Highly Efficient Photodynamic Hydrogel with AIE-Active Photosensitizers toward Methicillin-Resistant Staphylococcus aureus Ultrafast Imaging and Killing.

Methicillin-resistant Staphylococcus aureus (MRSA) causes great health hazards to society because most antibiotics are ineffective. Photodynamic treatment (PDT) has been proposed to combat MRSA due to the advantage of imaging-guided no-drug resistance therapy. However, the traditional photosensitizers for PDT are limited by aggregation-caused quenching for imaging and low photodynamic antibacterial efficiency. In this work, we synthesize a new aggregation-induced emission (AIE) photosensitizer (APNO), which can ultrafast distinguish between Gram-positive and Gram-negative bacteria within 3 s by AIE-active photosensitizer imaging. Meanwhile, APNO can generate antibacterial reactive oxygen species under light irradiation, which holds potential for antibacterial PDT. Then, APNO is loaded by PHEAA hydrogel to obtain a highly efficient photodynamic hydrogel (APNO@gel). In vitro results show complete inhibition of MRSA by APNO@gel under lower-power light irradiation. Transcriptome analysis is performed to investigate antibacterial mechanism of APNO@gel. Most importantly, APNO@gel also exhibits significant inhibition and killing ability of MRSA in the MRSA wound infection model, which will further promote rapid wound healing. Therefore, the photodynamic hydrogel provides a promising strategy toward MRSA ultrafast imaging and killing.

Automatic Detection of Scalp High-Frequency Oscillations Based on Deep Learning.

Scalp high-frequency oscillations (sHFOs) are a promising non-invasive biomarker of epilepsy. However, the visual marking of sHFOs is a time-consuming and subjective process, existing automatic detectors based on single-dimensional analysis have difficulty with accurately eliminating artifacts and thus do not provide sufficient reliability to meet clinical needs. Therefore, we propose a high-performance sHFOs detector based on a deep learning algorithm. An initial detection module was designed to extract candidate high-frequency oscillations. Then, one-dimensional (1D) and two-dimensional (2D) deep learning models were designed, respectively. Finally, the weighted voting method is used to combine the outputs of the two model. In experiments, the precision, recall, specificity and F1-score were 83.44%, 83.60%, 96.61% and 83.42%, respectively, on average and the kappa coefficient was 80.02%. In addition, the proposed detector showed a stable performance on multi-centre datasets. Our sHFOs detector demonstrated high robustness and generalisation ability, which indicates its potential applicability as a clinical assistance tool. The proposed sHFOs detector achieves an accurate and robust method via deep learning algorithm.

Predictive value of CD86 for the occurrence of sepsis (Sepsis-3) in patients with infection.

This prospective observational study explored the predictive value of CD86 in the early diagnosis of sepsis in the emergency department. The primary endpoint was the factors associated with a diagnosis of sepsis. The secondary endpoint was the factors associated with mortality among patients with sepsis. It enrolled inpatients with infection or high clinical suspicion of infection in the emergency department of a tertiary Hospital between September 2019 and June 2021. The patients were divided into the sepsis and non-sepsis groups according to the Sepsis-3 standard. The non-sepsis group included 56 patients, and the sepsis group included 65 patients (19 of whom ultimately died). The multivariable analysis showed that CD86% (odds ratio [OR] = 1.22, 95% confidence interval [CI]: 1.04-1.44, P = 0.015), platelet count (OR = 0.99, 95%CI: 0.986-0.997, P = 0.001), interleukin-10 (OR = 1.01, 95%CI: 1.004-1.025, P = 0.009), and procalcitonin (OR = 1.17, 95%CI: 1.01-1.37, P = 0.043) were independent risk factors for sepsis, while human leukocyte antigen (HLA%) (OR = 0.96, 05%CI: 0.935-0.995, P = 0.022), respiratory rate (OR = 1.16, 95%CI: 1.03-1.30, P = 0.014), and platelet count (OR = 1.01, 95%CI: 1.002-1.016, P = 0.016) were independent risk factors for death in patients with sepsis. The model for sepsis (CD86%, platelets, interleukin-10, and procalcitonin) and the model for death (HLA%, respiratory rate, and platelets) had an area under the curve (AUC) of 0.870 and 0.843, respectively. CD86% in the first 24 h after admission for acute infection was independently associated with the occurrence of sepsis in the emergency department.

