Repellent effects of Chinese cinnamon oil on nymphal ticks of Haemaphysalis longicornis, Rhipicephalus haemaphysaloides, and Hyalomma asiaticum.

The repellent activity of Chinese cinnamon oil (Cinnamomum cassia) on nymphal ticks (Haemaphysalis longicornis Neumann, Rhipicephalus haemaphysaloides Supino, and Hyalomma asiaticum Schulze and Schlottke) was evaluated in a sample Y-tube bioassay. The results were based on the vertical migration of ticks during the host-seek phase and showed a dose-dependent repellent effect of Chinese cinnamon oil on the tested nymphs after 6 h. For H. longicornis, R. haemaphysaloides, and H. asiaticum at the concentrations (vol/vol) of 3, 3, and 1.5%, the repellent percentages over time were 68-97, 69-94, and 69-93%, respectively, which indicated strong repellent activities against ticks, similar to the positive control DEET (N,N-diethyl-3-methylbenzamide). Chinese cinnamon oil exerted the strongest effect on H. asiaticum nymphs. To our knowledge, this is the first study to investigate the repellent effects of Chinese cinnamon oil on ticks. Chinese cinnamon oil has considerable potential and should be developed as a practical tick repellent.

Characterization of a single-domain von Willebrand factor type C protein (HaSVC) from the salivary gland of the tick Hyalomma asiaticum.

As a widely distributed arthropod and vector for various pathogens, Hyalomma asiaticum presents great risk and potential losses in animal husbandry. Effective measures, including the use of vaccines, are necessary for controlling ticks and tick-borne diseases. A concise understanding of the tick-host interaction associated molecules and pathways is required for vaccine development. In the present study, a protein containing a single-domain von Willebrand factor type C (HaSVC) was isolated from H. asiaticum and was subjected to functional identification. As a result, the full-length sequence of the HaSVC (506 bp) gene was obtained, which putatively encodes 100 amino acids with a predicted molecular mass of 11 kDa, excluding the 23-amino acid signal peptide. HaSVC contains 8 cysteines to form 4 disulfide bonds. The native HaSVC protein was detected in multiple tick organs. HaSVC neither attenuated the anti-coagulation process nor directly affected the blood feeding of adult ticks. However, the purified recombinant protein HaSVC (rHaSVC/GST) significantly increased the proliferation of mice spleen cells. This might suggest a regulatory function for HaSVC on inflammation, thus providing new information that may explain the "crosstalk" between ticks and hosts.

Engorgement of Rhipicephalus haemaphysaloides ticks blocked by silencing a protein inhibitor of apoptosis.

Inhibitors of apoptosis (IAPs) are regulators of cell death and may play a role in the salivary glands of ticks during blood-feeding. We cloned the open reading frame (ORF) sequence of the IAP gene in Rhipicephalus haemaphysaloides (RhIAP). The RhIAP ORF of 1887 bp encodes a predicted protein of 607 amino acids, which contains three baculovirus IAP repeat domains and a RING finger motif. A real-time PCR assay showed that RhIAP mRNA was expressed in all the tick developmental stages (eggs, larvae, nymphs, and adults) and in all tissues examined (midgut, ovary, salivary glands, fat body, and hemolymph). Western blot showed that the protein level of RhIAP in salivary glands increased during tick blood-feeding and decreased towards the end of tick engorgement. RhIAP gene silencing in vitro experiments with salivary glands demonstrated that RhIAP could be effectively knocked down within 48 h after dsRNA treatment, and as a consequence, salivary glands displayed apoptotic morphology. RhIAP gene silencing also inhibited tick blood-feeding and decreased the engorgement rate. These data suggest that RhIAP might be a suitable RNAi target for tick control.

A cysteine protease of Babesia microti and its interaction with tick cystatins.

