Absence of association between Pfnfs1 mutation and in vitro susceptibility to lumefantrine in Plasmodium falciparum.

Artemether-lumefantrine (AL) is the most widely used antimalarial drug for treating uncomplicated falciparum malaria. This study evaluated whether the K65Q mutation in the Plasmodium falciparum cysteine desulfurase IscS (Pfnfs1) gene was associated with alternated susceptibility to lumefantrine using clinical parasite samples from Ghana and the China-Myanmar border area. Parasite isolates from the China-Myanmar border had significantly higher IC50 values to lumefantrine than parasites from Ghana. In addition, the K65 allele was significantly more prevalent in the Ghanaian parasites (34.5%) than in the China-Myanmar border samples (6.8%). However, no difference was observed in the lumefantrine IC50 value between the Pfnfs1 reference K65 allele and the non reference 65Q allele in parasites from the two regions. These data suggest that the Pfnfs1 K65Q mutation may not be a reliable marker for reduced susceptibility to lumefantrine.

A Versatile Approach for the Synthesis of Antimicrobial Polymer Brushes on Natural Rubber/Graphene Oxide Composite Films via Surface-Initiated Atom-Transfer Radical Polymerization.

A simple strategy was adopted for the preparation of an antimicrobial natural rubber/graphene oxide (NR/GO) composite film modified through the use of zwitterionic polymer brushes. An NR/GO composite film with antibacterial properties was prepared using a water-based solution-casting method. The composited GO was dispersed uniformly in the NR matrix and compensated for mechanical loss in the process of modification. Based on the high bromination activity of α-H in the structure of cis-polyisoprene, the composite films were brominated on the surface through the use of N-bromosuccinimide (NBS) under the irradiation of a 40 W tungsten lamp. Polymerization was carried out on the brominated films using sulfobetaine methacrylate (SBMA) as a monomer via surface-initiated atom transfer radical polymerization (SI-ATRP). The NR/GO composite films modified using polymer brushes (PSBMAs) exhibited 99.99% antimicrobial activity for resistance to Escherichia coli and Staphylococcus aureus. A novel polymer modification strategy for NR composite materials was established effectively, and the enhanced antimicrobial properties expand the application prospects in the medical field.

Grafting photochromic spiropyran polymer brushes on graphene oxide surfaces via surface-initiated ring-opening metathesis polymerization.

A practical "grafting-from" strategy is described to grow photochromic polymer brushes bearing spiropyran (SP) functional groups on graphene oxide (GO) surfaces via surface-initiated ring-opening metathesis polymerization (SI-ROMP). The Grubbs II catalyst was fixed on the GO surface, and the norbornene derivatives functionalized using spiropyran were synthesized from this active site via the ROMP method. The results indicated that the spiropyran-modified polymer brushes were obtained on the GO surface in the form of thin films. The solubility of GO modified by spiropyran polymers (GO-SPs) in organic solvents was significantly improved. The GO-SPs exhibited excellent photochromic properties, including fast coloration/decoloration. The modified GO with an isomeric structure was colored in 90 s under ultraviolet irradiation and decolored in 360 s under white light. The fading kinetic rate in the dark was slow and the kinetic attenuation curve followed bi-exponential decay. The GO-SP composite materials took more than 2 h to return to thermodynamically stable forms. The reversible change in the water contact angle reached 8° after continuous cycling with ultraviolet and visible light. GO-SP maintained its photochromic performance and possessed excellent fatigue resistance after more than six successive UV/light cycles. This work describes a practical strategy for the preparation of photochromic polymer brush modified GO composite materials and extends the applications of GO in photochromic materials.

Alterations of ileal mucosa-associated microbiota in hypercholesterolemia patients.

Many metabolic diseases have been demonstrated to be associated with changes in the microbiome. However, no studies have yet been conducted to examine the characteristics of the mucosal microbiota of patients with hypercholesterolemia. We aimed to examine mucosa-associated microbiota in subjects with hypercholesterolemia. We conducted a case-control study, in which ileal mucosal samples were collected from 13 hypercholesterolemia patients and 13 controls for 16S rRNA gene sequencing. There were differences in the composition of ileal mucosal microbiota based on beta diversity between the hypercholesterolemia and control groups (P < 0.05). Compared with the control group, the phylum Bacteroidetes and the genera Bacteroides, Butyricicoccus, Parasutterella, Candidatus_Soleaferrea, and norank_f__norank_o__Izemoplasmatales were less abundant in the hypercholesterolemia group (P < 0.05), while the genus Anaerovibrio was enriched in the hypercholesterolemia group (P < 0.05). The relative abundance of Bacteroides was negatively correlated with total cholesterol and low-density lipoprotein cholesterol (P < 0.01). The relative abundance of Coprococcus was negatively correlated with triglycerides and body mass index (all P < 0.05). PICRUSt functional prediction analysis showed that pathways related to Glycerophospholipid metabolism, ABC transporters, Phosphotransferase system, and Biofilm formation - Escherichia coli, and infectious diseases of pathogenic Escherichia coli were enriched in the hypercholesterolemia group. This work suggests a potential role of ileal mucosal microbiota in the development of hypercholesterolemia.

