Diminished expression of GLS in CD4+ T cells serves as a prognostic indicator associated with cuproptosis in septic patients.

OBJECTIVES: Sepsis is defined as a life-threatening disease associated with a dysfunctional host immune response. Stratified identification of critically ill patients might significantly improve the survival rate. The present study sought to probe molecular markers associated with cuproptosis in septic patients to aid in stratification and improve prognosis. METHODS: We studied expression of cuproptosis-related genes (CRGs) using peripheral blood samples from septic patients. Further classification was made by examining levels of expression of these potential CRGs in patients. Coexpression networks were constructed using the weighted gene coexpression network analysis (WGCNA) method to identify crucial prognostic CRGs. Additionally, we utilized immune cell infiltration analysis to further examine the immune status of septic patients with different subtypes and its association with the CRGs. ScRNA-seq data were also analysed to verify expression of key CRGs among specific immune cells. Finally, immunoblotting, flow cytometry, immunofluorescence, and CFSE analysis were used to investigate possible regulatory mechanisms. RESULTS: We classified septic patients based on CRG expression levels and found significant differences in prognosis and gene expression patterns. Three key CRGs that may influence the prognosis of septic patients were identified. A decrease in GLS expression was subsequently verified in Jurkat cells, accompanied by a reduction in O-GlcNAc levels, and chelation of copper by TTM could not rescue the reduction in GLS and O-GLcNAc levels. Moreover, immoderate chelation of copper was detrimental to mitochondrial function, cell viability and cell proliferation as well as the immune status of the host. CONCLUSION: We have identified novel molecular markers associated with cuproptosis, which could potentially function as diagnostic indicators for septic patients. The reversible nature of the observed alterations in FDX1 and LIAS was demonstrated through copper chelation, while the correlation between copper and the observed changes in GLS requires further investigation.

Lower baseline testosterone level is related to earlier development of castration resistance in metastatic prostate cancer: a multi-center cohort study.

Purpose: In the era of concurrent combination therapy in metastatic hormone sensitive prostate cancer, the impact of the testosterone level before initiating androgen deprivation therapy on treatment outcome is still uncertain. We aimed to investigate its effect on time-to-castration-resistance in a metastatic hormone sensitive prostate cancer cohort. Methods: This is a multi-center retrospective study of 5 databases from China, Japan, Austria and Spain including 258 metastatic hormone sensitive prostate cancer patients with androgen deprivation therapy initiated between 2002 and 2021. Baseline testosterone was divided into high and low groups using 12 nmol/L as cutoff level. Primary outcome was time-to-castration-resistance. Secondary outcomes were survival functions. Kaplan-Meier method was employed to evaluate the correlation between baseline testosterone and time-to-castration-resistance. Subgroup analysis was performed to elucidate the effect of upfront combination-therapy and metastatic volume. Results: Median age was 72 years. Median follow-up time was 31 months. Median pre-treatment prostate-specific-antigen level was 161 ng/mL. Majority of case were graded as International-Society-of-Urological-Pathology grade 5 (63.6%). 57.8% patients had high volume disease and 69.0% received upfront combination treatment. 44.6% of the cohort developed castration-resistance. The low testosterone group demonstrated shorter mean-time-to-castration-resistance (19.0 vs 22.4 months, p=0.031). The variance was more significant in patients without combination therapy (13.2 vs 26.3 months, p=0.015). Cancer-specific and overall survival were inferior in the low baseline testosterone level group without receiving combination therapy (p=0.001). Conclusions: Lower pre-treatment testosterone level is correlated to shorter time-to-castration resistance and worse survival in metastatic prostate cancer patients without upfront combination therapy. Those with low baseline testosterone should be encouraged to adopt combination therapy to delay progression.

Mapping the widespread distribution and transmission dynamics of linezolid resistance in humans, animals, and the environment.