Babesiosis is a tick-borne protozoonosis caused by Babesia, which can cause fever, hemolytic anemia, hemoglobinuria, and even death. Babesia microti is a parasite found in rodents and can be pathogenic to humans. In this study, the full-length cDNA of a B. microti cysteine protease (BmCYP) was expressed and the recombinant rBmCYP protein analyzed and characterized. BmCYP is encoded by an ORF of 1.3 kb, with a predicted molecular weight of 50 kDa and a theoretical pI of 8.5. The amino acid sequence of BmCYP exhibits an identity of 32.9 to 35.2% with cysteine proteases of Babesia ovis, Babesia bovis, and Theileria, respectively. The results of the proteinase assays show that rBmCYP has cysteine protease enzymatic activity. In addition, we demonstrate that tick cystatins rRhcyst-1 and rRhcyst-2 were able to effectively inhibit the activity of rBmCYP; the inhibition rates were 57.2% and 30.9%, respectively. Tick cystatins Rhcyst-1 and Rhcyst-2 were differentially expressed in ticks that fed on Babesia-infected mice relative to non-infected control ticks. Our results suggest that BmCYP is a functional enzyme with cysteine protease enzymatic activity and may be involved in tick-B. microti interactions.

Molecular characterization of clathrin heavy chain (Chc) in Rhipicephalus haemaphysaloides and its effect on vitellogenin (Vg) expression via the clathrin-mediated endocytic pathway.

Clathrin plays an important role in arthropods, but its function in ticks has not been explored. Here, we describe the molecular characteristics of the clathrin heavy chain of the tick Rhipicephalus haemaphysaloides and its effects on yolk development. The open reading frame of the clathrin heavy chain (Chc) (Rh-Chc) gene consists of 5103 nucleotides encoding 670 amino acids, which is most closely related to that of Ixodes scapularis and relatively close to Homo sapiens and Drosophila melanogaster. Real-time qPCR revealed that Rh-Chc was expressed at all developmental stages and organs. After Rh-Chc is silenced, ticks did not feed and mortality rate was 100%. Moreover, Rh-Chc co-localized with Vitellogenin receptor (VgR) on oocyte membrane. Immunofluorescence showed that the expression of Vitellogenin (Vg) (Rh-Vg) was also closely related to Rh-Chc. Immunofluorescence showed that the expression of Vg was also closely related to Rh-Chc, Rh-Chc silencing slowed the development of oocytes in tick, and culture of ovary in vitro silenced Rh-Chc, the development of oocytes in ticks also slowed down. Overall, the results of this study indicated that Rh-Chc is a vital gene in the tick R. haemaphysaloides that plays an important role in its growth, development, and reproduction.

Stability of vincristine sulfate, doxorubicin hydrochloride and etoposide phosphate admixtures in polyisoprene elastomeric pump supporting transition of the EPOCH regimen to outpatient care.

BACKGROUND: The EPOCH regimen, consisting of vincristine sulfate, doxorubicin hydrochloride, and etoposide phosphate, is typically administered by continuous infusion over four days to oncology inpatients. If the EPOCH regimen was available to be administered through portable elastomeric pumps, chemotherapy could be transitioned to an outpatient setting, reducing inpatient bed days and overall healthcare costs. However, a lack of stability data for the admixtures in the elastomeric infusion devices currently prevents the transition of the regime to an outpatient setting. The purpose of this study is to determine the physical and chemical stability of the admixture in polyisoprene elastomeric pumps under different storage conditions to support the transition of the EPOCH regime to an outpatient setting. METHODS: The physico-chemical stability of three admixtures at a range of clinically relevant concentrations compounded in polyisoprene elastomeric infusors was determined when refrigerated at 2-6℃ over a 14-day period followed by 35℃ up to 7 days in the dark, and under standardized fluorescent light to simulate scenarios in clinical practice. RESULTS: All tested admixtures were compatible and the drugs were stable in the elastomeric infusors for up to 14 days when stored at 2-6℃ followed by 7 days at 35℃ in the dark, with nominal losses of <5%. The major degradant of etoposide phosphate was its active form etoposide. There was no degradation (<1% loss) found when the admixture was exposed to a standardized fluorescent light dose of 80 klux-h (25℃) for 10 h. The temperature and light conditions the infusors were exposed to during the stability study were more severe than the conditions determine during clinical administration. CONCLUSION: The extended stability of the three infusional admixtures compounded in elastomeric infusion pumps demonstrated herein permits advance preparation and storage of these drugs, reducing pharmacy compounding resources. The demonstrated stability at 35℃ and under light exposure, conditions more severe than those experienced during clinical practice, support continuous infusions for up to seven days from the elastomeric infusors without a loss of potency. The proven stability of the EPOCH regimens in the tested elastomeric infusion device supports the transition of treatment to an outpatient setting which will reduce inpatient bed days and overall healthcare costs.