Discovery of piperonyl-tethered sulfoximines as novel low bee-toxicity aphicides targeting Amelα1/ratß2 complex.

Nicotinic acetylcholine receptor (nAChR) is recognized as a significant insecticide target for neonicotinoids and some agonists. In this study, the nAChR α1 subunit from Apis mellifera was first found to be narrowly tuned to different bee toxicity insecticides, namely, sulfoxaflor (SFX) and flupyradifurone (FPF). Hence, novel sulfoximine derivatives 7a-h were rationally designed and synthesized by introducing a benzo[d][1,3]dioxole moiety into a unique sulfoximine skeleton based on the binding cavity characteristics of Amelα1/ratß2. The two electrode voltage clamp responses of 7a-h were obviously lower than that of SFX, indicating their potentially low bee toxicity. Besides, representative compounds 7b and 7g exhibited low bee toxicity (LD50 > 11.0 µg/bee at 48 h) revealed by acute contact toxicity bioassays. Molecular modelling results indicated that Ile152, Ala151, and Val160 from honeybee subunit Amelα1 and Lys144 and Trp80 from aphid subunit Mpα1 may be crucial for bee toxicity and aphicidal activity, respectively. These results clarify the toxic mechanism of agonist insecticides on nontargeted pollinators and reveal novel scaffold sulfoximine aphicidal candidates with low bee toxicity. These results will provide a new perspective on the rational design and highly effective development of novel eco-friendly insecticides based on the structure of the nAChR subunit.

Study of a Multiple Responses, High Deformation, and Programmable PLA-PPC/PVA-PDA Actuator.

The intelligent response actuators based on bilayer polymer can deform under the stimulation of temperature, humidity, light, and other external environment, which is the focus of research. However, achieving multiple responses, high deformation, and programmability is still one of the challenges for these actuators. Herein, a nondetachable bilayer structure, polylactic acid-polypropylene carbonate/polyvinyl alcohol-polydopamine (PLA-PPC/PVA-PDA) multiresponse programmable actuator is prepared by a simple scraping film method. Using PLA-PPC as the solvent-driven response layer, the effects of length, thickness, shape, and solvent vapor on the deformation of PLA-PPC/PVA-PDA actuators are studied. Among them, the high curvature of the film stimulated by ethyl acetate (EA) solution is 29.85 cm-1 . Using PVA-PDA as the response layer to water molecules and infrared (IR) light, the bilayer film shows excellent curling performance. Moreover, the dynamic processes of human clothing and biomimetic squid under solvent stimulation, the picture rolling motion under water molecule stimulation, the biomimetic flower blooming and merging under the synergistic of water molecules and IR light, and the deformation process of biomimetic mimosa under the competition between water molecules and IR light are simulated, which broadens the road for the development of intelligent driving materials.

First report of Pseudopithomyces chartarum causing leaf spot on Tetrapanax papyrifer in Guizhou, China.

Tetrapanax papyrifer (Hook.) K. Koch, widely utilized in clinical practices in traditional Chinese medicine, is a medicinal plant whose dried stem pich exhibits activities such as lactation induction, diuresis, and anti-inflammatory effects. The species is native to the southwest of China, such as Guizhou and Yunnan provinces. It thrives in sunlight and warmth and is planted in fertile valleys in the region (Zhang et al. 2023). In July 2021, a leaf spot-like disease was observed on approximately 15% of T. papyrifer (Big T. papyrifer) in a field in Shibing County (127.2°E, 25.2°N), Guizhou Province, China. The symptomatic leaves displayed irregular, watery dark brown lesions with black conidiomata in gray centers and surrounded by yellow halos. To identify the causal agent leading to the disease, 15 symptomatic leaves from five trees in one field were collected. These leaves underwent surface sterilization, which included 30s in 75% ethanol, 2 min in 3% NaOCl, and three times of washing with sterilized distilled water. Thereafter, small pieces of the symptomatic leaf tissues (0.2 × 0.2 cm) were plated on PDA and incubated at 25°C for seven days (Fang 2007). Three isolates were obtained based on the improved single spore isolation method proposed by Gong et al. (2010), and named as GUTC 321, GUTC 523 and GUTC 873. The fungal colonies on PDA were villiform, creamy-white, whorled, and sparse aerial mycelium on the surface with black, gregarious conidiomata. The conidia were ellipsoid, mid brown to dark brown, mainly with 3-4 transverse septa, usually divided by longitudinal septum, often constricted at the septa, 21.8 (12.6-34.5) × 13.9 (8.8-19.8) µm (n=50). The morphological features were consistent with the descriptions of Pseudopithomyces chartarum (Ariyawansa et al. 2015). All three isolates exhibited identical morphological properties. The potential pathogen was confirmed as P. chartarum by amplification and sequencing of the internal transcribed spacer regions (ITS), large subunit ribosomal (LSU) and translation elongation factor 1 alpha (TEF1) genes with primers ITS4/ITS5, LROR/LR7 and EF-983F/EF-2218R, respectively (Ariyawansa et al. 2015; Jayasiriet al. 2019). BLASTn analyses of the sequences showed 100% identity among the three isolates and a high homology (ITS, 99.8%: 598/599; LSU, 100%: 853/853; and TEF1, 100%: 871/871) with those of P. chartarum sequences in GenBank (MT123059, OK655822, and MK360080, respectively). The sequences of the genes from isolate GUTC321 were deposited in GenBank under accession numbers OP269599 (ITS), OP237015 (LSU), and OR069689 (TEF1). Phylogenetic analyses of the concatenated ITS-LSU-TEF1 sequence (2,685 bp) of GUTC 321 using PhyloSuite 1.2.2 with PartitionFinder model revealed that the isolate clustered closely with P. chartarum isolate CBS 329.86T (Cecilia 1986). The pathogenicity of GUTC 321 was tested thereafter on ten healthy T. papyrifer plants grown in pots in growth chamber. The plants were inoculated by spraying with spore suspension (106 spores mL-1) of GUTC 321 or sterile water (control) onto leaves that had been slightly injured with sterilized SiO2 (0.1-0.25 mm) until runoff. The plants were maintained at 25°C in the growth chamber, and monitored for symptom development. Local lesions began to appear on all GUTC 321-inoculated leaves, but not on those of the control plants, 48 hours after inoculation. Seven days after the inoculation, lesions similar to those observed on field plants occurred on GUTC321-inoculated plants but not on the control plants, the lesions observed only in inoculated leaves. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses (ITS, LSU and TEF1) from the infected leaves thus fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on T. papyrifer caused by P. chartarum in China. Considering the significance of T. papyrifer in Chinese medicine, approximate management measures need to be developed and applied to control the disease in the crop.

