5α-Epoxyalantolactone from Inula macrophylla attenuates cognitive deficits in scopolamine-induced Alzheimer's disease mice model.

Alzheimer's disease (AD) is a complex neurodegenerative condition. 5α-epoxyalantolactone (5α-EAL), a eudesmane-type sesquiterpene isolated from the herb of Inula macrophylla, has various pharmacological effects. This work supposed to investigate the improved impact of 5α-EAL on cognitive impairment. 5α-EAL inhibited the generation of nitric oxide (NO) in BV-2 cells stimulated with lipopolysaccharide (LPS) with an EC50 of 6.2 µM. 5α-EAL significantly reduced the production of prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α), while also inhibiting the production of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins. The ability of 5α-EAL to penetrate the blood-brain barrier (BBB) was confirmed via a parallel artificial membrane permeation assay. Scopolamine (SCOP)-induced AD mice model was employed to assess the improved impacts of 5α-EAL on cognitive impairment in vivo. After the mice were pretreated with 5α-EAL (10 and 30 mg/kg per day, i.p.) for 21 days, the behavioral experiments indicated that the administration of the 5α-EAL could alleviate the cognitive and memory impairments. 5α-EAL significantly reduced the AChE activity in the brain of SCOP-induced AD mice. In summary, these findings highlight the beneficial effects of the natural product 5α-EAL as a potential bioactive compound for attenuating cognitive deficits in AD due to its pharmacological profile.

High-Frequency Optical Coherence Elastography for Gingival Tissue Characterization: Variability in Stiffness and Response to Physiological Conditions.

Accurate measurement of gingiva's biomechanical properties in vivo has been an active field of research but remained an unmet challenge. Currently, there are no noninvasive tools that can accurately quantify tensile and shear moduli, which govern gingival health, with sufficiently high accuracy. This study presents the application of high-frequency optical coherence elastography (OCE) for characterizing gingival tissue in both porcine models and human subjects. Dynamic mechanical analysis, histology studies, and strain analysis are performed to support the OCE result. Our findings demonstrate substantial differences in tissue stiffness between supra-dental and inter-dental gingiva, validated by dynamic mechanical analysis and OCE. We confirmed the viscoelastic, nearly linear, and transverse-isotropic properties of gingiva in situ, establishing the reliability of OCE measurements. Further, we investigated the effects of tissue hydration, collagen degradation, and dehydration on gingival stiffness. These conditions showed a decrease and increase in stiffness, respectively. While preliminary, our study suggests OCE's potential in periodontal diagnosis and oral tissue engineering, offering real-time, millimeter-scale resolution assessments of tissue stiffness, crucial for clinical applications and biomaterial optimization in reconstructive surgeries.

ASS1 Enhances Anoikis Resistance via AMPK/CPT1A-mediated Fatty Acid Metabolism in Ovarian Cancer.

Metastasis is the leading cause of death in ovarian cancer (OC), with anoikis resistance being a crucial step for detached OC cells survival. Despite extensive research, targeting anoikis resistance remians a challenge. Here, we identify argininosuccinate synthase 1 (ASS1), a key enzyme in urea cycle, is markedly upregulated in OC cells in detached culture and is associated with increased anoikis resistance and metastasis. Disruption of the AMP/ATP balance by elevated ASS1 activates AMPK and its downstream factor, CPT1A. Then, ASS1 enhances FAO, leading to higher ATP generation and lipid utilization. Inhibition of CPT1A reverses ASS1-induced FAO. Our study gives some new functional insights into OC metabolism and represents a shift from traditional views, expanding ASS1's relevance beyond nitrogen metabolism to fatty acid metabolism. It uncovers how ASS1-induced FAO disrupts the AMP/ATP balance, leading to AMPK activation. By identifying the ASS1/AMPK/CPT1A axis as crucial for OC anoikis resistance and metastasis, our study opens up new avenues for therapeutic interventions.

Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus.

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, which include efficient genome editing, gene silencing, and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid. Although this plasmid coexists with pRN1 in its original host, early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa. islandicus. We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon. We identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (target N1). Mutated targets (N1a, N1b, and N1c) were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid interference assay. The results revealed that the original target triggered CRISPR immunity in this archaeon, whereas all three mutated targets did not, indicating that all the designed target mutations evaded host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD, and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability for replication. These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase. We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model.

Overexpression of Wild Soybean Expansin Gene GsEXLB14 Enhanced the Tolerance of Transgenic Soybean Hairy Roots to Salt and Drought Stresses.

