New methods for resolution of hydroxychloroquine by forming diastereomeric salt and adding chiral mobile phase agent on RP-HPLC.

Hydroxychloroquine (HCQ), 2-([4-([7-Chloro-4-quinolyl]amino)pentyl]ethylamino)ethanol, exhibited significant biological activity, while its side effects cannot be overlooked. The RP-HPLC enantio-separation was investigated for cost-effective and convenient optical purity analysis of HCQ. The thermodynamic resolution of Rac-HCQ, driven by enthalpy and entropy, was achieved on the C18 column using Carboxymethyl-ß-cyclodextrin (CM-ß-CD) as the chiral mobile phase agent (CMPA). The effects of CCM-ß-CD, pH, and triethylamine (TEA) V% on the enantio-separation process were explored. Under the optimum conditions at 24°C, the retention times for the two enantiomers were t R 1 = 29.39 min $$ {t}_{R1}=29.39\ \min $$ and t R 2 = 32.42 min $$ {t}_{R2}=32.42\ \min $$ , resulting in R s = 1.87 $$ {R}_s=1.87 $$ . The resolution via diastereomeric salt formation of Rac-HCQ was developed to obtain the active pharmaceutical ingredient of single enantiomer S-HCQ. Di-p-Anisoyl-L-Tartaric Acid (L-DATA) was proved effective as the resolution agent for Rac-HCQ. Surprisingly, it was found that refluxing time was a key fact affecting the resolution efficiency, which meant the kinetic dominate during the process of the resolution. Four factors-solvent volume, refluxing time, filtration temperature, and molar ratio-were optimized using the single-factor method and the response surface method. Two cubic models were established, and the reliability was subsequently verified. Under the optimal conditions, the less soluble salt of 2L-DATA:S-HCQ was obtained with a yield of 96.9% and optical purity of 63.0%. The optical purity of this less soluble salt increases to 99.0% with a yield of 74.2% after three rounds recrystallization.

ISG12a promotes immunotherapy of HBV-associated hepatocellular carcinoma through blocking TRIM21/AKT/ß-catenin/PD-L1 axis.

Hepatitis B virus (HBV) infection generally elicits weak type-I interferon (IFN) immune response in hepatocytes, covering the regulatory effect of IFN-stimulated genes. In this study, low level of IFN-stimulated gene 12a (ISG12a) predicted malignant transformation and poor prognosis of HBV-associated hepatocellular carcinoma (HCC), whereas high level of ISG12a indicated active NK cell phenotypes. ISG12a interacts with TRIM21 to inhibit the phosphorylation activation of protein kinase B (PKB, also known as AKT) and ß-catenin, suppressing PD-L1 expression to block PD-1/PD-L1 signaling, thereby enhancing the anticancer effect of NK cells. The suppression of PD-1-deficient NK-92 cells on HBV-associated tumors was independent of ISG12a expression, whereas the anticancer effect of PD-1-expressed NK-92 cells on HBV-associated tumors was enhanced by ISG12a and treatments of atezolizumab and nivolumab. Thus, tumor intrinsic ISG12a promotes the anticancer effect of NK cells by regulating PD-1/PD-L1 signaling, presenting the significant role of innate immunity in defending against HBV-associated HCC.

Single-cell transcriptome reveals a novel mechanism of C-Kit+-liver sinusoidal endothelial cells in NASH.

AIM: To understand how liver sinusoidal endothelial cells (LSECs) respond to nonalcoholic steatohepatitis (NASH). METHODS: We profiled single-LSEC from livers of control and MCD-fed mice. The functions of C-Kit+-LSECs were determined using coculture and bone marrow transplantation (BMT) methods. RESULTS: Three special clusters of single-LSEC were differentiated. C-Kit+-LSECs of cluster 0, Msr1+-LSECs of cluster 1 and Bmp4+Selp+-VECs of cluster 2 were revealed, and these cells with diverse ectopic expressions of genes participated in regulation of endothelial, fibrosis and lipid metabolism in NASH. The number of C-Kit+-primary LSECs isolated from MCD mice was lower than control mice. Immunofluorescence co-staining of CD31 and C-KIT showed C-Kit+-LSECs located in hepatic sinusoid were also reduced in NASH patients and MCD mice, compared to AIH patients and control mice respectively. Interestingly, lipotoxic hepatocytes/HSCs cocultured with C-Kit+-LSECs or the livers of MCD mice receipting of C-Kit+-BMCs (bone marrow cells) showed less steatosis, inflammation and fibrosis, higher expression of prolipolytic FXR and PPAR-α, lower expression of TNF-α and α-SMA. Furthermore, coculturing or BMT of C-Kit+-endothelial derived cells could increase the levels of hepatic mitochondrial LC3B, decrease the degree of mitochondrial damage and ROS production through activating Pink1-mediated mitophagy pathway in NASH. CONCLUSIONS: Hence, a novel transcriptomic view of LSECs was revealed to have heterogeneity and complexity in NASH. Importantly, a cluster of C-Kit+-LSECs was confirmed to recovery Pink1-related mitophagy and NASH progression.