First Report of Grey Spot on Tobacco caused by Alternaria alstroemeriae in China.

Tobacco (Nicotiana tabacum L.) is an important economic crop belonging to family Solanaceae and is widely cultivated in China (Basit 2021). From April to July in 2022, a foliar disease with symptoms similar to grey spot was extensively observed on tobacco in Guangxi Province (24°52' N, 111°23' E), China. Field surveys were conducted in 18 towns and the disease incidence was 0.89% to 6.95%. Symptomatic leaves displayed irregular, dark brown lesions surrounded by yellow halos and accompanied with black conidiomata in gray centers (Fig 1A-E). Symptomatic leaves were collected from 54 different tobacco plants. After surface sterilization (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on PDA and incubated at 25°C for 5 days (Fang 2007). Three single-spore isolates, GUCC BZ6-3, GUCC LJ3-4, and GUCC XH1-13 were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GUCC BZ6-3 was used for further study. The colonies on PDA were villiform, greyish (Fig 1F-G). Conidia were abundant, ovoid, with 2-6 transverse septa and 1-2 longitudinal septa 12.60 (9.43 to 14.76) × 4.30 (3.57 to 5.14) µm (n=50) (Fig 1H-S). The morphological features were consistent with Alternaria alstroemeriae E.G. Simmons & C.F. Hill (Simmons 2007; Nishikawa & Nakashima, 2013). The pathogen was confirmed to be A. alstroemeriae by amplification and sequencing of the ITS, GAPDH, LSU, TEF1, and RBP2 genes using primers ITS1/ITS4, gpd1/gpd2, LSU1Fd/LR5, EF1-728F/EF1-986R, and RPB2-5F2/fRPB2-7cR, respectively (Woudenberg 2013). The sequences of the PCR products were deposited in GenBank with accession numbers ON693856 (RBP2), ON714497 (ITS), ON694345 (GAPDH), ON931420 (TEF1) and ON714499 (LSU). BLAST searches of the obtained sequences revealed 99% (565/567 nucleotides), 99% (577/579 nucleotides), 99% (908/911 nucleotides), 99% (238/239 nucleotides), and 99% (751/753 nucleotides) homology with those of A. alstroemeriae in GenBank (MH863036, KP124154, MH874589, KP125072, and KP124765, respectively). Phylogenetic analyses of the sequence data consisted of Bayesian and Maximum likelihood analyses of the combined aligned dataset (MEGA 7.0 and PhyloSuite 1.2.2). The GUCC BZ6-3 in a well-supported cluster with A. alstroemeriae (Fig 2). The pathogen was thus identified as A. alstroemeriae based on morphological characterization and molecular analyses. The pathogenicity of GUCC BZ6-3 was tested through pot assay and carried out three times (Fang 2007). Ten healthy 30-day-old tobacco plants were inoculated by spraying a spore suspension (106 spores·ml-1) of strain GUCC BZ6-3 onto leaves until runoff, and the control leaves were sprayed with sterile water. The plants were maintained at 28°C with high relative humidity (95%) in a growth chamber. The symptoms developed on all inoculated leaves but not on the control. The lesions were first visible 48 h after inoculation, and typical lesions similar to those observed on field plants appeared after 7 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the noninoculated leaves. Results of pathogenicity experiments fulfilled Koch's postulates. To our knowledge, this is the first report of grey spot disease on tobacco caused by A. alstroemeriae in China. Our findings would be of great importance for the diagnosis and control of the emerging grey spot on tobacco.

