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Pathogens such as Staphylococcus aureus are able to survive in many types of host cells including phagocytes such as neutrophils and macrophages, thereby resulting in intracellular infections. Treatment of intracellular infections by conventional antimicrobials (e.g., antibiotics) is often ineffective due to low intracellular efficacy of the drugs. Thus, novel techniques which can enhance the activity of antimicrobials within cells are highly demanded. Our recent studies have shown that photochemical internalization (PCI) is a promising approach for improving the efficacy of antibiotics such as gentamicin against intracellular staphylococcal infection. In this chapter, we describe the protocols aiming to study the potential of PCI-antibiotic treatment for intracellular infections in vitro and in vivo using a RAW 264.7 cell infection model and a zebrafish embryo infection model. Proof of concept of this approach is demonstrated. The protocols are expected to prompt further development of PCI-antimicrobial based novel therapies for clinically challenging infectious diseases associated with intracellular survival of pathogens.

Two new aldoximes A–B (1−2), and two known analogues (3−4), were identified from ethanolic extracts of Glycosmis craibii. The structures of new compounds 1−2 were unambiguously elucidated based on spectroscopic methods. All isolated aldoximes were evaluated for their anti-inflammatory activity in vivo. Compounds 1−4 displayed remarkable inhibitory effects against nitric oxide (NO) production stimulated by lipopolysaccharide in mouse macrophage RAW 264.7 cells with the IC50 values ranging from 2.38 ± 0.08–8.43 ± 0.21 μM.

Lipopolysaccharide (LPS) is an endotoxin involved in a number of acute and chronic inflammatory syndromes. Although LPS‐induced signalling has been extensively studied, there are still mysteries remaining to be revealed. In the current study, we used high‐throughput phosphoproteomics to profile LPS‐initiated signalling and aimed to find novel mediators. A total of 448 phosphoproteins with 765 phosphorylation sites were identified, and we further validated that the phosphorylation of MARK2 on T208 was important for the regulation on LPS‐induced CXCL15 (human IL‐8 homolog), IL‐1β, IL‐6 and TNF‐α release, in which LKB1 had a significant contribution. In summary, induction of cytokines by LPS in mouse macrophage is regulated by LKB1‐MARK2 signals. Our study provides new clues for further exploring the underlying mechanisms of LPS‐induced diseases, and new therapeutic approaches concerning bacterial infection may be derived from these findings.

Aim:The present study aims to compare the cytoprotective effect of Cuminum cyminum L. (CC) extract and cuminaldehyde (CA) against lipotoxicity induced by oxidized low density lipoprotein (Ox-LDL) in mouse macrophage (RAW 264.7) cells.Objective:To assess comparative Cytoprotective potential of CC and CA against Ox-LDL induced cytotoxicity. ii) To study efficacy of CC and CA in preventing Ox-LDL induced apoptosisMethods:Protective effect of CC extract and CA aganist Ox-LDL induced cytotoxicity in RAW 264.7 cells was assessed by MTT assay. DCFDA stain was used to check the generation of ROS followed by analysis of apoptotic genes by quantitative RT-PCR.Results:CC extract was found to be non-toxic up to 300 μg/ml but CA showed significant toxicity from 50 to 300 μg/ml. Cells treated with Ox-LDL recorded 80 % decrement in cell viability as compared to the control cells. But Ox-LDL+CC treated group accounted for improved cell viability (88 %) which was comparable to that of control. However, Ox-LDL+CA treated cells did not record any improvement in cell viability (19 %). DCF-DA staining revealed that the presence of CC could minimize intracellular oxidative stress but similarly this was persistent in CA supplemented group. Furthermore, mRNA expression of apoptotic genes revealed that Ox-LDL induced upregulation of Bax and downregulation of Bcl-2 genes were not recorded in Ox-LDL+CC treated group.Conclusion:It can be concluded that CC extract efficiently prevented Ox-LDL induced lipotoxicity and apoptosis and has an anti-atherosclerotic potential. The failure of CA emphasizes the importance of naturally occurring polyherbal formulations over pure compounds in imparting bioactivity and for therapeutic applications.

