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The CRISPR/Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). The system forms an adaptive immune system in archaea and bacteria. The inherent defense mechanism enables these microorganisms to protect themselves against the invasion of foreign genetic material. The system functions of immune response including three main stages: adaptation, expression/maturation, and interference, each stage needs specific Cas proteins encoded by Cas gene located near the CRISPR sequences, along with other auxiliary proteins. In 2015, Zhang et al. reportedCas12a (Cpf1) as a member of the Class II type V CRISPR/Cas12a system, which possesses endonuclease activity. This finding holds great promise for its application in the field of biotechnology. In 2018, Doudna’s team first applied the CRISPR/Cas12a system for detecting HPV nucleic acid. The system comprises the following essential components in vitro detection: Cas12a, the crRNA sequence complementary to the target DNA, the PAM sequence, and the ssDNA reporter. Cas12a possesses a typical RuvC domain, displaying a canonical bilobed architecture that consists of a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe contains the REC1 and REC2 domains, and the NUC lobe includes RuvC, PAM-interacting (PI), Wedge (WED), and bridge helix (BH) domains. The mature crRNA for Cas12a has a length of 42-44 nt, consists of repeat sequence (19/20 nt) and spacer sequence (23-25 nt). The crRNA spacer sequence has been found to require a length of 18 nt to achieve complete cleavage activity in vitro. Additionally, mutation in the bases of crRNA can indeed affect the activity of Cas12a. The PAM sequence plays a critical role in the recognition and degradation of DNA by the CRISPR/Cas system, enabling the system to distinguish between self and non-self genomic materials. Cas12a can effectively target the spacer sequence downstream of a T-rich PAM sequence at the 5' end. LbCas12a and AsCas12a both recognize the PAM sequences of 5'-TTTN-3', while FnCas12a recognizes the PAM sequences of 5'-TTN-3'. All of these PAM sequences are located upstream on the non-template strand (NTS) at the 5' end. Cas12a (Cpf1), guided by the crRNA, binds to the target DNA by recognizing the PAM sequence. It exhibits the ability to induce arbitrary cleavage of ssDNA within the system while cleaving the target ssDNA or dsDNA. According to this feature, an array of nucleic acid detection methods has been developed for tumor detection and infection diagnostics, such as the DETECTR (RPA-CRISPR/Cas12a method) and HOLMES (PCR-CRISPR/Cas12a method) in 2018. Then, in 2019, Cas12aVDet (one-step detection method), where Cas12a protein was immobilized on the upper wall of the reaction tube. This not only prevented contamination from opening the tube but also reduced the detection reaction time. In 2021, the dWS-CRISPR (digital warm-start CRISPR) was developed as a one-pot detection method. It serves as an accurate approach for quantitatively detectingSARS-CoV-2 in clinical specimens. With the innovation of scientific technology, the high-sensitivity signal transduction technology has also been integrated with the CRISPR/Cas12a system, enabling direct detection of nucleic acids, and eliminating the need for nucleic acid amplification steps. Here, we elaborated the detection principles of CRISPR/Cas12a in in vitro detection. We discussed the different stages leading to the catalytic pathway of target DNA, and the practical applications of Cas12a in nucleic acid detection. These findings revealed a target interference mechanism that originates from the binding of Cas12a-guided RNA complex to complementary DNA sequences within PAM-dependent (dsDNA) regions. The crRNA-DNA binding activates Cas12a, enabling site-specific dsDNA cleavage and non-specific ssDNA trans-cleavage. The release of Cas12a ssDNase activity provides a novel approach to enhance the sensitivity and specificity of molecular diagnostic applications. Before these CRISPR/Cas12a-based nucleic acid detection methods can be introduced into clinical use, substantial work is still required to ensure the accuracy of diagnosis. Nevertheless, we believe that these innovative detection tools based on CRISPR/Cas will revolutionize future diagnostic technologies, particularly offering significant assistance in pathogen infection diagnosis for developing countries with relatively poor healthcare conditions and high prevalence of infectious diseases.