PZR suppresses innate immune response to RNA viral infection by inhibiting MAVS activation in interferon signaling mediated by RIG-I and MDA5.

RNA viral infections seriously endanger human health. Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2) suppresses innate immunity against influenza A virus, and pharmacological inhibition of SHP2 provokes hepatic innate immunity. SHP2 binds and catalyzes tyrosyl dephosphorylation of protein zero-related (PZR), but the regulatory effect of PZR on innate immune response to viral infection is unclear. In this study, the transcription and protein level of PZR in host cells were found to be decreased with RNA viral infection, and high level of PZR was uncovered to inhibit interferon (IFN) signaling mediated by RIG-I and MDA5. Through localizing in mitochondria, PZR targeted and interacted with MAVS (also known as IPS-1/VISA/Cardif), suppressing the aggregation and activation of MAVS. Specifically, Y263 residue in ITIM is critical for PZR to exert immunosuppression under RNA viral infection. Moreover, the recruited SHP2 by PZR that modified with tyrosine phosphorylation under RNA viral infection might inhibit phosphorylation activation of MAVS. In conclusion, PZR and SHP2 suppress innate immune response to RNA viral infection through inhibiting MAVS activation. This study reveals the regulatory mechanism of PZR-SHP2-MAVS signal axis on IFN signaling mediated by RIG-I and MDA5, which may provide new sight for developing antiviral drugs.

Establishment of cell culture model and humanized mouse model of chronic hepatitis B virus infection.

IMPORTANCE: Approximately 257 million people worldwide have been infected with hepatitis B virus (HBV), and HBV infection can cause chronic hepatitis, cirrhosis, and even liver cancer. The lack of suitable and effective infection models has greatly limited research in HBV-related fields for a long time, and it is still not possible to discover a method to completely and effectively remove the HBV genome. We have constructed a hepatocellular carcinoma cell line, HLCZ01, that can support the complete life cycle of HBV. This model can mimic the long-term stable infection of HBV in the natural state and can replace primary human hepatocytes for the development of human liver chimeric mice. This model will be a powerful tool for research in the field of HBV.

Carboxypeptidase A4 negatively regulates HGS-ETR1/2-induced pyroptosis by forming a positive feedback loop with the AKT signalling pathway.

Pyroptosis, a mode of inflammatory cell death, has recently gained significant attention. However, the underlying mechanism remains poorly understood. HGS-ETR1/2 is a humanized monoclonal antibody that can bind to DR4/5 on the cell membrane and induce cell apoptosis by activating the death receptor signalling pathway. In this study, by using morphological observation, fluorescence double staining, LDH release and immunoblot detection, we confirmed for the first time that HGS-ETR1/2 can induce GSDME-mediated pyroptosis in hepatocellular carcinoma cells. Our study found that both inhibition of the AKT signalling pathway and silencing of CPA4 promote pyroptosis, while the overexpression of CPA4 inhibits it. Furthermore, we identified a positive regulatory feedback loop is formed between CPA4 and AKT phosphorylation. Specifically, CPA4 modulates AKT phosphorylation by regulating the expression of the AKT phosphatase PP2A, while inhibition of the AKT signalling pathway leads to a decreased transcription and translation levels of CPA4. Our study reveals a novel mechanism of pyroptosis induced by HGS-ETR1/2, which may provide a crucial foundation for future investigations into cancer immunotherapy.

Analysis of Human microRNA Expression Profiling During Diquat-Induced Renal Proximal Tubular Epithelial Cell Injury.

