High-Sensitivity Enzyme-Free Fluorescence Probe Based on CRISPR/Cas13 and the Isothermal Amplification Strategy for Axl Sensing.

Axl is an important receptor tyrosine protein kinase that plays a key role in the development and progression of various diseases, such as cancer and inflammation. Developing a highly sensitive Axl detection method can help improve accuracy, better address-specific clinical needs, and guide personalized treatment. In this study, a CHA-CRISPR/Cas13 fluorescence probe was established using Axl-specific aptamers as a mediator to displace the polynucleotide chain (TA). Through TA construction, an entropy-driven nucleotide catalytic hairpin assembly system was created to cyclically release RNA that activates clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13 activity, triggering its cleavage activity. The activated CRISPR/Cas13 system cleaves the reporter labeled with BHQ1 and FAM at both ends, leading to the recovery of FAM fluorescence. Based on the optimization design using the free energy (△G) and secondary structure software simulation results of the nucleic acid sequence, the fluorescence intensity of the probe is proportional to the concentration of Axl. Results showed a good linear relationship between fluorescence intensity increment and log CAxl (CAxl in the range of 3.33-667 pM, r = 0.9907). The probe exhibited ultrahigh sensitivity with a detection limit of 0.84 pM. It was successfully applied in the detection of human serum samples, showing a higher Axl level in cervical cancer patients compared to breast cancer patients. The probe was also successfully applied in the imaging of various tumor cells, consistent with serum detection results. In conclusion, this probe represents an effective new method for detecting Axl, demonstrating outstanding specificity and sensitivity. It provides technological support for tumor diagnosis and shows the potential for detecting circulating tumor cells in blood through cell imaging.

Japanese spotted fever in an area endemic to SFTS virus: Case report and review of the literature.

RATIONALE: The geographic spread of Japanese spotted fever (JSF) in China is gradually expanding, particularly in regions where severe fever with thrombocytopenia syndrome (SFTS) is highly prevalent, with both diseases sharing similarities in epidemiology and clinical presentation. The microbiological diagnosis of JSF is challenging, compounded by low awareness among healthcare professionals in newly affected areas. Moreover, primary healthcare facilities without polymerase chain reaction (PCR) testing capabilities for SFTS often misdiagnose JSF as SFTS. PATIENT CONCERNS: All 3 patients had a history of working in the fields, with cold like symptoms in the early fever stages, but the fever did not improve after a few days. The accompanying symptoms were also very different. Physical examination revealed enlarged lymph nodes, different forms of rash, with or without eschar. Laboratory tests showed thrombocytopenia, eosinophilia, elevated lactate dehydrogenase, and transaminase, with 1 patient experiencing renal damage. It is worth noting that these 3 patients reside in an area where SFTS is endemic, and there have been no prior reports of JSF. They exhibited clinical symptoms and laboratory test results closely resembling those of SFTS. Therefore, they were initially misdiagnosed with SFTS in their local hospitals. DIAGNOSES: The 3 patients who arrived at our hospital 7 days after symptom onset and were subsequently diagnosed with JSF by metagenomic next-generation sequencing (mNGS). INTERVENTIONS: Doxycycline treatment for 1 week. OUTCOMES: The patients' symptoms quickly improved with no side effects, and the results of laboratory tests went back to normal. LESSONS: By comparing the clinical characteristics of JSF patients and SFTS patients comprehensively, we found that APTT and procalcitonin levels may be valuable in assisting in the identification of SFTS and JSF. In all areas where tick-borne diseases are endemic, include SFTS-epidemic areas, we recommend using the Weil-Felix test to screen for potential rickettsiosis in patients presenting with fever and thrombocytopenia with or without rash in primary healthcare settings, as well as simultaneous testing for the SFTS virus and spotted fever group rickettsioses sequence. Additionally, mNGS sequencing should be used to confirm the diagnosis and provide information for epidemiological investigations in patients who are suspected of having spotted fever group rickettsiosis.

Self-assembled hyaluronic acid-coated nanocomplexes for targeted delivery of curcumin alleviate acute kidney injury.

Acute kidney injury (AKI) is a pathological process with high morbidity, and drug resistance is easy to occur due to untargeted drug therapy. Curcumin can repair acute kidney injury. The expression of the CD44 receptor in renal tubular epithelial cells is abnormally elevated during AKI, and hyaluronic acid (HA) has the ability to bind specifically to the CD44 receptor. In this study, we developed a hyaluronic acid-coated liposome (HALP) nanocomplexes that targeted renal epithelial cells and its effect of relieving AKI was investigated. HALP was formed by self-assembly through the electrostatic interaction of curcumin-loaded cationic liposomes (LP) with hyaluronic acid and responds to the release of curcumin in the acidic microenvironment of lesions to treat AKI. HALP had good stability and biocompatibility. The in vitro results showed that compared to LP, HALP exhibited higher antioxidant, anti-inflammatory, and anti-apoptotic capacities. The AKI model suggested that HALP could not only target and accumulate in the injured kidney but also had an excellent ability to reduce the inflammatory response, which decreased tubular necrosis and restored kidney function.

Gentiana straminea Maxim. polysaccharide decolored via high-throughput graphene-based column and its anti-inflammatory activity.

