Chromones and biflavonoids from Garcinia pedunculata and Garcinia nujiangensis and their anti-inflammatory activity.

Plants of the Garcinia genus were rich in structurally diverse and naturally bioactive components, while limited studies have been reported for Garcinia pedunculata Roxb. and G. nujiangensis C. Y. Wu & Y. H. Li. Four previously undescribed compounds including three chromones, garpedunchromones A-C (1-3), and one biflavonoid, nujiangbiflavone A (14), along with fifteen known analogs (4-13, 15-19) were isolated from G. pedunculata and G. nujiangensis. The structures of the isolated compounds were determined based on their HRESIMS data, extensive NMR spectroscopic analyses, and ECD calculations. The chromone derivatives were isolated from Garcinia for the first time. Compound 14 was a rare biflavonoid with C-3─C-6″ linkage. The biological evaluation of these isolates against NO production was conducted in the LPS-induced RAW 264.7 cells, resulting in the identification of a series of potent NO inhibitors, of which garpedunchromone B (2) was the most active with an IC50 value of 18.11 ± 0.96 µM. In the network pharmacology studies, the potential targets of compounds and inflammation were obtained from PharmMapper and GeneCards database. GO and KEGG enrichment analysis revealed that the overlapped targets were closely related to the major pathogenic processes linked to inflammation. Garpedunchromone B and proteins binding sites were being predicted.

Cytogeography of Naturalized Solidago canadensis Populations in Europe.

Autopolyploidization has driven the successful invasion of Solidago canadensis in East Asia. However, it was believed that only diploid S. canadensis invaded Europe, whereas polyploids never did. Here, molecular identification, ploidy level, and morphological traits of ten S. canadensis populations collected in Europe were compared with previously identified S. canadensis populations from other continents and S. altissima populations. Furthermore, the ploidy-driven geographical differentiation pattern of S. canadensis in different continents was investigated. All ten European populations were identified as S. canadensis with five diploid and five hexaploid populations. Significant differences in morphological traits existed among diploids and polyploids (tetraploids and hexaploids), rather than between polyploids from different introduced ranges and between S. altissima and polyploidy S. canadensis. The invasive hexaploids and diploids had few differences in latitudinal distributions in Europe, which was similar to the native range but different from a distinct climate-niche differentiation in Asia. This may be attributed to the bigger difference in climate between Asia and Europe and North America. The morphological and molecular evidences proved the invasion of polyploid S. canadensis in Europe and suggest that S. altissima may be merged into a complex of S. canadensis species. Our study may be concluded that geographical and ecological niche differentiation of an invasive plant driven by ploidy depends on the degree of difference in the environmental factors between the introduced and native range, which provides new insight into the invasive mechanism.

Permethrin as a Potential Furin Inhibitor through a Novel Non-Competitive Allosteric Inhibition.

Furin is a potential target protein associated with numerous diseases; especially closely related to tumors and multiple viral infections including SARS-CoV-2. Most of the existing efficient furin inhibitors adopt a substrate analogous structure, and other types of small molecule inhibitors need to be discovered urgently. In this study, a high-throughput screening combining virtual and physical screening of natural product libraries was performed, coupled with experimental validation and preliminary mechanistic assays at the molecular level, cellular level, and molecular simulation. A novel furin inhibitor, permethrin, which is a derivative from pyrethrin I generated by Pyrethrum cinerariifolium Trev. was identified, and this study confirmed that it binds to a novel allosteric pocket of furin through non-competitive inhibition. It exhibits a very favorable protease-selective inhibition and good cellular activity and specificity. In summary, permethrin shows a new parent nucleus with a new mode of inhibition. It could be used as a highly promising lead compound against furin for targeting related tumors and various resistant viral infections, including SARS-CoV-2.

Knockout of ABC transporters by CRISPR/Cas9 contributes to reliable and accurate transporter substrate identification for drug discovery.