Background: We established a diquat-induced human kidney-2 cells (HK-2 cells) apoptosis model in this study to identify differentially expressed microRNAs (miRNAs) and signaling pathways involved in diquat poisoning via gene sequencing and bioinformatics analysis and explored the related therapeutic benefits. Methods: The effects of diquat on the viability and apoptosis of HK-2 cells were explored using the CCK-8 and Annexin V-FITC/PI double staining methods. Total RNAs were extracted using the TRizol method and detected by Illumina HiSeq 2500. Bioinformatics analysis was performed to explore differentially expressed (DE) miRNAs, their enriched biological processes, pathways, and potential target genes. The RT-qPCR method was used to verify the reliability of the results. Results: Diquat led to HK-2 cell injury and apoptosis played an important role, hence an HK-2 cell apoptosis model in diquat poisoning was established. Thirty-six DE miRNAs were screened in diquat-treated HK-2 cells. The enriched biological process terms were mainly cell growth, regulation of apoptotic signaling pathway, extrinsic apoptotic signaling pathway, and Ras protein signal transduction. The enriched cellular components were mainly cell-cell junction, cell-substrate junction, ubiquitin ligase complex, and protein kinase complex. The enriched molecular functions were mainly Ras GTPase binding, ubiquitin-like protein transferase activity, DNA-binding transcription factor binding, ubiquitin-protein transferase activity, nucleoside-triphosphatase regulator activity, transcription coactivator activity, and ubiquitin-like protein ligase binding. Signaling pathways such as MAPK, FoxO, Ras, PIK3-Akt, and Wnt were also enriched. Conclusion: These findings aid in understanding the mechanisms of diquat poisoning and the related pathways, where DE miRNAs serve as targets for gene therapy.

3ß-hydroxysteroid-Δ24 reductase dampens anti-viral innate immune responses by targeting K27 ubiquitination of MAVS and STING.

IMPORTANCE: The precise regulation of the innate immune response is essential for the maintenance of homeostasis. MAVS and STING play key roles in immune signaling pathways activated by RNA and DNA viruses, respectively. Here, we showed that DHCR24 impaired the antiviral response by targeting MAVS and STING. Notably, DHCR24 interacts with MAVS and STING and inhibits TRIM21-triggered K27-linked ubiquitination of MAVS and AMFR-triggered K27-linked ubiquitination of STING, restraining the activation of MAVS and STING, respectively. Together, this study elucidates how one cholesterol key enzyme orchestrates two antiviral signal transduction pathways.

Activation of AIM2 by hepatitis B virus results in antiviral immunity that suppresses hepatitis C virus during coinfection.

IMPORTANCE: Clinical data suggest that Hepatitis C virus (HCV) levels are generally lower in Hepatitis B virus (HBV) co-infected patients, but the mechanism is unknown. Here, we show that HBV, but not HCV, activated absent in melanoma-2. This in turn results in inflammasome-mediated cleavage of pro-IL-18, leading to an innate immune activation cascade that results in increased interferon-γ, suppressing both viruses.

Effect of cinnamon essential oil dietary supplementation on the growth, fatty acid composition, and meat quality of tilapia.

This study was conducted to investigate the effects of dietary supplementation of 0%, 0.2%, 0.35%, and 0.5% cinnamon essential oil on growth performance, fatty acid, and fillet quality of tilapia (Oreochromis niloticus). The results of growth experiments showed that the weight gain rate linearly and quadratically increased with increasing cinnamon essential oil doses (p < 0.05). The results of fatty acids experiments showed that the addition of essential oil significantly decreased the saturated fatty acids levels from 36.67% to 30.82% and increased the polyunsaturated fatty acids (PUFA) levels from 24.55% to 46.89%; especially, the n - 3 PUFA of 0.5% essential oil treatment showed the highest levels. Moreover, the n - 6 PUFA of 0.2% essential oil treatment were increased from 22.17% to 32.99%. The results of fillet quality experiments showed that the hardness and cohesiveness were linearly and quadratically increased with the increasing essential oil doses on days 4 and 7, respectively. The b* values linearly and quadratically decreased as the doses increased on day 7 (p < 0.05). The total volatile basic nitrogen levels were quadratically decreased with increasing cinnamon essential oil doses on day 7 (p < 0.05). In general, it can be concluded that cinnamon essential oil presented positive effects on the growth, nutritive values, and meat quality in tilapia.

