Variant allele frequency in circulating tumor DNA correlated with tumor disease burden and predicted outcomes in patients with advanced breast cancer.

PURPOSE: In patients with first-line advanced breast cancer (ABC), the correlation between ctDNA variant allele frequency (VAF) and tumor disease burden, and its prognostic value remains poorly investigated. METHODS: This study included patients with ABC diagnosed at Peking University Cancer Hospital who performed ctDNA test before receiving first-line treatment. Baseline plasma samples were collected for assessing ctDNA alterations and VAF with next-generation sequencing. The sum of tumor target lesion diameters (SLD) was measured with imaging methods according to RECIST 1.1 criteria. RESULTS: The final cohort included 184 patients. The median age of the cohort was 49.4 (IQR: 42.3-56.8) years. The median VAF was 15.6% (IQR: 5.4%-33.7%). VAF showed positive correlation with SLD in patients with relatively large tumor lesions (r = 0.314, p = 0.003), but not in patients with small tumor lesions (p = 0.226). VAF was associated with multiple metastasis sites (p = 0.001). Multivariate Cox regression analysis showed that high VAF was associated with shorter overall survival (OS) (HR: 3.519, 95% confidence interval (CI): 2.149-5.761), and first-line progression-free survival (PFS) (HR: 2.352, 95%CI: 1.462-3.782). Combined VAF and SLD improved prediction performance, both median OS and PFS of patients in VAF(H)/SLD(H) group were significantly longer than VAF(L)/SLD(L) group (mOS: 49.3 vs. 174.1 months; mPFS: 9.6 vs. 25.3 months). CONCLUSION: ctDNA VAF associated with tumor disease burden, and was a prognostic factor for patients with ABC. A combination of ctDNA test and radiographic imaging might enhance tumor burden evaluation, and improve prognosis stratification in patients with ABC.

Protists regulate microbially mediated organic carbon turnover in soil aggregates.

Soil protists, the major predator of bacteria and fungi, shape the taxonomic and functional structure of soil microbiome via trophic regulation. However, how trophic interactions between protists and their prey influence microbially mediated soil organic carbon turnover remains largely unknown. Here, we investigated the protistan communities and microbial trophic interactions across different aggregates-size fractions in agricultural soil with long-term fertilization regimes. Our results showed that aggregate sizes significantly influenced the protistan community and microbial hierarchical interactions. Bacterivores were the predominant protistan functional group and were more abundant in macroaggregates and silt + clay than in microaggregates, while omnivores showed an opposite distribution pattern. Furthermore, partial least square path modeling revealed positive impacts of omnivores on the C-decomposition genes and soil organic matter (SOM) contents, while bacterivores displayed negative impacts. Microbial trophic interactions were intensive in macroaggregates and silt + clay but were restricted in microaggregates, as indicated by the intensity of protistan-bacterial associations and network complexity and connectivity. Cercozoan taxa were consistently identified as the keystone species in SOM degradation-related ecological clusters in macroaggregates and silt + clay, indicating the critical roles of protists in SOM degradation by regulating bacterial and fungal taxa. Chemical fertilization had a positive effect on soil C sequestration through suppressing SOM degradation-related ecological clusters in macroaggregate and silt + clay. Conversely, the associations between the trophic interactions and SOM contents were decoupled in microaggregates, suggesting limited microbial contributions to SOM turnovers. Our study demonstrates the importance of protists-driven trophic interactions on soil C cycling in agricultural ecosystems.

New Phenanthro[9,10-d]oxazole-Based Fluorophores with Hybridized Local and Charge-Transfer Characteristics for Highly Efficient Blue Non-Doped OLEDs with Negligible Efficiency Roll-Off.

It is vital to develop highly efficient non-doped blue organic light-emitting diodes (OLEDs) with high color purity and low-efficiency roll-off for applications in display and lighting. Herein, two blue D-A fluorophores TPA-PO and TPA-DPO are designed and synthesized, in which phenanthro[9,10-d]oxazole (PO) acts as the acceptor and triphenylamine as the donor. TPA-PO and TPA-DPO display good thermal stability and efficient luminescence efficiency in neat film. Results based on photophysical property and theoretical calculation demonstrate that TPA-PO and TPA-DPO possess the hybridized local and charge-transfer (HLCT) feature, which can utilize the triplet exciton to achieve highly efficient electroluminance (EL). The non-doped OLEDs with TPA-PO/TPA-DPO as pure emissive layer show the uniform EL emission peak at 468 nm, corresponding to CIE coordinates of (0.168, 0.187) and (0.167, 0.167), respectively. The TPA-DPO-based non-doped OLEDs provide the maximum external quantum efficiency (EQE) of 7.99 % and high exciton utility efficiency of 48.4 %~72.6 %. Moreover, the TPA-DPO-based device exhibits low-efficiency roll-off, still maintaining the EQE of 6.03 % at the high luminance of 5000 cd m-2. Those findings state clearly that PO is a promising building block of blue fluorophore with a potential HLCT feature to be applied in non-doped OLEDs.