It is essential to explore the relationship between drugs and transporters in the process of drug development. Strong background signals in nonhuman MDCK or LLC-PK1 cells and overlapping interference of inhibitors or RNAi in human Caco-2 cells mean that an ideal alternative could be to knock out specific transporter genes in Caco-2 cells. However, the application of gene knockout (KO) to Caco-2 cells is challenging because it is still inefficient to obtain rapidly growing Caco-2 subclones with double-allele KO through long-term monoclonal cultivation. Herein, CRISPR/Cas9, a low cost but more efficient and precise gene editing technology, was utilized to singly or doubly knockout the P-gp, BCRP, and MRP2 genes in Caco-2 cells. By combining this with single cell expansion, rapidly growing transporter-deficient subclones were successfully screened and established. Bidirectional transport assays with probe substrates and three protease inhibitors indicated that more reliable and detailed data could be drawn easily with these KO Caco-2 models. The six robust KO Caco-2 subclones could contribute to efficient in vitro drug transport research.

Structural and biochemical insights into the association between ERAP1 polymorphism and autoimmune diseases.

Autoimmune diseases afflict nearly 10% of the world's population and have a serious impact on survival and quality of life. Unfortunately, the specific pathogenesis of almost all autoimmune diseases is still unclear, with more research findings identifying some key pathogenic genes at the genetic level and several pathogenic inflammatory factor phenotypes. ERAP1 has been suggested as a potential therapeutic target for several autoimmune diseases, especially MHC-Ⅰ related. How the structure and antigenic peptide processing function of ERAP1 affect the pathogenesis of these autoimmune diseases needs to be elucidated more clearly. Genetic studies on single nucleotide polymorphism of ERAP1 provide a good bridge to better understand the relationship and pattern between ERAP1 structure, function, and disease. However, existing reviews have focused on the genetic association of ERAP1 SNPs with autoimmune diseases, and no one has specifically addressed how ERAP1 gene polymorphisms embodied at the protein level specifically mediate antigenic peptide editing and the development of multiple autoimmune diseases. In this paper, we present a comprehensive review of these ERAP1 SNPs associated with multiple autoimmune diseases, in particular the polymorphisms affecting their protein structure and enzyme function, and attempt to unravel the underlying structural and biochemical mechanisms by which ERAP1 affects the pathogenesis of multiple autoimmune diseases through the SNP-protein structure-function-disease relationship. This study will provide theoretical help and ideas for understanding the relationship between ERAP1 and autoimmune diseases and for drug design targeting wild-type and mutant proteins with different polymorphisms.

Polyploidization-enhanced effective clonal reproduction endows the successful invasion of Solidago canadensis.

Clonality and ploidy levels are positively associated with plant invasiveness. However, there is still no consensus on whether polyploidization can promote the invasion of alien plants by enhancing clonality. Our recent long-term community succession study found that the more vigorous clone of introduced polyploid Solidago canadensis succeeded into mono-dominant community, which seems to be a positive correlationship between polyploidization and clonal reproduction. However, the formation process of clonal ramet and how polyploidization improves the clonal reproduction of S. canadensis remains unknown. Here, we compared clonal growth ability among diploids and polyploids of S. canadensis from native and introduced ranges in a common garden. Results showed that the rhizomes of S. canadensis originated from axillary buds of dense nodes at the basal stem of seedling and then produced into clonal ramets from the rhizomes. Diploids had denser nodes and more buds, developed more rhizomes per unit mass and produced more clonal propagules at the early growth stage compared with polyploids. However, the number of juvenile and secondary rhizomes, as well as the diameter and length of rhizomes in polyploid populations was significantly higher or greater than those of diploids, and those clonal traits in introduced polyploids were significantly higher than in native polyploids. Moreover, a phalanx growth form was observed in native and introduced diploid populations, which allocated about 3% and 5% of the total biomass to rhizomes, respectively, resulting in short and weak rhizomes. However, native and introduced polyploids allocated about 35% and 40%, respectively, of the total biomass to rhizomes, resulting in long and strong rhizomes, which were guerrilla growth forms. This study firstly shows that polyploidization enhanced the effective clonal reproduction of S. canadensis through pre-adaptation and rapid post-adaptation evolution, and consequently contributed to its successful invasion.

Efficient generation of a CYP3A4-T2A-luciferase knock-in HepaRG subclone and its optimized differentiation.

