WT) and a mutant lacking cold shock protein E (ΔcspE). CspE is an RNA chaperone which is crucial for survival of S. Typhimurium during bile stress. This study identifies transcriptional responses in bile-tolerant WT and bile-sensitive ΔcspE. Upregulation of several genes involved in nitrate metabolism was observed, including fnr, a global regulator of nitrate metabolism. Notably, Δfnr was susceptible to bile stress. Also, complementation with fnr lowered reactive oxygen species and enhanced the survival of bile-sensitive ΔcspE. Importantly, intracellular nitrite amounts were highly induced in bile-treated WT compared to ΔcspE. Also, the WT strain pre-treated with nitrate displayed better growth with bile. These results demonstrate that nitrate-dependent metabolism promotes adaptation of S. Typhimurium to bile.

Penetrating efficiency of supramolecular hydrogel eye drops: Electrostatic interaction surpasses ligand-receptor interaction

The low drug bioavailability of eye drops challenges the therapy of ocular disorders with high efficacy. One of solutions is to extend the corneal retention and enhance the penetration of drug into cornea. Here we synthesize two fluorophore-conjugated peptide based analogs rich in positive charges (i.e., NBD-FFKK) and with a specific ligand (i.e., NBD-FFRGD), respectively, to visualize their performances in vitro and in vivo. The peptides both can self-assemble into supramolecular hydrogels with the microstructure of nanofibers. The in vitro experiments exhibit that two peptides are both uniformly distributed in cytoplasm, and the intracellular amount of peptide rich in positive charges is significantly larger than that of peptide with a specific ligand. The living corneal fluorescence shows that two peptides enter the corneal stroma within 15 min, and the peptide rich in positive charges is accumulated more extensively throughout the entire cornea, revealing that the supramolecular hydrogel eye drops penetrate the cornea more efficiently via electrostatic interaction than that via ligand-receptor interaction. This work, as a comparative study of supramolecular hydrogel eye drops on penetrating efficiency, indicates a possible direction for the design of eye drops with efficient corneal penetration.

Mcl-1 Protein and Viral Infections: A Narrative Review.

MCL-1 is the prosurvival member of the Bcl-2 family. It prevents the induction of mitochondria-dependent apoptosis. The molecular mechanisms dictating the host cell viability gain importance in the context of viral infections. The premature apoptosis of infected cells could interrupt the pathogen replication cycle. On the other hand, cell death following the effective assembly of progeny particles may facilitate virus dissemination. Thus, various viruses can interfere with the apoptosis regulation network to their advantage. Research has shown that viral infections affect the intracellular amount of MCL-1 to modify the apoptotic potential of infected cells, fitting it to the "schedule" of the replication cycle. A growing body of evidence suggests that the virus-dependent deregulation of the MCL-1 level may contribute to several virus-driven diseases. In this work, we have described the role of MCL-1 in infections caused by various viruses. We have also presented a list of promising antiviral agents targeting the MCL-1 protein. The discussed results indicate targeted interventions addressing anti-apoptotic MCL1 as a new therapeutic strategy for cancers as well as other diseases. The investigation of the cellular and molecular mechanisms involved in viral infections engaging MCL1 may contribute to a better understanding of the regulation of cell death and survival balance.

Putrescine Upregulates Melanogenesis Through Modulation of MITF Transcription Factor in B16F1 Mouse Melanoma Cells.