As a type of cell-wall-relaxing protein that is widely present in plants, expansins have been shown to actively participate in the regulation of plant growth and responses to environmental stress. Wild soybeans have long existed in the wild environment and possess abundant resistance gene resources, which hold significant value for the improvement of cultivated soybean germplasm. In our previous study, we found that the wild soybean expansin gene GsEXLB14 is specifically transcribed in roots, and its transcription level significantly increases under salt and drought stress. To further identify the function of GsEXLB14, in this study, we cloned the CDS sequence of this gene. The transcription pattern of GsEXLB14 in the roots of wild soybean under salt and drought stress was analyzed by qRT-PCR. Using an Agrobacterium rhizogenes-mediated genetic transformation, we obtained soybean hairy roots overexpressing GsEXLB14. Under 150 mM NaCl- and 100 mM mannitol-simulated drought stress, the relative growth values of the number, length, and weight of transgenic soybean hairy roots were significantly higher than those of the control group. We obtained the transcriptomes of transgenic and wild-type soybean hairy roots under normal growth conditions and under salt and drought stress through RNA sequencing. A transcriptomic analysis showed that the transcription of genes encoding expansins (EXPB family), peroxidase, H+-transporting ATPase, and other genes was significantly upregulated in transgenic hairy roots under salt stress. Under drought stress, the transcription of expansin (EXPB/LB family) genes increased in transgenic hairy roots. In addition, the transcription of genes encoding peroxidases, calcium/calmodulin-dependent protein kinases, and dehydration-responsive proteins increased significantly. The results of qRT-PCR also confirmed that the transcription pattern of the above genes was consistent with the transcriptome. The differences in the transcript levels of the above genes may be the potential reason for the strong tolerance of soybean hairy roots overexpressing the GsEXLB14 gene under salt and drought stress. In conclusion, the expansin GsEXLB14 can be used as a valuable candidate gene for the molecular breeding of soybeans.

Boron-mediated one-pot access to salicylaldehydes via ortho-C-H hydroxylation of benzaldehydes.

A novel protocol has been devised for the ortho-C-H hydroxylation of benzaldehydes. Directed by a transient imine group, the borylation of benzaldehydes, sequentially followed by the hydroxylation, furnishes diverse salicylaldehydes in a one-pot manner. The resultant salicylaldehydes could be readily applied in the downstream synthesis to produce bioactive molecules.

Susceptibility gene identification and risk evaluation model construction by transcriptome-wide association analysis for salt sensitivity of blood pressure.

BACKGROUND: Salt sensitivity of blood pressure (SSBP) is an intermediate phenotype of hypertension and is a predictor of long-term cardiovascular events and death. However, the genetic structures of SSBP are uncertain, and it is difficult to precisely diagnose SSBP in population. So, we aimed to identify genes related to susceptibility to the SSBP, construct a risk evaluation model, and explore the potential functions of these genes. METHODS AND RESULTS: A genome-wide association study of the systemic epidemiology of salt sensitivity (EpiSS) cohort was performed to obtain summary statistics for SSBP. Then, we conducted a transcriptome-wide association study (TWAS) of 12 tissues using FUSION software to predict the genes associated with SSBP and verified the genes with an mRNA microarray. The potential roles of the genes were explored. Risk evaluation models of SSBP were constructed based on the serial P value thresholds of polygenetic risk scores (PRSs), polygenic transcriptome risk scores (PTRSs) and their combinations of the identified genes and genetic variants from the TWAS. The TWAS revealed that 2605 genes were significantly associated with SSBP. Among these genes, 69 were differentially expressed according to the microarray analysis. The functional analysis showed that the genes identified in the TWAS were enriched in metabolic process pathways. The PRSs were correlated with PTRSs in the heart atrial appendage, adrenal gland, EBV-transformed lymphocytes, pituitary, artery coronary, artery tibial and whole blood. Multiple logistic regression models revealed that a PRS of P < 0.05 had the best predictive ability compared with other PRSs and PTRSs. The combinations of PRSs and PTRSs did not significantly increase the prediction accuracy of SSBP in the training and validation datasets. CONCLUSIONS: Several known and novel susceptibility genes for SSBP were identified via multitissue TWAS analysis. The risk evaluation model constructed with the PRS of susceptibility genes showed better diagnostic performance than the transcript levels, which could be applied to screen for SSBP high-risk individuals.

Relationships Among Serological Indicators and In Vitro High-Throughput Drug Sensitivity Screening Results in Patients with Hepatocellular Carcinoma.