Effect of silencing the E74B gene on the development and metamorphosis of Helicoverpa armigera.

BACKGROUND: The growth and development transition of insects are mainly mediated by ecdysone. As one of the ecdysone-induced transcription factors, E74 is involved in many physiological processes of insect growth and development. However, E74 and its function in Helicoverpa armigera remains unclear. RESULTS: In this study, E74B, a subtype of the E74, was identified for the first time in H. armigera. Bioinformatics analysis showed that H. armigera E74B shared the highest homology with E74B in Bombyx mori, which belongs to the E26 transformation-specific (ETS) superfamily. The expression profile showed that the transcription level of HaE74B increased in the late stages of fourth to sixth instars compared with the early stages; it was also high in the pupa and midgut. Moreover, we investigated the function of HaE74B through RNA interference and 20E rescue experiments. The results showed silencing of E74B affected the molting and growth of larvae, resulting in the death of more than 60% of larvae. In addition, it also seriously affected the metamorphosis of H. armigera, which reduced the pupae rate, the eclosion rate of the pupae, and fecundity. Application of 20E partially restored the defects in the molting, development and pupae rate of H. armigera. CONCLUSION: Taken together, these results demonstrated that HaE74B plays a critical role in the growth, development, and metamorphosis of H. armigera, which serves as a molecular target and sets out a theoretical foundation for RNAi-mediated control of this key pest. © 2023 Society of Chemical Industry.

mRNA sequencing reveals the distinct gene expression and biological functions in cardiac fibroblasts regulated by recombinant fibroblast growth factor 2.

After myocardial injury, cardiac fibroblasts (CFs) differentiate into myofibroblasts, which express and secrete extracellular matrix (ECM) components for myocardial repair, but also promote myocardial fibrosis. Recombinant fibroblast growth factor 2 (FGF2) protein drug with low molecular weight can promote cell survival and angiogenesis, and it was found that FGF2 could inhibit the activation of CFs, suggesting FGF2 has great potential in myocardial repair. However, the regulatory role of FGF2 on CFs has not been fully elucidated. Here, we found that recombinant FGF2 significantly suppressed the expression of alpha smooth muscle actin (α-SMA) in CFs. Through RNA sequencing, we analyzed mRNA expression in CFs and the differently expressed genes regulated by FGF2, including 430 up-regulated genes and 391 down-regulated genes. Gene ontology analysis revealed that the differentially expressed genes were strongly enriched in multiple biological functions, including ECM organization, cell adhesion, actin filament organization and axon guidance. The results of gene set enrichment analysis (GSEA) show that ECM organization and actin filament organization are down-regulated, while axon guidance is up-regulated. Further cellular experiments indicate that the regulatory functions of FGF2 are consistent with the findings of the gene enrichment analysis. This study provides valuable insights into the potential therapeutic role of FGF2 in treating cardiac fibrosis and establishes a foundation for further research to uncover the underlying mechanisms of CFs gene expression regulated by FGF2.

EFFECTS OF SHENFU INJECTION ON SUBLINGUAL MICROCIRCULATION IN SEPTIC SHOCK PATIENTS: A RANDOMIZED CONTROLLED TRIAL.