BACKGROUND: The rise of linezolid resistance has been widely observed both in clinical and non-clinical settings. However, there were still data gaps regarding the comprehensive prevalence and interconnections of linezolid resistance genes across various niches. RESULTS: We screened for potential linezolid resistance gene reservoirs in the intestines of both humans and animals, in meat samples, as well as in water sources. A total of 796 bacteria strains out of 1538 non-duplicated samples were identified to be positive for at least one linezolid resistance gene, optrA, poxtA, cfr, and cfr(D). The prevalence of optrA reached 100% (95% CI 96.3-100%) in the intestines of pigs, followed by fish, ducks, and chicken at 77.5% (95% CI 67.2-85.3%), 62.0% (95% CI 52.2-70.9%), and 61.0% (95% CI 51.2-70.0%), respectively. The meat and water samples presented prevalences of 80.0% (95% CI 70.6-87.0%) and 38.0% (95% CI 25.9-51.9%), respectively. The unreported prevalence of the cfr(D) gene was also relatively higher at 13.0% (95% CI 7.8-21.0%) and 19.0% (95% CI 10.9-25.6%) for the feces samples of ducks and pigs, respectively. Enterococci were the predominant hosts for all genes, while several non-enterococcal species were also identified. Phylogenetic analysis revealed a significant genetic distance among linezolid resistance gene reservoirs, with polyclonal structures observed in strains within the same niche. Similar genetic arrays harboring assorted insertion sequences or transposons were shared by reservoirs displaying heterogeneous backgrounds, though large diversity in the genetic environment of linezolid resistance genes was also observed. CONCLUSIONS: The linezolid resistance genes were widespread among various niches. The horizontal transfer played a crucial role in driving the circulation of linezolid resistance reservoirs at the human-animal-environment interfaces. Video Abstract.

Drp1-dependent mitochondrial fragmentation mediates photoreceptor abnormalities in type 1 diabetic retina.

Recent studies have highlighted that retinal neurodegeneration precedes microvascular changes in diabetic retinopathy (DR), but the specific mechanisms remain unclear. Given the pivotal role of dysfunctional mitochondria and oxidative stress in early DR, our objective was to observe mitochondria-related alterations in the neural retina of type one diabetic mellitus mice with no evidence of DR (T1DM-NDR). We aimed to identify the key mitochondrial-related proteins contributing to mitochondrial injury. Our study revealed that T1DM-NDR mice exhibited outer retina thinning, including the ellipsoid zone, inner segment, and outer segment. Additionally, there was an impaired amplitude of the b-wave in electroretinogram (ERG) and a disorganized arrangement of the photoreceptor layer. In both the retina of DM mice and high glucose (HG)-treated 661w cells, mitochondria appeared swollen and fragmented, with disrupted cristae, disorganized or shortened branches in the mitochondrial network, and decreased mitochondrial membrane potential. Among the mitochondrial-related proteins, dynamin-related protein 1 (Drp1) was upregulated, and the ratio of phosphorylated Drp1 protein at serine 616 (S616) and serine 637 (S637) sites significantly increased in the retina of DM mice. The administration of Mdivi-1 ameliorated high-glucose-induced dysfunctional mitochondria, thereby protecting T1DM-NDR mice retina from morphological and functional injuries. Our findings suggest that hyperglycemia promotes Drp1-mediated mitochondrial dysfunction, which may be a significant factor in the development of DR. The inhibition of high-glucose-induced mitochondrial fission emerges as a potential and innovative intervention strategy for preventing DR.

[Effects of stress-induced protein Sestrin2 on necroptosis of dendritic cells induced by lipopolysaccharide].

OBJECTIVE: To investigate the effect of stress-induced protein Sestrin2 (SESN2) on necroptosis of mouse dendritic cell (DC) induced by lipopolysaccharide (LPS) combined with zVAD, a panaspartate-specific cysteine protease (caspase) inhibitor. METHODS: The DC2.4 cell line derived from the bone marrow of mouse in the 3rd to 10th generations was cultured. The cells were stimulated with LPS for 0 hour, 6 hours, 12 hours, and 24 hours, and grouped according to the stimulation time points. Western blotting was performed to determine the protein expression of SESN2 in each group. Overexpression empty lentivirus (NC), SESN2 gene overexpression RNA sequence lentivirus (SESN2 LV-RNA), small interfering empty lentivirus (NS), and SESN2 gene small interfering RNA sequence lentivirus (SESN2 siRNA) were transfected into DC2.4 cells. After 72 hours of transfection, cell fluorescence expression was observed under the inverted fluorescence microscope. Cells in each transfection group were stimulated with LPS for 24 hours. The blank control groups were set up and cultured with phosphate buffered saline (PBS) for 24 hours. Western blotting was performed to measure SESN2 protein expression. In the same groups as above, cells were stimulated with LPS+zVAD for 24 hours. The blank control groups were set up and cultured with PBS for 24 hours. Western blotting was used to determine the expression of mixed lineage kinase domain-like protein (MLKL) and phosphorylated-MLKL (p-MLKL). The p-MLKL levels and the number of positive cells were observed using laser scanning confocal microscopy. The necroptotic cell ratios were assessed by both flow cytometry and Hoechst staining. RESULTS: Compared to the LPS 0 hour group, the expression of SESN2 in the LPS 24 hours group showed a significant increase. Therefore, 24 hours was chosen as the subsequent stimulation time point. After successful lentivirus transduction and 24 hours of cultivation, the MLKL phosphorylation level in the SESN2 siRNA+LPS+zVAD group was significantly higher than that in the NS+LPS+zVAD group. The MLKL phosphorylation in the SESN2 LV-RNA+LPS+zVAD group was significantly lower than that in the NC+LPS+zVAD group. The MLKL phosphorylation levels in both the NS+LPS+zVAD group and the NC+LPS+zVAD group were obviously higher than those in the NS+PBS group and the NC+PBS group, respectively. Laser scanning confocal microscopy showed that the trends in quantity and fluorescence intensity of p-MLKL protein expressions were consistent with the above results. The results from flow cytometry analysis and Hoechst staining showed that the rates of cell necrotic apoptosis in SESN2 siRNA+LPS+zVAD group were significantly higher than those in NS+LPS+zVAD group [flow cytometry analysis: (30.800±1.153)% vs. (20.800±1.114)%, Hoechst staining: (75.267±0.451)% vs. (46.267±3.371)%, both P < 0.05], indicating that knocking down SESN2 further exacerbated the occurrence of necroptosis. The necrotic apoptosis rates in SESN2 LV-RNA+LPS+zVAD group were significantly lower than those in NC+LPS+zVAD group [flow cytometry analysis: (7.160±0.669)% vs. (19.240±2.322)%, Hoechst staining: (32.433±3.113)% vs. (48.567±4.128)%, both P < 0.05], indicating that overexpressing SESN2 reversed such response and markedly reduced the proportion of necroptotic cells compared to the corresponding empty vector group. CONCLUSIONS: SESN2 exhibits an inhibitory effect on necroptosis of DC in sepsis. Targeted SESN2 expression may regulate the process of DC-mediated immune response in sepsis.