Intramedullary administration of tranexamic acid reduces bleeding in proximal femoral nail antirotation surgery for intertrochanteric fractures in elderly individuals: A randomized controlled trial.

PURPOSE: Intertrochanteric fractures undergoing proximal femoral nail antirotation (PFNA) surgery are associated with significant hidden blood loss. This study aimed to explore whether intramedullary administration of tranexamic acid (TXA) can reduce bleeding in PFNA surgery for intertrochanteric fractures in elderly individuals. METHODS: A randomized controlled trial was conducted from January 2019 to December 2022. Patients aged over 60 years with intertrochanteric fractures who underwent intramedullary fixation surgery with PFNA were eligible for inclusion and grouped according to random numbers. A total of 249 patients were initially enrolled, of which 83 were randomly allocated to the TXA group and 82 were allocated to the saline group. The TXA group received intramedullary perfusion of TXA after the bone marrow was reamed. The primary outcomes were total peri-operative blood loss and post-operative transfusion rate. The occurrence of adverse events was also recorded. Continuous data was analyzed by unpaired t-test or Mann-Whitney U test, and categorical data was analyzed by Pearson Chi-square test. RESULTS: The total peri-operative blood loss (mL) in the TXA group was significantly lower than that in the saline group (577.23 ± 358.02 vs. 716.89 ± 420.30, p = 0.031). The post-operative transfusion rate was 30.67 % in the TXA group and 47.95 % in the saline group (p = 0.031). The extent of post-operative deep venous thrombosis and the 3-month mortality rate were similar between the 2 groups. CONCLUSION: We observed that intramedullary administration of TXA in PFNA surgery for intertrochanteric fractures in elderly individuals resulted in less peri-operative blood loss and decreased transfusion rate, without any adverse effects, and is, thus, recommended.

[Machine learning algorithms for identifying autism spectrum disorder through eye-tracking in different intention videos]. / ä¸åŒæ„å›¾åœºæ™¯çœ¼åŠ¨æ³¨è§†æ¨¡å¼æœºå™¨å­¦ä¹ ç®—æ³•è¯†åˆ«å­¤ç‹¬ç—‡è°±ç³»éšœç¢çš„ç ”ç©¶.

OBJECTIVES: To investigate the differences in visual perception between children with autism spectrum disorder (ASD) and typically developing (TD) children when watching different intention videos, and to explore the feasibility of machine learning algorithms in objectively distinguishing between ASD children and TD children. METHODS: A total of 58 children with ASD and 50 TD children were enrolled and were asked to watch the videos containing joint intention and non-joint intention, and the gaze duration and frequency in different areas of interest were used as original indicators to construct classifier-based models. The models were evaluated in terms of the indicators such as accuracy, sensitivity, and specificity. RESULTS: When using eight common classifiers, including support vector machine, linear discriminant analysis, decision tree, random forest, and K-nearest neighbors (with K values of 1, 3, 5, and 7), based on the original feature indicators, the highest classification accuracy achieved was 81.90%. A feature reconstruction approach with a decision tree classifier was used to further improve the accuracy of classification, and then the model showed the accuracy of 91.43%, the specificity of 89.80%, and the sensitivity of 92.86%, with an area under the receiver operating characteristic curve of 0.909 (P<0.001). CONCLUSIONS: The machine learning model based on eye-tracking data can accurately distinguish ASD children from TD children, which provides a scientific basis for developing rapid and objective ASD screening tools.

Multifocal Raman Spectrophotometer for Examining Drug-Induced and Chemical-Induced Cellular Changes in 3D Cell Spheroids.

Cell spheroids offer alternative in vitro cell models to monolayer cultured cells because they express complexities similar to those of in vivo tissues, such as cellular responses to drugs and chemicals. Raman spectroscopy emerged as a powerful analytical tool for detecting chemical changes in living cells because it nondestructively provides vibrational information regarding a target. Although multiple iterations are required in drug screening to determine drugs to treat cell spheroids and assess the inter-spheroid heterogeneity, current Raman applications used in spheroids analysis allow the observation of only a few spheroids owing to the low throughput of Raman spectroscopy. In this study, we developed a multifocal Raman spectrophotometer that enables simultaneous analysis of multiple spheroids in separate wells of a regular 96-well plate. By utilizing 96 focal spots excitation and parallel signal collection, our system can improve the throughput by approximately 2 orders of magnitude compared to a conventional single-focus Raman microscope. The Raman spectra of HeLa cell spheroids treated with anticancer drugs and HepG2 cell spheroids treated with free fatty acids were measured simultaneously, and concentration-dependent cellular responses were observed in both studies. Using the multifocal Raman spectrophotometer, we rapidly observed chemical changes in spheroids, and thus, this system can facilitate the application of Raman spectroscopy in analyzing the cellular responses of spheroids.

The newest Oxford Nanopore R10.4.1 full-length 16S rRNA sequencing enables the accurate resolution of species-level microbial community profiling.

