Electrochemical desulfurative formation of C-N bonds through selective activation of inert C(sp3)-S bonds.

In this study, we introduce an efficient, metal-free electrocatalytic desulfurative protocol for forming C-N bonds by selectively activating inert C(sp3)-S bonds of alkyl thioethers. This method offers a straightforward and environmentally friendly approach for modification of heterocyclic compounds from readily accessible thioethers. Preliminary mechanistic investigations suggest that the reaction proceeds via a carbocation intermediate. Furthermore, successful synthesis on a 10-gram scale was achieved in a continuous flow electrochemical reactor.

Joint Analysis of Genome-wide DNA Methylation and Transcription Sequencing Identifies the Role of BAX Gene in Heat Stress-Induced-Sertoli Cells Apoptosis.

The problem of male infertility is a global health crisis and poses a serious threat to the well-being of families. Under heat stress (HS), the reduction of Sertoli cells (SCs) inhibits energy transport and nutrient supply to germ cells, leading to spermatogenesis failure. DNA methylation of genes is a central epigenetic regulatory mechanism in mammalian reproduction. However, it remains unclear how DNA methylation regulates gene expression in heat-stressed SCs. In this study, we investigated whether the decrease in SC levels during HS could be related to epigenetic DNA modifications. The cells exposed to HS showed changes in differential methylation cytosines and regions (DMCs/DMRs) and differential expression genes (DEGs), but not in global DNA methylations. One of the most important biological processes affected by HS is cell apoptosis induced by the intrinsic apoptotic signaling pathway (GO: 2,001,244, P < 0.05) by enrichment in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The joint analysis showed that several gene expressions in RNA-seq and WGBS overlapped and the shortlisted genes BAX, HSPH1, HSF1B, and BAG were strongly correlated with stress response and apoptosis. Methylation-specific PCR (MSP) and flow cytometry (FCM) analyzes showed that reduced promoter methylation and enhanced gene expression of BAX with a consequence of apoptosis. The activity of BAX, as well as an increase in its expression, is likely to result in a reduction of SCs population which could further impair ATP supply and adversely affect membrane integrity. These findings provide novel insights into the molecular mechanisms through which stressors cause male reproductive dysfunction and a new molecular etiology of male infertility.

Rapid and sensitive detection of nucleic acids using an RAA-CRISPR/Cas12b one-pot detection assay (Rcod).

Rapid, sensitive and specific methods are crucial for nucleic acid detection. CRISPR/Cas12b has recently been widely used in nucleic acid detection. However, due to its thermophagic property, DNA isothermal recombinase-aided amplification (RAA) and subsequent CRISPR/Cas12b detection require two separate reactions, which is cumbersome and inconvenient and may cause aerosol pollution. In this study, we propose an RAA-CRISPR/Cas12b one-pot detection assay (Rcod) for Bordetella pertussis detection without additional amplification product transfer steps. The time from sample processing to response time was less than 30 min using nucleic acid extraction-free method, and the sensitivity reached 0.2 copies/µL. In this system, Alicyclobacillus acidoterrestris Cas12b protein (AacCas12b) exhibited strong and specific trans-cleavage activity at a constant temperature of 37 °C, while the cis-cleavage activity was weak. This characteristic reduces the interference of AacCas12b with nucleic acids in the system. Compared with real-time PCR, our Rcod system detected B. pertussis in 221 clinical samples with a sensitivity and specificity of 97.96 % and 99.19 %, respectively, with nucleic acid extraction-free method. The rapid, sensitive and specific Rcod system provides ideas for the establishment of CRISPR-based one-step nucleic acid detection and may aid the development of reliable point-of-care nucleic acid tests. IMPORTANCE: Pertussis is an acute respiratory infection caused by B. pertussis that is highly contagious and potentially fatal, and early diagnosis is essential for the treatment of whooping cough. In this study, we found that AacCas12b has high and strongly specific trans-cleavage activity at lower temperatures. A RAA-CRISPR/Cas12b one-step detection platform (Rcod) without interference with amplification was developed. In addition, the combination of Rcod and nucleic acid extraction-free method can quickly and accurately detect the qualitative detection of B. pertussis, and the detection results are visualized, which makes the pathogen nucleic acid detection and analysis process simpler, and provides a new method for the rapid clinical diagnosis of B. pertussis.