Ageing is a biochemical, metabolic and genetic physiological phenomenon. The suppression of melanin biosynthesis, evident in the greying of the hair, is a hallmark of ageing resulting from translation failure, reduced enzyme activity and cellular senescence. Putrescine, the smallest member of the polyamine family and an organic chemical, is present in living mammalian cells and plays a crucial role in regulating skin melanogenesis. Therefore, the purpose of this study is to explore the effect of putrescine on the signalling pathways of melanogenesis in melanoma cells. Melanin production capacity of putrescine was analysed using a tyrosinase activity assay. To assess the cell viability of B16F1 cells exposed to putrescine, a tetrazolium dye MTT assay was performed. The effect of putrescine on melanin synthesis in the presence of H2O2 was evaluated using various in vitro assays in B16F1 cells. The effect of putrescine on melanin production in B16F1 cells was determined using a specific melanin production assay. Gene expression was analysed using real-time polymerase chain reaction (RT-PCR). Furthermore, the effect of putrescine on the expression of proteins related to melanin production in the cells treated with H2O2 was analysed by immunofluorescence and Western blot analysis. Putrescine increased tyrosinase activity and showed no cytotoxicity in B16F1 cells. In addition, putrescine effectively scavenged H2O2, as shown by the reduction of intracellular H2O2 amounts in 2',7'-dichlorofluorescin diacetate analysis, and promoted melanin production in living cells. The stimulation of melanogenesis by putrescine was attributed to the increased expression of Mitf, Tyr, Trp-1 and Trp-2 genes. Immunofluorescence assays revealed that putrescine enhanced the expression of proteins associated with melanogenesis and upregulated TYR, TRP-1 and TRP-2 via the microphthalmia-associated transcription factor (MITF) and increased the expression of methionine sulfoxide reductases A (MSRA) and B (MSRB) in the cells treated with H2O2, effectively promoting melanogenesis. These results suggest that putrescine can be used to stimulate melanin synthesis. This is the first study to investigate the effect of putrescine on the signalling pathways of melanogenesis in B16F1 melanoma cells. The results confirm that putrescine can promote melanogenesis through the expression of TYR, TRP-1 and TRP-2 via the MITF in cells treated with H2O2. Putrescine can be used exclusively as a cosmetic product to prevent premature greying of hair.

Hedera helix (Wall Ivy) leaf ethanol extract shows cytotoxic and apoptotic effects in glioblastoma cells by generating reactive oxygen species

Aim: Glioblastoma, known for its aggressiveness, accounts for most malignant gliomas and the efficiency of its treatment is still not enough showing remarkably poor prognosis even though a complex treatment approach. Hedera helix is an evergreen plant mostly known for its effects on respiratory function, especially in chronic bronchial asthma, as well as its antimicrobial, anti-inflammatory, antioxidant, and potential antitumor properties, while its effect on glioblastoma cancer and the underlying mechanism has not been elucidated yet. The aim of this study was to examine the anti-cancer properties of Hedera helix (common ivy) in relation to its ability to induce cytotoxicity, apoptosis, and reactive oxygen species production in glioblastoma (U87) cells. Material and Methods: The leaves’ ethanol, methanol, and water extracts were analyzed for phenol, flavonoid, and antioxidant levels through photometric methods. Then, different concentrations of ethanol extract were applied to U87 cells for 24 hours and analyzed for cytotoxic, apoptotic, and ROS-generating effects by measuring the luminometric intracellular ATP amount, Acridine Orange/Ethidium Bromide double staining, and DCFH-DA methods, respectively. Results: Phenol, flavonoid, and antioxidant results of all three extracts show that ethanol was best for extraction. The results showed that Hedera helix ethanol extract had dose-dependent cytotoxic and apoptotic effects with increased intracellular ROS levels. Conclusion: These results concluded that high doses of Hedera helix ethanolic extract may exhibit anti-cancer effects through pro-oxidant activity.

Low-dose imiquimod induces melanogenesis in melanoma cells through an ROS-mediated pathway

BackgroundMelanogenesis is the process of melanin maturation which not only protects skin from UV radiation but also plays an important role in antigenicity of melanomas. Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) agonist that exhibits antiviral and anticancer activity. ObjectiveTo explore whether IMQ could induce melanogenesis in melanoma cells. MethodsThe mouse melanoma cell line B16F10, the mouse immortalized melanocyte Melan-A, and human melanoma cell lines MNT-1, C32 and A375 were utilized in this study. The pigmented level was observed by the centrifuged cell pellet. The intracellular and extracellular melanin levels were examined in the absorbance in NaOH-extracted cell lysate and cell-cultured medium, respectively. The expression of melanogenesis related proteins was examined by immunoblotting. The intracellular cyclic AMP amount was evaluated by the cAMP Glo assay kit. The activity of phosphodiesterase 4B (PDE4B) was investigated by CREB reporter assay with overexpressed PDE4B or not. ResultsWe demonstrated that a low dose of IMQ could trigger melanogenesis in B16F10 cells. IMQ induced microphthalmia-associated transcription factor (MITF) nuclear translocation, upregulated the expression of melanogenesis-related proteins, increased tyrosinase (TYR) activity, and led to pigmentation in B16F10 cells. Next, we found that IMQ-induced melanogenesis was activated by excessive intracellular cAMP accumulation, which was regulated through IMQ-mediated PDE4B inhibition. Finally, IMQ-induced ROS production was found to be involved in melanogenesis by its control of PDE4B activity. ConclusionsLow dose of IMQ could activate melanogenesis through the ROS/PDE4B/PKA pathway in melanoma cells.