Transcription factor 21 accelerates vascular calcification in mice by activating the IL-6/STAT3 signaling pathway and the interplay between VSMCs and ECs.

Vascular calcification is caused by the deposition of calcium salts in the intimal or tunica media layer of the aorta, which increases the risk of cardiovascular events and all-cause mortality. However, the mechanisms underlying vascular calcification are not fully clarified. Recently it has been shown that transcription factor 21 (TCF21) is highly expressed in human and mouse atherosclerotic plaques. In this study we investigated the role of TCF21 in vascular calcification and the underlying mechanisms. In carotid artery atherosclerotic plaques collected from 6 patients, we found that TCF21 expression was upregulated in calcific areas. We further demonstrated TCF21 expression was increased in an in vitro vascular smooth muscle cell (VSMC) osteogenesis model. TCF21 overexpression promoted osteogenic differentiation of VSMC, whereas TCF21 knockdown in VSMC attenuated the calcification. Similar results were observed in ex vivo mouse thoracic aorta rings. Previous reports showed that TCF21 bound to myocardin (MYOCD) to inhibit the transcriptional activity of serum response factor (SRF)-MYOCD complex. We found that SRF overexpression significantly attenuated TCF21-induced VSMC and aortic ring calcification. Overexpression of SRF, but not MYOCD, reversed TCF21-inhibited expression of contractile genes SMA and SM22. More importantly, under high inorganic phosphate (3 mM) condition, SRF overexpression reduced TCF21-induced expression of calcification-related genes (BMP2 and RUNX2) as well as vascular calcification. Moreover, TCF21 overexpression enhanced IL-6 expression and downstream STAT3 activation to facilitate vascular calcification. Both LPS and STAT3 could induce TCF21 expression, suggesting that the inflammation and TCF21 might form a positive feedback loop to amplify the activation of IL-6/STAT3 signaling pathway. On the other hand, TCF21 induced production of inflammatory cytokines IL-1ß and IL-6 in endothelial cells (ECs) to promote VSMC osteogenesis. In EC-specific TCF21 knockout (TCF21ECKO) mice, VD3 and nicotine-induced vascular calcification was significantly reduced. Our results suggest that TCF21 aggravates vascular calcification by activating IL-6/STAT3 signaling and interplay between VSMC and EC, which provides new insights into the pathogenesis of vascular calcification. TCF21 enhances vascular calcification by activating the IL-6-STAT3 signaling pathway. TCF21 inhibition may be a new potential therapeutic strategy for the prevention and treatment of vascular calcification.

HIF-1α-Overexpressing Mesenchymal Stem Cells Attenuate Colitis by Regulating M1-like Macrophages Polarization toward M2-like Macrophages.

A modified mesenchymal stem cell (MSC) transplantation is a highly effective and precise treatment for inflammatory bowel disease (IBD), with a significant curative effect. Thus, we aim to examine the efficacy of hypoxia-inducible factor (HIF)-1α-overexpressing MSC (HIF-MSC) transplantation in experimental colitis and investigate the immunity regulation mechanisms of HIF-MSC through macrophages. A chronic experimental colitis mouse model was established using 2,4,6-trinitrobenzene sulfonic acid. HIF-MSC transplantation significantly attenuated colitis in weight loss rate, disease activity index (DAI), colon length, and pathology score and effectively rebuilt the local and systemic immune balance. Macrophage depletion significantly impaired the benefits of HIF-MSCs on mice with colitis. Immunofluorescence analysis revealed that HIF-MSCs significantly decreased the number of M1-like macrophages and increased the number of M2-like macrophages in colon tissues. In vitro, co-culturing with HIF-MSCs significantly decreased the expression of pro-inflammatory factors, C-C chemokine receptor 7 (CCR-7), and inducible nitric oxide synthase (INOS) and increased the expression of anti-inflammatory factors and arginase I (Arg-1) in induced M1-like macrophages. Flow cytometry revealed that co-culturing with HIF-MSCs led to a decrease in the proportions of M1-like macrophages and an increase in that of M2-like macrophages. HIF-MSCs treatment notably upregulated the expression of downstream molecular targets of phosphatidylinositol 3-kinase-γ (PI3K-γ), including HIF-1α and p-AKT/AKT in the colon tissue. A selected PI3K-γ inhibitor, IPI549, attenuated these effects, as well as the effect on M2-like macrophage polarization and inflammatory cytokines in colitis mice. In vitro, HIF-MSCs notably upregulated the expression of C/EBPß and AKT1/AKT2, and PI3K-γ inhibition blocked this effect. Modified MSCs stably overexpressed HIF-1α, which effectively regulated macrophage polarization through PI3K-γ. HIF-MSC transplantation may be a potentially effective precision therapy for IBD.

[Genetic analysis of a Chinese pedigree affected with Mucopolysaccharidosis type â ¢A].