The long-read amplicon provides a species-level solution for the community. With the improvement of nanopore flowcells, the accuracy of Oxford Nanopore Technologies (ONT) R10.4.1 has been substantially enhanced, with an average of approximately 99%. To evaluate its effectiveness on amplicons, three types of microbiomes were analyzed by 16S ribosomal RNA (hereinafter referred to as "16S") amplicon sequencing using Novaseq, Pacbio sequel II, and Nanopore PromethION platforms (R9.4.1 and R10.4.1) in the current study. We showed the error rate, recall, precision, and bias index in the mock sample. The error rate of ONT R10.4.1 was greatly reduced, with a better recall in the case of the synthetic community. Meanwhile, in different types of environmental samples, ONT R10.4.1 analysis resulted in a composition similar to Pacbio data. We found that classification tools and databases influence ONT data. Based on these results, we conclude that the ONT R10.4.1 16S amplicon can also be used for application in environmental samples. IMPORTANCE The long-read amplicon supplies the community with a species-level solution. Due to the high error rate of nanopore sequencing early on, it has not been frequently used in 16S studies. Oxford Nanopore Technologies (ONT) introduced the R10.4.1 flowcell with Q20+ reagent to achieve more than 99% accuracy as sequencing technology advanced. However, there has been no published study on the performance of commercial PromethION sequencers with R10.4.1 flowcells on 16S sequencing or on the impact of accuracy improvement on taxonomy (R9.4.1 to R10.4.1) using 16S ONT data. In this study, three types of microbiomes were investigated by 16S ribosomal RNA (rRNA) amplicon sequencing using Novaseq, Pacbio sequel II, and Nanopore PromethION platforms (R9.4.1 and R10.4.1). In the mock sample, we displayed the error rate, recall, precision, and bias index. We observed that the error rate in ONT R10.4.1 is significantly lower, especially when deletions are involved. First and foremost, R10.4.1 and Pacific Bioscience platforms reveal a similar microbiome in environmental samples. This study shows that the R10.4.1 full-length 16S rRNA sequences allow for species identification of environmental microbiota.

Serum HBV RNA is associated with liver fibrosis regression in HBeAg-positive chronic hepatitis B patients treated with nucleos(t)ide analogues.

Noninvasive methods for assessing hepatic fibrosis are clinically necessary. This study aims to explore HBV markers correlated with liver fibrosis and capable of diagnosing significant fibrosis and predicting fibrosis regression. Seventy-four HBeAg-positive chronic hepatitis B (CHB) patients were enrolled and started on entecavir or adefovir therapy. Serum HBV RNA, HBV DNA, HBsAg and hepatitis B core-related antigen (HBcrAg) levels were measured at baseline and during treatment. Liver fibrosis was assessed at baseline and month 60 by liver biopsy. Fibrosis regression was defined as Ishak fibrosis score decreased ≥1-point. At baseline, HBsAg, HBcrAg and HBV RNA levels had a stronger correlation with Ishak fibrosis score (r = -.441, p = .002; r = -.469, p = .001; r = -.398, p = .001) than APRI and FIB-4 (r = .321 p = .006; r = .371, p = .001). HBsAg >4 log10 IU/ml plus HBcrAg >7 log10 IU/ml or HBsAg >4 log10 IU/ml plus HBV RNA >5 log10 copies/ml exhibited the same excellent diagnostic ability for significant fibrosis with the AUROC of 0.857. After 60 months of antiviral treatment, 66.7% of patients who suffered significant fibrosis at baseline achieved fibrosis regression, and an HBV RNA decline from baseline to month 6 greater than 0.63 log10 copies/ml could predict the fibrosis regression at month 60. In conclusion, serum HBsAg, HBcrAg and HBV RNA are potential markers for predicting significant liver fibrosis. HBV RNA measurement would be particularly useful for monitoring hepatic fibrosis changes in HBeAg-positive CHB patients.

Nucleos(t)ide analogues altered quasispecies composition of hepatitis B virus (HBV)-resistant mutations in serum HBV DNA and serum HBV RNA.

Serum hepatitis B virus (HBV) RNA is a new serological indicator reflecting viral replication with good clinical application prospects. This study aimed to clarify the dynamic changes of serum HBV RNA levels and the quasispecies of HBV RNA virus-like particles in nucleos(t)ide analogues (NAs)-experienced chronic hepatitis B (CHB) patients harboring NAs-resistant mutations and their identifiable effects on NAs resistance. We included CHB patients who were on long-term NAs treatment and with HBV DNA rebound. The longitudinally dynamics of serum HBV RNA levels were quantitatively detected, and the quasispecies differences between serum HBV DNA and serum HBV RNA were compared by high-throughput sequencing. The effect of NAs concentration pressure on altering the resistance mutations quasispecies proportion of HBV DNA and HBV RNA in cell supernatant was analyzed in vitro. A total of 447 serum samples from 36 CHB patients treated with NAs were collected. The median follow-up period was 47 months (about 4 years), and the longest follow-up period was 117 months (about 10 years). Our results showed that HBV RNA could reflect virological breakthrough in 23 (64%, 23/36) patients, and serum HBV RNA rebound earlier than HBV DNA in 12 (52%, 12/23) patients. However, serum HBV RNA remained at a consistently high level and did not fluctuate significantly with the HBV DNA rebound in 6 of 36 patients. In addition, serum HBV RNA was not consistently detectable in 7 of the 36 patients, and their serum HBV RNA was undetectable even after HBV DNA had rebounded. The proportion of drug-resistant mutations in HBV DNA was higher than that of HBV RNA by high-throughput sequencing. The results of in vitro experiments showed that the viral strains with drug-resistant mutation in HBV DNA in cell supernatants gradually become the dominant strains with the increase of NAs concentrations. Serum HBV RNA levels can reflect virological breakthrough in most NAs- treated CHB patients, but there are certain limitations. NAs alter the quasispecies composition of serum HBV DNA and serum HBV RNA, resulting in a higher detection rate of drug-resistant mutations in serum HBV DNA than in serum HBV RNA.