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Objective: To explore the molecular mechanism by which oral S2-Ag85DNA vaccines present intestinal antigens. The oral S2-Ag85 vaccine has been shown to protect the human body and effectively improve the titration of the vaccine by acting on intestinal mucosa cells and enhancing their immunogenicity. Method: Mice were immunized with the recombinant S2-Ag85 vaccine, and antibody secretion was then detected in the intestinal tissue. The molecular mechanisms of in vitro detection sensor molecules RIG-1, Pol III, and related conductor transductor molecules DAI, STING, AIM2, IRF3, and IRF7 were determined by separating intestinal IEC, DC, and IELC cells. Results: The S2-Ag85A vaccine was effective in activating dsDNA and RNA transduction pathways in intestinal cells and improving intestinal antigen presentation in mice.

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Ischemic heart diseases are one of the major causes of death worldwide. Effective restoration of blood flow can significantly improve patients’ quality of life and reduce mortality. However, reperfusion injury cannot be ignored. Flavonoids possess well-established antioxidant properties; They also have other benefits that may be relevant for ameliorating myocardial ischemia-reperfusion injury (MIRI). In this review, we focus on flavonoids with cardiovascular-protection function and emphasize their pharmacological effects. The main mechanisms of flavonoid pharmacological activities against MIRI involve the following aspects: a) antioxidant, b) anti-inflammatory, c) anti-platelet aggregation, d) anti-apoptosis, and e) myocardial-function regulation activities. We also summarized the effectiveness of flavonoids for MIRI.

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OBJECTIVE: To predict the monthly reported echinococcosis cases in China with the autoregressive integrated moving average (ARIMA) model, so as to provide a reference for prevention and control of echinococcosis. METHODS: SPSS 24.0 software was used to construct the ARIMA models based on the monthly reported echinococcosis cases of time series from 2007 to 2015 and 2007 to 2014, respectively, and the accuracies of the two ARIMA models were compared. RESULTS: The model based on the data of the monthly reported cases of echinococcosis in China from 2007 to 2015 was ARIMA (1, 0, 0) (1, 1, 0)12, the relative error among reported cases and predicted cases was -13.97%, AR (1) = 0.367 (t = 3.816, P < 0.001), SAR (1) = -0.328 (t = -3.361, P = 0.001), and Ljung-Box Q = 14.119 (df = 16, P = 0.590) . The model based on the data of the monthly reported cases of echinococcosis in China from 2007 to 2014 was ARIMA (1, 0, 0) (1, 0, 1)12, the relative error among reported cases and predicted cases was 0.56%, AR (1) = 0.413 (t = 4.244, P < 0.001), SAR (1) = 0.809 (t = 9.584, P < 0.001), SMA (1) = 0.356 (t = 2.278, P = 0.025), and Ljung-Box Q = 18.924 (df = 15, P = 0.217). CONCLUSIONS: The different time series may have different ARIMA models as for the same infectious diseases. It is needed to be further verified that the more data are accumulated, the shorter time of predication is, and the smaller the average of the relative error is. The establishment and prediction of an ARIMA model is a dynamic process that needs to be adjusted and optimized continuously according to the accumulated data, meantime, we should give full consideration to the intensity of the work related to infectious diseases reported (such as disease census and special investigation).

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Objective To predict the monthly reported echinococcosis cases in China with the autoregressive integrated mov-ing average(ARIMA)model,so as to provide a reference for prevention and control of echinococcosis. Methods SPSS 24.0 software was used to construct the ARIMA models based on the monthly reported echinococcosis cases of time series from 2007 to 2015 and 2007 to 2014,respectively,and the accuracies of the two ARIMA models were compared. Results The model based on the data of the monthly reported cases of echinococcosis in China from 2007 to 2015 was ARIMA(1,0,0)(1,1, 0)12,the relative error among reported cases and predicted cases was-13.97%,AR(1)=0.367(t=3.816,P<0.001),SAR (1)=-0.328(t=-3.361,P=0.001),and Ljung-Box Q=14.119(df=16,P=0.590).The model based on the data of the monthly reported cases of echinococcosis in China from 2007 to 2014 was ARIMA(1,0,0)(1,0,1)12,the relative error among reported cases and predicted cases was 0.56%,AR(1)=0.413(t=4.244,P<0.001),SAR(1)=0.809(t=9.584, P<0.001),SMA(1)=0.356(t=2.278,P=0.025),and Ljung-Box Q=18.924(df=15,P=0.217).Conclusions The different time series may have different ARIMA models as for the same infectious diseases.It is needed to be further verified that the more data are accumulated,the shorter time of predication is,and the smaller the average of the relative error is.The estab-lishment and prediction of an ARIMA model is a dynamic process that needs to be adjusted and optimized continuously accord-ing to the accumulated data,meantime,we should give full consideration to the intensity of the work related to infectious diseas-es reported(such as disease census and special investigation).