Molecular characterization, tissue-specific expression, and RNA knockdown of the putative heat shock cognate 70 protein from Rhipicephalus haemaphysaloides.

Heat shock cognate 70-kDa protein (RH-Hsc70) was identified from a cDNA library synthesized from the sialotranscriptomes of unfed and fed Rhipicephalus haemaphysaloides. The RH-Hsc70 open reading frame is 1950 bp long and encodes a protein that is 649 amino acids in length, with a predicted molecular weight of 71.1 kDa and a theoretical pI of 5.43. RH-Hsc70 exhibits 98% amino acid identity with Hsc70 in Haemaphysalis flava and 83% identity with Hsc70 in arthropods and mammals. RH-Hsc70 was mainly expressed in nymphs and adult ticks, not in larvae. Real-time quantitative PCR analysis indicated that RH-Hsc70 mRNA expression was induced by blood feeding in adult ticks. In addition, RH-Hsc70 gene expression was higher in the ovaries of fed adult ticks than that in the midguts, salivary glands, and fat bodies of unfed or fed adult ticks. RH-Hsc70 gene knockdown inhibited tick blood feeding, significantly decreased tick engorgement rate, and increased tick death rate. These data illustrate the importance of RH-Hsc70 in tick blood feeding and aging, which makes it a promising candidate for the development of anti-tick vaccines.

A Tick Cysteine Protease Inhibitor RHcyst-1 Exhibits Antitumor Potential.

BACKGROUND/AIMS: We previously identified a potent and tight-binding inhibitor of cysteine proteases from Rhipicephalus haemaphysaloides, RHcyst-1, which belongs to the cystatin type 1 family. Cathepsins, which are members of the cysteine protease family, participate in various pathological processes, including the initiation and development of cancers. The present study aimed to investigate the antitumor effects of RHcyst-1 and to explore the underlying mechanism of these effects. METHODS: Different tumor cells were treated with RHcyst-1 in vitro. Proliferation activity was evaluated using Cell Counting Kit-8, and migration and invasion were determined by wound healing and Transwell® invasion assays. In addition, a mouse tumor therapy model was established by inoculating the left forelimb of mice with B16-F10 cells, and tumor progression was evaluated by assessing tumor volume and survival. Flow cytometry was conducted to evaluate myeloid-derived suppressor cells (MDSCs), CD4+, and CD8+ T cell levels in PBMCs and spleens. Immunohistochemistry was performed to analyze immune cell infiltration and angiogenesis in the tumors. RESULTS: RHcyst-1 significantly inhibited the proliferation, migration, and invasion of all four different tumor cells in vitro. Additionally, it inhibited tumor growth and improved survival in vivo. A decrease and an increase in MDSCs levels were observed in PBMCs and in the spleen, respectively, after RHcyst-1 application. CONCLUSIONS: Tick RHcyst-1 has potential antitumor efficacy, and the observed antitumor activities may be partly attributable to changes in cathepsin expression and MDSCs levels in the PBMCs and spleens. The findings of the present study suggest that RHcyst-1 may have the potential to be utilized in cancer treatment.

Identification and anticoagulant activity of a novel Kunitz-type protein HA11 from the salivary gland of the tick Hyalomma asiaticum.

Kunitz/bovine pancreatic trypsin inhibitor proteins are abundant in the salivary glands of ticks and perform multiple functions in blood feeding, including inhibiting blood coagulation, regulating host blood supply and disrupting host angiogenesis. In this study, we identified a novel gene designated HA11 (Hyalomma asiaticum 11 kDa protein) from the salivary gland of the tick H. asiaticum. HA11 is encoded by a gene with an open reading frame of 306 bp that is translated into a deduced 101 amino acid 11 kDa protein that shares 27% sequence identity with a Kunitz-like protease inhibitor precursor in Amblyomma variegatum. Bioinformatic analysis confirmed HA11 as a member of the Kunitz-type family of inhibitors. Real time-PCR detected HA11 mRNA transcripts in tick larvae and nymphae stages, with levels highest in salivary gland tissue, and transcription was induced by blood feeding. HA11 anticoagulant activity was demonstrated by its ability to delay normal clotting of rabbit plasma in an activated partial thromboplastin time assay. Furthermore, RNA interference confirmed that HA11 influences H. asiaticum development and blood feeding, and the recombinant protein exerted low hemolytic activity. These results suggest HA11 is a novel Kunitz-type anticoagulant protein involved in tick blood feeding that may have potential as an anticoagulant drug or vaccine.