Metal-free photoinduced C(sp3)-H/C(sp3)-H cross-coupling to access α­tertiary amino acid derivatives.

The cross-dehydrogenative coupling (CDC) reaction is the most direct and efficient method for constructing α-tertiary amino acids (ATAAs), which avoids the pre-activation of C(sp3)-H substrates. However, the use of transition metals and harsh reaction conditions are still significant challenges for these reactions that urgently require solutions. This paper presents a mild, metal-free CDC reaction for the construction of ATAAs, which is compatible with various benzyl C-H substrates, functionalized C-H substrates, and alkyl substrates, with good regioselectivity. Notably, our method exhibits excellent functional group tolerance and late-stage applicability. According to mechanistic studies, the one-step synthesized and bench-stable N-alkoxyphtalimide generates a highly electrophilic trifluoro ethoxy radical that serves as a key intermediate in the reaction process and acts as a hydrogen atom transfer reagent. Therefore, our metal-free and additive-free method offers a promising strategy for the synthesis of ATAAs under mild conditions.

An extraction-free method for rapid detection of CYP2C19 * 2/3/17 polymorphisms in one tube using melting curve analysis.

Drug-metabolizing enzymes play an important role in the metabolism of drugs in vivo. Their activity is an important factor affecting the rate of drug metabolism, which directly determines the intensity and persistence of drug action. Patients taking medication can be divided into different metabolic types through detection of CYP2C19 drug-metabolizing enzyme gene polymorphisms, which can then be used for medication guidance for clopidogrel. Here, we describe a detection method based on real-time polymerase chain reaction (PCR). This method uses multicolor melting curve analysis to accurately identify different mutation sites and genotypes of CYP2C19 * 2, CYP2C19 * 3, and CYP2C19 * 17. The detection limit of plasmid samples was 1 copies µL-1 ; that of genomic samples was 0.1 ng µL-1 . The system can detect nine types of CYP2C19 * 2/3/17 at three sites in one tube, quickly achieving detection within 1 h. Combined with the sample release agent, sample extraction was completed in 5 s, achieving rapid diagnosis without extraction for timely diagnosis and treatment. Furthermore, the system is not limited to blood samples and can also be applied to oropharyngeal and saliva samples, increasing sampling diversity and convenience. When using clinical blood samples (n = 93), the detection system we established was able to quickly and accurately identify different genotypes, and the accuracy and effectiveness of the detection were confirmed by Sanger sequencing. Due to its accuracy, rapidity, simple operation, and low cost, detection technology based on real-time polymerase amplification combined with melting curve analysis is expected to become a powerful tool for detecting and guiding clopidogrel use in countries with limited resources.

Fast and visual detection of nucleic acids using a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM.

CRISPR-Cas12a (Cpf1) is widely used for pathogen detection. However, most Cas12a nucleic acid detection methods are limited by a PAM sequence requirement. Moreover, preamplification and Cas12a cleavage are separate. Here, we developed a one-step RPA-CRISPR detection (ORCD) system unrestricted by the PAM sequence with high sensitivity and specificity that offers one-tube, rapid, and visually observable detection of nucleic acids. In this system, Cas12a detection and RPA amplification are performed simultaneously, without separate preamplification and product transfer steps, and 0.2 copies/µL of DNA and 0.4 copies/µL of RNA can be detected. In the ORCD system, the activity of Cas12a is the key to the nucleic acid detection; specifically, reducing Cas12a activity increases the sensitivity of ORCD assay detection of the PAM target. Furthermore, by combining this detection technique with a nucleic acid extraction-free method, our ORCD system can be used to extract, amplify and detect samples within 30 min, as verified with tests of 82 Bordetella pertussis clinical samples with a sensitivity and specificity of 97.30% and 100% compared with PCR. We also tested 13 SARS-CoV-2 samples with RT-ORCD, and the results were consistent with RT-PCR.

