First Report of Alternaria alternata Causing Leaf Spot on Coffea arabica in China.

Yunnan Province is the major region for coffee (Coffea arabica) cultivation in China, contributing to over 98% of the national yield and total production value (Ma et al. 2022). In May 2023, brown spot symptoms were observed only on the leaves of coffee plants in a field located in Baoshan City (98°52'37.988400"E, 24°58'17.673600"N), Yunnan Province. Notably, brown and irregularly shaped spots initially started on the leaf bases. The spots enlarged and developed concentric rings with dark brown margins, which are often surrounded by yellow halos. Finally, the necrotic spots spread across the entire leaf and caused the leaf to curl and fall off. The incidence of the disease was approximately 3% of the coffee plants (n = 600). The symptomatic leaves collected from 10 plants were sectioned (5 × 5 mm), subjected to surface sterilization with 70% ethanol for 40 s, rinsed with sterile distilled water, air-dried, and transferred to potato dextrose agar (PDA). Fungi with grayish-white, cotton-like aerial mycelia grew after 7 days at 28°C. The older mycelia of these isolates displayed dark gray pigmentation. Single conidia were cultivated on PDA, and 15 morphologically similar monosporic isolates were ultimately obtained. Microscopic observation revealed that these isolates produced branched, septate, transparent and amber mycelium. Brown, elliptical or pear-shaped conidia with 2 to 4 transversal septa and 0 to 3 longitudinal septa, measuring 9.6 to 33.3 long × 6.0 to 15.0 µm wide (n = 30), were observed on potato carrot agar (PCA). Molecular identification of multiple genes, such as ITS (Schoch et al. 2012), RPB2 (O'Donnell et al. 2010) and GAPDH (Berbee et al. 1999), indicated consistent 100% identity among these isolates. Sequences of the representative isolates CFSY1-CFSY5 were deposited in GenBank (acc. nos.: OR351112, PP188577, PP188578, PP294863, PP294864, OR509742, PP215341-PP215344, OR509740 and PP239378-PP239381), revealing 98.35% - 100% homology with distinct Alternaria alternata strains previously deposited in GenBank (acc. nos.: PP110780, MN649031 and OR485338). The multigene phylogenetic analysis positioned isolates CFSY1-CFSY5 within a distinct cluster, alongside diverse A. alternata isolates. Based on morphological and molecular characterizations, the pathogen was identified as A. alternata. To verify its pathogenicity, a conidial suspension (1×106 conidia/mL) of isolate CFSY1 was sprayed on six leaves of three healthy one-year-old C. arabica seedlings. Subsequently, the inoculated seedlings were covered with plastic bags and placed in a growth chamber under controlled conditions (a 14 h daylight period and a 10 h dark period at 28°C). The experiment was repeated three times. After 20 days, typical brown spot symptoms analogous to those originally observed in the field appeared on the leaves in all inoculated plants. Reisolation, morphology identification and DNA sequencing substantiated Koch's postulates. In contrast, control plants treated with sterilized water remained asymptomatic, and no pathogen was reisolated from them. Significantly, A. alternata has been previously reported as the causal agent for leaf spot disease in a diverse variety of woody plant species in China, including Prunus avium (Ahmad et al. 2020), Magnolia grandiflora (Liu et al. 2019) and citrus (Wang et al. 2010). This study represents the first report of brown leaf spot caused by A. alternata specifically on C. arabica in China, enriching the contents of fungal pathogens under Chinese coffee cultivation conditions.

Endophytic bacterial communities in wild rice (Oryza officinalis) and their plant growth-promoting effects on perennial rice.