Microbial autotrophy explains large-scale soil CO2 fixation.

Microbial communities play critical roles in fixing carbon from the atmosphere and fixing it in the soils. However, the large-scale variations and drivers of these microbial communities remain poorly understood. Here, we conducted a large-scale survey across China and found that soil autotrophic organisms are critical for explaining CO2 fluxes from the atmosphere to soils. In particular, we showed that large-scale variations in CO2 fixation rates are highly correlated to those in autotrophic bacteria and phototrophic protists. Paddy soils, supporting a larger proportion of obligate bacterial and protist autotrophs, display four-fold of CO2 fixation rates over upland and forest soils. Precipitation and pH, together with key ecological clusters of autotrophic microbes, also played important roles in controlling CO2 fixation. Our work provides a novel quantification on the contribution of terrestrial autotrophic microbes to soil CO2 fixation processes at a large scale, with implications for global carbon regulation under climate change.

Efficacy and safety of PEG-rhG-CSF versus rhG-CSF in preventing chemotherapy-induced-neutropenia in early-stage breast cancer patients.

BACKGROUND: To compare the clinical value of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and pegylated rhG-CSF(PEG-rhG-CSF) in early-stage breast cancer (EBC) patients receiving adjuvant chemotherapy, compare the efficacy of PEG-rhG-CSF with different dose and explore the timing of rhG-CSF rescue treatment. METHODS: Patients in two PEG-rhG-CSF subgroups were given 3 mg or 6 mg PEG-rhG-CSF within 24 ~ 48 h after chemotherapy for preventing myelosuppression, while patients in the rhG-CSF group were given rhG-CSF. Observation indicators include the incidence of febrile neutropenia (FN) and grade 3/4 chemotherapy-induced-neutropenia (CIN), the overall levels and nadir values of white blood cells (WBC) and absolute neutrophil count (ANC), comparison of WBC and ANC curves over time, the incidence of CIN-related complications, the incidence of adverse events in each group and the timing of rescue treatment for rhG-CSF. RESULTS: There was no significant difference in the incidence of FN in the first cycle among the groups (P = 0.203). But the incidence of ≥ 3 grade CIN in two PEG-rhG-CSF subgroups was significantly lower than that in the rhG-CSF group (P < 0.001). The overall WBC and ANC levels in the PEG-rhG-CSF group were significantly higher than those in the rhG-CSF group (P < 0.001). In terms of CIN-related complications, less chemotherapy delay rate (1.1 vs. 7.5%, P = 0.092), less dose reduction rate (6.9 vs. 7.5%, P = 1.000), less antibiotic use rate (3.4 vs. 17.5%, P = 0.011) and less proportion of rhG-CSF rescue therapy (24.1 vs. 85.0%, P < 0.001) in the PEG-rhG-CSF group, and there were no significant differences between PEG-rhG-CSF subgroups. In the incidence of adverse events among the groups, there were no statistical differences. All patients undergoing rhG-CSF rescue treatment were mainly 4 grade (63.6%) and 3 grade (25.5%) CIN, and 10.9% of patients with 1 ~ 2 grade CIN who had high infection risk or had been infected. CONCLUSION: PEG-rhG-CSF has better efficacy and equal tolerance compared with rhG-CSF in preventing CIN in EBC patients receiving EC regimen. Moreover, a half-dose 3 mg PEG-rhG-CSF also had good efficacy. Last, patients with ≥ 3 grade CIN and others who have been assessed to be at high risk of infection or have co-infection should consider rhG-CSF or even antibiotic rescue treatment.

Phagotrophic Protists Modulate Copper Resistance of the Bacterial Community in Soil.