Effects of high tibial osteotomy combined with arthroscopy on local inflammation degree and gait activity index in patients with medial knee osteoarthritis.

Objective: To analyze the effects of high tibial osteotomy (HTO) combined with arthroscopy on the degree of inflammation and the gait activity indexes of patients with medial knee osteoarthritis. Methods: We collected the records of 112 patients with medial knee osteoarthritis treated in the Department of Orthopedics of our hospital from June 2019 to June 2021. We divided the data into two groups: the control group included those of 54 patients who had received simple HTO and the observation group included those of 58 patients who had undergone HTO combined with arthroscopy. We assessed clinical efficacies, degrees of inflammation and gait activity indexes of the two groups to compare treatment outcomes. Results: The percentage of excellent and good outcomes in the observation group (89.66%) was significantly higher than that in the control group (66.67%; p < 0.05). One year after the operation, the serum and synovial fluid levels of interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and interleukin-17 (IL-17) in the observation group were significantly lower than those in the control group (p < 0.05). Moreover, the double support phase was significantly lower in the observation group than in the control group, while the step length, speed and frequency were significantly higher in the observation group than in the control group (p < 0.05). Conclusions: HTO combined with Arthroscopy in patients with medial knee osteoarthritis improves the curative effect and the degree of inflammation, and it promotes the recovery of gait activity indices.

Cs2CO3 catalyzed direct aza-Michael addition of azoles to α,ß-unsaturated malonates.

A highly efficient method for the synthesis of azole derivatives via a direct aza-Michael addition of azoles to α,ß-unsaturated malonates using Cs2CO3 as a catalyst, has been successfully developed. A series of azole derivatives have been obtained in up to 94% yield and the reaction could be amplified to gram scale in excellent yield in the presence of 10 mol% of Cs2CO3.

Highly selective cross-coupling reactions of 1,1-dibromoethylenes with alkynylaluminums for the synthesis of aryl substituted conjugated enediynes and unsymmetrical 1,3-diynes.

A highly efficient method for the synthesis of aryl substituted conjugated enediynes and unsymmetrical 1,3-diynes via selective cross-coupling reactions of 1,1-dibromoethylenes with alkynylaluminums using the Pd(OAc)2-DPPE and Pd2(dba)3-TFP complexes as catalysts, respectively, has been successfully developed. Though the alkyl substituted conjugated enediynes and unsymmetrical 1,3-diynes were not obtained, this case is also remarkable as the same starting materials could selectively produce either aryl substituted conjugated enediynes or unsymmetrical 1,3-diynes in moderate to excellent yields (up to 99%) in the different Pd-phosphine catalytic systems.

Hemagglutinin-based DNA vaccines containing trimeric self-assembling nanoparticles confer protection against influenza.

Influenza viruses continue to threaten public health, and currently available vaccines provide insufficient immunity against seasonal and pandemic influenza. The use of recombinant trimeric hemagglutinin (HA) as an Ag provides an attractive alternative to current influenza vaccines. Aiming to develop an effective vaccine with rapid production, robust immunogenicity, and high protective efficiency, a DNA vaccine was designed by fusing influenza virus HA with self-assembled ferritin nanoparticles, denoted as HA-F. This candidate vaccine was prepared and purified in a 293-6E cell eukaryotic expression system. After BALB/c mice were immunized with 100 µg of HA-F DNA 3 times, HA-F elicited significant HA-specific humoral immunity and T cell immune responses. The HA-F DNA vaccine also conferred protection in mice against a lethal infection of homologous A/17/California/2009/38 (H1N1) virus. These results suggest that the HA-F DNA vaccine is a competitive vaccine candidate and presents a promising vaccination approach against influenza viruses.