Endophytic bacterial microbiomes of plants contribute to the physiological health of the host and its adaptive evolution and stress tolerance. Wild rice possesses enriched endophytic bacteria diversity, which is a potential resource for sustainable agriculture. Oryza officinalis is a unique perennial wild rice species in China with rich genetic resources. However, endophytic bacterial communities of this species and their plant growth-promoting (PGP) traits remain largely unknown. In this study, endophytic bacteria in the root, stem, and leaf tissues of O. officinalis were characterized using 16S rRNA gene Illumina sequencing. Culturable bacterial endophytes were also isolated from O. officinalis tissues and characterized for their PGP traits. The microbiome analysis showed a more complex structure and powerful function of the endophytic bacterial community in roots compared with those in other tissue compartments. Each compartment had its specific endophytic bacterial biomarkers, including Desulfomonile and Ruminiclostridium for roots; Lactobacillus, Acinetobacter, Cutibacterium and Dechloromonas for stems; and Stenotrophomonas, Chryseobacterium, Achromobacter and Methylobacterium for leaves. A total of 96 endophytic bacterial strains with PGP traits of phosphate solubilization, potassium release, nitrogen fixation, 1-aminocyclopropane-1-carboxylate (ACC) deaminase secretion, and siderophore or indole-3-acetic acid (IAA) production were isolated from O. officinalis. Among them, 11 strains identified as Enterobacter mori, E. ludwigii, E. cloacae, Bacillus amyloliquefaciens, B. siamensis, Pseudomonas rhodesiae and Kosakonia oryzae were selected for inoculation of perennial rice based on their IAA production traits. These strains showed promising PGP effects on perennial rice seedlings. They promoted plants to form a strong root system, stimulate biomass accumulation, and increase chlorophyll content and nitrogen uptake, which could fulfil the ecologically sustainable cultivation model of perennial rice. These results provide insights into the bacterial endosphere of O. officinalis and its application potential in perennial rice. There is the prospect of mining beneficial endophytic bacteria from wild rice species, which could rewild the microbiome of cultivated rice varieties and promote their growth.

First Report of Fusarium oxysporum Causing Leaf Wilt on Dinteranthus vanzylii (Green Stone Plant) in China.

Dinteranthus vanzylii is a low-growing species in the family Aizoaceae, native to southern Africa, with a pair of thick grey leaves covered with dark red spots and stripes. This stone-like succulent grows near the ground, which may protect it from water evaporation and herbivores. Dinteranthus vanzylii has become popular in China due to its attractive appearance and easy indoor cultivation. In September 2021, 7% of D. vanzylii (approximately 140 pots) showed leaf wilt symptoms in a commercial greenhouse located in Ningde (119°35'39.696″E, 27°23'30.556″N), Fujian Province, China. The diseased plants were shrivelling and eventually underwent necrosis. Their leaf tissues were rotting and carpeted with white mycelium. The leaf tissues of 10 symptomatic plants were cut into 0.5 cm2 pieces, surface-sterilized and placed on PDA medium. According to the colony morphology after 7 days of culture, 20 fungal isolates with abundant whitish aerial mycelium were divided into two types: 8 isolates produced lilac pigment whereas 12 did not. Both produced unicellular ovoid microconidia, sickled-shaped macroconidia with 3 - 4 septa and single or paired smooth, thick-walled chlamydospores on carnation leaf agar (CLA). Molecular identification based on DNA sequences from EF1-α (O'Donnell et al. 1998), RPB1 and RPB2 (O'Donnell et al. 2010) revealed 100% identity among isolates within each group; however, there were several base differences between two types. Sequences of representative isolates KMDV1 and KMDV2 were deposited in GenBank (acc. nos.: OP910243, OP910244, OR030448, OR030449, OR030450 and OR030451), which showed 99.10% - 99.74% identity with different F. oxysporum strains (GenBank acc. nos.: KU738441, LN828039, MN457050, MN457049, ON316742 and ON316741). Phylogenetic tree inferred from the concatenated EF1-α, RPB1 and RPB2 revealed that these isolates clustered with F. oxysporum. Thus, these isolates were identified as F. oxysporum. Using a root-drenching method, 10 one-year-old healthy D. vanzylii were inoculated in conidial suspensions (1*106 conidia/mL) of isolates KMDV1 and KMDV2 for 60 min, respectively. They were transplanted into pots with sterilized soil and incubated in a plant-growth chamber at 25°C and 60% relative humidity. Control plants were treated with sterilized water. The pathogenicity test was repeated three times. All plants inoculated with each isolate developed leaf wilt symptoms after 15 days and were dead after 20 - 30 days. However, no symptoms were observed in the control plants. Fusarium oxysporum was reisolated and confirmed based on morphology and EF1-α sequence analysis. No pathogens were isolated from the control plants. This is the first report of F. oxysporum causing leaf wilt disease on D. vanzylii in China. To date, several diseases have been reported on members of the Aizoaceae. For instance, collar and stem rot on Lampranthus sp. caused by Pythium aphanidermatum (Garibaldi et al. 2009), wilt on Lampranthus sp. and Tetragonia tetragonioides caused by Verticillium dahliae (Garibaldi et al. 2010; Garibaldi et al. 2013), and leaf spot on Sesuvium portulacastrum caused by Gibbago trianthemae (Chen et al., 2022). Our research could provide insight into fungal diseases on members of the Aizoaceae and contribute to their cultivation and management.