The association of islet autoantibodies with the neural retinal thickness and microcirculation in type 1 diabetes mellitus with no clinical evidence of diabetic retinopathy.

OBJECTIVE: To examine the association between islet autoantibodies (IAbs) and the retinal neurovascular changes in type 1 diabetes mellitus (T1DM) with no diabetic retinopathy (NDR). METHODS: This cross-sectional study measured the neural retinal structure and microvascular density of 118 NDR eyes using spectral-domain optical coherence tomography angiography. Retinal structure parameters included retinal thickness (RT), inner retinal thickness (iRT), retina never fibral layer thickness (RNFL thickness), ganglion cell complex thickness (GCC thickness), and loss volume of GCC. Microvascular parameters included vessel density of superficial capillary plexus (sVD), vessel density of deep capillary plexus, and vessel density of choroid capillary plexus. Comparison and correlation analyses of these OCTA parameters were made with various IAbs, including glutamic acid decarboxylase antibody (GADA), tyrosine phosphatase-related islet antigen 2 antibody (IA2A), and zinc transporter 8 antibody (ZnT8A). A general linear model was used to understand the association of IAbs with the retina parameters. RESULTS: The IAb positive (IAbs +) group, which included 85 patients, had thinner RT (235.20 ± 18.10 mm vs. 244.40 ± 19.90 mm at fovea, P = 0.021) and thinner iRT (120.10 ± 9.00 mm vs. 124.70 ± 6.90 mm at parafovea, P = 0.015), compared with the IAb negative (IAbs-) group comprising 33 patients. Furthermore, a more severe reduction of RT was demonstrated in the presence of multiple IAbs. Among the three IAbs, GADA was the most significant independent risk factor of all-round RT decrease (ß = -0.20 vs. -0.27 at fovea and parafovea, respectively, P < 0.05), while titers of IA2A negatively affect sVD in the parafovea (ß = -0.316, P = 0.003). CONCLUSIONS: IAbs are associated with neural retinal thinning and microcirculation reduction in T1DM patients before the clinical onset of diabetic retinopathy.

Identification macrophage signatures in prostate cancer by single-cell sequencing and machine learning.

BACKGROUND: The tumor microenvironment (TME) encompasses a variety of cells that influence immune responses and tumor growth, with tumor-associated macrophages (TAM) being a crucial component of the TME. TAM can guide prostate cancer in different directions in response to various external stimuli. METHODS: First, we downloaded prostate cancer single-cell sequencing data and second-generation sequencing data from multiple public databases. From these data, we identified characteristic genes associated with TAM clusters. We then employed machine learning techniques to select the most accurate TAM gene set and developed a TAM-related risk label for prostate cancer. We analyzed the tumor-relatedness of the TAM-related risk label and different risk groups within the population. Finally, we validated the accuracy of the prognostic label using single-cell sequencing data, qPCR, and WB assays, among other methods. RESULTS: In this study, the TAM_2 cell cluster has been identified as promoting the progression of prostate cancer, possibly representing M2 macrophages. The 9 TAM feature genes selected through ten machine learning methods and demonstrated their effectiveness in predicting the progression of prostate cancer patients. Additionally, we have linked these TAM feature genes to clinical pathological characteristics, allowing us to construct a nomogram. This nomogram provides clinical practitioners with a quantitative tool for assessing the prognosis of prostate cancer patients. CONCLUSION: This study has analyzed the potential relationship between TAM and PCa and established a TAM-related prognostic model. It holds promise as a valuable tool for the management and treatment of PCa patients.

