Cryptic piperazine derivatives activated by knocking out the global regulator LaeA in Aspergillus flavipes.

Genome sequencing on an intertidal zone-derived Aspergillus flavipes strain revealed its great potential to produce secondary metabolites. To activate the cryptic compounds of A. flavipes, the global regulator flLaeA was knocked out, leading to substantial up-regulation of the expression of two NRPS-like biosynthetic gene clusters in the ΔflLaeA mutant. With a scaled-up fermentation of the ΔflLaeA strain, five compounds, including two previously undescribed piperazine derivatives flavipamides A and B (1 and 2), along with three known compounds (3-5), were obtained by LC-MS guided isolation. The new compounds were elucidated by spectroscopic analysis and electronic circular dichroism (ECD) calculations, and the biosynthetic pathway was proposed on the bias of bioinformatic analysis and 13C isotope labeling evidence. This is the first report to access cryptic fungi secondary metabolites by inactivating global regulator LaeA and may provide a new approach to discovering new secondary metabolites by such genetic manipulation.

Discovery of 23,24-diols containing ergosterols with anti-neuroinflammatory activity from Penicillium citrinum TJ507.

Citristerones A-E (1-5), five new 23,24-diols containing ergosterols, along with three known analogues, were isolated from the endophytic fungus Penicillium citrinum TJ507 obtained from Hypericum wilsonii N. Robson. Their structures and absolute configurations were determined by NMR, HRESIMS, Snatzke's method, X-ray diffraction analyses and ECD calculation. Subsequently, the anti-neuroinflammatory effects of these isolates were screened using lipopolysaccharide (LPS)-induced BV-2 microglial cells, and citristerone B (2) showed outstanding anti-neuroinflammatory activity, with IC50 value of 0.60 ± 0.04 µM. Moreover, immunofluorescence and western blot analysis suggested that citristerone B not only reduced the release of nitric oxide (NO) and proinflammatory cytokines in LPS-induced BV-2 microglial cells, but also significantly inhibited the expression of TNF-α, iNOS and NF-κB, along with the production of cellular ROS.

An indole diketopiperazine alkaloid and a bisabolane sesquiterpenoid with unprecedented skeletons from Aspergillus fumigatus.

Fumitryprostatin A (1), the first example of an indole diketopiperazine alkaloid with a tricyclic 5/6/5 skeleton characterized by a dipyrrolo[1,2-a:1',2'-d]pyrazine-5,10-dione ring system decorated with a prenylated indole moiety, and fuminoid A (2), a sesquiterpenoid with a bicyclo[3.2.1]octane ring featuring a novel carbon skeleton via the transformation of the methyl, were isolated from the fungus Aspergillus fumigatus along with six known diketopiperazine alkaloids. The structure with the absolute configuration of 1 was determined based on spectroscopic analyses and X-ray crystallographic analysis, while the configuration of 2 was assigned tentatively by 13C NMR data with DP4+ probability analyses and ECD calculations. A plausible biosynthetic pathway for 1 was proposed starting from L-Trp and L-Pro via normal indole diketopiperazine. Compound 1 exhibited moderate cytotoxic activity with an IC50 value of 14.6 µM, while compound 8 exhibited moderate immunosuppressive activity in vitro.

Photocatalytic synthesis of azaheterocycle-fused piperidines and pyrrolidines via tandem difunctionalization of unactivated alkenes.

A variety of azaheterocycle-fused piperidines and pyrrolidines bearing CF3 and CHF2 functionalities were obtained using CF3SO2Na and CHF2SO2Na by visible light photocatalysis. This protocol involves a radical cascade cyclization via tandem tri- and difluoromethylation-arylation of pendent unactivated alkenes. Benzimidazole, imidazole, theophylline, purine, and indole serve as applicable anchors, thereby enriching the structural diversity of piperidine and pyrrolidine derivatives. This method features mild, additive-free and transition metal-free conditions.