[Preparation of mouse monoclonal antibodies against human adenovirus 55 Hexon (HAdV55 Hexon) protein].

Objective To prepare specific mouse monoclonal antibody (mAb) against human adenovirus type 55 Hexon protein (HAdV55 Hexon). Methods The Hexon genes of HAdV55, 3, 4, 7, 16 and 21 were chemically synthesized as templates for PCR amplification. The prokaryotic expression plasmids pET28a-HAdV55 Hexon and eukaryotic expression plasmids pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon were constructed respectively. The pET28a-HAdV55 Hexon plasmid was transformed into E. coli competent cell BL21 (DE3) and was induced by IPTG. After the purified inclusion body was denatured and renatured, Hexon55 protein was purified by tangential flow filtration system. pCAGGS-HAdV55 Hexon was used to immunize BALB/c mice by cupping, and HAdV55 Hexon protein was used to booster immunization. The anti-HAdV55 Hexon mAb was prepared by hybridoma technique and the titer and subclass were determined. The specificity of antibody was identified by Western blot using HEK293T cells transfected with pCAGGS-HAdV55 Hexon and by immunofluorescence assay (IFA) using BHK cells transfected with pCAGGS-HAdV55 Hexon. Both clones with high titer were selected, and the cross-reactivity of pCAGGS-HAdV3, 4, 7, 16, 21 and 55 Hexon transfected cells were analyzed by Western blot analysis and IFA. Results PET28a-HAdV55 Hexon and pCAGGS-HAdV55 Hexon, 3, 4, 7, 16 and 21 expression plasmids were successfully constructed. BL21 transformed with pET28a-HAdV55 Hexon was induced by IPTG. The HAdV55 Hexon protein was mainly expressed in the form of inclusion body. After denaturation and renaturation, the purified HAdV55 Hexon protein was obtained by ultrafiltration. Six hybridoma cell lines secreting HAdV55 Hexon mAb were obtained. The antibody subclass analysis showed that 2 strains were IgG2a subtypes and 4 strains were IgG2b. Two specific HAdV55 Hexon antibodies with high titer were obtained, and there was no cross-reactivity with HAdV3, 4, 7, 16, 21 Hexon. Conclusion The specific mice mAb against HAdV55 Hexon provides an experimental basis for establishing its antigen detection method.

A modified Fangji Huangqi decoction ameliorates pulmonary artery hypertension via phosphatidylinositide 3-kinases/protein kinase B-mediated regulation of proliferation and apoptosis of smooth muscle cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulmonary artery hypertension (PAH) is a progressive and fatal lung disease of multifactorial etiology, which arouses an enhanced interest in PAH disease therapy. Modified Fangji Huangqi decoction (MFJHQ), a traditional Chinese medicine (TCM) formula, has a crucial role in the treatment of PAH. However, the pharmacological roles and mechanisms of MFJHQ on PAH remain unknown. AIM OF THE STUDY: To investigate the effects and potential mechanism of MFJHQ on pulmonary vascular remodeling in PAH. MATERIAL AND METHODS: Ultra-performance liquid chromatography (UPLC) was employed to quantitate the principal components in MFJHQ. Rats were treated with MFJHQ by gavage for final 2 weeks in monocrotaline (MCT)-induced PAH rats. RNA-sequencing and network pharmacology analysis were performed to explore the potential mechanism. The primary rat pulmonary artery smooth muscle cells (PASMCs) were utilized to evaluate the regulatory effect of MFJHQ in vitro. RESULTS: Seven active components from MFJHQ were quantitated by UPLC. In rats with MCT-induced PAH, MFJHQ treatment significantly improved hemodynamic parameters, right ventricular hypertrophy index, lung function, and attenuated pulmonary vascular remodeling. Mechanistically, we further confirmed that MFJHQ inhibits MCT-induced phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt) pathway predicated by network pharmacology and RNA-sequencing analysis to reduce the proliferation of pulmonary arteries and promote pulmonary artery apoptosis in lung tissues. Additionally, MFJHQ hindered the proliferation and migration, and accelerated apoptosis in PDGF-BB-induced PASMCs in vitro, which can be enhanced by the presence of the PI3K inhibitor LY294002. CONCLUSIONS: Our results indicated that MFJHQ inhibited MCT-induced pulmonary vascular remodeling by decreasing proliferation and migration of PASMCs and promoting PASMC apoptosis through PI3K/Akt pathway, which provides a novel treatment option for PAH with multi-targeting mechanisms inspired by TCM theory.