Combined effect of dietary calcium consumption and physical activity on dental caries in children and adolescents: a study of the NHANES database.

BACKGROUND: Calcium (Ca) is a nutritional factor that associated with dental caries. A recent study showed that in the case of adequate Ca intake, a higher level of physical activity may contribute to bone mass accumulation. However, the combined effect between Ca intake and physical activity on caries experience is unclear. Herein, we aimed to explore the above combined effect on dental caries in children and adolescents. METHODS: Data of 5,917 children and adolescents were extracted from the National Health and Nutrition Examination Surveys (NHANES) database in 2015-2020 in this cross-sectional study. The NHANES assessed the dietary Ca intake through the 24-hour dietary recalls, and the physical activity level was self-reported using the questionnaires. Also, the dental caries was diagnosed according to the Decayed, Missing and Filled Teeth/Surfaces (DMFT/S) index. Weighted univariate and multivariate logistic regression analyses were utilized to screen the covariates and to investigate the associations of dietary Ca intake and physical activity with dental caries, respectively, and assess the combined effect between dietary Ca intake and physical activity on dental caries. The evaluation indexes were odd ratios (ORs) and 95% confidence intervals (CIs). Subgroup analyses of age, obesity, and total sugar intake were also performed. RESULTS: Among the eligible participants, 2,687 had caries experience. After adjusting for the covariates, we found that children and adolescents who not reach the recommendation level of Ca intake combined with physical activity less than 7 time in 1 week seemed to have higher odds of dental caries [OR = 1.77, 95%CI: (1.38-2.27)], compared with those who reached the standards. In addition, this potential combined effect was also found in age < 12 years old [OR = 1.62, 95%CI: (1.23-2.14)], non-obesity [OR = 1.88, 95%CI: (1.49-2.35)], and total sugar intake (all P < 0.05) subgroups. CONCLUSIONS: Ca intake and physical activity had a potential combined effect on dental caries in children and adolescents, but the causal relationships between them needed further clarification.

First Report of Alternaria alternata Causing Leaf blight on Epimedium sagittatum (Sieb. et Zucc.) Maxim. in China.

Epimedium sagittatum (Sieb.et Zucc.) Maxim. is an important material of traditional Chinese medicine because of the rich content of flavonoids that are used to treat osteoporosis, liver cancer, and sexual dysfunction (Liu et al. 2013). A leaf blight was observed on E. sagittatum in Zhumadian City, China (32°58'12" N, 114°37'48" E, continental monsoon climate) in June 2021. Survey indicated that about 18% of the plants were infected in a 266-ha commercial planting area. The initial symptoms were white patches with tan borders, irregular in outline, with small black particles visible on the center of the lesions. In a week or so, patches extended throughout the leaf, and then leaves withered. Thirty leaves with symptoms collected from five different sites were cut into 5×5 mm pieces, and then surface-sterilized with 75% ethanol for 15 s followed by rinsing with double distilled water (ddH2O) three times. The pieces were then disinfested with 0.1% HgCl2 solution for 30 s, and rinsed with ddH2O, then placed onto potato-dextrose agar medium (PDA) and incubated in the dark for 3 d at 28°C. Eight fungal isolates were purified; of these, only the isolate HY2-1 infected the host plant and was selected for further morphological characterization. The colonies of HY2-1 were olive green with loose aerial hyphae on PDA. Conidiophores were single or branched, producing brown conidia in short chains. Conidia were obclavate, obpyriform, or ellipsoidal, 15.9-47.3 µm × 7.6-16.6 µm (n=50) and pale brown or dark brown with a short cylindrical beak at the tip that contained 1-5 transverse septa and 0-4 longitudinal septa. Morphological characteristics of the isolate were identical with those of Alternaria species (Huang et al. 2022). For molecular identification, the internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al. 2012), major allergen Alt a 1(Alt a 1) and translation elongation factor 1-α gene (TEF) (Lawrence et al. 2013) were amplified and sequenced using the primers ITS4/5, GDF/GDR, Alt-F/R, and EF1-728F/986R, respectively. The results of the sequencing were uploaded to GenBank as ITS (OR418487), GAPDH (OR419792), Alt a 1 (OR419794), and TEF (OR419796), respectively. Phylogenetic analyses were performed by concatenating all the sequenced loci using the Bayesian method in Phylosuite (Zhang et al.2020). The phylogenetic tree indicated that the isolate belongs to the A. alternata clade with a bootstrap value of 75%. The pathogen was identified as A. alternata based on the morphological and molecular results. To satisfy Koch's postulates, a conidial suspension (106 conidia/mL) of the HY2-1 was prepared with ddH2O to infect the healthy plants. Ninety healthy leaves on 30 plants in pots were punctured using a sterilized needle, and then inoculated by spraying the conidial suspension on the wounded leaves in a greenhouse at 25°C and 80% relative humidity. The control plants were sprayed with ddH2O. The plants showed similar symptoms to the original infected plant 15 d after inoculation. The controls showed no symptoms. A pure culture of A. alternata was isolated and identified again as previously described. Leaf blight caused by A. alternata has been reported on Taro (Liu et al. 2020), Toona ciliata (Wang et al. 2023), etc. To our knowledge, this is the first report of E. sagittatum leaf blight caused by A. alternata in China. The results will help to develop effective control strategies for leaf blight on E. sagittatum.