The discovery of novel papillomaviruses in cats in Southwest China.

Feline viral diarrhea is a significant cause of death in kittens. In this study, 12 mammalian viruses were identified by metagenomic sequencing in diarrheal feces in 2019, 2020, and 2021, respectively. Interestingly, a novel of felis catus papillomavirus (FcaPV) was identified for the first time in China. Subsequently, we investigated the prevalence of FcaPV in 252 feline samples, including 168 diarrheal feces and 84 oral swabs, with a total of 57 (22.62%, 57/252) samples detected positive. Of the 57 positive samples, FcaPV genotype 3 (FcaPV-3) was detected at high prevalence rate (68.42%, 39/57), followed by FcaPV-4 (22.8%, 13/57), FcaPV-2 (17.54%, 10/57), and FcaPV-1 (1.75%, 1/55), while no FcaPV-5 and FcaPV-6. In addition, two novel putative FcaPVs were identified, which were the highest similarity to Lambdapillomavirus from Leopardus wiedii or canis familiaris, respectively. Therefore, this study was the first characterization of the viral diversity in feline diarrheal feces and the prevalence of FcaPV in Southwest China.

Lasiodiplodiapyrones A and B, Pyrone-Preussomerin Adducts with Highly Strained Polycyclic Ring Systems from Lasiodiplodia pseudotheobromae.

Lasiodiplodiapyrones A and B (1 and 2), two new preussomerin derivatives, possessing an unexpected 6-methyl-4H-furo[3,2-c]pyran-4-one moiety and a highly functionalized conjoint and complicated polycyclic ring system, along with two known congeners (3 and 4), were isolated from the fungus Lasiodiplodia pseudotheobromae. Their structures including absolute configurations were determined by spectroscopic analyses, Mosher's method, and ECD calculations. A biosynthetic pathway was proposed to explain the origin of lasiodiplodiapyrones A and B as well as their relationship with preussomerins. Compounds 1-4 showed suppressive effects on the production of NO with IC50 values of 4.8 ± 0.3, 8.5 ± 1.1, 5.9 ± 0.8, and 12.8 ± 1.3 µM, respectively.

Postoperative bone graft migration into the thecal sac and shifting down to the lower level after an endoscopic lumbar interbody fusion: a case report.

BACKGROUND: Postoperative bone graft migration (PBGM) is a fairly rare spinal postoperative complication. Its occurrence after endoscopic surgery has rarely been reported in the literature so far. This is a case report of a 52-year-old male occurring PBGM into the thecal sac in the 8th days after an endoscopic lumbar interbody fusion (ELIF), which can make surgeons more minded with such serious rare complication after BGM. CASE PRESENTATION: A 52-year-old male patient, underwent a L4-5 ELIF, presented with an acute radiculopathy on right leg and urinary incontinence in the 8th postoperative day. An emergency lumbar Computed Tomography(CT scan) and Magnetic Resonance Imaging (MRI) demonstrated bone graft migration into the thecal sac at the L4-5 level, and shifting down to the lower level. The revision surgery was performed at once successfully. Finally, the patient got well managed before discharge. CONCLUSION: Supported by this case report, we believe that PBGM into the thecal sac is a rare but horrible complication of ELIF. However, too much volume of bone graft and its posterior placement are more prone to developing this complication. Finally, we are not sure that the outcome presented in this study will be repeated in future cases.

Structural basis of Chikungunya virus inhibition by monoclonal antibodies.

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.

Two Pairs of New Bisabolane-Type Sesquiterpenoids from Aspergillus sydowii.