AIM: The purpose of this study was to analyze the relationship between serum indicators and high-throughput drug screening (HDS) results, aiming to achieve specific therapy for hepatocellular carcinoma (HCC). METHODS: This study recruited patients with HCC who underwent surgical resection at the Hepatobiliary Surgery Center of the First Affiliated Hospital of Chongqing Medical University from December 2019 to December 2021. HCC tissues were obtained from patients during surgery and subjected to in vitro cell culture, and then HDS testing was performed on the cultured tissue samples. We used Spearman's correlation analysis to examine the relationships between drug sensitivity results for anti-hepatocellular carcinoma drugs, other antitumor drugs, and serological indicators, the Neutrophil Lymphocyte Ratio (NLR), Platelet Lymphocyte Ratio (PLR), Systemic Immune Inflammatory Index (SII), Systemic Inflammatory Response Index (SIRI), Prognostic Nutritional Index (PNI), and Lymphocyte Monocyte Ratio (LMR). A significant correlation was considered when P<0.05 and |r|>0.40. Furthermore, linear regression analysis was conducted to elucidate the relationship between serological indicators and drug susceptibility, with significant results indicated by P<0.05 and R²≥0.50. RESULTS: In this study, 82 patients with HCC who had undergone hepatectomy and completed in vitro cell culture and HDS testing were evaluated. Using Spearman's correlation with a significance threshold of P<0.05 and |r|>0.40, we identified significant associations between serological indicators and specific drug regimens: NLR correlated with 5-Fluorouracil, 5- Fluorouracil+Calcium folinate (FOLFOX4), and Capecitabine + Cisplatin (XP); PLR with FOLFOX4; SII with XP, FOLFOX4, Doxorubicin + Oxaliplatin (ADM+L-OHP); and SIRI with XP and FOLFOX4. No correlations were found between PNI or LMR and any drug inhibition rates. A comprehensive evaluation using linear regression analysis-which included variables such as sex, age, hepatitis B virus and liver cirrhosis status, size and number of lesions, alphafetoprotein, total bilirubin, albumin, alanine aminotransferase, aspartate aminotransferase, and prothrombin time, alongside NLR, PLR, SII, and SIRI was conducted in relation to drug regimens. This analysis revealed that NLR, SII, and SIRI are significant predictors of FOLFOX4 inhibition rate, while NLR predicts the inhibition rate of XP effectively. However, no significant links were established between molecular targeted drugs, other antitumor drugs, and serological indicators. CONCLUSIONS: NLR, SII, and SIRI were correlated with FOLFOX4, and the higher the values of NLR, SII, and SIRI, the higher the in vitro inhibition of FOLFOX. Also, NLR was correlated with XP, and the higher the value of NLR, the higher the in vitro inhibition of XP.

Targeted discovery of unusual diterpenoids with anti-fungal activity from the root of Euphorbia lathyris.

Lathyrisone A (1), a diterpene with an undescribed tricyclic 6/6/6 fused carbon skeleton, along with spirolathyrisins B-D (3-5), three diterpenes with a rare [4.5.0] spirocyclic carbon skeleton, and one known compound (2) were isolated from the roots of Euphorbia lathyris. Their chemical structures were characterized by extensive spectroscopic analysis, X-ray crystallography, ECD and quantum chemistry calculation. A plausible biosynthetic pathway for compounds 1-5 was proposed, which suggested it is a competitive pathway for ingenol biosynthesis in the plant. The anti-fungal activities of these compounds were tested, especially, compound 2 showed stronger anti-fungal activities against Fusarium oxysporum and Alternaria alternata than the positive control fungicide thiophanate-methyl. The preliminary structure-activity relationship of compounds 1-5 was also discussed. These results not only expanded the chemical diversities of E. lathyris, but also provided a lead compound for the control of plant pathogens.

Chenodeoxycholic Acid-Modified Polyethyleneimine Nano-Composites Deliver Low-Density Lipoprotein Receptor Genes for Lipid-Lowering Therapy by Targeting the Liver.