Phenotypic and genomic characteristics of clinical IMP-producing Klebsiella spp. Isolates in China.

BACKGROUND: IMP-producing Klebsiella spp. (IMPKsp) strains have spread globally, including in China. Currently, the prevalence and genomic characterization of IMPKsp is largely unknown nationwide. Here we aimed to provide a general overview of the phenotypic and genomic characteristics of IMPKsp strains. METHODS: 61 IMPKsp strains were obtained from 13 provinces in China during 2016-2021. All strains were tested for their susceptibility to antimicrobial agents by the microdilution broth method and sequenced with Illumina next-generation sequencing. We performed conjugation experiments on thirteen representative strains which were also sequenced by Oxford nanopore sequencing technology to characterize blaIMP-encoding plasmids. RESULTS: We find that all IMPKsp strains display multidrug-resistant (MDR) phenotypes. All strains belong to 27 different STs. ST307 emerges as a principal IMP-producing sublineage. blaIMP-4 is found to be the major isoform, followed by blaIMP-38. Seven incompatibility types of blaIMP-encoding plasmids are identified, including IncHI5 (32/61, 52.5%), IncN-IncR (10/61, 16.4%), IncFIB(K)-HI1B (7/61, 11.5%), IncN (5/61, 8.2%), IncN-IncFII (2/61, 3.3%), IncFII (1/61, 1.6%) and IncP (1/61, 1.6%). The strains carrying IncHI5 and IncN plasmids belong to diverse ST types, indicating that these two plasmids may play an important role in the transmission of blaIMP genes among Klebsiella spp. strains. CONCLUSIONS: Our results highlight that multi-clonal transmission, multiple genetic environments and plasmid types play a major role in the dissemination process of blaIMP genes among Klebsiella spp. IncHI5 type plasmids have the potential to be the main vectors mediating the spread of the blaIMP genes in Klebsiella spp.

Antibiotic resistance occurs when bacteria evolve to withstand antibiotic drugs. We are aware that a bacteria called Klebsiella is rapidly becoming resistant to carbapenems, a class of broad-spectrum antibiotics. In this study, we conducted a genetic and microbiological surveillance study across 13 provinces of China to understand factors that contribute to the growing bacterial drug resistance. We find that the way the multiple bacterial types interact with each other and swap certain genetic material may be the main cause of growing resistance. These findings call for close monitoring of genetic evolution as a matter of public health management strategy.

Comparison of Short-term and Three-year Oncological Outcomes Between Robotic and Laparoscopic Gastrectomy for Gastric Cancer: A Large Multicenter Cohort Study.