Identification and functional study of a novel 2-cys peroxiredoxin (BmTPx-1) of Babesia microti.

Babesia microti is an emerging human pathogen and the primary causative agent of human babesiosis in many regions of the world. Although the peroxiredoxins (Prxs) or thioredoxin peroxidases (TPx) enzymes of this parasite have been sequenced and annotated, their biological properties remain largely unknown. Prxs are a family of antioxidant enzymes that protect biological molecules against metabolically produced reactive oxygen species (ROS) and reduce hydrogen peroxide (H2O2) to water in both eukaryotes and prokaryotes. In this study, TPx-1 cDNA was cloned from B. microti (designated BmTPx-1). Recombinant BmTPx-1 (rBmTPx-1) was expressed in Escherichia coli as a histidine fusion protein and purified using Ni-NTA His bind resin. To test the defense capacity of enzymatic antioxidants against the effect of ROS, a mixed-function oxidation system was utilized with the recombinant BmTPx-1 protein. A decreased ability of rBmTPx-1 to donate electrons to the thioredoxin (Trx)/TrxR reductase system was clarified by reaction with H2O2. These results suggest that BmTPx-1 has a great impact on protecting parasites from oxidative stress in the erythrocytic stage.

Identification of a thioredoxin reductase from Babesia microti during mammalian infection.

Babesia microti is the primary causative agent of human babesiosis worldwide and associated with increased human health risks and the safety of blood supply. The parasite replicates in the host's red blood cells, thus, in order to counteract the oxidative stress and toxic effects, parasites employ a thioredoxin (Trx) system to maintain a redox balance. Since thioredoxin reductase (TrxR) plays a critical role in the system, in this study, we report the cloning, expression, and functional characterization of a novel TrxR from B. microti (BmiTrxR). The complete gene BmiTrxR was obtained by amplifying the 5' and 3' regions of messenger RNA (mRNA) by RACE. The full-length complementary DNA (cDNA) of BmiTrxR was 1766 bp and contained an intact open reading frame with 1662 bp that encoded a polypeptide with 553 amino acids. Molecular weight of the predicted protein was 58.4 kDa with an isoelectric point of 6.95, similar to high molecular weight TrxR. The recombinant protein of BmiTrxR was expressed in a His-fused soluble form in Escherichia coli. The native protein BmiTrxR was identified with the mouse anti-BmiTrxR polyclonal serum by western blotting and IFAT. Moreover, the enzyme showed a disulfide reductase activity using DTNB as substrate and catalyzed the NADPH-dependent reduction of Trx. Auranofin, a known inhibitor of TrxR, completely abrogated the activity of the recombinant enzyme in vitro. These results not only contribute to the understanding of redox pathway in this parasite but also suggest that BmiTrxR could be a potential target for the development of novel strategies to control B. microti thus reducing the incidence of babesiosis.

RNA interference and the vaccine effect of a subolesin homolog from the tick Rhipicephalus haemaphysaloides.

Subolesin is a well-characterized protective antigen in many ticks and, thus, it is potentially useful in the development of a broad-spectrum vaccine or an autocidal gene silencing strategy to control tick infestations. A subolesin homolog was cloned from the tick Rhipicephalus haemaphysaloides, which is widespread in China, by rapid amplification of complementary DNA (cDNA) ends. Its full-length cDNA was 1386 base pairs (bp), containing a 483 bp open reading frame with a predicted molecular mass of 18.7 kilodaltons and an isoelectric point of 9.26. The subolesin protein had a typical nuclear localization signal in its amino-terminus. The full-length cDNA of R. haemaphysaloides showed 52 and 80% identities to those from Ixodes scapularis and R. microplus, respectively, whereas amino acid sequence alignments showed 80 and 97% identities, respectively. Native subolesin was recognized in the unfed tick midgut by an antibody against recombinant subolesin. Transcriptional analysis showed that subolesin was expressed in the tick's four developmental stages and in all of the tissues examined, except for the synganglion. The pathogen Babesia microti induced the subolesin transcript by fourfold. Subolesin gene silencing by RNA interference significantly decreased the larval engorgement rate, the attachment rate and body weight of engorged nymphs, and the body weight and attachment and engorgement rates of adults, as well as the egg weight per female tick. Vaccinating mice and rabbits with recombinant subolesin induced a significant protective effect, resulting in a reduction of blood feeding and oviposition. These results encourage further studies of using subolesin to control tick infestations in China.