Xuanfei Baidu Decoction regulates NETs formation via CXCL2/CXCR2 signaling pathway that is involved in acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening symptoms in Coronavirus Disease 2019 (COVID-19) patients. Xuanfei Baidu Decoction (XFBD) is a recommend first-line traditional Chinese medicine (TCM) formula therapeutic strategy for COVID-19 patients. Prior studies demonstrated the pharmacological roles and mechanisms of XFBD and its derived effective components against inflammation and infections through multiple model systems, which provided the biological explanations for its clinical use. Our previous work revealed that XFBD inhibited macrophages and neutrophils infiltration via PD-1/IL17A signaling pathway. However, the subsequent biological processes are not well elucidated. Here, we proposed a hypothesis that XFBD can regulate the neutrophils-mediated immune responses, including neutrophil extracellular traps (NETs) formation and the generation of platelet-neutrophil aggregates (PNAs) after XFBD administration in lipopolysaccharide (LPS)-induced ALI mice. The mechanism behind it was also firstly explained, that is XFBD regulated NETs formation via CXCL2/CXCR2 axis. Altogether, our findings demonstrated the sequential immune responses of XFBD after inhibiting neutrophils infiltration, as well as shedding light on exploiting the therapy of XFBD targeting neutrophils to ameliorate ALI during the clinical course.

Magnetic liposome as a dual-targeting delivery system for idiopathic pulmonary fibrosis treatment.

Idiopathic pulmonary fibrosis (IPF) is the most common form of idiopathic interstitial pneumonia, where M2 macrophages play an irreplaceable role in the anti-inflammatory progress. Targeting M2 macrophages and regulating their polarization may be a potential treatment strategy for IPF. Herein, we designed a magnetic liposome based dual-targeting delivery system for the IPF treatment, constructed by mannose-modified magnetic nanoparticles (MAN-MNPs) loaded on the surface of the liposome (MAN-MNPs@LP). The delivery system is capable of responding to a static magnetic field (SMF) and then recognizing in situ of M2 macrophages through the mannose receptor-dependent internalization. Firstly, a series of physical and chemical assays were used to characterize these nanoparticles. Subsequently, magnetic liposomes accumulation in the damaged lung with/without mannose modification and SMF were compared by in vivo imaging system. Finally, the reduction of M2 macrophages and inhibition of their polarization confirmed that the development of IPF was retarded due to the in situ release of encapsulated dexamethasone (Dex) in lungs under the SMF. Further investigation demonstrated that the expression of α-SMA and collagen deposition was reduced. Altogether, this dual-targeting delivery system can effectively deliver Dex into M2 macrophages in the lung, making it a novel and promising therapeutic system for the IPF treatment.

First Report of Fusarium oxysporum Causing Stem Rot on Mammillaria humboldtii in China.