Protist predation is a crucial biotic driver modulating bacterial populations and functional traits. Previous studies using pure cultures have demonstrated that bacteria with copper (Cu) resistance exhibited fitness advantages over Cu-sensitive bacteria under the pressure of protist predation. However, the impact of diverse natural communities of protist grazers on bacterial Cu resistance in natural environments remains unknown. Here, we characterized the communities of phagotrophic protists in long-term Cu-contaminated soils and deciphered their potential ecological impacts on bacterial Cu resistance. Long-term field Cu pollution increased the relative abundances of most of the phagotrophic lineages in Cercozoa and Amoebozoa but reduced the relative abundance of Ciliophora. After accounting for soil properties and Cu pollution, phagotrophs were consistently identified as the most important predictor of the Cu-resistant (CuR) bacterial community. Phagotrophs positively contributed to the abundance of a Cu resistance gene (copA) through influencing the cumulative relative abundance of Cu-resistant and -sensitive ecological clusters. Microcosm experiments further confirmed the promotion effect of protist predation on bacterial Cu resistance. Our results indicate that the selection by protist predation can have a strong impact on the CuR bacterial community, which broadens our understanding of the ecological function of soil phagotrophic protists.

Fruit rot causing by Athelia rolfsii (Sclerotium rolfsii) on jackfruit (Artocarpus heterophyllus) in China.

Artocarpus heterophyllus, known as jackfruit, was a tropical fruit and cultivated extensively as nutritional and medicinal properties in southern China in recent year. During July 2022, fruit rot was observed on the fruits at the bottom of jackfruit trees in an orchard in Zhanjiang, Guangdong (N21°9' 27" E110°17' 54") 3-4 days after typhoon. The incidence rate of fruit was about 0.3%. The initial symptom was white mycelia appearing on the surface of fruits. Mycelia with rhizomorphs spread rapidly over the fruits, formed white, often fan-shaped mats with the rapeseed size sclerotia. The infected fruits were water-soaked, quickly became rotten, and fell off. Sclerotia from disease fruits were incubated on PDA with 50 mg/L ampicillin at 25-28℃ in the dark for 2 days. Hyphae tips were transferred to get the purified isolates. Colonies with a radial growth rate of 23.2 mm/day had abundant aerial mycelia and profuse sclerotia on PDA. Hyphae of the isolates were transparent, branched, with clamp connections at septa, usually 2.9-8.3 µm (Ave. 5.8 µm) (n>30) wide. Aerial mycelia were whitish-cottony, with many narrow rhizomorphs. Spherical sclerotia developed at about 10 days after incubation, and gradually changed from white to tan-to-dark brown, and mature sclerotia were about 1.7 mm in size. The morphological characteristics was similar to those of Sclerotium rolfsii (teleomorph: Athelia rolfsii). To accurately identify the fungus, the internal transcribed spacer gene (ITS) and large subunit rRNA gene (LSU) of isolate CASS-BLM-1 were PCR amplified with primer pairs ITS1/ITS4 (White et al 1990) and V9G/LR5 (Klaubauf et al 2014). The amplicons were sequenced and deposited in GenBank with accession number OP535473 (ITS) and OP535474 (LSU). BLASTn results showed that the nucleotide sequences of ITS and LSU had high identity with corresponding sequences of A. rolfsii isolates CBS 191.62 (ITS: MH858139, 472/474(99.58%); LSU: MH869724, 882/885(99.66%)) (Vu et al 2019). Phylogenetic analysis based on ITS sequence data was obtained according to maximum likelihood method using MEGA analysis software, CASS-BLM1 was grouped in A. rolfsii clade with 100% bootstrap support value. Based on morphology and DNA sequences, the fungus was identified as A. rolfsii (anamorph: S. rolfsii). To fulfil Koch's postulates, healthy fruits on the tree and detached fruits were inoculated with 7-day-old sclerotia of isolate CASS-BLM1. Five unwound sites and five wound sites with a sterile needle were tested on each fruit and a sclerotium was put at each site. Fruits not inoculated with sclerotia were used as control the test was repeated three times. All fruit were enclosed in transparent plastic bags with sterile absorbent cotton moistened with sterile distilled water. The indoor and outdoor temperatures ranged from 25 to 30 ℃. Three days later, white mycelia were observed on all inoculation sites, and 5 days later, the inoculated fruits began to rot, while control fruits remained healthy. The same fungus with identical morphology and DNA sequences was re-isolated from the inoculated sites. Previously, A. rolfsii was reported to cause fruit rot disease on jackfruit in Bangladesh (Elahi et al 2021), this is the first report of A. rolfsii causing fruit rot on jackfruit in China. A. rolfsii is suitable for high temperature and humidity environment (Punja 1985), this report will help farmers to diagnose this disease, especially to strengthen the disease prevention during the typhoon season.

Erysiphe elevata causing powdery mildew on Eucalyptus urophylla × E. camaldulensis in China.