Two pairs of new bisabolane-type sesquiterpenoids, (+)-aspersydowin A (7S) [(+)-1], (-)-aspersydowin A (7R) [(-)-1], (+)-aspersydowin B (7S,11S) [(+)-2], (-)-aspersydowin B (7R,11R) [(-)-2], along with six known compounds (1-8) were isolated from the fungus Aspergillus sydowii. Compounds 1 and 2 are enantiomers resolved by the Chiralpak IC, using a hexane- propan-2-ol mobile phase. The structure of 1 and 2 with absolute configuration were assigned tentatively by 1D (1 H, 13 C, and DEPT) & 2D (HSQC, 1 H-1 H COSY, HMBC, and NOESY) NMR data analyses and ECD calculations. Compounds 1-8 were screened for the biological activities in vitro. The results showed that compounds 3, 4 and 8 exhibited immunosuppressive activities with IC50 values of 10.9, 17.6 and 13.4 µM, respectively.

Site-Specific Antibody Conjugation with Payloads beyond Cytotoxins.

As antibody-drug conjugates have become a very important modality for cancer therapy, many site-specific conjugation approaches have been developed for generating homogenous molecules. The selective antibody coupling is achieved through antibody engineering by introducing specific amino acid or unnatural amino acid residues, peptides, and glycans. In addition to the use of synthetic cytotoxins, these novel methods have been applied for the conjugation of other payloads, including non-cytotoxic compounds, proteins/peptides, glycans, lipids, and nucleic acids. The non-cytotoxic compounds include polyethylene glycol, antibiotics, protein degraders (PROTAC and LYTAC), immunomodulating agents, enzyme inhibitors and protein ligands. Different small proteins or peptides have been selectively conjugated through unnatural amino acid using click chemistry, engineered C-terminal formylglycine for oxime or click chemistry, or specific ligation or transpeptidation with or without enzymes. Although the antibody protamine peptide fusions have been extensively used for siRNA coupling during early studies, direct conjugations through engineered cysteine or lysine residues have been demonstrated later. These site-specific antibody conjugates containing these payloads other than cytotoxic compounds can be used in proof-of-concept studies and in developing new therapeutics for unmet medical needs.

Influence of Natural Aging on the Moisture Sorption Behaviour of Wooden Structural Components.

A greater understanding of moisture sorption behaviour of aged wooden structural components, which has a close relationship with dimensional stability, is required to effectively evaluate and preserve historical artefacts. This study focused on the effects of aging on Baotou beam samples from a Chinese historical wooden building. An analysis of the sorption isotherms and hysteresis loops of a naturally aged, decayed sample (AOS), an aged sound sample (AIS), and a reference sample (RS), using classical sorption isotherm models revealed that the moisture sorption behaviour of samples from the same growth ring in a Baotou beam can differ significantly. AOS showed higher hygroscopicity than AIS, and both these samples were more hygroscopic than RS. Furthermore, the mono/multilayer moisture contents of AOS were always higher than those of AIS and RS. In addition, Fourier transform infrared, second-derivative infrared, and two-dimensional correlation infrared spectroscopy were used to investigate chemical changes in the samples. The relative hemicellulose and lignin contents of the samples changed significantly with wood aging. Furthermore, AOS exhibited the highest calcium oxalate content, which may be associated with fungal infections. Overall, these results provide valuable insights into the effects of aging on wood samples and the dimensional stability of timber structures, which could inform future research on methods for the preservation or restoration of aging timber structures.

COVID-19 and primary wound healing: A new insights and advance.

With the outbreak and pandemic of coronavirus disease-2019 (COVID-19), a huge number of people died of it. Apart from lung injuries, multiple organs have been confirmed to be impaired. In COVID-19 time, primary wound healing processes always prolong, however, its possible underlying mechanisms are still unclear. Therefore, to overcome this clinical problem, clarifying its underlying mechanisms clearly is necessary and urgently needed. In this review, we summarized that COVID-19 can prolong primary wound healing by inducing excessive inflammation and oxidative stress, disturbing immune system and haematological system, as well as influencing the functions and viability of epidermal stem cells (ESCs). Otherwise, we summarized that the strict control measures of blocking up COVID-19 pandemic can also have side effects on primary wound healing process.

Integrated System for On-Site Rapid and Safe Screening of COVID-19.