Lipid-lowering drugs, especially statins, are extensively utilized in clinical settings for the prevention of hyperlipidemia. Nevertheless, prolonged usage of current lipid-lowering medications is associated with significant adverse reactions. Therefore, it is imperative to develop novel therapeutic agents for lipid-lowering therapy. In this study, a chenodeoxycholic acid and lactobionic acid double-modified polyethyleneimine (PDL) nanocomposite as a gene delivery vehicle for lipid-lowering therapy by targeting the liver, are synthesized. Results from the in vitro experiments demonstrate that PDL exhibits superior transfection efficiency compared to polyethyleneimine in alpha mouse liver 12 (AML12) cells and effectively carries plasmids. Moreover, PDL can be internalized by AML12 cells and rapidly escape lysosomal entrapment. Intravenous administration of cyanine5.5 (Cy5.5)-conjugated PDL nanocomposites reveals their preferential accumulation in the liver compared to polyethyleneimine counterparts. Systemic delivery of low-density lipoprotein receptor plasmid-loaded PDL nanocomposites into mice leads to reduced levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TC) in the bloodstream without any observed adverse effects on mouse health or well-being. Collectively, these findings suggest that low-density lipoprotein receptor plasmid-loaded PDL nanocomposites hold promise as potential therapeutics for lipid-lowering therapy.

Comprehensive analysis of next generation sequencing and ARMS-PCR for detecting EGFR exon 20 insertion (ex20ins) mutations in Chinese non-small cell lung cancer patients.

Background: Amivantamab (JNJ-372) and mobocertinib (TAK-788) have been reported to have favorable therapeutic effect for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutations. Thus, accurate detection of EGFR ex20ins mutations is crucial for subsequent individualized therapy. The aim of this study was to compare the two common methods of next generation sequencing (NGS) and amplification refractory mutation system polymerase chain reaction (ARMS-PCR) for detecting EGFR ex20ins mutations in Chinese NSCLC patients. Methods: We retrospectively analyzed EGFR mutations, especially for ex20ins, in 3,606 NSCLC patients detected by NGS and 1,785 patients by ARMS. Results: Among the 3,606 NGS patients, a total of 2,077 EGFR mutations and 95 EGFR ex20ins were identified, accounting for 57.6% and 2.6%, respectively. While 48.4% of EGFR mutations and 1.1% of ex20ins were detected in 1,785 ARMS patients, which were significantly lower than those of NGS (P<0.01). Thirty-four unique ex20ins variants were identified by NGS, and eight of them was reported for the first time. However, ARMS was designed to detect only several known EGFR ex20ins variants, and even did not include the most common variants in Chinese NSCLC patients. Conclusions: NGS is more advantageous and strongly recommended for the detection of EGFR ex20ins mutations. Considering the fast and cost-effective ARMS detection method, it is suggested that the primers design should be updated according to the characteristics of EGFR ex20ins mutations in Chinese NSCLC patients.

Exploring histone deacetylases in type 2 diabetes mellitus: pathophysiological insights and therapeutic avenues.

Diabetes mellitus is a chronic disease that impairs metabolism, and its prevalence has reached an epidemic proportion globally. Most people affected are with type 2 diabetes mellitus (T2DM), which is caused by a decline in the numbers or functioning of pancreatic endocrine islet cells, specifically the ß-cells that release insulin in sufficient quantity to overcome any insulin resistance of the metabolic tissues. Genetic and epigenetic factors have been implicated as the main contributors to the T2DM. Epigenetic modifiers, histone deacetylases (HDACs), are enzymes that remove acetyl groups from histones and play an important role in a variety of molecular processes, including pancreatic cell destiny, insulin release, insulin production, insulin signalling, and glucose metabolism. HDACs also govern other regulatory processes related to diabetes, such as oxidative stress, inflammation, apoptosis, and fibrosis, revealed by network and functional analysis. This review explains the current understanding of the function of HDACs in diabetic pathophysiology, the inhibitory role of various HDAC inhibitors (HDACi), and their functional importance as biomarkers and possible therapeutic targets for T2DM. While their role in T2DM is still emerging, a better understanding of the role of HDACi may be relevant in improving insulin sensitivity, protecting ß-cells and reducing T2DM-associated complications, among others.

Sodium halide solid state electrolyte of Na3YBr6 with low activation energy.

Halide solid-state electrolytes (SSEs) are considered promising candidates for practical applications in all-solid-state batteries (ASSBs), due to their outstanding high voltage stability and compatibility with electrode materials. However, Na+ halide SSEs suffer from low ionic conductivity and high activation energy, which limit their applications in sodium all-solid-state batteries. Here, sodium yttrium bromide solid-state electrolytes (Na3YBr6) with a low activation energy of 0.15 eV is prepared via solid state reaction. Structure characterization using X-ray diffraction reveals a monoclinic structure (P21/c) of Na3YBr6. First principle calculations reveal that the low migration activation energy comes from the larger size and vibration of Br- anions, both of which expand the Na+ ion migration channel and reduce its activation energy. The electrochemical window of Na3YBr6 is determined to be 1.43 to 3.35 V vs. Na/Na+, which is slightly narrower than chlorides. This work indicates bromides are a good catholyte candidate for sodium all solid-state batteries, due to their low ion migration activation energy and relatively high oxidation stability.