Eucalyptus urophylla × E. camaldulensis, named Chiwei eucalypt, is a hybrid species widely used in China. Many of its clones are cultivated for afforestation due to cold tolerance, high yield, high strength and disease resistance. Clone LH1 is planted extensively for its high stability and machinability in South China. In December 2021, severe powdery mildew signs were observed on clone LH1 in Zhanjiang, Guangdong (N28°8'29"; E110°17'5"). Whitish powder principally appeared on both abaxial and adaxial leaf surfaces. All plants were infected within about a week and above 90% leaves were diseased, which resulted in abnormal growth and shrinkage of leaves. Hyphae with single, lobed appressoria were hyaline, septate, branched, 3.3-6.8 µm (ave. 4.9 µm, n>50) wide. Conidiophores with a straight to flexuous foot-cell (14.7-46.1×5.4-9.7 µm, ave. 25.8×7.9 µm, n>30) were erect, hyaline, 2-septate, unbranched, 35.4-81.8 × 5.7-10.7 (ave. 56.7×8.7 µm, n>50). Conidia were solitary, hyaline, cylindrical to elliptical, 27.7-46.6 ×11.2-19.0 (ave. 35.7×16.6 µm, n>50). Chamothecia were not found on infected trees. The further identification was confirmed by partial sequences of internal transcribed spacer (ITS), large submit rRNA gene (LSU), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), and RNA polymerase II second largest subunit (RPB2) gene. A very small amount of mycelia and spores from voucher specimens CCAS-ASBF-1 and CCAS-ASBF-2 were deposited in the herbarium of Guangdong Ocean University. Specimens were PCR amplified and sequenced with primer pairs ITS1/ITS4 (White et al 1990), LROR/LR7 (Moncalvo et al 1995), PMGAPDH1/PMGAPDH3R, GSPM2/GSPM3R and PmRpb2_4/ PMRpb2_6R (Bradshaw, et al. 2022), respectively. BLASTn results showed that ITS (OP270019 and OQ380937), LSU (OP270018 and OQ380938), GAPDH, GS and RPB2 (OQ414445- OQ414450) were above 99% identical with those of E. elevata on Catalpa bignonioides (ITS: AY587013) (Cook et al 2004), Plumeria rubra (ITS: MH985631) (Yeh et al 2019), Cerbera manghas (ITS: MZ379159; LSU: MZ379160) (Mukhtar et al 2022), and Eucalyptus camaldulensis (LSU: LC177375-6) (Meebon et al. 2017), and above 99% identical with those of Erysiphe vaccinii FH00941201 on Vaccinium corymbosum (ITS: ON073869; RPB2: ON119159; GS: ON075687) and FH00112205 on V. vacillans (ITS: ON073870; GAPDH: ON075646) (Bradshaw et al 2022). This is the first sequence data for non rDNA of E. elevata. In an ITS tree phylogenetic analysis with Maximum likelihood (ML) method showed the fungus clustered in a highly supported clade with E. elevata and E. vaccinii. In a multi-locus tree, E. elevata grouped in a sister position to E. vaccinii FH00941201. Thus, the pathogen was identified as E. elevata based on morphology, DNA BLASTn and phylogenetic analysis (Braun and Cook 2012). Pathogenicity tests were conducted on healthy leaves of 1-year-old potted plants. Ten leaves were cleaned with sterile water, inoculated by gently dusting conidia from single lesion on the naturally infected leaves, and then covered with plastic bags containing wet absorbent cotton. Non-inoculated leaves served as controls. Symptoms developed on all inoculated leaves 3 to 5 days after inoculation, and the fungus was identical to the original fungus on the infected leaves, whereas control plants remained symptomless. This is the first report of powdery mildew caused by E. elevata on Eucalyptus sp. from China. This finding is helpful for land managers to diagnose and control the disease.

[Mechanism of Xuebijing Injection in treatment of sepsis-associated ARDS based on network pharmacology and in vitro experiment].

The aim of this study was to investigate the effect and molecular mechanism of Xuebijing Injection in the treatment of sepsis-associated acute respiratory distress syndrome(ARDS) based on network pharmacology and in vitro experiment. The active components of Xuebijing Injection were screened and the targets were predicted by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). The targets of sepsis-associated ARDS were searched against GeneCards, DisGeNet, OMIM, and TTD. Weishengxin platform was used to map the targets of the main active components in Xuebijing Injection and the targets of sepsis-associated ARDS, and Venn diagram was established to identify the common targets. Cytoscape 3.9.1 was used to build the "drug-active components-common targets-disease" network. The common targets were imported into STRING for the building of the protein-protein interaction(PPI) network, which was then imported into Cytoscape 3.9.1 for visualization. DAVID 6.8 was used for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment of the common targets, and then Weishe-ngxin platform was used for visualization of the enrichment results. The top 20 KEGG signaling pathways were selected and imported into Cytoscape 3.9.1 to establish the KEGG network. Finally, molecular docking and in vitro cell experiment were performed to verify the prediction results. A total of 115 active components and 217 targets of Xuebijing Injection and 360 targets of sepsis-associated ARDS were obtained, among which 63 common targets were shared by Xuebijing Injection and the disease. The core targets included interleukin-1 beta(IL-1ß), IL-6, albumin(ALB), serine/threonine-protein kinase(AKT1), and vascular endothelial growth factor A(VEGFA). A total of 453 GO terms were annotated, including 361 terms of biological processes(BP), 33 terms of cellular components(CC), and 59 terms of molecular functions(MF). The terms mainly involved cellular response to lipopolysaccharide, negative regulation of apoptotic process, lipopolysaccharide-mediated signaling pathway, positive regulation of transcription from RNA polyme-rase Ⅱ promoter, response to hypoxia, and inflammatory response. The KEGG enrichment revealed 85 pathways. After diseases and generalized pathways were eliminated, hypoxia-inducible factor-1(HIF-1), tumor necrosis factor(TNF), nuclear factor-kappa B(NF-κB), Toll-like receptor, and NOD-like receptor signaling pathways were screened out. Molecular docking showed that the main active components of Xuebijing Injection had good binding activity with the core targets. The in vitro experiment confirmed that Xuebijing Injection suppressed the HIF-1, TNF, NF-κB, Toll-like receptor, and NOD-like receptor signaling pathways, inhibited cell apoptosis and reactive oxygen species generation, and down-regulated the expression of TNF-α, IL-1ß, and IL-6 in cells. In conclusion, Xuebijing Injection can regulate apoptosis and response to inflammation and oxidative stress by acting on HIF-1, TNF, NF-κB, Toll-like receptor, and NOD-like receptor signaling pathways to treat sepsis-associated ARDS.