Mammillaria humboldtii found in Mexico is a short-globose ornamental cactus species of the Cactaceae family, which has gained increasing popularity in China. It is characterized by tuberculate stems, dimorphic areoles, small pink flowers and pitted seed cell walls. The populations of wild M. humboldtii are critically endangered and are now of international conservation concern. In July 2021, stem rot symptoms were observed on M. humboldtii in a commercial greenhouse located in Zhangzhou (117°39'44.0064″E, 24°28'3.7236″N), Fujian Province (southern China). The typical symptoms were water-soaking, rotting and wilting on the stem, eventually leading to necrosis of the plants within 20 to 30 days. The vascular system of infected stems and roots showed a reddish-brown discolouration. The disease affected approximately 10% of 1000 plants. Fungi were isolated from the diseased stems of 26 samples, which were chopped into small pieces (5 × 5 mm), surface-sterilized with 75% ethanol for 40 s, and placed onto potato dextrose agar (PDA). After seven days of dark culture at 28°C, morphologically similar fungal isolates with whitish aerial mycelium and purple pigment were observed. On carnation leaf agar (CLA), isolates produced sickle and slightly curved macroconidia with three to four septa, measuring 12.8 to 27.9 × 1.9 to 3.8 µm (n = 15), and unicellular, ovoid to elliptical microconidia measuring 3.8 to 7.7 × 1.4 to 2.5 µm (n = 30). Smooth walled chlamydospores were terminal or intercalary, single or in pairs, measuring 9.2 to 13.1 µm (n = 15) in diameter. For molecular identification, the internal transcribed spacer (ITS) region of rDNA (Schoch et al. 2012), translation elongation factor-1α (EF1-α) (Maryani et al. 2019) and gene coding endopolygalacturonase 1 (PG1) (Hirano et al. 2006) of the representative isolate FJMH7 were amplified, purified and sequenced. BLASTn analysis of the ITS, EF1-α and pg1 sequences (GenBank accession numbers: ON832660, ON843495 and ON843496) showed 100%, 99.70% and 98.96% identity with F. oxysporum (GenBank accession numbers: KX611626, OM801797 and KF437345), respectively. Phylogenetic analysis based on the the concatenated ITS and EF1-α sequences and pg1 genes placed isolate FJMH7 with F. oxysporum reference strains in the phylogenetic trees. Based on morphological identification and sequence analysis, this isolate was identified as F. oxysporum. For the pathogenicity assay, six 6-month-old healthy plants of Mammillaria humboldtii were inoculated by dipping roots in a conidial suspension (106 conidia/mL) of isolate FJMH7 cultured in Bilai's medium for three days. Six noninoculated plants treated with Bilai's medium served as a control. Plants were transplanted into pots filled with sterilized soils and placed in a glasshouse at 25°C. After 15 days, all the inoculated plants exhibited rot symptoms on stems, which were similar to those observed in the commercial greenhouses. All inoculated plants were dead 30 days after inoculation. Control plants did not show any symptoms. F. oxysporum was reisolated and confirmed based on morphology and sequencing. No fungi were reisolated from control plants. To fulfil Koch's postulates, the pathogenicity assay was repeated twice with the same results. To date, F. oxysporum isolates have been reported on golden barrel cactus (Echinocactus grusonii) (Polizzi et al. 2004), night-blooming cereus (Hylocereus undatus) (Wright et al. 2007), apple cactus (Cereus peruvianus monstruosus) (Garibaldi et al. 2011), Schlumbergera truncate (Lops et al. 2013), Astrophytum ornatum (Quezada-Salinas et al. 2017) and Nopalea cochenillifera (Santiago et al. 2018). To our knowledge, this is the first report of F. oxysporum on M. humboldtii in China, indicating that this pathogen could cause wilt and rot disease on different cactus hosts.

Salvianolic acid B dry powder inhaler for the treatment of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a serious and fatal pulmonary inflammatory disease with an increasing incidence worldwide. The drugs nintedanib and pirfenidone, are listed as conditionally recommended drugs in the "Evidence-Based Guidelines for the Diagnosis and Treatment of Idiopathic Pulmonary Fibrosis". However, these two drugs have many adverse reactions in clinical application. Salvianolic acid B (Sal B), a water-soluble component of Salvia miltiorrhiza, could alleviate bleomycin-induced peroxidative stress damage, and prevent or delay the onset of IPF by regulating inflammatory factors and fibrotic cytokines during the disease's progression. However, Sal B is poorly absorbed orally, and patient compliance is poor when administered intravenously. Therefore, there is an urgent need to find a new non-injection route of drug delivery. In this study, Sal B was used as model drug and l-leucine (LL) as excipient to prepare Sal B dry powder inhaler (Sal B-DPI) by spray drying method. Modern preparation evaluation methods were used to assess the quality of Sal B-DPI. Sal B-DPI is promising for the treatment of IPF, according to studies on pulmonary irritation evaluation, in vivo and in vitro pharmacodynamics, metabolomics, pharmacokinetics, and lung tissue distribution.

Xuanfei Baidu Decoction reduces acute lung injury by regulating infiltration of neutrophils and macrophages via PD-1/IL17A pathway.

The pathogenic hyper-inflammatory response has been revealed as the major cause of the severity and death of the Corona Virus Disease 2019 (COVID-19). Xuanfei Baidu Decoction (XFBD) as one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, shows unique advantages in the control of symptomatic transition from moderate to severe disease states. However, the roles of XFBD to against hyper-inflammatory response and its mechanism remain unclear. Here, we established acute lung injury (ALI) model induced by lipopolysaccharide (LPS), presenting a hyperinflammatory process to explore the pharmacodynamic effect and molecular mechanism of XFBD on ALI. The in vitro experiments demonstrated that XFBD inhibited the secretion of IL-6 and TNF-α and iNOS activity in LPS-stimulated RAW264.7 macrophages. In vivo, we confirmed that XFBD improved pulmonary injury via down-regulating the expression of proinflammatory cytokines such as IL-6, TNF-α and IL1-ß as well as macrophages and neutrophils infiltration in LPS-induced ALI mice. Mechanically, we revealed that XFBD treated LPS-induced acute lung injury through PD-1/IL17A pathway which regulates the infiltration of neutrophils and macrophages. Additionally, one major compound from XFBD, i.e. glycyrrhizic acid, shows a high binding affinity with IL17A. In conclusion, we demonstrated the therapeutic effects of XFBD, which provides the immune foundations of XFBD and fatherly support its clinical applications.