CRISPR/Cas9 has allowed development of better and easier-to-use ADME models than traditional methods by complete knockout or knock-in of genes. However, gene editing in HepaRG cells remains challenging because long-term monoclonal cultivation may alter their differentiation capacity to a large extent. Here, CRISPR/Cas9 was used to generate a CYP3A4-T2A-luciferase knock-in HepaRG subclone by Cas9-mediated homologous recombination and monoclonal cultivation. The knock-in HepaRG-#9 subclone retained a similar differentiation potential to wildtype HepaRG cells (HepaRG-WT). To further improve differentiation and expand the applications of knock-in HepaRG cells, two optimized differentiation procedures were evaluated by comparison with the standard differentiation procedure using the knock-in HepaRG-#9 subclone and HepaRG-WT. The results indicated that addition of forskolin (an adenylate cyclase activator) and SB431542 (a TGF-ß pathway inhibitor) to the first optimized differentiation procedure led to better differentiation consequence in terms of not only the initiation time for differentiation and morphological characterization, but also the mRNA levels of hepatocyte-specific genes. These data may contribute to more extensive applications of genetically modified HepaRG cells in ADME studies.

Validation Study to Determine the Accuracy of Widespread Promoterless EGFP Reporter at Assessing CRISPR/Cas9-Mediated Homology Directed Repair.

An accurate visual reporter system to assess homology-directed repair (HDR) is a key prerequisite for evaluating the efficiency of Cas9-mediated precise gene editing. Herein, we tested the utility of the widespread promoterless EGFP reporter to assess the efficiency of CRISPR/Cas9-mediated homologous recombination by fluorescence expression. We firstly established a promoterless EGFP reporter donor targeting the porcine GAPDH locus to study CRISPR/Cas9-mediated homologous recombination in porcine cells. Curiously, EGFP was expressed at unexpectedly high levels from the promoterless donor in porcine cells, with or without Cas9/sgRNA. Even higher EGFP expression was detected in human cells and those of other species when the porcine donor was transfected alone. Therefore, EGFP could be expressed at certain level in various cells transfected with the promoterless EGFP reporter alone, making it a low-resolution reporter for measuring Cas9-mediated HDR events. In summary, the widespread promoterless EGFP reporter could not be an ideal measurement for HDR screening and there is an urgent need to develop a more reliable, high-resolution HDR screening system to better explore strategies of increasing the efficiency of Cas9-mediated HDR in mammalian cells.

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform.

The increasing prevalence of SARS-CoV-2 variants with spike mutations has raised concerns owing to higher transmission rates, disease severity, and escape from neutralizing antibodies. Rapid and accurate detection of SARS-CoV-2 variants provides crucial information concerning the outbreaks of SARS-CoV-2 variants and possible lines of transmission. This information is vital for infection prevention and control. We used a Cas12a-based RT-PCR combined with CRISPR on-site rapid detection system (RT-CORDS) platform to detect the key mutations in SARS-CoV-2 variants, such as 69/70 deletion, N501Y, and D614G. We used type-specific CRISPR RNAs (crRNAs) to identify wild-type (crRNA-W) and mutant (crRNA-M) sequences of SARS-CoV-2. We successfully differentiated mutant variants from wild-type SARS-CoV-2 with a sensitivity of 10-17 M (approximately 6 copies/µL). The assay took just 10 min with the Cas12a/crRNA reaction after a simple RT-PCR using a fluorescence reporting system. In addition, a sensitivity of 10-16 M could be achieved when lateral flow strips were used as readouts. The accuracy of RT-CORDS for SARS-CoV-2 variant detection was 100% consistent with the sequencing data. In conclusion, using the RT-CORDS platform, we accurately, sensitively, specifically, and rapidly detected SARS-CoV-2 variants. This method may be used in clinical diagnosis.

Effect of insulin and an erythropoietin-derived peptide (ARA290) on established neuritic dystrophy and neuronopathy in Akita (Ins2 Akita) diabetic mouse sympathetic ganglia.