ABSTRACT: Background and Objective: The optimization of macrocirculatory hemodynamics is recommended by current sepsis guidelines. However, microcirculatory dysfunction is considered the cause of severe sepsis. In the present study, we designed to verify whether the application of Shenfu injection (SFI) restores microcirculation, thereby improving tissue perfusion and inhibiting organ dysfunction, resulting in improved outcomes. Design: We conducted a prospective, single-center, randomized, double-blind, placebo-controlled clinical trial. Intervention: Patients were randomly assigned to group receiving SFI (n = 20) or placebo (n = 20) for 5 days. We administered SFI or glucose injection for 5 days and blinded the investigators and clinical staff by applying light-proof infusion equipment that concealed therapy allocation. Measurements and Results: We measured the systemic dynamics and lactate levels, biomarkers of endothelial dysfunction, and inflammatory cytokines in the plasma. The parameters of sublingual microcirculation were assessed using side-stream dark-field imaging. Sequential Organ Failure Assessment (SOFA) score, Acute Physiology and Chronic Health Evaluation (APACHE) score, total dose, and duration of vasopressor use, emergency intensive care unit (EICU) stay, and 28-day mortality were evaluated. After treatment with SFI, the disturbance of the sublingual microcirculation was considerably alleviated, as indicated by the significant increase in total vessel density, perfused vessel density, and microvascular flow index. Moreover, the plasma biomarker levels of endothelial dysfunction, including Ang-2, Syn-1, and ET-1, were reversed after SFI treatment. Importantly, the SFI group had a more favorable prognosis than the control group in terms of the APACHE-II score, SOFA score, duration of vasopressor administration, and length of EICU stay. However, the difference in mortality at day 28 was not statistically different between the SFI (15%, 3/20) and placebo (25%, 5/20) groups ( P = 0.693). Conclusions : Shenfu injection provided apparent effects in improving sublingual microcirculatory perfusion in patients with septic shock, and this protection may be related with the inhibition of endothelial dysfunction and vasodilatory effects.

Clinical characteristics and related risk factors of disease severity in 101 COVID-19 patients hospitalized in Wuhan, China.

Coronavirus disease 2019 (COVID-19) broke out in December 2019. Due its high morbility and mortality, it is necessary to summarize the clinical characteristics of COVID-19 patients to provide more theoretical basis for future treatment. In the current study, we conducted a retrospective analysis of the clinical characteristics of COVID-19 patients and explored the risk factors for the severity of illness. A total of 101 COVID-19 patients hospitalized in Leishenshan Hospital (Wuhan, China) was classified into three sub-types: moderate (n = 47), severe (n = 36), and critical (n = 18); their clinical data were collected from the Electronic Medical Record. We showed that among the 101 COVID-19 patients, the median age was 62 years (IQR 51-74); 50 (49.5%) patients were accompanied by hypertension, while 25 (24.8%) and 22 (21.8%) patients suffered from diabetes and heart diseases, respectively, with complications. All patients were from Wuhan who had a definite history of exposure to the epidemic area. Multivariate logistic regression analysis revealed that older age, diabetes, chronic liver disease, percentage of neutrophils (N%) > 75%, CRP > 4 mg/L, D-dimer > 0.55 mg/L, IL-2R > 710 U/mL, IL-8 > 62 pg/mL, and IL-10 > 9.1 pg/mL were independent variables associated with severe COVID-19. In conclusion, we have identified the independent risk factors for the severity of COVID-19 pneumonia, including older age, diabetes, chronic liver disease, higher levels of N%, CRP, D-dimer, IL-2R, IL-8, and IL-10, providing evidence for more accurate risk prediction.

Hedgehog signalling controls sinoatrial node development and atrioventricular cushion formation.