OBJECTIVE: To explore the clinical and genetic characteristics of a patient with hypertrophic cardiomyopathy as the initial manifestation of Mucopolysaccharidosis type Ⅲ A (MPS Ⅲ A). METHODS: A female patient with MPS Ⅲ A who was admitted to the Affiliated Hospital of Jining Medical University in January 2022 and her family members (seven individuals from three generations) were selected as the study subjects. Clinical data of the proband were collected. Peripheral blood samples of the proband was collected and subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing. Heparan-N-sulfatase activity was determined for the disease associated with the variant site. RESULTS: The proband was a 49-year-old woman, for whom cardiac MRI has revealed significant thickening (up to 20 mm) of left ventricular wall and delayed gadolinium enhancement at the apical myocardium. Genetic testing revealed that she has harbored compound heterozygous variants in exon 17 of the SGSH gene, namely c.545G>A (p.Arg182His) and c.703G>A (p.Asp235Asn). Based on guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were predicted to be pathogenic (PM2_Supporting +PM3+PP1Strong+PP3+PP4; PS3+PM1+PM2_Supporting +PM3+PP3+PP4). Sanger sequencing confirmed that her mother was heterozygous for the c.545G>A (p.Arg182His) variant, whilst her father, sisters and her son were heterozygous for the c.703G>A (p.Asp235Asn) variant. Determination of blood leukocyte heparan-N-sulfatase activity suggested that the patient had a low level of 1.6 nmol/(g·h), whilst that of her father, elder and younger sisters and son were all in the normal range. CONCLUSION: The compound heterozygous variants of the SGSH gene probably underlay the MPS ⅢA in this patient, for which hypertrophic cardiomyopathy is an associated phenotype.

First Report of Anthracnose on Tetrapanax papyriferus Caused by Colletotrichum fructicola in China.

Tetrapanax papyriferus is an evergreen shrub native to China and traditionally used as a herbal medicine (Li et al., 2021). In September 2021, a serious leaf spot disease with symptoms similar to anthracnose was extensively observed on T. papyriferus in Shibing county (E 127°12'0", N 25°11'60"), Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou province, China. Field surveys were conducted in about 1000 T. papyriferus plants in Shibing in September 2021. The incidence of the leaf spot on leaves was 45% to 60%, significantly reducing the quality of medicinal materials. The symptoms began as small yellow spots, developing a brown center and dark brown to black margin, and eventually the diseased leaves were wiltered and rotted. Symptomatic leaves were collected from 20 trees. Symptomatic tissue from diseased leaves was surface desinfected (0.5 min in 75% ethanol and 1 min in 3% NaOCl, washed three times with sterilized distilled water), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days (Fang. 2007). Three single-spore isolates were obtained (GUTC37, GUTC310 and GUTC311) and deposited in the collection of the Plant Pathology Deparment, College of Agriculture, Guizhou University, China (GUCC) (with the accession numbers, GUCC220241, GUCC220242, GUCC220243 respectively). These isolates were identical in morphology and in the sequences of internal transcribed spacer region [ITS], glyceraldehy-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], actin [ACT], and calmodulin [CAL] genes (White et al. 1990; Carbone and Kohn 1999; Templeton et al. 1992). Therefore, the representative isolate GUTC37 was used for further analysis. The pathogenicity of GUTC37 was tested through a pot assay. Plants were inoculated by spraying a spore suspension (106 spores·ml-1) of isolated strains onto leaves until runoff, and the control leaves sprayed with sterile water. The inoculated plants were incubated in a growth chamber at 28 ℃ and 95% relative humidity for 10 days. Pathogenicity tests were repeated three times (Fang. 2007). The symptoms developed on the inoculated leaves, while control remained asymptomatic. The lesions were first visible 72 h after inoculation, and typical lesions like those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses from the infected leaves but not from the non-inoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white or greyish, aerial mycelium pale grey, dense, surface partly covered with orange conidial masses. The conidia were abundant, oval-ellipsoid, aseptate, and 13.89 (11.62 to 15.21) × 5.21 (4.39 to 5.65) µm (n=50). Appressorium were greyish green, nearly ovoid to cylindrical, 9.64 (6.62 to 14.61) × 6.33 (5.45-7.72) µm (n=50). The morphological features were consistent with the descriptions of Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Prihastuti et al. 2009). The pathogen was identified to be C. fructicola by amplification and sequencing of the five genes. The sequences of the PCR products were deposited in GenBank with accession numbers OP143657 (ITS), OP177868 (GAPDH), OP177865 (CHS-1), OP278677 (ACT) and OP177862 (CAL). BLAST searches of the obtained sequences revealed 100% (509/509 nucleotides), 99.63% (269/270 nucleotides), 99.31% (287/289 nucleotides), 99.29% (280/282 nucleotides), and 99.86% (728/729 nucleotides) homology with those of C. fructicola in GenBank (JX010165, JX010033, JX009866, FJ907426, and JX009676, respectively). Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUTC37 in a well-supported cluster with C. fructicola. To our knowledge, this is the first report of anthracnose on T. papyriferus caused by C. fructicola in Guizhou, China. This study provides valuable information for the identification and control of the anthracnose on T. papyriferus.

Genetic diversity of Plasmodium vivax populations from the China-Myanmar border identified by genotyping merozoite surface protein markers.