Carbon black (CB) has a wide range of industrial applications and recently has been used as the basic model for environmental health studies on airborne particulate matters (PM). Exposure characterization of CB is always the first and most important step towards a better understanding of its effects on human health. However, efforts were largely limited by the lack of valid methods capable of quantifying CB in biological samples. Here, we developed a new method based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for CB quantification in bio-samples, with a detection limit of 4.3 ng. The method is so economical and convenient that it can be performed in most biology labs. The application of the method was successfully demonstrated in three different cell models (mouse macrophage cells (Raw264.7), human epithelial cells (A549) and mouse mesenchymal stem cells (MSC)) and the results showed that the uptake rates decrease in the order of MSC > Raw264.7 > A549. The surprisingly highest uptake rate of MSC deserves further investigation. The novel method provides a complementary quantitative tool to the use of conventional methods such as radioactive and fluorescent labeling and may facilitate related toxicological studies.

A known polyoxygenated briarane, briaexcavatolide P (1), was isolated from a Formosan octocoral Briareum stechei. Moreover, the same species B. stechei, collected from Okinawan waters, yielded three chlorine-containing briaranes, including two new compounds, briastecholides B (2) and C (3) as well as a known analogue, briarenol R (4). The structures of 1–4 were established using spectroscopic methods. In addition, briarane 1 demonstrated anti-inflammatory activity in lipo-polysaccharide-induced RAW 264.7 mouse macrophage cells by suppressing the expression of inducible nitric oxide synthase (iNOS) protein.

Two new lignans (1–2), together with two known lignans (3–4), were isolated from the whole herb of Anemone vitifolia. The structures were identified based on extensive spectroscopic data analysis and comparison with reported data. All compounds of this plant were reported for the first time. In vitro test, all compounds were evaluated for their anti-inflammatory activity via calculating the production of nitric oxide (NO) in lipopolysaccharide (LPS)-induced mouse macrophage cells-RAW 264.7.

Manilkara zapota, usually known as Sapodilla, is a fairly slow-growing evergreen tropical tree which belongs to the genus Manilkara (Sapotaceae), indigenous to Central America, southern Mexico, and the Caribbean. The ripe fruits of M. zapota have been widely consumed as an uniquely flavored tropical fruit and verified to hold a variety of health benefits. In order to investigate the potential health-promoting chemical compositions from the fruits of M. zapota cultivated in Hainan Island of China, a systematic and in-depth phytochemical study on this fruit was accordingly implemented. In our current study, three new prenylated coumarins, manizapotins A-C (1-3), together with seven known prenylated coumarins (4-10), were separated from the fruits of M. zapota. The chemical structures of new prenylated coumarins 1-3 were unambiguously established by means of comprehensive spectroscopic analyses, and the known compounds 4-10 were determined by comparing their experimental spectral data with those described data in the literature. This is the first time to discover prenylated coumarins occurring in M. zapota. The potential anti-inflammatory effects and anti-HIV (human immunodeficiency virus) activities of all these separated prenylated coumarins were assessed. Prenylated coumarins 1-10 dispalyed remarkable inhibitory effects against nitric oxide production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells with the IC50 values equivalent to that of hydrocortisone in vitro. Meanwhile, prenylated coumarins 1-10 exhibited pronounced anti-HIV-1 reverse transcriptase activities with the EC50 values in range of 0.12-8.69 μM. These results suggest that appropriate and reasonable consumption of the fruits of M. zapota might assist people to prevent and reduce the occurrence of inflammatory diseases together with the infection of HIV. Furthermore, the discovery of these prenylated coumarins from the fruits of M. zapota holding pronounced anti-inflammatory effects along with anti-HIV activities could be of great significance to the research and development of new natural anti-inflammatory and anti-HIV agents.