The Akita mouse is a robust model of diabetic autonomic neuropathy which develops severe diabetes following beta cell death, which occurs reproducibly at 3-4 weeks of age, and maintains the diabetic state without therapy for as long as 11 additional months. Neuritic dystrophy and neuronopathy involving prevertebral sympathetic superior mesenteric and celiac ganglia begin to develop within the first two months of onset of diabetes and are progressive with time. We have examined the effect of insulin implants resulting in normoglycemia and injections of ARA290, a small erythropoietin peptide which has no effect on glycemic parameters, on the reversal of established neuritic dystrophy and neuronopathy. We have found that 4 weeks of insulin therapy beginning at 2 months of diabetes resulted in normalization of blood glucose, body weight and HbA1c. Insulin therapy successfully reversed established neuritic dystrophy and neuronopathy to control levels. Numbers of sympathetic neurons were not significantly changed in either 3 month diabetic or insulin-treated Akita mice. Treatment with ARA290 for 7 weeks beginning at 4 months of diabetes did not result in altered metabolic severity of diabetes as measured by blood glucose, body weight or HbA1c levels. ARA290 treatment was able to decrease neuritic dystrophy by 55-74% compared to untreated diabetics or in comparison to a separate group of diabetic animals representing the 4 month treatment onset point. Surprisingly, there was no effect of ARA290 on ganglionic neuron number or ongoing neuronopathy (pale/degenerating neurons) in diabetic Akita mice during this time period. The development of neuroprotective EPO-like peptides may provide a possible future therapy for this debilitating complication of diabetes; however, it appears that discrete elements may be differentially targeted by the diabetic state and may require selective therapy.

Altered epididymal sperm maturation and cytoplasmic droplet migration in subfertile male Alox15 mice.

Mammalian spermatozoa complete their morphogenesis and acquire their fertilizing potential in the epididymis. Prominent among the hallmarks of epididymal sperm maturation is the proximal-distal migration of the cytoplasmic droplet (CD), the last remnant of the spermatogenic cell cytoplasm, down the sperm flagellum. Failure to shed the CD has been associated with male infertility. Because of the presence of the organelle degradation enzyme 15-lipoxygenase (15LOX) in sperm CD, we hypothesize that subfertile male Alox15 mice lacking the 15Lox gene display sperm CD anomalies. Caput and cauda epididymal sperm samples from seven adult Alox15 and seven wild-type (wt) males of equal age were examined by differential interference contrast microscopy (DIC) and transmission electron microscopy (TEM). Compared with wt males, Alox15 males had significantly more spermatozoa with a retained CD in both caput (P = 0.004) and cauda (P = 0.005) epididymidis. TEM and DIC analyses revealed intact mitochondria present in the CDs of epididymal Alox15 spermatozoa. The CDs of wt spermatozoa, however, had a smooth appearance and contained only hollow membrane vesicles, with no intact mitochondria embedded in their CD matrix. Epithelial lesions, phagocytosis-like figures, and missing or aberrant apical blebs were observed in the caput epididymidis of Alox15 males. Thus, the process of epididymal sperm maturation and CD migration is altered in Alox15 males. Aberrant sperm maturation might contribute to the reduced fertility and smaller litter size of Alox15 mice, a rare example of subfertile mutants displaying normal spermatogenesis but altered epididymal sperm maturation.

Genome sequence of Cronobacter sakazakii BAA-894 and comparative genomic hybridization analysis with other Cronobacter species.

BACKGROUND: The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. METHODOLOGY/PRINCIPAL FINDINGS: We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 kb (51% GC) and 131 kb (56% GC). The genome was used to construct a 387,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10-17% absence of genes. CONCLUSIONS/SIGNIFICANCE: CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.

Neuritic dystrophy and neuronopathy in Akita (Ins2(Akita)) diabetic mouse sympathetic ganglia.

Diabetic autonomic neuropathy is a debilitating, poorly studied complication of diabetes. Our previous studies of non-obese diabetic (NOD) and related mouse models identified rapidly developing, dramatic pathology in prevertebral sympathetic ganglia; however, once diabetic, the mice did not survive for extended periods needed to examine the ability of therapeutic agents to correct established neuropathy. In the current manuscript we show that the Akita (Ins2(Akita)) mouse is a robust model of diabetic sympathetic autonomic neuropathy with unambiguous, spontaneous, rapidly-developing neuropathology which corresponds closely to the characteristic pathology of other rodent models and man. Akita mice diabetic for 2, 4 or 8 months of diabetes progressively developed markedly swollen axons and dendrites ("neuritic dystrophy") in the prevertebral superior mesenteric (SMG) and celiac ganglia (CG). Comparable changes failed to develop in the superior cervical ganglia (SCG) of the Akita mouse or in any ganglia of non-diabetic mice. Morphometric studies demonstrate an overall increase in presynaptic axon terminal cross sectional area, including those without any ultrastructural features of dystrophy. Neurons in Akita mouse prevertebral sympathetic ganglia show an unusual perikaryal alteration characterized by the accumulation of membranous aggregates and minute mitochondria and loss of rough endoplasmic reticulum. These changes result in the loss of a third of neurons in the CG over the course of 8 months of diabetes. The extended survival of diabetic mice and robust pathologic findings provide a clinically relevant paradigm that will facilitate the analysis of novel therapeutic agents on the reversal of autonomic neuropathy.