BACKGROUND: Parasite diversity and population structure influence malaria control measures. Malaria transmission at international borders affects indigenous residents and migrants, defying management efforts and resulting in malaria re-introduction. Here we aimed to determine the extent and distribution of genetic variations in Plasmodium vivax populations and the complexity of infections along the China-Myanmar border. METHODS: We collected clinical P. vivax samples from local and migrant malaria patients from Laiza and Myitsone, Kachin State, Myanmar, respectively. We characterized the polymorphisms in two P. vivax merozoite surface protein markers, Pvmsp-3α and Pvmsp-3ß, by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. We sought to determine whether these genetic markers could differentiate these two neighboring parasite populations. RESULTS: PCR revealed three major size variants for Pvmsp-3α and four for Pvmsp-3ß among the 370 and 378 samples, respectively. PCR-RFLP resolved 26 fragment-size alleles by digesting Pvmsp-3α with Alu I and Hha I and 28 alleles by digesting Pvmsp-3ß with Pst I. PCR-RFLP analysis of Pvmsp-3α found that infections in migrant laborers from Myitsone bore more alleles than did infections in residents of Laiza, while such difference was not evident from genotyping Pvmsp-3ß. Infections originating from these two places contained distinct but overlapping subpopulations of P. vivax. Infections from Myitsone had a higher multiplicity of infection as judged by the size of the Pvmsp-3α amplicons and alleles after Alu I/Hha I digestion. CONCLUSIONS: Migrant laborers from Myitsone and indigenous residents from Laiza harbored overlapping but genetically distinct P. vivax parasite populations. The results suggested a more diverse P. vivax population in Myitsone than in the border town of Laiza. PCR-RFLP of Pvmsp-3α offers a convenient method to determine the complexity of P. vivax infections and differentiate parasite populations.

Different In Vitro Drug Susceptibility Profile of Plasmodium falciparum Isolates from Two Adjacent Areas of Northeast Myanmar and Molecular Markers for Drug Resistance.

The Greater Mekong Subregion (GMS) is the epicenter of antimalarial drug resistance. We determined in vitro susceptibilities to 11 drugs of culture-adapted Plasmodium falciparum isolates from adjacent areas (Laiza and Muse) along the China−Myanmar border. Parasites from this region were highly resistant to chloroquine and pyrimethamine but relatively sensitive to other antimalarial drugs. Consistently, the Dd2-like pfcrt mutations were fixed or almost fixed in both parasite populations, and new mutations mediating piperaquine resistance were not identified. Similarly, several mutations related to pfdhfr and pfdhps were also highly prevalent. Despite their geographical proximity, malaria parasites from Laiza showed significantly higher in vitro resistance to artemisinin derivatives, naphthoquine, pyronaridine, lumefantrine, and pyrimethamine than parasites from Muse. Likewise, the pfdhfr N51I, pfdhps A581G, pfmrp1 H785N, and pfk13 F446I mutations were significantly more frequent in Laiza than in Muse (p < 0.05). For the pfmdr1 mutations, Y184F was found only in Laiza (70%), whereas F1226Y was identified only in Muse (31.8%). Parasite isolates from Laiza showed a median RSA value of 5.0%, significantly higher than the 2.4% in Muse. Altogether, P. falciparum parasite populations from neighboring regions in the GMS may diverge substantially in their resistance to several antimalarial drugs. This information about different parasite populations will guide antimalarial treatment policies to effectively manage drug resistance during malaria elimination.

Application of a low-cost, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay to detect Plasmodium falciparum imported from Africa.

BACKGROUND: Chinese citizens traveling abroad bring back imported malaria cases to China. Current malaria diagnostic tests, including microscopy and antigen-detecting rapid tests, cannot reliably detect low-density infections. To complement existing diagnostic methods, we aimed to develop a new loop-mediated isothermal amplification (LAMP) assay to detect and identify Plasmodium falciparum in Chinese travelers returning from Africa. METHODS: We developed a miniaturized LAMP assay to amplify the actin I gene of P. falciparum. Each reaction consumed only 25% of the reagents used in a conventional LAMP assay and the same amount of DNA templates used in nested PCR. We evaluated this LAMP assay's performance and compared it to microscopy and a nested PCR assay using 466 suspected malaria cases imported from Africa. We assessed the sensitivity of the new LAMP assay using cultured P. falciparum, clinical samples, and a plasmid construct, allowing unprecedented precision when quantifying the limit of detection. RESULTS: The new LAMP assay was highly sensitive and detected two more malaria cases than nested PCR. Compared to nested PCR, the sensitivity and specificity of the novel LAMP assay were 100% [95% confidence interval (CI) 98.5-100%] and 99.1% (95% CI 96.7-99.9%), respectively. When evaluated using serial dilutions of the plasmid construct, the detection limit of the new LAMP was as low as 102 copies/µL, 10-fold lower than PCR. The LAMP assay detected 0.01 parasites/µL of blood (equal to 0.04 parasites/µL of DNA) using cultured P. falciparum and 1-7 parasites/µL of blood (4-28 parasites/µL of DNA) in clinical samples, which is as good as or better than previously reported and commercially licensed assays. CONCLUSION: The novel LAMP assay based on the P. falciparum actin I gene was specific, sensitive, and cost-effective, as it consumes 1/4 of the reagents in a typical LAMP reaction.