Electron transfer bridge inducing polarization of nitrogen molecules for enhanced photocatalytic nitrogen fixation.

Ammonia (NH3) plays a crucial role in the production of fertilizers, medicines, fibers, etc., which are closely relevant to the development of human society. However, the inert and nonpolar properties of NN seriously hinder artificial nitrogen fixation under mild conditions. Herein, we introduce a novel strategy to enhance the photocatalytic efficiency of N2 fixation through the directional polarization of N2 by rare earth metal atoms, which act as a local "electron transfer bridge." This bridge facilitates the transfer of delocalized electrons to the distal N atom and redirects the polarization of adsorbed N2 molecules. Taking cerium doped BiOCl (Ce-BiOCl) as an example, our results reveal that the electrons transfer to the distal N atom through the cerium atom, resulting in absorbed nitrogen molecular polarization. Consequently, the polarized nitrogen molecules exhibit an easier trend for NN cleavage and the subsequent hydrogenation process, and exhibit a greatly enhanced photocatalytic ammonia production rate of 46.7 µmol g-1 h-1 in cerium doped BiOCl, nearly 4 times higher than that of pure BiOCl. The original concept of directional polarization of N2 presented in this work not only deepens our understanding of the N2 molecular activation mechanism but also broadens our horizons for designing highly efficient catalysts for N2 fixation.

The dual functions of KDM7A in HBV replication and immune microenvironment.

KDM7A (lysine demethylase 7A, also known as JHDM1D) is a histone demethylase, it is mainly involved in the intracellular post-translational modifications process. Recently, it has been proved that the histone demethylase members can regulate the replication of hepatitis B virus (HBV) and the expression of key molecules in the Janus-activated kinase-signal transducer and activator of the transcription (JAK/STAT) signaling pathway by chromatin modifying mechanisms. In our study, we identify novel roles of KDM7A in HBV replication and immune microenvironment through two subjects: pathogen and host. On the one hand, KDM7A is highly expressed in HBV-infected cells and promotes HBV replication in vitro and in vivo. Moreover, KDM7A interacts with HBV covalently closed circular DNA and augments the activity of the HBV core promoter. On the other hand, KDM7A can remodel the immune microenvironment. It inhibits the expression of interferon-stimulated genes (ISGs) through the IFN-γ/JAK2/STAT1 signaling pathway in both hepatocytes and macrophages. Further study shows that KDM7A interacts with JAK2 and STAT1 and affects their methylation. In general, we demonstrate the dual functions of KDM7A in HBV replication and immune microenvironment, and then we propose a new therapeutic target for HBV infection and immunotherapy. IMPORTANCE Histone lysine demethylase KDM7A can interact with covalently closed circular DNA and promote the replication of hepatitis B virus (HBV). The IFN-γ/JAK2/STAT1 signaling pathway in macrophages and hepatocytes is also downregulated by KDM7A. This study provides new insights into the mechanism of HBV infection and the remodeling of the immune microenvironment.

Directional Shunting of Photogenerated Carriers in POM@MOF for Promoting Nitrogen Adsorption and Oxidation.

The efficient catalysis of nitrogen (N2 ) into high-value N-containing products plays a crucial role in the N economic cycle. However, weak N2 adsorption and invalid N2 activation remain two major bottlenecks in rate-determining steps, leading to low N2 fixation performance. Herein, an effective dual active sites photocatalyst of polyoxometalates (POMs)-based metal-organic frameworks (MOFs) is highlighted via altering coordination microenvironment and inducing directional shunting of photogenerated carriers to facilitate N2 /catalyst interaction and enhance oxidation performance. MOFs create more open unsaturated metal cluster sites with unoccupied d orbital possessing Lewis acidity to accept electrons from the 3σg bonding orbital of N2 for storage by combining with POMs to replace bidentate linkers. POMs act as electron sponges donating electrons to MOFs, while the holes directional flow to POMs. The hole-rich POMs with strong oxidation capacity are easily involved in oxidizing adsorbed N2 . Taking UiO-66 (C48 H28 O32 Zr6 ) and Mo72 Fe30 ([Mo72 Fe30 O252 (CH3 COO)12 {Mo2 O7 (H2 O)}2 {H2 Mo2 O8 (H2 O)}(H2 O)91 ]·150H2 O) as an example, Mo72 Fe30 @UiO-66 shows twofold enhanced adsorption of N2 (250.5 cm3  g-1 ) than UiO-66 (122.9 cm3 g-1 ) at P/P0  = 1. And, the HNO3 yield of Mo72 Fe30 @UiO-66 is 702.4 µg g-1  h-1 , ≈7 times and 24 times higher than UiO-66 and Mo72 Fe30 . This work provides reliable value for the storage and relaying artificial N2 fixation.