Quantification of Nitric Oxide in Single Cells Using the Single-Probe Mass Spectrometry Technique.

Nitric oxide (NO) is a small molecule that plays important roles in biological systems and human diseases. The abundance of intracellular NO is tightly related to numerous biological processes. Due to cell heterogeneity, the intracellular NO amounts significantly vary from cell to cell, and therefore, any meaningful studies need to be conducted at the single-cell level. However, measuring NO in single cells is very challenging, primarily due to the extremely small size of single cells and reactive nature of NO. In the current studies, the quantitative reaction between NO and amlodipine, a compound containing the Hantzsch ester group, was performed in live cells. The product dehydro amlodipine was then detected by the Single-probe single-cell mass spectrometry technique to quantify NO in single cells. The experimental results indicated heterogeneous distributions of intracellular NO amounts in single cells with the existence of subpopulations.

Modeling heterogeneity in a cell culture using a coupled population balance-oxidative stress model

Population balance models (PBMs) were formulated to describe the evolution in time of the cell population in terms of growth and oxidative stress. Flow cytometry data was used to gain insight into the distribution of important quantities (e.g., cell size, intracellular concentrations of metabolites) over the entire cell population. A coupled population balance-oxidative stress model was developed to predict distributions in cell size and intracellular glutamate, ROS, NADPH and NADP+ concentrations in shake flask cultures of B. pertussis. The major advantage of using a PBM is that it accounts for the distributions and can predict the heterogeneity of the cell population with respect to experimental conditions that are averaged out in bulk models. When comparing the coupled population balance – oxidative stress model to the bulk oxidative stress model, it is apparent that the PBM provides much better predictions of the intracellular ROS concentration. We hypothesize that due to the nonlinear relations between cell growth and oxidative stress, intracellular and cell surface quantities can be better modeled with population balance models.

Nicotinamide mononucleotide impacts HIV-1 infection by modulating immune activation in T lymphocytes and humanized mice

HIV-1-associated immune activation drives CD4+ T cell depletion and the development of acquired immunodeficiency syndrome. We aimed to determine the role of nicotinamide mononucleotide (NMN), the direct precursor of nicotinamide adenine dinucleotide (NAD) co-enzyme, in CD4+ T cell modulation during HIV-1 infection. We examined HIV-1 integrated DNA or transcribed RNA, intracellular p24 protein, and T cell activation markers in CD4+ T cells including invitro HIV-1-infected cells, reactivated patient-derived cells, and in HIV-1-infected humanized mice, under NMN treatment. RNA-seq and CyTOF analyses were used for investigating the effect of NMN on CD4+ T cells. We found that NMN increased the intracellular NAD amount, resulting in suppressed HIV-1 p24 production and proliferation in infected CD4+ T cells, especially in activated CD25+CD4+ T cells. NMN also inhibited CD25 expression on reactivated resting CD4+ T cells derived from cART-treated people living with HIV-1 (PLWH). In HIV-1-infected humanized mice, the frequency of CD4+ T cells was reconstituted significantly by combined cART and NMN treatment as compared with cART or NMN alone, which correlated with suppressed hyperactivation of CD4+ T cells. Our results highlight the suppressive role of NMN in CD4+ T cell activation during HIV-1 infection. It warrants future clinical investigation of NMN as a potential treatment in combination with cART in PLWH. This work was supported by the Hong Kong Research Grants Council Theme-Based Research Scheme (T11-706/18-N), University Research Committee of The University of Hong Kong, the Collaborative Research with GeneHarbor (Hong Kong) Biotechnologies Limited and National Key R&D Program of China (Grant2021YFC2301900).