Lattice QCD Calculation of Electroweak Box Contributions to Superallowed Nuclear and Neutron Beta Decays.

We present the first lattice QCD calculation of the universal axial γW-box contribution □_{γW}^{VA} to both superallowed nuclear and neutron beta decays. This contribution emerges as a significant component within the theoretical uncertainties surrounding the extraction of |V_{ud}| from superallowed decays. Our calculation is conducted using two domain wall fermion ensembles at the physical pion mass. To construct the nucleon four-point correlation functions, we employ the random sparsening field technique. Furthermore, we incorporate long-distance contributions to the hadronic function using the infinite-volume reconstruction method. Upon performing the continuum extrapolation, we arrive at □_{γW}^{VA}=3.65(7)_{lat}(1)_{PT}×10^{-3}. Consequently, this yields a slightly higher value of |V_{ud}|=0.973 86(11)_{exp}(9)_{RC}(27)_{NS}, reducing the previous 2.1σ tension with the CKM unitarity to 1.8σ. Additionally, we calculate the vector γW-box contribution to the axial charge g_{A}, denoted as □_{γW}^{VV}, and explore its potential implications.

Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope.

TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.

DePARylation is critical for S phase progression and cell survival.

Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S-phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Euphorbia lathyris in China.

Euphorbia lathyris L. is a biennial herb in the Euphorbiaceae that has been used as a medicinal plant. It is distributed or cultivated worldwide, and the seeds of E. lathyris are the main source of ingenol, which is the precursor of Picato, the first medicine approved by USFDA for the treatment of solar keratosis (Abramovits et al. 2013). However, the production of E. lathyris can be severely hampered by the occurrence of plant diseases. Between 2020-2022 (specifically in October-November of each year), anthracnose-like symptoms were observed on E. lathyris in fields (E 118°49'50″, N 32°3'33″) in Nanjing, Jiangsu Province, China. The incidence of E. lathyris with disease symptoms was between 25%-30% (n = 100). The lesions on the leaves were evident initially as dark brown spots, which expanded into larger necrotic spots, finally resulting in leaves withering and dropping off. In severe cases, stem wilting was also observed. To determine the causal agent, we collected diseased leaf samples (n = 20) from different E. lathyris plants in the field (~ 1800 m2). After cleaning, the junctions of the diseased and healthy parts were excised and sterilized in 75% ethanol for 20-25 seconds, and rinsed with sterile water. After that, they were transferred onto potato sucrose agar (PSA) plates and placed at 25℃ for 3-4 days, until fungal growth was evident. The fungus was purified by recovering single conidia and growing them on PSA (Hu et al. 2015). A consistent fungal colony, based on morphological characteristics, was recovered from 17 samples. The colony color was initially white, green in the middle, and gradually changed into gray green as the colony matured. Conidia were transparent and cylindrical (22-28 µm × 7-9 µm, n = 50). Five loci informative (ITS, TUB, ACT, GAPDH, and CHS-1) (Weir et al. 2012) for Colletotrichum spp. identification were sequenced from two isolates ELC-1 and ELC-2 obtained from different plant individuals. Compared with a reference isolate (Colletotrichum gloeosporioides ZH3), the GAPDH, CHS-1, and TUB2 sequences of ELC-1 and ELC-2 showed 95% (263 bp out of 275 bp), 98% (295 bp out of 299 bp), and 99% (711 bp out of 712 bp and 717 bp out of 719 bp) similarity, respectively. The ITS sequence identities were 100% (577 bp out of 577 bp) and 99% (594 bp out of 597 bp), while the ACT sequence identities were 100% (281 bp out of 281 bp) and 98% (279 bp out of 284 bp). All sequences have been deposited in Genbank database (OR865865-OR865866 and OR873625-OR873632). After performing phylogenetic analysis with Mega 11, the pathogen was confirmed as C. gloeosporioides. To fulfil Koch's postulates, we sprayed six-week-old healthy plants with a conidia suspension of C. gloeosporioides (106 spores/mL) or sterile water (serve as control). The inoculated plants were placed at 25℃, 100% relative humidity, and 12-h photoperiod (Zhang et al. 2021). Six plants were inoculated for each treatment, and the experiment was repeated three times. After 6-8 days, the plants inoculated with C. gloeosporioides showed similar symptoms to those observed on diseased plants in the field, while the control plants remained healthy and free of disease. The pathogens were then re-isolated and identified as C. gloeosporioides. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on E. lathyris. Anthracnose may cause significant yield losses in E. lathyris production, and our results will provide experimental and theoretical basis for the management of the disease.