Programmable Proteolysis-Activated Transcription for Highly Sensitive Ratiometric Electrochemical Detection of Viral Protease.

Viral proteases play a crucial role in viral infection and are regarded as promising targets for antiviral drug development. Consequently, biosensing methods that target viral proteases have contributed to the study of virus-related diseases. This work presents a ratiometric electrochemical sensor that enables highly sensitive detection of viral proteases through the integration of target proteolysis-activated in vitro transcription and the DNA-functionalized electrochemical interface. In particular, each viral protease-mediated proteolysis triggers the transcription of multiple RNA outputs, leading to amplified ratiometric signals on the electrochemical interface. Using the NS3/4A protease of the hepatitis C virus as a model, this method achieves robust and specific NS3/4A protease sensing with sub-femtomolar sensitivity. The feasibility of this sensor was demonstrated by monitoring NS3/4A protease activities in virus-infected cell samples with varying viral loads and post-infection times. This study provides a new approach to analyzing viral proteases and holds the potential for developing direct-acting antivirals and novel therapies for viral infections.

Regulation of PKR-dependent RNA translation inhibition by TRIM21 upon virus infection or other stress.

The host always employs various ways to defend against viral infection and spread. However, viruses have evolved their own effective strategies, such as inhibition of RNA translation of the antiviral effectors, to destroy the host's defense barriers. Protein synthesis, commonly controlled by the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), is a basic cellular biological process among all species. In response to viral infection, in addition to inducing the transcription of antiviral cytokines by innate immunity, infected cells also inhibit the RNA translation of antiviral factors by activating the protein kinase R (PKR)-eIF2α signaling pathway. Regulation of innate immunity has been well studied; however, regulation of the PKR-eIF2α signaling pathway remains unclear. In this study, we found that the E3 ligase TRIM21 negatively regulates the PKR-eIF2α signaling pathway. Mechanistically, TRIM21 interacts with the PKR phosphatase PP1α and promotes K6-linked polyubiquitination of PP1α. Ubiquitinated PP1α augments its interaction with PKR, causing PKR dephosphorylation and subsequent translational inhibition release. Furthermore, TRIM21 can constitutively restrict viral infection by reversing PKR-dependent translational inhibition of various previously known and unknown antiviral factors. Our study highlights a previously undiscovered role of TRIM21 in regulating translation, which will provide new insights into the host antiviral response and novel targets for the treatment of translation-associated diseases in the clinic.

Primary malignant melanoma of the cervix: A comprehensive analysis of case reports in the Chinese population.

PURPOSE: Malignant melanoma is a tumor generated from the basal melanocytes of human epidermis. Primary malignant melanoma of the cervix (PMMC) is derived from cervical melanocytes. It is an uncommon disease, mostly occurring in perimenopausal women. PMMC has a bad prognosis and lacks a defined protocol or treatment standards. The aim of this study was to analyze the impact of different surgical procedures and different adjuvant treatment modalities on their prognosis and to find risk factors for their prognosis by integrating published case report data based on the Chinese population. METHODS AND RESULTS: This study included 165 patients with PMMC in the Chinese population. We used the Kaplan-Meier method to build the survival curve, and the log-rank test to examine the variations among the subgroups. Prognostic factors were examined utilizing the Cox proportional hazards regression model. We found that patients who underwent radical hysterectomy-based surgery, those who underwent lymphadenectomy, and those who underwent other treatments in addition to surgery had significantly better survival rates. The overall analysis, showed that age, and FIGO Stage II, III, and IV, increased the risk of death. Moreover, radical hysterectomy (RH), total hysterectomy (TAH), lymphadenectomy, and adjuvant therapy were correlated with a decreased mortality risk. CONCLUSION: After summarizing the current data, we recommend radical hysterectomy, and lymphadenectomy treatment for patients with PMMC. For patients who had already undergone surgery, other treatment options had a positive effect on prognosis. For patients who had already undergone surgery, other treatment options had a positive effect on prognosis; therefore patient-specific treatment options need to be further discussed.