Xuanfei Baidu Decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuanfei Baidu Decoction (XFBD), one of the "three medicines and three prescriptions" for the clinically effective treatment of COVID-19 in China, plays an important role in the treatment of mild and/or common patients with dampness-toxin obstructing lung syndrome. AIM OF THE STUDY: The present work aims to elucidate the protective effects and the possible mechanism of XFBD against the acute inflammation and pulmonary fibrosis. METHODS: We use TGF-ß1 induced fibroblast activation model and LPS/IL-4 induced macrophage inflammation model as in vitro cell models. The mice model of lung fibrosis was induced by BLM via endotracheal drip, and then XFBD (4.6 g/kg, 9.2 g/kg) were administered orally respectively. The efficacy and molecular mechanisms in the presence or absence of XFBD were investigated. RESULTS: The results proved that XFBD can effectively inhibit fibroblast collagen deposition, down-regulate the level of α-SMA and inhibit the migration of fibroblasts. IL-4 induced macrophage polarization was also inhibited and the secretions of the inflammatory factors including IL6, iNOS were down-regulated. In vivo experiments, the results proved that XFBD improved the weight loss and survival rate of the mice. The XFBD high-dose administration group had a significant effect in inhibiting collagen deposition and the expression of α-SMA in the lungs of mice. XFBD can reduce bleomycin-induced pulmonary fibrosis by inhibiting IL-6/STAT3 activation and related macrophage infiltration. CONCLUSIONS: Xuanfei Baidu Decoction protects against macrophages induced inflammation and pulmonary fibrosis via inhibiting IL-6/STAT3 signaling pathway.

Axl-/- neurons promote JEV infection by dampening the innate immunity.

Japanese encephalitis virus (JEV) causes the most commonly diagnosed viral encephalitis in Asia. JEV is a highly neurotropic flavivirus that can replicate efficiently in the brain. Axl belongs to the TAM (Tyro3, Axl, Mer) family, a group of tyrosine kinase receptors involved in the viral entry, micked as apoptotic bodies and regulation of innate immunity. However, the underlying mechanisms on its regulation in the neurons for JEV are unclear. Here, we found that Axl was upregulated in neurons after JEV infection. Unexpectedly, Axl deficient (Axl-/-) mice were more susceptible to JEV infection with increased viral loads in neurons. The RNA-sequencing analysis between the wild type neurons and Axl-/- neurons infected with JEV showed that many interferon-stimulated genes were downregulated in the Axl-/- neurons which innate immunity was attenuated largely. The rescue experiment in Axl-/- neurons indicated that Axl may be positively involved in the regulation of antiviral immunity. Taken together, our data demonstrated that Axl may play an antiviral role in JEV replication within neurons by modulating neuronal innate immunity.

Traditional Herbal Medicine Discovery for the Treatment and Prevention of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary artery remodeling that may subsequently culminate in right heart failure and premature death. Although there are currently both non-pharmacological (lung transplantation, etc.) and pharmacological (Sildenafil, Bosentan, and new oral drugs on trial) therapies available, PAH remains a serious and fatal pulmonary disease. As a unique medical treatment, traditional herbal medicine (THM) treatment has gradually exerted its advantages in treating PAH worldwide through a multi-level and multi-target approach. Additionally, the potential mechanisms of THM were deciphered, including suppression of proliferation and apoptosis of pulmonary artery smooth muscle cells, controlling the processes of inflammation and oxidative stress, and regulating vasoconstriction and ion channels. In this review, the effects and mechanisms of the frequently studied compound THM, single herbal preparations, and multiple active components from THM are comprehensively summarized, as well as their related mechanisms on several classical preclinical PAH models. It is worth mentioning that sodium tanshinone IIA sulfonate sodium and tetramethylpyrazine are under clinical trials and are considered the most promoting medicines for PAH treatment. Last, reverse pharmacology, a strategy to discover THM or THM-derived components, has also been proposed here for PAH. This review discusses the current state of THM, their working mechanisms against PAH, and prospects of reverse pharmacology, which are expected to facilitate the natural anti-PAH medicine discovery and development and its bench-to-bedside transformation.

Slow monomer vibrations in formic acid dimer: Stepping up the ladder with FTIR and Raman jet spectroscopy.