Gentiana straminea Maxim. exhibits various biological activities. However, the purification and functions of polysaccharides in Gentiana straminea Maxim. have never been reported. Herein, by proposing a flexible 3D graphene-based decoloration column (3DD column), Gentiana straminea Maxim. polysaccharide (GMP) was high-throughput obtained and its anti-inflammatory activity was investigated. Benefiting from the large macroporous network of 3D NH2-graphene oxide hydrogel with selective adsorption towards pigments, the 3DD column exhibits high decoloration ratio (96.41%). In addition, the 3DD column provides superior practical functionality as compared to the traditional approaches, which are time-consuming and need toxic solvents, and exhibiting widespread-application for the purification of polysaccharide from other common plant species. More importantly, the decolored GMP as a natural product has promising anti-inflammatory activity on RAW264.7 cells without negative impact on cell viability. Overall, this work reveals a new functional polysaccharides and provides a flexible approach for polysaccharide decoloration, exhibiting a promising prospect for natural polysaccharides in practical application of pharmaceutical.

Rickettsia japonica infections in Huanggang, China, in 2021.

Two patients from Huanggang, China, were diagnosed with spotted fever group (SFG) rickettsiosis-caused by spotted fever group rickettsiae (SFGR)-in 2021. This study aimed to investigate the clinical symptoms, laboratory examinations, epidemiological factors, and therapeutic responses in patients with SFG rickettsiosis-an emerging disease in this region. The patients showed a variety of clinical signs and symptoms, such as acute febrile illness with severe headache, myalgia, asthenia, anorexia, eschar, lymphadenopathy, and rash on the trunk and extremities. They exhibited increased neutrophil ratio, mild thrombocytopenia, liver dysfunction, and increased C-reactive protein and procalcitonin levels. Following treatment with doxycycline, the patients recovered completely. This is the first report of Rickettsia japonica infection in Huanggang City, Hubei Province, China. SFGR infection is a tick-borne disease, which can be effectively treated with doxycycline; however, it has a mortality rate of approximately 10% with delays in treatment. The Huanggang area is also a high-risk area for tick-borne severe fever with thrombocytopenia syndrome (SFTS). Therefore, SFTS and SFG rickettsiosis should be carefully diagnosed in this area and clinicians should be alert with respect to the possibility of infections with both SFTS and SFG rickettsiosis.

Metabolic regulation protects mice against Klebsiella pneumoniae lung infection.

PURPOSE: Klebsiella pneumoniae-caused pneumonia is a risk factor for development of lung injury. However, the current clinical isolates of K. pneumoniae are mostly multidrug-resistance and thus must be addressed with new treatments. One ideal approach is to enhance the innate immunity of the infected host through metabolic modulators. MATERIALS AND METHODS: We used GC/MS-based metabolomics to profile the metabolomes among Control, Dead and Survival groups. The key metabolites were administrated in mice, and the bacterial loads in lung and survival were measured. The effect of the key metabolites on macrophage phagocytosis was determined by flow cytometry. RESULTS: Compared with the mice that compromised from K. pneumoniae lung infection, mice that survived the infection displayed the varied metabolomic profile. The differential analysis of metabolome showed D-Glucose, Glutamine, L-Serine, Myo-inositol, Ethanedioic acid and Lactic acid related to the host surviving a K. pneumoniae lung infection. Further pathway enrichment analysis proposed that valine, leucine and isoleucine biosynthesis involved in outcome of lung infection. The follow-up data showed that exogenous L-Serine, L-Valine and L-Leucine could decline the load of K. pneumoniae in infected lung and increases the mouse survival. More interestingly, L-Serine, L-Valine and L-Leucine also were able to promote macrophage phagocytosis that is the natural way to promote hosts to clear lung pathogens. CONCLUSIONS: Our study establishes a novel strategy of identifying metabolic modulator from surviving host and emphasizes the feasibility of employing the metabolic modulator as a therapy for K. pneumoniae lung infection.

Relationship between carbachol hyperstimulation-induced pancreatic intracellular trypsinogen and NF-kappa B activation in rats in vitro.

The relationship between intracellular trypsinogen activation and NF-kappa B activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist (carbachol) hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc) and NF-kappa B inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The activity of NF-kappa B was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-kappa B in pancreatic acinar cells treated with high concentrations of carbachol (10(-3) mol/L) in vitro was significantly decreased as compared with control group (P<0.01). The addition of 10(-2) mol/L PDTC resulted in a significant decrease of NF-kappa B activities in pancreatic acinar cells after treated with high concentrations of carbachol (10(-3) mol/L) in vitro, but the intracellular trypsinogen activity was not obviously inhibited (P>0.05). It was concluded that intracellular trypsinogen activation is likely involved in the regulation of high concentrations of carbachol-induced NF-kappa B activation in pancreatic acinar cells in vitro. NF-kappa B activation is likely not necessary for high concentrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells.

AIM: To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell (MSC) in vitro, in order to find suitable resource of hepatocytes for bioartificial liver or liver transplantation. METHODS: The rat myeloid MSC was isolated and divided into three groups which were cultured by Friedensteion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor (HGF), respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS: After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured with cholestatic serum. Furthermore, on the 35th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF. CONCLUSION: Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.

[The preparation of sintered bovine cancellous bone and a study of its mechanical and chemical behavior and biocompatibility].

This article is devoted to a method for preparing a new bone plant material--sintered bovine cancellous bone with beta-TCP type, and to study the mechanical and chemical behavior of the material such as the diameter of the pores, the compress and bend intensity, and the component, and the rate of the porosity. The biocompatibility of the bone was assessed by hemolysis test, micronucleus test, systemic acute toxicity test, local irritation reaction, pyrogen test, and the test of planting in the body. The study indicate that the sintered bovine cancellous bone with beta-TCP type had certain intension, appropriate aperture, connective pore and credible biosecurity, it could be used clinically as bone graft material.