PvMSP-3α and PvMSP-3ß genotyping reveals higher genetic diversity in Plasmodium vivax parasites from migrant workers than residents at the China-Myanmar border.

BACKGROUND: The genetic diversity of malaria parasites traces the origin and spread of new variants and can be used to evaluate the effectiveness of malaria control measures. Therefore, this study aims to improve the understanding of the molecular epidemiology of Plasmodium vivax malaria at the China-Myanmar border by genotyping the PvMSP-3α and PvMSP-3ß genes. METHODS: Blood samples were collected from P. vivax malaria patients along the China-Myanmar border. The PvMSP-3α and PvMSP-3ß genes were amplified by polymerase chain reaction (PCR) and the genetic polymorphism and haplotype of the two genes were analyzed. RESULTS: A total of 422 blood samples were used for this study, of which 224 were analyzed at PvMSP-3α and 126 at PvMSP-3ß. Samples mainly were from young adults aged 18-45 years, although local patients were significantly younger than migrant laborers crossing the border at Tengchong (P < 0.0001). Molecular evolutionary analysis revealed that PvMSP-3α and PvMSP-3ß underwent diversifying natural selection, and intragenic recombination contributed to the diversity of the isolates. Based on the length of the genes, we identified three types of PvMSP-3α [1.9-2.0 kb (Type-A), 1.4-1.5 kb (Type-B), and 1.1-1.3 kb (Type-C)] and two types of PvMSP-3ß [1.7-2.2 kb (Type-A) and 1.4-1.5 kb (Type-B)]. Migrant laborers returning to China through Tengchong bore P. vivax infections displaying significantly higher genetic diversity than local residents. CONCLUSIONS: Both PvMSP-3 paralogs were subjected to diversifying selection in each sample population. Clustering of alleles supports ephemeral endemic differentiation of alleles, but the broader phylogeny suggests that alleles transit the globe, perhaps accelerated by movements of migrants such as those transiting Tengchong.

First report of leaf spot on Rhododendron delavayi caused by Pestalotiopsis scoparia in China.

Rhododendron delavayi is an evergreen shrub with large scarlet flowers that make it highly attractive as an ornamental species. The species is native to southwest China (Cai et al. 2015). From May to July in 2022, symptoms of leaf spot were observed on R. delavayi over a wide portion of the Baili Azalea Forest Area (N 27°10'-27°20', E 105°04'-106°04'), Guizhou Province, China. About 500 plants were surveyed and the incidence of leaf spot on R. delavayi leaves was 20 to 30%, significantly reducing their ornamental and economic value. The affected leaves had irregular, dark brown lesions with a clear blackish brown boundary and black conidiomata in a grayish center. To isolate the pathogen, 15 symptomatic leaves were collected from 10 plants. A few black dots were picked from the lesions with a sterilized needle, plated on water agar and incubated at 25℃ for 24 h to observe spore germination (Choi et al. 1999). Then the germinated spores were transferred onto PDA for further purification and morphological observation. Three single-spore isolates (GUDJ 61, GUDJ 62, GUDJ 63) that produced identical in morphology were obtained. The isolate GUDJ 61 was used for further study. Colonies on PDA grew velvety white on the upper surface and light yellow on the lower surface. Conidia were 5-celled, spindle- to ellipsoid-shaped, straight or slightly curved, 4-septate, and measured 39.0 ± 3.7 × 10.4 ± 0.79 µm (n=50). The morphological features were consistent with the description of Pestalotiopsis scoparia Maharachch., K.D. Hyde & Crous, (Maharachchikumbura et al. 2014). The pathogen was confirmed to be P. scoparia by amplification and sequencing of the internal transcribed spacer region (ITS), the partial ß-tubulin (TUB), and the partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b, and EF1-728F/EF-2, respectively. Sequences from PCR amplification were deposited in GenBank with accession numbers OP048045 (ITS), OP058111 (TUB) and OP058114 (TEF), respectively. BLAST searches of the sequences revealed 99% (549/552 nt), 99% (711/714 nt), and 82% (130/158 nt) homology with those of P. scoparia CBS 176.25T form GenBank (KM199330, KM199393, and KM199478), respectively. Phylogenetic analysis (MEGA 7.0) using the maximum likelihood method placed the isolate GUDJ 61 in a well-supported cluster with P. scoparia. The pathogen was thus identified as P. scoparia based on the morphological characterization and molecular analyses. The pathogenicity of GUDJ 61was tested through a pot assay. Ten healthy R. delavayi plants were scratched with a sterilized needle (0.45 mm in diameter) on three leaves per plant. Plants were inoculated by spraying a spore suspension (106 spores mL-1) of GUDJ 61 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 25°C and 75% relative humidity in a growth chamber. The pathogenicity test was repeated three times (Fang 2007). After 12 days, the treated leaves developed brown lesions similar to those in the field, while the control had no symptoms. The same fungus was reisolated from the infected leaves and identified based on a morphological characterization and molecular analyses. These results fulfilled Koch's postulates. To our knowledge, this is the first report of leaf spot on R. delavayi caused by P. scoparia in China. The fungal pathogen identification will provide valuable information for prevention and management of leaf spot disease associated with R. delavayi.