Functional analysis of a novel cysteine-rich antimicrobial peptide from the salivary glands of the tick Rhipicephalus haemaphysaloides.

Ticks encounter various microbes while sucking blood from an infected host and carrying these pathogens in themselves. Ticks can then transmit these pathogens to vertebrate hosts. The immune system of ticks can be stimulated to produce many bioactive molecules during feeding and pathogen invasion. Antimicrobial peptides (AMPs) are key effector molecules of a tick's immune response, as they can kill invading pathogenic microorganisms. In this study, we identified a novel cysteine-rich AMP, designated Rhamp1, in the salivary glands of unfed and fed female ticks (Rhipicephalus haemaphysaloides). Rhamp1 is encoded by a gene with an open reading frame of 333 bp, which in turn encodes a peptide of 12 kDa with a 22 amino acid residue signal peptide. The Rhamp1 protein had a pI of 8.6 and contained six conserved cysteine residues at the C-terminus. Rhamp1 shared 43% amino acid identity with a secreted cysteine-rich protein of another tick species, Ixodes scapularis. We cloned the Rhamp1 gene and attempted to express a recombinant protein using prokaryotic and eukaryotic systems, to determine its biological significance. Recombinant Rhamp1 was successfully expressed in both systems, yielding a glutathione S-transferase (GST)-tagged protein (36 kDa) from the prokaryotic system, and a polyhistidine-tagged Rhamp1 protein (14 kDa) from the eukaryotic system. Rhamp1 inhibited the activities of chymotrypsin (16%) and elastase (22%) and exerted low hemolytic activity. It also inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa (49%), Salmonella typhimurium (50%), and Escherichia coli (52%). Our findings suggest that Rhamp1 is a novel AMP in R. haemaphysaloides with the ability to inhibit proteinase activity.

Characterization of a secreted cystatin from the tick Rhipicephalus haemaphysaloides.

A novel cystatin, designated RHcyst-2, was isolated from the tick Rhipicephalus haemaphysaloides. The full-length cDNA of RHcyst-2 is 773 bp, including an intact open reading frame encoding an expected protein of 139 amino acids and consisting of a 23 amino acids signal peptide. Predicted RHcyst-2 mature protein molecular weight is about 13 kDa, isoelectric point is 4.96. A sequence analysis showed that it has significant homology with the known type 2 cystatins. The recombinant protein of RHcyst-2 was expressed in a glutathione S-transferase-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. The results showed that rRHcyst-2 can effectively inhibit the six cysteine proteases' enzyme activities. An investigation of the RHcyst-2 genes' expression profile by quantitative reverse transcription-PCR demonstrated that it was more richly transcribed in the embryo (egg) stage and mainly distributed in the mid-gut of adult ticks. Western blot analysis confirmed that RHcyst-2 was secreted into tick saliva.

Immunoglobulin G binding protein (IGBP) from Rhipicephalus haemaphysaloides: identification, expression, and binding specificity.

As the second most important human ectoparasite, ranked only after mosquitoes, the tick threatens the development of husbandry and even the health of humans worldwide. Immunoglobulin G binding proteins (IGBPs) are considered to be the major factors used by ticks to evade the host immune system and the damage caused by host antibodies. In this study, an IGBP-MB homologue was identified in the tick Rhipicephalus haemaphysaloides, which was predominantly detected in the salivary glands and hemolymph of male ticks. Recombinant IGBP (rIGBP/His) displayed significant binding activity to IgGs from rabbits and pigs, and bound to the F(ab)'2 but not the Fc fragment of rabbit IgG. Although the silencing of IGBP expression in ticks had no obvious effect on their blood-feeding and subsequent oviposition, antibodies raised to rIGBP/GST reduced the replete body weight (218.9 ± 20 mg in the control group vs. 142.5 ± 43.3 mg in the test group, P < 0.05 by Student's t test) and increased the mortality of the ticks. This study extends our understanding of the immunoevasive function of IGBPs and is a step towards the development of a vaccine against ticks.