Smoothened is a key receptor of the hedgehog pathway, but the roles of Smoothened in cardiac development remain incompletely understood. In this study, we found that the conditional knockout of Smoothened from the mesoderm impaired the development of the venous pole of the heart and resulted in hypoplasia of the atrium/inflow tract (IFT) and a low heart rate. The blockage of Smoothened led to reduced expression of genes critical for sinoatrial node (SAN) development in the IFT. In a cardiac cell culture model, we identified a Gli2-Tbx5-Hcn4 pathway that controls SAN development. In the mutant embryos, the endocardial-to-mesenchymal transition (EndMT) in the atrioventricular cushion failed, and Bmp signalling was downregulated. The addition of Bmp2 rescued the EndMT in mutant explant cultures. Furthermore, we analysed Gli2+ scRNAseq and Tbx5-/- RNAseq data and explored the potential genes downstream of hedgehog signalling in posterior second heart field derivatives. In conclusion, our study reveals that Smoothened-mediated hedgehog signalling controls posterior cardiac progenitor commitment, which suggests that the mutation of Smoothened might be involved in the aetiology of congenital heart diseases related to the cardiac conduction system and heart valves.

Clinical characteristics and risk factors for mortality in patients with coronavirus disease 2019 in intensive care unit: a single- center, retrospective, observational study in China.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a potentially life-threatening contagious disease which has spread all over the world. Risk factors associated with the clinical outcomes of COVID-19 pneumonia in intensive care unit (ICU) have not yet been well determined. METHODS: This was a retrospective, single-centered, observational study, in which 47 patients with confirmed COVID-19 were consecutively enrolled from February 24 to April 5, 2020. The patients were registered from the ICU of Leishenshan Hospital in Wuhan, China. Clinical characteristics and outcomes were collected and compared between survivors and non-survivors. Multivariable logistic regression was performed to analyze the risk factors of death in patients with COVID-19. RESULTS: The study cohort included 47 adult patients with an average age of 70.55±12.52 years, and 30 (63.8%) patients were men. Totally 15 (31.9%) patients died. When compared to survivors, nonsurvivors showed a higher proportion of septic shock [6 (40%) patients vs. 3 (9.4%) patients], disseminated intravascular coagulation [3 (21.4%) vs. 0], and had higher score of APACHE II (25.07±8.03 vs. 15.56±5.95), CURB-65 {3 [2-4] vs. 2 [1-3]}, Sequential Organ Failure Assessment (SOFA) {7 [5-9] vs. 3 [1-6]}, higher level of D-dimer {5.74 [2.32-18] vs 2.05 [1.09-4.00]} and neutrophil count {9.4 [7.68-14.54] vs. 5.32 [3.85-9.34]}. SOFA score (OR 1.47; 95% CI: 1.01-2.13; P=0.0042) and the lymphocyte count (OR 0.02; 95% CI: 0.00-0.86; P=0.042) on admission were independently risk factors for mortality. Patients with higher lymphocyte count (>0.63×109 /L) and lower SOFA score (≤4) on admission had a significantly better prognosis than those with lower lymphocyte count (≤0.63×109 /L) and higher SOFA score (>4) in overall survival. CONCLUSIONS: Higher SOFA score and lower lymphocyte count at admission were connected with poor prognosis of patients with COVID-19 in ICU. Lymphocyte count may serve as a promising prognostic biomarker.

The features comparison between patients in the ICU and general wards and between patients with different outcomes: a 2020 COVID-19 study.