Twelve sesquiterpene lactones were isolated from the whole plants of Vernonia cinerea. These included eight new compounds, vercinolides A-H (1-8), along with four known substances (9-12). The structures of the new compounds were determined by 1D and 2D NMR experiments and mass spectrometric methods. The absolute configurations of compounds 1-8 were determined by Mosher experiments and ECD data. Compounds 1-8 are the first examples of a new class of sesquiterpene lactones possessing a rare 4α,10α-ether ring and a 2,14-ether ring. Compounds 1-4, 6, 8, 10, and 12 were evaluated for their cytotoxic and anti-inflammatory activities. Compounds 10 and 12 exhibited inhibitory effects against nitric oxide production in lipopolysaccharide-activated RAW 264.7 mouse macrophage cells with IC50 values of 21 and 23 μM, respectively. Both compounds were inactive for HeLa cells (IC50 > 10 μM).

The investigation on the stems and leaves of Clausena sanki led to the isolation of a previously undescribed bisabolane sesquiterpene, clausemargic A (1), together with six known analogues (2-7). The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons with data reported in the literature. All known compounds (2-7) were isolated from C. sanki for the first time. All isolated compounds were evaluated for their anti-inflammatory activities via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. Compounds 1-7 showed significant inhibitory activities with IC50 values comparable to that of hydrocortisone.

Objectives: Differences have been observed in the susceptibility of macrophage cell lines to respiratory syncytial virus (RSV) infection. In this study, we evaluated whether the type of macrophage cell line and RSV strain used have an influence on the infectivity and production of progeny virus. Methods: Both human and murine macrophage-like cell lines were infected with different RSV strains, both lab strains as well as clinical isolates. The infection was evaluated after 24 and 72 h by immunofluorescence staining and microscopic analysis, and the production of new virus particles was determined by plaque assay. Results: Susceptibility of macrophages to RSV was influenced by the RSV strain used but was mostly dependent on the macrophage cell line. Numbers of infected cells and virus production were generally very low or absent in murine cell lines. In human cell lines, clear infection was observed associated with production of new virus particles. Conclusion: Differences in susceptibility of macrophage cell lines to RSV infection are primarily related to the species of origin of the cell line but are also influenced by the RSV strain.

Apoptotic pathways play an important role in Mycobacterium tuberculosis-infected macrophages. Sirt1 is a member of the deacetylase family that is known to promote apoptosis resistance in many mammalian cells. However, the apoptotic role of Sirt1 in the process of M. tuberculosis infection remains unclear. With the help of mouse macrophage samples, 129/Sv background mice, and PBMCs-derived macrophages from healthy controls and patients with tuberculosis, we have shown that M. tuberculosis infection reduced Sirt1 mRNA and protein expression, however, increased Bax mRNA and protein expression. Further, we found that resveratrol, a Sirt1 activator, inhibited M. tuberculosis-induced Bax expression. Thus, it seems that Sirt1 acts as a novel regulator of apoptosis signaling in M. tuberculosis infection via its effects on Bax. Sirt1 activation may therefore be a new candidate for the prevention and treatment of tuberculosis.

SummaryRecent studies have revealed diverse amino acid, post-translational and non-canonical modifications of proteins in diverse organisms and tissues. However, their unbiased detection and analysis remain hindered by technical limitations. Here, we present a spectral alignment method for the identification of protein modifications using high-resolution mass spectrometry proteomics. Termed SAMPEI for Spectral Alignment-based Modified PEptide Identification, this open-source algorithm is designed for the discovery of functional protein and peptide signaling modifications, without prior knowledge of their identities. Using synthetic standards and controlled chemical labeling experiments, we demonstrate its high specificity and sensitivity for the discovery of sub-stoichiometric protein modifications in complex cellular extracts. SAMPEI mapping of mouse macrophage differentiation revealed diverse post-translational protein modifications, including distinct forms of cysteine itaconatylation. SAMPEI’s robust parameterization and versatility are expected to facilitate the discovery of biological modifications of diverse macromolecules. SAMPEI is implemented as a Python package, and is available open-source from BioConda and GitHub (https://github.com/FenyoLab/SAMPEI).

As part of a bioprospecting program aimed at the discovery of anti-inflammatory agents from the Corydalis bungeana Turcz. (C. bungeana), five new hexahydrobenzophenanthridine alkaloids, corycaline A-E (1–5), along with four known alkaloids, were isolated from the whole plant of C. bungeana. Their structures including absolute configurations were elucidated on the basis of extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. The inhibitory activities of the nine compounds on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 mouse macrophage cells were determined; all tested compounds except 2 and 7 exhibited significant inhibitory effects with IC50 values in the range of 1.00–2.79 μM.