In an effort to extend the cold gas phase spectroscopic database of the cyclic formic acid dimer (FAD), we present and analyze the jet-cooled vibrational infrared and Raman spectrum of (HCOOH)2 in the monomer fingerprint region between 600 and 1500 cm-1. The present study bridges the gap between the intermolecular dimerization-induced and the carbonyl stretching fundamentals that have already been reexamined using jet-cooled or high-resolution spectroscopy. This completes the characterization of the jet-cooled vibrational (HCOOH)2 spectrum below the complex OH (CH) stretching fundamentals, and we report resonance-induced FAD combination/overtone transitions that will serve as a valuable reference for a theoretical modeling of its vibrational dynamics. As a by-product, several new formic acid trimer fundamentals are identified in the jet spectra and assigned with the help of second-order vibrational perturbation theory (VPT2). The polar formic acid dimer still eludes detection in a supersonic jet, but we are able to estimate an experimental upper-bound of the polar dimer-to-trimer-to-cyclic dimer intensity ratio to about 1:10:100 under typical expansion conditions. Using VPT2 with resonance treatment (VPT2+K), we reinvestigate the notorious ν22 resonance triad. Generally, we find that VPT2, which is, of course, inadequate for modeling the resonance-rich OH stretching spectrum of FAD, is performing very satisfactorily in predicting fundamental and two-quantum state term values for the slower modes below 1500 cm-1. As these modes are the building blocks for the ultrafast energy dissipation in the OH stretching region, the present work opens the door for its quantitative understanding.

Integrated Metabolomics and Transcriptomics Analyses Reveal Metabolic Landscape in Neuronal Cells during JEV Infection.

Japanese encephalitis virus (JEV) is a leading cause of viral encephalitis in endemic regions of Asia. The neurotropism of JEV and its high-efficiency replication in neurons are the key events for pathogenesis. Revealing the interplay between virus and host cells in metabolic facet is of great importance both for unraveling the pathogenesis mechanisms and providing novel antiviral targets. This study took advantage of the integration analysis of metabolomics and transcriptomics to depict the metabolic profiles of neurons during the early stage of JEV infection. Increased glycolysis and its branched pentose phosphate pathway (PPP) flux and impaired oxidative phosphorylation (OXPHOS) in glucose utilization, and the catabolic patterns of lipid metabolism were created to facilitate the biosynthesis of precursors needed for JEV replication in neurons. Pharmacological inhibitions of both glycolysis pathway and PPP in neurons suggested its indispensable role in maintaining the optimal propagation of JEV. In addition, analysis of metabolomic-transcriptomic regulatory network showed the pivotal biological function of lipid metabolism during JEV infection. Several pro-inflammatory lipid metabolites were significantly up-regulated and might partially be responsible for the progression of encephalitis. These unique metabolic reprogramming features might give deeper insight into JEV infected neurons and provide promising antiviral approaches targeting metabolism.

Combination therapy of tanshinone IIA and puerarin for pulmonary fibrosis via targeting IL6-JAK2-STAT3/STAT1 signaling pathways.

Efficient therapy of idiopathic pulmonary fibrosis (IPF) is still a major challenge. The current studies with single-target drug therapy are the pessimistic approaches due to the complex characteristics of IPF. Here, a combination therapy of Tanshinone IIA and Puerarin for IPF was proposed to alleviate IPF due to their antiinflammatory and anti-fibrotic effects. In vivo, the combination therapy could significantly attenuate the area of ground glass opacification that was presented by 85% percentile density score of the micro-CT images when compared to single conditions. In addition, the combination therapy enormously improved the survival rate and alleviated pathological changes in bleomycin (BLM)-induced IPF mice. By using a wide spectrum of infiltration biomarkers in immunofluorescence assay in pathological sections, we demonstrate that fewer IL6 related macrophage infiltration and fibrosis area after this combination therapy, and further proved that IL6-JAK2-STAT3/STAT1 is the key mechanism of the combination therapy. In vitro, combination therapy markedly inhibited the fibroblasts activation and migration which was induced by TGF-ß1 or/and IL6 through JAK2-STAT3/STAT1 signaling pathway. This study demonstrated that combination therapeutic effect of TanIIA and Pue on IPF may be related to the reduced inflammatory response targeting IL6, which could be an optimistic and effective approach for IPF.

QiShenYiQi ameliorates salt-induced hypertensive nephropathy by balancing ADRA1D and SIK1 expression in Dahl salt-sensitive rats.