Composition and Dynamics of Hexapod Communities on Yushan Bamboo (Yushania niitakayamensis) in the Subtropical Montane Areas of Taiwan.

Plant communities that colonize high-elevation zones generally have short growing seasons which lead to specialized adaptations in such zones. In montane areas of Taiwan, Yushan bamboo (YB, Yushania niitakayamensis) is dominant at elevations ranging from 2500 to 3300 m and grows in a grassland-like open habitat. In this study, hexapods were collected from YB bimonthly between 2009 and 2012 by using a sweeping net. The composition of and several bioindices for the hexapods were determined, and multivariate analyses were conducted to explore the dynamics and seasonal distribution of the hexapods. A total of 32,000 individuals belonging to 11 orders and 113 families were collected, with adult individuals being collected more frequently in warmer seasons (from June to October). Of the sampled individuals, 90% belonged to the orders Collembola (42%), Hemiptera (35%), and Hymenoptera (13%). The number of individuals belonging to Hemiptera were stable in all seasons, and the number of hymenopteran wasps was influenced by temperature and exhibited a stable dynamic pattern. The number of individuals belonging to Collembola fluctuated dramatically. The multivariate analyses revealed that the collected hexapods could be divided into two major family groups according to survey season (i.e., summer and winter groups). Several families were collected only in summer, but a few were collected only in winter. Eigenvalues obtained from a principal component analysis revealed that the families Chironomidae, Delphacidae, Entomobryidae, Hypogastruridae, Sminthuridae, and Thripidae (all dominant) were the major contributors to the winter group. These families were abundant all year, although some were more abundant during winter. The three dominant orders Collembola, Hemiptera, and Hymenoptera, each of which has a distinct community structure and dynamic pattern, may have their own adaptive mechanisms in the subtropical regions of Taiwan. Hemiptera individuals, which feed on YB, were most abundant in the adult stage in summer and in the nymphal stage in winter. The abundance of parasitic hymenopteran wasps, which had stable dynamic patterns, was associated with that of their host insects and temperature. The drastic fluctuations in the abundance of Collembola may have been caused by abiotic factors, such as precipitation and microhabitat factors. The early onset of spring and the late onset of winter might also affect the dynamics of the studied hexapods.

Construction of Asymmetrical Dual Jahn-Teller Sites for Photocatalytic CO2 Reduction.

Photocatalytic CO2 reduction (PCR) expresses great attraction to convert useless greenhouse gas into valuable chemical feedstock. However, the weak interactions between catalytic sites and PCR intermediates constrains the PCR activity and selectivity. Herein, we proposed a new strategy to match the intermediates due to the maximum orbital overlap of catalytic sites and C1 intermediates by establishing dual Jahn-Teller (J-T) sites, in which, the strongly asymmetric J-T sites can break the nonpolar CO2 molecules and self-adapt the different structure of C1 intermediates. Taking cobalt carbonate hydroxide as an example, the weakly symmetric dual cobalt (Co2 ) dual J-T sites, weakly asymmetric Fe&Co sites and strongly asymmetric Cu&Co sites were assembled. After illumination, the interaction between dual J-T sites and the CO2 molecules enhances J-T distortion, which further modulates the PCR activity and selectivity. As a result, the Cu&Co sites exhibited CO yield of 8137.9 µmol g-1 , about 2.3-fold and 4.2-fold higher than that of the Fe&Co and Co2 sites within 5-hour photoreaction, respectively. In addition, the selectivity achieved as high as 92.62 % than Fe&Co (88.67 %) and Co2 sites (55.33 %). This work provides a novel design concept for the construction of dual J-T sites to regulate the catalytic activity and selectivity.

Vsig4+ resident single-Kupffer cells improve hepatic inflammation and fibrosis in NASH.