BACKGROUND: The novel 2019 coronavirus (COVID-19) has largely abated in China; however, sporadic or imported cases are still a concern, while in other countries, the COVID-19 pandemic persists as a major health crisis. METHODS: All patients enrolled in this study were diagnosed with COVID-19 from February 21, 2020 to April 14, 2020 in Wuhan. We retrospectively analyzed the patients admitted to the ICU (137 patients) and general wards (114 patients) of Wuhan Leishenshan Hospital in China. The population characteristics, symptoms, and laboratory examination results between the patients in the ICU and those in the general wards were compared. Furthermore, the differences between the deceased patients in the ICU and those discharged from the ICU were compared. RESULTS: There were significant differences between the two groups in terms of symptoms, including fever, shortness of breath, no presence of complications, presence of 1 complication, and presence of 3 or more complications (P<0.05). There were also significant differences between the patients in terms of the laboratory examination results including elevated urea nitrogen, creatinine, direct bilirubin, aspartate aminotransferase, total protein, albumin, creatine kinase, lactate dehydrogenase, procalcitonin, erythrocyte sedimentation rate, white blood cells, C-reactive protein, prothrombin time, activated partial thromboplastin time, fibrinogen, D-dimer, interleukin 6, interleukin 8, interleukin 10, interleukin 2 receptor, tumor necrosis factor-α, troponin I, phosphokinase isoenzyme-MB, and B-type natriuretic peptide; and decreased platelets, lymphocyte absolute value, and eosinophil absolute value (<0.05). There were 45 patients who died in ICU and 57 improved and discharged patients. There were significant differences between the two groups in the number of patients that had 1 complication and 3 or more complications (P<0.05). There were also significant differences in the laboratory examination results between the patients including elevated urea nitrogen, total bilirubin, direct bilirubin, aspartate aminotransferase, procalcitonin, white blood cells, interleukin 8, interleukin 10, phosphokinase isoenzyme-MB, and B-type natriuretic peptide; and decreased platelets and eosinophil absolute value (P<0.05). CONCLUSIONS: Our findings highlight that the identified determinants may help to improve treatment of COVID-19 patients, to predict the risk of developing severe illness and to optimizing arrangement of health resources.

CRISPR-Assisted DNA Detection: A Novel dCas9-Based DNA Detection Technique.

Nucleic acid detection techniques are always critical to diagnosis, especially in the background of the present coronavirus disease 2019 pandemic. Simple and rapid detection techniques with high sensitivity and specificity are always urgently needed. However, current nucleic acid detection techniques are still limited by traditional amplification and hybridization. To overcome this limitation, here we developed CRISPR-Cas9-assisted DNA detection (CADD). In this detection, a DNA sample is incubated with a pair of capture single guide RNAs (sgRNAs; sgRNAa and sgRNAb) specific to a target DNA, dCas9, a signal readout-related probe, and an oligo-coated solid support beads or microplate at room temperature (RT) for 15 min. During this incubation, the dCas9-sgRNA-DNA complex is formed and captured on solid support by the capture sequence of sgRNAa, and the signal readout-related probe is captured by the capture sequence of sgRNAb. Finally, the detection result is reported by a fluorescent or colorimetric signal readout. This detection was verified by detecting DNA of bacteria, cancer cells, and viruses. In particular, by designing a set of sgRNAs specific to 15 high-risk human papillomaviruses (HPVs), the HPV infection in 64 clinical cervical samples was successfully detected by the method. All detections can be finished in 30 min at RT. This detection holds promise for rapid on-the-spot detection or point-of-care testing.

The moss flavone synthase I positively regulates the tolerance of plants to drought stress and UV-B radiation.

Flavonoids are extensively distributed secondary metabolites in land plants. They play a critical role in plant evolution from aquatic to terrestrial and plant adaption to ultraviolet radiation. However, the downstream branching pathway of flavonoids and its regulatory mechanism in bryophytes, which are the most ancient of terrestrial plants, remain unclear. Here, a type I flavone synthase (PnFNSI) was characterized from the Antarctic moss Pohlia nutans. PnFNSI was primarily distributed in the cytoplasm, as detected by subcellular localization. PnFNSI could catalyze the conversion of naringenin to apigenin with an optimal temperature between 15 and 20 °C in vitro. Overexpression of PnFNSI in Arabidopsis alleviated the growth restriction caused by naringenin and accumulated apigenin product. PnFNSI-overexpressing plants showed enhanced plant tolerance to drought stress and UV-B radiation. PnFNSI also increased the enzyme activities and gene transcription levels of reactive oxygen species (ROS) scavengers, protecting plants against oxidative stress. Moreover, overexpression of PnFNSI enhanced the flavone biosynthesis pathway in Arabidopsis. Therefore, this moss FNSI-type enzyme participates in flavone metabolism, conferring protection against drought stress and UV-B radiation.