OBJECTIVE: To compare the short-term and long-term outcomes between robotic gastrectomy (RG) and laparoscopic gastrectomy (LG) for gastric cancer. BACKGROUND: The clinical outcomes of RG over LG have not yet been effectively demonstrated. METHODS: This retrospective cohort study included 3599 patients with gastric cancer who underwent radical gastrectomy at eight high-volume hospitals in China from January 2015 to June 2019. Propensity score matching was performed between patients who received RG and LG. The primary end point was 3-year disease-free survival (DFS). RESULTS: After 1:1 propensity score matching, 1034 pairs of patients were enrolled in a balanced cohort for further analysis. The 3-year DFS in the RG and LG was 83.7% and 83.1% ( P =0.745), respectively, and the 3-year overall survival was 85.2% and 84.4%, respectively ( P =0.647). During 3 years of follow-up, 154 patients in the RG and LG groups relapsed (cumulative incidence of recurrence: 15.0% vs 15.0%, P =0.988). There was no significant difference in the recurrence sites between the 2 groups (all P >0.05). Sensitivity analysis showed that RG had comparable 3-year DFS (77.4% vs 76.7%, P =0.745) and overall survival (79.7% vs 78.4%, P =0.577) to LG in patients with advanced (pathologic T2-4a) disease, and the recurrence pattern within 3 years was also similar between the 2 groups (all P >0.05). RG had less intraoperative blood loss, lower conversion rate, and shorter hospital stays than LG (all P >0.05). CONCLUSIONS: For resectable gastric cancer, including advanced cases, RG is a safe approach with comparable 3-year oncological outcomes to LG when performed by experienced surgeons.

Quantitative comparison of three thyroidectomy approaches in neck muscles, voice, and swallowing functions.

OBJECTIVE: This study compares endoscopic thyroidectomy by gasless unilateral axillary approach (ETGUA) and sternocleidomastoid leading-edge approach (SLEA) with conventional open thyroidectomy (COT) in hemithyroidectomy. The main focus is on the protection of neck muscles (sternocleidomastoid, omohyoid, sternothyroid) and the postoperative function of voice and swallowing yielded through these common approaches. METHODS: A total of 302 patients who underwent hemithyroidectomy were enrolled and divided into three groups: ETGUA (n = 101), SLEA (n = 100), and COT (n = 101). Ultrasound was used to measure the thickness of bilateral neck muscles, including the sternocleidomastoid, omohyoid, and sternothyroid. The changes in thickness on the surgical side compared to the non-surgical side. Analyzed factors included muscle thickness changes, Swallowing Impairment Score (SIS), Voice Handicap Index (VHI), Scar Cosmesis Assessment and Rating (SCAR), Neck Injury Index (NII), surgery duration, drainage volume, hospitalization, and number of lymph nodes. RESULTS: The clinical characteristics among the three groups were consistent except for differences in sex, age, and BMI. Metrics such as sternocleidomastoid muscle, NII, hypocalcemia, postoperative PTH, transient hoarseness, and number of lymph nodes showed no significant differences among the three groups. However, significant differences were found in the duration of surgery, drainage volume, hospitalization period omohyoid muscle, Sternohyoid muscle, VHI, SIS, and SCAR (all p < 0.001). CONCLUSION: In comparison to COT, ETGUA and SLEA demonstrate superiority in protecting neck muscles and preserving voice and swallowing function without compromising surgical safety or radicality.

Standardized Iterative Genome Editing Method for Escherichia coli Based on CRISPR-Cas9.

The introduction of complex biosynthetic pathways into the hosts' chromosomes is gaining attention with the development of synthetic biology. While CRISPR-Cas9 has been widely employed for gene knock-in, the process of multigene insertion remains cumbersome due to laborious and empirical gene cloning procedures. To address this, we devised a standardized iterative genome editing system for Escherichia coli, harnessing the power of CRISPR-Cas9 and MetClo assembly. This comprehensive toolkit comprises two fundamental elements based on the Golden Gate standard for modular assembly of sgRNA or CRISPR arrays and donor DNAs. We achieved a gene insertion efficiency of up to 100%, targeting a single locus. Expression of tracrRNA using a strong promoter enhances multiplex genomic insertion efficiency to 7.3%, compared with 0.76% when a native promoter is used. To demonstrate the robust capabilities of this genome editing toolbox, we successfully integrated 5-10 genes from the coenzyme B12 biosynthetic pathway ranging from 5.3 to 8 Kb in length into the chromosome of E. coli chassis cells, resulting in 14 antibiotic-free, plasmid-free producers. Following an extensive screening process involving genes from diverse sources, cistronic design modifications, and chromosome repositioning, we obtained a recombinant strain yielding 1.49 mg L-1 coenzyme B12, the highest known titer achieved by using E. coli as the producer. Illuminating its user-friendliness, this genome editing system is an exceedingly versatile tool for expediently integrating complex biosynthetic pathway genes into hosts' genomes, thus facilitating pathway optimization for chemical production.

First Report of Colletotrichum fructicola Causing Anthracnose on Epimedium sagittatum (Sieb. et Zucc.) Maxim. in China.