In vitro susceptibility profile of Plasmodium falciparum clinical isolates from Ghana to antimalarial drugs and polymorphisms in resistance markers.

Drug resistance in Plasmodium falciparum compromises the effectiveness of antimalarial therapy. This study aimed to evaluate the extent of drug resistance in parasites obtained from international travelers returning from Ghana to guide the management of malaria cases. Eighty-two clinical parasite isolates were obtained from patients returning from Ghana in 2016-2018, of which 29 were adapted to continuous in vitro culture. Their geometric mean IC50 values to a panel of 11 antimalarial drugs, assessed using the standard SYBR Green-I drug sensitivity assay, were 2.1, 3.8, 1.0, 2.7, 17.2, 4.6, 8.3, 8.3, 19.6, 55.1, and 11,555 nM for artemether, artesunate, dihydroartemisinin, lumefantrine, mefloquine, piperaquine, naphthoquine, pyronaridine, chloroquine, quinine, and pyrimethamine, respectively. Except for chloroquine and pyrimethamine, the IC50 values for other tested drugs were below the resistance threshold. The mean ring-stage survival assay value was 0.8%, with four isolates exceeding 1%. The mean piperaquine survival assay value was 2.1%, all below 10%. Mutations associated with chloroquine resistance (pfcrt K76T and pfmdr1 N86Y) were scarce, consistent with the discontinuation of chloroquine a decade ago. Instead, the pfmdr1 86N-184F-1246D haplotype was predominant, suggesting selection by the extensive use of artemether-lumefantrine. No mutations in the pfk13 propeller domain were detected. The pfdhfr/pfdhps quadruple mutant IRNGK associated with resistance to sulfadoxine-pyrimethamine reached an 82% prevalence. In addition, five isolates had pfgch1 gene amplification but, intriguingly, increased susceptibilities to pyrimethamine. This study showed that parasites originating from Ghana were susceptible to artemisinins and the partner drugs of artemisinin-based combination therapies. Genotyping drug resistance genes identified the signature of selection by artemether-lumefantrine. Parasites showed substantial levels of resistance to the antifolate drugs. Continuous resistance surveillance is necessary to guide timely changes in drug policy.

Mesenchymal stem cell transplantation worsens intestinal inflammation and microenvironment in PI3Kγ-knockout mice.

Considering the possible interaction between mesenchymal stem cells (MSCs) and PI3Kγ-associated drugs, we evaluated the efficacy and action mechanism of MSCs in the treatment of colitis in PI3Kγ-/- mice. Trinitro-benzene-sulfonic acid enema was used to create a colitis model, and MSCs were transplanted through the caudal vein to treat colitis in wild-type and PI3Kγ-/- mice. We sequenced microbial 16S rRNA genes in the colonic mucosa of PI3Kγ-/- and wild-type mice and quantified colonic IgA, IL-2, IL-10, IL-17A, occludin, and serum IgA. MSC transplantation led to a more serious reduction in the weight of trinitro-benzene-sulfonic acid-administered PI3Kγ-/- mice than that in wild-type mice. The disease activity index, pathological scoring, number of taxa in the colon, Berger-Parker index, I-index, proportion of Proteobacteria, and IgA level in the blood were higher in PI3Kγ-/- mice than in wild-type mice after MSC transplantation. The occludin and IL-10 levels in the colon tissues decreased before and after MSC transplantation in PI3Kγ-/- mice, whereas they were increased in wild-type mice The IL-17 level decreased in both wild-type and PI3Kγ-/- mice, with knockout mice showing a greater decrease. Therefore, MSC transplantation in PI3Kγ-/- mice led to increased numbers of exogenous pathogenic microorganisms and enhanced colitis that was difficult to relieve.

The Mechanism of Chlorantraniliprole Resistance and Detoxification in Trichogramma chilonis (Hymenoptera: Trichogrammatidae).

Parasitic Trichogramma chilonis Ishii, an egg parasitoid of Grapholita molesta, is a critical agent for biological control of insect pests in crop plants. However, the efficiency of T. chilonis is influenced by its resistance to the common pesticide chlorantraniliprole. To elucidate the chlorantraniliprole detoxification mechanism, differentially expressed genes (DEGs) related to chlorantraniliprole resistance were studied at different developmental stages of the wasp. Individuals of T. chilonis were grouped and treated with chlorantraniliprole at different developmental stages. Untreated wasps were used as controls. Transcriptomic analysis identified the DEGs associated with chlorantraniliprole resistance and detoxification in T. chilonis. A total of 1,483 DEGs were associated with chlorantraniliprole resistance at all developmental stages. DEGs that correlated with chlorantraniliprole sensitivity of T. chilonis at different developmental stages were distinct and had various functions. The newly identified DEGs are involved in cytochrome P450- and glutathione metabolism-related pathways, which were predicted to contribute to chlorantraniliprole detoxification. Chlorantraniliprole detoxification by T. chilonis was associated with cytochrome P450- and glutathione-related pathways. Our findings may be useful for balancing chemical and biological control practices aimed to optimize agricultural production.