STAPLAg: a convenient early warning score for use in infected patients in the intensive care unit.

Sepsis is a life-threatening disease in the intensive care unit (ICU). The current diagnostic criteria for sequential organ failure assessment (SOFA) scores do not reflect the current understanding of sepsis. We developed a novel and convenient score to aid early prognosis.Retrospective multivariable regression analysis of 185 infected emergency ICU (EICU) patients was conducted to identify independent variables associated with death, to develop the new "STAPLAg" score; STAPLAg was then validated in an internal cohort (n = 106) and an external cohort (n = 78) and its predictive efficacy was compared with that of the initial SOFA score.Age, and initial serum albumin, sodium, PLR, troponin, and lactate tests in the emergency department were independent predictors of death in infected EICU patients, and were used to establish the STAPLAg score (area under the curve [AUC] 0.865). The initial SOFA score on admission was predictive of death (AUC 0.782). Applying the above categories to the derivation cohort yielded mortality risks of 7.7% for grade I, 56.3% for grade II, and 75.0% for grade III. Internal (AUC 0.884) and external (AUC 0.918) cohort validation indicated that the score had good predictive power.The STAPLAg score can be determined early in infected EICU patients, and exhibited better prognostic capacity than the initial SOFA score on admission in both internal and external cohorts. STAPLAg constitutes a new resource for use in the clinical diagnosis of sepsis and can also predict mortality in infected EICU patients. REGISTRATION NUMBER:: ChinCTR-PNC-16010288.

Fast DNA Extraction with Polyacrylamide Microspheres for Polymerase Chain Reaction Detection.

The fast and cost-effective DNA extraction is critical for all DNA-based detections. Here, we fabricated a new kind of polyacrylamide microsphere (PAMMP) in various sizes with two methods, spot polymerization (large size but low yield) and modified inverse microemulsion polymerization (small size but high yield). The fabricated PAMMPs have strong autofluorescence (fPAMMPs), including both visible fluorescence (VF) and near-infrared fluorescence (NIRF), which can remain very stable in various stringent conditions including strong acid and alkali and high temperature. The fabricated fPAMMPs were also highly positively charged, which could be used to effectively capture various biomolecules such as IRDye 800-labeled streptavidin and DNA. We thus developed a new method for rapid extraction (3-5 min) of DNA from various samples including bacteria, mammalian cells, plant and animal solid tissues, and human blood plasma using fPAMMPs. Moreover, the DNA captured on fPAMMPs (fPAMMP@DNA) could be effectively detected by both normal and quantitative PCR amplifications. Finally, we showed that NaBH4 treatment removed autofluorescence in fPAMMPs (PAMMPs), which could also be applied to DNA extraction and PCR detection. In conclusion, we here fabricated new kinds of fPAMMPs and PAMMPs, developed a new rapid DNA extraction method, and demonstrated their useful applications in PCR detection.

Tanshinone IIA Ameliorates Streptozotocin-Induced Diabetic Nephropathy, Partly by Attenuating PERK Pathway-Induced Fibrosis.