Erythropoietin and its carbamylated derivative prevent the development of experimental diabetic autonomic neuropathy in STZ-induced diabetic NOD-SCID mice.

Autonomic neuropathy is a significant diabetic complication resulting in increased morbidity and mortality. Studies of autopsied diabetic patients and several rodent models demonstrate that the neuropathologic hallmark of diabetic sympathetic autonomic neuropathy in prevertebral ganglia is the occurrence of synaptic pathology resulting in distinctive dystrophic neurites ("neuritic dystrophy"). Our prior studies show that neuritic dystrophy is reversed by exogenous IGF-I administration without altering the metabolic severity of diabetes, i.e. functioning as a neurotrophic substance. The description of erythropoietin (EPO) synergy with IGF-I function and the recent discovery of EPO's multifaceted neuroprotective role suggested it might substitute for IGF-I in treatment of diabetic autonomic neuropathy. Our current studies demonstrate EPO receptor (EPO-R) mRNA in a cDNA set prepared from NGF-maintained rat sympathetic neuron cultures which decreased with NGF deprivation, a result which demonstrates clearly that sympathetic neurons express EPO-R, a result confirmed by immunohistochemistry. Treatment of STZ-diabetic NOD-SCID mice have demonstrated a dramatic preventative effect of EPO and carbamylated EPO (CEPO, which is neuroprotective but not hematopoietic) on the development of neuritic dystrophy. Neither EPO nor CEPO had a demonstrable effect on the metabolic severity of diabetes. Our results coupled with reported salutary effects of EPO on postural hypotension in a few clinical studies of EPO-treated anemic diabetic and non-diabetic patients may reflect a primary neurotrophic effect of EPO on the sympathetic autonomic nervous system, rather than a primary hematopoietic effect. These findings may represent a major clinical advance since EPO has been widely and safely used in anemic patients due to a variety of clinical conditions.

Differential expression of genes encoding constitutive and inducible 20S proteasomal core subunits in the testis and epididymis of theophylline- or 1,3-dinitrobenzene-exposed rats.

Theophylline (THP) and 1,3-dinitrobenzene (DNB) are thought to induce infertility by incapacitating the nurturing Sertoli cells and causing germ cell apoptosis in the testicular seminiferous epithelium, respectively. We hypothesized that THP and DNB exposure would alter the expression of the genes within the ubiquitin-proteasome pathway (UPP), implicated in spermatogenesis and epididymal sperm quality control. Rats were fed 0 or 8000 ppm of THP and necropsied on Days 18, 30, and 42 or administered 0, 2, or 6 mg/kg DNB via oral gavage and necropsied on Day 7. Tissues were collected from the testis and the caput, corpus, and cauda regions of the epididymis for transcriptional profiling by semiquantitative RT-PCR, real-time RT-PCR, and histopathology. Target UPP genes included those encoding for constitutive the 20S proteasomal core subunits Psmb1 (beta1), Psmb2 (beta2), and Psmb5 (beta5); the inducible 20S core subunits Psmb9 (LMP2), Psmb8 (LMP7), and Psmb10 (LMP10); and Ube1 (ubiquitin-activating enzyme E1), Ube2d3 (ubiquitin-conjugating enzyme E2), and Uchl1 (ubiquitin C-terminal hydrolase PGP9.5). Spermatozoa were collected from the cauda epididymis for analysis by light microscopy and flow cytometric evaluation of sperm surface ubiquitin. These data show that reprotoxic exposure alters the tissue-specific expression of UPP genes in the testis and epididymis, which may contribute to the aberrant spermatogenesis and epididymal processing of both normal and defective spermatozoa. Transcriptional profiling and flow cytometric analysis of the UPP thus captures the prodromal effects of reproductive toxicity not captured by conventional histology and functional cytology. Complementing seminal analysis with these measures may be useful in screening drug-induced toxicity or environmental infertility.