The effect of obesity on the outcome of thoracic endovascular aortic repair: a systematic review and meta-analysis.

Background: Obesity is a well-known predictor for poor postoperative outcomes of vascular surgery. However, the association between obesity and outcomes of thoracic endovascular aortic repair (TEVAR) is still unclear. This systematic review and meta-analysis was performed to assess the roles of obesity in the outcomes of TEVAR. Methods: We systematically searched the Web of Science and PubMed databases to obtain articles regarding obesity and TEVAR that were published before July 2023. The odds ratio (OR) or hazard ratio (HR) was used to assess the effect of obesity on TEVAR outcomes. Body mass index (BMI) was also compared between patients experiencing adverse events after TEVAR and those not experiencing adverse events. The Newcastle-Ottawa Scale was used to evaluate the quality of the enrolled studies. Results: A total of 7,849 patients from 10 studies were included. All enrolled studies were high-quality. Overall, the risk of overall mortality (OR = 1.49, 95% CI [1.02-2.17], p = 0.04) was increased in obese patients receiving TEVAR. However, the associations between obesity and overall complications (OR = 2.41, 95% CI [0.84-6.93], p = 0.10) and specific complications were all insignificant, including stroke (OR = 1.39, 95% CI [0.56-3.45], p = 0.48), spinal ischemia (OR = 0.97, 95% CI [0.64-1.47], p = 0.89), neurological complications (OR = 0.13, 95% CI [0.01-2.37], p = 0.17), endoleaks (OR = 1.02, 95% CI [0.46-2.29], p = 0.96), wound complications (OR = 0.91, 95% CI [0.28-2.96], p = 0.88), and renal failure (OR = 2.98, 95% CI [0.92-9.69], p = 0.07). In addition, the patients who suffered from postoperative overall complications (p < 0.001) and acute kidney injury (p = 0.006) were found to have a higher BMI. In conclusion, obesity is closely associated with higher risk of mortality after TEVAR. However, TEVAR may still be suitable for obese patients. Physicians should pay more attention to the perioperative management of obese patients.

Towards modelling tick-virus interactions using the weakly pathogenic Sindbis virus: Evidence that ticks are competent vectors.

Most tick-borne viruses (TBVs) are highly pathogenic and require high biosecurity, which severely limits their study. We found that Sindbis virus (SINV), predominantly transmitted by mosquitoes, can replicate in ticks and be subsequently transmitted, with the potential to serve as a model for studying tick-virus interactions. We found that both larval and nymphal stages of Rhipicephalus haemaphysaloides can be infected with SINV-wild-type (WT) when feeding on infected mice. SINV replicated in two species of ticks (R. haemaphysaloides and Hyalomma asiaticum) after infecting them by microinjection. Injection of ticks with SINV expressing enhanced Green Fluorescent Protein (eGFP) revealed that SINV-eGFP specifically aggregated in the tick midguts for replication. During blood-feeding, SINV-eGFP migrated from the midguts to the salivary glands and was transmitted to a new host. SINV infection caused changes in expression levels of tick genes related to immune responses, substance transport and metabolism, cell growth and death. SINV mainly induced autophagy during the early stage of infection; with increasing time of infection, the level of autophagy decreased, while the level of apoptosis increased. During the early stages of infection, the transcript levels of immune-related genes were significantly upregulated, and then decreased. In addition, SINV induced changes in the transcription levels of some functional genes that play important roles in the interactions between ticks and tick-borne pathogens. These results confirm that the SINV-based transmission model between ticks, viruses, and mammals can be widely used to unravel the interactions between ticks and viruses.

Sesquiterpenoids from aged Artemisia argyi and their 3D-QSAR for anti-HBV activity.