PURPOSE: Tanshinone IIA (Tan IIA), a compound extracted from Salvia miltiorrhiza, can improve type II diabetes, while the molecular mechanisms underlying Tan IIA-mediated protective effects in diabetic nephropathy are unclear. This study explored the protective actions of Tan IIA on renal tissues in streptozotocin (STZ)-induced diabetic nephropathy. MATERIALS AND METHODS: Tan IIA (2, 4, 8 mg/kg/day) was daily administered to STZ-treated rats by intraperitoneal injection for 42 days. The morphologic pathology was evaluated by hematoxylin-eosin and Masson's trichrome staining, and transmission electron microscopy. The protein expression levels in renal tissues were evaluated by Western blotting and immunohistochemistry; the mRNA expression level was determined by quantitative real-time PCR. RESULTS: Tan IIA at 2 and 4 mg/kg attenuated the increase in the levels of uric acid and blood urea nitrogen and restored the reduction in the superoxide dismutase activity in the serum of the diabetic rats. Tan IIA at 2 and 4 mg/kg, but not 8 mg/kg, ameliorated the thickening of renal tubule in the diabetic rats; Tan IIA at 2 and 4 and 8 mg/kg attenuated the thickening of glomerular basement membrane and the collagen deposition in the renal tissues of the diabetic rats. Tan IIA treatment at 2, 4, 8 mg/kg decreased the expression levels of transforming growth factor-beta1, TSP-1, Grp78 and CHOP in the diabetic rats. Tan IIA at 2 and 4 and 8 mg/kg attenuated the increase in the protein levels of p-PERK, p-elf2α and ATF-4 from the renal tissues of diabetic rats, while the protein level of AFT-6 and the mRNA expression levels of XBP-1t, XBP-1s and p58IPK in the renal tissues were not affected by STZ or Tan IIA treatment. CONCLUSION: Tan IIA-mediated protective effects on the STZ-induced diabetic nephropathy may be associated with the reduced endoplasmic reticulum stress via attenuating PERK signaling activities.

Predictive role of monocyte-to-lymphocyte ratio in patients with Klebsiella pneumonia infection: A single-center experience.

The aim of the study is to explore whether monocyte-to-lymphocyte ratio (MLR) provides predictive value of the severity in patients with Klebsiella pneumonia infection (KPI).Patients in a tertiary medical center with Klebsiella pneumonia infection from 2014 to 2017 were recruited in this study. Patients with Klebsiella pneumonia infection were stratified into two groups based on the National Early Warning Score (NEWS). MLR was calculated by dividing monocytes count by lymphocytes count obtained from routine blood examination. The area under the curve (AUC) values was determined using the receiver-operating characteristic (ROC) curve. The correlation between the variables was tested with Pearson or Spearman correlation analysis. Ordinal logistic regression analysis was used to assess the relationship between MLR and the severity of Klebsiella pneumonia infection.One hundred fifty-two patients were finally enrolled for analysis. Among those, 43 (28.29%) cases had severe KPI. MLR was found to be an independent risk factor of the serious Klebsiella pneumonia infection (OR: 23.74, 95% CI: 5.41-104.11, P < .001). Besides, MLR was positively correlated with NEWS score (r = 0.57, P < .001). In the receiver-operating characteristic (ROC) curve analysis, MLR, with an optimal cut-off value of 0.665, predicted the severe coronary lesion with a sensitivity of 79.4% and specificity of 84.4%.MLR was an independent predictor of the severe Klebsiella pneumonia infection. Compared with neutrophil-to-lymphocyte ratio (NLR), MLR has a better performance to evaluate the severity of Klebsiella pneumonia infection.

Gene activation by a CRISPR-assisted trans enhancer.

The deactivated CRISPR/Cas9 (dCas9) is now the most widely used gene activator. However, current dCas9-based gene activators are still limited by their unsatisfactory activity. In this study, we developed a new strategy, the CRISPR-assisted trans enhancer, for activating gene expression at high efficiency by combining dCas9-VP64/sgRNA with the widely used strong CMV enhancer. In this strategy, CMV enhancer DNA was recruited to target genes in trans by two systems: dCas9-VP64/csgRNA-sCMV and dCas9-VP64-GAL4/sgRNA-UAS-CMV. The former recruited trans enhancer by annealing between two short complementary oligonucleotides at the ends of the sgRNA and trans enhancer. The latter recruited trans enhancer by binding between GAL4 fused to dCas9 and UAS sequence of trans enhancer. The trans enhancer activated gene transcription as the natural looped cis enhancer. The trans enhancer could activate both exogenous reporter genes and variant endogenous genes in various cells, with much higher activation efficiency than that of current dCas9 activators.