Since the outbreak of coronavirus disease 2019 (COVID-19), the epidemic has been spreading around the world for more than 2 years. Rapid, safe, and on-site detection methods of COVID-19 are in urgent demand for the control of the epidemic. Here, we established an integrated system, which incorporates a machine-learning-based Fourier transform infrared spectroscopy technique for rapid COVID-19 screening and air-plasma-based disinfection modules to prevent potential secondary infections. A partial least-squares discrimination analysis and a convolutional neural network model were built using the collected infrared spectral dataset containing 857 training serum samples. Furthermore, the sensitivity, specificity, and prediction accuracy could all reach over 94% from the results of the field test regarding 968 blind testing samples. Additionally, the disinfection modules achieved an inactivation efficiency of 99.9% for surface and airborne tested bacteria. The proposed system is conducive and promising for point-of-care and on-site COVID-19 screening in the mass population.

Transcriptome analyses shed light on floral organ morphogenesis and bract color formation in Bougainvillea.

BACKGROUND: Bougainvillea is a popular ornamental plant with brilliant color and long flowering periods. It is widely distributed in the tropics and subtropics. The primary ornamental part of the plant is its colorful and unusual bracts, rich in the stable pigment betalain. The developmental mechanism of the bracts is not clear, and the pathway of betalain biosynthesis is well characterized in Bougainvillea. RESULTS: At the whole-genome level, we found 23,469 protein-coding genes by assembling the RNA-Seq and Iso-Seq data of floral and leaf tissues. Genome evolution analysis revealed that Bougainvillea is related to spinach; the two diverged approximately 52.7 million years ago (MYA). Transcriptome analysis of floral organs revealed that flower development of Bougainvillea was regulated by the ABCE flower development genes; A-class, B-class, and E-class genes exhibited high expression levels in bracts. Eight key genes of the betalain biosynthetic pathway were identified by homologous alignment, all of which were upregulated concurrently with bract development and betalain accumulation during the bract initiation stage of development. We found 47 genes specifically expressed in stamens, including seven highly expressed genes belonging to the pentose and glucuronate interconversion pathways. BgSEP2b, BgSWEET11, and BgRD22 are hub genes and interacted with many transcription factors and genes in the carpel co-expression network. CONCLUSIONS: We assembled protein-coding genes of Bougainvilea, identified the floral development genes, and constructed the gene co-expression network of petal, stamens, and carpel. Our results provide fundamental information about the mechanism of flower development and pigment accumulation in Bougainvillea, and will facilitate breeding of cultivars with high ornamental value.

Detection and molecular characteristics of canine coronavirus in Chengdu city, Southwest China from 2020 to 2021.

Canine coronavirus (CCoV) is generally thought of as a mild, but highly contagious, enteritis of young dogs. This study was to investigate the molecular detection and characteristics of CCoV in Chengdu city, Southwest China. 218 canine fecal samples were collected from four animal hospitals and one animal shelter from 2020 to 2021. Fifty-nine CCoV-positive samples were detected by RT-PCR, including 40 CCoV-I, 25 CCoV-IIa, one CCoV-IIb and 10 untyped. To further analyze the genetic diversity of CCoV, we amplified ten complete spike (S) genes, including four CCoV-I and six CCoV-II strains. The amino acid sequence obtained in this study revealed 85.95% ± 12.55% homology with the reference strains. Moreover, in the N-terminal structural domain, there were two amino acid insertions (17QQ18) in two strains of CCoV-I and four amino acid insertions (95IGTN98) in CCoV-IIb strain. Interestingly, we identified that the S1/S2 cleavage site of the S protein of CCoV strains (SWU-SSX3 and SWU-SSX10) were consistent with feline coronavirus (FCoV). In the evolutionary tree, a strain of CCoV-I (SWU-SSX10) was found to be more closely related to FCoV, while SWU-SSX7 of CCoV-IIb was more closely related to coronavirus from the Chinese ferret badger. In addition, for the first time, recombination in a CCoV-IIb strain was found to occur between two subtypes occurring in the C domain of the S1 subunit, with a breakpoint starting at 2141 nt. The results enriched the epidemiological information of CCoV and provided an important reference for the prevention of CCoV in Chengdu city, Southwest China.