BACKGROUND: Hypertension is a leading risk factor for developing kidney disease. Current single-target antihypertensive drugs are not effective for hypertensive nephropathy, in part due to its less understood mechanism of pathogenesis. We recently showed that QiShenYiQi (QSYQ), a component-based cardiovascular Chinese medicine, is also effective for ischemic stroke. Given the important role of the brain-heart-kidney axis in blood pressure control, we hypothesized that QSYQ may contribute to blood pressure regulation and kidney protection in Dahl salt-sensitive hypertensive rats. METHODS: The therapeutic effects of QSYQ on blood pressure and kidney injury in Dahl salt-sensitive rats fed with high salt for 9 weeks were evaluated by tail-cuff blood pressure monitoring, renal histopathological examination and biochemical indicators in urine and serum. RNA-seq was conducted to identify QSYQ regulated genes in hypertensive kidney, and RT-qPCR, immunohistochemistry, and Western blotting analysis were performed to verify the transcriptomics results and validate the purposed mechanisms. RESULTS: QSYQ treatment significantly decreased blood pressure in Dahl salt-sensitive hypertensive rats, alleviated renal tissue damage, reduced renal interstitial fibrosis and collagen deposition, and improved renal physiological function. RNA-seq and subsequent bioinformatic analysis showed that the expression of ADRA1D and SIK1 genes were among the most prominently altered by QSYQ in salt-sensitive hypertensive rat kidney. RT-qPCR, immunohistochemistry and Western blotting results confirmed that the mRNA and protein expression levels of alpha-1D adrenergic receptor (ADRA1D) in the kidney tissue of the QSYQ-treated rats were markedly down-regulated, while the mRNA and protein levels of salt inducible kinase 1 (SIK1) were significantly increased. CONCLUSION: QSYQ not only lowered blood pressure, but also alleviated renal damage via reducing the expression of ADRA1D and increasing the expression of SIK1 in the kidney of Dahl salt-sensitive hypertensive rats.

Macrophage-Targeted Lung Delivery of Dexamethasone Improves Pulmonary Fibrosis Therapy via Regulating the Immune Microenvironment.

Idiopathic pulmonary fibrosis (IPF) is serious chronic lung disease with limited therapeutic approaches. Inflammation and immune disorders are considered as the main factors in the initiation and development of pulmonary fibrosis. Inspired by the key roles of macrophages during the processes of inflammation and immune disorders, here, we report a new method for direct drug delivery into the in-situ fibrotic tissue sites in vitro and in vivo. First, liposomes containing dexamethasone (Dex-L) are prepared and designed to entry into the macrophages in the early hours, forming the macrophages loaded Dex-L delivery system (Dex-L-MV). Chemokine and cytokine factors such as IL-6, IL-10, Arg-1 are measured to show the effect of Dex-L to the various subtypes of macrophages. Next, we mimic the inflammatory and anti-inflammatory microenvironment by co-culture of polarized/inactive macrophage and fibroblast cells to show the acute inflammation response of Dex-L-MV. Further, we confirm the targeted delivery of Dex-L-MV into the inflammatory sites in vivo, and surprisingly found that injected macrophage containing Dex can reduce the level of macrophage infiltration and expression of the markers of collagen deposition during the fibrotic stage, while causing little systematic toxicity. These data demonstrated the suitability and immune regulation effect of Dex-L-MV for the anti-pulmonary process. It is envisaged that these findings are a step forward toward endogenous immune targeting systems as a tool for clinical drug delivery.

Nano-Strategies for Improving the Bioavailability of Inhaled Pharmaceutical Formulations.

Pulmonary pharmaceutical formulations are targeted for the treatment of respiratory diseases. However, their application is limited due to the physiological characteristics of the lungs, such as branching structure, mucociliary and macrophages, as well as certain properties of the drugs like particle size and solubility. Nano-formulations can ameliorate particle sizes and improve drug solubility to enhance bioavailability in the lungs. The nano-formulations for lungs reviewed in this article can be classified into nanocarriers, no-carrier-added nanosuspensions and polymer-drug conjugates. Compared with conventional inhalation preparations, these novel pulmonary pharmaceutical formulations have their own advantages, such as increasing drug solubility for better absorption and less inflammatory reaction caused by the aggregation of insoluble drugs; prolonging pulmonary retention time and reducing drug clearance; improving the patient compliance by avoiding multiple repeated administrations. This review will provide the reader with some background information for pulmonary drug delivery and give an overview of the existing literature about nano-formulations for pulmonary application to explore nano-strategies for improving the bioavailability of pulmonary pharmaceutical formulations.