Cancer therapy with a CRISPR-assisted telomerase-activating gene expression system.

Cancer is caused by a series of alterations in genome and epigenome and exists in multiple complex forms, making it difficult to be prevented and/or treated. Telomerase, an enzyme responsible for the maintenance of telomere, is silent in most normal somatic cells but activated in 90% of cancer cells, making it an excellent target for cancer therapy. Therefore, various telomerase activity inhibitors have been developed to treat cancer but all failed due to side effects. Here we acted oppositely to develop a cancer gene therapy named telomerase-activating gene expression (Tage) system by utilizing the telomerase activity in cancer cells. The Tage system consisted of an effector gene expression vector that carried a 3' telomerase-recognizable stick end and an artificial transcription factor expression vector that could express dCas9-VP64 and an sgRNA targeting telomere repeat sequences. By using Cas9 as an effector gene, the Tage system effectively killed various cancer cells, including HepG2, HeLa, PANC-1, MDA-MB-453, A549, HT-29, SKOV-3, Hepa1-6, and RAW264.7, without affecting normal cells MRC-5, HL7702, and bone marrow mesenchymal stem cell (BMSC). More importantly, a four-base 3' stick end produced by the homothallic switching endonuclease in cells could be recognized by telomerase, allowing the Tage system to effectively kill cancer cells in vivo. The Tage system could effectively and safely realize its in vivo application by using adeno-associated virus (AAV) as gene vector. The virus-loaded Tage system could significantly and specifically kill cancer cells in mice by intravenous drug administration without side effects or toxicity.

CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method.

This study develops a new method for detecting and typing target DNA based on Cas9 nuclease, which was named as ctPCR, representing Cas9-sgRNA- or CRISPR-typing PCR. The technique can detect and type target DNA easily, rapidly, specifically, and sensitively. This technique detects target DNA in three steps: (1) amplifying target DNA with PCR by using a pair of universal primers (PCR1); (2) treating PCR1 products with a process referred to as CAT, representing Cas9 cutting, A tailing and T adaptor ligation; (3) amplifying the CAT-treated DNA with PCR by using a pair of general-specific primers (gs-primers) (PCR2). This method was verified by detecting HPV16 and HPV18 L1 gene in 13 different high-risk human papillomavirus (HPV) subtypes. This method was also verified by detecting the L1 and E6-E7 genes of two high-risk HPVs (HPV16 and 18) in cervical carcinoma cells and many clinical samples. In this method, PCR1 was performed to determine if the detected DNA sample contained the target DNA (such as virus infection), while PCR2 was performed to discriminate which genotypic target DNA was present in the detected DNA sample (such as virus subtypes). Based on these proof-of-concept experiments, this study provides a new CRISPR/Cas9-based DNA detection and typing method.

Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique.

This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing CRISPR- or Cas9/sgRNAs-associated reverse PCR. This technique detects target DNA in three steps: (1) cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; (2) ligating the cleaved DNA with DNA ligase; (3) amplifying target DNA with PCR. In the ligation step, the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA. In the PCR step, the ligated DNA was amplified with a pair of reverse primers. The technique was verified by detecting HPV16 and HPV18 L1 genes in nine different human papillomavirus (HPV) subtypes. The technique also detected the L1 and E6-E7 genes of two high-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells (HeLa and SiHa), in which no L1 and E6-E7 genes were detected in the HPV-negative cervical carcinoma cell, C-33a. By performing these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method. Especially, the CARP method developed by this study is ready for the clinical HPV detection, which was supported by the final clinical sample detection. Graphical abstract CRISPR-associated reverse PCR (CARP) can be used to detect and type target DNA in a simple three-step procedure, cutting, ligation, and amplification.