Optimization of the extraction process of flavonoids from Trollius ledebouri with natural deep eutectic solvents.

In recent years, natural deep eutectic solvents have been favored greatly due to their environment friendly, mild biological toxicity and simple biodegradability. Natural deep eutectic solvents gradually applied for the extracting bioactive compounds from natural products efficiently. In this study, 20 natural deep eutectic solvents were prepared and their physical and chemical properties were tested. The ultrasonic-assisted extraction method was used to extract flavonoids from Trollius ledebouri and high-performance liquid chromatography-ultraviolet was applied to examine two main bioactive flavonoids (orientin and vitexin). Compared with traditional solvents (water and 60% ethanol solution), natural deep eutectic solvents composed of L(-)-proline and levulinic acid (molar ratio 1:2) show a super extraction efficiency. On this basis, the response surface method was used to optimize the extraction temperature, extraction time, water contents, and solid-liquid ratio. As a consequence, the extraction temperature 60℃, extraction time 18 min, water content 14% (v/v), and the solid-liquid ratio 48 mL·g-1 were chosen as the best extraction process. This study shows that natural deep eutectic solvents can effectively extract flavonoids from T. ledebouri, laying a foundation for the further application of natural deep eutectic solvents to extract bioactive compounds from natural products.

Bu-Shen-Ning-Xin decoction alleviates premature ovarian insufficiency (POI) by regulating autophagy of granule cells through activating PI3K/AKT/mTOR pathway.

PURPOSE: To explore the therapeutic effects of Bu-Shen-Ning-Xin decoction (BSNXD) on POI and the underlying mechanism. METHODS: VCD was used to induce the in vivo and in vitro POI model. HE staining was used to evaluate the pathological state of ovarian tissues. ELISA was used to detect the production of hormones in the serum and granule cells (GCs). An immunohistochemical assay was used to determine the expression of ATG7 and p-AKT in the ovarian tissues. The number of oocytes in POI rats was counted. The mitochondrial membrane potential (MMP) in oocytes and GCs was detected by flow cytometry. A Western blot assay was used to measure the expression of AKT, p-AKT, p-mTOR, mTOR, S6K, p-S6K, ULK1, p-ULK1, Beclin-1, Bcl-2, LC3-II, LC3-I, ATG7, and cleaved Caspase3. The numbers of autophagosomes were detected by transmission electron microscope and autophagic flux assay. The CCK-8 assay was used to detect the cell viability. RESULTS: Decreased primary follicles in the ovarian tissues, elevated concentration of FSH, and LH, suppressed concentration of E2 and AMH in the serum, reduced number of oocytes, and mitochondrial dysfunction in oocytes induced by VCD were significantly reversed by BSNXD. Activated autophagy state and inhibited PI3K/AKT/mTOR pathway stimulated by VCD in both ovarian tissues and GCs were dramatically reversed by BSNXD. The protective effect of BSNXD on VCD-treated GCs was abolished by LY294002, an inhibitor of the PI3K/AKT/mTOR pathway. CONCLUSION: Our data revealed that BSNXD alleviated POI by regulating autophagy of granule cells through activating PI3K/AKT/mTOR pathway.

The Agronomic Traits, Alkaloids Analysis, FT-IR and 2DCOS-IR Spectroscopy Identification of the Low-Nicotine-Content Nontransgenic Tobacco Edited by CRISPR-Cas9.