Label-Free Monitoring of Drug-Induced Cytotoxicity and Its Molecular Fingerprint by Live-Cell Raman and Autofluorescence Imaging.

Simultaneous observation of drug distribution at the effector site and subsequent cell response are essential in the drug development process. However, few studies have visualized the drug itself and biomolecular interactions in living cells. Here, we used label-free Raman microscopy to investigate drug-induced cytotoxicity and visualize drug uptake and subcellular localization by its specific molecular fingerprint. A redox-sensitive Raman microscope detected the decrease of reduced cytochrome c (cyt c) after Actinomycin D (ActD) treatment in a time-dependent and dose-dependent format. Immunofluorescence staining of cyt c suggested that the release of cyt c was not the major cause. Combining Raman microscopy with conventional biological methods, we reported that the oxidization of cyt c is an early cytotoxicity marker prior to the release of cyt c. Moreover, as the spectral properties of ActD are sensitive to the surrounding environment, subcellular localization of ActD was visualized sensitively by the weak autofluorescence, and the intercalation of ActD into DNA was detected by shifted Raman peaks, allowing for parallel observation of drug uptake and the mechanism of action. In this research, we achieved simultaneous observation of cytotoxicity and cellular drug uptake by Raman microscopy, which could facilitate a precise understanding of pharmacological effects and predict potential drug toxicity in the future.

Identification of mutation patterns and circulating tumour DNA-derived prognostic markers in advanced breast cancer patients.

BACKGROUND: The correlations between circulating tumour DNA (ctDNA)-derived genomic markers and treatment response and survival outcome in Chinese patients with advanced breast cancer (ABC) have not been extensively characterized. METHODS: Blood samples from 141 ABC patients who underwent first-line standard treatment in Peking University Cancer Hospital were collected. A next-generation sequencing based liquid biopsy assay (PredicineCARE) was used to detect somatic mutations and copy number variations (CNVs) in ctDNA. A subset of matched blood samples and tumour tissue biopsies were compared to evaluate the concordance. RESULTS: Overall, TP53 (44.0%) and PIK3CA (28.4%) were the top two altered genes. Frequent CNVs included amplifications of ERBB2 (24.8%) and FGFR1 (8.5%) and deletions of CDKN2A (3.5%). PIK3CA/TP53 and FGFR1/2/3 variants were associated with drug resistance in hormone receptor-positive (HR +) and human epidermal growth factor receptor 2-positive (HER2 +) patients. The comparison of genomic variants across matched tumour tissue and ctDNA samples revealed a moderate to high concordance that was gene dependent. Triple-negative breast cancer (TNBC) patients harbouring TP53 or PIK3CA alterations had a shorter overall survival than those without corresponding mutations (P = 0.03 and 0.008). A high ctDNA fraction was correlated with a shorter progression-free survival (PFS) (P = 0.005) in TNBC patients. High blood-based tumor mutation burden (bTMB) was associated with a shorter PFS for HER2 + and TNBC patients (P = 0.009 and 0.05). Moreover, disease monitoring revealed several acquired genomic variants such as ESR1 mutations, CDKN2A deletions, and FGFR1 amplifications. CONCLUSIONS: This study revealed the molecular profiles of Chinese patients with ABC and the clinical validity of ctDNA-derived markers, including the ctDNA fraction and bTMB, for predicting treatment response, prognosis, and disease progression. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT03792529. Registered January 3rd 2019, https://clinicaltrials.gov/ct2/show/NCT03792529 .

Serum HBV RNA predicts HBeAg clearance and seroconversion in patients with chronic hepatitis B treated with nucleos(t)ide analogues.