BACKGROUND: The role of macrophages in the pathogenesis of nonalcoholic steatohepatitis (NASH) is complex and unclear. METHODS: Single-cell RNA sequencing was performed on nonparenchymal cells isolated from NASH and control mice. The expression of Vsig4+ macrophages was verified by qPCR, flow cytometry and immunohistochemistry. Primary hepatic macrophages were cocultured with primary hepatocytes or hepatic stellate cells (LX2) cells by Transwell to detect immunofluorescence and oil red O staining. RESULTS: Two main single macrophage subsets were identified that exhibited a significant change in cell percentage when NASH occurred: resident Kupffer cells (KCs; Cluster 2) and lipid-associated macrophages (LAMs; Cluster 13). Nearly 82% of resident single KCs in Cluster 2 specifically expressed Cd163, and an inhibited subgroup of Cd163+ resident single-KCs was suggested to be protective against NASH. Similar to Cd163, Vsig4 was both enriched in and specific to Cluster 2. The percentage of Vsig4+-KCs was significantly decreased in NASH in vivo and in vitro. Hepatocytes and hepatic stellate cells produced less lipid droplet accumulation, proinflammatory protein (TNF-α) and profibrotic protein (α-SMA) in response to coculture with Vsig4+-KCs than in those cocultured with lipotoxic KCs. CONCLUSIONS: A subgroup of Vsig4+ resident single-KCs was shown to improve hepatic inflammation and fibrosis in NASH.

First Report of Mucor irregularis Causing Flower Rot on Selenicereus undatus in China.

Dragon fruit (Selenicereus undatus (Haw.) D.R.Hunt is a famous tropical fruit (Korotkova et al. 2017). In May 2021, a flower rot disease was found on Dragon fruit in a field (21˚19'42''N, 110˚28'32''E), Zhanjiang, Guangdong Province, China. The incidence rate was approximately 30% (n=500 investigated plants from about 30 hectares). Flower rot was evident, and was light brown, watery, soft, and covered with white mycelia. The pathogen could continue to infect the fruit during the fruit ripening stage with about 20% rot rate. Ten samples of symptomatic flowers were collected in the field. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Three representative isolates (HUM-1,HUM-2, and HUM-3) by single-spore isolation were randomly selected for further study. Colonies on PDA were circular with massive aerial hyphae, white to ochraceous in color. Nonseptate hyphae were hyaline. Sporangiophores arose from hyphae. Sporangiospores were hyaline, smooth-walled, mostly subspherical to ellipsoidal, and measured 3.15 to 6.55 µm × 1.35 to 2.85 µm (n =50). Morphological characteristics of isolates were consistent with the description of Mucor irregularis (Lima et al. 2018). Molecular identification was done using the colony PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) used to amplify the internal transcribed spacer (ITS) region and large subunit (LSU) with ITS1/ITS4 and LR0R1/LR5 (Vilgalys et al. 1990). The amplicons were sequenced and the sequences were deposited in GenBank with accession numbers ITS, OL376751-OL376753, and LSU, OM672239-OM672241. BLAST analysis of these sequences revealed a 100% identity with M. irregularis in GenBank. The sequences were also concatenated for phylogenetic analysis by the maximum likelihood method. The isolates clustered with M. irregularis (the type strain CBS 103.93).The pathogenicity was tested through in vivo experiments. Nine healthy flowers of Dragon fruit were inoculated with 3-day-old mycelial plugs (5 × 5 mm) of isolates, while another five healthy flowers were treated with PDA plugs (controls). Those plugs were embedded inside the calyxes, and each flower was inoculated with one plug in one calyx. Besides, the inoculated and control flowers (n = 5) were sprayed with a spore suspension (1 × 105 per mL) of the three isolates individually and sterile distilled water, respectively, until run-off (Feng and Li. 2019). The plants were grown in pots in a greenhouse at 28°C, with relative humility approximately 80%. The test was repeated three times. After 3 days of incubation, rot symptoms developed on the inoculated flowers, which were similar to those observed on the naturally samples in the field. The control flowers remained healthy. The fungus was reisolated from the inoculated flowers and confirmed as M. irregularis by morphology and ITS analysis. M. irregularis was reported as a pathogen causing human skin diseases and post-harvest diseases of crop (Álvarez et al. 2011; Lima et al. 2018; Wang et al. 2022). This is the first report of M. irregularis causing flower rot of Dragon fruit and reduce yield in China. This research can provide a theoretical basis for the fruit industry to maintain yield.