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BACKGROUND: As the surgical treatment for intestinal fistulas nowadays needs to be improved, we are seeking a new kind of artificially synthesized hydrogel to replace fibrin gels as the sealing gel, which is imperative for both economic and social benefits.OBJECTIVE: To prepare a degradable antibacterial composite hydrogel and to detect the in vitro biological properties. METHODS: In this study, we combined soluble chitosan (S-Cts) with oxidized alginate (O-Alg) to prepare the injectable and degradable hydrogel under Schiff base reaction. Besides, nanosilver (nano-Ag) particles were added to obtain S-Cts/O-Alg/nano-Ag composite hydrogel. Gelation time, microstructure, swelling, degradation, and antibacterial properties of the composite hydrogel were observed and detected in simulated physiological environment. RESULTS AND CONCLUSION: The closer constituent contents of water-soluble chitosan and sodium alginate indicate the shorter gelation time, and the time could be controlled within the range of surgery. The variation in the constituent content of the two components can affect the hydrogel microstructure. The higher constituent content of water-soluble chitosan implicates the denser network of the hydrogel. The composite hydrogel has excellent swelling properties, and it degrades faster in the simulated intestinal fluid containing trypsin than in the PBS. Moreover, adding nanosilver particles can bring certain antibacterial properties. This hydrogel has better biocompatibility, biodegradability and antibacterial ability than natural macromolecules, and has certain research value and application prospect.

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BACKGROUND: As the surgical treatment for intestinal fistulas nowadays needs to be improved, we are seeking a new kind of artificially synthesized hydrogel to replace fibrin gels as the sealing gel, which is imperative for both economic and social benefits.OBJECTIVE: To prepare a degradable antibacterial composite hydrogel and to detect the in vitro biological properties. METHODS: In this study, we combined soluble chitosan (S-Cts) with oxidized alginate (O-Alg) to prepare the injectable and degradable hydrogel under Schiff base reaction. Besides, nanosilver (nano-Ag) particles were added to obtain S-Cts/O-Alg/nano-Ag composite hydrogel. Gelation time, microstructure, swelling, degradation, and antibacterial properties of the composite hydrogel were observed and detected in simulated physiological environment. RESULTS AND CONCLUSION: The closer constituent contents of water-soluble chitosan and sodium alginate indicate the shorter gelation time, and the time could be controlled within the range of surgery. The variation in the constituent content of the two components can affect the hydrogel microstructure. The higher constituent content of water-soluble chitosan implicates the denser network of the hydrogel. The composite hydrogel has excellent swelling properties, and it degrades faster in the simulated intestinal fluid containing trypsin than in the PBS. Moreover, adding nanosilver particles can bring certain antibacterial properties. This hydrogel has better biocompatibility, biodegradability and antibacterial ability than natural macromolecules, and has certain research value and application prospect.

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Objective To investigate the mechanism of 3-phosphoinositide-dependent protein kinase 1(PDK1)poly-ubiquitination.Methods Co-immunoprecipitation(Co-IP)and Western blot(WB)were used to analyze poly-ubiquitination of PDK1.It was confirmed that ubiquitin ligase smad ubiquitylation regulatory factor 1(Smurf1)inprove PDK1 poly-ubiquitination within MEF cells,site-directed mutagenesis and WB before PDK1 poly-ubiquitination sites were determined.Results We found that PDK1 could undergoes poly-ubiquitination,ubiquitin ligase Smurf1 was found to be a direct E3 ligase for PDK1 poly-ubiquitination and might rely on the ubiquitin ligase Smurf 1 K699 site activity.K304 was PDK1 poly-ubiquitination modification site point.Conclusion The ubiquitin ligase Smurf1 can promate poly-ubiquitination of PDK1.