This study evaluated the predictive value of serum HBV DNA, HBV RNA, HBcrAg, HBsAg, intrahepatic HBV DNA and cccDNA for HBeAg clearance and seroconversion during long-term treatment with nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB). A single centre, prospective cohort of CHB patients was used for this study. Serum HBV RNA levels were retrospectively measured at baseline, 6, 12, 24, 36, 48, 60, 72 and 84 months post-NAs treatment. Serum HBsAg and HBcrAg levels were quantified at baseline, month 6, 60 and 72. Histological samples from liver biopsy at baseline and month 60 were analysed for intrahepatic HBV DNA and cccDNA. Eighty-three HBeAg-positive patients were enrolled with a median follow-up time of 108 months (range 18-138 months). Of them, 53 (63.86%) patients achieved HBeAg clearance, and 37 (44.58%) achieved HBeAg seroconversion. Cox multivariate analysis showed that only baseline HBV RNA was independently associated with HBeAg clearance and seroconversion (<5.45 log10 copies/mL, HR = 5.06, 95% CI: 1.87-13.71, p = .001; HR = 3.38, 95% CI: 1.28-8.91, p = .01). The independent association with HBeAg clearance and seroconversion remained for HBV RNA levels at month 6 (<4.72 log10 copies/mL, HR = 4.16, 95% CI: 1.61-10.72, p = .003; HR = 6.52, 95% CI: 1.85-22.94, p = .003) and month 12 (<4.08 log10 copies/mL, HR = 3.68, 95% CI: 1.96-6.90, p < .001; HR = 2.79, 95% CI: 1.31-5.94, p = .008). The AUCs of baseline HBV RNA for predicting the HBeAg clearance (0.83, 95% CI: 0.70-0.96, 0.83, 95% CI: 0.70-0.96 and 0.82, 95% CI: 0.69-0.95 respectively) and seroconversion (0.89, 95% CI: 0.77-1.00; 0.81, 95% CI: 0.66-0.95 and 0.84, 95% CI: 0.71-0.98 respectively) at month 36, 60 and 84 were higher than those of HBV DNA, HBsAg and HBcrAg. In conclusion, lower serum HBV RNA at baseline, month 6 and 12 post-NAs treatment could predict HBeAg clearance and seroconversion during long-term NAs treatment.

Serum HBV DNA plus RNA reflecting cccDNA level before and during NAs treatment in HBeAg positive CHB patients.

Background & Aims: Correlations between serum viral markers and intrahepatic cccDNA in patients undergoing long-term nucleos(t)ide analogues (NAs) treatment haven't been fully explored. In this study, we evaluate the correlation between intrahepatic cccDNA and other serum viral markers and intrahepatic HBV DNA in HBeAg positive chronic hepatitis B (CHB) patients during 60-month treatment with NAs. Methods: Fifty-four HBeAg positive CHB patients received long-term NAs treatment were included in this study. Serial serum samples were regularly collected and quantitatively analyzed for HBsAg, HBV DNA, HBV RNA and HBcrAg. Histological samples from liver biopsy at baseline and month 60 were analyzed for intrahepatic HBV DNA and cccDNA. Results: At baseline, serum HBV DNA plus RNA was positively associated with intrahepatic cccDNA in multivariate regression analysis (ß=0.205, P<0.001). In the correlation analysis between cccDNA and serum viral markers, HBV DNA plus RNA had the highest correlation coefficient (r=0.698, P<0.001), followed by serum HBV DNA (r=0.641, P<0.001), HBV RNA (r=0.590, P<0.001), and HBcrAg (r=0.564, P<0.001). At month 60, correlations between these serum viral markers and cccDNA were not observed (P>0.05). Multivariate regression analysis showed that only the decreased HBV DNA plus RNA was positively associated with cccDNA decline (ß=0.172, P =0.006). Changes of HBV DNA plus RNA (r=0.525, P=0.001) was better correlated with cccDNA decline as compared to HBV RNA (r=0.384, P=0.008), HBV DNA (r=0.431, P=0.003), and HBsAg (r=0.342, P=0.029). Conclusions: Serum HBV DNA plus RNA better correlated with intrahepatic cccDNA than other viral makers before and during NAs treatment in HBeAg positive CHB patients.

Voltammetric biosensor for coronavirus spike protein using magnetic bead and screen-printed electrode for point-of-care diagnostics.

The rapid spread of the novel human coronavirus 2019 (COVID-19) and its morbidity have created an urgent need for rapid and sensitive diagnostics. The real-time polymerase chain reaction is the gold standard for detecting the coronavirus in various types of biological specimens. However, this technique is time consuming, labor intensive, and expensive. Screen-printed electrodes (SPEs) can be used as point-of-care devices because of their low cost, sensitivity, selectivity, and ability to be miniaturized. The ability to detect the spike protein of COVID-19 in serum, urine, and saliva was developed using SPE aided by magnetic beads (MBs) and a portable potentiostat. The antibody-peroxidase-loaded MBs were the captured and catalytic units for the electrochemical assays. The MBs enable simple washing and homogenous deposition on the working electrode using a magnet. The assembly of the immunological MBs and the electrochemical system increases the measuring sensitivity and speed. The physical and electrochemical properties of the layer-by-layer modified MBs were systematically characterized. The performance of these immunosensors was evaluated using spike protein in the range 3.12-200 ng mL-1. We achieved a limit of detection of 0.20, 0.31, and 0.54 ng mL-1 in human saliva, urine, and serum, respectively. A facile electrochemical method to detect COVID-19 spike protein was developed for quick point-of-care testing.