Artemisia argyi Levl. Et Vant, commonly known as "Chinese Mugwort," has been utilized in traditional Chinese medicine and cuisine for centuries. Aged Chinese Mugwort has been uncovered to possess superior quality and safety, and its ethyl acetate extract has been found to exhibit anti-hepatitis B virus (HBV) activity. In this study, twenty-five sesquiterpenoids were isolated and characterized from three-year-aged A. argyi. Among them, 14 previously undescribed sesquiterpenoids (1-14), featuring double bond oxidation or ring opening. It is hypothesized that during the aging process, sesquiterpenes undergo oxidative transformation of their double bonds to form alcohols due to external factors and inherent properties. The anti-HBV activity and cytotoxicity of all compounds were assessed in vitro using HepG 2.2.15 cells, and their structure-activity relationships were analyzed through three-dimensional quantitative structure-activity relationship (3D-QASR) techniques. The α-methylene-γ-lactone sesquiterpenoid derivatives were discovered to have potent inhibitory activity against HBV. This research may broaden the potential applications of Chinese Mugwort and offer further guidance for its development and utilization as functional food or traditional Chinese medicine.

Recent Advances in Microfluidics for the Early Detection of Plant Diseases in Vegetables, Fruits, and Grains Caused by Bacteria, Fungi, and Viruses.

In the context of global population growth expected in the future, enhancing the agri-food yield is crucial. Plant diseases significantly impact crop production and food security. Modern microfluidics offers a compact and convenient approach for detecting these defects. Although this field is still in its infancy and few comprehensive reviews have explored this topic, practical research has great potential. This paper reviews the principles, materials, and applications of microfluidic technology for detecting plant diseases caused by various pathogens. Its performance in realizing the separation, enrichment, and detection of different pathogens is discussed in depth to shed light on its prospects. With its versatile design, microfluidics has been developed for rapid, sensitive, and low-cost monitoring of plant diseases. Incorporating modules for separation, preconcentration, amplification, and detection enables the early detection of trace amounts of pathogens, enhancing crop security. Coupling with imaging systems, smart and digital devices are increasingly being reported as advanced solutions.

A comprehensive analysis of m6A/m7G/m5C/m1A-related gene expression and immune infiltration in liver ischemia-reperfusion injury by integrating bioinformatics and machine learning algorithms.

BACKGROUND: Liver ischemia-reperfusion injury (LIRI) is closely associated with immune infiltration, which commonly occurs after liver surgery, especially liver transplantation. Therefore, it is crucial to identify the genes responsible for LIRI and develop effective therapeutic strategies that target immune response. Methylation modifications in mRNA play various crucial roles in different diseases. This study aimed to identify potential methylation-related markers in patients with LIRI and evaluate the corresponding immune infiltration. METHODS: Two Gene Expression Omnibus datasets containing human liver transplantation data (GSE12720 and GSE151648) were downloaded for integrated analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to investigate the functional enrichment of differentially expressed genes (DEGs). Differentially expressed methylation-related genes (DEMRGs) were identified by overlapping DEG sets and 65 genes related to N6-methyladenosine (m6A), 7-methylguanine (m7G), 5-methylcytosine (m5C), and N1-methyladenosine (m1A). To evaluate the relationship between DEMRGs, a protein-protein interaction (PPI) network was utilized. The core DEMRGs were screened using three machine learning algorithms: least absolute shrinkage and selection operator, random forest, and support vector machine-recursive feature elimination. After verifying the diagnostic efficacy using the receiver operating characteristic curve, we validated the expression of the core DEMRGs in clinical samples and performed relative cell biology experiments. Additionally, the immune status of LIRI was comprehensively assessed using the single sample gene set enrichment analysis algorithm. The upstream microRNA and transcription factors of the core DEMRGs were also predicted. RESULTS: In total, 2165 upregulated and 3191 downregulated DEGs were identified, mainly enriched in LIRI-related pathways. The intersection of DEGs and methylation-related genes yielded 28 DEMRGs, showing high interaction in the PPI network. Additionally, the core DEMRGs YTHDC1, METTL3, WTAP, and NUDT3 demonstrated satisfactory diagnostic efficacy and significant differential expression and corresponding function based on cell biology experiments. Furthermore, immune infiltration analyses indicated that several immune cells correlated with all core DEMRGs in the LIRI process to varying extents. CONCLUSIONS: We identified core DEMRGs (YTHDC1, METTL3, WTAP, and NUDT3) associated with immune infiltration in LIRI through bioinformatics and validated them experimentally. This study may provide potential methylation-related gene targets for LIRI immunotherapy.