Differences between conventional wound closure and a closed suction irrigation method for the prevention of surgical site infection-a comparative study.

BACKGROUND: Wound infections can lead to high risks of postoperative septic complications. To date, the efficacy of a closed suction irrigation method of wound closure on the rate of postoperative wound infection in patients is poorly understood. Therefore, comparing this new strategy with conventional primary wound closure on the incidence of postoperative wound infection can provide value for clinical treatment. METHODS: Our study focused on retrospective research for patients with open abdominal wounds. All patients were referred by tertiary hospitals and registered in our hospital from January 2009 to July 2019. According to wound types, patients were divided into two groups, namely patients with wounds primarily closed as the control group and patients with wounds sutured by closed suction irrigation after primary closure as the study group. RESULTS: A total of 159 patients were collected in our present research, with 86 as study subjects and 73 as control subjects. The wound infection rate was significantly lower in the study group than the control group (P=0.017). The time interval for diagnosing wound infection from the primary operation was 4.895±0.875 days for the study group and 4.509±0.697 days for the control group. The length of hospital stay and the time taken for the wound to heal completely was shorter in patients in the study group compared to patients in the control group (8.046±2.110 vs. 8.983±2.887 days and 8.092±3.258 vs. 10.328±6.074 days, respectively). CONCLUSIONS: The closed suction irrigation method significantly reduced postoperative wound infection rates in patients with a high risk of postoperative septic complications. The discoveries of our investigation should be further validated in large scope randomized controlled trials.

Palladium-catalyzed cross-coupling reaction of alkenyl aluminums with 2-bromobenzo[b]furans.

Highly efficient and simple cross-coupling reactions of 2-bromobenzo[b]furans with alkenylaluminum reagents for the synthesis of 2-alkenylbenzo[b]furan derivatives using PdCl2 (3 mol%)/XantPhos (6 mol%) as catalyst are reported. Excellent yields (up to 97%) were obtained for a wide range of substrates at 80 °C for 4 h in DCE.

Unusual bleeding from hepaticojejunostomy controlled by side-to-side splenorenal shunt: A case report.

RATIONALE: Ectopic variceal bleeding due to hepaticojejunostomy (HJ) is unusual and difficult to manage. Reports on the use of side-to-side splenorenal shunt for severe bleeding from varices at HJ anastomosis are lacking. PATIENT CONCERNS: A 43-year-old man was admitted to our hospital with repeated episodes of hematemesis. He has a history of right hemihepatectomy with HJ reconstruction to the left hepatic duct for hilar cholangiocarcinoma. Two years after surgery, he presented with repeated episodes of hematemesis and underwent blood transfusion. DIAGNOSES: Imaging tests and endoscopic investigation failed to identify the bleeding source. When conservative management failed to control his bleeding, he underwent emergency laparotomy, which revealed hemorrhage from ectopic varices at the HJ anastomosis. INTERVENTIONS: To arrest the bleeding, a side-to-side venovenal anastomosis was created between the splenic and left renal veins to form a shunt for decompression of the varices at the HJ anastomosis. OUTCOMES: After the surgery, the patient's symptoms ceased, and a no bleeding in the digestive tract was noted at 2-year follow-up. LESSONS: The present patient is the first reported case of unusual bleeding from HJ controlled by a side-to-side splenorenal shunt. We believe this approach is a useful and effective surgical treatment for severe bleeding from varices at the HJ anastomosis.

MicroRNA-424 may function as a tumor suppressor in endometrial carcinoma cells by targeting E2F7.

MicroRNAs (miRNAs) are frequently dysregulated in human cancers and can act as potent oncogenes or tumor suppressor genes. Aberrant expression of miR-424 has been identified in some types of cancer, however, its expression and potential biologic role in endometrial cancer are remains to be determined. In the present study, we demonstrated that miR-424 was downregulated in human endometrial cancer and suppressed growth of the human Ishikawa and HEC-1B endometrial cancer cell lines. Bioinformatics analysis indicated that E2F7 was a putative target of miR-424. In a luciferase reporter system, we confirmed that E2F7 was a direct target gene of miR-424. Furthermore, knockdown of E2F7 inhibited Ishikawa and HEC-1B cell growth. These findings indicate that miR-424 targets the E2F7 transcript and suppresses endometrial cancer cell growth, suggesting that miR-424 has a tumor suppressive role in human endometrial cancer pathogenesis.