Testing multiplexed anti-ASFV CRISPR-Cas9 in reducing African swine fever virus.

African swine fever (ASF) is a highly fatal viral disease that poses a significant threat to domestic pigs and wild boars globally. In our study, we aimed to explore the potential of a multiplexed CRISPR-Cas system in suppressing ASFV replication and infection. By engineering CRISPR-Cas systems to target nine specific loci within the ASFV genome, we observed a substantial reduction in viral replication in vitro. This reduction was achieved through the concerted action of both Type II and Type III RNA polymerase-guided gRNA expression. To further evaluate its anti-viral function in vivo, we developed a pig strain expressing the multiplexable CRISPR-Cas-gRNA via germline genome editing. These transgenic pigs exhibited normal health with continuous expression of the CRISPR-Cas-gRNA system, and a subset displayed latent viral replication and delayed infection. However, the CRISPR-Cas9-engineered pigs did not exhibit a survival advantage upon exposure to ASFV. To our knowledge, this study represents the first instance of a living organism engineered via germline editing to assess resistance to ASFV infection using a CRISPR-Cas system. Our findings contribute valuable insights to guide the future design of enhanced viral immunity strategies. IMPORTANCE: ASFV is currently a devastating disease with no effective vaccine or treatment available. Our study introduces a multiplexed CRISPR-Cas system targeting nine specific loci in the ASFV genome. This innovative approach successfully inhibits ASFV replication in vitro, and we have successfully engineered pig strains to express this anti-ASFV CRISPR-Cas system constitutively. Despite not observing survival advantages in these transgenic pigs upon ASFV challenges, we did note a delay in infection in some cases. To the best of our knowledge, this study constitutes the first example of a germline-edited animal with an anti-virus CRISPR-Cas system. These findings contribute to the advancement of future anti-viral strategies and the optimization of viral immunity technologies.

Four lathyrane diterpenoids from the seeds of Euphorbia lathyris.

Four new diterpenoids, including three secolathyrane diterpenoids (1-3) and one lathyrane diterpenoid (4), together with seven known diterpenoids, were obtained in the shelled seeds of Euphorbia lathyris. In particular, 1-3 possess a rare split ring structure, and currently only one compound with the same skeleton has been identified in E. lathyris. Compound 4 furnishes an unprecedented oxygen bridge structure. The structures were identified using various spectral techniques, including NMR, HR-ESI-MS, single-crystal X-ray diffraction and calculated electronic circular dichroism (ECD). The biosynthetic pathway of 1-4 was inferred. Furthermore, the cytotoxic activities of all compounds (1-11) were measured on three human tumor cells. New compounds 2 and 3 showed moderate cytotoxic activities against U937 cells with IC50 values of 22.18 and 25.41 µM, respectively.

Temperature-Dependent Evaporative Anthropogenic VOC Emissions Significantly Exacerbate Regional Ozone Pollution.

The evaporative emissions of anthropogenic volatile organic compounds (AVOCs) are sensitive to ambient temperature. This sensitivity forms an air pollution-meteorology connection that has not been assessed on a regional scale. We parametrized the temperature dependence of evaporative AVOC fluxes in a regional air quality model and evaluated the impacts on surface ozone in the Beijing-Tianjin-Hebei (BTH) area of China during the summer of 2017. The temperature dependency of AVOC emissions drove an enhanced simulated ozone-temperature sensitivity of 1.0 to 1.8 µg m-3 K-1, comparable to the simulated ozone-temperature sensitivity driven by the temperature dependency of biogenic VOC emissions (1.7 to 2.4 µg m-3 K-1). Ozone enhancements driven by temperature-induced AVOC increases were localized to their point of emission and were relatively more important in urban areas than in rural regions. The inclusion of the temperature-dependent AVOC emissions in our model improved the simulated ozone-temperature sensitivities on days of ozone exceedance. Our results demonstrated the importance of temperature-dependent AVOC emissions on surface ozone pollution and its heretofore unrepresented role in air pollution-meteorology interactions.