Epimedium sagittatum (Sieb.et Zucc.) Maxim., belonging to the family Berberidaceae and genus Epimedium, is a perennial herb widely studied for its anti-osteoporosis, anti-cancer, and anti-sexual-dysfunction effects in Asian countries (Tan et al. 2016; Zhang et al. 2016). High levels of bioactive chemicals in Epimedium spp. has endowed it with important clinical and commercial values (Liu et al. 2013). In September 2021, a leaf disease was found in Zhumadian City, China (32°58'12" N, 114°37'48" E). Survey statistics indicated that disease prevalence in a 266-ha planting area was approximately 29.6%. The lesions appeared at the leaf tips, gradually enlarged, and were brown with a yellow halo. Further, the lesions were dry with distributed black spots. Thirty infected leaves collected from five sites within the planting base . The collected leaves were cut into 5×5 mm pieces , surface-sterilized in 75% alcohol for 15 s, triple washed with sterile ddH2O, disinfested with 0.1% HgCl2 solution for 30 s (Liu et al. 2021), triple washed again with sterilized ddH2O, and then placed onto PDA and incubated in the dark for 3 d at 28°C. Subsequently, five fungal strains were purified; among them, only the isolate HY3-2 infected the host plant and was selected for further morphological characterization. The colonies of HY3-2 initially appeared white, their mycelia became gray at the center after 4 d, and orange-red conidial clumps appeared in them after 7 d. Conidia (10.0-19.5 µm × 4.5-5.6 µm, n=50) were single celled, nearly spherical or stick-shaped and colorless. Morphological characteristics of the isolate were consistent with those of Colletotrichum species. Additionally, glycerol-3-phosphate dehydrogenase (gapdh), actin (act), calmodulin (cal), ß-tubulin 2 (tub2), and chitin synthase-1 (chs-1), (Weir et al. 2012) were amplified and sequenced using the primers GDF/GDR, ACT-512F/783R, CL1C/CL2C, T1/Bt2b, and CHS-79F/354R, respectively for molecular identification. The resulting sequences were deposited in GenBank: gapdh (ON351609), act (ON351608), tub2 (ON351610), chs-1 (ON532788), and cal (ON532787). Phylogenetic analyses were performed by concatenating all the sequenced loci using the Bayesian method (Zhang et al. 2020). The phylogenetic tree showed that the isolate belongs to C. fructicola clade with a credibility value of 85%.To satisfy Koch's postulates, a conidial suspension (106 conidia/mL) of the isolate HY3-2 were prepared with sterile ddH2O to infect the leaves. Ninety healthy leaves from 30 plants in pots were punctured using a sterilized needle (Huang et al. 2022), and inoculated by spraying the conidial suspension on the leaves in a greenhouse at 25°C and 80% relative humidity. In the control plants, the suspension was replaced with water. After 7 d, the inoculated plants showed symptoms similar to those of the original infected plant, whereas the control showed no symptoms. C. fructicola was isolated and identified again as previously described. A pathogenicity test was also conducted in the field using the same method as that used in the greenhouse in July 2022, the results of which were consistent with those of the greenhouse. In China, C. fructicola has been reported on Walnut (Wang et al. 2022), Punica granatum (Hu et al. 2023) and others. To our knowledge, this is the first report of C. fructicola causing anthracnose in E. sagittatum in China. This report provides an important basis for further disease control research.

Experimental study of modified Tavis-Cummings model with directly-coupled superconducting artificial atoms.

The Tavis-Cummings model is intensively investigated in quantum optics and has important applications in generation of multi-atom entanglement. Here, we employ a superconducting circuit quantum electrodynamic system to study a modified Tavis-Cummings model with directly-coupled atoms. In our device, three superconducting artificial atoms are arranged in a chain with direct coupling through fixed capacitors and strongly coupled to a transmission line resonator. By performing transmission spectrum measurements, we observe different anticrossing structures when one or two qubits are resonantly coupled to the resonator. In the case of the two-qubit Tavis-Cummings model without qubit-qubit interaction, we observe two dips at the resonance point of the anticrossing. The splitting of these dips is determined by Δ λ=2g12+g32, where g1 and g3 are the coupling strengths between Qubit 1 and the resonator, and Qubit 3 and the resonator, respectively. The direct coupling J12 between the two qubits results in three dressed states in the two-qubit Tavis-Cummings model at the frequency resonance point, leading to three dips in the transmission spectrum. In this case, the distance between the two farthest and asymmetrical dips, arising from the energy level splitting, is larger than in the previous case. The frequency interval between these two dips is determined by the difference in eigenvalues (Δ λ=ε 1+-ε 1-), obtained through numerical calculations. What we believe as novel and intriguing experimental results may potentially advance quantum optics experiments, providing valuable insights for future research.

A computed tomography-based comprehensive standardized adrenal tumor scoring model for predicting the perioperative outcomes of retroperitoneal laparoscopic adrenal surgery.