Laparoscopic liver resection under hemihepatic vascular inflow occlusion using the lowering of hilar plate approach.

BACKGROUND: With advances in technology, laparoscopic liver resection is widely accepted. Laparoscopic liver resection under hemihepatic vascular inflow occlusion has advantages over the conventional total hepatic inflow occlusion using the Pringle's maneuver, especially in patients with cirrhosis. METHOD: From November 2011 to August 2012, eight consecutive patients underwent laparoscopic liver resection under hemihepatic vascular inflow occlusion using the lowering of hilar plate approach with biliary bougie assistance. RESULTS: The types of liver resection included right hepatectomy (n=1), right posterior sectionectomy (n=1), left hepatectomy and common bile duct exploration (n=1), segment 4b resection (n=1), left lateral sectionectomy (n=2), and wedge resection (n=2). Four patients underwent right and 4 left hemihepatic vascular inflow occlusion. Four patients had cirrhosis. The mean operation time was 176.3 minutes. The mean time taken to achieve hemihepatic vascular inflow occlusion was 24.3 minutes. The mean duration of vascular inflow occlusion was 54.5 minutes. The mean intraoperative blood loss was 361 mL. No patient required blood transfusion. Postoperatively, one patient developed bile leak which healed with conservative treatment. No postoperative liver failure and mortality occurred. The mean hospital stay of the patients was 7 days. CONCLUSION: Our technique of hemihepatic vascular inflow vascular occlusion using the lowering of hilar plate approach was safe, and it improved laparoscopic liver resection by minimizing blood loss during liver parenchymal transection.

Nickel-catalyzed substitution reactions of propargyl halides with organotitanium reagents.

A simple and mild catalytic coupling reaction of propargyl halides with organotitanium reagents is reported. The reaction of propargyl bromide with organo-titanium reagents mediated by NiCl2 (2 mol%) and PCy3 (4 mol%) in CH2Cl2 afforded coupling product allenes in good to excellent yields (up to 95%) at room temperature. However, NiCl2(PPh3)2 was the best catalyst for substituted propargyl halides to yield allenes or alkynes preferentially. On the basis of the experimental results, a possible catalytic cycle has been proposed.

Room temperature and highly enantioselective additions of alkyltitanium reagents to aldehydes catalyzed by a titanium catalyst of (R)-H8-binol.

Three alkyltitanium reagents of RTi(O-i-Pr)3 (R = Cy (1a), i-Bu (1b), and n-Bu (1c)) were prepared in good yields. The high-resolution mass spectroscopy showed that and 1c in the gas phase are monomeric species. However, the solid state of 1a revealed a dimeric structure. Asymmetric additions of 1a-1c to aldehydes catalyzed by a titanium catalyst of (R)-H8 -BINOL were studied at room temperature. The reactions produced desired secondary alcohols in good yields with good to excellent enantioselectivities of up to 94% ee. Reactivity and enantioselectivity differences, in terms of steric bulkiness of the R nucleophiles, are herein described. The addition reactions of secondary c-hexyl to aldehydes were slower than the reactions of primary i-butyl or n-butyl nucleophiles. For the primary alkyls, lower enantioselectivities were obtained for products from addition reactions of the linear n-butyl as compared with the enantioselectivities of products from the addition reactions of the branched i-butyl group. The same stereochemistry of RTi(O-i-Pr)3 addition reactions as the addition reactions of organozinc, organoaluminum, Grignard, or organolithium reagents directly supports the argument of that titanium-catalyzed addition reactions of aldehydes involve an addition of an organotitanium nucleophile.