Two new aromatic lactone derivatives, homastenones A (1) and B (2), together with six known compounds (3–8), were isolated from the stems of Homalium stenophyllum. Their structures were established on the basis of extensive spectroscopic analyses. Among them, homastenones A (1) and B (2) are two novel aromatic lactone derivatives containing 12 carbon atoms on the carbon skeleton, and the known compounds (3–8) were isolated from this plant for the first time. All new compounds were evaluated for their inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. Compounds 1 and 2 exhibited inhibitory activities with IC50 values of 12.25 ± 0.45 and 25.36 ± 1.65 μM, respectively.

Susanto S, Sumarpo A, Parikesit AA, Putra ABNN, Ishida E, Tabuchi K, Sugahara T. 2018. Short Communication: Immunostimulatory effect of tempoyak (fermented durian) on inducing cytokine production (IL-6 and TNF-α) by RAW 264.7 cells. Biodiversitas 19: 318-322. Indonesia is known to be home to various fermented foods with many-reported usage as potential sources of probiotics. Tempoyak (fermented durian) is among one of the Indonesian fermented foods that are rarely studied for its bioactivities. This study was conducted to evaluate the potential bioactivities of tempoyak, particularly the immunostimulatory aspects. Water extract of tempoyak was prepared by suspending the freeze-dried tempoyak sample in distilled water. Immunostimulatory activity of tempoyak water extract was evaluated using mouse macrophage cell line RAW 264.7. ELISA was used to screen cytokine productions (IL-6 and TNF-α) by RAW 264.7 cells following treatment with tempoyak water extract. In addition, real-time RT-PCR was also used to determine IL-6 and TNF-α mRNA expression. We showed that water extract of tempoyak exerts immunostimulatory effects towards RAW 264.7 cells. This was observed from the increased production of IL-6 and TNF-α in a dose-dependent manner. This was also supported by increased IL-6 and TNF-α mRNA expression. Our finding suggests that tempoyak has immunostimulatory effects towards murine macrophage cell line RAW 264.7. However, further studies are needed to identify the specific compounds responsible for inducing immunostimulatory effects.

Chemical examination of a cultured octocoral, Sinularia sandensis, led to the isolation of a new constituent, (24S)-24-methylcholest-5-en-3β,4α-diol (1), together with two unusual sterols, 3β,4α-dihydroxyergosta-5,24(28)-diene (2) and gorgosterol (3). The structures of compounds 1−3 were established utilizing spectroscopic analysis and literature comparison. Additionally, the structure of 3 was identified by a single-crystal X-ray diffraction analysis directly, for the first time. Sterols 1 and 3 demonstrated slightly inhibition effects for the expression of inducible nitric oxide synthase (iNOS) protein in lipopolysaccharides (LPS)-induced RAW 264.7 mouse macrophage cells.

Two new lignans, mappianthines A (1) and B (2), along with ten known analogues (3-12), were isolated from the stems of Mappianthus iodoides. The structures with the absolute configurations of 1 and 2 were elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons their data with those reported in the literatures. All new compounds were evaluated for their anti-inflammatory activities via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. Compounds 1 and 2 exhibited inhibitory effects with IC50 values comparable to that of hydrocortisone.

Nine new humulane-derived sesquiterpenoids, ochracenes A–I (1–9), were isolated from the Antarctic fungus Aspergillus ochraceopetaliformis SCSIO 05702. Their structures including absolute configurations were elucidated on the basis of spectroscopic analysis, Mosher’s method, and electronic circular dichroism analysis. Compared with previous humulane-type sesquiterpenoids, ochracenes A–I (1–9) featured novel carbon skeletons with corresponding methyl migration, ring cleavage, and carbon loss. Two unprecedented 8,9-secocyclic sesquiterpenoids (2 and 3) exhibited inhibitory effects on lipopolysaccharide-induced NO release in RAW 264.7 mouse macrophage cell lines with IC50 values of 14.6 ± 0.5 and 18.3 ± 1.7 μM, respectively.