Background: Many imaging scoring models have been developed for tumor surgery to provide critical guidance for the selection of surgical methods. However, little research has been aimed at developing scoring models for adrenal tumors and retroperitoneal laparoscopic adrenal surgery (RLAS), which has become the primary technique for treating adrenal tumors. The study set out to establish a computed tomography (CT)-based adrenal tumor scoring model for predicting perioperative outcomes in patients with adrenal tumors who have undergone RLAS. Methods: The retrospective analysis included 306 patients with adrenal tumors diagnosed by preoperative unenhanced or enhanced CT from January 2014 to August 2018 in the First Affiliated Hospital of Fujian Medical University. CT images were used to quantify the tumor location and size; the relationships of the tumors with the surrounding organs and tissues, the large abdominal blood vessels, and the upper poles of the kidneys and renal hila; the adhesion of periadrenal fat (PF); and the tumor CT enhancement value. We conducted multivariate ordinal logistic regression analysis to screen variables and performed principal component analysis to construct a novel scoring model for RLAS. The perioperative outcomes of RLAS were evaluated according to postoperative length of stay, operative time (OT), intraoperative blood loss (IBL), and postoperative complications. Results: The final scoring model included tumor size; the relationships of the tumors with the surrounding organs and tissues, the large abdominal blood vessels, and the upper poles of the kidneys and renal hila; the tumor CT enhancement value; the adhesion of the PF; and the functional status of adrenal tumors. The total score had positive correlations with the OT (rs=0.431), IBL (rs=0.446), and postoperative length (rs=0.180) (all P values <0.001). Compared to any single metric, the total score provided better prediction of OT and IBL. The grading system for RLAS based on the scoring model also performed well in predicting the complexity and difficulty of RLAS. The coincidence rate for these factors was good (all P values <0.001). Conclusions: The developed model is feasible and repeatable in the prediction of the perioperative outcomes, complexity, and difficulty of RLAS.

Pharmacologically significant constituents collectively responsible for anti-sepsis action of XueBiJing, a Chinese herb-based intravenous formulation.

Sepsis, a life-threatening health issue, lacks effective medicine targeting the septic response. In China, treatment combining the intravenous herbal medicine XueBiJing with conventional procedures reduces the 28-day mortality of critically ill patients by modulating septic response. In this study, we identified the combined active constituents that are responsible for the XueBiJing's anti-sepsis action. Sepsis was induced in rats by cecal ligation and puncture (CLP). The compounds were identified based on their systemic exposure levels and anti-sepsis activities in CLP rats that were given an intravenous bolus dose of XueBiJing. Furthermore, the identified compounds in combination were assessed, by comparing with XueBiJing, for levels of primary therapeutic outcome, pharmacokinetic equivalence, and pharmacokinetic compatibility. We showed that a total of 12 XueBiJing compounds, unchanged or metabolized, circulated with significant systemic exposure in CLP rats that received XueBiJing. Among these compounds, hydroxysafflor yellow A, paeoniflorin, oxypaeoniflorin, albiflorin, senkyunolide I, and tanshinol displayed significant anti-sepsis activities, which involved regulating immune responses, inhibiting excessive inflammation, modulating hemostasis, and improving organ function. A combination of the six compounds, with the same respective doses as in XueBiJing, displayed percentage survival and systemic exposure in CLP rats similar to those by XueBiJing. Both the combination and XueBiJing showed high degrees of pharmacokinetic compatibility regarding interactions among the six active compounds and influences of other circulating XueBiJing compounds. The identification of XueBiJing's pharmacologically significant constituents supports the medicine's anti-sepsis use and provides insights into a polypharmacology-based approach to develop medicines for effective sepsis management.

Emergence of an ST1934:KL121 hypervirulent Klebsiella pneumoniae carrying a novel virulence-resistance hybrid plasmid with chromosomal integration of ICEKp1.

To identify the phenotypic and genomic characteristics of K. pneumoniae KP43 from bloodstream infection. KP43 was resistant to ticarcillin and tetracycline and was hypervirulent in the Galleria mellonella larvae infection model, positive for string test, and possessed high-level macrophage killing resistance. The hypervirulence phenotype was associated with the chromosome integration of ICEKp1 carrying iroBCDN-iroP, rmpADC, and peg-344, and a novel plasmid pKP43_vir_amr harboring iutAiucABCD. pKP43_vir_amr was an IncFIBκ/FII virulence-resistance hybrid conjugative plasmid which also carried antibiotic resistance genes. The emergence of such a strain and the spread of the novel virulence-resistance plasmid might pose a potential threat to public health.