In this study, the agricultural traits, alkaloids content and Fourier transform infrared spectroscopy (FT-IR) and two-dimensional correlation infrared spectroscopy (2DCOS-IR) analysis of the tobacco after Berberine Bridge Enzyme-Like Proteins (BBLs) knockout were investigated. The knockout of BBLs has limited effect on tobacco agricultural traits. After the BBLs knockout, nicotine and most alkaloids are significantly reduced, but the content of myosmine and its derivatives increases dramatically. In order to identify the gene editing of tobacco, principal component analysis (PCA) was performed on the FT-IR and 2DCOS-IR spectroscopy data. The results showed that FT-IR can distinguish between tobacco roots and leaves but cannot classify the gene mutation tobacco from the wild one. 2DCOS-IR can enhance the characteristics of the samples due to the increased apparent resolution of the spectra. Using the autopeaks in the synchronous map for PCA analysis, we successfully identified the mutants with an accuracy of over 90%.

Multi-institutional validation of novel models for predicting the prognosis of patients with huge hepatocellular carcinoma.

The population of patients with huge hepatocellular carcinoma (H-HCC diameter > 10.0 cm) is an odd group that is not well adjudicated in the current staging systems, whose prognosis after curative resection varies. We aimed to develop novel models to predict the long-term outcomes of patients with H-HCC without portal vein tumor thrombus after hepatectomy. There were 1076 H-HCC patients enrolled who underwent curative liver resection in five institutions in China. In total, 670 patients were recruited from our center and randomly divided into the training cohort (n = 502) and internal validation (n = 168) cohorts. Additionally, 406 patients selected from other four centers as the external validation cohort. Novel models were constructed based on independent preoperative and postoperative predictors of postsurgical recurrence (PSR) and postsurgical mortality (PSM) determined in multivariable cox regression analysis. The predictive accuracy and discriminative ability of the model were measured using Harrell's concordance index (C index) and calibration curve and compared with five conventional HCC staging systems. PSR model and PSM model were constructed based on tumor number, microscopic vascular invasion, tumor differentiation, preoperative alpha-fetoprotein level, albumin-bilirubin grade, liver segment invasion, neutrophil-to-lymphocyte ratio or platelet-to-neutrophil ratio, and surgical margin or intraoperative blood transfusion. The C-indexes were 0.84 (95% CI, 0.78-0.90) and 0.85 (95% CI, 0.78-0.91) for the PSR and PSM models, respectively, which were substantially higher than those of the five conventional HCC staging systems (0.63-0.75 for PSR; 0.66-0.77 for PSM). The two novel models achieved more accurate prognostic predictions of PSR and PSM for H-HCC patients after curative liver resection.

Prevalence and molecular characteristics of feline coronavirus in southwest China from 2017 to 2020.

Feline coronavirus (FCoV) is the causative agent of feline infectious peritonitis and diarrhoea in kittens worldwide. In this study, a total of 173 feline diarrhoeal faecal and ascetic samples were collected from 15 catteries and six veterinary hospitals in southwest China from 2017 to 2020. FCoV was detected in 80.35 % (139/173) of the samples using the RT-nPCR method; these included infections with 122 type I FCoV and 57 type II FCoV. Interestingly, 51 cases had co-infection with types I and II, the first such report in mainland China. To further analyse the genetic diversity of FCoV, we amplified 23 full-length spike (S) genes, including 18 type I and five type II FCoV. The type I FCoV and type II FCoV strains shared 85.5-98.7% and 97.4-98.9% nucleotide (nt) sequence identities between one another, respectively. The N-terminal domain (NTD) of 23 FCoV strains showed a high degree of variation (73.6-80.3 %). There was six type I FCoV strains with two amino acid insertions (159HL160) in the NTD. In addition, 18 strains of type I FCoV belonged to the Ie cluster, and five strains of type II FCoV were in the IIb cluster based on phylogenetic analysis. Notably, it was first time that two type I FCoV strains had recombination in the NTD, and the recombination regions was located 140-857 nt of the S gene. This study constitutes a systematic investigation of the current infection status and molecular characteristics of FCoV in southwest China.