Two new mexicanolide-type limonoids, trichiconnarones A ( 1 ) and B ( 2 ), along with six known analogues ( 3 – 8 ), were isolated from the fruits of Trichilia connaroides . The structures of 1 and 2 were elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons their data with those reported in the literatures. All new compounds were evaluated for their anti-inflammatory activities via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro . Compounds 1 and 2 exhibited inhibitory effects with IC 50 values comparable to that of hydrocortisone.

Ophiopogon japonicus (Linn. f.) Ker-Gawl (O. japonicas), mainly cultivated in Sichuan and Zhejiang province in China, has different bioactive components and therefore their pharmacological activities. To explain the different clinical efficacy of O. japonicas derived preparations, herein we report differences of pharmacological activities between Sichuan and Zhejiang O. japonicas and behind them the exact differences of bioactive components. Based on a LC/MS-IT-TOF method, the differences of bioactive components between Sichuan and Zhejiang O. japonicas extracts were analyzed and respective characteristic components were picked out. We determined 39 ophiopogonones and 71 ophiopogonins compounds in Sichuan and Zhejiang O. japonicas extracts and found the contents of these compositions have several times difference. Evidenced by experimental data of pharmacological activities in inhibiting cardiomyocyte damage induced by H2O2, mouse macrophage cell inflammation induced by lipopolysaccharide and cytotoxicity in vitro, Zhejiang O. japonicas extract had a stronger antioxidant and anti-inflammatory capacity than Sichuan O. japonicas extract, and the two O. japonicas extracts exhibited selective cytotoxicity on different cancer cell lines in vitro. These data shed light on the links between bioactive components and pharmacological activities of O. japonicas derived preparations. Thus, geographical origin of O. japonicas should be considered to be a key factor in efficacy studies and further clinical application.

A new indole alkaloid, 17-O-methyl-19-(Z)-naucline (1), together with seven known alkaloids (2–8), were isolated from the stems and leaves of Nauclea officinalis. The structure of 1 was elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons their data with those reported in the literature. 17-O-methyl-19-(Z)-naucline (1) showed significant inhibitory activity on nitric oxide production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro with an IC50 value of 3.6 μM.

The structural design parameters of a medical microrobot, such as the morphology and surface chemistry, should aim to minimize any physical interactions with the cells of the immune system. However, the same surface-borne design parameters are also critical for the locomotion performance of the microrobots. Understanding the interplay of such parameters targeting high locomotion performance and low immunogenicity at the same time is of paramount importance yet has so far been overlooked. Here, we investigated the interactions of magnetically steerable double-helical microswimmers with mouse macrophage cell lines and splenocytes, freshly harvested from mouse spleens, by systematically changing their helical morphology. We found that the macrophages and splenocytes can recognize and differentially elicit an immune response to helix turn numbers of the microswimmers that otherwise have the same size, bulk physical properties, and surface chemistries. Our findings suggest that the structural optimization of medical microrobots for the locomotion performance and interactions with the immune cells should be considered simultaneously because they are highly entangled and can demand a substantial design compromise from one another. Furthermore, we show that morphology-dependent interactions between macrophages and microswimmers can further present engineering opportunities for biohybrid microrobot designs. We demonstrate immunobots that can combine the steerable mobility of synthetic microswimmers and the immunoregulatory capability of macrophages for potential targeted immunotherapeutic applications.

Phytochemical and biological studies of the methanolic extracts from Ganoderma lucidum (Polyporaceae) have led to the identification and isolation of a new lanostane triterpenoid, ganosidone A (1), and its eight known derivatives (2‒9). The structure of new compound was determined by HREIMS, 1 D and 2 D NMR experiments and by comparing the acquired physicochemical data with the published values. All the compounds were evaluated for cancer chemopreventive potential based on their ability to inhibit nitric oxide (NO) production induced by lipopolysaccharides (LPS) in mouse macrophage RAW 264.7 cells in vitro. Notably, at a concentration of 50 μM, compounds 4 and 7 inhibited NO production by 86.5% and 88.2%, respectively.