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Objective: To compare the clinical features between very late stent thrombosis (VLST) and very late in-stent restenosis, to discuss the potential risk factors for VLST occurrence. Methods: Our research included in 2 groups: VLST group, 21 ACS patients with coronary angiography (CAG) confirmed VLST admitted in our hospital and Control group, 38 ACS patients with CAG confirmed very late in-stent restenosis at same period of time. Basic clinical data, laboratory tests and relevant examinations were compared between 2 groups; potential risk factors for VLST occurrence were studied by Logistic regression analysis. Results: ① There were 8 (38.1%) patients discontinued anti-platelet therapy in a month by themselves in VLST group and 5 (13.2%) in Control group, P=0.03. ② 13 (61.9%) patients presented as ST-segment elevation myocardial infarction (STEMI) in VLST group, while all (100%) patients presented as Non-ST-segment elevation ACS (NST-ACS) in Control group, P<0.001. ③ The age, gender, previous histories of hypertension, diabetes, MI, smoking and interventional therapy were similar between 2 groups, P>0.05. ④ Compared with Control group, VLST group had decreased LVEF, P=0.001, increased peak values of TnI and NT-pro BNP, elevated WBC and hs-CRP, all P<0.001. ⑤ The index of echocardiography, blood lipid profiles, glucose and creatinine were similar between 2 groups, P>0.05. ⑥ Logistic regression analysis showed that discontinued anti-platelet therapy, elevated NT-pro BNP and hs-CRP were the independent risk factors for VLST occurrence, P<0.05. Conclusion: VLST may have life-threatening clinical features, insisted anti-platelet therapy and improved cardiac function could reduce VLST occurrence.

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OBJECTIVE@#To determine the cellular compatibility of combined deproteinized bone(DPB) coated with hepatocyte growth factor (HGF), and to observe the adherent effect of osteoblasts in response to HGF.@*METHODS@#Osteoblasts were isolated from fetal rabbits. Osteoblasts were cultured with DPB coated with HGF and deproteinized bone as experimental group and contral group, respectively. The proliferation and alkalinephosphatase activity were tested. Their growth was examined by inverted phase contrast microscope and scanning electronmicroscope.@*RESULTS@#The osteoblasts were attached to the outside and inside surfaces and grew well. HGF/DPB could stimulate the alkalinephosphatase activity of the osteoblasts and improve the proliferation of the osteoblasts.@*CONCLUSION@#HGF/DPB has good biocompatibility and bone induction. HGF could improve the adherent effect of DPB on osteoblasts, and it could be used as scaffold material for the bone tissue engineering.

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<p><b>AIM</b>To investigate the preparation of diclofenac sodium pulsatile release pellets (DS-PRP), the release in vitro and the pharmacokinetics of the drug.</p><p><b>METHODS</b>Diclofenac sodium (DS) core pellets prepared by extrusion-spheronization technology were coated in a mini-fluidized bed spray coater with swelling material as the inner coating swelling layer and ethylcellulose aqueous dispersion as the outer coating controlled layer. The effects of formulation and medium on pulsatile release of DS were investigated under release rate test. Pharmacokinetic and bioavailability study in eight human subjects were performed by HPLC method.</p><p><b>RESULTS</b>The delayed-release time and release rate of DS from DS-PRP were influenced obviously by the swelling material, the concentration of SDS in medium, the coating level of the inner swelling layer and the outer controlled layer. In vitro, the delayed-release time T0.1 was 3.1 h, and the pulsed-release time T0.1-0.2 was 1.2 h. In vivo, the delayed-release time Tlag was 2.8 h, and the bioavailability was (91 +/- 12)%.</p><p><b>CONCLUSION</b>The release of drug from DS-PRP was shown to be in pulsed way both in vitro and in vivo.</p>

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